In vitro development rate of preimplantation rabbit embryos cultured with different levels of melatonin.

PubMed

Mehaisen, Gamal Mohamed Kamel; Saeed, Ayman Moustafa

2015-02-01

This study aimed to investigate the effect of melatonin supplementation at different levels in culture medium on embryo development in rabbits. Embryos of 2-4 cells, 8-16 cells and morula stages were recovered from nulliparous Red Baladi rabbit does by laparotomy technique 24, 48 and 72 h post-insemination, respectively. Normal embryos from each stage were cultured to hatched blastocyst stages in either control culture medium (TCM-199 + 20% fetal bovine serum) or control supplemented with melatonin at 10(-3) M, 10(-6) M or 10(-9) M. No effect of melatonin was found on development of embryos recovered at 24 h post-insemination. The high level of melatonin at 10(-3) M adversely affected the in vitro development rates of embryos recovered at 48 h post-insemination (52 versus 86, 87 and 80% blastocyst rate; 28 versus 66, 78 and 59% hatchability rate for 10(-3) M versus 10(-9) M, 10(-6) M and control, respectively, P< 0.05). At the morula stage, melatonin at 10-3 M significantly increased the in vitro development of embryos (92% for 10(-3) M versus 76% for control, P < 0.05), while the hatchability rate of these embryos was not improved by melatonin (16-30% versus 52% for melatonin groups versus control, P < 0.05). Results show that a moderate level of melatonin (10(-6) M) may improve the development and hatchability rates of preimplantation rabbit embryos. The addition of melatonin at a 10-3 M concentration enhances the development of rabbit morulae but may negatively affect the development of earlier embryos. More studies are needed to optimize the use of melatonin in in vitro embryo culture in rabbits.

Genetic mouse embryo assay: improving performance and quality testing for assisted reproductive technology (ART) with a functional bioassay.

PubMed

Gilbert, Rebecca S; Nunez, Brandy; Sakurai, Kumi; Fielder, Thomas; Ni, Hsiao-Tzu

2016-03-24

Growing concerns about safety of ART on human gametes, embryos, clinical outcomes and long-term health of offspring require improved methods of risk assessment to provide functionally relevant assays for quality control testing and pre-clinical studies prior to clinical implementation. The one-cell mouse embryo assay (MEA) is the most widely used for development and quality testing of human ART products; however, concerns exist due to the insensitivity/variability of this bioassay which lacks standardization and involves subjective analysis by morphology alone rather than functional analysis of the developing embryos. We hypothesized that improvements to MEA by the use of functional molecular biomarkers could enhance sensitivity and improve detection of suboptimal materials/conditions. Fresh one-cell transgenic mouse embryos with green fluorescent protein (GFP) expression driven by Pou6f1 or Cdx2 control elements were harvested and cultured to blastocysts in varied test and control conditions to compare assessment by standard morphology alone versus the added dynamic expression of GFP for screening and selection of critical raw materials and detection of suboptimal culture conditions. Transgenic mouse embryos expressing functionally relevant biomarkers of normal early embryo development can be used to monitor the developmental impact of culture conditions. This novel approach provides a superior MEA that is more meaningful and sensitive for detection of embryotoxicity than morphological assessment alone.

Latrunculin A treatment prevents abnormal chromosome segregation for successful development of cloned embryos.

PubMed

Terashita, Yukari; Yamagata, Kazuo; Tokoro, Mikiko; Itoi, Fumiaki; Wakayama, Sayaka; Li, Chong; Sato, Eimei; Tanemura, Kentaro; Wakayama, Teruhiko

2013-01-01

Somatic cell nuclear transfer to an enucleated oocyte is used for reprogramming somatic cells with the aim of achieving totipotency, but most cloned embryos die in the uterus after transfer. While modifying epigenetic states of cloned embryos can improve their development, the production rate of cloned embryos can also be enhanced by changing other factors. It has already been shown that abnormal chromosome segregation (ACS) is a major cause of the developmental failure of cloned embryos and that Latrunculin A (LatA), an actin polymerization inhibitor, improves F-actin formation and birth rate of cloned embryos. Since F-actin is important for chromosome congression in embryos, here we examined the relation between ACS and F-actin in cloned embryos. Using LatA treatment, the occurrence of ACS decreased significantly whereas cloned embryo-specific epigenetic abnormalities such as dimethylation of histone H3 at lysine 9 (H3K9me2) could not be corrected. In contrast, when H3K9me2 was normalized using the G9a histone methyltransferase inhibitor BIX-01294, the Magea2 gene-essential for normal development but never before expressed in cloned embryos-was expressed. However, this did not increase the cloning success rate. Thus, non-epigenetic factors also play an important role in determining the efficiency of mouse cloning.

An improved embryo-rescue protocol for hybrid progeny from seedless Vitis vinifera grapes × wild Chinese Vitis species.

PubMed

Li, Gui Rong; Ji, Wei; Wang, Gang; Zhang, Jian Xia; Wang, Yue Jin

A highly efficient technique of embryo rescue is critical when using stenospermocarpic Vitis vinifera cultivars (female parents) to breed novel, disease-resistant, seedless grape cultivars by hybridizing with wild Chinese Vitis species (male parents) having many disease-resistance alleles. The effects of various factors on the improvement of embryo formation, germination, and plantlet development for seven hybrid combinations were studied. The results indicated that Beichun and Shuangyou were the best male parents. The best sampling time for ovule inoculation differed among the female parents. When hybrid ovules were cultured on a double-phase medium with five different solid medium types, percent embryo formation was highest (11.3-28.3%) on a modified MM3 medium. Percentages of embryo germination (15.4-55.4%) and plantlet development (11.15-44.6%) were all highest when embryos were cultured on Woody Plant Medium + 5.7 μM indole-3-acetic acid + 4.4 μM 6-benzylaminopurine + 1.4 μM gibberellic acid + 2% sucrose + 0.05% casein hydrolysate + 0.3% activated charcoal + 0.7% agar. In the absence of other amino acids, the addition of proline significantly increased embryo formation (36.1%), embryo germination (64.6%), and plantlet development (90.5%). A highly efficient protocol has been developed for hybrid embryo rescue from seedless V. vinifera grapes × wild Chinese Vitis species that results in a significant improvement in breeding efficiency for new disease-resistant seedless grapes.

Enhance beef cattle improvement by embryo biotechnologies.

PubMed

Wu, B; Zan, L

2012-10-01

Embryo biotechnology has become one of the prominent high businesses worldwide. This technology has evolved through three major changes, that is, traditional embryo transfer (in vivo embryo production by donor superovulation), in vitro embryo production by ovum pick up with in vitro fertilization and notably current cloning technique by somatic cell nuclear transfer and transgenic animal production. Embryo biotechnology has widely been used in dairy and beef cattle industry and commercial bovine embryo transfer has become a large international business. Currently, many developed biotechnologies during the period from early oocyte stage to pre-implantation embryos can be used to create new animal breeds and accelerate genetic progression. Based on recent advances in embryo biotechnologies and authors current studies, this review will focus on a description of the application of this technology to beef cattle improvement and discuss how to use this technology to accelerate beef cattle breeding and production. The main topics of this presentation include the following: (i) how to increase calf production numbers from gametes including sperm and oocyte; (ii) multiple ovulation and embryo transfer breeding schemes; (iii) in vitro fertilization and intracytoplasm sperm injection in bovine; (iv) pronuclear development and transgenic animals; (v) sex selection from sperm and embryos; (vi) cloning and androgenesis; (vii) blastocyst development and embryonic stem cells; (viii) preservation of beef cattle genetic resources; and (ix) conclusions. © 2011 Blackwell Verlag GmbH.

In vitro development of cloned bovine embryos produced by handmade cloning using somatic cells from distinct levels of cell culture confluence.

PubMed

Gerger, R P C; Ribeiro, E S; Forell, F; Bertolini, L R; Rodrigues, J L; Ambrósio, C E; Miglino, M A; Mezzalira, A; Bertolini, M

2010-02-18

The relationship between the level of cell confluence near the plateau phase of growth and blastocyst yield following somatic cell cloning is not well understood. We examined the effect of distinct cell culture confluence levels on in vitro development of cloned bovine embryos. In vitro-matured bovine oocytes were manually bisected and selected by DNA staining. One or two enucleated hemi-cytoplasts were paired and fused with an adult skin somatic cell. Cultured skin cells from an adult Nellore cow harvested at three distinct culture confluence levels (70-80, 80-90, and >95%) were used for construction of embryos and hemi-embryos. After activation, structures were cultured in vitro as one embryo (1 x 100%) or as aggregates of two hemi-embryos (2 x 50%) per microwell. Fusion, cleavage and blastocyst rates were compared using the chi(2) test. The fusion rate for hemi-embryos (51.4%) was lower than for embryos (67.6%), with no influence of degree of cell confluence. However, blastocyst rates improved linearly (7.0, 17.5, and 29.4%) with increases in cell confluence. We conclude that degree of cell culture confluence significantly influences subsequent embryo development; use of a cell population in high confluence (>90%) for nuclear transfer significantly improved blastocyst yield after cloning.

In vitro production of small ruminant embryos: late improvements and further research.

PubMed

de Souza-Fabjan, Joanna Maria Gonçalves; Panneau, Barbara; Duffard, Nicolas; Locatelli, Yann; de Figueiredo, José Ricardo; Freitas, Vicente José de Figueirêdo; Mermillod, Pascal

2014-06-01

Beyond the potential use of in vitro production of embryos (IVP) in breeding schemes, embryos are also required for the establishment of new biotechnologies such as cloning and transgenesis. Additionally, the knowledge of oocyte and embryo physiology acquired through IVP techniques may stimulate the further development of other techniques such as marker assisted and genomic selection of preimplantation embryos, and also benefit assisted procreation in human beings. Efficient in vitro embryo production is currently a major objective for livestock industries, including small ruminants. The heterogeneity of oocytes collected from growing follicles by laparoscopic ovum pick up or in ovaries of slaughtered females, remains an enormous challenge for IVM success, and still limits the rate of embryo development. In addition, the lower quality of the IVP embryos, compared with their in vivo-derived counterparts, translates into poor cryosurvival, which restricts the wider use of this promising technology. Therefore, many studies have been reported in an attempt to determine the most suitable conditions for IVM, IVF, and in vitro development to maximize embryo production rate and quality. This review aims to present the current panorama of IVP production in small ruminants, describing important steps for its success, reporting the recent advances and also the main obstacles identified for its improvement and dissemination. Copyright © 2014 Elsevier Inc. All rights reserved.

[Embryos and embryo-like entities: problem of definition in the draft of the Swiss embryonic research law].

PubMed

Bürgin, M T; Bürkli, P

2002-11-01

At the end of May 2002, the draft of the Swiss "Federal Act on Research on Surplus Embryos and Embryonic Stem Cells" (EFG, Embryonic Research Act) reached the pre-legislative consultation stage. Under certain conditions, it would allow research on "surplus" embryos from in-vitro fertilization, and the derivation of embryonic stem cells from surplus embryos for research purposes. The EFG draft defines an embryo as "the developing organism from the point of nuclear fusion until the completion of organ development". New technological developments show that embryo-like entities can also be created without nuclear fusion having taken place. It remains unclear how to treat embryonic entities that don't fall under the draft's narrow definition of an embryo. Expanding this definition would be a welcome improvement.

A novel embryo culture media supplement that improves pregnancy rates in mice.

PubMed

Highet, A R; Bianco-Miotto, T; Pringle, K G; Peura, A; Bent, S; Zhang, J; Nottle, M B; Thompson, J G; Roberts, C T

2017-03-01

The preimplantation embryo in vivo is exposed to numerous growth factors in the female reproductive tract, which are not recapitulated in embryo culture media in vitro The IGF2 and plasminogen activator systems facilitate blastocyst development. We hypothesized that the addition of IGF2 in combination with urokinase plasminogen activator (uPA) and plasminogen could improve rates of blastocyst hatching and implantation in mice. B6BcF1 and CBAB6F2 mouse embryos were divided into one of four supplemented culture media treatment groups: (1) control (media only); (2) 12.5 nM IGF2; (3) 10 µg/mL uPA and 5 µg/mL plasminogen; or (4) a combination of IGF2, uPA and plasminogen treatments. Embryo development to blastocyst stage and hatching were assessed before transfer to pseudopregnant recipient females and implantation, pregnancy rates and postnatal growth were assessed. After 90.5 h of culture, IGF2 + U + P treatment increased the percentage of B6BcF1 embryos that were hatching/hatched and percentage developing to blastocyst stage compared with controls (P < 0.02). Following B6BcF1 embryo transfer, IGF2 + U + P treatment increased implantation sites at day 8 of pregnancy compared with controls (P < 0.05). Replication in the CBAB6F2 mouse strain showed significant improvements in pregnancy rates at days 8 and 18 but not in blastocyst development. No adverse effects were seen on gestational age, litter size or birthweight, or the reproductive capacity of offspring of IGF2 + U + P treated embryos. For embryos susceptible to detrimental effects of in vitro culture, IGF2, uPA and plasminogen supplementation of culture media can improve pregnancy success, but the effect of treatment is dependent on the mouse strain. © 2017 Society for Reproduction and Fertility.

NanoSMGT: transgene transmission into bovine embryos using halloysite clay nanotubes or nanopolymer to improve transfection efficiency.

PubMed

Campos, Vinicius Farias; de Leon, Priscila Marques Moura; Komninou, Eliza Rossi; Dellagostin, Odir Antônio; Deschamps, João Carlos; Seixas, Fabiana Kömmling; Collares, Tiago

2011-11-01

The objectives were to investigate whether: 1) nanotransfectants are more effective than other common transfection methods for SMGT; 2) NanoSMGT is able to transmit exogenous DNA molecules to bovine embryos; and 3) halloysite clay nanotubes (HCNs) can be used as a transfection reagent to improve transgene transmission. Four transfection systems were used: naked DNA (without transfectant), lipofection, nanopolymer, and halloysite clay nanotubes. Plasmid uptake by sperm and its transfer to embryos were quantified by conventional and real-time PCR, as well as EGFP expression by fluorescence microscopy. Furthermore, sperm motility and viability, and embryo development were investigated. Mean number of plasmids taken up was affected (P < 0.05) by transfection procedure, with the nanopolymer being the most effective transfectant (∼ 153 plasmids per spermatozoon). None of the treatments affected sperm motility or viability. The mean number of plasmids transmitted to four-cell stage embryos was higher (P < 0.05) in nanopolymer and HCNs than liposomes and naked DNA groups. The number of embryos carrying the transgene increased from 8-10% using naked DNA or liposomes to 40-45% using nanopolymer or HCN as transfectants (P < 0.05). There were no significant differences among transfection procedures regarding blastocyst formation rate of resulting embryos. However, no EGFP-expressing embryo was identified in any treatment. Therefore, nanotransfectants improved transgene transmission in bovine embryos without deleterious effects on embryo development. To our knowledge, this was the first time that bovine embryos carrying a transgene were produced by NanoSMGT. Copyright © 2011 Elsevier Inc. All rights reserved.

The effect of flurbiprofen on the development of anencephaly in early stage chicken embryos.

PubMed

Özeren, Ersin; Er, Uygur; Güvenç, Yahya; Demirci, Adnan; Arıkök, Ata Türker; Şenveli, Engin; Ergün, Rüçhan Behzat

2015-04-01

The study investigated the effect of flurbiprofen on the development of anencephaly in early stage chicken embryos. We looked at four groups with a total of 36 embryos. There was a control group, a normal saline group, a normal-dose group and a high-dose group with ten, ten, eight and eight eggs with embryo respectively. Two embryos in the control group, studied with light microscopy at 48 h, were consistent with 28-29 hours' incubation in the Hamburger-Hamilton System. They had open neural tubes. The other embryos in this group were considered normal. One embryo in the normal saline group was on the occlusion stage at 48 h. One embryo showed an open neural tube. They were compatible with 28-29 hours' incubation in the Hamburger-Hamilton system. The remaining eight embryos showed normal development. In the normal dose group, one embryo showed underdevelopment of the embryonic disc and the embryo was dead. In four embryos, the neural tubes were open. One cranial malformation was found that was complicated with anencephaly in one embryo. In two embryos the neural tubes were closed, as they showed normal development, and they reached their expected stages according to the Hamburger-Hamilton classification. There was no malformation or growth retardation. Four experimental embryos were anencephalic in the high dose group, and three embryos had open neural tubes. One embryo exhibited both anencephaly and a neural tube closure defect. None of the embryos in this group showed normal development. Even the usual therapeutic doses of flurbiprofen increased the risk of neural tube defect. Flurbiprofen was found to significantly increase the risk of anencephaly. The provision of improved technical materials and studies with larger sample sizes will reveal the stage of morphological disruption during the development of embryos.

A temporary immersion system improves in vitro regeneration of peach palm through secondary somatic embryogenesis

PubMed Central

Steinmacher, D. A.; Guerra, M. P.; Saare-Surminski, K.; Lieberei, R.

2011-01-01

Background and Aims Secondary somatic embryogenesis has been postulated to occur during induction of peach palm somatic embryogenesis. In the present study this morphogenetic pathway is described and a protocol for the establishment of cycling cultures using a temporary immersion system (TIS) is presented. Methods Zygotic embryos were used as explants, and induction of somatic embryogenesis and plantlet growth were compared in TIS and solid culture medium. Light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to describe in vitro morphogenesis and accompany morpho-histological alterations during culture. Key Results The development of secondary somatic embryos occurs early during the induction of primary somatic embryos. Secondary somatic embryos were observed to develop continually in culture, resulting in non-synchronized development of these somatic embryos. Using these somatic embryos as explants allowed development of cycling cultures. Somatic embryos had high embryogenic potential (65·8 ± 3·0 to 86·2 ± 5·0 %) over the period tested. The use of a TIS greatly improved the number of somatic embryos obtained, as well as subsequent plantlet growth. Histological analyses showed that starch accumulation precedes the development of somatic embryos, and that these cells presented high nucleus/cytoplasm ratios and high mitotic indices, as evidenced by DAPI staining. Morphological and SEM observations revealed clusters of somatic embryos on one part of the explants, while other parts grew further, resulting in callus tissue. A multicellular origin of the secondary somatic embryos is hypothesized. Cells in the vicinity of callus accumulated large amounts of phenolic substances in their vacuoles. TEM revealed that these cells are metabolically very active, with the presence of numerous mitochondria and Golgi apparatuses. Light microscopy and TEM of the embryogenic sector revealed cells with numerous amyloplasts, large nuclei and nucleoli, and numerous plasmodesmata. Plantlets were obtained and after 3 months in culture their growth was significantly better in TIS than on solid culture medium. However, during acclimatization the survival rate of TIS-grown plantlets was lower. Conclusions The present study confirms the occurrence of secondary somatic embryos in peach palm and describes a feasible protocol for regeneration of peach palm in vitro. Further optimizations include the use of explants obtained from adult palms and improvement of somatic embryo conversion rates. PMID:21355009

Influence of "Solcoseryl" during culture on the sex-dependent repair of bovine demi-embryos.

PubMed

Tominaga, K; Yoneda, K; Utsumi, K

1996-03-01

The purpose of this experiment was to determine the effect of culture conditions on the development of split embryos after bisection and on the sex ratio of resultant bovine demi-embryos. Embryos that had developed into blastocysts on days 6 1/2 to 7 or on days 7 1/2 to 8 from oocytes matured and fertilized in vitro were bisected in BMOC-3 medium supplemented with 33% calf serum. The medium also contained 0%, 0.1% or 1.0% Solcoseryl, a deproteinized hemodialysate product from calf blood. The demi-embryos were first cultured for 4 hours in the same medium in which they had been bisected and then co-cultured with cumulus cells in TCM199 supplemented with 1% calf serum for an additional 20 hr. The rate of production of good to excellent quality demi-embryos obtained from days 6 1/2 to 7 blastocysts was higher than from those on days 7 1/2 to 8. The rate was also significantly improved when blastocysts were bisected in medium containing 0.1% or 1.0% Solcoseryl, compared to the medium without Solcoseryl. Male embryos seemed to recover more rapidly than female embryos, as assessed by morphological quality at 4 hr, although the quality of female embryos had improved by 24 hr. The percentage of males after culture was higher in the medium without Solcoseryl than in its presence. Thus, addition of Solcoseryl at either 0.1% or 1.0% to BMOC-3 medium seemed to improve the production efficiency of good quality demi-embryos, but did not influence the sex ratio. It appears as if female demi-embryos required more time than male embryos to be repaired after bisection.

Developmental competence of equine oocytes and embryos obtained by in vitro procedures ranging from in vitro maturation and ICSI to embryo culture, cryopreservation and somatic cell nuclear transfer.

PubMed

Galli, C; Colleoni, S; Duchi, R; Lagutina, I; Lazzari, G

2007-03-01

Development of assisted reproductive technologies in horses has been relatively slow compared to other domestic species, namely ruminants and pigs. The scarce availability of abattoir ovaries and the lack of interest from horse breeders and breed associations have been the main reasons for this delay. Progressively though, the technology of oocyte maturation in vitro has been established followed by the application of ICSI to achieve fertilization in vitro. Embryo culture was initially performed in vivo, in the mare oviduct or in the surrogate sheep oviduct, to achieve the highest embryo development, in the range of 18-36% of the fertilised oocytes. Subsequently, the parallel improvement of in vitro oocyte maturation conditions and embryo culture media has permitted high rates of embryo development from in vitro matured and in vitro cultured ICSI embryos, ranging from 5 to 10% in the early studies to up to 38% in the latest ones. From 2003, with the birth of the first cloned equids, the technology of somatic cell nuclear transfer has also become established due to improvement of the basic steps of embryo production in vitro, including cryopreservation. Pregnancy and foaling rates are still estimated based on a small number of in vitro produced equine embryos transferred to recipients. The largest set of data on non-surgical embryo transfer of in vitro produced embryos, from ICSI of both abattoir and in vitro-matured Ovum Pick Up (OPU) oocytes, and from somatic cell nuclear transfer, has been obtained in our laboratory. The data demonstrate that equine embryos produced by OPU and then cryopreserved can achieve up to 69% pregnancy rate with a foaling rate of 83%. These percentages are reduced to 11 and 23%, respectively, for cloned embryos. In conclusion, extensive evidence exists that in vitro matured equine oocytes can efficiently develop into viable embryos and offspring.

Rabbit somatic cell cloning: effects of donor cell type, histone acetylation status and chimeric embryo complementation.

PubMed

Yang, Feikun; Hao, Ru; Kessler, Barbara; Brem, Gottfried; Wolf, Eckhard; Zakhartchenko, Valeri

2007-01-01

The epigenetic status of a donor nucleus has an important effect on the developmental potential of embryos produced by somatic cell nuclear transfer (SCNT). In this study, we transferred cultured rabbit cumulus cells (RCC) and fetal fibroblasts (RFF) from genetically marked rabbits (Alicia/Basilea) into metaphase II oocytes and analyzed the levels of histone H3-lysine 9-lysine 14 acetylation (acH3K9/14) in donor cells and cloned embryos. We also assessed the correlation between the histone acetylation status of donor cells and cloned embryos and their developmental potential. To test whether alteration of the histone acetylation status affects development of cloned embryos, we treated donor cells with sodium butyrate (NaBu), a histone deacetylase inhibitor. Further, we tried to improve cloning efficiency by chimeric complementation of cloned embryos with blastomeres from in vivo fertilized or parthenogenetic embryos. The levels of acH3K9/14 were higher in RCCs than in RFFs (P<0.05). Although the type of donor cells did not affect development to blastocyst, after transfer into recipients, RCC cloned embryos induced a higher initial pregnancy rate as compared to RFF cloned embryos (40 vs 20%). However, almost all pregnancies with either type of cloned embryos were lost by the middle of gestation and only one fully developed, live RCC-derived rabbit was obtained. Treatment of RFFs with NaBu significantly increased the level of acH3K9/14 and the proportion of nuclear transfer embryos developing to blastocyst (49 vs 33% with non-treated RFF, P<0.05). The distribution of acH3K9/14 in either group of cloned embryos did not resemble that in in vivo fertilized embryos suggesting that reprogramming of this epigenetic mark is aberrant in cloned rabbit embryos and cannot be corrected by treatment of donor cells with NaBu. Aggregation of embryos cloned from NaBu-treated RFFs with blastomeres from in vivo derived embryos improved development to blastocyst, but no cloned offspring were obtained. Two live cloned rabbits were produced from this donor cell type only after aggregation of cloned embryos with a parthenogenetic blastomere. Our study demonstrates that the levels of histone acetylation in donor cells and cloned embryos correlate with their developmental potential and may be a useful epigenetic mark to predict efficiency of SCNT in rabbits.

Latrunculin A Treatment Prevents Abnormal Chromosome Segregation for Successful Development of Cloned Embryos

PubMed Central

Terashita, Yukari; Yamagata, Kazuo; Tokoro, Mikiko; Itoi, Fumiaki; Wakayama, Sayaka; Li, Chong; Sato, Eimei; Tanemura, Kentaro; Wakayama, Teruhiko

2013-01-01

Somatic cell nuclear transfer to an enucleated oocyte is used for reprogramming somatic cells with the aim of achieving totipotency, but most cloned embryos die in the uterus after transfer. While modifying epigenetic states of cloned embryos can improve their development, the production rate of cloned embryos can also be enhanced by changing other factors. It has already been shown that abnormal chromosome segregation (ACS) is a major cause of the developmental failure of cloned embryos and that Latrunculin A (LatA), an actin polymerization inhibitor, improves F-actin formation and birth rate of cloned embryos. Since F-actin is important for chromosome congression in embryos, here we examined the relation between ACS and F-actin in cloned embryos. Using LatA treatment, the occurrence of ACS decreased significantly whereas cloned embryo-specific epigenetic abnormalities such as dimethylation of histone H3 at lysine 9 (H3K9me2) could not be corrected. In contrast, when H3K9me2 was normalized using the G9a histone methyltransferase inhibitor BIX-01294, the Magea2 gene—essential for normal development but never before expressed in cloned embryos—was expressed. However, this did not increase the cloning success rate. Thus, non-epigenetic factors also play an important role in determining the efficiency of mouse cloning. PMID:24205216

Glutathione and abscisic acid supplementation influences somatic embryo maturation and hormone endogenous levels during somatic embryogenesis in Podocarpus lambertii Klotzsch ex Endl.

PubMed

Fraga, Hugo Pacheco de Freitas; Vieira, Leila do Nascimento; Puttkammer, Catarina Corrêa; Dos Santos, Henrique Pessoa; Garighan, Julio de Andrade; Guerra, Miguel Pedro

2016-12-01

Here we propose a protocol for embryogenic cultures induction, proliferation and maturation for the Brazilian conifer Podocarpus lambertii, and investigated the effect of abscisic acid (ABA) and glutathione (GSH) supplementation on the maturation phase. ABA, zeatin (Z) and salicylic acid (SA) endogenous levels were quantified. Number of somatic embryos obtained in ABA-supplemented treatment was significant higher than in ABA-free treatment, showing the relevance of ABA supplementation during somatic embryos maturation. Histological analysis showed the stereotyped sequence of developmental stages in conifer somatic embryos, reaching the late torpedo-staged embryo. GSH supplementation in maturation culture medium improved the somatic embryos number and morphological features. GSH 0mM and GSH 0.1mM treatments correlated with a decreased ABA endogenous level during maturation, while GSH 0.5mM treatment showed constant levels. All treatments resulted in decreased Z endogenous levels, supporting the concept that cytokinins are important during the initial cell division but not for the later stages of embryo development. The lowest SA levels found in GSH 0.5mM treatment were coincident with early embryonic development, and this treatment resulted in the highest development of somatic embryos. Thus, a correlation between lower SA levels and improved somatic embryo formation can be hypothesized. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Efficient Term Development of Vitrified Ferret Embryos Using a Novel Pipette Chamber Technique1

PubMed Central

Sun, Xingshen; Li, Ziyi; Yi, Yaling; Chen, Juan; Leno, Gregory H.; Engelhardt, John F.

2008-01-01

Development of an efficient cryopreservation technique for the domestic ferret is key for the long-term maintenance of valuable genetic specimens of this species and for the conservation of related endangered species. Unfortunately, current cryopreservation procedures, such as slow-rate freezing and vitrification with open pulled straws, are inefficient. In this report, we describe a pipette tip-based vitrification method that significantly improves the development of thawed ferret embryos following embryo transfer (ET). Ferret embryos at the morula (MR), compact morula (CM), and early blastocyst (EB) stages were vitrified using an Eppendorf microloader pipette tip as the chamber vessel. The rate of in vitro development was significantly (P < 0.05) higher among embryos vitrified at the CM (93.6%) and EB (100%) stages relative to those vitrified at the MR stages (58.7%). No significant developmental differences were observed when comparing CM and EB vitrified embryos with nonvitrified control CM (100%) and EB (100%) embryos. In addition, few differences in the ultrastructure of intracellular lipid droplets or in microfilament structure were observed between control embryos and embryos vitrified at any developmental stage. Vitrified-thawed CM/EB embryos cultured for 2 or 16 h before ET resulted in live birth rates of 71.3% and 77.4%, respectively. These rates were not significantly different from the control live birth rate (79.2%). However, culture for 32 h (25%) or 48 h (7.8%) after vitrification significantly reduced the rate of live births. These data indicate that the pipette chamber vitrification technique significantly improves the live birth rate of transferred ferret embryos relative to current state-of-the-art methods.. PMID:18633142

Transfer of bovine demi-embryos with and without the zona pellucida.

PubMed

Warfield, S J; Seidel, G E; Elsden, R P

1987-09-01

Bisected bovine embryos with or without the zona pellucida were transferred to recipients nonsurgically in five field trials. Embryos were collected from superovulated donors 6.5 to 7.5 d after estrus; only embryos of good and excellent quality were bisected. Demi-embryos were transferred either within a zona pellucida, without a zona pellucida, without a zona pellucida, or in the third and fourth trials, without a zona but embedded in 7% gelatin. Pregnancies were diagnosed at 44 to 68 d of gestation. In a preliminary trial, 9/29 zona pellucida-intact demi-embryos developed into fetuses compared with 1/10 zona pellucida-free demi-embryos (P greater than .1). The proportion of zona-free demi-embryos developing to fetuses was not significantly different from the zona-intact group in the second trial either, 24/49 and 5/19, respectively. In trial 3, the proportion of zona pellucida-free demi-embryos developing was 8/25; of zona-enclosed embryos, 29/88; and of zona-free demi-embryos embedded in gelatin, 8/22 (P greater than .1). Similarly, in the fourth trial the rate of development of zona-free demi-embryos to fetuses was 5/12, that of zona-enclosed embryos was 32/81, and that of zona-free demi-embryos embedded in gelatin was 3/12 (P greater than .1). In trial 5, survival of zona-enclosed demi-embryos to fetuses was 40/105, and of zona-free demi-embryos, 46/109 (P greater than .1). Except for trial 2, half of the demi-embryos were twinned, one to each uterine horn; twinning did not significantly affect the proportion developing to fetuses for any of the demi-embryo groups. It is concluded that placing post-compaction demi-embryos into the zona pellucida for transfer does not improve pregnancy rates significantly.

The frequency of clinical pregnancy and implantation rate after cultivation of embryos in a medium with granulocyte macrophage colony-stimulating factor (GM-CSF) in patients with preceding failed attempts of ART.

PubMed

Tevkin, S; Lokshin, V; Shishimorova, M; Polumiskov, V

2014-10-01

The application in IVF practice of modern techniques can improve positive outcome of each cycle in the assisted reproductive technology (ART) programs and the effectiveness of treatment as a whole. There are embryos in the female reproductive tract in physiological medium which contain various cytokines and growth factors. It plays an important role in the regulation of normal embryonic development, improve implantation and subsequently optimizing the development of the fetus and the placenta. Granulocyte macrophage colony-stimulating factor (GM-CSF is one of the cytokines playing an important role in reproductive function. Addition of recombinant GM-CSF to the culture medium can makes closer human embryos culture to in vivo conditions and improve the efficacy ART cycles. The analysis of culture embryos in EmbryoGen medium has shown that fertilization rate embryo culture and transfer to patients with previous unsuccessful attempts increases clinical pregnancy rate compared to the control group 39.1 versus 27.8%, respectively. It is noted that the implantation rate (on 7 weeks' gestation) and progressive clinical pregnancy rate (on 12 weeks' gestation) were significantly higher in group embryos culture in EmbryoGen medium compared to standard combination of medium (ISM1+VA), and were 20.4 and 17.4% versus 11.6 and 9.1%, respectively.

Cosmetic micromanipulation of vitrified-warmed cleavage stage embryos does not improve ART outcomes: An ultrastructural study of fragments.

PubMed

Safari, Somayyeh; Khalili, Mohammad Ali; Barekati, Zeinab; Halvaei, Iman; Anvari, Morteza; Nottola, Stefania A

2017-09-01

The aim was to study the ultrastructure of cytoplasmic fragments along with the effect of cosmetic micromanipulation (CM) on the morphology and development of vitrified-warmed embryos as well as assisted reproductive technology (ART) outcomes. A total of 96 frozen embryo transfer (FET) cycles were included in this prospective randomized study. They were divided into three groups of CM (n=32), sham (n=32) and control (n=32). In the CM group, the vitrified- warmed embryos were subjected to fragments and coarse granules removal (cosmetic micromanipulation) after laser assisted zona hatching (LAH); sham group subjected only to LAH and no intervention was taken for the control group. Fragmented embryo was evaluated by transmission electron microscopy (TEM). Significant improvement was observed in the morphological parameters, such as fragmentation degrees, evenness of the blastomeres and embryo grade during the subsequent development, after applying cosmetic micromanipulation, when compared to sham or control groups (P=0.00001). However, there were no differences in the clinical outcomes amongst the three studied groups e.g. the rates of clinical, ongoing and multiple pregnancies, implantation, delivery and live birth. In fine structure view, fragments exhibited uniform cytoplasmic texture containing majority of organelles that were observed in normal blastomeres including mitochondria. In conclusion, application of cosmetic micromanipulation in low-grade vitrified-warmed embryos showed significant improvement on embryo morphology parameters; however, did not result in noticeable improvements in clinical outcomes of the patients undergoing ART program. In addition, embryo vitrification had no adverse effects on fine structure of the fragments. Copyright © 2017 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

The development of preimplantation mouse parthenogenones in vitro in absence of glucose: influence of the maternally inherited components.

PubMed

Mognetti, B; Leppens, G; Sakkas, D

1996-04-01

Mouse preimplantation embryo development is characterized by a switch from a dependence on the tricarboxylic acid cycle pre-compaction to a metabolism based on glycolysis post-compaction. In-view of this, the role of glucose in embryo culture medium has come under increased analysis and has lead to improved development of outbred mouse embryos in glucose free medium. Another type of embryo that has proven difficult to culture is the parthenogenetic (PN) mouse embryo. With this in mind we have investigated the effect of glucose deprivation on PN embryo development in vitro. Haploid and diploid PN embryos were grown in medium M16 with or without glucose (M16-G) and development, glycolytic rate, and methionine incorporation rates assessed. Haploid PN and normal embryo development to the blastocyst stage did not differ in either M16 or M16-G. In contrast, although diploid PN embryos formed blastocysts in M16 (28.3%), they had difficulty in undergoing the morula/blastocyst transition in M16-G (7.6%). There was no significant difference in mean cell numbers of haploid PN, diploid PN and normal embryos cultured in M16 and M16-G at the morula and blastocyst stage. Transfer of diploid PN embryos from M16-G to M16 at the four- to eight-cell stage dramatically increased blastocyst development. At the morula stage diploid PN embryos grown in M16-G exhibited a higher glucose metabolism and protein synthesis compared to those grown in M16 and to haploid PN embryos. Difficulties of diploid PN embryos in undergoing the morula/blastocyst transition in absence of glucose infer the existence of a link between the maternally inherited components and the preimplantation embryos dependence on glucose.

Trichostatin A specifically improves the aberrant expression of transcription factor genes in embryos produced by somatic cell nuclear transfer

PubMed Central

Inoue, Kimiko; Oikawa, Mami; Kamimura, Satoshi; Ogonuki, Narumi; Nakamura, Toshinobu; Nakano, Toru; Abe, Kuniya; Ogura, Atsuo

2015-01-01

Although mammalian cloning by somatic cell nuclear transfer (SCNT) has been established in various species, the low developmental efficiency has hampered its practical applications. Treatment of SCNT-derived embryos with histone deacetylase (HDAC) inhibitors can improve their development, but the underlying mechanism is still unclear. To address this question, we analysed gene expression profiles of SCNT-derived 2-cell mouse embryos treated with trichostatin A (TSA), a potent HDAC inhibitor that is best used for mouse cloning. Unexpectedly, TSA had no effect on the numbers of aberrantly expressed genes or the overall gene expression pattern in the embryos. However, in-depth investigation by gene ontology and functional analyses revealed that TSA treatment specifically improved the expression of a small subset of genes encoding transcription factors and their regulatory factors, suggesting their positive involvement in de novo RNA synthesis. Indeed, introduction of one of such transcription factors, Spi-C, into the embryos at least partially mimicked the TSA-induced improvement in embryonic development by activating gene networks associated with transcriptional regulation. Thus, the effects of TSA treatment on embryonic gene expression did not seem to be stochastic, but more specific than expected, targeting genes that direct development and trigger zygotic genome activation at the 2-cell stage. PMID:25974394

Effects of interval between fusion and activation, cytochalasin B treatment, and number of transferred embryos, on cloning efficiency in goats.

PubMed

Liu, J; Li, L L; Du, S; Bai, X Y; Zhang, H D; Tang, S; Zhao, M T; Ma, B H; Quan, F S; Zhao, X E; Zhang, Y

2011-10-01

To improve the efficiency of somatic cell nuclear transfer (SCNT) in goats, we evaluated the effects of the interval between fusion and activation (1 to 5 h), cytochalasin B (CB) treatment after electrofusion, and the number of transferred embryos on the in vivo and in vitro development of cloned caprine embryos. The majority of the reconstructed embryos had condensed chromosomes and metaphase-like chromosomes at 2 and 3 h after fusion; cleavage and blastocyst rates from those two groups were higher (P < 0.05) than those of embryos activated 1, 4, or 5 h after fusion. Treatment with CB between fusion and activation improved in vitro and in vivo development of nuclear transfer (NT) goat embryos by reducing the fragmentation rate (P < 0.05). Although there were no significant differences in NT efficiency, pregnancy rate and kids born per recipient were increased by transfer of 20 or 30 embryos per recipient compared with 10 embryos. We concluded that CB treatment for 2 to 3 h between fusion and activation was an efficient method for generating cloned goats by somatic cell NT. In addition, increasing the number of embryos transferred to each recipient resulted in more live offspring from fewer recipients. Copyright © 2011 Elsevier Inc. All rights reserved.

In vitro development of OPU-derived bovine embryos cultured either individually or in groups with the silk protein sericin and the viability of frozen-thawed embryos after transfer.

PubMed

Isobe, Tomohiro; Ikebata, Yoshihisa; Do, Lanh Thi Kim; Tanihara, Fuminori; Taniguchi, Masayasu; Otoi, Takeshige

2015-07-01

The optimization of single-embryo culture conditions is very important, particularly in the in vitro production of bovine embryos using the ovum pick-up (OPU) procedure. The purpose of this study was to examine the development of embryos derived from oocytes obtained by OPU that were cultured either individually or in groups in medium supplemented with or without sericin and to investigate the viability of the frozen-thawed embryos after a direct transfer. When two-cell-stage embryos were cultured either individually or in groups for 7 days in CR1aa medium supplemented with or without 0.5% sericin, the rates of development to blastocysts and freezable blastocysts were significantly lower for the embryos cultured individually without sericin than for the embryos cultured in groups with or without sericin. Moreover, the rate of development to freezable blastocysts of the embryos cultured individually with sericin was significantly higher than that of the embryos cultured without sericin. When the frozen-thawed embryos were transferred directly to recipients, the rates of pregnancy, abortion, stillbirth and normal calving in the recipients were similar among the groups, irrespective of the culture conditions and sericin supplementation. Our findings indicate that supplementation with sericin during embryo culture improves the quality of the embryos cultured individually but not the viability of the frozen-thawed embryos after transfer to recipients. © 2014 Japanese Society of Animal Science.

Near-infrared laser irradiation improves the development of mouse pre-implantation embryos.

PubMed

Yokoo, Masaki; Mori, Miho

2017-05-27

The aim of the present study was to assess the effects of near-infrared laser irradiation on the in vitro development of mouse embryos. Female ICR mice were superovulated with pregnant mare serum gonadotropin and human chorionic gonadotropin (hCG), and mated with male mice. Two-cell stage embryos were collected 40 h after administering hCG and cultured in M16 medium. Two-cell embryos (0 h after culture), 8-cell embryos (approx. 30 h after culture), morula (approx. 48 h after culture), and blastocysts (approx. 73 h after culture) were irradiated at 904 nm for 60 s. These embryos were cultured in a time-lapse monitoring system and the timing of blastocyst hatching was evaluated. Some of the irradiated blastocysts were transferred to the uterine horns of pseudopregnant recipients immediately after irradiation. Pregnancy rates, and offspring growth and fertility, were evaluated. Near-infrared laser irradiation increased the speed of in vitro mouse embryo development. In irradiated blastocysts, hatching was faster than in control (non-irradiated) blastocysts (18.4 vs. 28.2 h, P < 0.05). When 195 irradiated blastocysts were transferred to 18 pseudopregnant mice, all became pregnant and 92 (47.2%) normal-looking pups were born alive. When 182 control blastocysts were transferred to 17 pseudopregnant mice, 14 (82.4%) became pregnant and 54 (29.7%) normal-looking pups were born alive. The growth trajectories (up to 5 weeks) of offspring from irradiated blastocysts were similar to those from control blastocysts. Second generation offspring from transplanted animals were all fertile. These results indicate that near-infrared laser irradiation improves the quality of mouse embryo development in vitro, and increases the live birth rate without affecting the normality of the offspring. Thus, the near-infrared laser method may enhance the quality of embryos and contribute to improvements in reproductive technologies in mammals. Copyright © 2017 Elsevier Inc. All rights reserved.

Embryotrophic factor-3 from human oviductal cells enhances proliferation, suppresses apoptosis and stimulates the expression of the beta1 subunit of sodium-potassium ATPase in mouse embryos.

PubMed

Xu, J S; Lee, Y L; Lee, K F; Kwok, K L; Lee, W M; Luk, J M; Yeung, W S B

2004-12-01

Embrytrophic factor-3 (ETF-3) from human oviductal cells enhanced the development of mouse preimplantation embryos. This report studied the embryotrophic mechanisms of the molecule. Mouse embryos were incubated with ETF-3 for 24 h at different stages of development. ETF-3 treatment between 96 and 120 h post-HCG increased the cell count of blastocysts, whilst treatment between 72 and 96 h post-HCG enhanced the expansion and hatching of the blastocysts. ETF-3 increased the cell number of the embryos by suppressing apoptosis and increasing proliferation as determined by TUNEL and bromodeoxyuridine uptake assays, respectively. Real-time quantitative PCR showed that the in vivo developed and ETF-3-treated blastocysts had a significantly higher mRNA copy number of Na/K-ATPase-beta1, but not of hepsin, than that of blastocysts cultured in medium alone. The former gene was associated with cavitation of blastocysts while the latter was related to hatching of blastocyst. The beneficial effect of ETF-3 on blastocyst hatching was also seen when ETF-3-supplemented commercially available sequential culture medium for human embryo culture was used to culture mouse embryos. ETF-3 improves embryo development by enhancing proliferation, suppressing apoptosis and stimulating expression of genes related to blastocyst cavitation. Supplementating human embryo culture medium with ETF-3 may improve the success rate in clinical assisted reproduction.

Endometrial signals improve embryo outcome: functional role of vascular endothelial growth factor isoforms on embryo development and implantation in mice.

PubMed

Binder, N K; Evans, J; Gardner, D K; Salamonsen, L A; Hannan, N J

2014-10-10

Does vascular endothelial growth factor (VEGF) have important roles during early embryo development and implantation? VEGF plays key roles during mouse preimplantation embryo development, with beneficial effects on time to cavitation, blastocyst cell number and outgrowth, as well as implantation rate and fetal limb development. Embryo implantation requires synchronized dialog between maternal cells and those of the conceptus. Following ovulation, secretions from endometrial glands increase and accumulate in the uterine lumen. These secretions contain important mediators that support the conceptus during the peri-implantation phase. Previously, we demonstrated a significant reduction of VEGFA in the uterine cavity of women with unexplained infertility. Functional studies demonstrated that VEGF significantly enhanced endometrial epithelial cell adhesive properties and embryo outgrowth. Human endometrial lavages (n = 6) were obtained from women of proven fertility. Four-week old Swiss mice were superovulated and mated with Swiss males to obtain embryos for treatment with VEGF in vitro. Preimplantation embryo development was assessed prior to embryo transfer (n = 19-30/treatment group/output). Recipient F1 female mice (8-12 weeks of age) were mated with vasectomized males to induce pseudopregnancy and embryos were transferred. On Day 14.5 of pregnancy, uterine horns were collected for analysis of implantation rates as well as placental and fetal development (n = 14-19/treatment). Lavage fluid was assessed by western immunoblot analysis to determine the VEGF isoforms present. Mouse embryos were treated with either recombinant human (rh)VEGF, or VEGF isoforms 121 and 165. Preimplantation embryo development was quantified using time-lapse microscopy. Blastocysts were (i) stained for cell number, (ii) transferred to wells coated with fibronectin to examine trophoblast outgrowth or (iii) transferred to pseudo pregnant recipients to analyze implantation rates, placental and fetal development. Western blot analysis revealed the presence of VEGF121 and 165 isoforms in human uterine fluid. Time-lapse microscopy analysis revealed that VEGF (n = 22) and VEGF121 (n = 23) treatment significantly reduced the preimplantation mouse embryo time to cavitation (P < 0.05). VEGF and VEGF165 increased both blastocyst cell number (VEGF n = 27; VEGF165 n = 24: P < 0.001) and outgrowth (n = 15/treatment: 66 h, P < 0.001; 74, 90, 98 and 114 h, P < 0.01) on fibronectin compared with control. Furthermore, rhVEGF improved implantation rates and enhanced fetal limb development (P < 0.05). Due to the nature of this work, embryo development and implantation was only examined in the mouse. The absence or reduction in levels of VEGF during the preimplantation period likely affects key events during embryo development, implantation and placentation. The potential for improvement of clinical IVF outcomes by the addition of VEGF to human embryo culture media needs further investigation. This study was supported by a University of Melbourne Early Career Researcher Grant #601040, the NHMRC (L.A.S., Program grant #494802; Fellowship #1002028; N.J.H., Fellowship # 628927; J.E.; project grant #1047756) and L.A.S., Monash IVF Research and Education Foundation. N.K.B. was supported by an Australian Postgraduate Award. Work at PHI-MIMR Institute was also supported by the Victorian Government's Operational Infrastructure Support Program. There are no conflicts of interest to declare. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

[Cryopreservation of mouse embryos in ethylene glycol-based solutions: a search for the optimal and simple protocols].

PubMed

Luo, Ming-Jiu; Liu, Na; Miao, De-Qiang; Lan, Guo-Cheng; Suo-Feng; Chang, Zhong-Le; Tan, Jing-He

2005-09-01

Although ethylene glycol (EG) has been widely used for embryo cryopreservation in domestic animals, few attempts were made to use this molecule to freeze mouse and human embryos. In the few studies that used EG for slow-freezing of mouse and human embryos, complicated protocols for human embryos were used, and the protocols need to be simplified. Besides, freezing mouse morula with EG as a cryoprotectant has not been reported. In this paper, we studied the effects of embryo stages, EG concentration, duration and procedure of equilibration, sucrose supplementation and EG removal after thawing on the development of thawed mouse embryos, using the simple freezing and thawing procedures for bovine embryos. The blastulation and hatching rates (81.92% +/- 2.24% and 68.56% +/- 2.43%, respectively) of the thawed late compact morulae were significantly (P < 0.05) higher than those of embryos frozen-thawed at other stages. When mouse late compact morulae were frozen with different concentrations of EG, the highest rates of blastocyst formation and hatching were obtained with 1.8mol/L EG. The blastulation rate was significantly higher when late morulae were equilibrated in 1.8 mol/L EG for 10 min prior to freezing than when they were equilibrated for 30 min, and the hatching rate of embryos exposed to EG for 10 min was significantly higher than that of embryos exposed for 20 and 30 min. Both rates of blastocyst formation and hatching obtained with two-step equilibration were higher (P < 0.05) than with one-step equilibration in 1.8 mol/L EG. Addition of sucrose to the EG-based solution had no beneficial effects. On the contrary, an increased sucrose level (0.4 mol/L) in the solution impaired the development of the frozen-thawed embryos. In contrast, addition of 0.1 mol/L sucrose to the propylene glycol (PG)-based solution significantly improved the development of the frozen-thawed embryos. Elimination of the cryoprotectant after thawing did not improve the development of the thawed embryos. The cell numbers were less (P < 0.05) in blastocysts developed from the thawed morulae than in the in vivo derived ones. In summary, embryo stage, EG concentration, duration and procedure of equilibration and sucrose supplementation had marked effects on development of the thawed mouse embryos, and a protocol for cryopreservation of mouse embryos is recommended in which the late morulae are frozen in 1.8 mol/L EG using the simple freezing and thawing procedures of bovine embryos after a two-step equilibration and the embryos can be cultured or transferred without EG removal after thawing.

Metabolomic Assessment of Embryo Viability

PubMed Central

Uyar, Asli; Seli, Emre

2014-01-01

Preimplantation embryo metabolism demonstrates distinctive characteristics associated with the developmental potential of embryos. On this basis, metabolite content of culture media was hypothesized to reflect the implantation potential of individual embryos. This hypothesis was tested in consecutive studies reporting a significant association between culture media metabolites and embryo development or clinical pregnancy. The need for a noninvasive, reliable, and rapid embryo assessment strategy promoted metabolomics studies in vitro fertilization (IVF) in an effort to increase success rates of single embryo transfers. With the advance of analytical techniques and bioinformatics, commercial instruments were developed to predict embryo viability using spectroscopic analysis of surplus culture media. However, despite the initial promising results from proof-of-principal studies, recent randomized controlled trials using commercial instruments failed to show a consistent benefit in improving pregnancy rates when metabolomics is used as an adjunct to morphology. At present, the application of metabolomics technology in clinical IVF laboratory requires the elimination of factors underlying inconsistent findings, when possible, and development of reliable predictive models accounting for all possible sources of bias throughout the embryo selection process. PMID:24515909

Sodium butyrate improves the cloned yak embryo viability and corrects gene expression patterns.

PubMed

Xiong, Xian-rong; Lan, Dao-liang; Li, Jian; Wang, Yong; Zhong, Jin-cheng

2015-02-01

Interspecies somatic cell nuclear transfer (iSCNT), a powerful tool in basic scientific research, has been used widely to increase and preserve the population of endangered species. Yak (Bos grunniens) is one of these species. Development to term of interspecies cloned yak embryos has not been achieved, possibly due to abnormal epigenetic reprogramming. Previous studies have demonstrated that treatment of intraspecies cloned embryos with (NaBu) significantly improves nuclear-cytoplasmic reprogramming and viability in vitro. Therefore, in this study, we evaluated the effect of optimal NaBu concentration and exposure time on preimplantation development of yak iSCNT embryos and on the expression patterns of developmentally important genes. The results showed that 8-cell rate, blastocyst formation rate and total cell number increased significantly compared with their untreated counterparts when yak iSCNT embryos were treated with 5 nM NaBu for 12 h after activation, but that the 2-cell stage embryo rate was not significantly different. The treatment of NaBu also increased significantly the expression levels of Oct-4 and decreased the expression levels of HDAC-2, Dnmt-1 and IGF-1; the expression patterns of these genes were more similar to that of their bovine-yak in vitro fertilization (BY-IVF) counterparts. The results described above indicated that NaBu treatment improved developmental competence in vitro and 'corrected' the gene expression patterns of yak iSCNT embryos.

Cheetah interspecific SCNT followed by embryo aggregation improves in vitro development but not pluripotent gene expression.

PubMed

Moro, L N; Hiriart, M I; Buemo, C; Jarazo, J; Sestelo, A; Veraguas, D; Rodriguez-Alvarez, L; Salamone, D F

2015-07-01

The aim of this study was to evaluate the capacity of domestic cat (Dc, Felis silvestris) oocytes to reprogram the nucleus of cheetah (Ch, Acinonyx jubatus) cells by interspecies SCNT (iSCNT), by using embryo aggregation. Dc oocytes were in vitro matured and subjected to zona pellucida free (ZP-free) SCNT or iSCNT, depending on whether the nucleus donor cell was of Dc or Ch respectively. ZP-free reconstructed embryos were then cultured in microwells individually (Dc1X and Ch1X groups) or in couples (Dc2X and Ch2X groups). Embryo aggregation improved in vitro development obtaining 27.4, 47.7, 16.7 and 28.3% of blastocyst rates in the Dc1X, Dc2X, Ch1X and Ch2X groups, respectively (P<0.05). Moreover, aggregation improved the morphological quality of blastocysts from the Dc2X over the Dc1X group. Gene expression analysis revealed that Ch1X and Ch2X blastocysts had significantly lower relative expression of OCT4, CDX2 and NANOG than the Dc1X, Dc2X and IVF control groups. The OCT4, NANOG, SOX2 and CDX2 genes were overexpressed in Dc1X blastocysts, but the relative expression of these four genes decreased in the Dc2X, reaching similar relative levels to those of Dc IVF blastocysts. In conclusion, Ch blastocysts were produced using Dc oocytes, but with lower relative expression of pluripotent and trophoblastic genes, indicating that nuclear reprogramming could be still incomplete. Despite this, embryo aggregation improved the development of Ch and Dc embryos, and normalized Dc gene expression, which suggests that this strategy could improve full-term developmental efficiency of cat and feline iSCNT embryos. © 2015 Society for Reproduction and Fertility.

[Effect of TGF-beta1 on embryo implantation and development in mice in vitro].

PubMed

Luo, Shan; Yin, Hai-ning; Li, Shang-wei

2010-03-01

To investigate the role of TGF-beta1 in embryo implantation and development in vitro in mice. Mouse embryos at 2-cell stage were cultured in the media of M16 with exposure to different levels of TGF-beta1 (0, 1, 10 and 50 ng/mL). The percentage of embryos reaching fixed stages (early blastocyst, expanding blastocyst and hatched blastocyst) was monitored 68 h and 92 h after the culture. The expanding blastocys cultured for 68 h in M16 without TGF-beta1 and those with 10 ng/mL of TGF-beta1 were transferred to pseudopregnant mice. On the 6th day post transfer, the successful rates of implantation were counted. The level of IL-10/IFN-gamma in the serum and maternal-fetus interface of the mice was detected by ELISA on the 6th day post transfer. TGF-beta1 improved embryo growth in vitro. TGF-beta1 at a level of 10 ng/mL had the maximum impact, with 15.6%, 68.09%, 1.42% of embryos reaching early, expanding, and hatched stage, respectively, 68 h after culture, and 6.38%, 28.37%, 53.19% of embryos reaching early, expanding, and hatched stage, respectively, 92 h after culture. The promoting effect declined when TGF-beta1 reached 50 ng/mL. The successful rate of implantation of embryos cultured in M16 with TGF-beta1 was significantly higher than those cultured in M16 without TGF-beta1 (35. 2% vs. 17.19%, P < 0.05). The embryos cultured in M16 with TGF-beta1 had significantly lower level of IFN-gamma in the maternal-fetus interface than those cultured in M16 without TGF-beta1 [(30.89 +/- 11.31) pg/mL vs. (43.23 +/- 18. 09) pg/mL, P < 0.053. TGF-beta1 at an appropriate dose improves embryo implantation in mice in vitro. The mechanism may involve the improvement of the quality of embryos and their development, and decrease of IFN-gamma synthesis in maternal-fetal interface, a chemical that could cause Th2 bias.

Integration of microfluidics in animal in vitro embryo production.

PubMed

Wheeler, M B; Rubessa, M

2017-04-01

The in vitro production of livestock embryos is central to several areas of animal biotechnology. Further, the use of in vitro embryo manipulation is expanding as new applications emerge. ARTs find direct applications in increasing genetic quality of livestock, producing transgenic animals, cloning, artificial insemination, reducing disease transmission, preserving endangered germplasm, producing chimeric animals for disease research, and treating infertility. Whereas new techniques such as nuclear transfer and intracytoplasmic sperm injection are now commonly used, basic embryo culture procedures remain the limiting step to the development of these techniques. Research over the past 2 decades focusing on improving the culture medium has greatly improved in vitro development of embryos. However, cleavage rates and viability of these embryos is reduced compared with in vivo indicating that present in vitro systems are still not optimal. Furthermore, the methods of handling mammalian oocytes and embryos have changed little in recent decades. While pipetting techniques have served embryology well in the past, advanced handling and manipulation technologies will be required to efficiently implement and commercialize the basic biological advances made in recent years. Microfluidic systems can be used to handle gametes, mature oocytes, culture embryos, and perform other basic procedures in a microenvironment that more closely mimic in vivo conditions. The use of microfluidic technologies to fabricate microscale devices has being investigated to overcome this obstacle. In this review, we summarize the development and testing of microfabricated fluidic systems with feature sizes similar to the diameter of an embryo for in vitro production of pre-implantation mammalian embryos. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Improvement of vitrification of in vitro produced buffalo embryos with special reference to sex ratio following vitrification

PubMed Central

Mahmoud, K. Gh. M; Scholkamy, T. H; Darwish, S. F

2015-01-01

Cryopreservation and sexing of embryos are integrated into commercial embryo transfer technologies. To improve the effectiveness of vitrification of in vitro produced buffalo embryos, two experiments were conducted. The first evaluated the effect of exposure time (2 and 3 min) and developmental stage (morula and blastocysts) on the viability and development of vitrified buffalo embryos. Morphologically normal embryos and survival rates (re-expansion) significantly increased when vitrified morulae were exposed for 2 min compared to 3 min (P<0.001). On the other hand, morphologically normal and survival rates of blastocysts significantly increased when exposed for 3 min compared to 2 min (P<0.001). However, there were no significant differences between the two developmental stages (morulae and blastocystes) in the percentages of morphologically normal embryos and re-expansion rates after a 24 h culture. The second experiment aimed to evaluate the effect of viability on the sex ratio of buffalo embryos after vitrification and whether male and female embryos survived vitrification differently. A total number of 61 blastocysts were vitrified for 3 min with the same cryoprotectant as experiment 1. Higher percentages of males were recorded for live as compared to dead embryos; however, this difference was not significant. In conclusion, the post-thaw survival and development of in vitro produced morulae and blastocysts were found to be affected by exposure time rather than developmental stage. Survivability had no significant effect on the sex ratio of vitrified blastocysts; nevertheless, the number of surviving males was higher than dead male embryos. PMID:27175197

Growth and metabolism of murine and bovine embryos in bovine uterine flushing-supplemented culture media.

PubMed Central

Rondeau, M; Guay, P; Goff, A K; Cooke, G M

1996-01-01

The aim of this study was to compare the development and metabolic activity of cultured murine and bovine embryos in 2 standard media (HAM F-10 and RPMI) in the presence or absence of bovine uterine flushings. Murine morulae (n = 653) and day 7 bovine embryos (n = 273) were cultured for 18 h or 36 h in either HAM F-10 or RPMI in the presence or absence of bovine uterine flushings. After culture, the development, quality, and metabolic activity (glucose utilization or methionine uptake and incorporation) of embryos was assessed. It was found that HAM F-10 (without uterine flushings) was a more suitable medium than RPMI for optimal development and metabolism of murine and bovine embryos. Poor quality and development, as well as decreased metabolism, were evident after culture of murine embryos in RPMI; in contrast, this medium had no adverse effects on bovine embryos in culture. Supplementation of HAM F-10 with bovine uterine flushings improved the growth of murine embryos and the protein synthesis (as measured by an increased methionine incorporation) for both murine and bovine embryos. However, supplementation with bovine uterine flushings could not overcome deficiencies of an inappropriate medium (RPMI) for murine embryos. Supplementation of a well-defined culture medium with uterine flushings increased metabolism of embryos in culture, and thus might help to increase pregnancy rates after transfer of such embryos to recipient cows. PMID:8825988

Effect of steroid treatment of endometrial cells on blastocyst development during co-culture.

PubMed

Goff, A K; Smith, L C

1998-04-01

The objective of this study was to determine if treatment of endometrial cells with progesterone or progesterone plus estradiol would improve the development of bovine embryos to the blastocyst stage during co-culture. After IVF, bovine embryos were cultured with oviduct epithelial cells for 3 d. In Experiment 1 the embryos were cultured with a) oviduct epithelial cells; b) endometrial epithelial cells (EEC); c) EEC with 10 ng/ml progesterone (EEC + P); or d) EEC with 10 ng/ml progesterone and 10 pg/ml estradiol (EEC + PE) for 6 d. In Experiment 2 the embryos were cultured with a) oviduct epithelial cells; b) endometrial stromal cells (ESC); c) ESC with 10 ng/ml progesterone (ESC + P); or d) ESC with 10 ng/ml progesterone and 10 pg/ml estradiol (ESC + PE) for 6 d. Results from Experiment 1 showed that endometrial epithelial cells supported development to the blastocyst stage as effectively as the oviduct cells; however, the size of the blastocysts was smaller for the endometrial cells. There was no effect of steroid hormone treatment on development to the blastocyst stage or on the size of the blastocysts. Results from Experiment 2 showed that stromal cells supported development to the blastocyst stage as effectively as oviduct cells. The hatching rate was lower when the embryos were co-cultured with stromal cells than oviduct epithelial cells; but there was no effect of steroid treatment. These data show that untreated endometrial epithelial cells are as effective as oviduct cells in maintaining embryo development to the blastocyst stage. However, embryo development was not improved by steroid treatment of the cells.

In vitro development of donated frozen-thawed human embryos in a prototype static microfluidic device: a randomized controlled trial.

PubMed

Kieslinger, Dorit C; Hao, Zhenxia; Vergouw, Carlijn G; Kostelijk, Elisabeth H; Lambalk, Cornelis B; Le Gac, Séverine

2015-03-01

To compare the development of human embryos in microfluidic devices with culture in standard microdrop dishes, both under static conditions. Prospective randomized controlled trial. In vitro fertilization laboratory. One hundred eighteen donated frozen-thawed human day-4 embryos. Random allocation of embryos that fulfilled the inclusion criteria to single-embryo culture in a microfluidics device (n = 58) or standard microdrop dish (n = 60). Blastocyst formation rate and quality after 24, 28, 48, and 72 hours of culture. The percentage of frozen-thawed day-4 embryos that developed to the blastocyst stage did not differ significantly in the standard microdrop dishes and microfluidic devices after 28 hours of culture (53.3% vs. 58.6%) or at any of the other time points. The proportion of embryos that would have been suitable for embryo transfer was comparable after 28 hours of culture in the control dishes and microfluidic devices (90.0% vs. 93.1%). Furthermore, blastocyst quality was similar in the two study groups. This study shows that a microfluidic device can successfully support human blastocyst development in vitro under static culture conditions. Future studies need to clarify whether earlier stage embryos will benefit from the culture in microfluidic devices more than the tested day-4 embryos because many important steps in the development of human embryos already take place before day 4. Further improvements of the microfluidic device will include parallel culture of single embryos, application of medium refreshment, and built-in sensors. NTR3867. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Intrauterine air impairs embryonic postimplantation development in mice.

PubMed

Liu, Ruonan; Li, Yimeng; Miao, Yanping; Wei, Yanhui; Guan, Mo; Zhou, Rongyan; Li, Xiangyun

2017-12-01

Although most embryologists load air bubbles into the catheter along with embryos during embryo transfer, the effects of these air bubbles on embryo transfer success rate are not clear. Air bubbles were nonsurgically injected into unilateral uterine horns of mice to demonstrate the negative effects of intrauterine air bubbles on embryonic development. Our data showed that when air bubbles are nonsurgically injected into unilateral uterine horns of pregnant 4days mice the litter size is significantly decreased. Four days after the introduction of air, abnormal decidua and dead conceptuses were detected in the uterine horns receiving the air bubbles. In addition, intrauterine air also significantly impaired murine embryo transfer success rates, and induced an increase in endometrial capillary permeability and decidualization in mice on day 4 of pseudopregnancy. These results strongly indicated that the air bubbles loaded into embryo transfer catheters to bracket the embryo-containing medium may have negative effect on embryonic implantation and development. Intrauterine air impaired murine embryonic postimplantation development, and this provided some clues for improving embryo transfer techniques in human. Copyright © 2017 Elsevier B.V. All rights reserved.

Elucidating the origin of chromosomal aberrations in IVF embryos by preimplantation genetic analysis.

PubMed

Frumkin, Tsvia; Malcov, Mira; Yaron, Yuval; Ben-Yosef, Dalit

2008-01-30

Preimplantation genetic screening (PGS) has been proposed as a method for improving success rates in patients with repeated IVF failures. This approach is based on the hypothesis that such failures are the result of aneuploid embryos. It has been suggested that FISH analysis of blastomeres removed from preimplantation embryos represent the chromosomal constitution of the entire embryo. However, it is not yet clear whether it also represents the chromosomal constitution of the implanted embryo. PGS reanalysis on day 5 of embryos designated as "aneuploid" on day 3 may demonstrate a high rate of mosaicism for chromosomal aberration. Some of these mosaic embryos are capable of developing into normal embryos by "self-correction". Others, however, may accumulate additional chromosomal anomalies. It is therefore concluded that the chromosomal constitution of a preimplantation embryo may evolve during early cleavages. Meiotic and post zygotic mitotic errors may account for these chromosomal aberrations. This review will focus on elucidating the origin of chromosomal changes during preimplantation embryo development by studying their chromosomal constitution at different stages.

Fluorescence-based visualization of autophagic activity predicts mouse embryo viability

NASA Astrophysics Data System (ADS)

Tsukamoto, Satoshi; Hara, Taichi; Yamamoto, Atsushi; Kito, Seiji; Minami, Naojiro; Kubota, Toshiro; Sato, Ken; Kokubo, Toshiaki

2014-03-01

Embryo quality is a critical parameter in assisted reproductive technologies. Although embryo quality can be evaluated morphologically, embryo morphology does not correlate perfectly with embryo viability. To improve this, it is important to understand which molecular mechanisms are involved in embryo quality control. Autophagy is an evolutionarily conserved catabolic process in which cytoplasmic materials sequestered by autophagosomes are degraded in lysosomes. We previously demonstrated that autophagy is highly activated after fertilization and is essential for further embryonic development. Here, we developed a simple fluorescence-based method for visualizing autophagic activity in live mouse embryos. Our method is based on imaging of the fluorescence intensity of GFP-LC3, a versatile marker for autophagy, which is microinjected into the embryos. Using this method, we show that embryonic autophagic activity declines with advancing maternal age, probably due to a decline in the activity of lysosomal hydrolases. We also demonstrate that embryonic autophagic activity is associated with the developmental viability of the embryo. Our results suggest that embryonic autophagic activity can be utilized as a novel indicator of embryo quality.

Factors affecting the development of somatic cell nuclear transfer embryos in Cattle.

PubMed

Akagi, Satoshi; Matsukawa, Kazutsugu; Takahashi, Seiya

2014-01-01

Nuclear transfer is a complex multistep procedure that includes oocyte maturation, cell cycle synchronization of donor cells, enucleation, cell fusion, oocyte activation and embryo culture. Therefore, many factors are believed to contribute to the success of embryo development following nuclear transfer. Numerous attempts to improve cloning efficiency have been conducted since the birth of the first sheep by somatic cell nuclear transfer. However, the efficiency of somatic cell cloning has remained low, and applications have been limited. In this review, we discuss some of the factors that affect the developmental ability of somatic cell nuclear transfer embryos in cattle.

Dissection of culture media for embryos: the most important and less important components and characteristics.

PubMed

Gardner, David K

2008-01-01

Improvements in culture media formulations have led to an increase in the ability to maintain the mammalian embryo in culture throughout the preimplantation and pre-attachment period. Amino acids and specific macromolecules have been identified as being key medium components, whereas temporal dynamics have been recognised as important media characteristics. Furthermore, other laboratory factors that directly impact embryo development and viability have been identified. Such factors include the use of a reduced oxygen tension, an appropriate incubation system and an adequate prescreening of all contact supplies. With rigourous quality systems in place, it is possible to obtain in vivo rates of embryo development in vitro using new media formulations while maintaining high levels of embryo viability. The future of embryo culture will likely be based on novel culture chips capable of providing temporal dynamics while facilitating real-time analysis of embryo physiology.

Use of Embryos Extracted from Individual Cannabis sativa Seeds for Genetic Studies and Forensic Applications.

PubMed

Soler, Salvador; Borràs, Dionís; Vilanova, Santiago; Sifres, Alicia; Andújar, Isabel; Figàs, Maria R; Llosa, Ernesto R; Prohens, Jaime

2016-03-01

Legal limits on the psychoactive tetrahydrocannabinol (THC) content in Cannabis sativa plants have complicated genetic and forensic studies in this species. However, Cannabis seeds present very low THC levels. We developed a method for embryo extraction from seeds and an improved protocol for DNA extraction and tested this method in four hemp and six marijuana varieties. This embryo extraction method enabled the recovery of diploid embryos from individual seeds. An improved DNA extraction protocol (CTAB3) was used to obtain DNA from individual embryos at a concentration and quality similar to DNA extracted from leaves. DNA extracted from embryos was used for SSR molecular characterization in individuals from the 10 varieties. A unique molecular profile for each individual was obtained, and a clear differentiation between hemp and marijuana varieties was observed. The combined embryo extraction-DNA extraction methodology and the new highly polymorphic SSR markers facilitate genetic and forensic studies in Cannabis. © 2015 American Academy of Forensic Sciences.

Digital Microfluidic Dynamic Culture of Mammalian Embryos on an Electrowetting on Dielectric (EWOD) Chip

PubMed Central

Huang, Hong-Yuan; Shen, Hsien-Hua; Tien, Chang-Hung; Li, Chin-Jung; Fan, Shih-Kang; Liu, Cheng-Hsien; Hsu, Wen-Syang; Yao, Da-Jeng

2015-01-01

Current human fertilization in vitro (IVF) bypasses the female oviduct and manually inseminates, fertilizes and cultivates embryos in a static microdrop containing appropriate chemical compounds. A microfluidic microchannel system for IVF is considered to provide an improved in-vivo-mimicking environment to enhance the development in a culture system for an embryo before implantation. We demonstrate a novel digitalized microfluidic device powered with electrowetting on a dielectric (EWOD) to culture an embryo in vitro in a single droplet in a microfluidic environment to mimic the environment in vivo for development of the embryo and to culture the embryos with good development and live births. Our results show that the dynamic culture powered with EWOD can manipulate a single droplet containing one mouse embryo and culture to the blastocyst stage. The rate of embryo cleavage to a hatching blastocyst with a dynamic culture is significantly greater than that with a traditional static culture (p<0.05). The EWOD chip enhances the culture of mouse embryos in a dynamic environment. To test the reproductive outcome of the embryos collected from an EWOD chip as a culture system, we transferred embryos to pseudo-pregnant female mice and produced live births. These results demonstrate that an EWOD-based microfluidic device is capable of culturing mammalian embryos in a microfluidic biological manner, presaging future clinical application. PMID:25933003

Digital Microfluidic Dynamic Culture of Mammalian Embryos on an Electrowetting on Dielectric (EWOD) Chip.

PubMed

Huang, Hong-Yuan; Shen, Hsien-Hua; Tien, Chang-Hung; Li, Chin-Jung; Fan, Shih-Kang; Liu, Cheng-Hsien; Hsu, Wen-Syang; Yao, Da-Jeng

2015-01-01

Current human fertilization in vitro (IVF) bypasses the female oviduct and manually inseminates, fertilizes and cultivates embryos in a static microdrop containing appropriate chemical compounds. A microfluidic microchannel system for IVF is considered to provide an improved in-vivo-mimicking environment to enhance the development in a culture system for an embryo before implantation. We demonstrate a novel digitalized microfluidic device powered with electrowetting on a dielectric (EWOD) to culture an embryo in vitro in a single droplet in a microfluidic environment to mimic the environment in vivo for development of the embryo and to culture the embryos with good development and live births. Our results show that the dynamic culture powered with EWOD can manipulate a single droplet containing one mouse embryo and culture to the blastocyst stage. The rate of embryo cleavage to a hatching blastocyst with a dynamic culture is significantly greater than that with a traditional static culture (p<0.05). The EWOD chip enhances the culture of mouse embryos in a dynamic environment. To test the reproductive outcome of the embryos collected from an EWOD chip as a culture system, we transferred embryos to pseudo-pregnant female mice and produced live births. These results demonstrate that an EWOD-based microfluidic device is capable of culturing mammalian embryos in a microfluidic biological manner, presaging future clinical application.

Secondary SCNT doubles the pre-implantation development rate of reconstructed interspecies embryos by using cytoplasts of Sannen dairy goat ova.

PubMed

Zhang, Ai Min; Chen, Jian Quan; Sha, Hong Ying; Chen, Juan; Xu, Xu Jun; Wu, You Bin; Ge, Lai Xiang; Da, Hu Wei; Cheng, Guo Xiang

2007-10-01

The aim of this study was to investigate whether ova of Sannen goat could support the pre-implantation development of interspecies embryos constructed through somatic cell nucleus transfer (SCNT) embryos and whether secondary SCNT (SSCNT) could improve the pre-implantation development of those embryos. The primary SCNT (PSCNT) embryos were produced by using Sannen goat ovum cytoplasts as recipients and fibroblast cells, derived from human, rabbit and Boer goat skins, as nucleus donors. The blastomeres of 8 to 16 cells stage of PSCNT embryos were subsequently used as nucleus donor cells and Sannen goat ovum cytoplasts as recipients to evaluate the effect of SSCNT on the pre-implantation development rate of these reconstructed interspecies embryos. Our results indicate that the pre-implantation development rates of SSCNT embryos reconstructed using these three different blastomeres are almost twice of that of corresponding PSCNT embryos (human, 15.8% vs. 7.8%; rabbit, 27.9% vs. 12.5%; Boer goat 55.3% vs. 24.5%; P < 0.05 in all three cases). The time durations that embryos need for the serial events of remodeling and reprogramming to take place vary, depending on the animal species of nucleus donors. These data suggest that remodeling and reprogramming of donor nucleus may be enhanced by prolonged exposure of donor nucleus to maternal cytoplast. We conclude that Sannen goat cytoplast can support the pre-implantation development of embryos constructed with nuclei from various donors, including fibroblasts of human, rabbit and Boer goat; and the somatic nucleus derived from different species requires more time to achieve its reprogramming necessary for pre-implantation development.

Effect of Culture Conditions on Viability of Mouse and Rat Embryos Developed in Vitro

PubMed Central

Popova, Elena; Bader, Michael; Krivokharchenko, Alexander

2011-01-01

Currently in vitro culture of mouse preimplantation embryos has become a very important technique to investigate different mechanisms of early embryogenesis. However, there is a big difference in the preimplantation development between mammalian species. Despite close relatedness to mice, in vitro cultivation of rat preimplantation embryos is still delicate and needs further investigation and optimizations. In this study we have compared the in vitro developmental potential of mouse and rat embryos cultured at different culture conditions in parallel experiments. Interestingly, mouse zygotes developed in vitro until blastocyst stage even in inadequate medium without any phosphates and with low osmolarity which was formulated especially for cultivation of rat embryos. Rat parthenotes and zygotes developed in M16 medium formulated for mouse embryos only till 2-cell stage and further development is blocked completely at this stage. Moreover, developmental ability of rat embryos in vitro was significantly lower in comparison with mouse even in special rat mR1ECM medium. Mouse and rat embryos at 2-cell stage obtained in vivo developed until blastocyst stages significantly more efficiently compared to zygotes. Culture of mouse zygotes in glass capillaries resulted in a significantly higher rate of morula and blastocyst development compared with dishes. The Well-of-the-Well system resulted in a significant improvement when compared with dishes for the culture of rat zygotes only until morula stage. Reduced oxygen tension increased the developmental rate of rat but not mouse zygotes until blastocyst stage. This study demonstrates that development of early preimplantation embryos is altered by different culture conditions and show strong differences even between two related species such as mice and rats. Therefore, for understanding the fundamental mechanisms of early mammalian development it is very important to use embryos of various species. PMID:24710194

Simulated Microgravity Influences Bovine Oocyte In Vitro Fertilization and Preimplantation Embryo Development

USDA-ARS?s Scientific Manuscript database

The aim of this study was to investigate whether in vitro fertilization and preimplantation embryos exposed to a simulated microgravity environment in vitro would improve, or be deleterious to, their fertilization and embryonic development. A Rotating Cell Culture System™ (RCCS) bioreactor with a Hi...

Treatment of porcine donor cells and reconstructed embryos with the antioxidant melatonin enhances cloning efficiency.

PubMed

Pang, Yun-Wei; An, Lei; Wang, Peng; Yu, Yong; Yin, Qiu-Dan; Wang, Xiao-Hong; Xin-Zhang; Qian-Zhang; Yang, Mei-Ling; Min-Guo; Wu, Zhong-Hong; Tian, Jian-Hui

2013-05-01

This study was conducted to investigate the effect of melatonin during the culture of donor cells and cloned embryos on the in vitro developmental competence and quality of cloned porcine embryos. At concentrations of 10(-6 )M or 10(-8) M, melatonin significantly enhanced the proliferation of porcine fetal fibroblasts (PFFs), and the blastocyst rate was significantly increased in the 10(-10) M melatonin-treated donor cell group. Cloned embryo development was also improved in embryo culture medium that was supplemented with 10(-9) M or 10(-12) M melatonin. When both donor cells and cloned embryos were treated with melatonin, the cleavage rate and total cell number of blastocysts were not significantly affected; however, the blastocyst rate was increased significantly (20.0% versus 11.7%). TUNEL assays showed that combined melatonin treatment reduced the rate of apoptotic nuclei (3.6% versus 6.1%). Gene expression analysis of the apoptosis-related genes BAX, BCL2L1, and p53 showed that the expression of BCL2L1 was significantly elevated 2.7-fold relative to the control group, while the expression of BAX and p53 was significantly decreased by 3.7-fold and 23.2-fold, respectively. In addition, we detected the expression of two melatonin receptors (MT1 and MT2) in PFFs but not in porcine cloned embryos. We conclude that exogenous melatonin enhances the development of porcine cloned embryos and improves embryo quality by inhibiting p53-mediated apoptotic pathway. The proliferation of PFFs may be mediated by receptor binding, but the beneficial effects of melatonin on embryonic development may be receptor-independent, possibly through melatonin's ability to directly scavenge free radicals. © 2012 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd.

Improved quality of porcine embryos cultured with hyaluronan due to the modification of the mitochondrial membrane potential and reactive oxygen species level.

PubMed

Romek, Marek; Gajda, Barbara; Krzysztofowicz, Ewa; Kucia, Marcin; Uzarowska, Agnieszka; Smorag, Zdzislaw

2017-10-15

Although considerable progress has been made in pig embryo culture systems, the developmental competence and quality of the produced embryos are still lower than their in vivo-derived counterparts. Because hyaluronan (HA) regulates various cellular processes and possesses antioxidant properties, this glycosaminoglycan seems to be a promising supplement in culture media. However, until now, its beneficial influence on in vitro pig embryo development has been debatable. Hence, we aimed to investigate the effect of 0.25 mg/mL, 0.5 mg/mL and 1 mg/mL concentrations of HA on the developmental potential and quality of cultured porcine embryos. We found that 1 mg/mL HA supplementation significantly increased the obtained percentages of cleaved embryos to ∼95%, morulae to ∼87% and blastocysts to ∼77%. At 0.5 mg/mL and 1 mg/mL HA concentrations, we observed a significantly improved blastocyst quality, expressed as the total number of cells per blastocyst, number of cells in the inner cell mass, number of TUNEL-positive nuclei per blastocyst, the TUNEL index and the blastocyst diameter. Because the inner mitochondrial membrane potential (ΔΨm) and reactive oxygen species (ROS) level are important for proper embryo development, for the first time, we measured these two parameters in cultured embryos at various HA concentrations and during their development up to the expanded blastocyst stage. For blastocysts cultured with 1 mg/mL HA, the ΔΨm and ROS level were ∼1.6 and 2.7 times lower, respectively, than those of the control blastocysts. Both ΔΨm and the ROS level were increased in parallel during in vitro embryo development with and without HA, but this increase was less pronounced in the presence of HA. Hence, our quantitative data unequivocally show that supplementation of NCSU-23 culture medium with 1 mg/mL HA improves the developmental potential and quality of pig embryos. This effect results from a significant decrease in the ROS level induced by the HA-dependent ΔΨm reduction. Copyright © 2017 Elsevier Inc. All rights reserved.

Milrinone treatment of bovine oocytes during in vitro maturation benefits production of nuclear transfer embryos by improving enucleation rate and developmental competence.

PubMed

Naruse, Kenji; Iga, Kosuke; Shimizu, Manabu; Takenouchi, Naoki; Akagi, Satoshi; Somfai, Tamas; Hirao, Yuji

2012-01-01

In the production of cattle nuclear transfer embryos, the production efficiency is affected by the oocyte developmental competence and successful enucleation rate. This study investigated the effect of treating oocytes with milrinone, a phosphodiesterase inhibitor, on these two characteristics. When cumulus-oocyte complexes (COCs) were cultured for 19 h with 0, 50 or 100 μM of milrinone, the enucleation rate was significantly improved by 100 μM milrinone. However, milrinone treatment during in vitro maturation (IVM) also delayed meiotic progression by at least 2 h, which would affect the examination of enucleation rate and developmental competence of oocytes. Thus, in the second experiment, meiotic resumption was temporarily inhibited with butyrolactone I (BL-I; 100 μM, 18 h) to decrease the delayed maturation caused by milrinone; this enabled a more accurate comparison of the effects of milrinone after oocyte maturation. In nuclear transfer embryo production, oocytes treated with milrinone (100 μM, 20 h) showed a significantly higher rate of enucleation compared with that of control oocytes. This improved enucleation rate was associated with a closer location of the metaphase plate to the first polar body in the treated oocytes compared with that in control oocytes. Furthermore, milrinone improved the frequency of development to the blastocyst stage in the resulting embryos. In conclusion, milrinone supplementation during IVM improved enucleation rates by rendering the metaphase plate in close proximity to the first polar body, and this treatment also improved oocyte developmental competence. These benefits additively improved the yield of cloned embryos that developed to the blastocyst stage.

The cell agglutination agent, phytohemagglutinin-L, improves the efficiency of somatic nuclear transfer cloning in cattle (Bos taurus).

PubMed

Du, Fuliang; Shen, Perng-Chih; Xu, Jie; Sung, Li-Ying; Jeong, B-Seon; Lucky Nedambale, Tshimangadzo; Riesen, John; Cindy Tian, X; Cheng, Winston T K; Lee, Shan-Nan; Yang, Xiangzhong

2006-02-01

One of the several factors that contribute to the low efficiency of mammalian somatic cloning is poor fusion between the small somatic donor cell and the large recipient oocyte. This study was designed to test phytohemagglutinin (PHA) agglutination activity on fusion rate, and subsequent developmental potential of cloned bovine embryos. The toxicity of PHA was established by examining its effects on the development of parthenogenetic bovine oocytes treated with different doses (Experiment 1), and for different durations (Experiment 2). The effective dose and duration of PHA treatment (150 microg/mL, 20 min incubation) was selected and used to compare membrane fusion efficiency and embryo development following somatic cell nuclear transfer (Experiment 3). Cloning with somatic donor fibroblasts versus cumulus cells was also compared, both with and without PHA treatment (150 microg/mL, 20 min). Fusion rate of nuclear donor fibroblasts, after phytohemagglutinin treatment, was increased from 33 to 61% (P < 0.05), and from 59 to 88% (P < 0.05) with cumulus cell nuclear donors. The nuclear transfer (NT) efficiency per oocyte used was improved following PHA treatment, for both fibroblast (13% versus 22%) as well as cumulus cells (17% versus 34%; P < 0.05). The cloned embryos, both with and without PHA treatment, were subjected to vitrification and embryo transfer testing, and resulted in similar survival (approximately 90% hatching) and pregnancy rates (17-25%). Three calves were born following vitrification and embryo transfer of these embryos; two from the PHA-treated group, and one from non-PHA control group. We concluded that PHA treatment significantly improved the fusion efficiency of somatic NT in cattle, and therefore, increased the development of cloned blastocysts. Furthermore, within a determined range of dose and duration, PHA had no detrimental effect on embryo survival post-vitrification, nor on pregnancy or calving rates following embryo transfer.

Factors Affecting the Development of Somatic Cell Nuclear Transfer Embryos in Cattle

PubMed Central

AKAGI, Satoshi; MATSUKAWA, Kazutsugu; TAKAHASHI, Seiya

2014-01-01

Nuclear transfer is a complex multistep procedure that includes oocyte maturation, cell cycle synchronization of donor cells, enucleation, cell fusion, oocyte activation and embryo culture. Therefore, many factors are believed to contribute to the success of embryo development following nuclear transfer. Numerous attempts to improve cloning efficiency have been conducted since the birth of the first sheep by somatic cell nuclear transfer. However, the efficiency of somatic cell cloning has remained low, and applications have been limited. In this review, we discuss some of the factors that affect the developmental ability of somatic cell nuclear transfer embryos in cattle. PMID:25341701

Induction of autophagy improves embryo viability in cloned mouse embryos

PubMed Central

Shen, XingHui; Zhang, Na; Wang, ZhenDong; Bai, GuangYu; Zheng, Zhong; Gu, YanLi; Wu, YanShuang; Liu, Hui; Zhou, DongJie; Lei, Lei

2015-01-01

Autophagy is an essential cellular mechanism that degrades cytoplasmic proteins and organelles to recycle their components. Moreover, autophagy is essential for preimplantation development in mammals. Here we show that autophagy is also important for reprogramming in somatic cell nuclear transfer (SCNT). Our data indicate that unlike fertilized oocytes, autophagy is not triggered in SCNT embryos during 6 hours of activation. Mechanistically, the inhibited autophagic induction during SCNT activation is due to the cytochalasin B (CB) caused depolymerization of actin filaments. In this study, we induced autophagy during SCNT activation by rapamycin and pp242, which could restore the expected level of autophagy and significantly enhance the development of SCNT embryos to the blastocyst stage when compared with the control (68.5% and 68.7% vs. 41.5%, P < 0.05). Furthermore, the treatment of rapamycin and pp242 accelerates active DNA demethylation indicated by the conversion of 5 mC to 5 hmC, and treatment of rapamycin improves degradation of maternal mRNA as well. Thus, our findings reveal that autophagy is important for development of SCNT embryos and inhibited autophagic induction during SCNT activation might be one of the serious causes of low efficiency of SCNT. PMID:26643778

Developmental consequences of cryopreservation of mammalian oocytes and embryos.

PubMed

Smith, Gary D; Silva E Silva, Cristine Ane

2004-08-01

During the last three decades, significant advances have been made in successful cryopreservation of mammalian preimplantation embryos, and more recently oocytes. The ability to cryopreserve, thaw, and establish pregnancies with supernumerary preimplantation embryos has become an important tool in fertility treatment. Human oocyte cryopreservation has practical application in preserving fertility for individuals at risk of compromised egg quality due to cancer treatments or advanced maternal age. While oocyte/embryo cryopreservation success has increased over time, there is still room for improvement. Oocytes and embryos are susceptible to cryo-damage, which collectively entails cellular damage caused by mechanical, chemical, or thermal forces during the freeze-thaw process. Basic studies focused on understanding cellular structures, their composition, and more importantly their functions, in normal cell developments will continue to be critical in assessing, understanding, and correcting oocyte/embryo cryo-damage. This review will delineate many of the oocyte/embryo intracellular and extracellular structures that are or may be compromised during cryopreservation. A global theme presented throughout this review is that many structural components of the oocyte/embryo also have essential functional roles in development. Compromising these cellular structures, and thus their cellular homeostatic functions, can deleteriously influence initial cryo-survival or compromise subsequent normal development through effects on the oocyte and/or early embryo.

A transgene design for enhancing oil content in Arabidopsis and Camelina seeds

USDA-ARS?s Scientific Manuscript database

Increasing the oil yield is a major objective for oilseed crop improvement. Oil biosynthesis and accumulation are influenced by multiple genes involved in embryo and seed development. The LEAFY COTYLEDON1 (LEC1) is a master regulator of embryo development that also enhances the expression of genes i...

Manipulation of follicle development to ensure optimal oocyte quality and conception rates in cattle.

PubMed

Baruselli, P S; Sá Filho, M F; Ferreira, R M; Sales, J N S; Gimenes, L U; Vieira, L M; Mendanha, M F; Bó, G A

2012-08-01

Over the last several decades, a number of therapies have been developed that manipulate ovarian follicle growth to improve oocyte quality and conception rates in cattle. Various strategies have been proposed to improve the responses to reproductive biotechnologies following timed artificial insemination (TAI), superovulation (SOV) or ovum pickup (OPU) programmes. During TAI protocols, final follicular growth and size of the ovulatory follicle are key factors that may significantly influence oocyte quality, ovulation, the uterine environment and consequently pregnancy outcomes. Progesterone concentrations during SOV protocols influence follicular growth, oocyte quality and embryo quality; therefore, several adjustments to SOV protocols have been proposed depending on the animal category and breed. In addition, the success of in vitro embryo production is directly related to the number and quality of cumulus oocyte complexes harvested by OPU. Control of follicle development has a significant impact on the OPU outcome. This article discusses a number of key points related to the manipulation of ovarian follicular growth to maximize oocyte quality and improve conception rates following TAI and embryo transfer of in vivo- and in vitro-derived embryos in cattle. © 2012 Blackwell Verlag GmbH.

Manipulating the Mitochondrial Genome To Enhance Cattle Embryo Development

PubMed Central

Srirattana, Kanokwan; St. John, Justin C.

2017-01-01

The mixing of mitochondrial DNA (mtDNA) from the donor cell and the recipient oocyte in embryos and offspring derived from somatic cell nuclear transfer (SCNT) compromises genetic integrity and affects embryo development. We set out to generate SCNT embryos that inherited their mtDNA from the recipient oocyte only, as is the case following natural conception. While SCNT blastocysts produced from Holstein (Bos taurus) fibroblasts were depleted of their mtDNA, and oocytes derived from Angus (Bos taurus) cattle possessed oocyte mtDNA only, the coexistence of donor cell and oocyte mtDNA resulted in blastocysts derived from nondepleted cells. Moreover, the use of the reprogramming agent, Trichostatin A (TSA), further improved the development of embryos derived from depleted cells. RNA-seq analysis highlighted 35 differentially expressed genes from the comparison between blastocysts generated from nondepleted cells and blastocysts from depleted cells, both in the presence of TSA. The only differences between these two sets of embryos were the presence of donor cell mtDNA, and a significantly higher mtDNA copy number for embryos derived from nondepleted cells. Furthermore, the use of TSA on embryos derived from depleted cells positively modulated the expression of CLDN8, TMEM38A, and FREM1, which affect embryonic development. In conclusion, SCNT embryos produced by mtDNA depleted donor cells have the same potential to develop to the blastocyst stage without the presumed damaging effect resulting from the mixture of donor and recipient mtDNA. PMID:28500053

Effects of duration of electric pulse on in vitro development of cloned cat embryos with human artificial chromosome vector.

PubMed

Do, Ltk; Wittayarat, M; Terazono, T; Sato, Y; Taniguchi, M; Tanihara, F; Takemoto, T; Kazuki, Y; Kazuki, K; Oshimura, M; Otoi, T

2016-12-01

The current applications for cat cloning include production of models for the study of human and animal diseases. This study was conducted to investigate the optimal fusion protocol on in vitro development of transgenic cloned cat embryos by comparing duration of electric pulse. Cat fibroblast cells containing a human artificial chromosome (HAC) vector were used as genetically modified nuclear donor cells. Couplets were fused and activated simultaneously with a single DC pulse of 3.0 kV/cm for either 30 or 60 μs. Low rates of fusion and embryo development to the blastocyst stage were observed in the reconstructed HAC-transchromosomic embryos, when the duration of fusion was prolonged to 60 μs. In contrast, the prolongation of electric pulse duration improved the embryo development and quality in the reconstructed control embryos without HAC vector. Our results suggested that the optimal parameters of electric pulses for fusion in cat somatic cell nuclear transfer vary among the types used for donor cells. © 2016 Blackwell Verlag GmbH.

Improved Murine Blastocyst Quality and Development in a Single Culture Medium Compared to Sequential Culture Media

PubMed Central

Hennings, Justin M.; Zimmer, Randall L.; Nabli, Henda; Davis, J. Wade; Sutovsky, Peter; Sutovsky, Miriam; Sharpe-Timms, Kathy L.

2015-01-01

Objective: Validate single versus sequential culture media for murine embryo development. Design: Prospective laboratory experiment. Setting: Assisted Reproduction Laboratory. Animals: Murine embryos. Interventions: Thawed murine zygotes cultured for 3 or 5 days (d3 or d5) in single or sequential embryo culture media developed for human in vitro fertilization. Main Outcome Measures: On d3, zygotes developing to the 8 cell (8C) stage or greater were quantified using 4’,6-diamidino-2-phenylindole (DAPI), and quality was assessed by morphological analysis. On d5, the number of embryos reaching the blastocyst stage was counted. DAPI was used to quantify total nuclei and inner cell mass nuclei. Localization of ubiquitin C-terminal hydrolase L1 (UCHL1) and ubiquitin C-terminal hydrolase L3 (UCHL3) was reference points for evaluating cell quality. Results: Comparing outcomes in single versus to sequential media, the odds of embryos developing to the 8C stage on d3 were 2.34 time greater (P = .06). On d5, more embryos reached the blastocyst stage (P = <.0001), hatched, and had significantly more trophoblast cells (P = .005) contributing to the increased total cell number. Also at d5, localization of distinct cytoplasmic UCHL1 and nuclear UCHL3 was found in high-quality hatching blastocysts. Localization of UCHL1 and UCHL3 was diffuse and inappropriately dispersed throughout the cytoplasm in low-quality nonhatching blastocysts. Conclusions: Single medium yields greater cell numbers, an increased growth rate, and more hatching of murine embryos. Cytoplasmic UCHL1 and nuclear UHCL3 localization patterns were indicative of embryo quality. Our conclusions are limited to murine embryos but one might speculate that single medium may also be more beneficial for human embryo culture. Human embryo studies are needed. PMID:26668049

Improved Murine Blastocyst Quality and Development in a Single Culture Medium Compared to Sequential Culture Media.

PubMed

Hennings, Justin M; Zimmer, Randall L; Nabli, Henda; Davis, J Wade; Sutovsky, Peter; Sutovsky, Miriam; Sharpe-Timms, Kathy L

2016-03-01

Validate single versus sequential culture media for murine embryo development. Prospective laboratory experiment. Assisted Reproduction Laboratory. Murine embryos. Thawed murine zygotes cultured for 3 or 5 days (d3 or d5) in single or sequential embryo culture media developed for human in vitro fertilization. On d3, zygotes developing to the 8 cell (8C) stage or greater were quantified using 4',6-diamidino-2-phenylindole (DAPI), and quality was assessed by morphological analysis. On d5, the number of embryos reaching the blastocyst stage was counted. DAPI was used to quantify total nuclei and inner cell mass nuclei. Localization of ubiquitin C-terminal hydrolase L1 (UCHL1) and ubiquitin C-terminal hydrolase L3 (UCHL3) was reference points for evaluating cell quality. Comparing outcomes in single versus to sequential media, the odds of embryos developing to the 8C stage on d3 were 2.34 time greater (P = .06). On d5, more embryos reached the blastocyst stage (P = <.0001), hatched, and had significantly more trophoblast cells (P = .005) contributing to the increased total cell number. Also at d5, localization of distinct cytoplasmic UCHL1 and nuclear UCHL3 was found in high-quality hatching blastocysts. Localization of UCHL1 and UCHL3 was diffuse and inappropriately dispersed throughout the cytoplasm in low-quality nonhatching blastocysts. Single medium yields greater cell numbers, an increased growth rate, and more hatching of murine embryos. Cytoplasmic UCHL1 and nuclear UHCL3 localization patterns were indicative of embryo quality. Our conclusions are limited to murine embryos but one might speculate that single medium may also be more beneficial for human embryo culture. Human embryo studies are needed. © The Author(s) 2015.

Mutation of the XIST gene upregulates expression of X-linked genes but decreases the developmental rates of cloned male porcine embryos.

PubMed

Yang, Yang; Wu, Dan; Liu, Dewu; Shi, Junsong; Zhou, Rong; He, Xiaoyan; Quan, Jianping; Cai, Gengyuan; Zheng, Enqin; Wu, Zhenfang; Li, Zicong

2017-06-01

XIST is an X-linked, non-coding gene responsible for the cis induction of X-chromosome inactivation (XCI). Knockout of the XIST allele on an active X chromosome abolishes erroneous XCI and enhances the in vivo development of cloned mouse embryos by more than 10-fold. This study aimed to investigate whether a similar manipulation would improve cloning efficiency in pigs. A male, porcine kidney cell line containing an EGFP insert in exon 1 of the XIST gene, resulting in a knockout allele (XIST-KO), was generated by homologous recombination using transcription activator-like effector nucleases (TALENs). The expression of X-linked genes in embryos cloned from the XIST-KO kidney cells was significantly higher than in male embryos cloned from wild-type (WT) kidney cells, but remained lower than that of in vivo fertilization-produced counterparts. The XIST-KO cloned embryos also had a significantly lower blastocyst rate and a reduced full-term development rate compared to cloned WT embryos. These data suggested that while mutation of a XIST gene can partially rescue abnormal XCI, it cannot improve the developmental efficiency of cloned male porcine embryos-a deficiency that may be caused by incomplete rescue of abnormal XCI and/or by long-term drug selection of the XIST-KO nuclear donor cells, which might adversely affect the developmental efficiency of embryos created from them. © 2017 Wiley Periodicals, Inc.

Increasing of blastocyst rate and gene expression in co-culture of bovine embryos with adult adipose tissue-derived mesenchymal stem cells.

PubMed

Miranda, Moysés S; Nascimento, Hamilton S; Costa, Mayra P R; Costa, Nathália N; Brito, Karynne N L; Lopes, Cinthia T A; Santos, Simone S D; Cordeiro, Marcela S; Ohashi, Otávio M

2016-10-01

Despite advances in the composition of defined embryo culture media, co-culture with somatic cells is still used for bovine in vitro embryo production (IVEP) in many laboratories worldwide. Granulosa cells are most often used for this purpose, although recent work suggests that co-culture with stem cells of adult or embryonic origin or their derived biomaterials may improve mouse, cattle, and pig embryo development. In experiment 1, in vitro produced bovine embryos were co-cultured in the presence of two concentrations of bovine adipose tissue-derived mesenchymal cells (b-ATMSCs; 10 3 and 10 4 cells/mL), in b-ATMSC preconditioned medium (SOF-Cond), or SOF alone (control). In experiment 2, co-culture with 10 4 b-ATMSCs/mL was compared to the traditional granulosa cell co-culture system (Gran). In experiment 1, co-culture with 10 4 b-ATMSCs/mL improved blastocyst rates in comparison to conditioned and control media (p < 0.05). Despite that it did not show difference with 10 3 b-ATMSCs/mL (p = 0.051), group 10 4 b-ATMSCs/mL yielded higher results of blastocyst production. In experiment 2, when compared to group Gran, co-culture with 10 4 b-ATMSCs/mL improved not only blastocyst rates but also quality as assessed by increased total cell numbers and mRNA expression levels for POU5F1 and G6PDH (p < 0.05). Co-culture of bovine embryos with b-ATMSCs was more beneficial than the traditional co-culture system with granulosa cells. We speculate that the microenvironmental modulatory potential of MSCs, by means of soluble substances and exosome secretions, could be responsible for the positive effects observed. Further experiments must be done to evaluate if this beneficial effect in vitro also translates to an increase in offspring following embryo transfer. Moreover, this study provides an interesting platform to study the basic requirements during preimplantation embryo development, which, in turn, may aid the improvement of embryo culture protocols in bovine and other species.

Epigenetic disorders and altered gene expression after use of Assisted Reproductive Technologies in domestic cattle

PubMed Central

Urrego, Rodrigo; Rodriguez-Osorio, Nélida; Niemann, Heiner

2014-01-01

The use of Assisted Reproductive Technologies (ARTs) in modern cattle breeding is an important tool for improving the production of dairy and beef cattle. A frequently employed ART in the cattle industry is in vitro production of embryos. However, bovine in vitro produced embryos differ greatly from their in vivo produced counterparts in many facets, including developmental competence. The lower developmental capacity of these embryos could be due to the stress to which the gametes and/or embryos are exposed during in vitro embryo production, specifically ovarian hormonal stimulation, follicular aspiration, oocyte in vitro maturation in hormone supplemented medium, sperm handling, gamete cryopreservation, and culture of embryos. The negative effects of some ARTs on embryo development could, at least partially, be explained by disruption of the physiological epigenetic profile of the gametes and/or embryos. Here, we review the current literature with regard to the putative link between ARTs used in bovine reproduction and epigenetic disorders and changes in the expression profile of embryonic genes. Information on the relationship between reproductive biotechnologies and epigenetic disorders and aberrant gene expression in bovine embryos is limited and novel approaches are needed to explore ways in which ARTs can be improved to avoid epigenetic disorders. PMID:24709985

Insights on blastomere nuclearity.

PubMed

Gil, Mónica; D'Ommar, Gustavo; Póo, Maria E; Sosa, Anna; Piras, Marta; Piras, Romano; Rísquez, Francisco

2007-01-01

To analyze the results of our transferred embryos, especially those that "changed" their blastomere nuclearity from Multinucleated (MN) to Mono-nucleated during development. Pregnancies where at least one MN embryo was transferred were retrospectively evaluated and categorized in order to record and follow-up on the ones that were implanted. Embryos were classified as normal (when all blastomeres were mono-nucleated on day one and two of development), corrected (multinucleated embryos on day one that became mono-nucleated on day two) and non-corrected (multinucleated either on day one, on day two or both days). There were 633 transfer cycles analyzed. Thirty-three percent (206) had at least one embryo with a MN blastomere at a given stage of development. Pregnancy and implantation rates were 29.0% and 19.0% for the group of exclusively mono-nucleated embryo transfers, and 28.6% and 15.8% for the group with at least one MN embryo transferred. The pregnancy outcome for "corrected" and "non-corrected" embryos could be corroborated unequivocally in only 9 cases, with an outcome of 8 and 4 normal babies, respectively. Because the amount of data analyzed is not satisfactorily large, differences were not significantly different; however, a trend may exist showing that normal at term pregnancies obtained from corrected embryos are more likely to occur than those from non-corrected embryos. Nuclear observation on a daily basis should be one of the strategies used to select the best embryos for transferring, to improve implantation rates and avoid multiple pregnancies.

Scriptaid and 5-aza-2'deoxycytidine enhanced expression of pluripotent genes and in vitro developmental competence in interspecies Black-footed cat cloned embryos

USGS Publications Warehouse

Gómez, M. C.; Biancardi, M.N.; Jenkins, J.A.; Dumas, C.; Galiguis, J.; Wang, G.; Earle Pope, C.

2012-01-01

Somatic cell nuclear transfer offers the possibility of preserving endangered species including the black-footed cat, which is threatened with extinction. The effectiveness and efficiency of somatic cell nuclear transfer (SCNT) depends on a variety of factors, but 'inappropriate epigenetic reprogramming of the transplanted nucleus is the primary cause of the developmental failure of cloned embryos. Abnormal epigenetic events such as DNA methylation and histone modifications during SCNT perturb the expression of imprinted and pluripotent-related genes that, consequently, may result in foetal and neonatal abnormalities. We have demonstrated that pregnancies can be established after transfer of black-footed cat cloned embryos into domestic cat recipients, but none of the implanted embryos developed to term and the foetal failure has been associated to aberrant reprogramming in cloned embryos. There is growing evidence that modifying the epigenetic pattern of the chromatin template of both donor cells and reconstructed embryos with a combination of inhibitors of histone deacetylases and DNA methyltransferases results in enhanced gene reactivation and improved in vitro and in vivo developmental competence. Epigenetic modifications of the chromatin template of black-footed cat donor cells and reconstructed embryos with epigenetic-modifying compounds enhanced in vitro development, and regulated the expression of pluripotent genes, but these epigenetic modifications did not improve in vivo developmental competence.

Use of infrared imaging to predict the developmental stages and differences in chicken embryos exposed to different photoperiods

NASA Astrophysics Data System (ADS)

Frederick, Rebecca A.; Hsieh, Sheng-Jen; Palomares, Benjamin Giron

2012-06-01

Monitoring development of chicken embryos allows determination of when an egg is not developing and when eggs are close to hatching for more efficient production. Research has been conducted on the effects of temperature fluctuations and light exposure on embryo development; similarities between chicken and mammal embryos; and the use of MRI, tomography, and ultrasound to view specific areas and processes within the embryo. However, there has been little exploration of the use of infrared imaging as a non-destructive method for analyzing and predicting embryonic development. In this study, we built an automated loading system for image acquisition. Pilot experiments were conducted to determine the overall scanning time and scanning frequency. A batch of fertilized eggs was scanned each day as the embryos continued to grow. The captured images were analyzed and categorized into three stages: Stage 1 (days 1 to 7), Stage 2 (days 8 to 14), and Stage 3 (days 15 to 21). The temperature data abstracted from the captured images were divided into two groups. Group 1, consisting of two-thirds of the data, was used to construct a model. Group 2, consisting of one-third of the data, was used to evaluate the predictive accuracy of the model. A three-layer artificial neural network model was developed to predict embryo development stage given a temperature profile. Results suggest that the neural network model is sufficient to predict embryo development stage with good accuracy of 75%. Accuracy can likely be improved if more data sets for each development stage are available.

Improvement of porcine cloning efficiency by trichostain A through early-stage induction of embryo apoptosis.

PubMed

Ji, Qianqian; Zhu, Kongju; Liu, Zhiguo; Song, Zhenwei; Huang, Yuankai; Zhao, Haijing; Chen, Yaosheng; He, Zuyong; Mo, Delin; Cong, Peiqing

2013-03-15

Trichostain A (TSA), an inhibitor of histone deacetylases, improved developmental competence of SCNT embryos in many species, apparently by improved epigenetic reprogramming. The objective of the present study was to determine the effects of TSA-induced apoptosis in cloned porcine embryos. At various developmental stages, a comet assay and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining were used to detect apoptosis, and real-time polymerase chain reaction was used to assess expression of genes related to apoptosis and pluripotency. In this study, TSA significantly induced apoptosis (in a dose-dependent manner) at the one-, two-, and four-cell stages. However, in blastocyst stage embryos, TSA decreased the apoptotic index (P < 0.05). Expression levels of Caspase 3 were higher in TSA-treated versus control embryos at the two-cell stage (not statistically significant). The expression ratio of antiapoptotic Bcl-xl gene to proapoptotic Bax gene, an indicator of antiapoptotic potential, was higher in TSA-treated groups at the one-, two-, and four-cell and blastocyst stages. Furthermore, expression levels of pluripotency-related genes, namely, Oct4 and Nanog, were elevated at the morula stage (P < 0.05) in TSA treatment groups. We concluded that inducing apoptosis might be a mechanism by which TSA promotes development of reconstructed embryos. At the initial stage of apoptosis induction, abnormal cells were removed, thereby enhancing proliferation of healthy cells and improving embryo quality. Copyright © 2013 Elsevier Inc. All rights reserved.

Somatic embryogenesis in Hedychium bousigonianum

USDA-ARS?s Scientific Manuscript database

An efficient primary somatic embryo (SE) and secondary somatic embryo (SSE) production system was developed for the ornamental ginger Hedychium bousigonianum Pierre ex Gagnepain. Addition of two ethylene inhibitors, salicylic acid (SA) and silver nitrate (AgNO3), to the culture media improved the sy...

Effect of postactivation treatment with latrunculin A on in vitro and in vivo development of cloned embryos derived from kidney fibroblasts of an aged Clawn miniature boar.

PubMed

Himaki, Takehiro; Mizobe, Yamato; Tsuda, Kenichirou; Suetomo, Masashi; Yamakuchi, Hiroshi; Miyoshi, Kazuchika; Takao, Sonshin; Yoshida, Mitsutoshi

2012-01-01

The objective of this study was to examine the effect of postactivation treatment with latrunculin A (LatA), an actin polymerization inhibitor, on in vitro and in vivo development of somatic cell nuclear transfer (SCNT) embryos derived from kidney fibroblasts of an aged Clawn miniature boar (12 years old). After electric activation, SCNT embryos were treated with 0, 0.5 or 1 μM LatA and cultured in vitro. The rate of blastocyst formation was significantly higher (P<0.05) in SCNT embryos treated with 0.5 μM LatA (38%) than those in control (14%). When cloned embryos treated with 0.5 μM LatA were transferred into the oviducts of two recipient miniature gilts to assess their development in vivo, both recipients became pregnant; one maintained pregnancy to term, and a live piglet (weighing 220 g) was delivered by Caesarean section. The results of this study indicated that the postactivation treatment with LatA was effective in improving in vitro developmental capacity of SCNT miniature pig embryos derived from kidney fibroblasts of an aged animal and that miniature pig cloned embryos treated with LatA had the ability to develop to term.

In vitro culture of individual mouse preimplantation embryos: the role of embryo density, microwells, oxygen, timing and conditioned media.

PubMed

Kelley, Rebecca L; Gardner, David K

2017-05-01

Single embryo culture is suboptimal compared with group culture, but necessary for embryo monitoring, and culture systems should be improved for single embryos. Pronucleate mouse embryos were used to assess the effect of culture conditions on single embryo development. Single culture either before or after compaction reduced cell numbers (112.2 ± 3.1; 110.2 ± 3.5) compared with group culture throughout (127.0 ± 3.4; P < 0.05). Reduction of media volume from 20 µl to 2 µl increased blastocyst cell numbers in single embryos cultured in 5% oxygen (84.4 ± 3.2 versus 97.8 ± 2.8; P < 0.05), but not in 20% oxygen (55.2 ± 2.9 versus 57.1 ± 2.8). Culture in microwell plates for the EmbryoScope and Primo Vision time-lapse systems changed cleavage timings and increased inner cell mass cell number (24.1 ± 1.0; 23.4 ± 1.2) compared with a 2 µl microdrop (18.4 ± 1.0; P < 0.05). Addition of embryo-conditioned media to single embryos increased hatching rate and blastocyst cell number (91.5 ± 4.7 versus 113.1 ± 4.4; P < 0.01). Single culture before or after compaction is therefore detrimental; oxygen, media volume and microwells influence single embryo development; and embryo-conditioned media may substitute for group culture. Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Media composition: growth factors.

PubMed

Hegde, Aparna; Behr, Barry

2012-01-01

Despite the fact that the fundamental principle underlying the most common method of culture media constitution is that of mimicking the natural environment of the preimplantation embryo, one major difference that remains between current embryo culture media and in vivo conditions is the absence of growth factors in vitro. Numerous growth factors are known to be present in the in vivo environment of human and nonhuman preimplantation embryos, often with peak concentrations corresponding to when fertilization and preimplantation embryo growth would occur. Although these growth factors are found in very small concentrations, they have a profound effect on tissue growth and differentiation through attachment to factor-specific receptors on cell surfaces. Receptors for many different growth factors have also been detected in human preimplantation embryos. Preimplantation embryos themselves express many growth factors. The growth factors and receptors are metabolically costly to produce, and thus their presence in the environment of the preimplantation embryo and in the embryo respectively strongly implies that embryos are designed to encounter and respond to the corresponding factors. Studies of embryo coculture also indirectly suggest that growth factors can improve in vitro development. Several animal and human studies attest to a probable beneficial effect of addition of growth factors to culture media. However, there is still ambiguity regarding the exact role of growth factors in embryonic development, the optimal dose of growth factors to be added to culture media, the combinatorial effect and endocrine of growth factors in embryonic development.

A minimally invasive methodology based on morphometric parameters for day 2 embryo quality assessment.

PubMed

Molina, Inmaculada; Lázaro-Ibáñez, Elisa; Pertusa, Jose; Debón, Ana; Martínez-Sanchís, Juan Vicente; Pellicer, Antonio

2014-10-01

The risk of multiple pregnancy to maternal-fetal health can be minimized by reducing the number of embryos transferred. New tools for selecting embryos with the highest implantation potential should be developed. The aim of this study was to evaluate the ability of morphological and morphometric variables to predict implantation by analysing images of embryos. This was a retrospective study of 135 embryo photographs from 112 IVF-ICSI cycles carried out between January and March 2011. The embryos were photographed immediately before transfer using Cronus 3 software. Their images were analysed using the public program ImageJ. Significant effects (P < 0.05), and higher discriminant power to predict implantation were observed for the morphometric embryo variables compared with morphological ones. The features for successfully implanted embryos were as follows: four cells on day 2 of development; all blastomeres with circular shape (roundness factor greater than 0.9), an average zona pellucida thickness of 13 µm and an average of 17695.1 µm² for the embryo area. Embryo size, which is described by its area and the average roundness factor for each cell, provides two objective variables to consider when predicting implantation. This approach should be further investigated for its potential ability to improve embryo scoring. Copyright © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Supplementation with CTGF, SDF1, NGF, and HGF promotes ovine in vitro oocyte maturation and early embryo development.

PubMed

Wang, D H; Ren, J; Zhou, C J; Han, Z; Wang, L; Liang, C G

2018-05-17

The strategies for improving the in vitro maturation (IVM) of domestic animal oocytes focus on promoting nuclear and cytoplasmic maturation. The identification of paracrine factors and their supplementation in the culture medium represent effective approaches for oocyte maturation and embryo development. This study investigated the effects of paracrine factor supplementation including connective tissue growth factor (CTGF), nerve growth factor (NGF), hepatocyte growth factor (HGF), and stromal derived factor 1 (SDF1) on ovine oocytes and early parthenogenetic embryos using an in vitro culture system. First, we identified the optimal concentrations of CTGF (30 ng/mL), SDF1 (10 ng/mL), NGF (3 ng/mL), and HGF (100 ng/mL) for promoting oocyte maturation, which combined, induced nuclear maturation in 94.19% of oocytes. This combination also promoted cumulus cell expansion and inhibited oocyte/cumulus apoptosis, while enabling a larger proportion (33.04%) of embryos to develop into blastocysts than in the controls and prevented embryo apoptosis. These novel findings demonstrate that the paracrine factors CTGF, SDF1, NGF, and HGF facilitate ovine oocyte and early parthenogenetic embryo development in vitro. Thus, supplementation with these factors may help optimize the IVM of ovine oocytes and early parthenogenetic embryo development strategies. Copyright © 2018 Elsevier Inc. All rights reserved.

How does embryo manipulation fit into present and future pig reproduction?

PubMed

Polge, C

1985-01-01

Available techniques for the collection and direct transplantation of pig embryos are simple and efficient and could be used for the expansion of new lines, for increasing selection pressure in nucleus herds and for extracting healthy stock from a diseased source. However, the reduced viability of pig embryos during culture in vitro and the inability as yet to preserve them by deep-freezing impose limits to the use of embryo transplantation for the export or import of potential breeding stock. The efficiency of breeding schemes could be improved by the sexing of embryos and the possibility of producing genetically identical twins or quadruplets by micromanipulation of embryos should improve the efficiency of animal experimentation. Chimaerism may be used to rescue embryos of a non-viable genotype such as parthenotes or those derived by hybridization, but the greatest revolution in pig breeding may be brought about by the introduction of foreign cloned genes into eggs and the production of transgenic animals. Eggs at an appropriate stage for microinjection may be provided in the future by techniques for the maturation and fertilization of oocytes in vitro. Animal breeders should be aware of the potential impact of techniques for the manipulation of eggs and embryos on future developments in animal production.

Development of ovine embryos in synthetic oviductal fluid containing amino acids at oviductal fluid concentrations.

PubMed

Walker, S K; Hill, J L; Kleemann, D O; Nancarrow, C D

1996-09-01

The effects of supplementing synthetic oviductal fluid (SOF) with amino acids, at oviductal fluid concentrations, on the development of ovine in vitro-matured/in vitro-fertilized embryos was examined in three experiments. In the first, embryo development in SOF, SOF + 2% human serum (HS), SOF + 20% HS, and SOF + BSA, with and without amino acid supplementation, was examined. Development of zygotes to the blastocyst and hatching blastocyst stages was highest in medium containing 20% HS (64.8% and 54.4%, respectively) irrespective of amino acid supplementation. However, supplementation was significantly beneficial in all other media, with up to 42.1% of zygotes developing into hatching blastocysts. In these media, supplementation also significantly increased the mean number of nuclei per newly formed blastocyst (up to a mean of 70.8) and reduced the time during which blastocysts formed. Experiment 2 was an examination of the effect on embryo development of three amino acid preparations (oviduct amino acid concentrations vs. Eagle's Basal Medium (BME) essential + Minimum Essential Medium (MEM) nonessential vs. MEM essential + MEM nonessential concentrations) and the presence or absence of BSA. Both the amino acid and BSA treatments significantly influenced the percentage of zygotes that developed to the hatching blastocyst stage but not to the blastocyst stage. The preferred medium contained amino acids at oviductal fluid concentrations and BSA (54.5% hatching rate). The amino acid treatments did not significantly influence the mean number of nuclei per newly formed blastocyst, but the addition of BSA had a significant effect (70.7 +/- 1.14 vs. 75.7 +/- 1.13). In experiment 3, embryo development to Day 13 was examined after culture in SOF containing amino acids at oviductal fluid concentrations. Embryos were cultured in the presence of either BSA, polyvinyl alcohol (PVA), or no additional supplement and were transferred to recipient ewes on either Day 0 (after in vitro fertilization), 3, or 5. The addition of BSA or PVA had no significant effect, but significantly more embryos developed to Day 13 following transfer on Day 0 (60.0%) than on either Day 3 or 5 (overall 45.4%). It is concluded that SOF containing oviductal fluid concentrations of amino acids 1) facilitates the development of a high percentage (57.5%) of blastocysts, 2) improves embryo morphology compared with that observed in medium containing HS, 3) significantly improves hatching rates compared with those obtained in SOF containing commercially available preparations of amino acids, and 4) produces embryos with relatively high levels of viability to Day 13 of pregnancy.

Studies on Somatic Embryogenesis in Sweetpotato

NASA Technical Reports Server (NTRS)

Bennett, J. Rasheed; Prakash, C. S.

1997-01-01

The purpose of this study was to improve the somatic embryo (SE) system for plant production of sweetpotato Ipomoea batatas L.(Lam)l. Explants isolated from SE-derived sweet potato plants were compared with control (non SE-derived) plants for their competency for SE production. Leaf explants were cultured on Murashige-Skoog (MS) medium with 2,4-dichlorophenoxy acetic acid (0.2 mg/L) and 6-benzylaminopurine (2.5 mg/L) for 2 weeks in darkness and transferred to MS medium with abscisic acid (2.5 Explants isolated from those plants developed through somatic embryo-genesis produced new somatic embryos rapidly and in higher frequency than those isolated from control plants. They also appeared to grow faster in tissue culture than the control plants. Current studies in the laboratory are examining whether plants derived from a cyclical embryogenesis system (five cycles) would have any further positive impact on the rapidity and frequency of somatic embryo development. More detailed studies using electron microscopy are expected to show the point of origin of the embryos and to allow determination of their quality throughout the cyclical process. This study may facilitate improved plant micropropagation, gene transfer and germplasm conservation in sweet potato.

Glassfrog embryos hatch early after parental desertion.

PubMed

Delia, Jesse R J; Ramírez-Bautista, Aurelio; Summers, Kyle

2014-06-22

Both parental care and hatching plasticity can improve embryo survival. Research has found that parents can alter hatching time owing to a direct effect of care on embryogenesis or via forms of care that cue the hatching process. Because parental care alters conditions critical for offspring development, hatching plasticity could allow embryos to exploit variation in parental behaviour. However, this interaction of parental care and hatching plasticity remains largely unexplored. We tested the hypothesis that embryos hatch early to cope with paternal abandonment in the glassfrog Hyalinobatrachium fleischmanni (Centrolenidae). We conducted male-removal experiments in a wild population, and examined embryos' response to conditions with and without fathers. Embryos hatched early when abandoned, but extended development in the egg stage when fathers continued care. Paternal care had no effect on developmental rate. Rather, hatching plasticity was due to embryos actively hatching at different developmental stages, probably in response to deteriorating conditions without fathers. Our experimental results are supported by a significant correlation between the natural timing of abandonment and hatching in an unmanipulated population. This study demonstrates that embryos can respond to conditions resulting from parental abandonment, and provides insights into how variation in care can affect selection on egg-stage adaptations.

Glassfrog embryos hatch early after parental desertion

PubMed Central

Delia, Jesse R. J.; Ramírez-Bautista, Aurelio; Summers, Kyle

2014-01-01

Both parental care and hatching plasticity can improve embryo survival. Research has found that parents can alter hatching time owing to a direct effect of care on embryogenesis or via forms of care that cue the hatching process. Because parental care alters conditions critical for offspring development, hatching plasticity could allow embryos to exploit variation in parental behaviour. However, this interaction of parental care and hatching plasticity remains largely unexplored. We tested the hypothesis that embryos hatch early to cope with paternal abandonment in the glassfrog Hyalinobatrachium fleischmanni (Centrolenidae). We conducted male-removal experiments in a wild population, and examined embryos' response to conditions with and without fathers. Embryos hatched early when abandoned, but extended development in the egg stage when fathers continued care. Paternal care had no effect on developmental rate. Rather, hatching plasticity was due to embryos actively hatching at different developmental stages, probably in response to deteriorating conditions without fathers. Our experimental results are supported by a significant correlation between the natural timing of abandonment and hatching in an unmanipulated population. This study demonstrates that embryos can respond to conditions resulting from parental abandonment, and provides insights into how variation in care can affect selection on egg-stage adaptations. PMID:24789892

Embryos aggregation improves development and imprinting gene expression in mouse parthenogenesis.

PubMed

Bai, Guang-Yu; Song, Si-Hang; Wang, Zhen-Dong; Shan, Zhi-Yan; Sun, Rui-Zhen; Liu, Chun-Jia; Wu, Yan-Shuang; Li, Tong; Lei, Lei

2016-04-01

Mouse parthenogenetic embryonic stem cells (PgESCs) could be applied to study imprinting genes and are used in cell therapy. Our previous study found that stem cells established by aggregation of two parthenogenetic embryos at 8-cell stage (named as a2 PgESCs) had a higher efficiency than that of PgESCs, and the paternal expressed imprinting genes were observably upregulated. Therefore, we propose that increasing the number of parthenogenetic embryos in aggregation may improve the development of parthenogenetic mouse and imprinting gene expression of PgESCs. To verify this hypothesis, we aggregated four embryos together at the 4-cell stage and cultured to the blastocyst stage (named as 4aPgB). qPCR detection showed that the expression of imprinting genes Igf2, Mest, Snrpn, Igf2r, H19, Gtl2 in 4aPgB were more similar to that of fertilized blastocyst (named as fB) compared to 2aPgB (derived from two 4-cell stage parthenogenetic embryos aggregation) or PgB (single parthenogenetic blastocyst). Post-implantation development of 4aPgB extended to 11 days of gestation. The establishment efficiency of GFP-a4 PgESCs which derived from GFP-4aPgB is 62.5%. Moreover, expression of imprinting genes Igf2, Mest, Snrpn, notably downregulated and approached the level of that in fertilized embryonic stem cells (fESCs). In addition, we acquired a 13.5-day fetus totally derived from GFP-a4 PgESCs with germline contribution by 8-cell under zona pellucida (ZP) injection. In conclusion, four embryos aggregation improves parthenogenetic development, and compensates imprinting genes expression in PgESCs. It implied that a4 PgESCs could serve as a better scientific model applied in translational medicine and imprinting gene study. © 2016 Japanese Society of Developmental Biologists.

Impact of the Z potential technique on reducing the sperm DNA fragmentation index, fertilization rate and embryo development.

PubMed

Duarte, Carlos; Núñez, Víctor; Wong, Yat; Vivar, Carlos; Benites, Elder; Rodriguez, Urso; Vergara, Carlos; Ponce, Jorge

2017-12-01

In assisted reproduction procedures, we need to develop and enhance new protocols to optimize sperm selection. The aim of this study is to evaluate the ability of the Z potential technique to select sperm with intact DNA in non-normospermic patients and evaluate the impact of this selection on embryonic development. We analyzed a total of 174 human seminal samples with at least one altered parameter. We measured basal, post density gradients, and post density gradients + Z potential DNA fragmentation index. To evaluate the impact of this technique on embryo development, 54 cases were selected. The embryo development parameters evaluated were fertilization rate, cleavage rate, top quality embryos at the third day and blastocysts rate. We found significant differences in the study groups when we compared the sperm fragmentation index by adding the Z potential technique to density gradient selection vs. density gradients alone. Furthermore, there was no significant difference in the embryo development parameters between the low sperm fragmentation index group vs. the moderate and high sperm fragmentation index groups, when selecting sperms with this new technique. The Z potential technique is a very useful tool for sperm selection; it significantly reduces the DNA fragmentation index and improves the parameters of embryo development. This technique could be considered routine for its simplicity and low cost.

Should extended blastocyst culture include Day 7?

PubMed

Hammond, Elizabeth R; Cree, Lynsey M; Morbeck, Dean E

2018-06-01

Extended culture to the blastocyst stage is widely practised, improving embryo selection and promoting single embryo transfer. Selection of useable blastocysts typically occurs on Days 5 and 6 of embryo culture. Embryos not suitable for transfer, biopsy or cryopreservation after Day 6 are routinely discarded. Some embryos develop at a slower rate, however, forming blastocysts on Day 7 of culture. Day 7 blastocysts can be viable, they can be of top morphological grade, euploid and result in a healthy live birth. Since ending culture on Day 6 is current practice in most clinics, viable Day 7 blastocysts may be prematurely discarded. Although Day 7 blastocysts make up only 5% of useable blastocysts, those which are suitable for cryopreservation or biopsy are clinically significant. Overall, culturing embryos an additional day increases the number of useable embryos per IVF cycle and provides further opportunity for pregnancy for patients, especially those who have only a few or low-quality blastocysts.

Promotion of human early embryonic development and blastocyst outgrowth in vitro using autocrine/paracrine growth factors.

PubMed

Kawamura, Kazuhiro; Chen, Yuan; Shu, Yimin; Cheng, Yuan; Qiao, Jie; Behr, Barry; Pera, Renee A Reijo; Hsueh, Aaron J W

2012-01-01

Studies using animal models demonstrated the importance of autocrine/paracrine factors secreted by preimplantation embryos and reproductive tracts for embryonic development and implantation. Although in vitro fertilization-embryo transfer (IVF-ET) is an established procedure, there is no evidence that present culture conditions are optimal for human early embryonic development. In this study, key polypeptide ligands known to be important for early embryonic development in animal models were tested for their ability to improve human early embryo development and blastocyst outgrowth in vitro. We confirmed the expression of key ligand/receptor pairs in cleavage embryos derived from discarded human tri-pronuclear zygotes and in human endometrium. Combined treatment with key embryonic growth factors (brain-derived neurotrophic factor, colony-stimulating factor, epidermal growth factor, granulocyte macrophage colony-stimulating factor, insulin-like growth factor-1, glial cell-line derived neurotrophic factor, and artemin) in serum-free media promoted >2.5-fold the development of tri-pronuclear zygotes to blastocysts. For normally fertilized embryos, day 3 surplus embryos cultured individually with the key growth factors showed >3-fold increases in the development of 6-8 cell stage embryos to blastocysts and >7-fold increase in the proportion of high quality blastocysts based on Gardner's criteria. Growth factor treatment also led to a 2-fold promotion of blastocyst outgrowth in vitro when day 7 surplus hatching blastocysts were used. When failed-to-be-fertilized oocytes were used to perform somatic cell nuclear transfer (SCNT) using fibroblasts as donor karyoplasts, inclusion of growth factors increased the progression of reconstructed SCNT embryos to >4-cell stage embryos. Growth factor supplementation of serum-free cultures could promote optimal early embryonic development and implantation in IVF-ET and SCNT procedures. This approach is valuable for infertility treatment and future derivation of patient-specific embryonic stem cells.

Histone Deacetylase Inhibitor Improves the Development and Acetylation Levels of Cat–Cow Interspecies Cloned Embryos

PubMed Central

Wittayarat, Manita; Sato, Yoko; Do, Lanh Thi Kim; Morita, Yasuhiro; Chatdarong, Kaywalee; Techakumphu, Mongkol; Taniguchi, Masayasu

2013-01-01

Abstract Abnormal epigenetic reprogramming, such as histone acetylation, might cause low efficiency of interspecies somatic cell nuclear transfer (iSCNT). This study was conducted to evaluate the effects of trichostatin A (TSA) on the developmental competence and histone acetylation of iSCNT embryos reconstructed from cat somatic cells and bovine cytoplasm. The iSCNT cat and parthenogenetic bovine embryos were treated with various concentrations of TSA (0, 25, 50, or 100 nM) for 24 h, respectively, following fusion and activation. Treatment with 50 nM TSA produced significantly higher rates of cleavage and blastocyst formation (84.3% and 4.6%, respectively) of iSCNT embryos than the rates of non-TSA–treated iSCNT embryos (63.8% and 0%, respectively). Similarly, the treatment of 50 nM TSA increased the blastocyst formation rate of parthenogenetic bovine embryos. The acetylation levels of histone H3 lysine 9 (H3K9) in the iSCNT embryos with the treatment of 50 nM TSA were similar to those of in vitro–fertilized embryos and significantly higher (p<0.05) than those of non-TSA–treated iSCNT embryos (control), irrespective of the embryonic development stage (two-cell, four-cell, and eight-cell stages). These results indicated that the treatment of 50 nM TSA postfusion was beneficial for development to the blastocyst stage of iSCNT cat embryos and correlated with the increasing levels of acetylation at H3K9. PMID:23790014

Pluripotency maintenance in mouse somatic cell nuclear transfer embryos and its improvement by treatment with the histone deacetylase inhibitor TSA.

PubMed

Hai, Tang; Hao, Jie; Wang, Liu; Jouneau, Alice; Zhou, Qi

2011-02-01

Reprogramming of somatic cells to pluripotency can be achieved by nuclear transfer into enucleated oocytes (SCNT). A key event of this process is the demethylation of the Oct4 gene and its temporally and spatially regulated expression. Different studies have shown that it occurs abnormally in some SCNT embryos. TSA is a histone deacetylase inhibitor known to increase the efficiency of development to term of SCNT embryos, but its impact on the developmental features of SCNT embryos is poorly understood. Here, we have followed the fate of the pluripotent cells within SCNT embryos, from the late blastocyst to the early epiblast prior to gastrulation. Our data show a delay in development correlated with a defect in forming and maintaining a correct number of Oct4 expressing ICM and epiblast cells in SCNT embryos. As a consequence, during the outgrowth phase of embryonic stem cell derivation as well as during diapause in vivo, part of the SCNT blastocysts completely lose their ICM cells. Meanwhile, the others display a correctly reprogrammed ICM compatible with the derivation of ES cells and development of the epiblast. Our data also indicate that TSA favors the establishment of pluripotency in SCNT embryos.

Non-destructive monitoring of mouse embryo development and its qualitative evaluation at the molecular level using Raman spectroscopy

NASA Astrophysics Data System (ADS)

Ishigaki, Mika; Hashimoto, Kosuke; Sato, Hidetoshi; Ozaki, Yukihiro

2017-03-01

Current research focuses on embryonic development and quality not only by considering fundamental biology, but also by aiming to improve assisted reproduction technologies, such as in vitro fertilization. In this study, we explored the development of mouse embryo and its quality based on molecular information, obtained nondestructively using Raman spectroscopy. The detailed analysis of Raman spectra measured in situ during embryonic development revealed a temporary increase in protein content after fertilization. Proteins with a β-sheet structure—present in the early stages of embryonic development—are derived from maternal oocytes, while α-helical proteins are additionally generated by switching on a gene after fertilization. The transition from maternal to embryonic control during development can be non-destructively profiled, thus facilitating the in situ assessment of structural changes and component variation in proteins generated by metabolic activity. Furthermore, it was indicated that embryos with low-grade morphology had high concentrations of lipids and hydroxyapatite. This technique could be used for embryo quality testing in the future.

Big Animal Cloning Using Transgenic Induced Pluripotent Stem Cells: A Case Study of Goat Transgenic Induced Pluripotent Stem Cells.

PubMed

Song, Hui; Li, Hui; Huang, Mingrui; Xu, Dan; Wang, Ziyu; Wang, Feng

2016-02-01

Using of embryonic stem cells (ESCs) could improve production traits and disease resistance by improving the efficiency of somatic cell nuclear transfer (SCNT) technology. However, robust ESCs have not been established from domestic ungulates. In the present study, we generated goat induced pluripotent stem cells (giPSCs) and transgenic cloned dairy goat induced pluripotent stem cells (tgiPSCs) from dairy goat fibroblasts (gFs) and transgenic cloned dairy goat fibroblasts (tgFs), respectively, using lentiviruses that contained hOCT4, hSOX2, hMYC, and hKLF4 without chemical compounds. The giPSCs and tgiPSCs expressed endogenous pluripotent markers, including OCT4, SOX2, MYC, KLF4, and NANOG. Moreover, they were able to maintain a normal karyotype and differentiate into derivatives from all three germ layers in vitro and in vivo. Using SCNT, tgFs and tgiPSCs were used as donor cells to produce embryos, which were named tgF-Embryos and tgiPSC-Embryos. The fusion rates and cleavage rates had no significant differences between tgF-Embryos and tgiPSC-Embryos. However, the expression of IGF-2, which is an important gene associated with embryonic development, was significantly lower in tgiPSC-Embryos than in tgF-Embryos and was not significantly different from vivo-Embryos.

Effects of growth hormone on the ultrastructure of bovine preimplantation embryos.

PubMed

Kölle, Sabine; Stojkovic, Miodrag; Reese, Sven; Reichenbach, Horst-Dieter; Wolf, Eckhard; Sinowatz, Fred

2004-07-01

Growth hormone (GH) has recently been shown to promote the development of preimplantation embryos. The aim of our study was therefore to analyze the effects of GH on the morphology and ultrastructure of the cells of bovine preimplantation embryos produced by in vitro fertilization (IVF). In order to determine the physiologically optimal morphology of blastocysts, ex vivo embryos obtained by uterine flushing were also included in the study. As shown by transmission electron microscopy, treatment with GH induced the elimination of glycogen storage in cells of the inner cell mass of 7-day-old embryos. GH also stimulated the exocytosis of lipid vesicles in the inner cell mass and trophectoderm cells of these embryos. Quantitative analysis of micrographs demonstrated a higher volume density of embryonic mitochondria in 7-day-old embryos cultured with GH than in control embryos. Treatment with GH regularly resulted in an improvement of the ultrastructural features of embryos produced in vitro, thus resembling the morphology of ex vivo embryos. Scanning electron-microscopy studies demonstrated that GH altered the structure and the pore size of the zona pellucida of blastocysts. Our studies imply that GH can modulate carbohydrate, lipid, and energy metabolism and influence transportation processes in the early IVF embryo.

Neural network classification of sweet potato embryos

NASA Astrophysics Data System (ADS)

Molto, Enrique; Harrell, Roy C.

1993-05-01

Somatic embryogenesis is a process that allows for the in vitro propagation of thousands of plants in sub-liter size vessels and has been successfully applied to many significant species. The heterogeneity of maturity and quality of embryos produced with this technique requires sorting to obtain a uniform product. An automated harvester is being developed at the University of Florida to sort embryos in vitro at different stages of maturation in a suspension culture. The system utilizes machine vision to characterize embryo morphology and a fluidic based separation device to isolate embryos associated with a pre-defined, targeted morphology. Two different backpropagation neural networks (BNN) were used to classify embryos based on information extracted from the vision system. One network utilized geometric features such as embryo area, length, and symmetry as inputs. The alternative network utilized polar coordinates of an embryo's perimeter with respect to its centroid as inputs. The performances of both techniques were compared with each other and with an embryo classification method based on linear discriminant analysis (LDA). Similar results were obtained with all three techniques. Classification efficiency was improved by reducing the dimension of the feature vector trough a forward stepwise analysis by LDA. In order to enhance the purity of the sample selected as harvestable, a reject to classify option was introduced in the model and analyzed. The best classifier performances (76% overall correct classifications, 75% harvestable objects properly classified, homogeneity improvement ratio 1.5) were obtained using 8 features in a BNN.

An economic assessment of embryo diagnostics (Dx) - the costs of introducing non-invasive embryo diagnostics into IVF standard treatment practices.

PubMed

Fugel, Hans-Joerg; Connolly, Mark; Nuijten, Mark

2014-10-09

New techniques in assessing oocytes and embryo quality are currently explored to improve pregnancy and delivery rates per embryo transfer. While a better understanding of embryo quality could help optimize the existing "in vitro fertilization" (IVF) therapy schemes, it is essential to address the economic viability of such technologies in the healthcare setting. An Embryo-Dx economic model was constructed to assess the cost-effectiveness of 3 different IVF strategies from a payer's perspective; it compares Embryo-Dx with single embryo transfer (SET) to elective single embryo transfer (eSET) and to double embryo transfer (DET) treatment practices. The introduction of a new non-invasive embryo technology (Embryo-Dx) associated with a cost up to €460 is cost-effective compared to eSET and DET based on the cost per live birth. The model assumed that Embryo-Dx will improve ongoing pregnancy rate/realize an absolute improvement in live births of 9% in this case. This study shows that improved embryo diagnosis combined with SET may have the potential to reduce the cost per live birth per couple treated in IVF treatment practices. The results of this study are likely more sensitive to changes in the ongoing pregnancy rate and consequently the live birth rate than the diagnosis costs. The introduction of a validated Embryo-Dx technology will further support a move towards increased eSET procedures in IVF clinical practice and vice versa.

Cysteamine supplementation during in vitro maturation of slaughterhouse- and opu-derived bovine oocytes improves embryonic development without affecting cryotolerance, pregnancy rate, and calf characteristics.

PubMed

Merton, J S; Knijn, H M; Flapper, H; Dotinga, F; Roelen, B A J; Vos, P L A M; Mullaart, E

2013-09-01

Optimization of ovum pick up (OPU) followed by in vitro embryo production (IVP) is strongly driven by the needs of both beef and dairy cattle breeders to enhance genetic improvement. The rapidly growing use of genomic selection in cattle has increased the interest in using OPU-IVP technology to increase the number of embryos and offspring per donor, thus allowing enhanced selection intensity for the next generation. The aim of this study was to optimize embryo production through supplementation of cysteamine during in vitro maturation (IVM) and in vitro culture (IVC) of both slaughterhouse- and OPU-derived oocytes. The effects on embryo production and on embryo cryotolerance, post-transfer embryo survival, and calf characteristics, including gestation length, birth weight, perinatal mortality, and sex ratio were studied. In study 1, immature slaughterhouse-derived cumulus-oocyte complexes (COCs) were matured in IVM medium supplemented with or without 0.1 mM cysteamine, fertilized and cultured for 7 days in 0.5 ml SOFaaBSA. In study 2, cysteamine was present during both IVM (0.1 mM) and IVC (0.01, 0.05, 0.1 mM) from Days 1 to 4. In study 3, OPU-derived COCs were matured in medium supplemented with or without 0.1 mM cysteamine in a 2 × 2 factorial design (OPU week and cysteamine treatment). Embryos were evaluated for stage and grade on Day 7 and, depending on the number of transferable embryos and recipients available, the embryos were transferred either fresh or frozen-thawed at a later date. The presence of cysteamine during IVM significantly increased the embryo production rate with slaughterhouse-derived COCs (24.0% vs. 19.4%). The higher number of embryos at Day 7 was due to an increased number of blastocysts, whereas the distribution of embryos among different quality grades and cryotolerance was not affected. Embryo production rate was negatively affected when cysteamine was present during both the processes of IVM and IVC during Days 1 to 4 of culture (13.2%-19.3% vs. 26.4%). The presence of cysteamine during IVM of OPU-derived COCs also significantly increased the embryo production rate (34.4% vs. 23.4%). The higher number of embryos was again totally due to an increased number of blastocysts, whereas cryotolerance was not affected. The relative increase in embryo production rate was higher with OPU-derived oocytes compared with slaughterhouse-derived COCs (47% vs. 24%). This improvement resulted in a mean of 1.73 transferable embryos per OPU session compared with 1.06 in the absence of cysteamine. The presence of cysteamine did not affect pregnancy rate, gestation length, birth weight, perinatal mortality, and sex of calves born from either fresh or frozen-thawed embryos. This study reported that cysteamine supplementation during IVM greatly improved the efficiency and affectivity of an OPU-IVP program. Copyright © 2013 Elsevier Inc. All rights reserved.

Recent microfluidic devices for studying gamete and embryo biomechanics.

PubMed

Lai, David; Takayama, Shuichi; Smith, Gary D

2015-06-25

The technical challenges of biomechanic research such as single cell analysis at a high monetary cost, labor, and time for just a small number of measurements is a good match to the strengths of microfluidic devices. New scientific discoveries in the fertilization and embryo development process, of which biomechanics is a major subset of interest, is crucial to fuel the continual improvement of clinical practice in assisted reproduction. The following review will highlight some recent microfluidic devices tailored for gamete and embryo biomechanics where biomimicry arises as a major theme of microfluidic device design and function, and the application of fundamental biomechanic principles are used to improve outcomes of cryopreservation. Copyright © 2015 Elsevier Ltd. All rights reserved.

Where does New Zealand stand on permitting research on human embryos?

PubMed

Jones, D Gareth

2014-08-01

In many respects New Zealand has responded to the assisted reproductive technologies (ARTs) as positively as many comparable societies, such as Australia and the UK. Consequently, in vitro fertilisation (IVF) and pre-implantation genetic diagnosis (PGD) are widely available, as is non-commercial surrogacy utilising IVF. These developments have been made possible by the Human Assisted Reproductive Technology (HART) Act 2004, overseen by its two committees, the Advisory Committee on Assisted Reproductive Technology (ACART) and the Ethics Committee (ECART). However, New Zealand stands apart from many of these other societies by the lack of permission for scientists to conduct research using human embryos. There is no doubt this reflects strongly held viewpoints on the part of some that embryos should be protected and not exploited. Legitimate as this stance is, the resulting situation is problematic when IVF is already designated as an established procedure. This is because the development of IVF involved embryo research, and continuing improvements in procedures depend upon ongoing embryo research. While prohibition of research on human embryos gives the impression of protecting embryos, it fails to do this and also fails to enhance the health and wellbeing of children born using IVF. This situation will not be rectified until research is allowed on human embryos.

Osmotic measurements in whole megagametophytes and embryos of loblolly pine (Pinus taeda) during seed development.

PubMed

Pullman, Gerald S; Johnson, Shannon

2009-06-01

Water potential (Psi) and osmotic potential (Psis) were measured weekly through the sequence of seed development in megagametophytes of loblolly pine (Pinus taeda L.). A Wescor 5500XRS vapor pressure osmometer, modified with a cycle hold switch, was used to measure Psi for whole megagametophytes containing embryos. The Psi measurements for megagametophytes with embryos removed were also attempted but readings were distorted due to cell lysates from the cut surfaces. Six seasonal sets of megagametophyte Psi profiles were generated. Megagametophytes from most of the trees examined showed a consistent Psi pattern: low measurements of -1.0 to -0.75 MPa during early embryo development in late June to early July when embryo Stages 1-2 occur; an increase for one to several weeks to levels of -0.5 to -0.75 MPa, beginning at Stages 3-5 when apical dome formation occurs; followed by a steady drop from -0.85 to -1.7 to -2.0 MPa from Stage 6 onward from late August until just before cone seed release. The Psis was measured for supernatant from centrifuged frozen-thawed megagametophyte tissue (embryos removed). Megagametophyte Psis profiles were similar for seeds analyzed from two trees and resembled Psi observations starting low, rising around Stages 4-7 and then undergoing a major reduction indicating a strong solute accumulation beginning at Stages 7-9.1. Somatic embryos stop growth prematurely in vitro at Stages 8-9.1. The major change in the accumulation of megagametophyte solutes at Stages 8-9.1 correlates with the halt in somatic embryo maturation and suggests that identifying, quantifying and using the major natural soluble compounds that accumulate during mid- to late-stage seed development may be important to improve conifer somatic embryo maturation.

Expression of mRNA encoding leukaemia inhibitory factor (LIF) and its receptor (LIFRβ) in buffalo preimplantation embryos produced in vitro: markers of successful embryo implantation.

PubMed

Eswari, S; Sai Kumar, G; Sharma, G Taru

2013-05-01

Summary The objective of this study was to evaluate the effect of supplementation of recombinant leukaemia inhibitory factor (LIF) in culture media on blastocyst development, total cell number and blastocyst hatching rates and the reverse transcription-polymerase chain reaction analysis of preimplantation buffalo embryos to determine whether they contain the LIF-encoding mRNA and its beta receptor (LIFRβ) genes in different stages of preimplantation buffalo embryos. Cumulus-oocyte complexes retrieved from slaughterhouse buffalo ovaries were matured in vitro and fertilized using frozen buffalo semen. After 18 h of co-incubation with sperm, the presumptive zygotes were cultured in modified synthetic oviductal fluid without (control) or with rhLIF (100 ng/ml). There was no significant difference in the overall cleavage rate up to morula stage however the development of blastocysts, hatching rate and total cell numbers were significantly higher in the LIF-treated group than control. Transcripts for LIFRβ were detected from immature, in vitro-matured oocytes and in the embryos up to blastocyst stage, while transcripts for the LIF were detected from 8-16-cell stage up to blastocyst, which indicated that embryo-derived LIF can act in an autocrine manner on differentiation process and blastocyst formation. This study indicated that the addition of LIF to the embryo culture medium improved development of blastocysts, functional (hatching) and morphological (number of cells) quality of the blastocysts produced in vitro. The stage-specific expression pattern of LIF and LIFRβ mRNA transcripts in buffalo embryos indicated that LIF might play an important role in the preimplantation development and subsequent implantation of buffalo embryos.

The evolution of porcine embryo in vitro production.

PubMed

Grupen, Christopher G

2014-01-01

The in vitro production of porcine embryos has presented numerous challenges to researchers over the past four decades. Some of the problems encountered were specific to porcine gametes and embryos and needed the concerted efforts of many to overcome. Gradually, porcine embryo in vitro production systems became more reliable and acceptable rates of blastocyst formation were achieved. Despite the significant improvements, the problem of polyspermic fertilization has still not been adequately resolved and the embryo in vitro culture conditions are still considered to be suboptimal. Whereas early studies focused on increasing our understanding of the reproductive processes involved, the technology evolved to the point where in vitro-matured oocytes and in vitro-produced embryos could be used as research material for developing associated reproductive technologies, such as SCNT and embryo cryopreservation. Today, the in vitro procedures used to mature oocytes and culture embryos are integral to the production of transgenic pigs by SCNT. This review discusses the major achievements, advances, and knowledge gained from porcine embryo in vitro production studies and highlights the future research perspectives of this important technology. Copyright © 2014 Elsevier Inc. All rights reserved.

Media composition: salts and osmolality.

PubMed

Baltz, Jay M

2012-01-01

The main components of embryo culture media are salts, which dissociate into their component inorganic ions in aqueous solution. All embryo culture media contain the same six inorganic ions: Na(+), K(+), Cl(-), Ca(2+), Mg(2+), and SO(4)(2-), while most also contain PO(4)(2-). The salts that are used to formulate embryo culture media can be traced back to classic saline solutions, particularly Krebs-Ringer Bicarbonate (KRB), that were developed for somatic cells in the first half of the twentieth century. The salt and inorganic ion concentrations in the first successful defined mouse embryo culture medium, Whittens medium, were identical to those in KRB. These remained largely unchanged in embryo culture media for decades, with similar levels found in the standard mouse embryo culture medium, M16, formulated in the 1970s. Human embryos were initially cultured in undefined somatic cell media such as Earles and Hams F-10 with serum added. This changed in the mid-1980s, however, with the development of Quinns HTF, a defined medium specifically formulated for human embryo culture, in which the inorganic ion concentrations are similar to those in M16 and Whittens. While these media were useful both for experimental work and clinically, embryos suffered developmental blocks in all of them, with mouse embryos blocking at the 2-cell stage and human embryos at the 4- to 8-cell stage. Starting in the late 1980s, however, mouse embryo culture media were first developed that alleviated these developmental blocks. These media, CZB and KSOM, had much lower osmolalities than previous media, mainly due to lower inorganic ion concentrations. Indeed, lowering total inorganic ion concentration and osmolality proved key to understanding how media that supported complete preimplantation development in vitro can be formulated. A subsequent improvement was the addition of amino acids to culture media for both mouse and human embryos. At least in part, their beneficial effect during the cleavage stages of development is due to the presence in early preimplantation embryos of mechanisms for cell volume regulation that depend on the accumulation of amino acids as organic osmolytes to provide intracellular osmotic support. These amino acids, principally glycine, replace a portion of the intracellular inorganic ions that would otherwise be needed to maintain cell size, preventing the intracellular ionic strength from rising to deleterious levels and blocking development. Thus, the optimum salts levels, osmolality, and amino acid contents of culture media are not independent, but interact strongly because of their roles in cell volume regulation. In the absence of compounds that preimplantation embryos can use as organic osmolytes, embryos will develop only at lower osmolalities and salt concentrations in the medium. However, when organic osmolytes such as some amino acids are present, embryos will develop in culture at higher osmolarities that are similar to those they experience in tubal fluid in vivo.

Unraveling the association between genetic integrity and metabolic activity in pre-implantation stage embryos

PubMed Central

D’Souza, Fiona; Pudakalakatti, Shivanand M.; Uppangala, Shubhashree; Honguntikar, Sachin; Salian, Sujith Raj; Kalthur, Guruprasad; Pasricha, Renu; Appajigowda, Divya; Atreya, Hanudatta S.; Adiga, Satish Kumar

2016-01-01

Early development of certain mammalian embryos is protected by complex checkpoint systems to maintain the genomic integrity. Several metabolic pathways are modulated in response to genetic insults in mammalian cells. The present study investigated the relationship between the genetic integrity, embryo metabolites and developmental competence in preimplantation stage mouse embryos with the aim to identify early biomarkers which can predict embryonic genetic integrity using spent medium profiling by NMR spectroscopy. Embryos carrying induced DNA lesions (IDL) developed normally for the first 2.5 days, but began to exhibit a developmental delay at embryonic day 3.5(E3.5) though they were morphologically indistinguishable from control embryos. Analysis of metabolites in the spent medium on E3.5 revealed a significant association between pyruvate, lactate, glucose, proline, lysine, alanine, valine, isoleucine and thymine and the extent of genetic instability observed in the embryos on E4.5. Further analysis revealed an association of apoptosis and micronuclei frequency with P53 and Bax transcripts in IDL embryos on the E4.5 owing to delayed induction of chromosome instability. We conclude that estimation of metabolites on E3.5 in spent medium may serve as a biomarker to predict the genetic integrity in pre-implantation stage embryos which opens up new avenues to improve outcomes in clinical IVF programs. PMID:27853269

Genome-wide analysis reveals divergent patterns of gene expression during zygotic and somatic embryo maturation of Theobroma cacao L., the chocolate tree.

PubMed

Maximova, Siela N; Florez, Sergio; Shen, Xiangling; Niemenak, Nicolas; Zhang, Yufan; Curtis, Wayne; Guiltinan, Mark J

2014-07-16

Theobroma cacao L. is a tropical fruit tree, the seeds of which are used to create chocolate. In vitro somatic embryogenesis (SE) of cacao is a propagation system useful for rapid mass-multiplication to accelerate breeding programs and to provide plants directly to farmers. Two major limitations of cacao SE remain: the efficiency of embryo production is highly genotype dependent and the lack of full cotyledon development results in low embryo to plant conversion rates. With the goal to better understand SE development and to improve the efficiency of SE conversion we examined gene expression differences between zygotic and somatic embryos using a whole genome microarray. The expression of 28,752 genes was determined at 4 developmental time points during zygotic embryogenesis (ZE) and 2 time points during cacao somatic embryogenesis (SE). Within the ZE time course, 10,288 differentially expressed genes were enriched for functions related to responses to abiotic and biotic stimulus, metabolic and cellular processes. A comparison ZE and SE expression profiles identified 10,175 differentially expressed genes. Many TF genes, putatively involved in ethylene metabolism and response, were more strongly expressed in SEs as compared to ZEs. Expression levels of genes involved in fatty acid metabolism, flavonoid biosynthesis and seed storage protein genes were also differentially expressed in the two types of embryos. Large numbers of genes were differentially regulated during various stages of both ZE and SE development in cacao. The relatively higher expression of ethylene and flavonoid related genes during SE suggests that the developing tissues may be experiencing high levels of stress during SE maturation caused by the in vitro environment. The expression of genes involved in the synthesis of auxin, polyunsaturated fatty acids and secondary metabolites was higher in SEs relative to ZEs despite lack of lipid and metabolite accumulation. These differences in gene transcript levels associated with critical processes during seed development are consistent with the fact that somatic embryos do not fully develop the large storage cotyledons found in zygotic embryos. These results provide insight towards design of improved protocols for cacao somatic embryogenesis.

Genome-wide analysis reveals divergent patterns of gene expression during zygotic and somatic embryo maturation of Theobroma cacao L., the chocolate tree

PubMed Central

2014-01-01

Background Theobroma cacao L. is a tropical fruit tree, the seeds of which are used to create chocolate. In vitro somatic embryogenesis (SE) of cacao is a propagation system useful for rapid mass-multiplication to accelerate breeding programs and to provide plants directly to farmers. Two major limitations of cacao SE remain: the efficiency of embryo production is highly genotype dependent and the lack of full cotyledon development results in low embryo to plant conversion rates. With the goal to better understand SE development and to improve the efficiency of SE conversion we examined gene expression differences between zygotic and somatic embryos using a whole genome microarray. Results The expression of 28,752 genes was determined at 4 developmental time points during zygotic embryogenesis (ZE) and 2 time points during cacao somatic embryogenesis (SE). Within the ZE time course, 10,288 differentially expressed genes were enriched for functions related to responses to abiotic and biotic stimulus, metabolic and cellular processes. A comparison ZE and SE expression profiles identified 10,175 differentially expressed genes. Many TF genes, putatively involved in ethylene metabolism and response, were more strongly expressed in SEs as compared to ZEs. Expression levels of genes involved in fatty acid metabolism, flavonoid biosynthesis and seed storage protein genes were also differentially expressed in the two types of embryos. Conclusions Large numbers of genes were differentially regulated during various stages of both ZE and SE development in cacao. The relatively higher expression of ethylene and flavonoid related genes during SE suggests that the developing tissues may be experiencing high levels of stress during SE maturation caused by the in vitro environment. The expression of genes involved in the synthesis of auxin, polyunsaturated fatty acids and secondary metabolites was higher in SEs relative to ZEs despite lack of lipid and metabolite accumulation. These differences in gene transcript levels associated with critical processes during seed development are consistent with the fact that somatic embryos do not fully develop the large storage cotyledons found in zygotic embryos. These results provide insight towards design of improved protocols for cacao somatic embryogenesis. PMID:25030026

Time lapse imaging: is it time to incorporate this technology into routine clinical practice?

PubMed

Bhide, Priya; Maheshwari, Abha; Cutting, Rachel; Seenan, Susan; Patel, Anita; Khan, Khalid; Homburg, Roy

2017-06-01

Time-lapse imaging (TLI) systems for embryo incubation, assessment and selection are a novel technology available to in vitro fertilization (IVF) clinics. However, there is uncertainty about their clinical and cost-effectiveness and insufficient good quality evidence to warrant their routine use. Despite this, enthusiastic commercial marketing and slipping clinical equipoise have led to the widespread hasty introduction of this technology into practice, often at a considerable expense to the patient. We have reviewed the published literature and aim to summarize the strengths, weaknesses, opportunities and threats of these systems. These specialized incubators provide undisturbed embryo culture conditions and, by almost continuous monitoring of embryo development, generate morphokinetic parameters to aid embryo selection. They are thus hypothesized to improve outcomes following IVF. Although literature reports improved reproductive outcomes, these outcomes are largely surrogate and there is a paucity of studies reporting live births. The use of time lapse systems may reduce early pregnancy loss, increase elective single embryo transfers and limit multiple pregnancies through better embryo selection. However, the quality of the studies and hence the evidence so far, is low to moderate quality. We recommend further research producing robust high-quality evidence for and against the use of these systems.

Stenospermy and seed development in the "Brazilian seedless" variety of sugar apple (Annona squamosa).

PubMed

Dos Santos, Rayane C; Ribeiro, Leonardo M; Mercadante-Simões, Maria Olívia; Costa, Márcia R; Nietsche, Silvia; Pereira, Marlon C T

2014-12-01

Stenospermy was identified in naturally occurring sugar-apple (Annona squamosa) mutants with great potential for use in genetic improvement programs. However, to date, there have been no detailed studies of the development of aspermic fruit in this species. The aim of the present study was to characterize the anatomy of developing fruit in the 'Brazilian Seedless' mutant. Flower buds in pre-anthesis and developing fruits were subjected to common plant anatomy techniques. The abnormal ovules are unitegmic and orthotropic and have a long funiculus. There is evidence of fertilization, including the presence of embryos in early development and the proliferation of starch grains in the embryo sac. However, the embryos and embryo sac degenerate, although this does not affect pericarp development. Ovule abortion does not occur. The perisperm, which is formed from the peripheral layers of the nucellus, fills the cavity left by the embryo sac. The mature fruit contains numerous small sterile seeds with abundant perisperm and unlignified integument that is restricted to the micropylar region. The majority of perisperm cells are living and appear to be metabolically active in the periphery. Therefore, stenospermy leads to the formation of sterile seeds in A. squamosa, and the perisperm possibly play an important role in fruit development.

Effect of PMA-induced protein kinase C activation on development and apoptosis in early zebrafish embryos.

PubMed

Hrubik, Jelena; Glisic, Branka; Samardzija, Dragana; Stanic, Bojana; Pogrmic-Majkic, Kristina; Fa, Svetlana; Andric, Nebojsa

2016-12-01

Protein kinase C (PKC) isoforms have been implicated in several key steps during early development, but the consequences of xenobiotic-induced PKC activation during early embryogenesis are still unknown. In this study, zebrafish embryos were exposed to a range of phorbol 12-myristate 13-acetate (PMA) concentrations (0-200μg/L) at different time points after fertilization. Results showed that 200μgPMA/L caused development of yolk bags, cardiac edema, slow blood flow, pulsating blood flow, slow pulse, elongated heart, lack of tail fins, curved tail, and coagulation. PMA exposure decreased survival rate of the embryos starting within the first 24h and becoming more pronounced after prolonged exposure (96h). PMA increased the number of apoptotic cells in the brain region as demonstrated by acridine orange staining and caused up-regulation of caspase 9 (casp9) and p53 up-regulated modulator of apoptosis (puma) mRNA in whole embryos. PMA caused oxidative stress in the embryos as demonstrated by decreased mRNA expression of catalase and superoxide dismutase 2. Inhibition of Pkc with GF109203X improved overall survival rate, reduced apoptosis in the brain and decreased expression of casp9 and puma in the PMA-exposed embryos. However, Pkc inhibition neither prevented development of deformities nor reversed oxidative stress in the PMA-exposed embryos. These data suggest that direct over-activation of Pkc during early embryogenesis of zebrafish is associated with apoptosis and decreased survival rate of the embryos. Copyright © 2016 Elsevier Inc. All rights reserved.

RAPID COMMUNICATION: Nerve growth factor influences cleavage rate and embryo development in sheep.

PubMed

Crispo, M; Dos Santos-Neto, P C; Vilariño, M; Mulet, A P; de León, A; Barbeito, L; Menchaca, A

2016-10-01

Recent information about Nerve growth factor (NGF), a protein traditionally associated to the nervous system that regulates survival and maturation of developing neurons, suggests that it may exert action also on different levels in the reproductive system. The aim of this study was to evaluate the effect of NGF added during in vitro oocyte maturation, fertilization or in vitro embryo development in sheep. Nerve growth factor was supplemented to the culture medium at 0, 100, or 1,000 ng/mL, during either in vitro maturation (Exp. 1), in vitro fertilization (Exp. 2), or in vitro culture (Exp. 3). In addition, NGF mRNA expression was determined in cumulus cells and oocytes. Nerve growth factor induced early cleavage when added during oocyte maturation or fertilization, improved embryo development when added during fertilization, and had no significant effect when added during embryo culture. In general, the effect was more evident with 100 rather than 1,000 ng/mL (P < 0.05). Expression of endogenous NGF was not detected in oocytes, and increased in cumulus cells when 1,000 ng/mL of NGF was added during fertilization, but not during maturation and embryo culture. In conclusion, the addition of NGF during oocyte maturation and fertilization affects in vitro cleavage and embryo development in sheep. We suggest a possible effect of this growth factor on oocyte maturation and mainly on the fertilization process.

Desiccation and Cold Hardening of Date Palm Somatic Embryos Improve Germination.

PubMed

Shareef, Hussein J

2017-01-01

Embryogenic suspension cultures of date palm are ideal for mass propagation of somatic embryos; however, the low percentage of germination of somatic embryos (SE) remains an impediment. This chapter focuses on two important physical factors to improve germination of date palm somatic embryos: the use of partial desiccation (3 h) of somatic embryos and the exposure to low temperature (4 °C for 24 h). High germination percentage (41%) is achieved by desiccation for 3 h. Moreover, adding 0.3 g/L activated charcoal (AC) to the liquid medium further improves somatic embryo number and weight as well as the percentage of germination. Moreover, partial desiccation and low temperature exposure tend to increase proline content. This improved protocol for somatic embryo germination is potentially applicable for commercial micropropagation of date palm.

Melatonin protect the development of preimplantation mouse embryos from sodium fluoride-induced oxidative injury.

PubMed

Zhao, Jiamin; Fu, Beibei; Peng, Wei; Mao, Tingchao; Wu, Haibo; Zhang, Yong

2017-09-01

Recently study shows that melatonin can protect embryos from the culture environment oxidative stress. However, the protective effect of melatonin on the mouse development of preimplantation embryos under sodium fluoride (NaF) induced oxidative stress is still unclear. Here, we showed that exposure to NaF significantly increased the reactive oxygen species (ROS) level, decreased the blastocyst formation rates, and increased the fragmentation, apoptosis and retardation of blastocysts in the development of mouse preimplantation embryos. However, the protective of melatonin remarkable increased the of blastocyst formation rates, maintained mitochondrial function and total antioxidant capacity by clearing ROS. Importantly the data showed that melatonin improved the activity of enzymatic antioxidants, including glutathione(GSH), superoxide dismutase(SOD), and malonaldehyde (MDA), and increased the expression levels of antioxidative genes. Taken together, our results indicate that melatonin prevent NaF-induced oxidative damage to mouse preimplantation embryo through down regulation of ROS level, stabilization of mitochondrial function and modulation of the activity of antioxidases and antioxidant genes. Copyright © 2017 Elsevier B.V. All rights reserved.

BIX-01294 increases pig cloning efficiency by improving epigenetic reprogramming of somatic cell nuclei.

PubMed

Huang, Jiaojiao; Zhang, Hongyong; Yao, Jing; Qin, Guosong; Wang, Feng; Wang, Xianlong; Luo, Ailing; Zheng, Qiantao; Cao, Chunwei; Zhao, Jianguo

2016-01-01

Accumulating evidence suggests that faulty epigenetic reprogramming leads to the abnormal development of cloned embryos and results in the low success rates observed in all mammals produced through somatic cell nuclear transfer (SCNT). The aberrant methylation status of H3K9me and H3K9me2 has been reported in cloned mouse embryos. To explore the role of H3K9me2 and H3K9me in the porcine somatic cell nuclear reprogramming, BIX-01294, known as a specific inhibitor of G9A (histone-lysine methyltransferase of H3K9), was used to treat the nuclear-transferred (NT) oocytes for 14-16 h after activation. The results showed that the developmental competence of porcine SCNT embryos was significantly enhanced both in vitro (blastocyst rate 16.4% vs 23.2%, P<0.05) and in vivo (cloning rate 1.59% vs 2.96%) after 50 nm BIX-01294 treatment. BIX-01294 treatment significantly decreased the levels of H3K9me2 and H3K9me at the 2- and 4-cell stages, which are associated with embryo genetic activation, and increased the transcriptional expression of the pluripotency genes SOX2, NANOG and OCT4 in cloned blastocysts. Furthermore, the histone acetylation levels of H3K9, H4K8 and H4K12 in cloned embryos were decreased after BIX-01294 treatment. However, co-treatment of activated NT oocytes with BIX-01294 and Scriptaid rescued donor nuclear chromatin from decreased histone acetylation of H4K8 that resulted from exposure to BIX-01294 only and consequently improved the preimplantation development of SCNT embryos (blastocyst formation rates of 23.7% vs 21.5%). These results indicated that treatment with BIX-01294 enhanced the developmental competence of porcine SCNT embryos through improvements in epigenetic reprogramming and gene expression. © 2016 Society for Reproduction and Fertility.

Astaxanthin Normalizes Epigenetic Modifications of Bovine Somatic Cell Cloned Embryos and Decreases the Generation of Lipid Peroxidation.

PubMed

Li, R; Wu, H; Zhuo, W W; Mao, Q F; Lan, H; Zhang, Y; Hua, S

2015-10-01

Astaxanthin is an extremely common antioxidant scavenging reactive oxygen species (ROS) and blocking lipid peroxidation. This study was conducted to investigate the effects of astaxanthin supplementation on oocyte maturation, and development of bovine somatic cell nuclear transfer (SCNT) embryos. Cumulus-oocyte complexes were cultured in maturation medium with astaxanthin (0, 0.5, 1.0, or 1.5 mg/l), respectively. We found that 0.5 mg/l astaxanthin supplementation significantly increased the proportion of oocyte maturation. Oocytes cultured in 0.5 mg/l astaxanthin supplementation were used to construct SCNT embryos and further cultured with 0, 0.5, 1.0 or 1.5 mg/l astaxanthin. The results showed that the supplementation of 0.5 mg/l astaxanthin significantly improved the proportions of cleavage and blastulation, as well as the total cell number in blastocysts compared with the control group, yet this influence was not concentration dependent. Chromosomal analyses revealed that more blastomeres showed a normal chromosomal complement in 0.5 mg/l astaxanthin treatment group, which was similar to that in IVF embryos. The methylation levels located on the exon 1 of the imprinted gene H19 and IGF2, pluripotent gene OCT4 were normalized, and global DNA methylation, H3K9 and H4K12 acetylation were also improved significantly, which was comparable to that in vitro fertilization (IVF) embryos. Moreover, we also found that astaxanthin supplementation significantly decreased the level of lipid peroxidation. Our findings showed that the supplementation of 0.5 mg/l astaxanthin to oocyte maturation medium and embryo culture medium improved oocyte maturation, SCNT embryo development, increased chromosomal stability and normalized the epigenetic modifications, as well as inhibited overproduction of lipid peroxidation. © 2015 Blackwell Verlag GmbH.

Development of a new clinically applicable device for embryo evaluation which measures embryo oxygen consumption.

PubMed

Kurosawa, Hiroki; Utsunomiya, Hiroki; Shiga, Naomi; Takahashi, Aiko; Ihara, Motomasa; Ishibashi, Masumi; Nishimoto, Mitsuo; Watanabe, Zen; Abe, Hiroyuki; Kumagai, Jin; Terada, Yukihiro; Igarashi, Hideki; Takahashi, Toshifumi; Fukui, Atsushi; Suganuma, Ryota; Tachibana, Masahito; Yaegashi, Nobuo

2016-10-01

Does a new system-the chip-sensing embryo respiration monitoring system (CERMs)-enable evaluation of embryo viability for potential application in a clinical IVF setting? The system enabled the oxygen consumption rate of spheroids, bovine embryos and frozen-thawed human embryos to be measured, and this rate corresponded to the developmental potential of embryos. To date, no reliable and clinically suitable objective evaluation methods for embryos are available, which circumvent the differences in inter-observer subjective view. Existing systems such as the scanning electrochemical microscopy (SECM) technique, which enables the measurement of oxygen consumption rate in embryos, need improvement in usability before they can be applied to a clinical setting. This is a prospective original research study. The feasibility of measuring the oxygen consumption rate was assessed using CERMs for 9 spheroids, 9 bovine embryos and 30 redundant frozen-thawed human embryos. The endpoints for the study were whether CERMs could detect a dissolved oxygen gradient with high sensitivity, had comparable accuracy to the SECM measuring system with improved usability, and could predict the development of an embryo to a blastocyst by measuring the oxygen consumption rate. The relationship between the oxygen consumption rate and standard morphological evaluation was also examined. We developed a new CERMs, which enables the oxygen consumption rate to be measured automatically using an electrochemical method. The device was initially used for measuring a dissolved oxygen concentration gradient in order to calculate oxygen consumption rate using nine spheroids. Next, we evaluated data correlation between the CERMs and the SECM measuring systems using nine bovine embryos. Finally, the oxygen consumption rates of 30 human embryos, which were frozen-thawed on 2nd day after fertilization, were measured by CERMs at 6, 24, 48, 72 and 96 h after thawing with standard morphological evaluation. Furthermore, the developed blastocysts were scored using the blastocyst quality score (BQS), and the correlation with oxygen consumption rate was also assessed. The device enabled the oxygen consumption rate of an embryo to be measured automatically within a minute. The oxygen concentration gradient profile showed excellent linearity in a distance-dependent change. A close correlation in the oxygen consumption rates of bovine embryos was observed between the SECM measuring system and CERMs, with a determination coefficient of 0.8203 (P = 0.0008). Oxygen consumption rates of human embryos that have reached the blastocyst stage were significantly higher than those of arrested embryos at 48, 72 and 96 h after thawing (P = 0.039, 0.004 and 0.049, respectively). Thus, in vitro development of frozen-thawed human embryos to the blastocyst stage would be predicted at 48 h after thawing (day 4) by measuring the oxygen consumption using CERMs. Although a positive linear relationship between BQS and the oxygen consumption rate was observed [the determination coefficient was R(2) = 0.6537 (P = 0.008)], two blastocysts exhibited low oxygen consumption rates considering their relatively high BQS. This suggests that morphology and metabolism in human embryos might not correlate consistently. Transfer of the embryo and pregnancy evaluation was not performed. Thus, a correlation between oxygen consumption and the in vivo viability of embryos remains unknown. Clinical trials, including embryo transfer, would be desirable to determine a threshold value to elect clinically relevant, quality embryos for transfer. We utilized frozen-thawed human embryos in this study. The effect of these manipulations on the respiratory activity of the embryo is also unknown. Selection of quality embryos, especially in a single embryo transfer cycle, by CERMs may have an impact on obtaining better clinical outcomes, albeit with clinical trials being required. Furthermore, the early determination of quality embryos by CERMs may enable the omission of long-term in vitro embryo culture to the blastocyst stage. CERMs is scalable technology that can be integrated into incubators and/or other embryo evaluation systems, such as the time-lapse systems, due to its chip-based architecture. Thus, CERMS would enable automatic measurement of oxygen consumption, under 5% CO2, in the near future, in order to reduce oxidative stress from exposure to atmospheric air. This study was supported by grants from the Health and Labor Sciences Research Grant (H24-Hisaichiiki-Shitei-016). The authors have no conflicts of interest. Not applicable. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Dechorionation as a tool to improve the fish embryo toxicity test (FET) with the zebrafish (Danio rerio).

PubMed

Henn, Kirsten; Braunbeck, Thomas

2011-01-01

Prior to hatching, the zebrafish embryo is surrounded by an acellular envelope, the chorion. Despite repeated speculations, it could not be clarified unequivocally whether the chorion represents an effective barrier and, thus, protects the embryo from exposure to distinct chemicals. Potentially, there is a risk of generating false negative results in developmental toxicity studies due to limited permeability of the chorion for some compounds. The simplest way to exclude this is to remove the chorion and expose the "naked" embryo. In the context of ecotoxicity testing, standardized protocols do not exist for fish embryo dechorionation, and survival rates of dechorionated embryos have usually not been subjected to statistical analysis. Since reproducibly high survival rates are of fundamental importance for chemical toxicity assessment, the present study was designed to develop and optimize a dechorionation procedure. With appropriate modifications of the fish embryo test protocol, embryos can be dechorionated at 24h post-fertilization (hpf) with survival rates of ≥90%. However, for fish embryo tests with dechorionated embryos, the standard positive control test substance, 3,4-dichloroaniline, should be replaced by another compound, e.g., acetone, since 3,4-dichloroaniline exerts its effects during the first 24h of development. Dechorionation of younger stages (<24 hpf) is generally possible, however with lower survival rates. The effect of dechorionation was demonstrated with the cationic polymer Luviquat HM 552, which is blocked by the chorion non-dechorionated embryos due to its molecular weight of ~400,000 Dalton, but becomes strongly toxic after dechorionation. Copyright © 2010 Elsevier Inc. All rights reserved.

Inter-laboratory agreement on embryo classification and clinical decision: Conventional morphological assessment vs. time lapse.

PubMed

Martínez-Granados, Luis; Serrano, María; González-Utor, Antonio; Ortíz, Nereyda; Badajoz, Vicente; Olaya, Enrique; Prados, Nicolás; Boada, Montse; Castilla, Jose A

2017-01-01

The aim of this study is to determine inter-laboratory variability on embryo assessment using time-lapse platform and conventional morphological assessment. This study compares the data obtained from a pilot study of external quality control (EQC) of time lapse, performed in 2014, with the classical EQC of the Spanish Society for the Study of Reproductive Biology (ASEBIR) performed in 2013 and 2014. In total, 24 laboratories (8 using EmbryoScope™, 15 using Primo Vision™ and one with both platforms) took part in the pilot study. The clinics that used EmbryoScope™ analysed 31 embryos and those using Primo Vision™ analysed 35. The classical EQC was implemented by 39 clinics, based on an analysis of 25 embryos per year. Both groups were required to evaluate various qualitative morphological variables (cell fragmentation, the presence of vacuoles, blastomere asymmetry and multinucleation), to classify the embryos in accordance with ASEBIR criteria and to stipulate the clinical decision taken. In the EQC time-lapse pilot study, the groups were asked to determine, as well as the above characteristics, the embryo development times, the number, opposition and size of pronuclei, the direct division of 1 into 3 cells and/or of 3 into 5 cells and false divisions. The degree of agreement was determined by calculating the intra-class correlation coefficients and the coefficient of variation for the quantitative variables and the Gwet index for the qualitative variables. For both EmbryoScope™ and Primo Vision™, two periods of greater inter-laboratory variability were observed in the times of embryo development events. One peak of variability was recorded among the laboratories addressing the first embryo events (extrusion of the second polar body and the appearance of pronuclei); the second peak took place between the times corresponding to the 8-cell and morula stages. In most of the qualitative variables analysed regarding embryo development, there was almost-perfect inter-laboratory agreement among conventional morphological assessment (CMA), EmbryoScope™ and Primo Vision™, except for false divisions, vacuoles and asymmetry (users of all methods) and multinucleation (users of Primo Vision™), where the degree of agreement was lower. The inter-laboratory agreement on embryo classification according to the ASEBIR criteria was moderate-substantial (Gwet 0.41-0.80) for the laboratories using CMA and EmbryoScope™, and fair-moderate (Gwet 0.21-0.60) for those using Primo Vision™. The inter-laboratory agreement for clinical decision was moderate (Gwet 0.41-0.60) on day 5 for CMA users and almost perfect (Gwet 0.81-1) for time-lapse users. In conclusion, time-lapse technology does not improve inter-laboratory agreement on embryo classification or the analysis of each morphological variable. Moreover, depending on the time-lapse platform used, inter-laboratory agreement may be lower than that obtained by CMA. However, inter-laboratory agreement on clinical decisions is improved with the use of time lapse, regardless of the platform used.

Inter-laboratory agreement on embryo classification and clinical decision: Conventional morphological assessment vs. time lapse

PubMed Central

Serrano, María; González-Utor, Antonio; Ortíz, Nereyda; Badajoz, Vicente; Olaya, Enrique; Prados, Nicolás; Boada, Montse; Castilla, Jose A.

2017-01-01

The aim of this study is to determine inter-laboratory variability on embryo assessment using time-lapse platform and conventional morphological assessment. This study compares the data obtained from a pilot study of external quality control (EQC) of time lapse, performed in 2014, with the classical EQC of the Spanish Society for the Study of Reproductive Biology (ASEBIR) performed in 2013 and 2014. In total, 24 laboratories (8 using EmbryoScope™, 15 using Primo Vision™ and one with both platforms) took part in the pilot study. The clinics that used EmbryoScope™ analysed 31 embryos and those using Primo Vision™ analysed 35. The classical EQC was implemented by 39 clinics, based on an analysis of 25 embryos per year. Both groups were required to evaluate various qualitative morphological variables (cell fragmentation, the presence of vacuoles, blastomere asymmetry and multinucleation), to classify the embryos in accordance with ASEBIR criteria and to stipulate the clinical decision taken. In the EQC time-lapse pilot study, the groups were asked to determine, as well as the above characteristics, the embryo development times, the number, opposition and size of pronuclei, the direct division of 1 into 3 cells and/or of 3 into 5 cells and false divisions. The degree of agreement was determined by calculating the intra-class correlation coefficients and the coefficient of variation for the quantitative variables and the Gwet index for the qualitative variables. For both EmbryoScope™ and Primo Vision™, two periods of greater inter-laboratory variability were observed in the times of embryo development events. One peak of variability was recorded among the laboratories addressing the first embryo events (extrusion of the second polar body and the appearance of pronuclei); the second peak took place between the times corresponding to the 8-cell and morula stages. In most of the qualitative variables analysed regarding embryo development, there was almost-perfect inter-laboratory agreement among conventional morphological assessment (CMA), EmbryoScope™ and Primo Vision™, except for false divisions, vacuoles and asymmetry (users of all methods) and multinucleation (users of Primo Vision™), where the degree of agreement was lower. The inter-laboratory agreement on embryo classification according to the ASEBIR criteria was moderate-substantial (Gwet 0.41–0.80) for the laboratories using CMA and EmbryoScope™, and fair-moderate (Gwet 0.21–0.60) for those using Primo Vision™. The inter-laboratory agreement for clinical decision was moderate (Gwet 0.41–0.60) on day 5 for CMA users and almost perfect (Gwet 0.81–1) for time-lapse users. In conclusion, time-lapse technology does not improve inter-laboratory agreement on embryo classification or the analysis of each morphological variable. Moreover, depending on the time-lapse platform used, inter-laboratory agreement may be lower than that obtained by CMA. However, inter-laboratory agreement on clinical decisions is improved with the use of time lapse, regardless of the platform used. PMID:28841654

The effects of chemical and physical factors on mammalian embryo culture and their importance for the practice of assisted human reproduction.

PubMed

Wale, Petra L; Gardner, David K

2016-01-01

Although laboratory procedures, along with culture media formulations, have improved over the past two decades, the issue remains that human IVF is performed in vitro (literally 'in glass'). Using PubMed, electronic searches were performed using keywords from a list of chemical and physical factors with no limits placed on time. Examples of keywords include oxygen, ammonium, volatile organics, temperature, pH, oil overlays and incubation volume/embryo density. Available clinical and scientific evidence surrounding physical and chemical factors have been assessed and presented here. Development of the embryo outside the body means that it is constantly exposed to stresses that it would not experience in vivo. Sources of stress on the human embryo include identified factors such as pH and temperature shifts, exposure to atmospheric (20%) oxygen and the build-up of toxins in the media due to the static nature of culture. However, there are other sources of stress not typically considered, such as the act of pipetting itself, or the release of organic compounds from the very tissue culture ware upon which the embryo develops. Further, when more than one stress is present in the laboratory, there is evidence that negative synergies can result, culminating in significant trauma to the developing embryo. It is evident that embryos are sensitive to both chemical and physical signals within their microenvironment, and that these factors play a significant role in influencing development and events post transfer. From the viewpoint of assisted human reproduction, a major concern with chemical and physical factors lies in their adverse effects on the viability of embryos, and their long-term effects on the fetus, even as a result of a relatively brief exposure. This review presents data on the adverse effects of chemical and physical factors on mammalian embryos and the importance of identifying, and thereby minimizing, them in the practice of human IVF. Hence, optimizing the in vitro environment involves far more than improving culture media formulations. © The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Histone deacetylase inhibitor significantly improved the cloning efficiency of porcine somatic cell nuclear transfer embryos.

PubMed

Huang, Yongye; Tang, Xiaochun; Xie, Wanhua; Zhou, Yan; Li, Dong; Yao, Chaogang; Zhou, Yang; Zhu, Jianguo; Lai, Liangxue; Ouyang, Hongsheng; Pang, Daxin

2011-12-01

Valproic acid (VPA), a histone deacetylase inbibitor, has been shown to generate inducible pluripotent stem (iPS) cells from mouse and human fibroblasts with a significant higher efficiency. Because successful cloning by somatic cell nuclear transfer (SCNT) undergoes a full reprogramming process in which the epigenetic state of a differentiated donor nuclear is converted into an embryonic totipotent state, we speculated that VPA would be useful in promoting cloning efficiency. Therefore, in the present study, we examined whether VPA can promote the developmental competence of SCNT embryos by improving the reprogramming state of donor nucleus. Here we report that 1 mM VPA for 14 to 16 h following activation significantly increased the rate of blastocyst formation of porcine SCNT embryos constructed from Landrace fetal fibroblast cells compared to the control (31.8 vs. 11.4%). However, we found that the acetylation level of Histone H3 lysine 14 and Histone H4 lysine 5 and expression level of Oct4, Sox2, and Klf4 was not significantly changed between VPA-treated and -untreated groups at the blastocyst stage. The SCNT embryos were transferred to 38 surrogates, and the cloning efficiency in the treated group was significantly improved compared with the control group. Taken together, we have demonstrated that VPA can improve both in vitro and in vivo development competence of porcine SCNT embryos.

Omics in Reproductive Medicine: Application of Novel Technologies to Improve the IVF Success Rate.

PubMed

Nerenz, R D

Treatment for many infertile couples often consists of in vitro fertilization (IVF) but an estimated 70% of IVF cycles fail to produce a live birth. In an attempt to improve the live birth rate, the vast majority of IVF cycles performed in the United States involve the transfer of multiple embryos, a practice that increases the risk of multiple gestation pregnancy. This is a concern because multiple gestation pregnancies are associated with an increased incidence of maternal and fetal complications and significant cost associated with the care of preterm infants. As the ideal outcome of each IVF cycle is the birth of a single healthy baby, significant effort has focused on identifying embryos with the greatest developmental potential. To date, selection of euploid embryos using comprehensive chromosome screening (CCS) is the most promising approach while metabolomic and proteomic assessment of spent culture medium have the potential to noninvasively assess embryo viability. Endometrial gene expression profiling may help determine the optimal time to perform embryo transfer. While CCS has been implemented in some clinics, further development and optimization will be required before analysis of spent culture medium and endometrial gene expression profiling make the transition to clinical use. This review will describe efforts to identify embryos with the greatest potential to result in a healthy, live birth, with a particular emphasis on detection of embryo aneuploidy and metabolic profiling of spent embryo culture medium. Assessment of endometrial receptivity to identify the optimal time to perform embryo transfer will also be discussed. © 2016 Elsevier Inc. All rights reserved.

A multicenter prospective study to assess the effect of early cleavage on embryo quality, implantation, and live-birth rate.

PubMed

de los Santos, Maria José; Arroyo, Gemma; Busquet, Ana; Calderón, Gloria; Cuadros, Jorge; Hurtado de Mendoza, Maria Victoria; Moragas, Marta; Herrer, Raquel; Ortiz, Agueda; Pons, Carme; Ten, Jorge; Vilches, Miguel Angel; Figueroa, Maria José

2014-04-01

To investigate the impact of early cleavage (EC) on embryo quality, implantation, and live-birth rates. Prospective cross-sectional study. Multicenter study. Seven hundred embryo transfers and 1,028 early-stage human embryos. None. Implantation according to the presence of EC and embryo quality. The presence of EC is associated with embryo quality, especially in cycles with autologous oocytes. However, the use of EC as an additional criterion for selecting an embryo for transfer does not appear to significantly improve likelihood of implantation. Furthermore, embryos that presented EC had live-birth rates per implanted embryo similar to those that did not show any sign of cleavage. At least for conventional embryo culture and morphologic evaluations, the additional evaluation of EC in embryos may not be valuable to improve embryo implantation. Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Norway spruce embryogenesis: changes in carbohydrate profile, structural development and response to polyethylene glycol

PubMed Central

Hudec, Lukáš; Konrádová, Hana; Hašková, Anna; Lipavská, Helena

2016-01-01

Two unrelated, geographically distinct, highly embryogenic lines of Norway spruce (Picea abies (L.) Karst.) were analysed to identify metabolic traits characteristic for lines with good yields of high-quality embryos. The results were compared with corresponding characteristics of a poorly productive line (low embryo yield, scarce high-quality embryos). The following carbohydrate profiles and spectra during maturation, desiccation and germination were identified as promising characteristics for line evaluation: a gradual decrease in total soluble carbohydrates with an increasing sucrose : hexose ratio during maturation; accumulation of raffinose family oligosaccharides resulting from desiccation and their rapid degradation at the start of germination; and a decrease in sucrose, increase in hexoses and the appearance of pinitol with proceeding germination. We propose that any deviation from this profile in an embryonic line is a symptom of inferior somatic embryo development. We further propose that a fatty acid spectrum dominated by linoleic acid (18 : 2) was a common feature of healthy spruce somatic embryos, although it was quite different from zygotic embryos mainly containing oleic acid (18 : 1). The responses of the lines to osmotic stress were evaluated based on comparison of control (without osmoticum) and polyethylene glycol (PEG)-exposed (PEG 4000) variants. Although genetically distinct, both highly embryogenic lines responded in a very similar manner, with the only difference being sensitivity to high concentrations of PEG. At an optimum PEG concentration (3.75 and 5%), which was line specific, negative effects of PEG on embryo germination were compensated for by a higher maturation efficiency so that the application of PEG at an appropriate concentration improved the yield of healthy germinants per gram of initial embryonal mass and accelerated the process. Polyethylene glycol application, however, resulted in no improvement of the poorly productive line. PMID:27052433

Treatment of donor cell/embryo with different approaches to improve development after nuclear transfer.

PubMed

Mizutani, Eiji; Wakayama, Sayaka; Wakayama, Teruhiko

2015-01-01

The successful production of cloned animals by somatic cell nuclear transfer (SCNT) is a promising technology with many potential applications in basic research, medicine, and agriculture. However, the low efficiency and the difficulty of cloning are major obstacles to the widespread use of this technology. Since the first mammal cloned from an adult donor cell was born, many attempts have been made to improve animal cloning techniques, and some approaches have successfully improved its efficiency. Nuclear transfer itself is still difficult because it requires an accomplished operator with a practiced technique. Thus, it is very important to find simple and reproducible methods for improving the success rate of SCNT. In this chapter, we will review our recent protocols, which seem to be the simplest and most reliable method to date to improve development of SCNT embryos.

Interfacing Lab-on-a-Chip Embryo Technology with High-Definition Imaging Cytometry.

PubMed

Zhu, Feng; Hall, Christopher J; Crosier, Philip S; Wlodkowic, Donald

2015-08-01

To spearhead deployment of zebrafish embryo biotests in large-scale drug discovery studies, automated platforms are needed to integrate embryo in-test positioning and immobilization (suitable for high-content imaging) with fluidic modules for continuous drug and medium delivery under microperfusion to developing embryos. In this work, we present an innovative design of a high-throughput three-dimensional (3D) microfluidic chip-based device for automated immobilization and culture and time-lapse imaging of developing zebrafish embryos under continuous microperfusion. The 3D Lab-on-a-Chip array was fabricated in poly(methyl methacrylate) (PMMA) transparent thermoplastic using infrared laser micromachining, while the off-chip interfaces were fabricated using additive manufacturing processes (fused deposition modelling and stereolithography). The system's design facilitated rapid loading and immobilization of a large number of embryos in predefined clusters of traps during continuous microperfusion of drugs/toxins. It was conceptually designed to seamlessly interface with both upright and inverted fluorescent imaging systems and also to directly interface with conventional microtiter plate readers that accept 96-well plates. Compared with the conventional Petri dish assays, the chip-based bioassay was much more convenient and efficient as only small amounts of drug solutions were required for the whole perfusion system running continuously over 72 h. Embryos were spatially separated in the traps that assisted tracing single embryos, preventing interembryo contamination and improving imaging accessibility.

Effects of the histone deacetylase inhibitor 'Scriptaid' on the developmental competence of mouse embryos generated through round spermatid injection.

PubMed

Kong, Pengcheng; Yin, Mingru; Chen, Dongbao; Li, Shangang; Li, Yao; Xing, Fengying; Jiang, Manxi; Fang, Zhenfu; Lyu, Qifeng; Chen, Xuejin

2017-01-01

Can the histone deacetylase inhibitor Scriptaid improve the efficiency of the development of round spermatid injection (ROSI)-fertilized embryos in a mouse model? Treatment of ROSI mouse zygotes with Scriptaid increased the expression levels of several development-related genes at the blastocyst stage, resulting in more efficient in vitro development of the blastocyst and an increased birth rate of ROSI-derived embryos. The full-term development of embryos derived through ROSI is significantly lower than that following ICSI in humans and other species. Oocytes, spermatozoa and round spermatids were collected from BDF1 (C57BL/6 × DBA/2) mice. For in vitro development experiments, mouse ROSI-derived zygotes were treated with Scriptaid at different concentrations (0, 125, 250, 500 and 1000 nM) and for different exposure times (0, 6, 10, 16 or 24 h). Next, blastocysts of the optimal Scriptaid-treated group and the non-treated ROSI group were separately transferred into surrogate ICR mice to compare in vivo development with the ICSI group (control). Each experiment was repeated at least three times. Metaphase II (MII) oocytes, spermatozoa and round spermatids were obtained from sexually mature BDF1 female or male mice. The developmental potential of embryos among the three groups (the ICSI, ROSI and optimal Scriptaid-treated ROSI groups) was assessed based on the rates of obtaining zygotes, two-cell stage embryos, four-cell stage embryos, blastocysts and full-term offspring. In addition, the expression levels of development-related genes (Oct4, Nanog, Klf4 and Sox2) were analysed using real-time PCR, and the methylation states of imprinted genes (H19 and Snrpn) in these three groups were detected using methylation-specific PCR (MS-PCR) sequencing following bisulfite treatment. The in vitro experiments revealed that treating ROSI-derived zygotes with 250 nM Scriptaid for 10 h significantly improved the blastocyst formation rate (59%) compared with the non-treated group (38%) and further increased the birth rates of ROSI-derived embryos from 21% to 40% in vivo. Moreover, in ROSI-derived embryos, the expression of the Oct4, Nanog and Sox2 genes at the blastocyst stage was decreased, but the optimal Scriptaid treatment restored expression to a level similar to their ICSI counterparts. In addition, Scriptaid treatment moderately repaired the abnormal DNA methylation pattern in the imprinting control regions (ICRs) of H19 and Snrpn. N/A LIMITATIONS, REASONS FOR CAUTION: Because of the ethics regarding the use of human gametes for ROSI studies, the mouse model was used as an approach to explore the effects of Scriptaid on the developmental potential of ROSI-derived embryos. However, to determine whether these findings can be applied to humans, further investigation will be required. Scriptaid treatment provides a new means of improving the efficiency and safety of clinical human ROSI. The study was financially supported through grants from the National Key Research Program of China (No. 2016YFC1304800); the National Natural Science Foundation of China (Nos: 81170756, 81571486); the Natural Science Foundation of Shanghai (Nos: 15140901700, 15ZR1424900) and the Programme for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning. There are no conflicts of interest to declare. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Effects of heat stress on bovine preimplantation embryos produced in vitro

PubMed Central

SAKATANI, Miki

2017-01-01

Summer heat stress decreases the pregnancy rate in cattle and has been thought to be associated with the early embryonic death caused by the elevation of maternal body temperature. In vitro cultures have been widely used for the evaluation of effects of heat stress on oocytes, fertilization, preimplantation, and embryonic development. Susceptibility to heat stress is present in developmental stages from oocytes to cleavage-stage (before embryonic gene activation, EGA) embryos, leading to a consequent decrease in developmental competence. On the other hand, advanced-stage embryos such as morula or blastocysts have acquired thermotolerance. The mechanism for the developmental stage-dependent change in thermotolerance is considered to be the accumulation of antioxidants in embryos in response to heat-inducible production of reactive oxygen species. The supplementation of antioxidants to the culture media has been known to neutralize the detrimental effects of heat stress. Besides, EGA could be involved in acquisition of thermotolerance in later stages of embryos. Morulae or blastocysts can repair heat-induced unfolded proteins or prevent DNA damage occurring in processes such as apoptosis. Therefore, embryo transfer (ET) that can bypass the heat-sensitive stage could be a good solution to improve the pregnancy rate under heat stress. However, frozen-thawed ET could not improve the pregnancy rate as expected. Frozen-thawed blastocysts were more sensitive to heat stress and showed less proliferation upon heat exposure, compared to fresh blastocysts. Therefore, further research is required to improve the reduction in pregnancy rates due to summer heat stress. PMID:28496018

Effects of heat stress on bovine preimplantation embryos produced in vitro.

PubMed

Sakatani, Miki

2017-08-19

Summer heat stress decreases the pregnancy rate in cattle and has been thought to be associated with the early embryonic death caused by the elevation of maternal body temperature. In vitro cultures have been widely used for the evaluation of effects of heat stress on oocytes, fertilization, preimplantation, and embryonic development. Susceptibility to heat stress is present in developmental stages from oocytes to cleavage-stage (before embryonic gene activation, EGA) embryos, leading to a consequent decrease in developmental competence. On the other hand, advanced-stage embryos such as morula or blastocysts have acquired thermotolerance. The mechanism for the developmental stage-dependent change in thermotolerance is considered to be the accumulation of antioxidants in embryos in response to heat-inducible production of reactive oxygen species. The supplementation of antioxidants to the culture media has been known to neutralize the detrimental effects of heat stress. Besides, EGA could be involved in acquisition of thermotolerance in later stages of embryos. Morulae or blastocysts can repair heat-induced unfolded proteins or prevent DNA damage occurring in processes such as apoptosis. Therefore, embryo transfer (ET) that can bypass the heat-sensitive stage could be a good solution to improve the pregnancy rate under heat stress. However, frozen-thawed ET could not improve the pregnancy rate as expected. Frozen-thawed blastocysts were more sensitive to heat stress and showed less proliferation upon heat exposure, compared to fresh blastocysts. Therefore, further research is required to improve the reduction in pregnancy rates due to summer heat stress.

Time-lapse culture with morphokinetic embryo selection improves pregnancy and live birth chances and reduces early pregnancy loss: a meta-analysis.

PubMed

Pribenszky, Csaba; Nilselid, Anna-Maria; Montag, Markus

2017-11-01

Embryo evaluation and selection is fundamental in clinical IVF. Time-lapse follow-up of embryo development comprises undisturbed culture and the application of the visual information to support embryo evaluation. A meta-analysis of randomized controlled trials was carried out to study whether time-lapse monitoring with the prospective use of a morphokinetic algorithm for selection of embryos improves overall clinical outcome (pregnancy, early pregnancy loss, stillbirth and live birth rate) compared with embryo selection based on single time-point morphology in IVF cycles. The meta-analysis of five randomized controlled trials (n = 1637) showed that the application of time-lapse monitoring was associated with a significantly higher ongoing clinical pregnancy rate (51.0% versus 39.9%), with a pooled odds ratio of 1.542 (P < 0.001), significantly lower early pregnancy loss (15.3% versus 21.3%; OR: 0.662; P = 0.019) and a significantly increased live birth rate (44.2% versus 31.3%; OR 1.668; P = 0.009). Difference in stillbirth was not significant between groups (4.7% versus 2.4%). Quality of the evidence was moderate to low owing to inconsistencies across the studies. Selective application and variability were also limitations. Although time-lapse is shown to significantly improve overall clinical outcome, further high-quality evidence is needed before universal conclusions can be drawn. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Time-lapse cinematography-compatible polystyrene-based microwell culture system: a novel tool for tracking the development of individual bovine embryos.

PubMed

Sugimura, Satoshi; Akai, Tomonori; Somfai, Tamás; Hirayama, Muneyuki; Aikawa, Yoshio; Ohtake, Masaki; Hattori, Hideshi; Kobayashi, Shuji; Hashiyada, Yutaka; Konishi, Kazuyuki; Imai, Kei

2010-12-01

We have developed a polystyrene-based well-of-the-well (WOW) system using injection molding to track individual embryos throughout culture using time-lapse cinematography (TLC). WOW culture of bovine embryos following in vitro fertilization was compared with conventional droplet culture (control). No differences between control- and WOW-cultured embryos were observed during development to the blastocyst stage. Morphological quality and inner cell mass (ICM) and trophectoderm (TE) cell numbers were not different between control- and WOW-derived blastocysts; however, apoptosis in both the ICM and TE cells was reduced in WOW culture (P < 0.01). Oxygen consumption in WOW-derived blastocysts was closer to physiological level than that of control-derived blastocysts. Moreover, WOW culture improved embryo viability, as indicated by increased pregnancy rates at Days 30 and 60 after embryo transfer (P < 0.05). TLC monitoring was performed to evaluate the cleavage pattern and the duration of the first cell cycle of embryos from oocytes collected by ovum pickup; correlations with success of pregnancy were determined. Logistic regression analysis indicated that the cleavage pattern correlated with success of pregnancy (P < 0.05), but cell cycle length did not. Higher pregnancy rates (66.7%) were observed for animals in which transferred blastocysts had undergone normal cleavage, identified by the presence of two blastomeres of the same size without fragmentation, than among those with abnormal cleavage (33.3%). These results suggest that our microwell culture system is a powerful tool for producing and selecting healthy embryos and for identifying viability biomarkers.

Somatic embryogenesis in ferns: a new experimental system.

PubMed

Mikuła, Anna; Pożoga, Mariusz; Tomiczak, Karolina; Rybczyński, Jan J

2015-05-01

Somatic embryogenesis has never been reported in ferns. The study showed that it is much easier to evoke the acquisition and expression of embryogenic competence in ferns than in spermatophytes. We discovered that the tree fern Cyathea delgadii offers an effective model for the reproducible and rapid formation of somatic embryos on hormone-free medium. Our study provides cyto-morphological evidence for the single cell origin and development of somatic embryos. Somatic embryogenesis (SE) in both primary and secondary explants was induced on half-strength micro- and macro-nutrients Murashige and Skoog medium without the application of exogenous plant growth regulators, in darkness. The early stage of SE was characterized by sequential perpendicular cell divisions of an individual epidermal cell of etiolated stipe explant. These resulted in the formation of a linear pro-embryo. Later their development resembled that of the zygotic embryo. We defined three morphogenetic stages of fern somatic embryo development: linear, early and late embryonic leaf stage. The transition from somatic embryo to juvenile sporophyte was quick and proceeded without interruption caused by dormancy. Following 9 weeks of culture the efficiency of somatic embryogenesis reached 12-13 embryos per responding explant. Spontaneous formation of somatic embryos and callus production, which improved the effectiveness of the process sevenfold in 10-month-long culture, occurred without subculturing. The tendency for C. delgadii to propagate by SE in vitro makes this species an excellent model for studies relating to asexual embryogenesis and the endogenous hormonal regulation of that process and opens new avenues of experimentation.

Practical applications of new research information in the practice of bovine embryo transfer.

PubMed

Looney, C R; Pryor, J H

2010-01-01

For more than 40 years, practitioners have sought to improve all aspects of commercial bovine embryo transfer. The development of new technologies for this industry has been substantial, with recent focus on cryopreservation techniques and the in vitro production of embryos fertilised with sexed spermatozoa. When these and other new technologies are developed, the following questions remain: (1) is said technology regulated or does it require licensing; and (2) is it applicable and, if so, is it financially feasible? Computer access to published research and the advancement of data software programs conducive to the industry for data procurement have been essential for helping practitioners answer these questions by enhancing their ability to analyse and apply data. The focus of the present paper is to aid commercial embryo transfer practitioners in determining new technologies that are available and whether they can be implemented effectively, benefiting their programs.

Modification of mitochondrial function, cytoplasmic lipid content and cryosensitivity of bovine embryos by resveratrol.

PubMed

Abe, Takahito; Kawahara-Miki, Ryouka; Hara, Tomotaka; Noguchi, Tatsuo; Hayashi, Takeshi; Shirasuna, Koumei; Kuwayama, Takehito; Iwata, Hisataka

2017-10-18

Resveratrol is a potent activator of NAD-dependent deacetyltransferase sirtuin-1 (SIRT1) and affects lipid metabolism and ATP generation in somatic cells. In the present study, the effects of supplementing culture medium with resveratrol on lipid metabolism, ATP generation, and cryosensitivity of bovine in vitro produced embryos were investigated. Bovine early cleaved-stage embryos were cultured in medium containing 0 or 0.5 µM resveratrol for 1 or 5 days. Resveratrol treatment for both 1 day and 5 days increased the expression levels of SIRT1 and phosphorylated AMP-activated protein kinase (pAMPK) in the embryos. Furthermore, resveratrol treatment was effective to increase ATP generation and reduce lipid content of the embryos. The effects of resveratrol treatment were diminished by the SIRT1 inhibitor "EX527", and the reduced lipid content was reversed by treatment with etomoxir (a potent inhibitor of beta-oxidation). Blastocysts developed after resveratrol treatment showed low levels reactive oxygen species and increased cryotolerance. These results demonstrate that resveratrol improves in vitro development of bovine embryos, while reducing cytoplasmic lipid content through activation of beta-oxidation, thereby effective for production of bovine blastocysts with enhanced cryotolerance.

Comprehensive chromosome screening improves embryo selection: a meta-analysis.

PubMed

Dahdouh, Elias M; Balayla, Jacques; García-Velasco, Juan Antonio

2015-12-01

To study whether preimplantation genetic screening with comprehensive chromosome screening (PGS-CCS) improves clinical implantation rates (IR) and sustained IR (beyond 20 weeks) compared with routine care for embryo selection in IVF cycles. Meta-analysis of randomized controlled trials (RCTs) and observational studies (OSs). University-affiliated teaching hospital. Infertile couples undergoing IVF. PGS-CCS with the use of different genetic platforms performed on polar body (PB), cleavage embryo, or blastocyst following embryo biopsy. Clinical IR and sustained IR in RCTs as well as OSs comparing PGS-CCS and routine care were determined after a complete review of the literature. Pooled estimates of risk ratios (RRs) with their 95% confidence intervals (CIs) according to a fixed-effects model with the use of the Mantel-Haenszel method were calculated after the meta-analysis. Forest plots are provided for comparative purposes. Out of 763 citations identified, 29 articles met initial eligibility criteria and were further analyzed. Of these, only three RCTs and eight OSs met full inclusion criteria, allowing direct comparison of PGS-CCS and routine IVF care based on embryo morphology selection. In the RCTs, all embryo biopsies were performed on day 5-6 of embryo development. In the OSs, biopsies were performed on different stages of embryo development, including PB, day 3, or day 5-6. Meta-analysis of the RCTs (3 studies; n = 659) showed that PGS-CCS was associated with a significantly higher clinical IR, with a pooled RR of 1.29 (95% CI 1.15-1.45), as well as a significantly higher sustained IR, with a pooled RR of 1.39 (95% CI 1.21-1.60). Similar findings were shown in the OSs, where the pooled RR for clinical IR was 1.78 (95% CI 1.60-1.99; 7 studies; n = 2,993) and for sustained IR was 1.75 (95% CI 1.48-2.07; 4 studies; n = 1,124). Statistical heterogeneity (I(2)) was minimal for RCTs and substantial among OSs. PGS with the use of CCS technology increases clinical and sustained IRs, thus improving embryo selection, particularly in patients with normal ovarian reserve. Results from ongoing RCTs conducted on different patient populations (e.g., decreased ovarian reserve) and different embryo stage biopsy (e.g., PB, day 3) may further clarify the role of this technology. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Studies for Somatic Embryogenesis in Sweet Potato

NASA Technical Reports Server (NTRS)

Bennett, J. Rasheed; Prakash, C. S.

1997-01-01

The purpose of this study was to improve the somatic embryo (SE) system for plant production of sweet potato (Ipomoea batatas L(Lam)). Explants isolated from SE-derived sweet potato plants were compared with control (non SE-derived) plants for their competency for SE production. Leaf explants were cultured on Murashige-Skoog (MS) medium with 2,4-dichlorophenoxy acetic acid (0.2 mg/L) and 6-benzylaminopurine (2.5 mg/L) for 2 weeks in darkness and transferred to MS medium with abscisic acid (2.5 mg/L). Explants isolated from those plants developed through somatic embryogenesis produced new somatic embryos rapidly and in higher frequency than those isolated from control plants They also appeared to grow faster in tissue culture than the control plants. Current studies in the laboratory are examining whether plants derived from a cyclical embryogenesis system (five cycles) would have any further positive impact on the rapidity and frequency of somatic embryo development. More detailed studies using electron microscopy are expected to show the point of origin of the embryos and to allow determination of their quality throughout the cyclical process. This study may facilitate improved plant micropropagation, gene transfer and germplasm conservation in sweet potato.

Evaluating biomechanical properties of murine embryos using Brillouin microscopy and optical coherence tomography

NASA Astrophysics Data System (ADS)

Raghunathan, Raksha; Zhang, Jitao; Wu, Chen; Rippy, Justin; Singh, Manmohan; Larin, Kirill V.; Scarcelli, Giuliano

2017-08-01

Embryogenesis is regulated by numerous changes in mechanical properties of the cellular microenvironment. Thus, studying embryonic mechanophysiology can provide a more thorough perspective of embryonic development, potentially improving early detection of congenital abnormalities as well as evaluating and developing therapeutic interventions. A number of methods and techniques have been used to study cellular biomechanical properties during embryogenesis. While some of these techniques are invasive or involve the use of external agents, others are compromised in terms of spatial and temporal resolutions. We propose the use of Brillouin microscopy in combination with optical coherence tomography (OCT) to measure stiffness as well as structural changes in a developing embryo. While Brillouin microscopy assesses the changes in stiffness among different organs of the embryo, OCT provides the necessary structural guidance.

Demands for carbohydrates as major energy substrates depend on the preimplantation developmental stage in pig embryos: Differential use of fructose by parthenogenetic diploids before and after the 4-cell stage in the pig

PubMed Central

SHIBUTANI, Mihiro; LEE, Jibak; MIYANO, Takashi; MIYAKE, Masashi

2015-01-01

The embryo culture technique has been improving, but the detailed demands for energy substrates such as glucose, fructose, pyruvate and lactate of preimplantation embryos are still unclear. In the present study, the demands of pig preimplantation embryos at each different developmental stage were investigated by use of parthenogenetic diploids as a model of pig preimplantation embryos. Pig parthenogenetic diploids showed different use of glucose and fructose before and after the 4-cell stage. Although glucose supported the development of pig embryos throughout the preimplantation stages and even maintained the expansion and hatching of blastocysts, it suppressed development to the blastocyst stage when glucose coexisted with pyruvate and lactate from 4 h after activation, but not after 48 h (early 4-cell stage). Since ketohexokinase that metabolizes fructose was not expressed in 2-cell and 4-cell diploids, a medium that included only fructose as a major energy substrate did not support early cleavage of pig diploids beyond the 4-cell stage, and almost no diploids developed to the morula stage just as in a medium without carbohydrates. These results may explain the different suppressive effects on pig preimplantation development between glucose and fructose when pyruvate and lactate were present in a medium. In addition, 4-cell diploids that had been cultured in a medium with pyruvate and lactate developed to the expanded blastocyst stage without any carbohydrates as a major energy substrate. These results show that the demands for carbohydrates are different depending on the developmental stage in pig preimplantation embryos. PMID:25736264

Successful development of viable blastocysts from enhanced green fluorescent protein transgene-microinjected mouse embryos: comparison of culture media.

PubMed

Devgan, Vikram; Seshagiri, Polani B

2003-07-01

To improve efficiency of transgenesis, we compared M16 and CZB embryo culture media, supporting development to blastocysts of FVB/N mouse pronuclear-eggs, microinjected with enhanced green fluorescent protein (EGFP) transgene. When EGFP-injected-eggs were cultured (120 hr), blastocyst development was significantly (P < 0.03) higher in M16 medium (72.5 +/- 2.4%) than that in CZB (13.2 +/- 4.3%) or CZBG (CZB with 5.6 mM glucose at 48 hr culture) (62.1 +/- 3.7%) media. Blastocyst development of noninjected embryos was higher in M16 (92.0 +/- 2.6%) and CZBG (83.9 +/- 3.9%) media than in CZB (31.9 +/- 2.8%) medium (P < 0.0001). However, percentages of morulae at 72 hr were comparable in all treatments. Developed blastocysts were better in M16 than in CZB or CZBG media. Consistent with this, mean cell number per blastocyst, developed from injected embryos, was significantly (P < 0.002) higher in M16 medium (79.6), than those in CZB (31.3) or CZBG media (60.7); similar with noninjected embryos. Cell allocation to trophectoderm (TE) and inner cell mass (ICM), i.e., TE:ICM ratio, for injected blastocysts in M16 (3.0) was less than (P < 0.05) those in CZB (4.2) and CZBG (4.4) media; similar with noninjected blastocysts. Moreover, blastocysts, developed in M16 and CZBG media, hatched, attached, and exhibited trophoblast outgrowth; 18% of them showed EGFP-expression. Importantly, blastocysts from M16 medium produced live transgenic "green" pups (11%) following embryo transfer. Taken together, our results indicate that supplementation of glucose, at 48 hr of culture (CZBG), is required for morula to blastocyst transition; M16 medium, containing glucose from the beginning of culture, is superior to CZB or CZBG for supporting development of biologically viable blastocysts from EGFP-transgene-injected mouse embryos. Copyright 2003 Wiley-Liss, Inc.

Effects of trichostatin A on In vitro development and DNA methylation level of the satellite I region of swamp buffalo (Bubalus bubalis) cloned embryos.

PubMed

Srirattana, Kanokwan; Ketudat-Cairns, Mariena; Nagai, Takashi; Kaneda, Masahiro; Parnpai, Rangsun

2014-01-01

Trichostatin A (TSA), a histone deacetylase inhibitor, has been widely used to improve the cloning efficiency in several species. This brings our attention to investigation of the effects of TSA on developmental potential of swamp buffalo cloned embryos. Swamp buffalo cloned embryos were produced by electrical pulse fusion of male swamp buffalo fibroblasts with swamp buffalo enucleated oocytes. After fusion, reconstructed oocytes were treated with 0, 25 or 50 nM TSA for 10 h. The results showed that there was no significant difference in the rates of fusion (82-85%), cleavage (79-84%) and development to the 8-cell stage (59-65%) among treatment groups. The highest developmental rates to the morula and blastocyst stages of embryos were found in the 25 nM TSA-treated group (42.7 and 30.1%, respectively). We also analyzed the DNA methylation level in the satellite I region of donor cells and in in vitro fertilized (IVF) and cloned embryos using the bisulfite DNA sequencing method. The results indicated that the DNA methylation levels in cloned embryos were significantly higher than those of IVF embryos but approximately similar to those of donor cells. Moreover, there was no significant difference in the methylation level among TSA-treated and untreated cloned embryos. Thus, TSA treatments at 25 nM for 10 h could enhance the in vitro developmental potential of swamp buffalo cloned embryos, but no beneficial effect on the DNA methylation level was observed.

Effects of Trichostatin A on In Vitro Development and DNA Methylation Level of the Satellite I Region of Swamp Buffalo (Bubalus bubalis) Cloned Embryos

PubMed Central

SRIRATTANA, Kanokwan; KETUDAT-CAIRNS, Mariena; NAGAI, Takashi; KANEDA, Masahiro; PARNPAI, Rangsun

2014-01-01

Trichostatin A (TSA), a histone deacetylase inhibitor, has been widely used to improve the cloning efficiency in several species. This brings our attention to investigation of the effects of TSA on developmental potential of swamp buffalo cloned embryos. Swamp buffalo cloned embryos were produced by electrical pulse fusion of male swamp buffalo fibroblasts with swamp buffalo enucleated oocytes. After fusion, reconstructed oocytes were treated with 0, 25 or 50 nM TSA for 10 h. The results showed that there was no significant difference in the rates of fusion (82–85%), cleavage (79–84%) and development to the 8-cell stage (59–65%) among treatment groups. The highest developmental rates to the morula and blastocyst stages of embryos were found in the 25 nM TSA-treated group (42.7 and 30.1%, respectively). We also analyzed the DNA methylation level in the satellite I region of donor cells and in in vitro fertilized (IVF) and cloned embryos using the bisulfite DNA sequencing method. The results indicated that the DNA methylation levels in cloned embryos were significantly higher than those of IVF embryos but approximately similar to those of donor cells. Moreover, there was no significant difference in the methylation level among TSA-treated and untreated cloned embryos. Thus, TSA treatments at 25 nM for 10 h could enhance the in vitro developmental potential of swamp buffalo cloned embryos, but no beneficial effect on the DNA methylation level was observed. PMID:24909601

Stage selection and restricted oviposition period improves cryopreservation of dipteran embryos.

PubMed

Rajamohan, Arun; Rinehart, Joseph P; Leopold, Roger A

2015-04-01

Embryos of two dipteran species (Musca domestica and Lucilia sericata) were assessed for an effective sampling time that would result in the highest post-cryopreservation hatch rate, with a primary goal to define species-specific egg collection periods and the effects of manual stage selection on post cryopreservation yield. The effects of the time taken to collect eggs on, (a) the proportion of embryos reaching a specific developmental stage between 17 and 20 h of development, and (b) the post-cryopreservation hatch rate were assessed. Permeabilization treatment applied at any stage of embryonic development did not significantly reduce embryo viability. Eggs collected over longer durations significantly reduced the number of embryos available in a specific developmental stage amenable to cryopreservation. Hatch percentage after cryopreservation of the embryos of M. domestica collected over a 60 min period was 10.7 ± 8.7% compared to 31 ± 5% for the eggs collected for just 15 min. Similarly, percent hatch in L. sericata resulted in 17.0 ± 3.9 and <2% for 15 and 60 min samples, respectively. Significantly higher hatching rates were obtained for cryopreservation after manual selection of specific embryonic developmental stages from the dechorionated samples. Post-cryopreservation hatching rate for stage-selected M. domestica embryos was 86.5 ± 5.5% compared to 33.3 ± 4.5% for embryos staged only by an overall visual confirmation. In the case of L. sericata, the hatching percentage was 79.0 ± 11.1 for stage-selected embryos compared to 17.0 ± 3.9% without individual selection. Published by Elsevier Inc.

Treatment with proteasome inhibitor MG132 during cloning improves survival and pronuclear number of reconstructed rat embryos.

PubMed

Nakajima, Noriaki; Inomata, Tomo; Ito, Junya; Kashiwazaki, Naomi

2008-12-01

In several mammalian species including rats, successfully cloned animals have been generated using somatic cell nuclear transfer (SCNT). However, in the case of rats, additional treatment with MG132, a proteasome inhibitor, before enucleation of oocytes seems to be required for successful cloning because ovulated rat oocytes are spontaneously activated, and hence, their suppression is the key to successful cloning. A previous study on rats demonstrated that matured oocytes potentially possess lower cytostatic factor (CSF) activity compared to mouse oocytes, resulting in a low incidence of premature chromosome condensation in the reconstructed embryos after SCNT. It is known that mice having more than two pronuclei are generally observed in nuclear-transferred oocytes after induction of premature chromosome condensation, which implies successful reprogramming. This leads us to the hypothesis that MG132 treatment affects not only the inhibition of spontaneous activation but also the reprogramming and developmental ability of reconstructed rat embryos. If so, prolonged MG132 treatment during and/or after SCNT may further improve the survivability. However, the effect of MG132 treatment on reconstructed embryos after SCNT has been very limited in rats and other species. We show here that prolonged MG132 treatment during and after SCNT improves survival and the number of pronuclei in reconstructed rat embryos after activation. These reconstructed embryos treated before, during, and after SCNT showed significantly higher p34(cdc2) kinase activity involving CSF activity compared to that of the control embryos. On the other hand, p34(cdc2) kinase activity was not recovered in nuclear-transferred oocytes without MG132, which suggested that the enucleation had detrimental effects on the development of reconstructed oocytes. Taken together, MG132 treatment during SCNT increases survival and pronuclear numbers in reconstructed rat embryos via maintenance of high CSF activity. The data suggest that MG132 treatment is indispensable for at least rat SCNT.

The use of morphokinetics as a predictor of embryo implantation.

PubMed

Meseguer, Marcos; Herrero, Javier; Tejera, Alberto; Hilligsøe, Karen Marie; Ramsing, Niels Birger; Remohí, Jose

2011-10-01

Time-lapse observation presents an opportunity for optimizing embryo selection based on morphological grading as well as providing novel kinetic parameters, which may further improve accurate selection of viable embryos. The objective of this retrospective study was to identify the morphokinetic parameters specific to embryos that were capable of implanting. In order to compare a large number of embryos, with minimal variation in culture conditions, we have used an automatic embryo monitoring system. Using a tri-gas IVF incubator with a built-in camera designed to automatically acquire images at defined time points, we have simultaneously monitored up to 72 individual embryos without removing the embryos from the controlled environment. Images were acquired every 15 min in five different focal planes for at least 64 h for each embryo. We have monitored the development of transferred embryos from 285 couples undergoing their first ICSI cycle. The total number of transferred embryos was 522, of which 247 either failed to implant or fully implanted, with full implantation meaning that all transferred embryos in a treatment implanted. A detailed retrospective analysis of cleavage times, blastomere size and multinucleation was made for the 247 transferred embryos with either failed or full implantation. We found that several parameters were significantly correlated with subsequent implantation (e.g. time of first and subsequent cleavages as well as the time between cleavages). The most predictive parameters were: (i) time of division to 5 cells, t5 (48.8-56.6 h after ICSI); (ii) time between division to 3 cells and subsequent division to 4 cells, s2 (≤ 0.76 h) and (iii) duration of cell cycle two, i.e. time between division to 2 cells and division to 3 cells, cc2 (≤ 11.9 h). We also observed aberrant behavior such as multinucleation at the 4 cell stage, uneven blastomere size at the 2 cell stage and abrupt cell division to three or more cells, which appeared to largely preclude implantation. The image acquisition and time-lapse analysis system makes it possible to determine exact timing of embryo cleavages in a clinical setting. We propose a multivariable model based on our findings to classify embryos according to their probability of implantation. The efficacy of this classification will be evaluated in a prospective randomized study that ultimately will determine if implantation rates can be improved by time-lapse analysis.

Reprogramming towards totipotency is greatly facilitated by synergistic effects of small molecules

PubMed Central

Tajima, Yosuke; Yoshida, Koki; Oikawa, Mami; Azuma, Rika; Allen, George E.; Tsujikawa, Tomomi; Tsukaguchi, Tomomasa; Bradshaw, Charles R.; Jullien, Jerome; Yamagata, Kazuo; Matsumoto, Kazuya; Anzai, Masayuki; Imai, Hiroshi; Gurdon, John B.; Yamada, Masayasu

2017-01-01

ABSTRACT Animal cloning has been achieved in many species by transplanting differentiated cell nuclei to unfertilized oocytes. However, the low efficiencies of cloning have remained an unresolved issue. Here we find that the combination of two small molecules, trichostatin A (TSA) and vitamin C (VC), under culture condition with bovine serum albumin deionized by ion-exchange resins, dramatically improves the cloning efficiency in mice and 15% of cloned embryos develop to term by means of somatic cell nuclear transfer (SCNT). The improvement was not observed by adding the non-treated, rather than deionized, bovine serum. RNA-seq analyses of SCNT embryos at the two-cell stage revealed that the treatment with TSA and VC resulted in the upregulated expression of previously identified reprogramming-resistant genes. Moreover, the expression of early-embryo-specific retroelements was upregulated by the TSA and VC treatment. The enhanced gene expression was relevant to the VC-mediated reduction of histone H3 lysine 9 methylation in SCNT embryos. Our study thus shows a simply applicable method to greatly improve mouse cloning efficiency, and furthers our understanding of how somatic nuclei acquire totipotency. PMID:28412714

Interspecies somatic cell nucleus transfer with porcine oocytes as recipients: A novel bioassay system for assessing the competence of canine somatic cells to develop into embryos.

PubMed

Sugimura, S; Narita, K; Yamashiro, H; Sugawara, A; Shoji, T; Terashita, Y; Nishimori, K; Konno, T; Yoshida, M; Sato, E

2009-09-01

Interspecies somatic cell nucleus transfer (iSCNT) could be a useful bioassay system for assessing the ability of mammalian somatic cells to develop into embryos. To examine this possibility, we performed canine iSCNT using porcine oocytes, allowed to mature in vitro, as recipients. Canine fibroblasts from the tail tips and dewclaws of a female poodle (Fp) and a male poodle (Mp) were used as donors. We demonstrated that the use of porcine oocytes induced blastocyst formation in the iSCNT embryos cultured in porcine zygote medium-3. In Fp and Mp, the rate of blastocyst formation from cleaved embryos (Fp: 6.3% vs. 22.4%; and Mp: 26.1% vs. 52.4%) and the number of cells at the blastocyst stage (Fp: 30.7 vs. 60.0; and Mp: 27.2 vs. 40.1) were higher in the embryos derived from dewclaw cells than in those derived from tail-tip cells (P<0.05). The use of donor cells of any type in later passages decreased the rate of blastocyst formation. Treatment with trichostatin-A did not improve the rate of blastocyst formation from cleaved dewclaw cell-derived embryos but did so in the embryos derived from the tail-tip cells of Fp. Only blastocysts derived from dewclaw cells of Mp developed outgrowths. However, outgrowth formation was retrieved in the embryos derived from dewclaw cells of Fp by aggregation at the 4-cell stage. We inferred that iSCNT performed using porcine oocytes as recipients could represent a novel bioassay system for evaluating the developmental competence of canine somatic cells.

Differential developmental ability of embryos cloned from tissue-specific stem cells.

PubMed

Inoue, Kimiko; Noda, Shinichi; Ogonuki, Narumi; Miki, Hiromi; Inoue, Shinichi; Katayama, Kazufumi; Mekada, Kazuyuki; Miyoshi, Hiroyuki; Ogura, Atsuo

2007-05-01

Although cloning animals by somatic cell nuclear transfer is generally inefficient, the use of certain nuclear donor cell types may significantly improve or deteriorate outcomes. We evaluated whether two multipotent stem cell lines produced in vitro--neural stem cells (NSCs) and mesenchymal stem cells (MSCs)--could serve as nuclear donors for nuclear transfer cloning. Most (76%) NSC-derived embryos survived the two-cell-to-four-cell transition, the stage when the major zygotic gene activation occurs. Consistent with this observation, the expression patterns of zygotically active genes were better in NSC-derived embryos than in fibroblast clone embryos, which arrested at the two-cell stage more frequently. Embryo transfer experiments demonstrated that at least some of these NSC embryos had the ability to develop to term fetuses (1.6%, 3/189). In contrast, embryos reconstructed using MSCs showed a low rate of in vitro development and never underwent implantation in vivo. Chromosomal analysis of the donor MSCs revealed very frequent aneuploidy, which probably impaired the potential for development of their derived clones. This is the first demonstration that tissue-specific multipotent stem cells produced in vitro can serve as donors of nuclei for cloning mice; however, these cells may be prone to chromosomal aberrations, leading to high embryonic death rates. We found previously that hematopoietic stem cells (HSCs) are very inefficient donor cells because of their failure to activate the genes essential for embryonic development. Taken together, our data led us to conclude that tissue-specific stem cells in mice, namely NSCs, MSCs, and HSCs, exhibited marked variations in the ability to produce cloned offspring and that this ability varies according to both the epigenetic and genetic status of the original genomes. Disclosure of potential conflicts of interest is found at the end of this article.

Cotyledonary somatic embryos of Pinus pinaster Ait. most closely resemble fresh, maturing cotyledonary zygotic embryos: biological, carbohydrate and proteomic analyses.

PubMed

Morel, Alexandre; Trontin, Jean-François; Corbineau, Françoise; Lomenech, Anne-Marie; Beaufour, Martine; Reymond, Isabelle; Le Metté, Claire; Ader, Kevin; Harvengt, Luc; Cadene, Martine; Label, Philippe; Teyssier, Caroline; Lelu-Walter, Marie-Anne

2014-11-01

Cotyledonary somatic embryos (SEs) of maritime pine are routinely matured for 12 weeks before being germinated and converted to plantlets. Although regeneration success is highly dependent on SEs quality, the date of harvesting is currently determined mainly on the basis of morphological features. This empirical method does not provide any accurate information about embryo quality with respect to storage compounds (proteins, carbohydrates). We first analyzed SEs matured for 10, 12 and 14 weeks by carrying out biological (dry weight, water content) and biochemical measurements (total protein and carbohydrate contents). No difference could be found between collection dates, suggesting that harvesting SEs after 12 weeks is appropriate. Cotyledonary SEs were then compared to various stages, from fresh to fully desiccated, in the development of cotyledonary zygotic embryos (ZEs). We identified profiles that were similar using hierarchical ascendant cluster analysis (HCA). Fresh and dehydrated ZEs could be distinguished, and SEs clustered with fresh ZEs. Both types of embryo exhibited similar carbohydrate and protein contents and signatures. This high level of similarity (94.5 %) was further supported by proteome profiling. Highly expressed proteins included storage, stress-related, late embryogenesis abundant and energy metabolism proteins. By comparing overexpressed proteins in developing and cotyledonary SEs or ZEs, some (23 proteins) could be identified as candidate biomarkers for the late, cotyledonary stage. This is the first report of useful generic protein markers for monitoring embryo development in maritime pine. Our results also suggest that improvements of SEs quality may be achieved if the current maturation conditions are refined.

Culture medium composition affects the gene expression pattern and in vitro development potential of bovine somatic cell nuclear transfer (SCNT) embryos.

PubMed

Arias, María E; Ross, Pablo J; Felmer, Ricardo N

2013-01-01

Different culture systems have been studied that support development of somatic cell nuclear transfer (SCNT) embryos up to the blastocyst stage. However, the use of sequential and two-step culture systems has been less studied. The objective of the present study was to examine the developmental potential and quality of bovine SCNT embryos cultured in different two-step culture media based on KSOM, SOF and the macromolecules FBS and BSA (K-K/FBS, K-S/BSA and K-K/BSA, respectively). No differences were observed in the cleavage rate for any of the culture systems. However, there was a significant difference (P<0.01) in the rate of blastocyst development, with the K-K/ FBS culture system yielding a higher rate of blastocysts (28%) compared to other treatments (18 and 15%, for K-S/BSA and K-K/BSA, respectively). Although quality of embryos, as assessed by the total number of cells, was not different, the apoptosis index was significantly affected in the sequential culture system (K-S/BSA). Gene expression analysis showed alterations of DNMT1, IGF2, LIF, and PRDX6 genes in embryos cultured in K-S/FBS and of SOD2 in embryos cultured in K-K/BSA. In conclusion, we demonstrated that culture medium may affect not only the developmental potential of SCNT embryos but also, more importantly, the gene expression pattern and apoptotic index, presenting the possibility to manipulate the culture medium composition to modulate global gene expression and improve the overall efficiency of this technique.

Expression profile of genes as indicators of developmental competence and quality of in vitro fertilization and somatic cell nuclear transfer bovine embryos.

PubMed

Cánepa, Maria Jesús; Ortega, Nicolás Matías; Monteleone, Melisa Carolina; Mucci, Nicolas; Kaiser, German Gustavo; Brocco, Marcela; Mutto, Adrián

2014-01-01

Reproductive biotechnologies such as in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) enable improved reproductive efficiency of animals. However, the birth rate of in vitro-derived embryos still lags behind that of their in vivo counterparts. Thus, it is critical to develop an accurate evaluation and prediction system of embryo competence, both for commercial purposes and for scientific research. Previous works have demonstrated that in vitro culture systems induce alterations in the relative abundance (RA) of diverse transcripts and thus compromise embryo quality. The aim of this work was to analyze the RA of a set of genes involved in cellular stress (heat shock protein 70-kDa, HSP70), endoplasmic reticulum (ER) stress (immunoglobulin heavy chain binding protein, Bip; proteasome subunit β5, PSMB5) and apoptosis (BCL-2 associated X protein, Bax; cysteine aspartate protease-3, Caspase-3) in bovine blastocysts produced by IVF or SCNT and compare it with that of their in vivo counterparts. Poly (A) + mRNA was isolated from three pools of 10 blastocysts per treatment and analyzed by real-time RT-PCR. The RA of three of the stress indicators analyzed (Bax, PSMB5 and Bip) was significantly increased in SCNT embryos as compared with that of in vivo-derived blastocysts. No significant differences were found in the RA of HSP70 and Caspase-3 gene transcripts. This study could potentially complement morphological analyses in the development of an effective and accurate technique for the diagnosis of embryo quality, ultimately aiding to improve the efficiency of assisted reproductive techniques (ART).

Recent progress in bovine somatic cell nuclear transfer.

PubMed

Akagi, Satoshi; Geshi, Masaya; Nagai, Takashi

2013-03-01

Bovine somatic cell nuclear transfer (SCNT) embryos can develop to the blastocyst stage at a rate similar to that of embryos produced by in vitro fertilization. However, the full-term developmental rate of SCNT embryos is very low, owing to the high embryonic and fetal losses after embryo transfer. In addition, increased birth weight and postnatal mortality are observed at high rates in cloned calves. The low efficiency of SCNT is probably attributed to incomplete reprogramming of the donor nucleus and most of the developmental problems of clones are thought to be caused by epigenetic defects. Applications of SCNT will depend on improvement in the efficiency of production of healthy cloned calves. In this review, we discuss problems and recent progress in bovine SCNT. © 2013 Japanese Society of Animal Science.

[Influence of the Concentration of Dissolved Oxygen on Embryonic Development of the Common Toad (Bufo bufo)].

PubMed

Dmitrieva, E V

2015-01-01

Several series of experiments investigating the influence of dissolved oxygen concentrations on the growth rates and mortality in the embryogenesis of the common toad Bufo bufo were carried out. The experiments showed that, when the eggs develop singly, the lack of oxygen does not lead to an increase in mortality by the time of hatching and results only in a change in the dynamics of mortality: mortality occurs at an earlier stage of development than in the conditions of normal access to oxygen. Taking into account the combined effect of the density of eggs and the dissolved oxygen concentration, we increase the accuracy of analysis of the experimental results and improve the interpretation of the results. In the conditions of different initial density of eggs, the impact of the concentration of dissolved oxygen on mortality and rates of development of the common toad embryos is manifested in different ways. At high density, only a small percentage of embryos survives by the time of hatching, and the embryos are significantly behind in their development compared with the individuals that developed in normal oxygen conditions. The lack of oxygen dissolved in the water slows down the development of embryos of the common toad.

Effects of maternal dietary manganese and incubation temperature on hatchability, antioxidant status, and expression of heat shock proteins in chick embryos.

PubMed

Zhu, Y W; Lu, L; Li, W X; Zhang, L Y; Ji, C; Lin, X; Liu, H C; Odle, J; Luo, X G

2015-12-01

To investigate whether supplementing manganese (Mn) to the maternal diet could reduce the deleterious effect of heat stress on the developing embryo, the hatchability, antioxidant status, and expression of heat shock proteins (HSP) were evaluated in chick embryos under normal and high incubation temperatures. A completely randomized design ( = 6) with 2 maternal dietary Mn treatments (unsupplemented control basal diet versus the basal diet + 120 mg Mn/kg as inorganic Mn) × 2 incubation temperatures (normal, 37.8°C, versus high, 39.0°C) was used. High incubation temperature did not affect ( > 0.19) hatchability and embryo mortality and development but did increase ( < 0.05) activities of heart manganese superoxide dismutase (MnSOD) and liver copper zinc superoxide dismutase and liver MnSOD mRNA and protein levels in embryos. High incubation temperature also decreased ( < 0.003) HSP70 protein level in the heart but had no effects ( > 0.07) in the liver of embryos. Maternal diet with Mn supplementation not only increased ( < 0.05) the hatchability and Mn content ( < 0.001) in the yolk and embryonic tissues and the activity of MnSOD in the heart ( < 0.004) as well as relative liver weight ( < 0.05) under normal incubation temperature but also decreased ( ≤ 0.05) embryo mortality and HSP90 mRNA level in the liver and heart of embryos. Furthermore, under high incubation temperature, maternal diet Mn supplementation increased ( < 0.002) MnSOD protein expression in the liver of embryos but had no effect ( > 0.43) under normal incubation temperature. These results indicated that high incubation temperature induced self-protective responses of chick embryos with a modification of antioxidant status and a depression of HSP70 protein level. Maternal dietary supplementation of Mn could improve the hatchability as well as antioxidant ability to protect against heat challenge in embryos during incubation.

Embryo density and medium volume effects on early murine embryo development.

PubMed

Canseco, R S; Sparks, A E; Pearson, R E; Gwazdauskas, F C

1992-10-01

One-cell mouse embryos were used to determine the effects of drop size and number of embryos per drop for optimum development in vitro. Embryos were collected from immature C57BL6 female mice superovulated with pregnant mare serum gonadotropin and human chorionic gonadotropin and mated by CD1 males. Groups of 1, 5, 10, or 20 embryos were cultured in 5-, 10-, 20-, or 40-microliters drops of CZB under silicon oil at 37.5 degrees C in a humidified atmosphere of 5% CO2 and 95% air. Development score for embryos cultured in 10 microliters was higher than that of embryos cultured in 20 or 40 microliters. Embryos cultured in groups of 5, 10, or 20 had higher development scores than embryos cultured singly. The highest development score was obtained by the combination of 5 embryos per 10-microliters drop. The percentage of live embryos in 20 or 40 microliters was lower than that of embryos cultured in 10 microliters. Additionally, the percentage of live embryos cultured singly was lower than that of embryos cultured in groups. Our results suggest that a stimulatory interaction occurs among embryos possibly exerted through the secretion of growth factors. This effect can be diluted if the embryos are cultured in large drops or singly.

Significant Improvement in Cloning Efficiency of an Inbred Miniature Pig by Histone Deacetylase Inhibitor Treatment after Somatic Cell Nuclear Transfer1

PubMed Central

Zhao, Jianguo; Ross, Jason W.; Hao, Yanhong; Spate, Lee D.; Walters, Eric M.; Samuel, Melissa S.; Rieke, August; Murphy, Clifton N.; Prather, Randall S.

2009-01-01

The National Institutes of Health (NIH) miniature pig was developed specifically for xenotransplantation and has been extensively used as a large-animal model in many other biomedical experiments. However, the cloning efficiency of this pig is very low (<0.2%), and this has been an obstacle to the promising application of these inbred swine genetics for biomedical research. It has been demonstrated that increased histone acetylation in somatic cell nuclear transfer (SCNT) embryos, by applying a histone deacetylase (HDAC) inhibitor such as trichostatin A (TSA), significantly enhances the developmental competence in several species. However, some researchers also reported that TSA treatment had various detrimental effects on the in vitro and in vivo development of the SCNT embryos. Herein, we report that treatment with 500 nM 6-(1,3-dioxo-1H, 3H-benzo[de]isoquinolin-2-yl)-hexanoic acid hydroxyamide (termed scriptaid), a novel HDAC inhibitor, significantly enhanced the development of SCNT embryos to the blastocyst stage when NIH inbred fetal fibroblast cells (FFCs) were used as donors compared with the untreated group (21% vs. 9%, P < 0.05). Scriptaid treatment resulted in eight pregnancies from 10 embryo transfers (ETs) and 14 healthy NIH miniature pigs from eight litters, while no viable piglets (only three mummies) were obtained from nine ETs in the untreated group. Thus, scriptaid dramatically increased the cloning efficiency when using inbred genetics from 0.0% to 1.3%. In contrast, scriptaid treatment decreased the blastocyst rate in in vitro fertilization embryos (from 37% to 26%, P < 0.05). In conclusion, the extremely low cloning efficiency in the NIH miniature pig may be caused by its inbred genetic background and can be improved by alteration of genomic histone acetylation patterns. PMID:19386991

Significant improvement in cloning efficiency of an inbred miniature pig by histone deacetylase inhibitor treatment after somatic cell nuclear transfer.

PubMed

Zhao, Jianguo; Ross, Jason W; Hao, Yanhong; Spate, Lee D; Walters, Eric M; Samuel, Melissa S; Rieke, August; Murphy, Clifton N; Prather, Randall S

2009-09-01

The National Institutes of Health (NIH) miniature pig was developed specifically for xenotransplantation and has been extensively used as a large-animal model in many other biomedical experiments. However, the cloning efficiency of this pig is very low (<0.2%), and this has been an obstacle to the promising application of these inbred swine genetics for biomedical research. It has been demonstrated that increased histone acetylation in somatic cell nuclear transfer (SCNT) embryos, by applying a histone deacetylase (HDAC) inhibitor such as trichostatin A (TSA), significantly enhances the developmental competence in several species. However, some researchers also reported that TSA treatment had various detrimental effects on the in vitro and in vivo development of the SCNT embryos. Herein, we report that treatment with 500 nM 6-(1,3-dioxo-1H, 3H-benzo[de]isoquinolin-2-yl)-hexanoic acid hydroxyamide (termed scriptaid), a novel HDAC inhibitor, significantly enhanced the development of SCNT embryos to the blastocyst stage when NIH inbred fetal fibroblast cells (FFCs) were used as donors compared with the untreated group (21% vs. 9%, P < 0.05). Scriptaid treatment resulted in eight pregnancies from 10 embryo transfers (ETs) and 14 healthy NIH miniature pigs from eight litters, while no viable piglets (only three mummies) were obtained from nine ETs in the untreated group. Thus, scriptaid dramatically increased the cloning efficiency when using inbred genetics from 0.0% to 1.3%. In contrast, scriptaid treatment decreased the blastocyst rate in in vitro fertilization embryos (from 37% to 26%, P < 0.05). In conclusion, the extremely low cloning efficiency in the NIH miniature pig may be caused by its inbred genetic background and can be improved by alteration of genomic histone acetylation patterns.

New technique for fertilizing eggs of burbot, asp and ide under hatchery conditions.

PubMed

Kucharczyk, Dariusz; Nowosad, Joanna; Łuczyński, Marek J; Targońska, Katarzyna

2016-09-01

The development of a new protocol for egg fertilization may increase embryo survival and benefit the aquaculture process. In the present study, a new technique of partially adding sperm to activated eggs in the artificial fertilization of burbot (Lota lota), ide (Leuciscus idus) and asp (Aspius aspius) eggs was evaluated. If the same volume of sperm was divided into two or three parts and added to eggs in 30-60s intervals, it significantly improved embryo survival at the eyed-egg-stage of development. In the present study, the periodic addition of spermatozoa to eggs affected fertilization (ide and asp) and embryo survival rates (ide, asp and burbot) and might be successfully applied under hatchery conditions. Copyright © 2016 Elsevier B.V. All rights reserved.

A novel system for embryo-larval toxicity testing of pelagic fish: Applications for impact assessment of Deepwater Horizon crude oil.

PubMed

Stieglitz, John D; Mager, Edward M; Hoenig, Ronald H; Alloy, Matthew; Esbaugh, Andrew J; Bodinier, Charlotte; Benetti, Daniel D; Roberts, Aaron P; Grosell, Martin

2016-11-01

Key differences in the developmental process of pelagic fish embryos, in comparison to embryos of standard test fish species, present challenges to obtaining sufficient control survival needed to successfully perform traditional toxicity testing bioassays. Many of these challenges relate to the change in buoyancy, from positive to negative, of pelagic fish embryos that occurs just prior to hatch. A novel exposure system, the pelagic embryo-larval exposure chamber (PELEC), has been developed to conduct successful bioassays on the early life stages (ELSs; embryos/larvae) of pelagic fish. Using this unique recirculating upwelling system, it was possible to significantly improve control survival in pelagic fish ELS bioassays compared to commonly used static exposure methods. Results demonstrate that control performance of mahi-mahi (Coryphaena hippurus) embryos in the PELEC system, measured as percent survival after 96-hrs, significantly outperformed agitated static exposure and static exposure systems. Similar significant improvements in 72-hr control survival were obtained with yellowfin tuna (Thunnus albacares). The PELEC system was subsequently used to test the effects of photo-induced toxicity of crude oil to mahi-mahi ELSs over the course of 96-hrs. Results indicate a greater than 9-fold increase in toxicity of Deepwater Horizon (DWH) crude oil during co-exposure to ambient sunlight compared to filtered ambient sunlight, revealing the importance of including natural sunlight in 96-hr DWH crude oil bioassays as well as the PELEC system's potential application in ecotoxicological assessments. Copyright © 2016 Elsevier Ltd. All rights reserved.

Defining the Genomic Signature of Totipotency and Pluripotency during Early Human Development

PubMed Central

Galan, Amparo; Diaz-Gimeno, Patricia; Poo, Maria Eugenia; Valbuena, Diana; Sanchez, Eva; Ruiz, Veronica; Dopazo, Joaquin; Montaner, David; Conesa, Ana; Simon, Carlos

2013-01-01

The genetic mechanisms governing human pre-implantation embryo development and the in vitro counterparts, human embryonic stem cells (hESCs), still remain incomplete. Previous global genome studies demonstrated that totipotent blastomeres from day-3 human embryos and pluripotent inner cell masses (ICMs) from blastocysts, display unique and differing transcriptomes. Nevertheless, comparative gene expression analysis has revealed that no significant differences exist between hESCs derived from blastomeres versus those obtained from ICMs, suggesting that pluripotent hESCs involve a new developmental progression. To understand early human stages evolution, we developed an undifferentiation network signature (UNS) and applied it to a differential gene expression profile between single blastomeres from day-3 embryos, ICMs and hESCs. This allowed us to establish a unique signature composed of highly interconnected genes characteristic of totipotency (61 genes), in vivo pluripotency (20 genes), and in vitro pluripotency (107 genes), and which are also proprietary according to functional analysis. This systems biology approach has led to an improved understanding of the molecular and signaling processes governing human pre-implantation embryo development, as well as enabling us to comprehend how hESCs might adapt to in vitro culture conditions. PMID:23614026

Observation of human embryonic behavior in vitro by high-resolution time-lapse cinematography.

PubMed

Iwata, Kyoko; Mio, Yasuyuki

2016-07-01

Assisted reproductive technology (ART) has yielded vast amounts of information and knowledge on human embryonic development in vitro; however, still images provide limited data on dynamic changes in the developing embryos. Using our high-resolution time-lapse cinematography (hR-TLC) system, we were able to describe normal human embryonic development continuously from the fertilization process to the hatched blastocyst stage in detail. Our hR-TLC observation also showed the embryonic abnormality of a third polar body (PB)-like substance likely containing a small pronucleus being extruded and resulting in single-pronucleus (1PN) formation, while our molecular biological investigations suggested the possibility that some 1PN embryos could be diploid, carrying both maternal and paternal genomes. Furthermore, in some embryos the extruded third PB-like substance was eventually re-absorbed into the ooplasm resulting in the formation of an uneven-sized, two-PN zygote. In addition, other hR-TLC observations showed that cytokinetic failure was correlated with equal-sized, multi-nucleated blastomeres that were also observed in the embryo showing early initiation of compaction. Assessment combining our hR-TLC with molecular biological techniques enables a better understanding of embryonic development and potential improvements in ART outcomes.

Peering beneath the surface: novel imaging techniques to noninvasively select gametes and embryos for ART.

PubMed

Jasensky, Joshua; Swain, Jason E

2013-10-01

Embryo imaging has long been a critical tool for in vitro fertilization laboratories, aiding in morphological assessment of embryos, which remains the primary tool for embryo selection. With the recent emergence of clinically applicable real-time imaging systems to assess embryo morphokinetics, a renewed interest has emerged regarding noninvasive methods to assess gamete and embryo development as a means of inferring quality. Several studies exist that utilize novel imaging techniques to visualize or quantify intracellular components of gametes and embryos with the intent of correlating localization of organelles or molecular constitution with quality or outcome. However, the safety of these approaches varies due to the potential detrimental impact of light exposure or other variables. Along with complexity of equipment and cost, these drawbacks currently limit clinical application of these novel microscopes and imaging techniques. However, as evidenced by clinical incorporation of some real-time imaging devices as well as use of polarized microscopy, some of these imaging approaches may prove to be useful. This review summarizes the existing literature on novel imaging approaches utilized to examine gametes and embryos. Refinement of some of these imaging systems may permit clinical application and serve as a means to offer new, noninvasive selection tools to improve outcomes for various assisted reproductive technology procedures.

The effects of platelet lysate on maturation, fertilization and embryo development of NMRI mouse oocytes at germinal vesicle stage.

PubMed

Pazoki, Hassan; Eimani, Hussein; Farokhi, Farah; Shahverdi, Abdol-Hossein; Tahaei, Leila Sadat

2016-04-01

Improving in vitro maturation could increase the rate of pregnancy from oocytes matured in vitro. Consequently, patients will be prevented from using gonadotropin with its related side effects. In this study, the maturation medium was enriched by platelet lysate (PL), then maturation and subsequent developments were monitored. Oocytes at germinal vesicle stage with cumulus cells (cumulus-oocyte complex) and without cumulus cells (denuded oocytes) were obtained from mature female mice. The maturation medium was enriched by 5 and 10 % PL and 5 % PL + 5 % fetal bovine serum (FBS) as experimental groups; the control groups' media consisted of 5 and 10 % FBS. After 18 h, the matured oocytes were collected and, after fertilization, subsequent development was monitored. The rates of maturation, fertilization and 2-cell embryo development for the denuded oocyte groups in experimental media 5 % PL and 5 % PL + 5 % FBS were significantly higher than those of the control groups ( P < 0.05), while the results for the cumulus-oocyte complex groups were similar between the experimental groups and control groups. The results of this study indicated that platelet lysate could improve the maturation rate in the absence of granulosa cells compared to media with FBS. This extract also had positive effects on fertilization and embryo development.

Development of a security system for assisted reproductive technology (ART).

PubMed

Hur, Yong Soo; Ryu, Eun Kyung; Park, Sung Jin; Yoon, Jeong; Yoon, San Hyun; Yang, Gi Deok; Hur, Chang Young; Lee, Won Don; Lim, Jin Ho

2015-01-01

In the field of assisted reproductive technology (ART), medical accidents can result in serious legal and social consequences. This study was conducted to develop a security system (called IVF-guardian; IG) that could prevent mismatching or mix-ups in ART. A software program was developed in collaboration with outside computer programmers. A quick response (QR) code was used to identify the patients, gametes and embryos in a format that was printed on a label. There was a possibility that embryo development could be affected by volatile organic components (VOC) in the printing material and adhesive material in the label paper. Further, LED light was used as the light source to recognize the QR code. Using mouse embryos, the effects of the label paper and LED light were examined. The stability of IG was assessed when applied in clinical practice after developing the system. A total of 104 cycles formed the study group, and 82 cycles (from patients who did not want to use IG because of safety concerns and lack of confidence in the security system) to which IG was not applied comprised the control group. Many of the label paper samples were toxic to mouse embryo development. We selected a particular label paper (P touch label) that did not affect mouse embryo development. The LED lights were non-toxic to the development of the mouse embryos under any experimental conditions. There were no differences in the clinical pregnancy rates between the IG-applied group and the control group (40/104 = 38.5 % and 30/82 = 36.6 %, respectively). The application of IG in clinical practice did not affect human embryo development or clinical outcomes. The use of IG reduces the misspelling of patient names. Using IG, there was a disadvantage in that each treatment step became more complicated, but the medical staff improved and became sufficiently confident in ART to offset this disadvantage. Patients who received treatment using the IG system also went through a somewhat tedious process, but there were no complaints. These patients gained further confidence in the practitioners over the course of treatment.

The Histone Deacetylase Inhibitor, CI994, Improves Nuclear Reprogramming and In Vitro Developmental Potential of Cloned Pig Embryos.

PubMed

Jin, Long; Guo, Qing; Zhang, Guang-Lei; Xing, Xiao-Xu; Xuan, Mei-Fu; Luo, Qi-Rong; Luo, Zhao-Bo; Wang, Jun-Xia; Yin, Xi-Jun; Kang, Jin-Dan

2018-06-01

Epigenetic reprogramming and somatic cell nuclear transfer (SCNT) cloning efficiency were recently enhanced using histone deacetylase inhibitors (HDACis). In this study, we investigated the time effect of CI994, an HDACi, on the blastocyst formation rate, acetylation levels of H3K9 and H4K12, DNA methylation levels of anti-5-methylcytosine (5mC), and some mRNA expression of pluripotency-related genes in pig SCNT embryos. Treatment with 10 μM CI994 for 24 hours significantly improved the blastocyst formation rate of SCNT embryos in comparison with the untreated group (p < 0.05). Moreover, average fluorescence intensities of H3K9 and H4K12 in CI994-treated embryos were remarkably increased at the pseudo-pronuclear stage, but not at the blastocyst stage. The intensity of POU5F1 was higher in CI994-treated blastocysts than in control blastocysts, whereas that of 5mC did not differ between the two groups. The percentage of apoptotic cells in blastocysts was significantly higher in the untreated group than in the CI994-treated group. mRNA levels of POU5F1 and SOX2 were significantly increased in the CI994-treated group. These observations suggest that optimum exposure (10 μM for 24 hours) to CI994 after activation elevates the level of histone acetylation and subsequently improves the in vitro development of pig SCNT embryos.

Arrested human embryos are more likely to have abnormal chromosomes than developing embryos from women of advanced maternal age.

PubMed

Qi, Shu-Tao; Liang, Li-Feng; Xian, Ye-Xing; Liu, Jian-Qiao; Wang, Weihua

2014-01-01

Aneuploidy is one of the major factors that result in low efficiency in human infertility treatment by in vitro fertilization (IVF). The development of DNA microarray technology allows for aneuploidy screening by analyzing all 23 pairs of chromosomes in human embryos. All chromosome screening for aneuploidy is more accurate than partial chromosome screening, as errors can occur in any chromosome. Currently, chromosome screening for aneuploidy is performed in developing embryos, mainly blastocysts. It has not been performed in arrested embryos and/or compared between developing embryos and arrested embryos from the same IVF cycle. The present study was designed to examine all chromosomes in blastocysts and arrested embryos from the same cycle in patients of advanced maternal ages. Embryos were produced by routine IVF procedures. A total of 90 embryos (45 blastocysts and 45 arrested embryos) from 17 patients were biopsied and analyzed by the Agilent DNA array platform. It was found that 50% of the embryos developed to blastocyst stage; however, only 15.6% of the embryos (both blastocyst and arrested) were euploid, and most (84.4%) of the embryos had chromosomal abnormalities. Further analysis indicated that 28.9% of blastocysts were euploid and 71.1% were aneuploid. By contrast, only one (2.2%) arrested embryo was euploid while others (97.8%) were aneuploid. The prevalence of multiple chromosomal abnormalities in the aneuploid embryos was also higher in the arrested embryos than in the blastocysts. These results indicate that high proportions of human embryos from patients of advanced maternal age are aneuploid, and the arrested embryos are more likely to have abnormal chromosomes than developing embryos.

Revealing the secret life of pre-implantation embryos by time-lapse monitoring: A review

PubMed Central

Faramarzi, Azita; Khalili, Mohammad Ali; Micara, Giulietta; Agha-Rahimi, Azam

2017-01-01

High implantation success following in vitro fertilization cycles are achieved via the transfer of embryos with the highest developmental competence. Multiple pregnancies as a result of the transfer of several embryos per cycle accompany with various complication. Thus, single-embryo transfer (SET) is the preferred practice in assisted reproductive technique (ART) treatment. In order to improve the pregnancy rate for SET, embryologists need reliable biomarkers to aid their selection of embryos with the highest developmental potential. Time-lapse technology is a noninvasive alternative conventional microscopic assessment. It provides uninterrupted and continues the survey of embryo development to transfer day. Today, there are four time-lapse systems that are commercially available for ART centers. In world and Iran, the first time lapse babies were born in 2010 and 2015, respectively, conceived by SET. Here, we review the use of time-lapse monitoring in the observation of embryogenesis as well as its role in SET. Although, the findings from our review support common use of time-lapse monitoring in ART centers; but, future large studies assessing this system in well-designed trials are necessary. PMID:28744520

RepSox improves viability and regulates gene expression in rhesus monkey-pig interspecies cloned embryos.

PubMed

Zhu, Hai-Ying; Jin, Long; Guo, Qing; Luo, Zhao-Bo; Li, Xiao-Chen; Zhang, Yu-Chen; Xing, Xiao-Xu; Xuan, Mei-Fu; Zhang, Guang-Lei; Luo, Qi-Rong; Wang, Jun-Xia; Cui, Cheng-Du; Li, Wen-Xue; Cui, Zheng-Yun; Yin, Xi-Jun; Kang, Jin-Dan

2017-05-01

To investigate the effect of the small molecule, RepSox, on the expression of developmentally important genes and the pre-implantation development of rhesus monkey-pig interspecies somatic cell nuclear transfer (iSCNT) embryos. Rhesus monkey cells expressing the monomeric red fluorescent protein 1 which have a normal (42) chromosome complement, were used as donor cells to generate iSCNT embryos. RepSox increased the expression levels of the pluripotency-related genes, Oct4 and Nanog (p < 0.05), but not of Sox2 compared with untreated embryos at the 2-4-cell stage. Expression of the anti-apoptotic gene, Bcl2, and the pro-apoptotic gene Bax was also affected at the 2-4-cell stage. RepSox treatment also increased the immunostaining intensity of Oct4 at the blastocyst stage (p < 0.05). Although the blastocyst developmental rate was higher in the group treated with 25 µM RepSox for 24 h than in the untreated control group (2.4 vs. 1.2%, p > 0.05), this was not significant. RepSox can improve the developmental potential of rhesus monkey-pig iSCNT embryos by regulating the expression of pluripotency-related genes.

The influence of early embryo traits on human embryonic stem cell derivation efficiency.

PubMed

O'Leary, Thomas; Heindryckx, Björn; Lierman, Sylvie; Van der Jeught, Margot; Menten, Björn; Deforce, Dieter; Cornelissen, Ria; de Sousa Lopes, Susana Chuva; De Sutter, Petra

2011-05-01

Despite its prognostic value in in vitro fertilization, early embryo morphology is not reported on in the derivation of human embryonic stem cell (hESC) lines. Standard hESC derivation does rely on blastocyst development and its efficiency is highly correlated to inner cell mass (ICM) quality. Poor-quality embryos (PQEs) donated for hESC derivation may have a range of cleavage-stage abnormalities that are known to compromise further development. This study was implemented to determine whether specific PQEs traits influence the efficiency of good-quality ICMs to derive new hESC lines. We found that although the types of PQEs investigated were all able to make blastocysts with good-quality ICMs, the ICMs were unequal in their ability to derive hESCs. Good-quality ICMs from embryos with multiple poor-quality traits were unable to generate hESC lines, in contrast to good-quality ICMs from embryos with a single poor-quality trait. In addition, our data suggest a direct correlation between the number of ICM cells present in the blastocyst and its capacity to derive new hESC lines. This study is the first to demonstrate that ICM quality alone is an incomplete indicator of hESC derivation and that application of in vitro fertilization-based early embryo scoring can help predict hESC derivation efficiency. Experiments aiming to quantify, improve upon, or compare hESC derivation efficiency should thus take into consideration early embryo morphology scoring for the comparison of groups with equal developmental competence.

Somatic embryogenesis, scanning electron microscopy, histology and biochemical analysis at different developing stages of embryogenesis in six date palm (Phoenix dactylifera L.) cultivars.

PubMed

Aslam, Junaid; Khan, Saeed Ahmad; Cheruth, Abdul Jaleel; Mujib, Abdul; Sharma, Maheshwar Pershad; Srivastava, Prem Shanker

2011-10-01

An efficient somatic embryogenesis system has been established in six date palm (Phoenix dactylifera L.) cultivars (Barhee, Zardai, Khalasah, Muzati, Shishi and Zart). Somatic embryogenesis (SE) was growth regulators and cultivars dependent. Friable embryogenic callus was induced from excised shoot tips on MS medium supplemented with various auxins particularly 2,4-dichlorophenoxyacetic acid (2,4-D, 1.5 mg 1(-l)). Suspension culture increased embryogenesis potentiality. Only a-naphthaleneacetic acid (NAA, 0.5 mg 1(-1)) produced somatic embryos in culture. Somatic embryos germinated and converted into plantlets in N(6)-benzyladenine (BAP, 0.75 mg 1(-l)) added medium following a treatment with thidiazuron (TDZ, 1.0 mg 1(-l)) for maturation. Scanning electron microscopy showed early stages of somatic embryo particularly, globular types, and was in masses. Different developing stages of embryogenesis (heart, torpedo and cotyledonary) were observed under histological preparation of embryogenic callus. Biochemical screening at various stages of somatic embryogenesis (embryogenic callus, somatic embryos, matured, germinated embryos and converted plantlets) of date palm cultivars has been conducted and discussed in detail. The result discussed in this paper indicates that somatic embryos were produced in numbers and converted plantlets can be used as a good source of alternative propagation. Genetic modification to the embryo precursor cell may improve the fruit quality and yield further.

Somatic embryogenesis, scanning electron microscopy, histology and biochemical analysis at different developing stages of embryogenesis in six date palm (Phoenix dactylifera L.) cultivars

PubMed Central

Aslam, Junaid; Khan, Saeed Ahmad; Cheruth, Abdul Jaleel; Mujib, Abdul; Sharma, Maheshwar Pershad; Srivastava, Prem Shanker

2011-01-01

An efficient somatic embryogenesis system has been established in six date palm (Phoenix dactylifera L.) cultivars (Barhee, Zardai, Khalasah, Muzati, Shishi and Zart). Somatic embryogenesis (SE) was growth regulators and cultivars dependent. Friable embryogenic callus was induced from excised shoot tips on MS medium supplemented with various auxins particularly 2,4-dichlorophenoxyacetic acid (2,4-D, 1.5 mg 1−l). Suspension culture increased embryogenesis potentiality. Only a-naphthaleneacetic acid (NAA, 0.5 mg 1−1) produced somatic embryos in culture. Somatic embryos germinated and converted into plantlets in N6-benzyladenine (BAP, 0.75 mg 1−l) added medium following a treatment with thidiazuron (TDZ, 1.0 mg 1−l) for maturation. Scanning electron microscopy showed early stages of somatic embryo particularly, globular types, and was in masses. Different developing stages of embryogenesis (heart, torpedo and cotyledonary) were observed under histological preparation of embryogenic callus. Biochemical screening at various stages of somatic embryogenesis (embryogenic callus, somatic embryos, matured, germinated embryos and converted plantlets) of date palm cultivars has been conducted and discussed in detail. The result discussed in this paper indicates that somatic embryos were produced in numbers and converted plantlets can be used as a good source of alternative propagation. Genetic modification to the embryo precursor cell may improve the fruit quality and yield further. PMID:23961149

Methods for assessing the quality of mammalian embryos: How far we are from the gold standard?

PubMed

Rocha, José C; Passalia, Felipe; Matos, Felipe D; Maserati, Marc P; Alves, Mayra F; Almeida, Tamie G de; Cardoso, Bruna L; Basso, Andrea C; Nogueira, Marcelo F G

2016-08-01

Morphological embryo classification is of great importance for many laboratory techniques, from basic research to the ones applied to assisted reproductive technology. However, the standard classification method for both human and cattle embryos, is based on quality parameters that reflect the overall morphological quality of the embryo in cattle, or the quality of the individual embryonic structures, more relevant in human embryo classification. This assessment method is biased by the subjectivity of the evaluator and even though several guidelines exist to standardize the classification, it is not a method capable of giving reliable and trustworthy results. Latest approaches for the improvement of quality assessment include the use of data from cellular metabolism, a new morphological grading system, development kinetics and cleavage symmetry, embryo cell biopsy followed by pre-implantation genetic diagnosis, zona pellucida birefringence, ion release by the embryo cells and so forth. Nowadays there exists a great need for evaluation methods that are practical and non-invasive while being accurate and objective. A method along these lines would be of great importance to embryo evaluation by embryologists, clinicians and other professionals who work with assisted reproductive technology. Several techniques shows promising results in this sense, one being the use of digital images of the embryo as basis for features extraction and classification by means of artificial intelligence techniques (as genetic algorithms and artificial neural networks). This process has the potential to become an accurate and objective standard for embryo quality assessment.

Methods for assessing the quality of mammalian embryos: How far we are from the gold standard?

PubMed Central

Rocha, José C.; Passalia, Felipe; Matos, Felipe D.; Maserati Jr, Marc P.; Alves, Mayra F.; de Almeida, Tamie G.; Cardoso, Bruna L.; Basso, Andrea C.; Nogueira, Marcelo F. G.

2016-01-01

Morphological embryo classification is of great importance for many laboratory techniques, from basic research to the ones applied to assisted reproductive technology. However, the standard classification method for both human and cattle embryos, is based on quality parameters that reflect the overall morphological quality of the embryo in cattle, or the quality of the individual embryonic structures, more relevant in human embryo classification. This assessment method is biased by the subjectivity of the evaluator and even though several guidelines exist to standardize the classification, it is not a method capable of giving reliable and trustworthy results. Latest approaches for the improvement of quality assessment include the use of data from cellular metabolism, a new morphological grading system, development kinetics and cleavage symmetry, embryo cell biopsy followed by pre-implantation genetic diagnosis, zona pellucida birefringence, ion release by the embryo cells and so forth. Nowadays there exists a great need for evaluation methods that are practical and non-invasive while being accurate and objective. A method along these lines would be of great importance to embryo evaluation by embryologists, clinicians and other professionals who work with assisted reproductive technology. Several techniques shows promising results in this sense, one being the use of digital images of the embryo as basis for features extraction and classification by means of artificial intelligence techniques (as genetic algorithms and artificial neural networks). This process has the potential to become an accurate and objective standard for embryo quality assessment. PMID:27584609

Exploitation of genetic and physiological determinants of embryonic resistance to elevated temperature to improve embryonic survival in dairy cattle during heat stress.

PubMed

Hansen, P J

2007-09-01

Heat stress causes large reductions in fertility in lactating dairy cows. The magnitude and geographical extent of this problem is increasing because improvements in milk yield have made it more difficult for cows to regulate body temperature during warm weather. There have been efforts to improve fertility during heat stress by exploiting determinants of oocyte and embryonic responses to elevated temperature. Among these determinants are genotype, stage of development, and presence of cytoprotective molecules in the reproductive tract. One effective strategy for increasing pregnancy rate during heat stress is to use embryo transfer to bypass effects of elevated temperature on the oocyte and early embryo. Pregnancy success to embryo transfer in the summer can be further improved by exposure of embryos to insulin-like growth factor-I during culture before transfer. Among the cytoprotective molecules that have been examined for enhancing fertility during heat stress are bovine somatotropin and various antioxidants. To date, an effective method for delivery of these molecules to increase fertility during heat stress has not been identified. Genes in cattle exist for regulation of body temperature and for cellular resistance to elevated temperature. Although largely unidentified, the existence of these genes offers the possibility for their incorporation into dairy breeds through crossbreeding or on an individual-gene basis. In summary, physiological or genetic manipulation of the cow to improve embryonic resistance to elevated temperature is a promising approach for enhancing fertility of lactating dairy cows.

Establishment of the regeneration system for Vicia faba L.

PubMed

Bahgat, Shimaa; Shabban, Omer A; El-Shihy, Osama; Lightfoot, David A; El-Shemy, Hany A

2009-01-01

A reliable regeneration system for faba bean has been difficult to establish and therefore, the genetic improvement of Vicia faba L. was delayed. The paper describes a method of somatic embryo induction in callus of V. faba. Two Egyptian faba bean cultivars 'Giza 2' and '24 Hyto' were used. Callus was induced from epicotyls and shoot tips cultured on MS or Gamborg medium supplemented with 3% sucrose and 0.025% (w/v) for each of ascorbic and citric acid, 0.8% agar and different concentrations of 10 mg/l BAP, 0.5 mg/l of each NAA and 2,4-dichlorophenoxyacetic acid (M1) and 1 mg/l BAP and 0.5 mg/l NAA (M2) . The media with BAP, NAA and 2,4-D were optimal for embryogenic callus induction. Somatic embryos developed after transfer of the callus to 1/2 B5 medium with no plant growth regulators. There were various stages of somatic embryo development present including globular, heart-shaped, torpedo, and cotyledonary stages. Embryos developed into plantlets and plants were regenerated. RAPD analyses were performed to investigate the genetic stability of the regenerated plants obtained from different treatments and different explants. The cultivar Giza 2 exhibited more genetic stability than cultivar 24 Hyto. In conclusion, a regeneration system was established suitable for both gene transformation and the isolation of somaclonal mutants. The regeneration system will be used in order to improve the nutritional value of faba bean.

Desiccation Treatment and Endogenous IAA Levels Are Key Factors Influencing High Frequency Somatic Embryogenesis in Cunninghamia lanceolata (Lamb.) Hook

PubMed Central

Zhou, Xiaohong; Zheng, Renhua; Liu, Guangxin; Xu, Yang; Zhou, Yanwei; Laux, Thomas; Zhen, Yan; Harding, Scott A.; Shi, Jisen; Chen, Jinhui

2017-01-01

Cunninghamia lanceolata (Lamb.) Hook (Chinese fir) is an important tree, commercially and ecologically, in southern China. The traditional regenerating methods are based on organogenesis and cutting propagation. Here, we report the development of a high-frequency somatic embryogenesis (SE) regeneration system synchronized via a liquid culture from immature zygotic embryos. Following synchronization, PEM II cell aggregates were developmentally equivalent in appearance to cleaved zygotic embryos. Embryo and suspensor growth and subsequent occurrence of the apical and then the cotyledonary meristems were similar for zygotic and SE embryo development. However, SE proembryos exhibited a more reddish coloration than zygotic proembryos, and SE embryos were smaller than zygotic embryos. Mature somatic embryos gave rise to plantlets on hormone-free medium. For juvenile explants, low concentrations of endogenous indole-3-acetic acid in initial explants correlated with improved proembryogenic mass formation, and high SE competency. Analysis of karyotypes and microsatellites detected no major genetic variation in the plants regenerated via SE, and suggest a potential in the further development of this system as a reliable methodology for true-to-type seedling production. Treatment with polyethylene glycol (PEG) and abscisic acid (ABA) were of great importance to proembryo formation and complemented each other. ABA assisted the growth of embryonal masses, whereas PEG facilitated the organization of the proembryo-like structures. SOMATIC EMBRYOGENESIS RECEPTOR KINASE SERK) and the WUSCHEL homeobox (WOX) transcription factor served as molecular markers during early embryogenesis. Our results show that ClSERKs are conserved and redundantly expressed during SE. SERK and WOX transcript levels were highest during development of the proembryos and lowest in developed embryos. ClWOX13 expression correlates with the critical transition from proembryogenic masses to proembryos. Both SERK and WOX expression reveal their applicability in Chinese fir as markers of early embryogenesis. Overall, the findings provided evidence for the potential of this system in high fidelity Chinese fir seedlings production. Also, SE modification strategies were demonstrated and could be applied in other conifer species on the basis of our hormonal, morphological and molecular analyses. PMID:29259612

Overexpression of Tet3 in donor cells enhances goat somatic cell nuclear transfer efficiency.

PubMed

Han, Chengquan; Deng, Ruizhi; Mao, Tingchao; Luo, Yan; Wei, Biao; Meng, Peng; Zhao, Lu; Zhang, Qing; Quan, Fusheng; Liu, Jun; Zhang, Yong

2018-05-23

Ten-eleven translocation 3 (TET3) mediates active DNA demethylation of paternal genomes during mouse embryonic development. However, the mechanism of DNA demethylation in goat embryos remains unknown. In addition, aberrant DNA methylation reprogramming prevalently occurs in embryos cloned by somatic cell nuclear transfer (SCNT). In this study, we reported that TET3 is a key factor in DNA demethylation in goat pre-implantation embryos. Knockdown of Tet3 hindered DNA demethylation at the two- to four-cell stage in goat embryos and decreased Nanog expression in blastocysts. Overexpression of Tet3 in somatic cells can initiate DNA demethylation, reduce 5-methylcytosine level, increase 5-hydroxymethylcytosine level and promote the expression of key pluripotency genes. After SCNT, overexpression of Tet3 in donor cells corrected abnormal DNA hypermethylation of cloned embryos and significantly enhanced in vitro and in vivo developmental rate (P < 0.05). We conclude that overexpression of Tet3 in donor cells significantly improves goat SCNT efficiency. © 2018 Federation of European Biochemical Societies.

Preimplantation genetic haplotyping a new application for diagnosis of translocation carrier's embryos- preliminary observations of two robertsonian translocation carrier families.

PubMed

Shamash, Jana; Rienstein, Shlomit; Wolf-Reznik, Haike; Pras, Elon; Dekel, Michal; Litmanovitch, Talia; Brengauz, Masha; Goldman, Boleslav; Yonath, Hagith; Dor, Jehoshua; Levron, Jacob; Aviram-Goldring, Ayala

2011-01-01

Preimplantation genetic diagnosis using fluorescence in-situ hybridization (PGD-FISH) is currently the most common reproductive solution for translocation carriers. However, this technique usually does not differentiate between embryos carrying the balanced form of the translocation and those carrying the homologous normal chromosomes. We developed a new application of preimplantation genetic haplotyping (PGH) that can identify and distinguish between all forms of the translocation status in cleavage stage embryos prior to implantation. Polymorphic markers were used to identify and differentiate between the alleles that carry the translocation and those that are the normal homologous chromosomes. Embryos from two families of robertsonian translocation carriers were successfully analyzed using polymorphic markers haplotyping. Our preliminary results indicate that the PGH is capable of distinguishing between normal, balanced and unbalanced translocation carrier embryos. This method will improve PGD and will enable translocation carriers to avoid transmission of the translocation and the associated medical complications to offspring.

Gene expression of bovine embryos developing at the air-liquid interface on oviductal epithelial cells (ALI-BOEC).

PubMed

van der Weijden, Vera A; Chen, Shuai; Bauersachs, Stefan; Ulbrich, Susanne E; Schoen, Jennifer

2017-11-25

We recently developed an air-liquid interface long-term culture of differentiated bovine oviductal epithelial cells (ALI-BOEC). This ex vivo oviduct epithelium is capable of supporting embryo development in co-culture up to the blastocyst stage without addition of embryo culture medium. However, blastocyst rates in co-culture were markedly lower than in conventional in vitro embryo production procedures. In the present study, we assessed target gene expression of ALI-BOEC derived embryos to test their similarity to embryos from conventional in vitro embryo culture. We screened previously published data from developing bovine embryos and selected 41 genes which are either differentially expressed during embryo development, or reflect differences between various in vitro culture conditions or in vitro and in vivo embryos. Target gene expression was measured in 8-cell embryos and blastocysts using a 48.48 Dynamic Array™ on a Biomark HD instrument. For comparison with the ALI-BOEC system, we generated embryos by two different standard IVP protocols. The culture conditions lead to differential gene expression in both 8-cell embryos and blastocysts. Across the expression of all target genes the embryos developing on ALI-BOEC did not depart from conventional IVP embryos. These first results prove that gene expression in ALI-BOEC embryos is not largely aberrant. However, there was no clear indication for a more in vivo-like target gene expression of these embryos. This calls for further optimization of the ALI-BOEC system to increase its efficiency both quantitatively and qualitatively.

Seed development and maturation in early spring-flowering Galanthus nivalis and Narcissus pseudonarcissus continues post-shedding with little evidence of maturation in planta

PubMed Central

Newton, Rosemary J.; Hay, Fiona R.; Ellis, Richard H.

2013-01-01

Background and Aims Seeds of the moist temperate woodland species Galanthus nivalis and Narcissus pseudonarcissus, dispersed during spring or early summer, germinated poorly in laboratory tests. Seed development and maturation were studied to better understand the progression from developmental to germinable mode in order to improve seed collection and germination practices in these and similar species. Methods Phenology, seed mass, moisture content and ability to germinate and tolerate desiccation were monitored during seed development until shedding. Embryo elongation within seeds was investigated during seed development and under several temperature regimes after shedding. Key Results Seeds were shed at high moisture content (>59 %) with little evidence that dry mass accumulation or embryo elongation were complete. Ability to germinate developed prior to the ability of some seeds to tolerate enforced desiccation. Germination was sporadic and slow. Embryo elongation occurred post-shedding in moist environments, most rapidly at 20 °C in G. nivalis and 15 °C in N. pseudonarcissus. The greatest germination also occurred in these regimes, 78 and 48 %, respectively, after 700 d. Conclusions Seeds of G. nivalis and N. pseudonarcissus were comparatively immature at shedding and substantial embryo elongation occurred post-shedding. Seeds showed limited desiccation tolerance at dispersal. PMID:23478943

Mitochondrial DNA content in embryo culture medium is significantly associated with human embryo fragmentation.

PubMed

Stigliani, S; Anserini, P; Venturini, P L; Scaruffi, P

2013-10-01

Is the amount of cell-free DNA released by human embryos into culture medium correlated with embryo morphological features? The mitochondrial DNA (mtDNA) content of culture medium is significantly associated with the fragmentation rate on Days 2 and 3 of embryo development, whether the oocyte came from women ≤ 35 or >35 years old. Cellular fragmentation is often utilized as one of the morphological parameters for embryo quality assessment. The amount of cellular fragments is considered to be an important morphological parameter for embryo implantation potential. It has been hypothesized that fragments are apoptotic bodies or anuclear cytoplasmatic pieces of blastomeres, although no definitive conclusion has been drawn about their pathogenesis. Human fertilized oocytes were individually cultured from Day 1 to Days 2 and 3. A total of 800 samples (166 spent media from Day 2 and 634 from Day 3) were enrolled into the present study. Double-stranded DNA (dsDNA) was quantified in 800 spent embryo culture media by Pico Green dye fluorescence assay. After DNA purification, genomic DNA (gDNA) and mtDNA were profiled by specific quantitative PCR. Statistical analyses defined correlations among DNA contents, embryo morphology and maternal age. Different independent tests confirmed the presence of DNA into embryo culture medium and, for the first time, we demonstrate that both gDNA and mtDNA are detectable in the secretome. The amount of DNA is larger in embryos with bad quality cleavage compared with high-grade embryos, suggesting that the DNA profile of culture medium is an objective marker for embryo quality assessment. In particular, DNA profiles are significantly associated with fragmentation feature (total dsDNA: P = 0.0010; mtDNA; P = 0.0247) and advanced maternal age. It is necessary to establish whether DNA profiling of spent embryo culture medium is a robust onsite test that can improve the prediction of blastulation, implantation and/or pregnancy rate. The approach we are proposing may provide a novel, non-invasive, objective tool for embryo quality grading. The correlation between a high mtDNA concentration and the fragmentation rate of embryos is suggestive that fragments are mainly anuclear cytoplasmatic debris arising during cleavage. Therefore, blastomere shaping as an early event during in vitro development may play a homeostatic role and be related to embryo competence. This project was funded by Merck Serono (Grant for Fertility Innovation 2011). The sponsor had no role in study design, data collection, data analysis, data interpretation and writing of the paper. Authors declare no conflicts of interest. ClinicalTrials.gov Identifier: NCT01397136.

Clinical significance of intercellular contact at the four-cell stage of human embryos, and the use of abnormal cleavage patterns to identify embryos with low implantation potential: a time-lapse study.

PubMed

Liu, Yanhe; Chapple, Vincent; Feenan, Katie; Roberts, Peter; Matson, Phillip

2015-06-01

To investigate the clinical significance of intercellular contact point (ICCP) in four-cell stage human embryos and the effectiveness of morphology and abnormal cleavage patterns in identifying embryos with low implantation potential. Retrospective cohort study. Private IVF center. A total of 223 consecutive IVF and intracytoplasmic sperm injection treatment cycles, with all resulting embryos cultured in the Embryoscope, and a subset of 207 cycles analyzed for ICCP number where good-quality four-cell embryos were available on day 2 (n = 373 IVF and n = 392 intracytoplasmic sperm injection embryos). None. Morphologic score on day 3, embryo morphokinetic parameters, incidence of abnormal biological events, and known implantation results. Of 765 good-quality four-cell embryos, 89 (11.6%) failed to achieve six ICCPs; 166 of 765 (21.7%) initially had fewer than six ICCPs but were able to establish six ICCPs before subsequent division. Embryos with fewer than six ICCPs at the end of four-cell stage had a lower implantation rate (5.0% vs. 38.5%), with lower embryology performance in both conventional and morphokinetic assessments, compared with embryos achieving six ICCPs by the end of four-cell stage. Deselecting embryos with poor morphology, direct cleavage, reverse cleavage, and fewer than six ICCPs at the four-cell stage led to a significantly improved implantation rate (33.6% vs. 22.4%). Embryos with fewer than six ICCPs at the end of the four-cell stage show compromised subsequent development and reduced implantation potential. Deselection of embryos with poor morphology and abnormal cleavage revealed via time-lapse imaging could provide the basis of a qualitative algorithm for embryo selection. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Rotational imaging optical coherence tomography for full-body mouse embryonic imaging

PubMed Central

Wu, Chen; Sudheendran, Narendran; Singh, Manmohan; Larina, Irina V.; Dickinson, Mary E.; Larin, Kirill V.

2016-01-01

Abstract. Optical coherence tomography (OCT) has been widely used to study mammalian embryonic development with the advantages of high spatial and temporal resolutions and without the need for any contrast enhancement probes. However, the limited imaging depth of traditional OCT might prohibit visualization of the full embryonic body. To overcome this limitation, we have developed a new methodology to enhance the imaging range of OCT in embryonic day (E) 9.5 and 10.5 mouse embryos using rotational imaging. Rotational imaging OCT (RI-OCT) enables full-body imaging of mouse embryos by performing multiangle imaging. A series of postprocessing procedures was performed on each cross-section image, resulting in the final composited image. The results demonstrate that RI-OCT is able to improve the visualization of internal mouse embryo structures as compared to conventional OCT. PMID:26848543

Analysis of embryo morphokinetics, multinucleation and cleavage anomalies using continuous time-lapse monitoring in blastocyst transfer cycles.

PubMed

Desai, Nina; Ploskonka, Stephanie; Goodman, Linnea R; Austin, Cynthia; Goldberg, Jeffrey; Falcone, Tommaso

2014-06-20

Time-lapse imaging combined with embryo morphokinetics may offer a non-invasive means for improving embryo selection. Data from clinics worldwide are necessary to compare and ultimately develop embryo classifications models using kinetic data. The primary objective of this study was to determine if there were kinetic differences between embryos with limited potential and those more often associated with in vitro blastocyst formation and/or implantation. We also wanted to compare putative kinetic markers for embryo selection as proposed by other laboratories to what we were observing in our own laboratory setting. Kinetic data and cycle outcomes were retrospectively analyzed in patients age 39 and younger with 7 or more zygotes cultured in the Embryoscope. Timing of specific events from the point of insemination were determined using time-lapse (TL) imaging. The following kinetic markers were assessed: time to syngamy (tPNf), t2, time to two cells (c), 3c (t3), 4c ( t4), 5c (t5), 8c (t8), morula (tMor), start of blastulation (tSB); tBL, blastocyst (tBL); expanded blastocyst (tEBL). Durations of the second (cc2) and third (cc3) cell cycles, the t5-t2 interval as well as time to complete synchronous divisions s1, s2 and s3 were calculated. Incidence and impact on development of nuclear and cleavage anomalies were also assessed. A total of 648 embryos transferred on day 5 were analyzed. The clinical pregnancy and implantation rate were 72% and 50%, respectively. Morphokinetic data showed that tPNf, t2,t4, t8, s1, s2,s3 and cc2 were significantly different in embryos forming blastocysts (ET or frozen) versus those with limited potential either failing to blastulate or else forming poor quality blastocysts ,ultimately discarded. Comparison of embryo kinetics in cycles with all embryos implanting (KID+) versus no implantation (KID-) suggested that markers of embryo competence to implant may be different from ability to form a blastocyst. The incidence of multinucleation and reverse cleavage amongst the embryos observed was 25% and 7%, respectively. Over 40% of embryos exhibiting these characteristics did however form blastocysts meeting our criteria for freezing. These data provide us with a platform with which to potentially enhance embryo selection for transfer.

Analysis of embryo morphokinetics, multinucleation and cleavage anomalies using continuous time-lapse monitoring in blastocyst transfer cycles

PubMed Central

2014-01-01

Background Time-lapse imaging combined with embryo morphokinetics may offer a non-invasive means for improving embryo selection. Data from clinics worldwide are necessary to compare and ultimately develop embryo classifications models using kinetic data. The primary objective of this study was to determine if there were kinetic differences between embryos with limited potential and those more often associated with in vitro blastocyst formation and/or implantation. We also wanted to compare putative kinetic markers for embryo selection as proposed by other laboratories to what we were observing in our own laboratory setting. Methods Kinetic data and cycle outcomes were retrospectively analyzed in patients age 39 and younger with 7 or more zygotes cultured in the Embryoscope. Timing of specific events from the point of insemination were determined using time-lapse (TL) imaging. The following kinetic markers were assessed: time to syngamy (tPNf), t2, time to two cells (c), 3c (t3), 4c ( t4), 5c (t5), 8c (t8), morula (tMor), start of blastulation (tSB); tBL, blastocyst (tBL); expanded blastocyst (tEBL). Durations of the second (cc2) and third (cc3) cell cycles, the t5-t2 interval as well as time to complete synchronous divisions s1, s2 and s3 were calculated. Incidence and impact on development of nuclear and cleavage anomalies were also assessed. Results A total of 648 embryos transferred on day 5 were analyzed. The clinical pregnancy and implantation rate were 72% and 50%, respectively. Morphokinetic data showed that tPNf, t2,t4, t8, s1, s2,s3 and cc2 were significantly different in embryos forming blastocysts (ET or frozen) versus those with limited potential either failing to blastulate or else forming poor quality blastocysts ,ultimately discarded. Comparison of embryo kinetics in cycles with all embryos implanting (KID+) versus no implantation (KID-) suggested that markers of embryo competence to implant may be different from ability to form a blastocyst. The incidence of multinucleation and reverse cleavage amongst the embryos observed was 25% and 7%, respectively. Over 40% of embryos exhibiting these characteristics did however form blastocysts meeting our criteria for freezing. Conclusions These data provide us with a platform with which to potentially enhance embryo selection for transfer. PMID:24951056

Spent culture medium analysis from individually cultured bovine embryos demonstrates metabolomic differences.

PubMed

Perkel, Kayla J; Madan, Pavneesh

2017-12-01

Spent culture medium can provide valuable information regarding the physiological state of a bovine preimplantation embryos through non-invasive analysis of the sum/depleted metabolite constituents. Metabolomics has become of great interest as an adjunct technique to morphological and cleavage-rate assessment, but more importantly, in improving our understanding of metabolism. In this study, in vitro produced bovine embryos developing at different rates were evaluated using proton nuclear magnetic resonance (1H NMR). Spent culture medium from individually cultured embryos (2-cell to blastocyst stage) were divided into two groups based on their cleavage rate fast growing (FG) and slow growing (SG; developmentally delayed by 12-24 h), then analyzed by a 600 MHz NMR spectrometer. Sixteen metabolites were detected and investigated for sum/depletion throughout development. Data indicate distinct differences between the 4-cell SG and FG embryos for pyruvate (P < 0.05, n = 9) and at the 16-cell stage for acetate, tryptophan, leucine/isoleucine, valine and histidine. Overall sum/depletion levels of metabolites demonstrated that embryos produced glutamate, but consumed histidine, tyrosine, glycine, methionine, tryptophan, phenylalanine, lysine, arginine, acetate, threonine, alanine, pyruvate, valine, isoleucine/leucine, and lactate with an overall trend of higher consumption of these metabolites by FG groups. Principal component analysis revealed distinct clustering of the plain medium, SG, and FG group, signifying the uniqueness of the metabolomic signatures of each of these groups. This study is the first of its kind to characterize the metabolomic profiles of SG and FG bovine embryos produced in vitro using 1H NMR. Elucidating differences between embryos of varying developmental rates could contribute to a better understanding of embryonic health and physiology.

Vitamin C supplementation enhances compact morulae formation but reduces the hatching blastocyst rate of bovine somatic cell nuclear transfer embryos.

PubMed

Li, Qian; Wang, Yong-Sheng; Wang, Li-Jun; Zhang, Hui; Li, Rui-Zhe; Cui, Chen-Chen; Li, Wen-Zhe; Zhang, Yong; Jin, Ya-Ping

2014-08-01

Vitamin C, an antioxidant that reduces reactive oxygen species (ROS) in cells, is capable of significantly improving the developmental competence of porcine and mouse somatic cell nuclear transfer (SCNT) embryos, both in vitro and in vivo. In the present study, the effects of vitamin C on the developmental competence of bovine SCNT embryos were investigated. The results indicated that vitamin C (40 μg/mL) positively affected the scavenging of intracellular ROS, cleavage rate at 24 h (76.67 vs. 68.26%, p<0.05), compact morulae formation (60.83 vs. 51.30%, p<0.05), and the blastomere apoptosis index (3.70 ± 1.41 vs. 4.43% ± 1.65, p<0.05) of bovine SCNT embryos. However, vitamin C supplementation did not significantly affect the blastocyst formation rate and proportion of inner cell mass over total cells per blastocyst on day 7. Moreover, vitamin C supplementation obviously impaired the total cell numbers per blastocyst (97.20 ± 11.35 vs. 88.57 ± 10.43, p<0.05) on day 7 and the hatching blastocysts formation rate on day 9 (26.51 vs. 50.65%, p<0.05) compared with that of the untreated group. Vitamin C supplementation preferentially improved the viability of bovine SCNT embryos prior to the blastocyst stage, but did not enhance the formation and quality of blastocysts in vitro. In conclusion, the effect of vitamin C on the development of bovine SCNT embryos is complex, and vitamin C is not a suitable antioxidant chemical for the in vitro culture of bovine SCNT embryos.

Blastocyst development rate impacts outcome in cryopreserved blastocyst transfer cycles

PubMed Central

Levens, Eric D.; Whitcomb, Brian W.; Hennessy, Sasha; James, Aidita N.; Yauger, Belinda J.; Larsen, Frederick W.

2008-01-01

Structured Abstract Objective To assess cycle outcome among day 5 and day 6 cryopreserved frozen-thawed blastocyst embryo transfers (FBET). Design Retrospective cohort study. Setting Military-based ART center. Patients One hundred seventy-two non-donor, programmed cryopreserved embryo cycles. Interventions Fully expanded blastocysts on day 5 were cryopreserved on day 5 and those achieving this state on day 6 were cryopreserved on day 6. Leuprolide acetate was given for ovulation inhibition and endometrial supplementation was by oral and vaginal estradiol. Progesterone in oil was administered and blastocyst transfer occurred in the morning of the 6th day of progesterone. Main Outcome Measures Implantation, pregnancy and live birth rates. Results Fresh and frozen cycle characteristics were similar between groups. Day 5 FBET had significantly higher implantation rates (32.2% vs. 19.2%; p=0.01) which remained significant even when adjusting for co-variates (OR: 1.91; 95%CI: 1.00, 3.67). Live birth rates trended towards improvement after adjusting for co-variates (OR: 1.18; 95%CI: 0.61, 2.30). Conclusions Cryopreserved day 5 blastocysts have higher implantation rates and trend toward improved pregnancy outcomes compared to cryopreserved day 6 blastocysts. This suggests that embryo development rate may, in part, predict implantation and subsequent FBET outcomes although embryos not achieving the blastocyst stage until day 6 still demonstrate acceptable outcomes. PMID:18178191

Timing of first embryonic cleavage is a positive indicator of the in vitro developmental potential of porcine embryos derived from in vitro fertilization, somatic cell nuclear transfer and parthenogenesis.

PubMed

Isom, S Clay; Li, Rong Feng; Whitworth, Kristin M; Prather, Randall S

2012-03-01

Evidence in many species has suggested that those embryos that cleave earliest after fertilization are more developmentally competent than those that cleave relatively later after fertilization. Herein we document this phenomenon in porcine in vitro-fertilized (IVF), somatic cell nuclear transfer (SCNT), and parthenogenetic (PA) embryos. In vitro-matured pig oocytes were used to generate IVF, SCNT, and PA embryos. At 24 hr post-activation (or insemination; hpa/hpi), embryos were visually assessed, and cleaved embryos were moved into a new culture well. This process was repeated at 30 and 48 hpa/hpi. All embryos were allowed to develop 7 days in culture. For IVF embryos, 39.9%, 24.6%, and 10.5% of fast-, intermediate-, or slow-cleaving embryos, respectively, developed into blastocysts by day 7. For SCNT embryos, 31.8% of fast-, 5.7% of intermediate-, and 2.9% of late-cleaving embryos achieved the blastocyst stage of development. For PA embryos, the percentages of those cleaved embryos that developed to blastocyst were 59.3%, 36.7%, and 7.5% for early-, intermediate-, and late-cleaving embryos, respectively. Using RNA collected from early-, intermediate-, and late-cleaving embryos, real-time PCR was performed to assess the transcript levels of 14 different genes of widely varied function. The qPCR results suggest that maternal mRNA degradation may not proceed in an appropriate pattern in slow-cleaving embryos. These findings (1) confirm that, as observed in other species, earlier-cleaving porcine embryos are more successful at developing in culture than are slower-cleaving embryos, and (2) implicate mechanisms of maternal transcript destruction as potential determinants of oocyte/embryo quality. Copyright © 2011 Wiley Periodicals, Inc.

[Effect of phytohemagglutinin (PHA) from Yunnan white kidney bean on development of mouse embryos].

PubMed

Zhang, Lifen; Wang, Changmei; Yang, Mingjie; Zhang, Tian; Wang, Minkang

2011-06-01

To study the effect of different concentration of phytohemagglutinin (PHA) on mouse embryo development. In experiment 1, crude and purified PHA extracted from Yunnan white kidney bean with different concentration were added into M16 culture medium, the final concentration of PHA were: 50, 100, 200, 500, 1 000, 2 000 and 5 000 mg x L(-1) respectively. 2-cell stage embryos were collected and cultured in PHA containing or control medium for 72-96 h and their development were recorded. In experiment 2, different stage of embryos from 1-cell to blastocyst were treated by different concentrations of PHA same as experiment 1 and 10 000 mg x L(-1) in culture medium for 24 h before washing and cultured in M16 + PVA without PHA to blastocyst or hatching blastocyst stage. Low concentrations PHA at 50-100 mg x L(-1) promoted embryo development and increased the number of blastocyst stage embryos. In contrast, high concentrations of PHA (> 1 000 mg x L(-1)) blocked the embryos development from 1-cell to blastocyst stage and showed apoptosis morphology or death. Depending on the concentrations, PHA from white kidney bean shown promotion or inhibition on mouse embryo development. 1-cell stage embryo shown more sensitive to PHA treatment than that of later stage embryos. Pretreatment 24 h in PHA containing medium can influence the further development of embryos. Low concentrations of PHA is benefit to embryo development, but high concentrations of PHA (> 1 000 mg x L(-1)) will block of the development of embryos.

Fundamentals of human embryonic growth in vitro and the selection of high-quality embryos for transfer.

PubMed

Boiso, Irene; Veiga, Anna; Edwards, Robert G

2002-01-01

Knowledge of the nature of embryo growth, and the handling and scoring of quality in human embryos are significant aspects for embryologists in IVF clinics. This review describes the formation, growth and maturation of human oocytes, many aspects of fertilization in vitro, embryonic transcription during preimplantation stages, and the formation of polarities, timing controls, role of mitochondria and functions of endocrine and paracrine systems. Modern concepts are fully discussed, together with their significance in the practice of IVF. This knowledge is essential for the correct clinical care of human embryos growing in vitro, especially in view of their uncharacteristic tendency to vary widely in implantation potential. Underlying causes of such variation have not been identified. Stringent tests must be enforced to ensure human embryos develop under optimal conditions, and are scored for quality using the most advanced techniques. Optimal methods of culture are described, including methods such as co-culture introduced to improve embryo quality but less important today. Detailed attention is given to quality as assessed from embryonic characteristics determined by timers, polarities, disturbed embryo growth and anomalous cell cycles. Methods for classification are described. Approaches to single embryo transfers are described, including the use of sequential media to produce high-quality blastocysts. These approaches, and others involved in surgical methods to remove fragments, transfer ooplasm or utilize newer approaches such as preimplantation diagnosis of chromosomal complements in embryos are covered. New outlooks in this field are summarized.

Transcriptome analyses of rhesus monkey preimplantation embryos reveal a reduced capacity for DNA double-strand break repair in primate oocytes and early embryos

PubMed Central

Wang, Xinyi; Liu, Denghui; He, Dajian; Suo, Shengbao; Xia, Xian; He, Xiechao; Han, Jing-Dong J.; Zheng, Ping

2017-01-01

Preimplantation embryogenesis encompasses several critical events including genome reprogramming, zygotic genome activation (ZGA), and cell-fate commitment. The molecular basis of these processes remains obscure in primates in which there is a high rate of embryo wastage. Thus, understanding the factors involved in genome reprogramming and ZGA might help reproductive success during this susceptible period of early development and generate induced pluripotent stem cells with greater efficiency. Moreover, explaining the molecular basis responsible for embryo wastage in primates will greatly expand our knowledge of species evolution. By using RNA-seq in single and pooled oocytes and embryos, we defined the transcriptome throughout preimplantation development in rhesus monkey. In comparison to archival human and mouse data, we found that the transcriptome dynamics of monkey oocytes and embryos were very similar to those of human but very different from those of mouse. We identified several classes of maternal and zygotic genes, whose expression peaks were highly correlated with the time frames of genome reprogramming, ZGA, and cell-fate commitment, respectively. Importantly, comparison of the ZGA-related network modules among the three species revealed less robust surveillance of genomic instability in primate oocytes and embryos than in rodents, particularly in the pathways of DNA damage signaling and homology-directed DNA double-strand break repair. This study highlights the utility of monkey models to better understand the molecular basis for genome reprogramming, ZGA, and genomic stability surveillance in human early embryogenesis and may provide insights for improved homologous recombination-mediated gene editing in monkey. PMID:28223401

Effect of Embryo Density on In Vitro Development and Gene Expression in Bovine In Vitro-fertilized Embryos Cultured in a Microwell System

PubMed Central

SUGIMURA, Satoshi; AKAI, Tomonori; HASHIYADA, Yutaka; AIKAWA, Yoshio; OHTAKE, Masaki; MATSUDA, Hideo; KOBAYASHI, Shuji; KOBAYASHI, Eiji; KONISHI, Kazuyuki; IMAI, Kei

2012-01-01

Abstract To identify embryos individually during in vitro development, we previously developed the well-of-the-well (WOW) dish, which contains 25 microwells. Here we investigated the effect of embryo density (the number of embryos per volume of medium) on in vitro development and gene expression of bovine in vitro-fertilized embryos cultured in WOW dishes. Using both conventional droplet and WOW culture formats, 5, 15, and 25 bovine embryos were cultured in 125 µl medium for 168 h. The blastocysts at Day 7 were analyzed for number of cells and expression of ten genes (CDX2, IFN-tau, PLAC8, NANOG, OCT4, SOX2, AKR1B1, ATP5A1, GLUT1 and IGF2R). In droplet culture, the rates of formation of >4-cell cleavage embryos and blastocysts were significantly lower in embryos cultured at 5 embryos per droplet than in those cultured at 15 or 25 embryos per droplet, but not in WOW culture. In both droplet and WOW culture, developmental kinetics and blastocyst cell numbers did not differ among any groups. IFN-tau expression in embryos cultured at 25 embryos per droplet was significantly higher than in those cultured at 15 embryos per droplet and in artificial insemination (AI)-derived blastocysts. Moreover, IGF2R expression was significantly lower in the 25-embryo group than in the 5-embryo group and in AI-derived blastocysts. In WOW culture, these expressions were not affected by embryo density and were similar to those in AI-derived blastocysts. These results suggest that, as compared with conventional droplet culture, in vitro development and expression of IFN-tau and IGF2R in the microwell system may be insensitive to embryo density. PMID:23154384

Significant improvement of pig cloning efficiency by treatment with LBH589 after somatic cell nuclear transfer.

PubMed

Jin, Jun-Xue; Li, Suo; Gao, Qing-Shan; Hong, Yu; Jin, Long; Zhu, Hai-Ying; Yan, Chang-Guo; Kang, Jin-Dan; Yin, Xi-Jun

2013-10-01

The low success rate of animal cloning by somatic cell nuclear transfer (SCNT) associates with epigenetic aberrancy, including the abnormal acetylation of histones. Altering the epigenetic status by histone deacetylase inhibitors (HDACi) enhances the developmental potential of SCNT embryos. In the current study, we examined the effects of LBH589 (panobinostat), a novel broad-spectrum HDACi, on the nuclear reprogramming and development of pig SCNT embryos in vitro. In experiment 1, we compared the in vitro developmental competence of nuclear transfer embryos treated with different concentrations of LBH589. Embryos treated with 50 nM LBH589 for 24 hours showed a significant increase in the rate of blastocyst formation compared with the control or embryos treated with 5 or 500 nM LBH589 (32.4% vs. 11.8%, 12.1%, and 10.0%, respectively, P < 0.05). In experiment 2, we examined the in vitro developmental competence of nuclear transfer embryos treated with 50 nM LBH589 for various intervals after activation and 6-dimethylaminopurine. Embryos treated for 24 hours had higher rates of blastocyst formation than the other groups. In experiment 3, when the acetylation of H4K12 was examined in SCNT embryos treated for 6 hours with 50 nM LBH589 by immunohistochemistry, the staining intensities of these proteins in LBH589-treated SCNT embryos were significantly higher than in the control. In experiment 4, LBH589-treated nuclear transfer and control embryos were transferred into surrogate mothers, resulting in three (100%) and two (66.7%) pregnancies, respectively. In conclusion, LBH589 enhances the nuclear reprogramming and developmental potential of SCNT embryos by altering the epigenetic status and expression, and increasing blastocyst quality. Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.

Forskolin effect on the cryosurvival of in vitro-produced bovine embryos in the presence or absence of fetal calf serum.

PubMed

Paschoal, Daniela Martins; Sudano, Mateus José; Guastali, Midyan Daroz; Dias Maziero, Rosiára Rosária; Crocomo, Letícia Ferrari; Oña Magalhães, Luis Carlos; da Silva Rascado, Tatiana; Martins, Alicio; da Cruz Landim-Alvarenga, Fernanda

2014-05-01

The objective of this study was to assess the viability and cryotolerance of zebu embryos produced in vitro with or without the addition of fetal calf serum (FCS) and forskolin (F). Embryos produced in vivo were used as a control. Presumptive zygotes were cultured in modified synthetic oviductal fluid supplemented with amino acids (SOFaa), bovine serum albumin (BSA) and with (2.5%) or without (0%) FCS. On day 6 of growth, the embryos from each group were divided into treatments with or without 10 μM F to induce embryonic lipolysis, comprising a total of four experimental groups: 2.5% FCS, 0% FCS, 2.5% + F and 0% + F. For vitrification, embryos were exposed to vitrification solution 1 (5 M EG (ethylene glycol)) for 3 min and then transferred to vitrification solution 2 (7 M EG, 0.5 M galactose solution and 18% (w/v) Ficoll 70) before being introduced to liquid nitrogen. The presence of FCS in the culture medium resulted in the production of embryos with a similar rate of damaged cells compared with in vivo-produced embryos. After vitrification, the 2.5% FCS group had a significantly higher rate of damaged cells when compared with the other groups (P < 0.05). The results of this experiment indicated that the omission of FCS and the addition of forskolin do not have deleterious effect on embryo production rates. In addition, embryos produced in the presence of FCS had greater sensitivity to cryopreservation, but this effect was reversed when forskolin was added to the medium, which improved embryo survival without affecting embryo development and quality after vitrification.

Clinical utilisation of a rapid low-pass whole genome sequencing technique for the diagnosis of aneuploidy in human embryos prior to implantation.

PubMed

Wells, Dagan; Kaur, Kulvinder; Grifo, Jamie; Glassner, Michael; Taylor, Jenny C; Fragouli, Elpida; Munne, Santiago

2014-08-01

The majority of human embryos created using in vitro fertilisation (IVF) techniques are aneuploid. Comprehensive chromosome screening methods, applicable to single cells biopsied from preimplantation embryos, allow reliable identification and transfer of euploid embryos. Recently, randomised trials using such methods have indicated that aneuploidy screening improves IVF success rates. However, the high cost of testing has restricted the availability of this potentially beneficial strategy. This study aimed to harness next-generation sequencing (NGS) technology, with the intention of lowering the costs of preimplantation aneuploidy screening. Embryo biopsy, whole genome amplification and semiconductor sequencing. A rapid (<15 h) NGS protocol was developed, with consumable cost only two-thirds that of the most widely used method for embryo aneuploidy detection. Validation involved blinded analysis of 54 cells from cell lines or biopsies from human embryos. Sensitivity and specificity were 100%. The method was applied clinically, assisting in the selection of euploid embryos in two IVF cycles, producing healthy children in both cases. The NGS approach was also able to reveal specified mutations in the nuclear or mitochondrial genomes in parallel with chromosome assessment. Interestingly, elevated mitochondrial DNA content was associated with aneuploidy (p<0.05), a finding suggestive of a link between mitochondria and chromosomal malsegregation. This study demonstrates that NGS provides highly accurate, low-cost diagnosis of aneuploidy in cells from human preimplantation embryos and is rapid enough to allow testing without embryo cryopreservation. The method described also has the potential to shed light on other aspects of embryo genetics of relevance to health and viability. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Clinical utilisation of a rapid low-pass whole genome sequencing technique for the diagnosis of aneuploidy in human embryos prior to implantation

PubMed Central

Wells, Dagan; Kaur, Kulvinder; Grifo, Jamie; Glassner, Michael; Taylor, Jenny C; Fragouli, Elpida; Munne, Santiago

2014-01-01

Background The majority of human embryos created using in vitro fertilisation (IVF) techniques are aneuploid. Comprehensive chromosome screening methods, applicable to single cells biopsied from preimplantation embryos, allow reliable identification and transfer of euploid embryos. Recently, randomised trials using such methods have indicated that aneuploidy screening improves IVF success rates. However, the high cost of testing has restricted the availability of this potentially beneficial strategy. This study aimed to harness next-generation sequencing (NGS) technology, with the intention of lowering the costs of preimplantation aneuploidy screening. Methods Embryo biopsy, whole genome amplification and semiconductor sequencing. Results A rapid (<15 h) NGS protocol was developed, with consumable cost only two-thirds that of the most widely used method for embryo aneuploidy detection. Validation involved blinded analysis of 54 cells from cell lines or biopsies from human embryos. Sensitivity and specificity were 100%. The method was applied clinically, assisting in the selection of euploid embryos in two IVF cycles, producing healthy children in both cases. The NGS approach was also able to reveal specified mutations in the nuclear or mitochondrial genomes in parallel with chromosome assessment. Interestingly, elevated mitochondrial DNA content was associated with aneuploidy (p<0.05), a finding suggestive of a link between mitochondria and chromosomal malsegregation. Conclusions This study demonstrates that NGS provides highly accurate, low-cost diagnosis of aneuploidy in cells from human preimplantation embryos and is rapid enough to allow testing without embryo cryopreservation. The method described also has the potential to shed light on other aspects of embryo genetics of relevance to health and viability. PMID:25031024

Effects of polyethylene glycol and a synthetic ice blocker during vitrification of immature porcine oocytes on survival and subsequent embryo development.

PubMed

Santos, Elisa Caroline da Silva; Somfai, Tamas; Appeltant, Ruth; Dang-Nguyen, Thanh Quang; Noguchi, Junko; Kaneko, Hiroyuki; Kikuchi, Kazuhiro

2017-08-01

We evaluated the effects of polyethylene glycol (PEG) and Supercool X-1000 (SC) as supplements during the vitrification of immature cumulus-enclosed porcine oocytes in a solution based on 17.5% ethylene glycol + 17.5% propylene glycol. After warming, the oocytes were subjected to in vitro maturation, fertilization and embryo culture. In Experiment 1, equilibration and vitrification solutions were supplemented with or without 2% (w/v) PEG (PEG+ and PEG-, respectively). The survival rate, cleavage and blastocyst development were similar between PEG+ and PEG- groups; however, all values were lower than those in the non-vitrified control. In Experiment 2, vitrification solution was supplemented with or without 1% (v/v) SC (SC+ and SC-, respectively). The percentages of survival and blastocyst development were similar between SC+ and SC- groups but lower than those in the non-vitrified control. The percentage of cleavage in the SC- group was significantly lower than the control and the SC+ groups, which were in turn similar to one another. In both experiments, the cell numbers in blastocysts were not significantly different among the non-vitrified and vitrified groups. In conclusion, PEG did not improve oocyte survival and embryo development, whereas SC improved the ability of surviving oocytes to cleave but not to develop into blastocysts. © 2016 Japanese Society of Animal Science.

Localisation of stem cell factor, stanniocalcin-1, connective tissue growth factor and heparin-binding epidermal growth factor in the bovine uterus at the time of blastocyst formation.

PubMed

Muñoz, M; Martin, D; Carrocera, S; Alonso-Guervos, M; Mora, M I; Corrales, F J; Peynot, N; Giraud-Delville, C; Duranthon, V; Sandra, O; Gómez, E

2017-10-01

Early embryonic losses before implantation account for the highest rates of reproductive failure in mammals, in particular when in vitro-produced embryos are transferred. In the present study, we used molecular biology techniques (real-time quantitative polymerase chain reaction), classical immunohistochemical staining coupled with confocal microscopy and proteomic analysis (multiple reaction monitoring and western blot analysis) to investigate the role of four growth factors in embryo-uterine interactions during blastocyst development. Supported by a validated embryo transfer model, the study investigated: (1) the expression of stem cell factor (SCF), stanniocalcin-1 (STC1), connective tissue growth factor (CTGF) and heparin-binding epidermal growth factor-like growth factor (HB-EGF) in bovine uterine fluid; (2) the presence of SCF, STC1, CTGF and HB-EGF mRNA and protein in the bovine endometrium and embryos; and (3) the existence of reciprocal regulation between endometrial and embryonic expression of SCF, STC1, CTGF and HB-EGF. The results suggest that these growth factors most likely play an important role during preimplantation embryo development in cattle. The information obtained from the present study can contribute to improving the performance of in vitro culture technology in cattle and other species.

Effects of Two Types of Melatonin-Loaded Nanocapsules with Distinct Supramolecular Structures: Polymeric (NC) and Lipid-Core Nanocapsules (LNC) on Bovine Embryo Culture Model.

PubMed

Komninou, Eliza Rossi; Remião, Mariana Härter; Lucas, Caroline Gomes; Domingues, William Borges; Basso, Andrea Cristina; Jornada, Denise Soledade; Deschamps, João Carlos; Beck, Ruy Carlos Ruver; Pohlmann, Adriana Raffin; Bordignon, Vilceu; Seixas, Fabiana Kömmling; Campos, Vinicius Farias; Guterres, Silvia Stanisçuaski; Collares, Tiago

2016-01-01

Melatonin has been used as a supplement in culture medium to improve the efficiency of in vitro produced mammalian embryos. Through its ability to scavenge toxic oxygen derivatives and regulate cellular mRNA levels for antioxidant enzymes, this molecule has been shown to play a protective role against damage by free radicals, to which in vitro cultured embryos are exposed during early development. In vivo and in vitro studies have been performed showing that the use of nanocapsules as active substances carriers increases stability, bioavailability and biodistribution of drugs, such as melatonin, to the cells and tissues, improving their antioxidant properties. These properties can be modulated through the manipulation of formula composition, especially in relation to the supramolecular structures of the nanocapsule core and the surface area that greatly influences drug release mechanisms in biological environments. This study aimed to evaluate the effects of two types of melatonin-loaded nanocapsules with distinct supramolecular structures, polymeric (NC) and lipid-core (LNC) nanocapsules, on in vitro cultured bovine embryos. Embryonic development, apoptosis, reactive oxygen species (ROS) production, and mRNA levels of genes involved in cell apoptosis, ROS and cell pluripotency were evaluated after supplementation of culture medium with non-encapsulated melatonin (Mel), melatonin-loaded polymeric nanocapsules (Mel-NC) and melatonin-loaded lipid-core nanocapsules (Mel-LNC) at 10-6, 10-9, and 10-12 M drug concentrations. The highest hatching rate was observed in embryos treated with 10-9 M Mel-LNC. When compared to Mel and Mel-NC treatments at the same concentration (10-9 M), Mel-LNC increased embryo cell number, decreased cell apoptosis and ROS levels, down-regulated mRNA levels of BAX, CASP3, and SHC1 genes, and up-regulated mRNA levels of CAT and SOD2 genes. These findings indicate that nanoencapsulation with LNC increases the protective effects of melatonin against oxidative stress and cell apoptosis during in vitro embryo culture in bovine species.

Reduced blastocyst formation in reduced culture volume.

PubMed

De Munck, N; Santos-Ribeiro, S; Mateizel, I; Verheyen, G

2015-09-01

The aim of this prospective sibling oocyte study was to evaluate whether reduced culture volume improves blastocyst formation. Twenty-three patients with extended embryo culture until day 5 were selected for the study. After injection, 345 sibling oocytes were individually cultured in either 25 or 7 μl droplets of Origio cleavage medium under oil. On day 3 of development, embryos were transferred to droplets with the corresponding volume of Origio blastocyst culture medium. Fertilization and embryo quality on day 3 and day 5/6 were evaluated. No statistically significant difference (p = 0.326) in fertilization rate was observed (81.3 versus 83.0 %). There was no significant difference in terms of the number of excellent and good-quality embryos obtained on day 3 between both groups (p = 0.655). Embryo culture in 25 μl droplets led to more embryos with a higher cell number when compared to 7 μl culture (p = 0.024). On day 3, 132 and 131 embryos were considered for further culture until day 5/6. Blastulation rates were significantly higher in the 25 μl group (75.0 versus 61.6 %; p = 0.017) and significantly more day 5 embryos with excellent and good quality were found in this group (54.5 versus 40.5 %; p = 0.026). Finally, the utilization rates expressed per mature oocyte (41.4 versus 29.8 %; p = 0.043), per fertilized oocyte (50.7 versus 36.6 %; p = 0.023), and per day 3 embryo undergoing extended culture to day 5/6 (54.5 versus 39.7 %; p = 0.019) were all significantly higher in the 25 μl group. Reduced culture volume (7 μl) negatively impacts early development by reducing the cell number on day 3 and both blastocyst formation and quality.

Arabidopsis mitochondrial protein slow embryo development1 is essential for embryo development

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ju, Yan; Liu, Chunying; Lu, Wenwen

The plant seeds formation are crucial parts in reproductive process in seed plants as well as food source for humans. Proper embryo development ensure viable seed formation. Here, we showed an Arabidopsis T-DNA insertion mutant slow embryo development1 (sed1) which exhibited retarded embryogenesis, led to aborted seeds. Embryo without SED1 developed slower compared to normal one and could be recognized at early globular stage by its white appearance. In later development stage, storage accumulated poorly with less protein and lipid body production. In vitro culture did not rescue albino embryo. SED1 encoded a protein targeted to mitochondria. Transmission electron microscopic analysismore » revealed that mitochondria developed abnormally, and more strikingly plastid failed to construct grana in time in sed1/sed1 embryo. These data indicated that SED1 is indispensable for embryogenesis in Arabidopsis, and the mitochondria may be involved in the regulation of many aspects of seed development. -- Highlights: •Arabidopsis SED1 is essential for embryo development. •The sed1 embryo accumulates less storage and has abnormal ultrastructure. •SED1 localizes to the mitochondrion.« less

Activation of peroxisome proliferators-activated receptor δ (PPARδ) promotes blastocyst hatching in mice.

PubMed

Kang, Hee Jung; Hwang, Soo Jin; Yoon, Jung Ah; Jun, Jin Hyun; Lim, Hyunjung Jade; Yoon, Tae Ki; Song, Haengseok

2011-10-01

Prostaglandins participate in a variety of female reproductive processes, including ovulation, fertilization, embryo implantation and parturition. In particular, maternal prostacyclin (PGI(2)) is critical for embryo implantation and the action of PGI(2) is not mediated via its G-protein-coupled membrane receptor, IP, but its nuclear receptor, peroxisome-proliferator-activated receptor δ (PPARδ). Recently, several studies have shown that PGI(2) enhances blastocyst development and/or hatching rate in vitro, and subsequently implantation and live birth rates in mice. However, the mechanism by which PGI(2) improves preimplantation embryo development in vitro remains unclear. Using molecular, pharmacologic and genetic approaches, we show that PGI(2)-induced PPARδ activation accelerates blastocyst hatching in mice. mRNAs for PPARδ, retinoid X receptor (heterodimeric partners of PPARδ) and PGI(2) synthase (PGIS) are temporally induced after zygotic gene activation, and their expression reaches maximum levels at the blastocyst stage, suggesting that functional complex of PPARδ can be formed in the blastocyst. Carbaprostacyclin (a stable analogue of PGI(2)) and GW501516 (a PPARδ selective agonist) significantly accelerated blastocyst hatching but did not increase total cell number of cultured blastocysts. Whereas U51605 (a PGIS inhibitor) interfered with blastocyst hatching, GW501516 restored U51605-induced retarded hatching. In contrast to the improvement of blastocyst hatching by PPARδ agonists, PPAR antagonists significantly inhibited blastocyst hatching. Furthermore, deletion of PPARδ at early stages of preimplantation mouse embryos caused delay of blastocyst hatching, but did not impair blastocyst development. Taken together, PGI(2)-induced PPARδ activation accelerates blastocyst hatching in mice.

Embryo survival and birth rate after minimum volume vitrification or slow freezing of in vivo and in vitro produced ovine embryos.

PubMed

Dos Santos-Neto, P C; Cuadro, F; Barrera, N; Crispo, M; Menchaca, A

2017-10-01

The objective was to evaluate pregnancy outcomes and birth rate of in vivo derived vs. in vitro produced ovine embryos submitted to different cryopreservation methods. A total of 197 in vivo and 240 in vitro produced embryos were cryopreserved either by conventional freezing, or by vitrification with Cryotop or Spatula MVD methods on Day 6 after insemination/fertilization. After thawing/warming and transfer, embryo survival rate on Day 30 of gestation was affected by the source of the embryos (in vivo 53.3%, in vitro 20.8%; P < 0.05) and by the method of cryopreservation (conventional freezing 26.5%, Cryotop 52.0%, Spatula MVD 22.2%; P < 0.05). For in vivo derived embryos, survival rate after embryo transfer was 45.6% for conventional freezing, 67.1% for Cryotop, and 40.4% for Spatula MVD. For in vitro produced embryos, survival rate was 7.3% for conventional freezing, 38.7% for Cryotop, and 11.4% for Spatula MVD. Fetal loss from Day 30 to birth showed a tendency to be greater for in vitro (15.0%) rather than for in vivo produced embryos (5.7%), and was not affected by the cryopreservation method. Gestation length, weight at birth and lamb survival rate after birth were not affected by the source of the embryo, the cryopreservation method or stage of development (average: 150.5 ± 1.8 days; 4232.8 ± 102.8 g; 85.4%; respectively). This study demonstrates that embryo survival and birth rate of both in vivo and in vitro produced ovine embryos are improved by vitrification with the minimum volume Cryotop method. Copyright © 2017 Elsevier Inc. All rights reserved.

TSA and BIX-01294 Induced Normal DNA and Histone Methylation and Increased Protein Expression in Porcine Somatic Cell Nuclear Transfer Embryos

PubMed Central

Ding, Biao; Zuo, Xiaoyuan; Li, Hui; Ding, Jianping; Li, Yunsheng; Huang, Weiping; Zhang, Yunhai

2017-01-01

The poor efficiency of animal cloning is mainly attributed to the defects in epigenetic reprogramming of donor cells’ chromatins during early embryonic development. Previous studies indicated that inhibition of histone deacetylases or methyltransferase, such as G9A, using Trichostatin A (TSA) or BIX-01294 significantly enhanced the developmental efficiency of porcine somatic cell nuclear transfer (SCNT) embryos. However, potential mechanisms underlying the improved early developmental competence of SCNT embryos exposed to TSA and BIX-01294 are largely unclear. Here we found that 50 nM TSA or 1.0 μM BIX-01294 treatment alone for 24 h significantly elevated the blastocyst rate (P < 0.05), while further improvement was not observed under combined treatment condition. Furthermore, co-treatment or TSA treatment alone significantly reduced H3K9me2 level at the 4-cell stage, which is comparable with that in in vivo and in vitro fertilized counterparts. However, only co-treatment significantly decreased the levels of 5mC and H3K9me2 in trophectoderm lineage and subsequently increased the expression of OCT4 and CDX2 associated with ICM and TE lineage differentiation. Altogether, these results demonstrate that co-treatment of TSA and BIX-01294 enhances the early developmental competence of porcine SCNT embryos via improvements in epigenetic status and protein expression. PMID:28114389

TSA and BIX-01294 Induced Normal DNA and Histone Methylation and Increased Protein Expression in Porcine Somatic Cell Nuclear Transfer Embryos.

PubMed

Cao, Zubing; Hong, Renyun; Ding, Biao; Zuo, Xiaoyuan; Li, Hui; Ding, Jianping; Li, Yunsheng; Huang, Weiping; Zhang, Yunhai

2017-01-01

The poor efficiency of animal cloning is mainly attributed to the defects in epigenetic reprogramming of donor cells' chromatins during early embryonic development. Previous studies indicated that inhibition of histone deacetylases or methyltransferase, such as G9A, using Trichostatin A (TSA) or BIX-01294 significantly enhanced the developmental efficiency of porcine somatic cell nuclear transfer (SCNT) embryos. However, potential mechanisms underlying the improved early developmental competence of SCNT embryos exposed to TSA and BIX-01294 are largely unclear. Here we found that 50 nM TSA or 1.0 μM BIX-01294 treatment alone for 24 h significantly elevated the blastocyst rate (P < 0.05), while further improvement was not observed under combined treatment condition. Furthermore, co-treatment or TSA treatment alone significantly reduced H3K9me2 level at the 4-cell stage, which is comparable with that in in vivo and in vitro fertilized counterparts. However, only co-treatment significantly decreased the levels of 5mC and H3K9me2 in trophectoderm lineage and subsequently increased the expression of OCT4 and CDX2 associated with ICM and TE lineage differentiation. Altogether, these results demonstrate that co-treatment of TSA and BIX-01294 enhances the early developmental competence of porcine SCNT embryos via improvements in epigenetic status and protein expression.

[Association of human chorionic gonadotropin level in embryo culture media with early embryo development].

PubMed

Wang, Haiying; Zhang, Renli; Han, Dong; Liu, Caixia; Cai, Jiajie; Bi, Yanling; Wen, Anmin; Quan, Song

2014-06-01

To investigate the association of human chorionic gonadotropin (HCG) level on day 3 of embryo culture with embryo development. Spent culture media were collected from individually cultured embryos on day 3 of in vitro fertilization and embryo transfer (IVF-ET) cycles. HCG concentration in the culture media was measured using an ELISA kit and its association with embryo development was assessed. In the 163 samples of embryo culture media from 60 patients, HCG was positive in 153 sample (93.8%) with a mean level of 0.85 ± 0.43 mIU/ml. The concentration of hCG in the culture media increased gradually as the number of blastomeres increased (F=2.273, P=0.03), and decreased as the morphological grade of the embryo was lowered (F=3.900, P=0.02). ELISA is capable of detecting HCG levels in spent culture media of embryos on day 3 of in vitro culture. The concentration of HCG in spent culture media is positively correlated with the status of early embryo development and implantation rate and thus serves as a useful marker for embryo selection in IVF-ET procedure.

Zygotic and somatic embryo morphogenesis in Pinus pinaster: comparative histological and histochemical study.

PubMed

Tereso, Susana; Zoglauer, Kurt; Milhinhos, Ana; Miguel, Célia; Oliveira, M Margarida

2007-05-01

We compared morphogenesis and accumulation of storage proteins and starch in Pinus pinaster Ait. zygotic embryos with those in somatic embryos grown with different carbohydrate sources. The maturation medium for somatic embryos included 80 microM abscisic acid (ABA), 9 g l(-1) gellam gum and either glucose, sucrose or maltose at 44, 88, 175 or 263 mM in the presence or absence of 6% (w/v) polyethylene glycol (PEG) 4000 MW. Maturation medium containing 44 or 88 mM of a carbohydrate source produced only one or no cotyledonary somatic embryos per 0.6 g fresh mass of culture. The addition of PEG to the basal maturation medium resulted in a low yield of cotyledonary somatic embryos that generally showed incomplete development and anatomical abnormalities such as large intercellular spaces and large vacuoles. High concentrations of maltose also induced large intercellular spaces in the somatic embryonic cells, and 263 mM sucrose produced fewer and less developed cotyledonary somatic embryos compared with 175 mM sucrose, indicating that the effect of carbohydrate source is partially osmotic. Zygotic embryos had a lower dry mass than somatic embryos at the same stage of development. Starch granules followed a similar accumulation pattern in zygotic and somatic embryos. A low starch content was found in cotyledonary zygotic embryos and in somatic embryos developed in the presence of 175 mM maltose or 263 mM glucose. In zygotic embryos and in PEG-treated somatic embryos, protein bodies appeared later and were smaller and fewer than in well-developed somatic embryos grown without PEG. We propose that storage protein concentration might be a marker of embryo quality.

Biotechnology applied to fish reproduction: tools for conservation.

PubMed

de Siqueira-Silva, Diógenes Henrique; Saito, Taiju; Dos Santos-Silva, Amanda Pereira; da Silva Costa, Raphael; Psenicka, Martin; Yasui, George Shigueki

2018-04-29

This review discusses the new biotechnological tools that are arising and promising for conservation and enhancement of fish production, mainly regarding the endangered and the most economically important species. Two main techniques, in particular, are available to avoid extinction of endangered fish species and to improve the production of commercial species. Germ cell transplantation technology includes a number of approaches that have been studied, such as the transplantation of embryo-to-embryo blastomere, embryo-to-embryo differentiated PGC, larvae to larvae and embryo differentiated PGC, transplantation of spermatogonia from adult to larvae or between adults, and oogonia transplantation. However, the success of germ cell transplantation relies on the prior sterilization of fish, which can be performed at different stages of fish species development by means of several protocols that have been tested in order to achieve the best approach to produce a sterile fish. Among them, fish hybridization and triploidization, germline gene knockdown, hyperthermia, and chemical treatment deserve attention based on important results achieved thus far. This review currently used technologies and knowledge about surrogate technology and fish sterilization, discussing the stronger and the weaker points of each approach.

Goose embryonic development from oviposition through 16 hours of incubation.

PubMed

Lukaszewicz, E; Lason, M; Rosenberger, J; Kowalczyk, A; Bakst, M

2017-06-01

Normal tables provide an objective step-wise description of the morphological development of an embryo. Such tables have been described for the chicken, turkey, quail, and duck embryos, but there is no such staging table for goose embryos. As the goose has one of the longest incubation periods of all the poultry species and embryo mortality during incubation is relatively high, a normal table of goose embryo development would be useful in assessing the morpho-genetic status of the goose embryo before and during incubation. In this study, embryos were isolated from commercial White Koluda goose eggs stored no longer than four days in a cool room (18°C) prior to incubation and after 4, 8, 12, and 16 h of incubation. Embryo staging was based on the normal tables described for the chicken by Eyal-Giladi and Kochav (EGK) and Hamburger and Hamilton (HH). Goose embryos from unincubated eggs were at Stage X and XI EGK and after 16 h of incubation the majority of embryos were between Stages 2 and 4 HH. Our results suggest that while the stage of development of the embryo in the unincubated goose egg is similar to that reported for the chicken, although the diameter of goose embryo is slighter larger. Following incubation, a goose embryo advances more slowly than a chicken embryo up to 16 h of incubation. © 2016 Poultry Science Association Inc.

Influence of embryo handling and transfer method on pig cloning efficiency.

PubMed

Shi, Junsong; Zhou, Rong; Luo, Lvhua; Mai, Ranbiao; Zeng, Haiyu; He, Xiaoyan; Liu, Dewu; Zeng, Fang; Cai, Gengyuan; Ji, Hongmei; Tang, Fei; Wang, Qinglai; Wu, Zhenfang; Li, Zicong

2015-03-01

The somatic cell nuclear transfer (SCNT) technique could be used to produce genetically superior or genetically engineered cloned pigs that have wide application in agriculture and bioscience research. However, the efficiency of porcine SCNT currently is very low. Embryo transfer (ET) is a key step for the success of SCNT. In this study, the effects of several ET-related factors, including cloned embryo culture time, recipient's ovulation status, co-transferred helper embryos and ET position, on the success rate of pig cloning were investigated. The results indicated that transfer of cloned embryos cultured for a longer time (22-24h vs. 4-6h) into pre-ovulatory sows decreased recipient's pregnancy rate and farrowing rate, and use of pre-ovulatory and post-ovulatory sows as recipients for SCNT embryos cultured for 22-24h resulted in a similar porcine SCNT efficiency. Use of insemination-produced in vivo fertilized, parthenogenetically activated and in vitro fertilized embryos as helper embryos to establish and/or maintain pregnancy of SCNT embryos recipients could not improve the success rate of porcine SCNT. Transfer of cloned embryos into double oviducts of surrogates significantly increased pregnancy rate as well as farrowing rate of recipients, and the developmental rate of transferred cloned embryos, as compared to unilateral oviduct transfer. This study provided useful information for optimization of the embryo handling and transfer protocol, which will help to improve the ability to generate cloned pigs. Copyright © 2015 Elsevier B.V. All rights reserved.

Bayesian classification for the selection of in vitro human embryos using morphological and clinical data.

PubMed

Morales, Dinora Araceli; Bengoetxea, Endika; Larrañaga, Pedro; García, Miguel; Franco, Yosu; Fresnada, Mónica; Merino, Marisa

2008-05-01

In vitro fertilization (IVF) is a medically assisted reproduction technique that enables infertile couples to achieve successful pregnancy. Given the uncertainty of the treatment, we propose an intelligent decision support system based on supervised classification by Bayesian classifiers to aid to the selection of the most promising embryos that will form the batch to be transferred to the woman's uterus. The aim of the supervised classification system is to improve overall success rate of each IVF treatment in which a batch of embryos is transferred each time, where the success is achieved when implantation (i.e. pregnancy) is obtained. Due to ethical reasons, different legislative restrictions apply in every country on this technique. In Spain, legislation allows a maximum of three embryos to form each transfer batch. As a result, clinicians prefer to select the embryos by non-invasive embryo examination based on simple methods and observation focused on morphology and dynamics of embryo development after fertilization. This paper proposes the application of Bayesian classifiers to this embryo selection problem in order to provide a decision support system that allows a more accurate selection than with the actual procedures which fully rely on the expertise and experience of embryologists. For this, we propose to take into consideration a reduced subset of feature variables related to embryo morphology and clinical data of patients, and from this data to induce Bayesian classification models. Results obtained applying a filter technique to choose the subset of variables, and the performance of Bayesian classifiers using them, are presented.

In vivo characterization of the effects of abscisic acid and drying protocols associated with the acquisition of desiccation tolerance in alfalfa (Medicago sativa L.) somatic embryos.

PubMed

Sreedhar, Lekha; Wolkers, Willem F; Hoekstra, Folkert A; Bewley, J Derek

2002-04-01

Although somatic embryos of alfalfa (Medicago sativa L.) had acquired some tolerance to desiccation at the cotyledonary stage of development (22 d after plating), additional culturing in 20 microm abscisic acid (ABA) for 8 d induced greater desiccation tolerance, as determined by increased germination. Compared with fast drying, slow drying of the ABA-treated embryos improved desiccation tolerance. However, slow drying of non-ABA-treated embryos led to the complete loss of germination capacity, while some fast-dried embryos survived. An electron paramagnetic resonance spin probe technique and in vivo Fourier transform infrared microspectroscopy revealed that cellular membrane integrity and a-helical protein secondary structure were maintained during drying in embryos cultured in media enriched with 20 microM ABA, but not in embryos cultured in the absence of ABA. Slow-dried, non-ABA-treated embryos had low oligosaccharide to sucrose ratios, an increased proportion of beta-sheet protein secondary structures and broad membrane phase transitions extending over a temperature range of more than 60 degrees C, suggestive of irreversible phase separations. The spin probe study showed evidence of imbibitional damage, which could be alleviated by prehydration in humid air. These observations emphasize the importance of appropriate drying and prehydration protocols for the survival and storage of somatic embryos. It is suggested that ABA also plays a role in suppressing metabolism, thus increasing the level of desiccation tolerance; this is particularly evident under stressful conditions such as slow drying.

Characterization and quantification of proteins secreted by single human embryos prior to implantation.

PubMed

Poli, Maurizio; Ori, Alessandro; Child, Tim; Jaroudi, Souraya; Spath, Katharina; Beck, Martin; Wells, Dagan

2015-11-01

The use of in vitro fertilization (IVF) has revolutionized the treatment of infertility and is now responsible for 1-5% of all births in industrialized countries. During IVF, it is typical for patients to generate multiple embryos. However, only a small proportion of them possess the genetic and metabolic requirements needed in order to produce a healthy pregnancy. The identification of the embryo with the greatest developmental capacity represents a major challenge for fertility clinics. Current methods for the assessment of embryo competence are proven inefficient, and the inadvertent transfer of non-viable embryos is the principal reason why most IVF treatments (approximately two-thirds) end in failure. In this study, we investigate how the application of proteomic measurements could improve success rates in clinical embryology. We describe a procedure that allows the identification and quantification of proteins of embryonic origin, present in attomole concentrations in the blastocoel, the enclosed fluid-filled cavity that forms within 5-day-old human embryos. By using targeted proteomics, we demonstrate the feasibility of quantifying multiple proteins in samples derived from single blastocoels and that such measurements correlate with aspects of embryo viability, such as chromosomal (ploidy) status. This study illustrates the potential of high-sensitivity proteomics to measure clinically relevant biomarkers in minute samples and, more specifically, suggests that key aspects of embryo competence could be measured using a proteomic-based strategy, with negligible risk of harm to the living embryo. Our work paves the way for the development of "next-generation" embryo competence assessment strategies, based on functional proteomics. © 2015 The Authors. Published under the terms of the CC BY 4.0 license.

Eggshell structure in Caiman latirostris eggs improves embryo survival during nest inundation.

PubMed

Cedillo-Leal, César; Simoncini, Melina S; Leiva, Pamela M L; Larriera, Alejandro; Lang, Jeffrey W; Piña, Carlos I

2017-05-17

Egg inundation often results in poor hatching success in crocodylians. However, how tolerant eggs are to submergence, and/or how eggshell ultrastructure may affect embryo survival when inundated, are not well understood. In this study, our objective was to determine if embryo survival in Caiman latirostris is affected by eggshell surface roughness, when eggs are submerged under water. Tolerance to inundation was tested early (day 30) versus late (day 60) in development, using eight clutches (four per time treatments), subdivided into four groups: ( N = 9 per clutch per treatment; 9 × 4 = 36 eggs per group). 'Rough' eggshell represented the natural, unmodified eggshell surface structure. 'Smooth' eggshell surface structure was created by mechanically sanding the natural rough surface to remove surface columnar elements and secondary layer features, e.g. irregularities that result in 'roughness'. When inundated by submerging eggs under water for 10 h at day 30, 'smooth' eggshell structure resulted in more than twice as many dead embryos (16 versus 6, smooth versus rough; N = 36), and fewer than half as many healthy embryos (6 versus 13, smooth versus rough, respectively; N = 36). By contrast, at day 60, inundation resulted in very low hatching success, regardless of eggshell surface structure. Only two hatchlings survived the inundation, notably in the untreated group with intact, rough eggshells. Inundation produced a high rate of malformations (58% at day 30), but did not affect hatchling size. Our results indicate that eggshell roughness enhances embryo survival when eggs are inundated early in development, but not late in development. Apparently, the natural surface 'roughness' entraps air bubbles at the eggshell surface during inundation, thereby facilitating gas exchange through the eggshell even when the egg is submerged under water. © 2017 The Author(s).

Analysis of the Molecular Mechanisms of Reepithelialization in Drosophila Embryos

PubMed Central

Matsubayashi, Yutaka; Millard, Tom H.

2016-01-01

Significance: The epidermis provides the main barrier function of skin, and therefore its repair following wounding is an essential component of wound healing. Repair of the epidermis, also known as reepithelialization, occurs by collective migration of epithelial cells from around the wound edge across the wound until the advancing edges meet and fuse. Therapeutic manipulation of this process could potentially be used to accelerate wound healing. Recent Advances: It is difficult to analyze the cellular and molecular mechanisms of reepithelialization in human tissue, so a variety of model organisms have been used to improve our understanding of the process. One model system that has been especially useful is the embryo of the fruit fly Drosophila, which provides a simple, accessible model of the epidermis and can be manipulated genetically, allowing detailed analysis of reepithelialization at the molecular level. This review will highlight the key insights that have been gained from studying reepithelialization in Drosophila embryos. Critical Issues: Slow reepithelialization increases the risk of wounds becoming infected and ulcerous; therefore, the development of therapies to accelerate or enhance the process would be a great clinical advance. Improving our understanding of the molecular mechanisms that underlie reepithelialization will help in the development of such therapies. Future Directions: Research in Drosophila embryos has identified a variety of genes and proteins involved in triggering and driving reepithelialization, many of which are conserved in humans. These novel reepithelialization proteins are potential therapeutic targets and therefore findings obtained in Drosophila may ultimately lead to significant clinical advances. PMID:27274434

Photoautotrophic Culture of Coffea arabusta Somatic Embryos: Photosynthetic Ability and Growth of Different Stage Embryos

PubMed Central

AFREEN, F.; ZOBAYED, S. M. A.; KOZAI, T.

2002-01-01

Coffea arabusta somatic embryos were cultured and development of stomata, rate of CO2 fixation or production, chlorophyll content and chlorophyll fluorescence were studied in embryos at different stages of development. Cotyledonary and germinated embryos have photosynthetic capacity, although pretreatment at a high photosynthetic photon flux (PPF) (100 µmol m–2 s–1) for 14 d increased photosynthetic ability. Except in a very small number of cases, stomata did not develop fully in precotyledonary stage embryos and were absent in torpedo stage embryos. Low chlorophyll content (90–130 µg g–1 fresh mass) was noted in torpedo and precotyledonary stage embryos compared with cotyledonary and germinated embryos (300–500 µg g–1 fresh mass). Due to the absence of stomata and low chlorophyll content in the torpedo and precotyledonary stage embryos, the photosynthetic rate was low and, in some cases, CO2 production was observed. These data suggest that the cotyledonary stage is the earliest stage that can be cultured photoautotrophically to ensure plantlet development. When grown photoautotrophically (in a sugar‐free medium with CO2 enrichment in the culture headspace and high photosynthetic photon flux), torpedo and precotyledonary stage embryos lost 20–25 % of their initial dry mass after 60 d of culture. However, in cotyledonary and germinated embryos, the dry mass of each embryo increased by 10 and 50 %, respectively. By using a porous supporting material, growth (especially root growth) was increased in cotyledonary stage embryos. In addition, photoautotrophic conditions, high PPF (100–150 µmol m–2 s–1) and increased CO2 concentration (1100 µmol mol–1) were found to be necessary for the development of plantlets from cotyledonary stage embryos. PMID:12125763

Abscisic acid and osmoticum prevent germination of developing alfalfa embryos, but only osmoticum maintains the synthesis of developmental proteins.

PubMed

Xu, N; Coulter, K M; Derek Bewley, J

1990-10-01

Developing seeds of alfalfa (Medicago sativa L.) acquire the ability to germinate during the latter stages of development, the maturation drying phase. Isolated embryos placed on Murashige and Skoog medium germinate well during early and late development, but poorly during mid-development; however, when placed on water they germinate well only during the latter stage of development. Germination of isolated embryos is very slow and poor when they are incubated in the presence of surrounding seed structures (the endosperm or seed coat) taken from the mid-development stages. This inhibitory effect is also achieved by incubating embryos in 10(-5) M abscisic acid (ABA). Endogenous ABA attains a high level during mid-development, especially in the endosperm. Seeds developing in pods treated with fluridone (1-methyl-3-phenyl-5[3-(trifluoromethyl)-phenyl]-4(1H)-pyridinone) contain low levels of ABA during mid-development, and the endosperm and seed coat only weakly inhibit the germination of isolated embryos. However, intact seeds from fluridone-treated pods do not germinate viviparously, which is indicative that ABA alone is not responsible for maintaining seeds in a developing state. Application of osmoticum (e.g. 0.35 M sucrose) to isolated developing embryos prevents their germination. Also, in the developing seed in situ the osmotic potential is high. Thus internal levels of osmoticum may play a role in preventing germination of the embryo and maintaining development. Abscisic acid and osmoticum impart distinctly different metabolic responses on developing embryos, as demonstrated by their protein-synthetic capacity. Only in the presence of osmoticum do embryos synthesize proteins which are distinctly recognizable as those synthesized by developing embryos in situ, i.e. when inside the pod. Abscisic acid induces the synthesis of a few unique proteins, but these arise even in mature embryos treated with ABA. Thus while both osmoticum and ABA prevent precocious germination, their effects on the synthetic capacity of the developing embryo are quite distinct. Since seeds with low endogenous ABA do not germinate, osmotic regulation may be the more important of these two factors in controlling seed development.

Corticotrophin-releasing hormone and corticosterone impair development of preimplantation embryos by inducing oviductal cell apoptosis via activating the Fas system: an in vitro study.

PubMed

Tan, Xiu-Wen; Ji, Chang-Li; Zheng, Liang-Liang; Zhang, Jie; Yuan, Hong-Jie; Gong, Shuai; Zhu, Jiang; Tan, Jing-He

2017-08-01

What are the mechanisms by which corticotrophin-releasing hormone (CRH) and corticosterone impair the development of preimplantation embryos in the oviduct. CRH and corticosterone do not affect preimplantation embryos directly, but impair their development indirectly by triggering apoptosis of oviductal epithelial cells (OECs) through activation of the Fas system. Studies report that stress impairs embryo development with facilitated secretion of CRH and glucocorticoids. Although an in vivo study demonstrated that preimplantation stress impaired embryo development in conjunction with oviductal apoptosis and activation of the Fas system, whether CRH or glucocorticoids damage embryos directly or indirectly by way of oviductal cells remains to be clarified. Mice of Kunming strain, the generalized lymphoproliferative disorder (gld) mice with a germline mutation F273L in Fas ligand in a C57BL/6J genomic background and the wild-type C57BL/6J mice were used. Female mice were used 8-10 weeks after birth. While some female mice were killed 48 h after being injected with equine CG to collect oviducts and prepare OECs, others were killed to recover zygotes after mating with males following superovulation with eCG and hCG. The zygotes obtained were cultured with or without CRH or corticosterone (CRH/Cort) either in Chatot-Ziomek-Bavister (CZB) medium with or without OECs or in conditioned medium (CM) conditioned with OECs pretreated or not with CRH/Cort. Preimplantation development, levels of redox potential and apoptosis, and expression of CRH receptor 1 (CRHR1), glucocorticoid receptor (GR), Fas and 11β-hydroxysteroid dehydrogenase (HSD) were observed in embryos recovered at different times of in vitro culture. After culture of OECs with or without CRH/Cort, levels of redox potential and apoptosis, mRNA and protein expression of growth factors, and protein expression of CRHR1, GR and Fas were examined in OECs and the level of FasL was measured in CM. The gld mice were used to confirm a role for the Fas system in triggering apoptosis of embryos and oviducts. This study showed that blastocyst development was unaffected when mouse zygotes were cultured in CZB medium containing various concentrations of CRH/Cort but was impaired when embryos were cultured with CRH/Cort plus OECs or in CM conditioned with OECs pretreated with CRH/Cort (treatment CM). Culture in treatment-CM induced oxidative stress and apoptosis in embryos. Preimplantation embryos expressed GR and Fas at all stages and CRHR1 at the blastocyst stage only. Mouse 4-cell embryos and blastocysts expressed HSD2 but not HSD1. Culture of OECs with CRH/Cort increased their oxidative stress, apoptosis, CRHR1, Fas and FasL while decreasing their GR and growth factors. Blastocyst development in treatment-CM conditioned with OECs from gld mice harboring FasL mutations was superior to treatment-CM conditioned with wild-type mouse OECs. The results suggest that CRH/Cort impairs embryo development indirectly by inducing oviductal apoptosis via activating the Fas system. The insensitivity of preimplantation embryos to CRH and corticosterone is due to, respectively, a lack of CRHR and the exclusive expression of HSD2 that inactivate corticosterone. Not applicable. Although significant, the conclusions were drawn from limited results obtained using mice and thus they need further verification in other species. For example, bovine embryos express both HSD1 and HSD2 at all the preimplantation stages whereas mouse preimplantation embryos express HSD2 exclusively without HSD1. The data are important for our understanding of the mechanisms by which stress affects female reproduction in both human and animals, as early stages of pregnancy are considered more vulnerable to stress than the late stages. This study was supported by grants from the National Basic Research Program of China (Nos. 2014CB138503 and 2012CB944403), the China National Natural Science Foundation (Nos. 31272444 and 30972096) and the Animal breeding improvement program of Shandong Province. All authors declare that their participation in the study did not involve factual or potential conflicts of interests. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Direct embryo tagging and identification system by attachment of biofunctionalized polysilicon barcodes to the zona pellucida of mouse embryos.

PubMed

Novo, Sergi; Penon, Oriol; Barrios, Leonardo; Nogués, Carme; Santaló, Josep; Durán, Sara; Gómez-Matínez, Rodrigo; Samitier, Josep; Plaza, José Antonio; Pérez-García, Luisa; Ibáñez, Elena

2013-06-01

Is the attachment of biofunctionalized polysilicon barcodes to the outer surface of the zona pellucida an effective approach for the direct tagging and identification of cultured embryos? The results achieved provide a proof of concept for a direct embryo tagging system using biofunctionalized polysilicon barcodes, which could help to minimize the risk of mismatching errors (mix-ups) in human assisted reproduction technologies. Even though the occurrence of mix-ups is rare, several cases have been reported in fertility clinics around the world. Measures to prevent the risk of mix-ups in human assisted reproduction technologies are therefore required. Mouse embryos were tagged with 10 barcodes and the effectiveness of the tagging system was tested during fresh in vitro culture (n=140) and after embryo cryopreservation (n = 84). Finally, the full-term development of tagged embryos was evaluated (n =105). Mouse pronuclear embryos were individually rolled over wheat germ agglutinin-biofunctionalized polysilicon barcodes to distribute them uniformly around the ZONA PELLUCIDA surface. Embryo viability and retention of barcodes were determined during 96 h of culture. The identification of tagged embryos was performed every 24 h in an inverted microscope and without embryo manipulation to simulate an automatic reading procedure. Full-term development of the tagged embryos was assessed after their transfer to pseudo-pregnant females. To test the validity of the embryo tagging system after a cryopreservation process, tagged embryos were frozen at the 2-cell stage using a slow freezing protocol, and followed in culture for 72 h after thawing. Neither the in vitro or in vivo development of tagged embryos was adversely affected. The tagging system also proved effective during an embryo cryopreservation process. Global identification rates higher than 96 and 92% in fresh and frozen-thawed tagged embryos, respectively, were obtained when simulating an automatic barcode reading system, although these rates could be increased to 100% by simply rotating the embryos during the reading process. The direct embryo tagging developed here has exclusively been tested in mouse embryos. Its effectiveness in other species, such as the human, is currently being tested. The direct embryo tagging system developed here, once tested in human embryos, could provide fertility clinics with a novel tool to reduce the risk of mix-ups in human assisted reproduction technologies.

Epigenetic Alteration of Donor Cells with Histone Deacetylase Inhibitor m-Carboxycinnamic Acid Bishydroxymide Improves the In Vitro Developmental Competence of Buffalo (Bubalus bubalis) Cloned Embryos.

PubMed

Agrawal, Himanshu; Selokar, Naresh Lalaji; Saini, Monika; Singh, Manoj Kumar; Chauhan, Manmohan Singh; Palta, Prabhat; Singla, Suresh Kumar; Manik, Radhey Sham

2018-02-01

Epigenetic reprogramming is an indispensable process during the course of mammalian development, but aberrant in cloned embryos. The aim of this study was to examine the effect of donor cell treatment with histone deacetylase (HDAC) inhibitor m-carboxycinnamic acid bishydroxymide (CBHA) on cloned embryo development and establish its optimal concentration. Different concentrations of CBHA (2.5, 5.0, 10.0, and 20.0 μM) were used to treat buffalo adult fibroblast cells for 24 hours and effect on cell proliferation, gene expression, and histone modifications was analyzed. Based on these experiments, the best concentration was chosen to determine the effect of enhanced gene activation mark on developmental rates. Among the different concentrations, CBHA at higher concentration (20 μM) shows the sign of apoptosis and stress as indicated by proliferation rate and gene expression data. CBHA treatment significantly decreased the activity of HDACs and increased the level of gene activation mark H3K9ac and H3K4me3, but could not alter the level of H3K27ac. Based on these experiments, 5 μM CBHA was chosen for treatment of donor cells used for the production of cloned embryos. There was no significant difference in cleavage rate between the control and CBHA treatment group (98.5% ± 1.5% vs. 99.0% ± 1.0%), whereas, blastocyst rate markedly improved (46.65% ± 1.94% vs. 57.18% ± 2.68%). The level of H3K9ac and H3K27me3 did not differ significantly in cloned blastocyst produced from either control or CBHA-treated cells. Altogether, these results suggested that donor cell treatment with CBHA supports the reprogramming process and improves the cloned preimplantation development.

Human endometrial cell coculture reduces the endocrine disruptor toxicity on mouse embryo development

PubMed Central

2012-01-01

Backgrounds Previous studies suggested that endocrine disruptors (ED) are toxic on preimplantation embryos and inhibit development of embryos in vitro culture. However, information about the toxicity of endocrine disruptors on preimplantation development of embryo in human reproductive environment is lacking. Methods Bisphenol A (BPA) and Aroclor 1254 (polychlorinated biphenyls) were used as endocrine disruptors in this study. Mouse 2-cell embryos were cultured in medium alone or vehicle or co-cultured with human endometrial epithelial layers in increasing ED concentrations. Results At 72 hours the percentage of normal blastocyst were decreased by ED in a dose-dependent manner while the co-culture system significantly enhanced the rate and reduced the toxicity of endocrine disruptors on the embryonic development in vitro. Conclusions In conclusion, although EDs have the toxic effect on embryo development, the co-culture with human endometrial cell reduced the preimplantation embryo from it thereby making human reproductive environment protective to preimplantation embryo from the toxicity of endocrine disruptors. PMID:22546201

Proof of concept: preimplantation genetic screening without embryo biopsy through analysis of cell-free DNA in spent embryo culture media.

PubMed

Shamonki, Mousa I; Jin, Helen; Haimowitz, Zachary; Liu, Lian

2016-11-01

To assess whether preimplantation genetic screening (PGS) is possible by testing for free embryonic DNA in spent IVF media from embryos undergoing trophectoderm biopsy. Prospective cohort analysis. Academic fertility center. Seven patients undergoing IVF and 57 embryos undergoing trophectoderm biopsy for PGS. On day 3 of development, each embryo was placed in a separate media droplet. All biopsied embryos received a PGS result by array comparative genomic hybridization. Preimplantation genetic screening was performed on amplified DNA extracted from media and results were compared with PGS results for the corresponding biopsy. [1] Presence of DNA in spent IVF culture media. [2] Correlation between genetic screening result from spent media and corresponding biopsy. Fifty-five samples had detectable DNA ranging from 2-642 ng/μL after a 2-hour amplification. Six samples with the highest DNA levels underwent PGS, rendering one result with a derivative log ratio SD (DLRSD) of <0.85 (a quality control metric of oligonucleotide array comparative genomic hybridization). The fluid sample and trophectoderm results were identical demonstrating (45XY, -13). Three samples were reamplified 1 hour later and tested showing improving DLRSD. One of the three samples with a DLRSD of 0.85 demonstrated (46XY), consistent with the biopsy. Overnight DNA amplification showed DNA in all samples. We demonstrate two novel findings: the presence of free embryonic DNA in spent media and a result that is consistent with trophectoderm biopsy. Improvements in DNA collection, amplification, and testing may allow for PGS without biopsy in the future. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

No Relationship between Embryo Morphology and Successful Derivation of Human Embryonic Stem Cell Lines

PubMed Central

Ström, Susanne; Rodriguez-Wallberg, Kenny; Holm, Frida; Bergström, Rosita; Eklund, Linda; Strömberg, Anne-Marie; Hovatta, Outi

2010-01-01

Background The large number (30) of permanent human embryonic stem cell (hESC) lines and additional 29 which did not continue growing, in our laboratory at Karolinska Institutet have given us a possibility to analyse the relationship between embryo morphology and the success of derivation of hESC lines. The derivation method has been improved during the period 2002–2009, towards fewer xeno-components. Embryo quality is important as regards the likelihood of pregnancy, but there is little information regarding likelihood of stem cell derivation. Methods We evaluated the relationship of pronuclear zygote stage, the score based on embryo morphology and developmental rate at cleavage state, and the morphology of the blastocyst at the time of donation to stem cell research, to see how they correlated to successful establishment of new hESC lines. Results Derivation of hESC lines succeeded from poor quality and good quality embryos in the same extent. In several blastocysts, no real inner cell mass (ICM) was seen, but permanent well growing hESC lines could be established. One tripronuclear (3PN) zygote, which developed to blastocyst stage, gave origin to a karyotypically normal hESC line. Conclusion Even very poor quality embryos with few cells in the ICM can give origin to hESC lines. PMID:21217828

Maize embryogenesis.

PubMed

Fontanet, Pilar; Vicient, Carlos M

2008-01-01

Plant embryo development is a complex process that includes several coordinated events. Maize mature embryos consist of a well-differentiated embryonic axis surrounded by a single massive cotyledon called scutellum. Mature embryo axis also includes lateral roots and several developed leaves. In contrast to Arabidopsis, in which the orientation of cell divisions are perfectly established, only the first planes of cell division are predictable in maize embryos. These distinctive characteristics joined to the availability of a large collection of embryo mutants, well-developed molecular biology and tissue culture tools, an established genetics and its economical importance make maize a good model plant for grass embryogenesis. Here, we describe basic concepts and techniques necessary for studying maize embryo development: how to grow maize in greenhouses and basic techniques for in vitro embryo culture, somatic embryogenesis and in situ hybridization.

Polar body based aneuploidy screening is poorly predictive of embryo ploidy and reproductive potential.

PubMed

Salvaggio, C N; Forman, E J; Garnsey, H M; Treff, N R; Scott, R T

2014-09-01

Polar body (polar body) biopsy represents one possible solution to performing comprehensive chromosome screening (CCS). This study adds to what is known about the predictive value of polar body based testing for the genetic status of the resulting embryo, but more importantly, provides the first evaluation of the predictive value for actual clinical outcomes after embryo transfer. SNP array was performed on first polar body, second polar body, and either a blastomere or trophectoderm biopsy, or the entire arrested embryo. Concordance of the polar body-based prediction with the observed diagnoses in the embryos was assessed. In addition, the predictive value of the polar body -based diagnosis for the specific clinical outcome of transferred embryos was evaluated through the use of DNA fingerprinting to track individual embryos. There were 459 embryos analyzed from 96 patients with a mean maternal age of 35.3. The polar body-based predictive value for the embryo based diagnosis was 70.3%. The blastocyst implantation predictive value of a euploid trophectoderm was higher than from euploid polar bodies (51% versus 40%). The cleavage stage embryo implantation predictive value of a euploid blastomere was also higher than from euploid polar bodies (31% versus 22%). Polar body based aneuploidy screening results were less predictive of actual clinical outcomes than direct embryo assessment and may not be adequate to improve sustained implantation rates. In nearly one-third of cases the polar body based analysis failed to predict the ploidy of the embryo. This imprecision may hinder efforts for polar body based CCS to improve IVF clinical outcomes.

Water relations in culture media influence maturation of avocado somatic embryos.

PubMed

Márquez-Martín, Belén; Sesmero, Rafael; Quesada, Miguel A; Pliego-Alfaro, Fernando; Sánchez-Romero, Carolina

2011-11-15

Application of transformation and other biotechnological tools in avocado (Persea americana Mill.) is hampered by difficulties in obtaining mature somatic embryos capable of germination at an acceptable rate. In this work, we evaluated the effect of different compounds affecting medium water relations on maturation of avocado somatic embryos. Culture media were characterized with respect to gel strength, water potential and osmotic potential. Improved production of mature somatic embryos was achieved with gelling agent concentrations higher than those considered standard. The osmotic agents such as sorbitol and PEG did not have positive effects on embryo maturation. The number of w-o mature somatic embryos per culture was positively correlated with medium gel strength. Gel strength was significantly affected by gelling agent type as well as by gelling agent and PEG concentration. Medium water potential was influenced by sorbitol concentration; incorporation of PEG to a culture medium did not affect medium water potential. The highest maturation results were achieved on a medium gelled with 10 gl(-1) agar. Moreover, these somatic embryos had improved germination rates. These results corroborate the role of water restriction as a key factor controlling maturation of somatic embryos. Copyright © 2011 Elsevier GmbH. All rights reserved.

Administration of the nonsteroidal anti-inflammatory drug tolfenamic acid at embryo transfer improves maintenance of pregnancy and embryo survival in recipient mice.

PubMed

Schlapp, Geraldine; Goyeneche, Lucía; Fernández, Gabriel; Menchaca, Alejo; Crispo, Martina

2015-02-01

To evaluate the effect of the nonsteroidal anti-inflammatory drugs tolfenamic acid and flunixin meglumine in pregnancy rate and embryo survival of recipient mice subjected to embryo transfer. A total of 142 recipient females were transferred with 2,931 embryos and treated with a single injection of tolfenamic acid (1 mg/kg; n = 54 females with 1,129 embryos), flunixin meglumine (2.5 mg/kg; n = 46 females with 942 embryos), or bi-distilled water (10 mL/kg) as control group (n = 42 females with 860 embryos). Pregnancy was checked 2 weeks after embryo transfer, delivery was registered on the due date, and litter size was recorded on Day 7 after birth. Pregnancy rate of tolfenamic acid treated females was significantly higher than flunixin group (P < 0.05) and showed a tendency to be higher when compared to the control group (P = 0.06). The number of pups born from transferred embryos in pregnant females was significantly higher for both treatment groups compared to controls (P < 0.05). Number of pups from total transferred embryos was higher for both treatment groups (P < 0.05) when compared to controls. The use of tolfenamic acid at the time of embryo transfer improves both pregnancy rate and number of live pups in recipient mice, with optimal effects observed with flunixin meglumine. We suggest that the use of tolfenamic acid has beneficial effects on the maintenance of pregnancy and embryo survival in recipient mice, which should be taken into account for further studies in other mammalian females.

PreImplantation Factor (PIF) correlates with early mammalian embryo development-bovine and murine models

PubMed Central

2011-01-01

Background PreImplantation Factor (PIF), a novel peptide secreted by viable embryos is essential for pregnancy: PIF modulates local immunity, promotes decidual pro-adhesion molecules and enhances trophoblast invasion. To determine the role of PIF in post-fertilization embryo development, we measured the peptide's concentration in the culture medium and tested endogenous PIF's potential trophic effects and direct interaction with the embryo. Methods Determine PIF levels in culture medium of multiple mouse and single bovine embryos cultured up to the blastocyst stage using PIF-ELISA. Examine the inhibitory effects of anti-PIF-monoclonal antibody (mAb) added to medium on cultured mouse embryos development. Test FITC-PIF uptake by cultured bovine blastocysts using fluorescent microscopy. Results PIF levels in mouse embryo culture medium significantly increased from the morula to the blastocyst stage (ANOVA, P = 0.01). In contrast, atretic embryos medium was similar to the medium only control. Detectable - though low - PIF levels were secreted already by 2-cell stage mouse embryos. In single bovine IVF-derived embryos, PIF levels in medium at day 3 of culture were higher than non-cleaving embryos (control) (P = 0.01) and at day 7 were higher than day 3 (P = 0.03). In non-cleaving embryos culture medium was similar to medium alone (control). Anti-PIF-mAb added to mouse embryo cultures lowered blastocyst formation rate 3-fold in a dose-dependent manner (2-way contingency table, multiple groups, X2; P = 0.01) as compared with non-specific mouse mAb, and medium alone, control. FITC-PIF was taken-up by cultured bovine blastocysts, but not by scrambled FITC-PIF (control). Conclusions PIF is an early embryo viability marker that has a direct supportive role on embryo development in culture. PIF-ELISA use to assess IVF embryo quality prior to transfer is warranted. Overall, our data supports PIF's endogenous self sustaining role in embryo development and the utility of PIF- ELISA to detect viable embryos in a non-invasive manner. PMID:21569635

Analysis of global gene expression profiles to identify differentially expressed genes critical for embryo development in Brassica rapa.

PubMed

Zhang, Yu; Peng, Lifang; Wu, Ya; Shen, Yanyue; Wu, Xiaoming; Wang, Jianbo

2014-11-01

Embryo development represents a crucial developmental period in the life cycle of flowering plants. To gain insights into the genetic programs that control embryo development in Brassica rapa L., RNA sequencing technology was used to perform transcriptome profiling analysis of B. rapa developing embryos. The results generated 42,906,229 sequence reads aligned with 32,941 genes. In total, 27,760, 28,871, 28,384, and 25,653 genes were identified from embryos at globular, heart, early cotyledon, and mature developmental stages, respectively, and analysis between stages revealed a subset of stage-specific genes. We next investigated 9,884 differentially expressed genes with more than fivefold changes in expression and false discovery rate ≤ 0.001 from three adjacent-stage comparisons; 1,514, 3,831, and 6,633 genes were detected between globular and heart stage embryo libraries, heart stage and early cotyledon stage, and early cotyledon and mature stage, respectively. Large numbers of genes related to cellular process, metabolism process, response to stimulus, and biological process were expressed during the early and middle stages of embryo development. Fatty acid biosynthesis, biosynthesis of secondary metabolites, and photosynthesis-related genes were expressed predominantly in embryos at the middle stage. Genes for lipid metabolism and storage proteins were highly expressed in the middle and late stages of embryo development. We also identified 911 transcription factor genes that show differential expression across embryo developmental stages. These results increase our understanding of the complex molecular and cellular events during embryo development in B. rapa and provide a foundation for future studies on other oilseed crops.

Latrunculin A can improve the birth rate of cloned mice and simplify the nuclear transfer protocol by gently inhibiting actin polymerization.

PubMed

Terashita, Yukari; Wakayama, Sayaka; Yamagata, Kazuo; Li, Chong; Sato, Eimei; Wakayama, Teruhiko

2012-06-01

Although animal cloning is becoming more practicable, there are many abnormalities in cloned embryos, and the success rate of producing live animals by cloning has been low. Here, we focused on the procedure for preventing pseudo-second polar body extrusion from somatic cell nuclear transfer (SCNT)-derived oocytes. Typically, reconstructed oocytes are treated with cytochalasin B (CB), but here latrunculin A (LatA) was used instead of CB to prevent pseudo-second polar body extrusion by inhibiting actin polymerization. CB caps F-actin, LatA binds G-actin, and both drugs prevent their polymerization. When the localization of F-actin was examined using phalloidin staining, it was abnormally scattered in the cytoplasm of CB-treated 1-cell embryos, but this was not detected in LatA-treated or in vitro fertilization-derived control embryos. The spindle was larger in CB-treated oocytes than in LatA-treated or untreated control oocytes. LatA treatment also doubled the rate of full-term development after embryo transfer. These results suggest that cloning efficiency in mice can be improved by optimizing each step of the SCNT procedure. Moreover, by using LatA, we could simplify the procedure with a higher birth rate of cloned mice compared with our original method.

Brassica napus seed endosperm - metabolism and signaling in a dead end tissue.

PubMed

Lorenz, Christin; Rolletschek, Hardy; Sunderhaus, Stephanie; Braun, Hans-Peter

2014-08-28

Oilseeds are an important element of human nutrition and of increasing significance for the production of industrial materials. The development of the seeds is based on a coordinated interplay of the embryo and its surrounding tissue, the endosperm. This study aims to give insights into the physiological role of endosperm for seed development in the oilseed crop Brassica napus. Using protein separation by two-dimensional (2D) isoelectric focusing (IEF)/SDS polyacrylamide gel electrophoresis (PAGE) and protein identification by mass spectrometry three proteome projects were carried out: (i) establishment of an endosperm proteome reference map, (ii) proteomic characterization of endosperm development and (iii) comparison of endosperm and embryo proteomes. The endosperm proteome reference map comprises 930 distinct proteins, including enzymes involved in genetic information processing, carbohydrate metabolism, environmental information processing, energy metabolism, cellular processes and amino acid metabolism. To investigate dynamic changes in protein abundance during seed development, total soluble proteins were extracted from embryo and endosperm fractions at defined time points. Proteins involved in sugar converting and recycling processes, ascorbate metabolism, amino acid biosynthesis and redox balancing were found to be of special importance for seed development in B. napus. Implications for the seed filling process and the function of the endosperm for seed development are discussed. The endosperm is of key importance for embryo development during seed formation in plants. We present a broad study for characterizing endosperm proteins in the oilseed plant B. napus. Furthermore, a project on the biochemical interplay between the embryo and the endosperm during seed development is presented. We provide evidence that the endosperm includes a complete set of enzymes necessary for plant primary metabolism. Combination of our results with metabolome data will further improve systems-level understanding of the seed filling process and provide rational strategies for plant bioengineering. Copyright © 2014 Elsevier B.V. All rights reserved.

Development of a new screening assay to identify proteratogenic substances using zebrafish danio rerio embryo combined with an exogenous mammalian metabolic activation system (mDarT).

PubMed

Busquet, François; Nagel, Roland; von Landenberg, Friedrich; Mueller, Stefan O; Huebler, Nicole; Broschard, Thomas H

2008-07-01

The assessment of teratogenic effects of chemicals is generally performed using in vivo teratogenicity assays, for example, in rats or rabbits. We have developed an in vitro teratogenicity assay using the zebrafish Danio rerio embryo combined with an exogenous mammalian metabolic activation system (MAS), able to biotransform proteratogenic compounds. Cyclophosphamide (CPA) and ethanol were used as proteratogens to test the efficiency of this assay. Briefly, the zebrafish embryos were cocultured at 2 hpf (hours postfertilization) with the test material at varying concentrations, induced male rat liver microsomes and nicotinamide adenine dinucleotide phosphate (reduced) for 60 min at 32 degrees C under moderate agitation in Tris-buffer. The negative control (test material alone) and the MAS control (MAS alone) were incubated in parallel. For each test group, 20 eggs were used for statistical robustness. Afterward fish embryos were transferred individually into 24-well plates filled with fish medium for 48 h at 26 degrees C with a 12-h light cycle. Teratogenicity was scored after 24 and 48 hpf using morphological endpoints. No teratogenic effects were observed in fish embryos exposed to the proteratogens alone, that is, without metabolic activation. In contrast, CPA and ethanol induced abnormalities in fish embryos when coincubated with microsomes. The severity of malformations increased with increasing concentrations of the proteratogens. We conclude that the application of microsomes will improve and refine the D. rerio teratogenicity assay as a predictive and valuable alternative method to screen teratogenic substances.

[Traditional and modern approaches to culture of preimplantation mammalian embryos in vitro].

PubMed

Brusentsev, E Iu; Igonina, T N; Amstislavskiĭ, S Ia

2014-01-01

This review covers the basic principles and methods of in vitro culture of preimplantation mammalian embryos. The features of in vitro development of embryos of various species of animals with allowance for the composition of nutrient media are described, with special attention paid to those species that have traditionally been consideredas laboratory (i.e., mice, rats, and hamsters). The effects of suboptimal culturing conditions of preimplantation embryos on the formation of the phenotype of individuals developed from these embryos are discussed. New approaches to optimize the conditions of the development of preimplantation mammalian embryos in vitro are analyzed.

mRNA fragments in in vitro culture media are associated with bovine preimplantation embryonic development.

PubMed

Kropp, Jenna; Khatib, Hasan

2015-01-01

In vitro production (IVP) systems have been used to bypass problems of fertilization and early embryonic development. However, embryos produced by IVP are commonly selected for implantation based on morphological assessment, which is not a strong indicator of establishment and maintenance of pregnancy. Thus, there is a need to identify additional indicators of embryonic developmental potential. Previous studies have identified microRNA expression in in vitro culture media to be indicative of embryo quality in both bovine and human embryos. Like microRNAs, mRNAs have been shown to be secreted from cells into the extracellular environment, but it is unknown whether or not these RNAs are secreted by embryos. Thus, the objective of the present study was to determine whether mRNAs are secreted into in vitro culture media and if their expression in the media is indicative of embryo quality. In vitro culture medium was generated and collected from both blastocyst and degenerate (those which fail to develop from the morula to blastocyst stage) embryos. Small-RNA sequencing revealed that many mRNA fragments were present in the culture media. A total of 17 mRNA fragments were differentially expressed between blastocyst and degenerate conditioned media. Differential expression was confirmed by quantitative real-time PCR for fragments of mRNA POSTN and VSNL-1, in four additional biological replicates of media. To better understand the mechanisms of mRNA secretion into the media, the expression of a predicted RNA binding protein of POSTN, PUM2, was knocked down using an antisense oligonucleotide gapmer. Supplementation of a PUM2 gapmer significantly reduced blastocyst development and decreased secretion of POSTN mRNA into the media. Overall, differential mRNA expression in the media was repeatable and sets the framework for future study of mRNA biomarkers in in vitro culture media to improve predictability of reproductive performance.

Diamond Nanoparticles Modify Curcumin Activity: In Vitro Studies on Cancer and Normal Cells and In Ovo Studies on Chicken Embryo Model

PubMed Central

Strojny, Barbara; Grodzik, Marta; Sawosz, Ewa; Winnicka, Anna; Kurantowicz, Natalia; Jaworski, Sławomir; Kutwin, Marta; Urbańska, Kaja; Hotowy, Anna; Wierzbicki, Mateusz; Chwalibog, André

2016-01-01

Curcumin has been studied broadly for its wide range of biological activities, including anticancer properties. The major problem with curcumin is its poor bioavailability, which can be improved by the addition of carriers, such as diamond nanoparticles (DN). They are carbon allotropes, and are therefore biocompatible and easily taken up by cells. DN are non-toxic and have antiangiogenic properties with potential applications in cancer therapy. Their large surface makes them promising compounds in a drug delivery system for bioactive agents, as DN create bio-complexes in a fast and simple process of self-organisation. We investigated the cytotoxicity of such bio-complexes against liver cancer cells and normal fibroblasts, revealing that conjugation of curcumin with DN significantly improves its activity. The experiment performed in a chicken embryo model demonstrated that neither curcumin nor DN nor bio-complexes affect embryo development, even though DN can form deposits in tissues. Preliminary results confirmed the applicability of DN as an efficient carrier of curcumin, which improves its performance against cancer cells in vitro, yet is not toxic to an organism, which makes the bio-complex a promising anticancer agent. PMID:27736939

Diamond Nanoparticles Modify Curcumin Activity: In Vitro Studies on Cancer and Normal Cells and In Ovo Studies on Chicken Embryo Model.

PubMed

Strojny, Barbara; Grodzik, Marta; Sawosz, Ewa; Winnicka, Anna; Kurantowicz, Natalia; Jaworski, Sławomir; Kutwin, Marta; Urbańska, Kaja; Hotowy, Anna; Wierzbicki, Mateusz; Chwalibog, André

2016-01-01

Curcumin has been studied broadly for its wide range of biological activities, including anticancer properties. The major problem with curcumin is its poor bioavailability, which can be improved by the addition of carriers, such as diamond nanoparticles (DN). They are carbon allotropes, and are therefore biocompatible and easily taken up by cells. DN are non-toxic and have antiangiogenic properties with potential applications in cancer therapy. Their large surface makes them promising compounds in a drug delivery system for bioactive agents, as DN create bio-complexes in a fast and simple process of self-organisation. We investigated the cytotoxicity of such bio-complexes against liver cancer cells and normal fibroblasts, revealing that conjugation of curcumin with DN significantly improves its activity. The experiment performed in a chicken embryo model demonstrated that neither curcumin nor DN nor bio-complexes affect embryo development, even though DN can form deposits in tissues. Preliminary results confirmed the applicability of DN as an efficient carrier of curcumin, which improves its performance against cancer cells in vitro, yet is not toxic to an organism, which makes the bio-complex a promising anticancer agent.

Genetic transformation protocols using zygotic embryos as explants: an overview.

PubMed

Tahir, Muhammad; Waraich, Ejaz A; Stasolla, Claudio

2011-01-01

Genetic transformation of plants is an innovative research tool which has practical significance for the development of new and improved genotypes or cultivars. However, stable introduction of genes of interest into nuclear genomes depends on several factors such as the choice of target tissue, the method of DNA delivery in the target tissue, and the appropriate method to select the transformed plants. Mature or immature zygotic embryos have been a popular choice as explant or target tissue for genetic transformation in both angiosperms and gymnosperms. As a result, considerable protocols have emerged in the literature which have been optimized for various plant species in terms of transformation methods and selection procedures for transformed plants. This article summarizes the recent advances in plant transformation using zygotic embryos as explants.

In vitro embryo production in goats: Slaughterhouse and laparoscopic ovum pick up-derived oocytes have different kinetics and requirements regarding maturation media.

PubMed

Souza-Fabjan, Joanna Maria G; Locatelli, Yann; Duffard, Nicolas; Corbin, Emilie; Touzé, Jean-Luc; Perreau, Christine; Beckers, Jean François; Freitas, Vicente José F; Mermillod, Pascal

2014-05-01

A total of 3427 goat oocytes were used in this study to identify possible differences during in vitro embryo production from slaughterhouse or laparoscopic ovum pick up (LOPU) oocytes. In experiment 1, one complex, one semi-defined, and one simplified IVM media were compared using slaughterhouse oocytes. In experiment 2, we checked the effect of oocyte origin (slaughterhouse or LOPU) on the kinetics of maturation (18 vs. 22 vs. 26 hours) when submitted to semi-defined or simplified media. In experiment 3, we determined the differences in embryo development between slaughterhouse and LOPU oocytes when submitted to both media and then to IVF or parthenogenetic activation (PA). Embryos from all groups were vitrified, and their viability evaluated in vitro after thawing. In experiment 1, no difference (P > 0.05) was detected among treatments for maturation rate (metaphase II [MII]; 88% on average), cleavage (72%), blastocyst from the initial number of cumulus oocyte complexes (46%) or from the cleaved ones (63%), hatching rate (69%), and the total number of blastomeres (187). In experiment 2, there was no difference of MII rate between slaughterhouse oocytes cultured for 18 or 22 hours, whereas the MII rate increased significantly (P < 0.05) between 18 and 22 hours for LOPU oocytes in the simplified medium. Moreover, slaughterhouse oocytes cultured in simplified medium matured significantly faster than LOPU oocytes at 18 and 22 hours (P < 0.05). In experiment 3, cleavage rate was significantly greater (P < 0.001) in all four groups of embryos produced by PA than IVF. Interestingly, PA reached similar rates for slaughterhouse oocytes cultured in both media, but improved (P < 0.05) the cleavage rate of LOPU oocytes. Slaughterhouse oocytes had acceptable cleavage rate after IVF (∼67%), whereas LOPU oocytes displayed a lower one (∼38%), in contrast to cleavage after PA. The percentage of blastocysts in relation to cleaved embryos was not affected by the origin of the oocytes (P > 0.05). Therefore, slaughterhouse oocytes developed a greater proportion of blastocysts than LOPU ones, expressed as the percentage of total cumulus oocyte complexes entering to IVM. Vitrified-thawed blastocysts presented similar survival and hatching rates between the oocyte origin, media, or method of activation. In conclusion, slaughterhouse and LOPU derived oocytes may have different IVM kinetics and require different IVM and IVF conditions. Although the IVM and IVF systems still need improvements to enhance embryo yield, the in vitro development step is able to generate good quality embryos from LOPU-derived oocytes. Copyright © 2014 Elsevier Inc. All rights reserved.

Short communication: expression and alternative splicing of POU1F1 pathway genes in preimplantation bovine embryos.

PubMed

Laporta, J; Driver, A; Khatib, H

2011-08-01

Early embryo loss is a major contributing factor to cow infertility and that 70 to 80% of this loss occurs between d 8 and 16 postfertilization. However, little is known about the molecular mechanisms and the nature of genes involved in normal and abnormal embryonic development. Moreover, information is limited on the contributions of the genomes of dams and of embryos to the development and survival of preimplantation embryos. We hypothesized that proper gene expression level in the developing embryo is essential for embryo survival and pregnancy success. As such, the characterization of expression profiles in early embryos could lead to a better understanding of the mechanisms involved in normal and abnormal embryo development. To test this hypothesis, 2 d-8 embryo populations (degenerate embryos and blastocysts) that differed in morphology and developmental status were investigated. Expression levels of POU1F1 pathway genes were estimated in 4 sets of biological replicate pools of degenerate embryos and blastocysts. The OPN and STAT5A genes were found to be upregulated in degenerate embryos compared with blastocysts, whereas STAT5B showed similar expression levels in both embryo groups. Analysis of splice variants of OPN and STAT5A revealed expression patterns different from the total expression values of these genes. As such, measuring expression of individual transcripts should be considered in gene expression studies. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Laser assisted zona hatching does not lead to immediate impairment in human embryo quality and metabolism.

PubMed

Uppangala, Shubhashree; D'Souza, Fiona; Pudakalakatti, Shivanand; Atreya, Hanudatta S; Raval, Keyur; Kalthur, Guruprasad; Adiga, Satish Kumar

2016-12-01

Laser assisted zona hatching (LAH) is a routinely used therapeutic intervention in assisted reproductive technology for patients with poor prognosis. However, results are not conclusive in demonstrating the benefits of zona hatching in improving the pregnancy rate. Recent observations on LAH induced genetic instability in animal embryos prompted us to look into the effects of laser assisted zona hatching on the human preimplantation embryo quality and metabolic uptake using high resolution nuclear magnetic resonance (NMR) technology. This experimental prospective study included fifty embryos from twenty-five patients undergoing intra cytoplasmic sperm injection. Embryo quality assessment followed by profiling of spent media for the non-invasive evaluation of metabolites was performed using NMR spectroscopy 24 hours after laser treatment and compared with that of non-treated sibling embryos. Both cell number and embryo quality on day 3 of development did not vary significantly between the two groups at 24 hours post laser treatment interval. Time lapse monitoring of the embryos for 24 hours did not reveal blastomere fragmentation adjacent to the point of laser treatment. Similarly, principal component analysis of metabolites did not demonstrate any variation across the groups. These results suggest that laser assisted zona hatching does not affect human preimplantation embryo morphology and metabolism at least until 24 hours post laser assisted zona hatching. However, studies are required to elucidate laser induced metabolic and developmental changes at extended time periods. AH: assisted hatching; ART: assisted reproductive technology; DNA: deoxy-ribo nucleic acid; LAH: laser assisted hatching; MHz: megahertz; NMR: nuclear magnetic resonance; PCA: principal component analysis; PGD: preimplantation genetic diagnosis; TLM: time lapse monitoring.

Timing of The First Zygotic Cleavage Affects Post-Vitrification Viability of Murine Embryos Produced In Vivo

PubMed Central

Jusof, Wan-Hafizah Wan; Khan, Nor-Ashikin Mohamed Noor; Rajikin, Mohd Hamim; Satar, Nuraliza Abdul; Mustafa, Mohd-Fazirul; Jusoh, Norhazlin; Dasiman, Razif

2015-01-01

Background Timing of the first zygotic cleavage is an accurate predictor of embryo quality. Embryos that cleaved early (EC) have been shown to exhibit higher develop- mental viability compared to those that cleaved at a later period (LC). However, the vi- ability of EC embryos in comparison to LC embryos after vitrification is unknown. The present study aims to investigate the post-vitrification developmental viability of murine EC versus LC embryos. Materials and Methods In this experimental study, female ICR mice (6-8 weeks old) were superovulated and cohabited with fertile males for 24 hours. Afterwards, their ovi- ducts were excised and embryos harvested. Embryos at the 2-cell stage were catego- rized as EC embryos, while zygotes with two pronuclei were categorized as LC embryos. Embryos were cultured in M16 medium supplemented with 3% bovine serum albumin (BSA) in a humidified 5% CO2atmosphere. Control embryos were cultured until the blastocyst stage without vitrification. Experimental embryos at the 2-cell stage were vitri- fied for one hour using 40% v/v ethylene glycol, 18% w/v Ficoll-70 and 0.5 M sucrose as the cryoprotectant. We recorded the numbers of surviving embryos from the control and experimental groups and their development until the blastocyst stage. Results were analyzed using the chi-square test. Results A significantly higher proportion of EC embryos (96.7%) from the control group developed to the blastocyst stage compared with LC embryos (57.5%, P<0.0001). Similarly, in the experimental group, a significantly higher percentage of vitrified EC embryos (69.4%) reached the blastocyst stage compared to vitrified LC embryos (27.1%, P<0.0001). Conclusion Vitrified EC embryos are more vitrification tolerant than LC embryos. Prese- lection of EC embryos may be used as a tool for selection of embryos that exhibit higher developmental competence after vitrification. PMID:26246881

Silver nanoparticles induce developmental stage-specific embryonic phenotypes in zebrafish

NASA Astrophysics Data System (ADS)

Lee, Kerry J.; Browning, Lauren M.; Nallathamby, Prakash D.; Osgood, Christopher J.; Xu, Xiao-Hong Nancy

2013-11-01

Much is anticipated from the development and deployment of nanomaterials in biological organisms, but concerns remain regarding their biocompatibility and target specificity. Here we report our study of the transport, biocompatibility and toxicity of purified and stable silver nanoparticles (Ag NPs, 13.1 +/- 2.5 nm in diameter) upon the specific developmental stages of zebrafish embryos using single NP plasmonic spectroscopy. We find that single Ag NPs passively diffuse into five different developmental stages of embryos (cleavage, early-gastrula, early-segmentation, late-segmentation, and hatching stages), showing stage-independent diffusion modes and diffusion coefficients. Notably, the Ag NPs induce distinctive stage and dose-dependent phenotypes and nanotoxicity, upon their acute exposure to the Ag NPs (0-0.7 nM) for only 2 h. The late-segmentation embryos are most sensitive to the NPs with the lowest critical concentration (CNP,c << 0.02 nM) and highest percentages of cardiac abnormalities, followed by early-segmentation embryos (CNP,c < 0.02 nM), suggesting that disruption of cell differentiation by the NPs causes the most toxic effects on embryonic development. The cleavage-stage embryos treated with the NPs develop into a wide variety of phenotypes (abnormal finfold, tail/spinal cord flexure, cardiac malformation/edema, yolk sac edema, and acephaly). These organ structures are not yet developed in cleavage-stage embryos, suggesting that the earliest determinative events to create these structures are ongoing, and disrupted by NPs, which leads to the downstream effects. In contrast, the hatching embryos are most resistant to the Ag NPs, and majority of embryos (94%) develop normally, and none of them develop abnormally. Interestingly, early-gastrula embryos are less sensitive to the NPs than cleavage and segmentation stage embryos, and do not develop abnormally. These important findings suggest that the Ag NPs are not simple poisons, and they can target specific pathways in development, and potentially enable target specific study and therapy for early embryonic development.Much is anticipated from the development and deployment of nanomaterials in biological organisms, but concerns remain regarding their biocompatibility and target specificity. Here we report our study of the transport, biocompatibility and toxicity of purified and stable silver nanoparticles (Ag NPs, 13.1 +/- 2.5 nm in diameter) upon the specific developmental stages of zebrafish embryos using single NP plasmonic spectroscopy. We find that single Ag NPs passively diffuse into five different developmental stages of embryos (cleavage, early-gastrula, early-segmentation, late-segmentation, and hatching stages), showing stage-independent diffusion modes and diffusion coefficients. Notably, the Ag NPs induce distinctive stage and dose-dependent phenotypes and nanotoxicity, upon their acute exposure to the Ag NPs (0-0.7 nM) for only 2 h. The late-segmentation embryos are most sensitive to the NPs with the lowest critical concentration (CNP,c << 0.02 nM) and highest percentages of cardiac abnormalities, followed by early-segmentation embryos (CNP,c < 0.02 nM), suggesting that disruption of cell differentiation by the NPs causes the most toxic effects on embryonic development. The cleavage-stage embryos treated with the NPs develop into a wide variety of phenotypes (abnormal finfold, tail/spinal cord flexure, cardiac malformation/edema, yolk sac edema, and acephaly). These organ structures are not yet developed in cleavage-stage embryos, suggesting that the earliest determinative events to create these structures are ongoing, and disrupted by NPs, which leads to the downstream effects. In contrast, the hatching embryos are most resistant to the Ag NPs, and majority of embryos (94%) develop normally, and none of them develop abnormally. Interestingly, early-gastrula embryos are less sensitive to the NPs than cleavage and segmentation stage embryos, and do not develop abnormally. These important findings suggest that the Ag NPs are not simple poisons, and they can target specific pathways in development, and potentially enable target specific study and therapy for early embryonic development. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr03210h

Calcium-sensing receptor (CASR) is involved in porcine in vitro fertilisation and early embryo development.

PubMed

Liu, C; Liu, Y; Larsen, K; Hou, Y P; Callesen, H

2018-01-01

It has been demonstrated that extracellular calcium is necessary in fertilisation and embryo development but the mechanism is still not well understood. The present study mainly focussed on the extracellular calcium effector called the calcium-sensing receptor (CASR) and examined its expression in porcine gametes and embryos and its function during fertilisation and early embryo development. By using reverse transcription polymerase chain reaction, CASR was found to be expressed in porcine oocytes, spermatozoa and embryos at different developmental stages. Functionally, medium supplementation with a CASR agonist or an antagonist during in vitro fertilisation (IVF) and in vitro culture (IVC) was tested. During fertilisation, the presence of a CASR agonist increased sperm penetration rate and decreased polyspermy rate leading to an increased normal fertilisation rate. During embryo development, for the IVF embryos, agonist treatment during IVC significantly increased cleavage rate and blastocyst formation rate compared with the control group. Furthermore, parthenogenetically activated embryos showed similar results with lower cleavage and blastocyst formation rates in the antagonist group than in the other groups. It was concluded that CASR, as the effector of extracellular calcium, modulates porcine fertilisation and early embryo development.

[Effect of human oviductal embryotrophic factors on gene expression of mouse preimplantation embryos].

PubMed

Yao, Yuan-Qing; Lee, Kai-Fai; Xu, Jia-Seng; Ho, Pak-Chung; Yeung, Shu-Biu

2007-09-01

To investigate the effect of embryotrophic factors (ETF) from human oviductal cells on gene expression of mouse early developmental embryos and discuss the role of fallopian tube in early development of embryos. ETF was isolated from conditioned medium of human oviductal cell line by sequential liquid chromatographic systems. Mouse embryos were treated by ETF in vitro. Using differential display RT-PCR, the gene expression of embryos treated by ETF was compared with embryos without ETF treatment. The differentially expressed genes were separated, re-amplified, cloned and sequenced. Gene expression profiles of embryos with ETF treatment was different from embryos without this treatment. Eight differentially expressed genes were cloned and sequenced. These genes functioned in RNA degradation, synthesis, splicing, protein trafficking, cellular differentiation and embryo development. Embryotrophic factors from human oviductal cells affect gene expression of early developmental embryos. The human oviductal cells play wide roles in early developmental stages of embryos.

Polyamine levels during the development of zygotic and somatic embryos of Pinus radiata

Treesearch

Rakesh Minocha; Dale R. Smith; Cathie Reeves; Kevin D. Steele; Subhash C. Minocha

1999-01-01

Changes in the cellular content of three polyamines (putrescine, spermidine and spermine) were compared at different stages of development in zygotic and somatic embryos of Pinus radiata D. Don. During embryo development, both the zygotic and the somatic embryos showed a steady increase in spermidine content, with either a small decrease or no...

HSPC117 deficiency in cloned embryos causes placental abnormality and fetal death

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Yingying; State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080; Graduate University of Chinese Academy of Sciences, Beijing 100049

2010-07-02

Somatic cell nuclear transfer (SCNT) has been successfully used in many species to produce live cloned offspring, albeit with low efficiency. The low frequency of successful development has usually been ascribed to incomplete or inappropriate reprogramming of the transferred nuclear genome. Elucidating the genetic differences between normal fertilized and cloned embryos is key to understand the low efficiency of SCNT. Here, we show that expression of HSPC117, which encodes a hypothetical protein of unknown function, was absent or very low in cloned mouse blastocysts. To investigate the role of HSPC117 in embryo development, we knocked-down this gene in normal fertilizedmore » embryos using RNA interference. We assessed the post-implantation survival of HSPC117 knock-down embryos at 3 stages: E9 (prior to placenta formation); E12 (after the placenta was fully functional) and E19 (post-natal). Our results show that, although siRNA-treated in vivo fertilized/produced (IVP) embryos could develop to the blastocyst stage and implanted without any difference from control embryos, the knock-down embryos showed substantial fetal death, accompanied by placental blood clotting, at E12. Furthermore, comparison of HSPC117 expression in placentas of nuclear transfer (NT), intracytoplasmic sperm injection (ICSI) and IVP embryos confirmed that HSPC117 deficiency correlates well with failures in embryo development: all NT embryos with a fetus, as well as IVP and ICSI embryos, had normal placental HSPC117 expression while those NT embryos showing reduced or no expression of HSPC117 failed to form a fetus. In conclusion, we show that HSPC117 is an important gene for post-implantation development of embryos, and that HSPC117 deficiency leads to fetal abnormalities after implantation, especially following placental formation. We suggest that defects in HSPC117 expression may be an important contributing factor to loss of cloned NT embryos in vivo.« less

Effect of medium variations (zinc supplementation during oocyte maturation, perifertilization pH, and embryo culture protein source) on equine embryo development after intracytoplasmic sperm injection.

PubMed

Choi, Young-Ho; Gibbons, John R; Canesin, Heloísa S; Hinrichs, Katrin

2016-10-15

Prospective studies were conducted to help define procedural factors affecting in vitro embryo production via intracytoplasmic sperm injection (ICSI) of equine oocytes. In experiment 1, use of 10% fetal bovine serum as a protein source in embryo culture medium resulted in a higher blastocyst rate than did use of a combination of 3% fetal bovine serum, 3% equine preovulatory follicular fluid, and 4% human serum substitute (37% vs. 15%, respectively, P < 0.05). In experiment 2, the effect of zinc supplementation (0, 0.5, 1, or 1.5 μg/mL) during IVM was examined. There were no significant differences in rates of cleavage or blastocyst development (20%-31%). However, the proportion of blastocysts that developed on Day 7 for the added-zinc treatments was significantly higher than that for the control treatment (45% vs. 8%). In experiment 3, we tested whether use of high-pH medium (pH 8.0-8.4) during ICSI procedures would improve blastocyst rate when sperm with low cleavage rates after ICSI was used. When high-pH conditions were used for sperm preparation and also for the first 2 hours of incubation of injected oocytes after ICSI, the cleavage rate was unaffected but no blastocysts developed (0% vs. 24% for control). When high-pH conditions were used for sperm preparation only, the blastocyst rate was 37%. This was repeated using sperm from a second stallion; there was no significant difference in cleavage or blastocyst rates between sperm preparation in high pH vs. control medium. These findings add to our knowledge of factors affecting in vitro production of equine embryos. Copyright © 2016 Elsevier Inc. All rights reserved.

Use of the Coelomic Grafting Technique for Prolonged ex utero Cultivation of Late Preprimitive Streak-Stage Rabbit Embryos.

PubMed

Püschel, Bernd; Männer, Jörg

2016-01-01

Due to its morphological similarity with the early human embryo, the pregastrulation-stage rabbit may represent an appropriate mammalian model for studying processes involved in early human development. The usability of mammalian embryos for experimental studies depends on the availability of whole embryo culture methods facilitating prolonged ex utero development. While currently used culture methods yield high success rates for embryos from primitive streak stages onward, the success rate of extended cultivation of preprimitive streak-stage mammalian embryos is low for all previously established methods and for all studied species. This limits the usability of preprimitive streak-stage rabbit embryos in experimental embryology. We have tested whether the extraembryonic coelom of 4-day-old chick embryos may be used for prolonged ex utero culture of preprimitive streak-stage rabbit embryos (stage 2, 6.2 days post coitum). We found that, within this environment, stage 2 rabbit blastocysts can be cultured at decreasing success rates (55% after 1 day, 35% after 2 days, 15% after 3 days) up to a maximum of 72 h. Grafted blastocysts can continue development from the onset of gastrulation to early organogenesis and thereby form all structures characterizing age-matched controls (e.g. neural tube, somites, beating heart). Compared to normal controls, successfully cultured embryos developed at a slower rate and finally showed some structural and gross morphological anomalies. The method presented here was originally developed for whole embryo culture of mouse embryos by Gluecksohn-Schoenheimer in 1941. It is a simple and inexpensive method that may represent a useful extension to presently available ex utero culture systems for rabbit embryos. © 2016 S. Karger AG, Basel.

Effect of the association of IGF-I, IGF-II, bFGF, TGF-beta1, GM-CSF, and LIF on the development of bovine embryos produced in vitro.

PubMed

Neira, J A; Tainturier, D; Peña, M A; Martal, J

2010-03-15

This study examined the influence of the following growth factors and cytokines on early embryonic development: insulin-like growth factors I and II (IGF-I, IGF-II), basic fibroblast growth factor (bFGF), transforming growth factor (TGF-beta), granulocyte-macrophage colony-stimulating factor (GM-CSF), and leukemia inhibitory factor (LIF). Synthetic oviduct fluid (SOF) was used as the culture medium. We studied the development of bovine embryos produced in vitro and cultured until Day 9 after fertilization. TGF-beta1, bFGF, GM-CSF, and LIF used on their own significantly improved the yield of hatched blastocysts. IGF-I, bFGF, TGF-beta1, GM-CSF, and LIF significantly accelerated embryonic development, especially the change from the expanded blastocyst to hatched blastocyst stages. Use of a combination of these growth factors and cytokines (GF-CYK) in SOF medium produced higher percentages of blastocysts and hatched blastocysts than did use of SOF alone (45% and 22% vs. 24% and 12%; P<0.05) on Day 8 after in vitro fertilization and similar results to use of SOF+10% fetal calf serum (38% and 16%, at the same stages, respectively). The averages of total cells, inner cell mass cells, and trophectoderm cells of exclusively in vitro Day-8 blastocysts for pooled GF-CYK treatments were higher than those for SOF and similar to those for fetal calf serum. The presence of these growth factors and cytokines in the embryo culture medium therefore has a combined stimulatory action on embryonic development; in particular through an increase in hatching rate and in the number of cells of both the inner cell mass and trophoblast. These results are the first to demonstrate that use of a combination of recombinant growth factors and cytokine, as IGF-I, IGF-II, bFGF, TGF-beta1, LIF, and GM-CSF, produces similar results to 10% fetal calf serum for the development of in vitro-produced bovine embryos. This entirely synthetic method of embryo culture has undeniable advantages for the biosecurity of embryo transfer. Copyright 2010 Elsevier Inc. All rights reserved.

Random Walk of Single Gold Nanoparticles in Zebrafish Embryos Leading to Stochastic Toxic Effects on Embryonic Developments

PubMed Central

Browning, Lauren M.; Lee, Kerry J.; Huang, Tao; Nallathamby, Prakash D.; Lowman, Jill E.; Xu, Xiao-Hong Nancy

2010-01-01

We have synthesized and characterized stable (non-aggregation, non-photobleaching and non-blinking), nearly monodisperse and highly-purified Au nanoparticles, and used them to probe transport of cleavage-stage zebrafish embryos and to study their effects on embryonic development in real time. We found that single Au nanoparticles (11.6 ± 0.9 nm in diameter) passively diffused into chorionic space of the embryos via their chorionic-pore-canals and continued their random-walk through chorionic space and into inner mass of embryos. Diffusion coefficients of single nanoparticles vary dramatically (2.8×10-11 to 1.3×10-8 cm2/s) as nanoparticles diffuse through various parts of embryos, suggesting highly diverse transport barriers and viscosity gradients of embryos. The amount of Au nanoparticles accumulated in embryos increase with its concentration. Interestingly, their effects on embryonic development are not proportionally related to the concentration. Majority of embryos (74% on average) incubated chronically with 0.025-1.2 nM Au nanoparticles for 120 h developed to normal zebrafish, with some (24%) being dead and few (2%) deformed. We developed a new approach to image and characterize individual Au nanoparticles embedded in tissues using histology sample preparation methods and LSRP spectra of single nanoparticles. We found that Au nanoparticles in various parts of normally developed and deformed zebrafish, suggesting that random-walk of nanoparticles in embryos during their development might have led to stochastic effects on embryonic development. These results show that Au nanoparticles are much more biocompatible (less toxic) to the embryos than Ag nanoparticles that we reported previously, suggesting that they are better suited as biocompatible probes for imaging embryos in vivo. The results provide powerful evidences that biocompatibility and toxicity of nanoparticles highly depend on their chemical properties, and the embryos can serve as effective in-vivo assays to screen their biocompatibility. PMID:20644873

Effect of Medium Salt Concentration on Differentiation and Maturation of Somatic Embryos of Cassava (Manihot esculenta Crantz)

PubMed Central

GROLL, J.; MYCOCK, D. J.; GRAY, V. M.

2002-01-01

Culture of cassava somatic embryos on media with an altered macro‐ and micro‐nutrient salt concentration affected embryo development and germination capability. In the tests, quarter‐, half‐, full‐ or double‐strength Murashige and Skoog (MS) media were compared. The maximum number of somatic embryos differentiated from a proliferative nodular embryogenic callus (NEC) on either half‐ or full‐strength MS medium, and the greatest numbers of cotyledonary stage embryos were formed on full‐strength MS medium. Developed somatic embryos were then desiccated above a saturated K2SO4 solution for 10 d. After transfer to germination medium, embryos that had developed on half‐ and full‐strength MS medium yielded 8·3 and 8·6 germinants g–1 NEC tissue, respectively. For this important but often disregarded culture factor, either half‐ or full‐strength MS medium is recommended for both the differentiation and development of cassava somatic embryos that are capable of germination. PMID:12099540

Consequences of endogenous and exogenous WNT signaling for development of the preimplantation bovine embryo.

PubMed

Tribulo, Paula; Leão, Beatriz Caetano da Silva; Lehloenya, Khoboso C; Mingoti, Gisele Zoccal; Hansen, Peter J

2017-06-01

The specific role of WNT signaling during preimplantation development remains unclear. Here, we evaluated consequences of activation and inhibition of β-catenin (CTNNB1)-dependent and -independent WNT signaling in the bovine preimplantation embryo. Activation of CTNNB1-mediated WNT signaling by the agonist 2-amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine (AMBMP) and a glycogen synthase kinase 3 inhibitor reduced development to the blastocyst stage. Moreover, the antagonist of WNT signaling, dickkopf-related protein 1 (DKK1), alleviated the negative effect of AMBMP on development via reduction of CTNNB1. Based on labeling for phospho c-Jun N-terminal kinase, there was no evidence that DKK1 activated the planar cell polarity (PCP) pathway. Inhibition of secretion of endogenous WNTs did not affect development but increased number of cells in the inner cell mass (ICM). In contrast, DKK1 did not affect number of ICM or trophectoderm (TE) cells, suggesting that embryo-derived WNTs regulate ICM proliferation through a mechanism independent of CTNNB1. In addition, DKK1 did not affect the number of cells positive for the transcription factor yes-associated protein 1 (YAP1) involved in TE formation. In fact, DKK1 decreased YAP1. In contrast, exposure of embryos to WNT family member 7A (WNT7A) improved blastocyst development, inhibited the PCP pathway, and did not affect amounts of CTNNB1. Results indicate that embryo-derived WNTs are dispensable for blastocyst formation but participate in regulation of ICM proliferation, likely through a mechanism independent of CTNNB1. The response to AMBMP and WNT7A leads to the hypothesis that maternally derived WNTs can play a positive or negative role in regulation of preimplantation development. © The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction.

Embryo developmental events and the egg case of the Aleutian skate Bathyraja aleutica (Gilbert) and the Alaska skate Bathyraja parmifera (Bean).

PubMed

Hoff, G R

2009-02-01

Embryo development events were correlated with egg-case changes for the Aleutian skate Bathyraja aleutica and the Alaska skate Bathyraja parmifera. Yolk absorption underwent two phases: that of steady absorption during early development and that of rapid yolk absorption during the final development stages. Total length (L(T)) for 50% of the pre-hatching embryos egg-case jelly disappearance was 92.04 mm (range 81-102 mm) and 99.36 mm (range 81-100 mm) for B. aleutica and B. parmifera, respectively, allowing the inner chamber to open to seawater flow. The tail filament underwent three phases of growth: rapid elongation during early development (<100 mm embryo L(T)), stasis of tail filament length during the remainder of embryo development and rapid absorption soon after hatching. Complete tail filament development coincided with the disappearance of egg-case jelly. Clasper buds first developed at embryos >70 mm L(T) for both species and the sex ratio was 1:1 well before hatching. Egg cases that were devoid of an ova or developing embryo were c. 5.0 and 6.5% of the egg cases examined for B. aleutica and B. parmifera, respectively. Measurements showed that egg cases containing only egg jelly were smaller in both width and length than those possessing an ova. Embryo stages were punctuated with distinct events that correlated with egg case changes controlling the internal environment of the developing embryo.

Early morphological nuclear events and developmental capacity of embryos reconstructed with fetal fibroblasts at the M or G1 phase after intracytoplasmic nuclear injection in cattle.

PubMed

Ideta, Atsushi; Urakawa, Manami; Aoyagi, Yoshito; Saeki, Kazuhiro

2005-04-01

We examined morphological nuclear events during the first cell cycle of bovine embryos reconstructed with somatic cells at the M and G1 phases (M-embryos and G1-embryos, respectively) by intracytoplasmic nuclear injection, and the subsequent development of these embryos in vitro and in vivo. Bovine fetal fibroblasts (BFFs) at the M or G1 phase were directly injected into enucleated oocytes, and activated immediately. Only half (48%) of the M-embryos extruded polar body-like cells (PBCs) at 6 h post injection (hpi). At 15 to 19 hpi, 54% of the M-embryos formed a single pronucleus-like nucleus. Nuclear envelope-breakdown, premature chromosome condensation and single nuclear clusters were observed in most of the G1-embryos (88%) within 30 min following the nuclear injection. At 15 to 19 hpi, single pronucleus-like nuclei were formed in most G1-embryos (83%). The potential of G1-embryos to develop to blastocysts was significantly higher than that of M-embryos (31% vs 16%). Three of five recipients following transfer of blastocysts derived from the G1-embryos became pregnant on Day 30, and one recipient delivered a calf. Our results indicate that almost a half of the M-embryos failed to extrude PBCs and that the G1-embryos developed to blastocysts at a higher rate than the M-embryos.

Oxygen regulates amino acid turnover and carbohydrate uptake during the preimplantation period of mouse embryo development.

PubMed

Wale, Petra L; Gardner, David K

2012-07-01

Oxygen is a powerful regulator of preimplantation embryo development, affecting gene expression, the proteome, and energy metabolism. Even a transient exposure to atmospheric oxygen can have a negative impact on embryo development, which is greatest prior to compaction, and subsequent postcompaction culture at low oxygen cannot alleviate this damage. In spite of this evidence, the majority of human in vitro fertilization is still performed at atmospheric oxygen. One of the physiological parameters shown to be affected by the relative oxygen concentration, carbohydrate metabolism, is linked to the ability of the mammalian embryo to develop in culture and remain viable after transfer. The aim of this study was, therefore, to determine the effect of oxygen concentration on the ability of mouse embryos to utilize both amino acids and carbohydrates both before and after compaction. Metabolomic and fluorometric analysis of embryo culture media revealed that when embryos were exposed to atmospheric oxygen during the cleavage stages, they exhibited significantly greater amino acid utilization and pyruvate uptake than when cultured under 5% oxygen. In contrast, postcompaction embryos cultured in atmospheric oxygen showed significantly lower mean amino acid utilization and glucose uptake. These metabolic changes correlated with developmental compromise because embryos grown in atmospheric oxygen at all stages showed significantly lower blastocyst formation and proliferation. These findings confirm the need to consider both embryo development and metabolism in establishing optimal human embryo growth conditions and prognostic markers of viability, and further highlight the impact of oxygen on such vital parameters.

Forty years of embryo transfer in cattle: a review focusing on the journal Theriogenology, the growth of the industry in North America, and personal reminisces.

PubMed

Hasler, John F

2014-01-01

After the first successful transfer of mammalian embryos in 1890, it was approximately 60 years before significant progress was reported in the basic technology of embryo transfer (ET) in cattle. Starting in the early 1970s, technology had progressed sufficiently to support the founding of commercial ET programs in several countries. Today, well-established and reliable techniques involving superovulation, embryo recovery and transfer, cryopreservation, and IVF are utilized worldwide in hundreds, if not thousands, of commercial businesses located in many countries. The mean number of embryos produced via superovulation has changed little in 40 years, but there have been improvements in synchrony and hormonal protocols. Cryopreservation of in vivo-derived embryos is a reliable procedure, but improvements are needed for biopsied and in vitro-derived embryos. High pregnancy rates are achieved when good quality embryos are transferred into suitable recipients and low pregnancy rates are often owing to problems in recipient management and not technology per se. In the future, unanticipated disease outbreaks and the ever-changing economics of cattle and milk prices will continue to influence the ET industry. The issue of abnormal pregnancies involving in vitro embryos has not been satisfactorily resolved and the involvement of abnormal epigenetics associate with this technology merits continued research. Last, genomic testing of bovine embryos is likely to be available in the foreseeable future. This may markedly decrease the number of embryos that are actually transferred and stimulate the evolution of more sophisticated ET businesses. Copyright © 2014 Elsevier Inc. All rights reserved.

Tauroursodeoxycholic acid enhances the pre-implantation embryo development by reducing apoptosis in pigs.

PubMed

Kim, J-S; Song, B-S; Lee, K-S; Kim, D-H; Kim, S-U; Choo, Y-K; Chang, K-T; Koo, D-B

2012-10-01

Apoptosis is an important determinant of the normal development of pre-implantation embryos in vitro. Recently, endoplasmic reticulum (ER) stress-mediated apoptosis has been extensively investigated in a wide variety of diseases. Efficient functioning of the ER is essential for most cellular activities and survival. Tauroursodeoxycholic acid (TUDCA), an endogenous bile acid, has been reported to attenuate ER stress-mediated cell death by interrupting the classic pathways of apoptosis. Therefore, in this study, the anti-apoptotic effect of TUDCA on ER stress-induced apoptosis was examined in pre-implantation pig embryos. Also, tunicamycin was used to investigate the effects of ER stress on pig embryo development. After in vitro maturation and fertilization, presumptive pig embryos were cultured in NCSU-23 medium supplemented with TUDCA or TM for 6 days at 39 °C, 5% CO(2) in air. All data were analysed using one-way anova and Duncan's multiple range test in the statistical analysis system (SAS). In addition, we also determined the optimal TM and TUDCA concentrations. Samples were treated with TM at concentrations of 0, 1, 2 or 5 μm and with TUDCA at concentrations of 0, 100, 200 or 300 μm. When TM was used during in vitro culture, only 8.2% (8/97) of the embryos developed to the blastocyst stage when the treatment concentration was 1 μm compared with 27.4% (28/102) of the embryos in the control group (p < 0.05). In contrast, the frequency of blastocyst formation and the number of cells were higher when treated with 200 μm TUDCA compared with the control group (32.8% and 39.5 vs 22.2% and 35.6, p < 0.05). Moreover, the developmental rate to the blastocyst stage embryo in the group treated with TM and TUDCA was not significantly different from that of the control group (17.8%, 26/142 vs 24.9%, 36/145). Furthermore, the blastocyst cell number was enhanced (31.9 vs 36.9) and apoptosis reduced (TUNEL-positive nuclei number, 6.0 vs 3.2) by TUDCA treatment in pig embryos. In the real-time quantitative RT-PCR analysis, the expression of anti-apoptotic Bcl-XL gene was shown to be increased in the blastocyst stage because of TUDCA treatment, whereas expression of pro-apoptotic Bax was decreased. In addition, we also found that TUDCA decreased the rate of TM-induced apoptosis in the pre-implantation stage. Taken together, our results indicate that TUDCA improves the developmental competence of pig embryos by modulating ER stress-induced apoptosis during the pre-implantation stage. © 2011 Blackwell Verlag GmbH.

Protein Phosphorylation during Coconut Zygotic Embryo Development1

PubMed Central

Islas-Flores, Ignacio; Oropeza, Carlos; Hernández-Sotomayor, S.M. Teresa

1998-01-01

Evidence was obtained on the occurrence of protein threonine, serine, and tyrosine (Tyr) kinases in developing coconut (Cocos nucifera L.) zygotic embryos, based on in vitro phosphorylation of proteins in the presence of [γ-32P]ATP, alkaline treatment, and thin-layer chromatography analysis, which showed the presence of [32P]phosphoserine, [32P]phosphothreonine, and [32P]phosphotyrosine in [32P]-labeled protein hydrolyzates. Tyr kinase activity was further confirmed in extracts of embryos at different stages of development using antiphosphotyrosine monoclonal antibodies and the synthetic peptide derived from the amino acid sequence surrounding the phosphorylation site in pp60src (RR-SRC), which is specific for Tyr kinases. Anti-phosphotyrosine western blotting revealed a changing profile of Tyr-phosphorylated proteins during embryo development. Tyr kinase activity, as assayed using RR-SRC, also changed during embryo development, showing two peaks of activity, one during early and another during late embryo development. In addition, the use of genistein, a Tyr kinase inhibitor, diminished the ability of extracts to phosphorylate RR-SRC. Results presented here show the occurrence of threonine, serine, and Tyr kinases in developing coconut zygotic embryos, and suggest that protein phosphorylation, and the possible inference of Tyr phosphorylation in particular, may play a role in the coordination of the development of embryos in this species. PMID:9733545

Reduction of polyspermic penetration using biomimetic microfluidic technology during in vitro fertilization.

PubMed

Clark, Sherrie G; Haubert, Kathyrn; Beebe, David J; Ferguson, C Edward; Wheeler, Matthew B

2005-11-01

Efforts to improve the in vitro embryo production process in pigs have included modifying culture medium and number of spermatozoa inseminated in order to reduce the incidence of polyspermy. Polyspermy is a pathological condition which results in aberrant embryonic development. The microchannels are designed to more closely mimic the function of the oviduct and create a flow pattern of spermatozoa past the oocytes similar to the pattern in the oviduct. In vitro fertilization of porcine oocytes in the microchannels has produced a higher incidence of monospermic penetration (p<0.05) as compared to the oocytes fertilized in the traditional microdrop system with comparable penetration and male pronucleus formation rates. Additionally, cleavage rates of the embryos as well as development to the blastocyst stage are similar. Here we demonstrate that the biomimetic microchannel in vitro fertilization system can reduce polyspermy and, therefore, increase the number of potentially viable embryos without reducing the overall in vitro production efficiency.

Dietary propylene glycol and in vitro embryo production after ovum pick-up in heifers with different anti-Müllerian hormone profiles.

PubMed

Gamarra, G; Ponsart, C; Lacaze, S; Le Guienne, B; Humblot, P; Deloche, M-C; Monniaux, D; Ponter, A A

2015-11-01

Rapid genetic improvement in cattle requires the production of high numbers of embryos of excellent quality. Increasing circulating insulin and/or glucose concentrations improves ovarian follicular growth, which may improve the response to superovulation. The measurement of anti-Müllerian hormone (AMH) can help predict an animal's response to superovulation treatment. The aim of the present study was to investigate whether increasing circulating insulin concentrations, through propylene glycol (PG) drenches, could improve in vitro embryo production in oestrus-synchronised superovulated heifers with different AMH profiles. Holstein heifers were grouped according to pre-experimental AMH concentrations as low (L) or high (H). The PG drench increased circulating insulin and glucose concentrations and reduced β-hydroxybutyrate and urea concentrations compared with the control group. AMH was a good predictor of follicle and oocyte numbers at ovum pick-up (OPU), and of oocyte and embryo quality (AMH H>AMH L). PG in the AMH H group increased the number of follicles and blastocyst quality above that in the control group, but did not improve these parameters in the AMH L group. These results indicate that short-term oral PG supplementation modifies an animal's metabolic milieu and is effective in improving in vitro embryo production, after superovulation-OPU, more markedly in heifers with high rather than low AMH concentrations.

Selection of Norway spruce somatic embryos by computer vision

NASA Astrophysics Data System (ADS)

Hamalainen, Jari J.; Jokinen, Kari J.

1993-05-01

A computer vision system was developed for the classification of plant somatic embryos. The embryos are in a Petri dish that is transferred with constant speed and they are recognized as they pass a line scan camera. A classification algorithm needs to be installed for every plant species. This paper describes an algorithm for the recognition of Norway spruce (Picea abies) embryos. A short review of conifer micropropagation by somatic embryogenesis is also given. The recognition algorithm is based on features calculated from the boundary of the object. Only part of the boundary corresponding to the developing cotyledons (2 - 15) and the straight sides of the embryo are used for recognition. An index of the length of the cotyledons describes the developmental stage of the embryo. The testing set for classifier performance consisted of 118 embryos and 478 nonembryos. With the classification tolerances chosen 69% of the objects classified as embryos by a human classifier were selected and 31$% rejected. Less than 1% of the nonembryos were classified as embryos. The basic features developed can probably be easily adapted for the recognition of other conifer somatic embryos.

Polyamines and their biosynthetic enzymes during somatic embryo development in red spruce (Picea rubens Sarg.)

Treesearch

Rakesh Minocha; Subhash C. Minocha; Stephanie Long

2004-01-01

The major objective of this study was to determine if the observed changes in polyamines and their biosynthetic enzymes during somatic embryo development were specifically related to either the stage of the embryo development or to the duration of time spent on the maturation medium. Somatic embryos of red spruce (Picea rubens) at different...

Criteria to assess human oocyte quality after cryopreservation.

PubMed

Coticchio, G; Bonu, M A; Bianchi, V; Flamigni, C; Borini, A

2005-10-01

Oocyte cryopreservation certainly represents one of the most attractive developments in the field of assisted reproduction, with the aim of preserving female fertility and circumventing the ethical and legal drawbacks associated with embryo freezing. Despite the achievement of the first pregnancy from frozen oocytes dating back as early as 1987, since then fewer than 150 pregnancies have been reported. Over a long period of time, application of oocyte storage on a large scale has been prevented by various factors, namely poor post-thaw survival. Fertilization rates remained low even after the introduction of intracytoplasmic sperm injection. Modifications of slow-freezing protocols, mainly based on the increase of the concentration of sucrose used as non-penetrating cryoprotectant (CPA) and the replacement of sodium with choline, appear to have decisively improved survival rates to over 80%. Investigations at the cellular level on thawed oocytes are largely lacking. Fertilization rates have also benefited from protocol modifications, reaching values indistinguishable from those normally obtained with fresh material. Vitrification protocols have also been tested, giving rise to improvements whose reproducibility is still uncertain. Data on the dynamics of fertilization and preimplantation development of embryos derived from frozen oocytes are extremely scarce. At the moment, clinical efficiency of oocyte cryopreservation cannot be precisely assessed because of the lack of controlled studies, although it appears to be considerably lower than that achieved with embryo freezing. In summary, encouraging advances have been made in the field of oocyte cryopreservation, but presently no protocol can ensure standards of success and safety comparable to those guaranteed by embryo storage.

Acquisition of oocyte competence to develop as an embryo: integrated nuclear and cytoplasmic events.

PubMed

Conti, Marco; Franciosi, Federica

2018-05-01

Infertility affects ~7% of couples of reproductive age with little change in incidence in the last two decades. ART, as well as other interventions, have made major strides in correcting this condition. However, and in spite of advancements in the field, the age of the female partner remains a main factor for a successful outcome. A better understanding of the final stages of gamete maturation yielding an egg that can sustain embryo development and a pregnancy to term remains a major area for improvement in the field. This review will summarize the major cellular and molecular events unfolding at the oocyte-to-embryo transition. We will provide an update on the most important processes/pathways currently understood as the basis of developmental competence, including the molecular processes involved in mRNA storage, its recruitment to the translational machinery, and its degradation. We will discuss the hypothesis that the translational programme of maternal mRNAs plays a key role in establishing developmental competence. These regulations are essential to assemble the machinery that is used to establish a totipotent zygote. This hypothesis further supports the view that embryogenesis begins during oogenesis. A better understanding of the events required for developmental competence will guide the development of novel strategies to monitor and improve the success rate of IVF. Using this information, it will be possible to develop new biomarkers that may be used to better predict oocyte quality and in selection of the best egg for IVF.

Development of whole and demi-embryos of mice in culture and in vivo after supercooled storage.

PubMed

Fuku, E; Fiser, P S; Marcus, G J; Sasada, H; Downey, B R

1993-12-01

Demi-embryos (produced by destroying 1 or 2 blastomeres of 2- or 4-cell embryos, respectively) and intact mouse embryos were cultured to the blastocyst stage, stored at -5 degrees C for 48 h, then cultured for 24 h and transferred into pseudopregnant recipients. Supercooled storage did not impair the developmental potential of whole or demi-embryos in vitro, nor was there a difference between whole and demi-embryos with respect to growth in vitro. Similarly, there was no effect of supercooling on development of intact or demi embryos after transfer into pseudopregnant recipient mice, but fewer recipients of demi-embryos remained pregnant (P < 0.05). This was considered to be partly due to the lesser ability of demi-embryos to maintain luteal function and establish pregnancy.

Population trends and live birth rates associated with common ART treatment strategies.

PubMed

Chambers, Georgina M; Wand, Handan; Macaldowie, Alan; Chapman, Michael G; Farquhar, Cynthia M; Bowman, Mark; Molloy, David; Ledger, William

2016-11-01

Have ART live birth rates improved in Australia over the last 12 years? There were striking improvements in per-cycle live birth rates observed for frozen/thaw embryo transfers, blastocyst transfer and single embryo transfer (SET), while live birth rates following ICSI were lower than IVF for non-male factor infertility in most years. ART and associated techniques have become the predominant treatment of infertility over the past 30 years in most developed countries. However, there are differences in ART laboratory and clinical practices, and success rates worldwide. Australia has one of the highest ART utilization rates and lowest multiple birth rates in the world, thus providing a unique setting to investigate the contribution of common ART strategies in an unrestricted population of patients to ART success rates. A retrospective cohort study of 585 065 ART treatment cycles performed in Australia between 2002 and 2013 using the Australian and New Zealand Assisted Reproduction Database (ANZARD). An unrestricted population of all women who underwent autologous ART treatment between 2002 and 2013. Visual descriptive analysis was used to assess the trends in ART procedures by the calendar years. Adjusted odds ratios (aORs) of a live birth for four common ART techniques were calculated after controlling for important confounders including female age, infertility diagnosis, stage of the embryo (blastocyst versus cleavage stage), type of embryo (fresh versus thawed), fertilization method (IVF versus ICSI) and number of embryos transferred (SET versus multiple embryos). The overall live birth rate per embryo transfer increased from 19.2% in 2002 to 23.3% in 2013 (21.9-24.3% for fresh embryo transfers and 14.6-23.3% for frozen/thaw embryo transfers). This occurred concurrently with an increase in SET from 29.7% to 78.9%, and an increase in the average age of women undergoing treatment from 35.0 to 35.9 years. Individuals who had a frozen/thaw embryo transfer cycle in 2002 had 43% (aOR: 0.57, 95% CI: 0.53-0.61) reduced odds of a live birth compared with a fresh embryo transfer cycle. This contrasted with 16% (aOR: 0.84, 95% CI: 0.80-0.98) reduced odds of a live birth from frozen/thaw embryo transfer cycles in 2013. In 2013, the odds of blastocyst transfer resulting in a live birth were more than twice as great as for cleavage stage transfer (aOR 2.01, 95% CI: 1.92-2.11). The adjusted odds of live birth per SET compared with multiple embryo transfer increased significantly over the last 12 years, from a 38% reduced odds of a live birth follow SET in 2002 (aOR: 062, 95% CI: 0.57-0.67) compared to an 8% reduced odds in 2013 (aOR: 0.92, 95% CI: 0.87-0.98). The aOR of a live birth using ICSI compared to IVF in non-male factor patients was lower in most years bringing into question its widespread use. This is a retrospective cohort analysis and cannot confirm causality. High-level evidence on the effectiveness of particular ART techniques, particularly ICSI and blastocyst culture, requires prospective randomized controlled trials or detailed statistical analysis using large-scale data that counts for fertilization failure, embryo loss, prognostic factors and cycle characteristics. The most striking improvements in ART success rates in Australia have been observed for frozen/thaw embryo transfers, blastocyst transfer and SET. Further studies of the role of ICSI in non-male factor infertility and blastocyst transfer success rates that take into account embryo loss are needed. No funding was received to undertake this study. The authors declare that they do not have competing interests with this study. NA. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

An approach to successful freezing of demi-embryos derived from day-7 bovine embryos.

PubMed

Niemann, H; Brem, G; Sacher, B; Smidt, D; Kräusslich, H

1986-04-01

The developmental capacity of frozen/thawed bisected embryos (n = 33) derived from day-7 bovine embryos was investigated and compared to ordinary embryos after freezing and thawing (n = 28) and to freshly bisected embryos (n = 19). The freezing and thawing protocol was identical for ordinary and demi-embryos. The percentage of intact embryos classified as excellent, good, or poor after thawing was 92.9 and 96.3% for ordinary and demi-embryos, respectively. Pregnancy rates of 53.8 (8 15 ), 46.2 (6 13 ), and 47.5% (9 19 ) were obtained when frozen/thawed ordinary embryos and frozen/thawed demi-embryos classified as excellent or good and sealed with an additional zona pellucida from hatched pig blastocysts or freshly bisected embryos were transferred. One pair of identical twins resulted from the transfer of frozen/thawed demi-embryos sealed with an additional zona pellucida. Transfer of four frozen/thawed demi-embryos without an additional zona pellucida led to one pregnancy. In contrast, demi-embryos derived from frozen/thawed ordinary embryos (n = 8) as well as frozen/thawed demi-embryos classified as poor (n = 6) did not result in any pregnancies although two halves were transferred per recipient. It is concluded that sealing the punctured zona pellucida improves the developmental capacity of frozen/thawed demi-embryos derived from day-7 bovine embryos, and freezing demi-embryos is more efficient compared to the splitting of frozen/thawed ordinary embryos.

Unaltered timing of embryo development in women with polycystic ovarian syndrome (PCOS): a time-lapse study.

PubMed

Sundvall, Linda; Kirkegaard, Kirstine; Ingerslev, Hans Jakob; Knudsen, Ulla Breth

2015-07-01

Polycystic ovarian syndrome (PCOS) is a common cause of female infertility. Factors other than anovulation, such as low embryo quality have been suggested to contribute to the infertility in these women. This 2-year retrospective study used timelapse technology to investigate the PCOS-influence on timing of development in the pre-implantation embryo (primary endpoint). The secondary outcome measure was live birth rates after elective single-embryo transfer. In total, 313 embryos from 43 PCOS women, and 1075 embryos from 174 non-PCOS women undergoing assisted reproduction were included. All embryos were monitored until day 6. Differences in embryo kinetics were tested in a covariance regression model to account for potential confounding variables: female age, BMI, fertilization method and male infertility. Time to initiate compaction and reach the morula stage as well as the duration of the 4th cleavage division was significantly shorter in PCOS embryos compared with non-PCOS embryos. No other kinetic differences were found at any time-points annotated. The proportion of multi-nucleated cells at the 2-cell stage was significantly higher in PCOS embryos compared with non-PCOS embryos. The live birth rates were comparable between the two groups. The findings suggest that the causative factor for subfertility in PCOS is not related to timing of development in the pre-implantation embryo.

Effect of Short-Term Hypergravity Treatment on Mouse 2-Cell Embryo Development

NASA Astrophysics Data System (ADS)

Ning, Li-Na; Lei, Xiao-Hua; Cao, Yu-Jing; Zhang, Yun-Fang; Cao, Zhong-Hong; Chen, Qi; Duan, En-Kui

2015-11-01

Though there are numerous biological experiments, which have been performed in a space environment, to study the physiological effect of space travel on living organisms, while the potential effect of weightlessness or short-term hypergravity on the reproductive system in most species, particularly in mammalian is still controversial and unclear. In our previous study, we investigated the effect of space microgravity on the development of mouse 4-cell embryos by using Chinese SJ-8. .Unexpectedly, we did not get any developed embryo during the space-flight. Considering that the process of space experiment is quite different from most experiments done on earth in several aspects such as, the vibration and short-term hypergravity during the rock launching and landing. Thus we want to know whether the short-term hypergravity produced by the launch process affect the early embryo development in mice, and howthe early embryos respond to the hypergravity. In present study, we are mimicking the short-term hypergravity during launch by using a centrifuge to investigate its influence on the development of early embryo (2-cell) in mice. We also examined the actin filament distribution in 2-cell embryos by immunostaining to test their potential capacity of development under short-term hypergravity exposure. Our results showed that most 2-cell embryos in the hypergravity exposure groups developed into blastocysts with normal morphology after 72h cultured in vitro, and there is no obvious difference in the development rate of blastocyst formation compared to the control. Moreover, there were no statistically significant differences in birth rates after oviduct transfer of 2-cell mouse embryos exposed on short-term hypergravity compared with 1 g condition. In addition, the well-organized actin distribution appeared in 2-cell embryos after exposed on hypergravity and also in the subsequent developmental blastocysts. Taken together, our data shows that short-term exposure in hypergravity conditions does not affect the normal development and actin filament structures of mouse embryos.

Reversible neuronal and muscular toxicity of caffeine in developing vertebrates.

PubMed

Rodriguez, Rufino S; Haugen, Rebecca; Rueber, Alexandra; Huang, Cheng-Chen

2014-06-01

This study utilizes zebrafish embryos to understand the cellular and molecular mechanisms of caffeine toxicity in developing vertebrate embryos. By using a high concentration of caffeine, we observed almost all the phenotypes that have been described in humans and/or in other animal models, including neural tube closure defect, jittery, touch insensitivity, and growth retardation as well as a drastic coiled body phenotype. Zebrafish embryos exposed to 5mM caffeine exhibited high frequent movement, 10 moves/min comparing with around 3 moves/min in control embryos, within half an hour post exposure (HPE). They later showed twitching, uncoordinated movement, and eventually severe body curvature by 6HPE. Exposure at later stages resulted in the same phenotypes but more posteriorly. Surprisingly, when caffeine was removed before 6HPE, the embryos were capable of recovering but still exhibited mild curvature and shorter bodies. Longer exposure caused irreversible body curvature and lethality. These results suggest that caffeine likely targets the neuro-muscular physiology in developing embryos. Immunohistochemistry revealed that the motorneurons in treated embryos developed shorter axons, abnormal branching, and excessive synaptic vesicles. Developing skeletal muscles also appeared smaller and lacked the well-defined boundaries seen in control embryos. Finally, caffeine increases the expression of genes involved in synaptic vesicle migration. In summary, our results provide molecular understanding of caffeine toxicity on developing vertebrate embryos. Published by Elsevier Inc.

Birth of piglets from in vitro-produced, zona-intact porcine embryos vitrified in a closed system

PubMed Central

Men, Hongsheng; Zhao, Chongbei; Wei, Si; Murphy, Clifton N.; Spate, Lee; Liu, Yang; Walters, Eric M.; Samuel, Melissa S.; Prather, Randall S.; Critser, John K.

2011-01-01

As the importance of swine models in biomedical research increases, it is essential to develop low-cost, high-throughput systems to cryopreserve swine germplasm for maintenance of these models. However, porcine embryos are exceedingly sensitive to low temperature and successful cryopreservation is generally limited to the use of vitrification in open systems that allow direct contact of the embryos with liquid nitrogen (LN2). This creates a high risk of pathogen transmission. Therefore, cryopreservation of porcine embryos in a “closed” system is of very high importance. In this study, in vitro-produced (IVP) porcine embryos were used to investigate cryosurvival and developmental potential of embryos cryopreserved in a closed system. Optimal centrifugal forces to completely disassociate intracellular lipids from blastomeres were investigated using Day-4 embryos. Cryosurvival of delipidated embryos was investigated by vitrifying the embryos immediately after centrifugation, or after development to blastocysts. In this study, centrifugation for 30 min at 13,000 g was adequate to completely delipidate the embryos; furthermore, these embryos were able to survive cryopreservation at a rate comparable to those centrifuged for only 12 min. When delipidated embryos were vitrified at the blastocyst stage, there was no difference in survival between embryos vitrified using OPS and 0.25 mL straws. Some embryos vitrified by each method developed to term. These experiments demonstrated that porcine embryos can be cryopreserved in a closed system after externalizing their intracellular lipids. This has important implications for banking swine models of human health and disease. PMID:21458047

Choosing GnRH Antagonist Protocol Shows Improved Oocyte and Embryo Quality, Coherent with the Perifollicular Vascularity (PFV) in Assisted Reproductive Techniques

PubMed Central

Ramachandran, Amar; Kumar, Pratap

2015-01-01

Introduction The parent oocyte from which the embryo is derived, determines its quality and the perifollicular vascularity (PFV) determines the micro-environment of the developing ovum. The PFV correlates well with the follicular oxygenation, oocyte maturation and embryo viability. PFV is imaged with Power Doppler Ultrasound. Aim To study and compare the association of the PFV of follicles with the quality of the oocytes and embryos in agonist and antagonist protocol, employed in Assisted Reproductive techniques (ART). Study Design A prospective observational study was conducted on 75 patients, who were recruited for ART cycles, out of which 25 were given the Agonist protocol and the remaining 50 received the Antagonist protocol. Materials and Methods The patients underwent the stimulation protocol. The PFV of preovulatory follicles were studied with Transvaginal Power Doppler and graded. Each oocyte retrieved carried the same label of its parent follicle. Embryos were cultured. The embryologist was blinded. The oocyte and embryo quality were assessed and compared with the PFV of parent follicle. Results Follicles with grade 1 and 2 PFV were predominantly observed. The yield of oocytes was independent of PFV. The mean yield of good quality embryos in conjunction with the PFV of the parent follicle was found to be highly significant in both the groups. The antagonist group had statistically significant yield of mature oocytes and embryos, compared to the agonist group. Conclusion Antagonist protocol had favourable outcomes compared with the agonist protocol. The retrieval of oocytes, even the mature ones and the yield of high grade embryos were found higher. As the PFV increased, the yield and overall pregnancy rates were higher. PFV as assessed by Power Doppler is a useful non-invasive biomarker of embryo quality and can be employed in conjunction with other biomarkers in ART to predict successful outcome. PMID:26674932

Pro-NP™ protect against TiO2 nanoparticle-induced phototoxicity in zebrafish model: exploring potential application for skin care.

PubMed

Kim, Min-Sik; Stees, Melinda; Karuturi, Bala Vamsi K; Vijayaraghavalu, Sivakumar; Peterson, Richard E; Madsen, Gary L; Labhasetwar, Vinod

2017-06-01

Titanium dioxide nanoparticles (TiO 2 NPs) are used in sunscreen products to protect the skin from the sun's ultraviolet rays. However, following exposure to sunlight, the photocatalytic activity of TiO 2 NPs can produce an excess of reactive oxygen species (ROS), causing skin cell damage, triggering an inflammatory response. In zebrafish model, we evaluated how well Pro-NP™ (biodegradable NPs containing superoxide dismutase and catalase) could protect them from TiO 2 NP-induced photo-oxidative stress. We hypothesized that the antioxidant properties of Pro-NP™ would protect zebrafish embryos from the phototoxic effects of TiO 2 NPs, improving overall survival and growth. Dechorionated embryos were treated with TiO 2 NPs alone or co-treated with Pro-NP™, and then exposed to simulated sunlight. Pro-NP™ by itself caused no toxicity; however, for embryos exposed to 100 μg/ml TiO 2 NPs, zebrafish survival was reduced to ∼40% and at 500 μg/ml to ∼10%. In contrast, at 100 μg/ml TiO 2 NP, co-treatment with Pro-NP™ increased zebrafish survival in a dose-dependent manner. Co-treatment also improved percent of embryos hatching and resulted in normal growth of zebrafish. On the other hand, embryos treated with TiO 2 NPs alone developed deformities, had reduced pigmentation, and showed severely truncated growth. Pro-NP™ afforded a greater level of protection against TiO 2 NP-induced phototoxicity than other antioxidants (vitamin E or N-acetylcysteine) commonly used in topical skin care formulations. We conclude that Pro-NP™ exert significant protective effects against TiO 2 NP-induced phototoxicity and could be developed as a safe, effective skin care product, used alone or in combination with sunscreen products to protect the skin from sun's UV radiation.

Mammalian pre-implantation chromosomal instability: species comparison, evolutionary considerations, and pathological correlations.

PubMed

Carbone, Lucia; Chavez, Shawn L

2015-01-01

Pre-implantation embryo development in mammals begins at fertilization with the migration and fusion of the maternal and paternal pro-nuclei, followed by the degradation of inherited factors involved in germ cell specification and the activation of embryonic genes required for subsequent cell divisions, compaction, and blastulation. The majority of studies on early embryogenesis have been conducted in the mouse or non-mammalian species, often requiring extrapolation of the findings to human development. Given both conserved similarities and species-specific differences, however, even comparison between closely related mammalian species may be challenging as certain aspects, including susceptibility to chromosomal aberrations, varies considerably across mammals. Moreover, most human embryo studies are limited to patient samples obtained from in vitro fertilization (IVF) clinics and donated for research, which are generally of poorer quality and produced with germ cells that may be sub-optimal. Recent technical advances in genetic, epigenetic, chromosomal, and time-lapse imaging analyses of high quality whole human embryos have greatly improved our understanding of early human embryogenesis, particularly at the single embryo and cell level. This review summarizes the major characteristics of mammalian pre-implantation development from a chromosomal perspective, in addition to discussing the technological achievements that have recently been developed to obtain this data. We also discuss potential translation to clinical applications in reproductive medicine and conclude by examining the broader implications of these findings for the evolution of mammalian species and cancer pathology in somatic cells.

Metabolic and mitochondrial dysfunction in early mouse embryos following maternal dietary protein intervention.

PubMed

Mitchell, Megan; Schulz, Samantha L; Armstrong, David T; Lane, Michelle

2009-04-01

Dietary supply of nutrients, both periconception and during pregnancy, influence the growth and development of the fetus and offspring and their health into adult life. Despite the importance of research efforts surrounding the developmental origins of health and disease hypothesis, the biological mechanisms involved remain elusive. Mitochondria are of major importance in the oocyte and early embryo, particularly as a source of ATP generation, and perturbations in their function have been related to reduced embryo quality. The present study examined embryo development following periconception exposure of females to a high-protein diet (HPD) or a low-protein diet (LPD) relative to a medium-protein diet (MPD; control), and we hypothesized that perturbed mitochondrial metabolism in the mouse embryo may be responsible for the impaired embryo and fetal development reported by others. Although the rate of development to the blastocyst stage did not differ between diets, both the HPD and LPD reduced the number of inner cell mass cells in the blastocyst-stage embryo. Furthermore, mitochondrial membrane potential was reduced and mitochondrial calcium levels increased in the 2-cell embryo. Embryos from HPD females had elevated levels of reactive oxygen species and ADP concentrations, indicative of metabolic stress and, potentially, the uncoupling of oxidative phosphorylation, whereas embryos from LPD females had reduced mitochondrial clustering around the nucleus, suggestive of an overall quietening of metabolism. Thus, although periconception dietary supply of different levels of protein is permissive of development, mitochondrial metabolism is altered in the early embryo, and the nature of the perturbation differs between HPD and LPD exposure.

Application of Somatic Embryogenesis in Woody Plants.

PubMed Central

Guan, Yuan; Li, Shui-Gen; Fan, Xiao-Fen; Su, Zhen-Hong

2016-01-01

Somatic embryogenesis is a developmental process where a plant somatic cell can dedifferentiate to a totipotent embryonic stem cell that has the ability to give rise to an embryo under appropriate conditions. This new embryo can further develop into a whole plant. In woody plants, somatic embryogenesis plays a critical role in clonal propagation and is a powerful tool for synthetic seed production, germplasm conservation, and cryopreservation. A key step in somatic embryogenesis is the transition of cell fate from a somatic cell to embryo cell. Although somatic embryogenesis has already been widely used in a number of woody species, propagating adult woody plants remains difficult. In this review, we focus on molecular mechanisms of somatic embryogenesis and its practical applications in economic woody plants. Furthermore, we propose a strategy to improve the process of somatic embryogenesis using molecular means. PMID:27446166

Effect of MEM vitamins and forskolin on embryo development and vitrification tolerance of in vitro-produced pig embryos.

PubMed

Cuello, C; Gomis, J; Almiñana, C; Maside, C; Sanchez-Osorio, J; Gil, M A; Sánchez, A; Parrilla, I; Vazquez, J M; Roca, J; Martinez, E A

2013-01-30

The aims of this study were (1) to determine the effect of in vitro maturation (IVM) medium supplementation with MEM vitamins on in vitro embryo development and sensitivity to vitrification of Day 6 blastocysts and (2) to evaluate whether the addition of forskolin to in vitro culture (IVC) medium enhances blastocyst survival following Super Open Pulled Straw (SOPS) vitrification. Cumulus-oocyte complexes (COCs; n=4000) were matured with 0.0% or 0.05% (v/v) MEM vitamins. After 44h of IVM, the oocytes were in vitro fertilized, and presumptive zygotes were cultured. At Day 5 of IVC, embryos from both experimental groups were cultured for 24h with 0 or 10μM forskolin, achieving a 2×2 factorial design. The blastocyst formation rate was assessed on Day 6, and subsets of samples from the four experimental groups were vitrified (n=469) or kept fresh (n=546). Fresh and vitrified-warmed blastocysts were cultured for 24h prior to embryo survival and total blastocyst cell number assessment. The MEM vitamins increased (P<0.001) the blastocyst formation rate at Day 6, but they did not affect embryo survival after vitrification. In contrast, the addition of forskolin to the culture medium enhanced (P<0.05) the blastocyst vitrification tolerance. The total blastocyst cell number was similar among the groups. In conclusion, supplementation with 0.05% MEM vitamins improved the blastocyst formation rate, and the addition of 10μM forskolin to the culture medium increased survival in Day 6 in vitro-produced blastocysts after SOPS vitrification. Copyright © 2012 Elsevier B.V. All rights reserved.

O-polysaccharide is important for Salmonella Pullorum survival in egg albumen, and virulence and colonization in chicken embryos.

PubMed

Guo, Rongxian; Li, Zhuoyang; Jiao, Yang; Geng, Shizhong; Pan, Zhiming; Chen, Xiang; Li, Qiuchun; Jiao, Xinan

2017-10-01

The pathogen Salmonella Pullorum is the causative agent of persistent systemic infection of poultry, leading to economic losses in developing countries due to morbidity, mortality and reduction in egg production. These infections may result in vertical transmission to eggs or progeny. Limited information is available regarding the mechanisms involved in the survival of Salmonella Pullorum in egg albumen and developing chicken embryos. Hence, we investigated the role of O-polysaccharide in the contamination of eggs and the colonization of chicken embryos. Compared with the wild-type strain, the isogenic waaL mutant exhibited an O-antigen-deficient rough phenotype, and increased sensitivity to egg albumen and chicken serum, as well as reduced adherence to DF-1 cells. Infection with Salmonella Pullorum lacking O-polysaccharide resulted in significantly reduced embryo lethality and bacterial colonization. These results suggest that O-polysaccharide is essential for Salmonella Pullorum colonization in eggs, both post-lay and developing embryos. The chicken embryo infection model could be used to characterize the interaction between Salmonella Pullorum and developing embryos, and it will also contribute to the development of more rational vaccines to protect laying hens and embryos.

Crocetin improves the quality of in vitro-produced bovine embryos: Implications for blastocyst development, cryotolerance, and apoptosis.

PubMed

Zullo, G; De Canditiis, C; Pero, M E; Albero, G; Salzano, A; Neglia, G; Campanile, G; Gasparrini, B

2016-11-01

The aim of this work was to assess the effect of supplementation of bovine culture medium with the natural antioxidant crocetin on in vitro blastocyst development and quality. This was evaluated as cryotolerance, apoptosis index, and total cells number and allocation. Abattoir-derived oocytes were matured and fertilized in vitro according to standard procedure. Twenty hours after IVF, presumptive zygotes were cultured in synthetic oviduct fluid medium, supplemented with 0, 1, 2.5, and 5 μM crocetin (experiment 1) at 39 °C under humidified air with 5% CO2, 7% O2, and 88% N2. On Day 7, embryo yields were assessed and the blastocysts were vitrified by Cryotop method in 16.5% ethylene glycol, 16.5% DMSO, and 0.5 M sucrose. Finally, blastocysts produced on Day 8 in the absence (control) and presence of 1 μM crocetin were used for terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling and differential staining to evaluate, respectively, the apoptotic rate and the allocation of cells into inner cell mass (ICM) and trophectoderm (TE) lineages (experiment 2). Embryo development was higher in the 1 μM crocetin group compared to the control, both in terms of total embryo output (37.7 ± 4.2%, 52.9 ± 6.3%, 40.9 ± 7.6%, and 42.4 ± 8.7%, respectively, with 0, 1, 2.5, and 5 μM; P < 0.01) and grade 1 and 2 blastocysts (33.6 ± 4.9%, 46.1 ± 7.3%, 37.8 ± 7.9%, and 39.4 ± 7.9%, respectively, with 0, 1, 2.5, and 5 μM; P < 0.05). Moreover, the percentage of fast-developing embryos increased in 1 μM crocetin group compared to the control (23.4 ± 4.7%, 32.7 ± 6.6%, 27.2 ± 6.6%, and 30.1 ± 7.2%, respectively, with 0, 1, 2.5, and 5 μM; P < 0.05). In addition, the enrichment of culture medium with 1 μM crocetin improved embryo cryotolerance compared to the control, as indicated by higher hatching rates recorded after 48 hours postwarming culture (46.5% vs. 60.4%; P < 0.05). Furthermore, 1 μM crocetin decreased both the average number (9.9 ± 0.4 vs. 7.1 ± 0.3) and the percentage of apoptotic cells (7.1 ± 0.4 vs. 4.2 ± 0.2) in blastocysts compared to the control (P < 0.01). However, no differences were recorded in the average number of ICM, TE, and total cells between 1 μM crocetin and control groups. In conclusion, the enrichment of bovine culture medium with 1 μM crocetin increased both blastocyst yield and quality, as indicated by the improved chronology of embryo development, increased resistance to cryopreservation, and reduced incidence of apoptosis. Copyright © 2016 Elsevier Inc. All rights reserved.

Forskolin improves the cryosurvival of in vivo-derived porcine embryos at very early stages using two vitrification methods.

PubMed

Gomis, J; Cuello, C; Sanchez-Osorio, J; Gil, M A; Parrilla, I; Angel, M A; Vazquez, J M; Roca, J; Martinez, E A

2013-04-01

This study was aimed to determine the effect of forskolin on the viability of in vivo-derived porcine embryos vitrified by the superfine open pulled straw (SOPS) or solid surface vitrification (SSV) methods at the 2-cell, 4-cell, and blastocyst stages. Zygotes, 2- to 4-cell embryos, and morulae were obtained from superovulated sows. After collection, embryos were cultured for 24h with 0 or 10 μM forskolin and then vitrified using the SOPS and SSV method, or not vitrified (fresh controls). Fresh and vitrified-warmed 2-cells, 4-cells, and blastocysts were cultured for additional 96 h, 72 h and 24 h, respectively. At the end of the culture, embryos were evaluated for progression to the blastocyst stage and total cell number. The vitrification method did not affect any of the parameters evaluated for any embryo stage. Forskolin increased (P<0.01) the blastocyst formation and the final developmental stage of vitrified 2- and 4-cell embryos. However, these embryos exhibited lower (P<0.003) blastocyst formation rates than their fresh counterparts. The total cell number and hatching rate were similar in both groups (vitrified and fresh) of 2- and 4-cell embryos. Vitrified blastocysts exhibited viabilities, final developmental stages, hatching rates, and total cell numbers that were similar to those of their fresh counterparts, regardless of the addition of forskolin. In conclusion, the SOPS and SSV methods are suitable for the cryopreservation of in vivo-derived 2- to 4-cell porcine embryos. Pre-treatment with forskolin for 24h before vitrification improves the cryotolerance of 2- and 4-cell porcine embryos. Copyright © 2013 Elsevier Inc. All rights reserved.

Preimplantation death of xenomitochondrial mouse embryo harbouring bovine mitochondria

PubMed Central

Kawahara, Manabu; Koyama, Shiori; Iimura, Satomi; Yamazaki, Wataru; Tanaka, Aiko; Kohri, Nanami; Sasaki, Keisuke; Takahashi, Masashi

2015-01-01

Mitochondria, cellular organelles playing essential roles in eukaryotic cell metabolism, are thought to have evolved from bacteria. The organization of mtDNA is remarkably uniform across species, reflecting its vital and conserved role in oxidative phosphorylation (OXPHOS). Our objectives were to evaluate the compatibility of xenogeneic mitochondria in the development of preimplantation embryos in mammals. Mouse embryos harbouring bovine mitochondria (mtB-M embryos) were prepared by the cell-fusion technique employing the haemagglutinating virus of Japan (HVJ). The mtB-M embryos showed developmental delay at embryonic days (E) 3.5 after insemination. Furthermore, none of the mtB-M embryos could implant into the maternal uterus after embryo transfer, whereas control mouse embryos into which mitochondria from another mouse had been transferred developed as well as did non-manipulated embryos. When we performed quantitative PCR (qPCR) of mouse and bovine ND5, we found that the mtB-M embryos contained 8.3% of bovine mitochondria at the blastocyst stage. Thus, contamination with mitochondria from another species induces embryonic lethality prior to implantation into the maternal uterus. The heteroplasmic state of these xenogeneic mitochondria could have detrimental effects on preimplantation development, leading to preservation of species-specific mitochondrial integrity in mammals. PMID:26416548

The antifreeze protein type I (AFP I) increases seabream (Sparus aurata) embryos tolerance to low temperatures.

PubMed

Robles, V; Barbosa, V; Herráez, M P; Martínez-Páramo, S; Cancela, M L

2007-07-15

To date, all attempts at fish embryo cryopreservation have failed. One of the main reasons for this to occur is the high chilling sensitivity reported in fish embryos thus emphasizing the need for further testing of different methods and alternative cryoprotective agents (CPAs) in order to improve our chances to succeed in this purpose. In this work we have used the antifreeze protein type I (AFP I) as a natural CPA. This protein is naturally expressed in sub-arctic fish species, and inhibits the growth of ice crystals as well as recrystallization during thawing. Embryos from Sparus aurata were microinjected with AFP I at different developmental stages, 2 cells and blastula, into the blastomere-yolk interface and into the yolk sac, respectively. Control, punctured and microinjected embryos were subjected to chilling at two different temperatures, 0 degrees C (1h) and -10 degrees C (15min) when embryos reached 5-somite stage. Embryos were subjected to -10 degrees C chilling in a 3M DMSO extender to avoid ice crystal formation in the external solution. Survival after chilling was established as the percentage of embryos that hatch. To study the AFP I distribution in the microinjected embryos, a confocal microscopy study was done. Results demonstrate that AFP I can significantly improve chilling resistance at 0 degrees C, particularly in 2-cell microinjected embryos, displaying nearly 100% hatching rates. This fact is in agreement with the confocal microscopy observations which confirmed the presence of the AFP protein in embryonic cells. These results support the hypothesis that AFP protect cellular structures by stabilizing cellular membranes.

Applying embryo cryopreservation technologies to the production of domestic and black-footed cats.

PubMed

Pope, C E; Gómez, M C; Galiguis, J; Dresser, B l

2012-12-01

Our objectives were (i) compare in vitro development of early cleavage stage domestic cat embryos after cryopreservation by minimal volume vitrification vs a standard slow, controlled-rate method, (ii) determine viability of vitrified domestic cat embryos by oviductal transfer into synchronous recipients and (iii) evaluate in vivo survival of black-footed cat (BFC, Felis nigripes) embryos after intra- and inter-species transfer. In vitro-derived (IVM/IVF) cat embryos were used to evaluate in vitro development after controlled-rate cryopreservation vs vitrification vs controls. Blastocyst development was similar in both groups of cryopreserved embryos (22-26%), but it was lower (p < 0.05) than that of fresh embryos (50%). After embryo transfer, four of eight recipients of vitrified embryos established pregnancies--three of six (50%) and one of two (50%) that received embryos from in vivo- and in vitro-matured oocytes, respectively. Three male and two female kittens weighing from 51 to 124 g (mean = 88 g) were delivered on days 61-65 of gestation. In BFC, four intra-species embryo transfer procedures were carried out--two recipients received fresh day 2 embryos (n = 5, 8) and two recipients received embryos that had been cryopreserved on day 1 (n = 6) or 2 (n = 8). A 2-year-old recipient of cryopreserved embryos established pregnancy and delivered two live male kittens. Subsequently, five cryopreserved BFC embryos were transferred to a domestic cat recipient. On day 29, the recipient was determined to be pregnant and delivered naturally a live, healthy female BFC kitten on day 66. In summary, in vivo survival of vitrified domestic cat embryos was shown by the births of kittens after transfer into recipients. Also, we demonstrated that sperm and embryo cryopreservation could be combined with intra- and inter-species embryo transfer and integrated into the array of assisted reproductive techniques used successfully for propagation of a rare and vulnerable felid species, the black-footed cat. © 2012 Blackwell Verlag GmbH.

Transition of cell numbers in bovine preimplantation embryos: in vivo collected and in vitro produced embryos.

PubMed

Ushijima, Hitoshi; Akiyama, Kiyoshi; Tajima, Toshio

2008-08-01

The total cell numbers (TCNs) of bovine embryos collected from superovulated donors (VIVO embryos) were counted 0-9 d after ovulation to quantify the developmental process. Using numerical analysis of embryo development, we also compared the developmental process of VIVO embryos, in vitro-fertilized (IVF) embryos and nuclear transfer (NT) embryos obtained from enucleated oocytes and blastomere nuclei. The TCNs of embryos were measured using the air-dry method. Cleavage divisions (CD) of the embryos were obtained using logarithmic transformation of the TCN. The TCN of the VIVO embryos increased significantly (P<0.001) with time. The relationship between the CD of the VIVO embryos at 0-9 d after ovulation and age in days was described by a linear equation with a high correlation (y=1.03x+0.16, r=0.99), showing that CD occurs about once each day for all blastomeres. However, compared to the VIVO embryos, the TCN of the IVF embryos did not increase from 3-4 d nor after 7 d; the TCN of the NT embryos did not increase after 7 d (P>0.05). The results suggest a delay in development at these developmental stages. The slopes of regression lines of the IVF and NT embryos were significantly (P<0.001) smaller, indicating that quantification of the developmental process of VIVO embryos according to TCN and CD would be useful as criteria for numerical evaluation of the developmental process of bovine in vitro produced embryos.

Predictive Modeling of Implantation Outcome in an In Vitro Fertilization Setting: An Application of Machine Learning Methods.

PubMed

Uyar, Asli; Bener, Ayse; Ciray, H Nadir

2015-08-01

Multiple embryo transfers in in vitro fertilization (IVF) treatment increase the number of successful pregnancies while elevating the risk of multiple gestations. IVF-associated multiple pregnancies exhibit significant financial, social, and medical implications. Clinicians need to decide the number of embryos to be transferred considering the tradeoff between successful outcomes and multiple pregnancies. To predict implantation outcome of individual embryos in an IVF cycle with the aim of providing decision support on the number of embryos transferred. Retrospective cohort study. Electronic health records of one of the largest IVF clinics in Turkey. The study data set included 2453 embryos transferred at day 2 or day 3 after intracytoplasmic sperm injection (ICSI). Each embryo was represented with 18 clinical features and a class label, +1 or -1, indicating positive and negative implantation outcomes, respectively. For each classifier tested, a model was developed using two-thirds of the data set, and prediction performance was evaluated on the remaining one-third of the samples using receiver operating characteristic (ROC) analysis. The training-testing procedure was repeated 10 times on randomly split (two-thirds to one-third) data. The relative predictive values of clinical input characteristics were assessed using information gain feature weighting and forward feature selection methods. The naïve Bayes model provided 80.4% accuracy, 63.7% sensitivity, and 17.6% false alarm rate in embryo-based implantation prediction. Multiple embryo implantations were predicted at a 63.8% sensitivity level. Predictions using the proposed model resulted in higher accuracy compared with expert judgment alone (on average, 75.7% and 60.1%, respectively). A machine learning-based decision support system would be useful in improving the success rates of IVF treatment. © The Author(s) 2014.

Critical developmental windows for morphology and hematology revealed by intermittent and continuous hypoxic incubation in embryos of quail (Coturnix coturnix).

PubMed

Burggren, Warren W; Elmonoufy, Nourhan A

2017-01-01

Hypoxia during embryonic growth in embryos is frequently a powerful determinant of development, but at least in avian embryos the effects appear to show considerable intra- and inter-specific variation. We hypothesized that some of this variation may arise from different protocols that may or may not result in exposure during the embryo's critical window for hypoxic effects. To test this hypothesis, quail embryos (Coturnix coturnix) in the intact egg were exposed to hypoxia (~15% O2) during "early" (Day 0 through Day 5, abbreviated as D0-D5), "middle" (D6-D10) or "late" (D11-D15) incubation or for their entire 16-18 day incubation ("continuous hypoxia") to determine critical windows for viability and growth. Viability, body mass, beak and toe length, heart mass, and hematology (hematocrit and hemoglobin concentration) were measured on D5, D10, D15 and at hatching typically between D16 and D18 Viability rate was ~50-70% immediately following the exposure period in the early, middle and late hypoxic groups, but viability improved in the early and late groups once normoxia was restored. Middle hypoxia groups showed continuing low viability, suggesting a critical period from D6-D10 for embryo viability. The continuous hypoxia group experienced viability reaching <10% after D15. Hypoxia, especially during late and continuous hypoxia, also inhibited growth of body, beak and toe when measured at D15. Full recovery to normal body mass upon hatching occurred in all other groups except for continuous hypoxia. Contrary to previous avian studies, heart mass, hematocrit and hemoglobin concentration were not altered by any hypoxic incubation pattern. Although hypoxia can inhibit embryo viability and organ growth during most incubation periods, the greatest effects result from continuous or middle incubation hypoxic exposure. Hypoxic inhibition of growth can subsequently be "repaired" by catch-up growth if a final period of normoxic development is available. Collectively, these data indicate a critical developmental window for hypoxia susceptibility during the mid-embryonic period of development.

Fish embryo toxicity test: identification of compounds with weak toxicity and analysis of behavioral effects to improve prediction of acute toxicity for neurotoxic compounds.

PubMed

Klüver, Nils; König, Maria; Ortmann, Julia; Massei, Riccardo; Paschke, Albrecht; Kühne, Ralph; Scholz, Stefan

2015-06-02

The fish embryo toxicity test has been proposed as an alternative for the acute fish toxicity test, but concerns have been raised for its predictivity given that a few compounds have been shown to exhibit a weak acute toxicity in the fish embryo. In order to better define the applicability domain and improve the predictive capacity of the fish embryo test, we performed a systematic analysis of existing fish embryo and acute fish toxicity data. A correlation analysis of a total of 153 compounds identified 28 compounds with a weaker or no toxicity in the fish embryo test. Eleven of these compounds exhibited a neurotoxic mode of action. We selected a subset of eight compounds with weaker or no embryo toxicity (cyanazine, picloram, aldicarb, azinphos-methyl, dieldrin, diquat dibromide, endosulfan, and esfenvalerate) to study toxicokinetics and a neurotoxic mode of action as potential reasons for the deviating fish embryo toxicity. Published fish embryo LC50 values were confirmed by experimental analysis of zebrafish embryo LC50 according to OECD guideline 236. Except for diquat dibromide, internal concentration analysis did not indicate a potential relation of the low sensitivity of fish embryos to a limited uptake of the compounds. Analysis of locomotor activity of diquat dibromide and the neurotoxic compounds in 98 hpf embryos (exposed for 96 h) indicated a specific effect on behavior (embryonic movement) for the neurotoxic compounds. The EC50s of behavior for neurotoxic compounds were close to the acute fish toxicity LC50. Our data provided the first evidence that the applicability domain of the fish embryo test (LC50s determination) may exclude neurotoxic compounds. However, neurotoxic compounds could be identified by changes in embryonic locomotion. Although a quantitative prediction of acute fish toxicity LC50 using behavioral assays in fish embryos may not yet be possible, the identification of neurotoxicity could trigger the conduction of a conventional fish acute toxicity test or application of assessment factors while considering the very good fish embryo-acute fish toxicity correlation for other compounds.

PreImplantation factor (PIF) protects cultured embryos against oxidative stress: relevance for recurrent pregnancy loss (RPL) therapy.

PubMed

Goodale, Lindsay F; Hayrabedran, Soren; Todorova, Krassimira; Roussev, Roumen; Ramu, Sivakumar; Stamatkin, Christopher; Coulam, Carolyn B; Barnea, Eytan R; Gilbert, Robert O

2017-05-16

Recurrent pregnancy loss (RPL) affects 2-3% of couples. Despite a detailed work-up, the etiology is frequently undefined, leading to non-targeted therapy. Viable embryos and placentae express PreImplantation Factor (PIF). Maternal circulating PIF regulates systemic immunity and reduces circulating natural killer cells cytotoxicity in RPL patients. PIF promotes singly cultured embryos' development while anti-PIF antibody abrogates it. RPL serum induced embryo toxicity is negated by PIF. We report that PIF rescues delayed embryo development caused by <3 kDa RPL serum fraction likely by reducing reactive oxygen species (ROS). We reveal that protein disulfide isomerase/thioredoxin (PDI/TRX) is a prime PIF target in the embryo, rendering it an important ROS scavenger. The 16F16-PDI/TRX inhibitor drastically reduced blastocyst development while exogenous PIF increased >2 fold the number of embryos reaching the blastocyst stage. Mechanistically, PDI-inhibitor preferentially binds covalently to oxidized PDI over its reduced form where PIF avidly binds. PIF by targeting PDI/TRX at a distinct site limits the inhibitor's pro-oxidative effects. The >3kDa RPL serum increased embryo demise by three-fold, an effect negated by PIF. However, embryo toxicity was not associated with the presence of putative anti-PIF antibodies. Collectively, PIF protects cultured embryos both against ROS, and higher molecular weight toxins. Using PIF for optimizing in vitro fertilization embryos development and reducing RPL is warranted.

The first cell-fate decisions in the mouse embryo: destiny is a matter of both chance and choice.

PubMed

Zernicka-Goetz, Magdalena

2006-08-01

Development of the early mouse embryo has always been classified as regulative, meaning that when parts or blastomeres of the embryo are isolated they change their developmental fate and can even reconstruct the whole. However, regulative development does not mean that, in situ, these parts or blastomeres are equivalent; it does not mean that the early mammalian embryo is a ball of identical cells without any bias. Regulative development simply means that whatever bias the regions of the embryo might have they still remain flexible and can respond to experimental interference by changes of fate. This realization -- that regulative development and patterning can co-exist -- has led to a renaissance of interest in the first days of development of the mouse embryo, and several laboratories have provided evidence for some early bias. Now the challenge is to gain some understanding of the molecular basis of this bias.

Embryonic rather than extraembryonic tissues have more impact on the development of placental hyperplasia in cloned mice.

PubMed

Miki, H; Wakisaka, N; Inoue, K; Ogonuki, N; Mori, M; Kim, J-M; Ohta, A; Ogura, A

2009-06-01

Somatic cell cloning by nuclear transfer (NT) in mice is associated with hyperplastic placentas at term. To dissect the effects of embryonic and extraembryonic tissues on this clone-associated phenotype, we constructed diploid (2n) fused with (<-->) tetraploid (4n) chimeras from NT- and fertilization-derived (FD) embryos. Generally, the 4n cells contributed efficiently to all the extraembryonic tissues but not to the embryo itself. Embryos constructed by 2n NT<-->4n FD aggregation developed hyperplastic placentas (0.33+/-0.22 g) with a predominant contribution by NT-derived cells. Even when the population of FD-derived cells in placentas was increased using multiple FD embryos (up to four) for aggregation, most placentas remained hyperplastic (0.36+/-0.13 g). By contrast, placentas of the reciprocal combination, 2n FD<-->4n NT, were less hyperplastic (0.15+/-0.02 g). These nearly normal-looking placentas had a large proportion of NT-derived cells. Thus, embryonic rather than extraembryonic tissues had more impact on the onset of placental hyperplasia, and that the abnormal placentation in clones occurs in a noncell-autonomous manner. These findings suggest that for improvement of cloning efficiency we should understand the mechanisms regulating placentation, especially those of embryonic origin that might control the proliferation of trophoblastic lineage cells.

[Study on physiological and germination characteristics of Tulipa edulis seed].

PubMed

Wu, Zhengjun; Zhu, Zaibiao; Guo, Qiaosheng; Ma, Hongliang; Xu, Hongjian; Miao, Yuanyuan

2012-03-01

Current study was conducted to investigate the seed physiological characteristics of Tulipa edulis and improve germination rate. Anatomical characteristics was observed. Seed water absorption curve was tested by soaking method. Dynamic of embryo development and germination rate as well as germination index under different conditions were recorded. And the biological test of cabbage seed was used for detecting the germination inhibitors. The embryo rate of newly matured seeds was about 10%, and there was no obstacle of water absorption on testa of T. edulis. The optimum method for embryo development was exposure to 300 mg x L(-1) gibberellin solution for 24 hours, and stratification at 25 degrees C for 70 days followed by stratification at 5 degrees C for 40 days. The germintion rate and germination index of dormancy-broken seeds under the dark environment at 10 degrees C and 15 degrees C were significantly higher than those under other conditions. Additionally, there were some germination inhibitory substances in dry seeds. The seed of T. edulis can be classified as having complex morphophysiological dormancy, and the morphological embryo dormancy played a leading role. Warm and cold stratification resulted in a fast dormancy breaking effect, and a high germination rate more than 90% could be obtained under the optimum conditions.

Association between mitochondrial DNA haplotype compatibility and increased efficiency of bovine intersubspecies cloning.

PubMed

Yan, Hao; Yan, Zhonghai; Ma, Qingwen; Jiao, Fei; Huang, Shuzhen; Zeng, Fanyi; Zeng, Yitao

2011-01-01

Reconstructed embryos derived from intersubspecies somatic cell nuclear transfer (SCNT) have poorer developmental potential than those from intrasubspecies SCNT. Based on our previous study that Holstein dairy bovine (HD) mitochondrial DNA (mtDNA) haplotype compatibility between donor karyoplast and recipient cytoplast is crucial for SCNT embryo development, we performed intersubspecies SCNT using HD as donor karyoplast and Luxi yellow heifer (LY) as recipient cytoplast according to mtDNA haplotypes determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. The results demonstrated that intersubspecies mtDNA homotype SCNT embryos had higher pre- and post-implantation developmental competence than intrasubspecies mtDNA heterotype embryos as well as improved blastocyst reprogramming status, including normal H3K9 dimethylation pattern and promoter hypomethylation of pluripotent genes such as Oct4 and Sox2, suggesting that intersubspecies SCNT using LY oocytes maintains HD cloning efficiency and may reprogram HD nuclei to develop into a normal cloned animal ultimately. Our results indicated that karyoplast-cytoplast interactions and mtDNA haplotype compatibility may affect bovine intersubspecies SCNT efficiency. This study on bovine intersubspecies SCNT is valuable for understanding the mechanisms of mtDNA haplotype compatibility between karyoplast and cytoplast impacting the bovine SCNT efficiency, and provides an alternative and economic resource for HD cloning. Copyright © 2011. Published by Elsevier Ltd.

Optimization of CRISPR/Cas9 genome editing for loss-of-function in the early chick embryo.

PubMed

Gandhi, Shashank; Piacentino, Michael L; Vieceli, Felipe M; Bronner, Marianne E

2017-12-01

The advent of CRISPR/Cas9 has made genome editing possible in virtually any organism, including those not previously amenable to genetic manipulations. Here, we present an optimization of CRISPR/Cas9 for application to early avian embryos with improved efficiency via a three-fold strategy. First, we employed Cas9 protein flanked with two nuclear localization signal sequences for improved nuclear localization. Second, we used a modified guide RNA (gRNA) scaffold that obviates premature termination of transcription and unstable Cas9-gRNA interactions. Third, we used a chick-specific U6 promoter that yields 4-fold higher gRNA expression than the previously utilized human U6. For rapid screening of gRNAs for in vivo applications, we also generated a chicken fibroblast cell line that constitutively expresses Cas9. As proof of principle, we performed electroporation-based loss-of-function studies in the early chick embryo to knock out Pax7 and Sox10, key transcription factors with known functions in neural crest development. The results show that CRISPR/Cas9-mediated deletion causes loss of their respective proteins and transcripts, as well as predicted downstream targets. Taken together, the results reveal the utility of this optimized CRISPR/Cas9 method for targeted gene knockout in chicken embryos in a manner that is reproducible, robust and specific. Copyright © 2017 Elsevier Inc. All rights reserved.

The effect of the number of transferred embryos, the interval between nuclear transfer and embryo transfer, and the transfer pattern on pig cloning efficiency.

PubMed

Rim, Chol Ho; Fu, Zhixin; Bao, Lei; Chen, Haide; Zhang, Dan; Luo, Qiong; Ri, Hak Chol; Huang, Hefeng; Luan, Zhidong; Zhang, Yan; Cui, Chun; Xiao, Lei; Jong, Ui Myong

2013-12-01

To improve the efficiency of producing cloned pigs, we investigated the influence of the number of transferred embryos, the culturing interval between nuclear transfer (NT) and embryo transfer, and the transfer pattern (single oviduct or double oviduct) on cloning efficiency. The results demonstrated that transfer of either 150-200 or more than 200NT embryos compared to transfer of 100-150 embryos resulted in a significantly higher pregnancy rate (48 ± 16, 50 ± 16 vs. 29 ± 5%, p<0.05) and average litter size (4.1 ± 2.3, 7 ± 3.6 vs. 2.5 ± 0.5). In vitro culture of reconstructed embryos for a longer time (40 h vs. 20 h) resulted in higher (p<0.05) pregnancy rate (44 ± 9 vs. 31 ± 3%) and delivery rate (44 ± 9 vs. 25 ± 9%). Furthermore, double oviductal transfer dramatically increased pregnancy rate (83 ± 6 vs. 27+8%, p<0.05), delivery rate (75 ± 2 vs. 27+8%, p<0.05) and average litter size (6.5 ± 2.8 vs. 2.6 ± 1.2) compared to single oviductal transfer. Our study demonstrated that an improvement in pig cloning efficiency is achieved by adjusting the number and in vitro culture time of reconstructed embryos as well as the embryo transfer pattern. Copyright © 2013 Elsevier B.V. All rights reserved.

Assessing embryo development using swept source optical coherence tomography

NASA Astrophysics Data System (ADS)

Caujolle, S.; Cernat, R.; Silvestri, G.; Marques, M. J.; Bradu, A.; Feuchter, T.; Robinson, G.; Griffin, D.; Podoleanu, A.

2018-03-01

A detailed assessment of embryo development would assist biologists with selecting the most suitable embryos for transfer leading to higher pregnancy rates. Currently, only low resolution microscopy is employed to perform this assessment. Although this method delivers some information on the embryo surface morphology, no specific details are shown related to its inner structure. Using a Master-Slave Swept-Source Optical Coherence Tomography (SS-OCT), images of bovine embryos from day 7 after fertilization were collected from different depths. The dynamic changes inside the embryos were examined, in detail and in real-time from several depths. To prove our ability to characterize the morphology, a single embryo was imaged over 26 hours. The embryo was deprived of its life support environment, leading to its death. Over this period, clear morphological changes were observed.

Early embryo development in Fucus distichus is auxin sensitive

NASA Technical Reports Server (NTRS)

Basu, Swati; Sun, Haiguo; Brian, Leigh; Quatrano, Ralph L.; Muday, Gloria K.

2002-01-01

Auxin and polar auxin transport have been implicated in controlling embryo development in land plants. The goal of these studies was to determine if auxin and auxin transport are also important during the earliest stages of development in embryos of the brown alga Fucus distichus. Indole-3-acetic acid (IAA) was identified in F. distichus embryos and mature tissues by gas chromatography-mass spectroscopy. F. distichus embryos accumulate [(3)H]IAA and an inhibitor of IAA efflux, naphthylphthalamic acid (NPA), elevates IAA accumulation, suggesting the presence of an auxin efflux protein complex similar to that found in land plants. F. distichus embryos normally develop with a single unbranched rhizoid, but growth on IAA leads to formation of multiple rhizoids and growth on NPA leads to formation of embryos with branched rhizoids, at concentrations that are active in auxin accumulation assays. The effects of IAA and NPA are complete before 6 h after fertilization (AF), which is before rhizoid germination and cell division. The maximal effects of IAA and NPA are between 3.5 and 5 h AF and 4 and 5.5 h AF, respectively. Although, the location of the planes of cell division was significantly altered in NPA- and IAA-treated embryos, these abnormal divisions occurred after abnormal rhizoid initiation and branching was observed. The results of this study suggest that auxin acts in the formation of apical basal patterns in F. distichus embryo development.

Nuclear-mitochondrial incompatibility in interorder rhesus monkey-cow embryos derived from somatic cell nuclear transfer.

PubMed

Kwon, Daekee; Koo, Ok-Jae; Kim, Min-Jung; Jang, Goo; Lee, Byeong Chun

2016-10-01

Monkey interorder somatic cell nuclear transfer (iSCNT) using enucleated cow oocytes yielded poor blastocysts development and contradictory results among research groups. Determining the reason for this low blastocyst development is a prerequisite for optimizing iSCNT in rhesus monkeys. The aim of this study was to elucidate nuclear-mitochondrial incompatibility of rhesus monkey-cow iSCNT embryos and its relationship to low blastocyst development. Cytochrome b is a protein of complex III of the electron transport chain (ETC). According to meta-analysis of amino acid sequences, the homology of cytochrome b is 75 % between rhesus monkeys and cattle. To maintain the function of ETC after iSCNT, 4n iSCNT embryos were produced by fusion of non-enucleated cow oocytes and rhesus monkey somatic cells. The blastocyst development rate of 4n iSCNT embryos was higher than that of 2n embryos (P < 0.01). Formation of reactive oxygen species (ROS) is an indirect indicator of ETC activity of cells. The ROS levels of 4n iSCNT embryos was higher than that of 2n embryos (P < 0.01). Collectively, rhesus monkey iSCNT embryos reconstructed with cow oocytes have nuclear-mitochondrial incompatibility due to fundamental species differences between rhesus monkeys and cattle. Nuclear-mitochondrial incompatibility seems to correlate with low ETC activity and extremely low blastocyst development of rhesus monkey-cow iSCNT embryos.

Elective single-embryo transfer.

PubMed

2012-04-01

As in vitro fertilization implantation rates have improved, the practice of transfering multiple embryos must be evaluated. The purpose of this document is to reassess the literature on elective single-embryo transfer, to provide guidance for patient selection, and to discuss barriers to utilization. Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Maternally derived trypsin may have multiple functions in the early development of turbot (Scopthalmus maximus).

PubMed

Chi, Liang; Liu, Qinghua; Xu, Shihong; Xiao, Zhizhong; Ma, Daoyuan; Li, Jun

2015-10-01

Trypsin is an important serine protease that is considered to be involved in digestion of protein in teleost fish. Nevertheless, studies on trypsin/trypsinogen in fish embryos are very limited. In this study, the trypsinogen of turbot (Scophthalmus maximus) (tTG) was identified and the expression patterns and activity of trypsinogen/trypsin were investigated. The results showed that the tTG mRNA was evenly distributed in the oocytes and was also expressed along the yolk periphery in early embryos. At later embryo stages and 1 days after hatching (dph), the tTG mRNA concentrated at the alimentary tract and head. Quantitative expression analysis showed that the tTG transcripts decreased after fertilization until the gastrula stage, then increased with the embryo and larvae development. This result was also confirmed by the specific activity analysis of trypsin and in-situ-hybridization (ISH). All of the results indicated that tTG in early embryo stages was maternally derived and expressed by itself after gastrula stages. Additionally, location of tTG mRNA in embryos and larvae was investigated; we considered that trypsin may have multiple functions during the embryo development process. Based on our results regarding trypsinogen in embryos and early development, we concluded that the trypsin/trypsinogen in turbot embryos was inherited from a maternal source and we suggested that trypsin in early development has multiple functions in the process of development. Copyright © 2015 Elsevier Inc. All rights reserved.

Sex determination of duck embryos: observations on syrinx development

USGS Publications Warehouse

Wilson, Robert E.; Sonsthagen, Sarah A.; Franson, J. Christian

2013-01-01

Ducks exhibit sexual dimorphism in vocal anatomy. Asymmetrical ossification of the syrinx (bulla syringealis) is discernable at about 10 days of age in male Pekin duck (Anas platyrhynchos domestica) embryos, but information is lacking on the early development of the bulla in wild ducks. To evaluate the reliability of this characteristic for sexing developing embryos, we examined the syrinx of dead embryos and compared results with molecular sexing techniques in high arctic nesting Common Eiders (Somateria mollissima). Embryos 8 days or older were accurately (100%) sexed based on the presence/absence of a bulla, 2 days earlier than Pekin duck. The use of the tracheal bulla can be a valuable technique when sex identification of embryos or young ducklings is required.

Growth and development of cultured carrot cells and embryos under spaceflight conditions

NASA Technical Reports Server (NTRS)

Krikorian, A. D.; Dutcher, F. R.; Quinn, C. E.; Steward, F. C.

1981-01-01

Morphogenetically competent proembryonic cells and well-developed somatic embryos of carrot at two levels of organization were exposed for 18.5 days to a hypogravity environment aboard the Soviet Biosatellite Cosmos 1129. It was confirmed that cultured totipotent cells of carrot can give rise to embryos with well-developed roots and minimally developed shoots. It was also shown that the space hypogravity environment could support the further growth of already-organized, later somatic embryonic stages and give rise to fully developed embryo-plantlets with roots and shoots.

Privileged Communication Embryonic Development Following Somatic Cell Nuclear Transfer Impeded by Persisting Histone Methylation

PubMed Central

Matoba, Shogo; Liu, Yuting; Lu, Falong; Iwabuchi, Kumiko A.; Shen, Li; Inoue, Azusa; Zhang, Yi

2014-01-01

SUMMARY Mammalian oocytes can reprogram somatic cells into a totipotent state enabling animal cloning through somatic cell nuclear transfer (SCNT). However, the majority of SCNT embryos fail to develop to term due to undefined reprogramming defects. Here we identify histone H3 lysine 9 trimethylation (H3K9me3) of donor cell genome as a major epigenetic barrier for efficient reprogramming by SCNT. Comparative transcriptome analysis identified reprogramming resistant regions (RRRs) that are expressed normally at 2-cell mouse embryos generated by IVF but not SCNT. RRRs are enriched for H3K9me3 in donor somatic cells, and its removal by ectopic expression of the H3K9me3 demethylase Kdm4d not only reactivates the majority of RRRs, but also greatly improves SCNT efficiency. Furthermore, use of donor somatic nuclei depleted of H3K9 methyltransferases markedly improves SCNT efficiency. Our study thus identifies H3K9me3 as a critical epigenetic barrier in SCNT-mediated reprogramming and provides a promising approach for improving mammalian cloning efficiency. PMID:25417163

The Theobroma cacao B3 domain transcription factor TcLEC2 plays a duel role in control of embryo development and maturation.

PubMed

Zhang, Yufan; Clemens, Adam; Maximova, Siela N; Guiltinan, Mark J

2014-04-24

The Arabidopsis thaliana LEC2 gene encodes a B3 domain transcription factor, which plays critical roles during both zygotic and somatic embryogenesis. LEC2 exerts significant impacts on determining embryogenic potential and various metabolic processes through a complicated genetic regulatory network. An ortholog of the Arabidopsis Leafy Cotyledon 2 gene (AtLEC2) was characterized in Theobroma cacao (TcLEC2). TcLEC2 encodes a B3 domain transcription factor preferentially expressed during early and late zygotic embryo development. The expression of TcLEC2 was higher in dedifferentiated cells competent for somatic embryogenesis (embryogenic calli), compared to non-embryogenic calli. Transient overexpression of TcLEC2 in immature zygotic embryos resulted in changes in gene expression profiles and fatty acid composition. Ectopic expression of TcLEC2 in cacao leaves changed the expression levels of several seed related genes. The overexpression of TcLEC2 in cacao explants greatly increased the frequency of regeneration of stably transformed somatic embryos. TcLEC2 overexpressing cotyledon explants exhibited a very high level of embryogenic competency and when cultured on hormone free medium, exhibited an iterative embryogenic chain-reaction. Our study revealed essential roles of TcLEC2 during both zygotic and somatic embryo development. Collectively, our evidence supports the conclusion that TcLEC2 is a functional ortholog of AtLEC2 and that it is involved in similar genetic regulatory networks during cacao somatic embryogenesis. To our knowledge, this is the first detailed report of the functional analysis of a LEC2 ortholog in a species other then Arabidopsis. TcLEC2 could potentially be used as a biomarker for the improvement of the SE process and screen for elite varieties in cacao germplasm.

9 CFR 98.11 - Definitions.

Code of Federal Regulations, 2013 CFR

2013-01-01

... EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth... Health Inspection Service of the United States Department of Agriculture. Collection of embryos. Embryos removed from a single donor dam in one operation. Embryo. The initial stages of development of an animal...

9 CFR 98.11 - Definitions.

Code of Federal Regulations, 2012 CFR

2012-01-01

... EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth... Health Inspection Service of the United States Department of Agriculture. Collection of embryos. Embryos removed from a single donor dam in one operation. Embryo. The initial stages of development of an animal...

9 CFR 98.11 - Definitions.

Code of Federal Regulations, 2014 CFR

2014-01-01

... EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth... Health Inspection Service of the United States Department of Agriculture. Collection of embryos. Embryos removed from a single donor dam in one operation. Embryo. The initial stages of development of an animal...

Biotechnological advances in mango (Mangifera indica L.) and their future implication in crop improvement: a review.

PubMed

Krishna, Hare; Singh, S K

2007-01-01

Biotechnology can complement conventional breeding and expedite the mango improvement programmes. Studies involving in vitro culture and selection, micropropagation, embryo rescue, genetic transformation, marker-assisted characterization and DNA fingerprinting, etc. are underway at different centers worldwide. In vitro culture and somatic embryogenesis of several different genotypes have been achieved. The nucellus excised from immature fruitlets is the appropriate explant for induction of embryogenic cultures. High frequency somatic embryogenesis has been achieved in some genotypes; however, some abnormalities can occur during somatic embryo germination. Embryo rescue from young and dropped fruitlets can improve the hybridization success in a limited flowering season. Protocols for protoplast culture and regeneration have also been developed. In vitro selections for antibiotic tolerance and fungal toxin resistance have been very promising for germplasm screening. Genetic transformation using Agrobacterium tumefaciens has been reported. Genes that are involved with fruit ripening have been cloned and there have been attempts to deliver these genes into plants. DNA fingerprinting and studies on genetic diversity of mango cultivars and Mangifera species are also being conducted at several research stations. The purpose of this review is to focus upon contemporary information on biotechnological advances made in mango. It also describes some ways of overcoming the problems encountered during in vitro propagation of mango.

Auxin polar transport is essential for the development of zygote and embryo in Nicotiana tabacum L. and correlated with ABP1 and PM H+-ATPase activities

PubMed Central

Chen, Dan; Ren, Yujun; Deng, Yingtian; Zhao, Jie

2010-01-01

Auxin is an important plant growth regulator, and plays a key role in apical–basal axis formation and embryo differentiation, but the mechanism remains unclear. The level of indole-3-acetic acid (IAA) during zygote and embryo development of Nicotiana tabacum L. is investigated here using the techniques of GC-SIM-MS analysis, immunolocalization, and the GUS activity assay of DR5::GUS transgenic plants. The distribution of ABP1 and PM H+-ATPase was also detected by immunolocalization, and this is the first time that integral information has been obtained about their distribution in the zygote and in embryo development. The results showed an increase in IAA content in ovules and the polar distribution of IAA, ABP1, and PM H+-ATPase in the zygote and embryo, specifically in the top and basal parts of the embryo proper (EP) during proembryo development. For information about the regulation mechanism of auxin, an auxin transport inhibitor TIBA (2,3,5-triiodobenzoic acid) and exogenous IAA were, respectively, added to the medium for the culture of ovules at the zygote and early proembryo stages. Treatment with a suitable IAA concentration promoted zygote division and embryo differentiation, while TIBA treatment obviously suppressed these processes and caused the formation of abnormal embryos. The distribution patterns of IAA, ABP1, and PM H+-ATPase were also disturbed in the abnormal embryos. These results indicate that the polar distribution and transport of IAA begins at the zygote stage, and affects zygote division and embryo differentiation in tobacco. Moreover, ABP1 and PM H+-ATPase may play roles in zygote and embryo development and may also be involved in IAA signalling transduction. PMID:20348352

Silver nanoparticles induce developmental stage-specific embryonic phenotypes in zebrafish.

PubMed

Lee, Kerry J; Browning, Lauren M; Nallathamby, Prakash D; Osgood, Christopher J; Xu, Xiao-Hong Nancy

2013-12-07

Much is anticipated from the development and deployment of nanomaterials in biological organisms, but concerns remain regarding their biocompatibility and target specificity. Here we report our study of the transport, biocompatibility and toxicity of purified and stable silver nanoparticles (Ag NPs, 13.1 ± 2.5 nm in diameter) upon the specific developmental stages of zebrafish embryos using single NP plasmonic spectroscopy. We find that single Ag NPs passively diffuse into five different developmental stages of embryos (cleavage, early-gastrula, early-segmentation, late-segmentation, and hatching stages), showing stage-independent diffusion modes and diffusion coefficients. Notably, the Ag NPs induce distinctive stage and dose-dependent phenotypes and nanotoxicity, upon their acute exposure to the Ag NPs (0-0.7 nM) for only 2 h. The late-segmentation embryos are most sensitive to the NPs with the lowest critical concentration (CNP,c < 0.02 nM) and highest percentages of cardiac abnormalities, followed by early-segmentation embryos (CNP,c < 0.02 nM), suggesting that disruption of cell differentiation by the NPs causes the most toxic effects on embryonic development. The cleavage-stage embryos treated with the NPs develop into a wide variety of phenotypes (abnormal finfold, tail/spinal cord flexure, cardiac malformation/edema, yolk sac edema, and acephaly). These organ structures are not yet developed in cleavage-stage embryos, suggesting that the earliest determinative events to create these structures are ongoing, and disrupted by NPs, which leads to the downstream effects. In contrast, the hatching embryos are most resistant to the Ag NPs, and majority of embryos (94%) develop normally, and none of them develop abnormally. Interestingly, early-gastrula embryos are less sensitive to the NPs than cleavage and segmentation stage embryos, and do not develop abnormally. These important findings suggest that the Ag NPs are not simple poisons, and they can target specific pathways in development, and potentially enable target specific study and therapy for early embryonic development.

Transcriptional regulators TRIM28, SETDB1, and TP53 are aberrantly expressed in porcine embryos produced by in vitro fertilization in comparison to in vivo- and somatic-cell nuclear transfer-derived embryos

PubMed Central

Hamm, Jennifer; Tessanne, Kim; Murphy, Clifton N; Prather, Randall S

2014-01-01

In vitro embryo production is important for research in animal reproduction, embryo transfer, transgenics, and cloning. Yet, in vitro-fertilized (IVF) embryos are generally developmentally delayed and are inferior to in vivo-derived (IVV) embryos; this discrepancy is likely a result of aberrant gene expression. Transcription of three genes implicated to be important in normal preimplantation embryo development, TRIM28, SETDB1, and TP53, was determined by quanitative PCR in IVF, somatic-cell nuclear transfer (SCNT), parthenogenetic, and IVV porcine oocytes and embryos. There was no difference in TRIM28 or SETDB1 abundance between oocytes matured in vitro versus in vivo (P > 0.05), whereas TP53 levels were higher in in vitro-matured oocytes. TRIM28 increased from metaphase-II oocytes to the 4-cell and blastocyst stages in IVF embryos, whereas IVV embryos showed a reduction in TRIM28 abundance from maturation throughout development. The relative abundance of TP53 increased by the blastocyst stage in all treatment groups, but was higher in IVF embryos compared to IVV and SCNT embryos. In contrast, SETDB1 transcript levels decreased from the 2-cell to blastocyst stage in all treatments. For each gene analyzed, SCNT embryos of both hard-to-clone and easy-to-clone cell lines were more comparable to IVV than IVF embryos. Knockdown of TRIM28 also had no effect on blastocyst development or expression of SETDB1 or TP53. Thus, TRIM28, SETDB1, and TP53 are dynamically expressed in porcine oocytes and embryos. Furthermore, TRIM28 and TP53 abundances in IVV and SCNT embryos are similar, but different from quantities in IVF embryos. Mol. Reprod. Dev. 81: 552–556, 2014. © 2014 The Authors. Published by Wiley Periodicals, Inc. PMID:24659575

Relationships between oxygen consumption rate, viability, and subsequent development of in vivo-derived porcine embryos.

PubMed

Sakagami, N; Nishida, K; Akiyama, K; Abe, H; Hoshi, H; Suzuki, C; Yoshioka, K

2015-01-01

Oxygen consumption rate of in vivo-derived porcine embryos was measured, and its value as an objective method for the assessment of embryo quality was evaluated. Embryos were surgically collected 5 or 6 days after artificial insemination (AI), and oxygen consumption rate of embryos was measured using an embryo respirometer. The average oxygen consumption rate (F × 10(14)/mol s(-1)) of the embryos that developed to the compacted morula stage on Day 5 (Day 0 = the day of artificial insemination) was 0.58 ± 0.03 (mean ± standard error of the mean). The Day-6 embryos had consumption rates of 0.56 ± 0.13, 0.87 ± 0.06, and 1.13 ± 0.07 at the early blastocyst, blastocyst, and expanded blastocyst stages, respectively, showing a gradual increase as the embryos developed. Just after collection, the average oxygen consumption rates of embryos that hatched and of those that did not hatch after culture were 0.60 ± 0.04 and 0.50 ± 0.04 for Day 5 (P = 0.08) and 1.05 ± 0.09 and 0.77 ± 0.05 for Day 6 (P < 0.05), respectively. The value and probability of discrimination by measuring the oxygen consumption rates of embryos to predict their hatching ability after culture were 0.56 and 63.6% for Day-5 embryos and 0.91 and 68.4% for Day-6 blastocysts, respectively. When Day-5 embryos were classified based on the oxygen consumption rate and then transferred non-surgically to recipient sows, three of the seven sows, to which embryos having a high oxygen consumption rate (≥ 0.59) were transferred, became pregnant and farrowed a total of 20 piglets. However, none of the four sows, to which embryos having low oxygen consumption rate (< 0.59) were transferred, became pregnant. These results suggest that the viability of in vivo-derived porcine embryos and subsequent development can be estimated by measuring the oxygen consumption rate. Copyright © 2015 Elsevier Inc. All rights reserved.

Diffusion of small molecules into medaka embryos improved by electroporation

PubMed Central

2013-01-01

Background Diffusion of small molecules into fish embryos is essential for many experimental procedures in developmental biology and toxicology. Since we observed a weak uptake of lithium into medaka eggs we started a detailed analysis of its diffusion properties using small fluorescent molecules. Results Contrary to our expectations, not the rigid outer chorion but instead membrane systems surrounding the embryo/yolk turned out to be the limiting factor for diffusion into medaka eggs. The consequence is a bi-phasic uptake of small molecules first reaching the pervitelline space with a diffusion half-time in the range of a few minutes. This is followed by a slow second phase (half-time in the range of several hours) during which accumulation in the embryo/yolk takes place. Treatment with detergents improved the uptake, but strongly affected the internal distribution of the molecules. Testing electroporation we could establish conditions to overcome the diffusion barrier. Applying this method to lithium chloride we observed anterior truncations in medaka embryos in agreement with its proposed activation of Wnt signalling. Conclusions The diffusion of small molecules into medaka embryos is slow, caused by membrane systems underneath the chorion. These results have important implications for pharmacologic/toxicologic techniques like the fish embryo test, which therefore require extended incubation times in order to reach sufficient concentrations in the embryos. PMID:23815821

Efficiency of porcine somatic cell nuclear transfer – a retrospective study of factors related to embryo recipient and embryos transferred

PubMed Central

Huang, Yongye; Ouyang, Hongsheng; Yu, Hao; Lai, Liangxue; Pang, Daxin; Li, Zhanjun

2013-01-01

Summary The successful generation of pigs via somatic cell nuclear transfer depends on reducing risk factors in several aspects. To provide an overview of some influencing factors related to embryo transfer, the follow-up data related to cloned pig production collected in our laboratory was examined. (i) Spring showed a higher full-term pregnancy rate compared with winter (33.6% vs 18.6%, P = 0.006). Furthermore, a regression equation can be drawn between full-term pregnancy numbers and pregnancy numbers in different months (y = 0.692x−3.326). (ii) There were no significant differences detected in the number of transferred embryos between surrogate sows exhibiting full-term development compared to those that did not. (iii) Non-ovulating surrogate sows presented a higher percentage of full-term pregnancies compared with ovulating sows (32.0% vs 17.5%, P = 0.004; respectively). (iv) Abortion was most likely to take place between Day 27 to Day 34. (v) Based on Life Table Survival Analysis, delivery in normally fertilized and surrogate sows is expected to be completed before Day 117 or Day 125, respectively. Additionally, the length of pregnancy in surrogate sows was negatively correlated with the average litter size, which was not found for normally fertilized sows. In conclusion, performing embryo transfer in appropriate seasons, improving the quality of embryos transferred, optimizing the timing of embryo transfer, limiting the occurrence of abortion, combined with ameliorating the management of delivery, is expected to result in the harvest of a great number of surviving cloned piglets. PMID:24244859

Effects of high gravity on amphibian development.

PubMed

Kashiwagi, Akihiko; Hanada, Hideki; Kawakami, Satomi; Kubo, Hideo; Shinkai, Tadashi; Fujii, Hirotada; Kashiwagi, Keiko

2003-10-01

In order to clarify the possible effects of high gravity environments on eggs and developing embryos, Rana rugosa and Xenopus laevis fertilized eggs and early embryos were raised in 2 G, 5 G, 7 G and 10 G up to the hatched tadpole stage. The results showed that: (1) High gravity significantly retarded the development of eggs and embryos beginning treatment before the blastula stage and induced various abnormalities, including two heads and microcephally suggesting that high gravity is apt to disrupt the animal-vegital axis. On the other hand, embryos beginning treatment after the gastrula stage showed a striking increase in the number of normal-appearing feeding tadpoles. (2) Autopsy revealed that brains, notochords and muscles were reduced in development and differentiation for embryos and tadpoles developed in high gravity. (3) It seems likely that the system for hydrogen peroxide detoxification develops abnormally in high gravity-treated embryos and tadpoles, which probably results in oxidative stress, leading to considerable cell damage.

Embryo quality and implantation rate in two different culture media: ISM1 versus Universal IVF Medium.

PubMed

Xella, Susanna; Marsella, Tiziana; Tagliasacchi, Daniela; Giulini, Simone; La Marca, Antonio; Tirelli, Alessandra; Volpe, Annibale

2010-04-01

To compare the outcome of two different culture media marketed by the MediCult AS Company (Jyllinge, Denmark)-Universal IVF Medium and ISM1 Medium culture-which, in addition to glucose, pyruvate, and energy-providing components, also contain amino acids, nucleotides, vitamins, and cholesterol. Laboratory and retrospective clinical study. University teaching hospital. A total of 726 patients, undergoing IVF-intracytoplasmic sperm injection procedure, comparable in mean age range, oocyte retrieval, and infertility indication, were included in the study. Laboratory quality and standard procedures were maintained unaffected. Oocyte retrieval, different embryo culture media. Embryo quality, ongoing pregnancy, and implantation rate. The frequency of good-quality embryos (79% vs. 74%) and the percentages of ongoing pregnancy (27.5% vs. 18%) and implantation rate (15% vs. 10%) were significantly higher in the group treated with ISM1 Medium rather than Universal IVF Medium. ISM1 Medium culture seems to improve the performance of embryonic growth and development, as well as increasing the percentage of pregnancy. Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Pretreatment of bovine sperm with dithiobutylamine (DTBA) significantly improves embryo development after ICSI

PubMed Central

SUTTIROJPATTANA, Tayita; SOMFAI, Tamas; MATOBA, Satoko; NAGAI, Takashi; PARNPAI, Rangsun; GESHI, Masaya

2016-01-01

We assessed the effect of pretreating sperm with dithiobutylamine (DTBA) to improve embryo development by intracytoplasmic sperm injection (ICSI) in cows. Acridine Orange staining revealed that when applied at different concentrations (2.5, 5, and 10 mM) and exposure times (5 min, 20 min, 1 h, and 2 h), DTBA reduced disulfide bonds in spermatozoa with the highest efficacy at 5 mM for 5 min. DTBA enhanced the percentage of spermatozoa with free protamine thiol groups compared with untreated spermatozoa (control) (P < 0.05); however, this result did not differ from that of dithiothreitol (DTT) treatment. The percentage of live spermatozoa after DTBA treatment was identical to that in the control, but significantly higher than that after DTT treatment (P < 0.05). After ICSI, DTBA treatment tended to improve male pronuclear formation rate (P = 0.071) compared with non-treated sperm injection. Blastocyst formation rate was significantly improved by DTBA treatment compared with that in DTT, control, and sham injection groups (P < 0.05). Blastocyst quality in terms of cell numbers and ploidy was not different among these groups. In conclusion, DTBA increases the efficacy of blastocyst production by ICSI even if DTT treatment does not work. PMID:27523189

POST-HARVEST EMBRYO DEVELOPMENT IN GINSENG SEEDS INCREASES DESICCATION SENSITIVITY AND NARROWS THE HYDRATION WINDOW FOR CRYOPRESERVATION.

PubMed

Han, E; Popova, E; Cho, G; Park, S; Lee, S; Pritchard, H W; Kim, H H

Despite its self-pollinating characteristics, Korean ginseng germplasm is mainly maintained in clonal gene banks as there is no defined approach to the long-term conservation of its seed, including the most appropriate stage of embryo development for storage. The aim of this study was to reveal the effect of embryo development on desiccation tolerance and cryopreservation success in ginseng seeds. Seeds of Korean ginseng (Panax ginseng C.A. Meyer) at three post-harvest stages (immediately after harvesting and following treatments to enable internal growth of the embryo) were desiccated and cryopreserved. The hydration window for the >80% dehiscence and germination of cryopreserved ginseng seeds varied with embryo developmental stage: 3-9% moisture content (MC) for both unpulped and undehisced seeds when the embryo was 0.1 the length of the endosperm, 7-10% MC for dehisced seeds (0.5 embryo:endosperm) and 9-11% MC for seeds with fully developed embryos (0.9 embryo:endosperm). Whilst dried (4-8% moisture content) and undehisced seeds within fruits (unpulped seeds) lost more than half their viability during 1 year's storage at room temperature, cryopreservation enabled germination levels of c. 90%. Overall, 432 accessions of Korean ginseng landraces have been cryopreserved using undehisced seeds with or without fruits. Post-harvest treatment of Korean ginseng seeds to enable embryo development decreases tolerance of very low MCs, and thus narrows the hydration window for cryopreservation. Fresh-harvested and unpulped seeds that have been dried to c. 5% MC are recommended for long-term cryogenic storage.

Comparison of in vitro developmental competence of cloned caprine embryos using donor karyoplasts from adult bone marrow mesenchymal stem cells vs ear fibroblast cells.

PubMed

Kwong, P J; Nam, H Y; Wan Khadijah, W E; Kamarul, T; Abdullah, R B

2014-04-01

The aim of this study was to produce cloned caprine embryos using either caprine bone marrow-derived mesenchymal stem cells (MSCs) or ear fibroblast cells (EFCs) as donor karyoplasts. Caprine MSCs were isolated from male Boer goats of an average age of 1.5 years. To determine the pluripotency of MSCs, the cells were induced to differentiate into osteocytes, chondrocytes and adipocytes. Subsequently, MSCs were characterized through cell surface antigen profiles using specific markers, prior to their use as donor karyoplasts for nuclear transfer. No significant difference (p > 0.05) in fusion rates was observed between MSCs (87.7%) and EFCs (91.3%) used as donor karyoplasts. The cleavage rate of cloned embryos derived with MSCs (87.0%) was similar (p > 0.05) to those cloned using EFCs (84.4%). However, the in vitro development of MSCs-derived cloned embryos (25.3%) to the blastocyst stage was significantly higher (p < 0.05) than those derived with EFCs (20.6%). In conclusion, MSCs could be reprogrammed by caprine oocytes, and production of cloned caprine embryos with MSCs improved their in vitro developmental competence, but not in their fusion and cleavage rate as compared to cloning using somatic cells such as EFCs. © 2014 Blackwell Verlag GmbH.

Analysis of compaction initiation in human embryos by using time-lapse cinematography.

PubMed

Iwata, Kyoko; Yumoto, Keitaro; Sugishima, Minako; Mizoguchi, Chizuru; Kai, Yoshiteru; Iba, Yumiko; Mio, Yasuyuki

2014-04-01

To analyze the initiation of compaction in human embryos in vitro by using time-lapse cinematography (TLC), with the goal of determining the precise timing of compaction and clarifying the morphological changes underlying the compaction process. One hundred and fifteen embryos donated by couples with no further need for embryo-transfer were used in this study. Donated embryos were thawed and processed, and then their morphological behavior during the initiation of compaction was dynamically observed via time-lapse cinematography (TLC) for 5 days. Although the initiation of compaction occurred throughout the period from the 4-cell to 16-cell stage, 99 (86.1 %) embryos initiated compaction at the 8-cell stage or later, with initiation at the 8-cell stage being most frequent (22.6 %). Of these 99 embryos, 49.5 % developed into good-quality blastocysts. In contrast, of the 16 (13.9 %) embryos that initiated compaction prior to the 8-cell stage, only 18.8 % developed into good-quality blastocysts. Embryos that initiated compaction before the 8-cell stage showed significantly higher numbers of multinucleated blastomeres, due to asynchronism in nuclear division at the third mitotic division resulting from cytokinetic failure. The initiation of compaction primarily occurs at the third mitotic division or later in human embryos. Embryos that initiate compaction before the 8-cell stage are usually associated with aberrant embryonic development (i.e., cytokinetic failure accompanied by karyokinesis).

Early Activation of MAPK and Apoptosis in Nutritive Embryos of Calyptraeid Gastropods.

PubMed

Lesoway, Maryna P; Collin, Rachel; Abouheif, Ehab

2017-07-01

Investigation of alternative phenotypes, different morphologies produced by a single genome, has contributed novel insights into development and evolution. Yet, the mechanisms underlying developmental switch points between alternative phenotypes remain poorly understood. The calyptraeid snails Crepidula navicella and Calyptraea lichen produce two phenotypes: viable and nutritive embryos, where nutritive embryos arrest their development after gastrulation and are ingested by their viable siblings as a form of intracapsular nutrition. Here, we investigate the activity of mitogen-activated protein kinase (MAPK, ERK1/2) and apoptosis during early cleavage. MAPK and apoptosis, found in a previous transcriptomic study, are known to be involved in organization of other spiralian embryos and nutritive embryo development, respectively. In the model Crepidula fornicata, MAPK activation begins at the 16-cell stage. In contrast, we discovered in C. navicella and C. lichen that many embryos begin MAPK activation at the one-cell stage. A subset of embryos shows a similar pattern of MAPK activation to C. fornicata at later stages. In all stages where MAPK is detected, the activation pattern is highly variable, frequently occurring in all quadrants or in multiple tiers of cells. We also detected apoptosis in cleaving embryos, while C. fornicata and Crepidula lessoni, which do not produce nutritive embryos, show no signs of apoptosis during cleavage. Our results show that MAPK and apoptosis are expressed during early development in species with nutritive embryos, and raises the possibility that these processes may play a role and even interact with one another in producing the nutritive embryo phenotype. © 2017 Wiley Periodicals, Inc.

Expression of renin–angiotensin system components in the early bovine embryo

PubMed Central

Pijacka, Wioletta; Hunter, Morag G; Broughton Pipkin, Fiona; Luck, Martin R

2012-01-01

The renin–angiotensin system (RAS), mainly associated with the regulation of blood pressure, has been recently investigated in female reproductive organs and the developing foetus. Angiotensin II (Ang II) influences oviductal gamete movements and foetal development, but there is no information about RAS in the early embryo. The aim of this study was to determine whether RAS components are present in the pre-implantation embryo, to determine how early they are expressed and to investigate their putative role at this stage of development. Bovine embryos produced in vitro were used for analysis of RAS transcripts (RT-PCR) and localisation of the receptors AGTR1 and AGTR2 (immunofluorescent labelling). We also investigated the effects of Ang II, Olmesartan (AGTR1 antagonist) and PD123319 (AGTR2 antagonist) on oocyte cleavage, embryo expansion and hatching. Pre-implanted embryos possessed AGTR1 and AGTR2 but not the other RAS components. Both receptors were present in the trophectoderm and in the inner cell mass of the blastocyst. AGTR1 was mainly localised in granular-like structures in the cytoplasm, suggesting its internalisation into clathrin-coated vesicles, and AGTR2 was found mainly in the nuclear membrane and in the mitotic spindle of dividing trophoblastic cells. Treating embryos with PD123319 increased the proportion of hatched embryos compared with the control. These results, the first on RAS in the early embryo, suggest that the pre-implanted embryo responds to Ang II from the mother rather than from the embryo itself. This may be a route by which the maternal RAS influences blastocyst hatching and early embryonic development. PMID:23781300

Early embryonic survival and embryo development in two lines of rabbits divergently selected for uterine capacity.

PubMed

Peiró, R; Santacreu, M A; Climent, A; Blasco, A

2007-07-01

The aim of this work is to study early embryo survival and development in 2 lines divergently selected for high and low uterine capacity throughout 10 generations. A total of 162 female rabbits from the high line and 133 from the low line were slaughtered at 25, 48, or 62 h of gestation. There were no differences in ovulation rate and fertilization rate between lines in any of the 3 stages of gestation. Embryo survival, estimated as the number of normal embryos recovered at a constant ovulation rate, was similar in both lines at 25 and 48 h. Embryo survival was greater in the high line [D (posterior mean of the difference between the high and low lines) = 0.57 embryos] at 62 h of gestation. There was no difference in embryonic stage of development at 25 h, but at 48 and 62 h of gestation, the high line, compared with the low line, had a greater percentage of early morulae (83 vs. 72%) and compacted morulae (55 vs. 38%). Divergent selection for uterine capacity appeared to modify embryo development, at least from 48 h of gestation, and embryo survival from 62 h.

The effects of superovulation of donor sows on ovarian response and embryo development after nonsurgical deep-uterine embryo transfer.

PubMed

Angel, M A; Gil, M A; Cuello, C; Sanchez-Osorio, J; Gomis, J; Parrilla, I; Vila, J; Colina, I; Diaz, M; Reixach, J; Vazquez, J L; Vazquez, J M; Roca, J; Martinez, E A

2014-04-01

This study aimed to evaluate the effectiveness of superovulation protocols in improving the efficiency of embryo donors for porcine nonsurgical deep-uterine (NsDU) embryo transfer (ET) programs. After weaning (24 hours), purebred Duroc sows (2-6 parity) were treated with 1000 IU (n = 27) or 1500 IU (n = 27) of eCG. Only sows with clear signs of estrus 4 to 72 hours after eCG administration were treated with 750 IU hCG at the onset of estrus. Nonhormonally treated postweaning estrus sows (n = 36) were used as a control. Sows were inseminated and subjected to laparotomy on Days 5 to 6 (Day 0 = onset of estrus). Three sows (11.1%) treated with the highest dosage of eCG presented with polycystic ovaries without signs of ovulation. The remaining sows from nonsuperovulated and superovulated groups were all pregnant, with no differences in fertilization rates among groups. The number of CLs and viable embryos was higher (P < 0.05) in the superovulated groups compared with the controls and increased (P < 0.05) with increasing doses of eCG. There were no differences among groups in the number of oocytes and/or degenerated embryos. The number of transferable embryos (morulae and unhatched blastocysts) obtained in pregnant sows was higher (P < 0.05) in the superovulated groups than in the control group. In all groups, there was a significant correlation between the number of CLs and the number of viable and transferable embryos, but the number of CLs and the number of oocytes and/or degenerated embryos were not correlated. A total of 46 NsDU ETs were performed in nonhormonally treated recipient sows, with embryos (30 embryos per transfer) recovered from the 1000-IU eCG, 1500-IU eCG, and control groups. In total, pregnancy and farrowing rates were 75.1% and 73.2%, respectively, with a litter size of 9.4 ± 0.6 piglets born, of which 8.8 ± 0.5 were born alive. There were no differences for any of the reproductive parameters evaluated among groups. In conclusion, our results demonstrated the efficiency of eCG superovulation treatments in decreasing the donor-to-recipient ratio. Compared with nonsuperovulated sows, the number of transferable embryos was increased in superovulated sows without affecting their quality and in vivo capacity to develop to term after transfer. The results from this study also demonstrate the effectiveness of the NsDU ET procedure used, making possible the commercial use of ET technology by the pig industry. Copyright © 2014 Elsevier Inc. All rights reserved.

Nucleoli from two-cell embryos support the development of enucleolated germinal vesicle oocytes in the pig.

PubMed

Kyogoku, Hirohisa; Ogushi, Sugako; Miyano, Takashi

2012-11-01

Recent research has shown that nucleoli of oocytes at the germinal vesicle (GV) stage (GV nucleoli) are not necessary for oocyte maturation but are essential for early embryonic development. Nucleoli of 2-cell embryos (2-cell nucleoli) have morphology similar to that of nucleoli in oocytes at the GV stage. In this study, we examined the ability of 2-cell nucleoli to substitute for GV nucleoli in terms of supporting early embryonic development by nucleolus aspiration (enucleolation) and transfer into metaphase II (MII) oocytes or 2-cell embryos that were derived from enucleolated oocytes at the GV stage in the pig. When 2-cell embryos were centrifuged to move the lipid droplets to one side of the blastomere, multiple nucleoli in the nucleus fused into a single nucleolus. The nucleoli were then aspirated from the 2-cell embryos by micromanipulation. The injection of 2-cell nucleoli to GV enucleolated oocytes at the MII stage rescued the embryos from the early embryonic arrest, and the resulting oocytes developed to blastocysts. However, the injection of 2-cell and GV nucleoli to 2-cell embryos derived from GV enucleolated oocytes rarely restored the development to blastocysts. These results indicate that 2-cell nucleoli support early embryonic development as GV nucleoli and that the presence of nucleoli is essential for pig embryos before the 2-cell stage.

Characterizing early embryonic development of Brown Tsaiya Ducks (Anas platyrhynchos) in comparison with Taiwan Country Chicken (Gallus gallus domestics)

PubMed Central

Lumsangkul, Chompunut; Fan, Yang-Kwang; Chang, Shen-Chang; Ju, Jyh-Cherng

2018-01-01

Avian embryos are among the most convenient and the primary representatives for the study of classical embryology. It is well-known that the hatching time of duck embryos is approximately one week longer than that of chicken embryos. However, the key features associated with the slower embryonic development in ducks have not been adequately described. This study aimed to characterize the pattern and the speed of early embryogenesis in Brown Tsaiya Ducks (BTD) compared with those in Taiwan Country Chicken (TCC) by using growth parameters including embryonic crown-tail length (ECTL), primitive streak formation, somitogenesis, and other development-related parameters, during the first 72 h of incubation. Three hundred and sixty eggs from BTD and TCC, respectively, were incubated at 37.2°C, and were then dissected hourly to evaluate their developmental stages. We found that morphological changes of TCC embryos shared a major similarity with that of the Hamburger and Hamilton staging system during early chick embryogenesis. The initial primitive streak in TCC emerged between 6 and 7 h post-incubation, but its emergence was delayed until 10 to 13 h post-incubation in BTD. Similarly, the limb primordia (wing and limb buds) were observed at 51 h post-incubation in TCC embryos compared to 64 h post-incubation in BTD embryos. The allantois first appeared around 65 to 68 h in TCC embryos, but it was not observed in BTD embryos. At the 72 h post-incubation, 40 somites were clearly formed in TCC embryos while only 32 somites in BTD embryos. Overall, the BTD embryos developed approximately 16 h slower than the chicken embryo during the first 72 h of development. To our best knowledge, this is the first study to describe two distinct developmental time courses between TCC and BTD, which would facilitate future embryogenesis-related studies of the two important avian species in Taiwan. PMID:29742160

Plantform Bioreactor for Mass Micropropagation of Date Palm.

PubMed

Almusawi, Abdulminam H A; Sayegh, Abdullah J; Alshanaw, Ansam M S; Griffis, John L

2017-01-01

A novel protocol for the commercial production of date palm through micropropagation is presented. This protocol includes the use of a semisolid medium alternation or in combination with a temporary immersion system (TIS, Plantform bioreactor) in date palm micropropagation. The use of the Plantform bioreactor for date palm results in an improved multiplication rate, reduced micropropagation time, and improved weaning success. It also reduces the cost of saleable units and thus improves economic return for commercial micropropagation. The use of the Plantform bioreactor successfully addresses other hindrances that can occur during the scale-up of date palm micropropagation, including asynchrony of somatic embryos, limited maturation of somatic embryos, and highly variable germination frequencies of embryos.

[Polarized light microscopy for evaluation of oocytes as a prognostic factor in the evolution of a cycle in assisted reproduction].

PubMed

González-Ortega, C; Cancino-Villarreal, P; Alonzo-Torres, V E; Martínez-Robles, I; Pérez-Peña, E; Gutiérrez-Gutiérrez, A M

2016-04-01

Identification of the best embryos to transfer is a key element for success in assisted reproduction. In the last decade, several morphological criteria of oocytes and embryos were evaluated with regard to their potential for predicting embryo viability. The introduction of polarization light microscopy systems has allowed the visualization of the meiotic spindle and the different layers of the zona pellucida in human oocytes on the basis of birefringence in a non-destructive way. Conflicting results have been reported regarding the predictive value in ICSI cycles. To assess the predictive ability of meiotic spindle and zona pellucida of human oocytes to implant by polarized microscopy in ICSI cycles. Prospective and observational clinical study. 903 oocytes from 94 ICSI cycles were analyzed with polarized microscopy. Meiotic spindle visualization and zona pellucida birefringence values by polarized microscopy were correlated with ICSI cycles results. Meiotic spindle visualization and birefringence values of zona pellucida decreased in a direct basis with increasing age. In patients aged over the 35 years, the percentage of a visible spindle and mean zona pellucida birefringence was lower than in younger patients. Fertilization rate were higher in oocytes with visible meiotic spindle (81.3% vs. 64%; p < 0.0001), as well as embryo quality (47.4% vs. 39%; p=0.01). Fertilization rate was higher in oocytes with positive values of birefringence (77.5 % vs. 68.5% p=0.005) with similar embryo quality. Conception cycles showed oocytes with higher mean value of zona birefringence and visible spindle vs. no-conception cycles (p<0.05). Polarized light microscopy improves oocyte selection, which significantly impacts in the development of embryos with greater implantation potential. The use of polarized light microscopy with sperm selection methods, blastocyst culture and deferred embryo transfers will contribute to transfer fewer embryos without diminishing rates of live birth and single embryo transfer will be more feasible.

Use of assisted reproduction for the improvement of milk production in dairy camels (Camelus dromedarius).

PubMed

Nagy, P; Skidmore, J A; Juhasz, J

2013-01-10

Despite their production potential and ability to survive on marginal resources in extreme conditions, dromedaries have not been exploited as an important food source. Camels have not been specifically selected for milk production, and genetic improvement has been negligible. High individual variation in milk production both within the population and within breeds provides a good base for selection and genetic progress. In this paper, we discuss the possibilities and constraints of selective breeding for milk production in camels, and include a summary of the use of embryo transfer at the world's first camel dairy farm. Embryo transfer is an integral part of the breeding strategy at the camel dairy farm because it increases selection intensity and decreases the generation interval. Using high milk-producing camels as donors and low producing camels as recipients, 146 embryos were recovered (6.1±1.0embryos/donor; range: 0-18). Embryos were transferred non-surgically into 111 recipients (83 single and 28 twin embryo transfers). Pregnancy rate at 21 days and 5 months was 55% (61/111) and 45% (50/111), respectively. Finally, a total of 46 recipients delivered a live calf. These results document the utility of embryo transfer using high milk producing dromedaries as donors. Copyright © 2012 Elsevier B.V. All rights reserved.

Tissue culture and associated biotechnological interventions for the improvement of coconut (Cocos nucifera L.): a review.

PubMed

Nguyen, Quang Thien; Bandupriya, H D Dharshani; López-Villalobos, Arturo; Sisunandar, S; Foale, Mike; Adkins, Steve W

2015-11-01

The present review discusses not only advances in coconut tissue culture and associated biotechnological interventions but also future research directions toward the resilience of this important palm crop. Coconut (Cocos nucifera L.) is commonly known as the 'tree of life'. Every component of the palm can be used to produce items of value and many can be converted into industrial products. Coconut cultivation faces a number of acute problems that reduce its productivity and competitiveness. These problems include various biotic and abiotic challenges as well as an unstable market for its traditional oil-based products. Around 10 million small-holder farmers cultivate coconut palms worldwide on c. 12 million hectares of land, and many more people own a few coconut palms that contribute to their livelihoods. Inefficiency in the production of seedlings for replanting remains an issue; however, tissue culture and other biotechnological interventions are expected to provide pragmatic solutions. Over the past 60 years, much research has been directed towards developing and improving protocols for (i) embryo culture; (ii) clonal propagation via somatic embryogenesis; (iii) homozygote production via anther culture; (iv) germplasm conservation via cryopreservation; and (v) genetic transformation. Recently other advances have revealed possible new ways to improve these protocols. Although effective embryo culture and cryopreservation are now possible, the limited frequency of conversion of somatic embryos to ex vitro seedlings still prevents the large-scale clonal propagation of coconut. This review illustrates how our knowledge of tissue culture and associated biotechnological interventions in coconut has so far developed. Further improvement of protocols and their application to a wider range of germplasm will continue to open up new horizons for the collection, conservation, breeding and productivity of coconut.

The Development of Motor Coordination in Drosophila Embryos

PubMed Central

Crisp, Sarah; Evers, Jan Felix; Fiala, André; Bate, Michael

2012-01-01

We use non-invasive muscle imaging to study onset of motor activity and emergence of coordinated movement in Drosophila embryos. Earliest movements are myogenic and neurally controlled muscle contractions first appear with the onset of bursting activity 17 hours after egg laying. Initial episodes of activity are poorly organised and coordinated crawling sequences only begin to appear after a further hour of bursting. Thus network performance improves during this first period of activity. The embryo continues to exhibit bursts of crawling like sequences until shortly before hatching, while other reflexes also mature. Bursting does not begin as a reflex response to sensory input but appears to reflect the onset of spontaneous activity in the motor network. It does not require GABA-ergic transmission, and using a light activated channel to excite the network we demonstrate activity dependent depression that may cause burst termination. PMID:18927150

Viability and DNA fragmentation of rainbow trout embryos (Oncorhynchus mykiss) obtained from eggs stored at 4 °C.

PubMed

Ubilla, A; Valdebenito, I; Árias, M E; Risopatrón, J

2016-05-01

In vitro storage of salmonid eggs leads to aging of the cells causing a decline in quality and reducing their capacity to develop and produce embryos. The quality of salmonid embryos is assessed by morphologic analyses; however, data on the application of biomarkers to determine the cell viability and DNA integrity of embryos in these species are limited. The aim of this study was to evaluate the effect on embryo development, viability and DNA fragmentation in the embryonic cells of in vitro storage time at 4 °C of rainbow trout (Oncorhynchus mykiss) eggs. The embryos were obtained by IVF from eggs stored for 0 (control), 48, and 96 hours at 4 °C. At 72 hours after fertilization, dechorionated embryos were examined to determine percentages of developed embryos (embryos with normal cell division morphology), viability (LIVE/DEAD sperm viability kit), and DNA integrity (terminal deoxynucleotidyl transferase [TdT] dUTP nick-end labeling assay). The percentage of developing embryos decreased (P < 0.05) with storage time of the eggs (95.10 ± 2.55; 88.14 ± 4.50; 79.99 ± 6.60 for 0, 48, and 96 hours, respectively). Similarly, cell viability decreased (P < 0.05; 96.07 ± 7.15; 80.42 ± 8.55; 77.47 ± 7.88 for 0, 48, and 96 hours, respectively), and an increase (P < 0.05) in DNA fragmentation in the embryos was observed at 96-hour storage. A positive correlation was found between cell DNA fragmentation and storage time (r = 0.8173; P < 0.0001). The results revealed that terminal deoxynucleotidyl transferase [TdT] dUTP nick-end labeling assay technique is reliable mean to assess the state of the DNA in salmonid embryos and that in vitro eggs storage for 96h reduces embryo development and cell DNA integrity. DNA integrity evaluation constitutes a biomarker of the quality of the ova and resulting embryos so as to predict their capacity to produce good-quality embryos in salmonids, particularly under culture conditions. Copyright © 2016 Elsevier Inc. All rights reserved.

Release of somatic embryogenic potential from excised zygotic embryos of carrot and maintenance of proembryonic cultures in hormone-free medium.

PubMed

Smith, D L; Krikorian, A D

1989-01-01

Excised zygotic embryos, mericarps ("seeds") and hypocotyls of seedlings of cultivated carrot Daucus carota cv. Scarlet Nantes were evaluated for their ability to generate somatic embryos on a semisolid hormone-free nutrient medium. Neither intact zygotic embryos nor hypocotyls ever produced somatic embryos. However, mericarps and broken zygotic embryos were excellent sources for somatic embryo production (response levels as high as 86%). Somatic embryo formation was highest from cotyledons, but was also observed on isolated hypocotyls and root tips of mature zygotic embryos. On media containing unreduced nitrogen, somatic embryo formation led to the generation of vigorous cultures comprised entirely of somatic embryos at various stages of development which in turn proliferated still other somatic embryos. However, a medium was devised which when 1-5 mM NH4+ was the sole nitrogen source, led only to a proliferation of globular proembryos. Sustained subculturing of these proembryos at 2-3 week intervals enabled establishment of highly uniform cultures in which no further development into more mature stages of embryonic development occurred. These have been maintained, without decline, as morphogenetically competent proembryonic globules for over ten months. A basal medium containing from 1-5 mM NH4+ as the sole nitrogen source appears not to be inductive to somatic proembryo formation. Instead, such a medium is best thought of as permissive to the expression of embryogenically determined cells within zygotic embryos. By excising and breaking or wounding zygotic embryos, constituent cells are probably released from positional or chemical restraints and thus are able to express their innate embryogenic potential. Once a proembryonic culture is established, this medium containing 1-5 mM NH4+ as the sole nitrogen source provides a nonpermissive environment to the development and growth of later embryonic stages, but it does allow the continued formation and multiplication of globular somatic proembryos. The sequence of events leading from excised broken zygotic embryos to the formation of somatic embryos and the maintenance of somatic proembryos are demonstrated by scanning electron microscopy and histological preparations. Germination levels from intact zygotic embryos on media with varying levels and ratios of unreduced vs. reduced inorganic nitrogen were determined as well and provided baseline or control data on the type of response obtained from nonwounded material.

Release of somatic embryogenic potential from excised zygotic embryos of carrot and maintenance of proembryonic cultures in hormone-free medium

NASA Technical Reports Server (NTRS)

Smith, D. L.; Krikorian, A. D.

1989-01-01

Excised zygotic embryos, mericarps ("seeds") and hypocotyls of seedlings of cultivated carrot Daucus carota cv. Scarlet Nantes were evaluated for their ability to generate somatic embryos on a semisolid hormone-free nutrient medium. Neither intact zygotic embryos nor hypocotyls ever produced somatic embryos. However, mericarps and broken zygotic embryos were excellent sources for somatic embryo production (response levels as high as 86%). Somatic embryo formation was highest from cotyledons, but was also observed on isolated hypocotyls and root tips of mature zygotic embryos. On media containing unreduced nitrogen, somatic embryo formation led to the generation of vigorous cultures comprised entirely of somatic embryos at various stages of development which in turn proliferated still other somatic embryos. However, a medium was devised which when 1-5 mM NH4+ was the sole nitrogen source, led only to a proliferation of globular proembryos. Sustained subculturing of these proembryos at 2-3 week intervals enabled establishment of highly uniform cultures in which no further development into more mature stages of embryonic development occurred. These have been maintained, without decline, as morphogenetically competent proembryonic globules for over ten months. A basal medium containing from 1-5 mM NH4+ as the sole nitrogen source appears not to be inductive to somatic proembryo formation. Instead, such a medium is best thought of as permissive to the expression of embryogenically determined cells within zygotic embryos. By excising and breaking or wounding zygotic embryos, constituent cells are probably released from positional or chemical restraints and thus are able to express their innate embryogenic potential. Once a proembryonic culture is established, this medium containing 1-5 mM NH4+ as the sole nitrogen source provides a nonpermissive environment to the development and growth of later embryonic stages, but it does allow the continued formation and multiplication of globular somatic proembryos. The sequence of events leading from excised broken zygotic embryos to the formation of somatic embryos and the maintenance of somatic proembryos are demonstrated by scanning electron microscopy and histological preparations. Germination levels from intact zygotic embryos on media with varying levels and ratios of unreduced vs. reduced inorganic nitrogen were determined as well and provided baseline or control data on the type of response obtained from nonwounded material.

Pretreatment with coenzyme Q10 improves ovarian response and embryo quality in low-prognosis young women with decreased ovarian reserve: a randomized controlled trial.

PubMed

Xu, Yangying; Nisenblat, Victoria; Lu, Cuiling; Li, Rong; Qiao, Jie; Zhen, Xiumei; Wang, Shuyu

2018-03-27

Management of women with reduced ovarian reserve or poor ovarian response (POR) to stimulation is one of the major challenges in reproductive medicine. The primary causes of POR remain elusive and oxidative stress was proposed as one of the important contributors. It has been suggested that focus on the specific subpopulations within heterogeneous group of poor responders could assist in evaluating optimal management strategies for these patients. This study investigated the effect of anti-oxidant treatment with coenzyme Q10 (CoQ10) on ovarian response and embryo quality in young low-prognosis patients with POR. This prospective, randomized controlled study included 186 consecutive patients with POR stratified according to the POSEIDON classification group 3 (age < 35, poor ovarian reserve parameters). The participants were randomized to the CoQ10 pre-treatment for 60 days preceding IVF-ICSI cycle or no pre-treatment. The number of high quality embryos was a primary outcome measure. A total of 169 participants were evaluated (76 treated with CoQ10 and 93 controls); 17 women were excluded due to low compliance with CoQ10 administration. The baseline demographic and clinical characteristics were comparable between the groups. CoQ10 pretreatment resulted in significantly lower gonadotrophin requirements and higher peak E2 levels. Women in CoQ10 group had increased number of retrieved oocytes (4, IQR 2-5), higher fertilization rate (67.49%) and more high-quality embryos (1, IQR 0-2); p < 0.05. Significantly less women treated with CoQ10 had cancelled embryo transfer because of poor embryo development than controls (8.33% vs. 22.89%, p = 0.04) and more women from treatment group had available cryopreserved embryos (18.42% vs. 4.3%, p = 0.012). The clinical pregnancy and live birth rates per embryo transfer and per one complete stimulation cycle tended to be higher in CoQ10 group but did not achieve statistical significance. Pretreatment with CoQ10 improves ovarian response to stimulation and embryological parameters in young women with poor ovarian reserve in IVF-ICSI cycles. Further work is required to determine whether there is an effect on clinical treatment endpoints.

Early Embryo Development in Fucus distichus Is Auxin Sensitive1

PubMed Central

Basu, Swati; Sun, Haiguo; Brian, Leigh; Quatrano, Ralph L.; Muday, Gloria K.

2002-01-01

Auxin and polar auxin transport have been implicated in controlling embryo development in land plants. The goal of these studies was to determine if auxin and auxin transport are also important during the earliest stages of development in embryos of the brown alga Fucus distichus. Indole-3-acetic acid (IAA) was identified in F. distichus embryos and mature tissues by gas chromatography-mass spectroscopy. F. distichus embryos accumulate [3H]IAA and an inhibitor of IAA efflux, naphthylphthalamic acid (NPA), elevates IAA accumulation, suggesting the presence of an auxin efflux protein complex similar to that found in land plants. F. distichus embryos normally develop with a single unbranched rhizoid, but growth on IAA leads to formation of multiple rhizoids and growth on NPA leads to formation of embryos with branched rhizoids, at concentrations that are active in auxin accumulation assays. The effects of IAA and NPA are complete before 6 h after fertilization (AF), which is before rhizoid germination and cell division. The maximal effects of IAA and NPA are between 3.5 and 5 h AF and 4 and 5.5 h AF, respectively. Although, the location of the planes of cell division was significantly altered in NPA- and IAA-treated embryos, these abnormal divisions occurred after abnormal rhizoid initiation and branching was observed. The results of this study suggest that auxin acts in the formation of apical basal patterns in F. distichus embryo development. PMID:12226509

Cinematographic analysis of bovine embryo development in serum-free oviduct-conditioned medium.

PubMed

Grisart, B; Massip, A; Dessy, F

1994-07-01

Development of bovine embryos produced in vitro from the one-cell to the blastocyst stage in serum-free oviduct-conditioned medium was investigated for 8 days consecutively by time-lapse cinematography. Three movies were analysed (130 embryos). The following observations were made. (1) Development under cine-recording conditions was similar to that in a classical incubator. (2) The highest proportion of embryos at the two-cell, three-four-cell, five-eight-cell, 9-16-cell, morula and blastocyst stages were recorded at 34, 46, 61, 115, 149 and 192 h after insemination, respectively. Cleavage asynchrony between blastomeres within individual embryos started at the two-cell stage. (3) The duration of the first three cell cycles was 35 h, 14 h and 11-62 h, respectively. (4) Detailed analysis of 13 embryos revealed that developmental arrest ('Lag-phase') occurred at the four-cell (1 of 13), five-cell (2 of 13), six-cell (3 of 13), seven-cell (3 of 13) or eight-cell stage (4 of 13); this phase lasted about 59 h. Embryos arrested at the eight-cell stage developed into morula-blastocysts (3 of 4) at a higher rate than did those arrested at earlier stages (2 of 9). (5) The faster the embryos cleaved into early stages (two-cell, three-four-cell and five-eight-cell), the higher the probability that they developed into morula-blastocyst: 70% of the embryos reaching the two-cell stage before 30-31 h after insemination developed into morula-blastocyst.(ABSTRACT TRUNCATED AT 250 WORDS)

Embryotoxic cytokines-Potential roles in embryo loss and fetal programming.

PubMed

Robertson, Sarah A; Chin, Peck-Yin; Femia, Joseph G; Brown, Hannah M

2018-02-01

Cytokines in the reproductive tract environment at conception mediate a dialogue between the embryo and maternal tissues to profoundly influence embryo development and implantation success. Through effects on gene expression and the cell stress response, cytokines elicit an epigenetic impact with consequences for placental development and fetal growth, which in turn affect metabolic phenotype and long-term health of offspring. There is substantial evidence demonstrating that pro-survival cytokines, such as GM-CSF, CSF1, LIF, HB-EGF and IGFII, support embryos to develop optimally. Less attention has been paid to cytokines that adversely impact embryo development, including the pro-inflammatory cytokines TNF, TRAIL and IFNG. These agents elicit cell stress, impair cell survival and retard blastocyst development, and at sufficiently high concentrations, can cause embryo demise. Experiments in mice suggest these so-called 'embryotoxic' cytokines can harm embryos through pro-apoptotic and adverse programming effects, as well as indirectly suppressing uterine receptivity through the maternal immune response. Embryotrophic factors may mitigate against and protect from these adverse effects. Thus, the balance between embryotrophic and embryotoxic cytokines can impart effects on embryo development and implantation, and has the potential to contribute to endometrial 'biosensor' function to mediate embryo selection. Embryotoxic cytokines can be elevated in plasma and reproductive tract tissues in inflammatory conditions including infection, diabetes, obesity, PCOS and endometriosis. Studies are therefore warranted to investigate whether excessive embryotoxic cytokines contribute to infertility and recurrent implantation failure in women, and compromised reproductive performance in livestock animals. Copyright © 2018 Elsevier B.V. All rights reserved.

Using game theory to investigate the epigenetic control mechanisms of embryo development. Comment on: ;Epigenetic game theory: How to compute the epigenetic control of maternal-to-zygotic transition; by Qian Wang et al.

NASA Astrophysics Data System (ADS)

Zhang, Le; Zhang, Shaoxiang

2017-03-01

A body of research [1-7] has already shown that epigenetic reprogramming plays a critical role in maintaining the normal development of embryos. However, the mechanistic quantitation of the epigenetic interactions between sperms and oocytes and the related impact on embryo development are still not clear [6,7]. In this study, Wang et al., [8] develop a modeling framework that addresses this question by integrating game theory and the latest discoveries of the epigenetic control of embryo development.

EMAGE mouse embryo spatial gene expression database: 2010 update

PubMed Central

Richardson, Lorna; Venkataraman, Shanmugasundaram; Stevenson, Peter; Yang, Yiya; Burton, Nicholas; Rao, Jianguo; Fisher, Malcolm; Baldock, Richard A.; Davidson, Duncan R.; Christiansen, Jeffrey H.

2010-01-01

EMAGE (http://www.emouseatlas.org/emage) is a freely available online database of in situ gene expression patterns in the developing mouse embryo. Gene expression domains from raw images are extracted and integrated spatially into a set of standard 3D virtual mouse embryos at different stages of development, which allows data interrogation by spatial methods. An anatomy ontology is also used to describe sites of expression, which allows data to be queried using text-based methods. Here, we describe recent enhancements to EMAGE including: the release of a completely re-designed website, which offers integration of many different search functions in HTML web pages, improved user feedback and the ability to find similar expression patterns at the click of a button; back-end refactoring from an object oriented to relational architecture, allowing associated SQL access; and the provision of further access by standard formatted URLs and a Java API. We have also increased data coverage by sourcing from a greater selection of journals and developed automated methods for spatial data annotation that are being applied to spatially incorporate the genome-wide (∼19 000 gene) ‘EURExpress’ dataset into EMAGE. PMID:19767607

Supplementation with nanomolar concentrations of verbascoside during in vitro maturation improves embryo development by protecting the oocyte against oxidative stress: a large animal model study.

PubMed

Martino, Nicola Antonio; Ariu, Federica; Bebbere, Daniela; Uranio, Manuel Filioli; Chirico, Adriana; Marzano, Giuseppina; Sardanelli, Anna Maria; Cardinali, Angela; Minervini, Fiorenza; Bogliolo, Luisa; Dell'Aquila, Maria Elena

2016-10-01

The effects of verbascoside (VB), added at nanomolar concentrations during in vitro maturation (IVM) of juvenile sheep oocytes, on in vitro embryo development and its mechanisms of action at the oocyte level were analyzed. Developmental rates, after IVM in the presence/absence of VB (1nM for 24h; 1nM for 2h; 10nM for 2h), were evaluated. The bioenergetic/oxidative status of oocytes matured after IVM in the presence/absence of 1nM VB for 24h was assessed by confocal analysis of mitochondria and reactive oxygen species (ROS), lipid peroxidation (LPO) assay, and quantitative PCR of bioenergy/redox-related genes. The addition of 1nM VB during 24h IVM significantly increased blastocyst formation and quality. Verbascoside reduced oocyte ROS and LPO and increased mitochondria/ROS colocalization while keeping mitochondria activity and gene expression unchanged. In conclusion, supplementation with nanomolar concentrations of VB during IVM, in the juvenile sheep model, promotes embryo development by protecting the oocyte against oxidative stress. Copyright © 2016 Elsevier Inc. All rights reserved.

Incubation media affect the survival, pathway and time of embryo development in Neotropical annual fish Austrolebias nigrofasciatus (Rivulidae).

PubMed

da Fonseca, A P; Volcan, M V; Robaldo, R B

2018-01-01

To analyse the survival, pathway and time of embryo development in the annual fish Austrolebias nigrofasciatus eggs were monitored in four liquid media and two damp media under experimental conditions for 130 days until their development was complete. Eggs kept in the same breeding water from oviposition remained in diapause I (DI) during all experiments. In constrast, up to the stage prior to entering diapause II (DII), the other media had no influence on development. Embryos at this stage (DII), however, show longer development time when treated in medium with water and powdered coconut shell so that about 80% of embryos remained in DII at 100 days. In contrast, all other treatments had a significantly lower proportion of embryos remaining in DII. When treated with Yamamoto's solution in humid media, embryos showed the fastest development. The first fully developed embryos (DIII) were seen at 27 days after oviposition. It took an average of 46-58 days for 50% of eggs in each treatment to reach DIII. Compared with other studies, survival in all incubation media was high at between 70 and 98%. Taken together, it can be concluded that all incubation media were found to be viable for maintaining embryos. Altering developmental trajectories through the manipulation of diapauses in different media makes this species a potential model organism for laboratory studies. © 2017 The Fisheries Society of the British Isles.

Fertilization and embryo recovery rates in superovulated chios ewes after laparoscopic intrauterine insemination.

PubMed

Lymberopoulos, A G; Amiridis, G S; Kühholzer, B; Besenfelder, U; Christodoulou, V; Vainas, E; Brem, G

2001-06-01

Forty superovulated dairy ewes of the Greek Chios breed were used in an experiment to evaluate the efficiency of laparoscopic intrauterine insemination on fertilization and embryo recovery rates as well as embryo quality. Estrus was synchronized by intravaginal progestagen impregnated sponges and superovulation was induced by administration of 8.8 mg o-FSH i.m. following a standard 8 dose protocol. A small volume (0.3 mL) of diluted fresh ram semen was deposited in each uterine horn 24 to 28 h after onset of the estrus by a laparoscopic technique. The animals were allocated randomly into two groups (Group A and B) of 20 animals each. In Group A, embryos were recovered 18 to 24 h after the intrauterine insemination and in Group B on Day 6. The average number of corpora lutea was 12.8 +/- 1.2 and 11.5 +/- 1.1 (+/- SEM); the overall embryo recovery was 66.4% and 57% and the percentage of recovered fertilized ova was 81% and 82.8% in Groups A and B, respectively. More fertilized ova were collected per ewe from Group A (P < or = 0.1). Results indicated that in Chios breed, superovulation using homologous FSH combined with laparoscopic AI leads to good ovarian response with satisfactory results in fertilization, embryo recovery and quality of embryos. This could lead to improved and more efficient methods for obtaining large numbers of high quality oocytes and embryos for embryo transfer programs which could contribute to genetic improvement and increase of the population size.

Synthetic profiles of polypeptides of human oocytes and normal and abnormal preimplantation embryos.

PubMed

Capmany, G; Bolton, V N

1999-09-01

There is considerable variation in the rate of development in vitro of individual preimplantation human embryos. The relationship between the rate of development and patterns of polypeptide synthesis in individual embryos was examined using SDS-PAGE and autoradiography. After incubation in [35S]methionine, 19 polypeptide bands were identified that change between fertilization and the morula stage. Although changes in two of the bands occurred in embryos that were developing normally and in ageing oocytes, and are thus independent of fertilization, the changes identified in the remaining 17 bands occurred only after fertilization. In embryos that were developing abnormally, as assessed by delayed cleavage, cleavage arrest or extensive fragmentation, the alteration in polypeptide synthetic profiles increased with increasing abnormality.

Expression of microRNAs in bovine and human pre-implantation embryo culture media

PubMed Central

Kropp, Jenna; Salih, Sana M.; Khatib, Hasan

2014-01-01

MicroRNAs (miRNA) are short non-coding RNAs which act to regulate expression of genes driving numerous cellular processes. These RNAs are secreted within exosomes from cells into the extracellular environment where they may act as signaling molecules. In addition, they are relatively stable and are specifically expressed in association to certain cancers making them strong candidates as biological markers. Moreover, miRNAs have been detected in body fluids including urine, milk, saliva, semen, and blood plasma. However, it is unknown whether they are secreted by embryonic cells into the culture media. Given that miRNAs are expressed throughout embryonic cellular divisions and embryonic genome activation, we hypothesized that they are secreted from the embryo into the extracellular environment and may play a role in the developmental competence of bovine embryos. To test this hypothesis, bovine embryos were cultured individually from day 5 to day 8 of development in an in vitro fertilization system and gene expression of 5 miRNAs was analyzed in both embryos and culture media. Differential miRNA gene expression was observed between embryos that developed to the blastocyst stage and those that failed to develop from the morula to blastocyst stage, deemed degenerate embryos. MiR-25, miR-302c, miR-196a2, and miR-181a expression was found to be higher in degenerate embryos compared to blastocyst embryos. Interestingly, these miRNAs were also found to be expressed in the culture media of both bovine and human pre-implantation embryos. Overall, our results show for the first time that miRNAs are secreted from pre-implantation embryos into culture media and that miRNA expression may correlate with developmental competence of the embryo. Expression of miRNAs in in vitro culture media could allow for the development of biological markers for selection of better quality embryos and for subsequent successful pregnancy. PMID:24795753

Is it time for a paradigm shift in understanding embryo selection?

PubMed

Gleicher, Norbert; Kushnir, Vitaly A; Barad, David H

2015-01-11

Embryo selection has been an integral feature of in vitro fertilization (IVF) almost since its inception. Since the advent of extended blastocyst stage embryo culture, and especially with increasing popularity of elective single embryo transfer (eSET), the concept of embryo selection has increasingly become a mainstay of routine IVF. We here, however, argue that embryo selection via blastocyst stage embryo transfer (BSET), as currently practiced, at best improves IVF outcomes only for a small minority of patients undergoing IVF cycles. For a large majority BSET is either ineffective or, indeed, may actually be harmful by decreasing IVF pregnancy chances. Overall, only a small minority of patients, thus, benefit from prolonged embryo culture, while BSET, as a tool to enhance IVF outcomes, is increasingly utilized as routine care in IVF for all patients. Since newer methods of embryo selection, like preimplantation genetic screening (PGS) and closed system embryo incubation with time-lapse photography are practically dependent on BSET, these concepts of embryo selection, currently increasingly adopted in mainstream IVF, require reconsideration. They, automatically, transfer the downsides of BSET, including decreases in IVF pregnancy chances in some patients, to these new procedures, and in addition raise serious questions about cost-effectiveness.

A Cascade of Sequentially Expressed Sucrose Transporters in the Seed Coat and Endosperm Provides Nutrition for the Arabidopsis Embryo[OPEN

PubMed Central

Chen, Li-Qing; Lin, I Winnie; Qu, Xiao-Qing; Sosso, Davide; McFarlane, Heather E.; Londoño, Alejandra; Samuels, A. Lacey; Frommer, Wolf B.

2015-01-01

Developing plant embryos depend on nutrition from maternal tissues via the seed coat and endosperm, but the mechanisms that supply nutrients to plant embryos have remained elusive. Sucrose, the major transport form of carbohydrate in plants, is delivered via the phloem to the maternal seed coat and then secreted from the seed coat to feed the embryo. Here, we show that seed filling in Arabidopsis thaliana requires the three sucrose transporters SWEET11, 12, and 15. SWEET11, 12, and 15 exhibit specific spatiotemporal expression patterns in developing seeds, but only a sweet11;12;15 triple mutant showed severe seed defects, which include retarded embryo development, reduced seed weight, and reduced starch and lipid content, causing a “wrinkled” seed phenotype. In sweet11;12;15 triple mutants, starch accumulated in the seed coat but not the embryo, implicating SWEET-mediated sucrose efflux in the transfer of sugars from seed coat to embryo. This cascade of sequentially expressed SWEETs provides the feeding pathway for the plant embryo, an important feature for yield potential. PMID:25794936

Prognostic factors for patients undergoing vitrified-warmed human embryo transfer cycles: a retrospective cohort study.

PubMed

Takahashi, Toshifumi; Hasegawa, Ayumi; Igarashi, Hideki; Amita, Mitsuyoshi; Matsukawa, Jun; Takehara, Isao; Suzuki, Satoko; Nagase, Satoru

2017-06-01

We examined the prognostic factors for pregnancy in 210 vitrified-warmed embryo transfer (ET) cycles in 121 patients. The univariate analysis showed that age, gravida, the number of cycles associated with infertility caused by endometriosis, the number of previous assisted reproductive technology (ART) treatment cycles, and the number of ICSI procedures were significantly lower in pregnant cycles compared with non-pregnant cycles. The percentages of ET using at least one intact embryo and of ET using at least one embryo that had developed further after warming were significantly higher in pregnant cycles compared with non-pregnant cycles. Multivariate logistic regression analysis showed that previous ART treatment cycles, ET with at least one intact embryo, and ET using at least one embryo that had developed further were independent prognostic factors for pregnancy in vitrified-warmed ET cycles. We conclude that fewer previous ART treatment cycles, ET using at least one intact embryo, and ET with embryos that have developed further after warming might be favourable prognostic factors for pregnancy in vitrified-warmed ET cycles.

Influence of cell loss after vitrification or slow-freezing on further in vitro development and implantation of human Day 3 embryos.

PubMed

Van Landuyt, L; Van de Velde, H; De Vos, A; Haentjens, P; Blockeel, C; Tournaye, H; Verheyen, G

2013-11-01

Is the effect of cell loss on further cleavage and implantation different for vitrified than for slowly frozen Day 3 embryos? Vitrified embryos develop better overnight than slowly frozen embryos, regardless of the number of cells lost, but have similar implantation potential if further cleavage occurs overnight. After slow-freezing, similar implantation rates have been obtained for intact 4-cell embryos or 4-cell embryos with 1 cell damaged. For slowly frozen Day 3 embryos, lower implantation rates have been observed when at least 25% of cells were lost. Other studies reported similar implantation potential for 7- to 8-cell embryos with 0, 1 or 2 cells damaged. No data are available on further development of vitrified embryos in relation to cell damage. Survival and overnight cleavage were retrospectively assessed for 7664 slowly frozen Day 3 embryos (study period: January 2004-December 2008) and 1827 vitrified embryos (study period: April 2010-September 2011). Overnight cleavage was assessed according to cell stage at cryopreservation and post-thaw cell loss for both protocols. The relationship between cell loss and implantation rate was analysed in a subgroup of single-embryo transfers (SETs) with 780 slowly frozen and 294 vitrified embryos. Embryos with ≥6 blastomeres and ≤20% fragmentation were cryopreserved using slow controlled freezing [with dimethyl sulphoxide (DMSO) as cryoprotectant] or closed vitrification [with DMSO-ethylene glycol (EG)-sucrose (S) as cryoprotectants]. Only embryos with ≥50% of cells intact after thawing were cultured overnight and were only transferred if further cleaved. For each outcome, logistic regression analysis was performed. Survival was 94 and 64% after vitrification and slow-freezing respectively. Logistic regression analysis showed that overnight cleavage of surviving embryos was higher after vitrification than after slow-freezing (P < 0.001) and decreased according to the degree of cell damage (P < 0.001). If the embryo continued to cleave after thawing, there was no effect of the number of cells lost or the cryopreservation method on its implantation potential. The implantation rates of embryos with 0, 1 or 2 cells damaged were, respectively, 21% (n = 114), 21% (n = 28) and 20% (n = 12) after slow-freezing and 20% (n = 50), 21% (n = 5) and 27% (n = 4) after vitrification. This analysis is retrospective and study periods for vitrification and slow-freezing are different. The number of SETs with vitrified embryos is limited. However, a large number of vitrified embryos were available to analyse the further cleavage of surviving embryos. Although it is not proved that vitrified embryos are more viable than slowly frozen embryos in terms of pregnancy outcome, vitrification yields higher survival rates, better overnight development and higher transfer rates per embryo warmed. This increases the number of frozen transfer cycles originating from a single treatment and might result in a better cumulative clinical outcome. Based on the present data, the policy to warm an extra embryo before overnight culture depends on the cell stage at cryopreservation and the cell damage after warming. For 8-cell embryos, up to two cells may be damaged compared with only one cell for 6- to 7-cell embryos, before an additional embryo is warmed. none.

Comparison of effects of albendazole sulfoxide on in vitro produced bovine embryos and rat embryos.

PubMed

Piscopo, S E; Smoak, I W

1997-09-01

To evaluate and compare effects of albendazole sulfoxide (ABZSO) on rat embryos and bovine embryos produced in vitro. In vitro produced bovine embryos. Rat embryos recovered from naturally bred Sprague-Dawley rats. 4- and 8-cell bovine embryos were randomly allocated to ABZSO or vehicle control groups. After 48 hours, embryos were evaluated for cell number and blastomere morphology. Rat embryos of similar stages, flushed from the uterine tube on gestational day 2-5, were randomly allocated to treatment or control groups. After 24 hours, embryos were evaluated as described previously. 44% of control bovine embryos divided in culture (> or = 16-cell stage). Fifteen percent of the controls had morphologic abnormalities, including disparity in blastomere size and cytoplasmic vacuoles and stippling. Treated (> or = 1 microgram of ABZSO/ml) bovine embryos differed (P < 0.0001) from controls, with 4% development and 93% abnormal morphology. Forty-five percent of control rat embryos divided in culture. Treated (> or = 500 ng of ABZSO/ml) rat embryos differed (P < 0.0003) from controls with regard to ability to divide. There were no consistent morphologic abnormalities in rat embryos. In vitro produced bovine embryos were susceptible to ABZSO at a concentration > or = 1 microgram/ ml, resulting in decreased ability to divide and presence of gross morphologic abnormalities. Rat embryos produced in vivo and exposed in vitro to ABZSO at a concentration > or = 500 ng/ml had decreased ability to divide in culture. Despite severe effects of ABZSO (> or = 1 microgram/ml) on bovine embryo development in vitro, it is beyond the scope of this study to speculate whether a therapeutic dosage of albendazole (10 mg/kg of body weight) would result in necessary concentrations of ABZSO in vivo to disrupt embryogenesis.

Quantifying Three-Dimensional Morphology and RNA from Individual Embryos

PubMed Central

Green, Rebecca M.; Leach, Courtney L.; Hoehn, Natasha; Marcucio, Ralph S.; Hallgrímsson, Benedikt

2017-01-01

Quantitative analysis of morphogenesis aids our understanding of developmental processes by providing a method to link changes in shape with cellular and molecular processes. Over the last decade many methods have been developed for 3D imaging of embryos using microCT scanning to quantify the shape of embryos during development. These methods generally involve a powerful, cross-linking fixative such as paraformaldehyde to limit shrinkage during the CT scan. However, the extended time frames that these embryos are incubated in such fixatives prevent use of the tissues for molecular analysis after microCT scanning. This is a significant problem because it limits the ability to correlate variation in molecular data with morphology at the level of individual embryos. Here, we outline a novel method that allows RNA, DNA or protein isolation following CT scan while also allowing imaging of different tissue layers within the developing embryo. We show shape differences early in craniofacial development (E11.5) between common mouse genetic backgrounds, and demonstrate that we are able to generate RNA from these embryos after CT scanning that is suitable for downstream RT-PCR and RNAseq analyses. PMID:28152580

Efficient harvesting methods for early-stage snake and turtle embryos.

PubMed

Matsubara, Yoshiyuki; Kuroiwa, Atsushi; Suzuki, Takayuki

2016-04-01

Reptile development is an intriguing research target for understating the unique morphogenesis of reptiles as well as the evolution of vertebrates. However, there are numerous difficulties associated with studying development in reptiles. The number of available reptile eggs is usually quite limited. In addition, the reptile embryo is tightly adhered to the eggshell, making it a challenge to isolate reptile embryos intact. Furthermore, there have been few reports describing efficient procedures for isolating intact embryos especially prior to pharyngula stage. Thus, the aim of this review is to present efficient procedures for obtaining early-stage reptilian embryos intact. We first describe the method for isolating early-stage embryos of the Japanese striped snake. This is the first detailed method for obtaining embryos prior to oviposition in oviparous snake species. Second, we describe an efficient strategy for isolating early-stage embryos of the soft-shelled turtle. © 2016 Japanese Society of Developmental Biologists.

Shortening gametes co-incubation time improves live birth rate for couples with a history of fragmented embryos.

PubMed

Le Bras, Anne; Hesters, Laetitia; Gallot, Vanessa; Tallet, Cathie; Tachdjian, Gerard; Frydman, Nelly

2017-10-01

Short gamete co-incubation (SGCO) consists in decreasing the duration of contact between oocytes and sperm from the standard overnight insemination (SOI) toward 2 hours. However, the effectiveness of this technique to improve in vitro fertilization and embryo transfer (IVF-ET) outcomes remains controversial. Our study was designed to evaluate the efficiency of SGCO in a poor prognosis population with a history of fragmented embryos defined by the presence of at least 50% of the embryos with more than 25% of cytoplasmic fragments. From January 2010 to January 2014, 97 couples were included in a SGCO protocol. We separated women into 2 subgroups: younger and older than 35 years. Compared to SOI, after SGCO, 2-cell stage embryos were higher in all women (p<0.001) and less fragmented in women over 35 years (p<0.05). On day 2, top quality embryos obtained and transferred were higher with SCGO than with SOI, independently of the age of the women (p<0.001). Moreover, the number of embryos with less than 25% of fragmentation was higher after SGCO than SOI (p<0.001) whereas the number of multinucleated embryos was lower (p<0.001). We observed that after fresh ET, independently of the age of the women, the clinical pregnancy rate was 3 times higher after SGCO than after SOI. However, the live-birth rate was 4 times higher with SGCO than with SOI in women above 35 years but 3 times higher with SGCO than with SOI in women younger than 35 years. The present results indicate that for a particular indication, reducing the time of oocytes and sperm co-incubation may improve IVF-ET outcomes in terms of live-birth rate. AMH: anti mullerian hormone; COC: cumulus-oocytes complex; E2: estradiol; ET: embryo transfer; FET: frozen embryo transfer; FSH: follicle stimulating hormone; GnRH: gonadotrophin releasing hormone; hCG: human chorionic gonadotropin hormone; hMG: human menopausal gonadotropin hormone; IRB: institutional review board; IVF: in vitro fertilization; IVF-ET: in vitro fertilization and embryo transfer; MNB: multinucleated blastomere; mRNA: messanger ribonucleic acid; OC: oocyte retrieval; O2: oxygen; ROS: reactive oxygen species; SGCO: short gamete co-incubation; SOI: standard overnight insemination.

Chick ex ovo culture and ex ovo CAM assay: how it really works.

PubMed

Dohle, Daniel S; Pasa, Susanne D; Gustmann, Sebastian; Laub, Markus; Wissler, Josef H; Jennissen, Herbert P; Dünker, Nicole

2009-11-30

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study angiogenesis but also to study tumorigenesis. After the chick chorioallantoic membrane (CAM) has developed, its blood vessel network can be easily accessed, manipulated and observed and therefore provides an optimal setting for angiogenesis assays. Since the lymphoid system is not fully developed until late stages of incubation, the chick embryo serves as a naturally immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition to nurturing developing allo- and xenografts, the CAM blood vessel network provides a uniquely supportive environment for tumor cell intravasation, dissemination, and vascular arrest and a repository where arrested cells extravasate to form micro metastatic foci. For experimental purposes, in most of the recent studies the CAM was exposed by cutting a window through the egg shell and experiments were carried out in ovo, resulting in significant limitations in the accessibility of the CAM and possibilities for observation and photo documentation of effects. When shell-less cultures of the chick embryo were used(1-4), no experimental details were provided and, if published at all, the survival rates of these cultures were low. We refined the method of ex ovo culture of chick embryos significantly by introducing a rationally controlled extrusion of the egg content. These ex ovo cultures enhance the accessibility of the CAM and chick embryo, enabling easy in vivo documentation of effects and facilitating experimental manipulation of the embryo. This allows the successful application to a large number of scientific questions: (1) As an improved angiogenesis assay(5,6), (2) an experimental set up for facilitated injections in the vitreous of the chick embryo eye(7-9), (3) as a test environment for dissemination and intravasation of dispersed tumor cells from established cell lines inoculated on the CAM(10-12), (4) as an improved sustaining system for successful transplantation and culture of limb buds of chicken and mice(13) as well as (5) for grafting, propagation, and re-grafting of solid primary tumor tissue obtained from biopsies on the surface of the CAM(14). In this video article we describe the establishment of a refined chick ex ovo culture and CAM assay with survival rates over 50%. Besides we provide a step by step demonstration of the successful application of the ex ovo culture for a large number of scientific applications.

Simple and efficient production of embryonic stem cell-embryo chimeras by coculture.

PubMed Central

Wood, S A; Pascoe, W S; Schmidt, C; Kemler, R; Evans, M J; Allen, N D

1993-01-01

A method for the production of embryonic stem (ES) cell-embryo chimeras was developed that involves the simple coculture of eight-cell embryos on a lawn of ES cells. After coculture, the embryos with ES cells attached are transferred to normal embryo culture medium and allowed to develop to the blastocyst stage before reimplantation into foster mothers. Although the ES cells initially attach to the outside of the embryos, they primarily colonize the inner cell mass and its derivatives. This method results in the efficient production of chimeras with high levels of chimerism including the germ line. As embryos are handled en masse and manipulative steps are minimal, this method should greatly reduce the time and effort required to produce chimeric mice. Images Fig. 1 Fig. 2 PMID:8506303

Cooling strategies for brazilian flounder Paralichthys orbignyanus embryos.

PubMed

Varela, A S; Cardoso, T F; Fernandes E Silva, E; Goularte, K L; Okamoto, M H; Sampaio, L A; Jardim, R D; Corcini, C D

Paralichthys orbignyanus is the species of the greatest potential for marine and estuarine fish farming in southern Brazil. Consequently, embryo cryopreservation becomes an important tool for increasing their production. To evaluate the effects of cooling protocols on the viability of embryos of P. orbignyanus at two stages of development (neurula and early differentiation of the tail). Control embryos were maintained at 23 degree C and treated embryos were cooled to 15 degree C, 10 degree C and 5 degree C at rapid, moderate and slow cooling rates. Then embryos were maintained at these different temperatures for 30, 60 and 90 min and the loss of viability assessed as hatching rates (HR) and morphologically normal larvae (MNL). The average HR for embryos following cooling was higher for those at the tail stage compared to the neurula stage (P<0.05). In both stages there was no statistical difference between the HR of control embryos and those exposed to rapid cooling. Also for tail stage embryos, there was no difference between MNL of control and rapidly cooled embryos. As first steps in the development of cryopreservation methods for P. orbignyanus embryos, the use of a rapid cooling and holding at 5 degree C for 30 min are recommended.

Oxidative Damage to Rhesus Macaque Spermatozoa Results in Mitotic Arrest and Transcript Abundance Changes in Early Embryos1

PubMed Central

Burruel, Victoria; Klooster, Katie L.; Chitwood, James; Ross, Pablo J.; Meyers, Stuart A.

2013-01-01

ABSTRACT Our objective was to determine whether oxidative damage of rhesus macaque sperm induced by reactive oxygen species (ROS) in vitro would affect embryo development following intracytoplasmic sperm injection (ICSI) of metaphase II (MII) oocytes. Fresh rhesus macaque spermatozoa were treated with ROS as follows: 1 mM xanthine and 0.1 U/ml xanthine oxidase (XXO) at 37°C and 5% CO2 in air for 2.25 h. Sperm were then assessed for motility, viability, and lipid peroxidation. Motile ROS-treated and control sperm were used for ICSI of MII oocytes. Embryo culture was evaluated for 3 days for development to the eight-cell stage. Embryos were fixed and stained for signs of cytoplasmic and nuclear abnormalities. Gene expression was analyzed by RNA-Seq in two-cell embryos from control and treated groups. Exposure of sperm to XXO resulted in increased lipid peroxidation and decreased sperm motility. ICSI of MII oocytes with motile sperm induced similar rates of fertilization and cleavage between treatments. Development to four- and eight-cell stage was significantly lower for embryos generated with ROS-treated sperm than for controls. All embryos produced from ROS-treated sperm demonstrated permanent embryonic arrest and varying degrees of degeneration and nuclear fragmentation, changes that are suggestive of prolonged senescence or apoptotic cell death. RNA-Seq analysis of two-cell embryos showed changes in transcript abundance resulting from sperm treatment with ROS. Differentially expressed genes were enriched for processes associated with cytoskeletal organization, cell adhesion, and protein phosphorylation. ROS-induced damage to sperm adversely affects embryo development by contributing to mitotic arrest after ICSI of MII rhesus oocytes. Changes in transcript abundance in embryos destined for mitotic arrest is evident at the two-cell stage of development. PMID:23904511

New data about the suspensor of succulent angiosperms: Ultrastructure and cytochemical study of the embryo-suspensor of Sempervivum arachnoideum L. and Jovibarba sobolifera (Sims) Opiz.

PubMed

Kozieradzka-Kiszkurno, Małgorzata; Płachno, Bartosz Jan; Bohdanowicz, Jerzy

2012-07-01

The development of the suspensor in two species - Sempervivum arachnoideum and Jovibarba sobolifera - was investigated using cytochemical methods, light and electron microscopy. Cytological processes of differentiation in the embryo-suspensor were compared with the development of embryo-proper. The mature differentiated suspensor consists of a large basal cell and three to four chalazal cells. The basal cell produces haustorial branched invading ovular tissues. The walls of the haustorium and the micropylar part of the basal cell form the wall ingrowths typical for a transfer cells. The ingrowths also partially cover the lateral wall and the chalazal wall separating the basal cell from the other embryo cells. The dense cytoplasm filling the basal cell is rich in: numerous polysomes lying free or covering rough endoplasmic reticulum (RER), active dictyosomes, microtubules, bundles of microfilaments, microbodies, mitochondria, plastids and lipid droplets. Cytochemical tests (including proteins, insoluble polysaccharides and lipids are distributed in the suspensor during different stages of embryo development) showed the presence of high amounts of macromolecules in the suspensor cells, particularly during the globular and heart-shaped phases of embryo development. The protein bodies and lipid droplets are the main storage products in the cells of the embryo-proper. The results of Auramine 0 indicate that a cuticular material is present only on the surface walls of the embryo-proper, but is absent from the suspensor cell wall. The ultrastructural features and cytochemical tests indicate that in the two species - S. arachnoideum and J. sobolifera - the embryo-suspensor is mainly involved in the absorption and transport of metabolites from the ovular tissues to the developing embryo-proper.

The relevance and use of mouse embryo bioassays for quality control in an assisted reproductive technology program.

PubMed

Scott, L F; Sundaram, S G; Smith, S

1993-09-01

To define both the limits of a mouse embryo bioassay for quality control in an assisted reproductive technology (ART) program and the areas where it can be effectively used. Embryos at the pronuclear and two-cell stage from three different strains of mice were used to assess the effectiveness of this assay for media quality control using five different media routinely used in ART. Pronuclear and two-cell embryos from CD-1 mice were used to test the ability of a mouse embryo bioassay to control for water quality, contaminants in the culture system, and fluctuations in the environmental conditions using a medium, culture system, and scoring technique that were optimized for this strain. The mouse embryo bioassay is not effective in differentiating media appropriate for supporting human embryo development since the development of mouse embryos in vitro is strain, stage, and media related. However, CD-1 embryos were shown to be sensitive to variations in water quality, pH, temperature, incubator conditions, and contaminants in the system when grown in a protein-free medium optimized for their development. Both total blastocyst number and the cell count in the blastocysts were affected. Pronuclear embryos were more sensitive to perturbations in the culture system than two-cell embryos. A mouse embryo bioassay can be effectively used as a means of quality control of water, chemicals, and contact materials and for technique standardization and training in an assisted reproduction program. All the conditions of the test should be defined, pronuclear embryos should be used, and the end point should be fully expanded blastocysts and/or cell numbers in these blastocysts where appropriate.

Enhanced Indirect Somatic Embryogenesis of Date Palm Using Low Levels of Seawater.

PubMed

Taha, Rania A

2017-01-01

Date palm tolerates salinity, drought, and high temperatures. Arid and semiarid zones, especially the Middle East region, need a huge number of date palms for cultivation. To meet this demand, tissue culture techniques have great potential for mass production of plantlets, especially using the indirect embryogenesis technique; any improvement of these techniques is a worthy objective. Low levels of salinity can enhance growth and development of tolerant plants. A low level of seawater, a natural source of salinity, reduces the time required for micropropagation processes of date palm cv. Malkaby when added to MS medium. Medium containing seawater at 500 ppm total dissolved solid (TDS) (12.2 mL/L) improves callus proliferation, whereas 1500 ppm (36.59 mL/L) enhances plant regeneration including multiplication of secondary embryos, embryo germination, and rooting.

Impact of PCOS on early embryo cleavage kinetics.

PubMed

Wissing, M L; Bjerge, M R; Olesen, A I G; Hoest, T; Mikkelsen, A L

2014-04-01

This study investigated whether polycystic ovary syndrome (PCOS) affected early embryo development assessed by time-lapse analysis of embryo kinetics from fertilization to the blastocyst stage. This was a prospective cohort study of two pronuclei (2PN) embryos from 25 hyperandrogenic PCOS patients (110 2PN embryos), 26 normoandrogenic PCOS patients (140 2PN embryos) and 20 healthy, regularly cycling women (controls, 97 2PN embryos). Patients underwent the same baseline evaluation and the same ovarian stimulation from April 2010 to February 2013. Oocytes were fertilized by intracytoplasmic sperm injection and incubated in an EmbryoScope with pictures taken every 20 min in seven focal planes. Time to 2PN breakdown, first cleavage and cleavage to 3, 4, 5, 6, 7 and 8 cells, morula and blastocyst (t₂, t₃, t₄, t₅, t₆, t₇, t₈, t(M), t(B)) were annotated. Differences in embryo kinetics between groups were assessed by mixed modelling. Compared with controls, embryos from hyperandrogenic PCOS patients were significantly delayed at 2PN breakdown, t₂, t₃, t₄ and t₇ but not at t₅, t₆, t₈, t(M) or t(B). Embryos from hyperandrogenic PCOS women had developed slower from fertilization to the 8-cell stage compared with embryos from controls. Copyright © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Nuclear Reprogramming: Kinetics of Cell Cycle and Metabolic Progression as Determinants of Success

PubMed Central

Balbach, Sebastian Thomas; Esteves, Telma Cristina; Houghton, Franchesca Dawn; Siatkowski, Marcin; Pfeiffer, Martin Johannes; Tsurumi, Chizuko; Kanzler, Benoit; Fuellen, Georg; Boiani, Michele

2012-01-01

Establishment of totipotency after somatic cell nuclear transfer (NT) requires not only reprogramming of gene expression, but also conversion of the cell cycle from quiescence to the precisely timed sequence of embryonic cleavage. Inadequate adaptation of the somatic nucleus to the embryonic cell cycle regime may lay the foundation for NT embryo failure and their reported lower cell counts. We combined bright field and fluorescence imaging of histone H2b-GFP expressing mouse embryos, to record cell divisions up to the blastocyst stage. This allowed us to quantitatively analyze cleavage kinetics of cloned embryos and revealed an extended and inconstant duration of the second and third cell cycles compared to fertilized controls generated by intracytoplasmic sperm injection (ICSI). Compared to fertilized embryos, slow and fast cleaving NT embryos presented similar rates of errors in M phase, but were considerably less tolerant to mitotic errors and underwent cleavage arrest. Although NT embryos vary substantially in their speed of cell cycle progression, transcriptome analysis did not detect systematic differences between fast and slow NT embryos. Profiling of amino acid turnover during pre-implantation development revealed that NT embryos consume lower amounts of amino acids, in particular arginine, than fertilized embryos until morula stage. An increased arginine supplementation enhanced development to blastocyst and increased embryo cell numbers. We conclude that a cell cycle delay, which is independent of pluripotency marker reactivation, and metabolic restraints reduce cell counts of NT embryos and impede their development. PMID:22530006

Erythro-Myeloid Progenitors: “definitive” hematopoiesis in the conceptus prior to the emergence of hematopoietic stem cells

PubMed Central

Frame, Jenna M.; McGrath, Kathleen E.; Palis, James

2013-01-01

Erythro-myeloid progenitors (EMP) serve as a major source of hematopoiesis in the developing conceptus prior to the formation of a permanent blood system. In this review, we summarize the current knowledge regarding the emergence, fate, and potential of this hematopoietic stem cell (HSC)-independent wave of hematopoietic progenitors, focusing on the murine embryo as a model system. A better understanding of the temporal and spatial control of hematopoietic emergence in the embryo will ultimately improve our ability to derive hematopoietic stem and progenitor cells from embryonic stem cells and induced pluripotent stem cells to serve therapeutic purposes. PMID:24095199

Role of nucleation-promoting factors in mouse early embryo development.

PubMed

Wang, Qiao-Chu; Liu, Jun; Wang, Fei; Duan, Xing; Dai, Xiao-Xin; Wang, Teng; Liu, Hong-Lin; Cui, Xiang-Shun; Sun, Shao-Chen; Kim, Nam-Hyung

2013-06-01

During mitosis nucleation-promoting factors (NPFs) bind to the Arp2/3 complex and activate actin assembly. JMY and WAVE2 are two critical members of the NPFs. Previous studies have demonstrated that NPFs promote multiple processes such as cell migration and cytokinesis. However, the role of NPFs in development of mammalian embryos is still unknown. Results of the present study show that the NPFs JMY and WAVE2 are critical for cytokinesis during development of mouse embryos. Both JMY and WAVE2 are expressed in mouse embryos. After injection of JMY or WAVE2 siRNA, all embryos failed to develop to the morula or blastocyst stages. Moreover, using fluorescence intensity analysis, we found that the expression of actin decreased, and multiple nuclei were observed within a single cell indicating that NPFs-induced actin reduction caused the failure of cell division. In addition, injection of JMY and WAVE2 siRNA also caused ARP2 degradation, indicating that involvement of NPFs in development of mouse embryos is mainly through regulation of ARP2/3-induced actin assembly. Taken together, these data suggested that WAVE2 and JMY are involved in development of mouse embryos, and their regulation may be through a NPFs-Arp2/3-actin pathway.

Effect of sperm entry on blastocyst development after in vitro fertilization and intracytoplasmic sperm injection - mouse model.

PubMed

Piotrowska-Nitsche, Karolina; Chan, Anthony W S

2013-01-01

To investigate whether in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), influence the embryo's development and its quality using the mouse as a model. Assisted fertilization was performed using ICSI and IVF. Fluorescent beads were adhered to the fertilization cone or place of previous sperm injection in the natural mated (NM), IVF and ICSI embryos, respectively. Embryo examination was carried out at the two-cell and blastocyst stage to determine the position of fluorescent bead. Protein expression was detected by fluorescence immunocytochemical staining and confocal microscopic imaging of blastocysts. IVF and ICSI embryos developed at rates comparable to NM group. Embryos show similar expression patterns of two transcription factors, Oct4 and Cdx2. The most preferred place for spermatozoa attachment was the equatorial site of the egg, whether fertilization occurred in vitro or under natural conditions. We also link the sperm entry position (SEP) to embryo morphology and the number of cells at the blastocyst stage, with no influence of the method of fertilization. IVF and ICSI, do not compromise in vitro pre-implantation development. Additional data, related to sperm entry, could offer further criteria to predict embryos that will implant successfully. Based on embryo morphology, developmental rate and protein expression level of key transcription factors, our results support the view that ART techniques, such as IVF and ICSI, do not perturb embryonic development or quality.

Cryotolerance of Day 2 or Day 6 in vitro produced ovine embryos after vitrification by Cryotop or Spatula methods.

PubMed

Dos Santos Neto, P C; Vilariño, M; Barrera, N; Cuadro, F; Crispo, M; Menchaca, A

2015-02-01

This study was conducted to evaluate the cryotolerance of in vitro produced ovine embryos submitted to vitrification at different developmental stages using two methods of minimum volume and rapid cooling rate. Embryos were vitrified at early stage (2 to 8-cells) on Day 2 or at advanced stage (morulae and blastocysts) on Day 6 after in vitro fertilization. Vitrification procedure consisted of the Cryotop (Day 2, n=165; Day 6, n=174) or the Spatula method (Day 2, n=165; Day 6, n=175). Non vitrified embryos were maintained in in vitro culture as a control group (n=408). Embryo survival was determined at 3h and 24h after warming, development and hatching rates were evaluated on Day 6 and Day 8 after fertilization, and total cell number was determined on expanded blastocysts. Embryo survival at 24h after warming increased as the developmental stage progressed (P<0.05) and was not affected by the vitrification method. The ability for hatching of survived embryos was not affected by the stage of the embryos at vitrification or by the vitrification method. Thus, the proportion of hatching from vitrified embryos was determined by the survival rate and was lower for Day 2 than Day 6 vitrified embryos. The percentage of blastocysts on Day 8 was lower for the embryos vitrified on Day 2 than Day 6 (P<0.05), and was lower for both days of vitrification than for non-vitrified embryos (P<0.05). No interaction of embryo stage by vitrification method was found (P=NS) and no significant difference was found in the blastocyst cell number among vitrified and non-vitrified embryos. In conclusion, both methods using minimum volume and ultra-rapid cooling rate allow acceptable survival and development rates in Day 2 and Day 6 in vitro produced embryos in sheep. Even though early stage embryos showed lower cryotolerance, those embryos that survive the vitrification-warming process show high development and hatching rates, similar to vitrification of morulae or blastocysts. Copyright © 2014 Elsevier Inc. All rights reserved.

A three-dimensional culture system using alginate hydrogel prolongs hatched cattle embryo development in vitro.

PubMed

Zhao, Shuan; Liu, Zhen-Xing; Gao, Hui; Wu, Yi; Fang, Yuan; Wu, Shuai-Shuai; Li, Ming-Jie; Bai, Jia-Hua; Liu, Yan; Evans, Alexander; Zeng, Shen-Ming

2015-07-15

No successful method exists to maintain the three-dimensional architecture of hatched embryos in vitro. Alginate, a linear polysaccharide derived from brown algae, has characteristics that make it an ideal material as a three-dimensional (3D) extracellular matrix for in vitro cell, tissue, or embryo culture. In this study, alginate hydrogel was used for IVC of posthatched bovine embryos to observe their development under the 3D system. In vitro-fertilized and parthenogenetically activated posthatched bovine blastocysts were cultured in an alginate encapsulation culture system (AECS), an alginate overlay culture system (AOCS), or control culture system. After 18 days of culture, the survival rate of embryos cultured in AECS was higher than that in the control group (P < 0.05), and the embryos were expanded and elongated in AECS with the maximal length of 1.125 mm. When the AECS shrinking embryos were taken out of the alginate beads on Day 18 and cultured in the normal culture system, 9.09% of them attached to the bottoms of the plastic wells and grew rapidly, with the largest area of an attached embryo being 66.00 mm(2) on Day 32. The embryos cultured in AOCS developed monovesicular or multivesicular morphologies. Total cell number of the embryos cultured in AECS on Day 19 was significantly higher than that of embryos on Day 8. Additionally, AECS and AOCS supported differentiation of the embryonic cells. Binuclear cells were visible in Day-26 adherent embryos, and the messenger RNA expression patterns of Cdx2 and Oct4 in AOCS-cultured embryos were similar to those in vivo embryos, whereas IFNT and ISG15 messenger RNA were still expressed in Day-26 and Day-32 prolong-cultured embryos. In conclusion, AECS and AOCS did support cell proliferation, elongation, and differentiation of hatched bovine embryos during prolonged IVC. The culture system will be useful to further investigate the molecular mechanisms controlling ruminant embryo elongation and implantation. Copyright © 2015 Elsevier Inc. All rights reserved.

Production of rhesus monkey cloned embryos expressing monomeric red fluorescent protein by interspecies somatic cell nuclear transfer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu, Hai-Ying; Kang, Jin-Dan; Li, Suo

2014-02-21

Highlights: • Rhesus monkey cells were electroporated with a plasmid containing mRFP1, and an mRFP1-expressing cell line was generated. • For the first time, mRFP1-expressing rhesus monkey cells were used as donor cells for iSCNT. • The effect of VPA on the development of embryos cloned using iSCNT was determined. - Abstract: Interspecies somatic cell nuclear transfer (iSCNT) is a promising method to clone endangered animals from which oocytes are difficult to obtain. Monomeric red fluorescent protein 1 (mRFP1) is an excellent selection marker for transgenically modified cloned embryos during somatic cell nuclear transfer (SCNT). In this study, mRFP-expressing rhesusmore » monkey cells or porcine cells were transferred into enucleated porcine oocytes to generate iSCNT and SCNT embryos, respectively. The development of these embryos was studied in vitro. The percentage of embryos that underwent cleavage did not significantly differ between iSCNT and SCNT embryos (P > 0.05; 71.53% vs. 80.30%). However, significantly fewer iSCNT embryos than SCNT embryos reached the blastocyst stage (2.04% vs. 10.19%, P < 0.05). Valproic acid was used in an attempt to increase the percentage of iSCNT embryos that developed to the blastocyst stage. However, the percentages of embryos that underwent cleavage and reached the blastocyst stage were similar between untreated iSCNT embryos and iSCNT embryos treated with 2 mM valproic acid for 24 h (72.12% vs. 70.83% and 2.67% vs. 2.35%, respectively). These data suggest that porcine-rhesus monkey interspecies embryos can be generated that efficiently express mRFP1. However, a significantly lower proportion of iSCNT embryos than SCNT embryos reach the blastocyst stage. Valproic acid does not increase the percentage of porcine-rhesus monkey iSCNT embryos that reach the blastocyst stage. The mechanisms underling nuclear reprogramming and epigenetic modifications in iSCNT need to be investigated further.« less

Somatic embryogenesis and massive shoot regeneration from immature embryo explants of tef.

PubMed

Gugsa, Likyelesh; Kumlehn, Jochen

2011-01-01

Tef (Eragrostis tef) provides a major source of human nutrition in the Horn of Africa, but biotechnology has had little impact on its improvement to date. Here, we report the elaboration of an in vitro regeneration protocol, based on the use of immature zygotic embryos as explant. Explant size was an important determinant of in vitro regeneration efficiency, as was the formulation of the culture medium. Optimal results were obtained by culturing 0.2-0.35 mm embryo explants on a medium containing KBP minerals, 9.2-13.8 μM 2,4-dichlorophenoxyacetic acid, 6 mM glutamine, and 0.5% Phytagel. Although this protocol was effective for both the improved cultivar "DZ-01-196" and the landrace "Fesho", the former produced consistently more embryogenic tissue and a higher number of regenerants. An average of more than 2,800 shoots could be obtained from each "DZ-01-196" explant after 12 weeks of in vitro culture. These shoots readily formed roots, and plantlets transferred to soil were able to develop into morphologically normal, fertile plants. This regeneration and multiplication system should allow for the application of a range of biotechnological methods to tef.

Is the hypothesis of preimplantation genetic screening (PGS) still supportable? A review.

PubMed

Gleicher, Norbert; Orvieto, Raoul

2017-03-27

The hypothesis of preimplantation genetic diagnosis (PGS) was first proposed 20 years ago, suggesting that elimination of aneuploid embryos prior to transfer will improve implantation rates of remaining embryos during in vitro fertilization (IVF), increase pregnancy and live birth rates and reduce miscarriages. The aforementioned improved outcome was based on 5 essential assumptions: (i) Most IVF cycles fail because of aneuploid embryos. (ii) Their elimination prior to embryo transfer will improve IVF outcomes. (iii) A single trophectoderm biopsy (TEB) at blastocyst stage is representative of the whole TE. (iv) TE ploidy reliably represents the inner cell mass (ICM). (v) Ploidy does not change (i.e., self-correct) downstream from blastocyst stage. We aim to offer a review of the aforementioned assumptions and challenge the general hypothesis of PGS. We reviewed 455 publications, which as of January 20, 2017 were listed in PubMed under the search phrase < preimplantation genetic screening (PGS) for aneuploidy>. The literature review was performed by both authors who agreed on the final 55 references. Various reports over the last 18 months have raised significant questions not only about the basic clinical utility of PGS but the biological underpinnings of the hypothesis, the technical ability of a single trophectoderm (TE) biopsy to accurately assess an embryo's ploidy, and suggested that PGS actually negatively affects IVF outcomes while not affecting miscarriage rates. Moreover, due to high rates of false positive diagnoses as a consequence of high mosaicism rates in TE, PGS leads to the discarding of large numbers of normal embryos with potential for normal euploid pregnancies if transferred rather than disposed of. We found all 5 basic assumptions underlying the hypothesis of PGS to be unsupported: (i) The association of embryo aneuploidy with IVF failure has to be reevaluated in view how much more common TE mosaicism is than has until recently been appreciated. (ii) Reliable elimination of presumed aneuploid embryos prior to embryo transfer appears unrealistic. (iii) Mathematical models demonstrate that a single TEB cannot provide reliable information about the whole TE. (iv) TE does not reliably reflect the ICM. (v) Embryos, likely, still have strong innate ability to self-correct downstream from blastocyst stage, with ICM doing so better than TE. The hypothesis of PGS, therefore, no longer appears supportable. With all 5 basic assumptions underlying the hypothesis of PGS demonstrated to have been mistaken, the hypothesis of PGS, itself, appears to be discredited. Clinical use of PGS for the purpose of IVF outcome improvements should, therefore, going forward be restricted to research studies.

Additional mitochondrial DNA influences the interactions between the nuclear and mitochondrial genomes in a bovine embryo model of nuclear transfer.

PubMed

Srirattana, Kanokwan; St John, Justin C

2018-05-08

We generated cattle embryos using mitochondrial supplementation and somatic cell nuclear transfer (SCNT), named miNT, to determine how additional mitochondrial DNA (mtDNA) modulates the nuclear genome. To eliminate any confounding effects from somatic cell mtDNA in intraspecies SCNT, donor cell mtDNA was depleted prior to embryo production. Additional oocyte mtDNA did not affect embryo development rates but increased mtDNA copy number in blastocyst stage embryos. Moreover, miNT-derived blastocysts had different gene expression profiles when compared with SCNT-derived blastocysts. Additional mtDNA increased expression levels of genes involved in oxidative phosphorylation, cell cycle and DNA repair. Supplementing the embryo culture media with a histone deacetylase inhibitor, Trichostatin A (TSA), had no beneficial effects on the development of miNT-derived embryos, unlike SCNT-derived embryos. When compared with SCNT-derived blastocysts cultured in the presence of TSA, additional mtDNA alone had beneficial effects as the activity of glycolysis may increase and embryonic cell death may decrease. However, these beneficial effects were not found with additional mtDNA and TSA together, suggesting that additional mtDNA alone enhances reprogramming. In conclusion, additional mtDNA increased mtDNA copy number and expression levels of genes involved in energy production and embryo development in blastocyst stage embryos emphasising the importance of nuclear-mitochondrial interactions.

Climatic factors affecting quantity and quality grade of in vivo derived embryos of cattle.

PubMed

Chinchilla-Vargas, Josué; Jahnke, Marianna M; Dohlman, Tyler M; Rothschild, Max F; Gunn, Patrick J

2018-05-01

The present study investigated the effects of climatic variables on the quality grade and quantity of in vivo derived cattle embryos in the Midwestern United States. Climatic information included greatest and least daily temperature, average daily wind speed and average temperature-humidity index for each of the 765 records. The response variables included the number of ovarian structures, viable embryos, quality grade 1 embryos, quality grade 2 embryos, quality grade 3 embryos, freezable embryos (sum of quality grade 1 and quality grade 2 embryos), transferable embryos (sum of quality grade 1-3 embryos), degenerate embryos and unfertilized ova. Measures for variables among the breeds of donors and sires grouped by geographical origin were compared. A negative effect of greater temperatures during the early embryonic development stage tended (P < 0.10) to be associated with a decrease in the quality of embryos recovered. Interestingly, the greater the Temperature-Humidity Index (THI) during the early ovarian antral follicular development stage 40-45 days prior to ovulation was associated with a tendency for greater numbers of total number of freezable and transferable embryos recovered per uterine flushing (P < 0.10). Increased wind speed at the early antral follicular phase 40-45 days prior to ovulation was associated with an increase in the percentage of quality grade 1 embryos recovered (P < 0.05). Wind speed during the estrous synchronization period was also associated with a lesser number of embryos recovered (P < 0.05). This retrospective study confirms that climatic variables have significant effects on the in vivo production of cattle embryos and that wind speed should be considered in future analyses of factors affecting embryo quality. Copyright © 2018 Elsevier B.V. All rights reserved.

Effect of ambient light exposure of media and embryos on development and quality of porcine parthenogenetically activated embryos.

PubMed

Li, Rong; Liu, Ying; Pedersen, Hanne Skovsgaard; Callesen, Henrik

2015-06-01

Light exposure is a common stress factor during in vitro handling of oocytes and embryos that originates from both microscope and ambient light. In the current study, the effect of two types of ambient light (daylight and laboratory light) on porcine parthenogenetically activated (PA) embryos was tested in two experiments: (1) ambient light on medium subsequently used for embryo in vitro development; and (2) ambient light exposure on activated oocytes before in vitro development. The results from Experiment 1 showed that exposure of culture medium to both types of ambient light decreased the percentage of blastocysts that showed good morphology, only after 24 h exposure. The results from Experiment 2 revealed a reduction in both blastocyst formation and quality when activated oocytes were exposed to both types of ambient light. This effect was seen after only 1 h exposure and increased with time. In conclusion, exposure to ambient light can be harmful to embryo development, both when medium is exposed for a long period of time and, to a greater extent, when the embryo itself is exposed for >1 h. In practice, it is therefore recommended to protect both culture medium and porcine embryos against ambient light during in vitro handling in the laboratory.

Development of a green fluorescent protein metastatic-cancer chick-embryo drug-screen model.

PubMed

Bobek, Vladimir; Plachy, Jiri; Pinterova, Daniela; Kolostova, Katarina; Boubelik, Michael; Jiang, Ping; Yang, Meng; Hoffman, Robert M

2004-01-01

The chick-embryo model has been an important tool to study tumor growth, metastasis, and angiogenesis. However, an imageable model with a genetic fluorescent tag in the growing and spreading cancer cells that is stable over time has not been developed. We report here the development of such an imageable fluorescent chick-embryo metastatic cancer model with the use of green fluorescent protein (GFP). Lewis lung carcinoma cells, stably expressing GFP, were injected on the 12th day of incubation in the chick embryo. GFP-Lewis lung carcinoma metastases were visualized by fluorescence, after seven days additional incubation, in the brain, heart, and sternum of the developing chick embryo, with the most frequent site being the brain. The combination of streptokinase and gemcitabine was evaluated in this GFP metastatic model. Twelve-day-old chick embryos were injected intravenously with GFP-Lewis lung cancer cells, along with these two agents either alone or in combination. The streptokinase-gemcitabine combination inhibited metastases at all sites. The effective dose of gemcitabine was found to be 10 mg/kg and streptokinase 2000 IU per embryo. The data in this report suggest that this new stably fluorescent imageable metastatic-cancer chick-embryo model will enable rapid screening of new antimetastatic agents.

Novel embryo selection techniques to increase embryo implantation in IVF attempts.

PubMed

Sigalos, George Α; Triantafyllidou, Olga; Vlahos, Nikos F

2016-11-01

The final success of an IVF attempt depends on several steps and decisions taken during the ovarian stimulation, the oocyte retrieval, the embryo culture and the embryo transfer. The final selection of the embryos most likely to implant is the final step in this process and the responsibility of the lab. Apart from strict morphologic criteria that historically have been used in embryo selection, additional information on genetic, metabolomic and morphokinetic characteristics of the embryo is recently combined to morphology to select the embryo most likely to produce a pregnancy. In this manuscript, we review the most recent information on the current methods used for embryo selection presenting the predictive capability of each one. A literature search was performed on Pubmed, Medline and Cochrane Database of Systematic Reviews for published studies using appropriate key words and phrases with no limits placed on time. It seems that the combination of morphologic criteria in conjunction to embryo kinetics as documented by time-lapse technology provides the most reliable information on embryo quality. Blastocyst biopsy with subsequent comprehensive chromosome analysis allows the selection of the euploid embryos with the higher implantation potential. Embryo time-lapse imaging and blastocyst biopsy combined to comprehensive chromosome analysis are the most promising technologies to increase pregnancy rates and reduce the possibility of multiple pregnancies. However, further studies will demonstrate the capability of routinely using these technologies to significantly improve IVF outcomes.

A mathematical model of in vivo bovine blastocyst developmental to gestational Day 15.

PubMed

Shorten, P R; Donnison, M; McDonald, R M; Meier, S; Ledgard, A M; Berg, D

2018-06-20

Bovine embryo growth involves a complex interaction between the developing embryo and the growth-promoting potential of the uterine environment. We have previously established links between embryonic factors (embryo stage, embryo gene expression), maternal factors (progesterone, body condition score), and embryonic growth to 8 d after bulk transfer of Day 7 in vitro-produced blastocysts. In this study we recovered blastocysts on Days 7 and 15 after artificial insemination to test the hypothesis that in vivo and in vitro embryos follow a similar growth program. We conducted our study using 4 commercial farms and repeated our study over 2 yr (2014, 2015), with data available from 2 of the 4 farms in the second year. Morphological and gene expression measurements (196 candidate genes) of the Day 7 embryos were measured and the progesterone concentration of the cows were measured throughout the reproductive cycle as a reflection of the state of the uterine environment. These data were also used to assess the interaction between the uterine environment and the developing embryo and to examine how well Day 7 embryo stage can be predicted from the Day 7 gene expression profile. Progesterone was not a strong predictor of in vivo embryo growth to Day 15. This contrasts with a range of Day 7 embryo transfer studies which demonstrated that progesterone is a very good predictor of embryo growth to Day 15. Our analysis demonstrates that in vivo embryos are 3 times less sensitive to progesterone than in vitro-transferred embryos (up to Day 15). This highlights that caution must be applied when extrapolating the results of in vitro embryo transfer studies to the in vivo situation. The similar variance in measured and predicted (based on Day 15 length) Day 7 embryo stage indicate low stochastic perturbations for in vivo embryo growth (large stochastic growth effects would generate a significantly larger standard deviation in measured embryo length on Day 15). We also identified that Day 7 embryo stage could be predicted based on the Day 7 gene expression profile (58% overall success rate for classification of 5 embryo stages). Our analysis also associated genes with each developmental stage and demonstrates the high level of temporal regulation of genes that occurs during early embryonic development. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Birth of normal infants after transfer of embryos that were twice vitrified/warmed at cleavage stages: report of two cases.

PubMed

Valle, Marcello; Guimarães, Fernando; Cavagnoli, Melissa; Sampaio, Marcos; Geber, Selmo

2012-12-01

The role of cryopreservation in assisted reproductive technology programs has increased within the last years allowing the transfer of a limited number of embryos and the storage of the remaining for future use. The reduction in the number of transferred embryos decreases the frequency of multiple pregnancy rates and of ovarian hyperstimulation syndrome while the cumulative pregnancy rate can be maximized. Moreover, as not all embryos will survive the warming process more cleavage stage embryos are warmed to improve selection for transfer. Therefore, surplus good quality cleavage stage embryos and/or blastocysts must be re-vitrified for further transfer to achieve pregnancy. To our knowledge, there have been no reports demonstrating that human embryos can be successfully vitrified/warmed twice at the cleavage stage. Thus we report two successful pregnancies and deliveries of healthy babies after transfer of embryos that were twice vitrified/warmed at 2-4 cells stage. Copyright © 2012 Elsevier Inc. All rights reserved.

Loss of maternal CTCF is associated with peri-implantation lethality of Ctcf null embryos.

PubMed

Moore, James M; Rabaia, Natalia A; Smith, Leslie E; Fagerlie, Sara; Gurley, Kay; Loukinov, Dmitry; Disteche, Christine M; Collins, Steven J; Kemp, Christopher J; Lobanenkov, Victor V; Filippova, Galina N

2012-01-01

CTCF is a highly conserved, multifunctional zinc finger protein involved in critical aspects of gene regulation including transcription regulation, chromatin insulation, genomic imprinting, X-chromosome inactivation, and higher order chromatin organization. Such multifunctional properties of CTCF suggest an essential role in development. Indeed, a previous report on maternal depletion of CTCF suggested that CTCF is essential for pre-implantation development. To distinguish between the effects of maternal and zygotic expression of CTCF, we studied pre-implantation development in mice harboring a complete loss of function Ctcf knockout allele. Although we demonstrated that homozygous deletion of Ctcf is early embryonically lethal, in contrast to previous observations, we showed that the Ctcf nullizygous embryos developed up to the blastocyst stage (E3.5) followed by peri-implantation lethality (E4.5-E5.5). Moreover, one-cell stage Ctcf nullizygous embryos cultured ex vivo developed to the 16-32 cell stage with no obvious abnormalities. Using a single embryo assay that allowed both genotype and mRNA expression analyses of the same embryo, we demonstrated that pre-implantation development of the Ctcf nullizygous embryos was associated with the retention of the maternal wild type Ctcf mRNA. Loss of this stable maternal transcript was temporally associated with loss of CTCF protein expression, apoptosis of the developing embryo, and failure to further develop an inner cell mass and trophoectoderm ex vivo. This indicates that CTCF expression is critical to early embryogenesis and loss of its expression rapidly leads to apoptosis at a very early developmental stage. This is the first study documenting the presence of the stable maternal Ctcf transcript in the blastocyst stage embryos. Furthermore, in the presence of maternal CTCF, zygotic CTCF expression does not seem to be required for pre-implantation development.

Bessel beam fluorescence lifetime tomography of live embryos (Conference Presentation)

NASA Astrophysics Data System (ADS)

Xu, Dongli; Peng, Leilei

2016-03-01

Optical tomography allows isotropic 3D imaging of embryos. Scanning-laser optical tomography (SLOT) has superior light collecting efficiency than wide-field optical tomography, making it ideal for fluorescence imaging of live embryos. We previously reported an imaging system that combines SLOT with a novel Fourier-multiplexed fluorescence lifetime imaging (FmFLIM) technique named FmFLIM-SLOT. FmFLIM-SLOT performs multiplexed FLIM-FRET readout of multiple FRET sensors in live embryos. Here we report a recent effort on improving the spatial resolution of the FmFLIM-SLOT system in order to image complex biochemical processes in live embryos at the cellular level. Optical tomography has to compromise between resolution and the depth of view. In SLOT, the commonly-used focused Gaussian beam diverges quickly from the focal plane, making it impossible to achieve high resolution imaging in a large volume specimen. We thus introduce Bessel beam laser-scanning tomography, which illuminates the sample with a spatial-light-modulator-generated Bessel beam that has an extended focal depth. The Bessel beam is scanned across the whole specimen. Fluorescence projection images are acquired at equal angular intervals as the sample rotates. Reconstruction artifacts due to annular-rings of the Bessel beam are removed by a modified 3D filtered back projection algorithm. Furthermore, in combination of Fourier-multiplexing fluorescence lifetime imaging (FmFLIM) method, the Bessel FmFLIM-SLOT system is capable of perform 3D lifetime imaging of live embryos at cellular resolution. The system is applied to in-vivo imaging of transgenic Zebrafish embryos. Results prove that Bessel FmFLIM-SLOT is a promising imaging method in development biology research.

Cell wall invertase as a regulator in determining sequential development of endosperm and embryo through glucose signaling early in seed development.

PubMed

Wang, Lu; Liao, Shengjin; Ruan, Yong-Ling

2013-01-01

Seed development depends on coordination among embryo, endosperm and seed coat. Endosperm undergoes nuclear division soon after fertilization, whereas embryo remains quiescent for a while. Such a developmental sequence is of great importance for proper seed development. However, the underlying mechanism remains unclear. Recent results on the cellular domain- and stage-specific expression of invertase genes in cotton and Arabidopsis revealed that cell wall invertase may positively and specifically regulate nuclear division of endosperm after fertilization, thereby playing a role in determining the sequential development of endosperm and embryo, probably through glucose signaling.

Paternal Diet-Induced Obesity Retards Early Mouse Embryo Development, Mitochondrial Activity and Pregnancy Health

PubMed Central

Binder, Natalie K.; Hannan, Natalie J.; Gardner, David K.

2012-01-01

Worldwide, 48% of adult males are overweight or obese. An association between infertility and excessive body weight is now accepted, although focus remains primarily on females. It has been shown that parental obesity results in compromised embryo development, disproportionate changes in embryo metabolism and reduced blastocyst cell number. The aim of this study was to determine whether paternal obesity has negative effects on the resultant embryo. Specifically, using in vitro fertilisation (IVF), we wanted to isolate the functional effects of obesity on sperm by examining the subsequent embryo both pre- and post-implantation. Epididymal sperm was collected from age matched normal and obese C57BL/6 mice and cryopreserved for subsequent IVF with oocytes collected from Swiss females (normal diet/weight). Obesity was induced in male mice by feeding a high fat diet of 22% fat for 10 weeks. Resultant embryos were cultured individually and development monitored using time-lapse microscopy. Paternal obesity resulted in a significant delay in preimplantation embryo development as early as syngamy (P<0.05). Metabolic parameters were measured across key developmental stages, demonstrating significant reduction in mitochondrial membrane potential (P<0.01). Blastocysts were stained to determine trophectoderm (TE) and inner cell mass (ICM) cell numbers, revealing significant differences in the ratio of cell allocation to TE and ICM lineages (P<0.01). Functional studies examining blastocyst attachment, growth and implantation demonstrated that blastocysts derived from sperm of obese males displayed significantly reduced outgrowth on fibronectin in vitro (P<0.05) and retarded fetal development in vivo following embryo transfer (P<0.05). Taken together, these data clearly demonstrate that paternal obesity has significant negative effects on the embryo at a variety of key early developmental stages, resulting in delayed development, reduced placental size and smaller offspring. PMID:23300638

Embryoids, organoids and gastruloids: new approaches to understanding embryogenesis

PubMed Central

2017-01-01

ABSTRACT Cells have an intrinsic ability to self-assemble and self-organize into complex and functional tissues and organs. By taking advantage of this ability, embryoids, organoids and gastruloids have recently been generated in vitro, providing a unique opportunity to explore complex embryological events in a detailed and highly quantitative manner. Here, we examine how such approaches are being used to answer fundamental questions in embryology, such as how cells self-organize and assemble, how the embryo breaks symmetry, and what controls timing and size in development. We also highlight how further improvements to these exciting technologies, based on the development of quantitative platforms to precisely follow and measure subcellular and molecular events, are paving the way for a more complete understanding of the complex events that help build the human embryo. PMID:28292844

Effect of exogenous transforming growth factor β1 (TGF-β1) on early bovine embryo development.

PubMed

Barrera, Antonio D; García, Elina V; Miceli, Dora C

2018-06-08

SummaryDuring preimplantation development, embryos are exposed and have the capacity to respond to different growth factors present in the maternal environment. Among these factors, transforming growth factor β1 (TGF-β1) is a well known modulator of embryonic growth and development. However, its action during the first stages of development, when the embryo transits through the oviduct, has not been yet elucidated. The objective of the present study was to examine the effect of early exposure to exogenous TGF-β1 on embryo development and expression of pluripotency (OCT4, NANOG) and DNA methylation (DNMT1, DNMT3A, DNMT3B) genes in bovine embryos produced in vitro. First, gene expression analysis of TGF-β receptors confirmed a stage-specific expression pattern, showing greater mRNA abundance of TGFBR1 and TGFBR2 from the 2- to the 8-cell stage, before embryonic genome activation. Second, embryo culture for the first 48 h in serum-free CR1aa medium supplemented with 50 or 100 ng/ml recombinant TGF-β1 did not affect the cleavage and blastocyst rate (days 7 and 8). However, RT-qPCR analysis showed a significant increase in the relative abundance of NANOG and DNMT3A in the 8-cell stage embryos and expanded blastocysts (day 8) derived from TGF-β1 treated embryos. These results suggest an early action of exogenous TGF-β1 on the bovine embryo, highlighting the importance to provide a more comprehensive understanding of the role of TGF-β signalling during early embryogenesis.

Hybrid embryos produced by transferring panda or cat somatic nuclei into rabbit MII oocytes can develop to blastocyst in vitro.

PubMed

Wen, Duan-Cheng; Bi, Chun-Ming; Xu, Ying; Yang, Cai-Xia; Zhu, Zi-Yu; Sun, Qing-Yuan; Chen, Da-Yuan

2005-08-01

The developmental potential of hybrid embryos produced by transferring panda or cat fibroblasts into nucleated rabbit oocytes was assessed. Both the panda-rabbit and the cat-rabbit hybrid embryos were able to form blastocysts in vitro. However, the rates of attaining the two-cell, four-cell, eight-cell, morula, or blastocyst stages for panda-rabbit hybrids were significantly greater than those of cat-rabbit hybrids (P<0.05). Transferring the rabbit fibroblasts into nucleated rabbit oocytes, 31.0% of the blastocyst rate was obtained, which was significantly higher than that of both the panda-rabbit and the cat-rabbit hybrid embryos (P<0.05). Whether or not the second polar body (PB2) was extruded from the one-cell hybrid embryos (both panda-rabbit and cat-rabbit hybrids) significantly affected their developmental capacity. Embryos without an extruded PB2 showed a higher capacity to develop into blastocysts (panda-rabbit: 19.2%; cat-rabbit: 4.3%), while embryos with extruded PB2 could only develop to the morula stage. The hybrid embryos formed pronucleus-like structures (PN) in 2-4 hr after activation, and the number of PN in one-cell embryos varied from one to five. Tracking of the nucleus in the egg after fusion revealed that the somatic nucleus could approach and aggregate with the oocyte nucleus spontaneously. Chromosome analysis of the panda-rabbit blastocysts showed that the karyotype of the hybrid embryos (2n=86) consisted of chromosomes from both the panda (2n=42) and the rabbit (2n=44). The results demonstrate that (1) it is possible to produce genetic hybrid embryos by interspecies nuclear transfer; (2) the developmental potential of the hybrid embryos is highly correlated to the donor nucleus species; and (3) the hybrid genome is able to support the complete preimplantation embryonic development of the hybrids. Copyright (c) 2005 Wiley-Liss, Inc.

Optimized ex-ovo culturing of chick embryos to advanced stages of development.

PubMed

Cloney, Kellie; Franz-Odendaal, Tamara Anne

2015-01-24

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos model organisms, such as the chicken, are often used. The chicken is an excellent model organism due to its low cost and minimal maintenance, however they present observational challenges because they are enclosed in an opaque eggshell. In order to properly view the embryo as it develops, the shell must be windowed or removed. Both windowing and ex ovo techniques have been developed to assist researchers in the study of embryonic development. However, each of the methods has limitations and challenges. Here, we present a simple, optimized ex ovo culture technique for chicken embryos that enables the observation of embryonic development from stage HH 19 into late stages of development (HH 40), when many organs have developed. This technique is easy to adopt in both undergraduate classes and more advanced research laboratories where embryo manipulations are conducted.

An Arabidopsis thaliana embryo arrest mutant exhibiting germination potential

USDA-ARS?s Scientific Manuscript database

The ability to initiate radicle elongation, or germination potential, occurs in developing embryos before the completion of seed maturation. Green embryos after walking-stick stage in developing Arabidopsis thaliana seeds germinate when excised from seeds and incubated in MS media containing 1 % suc...

NMR studies of preimplantation embryo metabolism in human assisted reproductive techniques: a new biomarker for assessment of embryo implantation potential.

PubMed

Pudakalakatti, Shivanand M; Uppangala, Shubhashree; D'Souza, Fiona; Kalthur, Guruprasad; Kumar, Pratap; Adiga, Satish Kumar; Atreya, Hanudatta S

2013-01-01

There has been growing interest in understanding energy metabolism in human embryos generated using assisted reproductive techniques (ART) for improving the overall success rate of the method. Using NMR spectroscopy as a noninvasive tool, we studied human embryo metabolism to identify specific biomarkers to assess the quality of embryos for their implantation potential. The study was based on estimation of pyruvate, lactate and alanine levels in the growth medium, ISM1, used in the culture of embryos. An NMR study involving 127 embryos from 48 couples revealed that embryos transferred on Day 3 (after 72 h in vitro culture) with successful implantation (pregnancy) exhibited significantly (p < 10(-5) ) lower pyruvate/alanine ratios compared to those that failed to implant. Lactate levels in media were similar for all embryos. This implies that in addition to lactate production, successfully implanted embryos use pyruvate to produce alanine and other cellular functions. While pyruvate and alanine individually have been used as biomarkers, the present study highlights the potential of combining them to provide a single parameter that correlates strongly with implantation potential. Copyright © 2012 John Wiley & Sons, Ltd.

Dynamic Subcellular Localization of Iron during Embryo Development in Brassicaceae Seeds

PubMed Central

Ibeas, Miguel A.; Grant-Grant, Susana; Navarro, Nathalia; Perez, M. F.; Roschzttardtz, Hannetz

2017-01-01

Iron is an essential micronutrient for plants. Little is know about how iron is loaded in embryo during seed development. In this article we used Perls/DAB staining in order to reveal iron localization at the cellular and subcellular levels in different Brassicaceae seed species. In dry seeds of Brassica napus, Nasturtium officinale, Lepidium sativum, Camelina sativa, and Brassica oleracea iron localizes in vacuoles of cells surrounding provasculature in cotyledons and hypocotyl. Using B. napus and N. officinale as model plants we determined where iron localizes during seed development. Our results indicate that iron is not detectable by Perls/DAB staining in heart stage embryo cells. Interestingly, at torpedo development stage iron localizes in nuclei of different cells type, including integument, free cell endosperm and almost all embryo cells. Later, iron is detected in cytoplasmic structures in different embryo cell types. Our results indicate that iron accumulates in nuclei in specific stages of embryo maturation before to be localized in vacuoles of cells surrounding provasculature in mature seeds. PMID:29312417

The effect of MRN complex and ATM kinase inhibitors on Zebrafish embryonic development

NASA Astrophysics Data System (ADS)

Kumaran, Malina; Fazry, Shazrul

2018-04-01

Zebrafish is an ideal animal model to study developmental biology due to its transparent embryos and rapid development stages of embryogenesis. Here we investigate the role of DNA damage proteins, specifically Mre11/Rad50/NBN (MRN) complex and ataxia-telangiectasia mutated (ATM) kinase during embryogenesis by inhibiting its function using specific MRN complex (Mirin) and ATM Kinase inhibitors (Ku60019 and Ku55933). Zebrafish embryos at midblastula transition (MBT) stage are treated with Mirin, Ku60019 and Ku55933. The embryonic development of the embryos was monitored at 24 hours-post fertilisation (hpf), 48 hpf and 72 hpf. We observed that at the lowest concentrations (3 µM of Mirin, 1.5 nM of Ku60019 and 3 nM of Ku55933), the inhibitors treated embryos have 100% survivability. However, with increasing inhibitor concentration, the survivability drops. Control or mock treatment of all embryos shows 100 % survivability rate. This study suggests that DNA damage repair proteins may be crucial for normal zebrafish embryo development and survival.

Carbonic anhydrase activity in developing sea urchin embryos with special reference to calcification of spicules.

PubMed

Mitsunaga, K; Akasaka, K; Shimada, H; Fujino, Y; Yasumasu, I; Numanoi, H

1986-06-01

Eggs and embryos of the sea urchins Anthocidaris crassispina and Hemicentrotus pulcherrimus did not exhibit significant changes in carbonic anhydrase activity during early development. Acetazolamide inhibited enzyme activity in homogenates of embryos and inhibited the formation of calcified spicules in a culture of micromeres at concentrations between 40 and 100 microM. Acetazolamide allowed intact embryos to develop to quasi-normal plutei but inhibited calcium deposition in the spicules. It is suggested that carbonic anhydrase contributes to CaCO3 deposition in the spicule.

The ovine uterus as a host for in vitro-produced bovine embryos.

PubMed

Rexroad, C E; Powell, A M

1999-07-15

A series of experiments were conducted to determine whether bovine blastocysts would develop beyond the blastocyst stage in the ovine uterine environment. In Experiment 1, in vitro matured, fertilized and cultured (IVM/IVF/IVC) expanded bovine blastocysts were transferred into uteri of ewes on Day 7 or 9 of the estrous cycle and collected on Day 14 or 15 to determine if the bovine blastocysts would elongate and form an embryonic disk. Springtime trials with ewes that were synchronized with a medroxyprogesterone acetate (MAP) sponge resulted in a 78% blastocyst recovery rate, and 68% of the recovered spherical or elongated embryos had embryonic disks. In Experiment 2, transfer of 4-cell bovine embryos to the oviducts of ewes at Day 3 resulted in a lower recovery (47 vs 80%) than the transfer of blastocysts at Day 7 when embryos were recovered at Day 14. However, the percentage of embryos containing embryonic disks was higher for embryos transferred at the 4-cell stage (71%) than for embryos transferred as blastocysts (50%). In Experiment 3, IVF embryos from super-ovulated cows or Day 8 in vitro produced embryos transferred to cows were collected at Day 14 and were found to be similar in size to those produced by transfer to ewes in Experiment 2. In Experiment 4, the transfer of bovine blastocysts to ewes did not prolong the ovine estrous cycle. In Experiment 5, extension of the ovine estrous cycle by administration of a MAP releasing intravaginal device allowed bovine embryos to elongate extensively and to become filamentous. In Experiment 6, uterine flushings on Day 14 or Day 16 contained elevated levels of interferon-tau when bovine blastocyst were transferred on Day 7. Transfer of bovine embryos to the reproductive tract of a ewe allows some embryos to develop normally to advanced perimplantation stages and may be a useful tool for studying critical stages of embryo development and the developmental capacity of experimental embryos.

Early detection and staging of spontaneous embryo resorption by ultrasound biomicroscopy in murine pregnancy.

PubMed

Flores, Luis E; Hildebrandt, Thomas B; Kühl, Anja A; Drews, Barbara

2014-05-10

Embryo resorption is a major problem in human medicine, agricultural animal production and in conservation breeding programs. Underlying mechanisms have been investigated in the well characterised mouse model. However, post mortem studies are limited by the rapid disintegration of embryonic structures. A method to reliably identify embryo resorption in alive animals has not been established yet. In our study we aim to detect embryos undergoing resorption in vivo at the earliest possible stage by ultra-high frequency ultrasound. In a longitudinal study, we monitored 30 pregnancies of wild type C57BI/6 mice using ultra-high frequency ultrasound (30-70 MHz), so called ultrasound biomicroscopy (UBM). We compared the sonoembryology of mouse conceptuses under spontaneous resorption and neighbouring healthy conceptuses and correlated the live ultrasound data with the respective histology. The process of embryo resorption comprised of four stages: first, the conceptus exhibited growth retardation, second, bradycardia and pericardial edema were observed, third, further development ceased and the embryo died, and finally embryo remnants were resorbed by maternal immune cells. In early gestation (day 7 and 8), growth retardation was characterized by a small embryonic cavity. The embryo and its membranes were ill defined or did not develop at all. The echodensity of the embryonic fluid increased and within one to two days, the embryo and its cavity disappeared and was transformed into echodense tissue surrounded by fluid filled caverns. In corresponding histologic preparations, fibrinoid material interspersed with maternal granulocytes and lacunae filled with maternal blood were observed. In later stages (day 9-11) resorption prone embryos were one day behind in their development compared to their normal siblings. The space between Reichert's membrane and inner yolk sac membrane was enlarged The growth retarded embryos exhibited bradycardia and ultimately cessation of heart beat. Corresponding histology showed apoptotic cells in the embryo while the placenta was still intact. In the subsequent resorption process first the embryo and then its membranes disappeared. Our results provide a temporal time course of embryo resorption. With this method, animals exhibiting embryo resorption can be targeted, enabling the investigation of underlying mechanisms before the onset of total embryo disintegration.

Production of monozygotic twin calves using the blastomere separation technique and Well of the Well culture system.

PubMed

Tagawa, M; Matoba, S; Narita, M; Saito, N; Nagai, T; Imai, K

2008-03-15

The present study was conducted to establish a simple and efficient method of producing monozygotic twin calves using the blastomere separation technique. To produce monozygotic twin embryos from zona-free two- and eight-cell embryos, blastomeres were separated mechanically by pipetting to form two demi-embryos; each single blastomere from the two-cell embryo and tetra-blastomeres from the eight-cell embryo were cultured in vitro using the Well of the Well culture system (WOW). This culture system supported the successful arrangement of blastomeres, resulting in their subsequent aggregation to form a demi-embryo developing to the blastocyst stage without a zona pellucida. There was no significant difference in the development to the blastocyst stage between blastomeres separated from eight-cell (72.0%) and two-cell (62.0%) embryos. The production rates of the monozygotic pair blastocysts and transferable paired blastocysts for demi-embryos obtained from eight-cell embryos (64.0 and 45.0%, respectively) were higher than those for demi-embryos obtained from two-cell embryos (49.0 and 31.0%, P<0.05). The separated demi-embryos obtained from eight-cell embryos produced by IVM/IVF of oocytes collected by ovum pick-up (OPU) from elite cows and cultured in wells tended to have a higher pregnancy rate (78.9% vs. 57.1%) and similar monozygotic twinning rate (40.0% vs. 33.3%) compared with monozygotic twin blastocysts obtained by the conventional bisection of in vivo derived blastocysts. In conclusion, producing twins by separation of blastomeres in OPU-IVF embryos, followed by the WOW culture system, yielded viable monozygotic demi-embryos, resulting in high rates of pregnancy and twinning rates after embryo transfer.

Splitting of IVP bovine blastocyst affects morphology and gene expression of resulting demi-embryos during in vitro culture and in vivo elongation.

PubMed

Velasquez, Alejandra E; Castro, Fidel O; Veraguas, Daniel; Cox, Jose F; Lara, Evelyn; Briones, Mario; Rodriguez-Alvarez, Lleretny

2016-02-01

Embryo splitting might be used to increase offspring yield and for molecular analysis of embryo competence. How splitting affects developmental potential of embryos is unknown. This research aimed to study the effect of bovine blastocyst splitting on morphological and gene expression homogeneity of demi-embryos and on embryo competence during elongation. Grade I bovine blastocyst produced in vitro were split into halves and distributed in nine groups (3 × 3 setting according to age and stage before splitting; age: days 7-9; stage: early, expanded and hatched blastocysts). Homogeneity and survival rate in vitro after splitting (12 h, days 10 and 13) and the effect of splitting on embryo development at elongation after embryo transfer (day 17) were assessed morphologically and by RT-qPCR. The genes analysed were OCT4, SOX2, NANOG, CDX2, TP1, TKDP1, EOMES, and BAX. Approximately 90% of split embryos had a well conserved defined inner cell mass (ICM), 70% of the halves had similar size with no differences in gene expression 12 h after splitting. Split embryos cultured further conserved normal and comparable morphology at day 10 of development; this situation changes at day 13 when embryo morphology and gene expression differed markedly among demi-embryos. Split and non-split blastocysts were transferred to recipient cows and were recovered at day 17. Fifty per cent of non-split embryos were larger than 100 mm (33% for split embryos). OCT4, SOX2, TP1 and EOMES levels were down-regulated in elongated embryos derived from split blastocysts. In conclusion, splitting day-8 blastocysts yields homogenous demi-embryos in terms of developmental capability and gene expression, but the initiation of the filamentous stage seems to be affected by the splitting.

Embryo transfer after 2 or 5 days of IVF culture: a retrospective comparison.

PubMed

Lundqvist, Monalill; Rova, Karin; Simberg, Niklas; Lundkvist, Orjan

2002-02-01

To determine whether prolongation of embryo culture in vitro from day 2 to day 5 after ovum pick-up (OPU) and fertilization can improve the results of in vitro fertilization (IVF), and the morphology of the spare embryos on day 2 can predict the developmental capacity during prolonged culture. We also wanted to consider this as a strategy to avoid twin pregnancies if it could be possible to transfer only one blastocyst at a time in the future. A retrospective analysis with embryo transfer timed according to the weekday of OPU. Embryo transfer was performed on day 2 in 103 cases and on day 5 in 120 cases. Only one cycle per couple was included. The pregnancy rates per embryo transfer on day 2 (27/103, 26%) and day 5 (36/120, 30%) were similar. There were significantly more miscarriages in the day 5 (50%) than in the day 2 group (22%, p = 0.02), but there was no significant difference in the baby take home rate (20% in day 2 group, 15% in day 5 group). The morphological appearance of the embryos on day 2 was poorly correlated to the developmental potential during prolonged culture in vitro. On day 5, transfer of one or two blastocysts resulted in a pregnancy rate that tended to be higher than that after transfer of morulae only. Prolongation of embryo culture from day 2 to day 5 did not improve the clinical outcome of the IVF treatment when measured as baby take home rate. Therefore, for the time being, this strategy does not increase our chances to move towards single embryo transfer.

Glutamine supplementation enhances development of in vitro-produced porcine embryos and increases leucine consumption from the medium.

PubMed

Chen, Paula R; Redel, Bethany K; Spate, Lee D; Ji, Tieming; Salazar, Shirley Rojas; Prather, Randall S

2018-05-31

Improper composition of culture medium contributes to reduced viability of in vitro-produced embryos. Glutamine (Gln) is a crucial amino acid for preimplantation embryos as it supports proliferation and is involved in many different biosynthetic pathways. Previous transcriptional profiling revealed several upregulated genes related to Gln transport and metabolism in in vitro-produced porcine blastocysts compared to in vivo-produced counterparts, indicating a potential deficiency in the culture medium. Therefore, the objective of this study was to determine the effects of Gln supplementation on in vitro-produced porcine embryo development, gene expression, and metabolism. Cleaved embryos were selected and cultured in MU2 medium supplemented with 1 mM Gln (control), 3.75 mM Gln (+Gln), 3.75 mM GlutaMAX (+Max), or 3.75 mM alanine (+Ala) until day 6. Embryos cultured with +Gln or +Max had increased development to the blastocyst stage and total number of nuclei compared to the control (P < 0.05). Moreover, expression of misregulated transcripts involved in glutamine and glutamate transport and metabolism were corrected when embryos were cultured with +Gln or +Max. Metabolomics analysis revealed increased production of glutamine and glutamate into the medium by embryos cultured with +Max and increased consumption of leucine by embryos cultured with +Gln or +Max. As an indicator of cellular health, mitochondrial membrane potential was increased when embryos were cultured with +Max which was coincident with decreased apoptosis in these blastocysts. Lastly, two embryo transfers by using embryos cultured with +Max resulted in viable piglets, confirming that this treatment is consistent with in vivo developmental competence.

Allele-specific expression of the MAOA gene and X chromosome inactivation in in vitro produced bovine embryos.

PubMed

Ferreira, A R; Machado, G M; Diesel, T O; Carvalho, J O; Rumpf, R; Melo, E O; Dode, M A N; Franco, M M

2010-07-01

During embryogenesis, one of the two X chromosomes is inactivated in embryos. The production of embryos in vitro may affect epigenetic mechanisms that could alter the expression of genes related to embryo development and X chromosome inactivation (XCI). The aim of this study was to understand XCI during in vitro, pre-implantation bovine embryo development by characterizing the allele-specific expression pattern of the X chromosome-linked gene, monoamine oxidase A (MAOA). Two pools of ten embryos, comprised of the 4-, 8- to 16-cell, morula, blastocyst, and expanded blastocyst stages, were collected. Total RNA from embryos was isolated, and the RT-PCR-RFLP technique was used to observe expression of the MAOA gene. The DNA amplicons were also sequenced using the dideoxy sequencing method. MAOA mRNA was detected, and allele-specific expression was identified in each pool of embryos. We showed the presence of both the maternal and paternal alleles in the 4-, 8- to 16-cell, blastocyst and expanded blastocyst embryos, but only the maternal allele was present in the morula stage. Therefore, we can affirm that the paternal X chromosome is totally inactivated at the morula stage and reactivated at the blastocyst stage. To our knowledge, this is the first report of allele-specific expression of an X-linked gene that is subject to XCI in in vitro bovine embryos from the 4-cell to expanded blastocyst stages. We have established a pattern of XCI in our in vitro embryo production system that can be useful as a marker to assist the development of new protocols for in vitro embryo production. (c) 2010 Wiley-Liss, Inc.

Natural Selection of Human Embryos: Decidualizing Endometrial Stromal Cells Serve as Sensors of Embryo Quality upon Implantation

PubMed Central

Teklenburg, Gijs; Salker, Madhuri; Molokhia, Mariam; Lavery, Stuart; Trew, Geoffrey; Aojanepong, Tepchongchit; Mardon, Helen J.; Lokugamage, Amali U.; Rai, Raj; Landles, Christian; Roelen, Bernard A. J.; Quenby, Siobhan; Kuijk, Ewart W.; Kavelaars, Annemieke; Heijnen, Cobi J.; Regan, Lesley; Brosens, Jan J.; Macklon, Nick S.

2010-01-01

Background Pregnancy is widely viewed as dependent upon an intimate dialogue, mediated by locally secreted factors between a developmentally competent embryo and a receptive endometrium. Reproductive success in humans is however limited, largely because of the high prevalence of chromosomally abnormal preimplantation embryos. Moreover, the transient period of endometrial receptivity in humans uniquely coincides with differentiation of endometrial stromal cells (ESCs) into highly specialized decidual cells, which in the absence of pregnancy invariably triggers menstruation. The role of cyclic decidualization of the endometrium in the implantation process and the nature of the decidual cytokines and growth factors that mediate the crosstalk with the embryo are unknown. Methodology/Principal Findings We employed a human co-culture model, consisting of decidualizing ESCs and single hatched blastocysts, to identify the soluble factors involved in implantation. Over the 3-day co-culture period, approximately 75% of embryos arrested whereas the remainder showed normal development. The levels of 14 implantation factors secreted by the stromal cells were determined by multiplex immunoassay. Surprisingly, the presence of a developing embryo had no significant effect on decidual secretions, apart from a modest reduction in IL-5 levels. In contrast, arresting embryos triggered a strong response, characterized by selective inhibition of IL-1β, -6, -10, -17, -18, eotaxin, and HB-EGF secretion. Co-cultures were repeated with undifferentiated ESCs but none of the secreted cytokines were affected by the presence of a developing or arresting embryo. Conclusions Human ESCs become biosensors of embryo quality upon differentiation into decidual cells. In view of the high incidence of gross chromosomal errors in human preimplantation embryos, cyclic decidualization followed by menstrual shedding may represent a mechanism of natural embryo selection that limits maternal investment in developmentally impaired pregnancies. PMID:20422011

Enhanced induction of microspore embryogenesis after n-butanol treatment in wheat (Triticum aestivum L.) anther culture.

PubMed

Soriano, M; Cistué, L; Castillo, A M

2008-05-01

The aim of this study was the improvement of embryo production in wheat anther culture. Three butanol alcohols, n-butanol, sec-butanol and tert-butanol, were evaluated for their effect on microspore embryogenesis in two spring cultivars of wheat, Pavon and Caramba. Application of n-butanol, at 0.1 and 0.2% (v/v) in the induction media for 5 h, highly improved embryo production in both cultivars. Sec- and tert-butanol performed similarly to control plates. Regeneration ability was unaffected by any butyl-alcohol treatment. As a consequence of the higher embryo production after n-butanol treatment, the number of green regenerated plants increased up to five times in cultivar Pavon and up to three times in cultivar Caramba. The percentage of green plants was improved or unaffected by the treatment. Doubled haploid plant production was between 2 and 4 times higher after n-butanol treatment than in control plates. Therefore, n-butanol was successfully applied in the production of wheat doubled haploids. This primary alcohol is known as an activator of phospholipase D and has been previously reported to disrupt cortical microtubules and detach them from the plasma membrane in plants. Its effects on androgenetic induction could confirm the importance of microtubule regulation in plant cell fate, specifically in microspore development. A possible implication of phospholipase D is discussed.

Somatic Embryogenesis in Peach Palm Using the Thin Cell Layer Technique: Induction, Morpho-histological Aspects and AFLP Analysis of Somaclonal Variation

PubMed Central

Steinmacher, D. A.; Krohn, N. G.; Dantas, A. C. M.; Stefenon, V. M.; Clement, C. R.; Guerra, M. P.

2007-01-01

Background and Aims The thin cell layer (TCL) technique is based on the use of very small explants and has allowed enhanced in vitro morphogenesis in several plant species. The present study evaluated the TCL technique as a procedure for somatic embryo production and plantlet regeneration of peach palm. Methods TCL explants from different positions in the shoot apex and leaf sheath of peach palm were cultivated in MS culture medium supplemented with 0–600 µm Picloram in the presence of activated charcoal. The production of primary calli and embryogenic calli was evaluated in these different conditions. Histological and amplified fragment length polymorphism (AFLP) analyses were conducted to study in vitro morphogenetic responses and genetic stability, respectively, of the regenerated plantlets. Key Results Abundant primary callus induction was observed from TCLs of the shoot meristem in culture media supplemented with 150–600 µm Picloram (83–97 %, respectively). The production of embryogenic calli depends on Picloram concentration and explant position. The best response observed was 43 % embryogenic callus production from shoot meristem TCL on 300 µm Picloram. In maturation conditions, 34 ± 4 somatic embryos per embryogenic callus were obtained, and 45·0 ± 3·4 % of these fully developed somatic embryos were converted, resulting in plantlets ready for acclimatization, of which 80 % survived. Histological studies revealed that the first cellular division events occurred in cells adjacent to vascular tissue, resulting in primary calli, whose growth was ensured by a meristematic zone. A multicellular origin of the resulting somatic embryos arising from the meristematic zone is suggested. During maturation, histological analyses revealed bipolarization of the somatic embryos, as well as the development of new somatic embryos. AFLP analyses revealed that 92 % of the regenerated plantlets were true to type. The use of TCL explants considerably improves the number of calli and somatic embryos produced in comparison with previously described protocols for in vitro regeneration of peach palm. Conclusions The present study suggests that the TCL somatic embryogenesis protocol developed is feasible, although it still requires further optimization for in vitro multiplication of peach palm, especially the use of similar explants obtained from adult palm trees. PMID:17670751

The role of cGMP as a mediator of lipolysis in bovine oocytes and its effects on embryo development and cryopreservation.

PubMed

Schwarz, Kátia R L; de Castro, Fernanda C; Schefer, Letícia; Botigelli, Ramon C; Paschoal, Daniela M; Fernandes, Hugo; Leal, Cláudia L V

2018-01-01

This study aimed to determine the influence of cyclic guanosine 3'5'-monophosphate (cGMP) and cGMP-dependent kinase (PKG) during in vitro maturation (IVM) on lipolysis-related parameters in bovine cumulus-oocyte complexes (COCs), and on embryo development and cryosurvival. COCs were matured with cGMP/PKG modulators and assessed for metaphase II rates (MII), cGMP levels, lipid content in oocytes (OO), transcript abundance for genes involved in lipolysis (ATGL) and lipid droplets (PLIN2) in cumulus cells (CC) and OO, and presence of phosphorylated (active) hormone sensitive lipase (HSLser563) in OO. Embryo development, lipid contents and survival to vitrification were also assessed. Phosphodiesterase 5 inhibition (PDE5; cGMP-hydrolyzing enzyme) with 10-5M sildenafil (SDF) during 24 h IVM increased cGMP in COCs (56.9 vs 9.5 fMol/COC in untreated controls, p<0.05) and did not affect on maturation rate (84.3±6.4% MII). Fetal calf serum (FCS) in IVM medium decreased cGMP in COCs compared to bovine serum albumin (BSA) + SDF (19.6 vs 66.5 fMol/COC, respectively, p<0.05). FCS increased lipid content in OO (40.1 FI, p<0.05) compared to BSA (34.6 FI), while SDF decreased (29.8 and 29.6 FI, with BSA or FCS, respectively p<0.05). PKG inhibitor (KT5823) reversed this effect (38.9 FI, p<0.05). ATGL and PLIN2 transcripts were detected in CC and OO, but were affected by cGMP and PKG only in CC. HSLser563 was detected in OO matured with or without modulators. Reduced lipid content in embryos were observed only when SDF was added during IVM and IVC (27.6 FI) compared to its use in either or none of the culture periods (34.2 FI, p<0.05). Survival to vitrification was unaffected by SDF. In conclusion, cGMP and PKG are involved in lipolysis in OO and possibly in CC and embryos; serum negatively affects this pathway, contributing to lipid accumulation, and cGMP modulation may reduce lipid contents in oocytes and embryos, but without improving embryo cryotolerance.

Post-thaw motility of frozen boar sperm does not predict success with in vitro fertilization

USDA-ARS?s Scientific Manuscript database

Using cryopreserved boar sperm rather than liquid semen for in vitro fertilization (IVF) allows improved IVF consistency. However, cryopreservation of boar sperm results in reduced post-thaw motility, fertilization and embryo development. Boars are often screened on an individual basis prior to use ...

Effects of exogenous melatonin on in vivo embryo viability and oocyte competence of undernourished ewes after weaning during the seasonal anestrus.

PubMed

Vázquez, M I; Abecia, J A; Forcada, F; Casao, A

2010-09-01

This study investigated the effects of exogenous melatonin on embryo viability and oocyte competence in post-partum undernourished ewes during the seasonal anestrus. At parturition (mid-Feb), 36 adult Rasa Aragonesa ewes were assigned to one of two groups: treated (+MEL) or not treated (-MEL) with a subcutaneous implant of melatonin (Melovine(R), CEVA) on the day of lambing. After 45 d of suckling, lambs were weaned, ewes were synchronized using intravaginal pessaries, and fed to provide 1.5x (Control, C) or 0.5x (Low, L) times daily maintenance requirements. Thus, ewes were divided into four groups: C-MEL, C+MEL, L-MEL, and L+MEL. At estrus (Day=0), ewes were mated. At Day 5 after estrus, embryos were recovered by mid-ventral laparotomy and classified based on their developmental stage and morphology. After embryo collection, ovaries were recovered and oocytes were classified and selected for use in in vitro fertilization (IVF). Neither diet nor melatonin treatment had a significant effect on ovulation rate and on the number of ova recovered per ewe. Melatonin treatment significantly improved the number of fertilized embryos/corpus luteum (CL) (-MEL: 0.35 +/- 0.1, +MEL: 0.62 +/- 0.1; P = 0.08), number of viable embryos/CL (-MEL: 0.23 +/- 0.1, +MEL: 0.62 +/- 0.1; P < 0.01), viability rate (-MEL: 46.6%, +MEL: 83.9%; P < 0.05), and pregnancy rate (-MEL: 26.3%, +MEL: 76.5%; P < 0.05). In particular, exogenous melatonin improved embryo viability in undernourished ewes (L-MEL: 40%, L+MEL: 100%, P < 0.01). Neither nutrition nor exogenous melatonin treatments significantly influenced the competence of oocytes during IVF. Treatment groups did not differ significantly in the number of healthy oocytes used for IVF, number of cleaved embryos, or number of blastocysts and, consequently, the groups had similar cleavage and blastocyst rates. In conclusion, melatonin treatments improved ovine embryo viability during anestrus, particularly in undernourished post-partum ewes, although the effects of melatonin did not appear to be mediated at the oocyte competence level. Copyright 2010 Elsevier Inc. All rights reserved.

Transcriptomic analysis highlights epigenetic and transcriptional regulation during zygotic embryo development of Pinus pinaster.

PubMed

de Vega-Bartol, José J; Simões, Marta; Lorenz, W Walter; Rodrigues, Andreia S; Alba, Rob; Dean, Jeffrey F D; Miguel, Célia M

2013-08-30

It is during embryogenesis that the plant body plan is established and the meristems responsible for all post-embryonic growth are specified. The molecular mechanisms governing conifer embryogenesis are still largely unknown. Their elucidation may contribute valuable information to clarify if the distinct features of embryo development in angiosperms and gymnosperms result from differential gene regulation. To address this issue, we have performed the first transcriptomic analysis of zygotic embryo development in a conifer species (Pinus pinaster) focusing our study in particular on regulatory genes playing important roles during plant embryo development, namely epigenetic regulators and transcription factors. Microarray analysis of P. pinaster zygotic embryogenesis was performed at five periods of embryo development from early developing to mature embryos. Our results show that most changes in transcript levels occurred in the first and the last embryo stage-to-stage transitions, namely early to pre-cotyledonary embryo and cotyledonary to mature embryo. An analysis of functional categories for genes that were differentially expressed through embryogenesis highlighted several epigenetic regulation mechanisms. While putative orthologs of transcripts associated with mechanisms that target transposable elements and repetitive sequences were strongly expressed in early embryogenesis, PRC2-mediated repression of genes seemed more relevant during late embryogenesis. On the other hand, functions related to sRNA pathways appeared differentially regulated across all stages of embryo development with a prevalence of miRNA functions in mid to late embryogenesis. Identification of putative transcription factor genes differentially regulated between consecutive embryo stages was strongly suggestive of the relevance of auxin responses and regulation of auxin carriers during early embryogenesis. Such responses could be involved in establishing embryo patterning. Later in development, transcripts with homology to genes acting on modulation of auxin flow and determination of adaxial-abaxial polarity were up-regulated, as were putative orthologs of genes required for meristem formation and function as well as establishment of organ boundaries. Comparative analysis with A. thaliana embryogenesis also highlighted genes involved in auxin-mediated responses, as well as epigenetic regulation, indicating highly correlated transcript profiles between the two species. This is the first report of a time-course transcriptomic analysis of zygotic embryogenesis in a conifer. Taken together our results show that epigenetic regulation and transcriptional control related to auxin transport and response are critical during early to mid stages of pine embryogenesis and that important events during embryogenesis seem to be coordinated by putative orthologs of major developmental regulators in angiosperms.

Transcriptomic analysis highlights epigenetic and transcriptional regulation during zygotic embryo development of Pinus pinaster

PubMed Central

2013-01-01

Background It is during embryogenesis that the plant body plan is established and the meristems responsible for all post-embryonic growth are specified. The molecular mechanisms governing conifer embryogenesis are still largely unknown. Their elucidation may contribute valuable information to clarify if the distinct features of embryo development in angiosperms and gymnosperms result from differential gene regulation. To address this issue, we have performed the first transcriptomic analysis of zygotic embryo development in a conifer species (Pinus pinaster) focusing our study in particular on regulatory genes playing important roles during plant embryo development, namely epigenetic regulators and transcription factors. Results Microarray analysis of P. pinaster zygotic embryogenesis was performed at five periods of embryo development from early developing to mature embryos. Our results show that most changes in transcript levels occurred in the first and the last embryo stage-to-stage transitions, namely early to pre-cotyledonary embryo and cotyledonary to mature embryo. An analysis of functional categories for genes that were differentially expressed through embryogenesis highlighted several epigenetic regulation mechanisms. While putative orthologs of transcripts associated with mechanisms that target transposable elements and repetitive sequences were strongly expressed in early embryogenesis, PRC2-mediated repression of genes seemed more relevant during late embryogenesis. On the other hand, functions related to sRNA pathways appeared differentially regulated across all stages of embryo development with a prevalence of miRNA functions in mid to late embryogenesis. Identification of putative transcription factor genes differentially regulated between consecutive embryo stages was strongly suggestive of the relevance of auxin responses and regulation of auxin carriers during early embryogenesis. Such responses could be involved in establishing embryo patterning. Later in development, transcripts with homology to genes acting on modulation of auxin flow and determination of adaxial-abaxial polarity were up-regulated, as were putative orthologs of genes required for meristem formation and function as well as establishment of organ boundaries. Comparative analysis with A. thaliana embryogenesis also highlighted genes involved in auxin-mediated responses, as well as epigenetic regulation, indicating highly correlated transcript profiles between the two species. Conclusions This is the first report of a time-course transcriptomic analysis of zygotic embryogenesis in a conifer. Taken together our results show that epigenetic regulation and transcriptional control related to auxin transport and response are critical during early to mid stages of pine embryogenesis and that important events during embryogenesis seem to be coordinated by putative orthologs of major developmental regulators in angiosperms. PMID:23987738

Baicalin increases developmental competence of mouse embryos in vitro by inhibiting cellular apoptosis and modulating HSP70 and DNMT expression

PubMed Central

QI, Xiaonan; LI, Huatao; CONG, Xia; WANG, Xin; JIANG, Zhongling; CAO, Rongfeng; TIAN, Wenru

2016-01-01

Scutellaria baicalensis has been effectively used in Chinese traditional medicine to prevent miscarriages. However, little information is available on its mechanism of action. This study is designed specifically to reveal how baicalin, the main effective ingredient of S. baicalensis, improves developmental competence of embryos in vitro, using the mouse as a model. Mouse pronuclear embryos were cultured in KSOM medium supplemented with (0, 2, 4 and 8 μg/ml) baicalin. The results demonstrated that in vitro culture conditions significantly decreased the blastocyst developmental rate and blastocyst quality, possibly due to increased cellular stress and apoptosis. Baicalin (4 µg/ml) significantly increased 2- and 4-cell cleavage rates, morula developmental rate, and blastocyst developmental rate and cell number of in vitro-cultured mouse embryos. Moreover, baicalin increased the expression of Gja1, Cdh1, Bcl-2, and Dnmt3a genes, decreased the expression of Dnmt1 gene, and decreased cellular stress and apoptosis as it decreased the expression of HSP70, CASP3, and BAX and increased BCL-2 expression in blastocysts cultured in vitro. In conclusion, baicalin improves developmental competence of in vitro-cultured mouse embryos through inhibition of cellular apoptosis and HSP70 expression, and improvement of DNA methylation. PMID:27478062

Chapter 1 Historical Background on Gamete and Embryo Cryopreservation.

PubMed

Ali, Jaffar; AlHarbi, Naif H; Ali, Nafisa

2017-01-01

This chapter describes the development of the science of cryopreservation of gametes and embryos of various species including human. It attempts to record in brief the main contributions of workers in their attempts to cryopreserve gametes and embryos. The initial difficulties faced and subsequent developments and triumphs leading to present-day state of the art are given in a concise manner. The main players and their contributions are mentioned and the authors' aim is to do justice to them. This work also attempts to ensure that credit is correctly attributed for significant advances in gamete and embryo cryopreservation. In general this chapter has tried to describe the historical development of the science of cryopreservation of gametes and embryos as accurately as possible without bias or partiality.

In vitro development of canine somatic cell nuclear transfer embryos in different culture media.

PubMed

Kim, Dong-Hoon; No, Jin-Gu; Choi, Mi-Kyung; Yeom, Dong-Hyeon; Kim, Dong-Kyo; Yang, Byoung-Chul; Yoo, Jae Gyu; Kim, Min Kyu; Kim, Hong-Tea

2015-01-01

The objective of the present study was to investigate the effects of three different culture media on the development of canine somatic cell nuclear transfer (SCNT) embryos. Canine cloned embryos were cultured in modified synthetic oviductal fluid (mSOF), porcine zygote medium-3 (PZM-3), or G1/G2 sequential media. Our results showed that the G1/G2 media yielded significantly higher morula and blastocyst development in canine SCNT embryos (26.1% and 7.8%, respectively) compared to PZM-3 (8.5% and 0%or mSOF (2.3% and 0%) media. In conclusion, this study suggests that blastocysts can be produced more efficiently using G1/G2 media to culture canine SCNT embryos.

Persons and their bodies: how we should think about human embryos.

PubMed

McLachlan, Hugh V

2002-01-01

The status of human embryos is discussed particularly in the light of the claim by Fox, in Health Care Analysis 8 that it would be useful to think of them in terms of cyborg metaphors. It is argued that we should consider human embryos for what they are--partially formed human bodies--rather than for what they are like in some respects (and unlike in others)--cyborgs. However to settle the issue of the status of the embryo is not to answer the moral questions which arise concerning how embryos should be treated. Since persons rather than bodies have rights, embryos do not have rights. However, whether or not embryos have rights, people can have duties concerning them. Furthermore, the persons whose fully developed bodies embryos will, might (or might have) become can have rights. Contrary to what is often assumed, it is not merely persons who have (or have had) living, developed human bodies who have moral rights: so it is argued in this paper.

Pattern of Variations in Abscisic Acid Content in Suspensors, Embryos, and Integuments of Developing Phaseolus coccineus Seeds 1

PubMed Central

Perata, Pierdomenico; Picciarelli, Piero; Alpi, Amedeo

1990-01-01

Free abscisic acid (ABA) content in suspensors, embryos, and integuments was determined during seed development of Phaseolus coccineus. A highly specific and sensitive solid-phase radioimmunoassay based on a monocional antibody raised against free (S)-ABA was used for ABA quantification. Very small amounts of ABA were detected in the suspensor during initial stages of development; later two peaks of ABA occurred. Levels of ABA in the embryo and integument show a coincident triphasic distribution: two maxima in ABA content occurred when the embryo was 11 to 12 and 15 to 16 millimeters in length; later, when the embryo was 19 to 20 millimeters long, a further increase was observed. The role of ABA in runner bean seeds is discussed in relation to the development of the different seed tissues. PMID:16667915

Screening the Toxicity of Selected Personal Care Products Using Embryo Bioassays: 4-MBC, Propylparaben and Triclocarban

PubMed Central

Torres, Tiago; Cunha, Isabel; Martins, Rosário; Santos, Miguel M.

2016-01-01

Recently, several emerging pollutants, including Personal Care Products (PCPs), have been detected in aquatic ecosystems, in the ng/L or µg/L range. Available toxicological data is limited, and, for certain PCPs, evidence indicates a potential risk for the environment. Hence, there is an urgent need to gather ecotoxicological data on PCPs as a proxy to improve risk assessment. Here, the toxicity of three different PCPs (4-Methylbenzylidene Camphor (4-MBC), propylparaben and triclocarban) was tested using embryo bioassays with Danio rerio (zebrafish) and Paracentrotus lividus (sea urchin). The No Observed Effect Concentration (NOEC) for triclocarban was 0.256 µg/L for sea urchin and 100 µg/L for zebrafish, whereas NOEC for 4-MBC was 0.32 µg/L for sea urchin and 50 µg/L for zebrafish. Both PCPs impacted embryo development at environmentally relevant concentrations. In comparison with triclocarban and 4-MBC, propylparaben was less toxic for both sea urchin (NOEC = 160 µg/L) and zebrafish (NOEC = 1000 µg/L). Overall, this study further demonstrates the sensitivity of embryo bioassays as a high-throughput approach for testing the toxicity of emerging pollutants. PMID:27775672

Screening the Toxicity of Selected Personal Care Products Using Embryo Bioassays: 4-MBC, Propylparaben and Triclocarban.

PubMed

Torres, Tiago; Cunha, Isabel; Martins, Rosário; Santos, Miguel M

2016-10-21

Recently, several emerging pollutants, including Personal Care Products (PCPs), have been detected in aquatic ecosystems, in the ng/L or µg/L range. Available toxicological data is limited, and, for certain PCPs, evidence indicates a potential risk for the environment. Hence, there is an urgent need to gather ecotoxicological data on PCPs as a proxy to improve risk assessment. Here, the toxicity of three different PCPs (4-Methylbenzylidene Camphor (4-MBC), propylparaben and triclocarban) was tested using embryo bioassays with Danio rerio (zebrafish) and Paracentrotus lividus (sea urchin). The No Observed Effect Concentration (NOEC) for triclocarban was 0.256 µg/L for sea urchin and 100 µg/L for zebrafish, whereas NOEC for 4-MBC was 0.32 µg/L for sea urchin and 50 µg/L for zebrafish. Both PCPs impacted embryo development at environmentally relevant concentrations. In comparison with triclocarban and 4-MBC, propylparaben was less toxic for both sea urchin (NOEC = 160 µg/L) and zebrafish (NOEC = 1000 µg/L). Overall, this study further demonstrates the sensitivity of embryo bioassays as a high-throughput approach for testing the toxicity of emerging pollutants.

Limiting factors to encapsulation: the combined effects of dissolved protein and oxygen availability on embryonic growth and survival of species with contrasting feeding strategies.

PubMed

Brante, Antonio; Fernández, Miriam; Viard, Frédérique

2009-07-01

Encapsulation is a common strategy among marine invertebrate species. It has been shown that oxygen and food availability independently constrain embryo development during intracapsular development. However, it is unclear how embryos of species with different feeding strategies perceive these two constraints when operating jointly. In the present study, we examined the relative importance of dissolved albumen, as a food source, oxygen condition and their interaction on embryonic growth and the survival of two calyptraeid species, Crepidula coquimbensis and Crepidula fornicata, exhibiting different embryo feeding behaviours (i.e. presence vs absence of intracapsular cannibalism). Two oxygen condition treatments (normoxia and hypoxia) and three albumen concentrations (0, 1 and 2 mg l(-1)) were studied. In addition, albumen intake by embryos was observed using fluorescence microscopy. Our study shows that embryos of both species incorporated dissolved albumen but used a different set of embryonic organs. We observed that embryo growth rates in C. coquimbensis were negatively affected only by hypoxic conditions. Conversely, a combination of low albumen concentration and oxygen availability slowed embryo growth in C. fornicata. These findings suggest that oxygen availability is a limiting factor for the normal embryo development of encapsulated gastropod species, regardless of feeding behaviour or developmental mode. By contrast, the effect of dissolved albumen as an alternative food source on embryo performance may depend on the feeding strategy of the embryos.

Ice nucleating agents allow embryo freezing without manual seeding.

PubMed

Teixeira, Magda; Buff, Samuel; Desnos, Hugo; Loiseau, Céline; Bruyère, Pierre; Joly, Thierry; Commin, Loris

2017-12-01

Embryo slow freezing protocols include a nucleation induction step called manual seeding. This step is time consuming, manipulator dependent and hard to standardize. It requires access to samples, which is not always possible within the configuration of systems, such as differential scanning calorimeters or cryomicroscopes. Ice nucleation can be induced by other methods, e.g., by the use of ice nucleating agents. Snomax is a commercial preparation of inactivated proteins extracted from Pseudomonas syringae. The aim of our study was to investigate if Snomax can be an alternative to manual seeding in the slow freezing of mouse embryos. The influence of Snomax on the pH and osmolality of the freezing medium was evaluated. In vitro development (blastocyst formation and hatching rates) of fresh embryos exposed to Snomax and embryo cryopreserved with and without Snomax was assessed. The mitochondrial activity of frozen-thawed blastocysts was assessed by JC-1 fluorescent staining. Snomax didn't alter the physicochemical properties of the freezing medium, and did not affect embryo development of fresh embryos. After cryopreservation, the substitution of manual seeding by the ice nucleating agent (INA) Snomax did not affect embryo development or embryo mitochondrial activity. In conclusion, Snomax seems to be an effective ice nucleating agent for the slow freezing of mouse embryos. Snomax can also be a valuable alternative to manual seeding in research protocols in which manual seeding cannot be performed (i.e., differential scanning calorimetry and cryomicroscopy). Copyright © 2017 Elsevier Inc. All rights reserved.

In vitro survival of fresh and frozen/thawed bovine demi-embryos.

PubMed

Lucas-Hahn, A; Niemann, H

1991-10-01

Three experiments were conducted to investigate the effects of type of culture medium in freshly bisected bovine embryos and the effects of agar embedding and of 1.2 propanediol (PROH) as the cryoprotectant in frozen/thawed bisected bovine embryos on development in vitro. A total of 265 bovine embryos were used as controls or were microsurgically bisected and were cultured in vitro for 48 hours and development was determined 24 and 48 hours after the onset of culture. Whitten's medium supported more (P<0.05) intact and demi-embryos to grow to expanded blastocysts (92.9 and 73.1%, respectively) compared with Ham's F10 (43.8 and 26.3%, respectively) and PBS (53.8 and 12.5%, respectively). Embedding in agar and culture in Whitten's medium resulted in a higher (P<0.05) percentage of in vitro development of frozen/thawed demi-embryos after 24 hours than the freezing of nonembedded demi-embryos (44.1 versus 19.6%, respectively). This difference disappeared, however, after a 48 hours culture period (17.6 versus 11.8%, respectively). Following freezing in PROH, survival rates of 40 and 28%, respectively after 24 hours of culture were obtained for intact and demi-embryos. The respective percentages after 48 hours were 8.6 and 16%. Since neither embedding in agar nor the use of PROH as the cryoprotectant resulted in high survival rates of frozen/thawed demi-embryos in vitro, new freezing procedures are needed to overcome the sensitivity of demi-embryos to freezing and thawing.

Post-fusion treatment with MG132 increases transcription factor expression in somatic cell nuclear transfer embryos in pigs.

PubMed

You, Jinyoung; Lee, Joohyeong; Kim, Jinyoung; Park, Junhong; Lee, Eunsong

2010-02-01

The objective of this study was to examine the effect of post-fusion treatment of somatic cell nuclear transfer (SCNT) oocytes with the proteasomal inhibitor MG132 on maturation promoting factor (MPF) activity, nuclear remodeling, embryonic development, and gene expression of cloned pig embryos. Immediately after electrofusion, SCNT oocytes were treated with MG132 and/or caffeine for 2 hr, vanadate for 0.5 hr, or vanadate for 0.5 hr followed by MG132 for 1.5 hr. Of the MG132 concentrations tested (0-5 microM), the 1 microM concentration showed a higher rate of blastocyst formation (25.9%) than 0 (14.2%), 0.5 (16.9%), and 5 microM (16.9%). Post-fusion treatment with MG132, caffeine, and both MG132 and caffeine improved blastocyst formation (22.1%, 21.4%, and 24.4%, respectively), whereas vanadate treatment inhibited blastocyst formation (6.5%) compared to the control (11.1%). When examined 2 hr after fusion and 1 hr after activation, MPF activity remained at a higher (P < 0.05) level in SCNT oocytes that were treated post-fusion with caffeine and/or MG132, but it was decreased by vanadate. The rate of oocytes showing premature chromosome condensation was not altered by MG132 but was decreased by vanadate treatment. In addition, formation of single pronuclei was increased by MG132 compared to control and vanadate treatment. MG132-treated embryos showed increased expression of POU5F1, DPPA2, DPPA3, DPPA5, and NDP52l1 genes compared to control embryos. Our results demonstrate that post-fusion treatment of SCNT oocytes with MG132 prevents MPF degradation and increases expression of transcription factors in SCNT embryos, which are necessary for normal development of SCNT embryos. (c) 2009 Wiley-Liss, Inc.

Are there symplastic connections between the endosperm and embryo in some angiosperms?--a lesson from the Crassulaceae family.

PubMed

Kozieradzka-Kiszkurno, Małgorzata; Płachno, Bartosz Jan

2012-10-01

It is believed that there is symplastic isolation between the embryo (new sporophyte) and the endosperm (maternal-parental origin tissue, which nourishes the embryo) in angiosperms. However, in embryological literature there are rare examples in which plasmodesmata between the embryo suspensor and endosperm cells have been recorded (three species from Fabaceae). This study was undertaken in order to test the hypothesis that plasmodesmata between the embryo suspensor and the endosperm are not so rare but also occur in other angiosperm families; in order to check this, we used the Crassulaceae family because embryogenesis in Crassulaceae has been studied extensively at an ultrastructure level recently and also we tread members of this family as model for suspensor physiology and function studies. These plasmodesmata even occurred between the basal cell of the two-celled proembryo and endosperm cells. The plasmodesmata were simple at this stage of development. During the development of the embryo proper and the suspensor, the structure of plasmodesmata changes. They were branched and connected with electron-dense material. Our results suggest that in Crassulaceae with plasmodesmata between the endosperm and suspensor, symplastic connectivity at this cell-cell boundary is still reduced or blocked at a very early stage of embryo development (before the globular stage). The occurrence of plasmodesmata between the embryo suspensor and endosperm cells suggests possible symplastic transport between these different organs, at least at a very early stage of embryo development. However, whether this transport actually occurs needs to be proven experimentally. A broader analysis of plants from various families would show whether the occurrence of plasmodesmata between the embryo suspensor and the endosperm are typical embryological characteristics and if this is useful in discussions about angiosperm systematic and evolution.

Critical developmental windows for morphology and hematology revealed by intermittent and continuous hypoxic incubation in embryos of quail (Coturnix coturnix)

PubMed Central

Elmonoufy, Nourhan A.

2017-01-01

Hypoxia during embryonic growth in embryos is frequently a powerful determinant of development, but at least in avian embryos the effects appear to show considerable intra- and inter-specific variation. We hypothesized that some of this variation may arise from different protocols that may or may not result in exposure during the embryo’s critical window for hypoxic effects. To test this hypothesis, quail embryos (Coturnix coturnix) in the intact egg were exposed to hypoxia (~15% O2) during “early” (Day 0 through Day 5, abbreviated as D0-D5), “middle” (D6-D10) or “late” (D11-D15) incubation or for their entire 16–18 day incubation (“continuous hypoxia”) to determine critical windows for viability and growth. Viability, body mass, beak and toe length, heart mass, and hematology (hematocrit and hemoglobin concentration) were measured on D5, D10, D15 and at hatching typically between D16 and D18 Viability rate was ~50–70% immediately following the exposure period in the early, middle and late hypoxic groups, but viability improved in the early and late groups once normoxia was restored. Middle hypoxia groups showed continuing low viability, suggesting a critical period from D6-D10 for embryo viability. The continuous hypoxia group experienced viability reaching <10% after D15. Hypoxia, especially during late and continuous hypoxia, also inhibited growth of body, beak and toe when measured at D15. Full recovery to normal body mass upon hatching occurred in all other groups except for continuous hypoxia. Contrary to previous avian studies, heart mass, hematocrit and hemoglobin concentration were not altered by any hypoxic incubation pattern. Although hypoxia can inhibit embryo viability and organ growth during most incubation periods, the greatest effects result from continuous or middle incubation hypoxic exposure. Hypoxic inhibition of growth can subsequently be “repaired” by catch-up growth if a final period of normoxic development is available. Collectively, these data indicate a critical developmental window for hypoxia susceptibility during the mid-embryonic period of development. PMID:28926567

JPRS Report, China

DTIC Science & Technology

1988-02-05

China has decided to use calf embryo transplants, in conjunction with artificial insemination , to improve meat and milk production from its native...oxen. Though artificial insemination will play the main role in improving herds, embryo transplants are also intended. Hybrids will play a key role...projects offering good returns; in reality, however, "trading" in funds is largely artificially restricted to "adjustments within the system

Using game theory to investigate the epigenetic control mechanisms of embryo development: Comment on: "Epigenetic game theory: How to compute the epigenetic control of maternal-to-zygotic transition" by Qian Wang et al.

PubMed

Zhang, Le; Zhang, Shaoxiang

2017-03-01

A body of research [1-7] has already shown that epigenetic reprogramming plays a critical role in maintaining the normal development of embryos. However, the mechanistic quantitation of the epigenetic interactions between sperms and oocytes and the related impact on embryo development are still not clear [6,7]. In this study, Wang et al., [8] develop a modeling framework that addresses this question by integrating game theory and the latest discoveries of the epigenetic control of embryo development. Copyright © 2017 Elsevier B.V. All rights reserved.

Endometrium as an early sensor of in vitro embryo manipulation technologies

PubMed Central

Mansouri-Attia, Nadéra; Sandra, Olivier; Aubert, Julie; Degrelle, Séverine; Everts, Robin E.; Giraud-Delville, Corinne; Heyman, Yvan; Galio, Laurent; Hue, Isabelle; Yang, Xiangzhong; Tian, X. Cindy; Lewin, Harris A.; Renard, Jean-Paul

2009-01-01

Implantation is crucial for placental development that will subsequently impact fetal growth and pregnancy success with consequences on postnatal health. We postulated that the pattern of genes expressed by the endometrium when the embryo becomes attached to the mother uterus could account for the final outcome of a pregnancy. As a model, we used the bovine species where the embryo becomes progressively and permanently attached to the endometrium from day 20 of gestation onwards. At that stage, we compared the endometrial genes profiles in the presence of an in vivo fertilized embryo (AI) with the endometrial patterns obtained in the presence of nuclear transfer (SCNT) or in vitro fertilized embryos (IVF), both displaying lower and different potentials for term development. Our data provide evidence that the endometrium can be considered as a biological sensor able to fine-tune its physiology in response to the presence of embryos whose development will become altered much later after the implantation process. Compared with AI, numerous biological functions and several canonical pathways with a major impact on metabolism and immune function were found to be significantly altered in the endometrium of SCNT pregnancies at implantation, whereas the differences were less pronounced with IVF embryos. Determining the limits of the endometrial plasticity at the onset of implantation should bring new insights on the contribution of the maternal environment to the development of an embryo and the success of pregnancy. PMID:19297625

Identification and characterization of an oocyte factor required for development of porcine nuclear transfer embryos

PubMed Central

Miyamoto, Kei; Nagai, Kouhei; Kitamura, Naoya; Nishikawa, Tomoaki; Ikegami, Haruka; Binh, Nguyen T.; Tsukamoto, Satoshi; Matsumoto, Mai; Tsukiyama, Tomoyuki; Minami, Naojiro; Yamada, Masayasu; Ariga, Hiroyoshi; Miyake, Masashi; Kawarasaki, Tatsuo; Matsumoto, Kazuya; Imai, Hiroshi

2011-01-01

Nuclear reprogramming of differentiated cells can be induced by oocyte factors. Despite numerous attempts, these factors and mechanisms responsible for successful reprogramming remain elusive. Here, we identify one such factor, necessary for the development of nuclear transfer embryos, using porcine oocyte extracts in which some reprogramming events are recapitulated. After incubating somatic nuclei in oocyte extracts from the metaphase II stage, the oocyte proteins that were specifically and abundantly incorporated into the nuclei were identified by mass spectrometry. Among 25 identified proteins, we especially focused on a multifunctional protein, DJ-1. DJ-1 is present at a high concentration in oocytes from the germinal vesicle stage until embryos at the four-cell stage. Inhibition of DJ-1 function compromises the development of nuclear transfer embryos but not that of fertilized embryos. Microarray analysis of nuclear transfer embryos in which DJ-1 function is inhibited shows perturbed expression of P53 pathway components. In addition, embryonic arrest of nuclear transfer embryos injected with anti–DJ-1 antibody is rescued by P53 inhibition. We conclude that DJ-1 is an oocyte factor that is required for development of nuclear transfer embryos. This study presents a means for identifying natural reprogramming factors in mammalian oocytes and a unique insight into the mechanisms underlying reprogramming by nuclear transfer. PMID:21482765

A Technique for Facile and Precise Transfer of Mouse Embryos

PubMed Central

Sarvari, Ali; Naderi, Mohammad Mehdi; Sadeghi, Mohammad Reza; Akhondi, Mohammad Mehdi

2013-01-01

Background Successful Embryo Transfer (ET) technique is a fateful step of all efforts to achieve live births from in vitro produced embryos in assisted reproductive techniques or in knockout, transgenic or cloned animal projects. Small reproductive tract of mice and limitation of current techniques may not well satisfy the requirements for mass production of genetically modified mice. Genetic abnormalities of embryos, receptivity and uterine contractions, expulsion of embryos, blood, mucus or bacterial contamination on the transfer pipette tip, technical problems and even animal strain may affect embryo transfer outcome. Methods In this study, two techniques of embryo transfer in mice were compared. In conventional technique the oviduct wall was punctured with a 30-gauge needle and the loaded Pasteur pipette with embryos and medium was inserted into the hole. In new technique, embryos that were loaded in modified micropipette with minimal medium were transferred directly to the oviduct by manual piston micro-pump easily. Embryo viability was evaluated considering the percentage of live healthy newborns. Results Results of the two techniques were compared by t-test within the NPAR1WAY procedure of SAS software (ver. 9.2). The average live birth rates in the novel methods was significantly higher (42.4%) than the conventional method (21.7%, p<0.05). Conclusion In conclusion, using new embryo transfer technique improved birth rate by preventing embryos expulsion from the oviduct, saving time and easy transfer of embryos with minimum volume of medium. PMID:23626878

Normal calves produced after transfer of embryos cultured in a chemically defined medium supplemented with epidermal growth factor and insulin-like growth factor I following ovum pick up and in vitro fertilization in Japanese black cows.

PubMed

Sakagami, Nobutada; Umeki, Hidenobu; Nishino, Osamu; Uchiyama, Hiroko; Ichikawa, Kyoko; Takeshita, Kazuhisa; Kaneko, Etsushi; Akiyama, Kiyoshi; Kobayashi, Shuji; Tamada, Hiromichi

2012-01-01

The objective of this study was to examine whether high concentrations of epidermal growth factor (EGF) and/or insulin-like growth factor I (IGF-I) would have a beneficial effect on bovine embryo development in vitro and to obtain normal calves by using an ovum pick up method and embryo culture in a chemically defined medium. When compared with controls, EGF (100 or 200 ng/ml) or IGF-I (50 or 100 ng/ml) significantly increased the rate of embryos that developed into blastocysts during an 8-day culture after the in vitro fertilization of oocytes obtained from ovaries from a slaughterhouse. IGF-I induced a dose-dependent increase in cell number in both the inner cell mass and the trophectoderm, whereas EGF stimulated proliferation only in the inner cell mass. A combination of EGF (100 ng/ml) and IGF-I (50 ng/ml) produced an additive effect, and embryos developed into blastocysts at a comparatively high rate (27.9%) compared with controls (12.0%). A similar rate of development was achieved using a combination of EGF and IGF-I in the culture of embryos following ovum pick up by ultrasound-guided transvaginal follicular aspiration and in vitro fertilization, and 5 blastocysts that developed after the culture were transferred into uteri; two embryos implanted, and normal calves were born. These results suggest that the combined use of EGF and IGF-I makes bovine embryo culture in a chemically defined medium a practical and useful procedure for producing blastocysts, and its application to embryo culture following ovum pick up and in vitro fertilization could be useful for producing normal calves.

Pleurodeles waltl, amphibian, Urodele, is a suitable biological model for embryological and physiological space experiments on a vertebrate

NASA Astrophysics Data System (ADS)

Gualandris-Parisot, L.; Husson, D.; Foulquier, F.; Kan, P.; Davet, J.; Aimar, C.; Dournon, C.; Duprat, A. M.

2001-01-01

Pleurodeles waltl (amphibian, Urodele) is an appropriate biological model for space experiments on a vertebrate. One reason for interest in this animal concerns the study of the effects of absence of gravity on embryonic development. First, after mating (on Earth) the females retain live, functional sperm in their cloacum for up to 5 months, allowing normal in vivo fertilisation after hormonal stimulation. Second, their development is slow, which allows analyses of all the key stages of ontogenesis from the oocyte to swimming tailbud embryos or larvae. We have performed detailed studies and analyses of the effects of weightlessness on amphibian Pleurodeles embryos, fertilised and allowed to develop until the swimming larvae stage. These experiments were performed in space during three missions on the MIR-station: FERTILE I, FERTILE II and NEUROGENESIS respectively in 1996, 1998 and 1999. We show that in microgravity abnormalities appeared at specific stages of development compared to 1g-centrifuge control embryos and 1g-ground control embryos. In this report we describe abnormalities occurring in the central nervous system. These modifications occur during the neurulation process (delay in the closure of the neural tube and failure of closure of this tube in the cephalic area) and at the early tailbud stage (microcephaly observed in 40% of the microgravity-embryos). However, if acephalic and microcephalic embryos are not taken into account, these abnormalities did not disturb further morphological, biochemical and functional development and the embryos were able to regulate and a majority of normal hatching and swimming larvae were obtained in weightlessness with a developmental time-course equivalent to that of 1g-centrifuge control embryos (on the MIR station) and 1g-ground control embryos.

Combinatorial effects of zinc deficiency and arsenic exposure on zebrafish (Danio rerio) development

PubMed Central

Truong, Lisa; Barton, Carrie L.; Chase, Tyler T.; Gonnerman, Greg D.; Wong, Carmen P.; Tanguay, Robert L.; Ho, Emily

2017-01-01

Zinc deficiency and chronic low level exposures to inorganic arsenic in drinking water are both significant public health concerns that affect millions of people including pregnant women. These two conditions can co-exist in the human population but little is known about their interaction, and in particular, whether zinc deficiency sensitizes individuals to arsenic exposure and toxicity, especially during critical windows of development. To address this, we utilized the Danio rerio (zebrafish) model to test the hypothesis that parental zinc deficiency sensitizes the developing embryo to low-concentration arsenic toxicity, leading to altered developmental outcomes. Adult zebrafish were fed defined zinc deficient and zinc adequate diets and were spawned resulting in zinc adequate and zinc deficient embryos. The embryos were treated with environmentally relevant concentrations of 0, 50, and 500 ppb arsenic. Arsenic exposure significantly reduced the amount of zinc in the developing embryo by ~7%. The combination of zinc deficiency and low-level arsenic exposures did not sensitize the developing embryo to increased developmental malformations or mortality. The combination did cause a 40% decline in physical activity of the embryos, and this decline was significantly greater than what was observed with zinc deficiency or arsenic exposure alone. Significant changes in RNA expression of genes that regulate zinc homeostasis, response to oxidative stress and insulin production (including zip1, znt7, nrf2, ogg1, pax4, and insa) were found in zinc deficient, or zinc deficiency and arsenic exposed embryos. Overall, the data suggests that the combination of zinc deficiency and arsenic exposure has harmful effects on the developing embryo and may increase the risk for developing chronic diseases like diabetes. PMID:28837703

Improvement of transgenic cloning efficiencies by culturing recipient oocytes and donor cells with antioxidant vitamins in cattle.

PubMed

Wongsrikeao, Pimprapar; Nagai, Takashi; Agung, Budiyanto; Taniguchi, Masayasu; Kunishi, Miho; Suto, Shizuyo; Otoi, Takeshige

2007-06-01

The present study was conducted to investigate effects of antioxidants during maturation culture of recipient oocytes and/or culture of gene-transfected donor cells on the meiotic competence of recipient oocytes, and the developmental competence and quality of the reconstructed embryos after nuclear transfer (NT) in cattle. Gene-transfected donor cells had negative effects on the proportions of blastocyst formation, total cell numbers, and DNA fragmentation indices of reconstructed embryos. Supplementation of either vitamin E (alpha-tocopherol: 100 microM) or vitamin C (ascorbic acid: 100 microM) during maturation culture significantly enhanced the cytoplasmic maturation of oocytes and subsequent development of embryos reconstructed with the oocytes and gene-transfected donor cells, but did not have synergistic effects. The supplementation of vitamin E during maturation culture of recipient oocytes increased the proportions of fusion and blastocyst formation of gene-transfected NT embryos, in which the proportions were similar to those of nontransfected NT embryos. When the gene-transfected donor cells that had been cultured with 0, 50, or 100 microM of vitamin E were transferred into recipient oocytes matured with vitamin E (100 microM), 50 microM of vitamin E increased the proportion of blastocyst formation and reduced the index of DNA fragmentation of blastocysts. In conclusion, gene-transfected donor cells have negatively influenced the NT outcome. Supplementation of vitamin E during both recipient oocyte maturation and donor cell culture enhanced the blastocyst formation and efficiently blocked DNA damage in transgenic NT embryos. (c) 2006 Wiley-Liss, Inc.

Effects of Histone Deacetylase Inhibitor Oxamflatin on In Vitro Porcine Somatic Cell Nuclear Transfer Embryos

PubMed Central

Hou, Liming; Ma, Fanhua; Yang, Jinzeng; Riaz, Hasan; Wang, Yongliang; Wu, Wangjun; Xia, Xiaoliang; Ma, Zhiyuan; Zhou, Ying; Zhang, Lin; Ying, Wenqin; Xu, Dequan; Zuo, Bo; Ren, Zhuqing

2014-01-01

Abstract Low cloning efficiency is considered to be caused by the incomplete or aberrant epigenetic reprogramming of differentiated donor cells in somatic cell nuclear transfer (SCNT) embryos. Oxamflatin, a novel class of histone deacetylase inhibitor (HDACi), has been found to improve the in vitro and full-term developmental potential of SCNT embryos. In the present study, we studied the effects of oxamflatin treatment on in vitro porcine SCNT embryos. Our results indicated that the rate of in vitro blastocyst formation of SCNT embryos treated with 1 μM oxamflatin for 15 h postactivation was significantly higher than all other treatments. Treatment of oxamflatin decreased the relative histone deacetylase (HDAC) activity in cloned embryos and resulted in hyperacetylation levels of histone H3 at lysine 9 (AcH3K9) and histone H4 at lysine 5 (AcH4K5) at pronuclear, two-cell, and four-cell stages partly through downregulating HDAC1. The suppression of HDAC6 through oxamflatin increased the nonhistone acetylation level of α-tubulin during the mitotic cell cycle of early SCNT embryos. In addition, we demonstrated that oxamflatin downregulated DNA methyltransferase 1 (DNMT1) expression and global DNA methylation level (5-methylcytosine) in two-cell-stage porcine SCNT embryos. The pluripotency-related gene POU5F1 was found to be upregulated in the oxamflatin-treated group with a decreased DNA methylation tendency in its promoter regions. Treatment of oxamflatin did not change the locus-specific DNA methylation levels of Sus scrofa heterochromatic satellite DNA sequences at the blastocyst stage. Meanwhile, our findings suggest that treatment with HDACi may contribute to maintaining the stable status of cytoskeleton-associated elements, such as acetylated α-tubulin, which may be the crucial determinants of donor nuclear reprogramming in early SCNT embryos. In summary, oxamflatin treatment improves the developmental potential of porcine SCNT embryos in vitro. PMID:24960409

Improving the quality of miniature pig somatic cell nuclear transfer blastocysts: aggregation of SCNT embryos at the four-cell stage.

PubMed

Terashita, Y; Sugimura, S; Kudo, Y; Amano, R; Hiradate, Y; Sato, E

2011-04-01

Miniature pigs share many similar characteristics such as anatomy, physiology and body size with humans and are expected to become important animal models for therapeutic cloning using embryonic stem cells (ESCs) derived by somatic cell nuclear transfer (SCNT). In the present study, we observed that miniature pig SCNT blastocysts possessed a lower total number of nuclei and a lower percentage of POU5F1-positive cells than those possessed by in vitro fertilized (IVF) blastocysts. To overcome these problems, we evaluated the applicability of aggregating miniature pig SCNT embryos at the four-cell stage. We showed that (i) aggregation of two or three miniature pig SCNT embryos at the four-cell stage improves the total number of nuclei and the percentage of POU5F1-positive cells in blastocysts, and (ii) IVF blastocysts with low cell numbers induced by the removal of two blastomeres at the four-cell stage did not exhibit a decrease in the percentage of POU5F1-positive cells. These results suggest that the aggregation of miniature pig SCNT embryos at the four-cell stage can be a useful technique for improving the quality of miniature pig SCNT blastocysts and indicating that improvement in the percentage of POU5F1-positive cells in aggregated SCNT embryos is not simply the consequence of increased cell numbers. © 2010 Blackwell Verlag GmbH.

Laser fusion of mouse embryonic cells and intra-embryonic fusion of blastomeres without affecting the embryo integrity.

PubMed

Krivokharchenko, Alexander; Karmenyan, Artashes; Sarkisov, Oleg; Bader, Michael; Chiou, Arthur; Shakhbazyan, Avetik

2012-01-01

Manipulation with early mammalian embryos is the one of the most important approach to study preimplantation development. Artificial cell fusion is a research tool for various biotechnological experiments. However, the existing methods have various disadvantages, first of them impossibility to fuse selected cells within multicellular structures like mammalian preimplantation embryos. In our experiments we have successfully used high repetition rate picosecond near infrared laser beam for fusion of pairs of oocytes and oocytes with blastomeres. Fused cells looked morphologically normal and keep their ability for further divisions in vitro. We also fused two or three blastomeres inside four-cell mouse embryos. The presence of one, two or three nuclei in different blastomeres of the same early preimplantation mouse embryo was confirmed under UV-light after staining of DNA with the vital dye Hoechst-33342. The most of established embryos demonstrated high viability and developed in vitro to the blastocyst stage. We demonstrated for the first time the use of laser beam for the fusion of various embryonic cells of different size and of two or three blastomeres inside of four-cell mouse embryos without affecting the embryo's integrity and viability. These embryos with blastomeres of various ploidy maybe unique model for numerous purposes. Thus, we propose laser optical manipulation as a new tool for investigation of fundamental mechanisms of mammalian development.

Whole embryo culture: a "New" technique that enabled decades of mechanistic discoveries.

PubMed

Ellis-Hutchings, Robert G; Carney, Edward W

2010-08-01

Denis New's development of the rodent whole embryo culture (WEC) method in the early 1960s was a groundbreaking achievement that gave embryologists and teratologists an unprecedented degree of access to the developing postimplantation rodent embryo. In the five decades since its development, WEC has enabled detailed investigations into the regulation of normal embryo development as well as a plethora of research on mechanisms of teratogenesis as induced by a wide range of agents. In addition, WEC is one of the few techniques that has been validated for use in teratogenicity screening of drugs and chemicals. In this review, we retrace the steps leading to New's development of WEC, and highlight many examples in which WEC played a crucial role leading to important discoveries in teratological research. The impact of WEC on the field of teratology has been enormous, and it is anticipated that WEC will remain a preferred tool for teratologists and embryologists seeking to interrogate embryo development for many years to come. Copyright 2010 Wiley-Liss, Inc.

Development of Embryonic Market Squid, Doryteuthis opalescens, under Chronic Exposure to Low Environmental pH and [O2].

PubMed

Navarro, Michael O; Kwan, Garfield T; Batalov, Olga; Choi, Chelsea Y; Pierce, N Tessa; Levin, Lisa A

2016-01-01

The market squid, Doryteuthis opalescens, is an important forage species for the inshore ecosystems of the California Current System. Due to increased upwelling and expansion of the oxygen minimum zone in the California Current Ecosystem, the inshore environment is expected to experience lower pH and [O2] conditions in the future, potentially impacting the development of seafloor-attached encapsulated embryos. To understand the consequences of this co-occurring environmental pH and [O2] stress for D. opalescens encapsulated embryos, we performed two laboratory experiments. In Experiment 1, embryo capsules were chronically exposed to a treatment of higher (normal) pH (7.93) and [O2] (242 μM) or a treatment of low pH (7.57) and [O2] (80 μM), characteristic of upwelling events and/or La Niña conditions. The low pH and low [O2] treatment extended embryo development duration by 5-7 days; embryos remained at less developed stages more often and had 54.7% smaller statolith area at a given embryo size. Importantly, the embryos that did develop to mature embryonic stages grew to sizes that were similar (non-distinct) to those exposed to the high pH and high [O2] treatment. In Experiment 2, we exposed encapsulated embryos to a single stressor, low pH (7.56) or low [O2] (85 μM), to understand the importance of environmental pH and [O2] rising and falling together for squid embryogenesis. Embryos in the low pH only treatment had smaller yolk reserves and bigger statoliths compared to those in low [O2] only treatment. These results suggest that D. opalescens developmental duration and statolith size are impacted by exposure to environmental [O2] and pH (pCO2) and provide insight into embryo resilience to these effects.

Development of Embryonic Market Squid, Doryteuthis opalescens, under Chronic Exposure to Low Environmental pH and [O2

PubMed Central

Navarro, Michael O.; Kwan, Garfield T.; Batalov, Olga; Choi, Chelsea Y.; Pierce, N. Tessa; Levin, Lisa A.

2016-01-01

The market squid, Doryteuthis opalescens, is an important forage species for the inshore ecosystems of the California Current System. Due to increased upwelling and expansion of the oxygen minimum zone in the California Current Ecosystem, the inshore environment is expected to experience lower pH and [O2] conditions in the future, potentially impacting the development of seafloor-attached encapsulated embryos. To understand the consequences of this co-occurring environmental pH and [O2] stress for D. opalescens encapsulated embryos, we performed two laboratory experiments. In Experiment 1, embryo capsules were chronically exposed to a treatment of higher (normal) pH (7.93) and [O2] (242 μM) or a treatment of low pH (7.57) and [O2] (80 μM), characteristic of upwelling events and/or La Niña conditions. The low pH and low [O2] treatment extended embryo development duration by 5–7 days; embryos remained at less developed stages more often and had 54.7% smaller statolith area at a given embryo size. Importantly, the embryos that did develop to mature embryonic stages grew to sizes that were similar (non-distinct) to those exposed to the high pH and high [O2] treatment. In Experiment 2, we exposed encapsulated embryos to a single stressor, low pH (7.56) or low [O2] (85 μM), to understand the importance of environmental pH and [O2] rising and falling together for squid embryogenesis. Embryos in the low pH only treatment had smaller yolk reserves and bigger statoliths compared to those in low [O2] only treatment. These results suggest that D. opalescens developmental duration and statolith size are impacted by exposure to environmental [O2] and pH (pCO2) and provide insight into embryo resilience to these effects. PMID:27936085

Role of glucose in mouse preimplantation embryo development.

PubMed

Martin, K L; Leese, H J

1995-04-01

Mouse preimplantation embryos consume pyruvate preferentially during the early developmental stages, before glucose becomes the predominant energy substrate in the blastocyst. To investigate the importance of the switch to glucose utilization at the later developmental stages, mouse embryos from F1 hybrid mice (CBA/Ca x C57BL/6) were cultured from the one- and two-cell stages (22 and 46 h post hCG, respectively) for 5 days in a modified medium, M16, containing 0.33 mM pyruvate and 5 or 23 mM D + L-lactate, in the presence and absence of 1 mM glucose (M16 + G and M16 - G, respectively). Nutrient uptakes were also determined over this time. Some embryos cultured in M16 - G were transferred to M16 + G at 94 or 118 h post hCG. Embryos cultured from the two-cell stage in M16 + G exhibited the characteristic fall in pyruvate consumption between the morula and the blastocyst stage; those cultured from the two-cell stage in M16 - G compensated for the lack of glucose by consuming increasing amounts of pyruvate, from 2.78 pmol/embryo/h at 58 h post hCG to 5.21 pmol/embryo/h at 154 h post hCG. However, the percentage of embryos developing to the blastocyst stage, the hatching rate, and blastocyst cell numbers (50.6 +/- 2.5 [28] vs. 105 +/- 3.8 [37]) were all lower in this group. When exposed to glucose at 94 or 118 h post hCG, embryos cultured from the two-cell stage in M16 - G readily consumed glucose in preference to pyruvate, although the characteristic fall in pyruvate consumption was not observed. One-cell embryos cultured continuously in M16 - G were only able to develop to the morula stage, after which time they degenerated. In these embryos pyruvate was readily consumed between 22 and 94 h post hCG, before falling from 2.77 pmol/embryo/h at 83 h post hCG to 0.045 pmol/embryo/h at 130 h post hCG. Transfer of these embryos to M16 + G at 94 and 118 h post hCG did not support development to the hatching blastocyst stage.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronology of early embryonic development and embryo uterine migration in alpacas.

PubMed

Picha, Y; Tibary, A; Memon, M; Kasimanickam, R; Sumar, J

2013-03-01

The objectives were to: (1) describe the chronology of early embryonic development from ovulation to entry into the uterus; and (2) to determine the timing of embryo migration to the left uterine horn when ovulation occurred from the right ovary. The experiment was conducted in Peru. Females (n = 132) were randomly assigned to 15 experimental groups. All females were mated to an intact male, given 50 μg GnRH im (Cystorelin) and ovulation time determined by transrectal ultrasonography, conducted every 6 hours, starting 24 hours postmating. Animals were slaughtered at a specific intervals postovulation and reproductive tracts were recovered and subjected to oviductal and uterine flushing for females slaughtered between 1 and 6 days postovulation (dpo; Day 0 = ovulation) and uterine flushing for females slaughtered from 7 to 15 dpo for recovery of oocytes/embryos. Season of mating did not influence the interval from mating to ovulation (winter: 29 ± 6 hours vs. summer: 30 ± 6 hours; P = 0.49). Ovulation rates for females mated during winter and summer were 92% versus 100%, respectively (P = 0.05). Fertilization rates for winter and summer mated females were 72% and 82% (P = 0.29). Unfertilized ova were not retained in the uterine tube. All embryos collected were in the uterine tube ipsilateral to the side of ovulation between 1 and 5 dpo. Embryos reached the uterus on 6 dpo. Embryos began to elongate on 9 dpo; at this time, 83% of embryos derived from right-ovary ovulations were collected from the left uterine horn. Embryos occupied the entire uterine cavity by 10 dpo. In conclusion, we characterized early embryo development and location of embryo during its early developmental stages in alpaca. This was apparently the first report regarding chronology of embryo development and migration to the left horn in alpaca which merits further investigation regarding its role in maternal recognition of pregnancy. Copyright © 2013 Elsevier Inc. All rights reserved.

Barcode tagging of human oocytes and embryos to prevent mix-ups in assisted reproduction technologies.

PubMed

Novo, Sergi; Nogués, Carme; Penon, Oriol; Barrios, Leonardo; Santaló, Josep; Gómez-Martínez, Rodrigo; Esteve, Jaume; Errachid, Abdelhamid; Plaza, José Antonio; Pérez-García, Lluïsa; Ibáñez, Elena

2014-01-01

Is the attachment of biofunctionalized polysilicon barcodes to the outer surface of the zona pellucida an effective approach for the direct tagging and identification of human oocytes and embryos during assisted reproduction technologies (ARTs)? The direct tagging system based on lectin-biofunctionalized polysilicon barcodes of micrometric dimensions is simple, safe and highly efficient, allowing the identification of human oocytes and embryos during the various procedures typically conducted during an assisted reproduction cycle. Measures to prevent mismatching errors (mix-ups) of the reproductive samples are currently in place in fertility clinics, but none of them are totally effective and several mix-up cases have been reported worldwide. Using a mouse model, our group has previously developed an effective direct embryo tagging system which does not interfere with the in vitro and in vivo development of the tagged embryos. This system has now been tested in human oocytes and embryos. Fresh immature and mature fertilization-failed oocytes (n = 21) and cryopreserved day 1 embryos produced by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) (n = 205) were donated by patients (n = 76) undergoing ARTs. In vitro development rates, embryo quality and post-vitrification survival were compared between tagged (n = 106) and non-tagged (control) embryos (n = 99). Barcode retention and identification rates were also calculated, both for embryos and for oocytes subjected to a simulated ICSI and parthenogenetic activation. Experiments were conducted from January 2012 to January 2013. Barcodes were fabricated in polysilicon and biofunctionalizated with wheat germ agglutinin lectin. Embryos were tagged with 10 barcodes and cultured in vitro until the blastocyst stage, when they were either differentially stained with propidium iodide and Hoechst or vitrified using the Cryotop method. Embryo quality was also analyzed by embryo grading and time-lapse monitoring. Injected oocytes were parthenogenetically activated using ionomycin and 6-dimethylaminopurine. Blastocyst development rates of tagged (27/58) and non-tagged embryos (24/51) were equivalent, and no significant differences in the timing of key morphokinetic parameters and the number of inner cell mass cells were detected between the two groups (tagged: 24.7 ± 2.5; non-tagged: 22.3 ± 1.9), indicating that preimplantation embryo potential and quality are not affected by the barcodes. Similarly, re-expansion rates of vitrified-warmed tagged (19/21) and non-tagged (16/19) blastocysts were similar. Global identification rates of 96.9 and 89.5% were obtained in fresh (mean barcode retention: 9.22 ± 0.13) and vitrified-warmed (mean barcode retention: 7.79 ± 0.35) tagged embryos, respectively, when simulating an automatic barcode reading process, though these rates were increased to 100% just by rotating the embryos during barcode reading. Only one of the oocytes lost one barcode during intracytoplasmic injection (100% identification rate) and all oocytes retained all the barcodes after parthenogenetic activation. Although the direct embryo tagging system developed is effective, it only allows the identification and traceability of oocytes destined for ICSI and embryos. Thus, the traceability of all reproductive samples (oocytes destined for IVF and sperm) is not yet ensured. The direct embryo tagging system developed here provides fertility clinics with a novel tool to reduce the risk of mix-ups in human ARTs. The system can also be useful in research studies that require the individual identification of oocytes or embryos and their individual tracking. This study was supported by the Sociedad Española de Fertilidad, the Spanish Ministry of Education and Science (TEC2011-29140-C03) and the Generalitat de Catalunya (2009SGR-00282 and 2009SGR-00158). The authors do not have any competing interests.

East Europe Report

DTIC Science & Technology

1987-01-08

the basis of improving the method of embryo transplants, for exchanging genetic material, for training personnel and for developing the necessary...its NATO allies. 65 The Soviet Government declaration came as further evidence of the Soviet Union’s concern over the destinies of civilisation and...events and developments in the destinies of our two peoples. Both our peoples suffered under foreign domination, both our peoples were robbed by

Perspectives for artificial insemination and genomics to improve global swine populations.

PubMed

Gerrits, Roger J; Lunney, Joan K; Johnson, Lawrence A; Pursel, Vernon G; Kraeling, Robert R; Rohrer, Gary A; Dobrinsky, John R

2005-01-15

Civilizations throughout the world continue to depend on pig meat as an important food source. Approximately 40% of the red meat consumed annually worldwide (94 million metric tons) is pig meat. Pig numbers (940 million) and consumption have increased consistent with the increasing world population (FAO 2002). In the past 50 years, research guided genetic selection and nutrition programs have had a major impact on improving carcass composition and efficiency of production in swine. The use of artificial insemination (AI) in Europe has also had a major impact on pig improvement in the past 35 years and more recently in the USA. Several scientific advances in gamete physiology and/or manipulation have been successfully utilized while others are just beginning to be applied at the production level. Semen extenders that permit the use of fresh semen for more than 5 days post-collection are largely responsible for the success of AI in pigs worldwide. Transfer of the best genetics has been enabled by use of AI with fresh semen, and to some extent, by use of AI with frozen semen over the past 25 years. Sexed semen, now a reality, has the potential for increasing the rate of genetic progress in AI programs when used in conjunction with newly developed low sperm number insemination technology. Embryo cryopreservation provides opportunities for international transport of maternal germplasm worldwide; non-surgical transfer of viable embryos in practice is nearing reality. While production of transgenic animals has been successful, the low level of efficiency in producing these animals and lack of information on multigene interactions limit the use of the technology in applied production systems. Technologies based on research in functional genomics, proteomics and cloning have significant potential, but considerable research effort will be required before they can be utilized for AI in pig production. In the past 15 years, there has been a coordinated worldwide scientific effort to develop the genetic linkage map of the pig with the goal of identifying pigs with genetic alleles that result in improved growth rate, carcass quality, and reproductive performance. Molecular genetic tests have been developed to select pigs with improved traits such as removal of the porcine stress (RYR1) syndrome, and selection for specific estrogen receptor (ESR) alleles. Less progress has been made in developing routine tests related to diseases. Major research in genomics is being pursued to improve the efficiency of selection for healthier pigs with disease resistance properties. The sequencing of the genome of the pig to identify new genes and unique regulatory elements holds great promise to provide new information that can be used in pig production. AI, in vitro embryo production and embryo transfer will be the preferred means of implementing these new technologies to enhance efficiency of pig production in the future.

Strain preservation of experimental animals: vitrification of two-cell stage embryos for multiple mouse strains.

PubMed

Eto, Tomoo; Takahashi, Riichi; Kamisako, Tsutomu

2015-04-01

Strain preservation of experimental animals is crucial for experimental reproducibility. Maintaining complete animal strains, however, is costly and there is a risk for genetic mutations as well as complete loss due to disasters or illness. Therefore, the development of effective vitrification techniques for cryopreservation of multiple experimental animal strains is important. We examined whether a vitrification method using cryoprotectant solutions, P10 and PEPeS, is suitable for preservation of multiple inbred and outbred mouse strains. First, we investigated whether our vitrification method using cryoprotectant solutions was suitable for two-cell stage mouse embryos. In vitro development of embryos exposed to the cryoprotectant solutions was similar to that of fresh controls. Further, the survival rate of the vitrified embryos was extremely high (98.1%). Next, we collected and vitrified two-cell stage embryos of 14 mouse strains. The average number of embryos obtained from one female was 7.3-33.3. The survival rate of vitrified embryos ranged from 92.8% to 99.1%, with no significant differences among mouse strains. In vivo development did not differ significantly between fresh controls and vitrified embryos of each strain. For strain preservation using cryopreserved embryos, two offspring for inbred lines and one offspring for outbred lines must be produced from two-cell stage embryos collected from one female. The expected number of surviving fetuses obtained from embryos collected from one female of either the inbred or outbred strains ranged from 2.9 to 19.5. The findings of the present study indicated that this vitrification method is suitable for strain preservation of multiple mouse strains. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Attitudes of Infertile Couples, Fertility Clinic Staff and Researchers toward Personhood of The Human Embryo in Iran

PubMed Central

Kayssan, Marjaneh; Dolatian, Mahrokh; Omani Samani, Reza; Maroufizadeh, Saman

2017-01-01

Objective After the introduction of assisted reproductive techniques, human embryos were officially introduced into laboratories and now thousands of them are cryopreserved in such settings. Embryonic stem cells and the future application of such cells in the treatment of disease opened the door to further research on human embryos. These developments raise many ethical issues, some of which have religious aspects. The main question is: what is the embryo? Should we consider it a human being? Thus, the purpose of this study was to investigate attitudes towards the personhood of the embryo. Materials and Methods In this cross sectional study, 203 infertile patients (n=406), 54 clinic staff and 49 embryo researchers, selected using convenience sampling at the Royan Institute, completed a questionnaire on personhood of human embryo. The questionnaire had been developed following qualitative research and had satisfied face and content validity tests. Results At the pre-implantation stage the majority of participants in all three groups considered the human embryo as "not a human being". Also, at the post-implantation stage of development, the majority of infertile couples and clinic staff considered the embryo as "not a human being" but, half the researchers (51%) considered the embryo in this stage as a "potential human". Half of the infertile couples considered the human fetus before ensoulment time (19th week of pregnancy according to the Shiite Islamic scholars) as "not-human being", while more than half of researchers (55.1%) considered it as a "potential human". Conclusion Ensoulment time is a major and important border for personhood. Most infertile couples and clinic staff consider the human embryo as "not a human being" but majority of all study participants considered the human fetus to be a complete human after ensoulment time. PMID:28670524

Effects of in vitro fertilization and embryo culture on TRP53 and Bax expression in B6 mouse embryos.

PubMed

Chandrakanthan, Vashe; Li, Aiqing; Chami, Omar; O'Neill, Christopher

2006-11-21

In the mouse, embryo culture results in a characteristic phenotype of retarded embryo preimplantation development and reduced numbers of cells within embryos. The expression of TRP53 is central to the regulation of the cell's capacity to proliferate and survive. In this study we found that Trp53 mRNA is expressed throughout the preimplantation stage of development. Levels of TRP53 protein expression were low during the cleavage stages and increased at the morula and blastocyst stages in B6 embryos collected from the reproductive tract. Embryos collected at the zygote stage and cultured for 96 h also showed low levels of TRP53 expression at precompaction stages. There were higher levels of TRP53 in cultured morula and the level in cultured blastocysts was clearly increased above blastocysts collected directly from the uterus. Immunolocalization of TRP53 showed that its increased expression in cultured blastocysts corresponded with a marked accumulation of TRP53 within the nuclei of embryonic cells. This pattern of expression was enhanced in embryos produced by in vitro fertilization and subjected to culture. The TRP53 was transcriptionally active since culture also induced increased expression of Bax, yet this did not occur in embryos lacking Trp53 (Trp53-/-). The rate of development of Trp53-/- zygotes to the blastocyst stage was not different to wildtype controls when embryos were cultured in groups of ten but was significantly faster when cultured individually. The results show that zygote culture resulted in the accumulation of transcription activity of TRP53 in the resulting blastocysts. This accounts for the adverse effects of culture of embryos individually, but does not appear to be the sole cause of the retarded preimplantation stage growth phenotype associated with culture in vitro.

Based serum metabolomics analysis reveals simultaneous interconnecting changes during chicken embryonic development.

PubMed

Peng, M L; Li, S N; He, Q Q; Zhao, J L; Li, L L; Ma, H T

2018-05-28

Metabolic disorder is a major health problem and is associated with a number of metabolic diseases. Due to native hyperglycaemia and resistance to exogenous insulin, chickens as a model had used in the studies of adipose tissue biology, metabolism and obesity. But no detailed information is available about the comprehensive changes of serum metabolites at different stages of chicken embryonic development. This study employed LC/MS-QTOF to determine the changes of major functional metabolites at incubation day 14 (E14d), 19 (E19d) and hatching day 1 (H1d), and the associated pathways of differential metabolites during chicken embryonic development were analysed using Metabolite Set Enrichment Analysis method. Results showed that 39 metabolites were significantly changed from E14d to E19d and 68 metabolites were significantly altered from E19d to H1d in chicken embryos. Protein synthesis was promoted by increasing the concentrations of L-glutamine and threonine, and gonadal development was promoted through increasing oestrone content from E14d to E19d in chicken embryos, which indicated that serum glutamine, threonine and oestrone contents may be considered as the candidate indicators for assessment of early embryonic development. 2-oxoglutaric acid mainly contributed to enhancing the citric cycle, and it plays an important role in improving the growth of chicken embryos at the late development; the decreasing of L-glutamine, L-isoleucine and L-leucine contents from E19d to H1d in chicken embryonic development implied their possible functions as the feed additive during early posthatch period of broiler chickens to satisfy the growth. These results provided insights into understand the roles of serum metabolites at different developmental stages of chicken embryos, it also provides available information for chicken as a model to study metabolic disease or human obesity. © 2018 Blackwell Verlag GmbH.

3P: Personalized Pregnancy Prediction in IVF Treatment Process

NASA Astrophysics Data System (ADS)

Uyar, Asli; Ciray, H. Nadir; Bener, Ayse; Bahceci, Mustafa

We present an intelligent learning system for improving pregnancy success rate of IVF treatment. Our proposed model uses an SVM based classification system for training a model from past data and making predictions on implantation outcome of new embryos. This study employs an embryo-centered approach. Each embryo is represented with a data feature vector including 17 features related to patient characteristics, clinical diagnosis, treatment method and embryo morphological parameters. Our experimental results demonstrate a prediction accuracy of 82.7%. We have obtained the IVF dataset from Bahceci Women Health, Care Centre, in Istanbul, Turkey.

Complete method to obtain, culture, and transfer mouse blastocysts nonsurgically to study implantation and development.

PubMed

Moreno-Moya, Juan Manuel; Ramírez, Leslie; Vilella, Felipe; Martínez, Sebastián; Quiñonero, Alicia; Noguera, Inmaculada; Pellicer, Antonio; Simón, Carlos

2014-03-01

To illustrate an efficient, complete, step-by-step protocol for studying implantation in mice. Video presentation of an animal model for research in reproductive biology. Mouse (Mus musculus). A nonsurgical embryo transfer system very similar to that used for human embryo transfer. The protocols with recipient and donor mice are performed in parallel in the same week. For the donor mice: the first step is ovarian stimulation, followed by ovulation induction and mating; finally, the mice are sacrificed, and the embryos are collected and cultured. For recipient mice: first estrous synchrony is induced, followed by mating with a vasectomized male, visualization of the vaginal plug, and nonsurgical transfer of the embryos. Finally (optionally), the implantation sites can be visualized on day 7.5 of development. (All animal experiments were performed with the approval of the institutional review board.) Implantation is an essential step in human reproduction although, because of technical and ethics considerations, still relatively little is known about human implantation and early development. Conversely, mouse models are well established and can be used for preliminary experiments. However, there are various bottlenecks in the procedure for obtaining and transferring murine embryos, which makes experimentation with this model more difficult. These difficulties include pseudopregnancy, ovarian hyperstimulation, and embryo collection, culture, and transfer. We have proposed a complete, efficient method for obtaining, culturing, and transferring mouse blastocysts that can be easily applied in research. Potential applications include testing new media components that do not affect preimplantation but do affect implantation and early development. The embryo transfer method proposed here has been demonstrated to achieve embryo implantation easier and faster than, and in approximately similar rates as other traditional surgery methods. This workflow is the first set of complete step-by-step instructions available that incorporate advances such as nonsurgical mouse embryo transfer. This will facilitate research into different reproduction events such as embryo development, embryo implantation, or contraception. Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Time to take human embryo culture seriously.

PubMed

Sunde, Arne; Brison, Daniel; Dumoulin, John; Harper, Joyce; Lundin, Kersti; Magli, M Cristina; Van den Abbeel, Etienne; Veiga, Anna

2016-10-01

Is it important that end-users know the composition of human embryo culture media? We argue that there is as strong case for full transparency concerning the composition of embryo culture media intended for human use. Published data suggest that the composition of embryo culture media may influence the phenotype of the offspring. A review of the literature was carried out. Data concerning the potential effects on embryo development of culture media were assessed and recommendations for users made. The safety of ART procedures, especially with respect to the health of the offspring, is of major importance. There are reports from the literature indicating a possible effect of culture conditions, including culture media, on embryo and fetal development. Since the introduction of commercially available culture media, there has been a rapid development of different formulations, often not fully documented, disclosed or justified. There is now evidence that the environment the early embryo is exposed to can cause reprogramming of embryonic growth leading to alterations in fetal growth trajectory, birthweight, childhood growth and long-term disease including Type II diabetes and cardiovascular problems. The mechanism for this is likely to be epigenetic changes during the preimplantation period of development. In the present paper the ESHRE working group on culture media summarizes the present knowledge of potential effects on embryo development related to culture media, and makes recommendations. There is still a need for large prospective randomized trials to further elucidate the link between the composition of embryo culture media used and the phenotype of the offspring. We do not presently know if the phenotypic changes induced by in vitro embryo culture represent a problem for long-term health of the offspring. Published data indicate that there is a strong case for demanding full transparency concerning the compositions of and the scientific rationale behind the composition of embryo culture media. This work was funded by The European Society for Human Reproduction and Embryology. No competing interests to declare. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Exposure to high ambient temperatures alters embryology in rabbits

NASA Astrophysics Data System (ADS)

García, M. L.; Argente, M. J.

2017-09-01

High ambient temperatures are a determining factor in the deterioration of embryo quality and survival in mammals. The aim of this study was to evaluate the effect of heat stress on embryo development, embryonic size and size of the embryonic coats in rabbits. A total of 310 embryos from 33 females in thermal comfort zone and 264 embryos of 28 females in heat stress conditions were used in the experiment. The traits studied were ovulation rate, percentage of total embryos, percentage of normal embryos, embryo area, zona pellucida thickness and mucin coat thickness. Traits were measured at 24 and 48 h post-coitum (hpc); mucin coat thickness was only measured at 48 hpc. The embryos were classified as zygotes or two-cell embryos at 24 hpc, and 16-cells or early morulae at 48 hpc. The ovulation rate was one oocyte lower in heat stress conditions than in thermal comfort. Percentage of normal embryos was lower in heat stress conditions at 24 hpc (17.2%) and 48 hpc (13.2%). No differences in percentage of zygotes or two-cell embryos were found at 24 hpc. The embryo development and area was affected by heat stress at 48 hpc (10% higher percentage of 16-cells and 883 μm2 smaller, respectively). Zona pellucida was thicker under thermal stress at 24 hpc (1.2 μm) and 48 hpc (1.5 μm). No differences in mucin coat thickness were found. In conclusion, heat stress appears to alter embryology in rabbits.

Demi-embryo production from hatching of zona-drilled bovine and rabbit blastocysts.

PubMed

Skrzyszowska, M; Smorag, Z; Katska, L

1997-09-01

It is known that the pregnancy rate resulting after transfer of bisected embryos is lower than after transfer of whole embryos. The main reason is the reduced cell number in the demi-embryo which is less than 1 2 of that in the intact embryo, since a number of blastomeres is damaged as a result of the procedure used in conventional embryo splitting. The aim of our experiment was to develop a non-invasive procedure which would limit cell losses during microsurgery. The experiment was carried out on bovine IVM-IVF embryos at middle, late and expanded blastocyst stage and rabbit embryos at late blastocyst stage cultured in vitro from in vivo produced zygotes. The zona pellucida of these embryos was drilled on the line between the inner cell mass and the trophoblast using a glass microneedle (

PGF₂α levels in Day 8 blood plasma are increased by the presence of one or more embryos in the uterus.

PubMed

Gomez, E; Martin, D; Carrocera, S; Muñoz, M

2015-08-01

In cattle, the detection of very early endometrial responses is considered to be hampered by the presence of only a single embryo. Therefore, we have previously developed a model of multiple embryo transfer to circumvent this hindrance. In this work, we analysed embryo-maternal interactions in the bovine uterus on day 8 of development while comparing the presence of multiple v. single embryos using embryo transfer and artificial insemination, respectively. Concentration of proteins (β-actin, NFkB, clusterin and immunoproteosome 20S β5i subunit-i20S), by western blot, and hexoses (glucose and fructose) were measured in paired samples of uterine fluid (UF) from the same animal with and without embryos in the uterus and were compared with UF obtained after artificial insemination. Prostaglandin (PG) F2 α and PGE2 concentrations were also analysed in blood plasma. The four proteins analysed and hexoses were unaffected by the presence of one or more embryos in the uterus. However, blood PGF2 α showed similar, significant increases with one or more embryos over cyclic animals; such changes were not observed in blood PGE2. Although multiple embryo transfer may appear to be non-physiological, we showed that the uterus, at the very early embryonic stages, does exhibit physiological reactions. Multiple embryo transfer can, therefore, be used for studies of very early embryo-maternal interactions in vivo in monotocous species.

Patterning in time and space: HoxB cluster gene expression in the developing chick embryo.

PubMed

Gouveia, Analuce; Marcelino, Hugo M; Gonçalves, Lisa; Palmeirim, Isabel; Andrade, Raquel P

2015-01-01

The developing embryo is a paradigmatic model to study molecular mechanisms of time control in Biology. Hox genes are key players in the specification of tissue identity during embryo development and their expression is under strict temporal regulation. However, the molecular mechanisms underlying timely Hox activation in the early embryo remain unknown. This is hindered by the lack of a rigorous temporal framework of sequential Hox expression within a single cluster. Herein, a thorough characterization of HoxB cluster gene expression was performed over time and space in the early chick embryo. Clear temporal collinearity of HoxB cluster gene expression activation was observed. Spatial collinearity of HoxB expression was evidenced in different stages of development and in multiple tissues. Using embryo explant cultures we showed that HoxB2 is cyclically expressed in the rostral presomitic mesoderm with the same periodicity as somite formation, suggesting a link between timely tissue specification and somite formation. We foresee that the molecular framework herein provided will facilitate experimental approaches aimed at identifying the regulatory mechanisms underlying Hox expression in Time and Space.

Patterning in time and space: HoxB cluster gene expression in the developing chick embryo

PubMed Central

Gouveia, Analuce; Marcelino, Hugo M; Gonçalves, Lisa; Palmeirim, Isabel; Andrade, Raquel P

2015-01-01

The developing embryo is a paradigmatic model to study molecular mechanisms of time control in Biology. Hox genes are key players in the specification of tissue identity during embryo development and their expression is under strict temporal regulation. However, the molecular mechanisms underlying timely Hox activation in the early embryo remain unknown. This is hindered by the lack of a rigorous temporal framework of sequential Hox expression within a single cluster. Herein, a thorough characterization of HoxB cluster gene expression was performed over time and space in the early chick embryo. Clear temporal collinearity of HoxB cluster gene expression activation was observed. Spatial collinearity of HoxB expression was evidenced in different stages of development and in multiple tissues. Using embryo explant cultures we showed that HoxB2 is cyclically expressed in the rostral presomitic mesoderm with the same periodicity as somite formation, suggesting a link between timely tissue specification and somite formation. We foresee that the molecular framework herein provided will facilitate experimental approaches aimed at identifying the regulatory mechanisms underlying Hox expression in Time and Space. PMID:25602523

Can a genetically-modified organism-containing diet influence embryo development? A preliminary study on pre-implantation mouse embryos.

PubMed

Cisterna, B; Flach, F; Vecchio, L; Barabino, S M L; Battistelli, S; Martin, T E; Malatesta, M; Biggiogera, M

2008-01-01

In eukaryotic cells, pre-mRNAs undergo several transformation steps to generate mature mRNAs. Recent studies have demonstrated that a diet containing a genetically modified (GM) soybean can induce modifications of nuclear constituents involved in RNA processing in some tissues of young, adult and old mice. On this basis, we have investigated the ultrastructural and immunocytochemical features of pre-implantation embryos from mice fed either GM or non- GM soybean in order to verify whether the parental diet can affect the morpho-functional development of the embryonic ribonucleoprotein structural constituents involved in pre-mRNA pathways. Morphological observations revealed that the general aspect of embryo nuclear components is similar in the two experimental groups. However, immunocytochemical and in situ hybridization results suggest a temporary decrease of pre-mRNA transcription and splicing in 2-cell embryos and a resumption in 4-8-cell embryos from mice fed GM soybean; moreover, pre-mRNA maturation seems to be less efficient in both 2-cell and 4-8-cell embryos from GM-fed mice than in controls. Although our results are still preliminary and limited to the pre-implantation phases, the results of this study encourage deepening on the effects of food components and/or contaminants on embryo development.

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana.

PubMed

Kurihara, Daisuke; Kimata, Yusuke; Higashiyama, Tetsuya; Ueda, Minako

2017-09-11

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

Radial extracorporeal shock wave treatment harms developing chicken embryos

PubMed Central

Kiessling, Maren C.; Milz, Stefan; Frank, Hans-Georg; Korbel, Rüdiger; Schmitz, Christoph

2015-01-01

Radial extracorporeal shock wave treatment (rESWT) has became one of the best investigated treatment modalities for cellulite, including the abdomen as a treatment site. Notably, pregnancy is considered a contraindication for rESWT, and concerns have been raised about possible harm to the embryo when a woman treated with rESWT for cellulite is not aware of her pregnancy. Here we tested the hypothesis that rESWT may cause serious physical harm to embryos. To this end, chicken embryos were exposed in ovo to various doses of radial shock waves on either day 3 or day 4 of development, resembling the developmental stage of four- to six-week-old human embryos. We found a dose-dependent increase in the number of embryos that died after radial shock wave exposure on either day 3 or day 4 of development. Among the embryos that survived the shock wave exposure a few showed severe congenital defects such as missing eyes. Evidently, our data cannot directly be used to draw conclusions about potential harm to the embryo of a pregnant woman treated for cellulite with rESWT. However, to avoid any risks we strongly recommend applying radial shock waves in the treatment of cellulite only if a pregnancy is ruled out. PMID:25655309

High-frequency plant regeneration through cyclic secondary somatic embryogenesis in black pepper (Piper nigrum L.).

PubMed

Nair, R Ramakrishnan; Dutta Gupta, S

2006-01-01

A high-frequency plantlet regeneration protocol was developed for black pepper (Piper nigrum L.) through cyclic secondary somatic embryogenesis. Secondary embryos formed from the radicular end of the primary somatic embryos which were originally derived from micropylar tissues of germinating seeds on growth regulator-free SH medium in the absence of light. The process of secondary embryogenesis continued in a cyclic manner from the root pole of newly formed embryos resulting in clumps of somatic embryos. Strength of the medium and sucrose concentration influenced the process of secondary embryogenesis and fresh weight of somatic embryo clumps. Full-strength SH medium supplemented with 1.5% sucrose produced significantly higher fresh weight and numbers of secondary somatic embryos while 3.0 and 4.5% sucrose in the medium favored further development of proliferated embryos into plantlets. Ontogeny of secondary embryos was established by histological analysis. Secondary embryogenic potential was influenced by the developmental stage of the explanted somatic embryo and stages up to "torpedo" were more suitable. A single-flask system was standardized for proliferation, maturation, germination and conversion of secondary somatic embryos in suspension cultures. The system of cyclic secondary somatic embryogenesis in black pepper described here represents a permanent source of embryogenic material that can be used for genetic manipulations of this crop species.

The Theobroma cacao B3 domain transcription factor TcLEC2 plays a duel role in control of embryo development and maturation

PubMed Central

2014-01-01

Background The Arabidopsis thaliana LEC2 gene encodes a B3 domain transcription factor, which plays critical roles during both zygotic and somatic embryogenesis. LEC2 exerts significant impacts on determining embryogenic potential and various metabolic processes through a complicated genetic regulatory network. Results An ortholog of the Arabidopsis Leafy Cotyledon 2 gene (AtLEC2) was characterized in Theobroma cacao (TcLEC2). TcLEC2 encodes a B3 domain transcription factor preferentially expressed during early and late zygotic embryo development. The expression of TcLEC2 was higher in dedifferentiated cells competent for somatic embryogenesis (embryogenic calli), compared to non-embryogenic calli. Transient overexpression of TcLEC2 in immature zygotic embryos resulted in changes in gene expression profiles and fatty acid composition. Ectopic expression of TcLEC2 in cacao leaves changed the expression levels of several seed related genes. The overexpression of TcLEC2 in cacao explants greatly increased the frequency of regeneration of stably transformed somatic embryos. TcLEC2 overexpressing cotyledon explants exhibited a very high level of embryogenic competency and when cultured on hormone free medium, exhibited an iterative embryogenic chain-reaction. Conclusions Our study revealed essential roles of TcLEC2 during both zygotic and somatic embryo development. Collectively, our evidence supports the conclusion that TcLEC2 is a functional ortholog of AtLEC2 and that it is involved in similar genetic regulatory networks during cacao somatic embryogenesis. To our knowledge, this is the first detailed report of the functional analysis of a LEC2 ortholog in a species other then Arabidopsis. TcLEC2 could potentially be used as a biomarker for the improvement of the SE process and screen for elite varieties in cacao germplasm. PMID:24758406

Colony-stimulating factor 2 acts from days 5 to 7 of development to modify programming of the bovine conceptus at day 86 of gestation†.

PubMed

Siqueira, Luiz G; Tribulo, Paula; Chen, Zhiyuan; Denicol, Anna C; Ortega, M Sofia; Negrón-Pérez, Veronica M; Kannampuzha-Francis, Jasmine; Pohler, Ky G; Rivera, Rocio M; Hansen, Peter J

2017-04-01

Colony-stimulating factor 2 (CSF2) is an embryokine that improves competence of the embryo to establish pregnancy and which may participate in developmental programming. We tested whether culture of bovine embryos with CSF2 alters fetal development and alleviates abnormalities associated with in vitro production (IVP) of embryos. Pregnancies were established by artificial insemination (AI), transfer of an IVP embryo (IVP), or transfer of an IVP embryo treated with 10 ng/ml CSF2 from day 5 to 7 of development (CSF2). Pregnancies were produced using X-sorted semen. Female singleton conceptuses were collected on day 86 of gestation. There were few morphological differences between groups, although IVP and CSF2 fetuses were heavier than AI fetuses. Bicarbonate concentration in allantoic fluid was lower for IVP than for AI or CSF2. Expression of 92 genes in liver, placenta, and muscle was determined. The general pattern for liver and placenta was for IVP to alter expression and for CSF2 to sometimes reverse this effect. For muscle, CSF2 affected gene expression but did not generally reverse effects of IVP. Levels of methylation for each of the three tissues at 12 loci in the promoter of insulin-like growth factor 2 (IGF2) and five in the promoter of growth factor receptor bound protein 10 were unaffected by treatment except for CSF2 effects on two CpG for IGF2 in placenta and muscle. In conclusion, CSF2 can act as a developmental programming agent but alone is not able to abolish the adverse effects of IVP on fetal characteristics. © The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please journals.permissions@oup.com.

Fresh embryo transfer versus frozen embryo transfer in in vitro fertilization cycles: a systematic review and meta-analysis.

PubMed

Roque, Matheus; Lattes, Karinna; Serra, Sandra; Solà, Ivan; Geber, Selmo; Carreras, Ramón; Checa, Miguel Angel

2013-01-01

To examine the available evidence to assess if cryopreservation of all embryos and subsequent frozen embryo transfer (FET) results in better outcomes compared with fresh transfer. Systematic review and meta-analysis. Centers for reproductive care. Infertility patient(s). An exhaustive electronic literature search in MEDLINE, EMBASE, and the Cochrane Library was performed through December 2011. We included randomized clinical trials comparing outcomes of IVF cycles between fresh and frozen embryo transfers. The outcomes of interest were ongoing pregnancy rate, clinical pregnancy rate, and miscarriage. We included three trials accounting for 633 cycles in women aged 27-33 years. Data analysis showed that FET resulted in significantly higher ongoing pregnancy rates and clinical pregnancy rates. Our results suggest that there is evidence that IVF outcomes may be improved by performing FET compared with fresh embryo transfer. This could be explained by a better embryo-endometrium synchrony achieved with endometrium preparation cycles. Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Effect of early addition of bone morphogenetic protein 5 (BMP5) to embryo culture medium on in vitro development and expression of developmentally important genes in bovine preimplantation embryos.

PubMed

García, Elina V; Miceli, Dora C; Rizo, Gabriela; Valdecantos, Pablo A; Barrera, Antonio D

2015-09-01

Previous studies have reported that bone morphogenetic protein 5 (BMP5) is differentially expressed in the isthmus of bovine oviducts and it is present in the oviductal fluid. However, the specific action of this factor is unknown. To evaluate whether BMP5 exerts some effect during early bovine embryo development, gene expression of BMP5, BMP receptors, and the effect of exogenous BMP5 on in vitro development and expression of developmentally important genes were assessed. In experiment 1, pools of embryos at two-cell, four-cell, eight-cell, and blastocyst stages, derived from in vitro fertilization, were collected for analysis of BMP5 and BMP receptors (BMPR1A, BMPR1B, and BMPR2) messenger RNA (mRNA) expression. On the basis of previous results, in experiment 2, presumptive zygotes were cultured for the first 48 hours after insemination in CR1aa medium assaying three different treatments: (1) control (CR1aa); (2) vehicle control (CR1aa + 0.04 mM HCl), and (3) BMP5 treatment (CR1aa + 100 ng/mL of BMP5). The cleavage rate was evaluated 48 hours after insemination (Day 2), and then, embryos were transferred to CR1aa + 10% fetal bovine serum. The blastocyst rate was determined on Day 7. In experiment 3, pools of embryos at two-cell, four-cell, eight-cell, and blastocyst stages, derived from control and BMP5-treated groups, were collected for analysis of ID2 (BMP target gene), OCT4, NANOG, and SOX2 (pluripotency genes) mRNA expression. BMP5 transcripts were not detectable in any of the embryonic stages examined, whereas the relative mRNA abundance of the three BMP receptors analyzed was greater in early embryo development stages before maternal-embryonic transition, raising the possibility of a direct effect of exogenous BMPs on the embryo during the first developmental period. Although early addition of 100 ng/mL of BMP5 to the embryo culture medium had no effect on the cleavage rate, a significantly higher proportion of cleaved embryos developed to the blastocyst stage in the BMP5 group. Moreover, reverse transcription quantitative real-time polymerase chain reaction analysis showed a significant increase in the relative abundance of SOX2 in two-cell stage embryos, ID2 and OCT4 in eight-cell stage embryos, and NANOG and OCT4 in blastocysts derived from BMP5-treated embryos. In conclusion, our results report that early addition of BMP5 to the embryo culture medium had a positive effect on the blastocyst rate and affected the relative expression of BMP target and pluripotency genes, suggesting that BMP5 could play an important role in the preimplantation development of bovine embryos. Copyright © 2015 Elsevier Inc. All rights reserved.

Zebularine and scriptaid significantly improve epigenetic reprogramming of yak fibroblasts and cloning efficiency.

PubMed

Xiong, Xianrong; Lan, Daoliang; Li, Jian; Zhong, Jincheng; Zi, Xiangdong; Ma, Li; Wang, Yong

2013-08-01

Abnormal epigenetic reprogramming of the donor nucleus after somatic cell nuclear transfer (SCNT) is thought to be the main cause of low cloning efficiency. Following SCNT, the donor nucleus often fails to express early embryonic genes and establish a normal embryonic pattern of chromatin modification. Therefore, in this study, we have attempted to improve epigenetic reprogramming of the donor nucleus and cloned embryos with Zebularine and Scriptaid. Yak fibroblasts were treated with 20 μM Zebularine alone or 20 μM Zebularine plus 0.5 μM Scriptaid for 24 h, whereas yak cloned embryos were treated exclusively with 0.5 μM Scriptaid for 12 h. There was no effect on cellular viability and proliferation after drug treatment. The treatment of fibroblasts with Zebularine or Zebularine plus Scriptaid increased histone acetylation of histone 3 lysine 9 (H3K9), but decreased the level of DNA methylation of Oct-4 and Sox-2 promoter regions. When donor cells were used after Zebularine plus Scriptaid treatment to reconstruct cloned embryos and then treated with Scriptaid, the developmental competence and cryosurvival of embryos were improved significantly. In addition, the relative expression of Oct-4 and Sox-2 were increased significantly. The expression levels of Dnmt-1 and Hdac-1 were significantly decreased when fibroblasts and cloned embryos were treated with Zebularine or Scriptaid. This work provides functional evidence that treatment with Zebularine and Scriptaid modifies the epigenetic status of yak fibroblasts, subsequently enhancing in vitro developmental potential and the quality of yak cloned embryos.

Generation of a transgenic cashmere goat using the piggyBac transposition system.

PubMed

Bai, Ding-Ping; Yang, Ming-Ming; Qu, Lei; Chen, Yu-Lin

2017-04-15

The development of transgenic technologies in the Cashmere goat (Capra hircus) has the potential to improve the quality of the meat and wool. The piggyBac (PB) transposon system is highly efficient and can be used to transpose specific target genes into the genome. Here, we developed a PB transposon system to produce transgenic Cashmere goat fetal fibroblasts (GFFs) with the enhanced green fluorescent protein (EGFP). We then used the genetically modified GFFs as nuclear donors to generate transgenic embryos by somatic cell nuclear transfer (SCNT). The embryos (n = 40) were implanted into female goats (n = 20). One transgenic kid that expressed EGFP throughout the surface features of its body was born. This result demonstrated the usefulness of PB transposon system in generating transgenic Cashmere goats. Copyright © 2017 Elsevier Inc. All rights reserved.

Femtosecond laser surgery of two-cell mouse embryos: effect on viability, development, and tetraploidization

NASA Astrophysics Data System (ADS)

Osychenko, Alina A.; Zalessky, Alexandr D.; Kostrov, Andrey N.; Ryabova, Anastasia V.; Krivokharchenko, Alexander S.; Nadtochenko, Viktor A.

2017-12-01

The effect of the laser pulse energy and total expose of the energy incident on the embryo blastomere fusion probability was investigated. The probability of the four different events after laser pulse was determined: the fusion of two blastomeres with the following formation of tetraploid embryo, the destruction of the first blastomere occurs, the second blastomere conservation remains intact, the destruction and the death of both cells; two blastomeres were not fused, and no morphological changes occurred. We report on viability and quality of the embryo after laser surgery as a function of the laser energy incident. To characterize embryo quality, the probability of the blastocyst stage achievement was estimated and the blastocyst cells number was calculated. Blastocoel formation is the only event of morphogenesis in the preimplantation development of mammals, so we assumed it as an indicator of the time of embryonic "clocks" and observed it among fused and control embryos. The blastocoel formation time is the same for fused and control embryos. It indicates that embryo clocks were not affected due to blastomere fusion. Thus, the analysis of the fluorescence microscopic images of nuclei in the fused embryo revealed that nuclei fusion does not occur after blastomere fusion.

Adjustments in cholinergic, adrenergic and purinergic control of cardiovascular function in snapping turtle embryos (Chelydra serpentina) incubated in chronic hypoxia.

PubMed

Eme, John; Rhen, Turk; Crossley, Dane A

2014-10-01

Adenosine is an endogenous nucleoside that acts via G-protein coupled receptors. In vertebrates, arterial or venous adenosine injection causes a rapid and large bradycardia through atrioventricular node block, a response mediated by adenosine receptors that inhibit adenylate cyclase and decrease cyclic AMP concentration. Chronic developmental hypoxia has been shown to alter cardioregulatory mechanisms in reptile embryos, but adenosine's role in mediating these responses is not known. We incubated snapping turtle embryos under chronic normoxic (N21; 21 % O2) or chronic hypoxic conditions (H10; 10 % O2) beginning at 20 % of embryonic incubation. H10 embryos at 90 % of incubation were hypotensive relative to N21 embryos in both normoxic and hypoxic conditions. Hypoxia caused a hypotensive bradycardia in both N21 and H10 embryos during the initial 30 min of exposure; however, f H and P m both trended towards increasing during the subsequent 30 min, and H10 embryos were tachycardic relative to N21 embryos in hypoxia. Following serial ≥1 h exposure to normoxic and hypoxic conditions, a single injection of adenosine (1 mg kg(-1)) was given. N21 and H10 embryos responded to adenosine injection with a rapid and large hypotensive bradycardia in both normoxia and hypoxia. Gene expression for adenosine receptors were quantified in cardiac tissue, and Adora1 mRNA was the predominant receptor subtype with transcript levels 30-82-fold higher than Adora2A or Adora2B. At 70 % of incubation, H10 embryos had lower Adora1 and Adora2B expression compared to N21 embryos. Expression of Adora1 and Adora2B decreased in N21 embryos during development and did not differ from H10 embryos at 90 % of incubation. Similar to previous results in normoxia, H10 embryos in hypoxia were chronically tachycardic compared to N21 embryos before and after complete cholinergic and adrenergic blockade. Chronic hypoxia altered the development of normal cholinergic and adrenergic tone, as well as adenosine receptor mRNA levels. This study demonstrates that adenosine may be a major regulator of heart rate in developing snapping turtle embryos, and that chronic hypoxic incubation alters the response to hypoxic exposure.

Early detection and staging of spontaneous embryo resorption by ultrasound biomicroscopy in murine pregnancy

PubMed Central

2014-01-01

Background Embryo resorption is a major problem in human medicine, agricultural animal production and in conservation breeding programs. Underlying mechanisms have been investigated in the well characterised mouse model. However, post mortem studies are limited by the rapid disintegration of embryonic structures. A method to reliably identify embryo resorption in alive animals has not been established yet. In our study we aim to detect embryos undergoing resorption in vivo at the earliest possible stage by ultra-high frequency ultrasound. Methods In a longitudinal study, we monitored 30 pregnancies of wild type C57BI/6 mice using ultra-high frequency ultrasound (30-70 MHz), so called ultrasound biomicroscopy (UBM). We compared the sonoembryology of mouse conceptuses under spontaneous resorption and neighbouring healthy conceptuses and correlated the live ultrasound data with the respective histology. Results The process of embryo resorption comprised of four stages: first, the conceptus exhibited growth retardation, second, bradycardia and pericardial edema were observed, third, further development ceased and the embryo died, and finally embryo remnants were resorbed by maternal immune cells. In early gestation (day 7 and 8), growth retardation was characterized by a small embryonic cavity. The embryo and its membranes were ill defined or did not develop at all. The echodensity of the embryonic fluid increased and within one to two days, the embryo and its cavity disappeared and was transformed into echodense tissue surrounded by fluid filled caverns. In corresponding histologic preparations, fibrinoid material interspersed with maternal granulocytes and lacunae filled with maternal blood were observed. In later stages (day 9–11) resorption prone embryos were one day behind in their development compared to their normal siblings. The space between Reichert’s membrane and inner yolk sac membrane was enlarged The growth retarded embryos exhibited bradycardia and ultimately cessation of heart beat. Corresponding histology showed apoptotic cells in the embryo while the placenta was still intact. In the subsequent resorption process first the embryo and then its membranes disappeared. Conclusions Our results provide a temporal time course of embryo resorption. With this method, animals exhibiting embryo resorption can be targeted, enabling the investigation of underlying mechanisms before the onset of total embryo disintegration. PMID:24886361

Biosensors for detecting stress in developing embryos

NASA Astrophysics Data System (ADS)

Purdey, Malcolm S.; Saini, Avishkar; McLennan, Hanna J.; Pullen, Benjamin J.; Schartner, Erik P.; Sutton-McDowall, Melanie L.; Thompson, Jeremy G.; Monro, Tanya M.; Nicholls, Stephen J.; Abell, Andrew D.

2016-12-01

Reactive Oxygen Species (ROS) cause DNA damage and defective function in sperm and also affects the developmental competence of embryos. It is therefore critical to monitor ROS in sperm, oocytes and developing embryos. In particular, hydrogen peroxide (H2O2) is a ROS important to normal cell function and signalling as well as its role in oxidative stress. Here we report the development of a fluorescent sensor for H2O2 using carboxyperoxyfluor-1 (CPF1) in solution and attached to a glass slide or multi-mode optical fibre. CPF1 increases in fluorescence upon reaction with H2O2 to non-invasively detect H2O2 near developing embryos. These probes are constructed by immobilising CPF1 to the optical fibre tip a polyacrylamide layer. Also reported is a new dual optical fibre sensor for detecting both H2O2 and pH that is functional at biologically concentrations of H2O2 and can sense pH to 0.1 units. This research shows promise for the use of optical fibre sensors for monitoring the health of developing embryos. Furthermore, these sensors are applicable for use beyond embryos such as detecting stress in endothelial cells involved in cardiovascular dysfunction.

Effects of the popular food additive sodium benzoate on neural tube development in the chicken embryo.

PubMed

Emon, Selin Tural; Orakdogen, Metin; Uslu, Serap; Somay, Hakan

2015-01-01

Many more additives have been introduced with the development of processed foods. Neural tube defects are congenital malformations of the central nervous system. More than 300 000 children are born with neural tube defects every year and surviving children remain disabled for life. Sodium benzoate is used intensively in our daily lives. We therefore aimed to evaluate the effects of sodium benzoate on neural tube defects in chicken embryos. Fertile, specific pathogen-free eggs were used. The study was conducted on five groups. After 30 hours of incubation, the eggs were opened under 4x optical magnification. The embryonic disc was identified and sodium benzoate solution was injected. Eggs were closed with sterile adhesive strips and incubation was continued till the end of the 72nd hour. All eggs were then reopened and embryos were dissected from embryonic membranes and evaluated histopathologically. We found that the development of all embryos was consistent with the stage. We detected neural tube obstruction in one embryo. Neural tube defects were not detected in any embryos. This study showed that sodium benzoate as one of the widely used food preservatives has no effect to neural tube defect development in chicken embryos even at high doses.

Breeding for seedless grapes using Chinese wild Vitis spp. II. In vitro embryo rescue and plant development.

PubMed

Ji, Wei; Wang, Yuejin

2013-12-01

Since 1982, the embryo rescue technique has been widely applied to embryo germination of stenospermic grapes in cross-breeding programmes. This project aimed to: (1) use embryos to breed new seedless cultivars of Vitis vinifera as the female parents utilising wild Chinese Vitis spp. as the male parents; and (2) develop an efficient method for in vitro embryo rescue and plant development. Among the different genotypes, the productions of hybrid plants were significantly different, ranged from 21.1% ('Ruby Seedless' × 'Beichun') to only 1.1% ('Pink Seedless' × 'Beichun'), except for the combinations from which no surviving seedlings were obtained. We collected hybridisation fruits from 28 June to 3 August, and obtained their best sampling times described within days after flowering. The highest rates of embryo formation (24.3%) and plant development (91.4%) were found when ovules of 'Ruby Seedless' were cultured in MM4 + 500 mg L(-1) mashed banana. Seven new hybrids of V. vinifera with wild Chinese Vitis spp. were obtained. As a result of early nuclear-free character identification, 17 seedless grape lines were obtained. An efficient system of seedless grape breeding through embryo rescue was also established. © 2013 Society of Chemical Industry.

Hypoxic alligator embryos: chronic hypoxia, catecholamine levels and autonomic responses of in ovo alligators.

PubMed

Eme, John; Altimiras, Jordi; Hicks, James W; Crossley, Dane A

2011-11-01

Hypoxia is a naturally occurring environmental challenge for embryonic reptiles, and this is the first study to investigate the impact of chronic hypoxia on the in ovo development of autonomic cardiovascular regulation and circulating catecholamine levels in a reptile. We measured heart rate (f(H)) and chorioallantoic arterial blood pressure (MAP) in normoxic ('N21') and hypoxic-incubated ('H10'; 10% O(2)) American alligator embryos (Alligator mississippiensis) at 70, 80 and 90% of development. Embryonic alligator responses to adrenergic blockade with propranolol and phentolamine were very similar to previously reported responses of embryonic chicken, and demonstrated that embryonic alligator has α and β-adrenergic tone over the final third of development. However, adrenergic tone originates entirely from circulating catecholamines and is not altered by chronic hypoxic incubation, as neither cholinergic blockade with atropine nor ganglionic blockade with hexamethonium altered baseline cardiovascular variables in N21 or H10 embryos. In addition, both atropine and hexamethonium injection did not alter the generally depressive effects of acute hypoxia - bradycardia and hypotension. However, H10 embryos showed significantly higher levels of noradrenaline and adrenaline at 70% of development, as well as higher noradrenaline at 80% of development, suggesting that circulating catecholamines reach maximal levels earlier in incubation for H10 embryos, compared to N21 embryos. Chronically elevated levels of catecholamines may alter the normal balance between α and β-adrenoreceptors in H10 alligator embryos, causing chronic bradycardia and hypotension of H10 embryos measured in normoxia. Copyright © 2011 Elsevier Inc. All rights reserved.

Absence of nucleolus formation in raccoon dog-porcine interspecies somatic cell nuclear transfer embryos results in embryonic developmental failure.

PubMed

Jeon, Yubyeol; Nam, Yeong-Hee; Cheong, Seung-A; Kwak, Seong-Sung; Lee, Eunsong; Hyun, Sang-Hwan

2016-08-25

Interspecies somatic cell nuclear transfer (iSCNT) can be a solution for preservation of endangered species that have limited oocytes. It has been reported that blastocyst production by iSCNT is successful even if the genetic distances between donors and recipients are large. In particular, domestic pig oocytes can support the development of canine to porcine iSCNT embryos. Therefore, we examined whether porcine oocytes may be suitable recipient oocytes for Korean raccoon dog iSCNT. We investigated the effects of trichostatin A (TSA) treatment on iSCNT embryo developmental patterns and nucleolus formation. Enucleated porcine oocytes were fused with raccoon dog fibroblasts by electrofusion and cleavage, and blastocyst development and nucleolus formation were evaluated. To our knowledge, this study is the first in which raccoon dog iSCNT was performed using porcine oocytes; we found that 68.5% of 158 iSCNT embryos had the ability to cleave. However, these iSCNT embryos did not develop past the 4-cell stage. Treatment with TSA did not affect iSCNT embryonic development; moreover, the nuclei failed to form nucleoli at 48 and 72 h post-activation (hpa). In contrast, pig SCNT embryos of the control group showed 18.8% and 87.9% nucleolus formation at 48 and 72 hpa, respectively. Our results demonstrated that porcine cytoplasts efficiently supported the development of raccoon dog iSCNT embryos to the 4-cell stage, the stage of porcine embryonic genome activation (EGA); however, these embryos failed to reach the blastocyst stage and showed defects in nucleolus formation.

Micro-magnetic resonance imaging study of live quail embryos during embryonic development.

PubMed

Duce, Suzanne; Morrison, Fiona; Welten, Monique; Baggott, Glenn; Tickle, Cheryll

2011-01-01

Eggs containing live Japanese quail embryos were imaged using micro-magnetic resonance imaging (μMRI) at 24-h intervals from Day 0 to 8, the period during which the main body axis is being laid down and organogenesis is taking place. Considerable detail of non-embryonic structures such as the latebra was revealed at early stages but the embryo could only be visualized around Day 3. Three-dimensional (3D) changes in embryo length and volume were quantified and also changes in volume in the extra- and non-embryonic components. The embryo increased in length by 43% and nearly trebled in volume between Day 4 and Day 5. Although the amount of yolk remained fairly constant over the first 5 days, the amount of albumen decreases significantly and was replaced by extra-embryonic fluid (EEF). ¹H longitudinal (T₁) and transverse (T₂) relaxation times of different regions within the eggs were determined over the first 6 days of development. The T₂ measurements mirrored the changes in image intensity observed, which can be related to the aqueous protein concentrations. In addition, a comparison of the development of Day 0 to 3 quail embryos exposed to radiofrequency (rf) pulses, 7 T static magnetic fields and magnetic field gradients for an average of 7 h with the development of control embryos did not reveal any gross changes, thus confirming that μMRI is a suitable tool for following the development of live avian embryos over time from the earliest stages. Copyright © 2011 Elsevier Inc. All rights reserved.

Effects of multi-well plate incubation on embryo-larval development in the fathead minnow (Pimephales promelas).

PubMed

Marentette, Julie R; Sullivan, Cheryl A; Lavalle, Christine; Shires, Kallie; Parrott, Joanne L

2015-01-01

Fathead minnow embryos and larvae are frequently used in toxicology, including short-term embryo-only tests which often use small volumes of test solution. The effect that such conditions may have on fathead minnow development has yet to be explicitly described. Here we compared rates of embryonic development in fathead minnow embryos reared under standard light and temperature conditions with a range of possible methods. All methods yielded excellent control survival. We demonstrated that fathead minnow embryos incubated in a range of small volumes in multi-well plates (500 μL to 2 mL per embryo) did not substantially vary in developmental rate, but flexed less frequently as embryos, hatched smaller, later and with larger yolk-sacs, and initiated feeding later than embryos reared in an excess of solution (20 mL per embryo) with or without supplemental aeration. Faster hatch and growth were promoted with an orbital shaker, but growth benefits were not sustained into the larval stage. Developmental differences persisted in larvae reared to 20 days post-fertilization when monitoring ceased, but growth differences did not magnify and in some measurements partially resolved. To our knowledge we are the first to report effects of incubation in multi-well plates in any fish taxa. As our data revealed that the eleutheroembryonic stage for fathead minnow may be prolonged in multi-well plates, this may allow the use of longer toxicity tests using fathead minnow embryos without conflicting with existing animal welfare legislation in many countries. Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.

Alteration of development and gene expression induced by in ovo-nanoinjection of 3-hydroxybenzo[c]phenanthrene into Japanese medaka (Oryzias latipes) embryos.

PubMed

Chen, Kun; Tsutsumi, Yuki; Yoshitake, Shuhei; Qiu, Xuchun; Xu, Hai; Hashiguchi, Yasuyuki; Honda, Masato; Tashiro, Kosuke; Nakayama, Kei; Hano, Takeshi; Suzuki, Nobuo; Hayakawa, Kazuichi; Shimasaki, Yohei; Oshima, Yuji

2017-01-01

Benzo[c]phenanthrene (BcP) is a highly toxic polycyclic aromatic hydrocarbon (PAHs) found throughout the environment. In fish, it is metabolized to 3-hydroxybenzo[c]phenanthrene (3-OHBcP). In the present study, we observed the effects of 1nM 3-OHBcP on the development and gene expression of Japanese medaka (Oryzias latipes) embryos. Embryos were nanoinjected with the chemical after fertilization. Survival, developmental stage, and heart rate of the embryos were observed, and gene expression differences were quantified by messenger RNA sequencing (mRNA-Seq). The exposure to 1nM 3-OHBcP accelerated the development of medaka embryos on the 1st, 4th, and 6th days post fertilization (dpf), and increased heart rates significantly on the 5th dpf. Physical development differences of exposed medaka embryos were consistent with the gene expression profiles of the mRNA-Seq results for the 3rd dpf, which show that the expression of 780 genes differed significantly between the solvent control and 1nM 3-OHBcP exposure groups. The obvious expression changes in the exposure group were found for genes involved in organ formation (eye, muscle, heart), energy supply (ATPase and ATP synthase), and stress-response (heat shock protein genes). The acceleration of development and increased heart rate, which were consistent with the changes in mRNA expression, suggested that 3-OHBcP affects the development of medaka embryos. The observation on the developmental stages and heart beat, in ovo-nanoinjection and mRNA-Seq may be efficient tools to evaluate the effects of chemicals on embryos. Copyright © 2016 Elsevier B.V. All rights reserved.

Inter-laboratory assessment of a harmonized zebrafish developmental toxicology assay - progress report on phase I.

PubMed

Gustafson, A-L; Stedman, D B; Ball, J; Hillegass, J M; Flood, A; Zhang, C X; Panzica-Kelly, J; Cao, J; Coburn, A; Enright, B P; Tornesi, M B; Hetheridge, M; Augustine-Rauch, K A

2012-04-01

This report provides a progress update of a consortium effort to develop a harmonized zebrafish developmental toxicity assay. Twenty non-proprietary compounds (10 animal teratogens and 10 animal non-teratogens) were evaluated blinded in 4 laboratories. Zebrafish embryos from pond-derived and cultivated strain wild types were exposed to the test compounds for 5 days and subsequently evaluated for lethality and morphological changes. Each of the testing laboratories achieved similar overall concordance to the animal data (60-70%). Subsequent optimization procedures to improve the overall concordance focused on compound formulation and test concentration adjustments, chorion permeation and number of replicates. These optimized procedures were integrated into a revised protocol and all compounds were retested in one lab using embryos from pond-derived zebrafish and achieved 85% total concordance. To further assess assay performance, a study of additional compounds is currently in progress at two laboratories using embryos from pond-derived and cultivated-strain wild type zebrafish. Copyright © 2011 Elsevier Inc. All rights reserved.

Dissection and lateral mounting of zebrafish embryos: analysis of spinal cord development.

PubMed

Beck, Aaron P; Watt, Roland M; Bonner, Jennifer

2014-02-28

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue.

Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development

PubMed Central

Beck, Aaron P.; Watt, Roland M.; Bonner, Jennifer

2014-01-01

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue. PMID:24637734

Effect of alcohol exposure on fetal brain development

NASA Astrophysics Data System (ADS)

Sudheendran, Narendran; Bake, Shameena; Miranda, Rajesh C.; Larin, Kirill V.

2013-02-01

Alcohol consumption during pregnancy can be severely damage to the brain development in fetuses. This study investigates the effects of maternal ethanol consumption on brain development in mice embryos. Pregnant mice at gestational day 12.5 were intragastrically gavaged with ethanol (3g/Kg bwt) twice daily for three consecutive days. On gestational day 14.5, fetuses were collected and fixed in 4% paraformaldehyde and imaged using a swept-source optical coherence tomography (SSOCT) system. 3D images of the mice embryo brain were obtained and the volumes of the left and right ventricles of the brain were measured. The average volumes of the left and the right volumes of 5 embryos each alcohol-exposed and control embryos were measured to be 0.35 and 0.15 mm3, respectively. The results suggest that the left and right ventricle volumes of brain are much larger in the alcohol-exposed embryos as compared to control embryos indicating alcohol-induced developmental delay.

Use of micro computed-tomography and 3D printing for reverse engineering of mouse embryo nasal capsule

NASA Astrophysics Data System (ADS)

Tesařová, M.; Zikmund, T.; Kaucká, M.; Adameyko, I.; Jaroš, J.; Paloušek, D.; Škaroupka, D.; Kaiser, J.

2016-03-01

Imaging of increasingly complex cartilage in vertebrate embryos is one of the key tasks of developmental biology. This is especially important to study shape-organizing processes during initial skeletal formation and growth. Advanced imaging techniques that are reflecting biological needs give a powerful impulse to push the boundaries of biological visualization. Recently, techniques for contrasting tissues and organs have improved considerably, extending traditional 2D imaging approaches to 3D . X-ray micro computed tomography (μCT), which allows 3D imaging of biological objects including their internal structures with a resolution in the micrometer range, in combination with contrasting techniques seems to be the most suitable approach for non-destructive imaging of embryonic developing cartilage. Despite there are many software-based ways for visualization of 3D data sets, having a real solid model of the studied object might give novel opportunities to fully understand the shape-organizing processes in the developing body. In this feasibility study we demonstrated the full procedure of creating a real 3D object of mouse embryo nasal capsule, i.e. the staining, the μCT scanning combined by the advanced data processing and the 3D printing.

Preimplantation development of somatic cell cloned embryos in the common marmoset (Callithrix jacchus).

PubMed

Sotomaru, Yusuke; Hirakawa, Reiko; Shimada, Akiko; Shiozawa, Seiji; Sugawara, Ayako; Oiwa, Ryo; Nobukiyo, Asako; Okano, Hideyuki; Tamaoki, Norikazu; Nomura, Tatsuji; Hiyama, Eiso; Sasaki, Erika

2009-12-01

The somatic cell nuclear transfer technique has been applied to various mammals to produce cloned animals; however, a standardized method is not applicable to all species. We aimed here to develop optimum procedures for somatic cell cloning in nonhuman primates, using common marmosets. First, we confirmed that parthenogenetic activation of in vitro matured oocytes was successfully induced by electrical stimulation (three cycles of 150 V/mm, 50 microsec x 2, 20 min intervals), and this condition was applied to the egg activation procedure in the subsequent experiments. Next, nuclear transfer to recipient enucleated oocytes was performed 1 h before, immediately after, or 1 h after egg activation treatment. The highest developmental rate was observed when nuclear transfer was performed 1 h before activation, but none of the cloned embryos developed beyond the eight-cell stage. To investigate the causes of the low developmental potential of cloned embryos, a study was performed to determine whether the presence of metaphase II (MII) chromosome in recipient ooplasm has an effect on developmental potential. As a result, only tetraploid cloned embryos produced by transferring a donor cell into a recipient bearing the MII chromosome developed into blastocysts (66.7%). In contrast, neither parthenogenetic embryos nor cloned embryos (whether diploid or tetraploid) produced using enucleated oocytes developed past the eight-cell stage. These results suggest that MII chromosome, or cytoplasm proximal to the MII chromosome, plays a major role in the development of cloned embryos in common marmosets.

Rapid and simple method for in vivo ex utero development of mouse embryo explants.

PubMed

Gonçalves, André B; Thorsteinsdóttir, Sólveig; Deries, Marianne

2016-01-01

The in utero development of mammals drastically reduces the accessibility of the mammalian embryo and therefore limits the range of experimental manipulation that can be done to study functions of genes or signaling pathways during embryo development. Over the past decades, tissue and organ-like culture methods have been developed with the intention of reproducing in vivo situations. Developing accessible and simple techniques to study and manipulate embryos is an everlasting challenge. Herein, we describe a reliable and quick technique to culture mid-gestation explanted mouse embryos on top of a floating membrane filter in a defined medium. Viability of the cultured tissues was assessed by apoptosis and proliferation analysis showing that cell proliferation is normal and there is only a slight increase in apoptosis after 12h of culture compared to embryos developing in utero. Moreover, differentiation and morphogenesis proceed normally as assessed by 3D imaging of the transformation of the myotome into deep back muscles. Not only does muscle cell differentiation occur as expected, but so do extracellular matrix organization and the characteristic splitting of the myotome into the three epaxial muscle groups. Our culture method allows for the culture and manipulation of mammalian embryo explants in a very efficient way, and it permits the manipulation of in vivo developmental events in a controlled environment. Explants grown under these ex utero conditions simulate real developmental events that occur in utero. Copyright © 2016 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Commonly used fertility drugs, a diet supplement, and stress force AMPK-dependent block of stemness and development in cultured mammalian embryos.

PubMed

Bolnick, Alan; Abdulhasan, Mohammed; Kilburn, Brian; Xie, Yufen; Howard, Mindie; Andresen, Paul; Shamir, Alexandra M; Dai, Jing; Puscheck, Elizabeth E; Rappolee, Daniel A

2016-08-01

The purpose of the present study is to test whether metformin, aspirin, or diet supplement (DS) BioResponse-3,3'-Diindolylmethane (BR-DIM) can induce AMP-activated protein kinase (AMPK)-dependent potency loss in cultured embryos and whether metformin (Met) + Aspirin (Asa) or BR-DIM causes an AMPK-dependent decrease in embryonic development. The methods used were as follows: culture post-thaw mouse zygotes to the two-cell embryo stage and test effects after 1-h AMPK agonists' (e.g., Met, Asa, BR-DIM, control hyperosmotic stress) exposure on AMPK-dependent loss of Oct4 and/or Rex1 nuclear potency factors, confirm AMPK dependence by reversing potency loss in two-cell-stage embryos with AMPK inhibitor compound C (CC), test whether Met + Asa (i.e., co-added) or DS BR-DIM decreases development of two-cell to blastocyst stage in an AMPK-dependent (CC-sensitive) manner, and evaluate the level of Rex1 and Oct4 nuclear fluorescence in two-cell-stage embryos and rate of two-cell-stage embryo development to blastocysts. Met, Asa, BR-DIM, or hyperosmotic sorbitol stress induces rapid ~50-85 % Rex1 and/or Oct4 protein loss in two-cell embryos. This loss is ~60-90 % reversible by co-culture with AMPK inhibitor CC. Embryo development from two-cell to blastocyst stage is decreased in culture with either Met + Asa or BR-DIM, and this is either >90 or ~60 % reversible with CC, respectively. These experimental designs here showed that Met-, Asa-, BR-DIM-, or sorbitol stress-induced rapid potency loss in two-cell embryos is AMPK dependent as suggested by inhibition of Rex1 and/or Oct4 protein loss with an AMPK inhibitor. The DS BR-DIM or fertility drugs (e.g., Met + Asa) that are used to enhance maternal metabolism to support fertility can also chronically slow embryo growth and block development in an AMPK-dependent manner.

The effect of soluble uterine factors on porcine embryo development within a three-dimensional alginate matrix system

USDA-ARS?s Scientific Manuscript database

Between day 10 and 12 of gestation in the pig, the embryo undergoes a dramatic morphological change, known as elongation. During elongation the embryo produces and secretes estrogen, which serves as a key signal for maternal recognition of pregnancy. The uterine environment prepares for embryo elong...

Physiological and molecular determinants of embryo implantation

PubMed Central

Zhang, Shuang; Lin, Haiyan; Kong, Shuangbo; Wang, Shumin; Wang, Hongmei; Wang, Haibin; Armant, D. Randall

2014-01-01

Embryo implantation involves the intimate interaction between an implantation-competent blastocyst and a receptive uterus, which occurs in a limited time period known as the window of implantation. Emerging evidence shows that defects originating during embryo implantation induce ripple effects with adverse consequences on later gestation events, highlighting the significance of this event for pregnancy success. Although a multitude of cellular events and molecular pathways involved in embryo-uterine crosstalk during implantation have been identified through gene expression studies and genetically engineered mouse models, a comprehensive understanding of the nature of embryo implantation is still missing. This review focuses on recent progress with particular attention to physiological and molecular determinants of blastocyst activation, uterine receptivity, blastocyst attachment and uterine decidualization. A better understanding of underlying mechanisms governing embryo implantation should generate new strategies to rectify implantation failure and improve pregnancy rates in women. PMID:23290997

Sperm-derived factors enhance the in vitro developmental potential of haploid parthenotes.

PubMed

Nair, Ramya; Aboobacker, Shahin; Mutalik, Srinivas; Kalthur, Guruprasad; Adiga, Satish Kumar

2017-12-01

Parthenotes are characterized by poor in vitro developmental potential either due to the ploidy status or the absence of paternal factors. In the present study, we demonstrate the beneficial role of sperm-derived factors (SDF) on the in vitro development of mouse parthenotes. Mature (MII) oocytes collected from superovulated Swiss albino mice were activated using strontium chloride (SrCl2) in the presence or absence of various concentrations of SDF in M16 medium. The presence of SDF in activation medium did not have any significant influence on the activation rate. However, a significant increase in the developmental potential of the embryos and increased blastocyst rate (P < 0.01) was observed at 50 µg/ml concentration. Furthermore, the activated oocytes from this group exhibited early cleavage and cortical distribution of cortical granules that was similar to that of normally fertilized zygotes. Culturing 2-cell stage parthenotes in the presence of SDF significantly improved the developmental potential (P < 0.05) indicating that they also play a significant role in embryo development. In conclusion, artificial activation of oocytes with SDF can improve the developmental potential of parthenotes in vitro.

Application of assisted reproduction for population management in felids: the potential and reality for conservation of small cats.

PubMed

Swanson, William F

2006-07-01

Assisted reproductive technology (ART), using the primary applied tools of AI, ET, and sperm and embryo cryopreservation, has been promoted over the past decades for its potential to conserve endangered wildlife, including felids. However, if the goal is efficient, consistent production of viable offspring for population management, then the 'potential' of ART has yet to become 'reality' for any non-domestic cat species. For the five small-sized felids (i.e., Brazilian ocelot, fishing cat, Pallas' cat, Arabian sand cat, black-footed cat) managed by Species Survival Plans (SSPs) in North American zoos, achieving this potential may be an absolute necessity if genetically viable captive populations are to be maintained into the next century. Modeling programs suggest that current SSP populations are not sustainable without periodic introduction of new founders and improved demographic parameters, including longer generation intervals and larger population sizes. ART provides the means to address each of these management challenges. In each small cat SSP species, fecal hormone metabolite assays and seminal analysis have proven useful for characterizing basal reproductive parameters, a necessary prerequisite to developing ART. Of the five SSP species, ART has been used to produce living offspring only in the ocelot, including after AI with frozen-thawed spermatozoa and following transfer of frozen-thawed IVF embryos. The true efficacy of these techniques, however, is still unknown. To improve the applicability of ART for population management, priorities for immediate research include further investigation of ovarian stimulation protocols, sperm and embryo cryopreservation methods, embryo culture systems, and fetal and neonatal viability following ART.

Successful pregnancies from vitrified embryos in the dromedary camel: Avoidance of a possible toxic effect of sucrose on embryos.

PubMed

Herrid, M; Billah, M; Skidmore, J A

2017-12-01

Successful embryo cryopreservation facilitates the wider application of assisted reproduction technologies and also provides a useful method for gene banking of valuable genetics. Unfortunately attempts to establish an effective cryopreservation protocol for camelid embryos have been unsuccessful. In the current study, a modified vitrification protocol with three steps was investigated, whereby embryos were exposed to solutions containing increasing amounts of glycerol and ethylene glycol for fixed time periods. Embryos were then loaded into an Open Pull Straw (OPS) and plunged directly into liquid nitrogen for storage. Three experiments were designed to investigate the effect of 1) artificial shrinkage (AS) of embryos, 2) the addition of sucrose to the vitrification solutions, and 3) the replacement of sucrose by galactose in the warming solution, on the outcome of vitrification. The results showed that neither AS of hatched embryos prior to vitrification, nor the addition of sucrose into vitrification solutions improves the outcome of vitrification, while replacement of sucrose with galactose in warming solution increases the survival and developmental rates of vitrified embryos in culture. Transfer of vitrified embryos that were warmed in galactose resulted in a pregnancy rate of 42.8% per embryo or 46.1% per recipient. Collectively, these results suggest a possible species-specific toxic effect of sucrose on camel embryos, and that avoiding its use either in vitrification or warming solution is critical for establishing an effective protocol. This study may also be applicable to the vitrification of embryos of other camelid species including alpaca and llamas. Copyright © 2017 Elsevier B.V. All rights reserved.

Characteristics of plastids responsible for starch synthesis in developing pea embryos.

PubMed

Smith, A M; Quinton-Tulloch, J; Denyer, K

1990-03-01

The nature of the starch-synthesising plastids in developing pea (Pisum sativum L.) embryos has been investigated. Chlorophyll and starch were distributed throughout the cotyledon during development. Chlorophyll content increased initially, then showed little change up to the point of drying out of the embryo. Starch content per embryo increased dramatically throughout development. The chlorophyll content per unit volume was highest on the outer edge of the cotyledon, while the starch content was highest on inner face. Nycodenz gradients, which fractionated mechanically-prepared plastids according to their starch content, failed to achieve any significant separation of plastids rich in starch and ADP-glucose pyrophosphorylase from those rich in chlorophyll and a Calvin-cycle marker enzyme, NADP-glyceraldehyde-3-phosphate dehydrogenase. However, material that was not sufficiently dense to enter the gradients was enriched in activity of the Calvin-cycle marker enzyme relative to that of ADP-glucose pyrophosphorylase. Nomarski and epi-fluorescence microscopy showed that intact, isolated plastids, including those with very large starch grains, invariably contained chlorophyll in stromal structures peripheral to the starch grain. We suggest that the starch-storing plastids of developing pea embryos are derived directly from chloroplasts, and retain chloroplast-like characteristics throughout their development. Developing pea embryos also contain chloroplasts which store little or no starch. These are probably located primarily on the outer edge of the cotyledons where there is sufficient light for photosynthesis at some stages of development.

Normalizing gene expression by quantitative PCR during somatic embryogenesis in two representative conifer species: Pinus pinaster and Picea abies.

PubMed

de Vega-Bartol, José J; Santos, Raquen Raissa; Simões, Marta; Miguel, Célia M

2013-05-01

Suitable internal control genes to normalize qPCR data from different stages of embryo development and germination were identified in two representative conifer species. Clonal propagation by somatic embryogenesis has a great application potentiality in conifers. Quantitative PCR (qPCR) is widely used for gene expression analysis during somatic embryogenesis and embryo germination. No single reference gene is universal, so a systematic characterization of endogenous genes for concrete conditions is fundamental for accuracy. We identified suitable internal control genes to normalize qPCR data obtained at different steps of somatic embryogenesis (embryonal mass proliferation, embryo maturation and germination) in two representative conifer species, Pinus pinaster and Picea abies. Candidate genes included endogenous genes commonly used in conifers, genes previously tested in model plants, and genes with a lower variation of the expression along embryo development according to genome-wide transcript profiling studies. Three different algorithms were used to evaluate expression stability. The geometric average of the expression values of elongation factor-1α, α-tubulin and histone 3 in P. pinaster, and elongation factor-1α, α-tubulin, adenosine kinase and CAC in P. abies were adequate for expression studies throughout somatic embryogenesis. However, improved accuracy was achieved when using other gene combinations in experiments with samples at a single developmental stage. The importance of studies selecting reference genes to use in different tissues or developmental stages within one or close species, and the instability of commonly used reference genes, is highlighted.

Diploid, but not haploid, human embryonic stem cells can be derived from microsurgically repaired tripronuclear human zygotes

PubMed Central

Fan, Yong; Li, Rong; Huang, Jin; Yu, Yang; Qiao, Jie

2013-01-01

Human embryonic stem cells have shown tremendous potential in regenerative medicine, and the recent progress in haploid embryonic stem cells provides new insights for future applications of embryonic stem cells. Disruption of normal fertilized embryos remains controversial; thus, the development of a new source for human embryonic stem cells is important for their usefulness. Here, we investigated the feasibility of haploid and diploid embryo reconstruction and embryonic stem cell derivation using microsurgically repaired tripronuclear human zygotes. Diploid and haploid zygotes were successfully reconstructed, but a large proportion of them still had a tripolar spindle assembly. The reconstructed embryos developed to the blastocyst stage, although the loss of chromosomes was observed in these zygotes. Finally, triploid and diploid human embryonic stem cells were derived from tripronuclear and reconstructed zygotes (from which only one pronucleus was removed), but haploid human embryonic stem cells were not successfully derived from the reconstructed zygotes when two pronuclei were removed. Both triploid and diploid human embryonic stem cells showed the general characteristics of human embryonic stem cells. These results indicate that the lower embryo quality resulting from abnormal spindle assembly contributed to the failure of the haploid embryonic stem cell derivation. However, the successful derivation of diploid embryonic stem cells demonstrated that microsurgical tripronuclear zygotes are an alternative source of human embryonic stem cells. In the future, improving spindle assembly will facilitate the application of triploid zygotes to the field of haploid embryonic stem cells. PMID:23255130