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Sample records for 4-cell stage embryos

  1. Human embryonic stem cell lines derived from single blastomeres of two 4-cell stage embryos

    PubMed Central

    Geens, Mieke; Mateizel, Ileana; Sermon, Karen; De Rycke, Martine; Spits, Claudia; Cauffman, Greet; Devroey, Paul; Tournaye, Herman; Liebaers, Inge; Van de Velde, Hilde

    2009-01-01

    BACKGROUND Recently, we demonstrated that single blastomeres of a 4-cell stage human embryo are able to develop into blastocysts with inner cell mass and trophectoderm. To further investigate potency at the 4-cell stage, we aimed to derive pluripotent human embryonic stem cells (hESC) from single blastomeres. METHODS Four 4-cell stage embryos were split on Day 2 of preimplantation development and the 16 blastomeres were individually cultured in sequential medium. On Day 3 or 4, the blastomere-derived embryos were plated on inactivated mouse embryonic fibroblasts (MEFs). RESULTS Ten out of sixteen blastomere-derived morulae attached to the MEFs, and two produced an outgrowth. They were mechanically passaged onto fresh MEFs as described for blastocyst ICM-derived hESC, and shown to express the typical stemness markers by immunocytochemistry and/or RT–PCR. In vivo pluripotency was confirmed by the presence of all three germ layers in the teratoma obtained after injection in immunodeficient mice. The first hESC line displays a mosaic normal/abnormal 46, XX, dup(7)(q33qter), del(18)(q23qter) karyotype. The second hESC line displays a normal 46, XY karyotype. CONCLUSION We report the successful derivation and characterization of two hESC lines from single blastomeres of four split 4-cell stage human embryos. These two hESC lines were derived from distinct embryos, proving that at least one of the 4-cell stage blastomeres is pluripotent. PMID:19633307

  2. Zebrafish vasa homologue RNA is localized to the cleavage planes of 2- and 4-cell-stage embryos and is expressed in the primordial germ cells.

    PubMed

    Yoon, C; Kawakami, K; Hopkins, N

    1997-08-01

    Identification and manipulation of the germ line are important to the study of model organisms. Although zebrafish has recently emerged as a model for vertebrate development, the primordial germ cells (PGCs) in this organism have not been previously described. To identify a molecular marker for the zebrafish PGCs, we cloned the zebrafish homologue of the Drosophila vasa gene, which, in the fly, encodes a germ-cell-specific protein. Northern blotting revealed that zebrafish vasa homologue (vas) transcript is present in embryos just after fertilization, and hence it is probably maternally supplied. Using whole-mount in situ hybridization, we investigated the expression pattern of vas RNA in zebrafish embryos from the 1-cell stage to 10 days of development. Here we present evidence that vas RNA is a germ-cell-specific marker, allowing a description of the zebrafish PGCs for the first time. Furthermore, vas transcript was detected in a novel pattern, localized to the cleavage planes in 2- and 4-cell-stage embryos. During subsequent cleavages, the RNA is segregated as subcellular clumps to a small number of cells that may be the future germ cells. These results suggest new ways in which one might develop techniques for the genetic manipulation of zebrafish. Furthermore, they provide the basis for further studies on this novel RNA localization pattern and on germ-line development in general.

  3. Heterogeneity in Oct4 and Sox2 Targets Biases Cell Fate in 4-Cell Mouse Embryos.

    PubMed

    Goolam, Mubeen; Scialdone, Antonio; Graham, Sarah J L; Macaulay, Iain C; Jedrusik, Agnieszka; Hupalowska, Anna; Voet, Thierry; Marioni, John C; Zernicka-Goetz, Magdalena

    2016-03-24

    The major and essential objective of pre-implantation development is to establish embryonic and extra-embryonic cell fates. To address when and how this fundamental process is initiated in mammals, we characterize transcriptomes of all individual cells throughout mouse pre-implantation development. This identifies targets of master pluripotency regulators Oct4 and Sox2 as being highly heterogeneously expressed between blastomeres of the 4-cell embryo, with Sox21 showing one of the most heterogeneous expression profiles. Live-cell tracking demonstrates that cells with decreased Sox21 yield more extra-embryonic than pluripotent progeny. Consistently, decreasing Sox21 results in premature upregulation of the differentiation regulator Cdx2, suggesting that Sox21 helps safeguard pluripotency. Furthermore, Sox21 is elevated following increased expression of the histone H3R26-methylase CARM1 and is lowered following CARM1 inhibition, indicating the importance of epigenetic regulation. Therefore, our results indicate that heterogeneous gene expression, as early as the 4-cell stage, initiates cell-fate decisions by modulating the balance of pluripotency and differentiation.

  4. Heterogeneity in Oct4 and Sox2 Targets Biases Cell Fate in 4-Cell Mouse Embryos.

    PubMed

    Goolam, Mubeen; Scialdone, Antonio; Graham, Sarah J L; Macaulay, Iain C; Jedrusik, Agnieszka; Hupalowska, Anna; Voet, Thierry; Marioni, John C; Zernicka-Goetz, Magdalena

    2016-03-24

    The major and essential objective of pre-implantation development is to establish embryonic and extra-embryonic cell fates. To address when and how this fundamental process is initiated in mammals, we characterize transcriptomes of all individual cells throughout mouse pre-implantation development. This identifies targets of master pluripotency regulators Oct4 and Sox2 as being highly heterogeneously expressed between blastomeres of the 4-cell embryo, with Sox21 showing one of the most heterogeneous expression profiles. Live-cell tracking demonstrates that cells with decreased Sox21 yield more extra-embryonic than pluripotent progeny. Consistently, decreasing Sox21 results in premature upregulation of the differentiation regulator Cdx2, suggesting that Sox21 helps safeguard pluripotency. Furthermore, Sox21 is elevated following increased expression of the histone H3R26-methylase CARM1 and is lowered following CARM1 inhibition, indicating the importance of epigenetic regulation. Therefore, our results indicate that heterogeneous gene expression, as early as the 4-cell stage, initiates cell-fate decisions by modulating the balance of pluripotency and differentiation. PMID:27015307

  5. 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.

  6. Embryo apoptosis identification: Oocyte grade or cleavage stage?

    PubMed Central

    Bakri, Noraina Mohd; Ibrahim, Siti Fatimah; Osman, Nurul Atikah; Hasan, Nurhaslina; Jaffar, Farah Hanan Fathihah; Rahman, Zulaiha Abdul; Osman, Khairul

    2015-01-01

    Apoptosis is a programed cell death that is vital for tissue homeostasis. However, embryo apoptosis had been known to be related to embryo fragmentation which should be avoided in in vitro fertilization (IVF). The purpose of this study was to evaluate the relationship of embryo apoptosis with the grade of immature oocytes and cleavage stage of in vitro produced (IVP) cattle embryos. This study consisted of 345 oocytes collected through ovary slicing. Immature oocytes were graded as A, B and C. This grading was based on cumulus cell thickness and compactness. All oocytes then underwent an in vitro maturation (IVM) procedure. An IVF was done 24 h after IVM culture. Prior to staining, stage of cleaved embryos was determined and classified as either 2, 4, 8 or >8-cell embryo stage. Apoptosis status of cleaved IVP embryos was determined by using annexin V-FITC staining technique at 48 and 72 h post insemination (hpi). Apoptosis status for each embryo was classified as either early or late. The result showed that there was no significant difference (p > 0.05) of apoptosis status among grade A, B and C embryos. All grades of oocytes showed embryo apoptosis where 1.5% late apoptosis for grade A, 4.5% and 10.4% of early and late apoptosis for grade B and grade C. Early apoptosis was not seen in grade A embryo. We also noted no significant difference (p > 0.05) of apoptosis status between 2, 4, 8 and >8-cell embryo stage. Early apoptosis was also not seen in >8-cell stage. Even though there were no differences in apoptosis expression between the three classes, the cleavage rate of grade A oocytes was significantly higher (p < 0.01) than grade B and grade C. In conclusion, the apoptosis expression in the embryo can occur regardless of the oocyte quality and the cleavage stage of the embryo produced. PMID:26858565

  7. Stage-dependent uptake of cadmium by Bufo arenarum embryos

    SciTech Connect

    Preez-Coll, C.S.; Herkovits, J.

    1996-04-01

    Over the last several years, environmental contamination with cadmium has significantly increased because of its extensive use In anthropogenic activities. This heavy metal is a very toxic xenobiotic producing reproductive and developmental impairments in a wide spectrum of organisms. Within the life cycle of organisms, the embryo is the most sensitive period to adverse conditions. Moreover, stage-dependent susceptibilities to toxic agents in amphibian embryos treated with lead, cadmium and aluminium were described. In the case of cadmium, this differential sensitivity could be related to changes in the metal accumulation through development or in the induction of defense mechanisms against cadmium toxicity, such as metallothionein (Mt) synthesis, which seems to be developmentally regulated. In the case of the toad Bufo arenarum, susceptibility to cadmium seems to follow a biphasic pattern during embryonic development. From the two-cell stage to the neurula stage an increase in susceptibility occurs, whereas from the last stage onwards a gradual increase in the resistance against this heavy metal seems to be achieved. This stage reports the uptake profile of cadmium at different post-hatching stages. 20 refs., 3 figs.

  8. Effect of Maternal Age on the Ratio of Cleavage and Mitochondrial DNA Copy Number in Early Developmental Stage Bovine Embryos

    PubMed Central

    TAKEO, Shun; GOTO, Hiroya; KUWAYAMA, Takehito; MONJI, Yasunori; IWATA, Hisataka

    2012-01-01

    Abstract Age-associated deterioration in both the quality and quantity of mitochondria occurs in older women. The main aim of this study was to examine the effect of age on mitochondrial DNA copy number (mtDNA number) in early developmental stage bovine embryos as well as the dynamics of mtDNA number during early embryo development. Real-time PCR was used to determine mtDNA number. In vitro-produced embryos 48 h after insemination derived from Japanese black cows, ranging in age from 25 to 209 months were categorized based on their cleavage status. There was an overall negative relationship between the age of the cow and cleavage status, to the extent that the ratio of embryos cleaved over the 4-cell stage was greater in younger cows. The mtDNA number did not differ among the cleaved status of embryos. In the next experiment, oocytes collected from each donor cow were divided into 2 groups containing 10 oocytes each, in order to compare the mtDNA number of mature oocytes and early developmental stage embryos within individuals. Upon comparing the mtDNA number between oocytes at the M2 stage and early developmental stage 48 h post insemination, mtDNA number was found to decrease in most cows, but was found to increase in some cows. In conclusion, age affects the cleaving ability of oocytes, and very old cows (> 180 months) tend to have lower mtDNA numbers in their oocytes. The change in mtDNA number during early development varied among individual cows, although overall, it showed a tendency to decrease. PMID:23269452

  9. PEI1, an embryo-specific zinc finger protein gene required for heart-stage embryo formation in Arabidopsis.

    PubMed Central

    Li, Z; Thomas, T L

    1998-01-01

    We used virtual subtraction, a new gene isolation strategy, to isolate several genes of interest that are expressed in Arabidopsis embryos. These genes have demonstrated biological properties or have the potential to be involved in important biological processes. One gene isolated by virtual subtraction is PEI. It encodes a protein containing a Cys3His zinc finger domain associated with a number of animal and fungal transcription factors. In situ hybridization results showed that PEI1 is expressed throughout the embryo from globular to late cotyledon stage. Transgenic Arabidopsis plants expressing a PEI1 antisense gene produced white seeds in which embryo development did not progress through heart stage. Aberrant embryos failed to form cotyledons, but the embryonic root appeared to be normal. Aberrant embryos did not turn green, and the expression of genes involved in photomorphogenesis was drastically attenuated. In culture, aberrant embryos did not form true leaves, but root formation was apparently normal. These results suggest that PEI1 is an embryo-specific transcription factor that plays an important role during Arabidopsis embryogenesis, functioning primarily in the apical domain of the embryo. PMID:9501112

  10. Cryopreservation of In Vitro-Produced Early-Stage Porcine Embryos in a Closed System

    PubMed Central

    Men, Hongsheng; Spate, Lee D.; Murphy, Clifton N.; Prather, Randall S.

    2015-01-01

    Abstract Cryostorage of porcine embryos in a closed pathogen-free system is essential for the maintenance and safeguard of swine models. Previously, we reported a protocol for the successful cryopreservation of porcine embryos at the blastocyst stage in 0.25 mL ministraws. In this experiment, we aimed at developing a protocol to apply the same concept for the cryopreservation of early-stage porcine embryos. Porcine embryos from day 2 through day 4 were delipidated by using a modified two-step centrifugation method and were then cryopreserved in sealed 0.25 mL straws by using a slow cooling method. Control groups included open pulled straw (OPS) vitrified embryos after delipidation and noncryopreserved embryos without delipidation. There were no significant differences in cryosurvival between embryos frozen in 0.25 mL straws and OPS vitrified embryos across all the stages (two cell to morula) examined (p>0.05). Similarly, in all groups examined, the blastocyst rates were not different between the two cryopreserved groups. However, the blastocyst rates from the cryopreserved groups were significantly lower than the noncryopreserved controls (p<0.05). This experiment demonstrated that early-stage porcine embryos can survive cryopreservation in a closed system by using a slow cooling method at a comparable rate to those vitrified by using an ultrarapid cooling method (p>0.05). However, the developmental competence was significantly reduced after cryopreservation compared to noncryopreserved embryos. Further research is needed to optimize the protocol to improve the developmental potential of cryopreserved early-stage porcine embryos in sealed straws. PMID:26309801

  11. Application of hollow fiber vitrification for cryopreservation of bovine early cleavage stage embryos and porcine morula-blastomeres

    PubMed Central

    UCHIKURA, Ayuko; MATSUNARI, Hitomi; NAKANO, Kazuaki; HATAE, Shota; NAGASHIMA, Hiroshi

    2016-01-01

    A novel hollow fiber vitrification (HFV) method was applied to materials that have previously been difficult to cryopreserve, thereby expanding the potential application of this method. The results showed that zona-free porcine morulae and their isolated blastomeres remained viable even after vitrification. The rate of development to blastocysts after vitrification was similar for zona-free and zona-intact morulae (21/23, 91.3% for both). Vitrified blastomeres had a developmental potential equal to that of non-vitrified blastomeres (blastocyst formation rate after reaggregation: 16/17, 94.1% for both). The HFV method was also effective for the cryopreservation of in vitro matured/fertilized bovine embryos at the 2- to 4-cell, 8- to 16-cell and morula stages. The blastocyst formation rates of vitrified embryos (66.1–82.5%) were similar to those of non-vitrified embryos (74.5–82.5%). These results indicate that this novel HFV method is an effective tool for embryo cryopreservation that can enhance current practices in reproductive biology. PMID:26875691

  12. Live imaging reveals spatial separation of parental chromatin until the four-cell stage in Caenorhabditis elegans embryos.

    PubMed

    Bolková, Jitka; Lanctôt, Christian

    2016-01-01

    The parental genomes are initially spatially separated in each pronucleus after fertilization. Here we have used green-to-red photoconversion of Dendra2-H2B-labeled pronuclei to distinguish maternal and paternal chromatin domains and to track their spatial distribution in living Caenorhabditis elegans embryos starting shortly after fertilization. Intermingling of the parental chromatin did not occur until after the division of the AB and P1 blastomeres, at the 4-cell stage. Unexpectedly, we observed that the intermingling of chromatin did not take place during mitosis or during chromatin decondensation, but rather ∼ 3-5 minutes into the cell cycle. Furthermore, unlike what has been observed in mammalian cells, the relative spatial positioning of chromatin domains remained largely unchanged during prometaphase in the early C. elegans embryo. Live imaging of photoconverted chromatin also allowed us to detect a reproducible 180° rotation of the nuclei during cytokinesis of the one-cell embryo. Imaging of fluorescently-labeled P granules and polar bodies showed that the entire embryo rotates during the first cell division. To our knowledge, we report here the first live observation of the initial separation and subsequent mixing of parental chromatin domains during embryogenesis. PMID:26934289

  13. New observations regarding staging turkey embryos from oviposition through primitive streak formation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The normal developmental sequence of the turkey embryo from the initial cleavage divisions through hypoblast formation has been described previously in eleven separate stages based on the progressive morphological differentiation of the embryo (Gupta and Bakst, 1993). However, in recent preliminar...

  14. Confocal laser scanning microscopy of apoptosis in organogenesis-stage mouse embryos

    EPA Science Inventory

    Confocal laser scanning microscopy combined with a vital stain has been used to study apoptosis in organogenesis-stage mouse embryos. In order to achieve optical sectioning through embryos, it was necessary to use low power objectives and to prepare the sample appropriately. Mous...

  15. The expression and localization of Crb3 in developmental stages of the mice embryos and in different organs of 1-week-old female mice.

    PubMed

    Yin, Y; Sheng, J; Hu, R; Yang, Y; Qing, S

    2014-10-01

    Crumbs homolog 3 (Crb3) is a member of the Crumbs family of proteins. This protein may play a role in epithelial cell polarity and is associated with tight junctions at the apical surface of epithelial cells. Alternative transcriptional splice variants that encode different Crb3 isoforms have been characterized. The expression of Crb3 mRNA and protein was observed in the pre-implantation mouse embryos and different organs of 1-week-old mouse, and Crb3 expression was primarily observed in the cytoplasm. Crb3 was expressed in a unique temporal pattern in pre-implantation embryos. The main characteristic of Crb3 expression was that the positive signals were stronger in the mature oocytes and zygotes than in the 2-cell, 4-cell, and 8-cell stages and the morula, but a similar level of high expression was observed in blastocysts. Therefore, the Crb3 expression signal during the course of development process grew gradually stronger from the 2-cell stage to blastocyst. In addition, Crb3 protein was widely distributed in each stage of the post-implantation embryos. Crb3 expression was observed in the inner cell mass, trophoblast cells and endoderm of E4.5d embryos; in the chorion, amnion, trophoblast cells, yolk sac endoderm and embryo ectoderm of E7.5d embryos; in the amnion and limb bud of E8.0d embryos; and in the semicircular canal epithelium, retina, lens vesicle and liver tissue of E13.5d embryos. Crb3 was expressed at different levels in different organs of 1-week-old mouse with the strengths in the following order: kidney > small intestine > stomach > uterus > liver > skeletal muscle > cerebellum > brain. The presence of Crb3 in many organs and the regularity of Crb3 distribution in the process of mouse embryonic development indicate that Crb3 protein plays an important role in establishing and maintaining the polarity of mouse embryos. PMID:25131306

  16. 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.

  17. 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.

  18. Pentachlorophenol exposure causes Warburg-like effects in zebrafish embryos at gastrulation stage

    SciTech Connect

    Xu, Ting; Zhao, Jing; Hu, Ping; Dong, Zhangji; Li, Jingyun; Zhang, Hongchang; Yin, Daqiang; Zhao, Qingshun

    2014-06-01

    Pentachlorophenol (PCP) is a prevalent pollutant in the environment and has been demonstrated to be a serious toxicant to humans and animals. However, little is known regarding the molecular mechanism underlying its toxic effects on vertebrate early development. To explore the impacts and underlying mechanisms of PCP on early development, zebrafish (Danio rerio) embryos were exposed to PCP at concentrations of 0, 20 and 50 μg/L, and microscopic observation and cDNA microarray analysis were subsequently conducted at gastrulation stage. The morphological observations revealed that PCP caused a developmental delay of zebrafish embryos in a concentration-dependent manner. Transcriptomic data showed that 50 μg/L PCP treatment resulted in significant changes in gene expression level, and the genes involved in energy metabolism and cell behavior were identified based on gene functional enrichment analysis. The energy production of embryos was influenced by PCP via the activation of glycolysis along with the inhibition of oxidative phosphorylation (OXPHOS). The results suggested that PCP acts as an inhibitor of OXPHOS at 8 hpf (hours postfertilization). Consistent with the activated glycolysis, the cell cycle activity of PCP-treated embryos was higher than the controls. These characteristics are similar to the Warburg effect, which occurs in human tumors. The microinjection of exogenous ATP confirmed that an additional energy supply could rescue PCP-treated embryos from the developmental delay due to the energy deficit. Taken together, our results demonstrated that PCP causes a Warburg-like effect on zebrafish embryos during gastrulation, and the affected embryos had the phenotype of developmental delay. - Highlights: • We treat zebrafish embryos with PCP at gastrula stage. • PCP acts as an oxidative phosphorylation inhibitor, not an uncoupler, in gastrulation. • Exogenous ATP injection will rescue the development of effected embryos. • The transcriptome of PCP

  19. A study on cryoprotectant solution suitable for vitrification of rat two-cell stage embryos.

    PubMed

    Eto, Tomoo; Takahashi, Riichi; Kamisako, Tsutomu; Hioki, Kyoji; Sotomaru, Yusuke

    2014-02-01

    The present study was performed to develop a suitable cryoprotectant solution for cryopreservation of rat two-cell stage embryos. First, we examined the cell permeability of several cryoprotectants; propylene glycol had the fastest permeability compared to dimethyl sulfoxide, ethylene glycol, and glycerol. Embryos were then exposed to a solution containing propylene glycol to evaluate its effects on fetal development. As the development was similar to that of fresh embryos, P10 (10% v/v propylene glycol in PB1) was used as a pretreatment solution. Next, the effects of the vitrification solution components (sucrose, propylene glycol, ethylene glycol, and Percoll) were examined by observing the vitrification status; 10% v/v propylene glycol, 30% v/v ethylene glycol, 0.3 mol sucrose, and 20% v/v Percoll in PB1 (PEPeS) was the minimum essential concentration for effective vitrification without the formation of ice crystals or freeze fractures. A new vitrification method using P10 and PEPeS was tested using rat embryos. The survival rate of vitrified embryos after exposure to P10 for 120, 300, or 600 s ranged from 95.9% to 98.3%. The fetal developmental rate ranged from 57.7% to 65.2%, which was not significantly different from that of fresh embryos. The experimental results indicated that vitrification using a combination of P10 and PEPeS was suitable for cryopreservation of rat early stage embryos. PMID:24462541

  20. Intrafallopian transfer of gametes and early stage embryos for in vivo culture in cattle.

    PubMed

    Wetscher, F; Havlicek, V; Huber, T; Gilles, M; Tesfaye, D; Griese, J; Wimmers, K; Schellander, K; Müller, M; Brem, G; Besenfelder, U

    2005-07-01

    It may be possible to avoid inadequate in vitro culture conditions by incubating gametes or embryos in the oviducts for a short time. Ideally, an optimized procedure should be devised, combining in vitro and in vivo systems, in order to achieve synchronization in cattle. We transferred gametes as well as embryos in various stages of development and placed them into the oviducts. Embryos were recovered on Day 7 by flushing of oviducts and uterine horns. Blastocyst rates were determined on Day 7 and on Day 8. Experimental designs included transfer of in vitro matured cumulus oocyte complexes into previously inseminated heifers (COCs group), transfer of in vitro matured COCs simultaneously with capacitated spermatozoa (GIFTs group), transfer of four to eight cell stage embryos developed in vitro after IVM/IVF (Cleaved Stages group) and a group of solely in vitro produced embryos (IVP control group). Our results indicate that in vivo culture of IVM/IVF embryos in the homologous bovine oviduct has a positive influence on subsequent pre-implantation development. In addition, we have evidence that in vitro maturation and in vivo fertilization cannot be synchronized. PMID:15935840

  1. 4D atlas of the mouse embryo for precise morphological staging.

    PubMed

    Wong, Michael D; van Eede, Matthijs C; Spring, Shoshana; Jevtic, Stefan; Boughner, Julia C; Lerch, Jason P; Henkelman, R Mark

    2015-10-15

    After more than a century of research, the mouse remains the gold-standard model system, for it recapitulates human development and disease and is quickly and highly tractable to genetic manipulations. Fundamental to the power and success of using a mouse model is the ability to stage embryonic mouse development accurately. Past staging systems were limited by the technologies of the day, such that only surface features, visible with a light microscope, could be recognized and used to define stages. With the advent of high-throughput 3D imaging tools that capture embryo morphology in microscopic detail, we now present the first 4D atlas staging system for mouse embryonic development using optical projection tomography and image registration methods. By tracking 3D trajectories of every anatomical point in the mouse embryo from E11.5 to E14.0, we established the first 4D atlas compiled from ex vivo 3D mouse embryo reference images. The resulting 4D atlas comprises 51 interpolated 3D images in this gestational range, resulting in a temporal resolution of 72 min. From this 4D atlas, any mouse embryo image can be subsequently compared and staged at the global, voxel and/or structural level. Assigning an embryonic stage to each point in anatomy allows for unprecedented quantitative analysis of developmental asynchrony among different anatomical structures in the same mouse embryo. This comprehensive developmental data set offers developmental biologists a new, powerful staging system that can identify and compare differences in developmental timing in wild-type embryos and shows promise for localizing deviations in mutant development.

  2. Effects of cigarette smoke exposure on early stage embryos in the rat

    SciTech Connect

    Tachi, Norihide; Aoyama, Mitsuko )

    1989-09-01

    It is well recognized that cigarette smoking in pregnant women exerts many deleterious effects on their progenies; intrauterine growth retardation, and increases in perinatal mortality and premature births. The fetal growth retardation also has been reported in animals exposed to cigarette smoke. The authors previously demonstrated that cigarette smoke exposure in pregnant rats retarded the growth of fetuses from mid to late stages of pregnancy. In addition, the weight of uteri containing embryos in animals inhaling the smoke was smaller, although not significant, than that in the control on day 7 of pregnancy. Based on these findings, it was suggested that the growth of embryos in early stage seemed to be harmfully affected as well as during mid and late stages of pregnancy. However, since the uterine weight in early pregnancy was measured in the previous study instead of the direct observation of early stage embryos, it remained unclear whether the early development of embryos was really influenced by cigarette smoke exposure or not. The present study was designed to observe the effects of cigarette smoke inhalation by pregnant rats on early development of embryos from fertilization to implantation.

  3. Transcriptome profiles of embryos before and after cleavage in Eriocheir sinensis: identification of developmental genes at the earliest stages

    NASA Astrophysics Data System (ADS)

    Hui, Min; Cui, Zhaoxia; Liu, Yuan; Song, Chengwen

    2016-09-01

    In crab, embryogenesis is a complicated developmental program marked by a series of critical events. RNA-Sequencing technology offers developmental biologists a way to identify many more developmental genes than ever before. Here, we present a comprehensive analysis of the transcriptomes of Eriocheir sinensis oosperms (Os) and embryos at the 2-4 cell stage (Cs), which are separated by a cleavage event. A total of 18 923 unigenes were identified, and 403 genes matched with gene ontology (GO) terms related to developmental processes. In total, 432 differentially expressed genes (DEGs) were detected between the two stages. Nine DEGs were specifically expressed at only one stage. These DEGs may be relevant to stage-specific molecular events during development. A number of DEGs related to `hedgehog signaling pathway', `wnt signaling pathway' `germplasm', `nervous system', `sensory perception' and `segment polarity' were identified as being up-regulated at the Cs stage. The results suggest that these embryonic developmental events begin before the early cleavage event in crabs, and that many of the genes expressed in the two transcriptomes might be maternal genes. Our study provides ample information for further research on the molecular mechanisms underlying crab development.

  4. Measurement of in vivo stress resultants in neurulation-stage amphibian embryos.

    PubMed

    Benko, Richard; Brodland, G Wayne

    2007-04-01

    In order to obtain the first quantitative measurements of the in vivo stresses in early-stage amphibian embryos, we developed a novel instrument that uses a pair of parallel wires that are glued to the surface of an embryo normal to the direction in which the stress is to be determined. When a slit is made parallel to the wires and between them, tension in the surrounding tissue causes the slit to open. Under computer control, one of the wires is moved so as to restore the original wire spacing, and the steady-state closure force is determined from the degree of wire flexure. A cell-level finite element model is used to convert the wire bending force to an in-plane stress since the wire force is not proportional to the slit length. The device was used to measure stress resultants (force carried per unit of slit length) on the dorsal, ventral and lateral aspects of neurulation-stage axolotl (Ambystoma mexicanum) embryos. The resultants were anisotropic and varied with location and developmental stage, with values ranging from -0.17 mN/m to 1.92 mN/m. In general, the resultants could be decomposed into patterns associated with internal pressure in the embryo, bending of the embryo along its mid-sagittal plane and neural tube closure. The patterns of stress revealed by the experiments support a number of current theories about the mechanics of neurulation. PMID:17237990

  5. Stage-dependent toxicity of bisphenol a on Rhinella arenarum (anura, bufonidae) embryos and larvae.

    PubMed

    Wolkowicz, Ianina R Hutler; Herkovits, Jorge; Pérez Coll, Cristina S

    2014-02-01

    The acute and chronic toxicity of bisphenol A (BPA) was evaluated on the common South American toad Rhinella arenarum embryos and larvae by means of continuous and pulse exposure treatments. Embryos were treated continuously from early blastula (S.4) up to complete operculum (S.25), during early larval stages and by means of 24 h pulse exposures of BPA in concentrations ranging between 1.25 and 40 mg L(-1) , in order to evaluate the susceptibility to this compound in different developmental stages. For lethal effects, S.25 was the most sensitive and gastrula was the most resistant to BPA. The Teratogenic Index for neurula, the most sensitive embryonic stage for sublethal effects was 4.7. The main morphological alterations during early stages were: delayed or arrested development, reduced body size, persistent yolk plug, microcephaly, axial/tail flexures, edemas, blisters, waving fin, underdeveloped gills, mouth malformations, and cellular dissociation. BPA caused a remarkable narcotic effect from gill circulation stage (S.20) onwards in all the organisms exposed after 3 h of treatment with 10 mg L(-1) BPA. After recovering, the embryos exhibited scarce response to stimuli, erratic or circular swimming, and spasmodic contractions from 5 mg L(-1) onwards. Our results highlight the lethal and sublethal effectsof BPA on R. arenarum embryos and larvae, in the last case both at structural and functional levels.

  6. Effects of amino acids on development in vitro of cleavage-stage bovine embryos into blastocysts.

    PubMed

    Pinyopummintr, T; Bavister, B D

    1996-01-01

    Effects of amino acids on early bovine embryo development in vitro were examined using a chemically-defined, protein-free culture medium. Bovine embryos produced in vitro were cultured from 18 h to 72 h post insemination in a simple medium containing lactate as the only energy source except for the amino acid treatments. Subsequently, embryos were transferred to TCM-199 supplemented with serum for blastocyst development to substantiate their developmental competence. Treatments were: (1) non-essential amino acids from TCM-199 (NEA); (2) essential amino acids from TCM-199 (EA); (3) NEA+EA; (4) Eagle's minimum essential medium amino acids (MEM AA); (5) 11 amino acids present in HECM-6 (11 AA); and (6) 0.2 mM glutamine (GLN). A higher proportion of embryos (percentage of inseminated ova) cleaved to the > or = 8-cell stage by 72 h post insemination in NEA (56.7%), EA (41.2%), 11 AA (40.3%) and GLN (51.1%) than in either NEA+EA (30.0%) or MEM AA (33.1%). However, after transfer to complex medium, embryos that had developed in EA, as well as those in MEM AA or NEA+EA, produced significantly fewer blastocysts (37.1%, 34.4% and 25.6% respectively) than those in NEA (56.7%), GLN (48.9%) or 11 AA (37.7%). The ability of blastocysts to hatch from their zonae pellucidae was also affected by amino acid treatment during cleavage stages. The present study indicated that the addition of NEA or GLN or 11 AA to a chemically-defined culture medium during the cleavage phase of bovine embryo development increases their subsequent ability to reach the blastocyst stage. These data have implications for understanding the nutritional needs of bovine embryos produced in vitro and for optimizing the composition of culture media to support their development.

  7. Fate mapping of gallbladder progenitors in posteroventral foregut endoderm of mouse early somite-stage embryos.

    PubMed

    Uemura, Mami; Igarashi, Hitomi; Ozawa, Aisa; Tsunekawa, Naoki; Kurohmaru, Masamichi; Kanai-Azuma, Masami; Kanai, Yoshiakira

    2015-05-01

    In early embryogenesis, the posteroventral foregut endoderm gives rise to the budding endodermal organs including the liver, ventral pancreas and gallbladder during early somitogenesis. Despite the detailed fate maps of the liver and pancreatic progenitors in the mouse foregut endoderm, the exact location of the gallbladder progenitors remains unclear. In this study, we performed a DiI fate-mapping analysis using whole-embryo cultures of mouse early somite-stage embryos. Here, we show that the majority of gallbladder progenitors in 9-11-somite-stage embryos are located in the lateral-most domain of the foregut endoderm at the first intersomite junction level along the anteroposterior axis. This definition of their location highlights a novel entry point to understanding of the molecular mechanisms of initial specification of the gallbladder.

  8. (14)C METHANOL INCORPORATION INTO DNA AND SPECIFIC PROTEINS OF ORGANOGENESIS STAGE MOUSE EMBRYOS IN VITRO

    EPA Science Inventory

    Methanol (MeOH), a widely used industrial solvent and alternative motor fuel, has been shown to be mutagenic and teratogenic. We have demonstrated that methanol is teratogenic in mice in vivo and causes dysmorphogenesis in cultured organogenesis stage mouse embryos. Methanol is ...

  9. INCREASED APOPTOSIS IN ORGANOGENESIS-STAGED MOUSE EMBRYOS INDUCED BY DISINFECTION BY-PRODUCTS

    EPA Science Inventory

    Increased apoptosis in organogenesis-staged mouse embryos induced by disinfection by-products. Sid Hunter1,2, Ellen Rogers1 and Keith Ward2, 1 Developmental Biology Branch, Reproductive Toxicology Division, NHEERL, US EPA, RTP, NC; 2 Curriculum in Toxicology, UNC Chapel Hill, Cha...

  10. A small set of extra-embryonic genes defines a new landmark for bovine embryo staging.

    PubMed

    Degrelle, Séverine A; Lê Cao, Kim-Anh; Heyman, Yvan; Everts, Robin E; Campion, Evelyne; Richard, Christophe; Ducroix-Crépy, Céline; Tian, X Cindy; Lewin, Harris A; Renard, Jean-Paul; Robert-Granié, Christèle; Hue, Isabelle

    2011-01-01

    Axis specification in mouse is determined by a sequence of reciprocal interactions between embryonic and extra-embryonic tissues so that a few extra-embryonic genes appear as 'patterning' the embryo. Considering these interactions as essential, but lacking in most mammals the genetically driven approaches used in mouse and the corresponding patterning mutants, we examined whether a molecular signature originating from extra-embryonic tissues could relate to the developmental stage of the embryo proper and predict it. To this end, we have profiled bovine extra-embryonic tissues at peri-implantation stages, when gastrulation and early neurulation occur, and analysed the subsequent expression profiles through the use of predictive methods as previously reported for tumour classification. A set of six genes (CALM1, CPA3, CITED1, DLD, HNRNPDL, and TGFB3), half of which had not been previously associated with any extra-embryonic feature, appeared significantly discriminative and mainly dependent on embryonic tissues for its faithful expression. The predictive value of this set of genes for gastrulation and early neurulation stages, as assessed on naive samples, was remarkably high (93%). In silico connected to the bovine orthologues of the mouse patterning genes, this gene set is proposed as a new trait for embryo staging. As such, this will allow saving the bovine embryo proper for molecular or cellular studies. To us, it offers as well new perspectives for developmental phenotyping and modelling of embryonic/extra-embryonic co-differentiation.

  11. Blastomere Removal from Cleavage-Stage Mouse Embryos Alters Steroid Metabolism During Pregnancy1

    PubMed Central

    Sugawara, Atsushi; Sato, Brittany; Bal, Elise; Collier, Abby C.; Ward, Monika A.

    2012-01-01

    ABSTRACT Preimplantation genetic diagnosis (PGD) is a genetic screening of embryos conceived with assisted reproduction technologies (ART). A single blastomere from an early-stage embryo is removed and molecular analyses follow to identify embryos carrying genetic defects. PGD is considered highly successful for detecting genetic anomalies, but the effects of blastomere biopsy on fetal development are understudied. We aimed to determine whether single blastomere removal affects steroid homeostasis in the maternal-placental-fetal unit during mouse pregnancy. Embryos generated by in vitro fertilization (IVF) were biopsied at the four-cell stage, cultured to morula/early blastocyst, and transplanted into the oviducts of surrogate mothers. Nonbiopsied embryos from the same IVF cohorts served as controls. Cesarean section was performed at term, and maternal and fetal tissues were collected. Embryo biopsy affected the levels of steroids (estradiol, estrone, and progesterone) in fetal and placental compartments but not in maternal tissues. Steroidogenic enzyme activities (3beta-hydroxysteroid dehydrogenase, cytochrome P450 17alpha-hydroxylase, and cytochrome P450 19) were unaffected but decreased activities of steroid clearance enzymes (uridine diphosphate-glucuronosyltransferase and sulfotransferase) were observed in placentas and fetal livers. Although maternal body, ovarian, and placental weights did not differ, the weights of fetuses derived from biopsied embryos were lower than those of their nonbiopsied counterparts. The data demonstrate that blastomere biopsy deregulates steroid metabolism during pregnancy. This may have profound effects on several aspects of fetal development, of which low birth weight is only one. If a similar phenomenon occurs in humans, it may explain low birth weights associated with PGD/ART and provide a plausible target for improving PGD outcomes. PMID:22517623

  12. Dual modality optical coherence and whole-body photoacoustic tomography imaging of chick embryos in multiple development stages

    PubMed Central

    Liu, Mengyang; Maurer, Barbara; Hermann, Boris; Zabihian, Behrooz; Sandrian, Michelle G.; Unterhuber, Angelika; Baumann, Bernhard; Zhang, Edward Z.; Beard, Paul C.; Weninger, Wolfgang J.; Drexler, Wolfgang

    2014-01-01

    Chick embryos are an important animal model for biomedical studies. The visualization of chick embryos, however, is limited mostly to postmortem sectional imaging methods. In this work, we present a dual modality optical imaging system that combines swept-source optical coherence tomography and whole-body photoacoustic tomography, and apply it to image chick embryos at three different development stages. The explanted chick embryos were imaged in toto with complementary contrast from both optical scattering and optical absorption. The results serve as a prelude to the use of the dual modality system in longitudinal whole-body monitoring of chick embryos in ovo. PMID:25401028

  13. Peculiarities [correction of Pfculiarities] of lipid accumulation in Brassica rapa L. embryos on different stages development under altered gravity.

    PubMed

    Popova, Antonina; Kononko, Anna; Ivanenko, Galina

    2004-07-01

    Accumulation of lipid inclusions in Brassica rapa embryos generated under slow horizontal clinorotation and in the laboratory control were analyzed by histochemical methods. The research of lipid accumulation was carried out on consecutive stages of the embryo development, from the moment of two-cellular proembryo formation up to the stages of their full differentiation (21-22-day-old embryos). Accumulation of lipid drops was revealed for the first time at early stages of embryogenesis in this species, beginning from 3-day-old embryos (ball-like stage of embryo development) under clinorotation and in the laboratory control. The quantity of lipid inclusion was estimated by morphometrical analysis. Statistically significant differences between the clinorotation and laboratory control variants in quantity of lipid drops per cell were revealed from 6-day-old embryos (heart-shaped stage). Especially pronounced differences were noted in differentiated embryos (beginning from 12-day-old embryos) under horizontal clinorotation in comparison with the laboratory control. The registered differences testify about influence of altered gravity conditions on lipid accumulation in Brassica rapa embryos.

  14. Pentachlorophenol exposure causes Warburg-like effects in zebrafish embryos at gastrulation stage.

    PubMed

    Xu, Ting; Zhao, Jing; Hu, Ping; Dong, Zhangji; Li, Jingyun; Zhang, Hongchang; Yin, Daqiang; Zhao, Qingshun

    2014-06-01

    Pentachlorophenol (PCP) is a prevalent pollutant in the environment and has been demonstrated to be a serious toxicant to humans and animals. However, little is known regarding the molecular mechanism underlying its toxic effects on vertebrate early development. To explore the impacts and underlying mechanisms of PCP on early development, zebrafish (Danio rerio) embryos were exposed to PCP at concentrations of 0, 20 and 50 μg/L, and microscopic observation and cDNA microarray analysis were subsequently conducted at gastrulation stage. The morphological observations revealed that PCP caused a developmental delay of zebrafish embryos in a concentration-dependent manner. Transcriptomic data showed that 50 μg/L PCP treatment resulted in significant changes in gene expression level, and the genes involved in energy metabolism and cell behavior were identified based on gene functional enrichment analysis. The energy production of embryos was influenced by PCP via the activation of glycolysis along with the inhibition of oxidative phosphorylation (OXPHOS). The results suggested that PCP acts as an inhibitor of OXPHOS at 8 hpf (hours postfertilization). Consistent with the activated glycolysis, the cell cycle activity of PCP-treated embryos was higher than the controls. These characteristics are similar to the Warburg effect, which occurs in human tumors. The microinjection of exogenous ATP confirmed that an additional energy supply could rescue PCP-treated embryos from the developmental delay due to the energy deficit. Taken together, our results demonstrated that PCP causes a Warburg-like effect on zebrafish embryos during gastrulation, and the affected embryos had the phenotype of developmental delay. PMID:24642059

  15. Developmental arrest at early stages of Chinese hamster embryos homozygous for chromosomal rearrangements

    SciTech Connect

    Sonta, S.; Yamada, M.; Iida, T.; Ohashi, H. )

    1991-03-01

    Forty-three Chinese hamster stocks with autosomal rearrangements produced by X-irradiation were used. These rearrangements, 38 reciprocal translocations and 5 inversions, were chromosomally balanced. Heterozygotes for these rearrangements were all fertile and morphologically normal in both sexes except for one line with growth retardation. By crossing male and female heterozygotes for the same rearrangements, homozygotes were obtained in 37 lines. In the remaining 6 lines (5 with reciprocal translocations and 1 with an inversion), no homozygotes were viable. These 6 lines revealed arrested development of homozygous embryos at the two-cell stage, around the eight-cell stage, and after implantation, respectively. The bands of the breakpoints of rearrangements associated with lethality of homozygous embryos were different for each rearrangement. These results suggest that abnormal expression including embryonic lethality in homozygotes may be due to an influence of genes at the breakpoints.

  16. A comparative study on expression profile of developmentally important genes during pre-implantation stages in buffalo hand-made cloned embryos derived from adult fibroblasts and amniotic fluid derived stem cells.

    PubMed

    Em, Sadeesh; Shah, Fozia; Kataria, Meena; Yadav, P S

    2016-08-01

    Abnormal gene expression in somatic cell nuclear transfer embryos due to aberrant epigenetic modifications of the donor nucleus may account for much of the observed diminished viability and developmental abnormalities. The present study compared the developmentally important gene expression pattern at 4-cell, 8- to 16-cell, morula, and blastocyst stages of buffalo nuclear transfer (NT) embryos from adult fibroblasts (AFs) and amniotic fluid stem cells (AFSCs). In vitro fertilized embryos were used as control embryos. Alterations in the expression pattern of genes implicated in transcription and pluripotency (OCT4, STAT3, NANOG), DNA methylation (DNMT1, DNMT3A), histone deacetylation (HDAC2), growth factor signaling, and imprinting (IGF2, IGF2R), apoptosis (BAX, BCL2), oxidative stress (MnSOD), metabolism (GLUT1) regulation were observed in cloned embryos. The expression of transcripts in AFSC-NT embryos more closely followed that of the in vitro fertilized embryos compared with AF-NT embryos. It is concluded that AFSCs with a relatively undifferentiated genome may serve as suitable donors which could be reprogrammed more efficiently to reactivate expression of early embryonic genes in buffalo NT.

  17. Molecular asymmetry in the 8-cell stage Xenopus tropicalis embryo described by single blastomere transcript sequencing

    PubMed Central

    De Domenico, Elena; Owens, Nick D.L.; Grant, Ian M.; Gomes-Faria, Rosa; Gilchrist, Michael J.

    2015-01-01

    Correct development of the vertebrate body plan requires the early definition of two asymmetric, perpendicular axes. The first axis is established during oocyte maturation, and the second is established by symmetry breaking shortly after fertilization. The physical processes generating the second asymmetric, or dorsal–ventral, axis are well understood, but the specific molecular determinants, presumed to be maternal gene products, are poorly characterized. Whilst enrichment of maternal mRNAs at the animal and vegetal poles in both the oocyte and the early embryo has been studied, little is known about the distribution of maternal mRNAs along either the dorsal–ventral or left–right axes during the early cleavage stages. Here we report an unbiased analysis of the distribution of maternal mRNA on all axes of the Xenopus tropicalis 8-cell stage embryo, based on sequencing of single blastomeres whose positions within the embryo are known. Analysis of pooled data from complete sets of blastomeres from four embryos has identified 908 mRNAs enriched in either the animal or vegetal blastomeres, of which 793 are not previously reported as enriched. In contrast, we find no evidence for asymmetric distribution along either the dorsal–ventral or left–right axes. We confirm that animal pole enrichment is on average distinctly lower than vegetal pole enrichment, and that considerable variation is found between reported enrichment levels in different studies. We use publicly available data to show that there is a significant association between genes with human disease annotation and enrichment at the animal pole. Mutations in the human ortholog of the most animally enriched novel gene, Slc35d1, are causative for Schneckenbecken dysplasia, and we show that a similar phenotype is produced by depletion of the orthologous protein in Xenopus embryos. PMID:26100918

  18. Karyomapping identifies second polar body DNA persisting to the blastocyst stage: implications for embryo biopsy.

    PubMed

    Ottolini, Christian S; Rogers, Shaun; Sage, Karen; Summers, Michael C; Capalbo, Antonio; Griffin, Darren K; Sarasa, Jonas; Wells, Dagan; Handyside, Alan H

    2015-12-01

    Blastocyst biopsy is now widely used for both preimplantation genetic screening (PGS) and preimplantation genetic diagnosis (PGD). Although this approach yields good results, variable embryo quality and rates of development remain a challenge. Here, a case is reported in which a blastocyst was biopsied for PGS by array comparative genomic hybridization on day 6 after insemination, having hatched completely. In addition to a small trophectoderm sample, excluded cell fragments from the subzonal space from this embryo were also sampled. Unexpectedly, the array comparative genomic hybridization results from the fragments and trophectoderm sample were non-concordant: 47,XX,+19 and 46,XY, respectively. DNA fingerprinting by short tandem repeat and amelogenin analysis confirmed the sex chromosome difference but seemed to show that the two samples were related but non-identical. Genome-wide single nucleotide polymorphism genotyping and karyomapping identified that the origin of the DNA amplified from the fragments was that of the second polar body corresponding to the oocyte from which the biopsied embryo developed. The fact that polar body DNA can persist to the blastocyst stage provides evidence that excluded cell fragments should not be used for diagnostic purposes and should be avoided when performing embryo biopsies as there is a risk of diagnostic errors.

  19. Development of the coronary arteries in staged human embryos (the Paris Embryological Collection revisited).

    PubMed

    Mandarim-de-Lacerda, C A

    1990-03-01

    Twenty seven human embryos from stages 15 to 23 (postsomitic period), belonging to the collection of the "UFR Biomédicale des Saints-Pères, Université René Descartes Paris V", were studied. Details of the aorticopulmonary cleavage were analysed specially aortic valve development and origin of the coronary artery. At stage 18 the aortic valve was clearly distinguished (cup-shaped) presenting semilunar valves and aortic sinus (Valsalvae); at this stage the left coronary artery was detected in 66.7 per cent of the cases as an endothelial epicardial invagination. At stage 19, the left and right coronary arteries were detected simultaneously in 100 per cent of the cases. At stage 20, the coronary arteries showed greater structural complexity with a coat of mesenchymal cells. These results agree with previous data from different embryological collections. These findings suggest that the left coronary artery has a tendency to develop earlier than the right. We found no evidence of the coronary origin from the aortic lumen. This work provides additional information about the embryological development of the heart, obtained from the analyses of a French collection of human embryos.

  20. The initial appearance of the cranial nerves and related neuronal migration in staged human embryos.

    PubMed

    Müller, Fabiola; O'Rahilly, Ronan

    2011-01-01

    The initial development of the cranial nerves was studied in 245 human embryos of stages 10-23 (4-8 postfertilizational weeks). Significant findings in the human embryo include the following. (1) Neuronal migration is a characteristic feature in the development of all the cranial nerves at stages 13-18, with the exception of the somatic efferent group. (2) The somatic efferent and the visceral efferent neurons are arranged respectively in ventrolateral and ventromedial columns (stages 13-17). (3) The ventrolateral column gives rise to somatic efferent nuclei; the neurons of the hypoglossal nerve develop rapidly and show a segmental organization as four roots that innervate three of the four occipital somites (stage 13); the abducent nucleus becomes displaced rostrally by a change in the rhombomeric pattern at stage 16. (4) The ventromedial column, originally continuous in rhombomeres 2-7, gives rise to visceral efferent and pharyngeal efferent nuclei. (5) All the 'true' cranial nerves (III-XII) are recognizable by stage 16. (6) In a primary migration the visceral efferent neurons proceed mediolaterally and accumulate dorsolaterally as nuclei (stages 13, 14); they differentiate into salivatory nuclei (stages 16, 17). (7) A secondary migration involves the pharyngeal efferent neurons (of nerves V and IX-XI), which also proceed mediolaterally and then form ventrolateral nuclei (stages 17, 18). (8) The facial complex shows a distinctive development in that its neural crest arises from the lateral wall of the neural folds/tube. Moreover, the migration of its pharyngeal efferent neurons is delayed, which may be related to the formation of the internal genu, and the motor nucleus begins to appear only at stage 23. (9) The sequence of appearance of afferent constituents is: cranial ganglia (stage 12), mesencephalic trigeminal nucleus (stage 15), vestibular nuclei (stages 18-22), and cochlear nuclei (stage 19). The unsatisfactory term special is avoided and the term

  1. Activin/Nodal signalling before implantation: setting the stage for embryo patterning

    PubMed Central

    Papanayotou, Costis; Collignon, Jérôme

    2014-01-01

    Activins and Nodal are members of the transforming growth factor beta (TGF-β) family of growth factors. Their Smad2/3-dependent signalling pathway is well known for its implication in the patterning of the embryo after implantation. Although this pathway is active early on at preimplantation stages, embryonic phenotypes for loss-of-function mutations of prominent components of the pathway are not detected before implantation. It is only fairly recently that an understanding of the role of the Activin/Nodal signalling pathway at these stages has started to emerge, notably from studies detailing how it controls the expression of target genes in embryonic stem cells. We review here what is currently known of the TGF-β-related ligands that determine the activity of Activin/Nodal signalling at preimplantation stages, and recent advances in the elucidation of the Smad2/3-dependent mechanisms underlying developmental progression. PMID:25349448

  2. Activin/Nodal signalling before implantation: setting the stage for embryo patterning.

    PubMed

    Papanayotou, Costis; Collignon, Jérôme

    2014-12-01

    Activins and Nodal are members of the transforming growth factor beta (TGF-β) family of growth factors. Their Smad2/3-dependent signalling pathway is well known for its implication in the patterning of the embryo after implantation. Although this pathway is active early on at preimplantation stages, embryonic phenotypes for loss-of-function mutations of prominent components of the pathway are not detected before implantation. It is only fairly recently that an understanding of the role of the Activin/Nodal signalling pathway at these stages has started to emerge, notably from studies detailing how it controls the expression of target genes in embryonic stem cells. We review here what is currently known of the TGF-β-related ligands that determine the activity of Activin/Nodal signalling at preimplantation stages, and recent advances in the elucidation of the Smad2/3-dependent mechanisms underlying developmental progression.

  3. Teratogenic effects of zinc on embryo-larval stages of the fathead minnow

    SciTech Connect

    Ramey, B.A.

    1988-10-01

    The effects of zinc on embryos and larvae of the fathead minnow were studied using standard 8-day embryo-larval bioassay techniques to determine if there was a period in embryonic development which would be the most sensitive. Virtually all larvae at all stages were abnormal at 1.0 mg Zn/L, displaying edema and curvature of the vertebral axis. Although LC/sub 50/ values for the five developmental stages ranged from 0.13 to 0.62 mg/L, there was not a significantly most sensitive stage. When larvae were exposed to 1.0 mg Zn/L only during the 96 hour after hatching, all animals were anomalous. If zinc exposure occurred only during the prehatch period and larvae were placed in control water posthatch, only 5% of the larvae were abnormal. At sublethal concentrations the chorion may impart a protective mechanism to the teratogenic effects of zinc, but this protection may be lost after hatching.

  4. Automated 3-D reconstruction of the surface of live early-stage amphibian embryos.

    PubMed

    Bootsma, Gregory J; Brodland, G Wayne

    2005-08-01

    Although three-dimensional (3-D) reconstructions of the surfaces of live embyos are vital to understanding embryo development, morphogenetic tissue movements and other factors have prevented the automation of this task. Here, we report an integrated set of software algorithms that overcome these challenges, making it possible to completely automate the reconstruction of embryo surfaces and other textured surfaces from multiview images. The process involves: 1) building accurate point correspondences using a robust deformable template block matching algorithm; 2) removing outliers using fundamental matrix calculations in conjunction with a RANSAC algorithm; 3) generating 3-D point clouds using a bundle adjustment algorithm that includes camera position and distortion corrections; 4) meshing the point clouds into triangulated surfaces using a Tight Cocone algorithm that produces water tight models; 5) refining surfaces using midpoint insertion and Laplacian smoothing algorithms; and 6) repeating these steps until a measure of convergence G, the rms difference between successive reconstructions, is below a specified threshold. Reconstructions were made of 2.2-mm diameter, neurulation-stage axolotl (amphibian) embryos using 44 multiview images collected with a robotic microscope. A typical final model (sixth iteration) contained 3787 points and 7562 triangles and had an error measure of G = 5.9 microm.

  5. Glutathione transferase isoenzymes from Bufo bufo embryos at an early developmental stage.

    PubMed Central

    Di Ilio, C; Aceto, A; Bucciarelli, T; Dragani, B; Angelucci, S; Miranda, M; Poma, A; Amicarelli, F; Barra, D; Federici, G

    1992-01-01

    Six forms of glutathione transferase (GST) were resolved from the cytosolic fraction of Bufo bufo embryos at developmental stage 4 by GSH-Sepharose affinity chromatography followed by f.p.l.c. chromatofocusing in the 9-6 pH range. They have apparent isoelectric points at pH 8.37 (GST I), 8.22 (GST II), 8.10 (GST III), 7.84 (GST IV), 7.37 (GST V) and 7.12 (GST VI), and each displayed an apparent subunit molecular mass of 23 kDa by SDS/PAGE. The Bufo bufo embryo enzymes showed very similar structural, catalytic and immunological properties, as indicated by their substrate-specificities, inhibition characteristics, c.d. spectra, h.p.l.c. elution profiles and immunological reactivities, as well as by their N-terminal amino acid sequences. Although Bufo bufo embryo GSTs do not correspond to any other known GSTs, the results of our experiments indicate that amphibian GSTs could be included in the Pi family of GSTs. This conclusion is supported by the analysis of c.d. spectra, and by the fact that mammalian Pi class GSTs and amphibian GSTs showed about 80% identity in their N-terminal amino acid sequences. Furthermore, antisera prepared against Bufo bufo GST III cross-reacted in immunoblotting analysis with Pi class GSTs, and vice versa. Images Fig. 2. Fig. 3. PMID:1567369

  6. The activation of DNA damage detection and repair responses in cleavage-stage rat embryos by a damaged paternal genome.

    PubMed

    Grenier, Lisanne; Robaire, Bernard; Hales, Barbara F

    2012-06-01

    Male germ cell DNA damage, after exposure to radiation, exogenous chemicals, or chemotherapeutic agents, is a major cause of male infertility. DNA-damaged spermatozoa can fertilize oocytes; this is of concern because there is limited information on the capacity of early embryos to repair a damaged male genome or on the fate of these embryos if repair is inadequate. We hypothesized that the early activation of DNA damage response in the early embryo is a critical determinant of its fate. The objective of this study was to assess the DNA damage response and mitochondrial function as a measure of the energy supply for DNA repair and general health in cleavage-stage embryos sired by males chronically exposed to an anticancer alkylating agent, cyclophosphamide. Male rats were treated with saline or cyclophosphamide (6 mg/kg/day) for 4 weeks and mated to naturally cycling females. Pronuclear two- and eight-cell embryos were collected for immunofluorescence analysis of mitochondrial function and biomarkers of the DNA damage response: γH2AX foci, 53BP1 reactivity, and poly(ADP-ribose) polymer formation. Mitochondrial activities did not differ between embryos sired by control- and cyclophosphamide-exposed males. At the two-cell stage, there was no treatment-related increase in DNA double-strand breaks; by the eight-cell stage, a significant increase was noted, as indicated by increased medium and large γH2AX foci. This was accompanied by a dampened DNA repair response, detected as a decrease in the nuclear intensity of poly(ADP-ribose) polymers. The micronuclei formed in cyclophosphamide-sired embryos contained large γH2AX foci and enhanced poly(ADP-ribose) polymer and 53BP1 reactivity compared with their nuclear counterparts. Thus, paternal cyclophosphamide exposure activated a DNA damage response in cleavage-stage embryos. Furthermore, this damage response may be useful in assessing embryo quality and developmental competence. PMID:22454429

  7. Stage-dependent ethoxyresorufin-O-deethylase (EROD) in vivo activity in medaka (Oryzias latipes) embryos.

    PubMed

    González-Doncel, Miguel; Carbonell, Gregoria; San Segundo, Laura; Sastre, Salvador; Beltrán, Eulalia M; Fernández-Torija, Carlos

    2015-09-01

    Using medaka (Oryzias latipes) embryos, this study aimed to quantitatively characterize the stage-dependent in vivo ethoxyresorufin-O-deethylase (EROD) as indicator of cytochrome P4501A (CYP1A) activity. Embryos were challenged for 24-h to an agonist (β-naphthoflavone [BNF], 2.5, 5, 10, and 20 μg L(-1)) or to its combination (2.5 μg L(-1)) with an antagonist (α-naphthoflavone [ANF], 25, 50, 100, and 200 μg L(-1)), initiated at four different developmental time points (1, 3, 6, and 9 d post-fertilization [dpf]). Respective induction and competitive inhibition were evaluated over fluorescent images of whole embryo (nonorgan-specific [NOS] EROD activity) and gallbladder (organ-specific [OS] EROD activity). Both flavonoids showed signs of stability in solution. Generally speaking, the mean fluorescence intensity (MFI) values for NOS EROD increased with BNF concentration and exposure challenge. BNF co-exposure with ⩾50 μg ANF L(-1) during the 1-2 and 3-4 dpf challenges lowered NOS EROD to undetectably induced levels. Significant increments in MFIs for OS-EROD were seen from exposures to ⩾2.5 μg BNF L(-1), peaking during the 6-7 dpf challenge regardless of BNF concentration. The simultaneous BNF/ANF incubation showed competitive inhibition for OS EROD activity, although levels were generally detectably induced during all challenges and at all ANF concentrations. The morphometric in vivo gallbladder analysis indicated significant dilation in the 10 dpf-old embryos co-exposed to BNF and 200 μg ANF L(-1). This quantitative approach can be used successfully at 4 dpf at the NOS-EROD or OS-EROD levels, although the NOS-EROD response was sensitive enough to induction or inhibition, even at 2 dpf.

  8. Open pulled straw vitrification of goat embryos at various stages of development.

    PubMed

    Al Yacoub, A N; Gauly, M; Holtz, W

    2010-05-01

    This investigation addresses the question whether it is possible to apply the open pulled straw (OPS) vitrification method, found to be effective for cryopreserving caprine (Capra aegagrus hircus) blastocysts, to other embryonal stages. Morulae, blastocysts and hatched blastocysts were cryopreserved by way of OPS vitrification and blastocysts and hatched blastocysts by conventional freezing. Morulae were not included with conventional freezing because in our experience the survival rate is very low. To assess the viability of the cryopreserved embryos, they were transferred to synchronized does; in most cases, two embryos per doe. After OPS vitrification, of nine does receiving morulae, not a single one became pregnant; of 11 does receiving blastocysts, nine (82%) became pregnant (all of which kidded and gave birth to, on average, 1.8 kids); and of nine does receiving hatched blastocysts, three (33%) became pregnant (two of which [22%] kidded, giving birth to a single kid each). After conventional freezing, of 10 does receiving blastocysts, five became pregnant (four of which [40%] carried to term and gave birth to a pair of twins each); and of nine does receiving hatched blastocysts, three (33%) became pregnant (and gave birth to a single kid each). Embryo survival (kids born/embryos transferred) after vitrification for morulae, blastocysts, and hatched blastocysts was 0, 70% (16 of 23), and 13% (2 of 16), respectively, and after conventional freezing for blastocysts and hatched blastocysts was 42% (8 of 19) and 19% (3 of 16), respectively. The difference in pregnancy and kidding rate between vitrified and conventionally frozen blastocysts was significant, and so was the difference in pregnancy rate between hatched and nonhatched blastocysts, regardless whether OPS-vitrified or conventionally frozen. The results of the current study indicate that OPS vitrification is a very effective means of cryopreserving caprine blastocysts. Unfortunately, the superiority of

  9. 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.

  10. Experimental model for determining developmental stage of chicken embryo using infrared images and artificial neural networks

    NASA Astrophysics Data System (ADS)

    Jung, Seung Kwon "Paul"; Hsieh, Sheng-Jen "Tony"; Chen, Che-Hao

    2013-05-01

    Development of a chicken embryo is conventionally assumed to follow a set growth pattern over the course of 21 days. However, despite identical incubation settings, many factors may contribute to an egg developing at a different rate from those around it. Being able to determine an embryo's actual development instead of relying on chronological assumptions of normal growth should prove to be a useful tool in the poultry industry for responding early to abnormal development and improving hatch rates. Previous studies have used infrared imaging to enhance candling observation, but relatively little has been done to implement infrared imaging in problem-solving. The purpose of this research is to construct a quantitative model for predicting the development stage and early viability of a chicken embryo during incubation. It may be noted that a similar project was conducted previously using different input parameters. This study seeks to improve upon the results from the earlier project. In this project, infrared images of eggs were processed to calculate air cell volumes and cooling rates, and daily measurements of egg weight and ambient temperature were compiled. Artificial neural networks (ANNs) were "trained" using multiple input parameters to recognize patterns in the data. Various training functions and topologies were evaluated in order to optimize prediction rates and consistency. The prediction rates obtained for the ANNs were around 81% for development stage and around 92% for viability. It is recommended for future research to expand the potential combinations of input parameters used in order to increase this model's versatility in the field.

  11. Expression of growth factor ligand and receptor genes in preimplantation stage water buffalo (Bubalus bubalis) embryos and oviduct epithelial cells.

    PubMed

    Daliri, M; Rao, K B; Kaur, G; Garg, S; Patil, S; Totey, S M

    1999-09-01

    The temporal pattern of expression of genes for several growth factor ligands and receptors was examined in preimplantation water buffalo embryos and oviduct epithelial cells using RT-PCR. The identity of the resulting PCR products was confirmed by their expected size, restriction analysis, Southern blot hybridization and nucleotide sequence analysis. Preimplantation stage embryos from the one-cell to the blastocyst stage were derived after maturation, fertilization and culture of oocytes in vitro. Expression of members of the insulin-like growth factor (IGF) family was observed predominantly in preimplantation stage embryos and oviduct epithelial cells. Similarly, transcripts encoding insulin and IGF-I receptors were detected at each stage of embryonic development. The mRNA transcript of the IGF-I receptor was not detected in oviduct epithelial cells, but a prominent band corresponding to the insulin receptor was observed. Insulin and IGF-II mRNA were expressed as maternal transcripts that were not detected at the two- to four-cell stage but were present as zygotic transcripts at the eight-cell stage. Transcripts encoding IGF-I were detected in oviduct epithelial cells, but were not observed in any of the preimplantation stage embryos. Transforming growth factor (TGF) alpha and beta and epidermal growth factor mRNA transcripts were not detected in any of the preimplantation stage embryos. These results indicate that IGF-I acts via a paracrine mechanism to promote growth and development of preimplantation water buffalo embryos. Similarly, IGF-II appears to act through a heterologous autocrine mechanism via the IGF-I or the insulin receptor. Furthermore, the presence of TGF-alpha in oviduct epithelial cells indicates that it may have a critical role during development.

  12. Transcript profiling of individual twin blastomeres derived by splitting two-cell stage murine embryos.

    PubMed

    Roberts, R Michael; Katayama, Mika; Magnuson, Scott R; Falduto, Michael T; Torres, Karen E O

    2011-03-01

    In invertebrates and amphibians, informational macromolecules in egg cytoplasm are organized to provide direction to the formation of embryonic lineages, but it is unclear whether vestiges of such prepatterning exist in mammals. Here we examined whether twin blastomeres from 2-cell stage mouse embryos differ in mRNA content. mRNA from 26 blastomeres derived from 13 embryos approximately mid-way through their second cell cycle was subjected to amplification. Twenty amplified samples were hybridized to arrays. Of those samples that hybridized successfully, 12 samples in six pairs were used in the final analysis. Probes displaying normalized values >0.25 (n = 4573) were examined for consistent bias in expression within blastomere pairs. Although transcript content varied between both individual embryos and twin blastomeres, no consistent asymmetries were observed for the majority of genes, with only 178 genes displaying a >1.4-fold difference in expression across all six pairs. Although class discovery clustering showed that blastomere pairs separated into two distinct groups in terms of their differentially expressed genes, when the data were tested for significance of asymmetrical expression, only 39 genes with >1.4-fold change ratios in six of six blastomere pairs passed the two-sample t-test (P < 0.05). Transcripts encoding proteins implicated in RNA processing and cytoskeletal organization were among the most abundant, differentially distributed mRNA, suggesting that a stochastically based lack of synchrony in cell cycle progression between the two cells might explain at least some and possibly all of the asymmetries in transcript composition.

  13. Chemical exposure of embryos during the preimplantation stages of pregnancy: mortality rate and intrauterine development.

    PubMed

    Fabro, S; McLachlan, J A; Dames, N M

    1984-04-01

    Exposure of CD-1 mouse embryos at the eight- to 16-cell stage for 1 hour to methylmethanesulfonate (MMS; 0.25, 0.5, and 1.0 mM) produced DNA breakage and interfered with embryonic development in a dose-related manner. MMS-exposed blastocysts were transferred to oviducts of untreated recipient female mice, and the conceptuses were allowed to develop to term. MMS exposure resulted in an increased intrauterine death rate, although the number of implantation sites was not decreased. Surviving MMS-treated offspring showed intrauterine growth retardation, but there was no increase in the incidence of gross abnormalities. Intrauterine growth retardation, without an increase in gross abnormalities, was also observed in the offspring of pregnant New Zealand White rabbits dosed during the preimplantation stages of pregnancy with an "environmental cocktail" composed of ethanol, nicotine, caffeine, sodium salicylate, and dichloro-diphenyl-trichloro-ethane (DDT). When the compounds were tested individually, nicotine and DDT were the only two that produced intrauterine growth retardation. DDT-treated 8-day rabbit conceptuses were smaller than controls and showed abnormal persistence of preimplantation proteins in the yolk sac fluid. These results suggest that exposure to chemicals during the preimplantation stages of pregnancy may result in a cessation of growth and development before implantation or during later intrauterine development. Damage can be repaired but it may result in offspring that show intrauterine growth retardation without gross abnormalities. PMID:6711631

  14. Cyclin D and cdk4 Are Required for Normal Development beyond the Blastula Stage in Sea Urchin Embryos

    PubMed Central

    Moore, Jennifer C.; Sumerel, Jan L.; Schnackenberg, Bradley J.; Nichols, Jason A.; Wikramanayake, Athula; Wessel, Gary M.; Marzluff, William F.

    2002-01-01

    cdk4 mRNA and protein are constitutively expressed in sea urchin eggs and throughout embryonic development. In contrast, cyclin D mRNA is barely detectable in eggs and early embryos, when the cell cycles consist of alternating S and M phases. Cyclin D mRNA increases dramatically in embryos at the early blastula stage and remains at a constant level throughout embryogenesis. An increase in cdk4 kinase activity occurs concomitantly with the increase in cyclin D mRNA. Ectopic expression of cyclin D mRNA in eggs arrests development before the 16-cell stage and causes eventual embryonic death, suggesting that activation of cyclin D/cdk4 in cleavage cell cycles is lethal to the embryo. In contrast, blocking cyclin D or cdk4 expression with morpholino antisense oligonucleotides results in normal development of early gastrula-stage embryos but abnormal, asymmetric larvae. These results suggest that in sea urchins, cyclin D and cdk4 are required for normal development and perhaps the patterning of the developing embryo, but may not be directly involved in regulating entry into the cell cycle. PMID:12052892

  15. Two-staged nuclear transfer can enhance the developmental ability of goat-sheep interspecies nuclear transfer embryos in vitro.

    PubMed

    Ma, Li-Bing; Cai, Lu; Li, Jia-Jia; Chen, Xiu-Li; Ji, Feng-Yu

    2011-02-01

    The technique of interspecies somatic cell nuclear transfer, in which interspecies cloned embryos can be reconstructed by using domestic animal oocytes as nuclear recipients and endangered animal or human somatic cells as nuclear donors, can afford more opportunities in endangered animal rescue and human tissue transplantation, but the application of this technique is limited by extremely low efficiency which may be attributed to donor nucleus not fully reprogrammed by xenogenic cytoplasm. In this study, goat fetal fibroblasts (GFFs) were used as nuclear donors, in vitro-matured sheep oocytes were used as nuclear recipients, and a two-stage nuclear transfer procedure was performed to improve the developmental ability of goat-sheep interspecies clone embryos. In the first stage nuclear transfer (FSNT), GFFs were injected into the ooplasm of enucleated sheep metaphase-II oocytes, then non-activated reconstructed embryos were cultured in vitro, so that the donor nucleus could be exposed to the ooplasm for a period of time. Subsequently, in the second stage nuclear transfer, FSNT-derived non-activated reconstructed embryo was centrifuged, and the donor nucleus was then transferred into another freshly enucleated sheep oocyte. Compared with the one-stage nuclear transfer, two-stage nuclear transfer could significantly enhance the blastocyst rate of goat-sheep interspecies clone embryos, and this result indicated that longtime exposure to xenogenic ooplasm benefits the donor nucleus to be reprogrammed. The two-stage nuclear transfer procedure has two advantages, one is that the donor nucleus can be exposed to the ooplasm for a long time, the other is that the problem of oocyte aging can be solved. PMID:21082282

  16. The Chromosomes of Turkey Embryos during Early Stages of Parthenogenetic Development

    PubMed Central

    Harada, Ko; Buss, Edward G.

    1981-01-01

    In the early stages of parthenogenetic development in turkey eggs, many blastoderms are mosaics of haploid, diploid and polyploid cells. The genome composition of these blastoderms can be identified by C-banding. They may be generally described as either A-Z/2A-ZZ/nA-nZ or A-W/2A-WW/nA-nW and are found in a nearly 1:1 ratio. The blastoderms showing the W body (W+) become lethal within two days of incubation. The haploid cell proportion decreases rapidly during the early stage of development, and, as haploid cells decrease, the proportion of polyploid cells appears to increase. At six days of incubation, various kinds of parthenogenetic development can be observed. Their genome compositions are either diploid (2A-ZZ) or mosaic (A-Z/2A-ZZ). These findings suggest that diploid parthenogenesis occurs by either suppression of meiosis II or chromosome doubling some time after the first cleavage division. The frequent occurrence of mosaic blastoderms indicates that the majority, if not all, of the parthenogenetic embryos initiate their development in haploid ova. PMID:7327389

  17. Analysis of cerebro-spinal fluid protein composition in early developmental stages in chick embryos.

    PubMed

    Gato, A; Martín, P; Alonso, M I; Martín, C; Pulgar, M A; Moro, J A

    2004-04-01

    Foetal cerebro-spinal fluid (CSF) has a very high protein concentration when compared to adult CSF, and in many species five major protein fractions have been described. However, the protein concentration and composition in CSF during early developmental stages remains largely unknown. Our results show that in the earliest stages (18 to 30 H.H.) of chick development there is a progressive increase in CSF protein concentration until foetal values are attained. In addition, by performing electrophoretic separation and high-sensitivity silver staining, we were able to identify a total of 21 different protein fractions in the chick embryo CSF. In accordance with the developmental pattern of their concentration, these can be classified as follows: A: high-concentration fractions which corresponded with the ones described in foetal CSF by other authors; B: low-concentration fractions which remained stable throughout the period studied; C: low-concentration fractions which show changes during this period. The evolution and molecular weight of the latter group suggest the possibility of an important biological role. Our data demonstrate that all the CSF protein fractions are present in embryonic serum; this could mean that the specific transport mechanisms in neuroepithelial cells described in the foetal period evolve in very early stages of development. In conclusion, this paper offers an accurate study of the protein composition of chick embryonic CSF, which will help the understanding of the influences on neuroepithelial stem cells during development and, as a result, the appropriate conditions for the in vitro study of embryonic/foetal nervous tissue cells. PMID:15039986

  18. Effects of Nitrite on Development of Embryos and Early Larval Stages of the Zebrafish (Danio rerio)

    PubMed Central

    Simmons, Alison E.; Karimi, Ida; Talwar, Mayank

    2012-01-01

    Abstract Epidemiological studies suggest that high nitrate levels in food and water may cause birth defects or spontaneous abortions in humans. Experimental mammalian studies show that high nitrite levels adversely affect reproductive outcomes, but have not shown congenital malformations. Consequently, the teratogenic potential of nitrite is unclear. In this study, the effects of nitrite on development of zebrafish embryos and early larval stages were investigated. Eggs were exposed to ethanol (a known teratogen), nitrite, or nitrate for 24 or 96 hours, and larvae examined at 120 hours. Sublethal exposure to 300 mM ethanol for 24 hours caused severe pericardial and yolk sac edema, craniofacial and axial malformations, and swim bladder noninflation. The 96 hour LC50 for nitrite was 411 mg/L. Less severe edema, craniofacial (but not axial) malformations, swim bladder noninflation, and immobility were observed after sublethal exposure to nitrite between 10 and 300 mg/L for 96 hours. Exposure to nitrite for 24 hours at concentrations as high as 2000 mg/L was not lethal. Only axial malformations and swim bladder noninflation were observed at 1500 mg/L. The results demonstrate that sublethal nitrite concentrations cause developmental defects. The type and magnitude of these defects differed after 24 and 96 hours of exposure. PMID:22823424

  19. Effects of cadmium-enriched sediment on fish and amphibian embryo-larval stages

    SciTech Connect

    Francis, P.C.; Birge, W.J.; Black, J.A.

    1984-08-01

    Aquatic toxicity tests were conducted to evaluate the effects of cadmium-enriched sediment on embryo-larval stages of the goldfish (Carassius auratus), leopard frog (Rana pipiens), and largemouth bass (Micropterus salmoides). Natural stream sediment was collected and enriched with cadmium to nominal concentrations of 1.0, 10.0, 100, and 1000 mg/kg. Enriched sediments were placed in Pyrex dishes and covered with 350 ml of reconstituted water. Fertilized eggs were placed in the dishes and maintained through 4 days posthatching, giving a total exposure time of 6 to 7 days. For all tests the cadmium concentrations ranged from 1.1 to 76.5 micrograms/liter in water above sediments containing 1 to 1000 mg Cd/kg, respectively. Although low frequencies of mortality were observed in all tests, goldfish, leopard frog, and bass exposed to sediments enriched to 1000 mg Cd/kg accumulated 4.61, 12.55, and 60.0 micrograms Cd/g, respectively. No significant correlations were found between mortality of the goldfish and leopard frog and the cadmium concentrations in either water or sediment. However, all three species showed strong correlations between cadmium concentrations in water and tissue, sediment and tissue, and water and sediment. Tissue cadmium concentrations were related to the length of time test organisms were in direct contact with cadmium-enriched sediment.

  20. Proteomic analysis of early-stage embryos: implications for egg quality in hapuku (Polyprion oxygeneios).

    PubMed

    Kohn, Yair Y; Symonds, Jane E; Kleffmann, Torsten; Nakagawa, Shinichi; Lagisz, Malgorzata; Lokman, P Mark

    2015-12-01

    In order to develop biomarkers that may help predict the egg quality of captive hapuku (Polyprion oxygeneios) and provide potential avenues for its manipulation, the present study (1) sequenced the proteome of early-stage embryos using isobaric tag for relative and absolute quantification analysis, and (2) aimed to establish the predictive value of the abundance of identified proteins with regard to egg quality through regression analysis. Egg quality was determined for eight different egg batches by blastomere symmetry scores. In total, 121 proteins were identified and assigned to one of nine major groups according to their function/pathway. A mixed-effects model analysis revealed a decrease in relative protein abundance that correlated with (decreasing) egg quality in one major group (heat-shock proteins). No differences were found in the other protein groups. Linear regression analysis, performed for each identified protein separately, revealed seven proteins that showed a significant decrease in relative abundance with reduced blastomere symmetry: two correlates that have been named in other studies (vitellogenin, heat-shock protein-70) and a further five new candidate proteins (78 kDa glucose-regulated protein, elongation factor-2, GTP-binding nuclear protein Ran, iduronate 2-sulfatase and 6-phosphogluconate dehydrogenase). Notwithstanding issues associated with multiple statistical testing, we conclude that these proteins, and especially iduronate 2-sulfatase and the generic heat-shock protein group, could serve as biomarkers of egg quality in hapuku.

  1. Stage-dependent teratogenic and lethal effects exerted by ultraviolet B radiation on Rhinella (Bufo) arenarum embryos.

    PubMed

    Castañaga, Luis A; Asorey, Cynthia M; Sandoval, María T; Pérez-Coll, Cristina S; Argibay, Teresa I; Herkovits, Jorge

    2009-02-01

    The adverse effects of ultraviolet B radiation from 547.2 to 30,096 J/m2 on morphogenesis, cell differentiation, and lethality of amphibian embryos at six developmental stages were evaluated from 24 up to 168 h postexposure. The ultraviolet B radiation lethal dose 10, 50, and 90 values were obtained for all developmental stages evaluated. The lethal dose 50 values, considered as the dose causing lethality in the 50% of the organisms exposed, in J/m2 at 168 h postexposure, ranged from 2,307 to 18,930; gill circulation and blastula were the most susceptible and resistant stages, respectively. Ultraviolet B radiation caused malformations in all developmental stages but was significantly more teratogenic at the gill circulation and complete operculum stages. Moreover, at the gill circulation stage, even the lowest dose (547.2 J/m2) resulted in malformations to 100% of embryos. The most common malformations were persistent yolk plug, bifid spine, reduced body size, delayed development, asymmetry, microcephaly and anencephaly, tail and body flexures toward the irradiated side, agenesia or partial gill development, abnormal pigment distribution, and hypermotility. The stage-dependent susceptibility to ultraviolet B radiation during amphibian embryogenesis could be explained in the framework of evoecotoxicology, considering ontogenic features as biomarkers of environmental signatures of living forms ancestors during the evolutionary process. The stage-dependent susceptibility to ultraviolet B radiation on Rhinella (Bufo) arenarum embryos for both lethal and teratogenic effects could contribute to a better understanding of the role of the increased ultraviolet B radiation on worldwide amphibian populations decline.

  2. The precise timing of embryo splitting for monozygotic dichorionic diamniotic twins: when does embryo splitting for monozygotic dichorionic diamniotic twins occur? Evidence for splitting at the morula/blastocyst stage from studies of in vitro fertilization.

    PubMed

    Kyono, Koichi

    2013-08-01

    There is a long-held credo, as illustrated in Langman's Medical Embryology (11th ed., Sadler, 2010), that dichorionic diamniotic (DD) twins develop after embryo splitting in the early stages of embryonic development. However, from our clinical experiences of the examination of data from single-embryo transfers in 16 fertility clinics in Japan and from various reports, the majority of occurrences of DD twins have been found in the blastocyst stages.

  3. Effects of Ionizing Radiation on Embryos of the Tardigrade Milnesium cf. tardigradum at Different Stages of Development

    PubMed Central

    Beltrán-Pardo, Eliana; Jönsson, K. Ingemar; Wojcik, Andrzej; Haghdoost, Siamak; Harms-Ringdahl, Mats; Bermúdez-Cruz, Rosa M.; Bernal Villegas, Jaime E.

    2013-01-01

    Tardigrades represent one of the most desiccation and radiation tolerant animals on Earth, and several studies have documented their tolerance in the adult stage. Studies on tolerance during embryological stages are rare, but differential effects of desiccation and freezing on different developmental stages have been reported, as well as dose-dependent effect of gamma irradiation on tardigrade embryos. Here, we report a study evaluating the tolerance of eggs from the eutardigrade Milnesium cf. tardigradum to three doses of gamma radiation (50, 200 and 500 Gy) at the early, middle, and late stage of development. We found that embryos of the middle and late developmental stages were tolerant to all doses, while eggs in the early developmental stage were tolerant only to a dose of 50 Gy, and showed a declining survival with higher dose. We also observed a delay in development of irradiated eggs, suggesting that periods of DNA repair might have taken place after irradiation induced damage. The delay was independent of dose for eggs irradiated in the middle and late stage, possibly indicating a fixed developmental schedule for repair after induced damage. These results show that the tolerance to radiation in tardigrade eggs changes in the course of their development. The mechanisms behind this pattern are unknown, but may relate to changes in mitotic activities over the embryogenesis and/or to activation of response mechanisms to damaged DNA in the course of development. PMID:24039737

  4. Effects of ionizing radiation on embryos of the tardigrade Milnesium cf. tardigradum at different stages of development.

    PubMed

    Beltrán-Pardo, Eliana; Jönsson, K Ingemar; Wojcik, Andrzej; Haghdoost, Siamak; Harms-Ringdahl, Mats; Bermúdez-Cruz, Rosa M; Bernal Villegas, Jaime E

    2013-01-01

    Tardigrades represent one of the most desiccation and radiation tolerant animals on Earth, and several studies have documented their tolerance in the adult stage. Studies on tolerance during embryological stages are rare, but differential effects of desiccation and freezing on different developmental stages have been reported, as well as dose-dependent effect of gamma irradiation on tardigrade embryos. Here, we report a study evaluating the tolerance of eggs from the eutardigrade Milnesium cf. tardigradum to three doses of gamma radiation (50, 200 and 500 Gy) at the early, middle, and late stage of development. We found that embryos of the middle and late developmental stages were tolerant to all doses, while eggs in the early developmental stage were tolerant only to a dose of 50 Gy, and showed a declining survival with higher dose. We also observed a delay in development of irradiated eggs, suggesting that periods of DNA repair might have taken place after irradiation induced damage. The delay was independent of dose for eggs irradiated in the middle and late stage, possibly indicating a fixed developmental schedule for repair after induced damage. These results show that the tolerance to radiation in tardigrade eggs changes in the course of their development. The mechanisms behind this pattern are unknown, but may relate to changes in mitotic activities over the embryogenesis and/or to activation of response mechanisms to damaged DNA in the course of development.

  5. Effects of ionizing radiation on embryos of the tardigrade Milnesium cf. tardigradum at different stages of development.

    PubMed

    Beltrán-Pardo, Eliana; Jönsson, K Ingemar; Wojcik, Andrzej; Haghdoost, Siamak; Harms-Ringdahl, Mats; Bermúdez-Cruz, Rosa M; Bernal Villegas, Jaime E

    2013-01-01

    Tardigrades represent one of the most desiccation and radiation tolerant animals on Earth, and several studies have documented their tolerance in the adult stage. Studies on tolerance during embryological stages are rare, but differential effects of desiccation and freezing on different developmental stages have been reported, as well as dose-dependent effect of gamma irradiation on tardigrade embryos. Here, we report a study evaluating the tolerance of eggs from the eutardigrade Milnesium cf. tardigradum to three doses of gamma radiation (50, 200 and 500 Gy) at the early, middle, and late stage of development. We found that embryos of the middle and late developmental stages were tolerant to all doses, while eggs in the early developmental stage were tolerant only to a dose of 50 Gy, and showed a declining survival with higher dose. We also observed a delay in development of irradiated eggs, suggesting that periods of DNA repair might have taken place after irradiation induced damage. The delay was independent of dose for eggs irradiated in the middle and late stage, possibly indicating a fixed developmental schedule for repair after induced damage. These results show that the tolerance to radiation in tardigrade eggs changes in the course of their development. The mechanisms behind this pattern are unknown, but may relate to changes in mitotic activities over the embryogenesis and/or to activation of response mechanisms to damaged DNA in the course of development. PMID:24039737

  6. Automatic segmentation of zona pellucida and its application in cleavage-stage embryo biopsy position selection.

    PubMed

    Wang, Zenan; Ang, Wei Tech; Tan, Steven Yih Min; Latt, Win Tun

    2015-08-01

    A very important step of Pre-implantation genetic diagnosis (PGD) is embryo biopsy, in which process the zona pellucida (ZP) is cut open partially and a part of cellular material is extracted from the embryo. Recognition of the ZP is necessary not only for embryo biopsy, but also for other applications such as zona pellucida thickness variation (ZPTV), embryo dissection, etc. The ZP opening position is closely related to the cell survival rate after the biopsy. Selection of an unsuitable position may cause blastomere lysis after the ZP opening. Normal procedures of ZP recognition and biopsy position selection involve a skilled human embryologist. In order to make the process automatic, we introduce an automatic segmentation method for ZP recognition by using edge detection and ellipse fitting with a value adjustment algorithm in this paper. An application of ZP recognition in embryo biopsy position selection is also introduced. Our ZP recognition algorithm was able to correctly segment 43 out of 45 sample embryo images, achieving a success rate of 96%. Its application in embryo biopsy position selection achieved a success rate of 93%. PMID:26737136

  7. The early-stage diagnosis of albinic embryos by applying optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Yang, Bor-Wen; Wang, Shih-Yuan; Wang, Yu-Yen; Cai, Jyun-Jhang; Chang, Chung-Hao

    2013-09-01

    Albinism is a kind of congenital disease of abnormal metabolism. Poecilia reticulata (guppy fish) is chosen as the model to study the development of albinic embryos as it is albinic, ovoviviparous and with short life period. This study proposed an imaging method for penetrative embryo investigation using optical coherence tomography. By imaging through guppy mother’s reproduction purse, we found the embryo’s eyes were the early-developed albinism features. As human’s ocular albinism typically appear at about four weeks old, it is the time to determine if an embryo will grow into an albino.

  8. Morphological and Gene Expression Changes in Cattle Embryos from Hatched Blastocyst to Early Gastrulation Stages after Transfer of In Vitro Produced Embryos

    PubMed Central

    van Leeuwen, Jessica; Berg, Debra K.; Pfeffer, Peter L.

    2015-01-01

    A detailed morphological staging system for cattle embryos at stages following blastocyst hatching and preceding gastrulation is presented here together with spatiotemporal mapping of gene expression for BMP4, BRACHYURY, CERBERUS1 (CER1), CRIPTO, EOMESODERMIN, FURIN and NODAL. Five stages are defined based on distinct developmental events. The first of these is the differentiation of the visceral hypoblast underlying the epiblast, from the parietal hypoblast underlying the mural trophoblast. The second concerns the formation of an asymmetrically positioned, morphologically recognisable region within the visceral hypoblast that is marked by the presence of CER1 and absence of BMP4 expression. We have termed this the anterior visceral hypoblast or AVH. Intra-epiblast cavity formation and the disappearance of the polar trophoblast overlying the epiblast (Rauber’s layer) have been mapped in relation to AVH formation. The third chronological event involves the transition of the epiblast into the embryonic ectoderm with concomitant onset of posterior NODAL, EOMES and BRACHYURY expression. Lastly, gastrulation commences as the posterior medial embryonic ectoderm layer thickens to form the primitive streak and cells ingress between the embryonic ectoderm and hypoblast. At this stage a novel domain of CER1 expression is seen whereas the AVH disappears. Comparison with the mouse reveals that while gene expression patterns at the onset of gastrulation are well conserved, asymmetry establishment, which relies on extraembryonic tissues such as the hypoblast and trophoblast, has diverged in terms of both gene expression and morphology. PMID:26076128

  9. Development of the arterial pattern in the upper limb of staged human embryos: normal development and anatomic variations

    PubMed Central

    RODRÍGUEZ-NIEDENFÜHR, M.; BURTON, G. J.; DEU, J.; SAÑUDO, J. R.

    2001-01-01

    A total of 112 human embryos (224 upper limbs) between stages 12 and 23 of development were examined. It was observed that formation of the arterial system in the upper limb takes place as a dual process. An initial capillary plexus appears from the dorsal aorta during stage 12 and develops at the same rate as the limb. At stage 13, the capillary plexus begins a maturation process involving the enlargement and differentiation of selected parts. This remodelling process starts in the aorta and continues in a proximal to distal sequence. By stage 15 the differentiation has reached the subclavian and axillary arteries, by stage 17 it has reached the brachial artery as far as the elbow, by stage 18 it has reached the forearm arteries except for the distal part of the radial, and finally by stage 21 the whole arterial pattern is present in its definitive morphology. This differentiation process parallels the development of the skeletal system chronologically. A number of arterial variations were observed, and classified as follows: superficial brachial (7.7%), accessory brachial (0.6%), brachioradial (14%), superficial brachioulnar (4.7%), superficial brachioulnoradial (0.7%), palmar pattern of the median (18.7%) and superficial brachiomedian (0.7%) arteries. They were observed in embryos belonging to stages 17–23 and were not related to a specific stage of development. Statistical comparison with the rates of variations reported in adults did not show significant differences. It is suggested that the variations arise through the persistence, enlargement and differentiation of parts of the initial network which would normally remain as capillaries or even regress. PMID:11693301

  10. Comparison between Cleavage Stage versus Blastocyst Stage Embryo Transfer in an Egyptian Cohort Undergoing in vitro Fertilization: A Possible Role for Laser Assisted Hatching

    PubMed Central

    Hendawy, Sherif F.; Raafat, TA

    2011-01-01

    Background Extended in vitro embryo culture and blastocyst transfer have emerged as essential components of the advanced reproductive technology armamentarium, permitting selection of more advanced embryos considered best suited for transfer. Aim of study The aim of this study was to compare between cleavage stage and blastocyst stage embryo transfer in patients undergoing intracytoplasmic sperm injection, and to assess the role of assisted hatching technique in patients undergoing blastocyst transfer. Patients and methods This study was carried out on two groups. Group I: 110 patients who underwent 120 cycles of intracytoplasmic sperm injection with day 2–3 embryo transfer—for unexplained infertility or male factor within the previous 3 years. Their data obtained retrospectively from medical records. Group II: 46 age matched infertile female patients undergoing 51 intracytoplasmic sperm injection cycles for similar causes. Patients in Group II were further subdivided into 2 equal subgroups; Group IIa (23 patients), which had laser assisted hatching and Group IIb (23 patients), which did not have assisted hatching. All patients had an infertility workup including basal hormonal profile, pelvic ultrasound, hysterosalpingogram and/or laparoscope and semen analysis of the patient’s partner. All patients underwent controlled ovarian hyperstimulation: Using long protocol of ovulation induction. Laser assisted hatching was done for blastocysts of 23 patients. Results Comparison between both groups as regards the reproductive outcome showed a significant difference in pregnancy and implantation rates, both being higher in group II (P < 0.05) Comparison between both subgroups as regards the reproductive outcome showed a highly significant difference in pregnancy and implantation rates, both being higher in Group IIa (P < 0.01). There was also a significantly higher rate of multiple pregnancies among Group IIa (P < 0.05). Conclusion Blastocyst transfer is a successful

  11. Production of chimeric embryos by aggregation of bovine egfp eight-cell stage blastomeres with two-cell fused and asynchronic embryos.

    PubMed

    Hiriart, M I; Bevacqua, R J; Canel, N G; Fernández-Martín, R; Salamone, D F

    2013-09-01

    Embryo disaggregation allows the production of two to four identical offspring from a single cow embryo. In addition, embryo complementation has become the technique of choice to demonstrate the totipotency of embryonic stem cells and induced pluripotent stem cells. Therefore, the aim of this study was to generate a new and simple method by aggregation in the well-of-the-well system to direct each single enhanced green fluorescent protein (egfp) eight-cell blastomere derived from bovine in vitro fertilization embryos to the inner cell mass (ICM) of chimeras produced with fused and asynchronic embryos. To this end, the best conditions to generate in vitro fertilization-fused embryos were determined. Then, the fused (F) and nonfused (NF) embryos were aggregated in two distinct conditions: synchronically (S), with both transgenic and F embryos produced on the same day, and asynchronically (AS), with transgenic embryos produced one day before F embryos. The highest fusion and blastocysts rates were obtained with two pulses of 40 V. The 2ASF and 2ASNF groups showed the best number of blastocysts expressing the EGFP protein (48% and 41%, respectively). Furthermore, the 2ASF group induced the highest localization rates of the egfp-expressing blastomere in the ICM (6/13, 46% of ICM transgene-expressing blastocysts). This technique will have great application for multiplication of embryos of high genetic value or transgenic embryos and also with the generation of truly bovine embryonic stem cells and induced pluripotent stem cells.

  12. [Pregnancy and birth of monozygous female twins following in vitro fertilization and embryo transfer].

    PubMed

    Mettler, L; Riedel, H H; Grillo, M; Michelmann, H W; Baukloh, V; Weisner, D; Semm, K; Bastert, G; Hack, H J

    1984-10-01

    The rate of identical twins after in vitro fertilisation and embryo replacement is higher than after physiological conception. The present paper reports on the first delivery of twins (identical twins) after in vitro fertilisation and embryo replacement. The in vitro fertilisation and embryo replacement of one 4-cell stage resulted in a twin pregnancy. The difficult pregnancy, early delivery and intense perinatal care of the children deserves special consideration. In addition the paper gives a survey on the present number of deliveries after in vitro fertilisation and embryo replacement given at the 3rd World Congress on In Vitro Fertilisation and Embryo Transfer at Helsinki in May 1984.

  13. Stage selection and restricted oviposition period improves cryopreservation of Dipteran embryos

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 proportion. Additionally, the effects of cryopreservation pretreatment viz. permeabilization, on the embryonic age and the...

  14. Actomyosin stiffens the vertebrate embryo during crucial stages of elongation and neural tube closure

    PubMed Central

    Zhou, Jian; Kim, Hye Young; Davidson, Lance A.

    2009-01-01

    Summary Physical forces drive the movement of tissues within the early embryo. Classical and modern approaches have been used to infer and, in rare cases, measure mechanical properties and the location and magnitude of forces within embryos. Elongation of the dorsal axis is a crucial event in early vertebrate development, yet the mechanics of dorsal tissues in driving embryonic elongation that later support neural tube closure and formation of the central nervous system is not known. Among vertebrates, amphibian embryos allow complex physical manipulation of embryonic tissues that are required to measure the mechanical properties of tissues. In this paper, we measure the stiffness of dorsal isolate explants of frog (Xenopus laevis) from gastrulation to neurulation and find dorsal tissues stiffen from less than 20 Pascal (Pa) to over 80 Pa. By iteratively removing tissues from these explants, we find paraxial somitic mesoderm is nearly twice as stiff as either the notochord or neural plate, and at least 10-fold stiffer than the endoderm. Stiffness measurements from explants with reduced fibronectin fibril assembly or disrupted actomyosin contractility suggest that it is the state of the actomyosin cell cortex rather than accumulating fibronectin that controls tissue stiffness in early amphibian embryos. PMID:19168681

  15. Molecular cloning of five individual stage- and tissue-specific mRNA sequences from sea urchin pluteus embryos.

    PubMed

    Fregien, N; Dolecki, G J; Mandel, M; Humphreys, T

    1983-06-01

    Five developmentally regulated sea urchin mRNA sequences which increase in abundance between the blastula and pluteus stages of development were isolated by molecular cloning of cDNA. The regulated sequences all appeared in moderately abundant mRNA molecules of pluteus cells and represented 4% of the clones tested. There were no regulated sequences detected in the 40% of the clones which hybridized to the most abundant mRNA, and the screening procedures were inadequate to detect possible regulation in the 20 to 30% of the clones presumably derived from rare-class mRNA. The reaction of 32P[cDNA] from blastula and pluteus mRNA to dots of the cloned DNAs on nitrocellulose filters indicated that the mRNAs complementary to the different cloned pluteus-specific sequences were between 3- and 47-fold more prevalent at the pluteus stage than at the blastula stage. Polyadenylated RNA from different developmental stages was transferred from electrophoretic gels to nitrocellulose filters and reacted to the different cloned sequences. The regulated mRNAs were undetectable in the RNA of 3-h embryos, became evident at the hatching blastula stage, and reached a maximum in abundance by the gastrula or pluteus stage. Certain of the clones reacted to two sizes of mRNA which did not vary coordinately with development. Transfers of RNA isolated from each of the three cell layers of pluteus embryos that were reacted to the cloned sequences revealed that two of the sequences were found in the mRNA of all three layers, two were ectoderm specific, and one was endoderm specific. Four of the regulated sequences were complementary to one or two major bands and one to at least 50 bands on Southern transfers of restriction endonuclease-digested total sea urchin DNA. PMID:6688291

  16. The temperature and type of intracellular ice formation in preimplantation mouse embryos as a function of the developmental stage.

    PubMed

    Seki, Shinsuke; Mazur, Peter

    2010-06-01

    Our studies the past 5 yr have concentrated on intracellular ice formation (IIF) in mature mouse oocytes at the metaphase stage of meiosis II. Here we report an analogous investigation of the temperature of intracellular ice nucleation in preimplantation embryo stages from one-cell to early morula suspended in 1 M ethylene glycol/PBS and cooled at 20 degrees C/min to -70 degrees C. Physical modeling indicates that oocytes and preimplantation embryos undergo very little osmotic shrinkage at that cooling rate. As a consequence, their interior becomes increasingly supercooled until the supercooling is abruptly terminated by IIF. Four categories of IIF were observed. The first two were 1) those undergoing IIF at temperatures well below the temperature of external ice formation (EIF; -7.2 degrees C) vs. 2) those undergoing IIF within 1 degrees C of the EIF temperature. The other two categories were those multicellular stages in which 3) all the blastomeres underwent IIF simultaneously vs. 4) those in which blastomeres underwent IIF sequentially. Embryos in categories 1 and 3 constituted the majority (80-90%), and for them, the mean IIF temperatures of one-cell, two-cell, four- to six-cell, and early eight-cell ranged from -37 degrees C to -43 degrees C, temperatures that indicate that IIF is a consequence of homogeneous nucleation. However, the IIF nucleation temperature of early morulae in categories 1 and 3 was markedly higher; namely, -23.1 +/- 1.5 degrees C. This marked rise in nucleation temperature coincides with the appearance of aquaporin 3 and gap junctions in early morulae (compacted eight-cell), and is presumably causally related.

  17. Optimizing sweet potato [Ipomoea batatas (L.) Lam.] root and plantlet formation by selection of proper embryo developmental stage and size, and gel type for fluidized sowing.

    PubMed

    Schultheis, J R; Cantliffe, D J; Chee, R P

    1990-11-01

    Potassium starch polyacrylamide, potassium acrylate, a copolymer of potassium acrylate and acrylamide, and hydroxyethylcellulose carrier gels were tested to find a fluid drilling material suited for synthetic seeding of sweet potato (Ipomoea batatas (L.) Lam.) somatic embryos. Somatic embryo developmental stage and size, and maturation (incubation) time were also evaluated to improve plantlet formation. All embryos suspended in the fluidized hydroxyethylcellulose gel were viable after six days and 7% developed into plantlets after two weeks. Up to 97% of the somatic embryos suspended in acrylate and/or acrylamide gels died within six days. Root development was at least 10% and plantlet development at least 30% greater when embryos were subcultured on basal medium for 16 instead of 25 days prior to placement and suspension in hydroxyethylcellulose gel. Up to 25% more plantlets were obtained from embryos at the elongated torpedo stage than those at the cotyledonary or torpedo stages of development. When suspended in hydroxyethylcellulose gel embryo length had no effect on the percentage of plantlets obtained. PMID:24227054

  18. Par-aPKC-dependent and -independent mechanisms cooperatively control cell polarity, Hippo signaling, and cell positioning in 16-cell stage mouse embryos.

    PubMed

    Hirate, Yoshikazu; Hirahara, Shino; Inoue, Ken-Ichi; Kiyonari, Hiroshi; Niwa, Hiroshi; Sasaki, Hiroshi

    2015-10-01

    In preimplantation mouse embryos, the Hippo signaling pathway plays a central role in regulating the fates of the trophectoderm (TE) and the inner cell mass (ICM). In early blastocysts with more than 32 cells, the Par-aPKC system controls polarization of the outer cells along the apicobasal axis, and cell polarity suppresses Hippo signaling. Inactivation of Hippo signaling promotes nuclear accumulation of a coactivator protein, Yap, leading to induction of TE-specific genes. However, whether similar mechanisms operate at earlier stages is not known. Here, we show that slightly different mechanisms operate in 16-cell stage embryos. Similar to 32-cell stage embryos, disruption of the Par-aPKC system activated Hippo signaling and suppressed nuclear Yap and Cdx2 expression in the outer cells. However, unlike 32-cell stage embryos, 16-cell stage embryos with a disrupted Par-aPKC system maintained apical localization of phosphorylated Ezrin/Radixin/Moesin (p-ERM), and the effects on Yap and Cdx2 were weak. Furthermore, normal 16-cell stage embryos often contained apolar cells in the outer position. In these cells, the Hippo pathway was strongly activated and Yap was excluded from the nuclei, thus resembling inner cells. Dissociated blastomeres of 8-cell stage embryos form polar-apolar couplets, which exhibit different levels of nuclear Yap, and the polar cell engulfed the apolar cell. These results suggest that cell polarization at the 16-cell stage is regulated by both Par-aPKC-dependent and -independent mechanisms. Asymmetric cell division is involved in cell polarity control, and cell polarity regulates cell positioning and most likely controls Hippo signaling.

  19. Establishment of goat embryonic stem cells from in vivo produced blastocyst-stage embryos.

    PubMed

    Behboodi, E; Bondareva, A; Begin, I; Rao, K; Neveu, N; Pierson, J T; Wylie, C; Piero, F D; Huang, Y J; Zeng, W; Tanco, V; Baldassarre, H; Karatzas, C N; Dobrinski, I

    2011-03-01

    Embryonic stem (ES) cells with the capacity for germ line transmission have only been verified in mouse and rat. Methods for derivation, propagation, and differentiation of ES cells from domestic animals have not been fully established. Here, we describe derivation of ES cells from goat embryos. In vivo-derived embryos were cultured on goat fetal fibroblast feeders. Embryos either attached to the feeder layer or remained floating and expanded in culture. Embryos that attached showed a prominent inner cell mass (ICM) and those that remained floating formed structures resembling ICM disks surrounded by trophectodermal cells. ICM cells and embryonic disks were isolated mechanically, cultured on feeder cells in the presence of hLIF, and outgrown into ES-like colonies. Two cell lines were cultured for 25 passages and stained positive for alkaline phosphatase, POU5F1, NANOG, SOX2, SSEA-1, and SSEA-4. Embryoid bodies formed in suspension culture without hLIF. One cell line was cultured for 2 years (over 120 passages). This cell line differentiated in vitro into epithelia and neuronal cells, and could be stably transfected and selected for expression of a fluorescent marker. When cells were injected into SCID mice, teratomas were identified 5-6 weeks after transplantation. Expression of known ES cell markers, maintenance in vitro for 2 years in an undifferentiated state, differentiation in vitro, and formation of teratomas in immunodeficient mice provide evidence that the established cell line represents goat ES cells. This also is the first report of teratoma formation from large animal ES cells.

  20. [Comparative Characteristics of the Lipid and Fatty Acid Status of Eyed-Stage Atlantic Salmon Embryos Reared in Natural and Artificial Environments].

    PubMed

    Nemova, N N; Nefedova, Z A; Murzina, S A; Veselov, A E; Ripatti, P

    2015-01-01

    The lipid status of Atlantic salmon (Salmo salar L.) embryos has been compared between embryos developing in incubator nests installed in the natural environment (UmbaRiver, Kola Peninsula) and those developing in a laboratory (in aquaria). The developmental stage of eye pigmentation selected for comparison is characterized by the highest sensitivity of the embryos to environmental factors. The content of certain classes of total lipids and fatty acids was different between the two groups of embryos, and the ratios between the relative content values for some of the compounds were different as well. The difference may be due to environmental factors (temperature, oxygen levels, running water in the nests, etc.) affecting the developing embryos, and they may determine the subsequent embryonic and postembryonic development of the fish. PMID:26852477

  1. Time-lapse monitoring reveals that vitrification increases the frequency of contraction during the pre-hatching stage in mouse embryos

    PubMed Central

    SHIMODA, Yuki; KUMAGAI, Jin; ANZAI, Mibuki; KABASHIMA, Katsuya; TOGASHI, Kazue; MIURA, Yasuko; SHIRASAWA, Hiromitsu; SATO, Wataru; KUMAZAWA, Yukiyo; TERADA, Yukihiro

    2016-01-01

    Contraction during the blastocyst stage is observed during embryonic development of various mammals, including humans, but the physiological role of this process is not well understood. Using time-lapse monitoring (TLM), we studied the influence of vitrification and contractions on embryonic development in mice. Mouse embryos were cultured at the 2-cell stage. At the 8-cell stage, embryos were randomly divided into a fresh group (FG) and vitrified group (VG) and observed for up to 144 h. Strong contractions (i.e., contractions causing a decrease in volume of more than 20% and expansion of the perivitelline space) occurred significantly more often in unhatched embryos than hatching embryos in both groups. Regarding hatching embryos, contractions in the pre-hatching stage were significantly more frequent in the VG than the FG. Furthermore, mRNA expression levels of genes related to contractions were determined at three time points, the 8-cell stage, early blastocyst stage, and 20 h after blastocoel formation, with quantitative reverse transcription-polymerase chain reaction. There was no significant difference in Hspa1a expression between the FG and VG, but Hspa1a overexpression was observed just after thawing and tended to decrease gradually thereafter in some blastocysts. Furthermore, in the VG, Atp1a1 tended to show higher expression in the strong contraction group than in the weak contraction group. Overall, vitrification is an excellent method for cryopreservation but could increase contractions in the pre-hatching stage and may increase energy demands of the embryo. Observation of contraction by TLM may improve the evaluation of embryo quality. PMID:26806421

  2. Expression pattern of Chlamys farreri sox2 in eggs, embryos and larvae of various stages

    NASA Astrophysics Data System (ADS)

    Liang, Shaoshuai; Ma, Xiaoshi; Han, Tiantian; Yang, Dandan; Zhang, Zhifeng

    2015-08-01

    The SOX2 protein is an important transcription factor functioning during the early development of animals. In this study, we isolated a full-length cDNA sequence of scallop Chlamys farreri sox2, Cf-sox2 which was 2194 bp in length with a 981 bp open reading frame encoding 327 amino acids. With real-time PCR analysis, it was detected that Cf-sox2 was expressed in unfertilized oocytes, fertilized eggs and all the tested embryos and larvae. The expression level increased significantly ( P < 0.01) in embryos from 2-cell to blastula, and then decreased significantly ( P < 0.01) and reached the minimum in umbo larva. Moreover, location of the Cf-sox2 expression was revealed using whole mount in situ hybridization technique. Positive hybridization signal could be detected in the central region of unfertilized oocytes and fertilized eggs, and then strong signals dispersed throughout the embryos from 2-cell to gastrula. During larval development, the signals were concentrated and strong signals were restricted to 4 regions of viscera mass in veliger larva. In umbo larva, weak signals could be detected in regions where presumptive visceral and pedal ganglia may be formed. The expression pattern of Cf-sox2 during embryogenesis was similar to that of mammal sox2, which implied that Cf-SOX2 may participate in the regulation of early development of C. farreri.

  3. Monoclonal antibody against dnmt1 arrests the cell division of xenopus early-stage embryos.

    PubMed

    Hashimoto, Hideharu; Suetake, Isao; Tajima, Shoji

    2003-06-10

    DNA methylation plays a crucial role in embryogenesis, and Dnmt1 is known to be a key enzyme in the maintenance of DNA methylation. Dnmt1 is highly accumulated in mature oocytes and eggs. To analyze the function of the maternally accumulated Dnmt1, we injected monoclonal antibodies that specifically recognize the amino terminus of Xenopus Dnmt1 into Xenopus laevis embryos. The monoclonal antibodies inhibited the cell division of the embryos before the midblastula transition. Monoclonal antibody neither inhibited DNA methylation activity of Dnmt1 in vitro nor affected its stability in embryos. In addition, injection of alpha-amanitin, an inhibitor of transcription, did not rescue the cell division arrest. The results suggest that the inhibition of cell division by monoclonal antibodies was due neither to the direct inhibition of DNA methylation activity of Dnmt1 nor to aberrant transcription before the midblastula transition. The morphology of chromatin of the arrested cells showed that the cell cycle was arrested at interphase. This was supported by the biochemical analysis in which the arrested cells demonstrated low histone H1 kinase activity, which indicated that the cells had not entered M phase. Dnmt1 may have an important function other than DNA methylation activity for early embryogenesis in Xenopus laevis. PMID:12749854

  4. Calcium waves along the cleavage furrows in cleavage-stage Xenopus embryos and its inhibition by heparin

    PubMed Central

    1996-01-01

    Calcium signaling is known to be associated with cytokinesis; however, the detailed spatio-temporal pattern of calcium dynamics has remained unclear. We have studied changes of intracellular free calcium in cleavage-stage Xenopus embryos using fluorescent calcium indicator dyes, mainly Calcium Green-1. Cleavage formation was followed by calcium transients that localized to cleavage furrows and propagated along the furrows as calcium waves. The calcium transients at the cleavage furrows were observed at each cleavage furrow at least until blastula stage. The velocity of the calcium waves at the first cleavage furrow was approximately 3 microns/s, which was much slower than that associated with fertilization/egg activation. These calcium waves traveled only along the cleavage furrows and not in the direction orthogonal to the furrows. These observations imply that there exists an intracellular calcium-releasing activity specifically associated with cleavage furrows. The calcium waves occurred in the absence of extracellular calcium and were inhibited in embryos injected with heparin an inositol 1,4,5-trisphosphate (InsP3) receptor antagonist. These results suggest that InsP3 receptor-mediated calcium mobilization plays an essential role in calcium wave formation at the cleavage furrows. PMID:8858172

  5. mTOR-rictor is the Ser473 kinase for AKT1 in mouse one-cell stage embryos.

    PubMed

    Zhang, Zhe; Zhang, Guojun; Xu, Xiaoyan; Su, Wenhui; Yu, Bingzhi

    2012-02-01

    Mammalian target of rapamycin (mTOR) controls cell growth and proliferation via the raptor-mTOR (TORC1) and rictor-mTOR (TORC2) protein complexes. The mTORC2 containing mTOR and rictor is thought to be rapamycin insensitive and it is recently shown that both rictor and mTORC2 are essential for the development of both embryonic and extra embryonic tissues. To explore rictor function in the early development of mouse embryos, we disrupted the expression of rictor, a specific component of mTORC2, in mouse fertilized eggs by using rictor shRNA. Our results showed that one-cell stage eggs that were lack of rictor could not enter into the two-cell stage normally. Recent biochemical studies suggests that TORC2 is the elusive PDK2 (3'-phosphoinositide-dependent kinase 2) for AKT/PKB Ser473 phosphorylation, which is deemed necessary for AKT function, so we microinjected AKT-S473A into mouse fertilized eggs to investigate whether AKT-S473A is downstream effector of mTOR.rictor to regulate the mitotic division. Our findings revealed that the rictor induced phosphorylation of AKT in Ser473 is required for TORC2 function in early development of mouse embryos. PMID:22057724

  6. Deprenyl enhances the teratogenicity of hydroxyurea in organogenesis stage mouse embryos.

    PubMed

    Schlisser, Ava E; Hales, Barbara F

    2013-08-01

    Hydroxyurea, an antineoplastic drug, is a model teratogen. The administration of hydroxyurea to CD1 mice on gestation day 9 induces oxidative stress, increasing the formation of 4-hydroxy-2-nonenal adducts to redox-sensitive proteins such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the caudal region of the embryo. GAPDH catalytic activity is reduced, and its translocation into the nucleus is increased. Because the nuclear translocation of GAPDH is associated with oxidative stress-induced cell death, we hypothesized that this translocation plays a role in mediating the teratogenicity of hydroxyurea. Deprenyl (also known as selegiline), a drug used as a neuroprotectant in Parkinson's disease, inhibits the nuclear translocation of GAPDH. Hence, timed pregnant CD1 mice were treated with deprenyl (10mg/kg) on gestation day 9 followed by the administration of hydroxyurea (400 or 600mg/kg). Deprenyl treatment significantly decreased the hydroxyurea-induced nuclear translocation of GAPDH in the caudal lumbosacral somites. Deprenyl enhanced hydroxyurea-mediated caudal malformations, inducing specifically limb reduction, digit anomalies, tail defects, and lumbosacral vertebral abnormalities. Deprenyl did not augment the hydroxyurea-induced inhibition of glycolysis or alter the ratio of oxidized to reduced glutathione. However, it did dramatically increase cleaved caspase-3 in embryos. These data suggest that nuclear GAPDH plays an important, region-specific, role in teratogen-exposed embryos. Deprenyl exacerbated the developmental outcome of hydroxyurea exposure by a mechanism that is independent of oxidative stress. Although the administration of deprenyl alone did not affect pregnancy outcome, this drug may have adverse consequences when combined with exposures that increase the risk of malformations.

  7. A mean field Ising model for cortical rotation in amphibian one-cell stage embryos.

    PubMed

    Tuszynski, Jack A; Gordon, Richard

    2012-09-01

    We propose a new physical mechanism of cortical rotation generation in one-cell embryos of amphibians based on a phase transition in the ensemble of microtubules localized to the cortical region of the cell interior. Microtubules, protein polymers formed from tubulin heterodimers, are highly negatively charged, which results in strong electrostatic interactions over tens of nanometers, even in the presence of counterions that partially screen electrostatic interactions. A simplified model that offers a plausible representation of these effects is based on the Ising Hamiltonian, which has been robustly applied to explain a wide range of order-disorder transitions in physics, chemistry and other sciences. An Ising model phase transition, especially with the supercooperative flow alignment effect of global rotation of the cortex, provides an alternative to models of cortical rotation based on microtubule polymerization or motor molecules. Insofar as there is any reality to the concept that microtubules are involved in consciousness, we propose that cortical rotation in the one-cell embryo is a better place to look for the purported microtubule entanglement or coherence properties than the adult brain. PMID:22626532

  8. Necropsy findings in American alligator late-stage embryos and hatchlings from northcentral Florida lakes contaminated with organochlorine pesticides

    USGS Publications Warehouse

    Sepulveda, M.S.; Del, Piero F.; Wiebe, J.J.; Rauschenberger, H.R.; Gross, T.S.

    2006-01-01

    Increased American alligator (Alligator mississippiensis) embryo and neonatal mortality has been reported from several northcentral Florida lakes contaminated with old-use organochlorine pesticides (OCPs). However, a clear relationship among these contaminants and egg viability has not been established, suggesting the involvement of additional factors in these mortalities. Thus, the main objective of this study was to determine the ultimate cause of mortality of American alligator late-stage embryos and hatchlings through the conduction of detailed pathological examinations, and to evaluate better the role of OCPs in these mortalities. Between 2000 and 2001, 236 dead alligators were necropsied at or near hatching (after ???65 days of artificial incubation and up to 1 mo of age posthatch). Dead animals were collected from 18 clutches ranging in viability from 0% to 95%. Total OCP concentrations in yolk ranged from ???100 to 52,000 ??g/kg, wet weight. The most common gross findings were generalized edema (34%) and organ hyperemia (29%), followed by severe emaciation (14%) and gross deformities (3%). Histopathologic examination revealed lesions in 35% of the animals, with over half of the cases being pneumonia, pulmonary edema, and atelectasis. Within and across clutches, dead embryos and hatchlings compared with their live cohorts were significantly smaller and lighter. Although alterations in growth and development were not related to yolk OCPs, there was an increase in prevalence of histologic lesions in clutches with high OCPs. Overall, these results indicate that general growth retardation and respiratory abnormalities were a major contributing factor in observed mortalities and that contaminants may increase the susceptibility of animals to developing certain pathologic conditions. ?? Wildlife Disease Association 2006.

  9. Genome-Wide Dissection of the MicroRNA Expression Profile in Rice Embryo during Early Stages of Seed Germination

    PubMed Central

    He, Dongli; Wang, Qiong; Wang, Kun; Yang, Pingfang

    2015-01-01

    The first 24 hours after imbibition (HAI) is pivotal for rice seed germination, during which embryo cells switch from a quiescent state to a metabolically active state rapidly. MicroRNAs (miRNAs) have increasingly been shown to play important roles in rice development. Nevertheless, limited knowledge about miRNA regulation has been obtained in the early stages of rice seed germination. In this study, the small RNAs (sRNAs) from embryos of 0, 12, and 24 HAI rice seeds were sequenced to investigate the composition and expression patterns of miRNAs. The bioinformatics analysis identified 289 miRNA loci, including 59 known and 230 novel miRNAs, and 35 selected miRNAs were confirmed by stem-loop real-time RT-PCR. Expression analysis revealed that the dry and imbibed seeds have unique miRNA expression patterns compared with other tissues, particularly for the dry seeds. Using three methods, Mireap, psRNATarget and degradome analyses, 1197 potential target genes of identified miRNAs involved in various molecular functions were predicted. Among these target genes, 39 had significantly negative correlations with their corresponding miRNAs as inferred from published transcriptome data, and 6 inversely expressed miRNA-target pairs were confirmed by 5ʹ-RACE assay. Our work provides an inventory of miRNA expression profiles and miRNA-target interactions in rice embryos, and lays a foundation for further studies of miRNA-mediated regulation in initial seed germination. PMID:26681181

  10. [Rudimentary stages of the extremities of Scelotes gronovii (Daudin) embryos, a South African Scincidea reptile].

    PubMed

    Raynaud, A; Van den Elzen, P

    1976-01-01

    The development of the limbs has been studied in 15 embryos of Scelotes gronovii, found in 8 ovoviviparous females collected at Saldanha Bay, in South Africa, Cape Province. This study leads to the following constatations: In all the young embryos of this species, their appears anlagen of anterior and of posterior limb-buds. The primordia of forelimb-buds retrogress early and disappear, whereas the primordia of hind limb-buds transform into rudimentary limbs which persist in adult. Histological study of the anlagen of fore limb buds establish that 7 somites (S6 to S12, S1 being the first post-otic somite) send ventral processes in the mesoblast of the anlage. These processes follow a sinuous pathway in the limb-bud, and are bent towards the basal cell layer of the somatopleural mesoderm. On the apical part of the limb-bud lie a wholly rudimentary epiblastic ridge, which disappears early. On the apical part of the hind limb-bud an ectodermic ridge is present, well differentiated which transforms soon in an apical fold; and the anlage of the hind limb produced a short conical appendage with short femur, tibia and fibula and one terminal finger. A comparison was made of the main steps of the development of the limbs in three species of Scelotes with rudimentary limbs. Scelotes inornatus, Scelotes brevipes and Scelotes gronovii. In these three species the ectodermal apical ridge of the fore limb-buds is rudimentary or incompletely differentiated; it never transforms into an ectodermal fold and its retrogresses rapidly; and in these species an early arrest of development and an involution of the primordia of the limb-buds occurs. These fact corroborate the anterior observations made on embryos of Anguis fragilis and of Ophisaurus apodus and they strengthen the interpretation postulating that the spontaneous retrogression of the apical ridge is an essential factor in the morphogenetic events involved in the arrest of development of the limb bud in the snake-like Reptiles.

  11. Characterization of the finch embryo supports evolutionary conservation of the naive stage of development in amniotes.

    PubMed

    Mak, Siu-Shan; Alev, Cantas; Nagai, Hiroki; Wrabel, Anna; Matsuoka, Yoko; Honda, Akira; Sheng, Guojun; Ladher, Raj K

    2015-09-11

    Innate pluripotency of mouse embryos transits from naive to primed state as the inner cell mass differentiates into epiblast. In vitro, their counterparts are embryonic (ESCs) and epiblast stem cells (EpiSCs), respectively. Activation of the FGF signaling cascade results in mouse ESCs differentiating into mEpiSCs, indicative of its requirement in the shift between these states. However, only mouse ESCs correspond to the naive state; ESCs from other mammals and from chick show primed state characteristics. Thus, the significance of the naive state is unclear. In this study, we use zebra finch as a model for comparative ESC studies. The finch blastoderm has mESC-like properties, while chick blastoderm exhibits EpiSC features. In the absence of FGF signaling, finch cells retained expression of pluripotent markers, which were lost in cells from chick or aged finch epiblasts. Our data suggest that the naive state of pluripotency is evolutionarily conserved among amniotes.

  12. Derivation of Porcine Embryonic Stem-Like Cells from In Vitro-Produced Blastocyst-Stage Embryos

    PubMed Central

    Hou, Dao-Rong; Jin, Yong; Nie, Xiao-Wei; Zhang, Man-Ling; Ta, Na; Zhao, Li-Hua; Yang, Ning; Chen, Yuan; Wu, Zhao-Qiang; Jiang, Hai-Bin; Li, Yan-Ru; Sun, Qing-Yuan; Dai, Yi-Fan; Li, Rong-Feng

    2016-01-01

    Efficient isolation of embryonic stem (ES) cells from pre-implantation porcine embryos has remained a challenge. Here, we describe the derivation of porcine embryonic stem-like cells (pESLCs) by seeding the isolated inner cell mass (ICM) from in vitro-produced porcine blastocyst into α-MEM with basic fibroblast growth factor (bFGF). The pESL cells kept the normal karyotype and displayed flatten clones, similar in phenotype to human embryonic stem cells (hES cells) and rodent epiblast stem cells. These cells exhibited alkaline phosphatase (AP) activity and expressed pluripotency markers such as OCT4, NANOG, SOX2, SSEA-4, TRA-1-60, and TRA-1-81 as determined by both immunofluorescence and RT-PCR. Additionally, these cells formed embryoid body (EB), teratomas and also differentiated into 3 germ layers in vitro and in vivo. Microarray analysis showed the expression of the pluripotency markers, PODXL, REX1, SOX2, KLF5 and NR6A1, was significantly higher compared with porcine embryonic fibroblasts (PEF), but expression of OCT4, TBX3, REX1, LIN28A and DPPA5, was lower compared to the whole blastocysts or ICM of blastocyst. Our results showed that porcine embryonic stem-like cells can be established from in vitro-produced blastocyst-stage embryos, which promote porcine naive ES cells to be established. PMID:27173828

  13. Derivation of Porcine Embryonic Stem-Like Cells from In Vitro-Produced Blastocyst-Stage Embryos.

    PubMed

    Hou, Dao-Rong; Jin, Yong; Nie, Xiao-Wei; Zhang, Man-Ling; Ta, Na; Zhao, Li-Hua; Yang, Ning; Chen, Yuan; Wu, Zhao-Qiang; Jiang, Hai-Bin; Li, Yan-Ru; Sun, Qing-Yuan; Dai, Yi-Fan; Li, Rong-Feng

    2016-01-01

    Efficient isolation of embryonic stem (ES) cells from pre-implantation porcine embryos has remained a challenge. Here, we describe the derivation of porcine embryonic stem-like cells (pESLCs) by seeding the isolated inner cell mass (ICM) from in vitro-produced porcine blastocyst into α-MEM with basic fibroblast growth factor (bFGF). The pESL cells kept the normal karyotype and displayed flatten clones, similar in phenotype to human embryonic stem cells (hES cells) and rodent epiblast stem cells. These cells exhibited alkaline phosphatase (AP) activity and expressed pluripotency markers such as OCT4, NANOG, SOX2, SSEA-4, TRA-1-60, and TRA-1-81 as determined by both immunofluorescence and RT-PCR. Additionally, these cells formed embryoid body (EB), teratomas and also differentiated into 3 germ layers in vitro and in vivo. Microarray analysis showed the expression of the pluripotency markers, PODXL, REX1, SOX2, KLF5 and NR6A1, was significantly higher compared with porcine embryonic fibroblasts (PEF), but expression of OCT4, TBX3, REX1, LIN28A and DPPA5, was lower compared to the whole blastocysts or ICM of blastocyst. Our results showed that porcine embryonic stem-like cells can be established from in vitro-produced blastocyst-stage embryos, which promote porcine naive ES cells to be established. PMID:27173828

  14. Morphological and morphometric study of early-cleavage mice embryos resulting from in vitro fertilization at different cleavage stages after vitrification

    PubMed Central

    Homayoun, H.; Zahiri, Sh.; Hemayatkhah Jahromi, V.; Hassanpour Dehnavi, A.

    2016-01-01

    The aim of this study was to examine the possible morphological and morphometric changes resulting from vitrification of embryos at the cleavage stage. In this study, 30 mice early-cleavage embryos at different stages of cleavage, resulting from in vitro fertilization (IVF) techniques, were examined before and after vitrification. Digital images were taken from embryos before and after vitrification. Zona pellucida thickness, differences in zona pellucida thickness, and diameter and volume of blastomeres and embryos as morphometric parameters and current rating of appearance of embryos as morphological parameters, have been studied. According to our findings, there were significant mean differences in all morphometric parameters of the two groups except in the zona pellucid thickness (P≤0.05). With regard to the morphological parameter, the decrease in embryo quality was observed but it was not significant. According to the results, although little quantitative change observed is not necessarily synonymous with harmful intracellular damage, it seems that it is better to examine vitrification method more accurately. Because by making subtle changes in concentration and type of consumed solutions or techniques used, the changes may be minimized. PMID:27656231

  15. Morphological and morphometric study of early-cleavage mice embryos resulting from in vitro fertilization at different cleavage stages after vitrification.

    PubMed

    Homayoun, H; Zahiri, Sh; Hemayatkhah Jahromi, V; Hassanpour Dehnavi, A

    2016-01-01

    The aim of this study was to examine the possible morphological and morphometric changes resulting from vitrification of embryos at the cleavage stage. In this study, 30 mice early-cleavage embryos at different stages of cleavage, resulting from in vitro fertilization (IVF) techniques, were examined before and after vitrification. Digital images were taken from embryos before and after vitrification. Zona pellucida thickness, differences in zona pellucida thickness, and diameter and volume of blastomeres and embryos as morphometric parameters and current rating of appearance of embryos as morphological parameters, have been studied. According to our findings, there were significant mean differences in all morphometric parameters of the two groups except in the zona pellucid thickness (P≤0.05). With regard to the morphological parameter, the decrease in embryo quality was observed but it was not significant. According to the results, although little quantitative change observed is not necessarily synonymous with harmful intracellular damage, it seems that it is better to examine vitrification method more accurately. Because by making subtle changes in concentration and type of consumed solutions or techniques used, the changes may be minimized. PMID:27656231

  16. Neurotoxicological effects of nicotine on the embryonic development of cerebellar cortex of chick embryo during various stages of incubation.

    PubMed

    El-Beltagy, Abd El-Fattah B M; Abou-El-Naga, Amoura M; Sabry, Dalia M

    2015-10-01

    Long-acting nicotine is known to exert pathological effects on almost all tissues including the cerebellar cortex. The present work was designed to elucidate the effect of nicotine on the development of cerebellar cortex of chick embryo during incubation period. The fertilized eggs of hen (Gallus gallus domesticus) were injected into the air space by a single dose of long acting nicotine (1.6 mg/kg/egg) at the 4th day of incubation. The embryos were taken out of the eggs on days 8, 12 and 16 of incubation. The cerebellum of the control and treated embryos at above ages were processed for histopathological examination. The TEM were examined at 16th day of incubation. The results of the present study revealed that, exposure to long-acting nicotine markedly influence the histogenesis of cerebellar cortex of chick embryo during the incubation period. At 8th day of incubation, nicotine delayed the differentiation of the cerebellar analge; especially the external granular layer (EGL) and inner cortical layer (ICL). Furthermore, at 12th day of incubation, the cerebellar foliation was irregular and the Purkinje cells not recognized. By 16th day of incubation, the cerebellar foliations were irregular with interrupted cerebellar cortex and irregular arrangement of Purkinje cells. Immunohistochemical analysis for antibody P53 protein revealed that the cerebellar cortex in all stages of nicotine treated groups possessed a moderate to weak reaction for P53 protein however; this reaction was markedly stronger in the cerebellar cortex of control groups. Moreover, the flow cytometric analysis confirmed that the percentage of apoptosis in control group was significantly higher compared with that of nicotine treated group. At the TEM level, the cerebellar Purkinje cells of 16th day of treated groups showed multiple subcellular alterations in compared with those of the corresponding control group. Such changes represented by appearing of vacuolated mitochondria, cisternal

  17. EVALUATING THE EFFECTS OF FLY ASH EXPOSURE ON FISH EARLY LIFE STAGES: FATHEAD MINNOW EMBRYO-LARVAL TESTS

    SciTech Connect

    Greeley Jr, Mark Stephen; Elmore, Logan R; McCracken, Kitty

    2012-05-01

    On December 22, 2008, a dike containing fly ash and bottom ash in an 84-acre complex of the Tennessee Valley Authority's (TVA) Kingston Steam Plant in East Tennessee failed and released a large quantity of ash into the adjacent Emory River. Ash deposits extended as far as 4 miles upstream (Emory River mile 6) of the Plant, and some ash was carried as far downstream as Tennessee River mile 564 ({approx}4 miles downstream of the Tennessee River confluence with the Clinch River). A byproduct of coal burning power plants, fly ash contains a variety of metals and other elements which, at sufficient concentrations and in specific forms, can be toxic to biological systems. The effects of fly ash contamination on exposed fish populations depend on the magnitude and duration of exposure, with the most significant risk considered to be the effects of specific ash constituents, especially selenium, on fish early life stages. Uptake by adult female fish of fly ash constituents through the food chain and subsequent maternal transfer of contaminants to the developing eggs is thought to be the primary route of selenium exposure to larval fish (Woock and others 1987, Coyle and others 1993, Lemly 1999, Moscatello and others 2006), but direct contact of the fertilized eggs and developing embryos to ash constituents in river water and sediments is also a potential risk factor (Woock and others 1987, Coyle and others 1993, Jezierska and others 2009). To address the risk of fly ash from the Kingston spill to the reproductive health of downstream fish populations, ORNL has undertaken a series of studies in collaboration with TVA including: (1) a field study of the bioaccumulation of fly ash constituents in fish ovaries and the reproductive condition of sentinel fish species in reaches of the Emory and Clinch Rivers affected by the fly ash spill; (2) laboratory tests of the potential toxicity of fly ash from the spill area on fish embryonic and larval development (reported in the current

  18. Susceptibility of zona-intact and zona-free in vitro-produced bovine embryos at different stages of development to infection with bovine herpesvirus-1.

    PubMed

    Vanroose, G; Nauwynck, H; Van Soom, A; Vanopdenbosch, E; de Kruif, A

    1997-05-01

    The aim of the present study was to determine if BHV-1 is able to replicate within in vitro produced embryos and to investigate the degree to which the zona pellucida (ZP) is able to protect in vitro produced embryos against infection with BHV-1. Both ZP-intact and ZP-free matured oocytes, zygotes (1 d post insemination; 1dpi), 8-cell stage embryos (3 dpi), morulae (6 dpi) were incubated for 1 h in 1 ml of MEM containing 10(7.7) TCID(50)/ml BHV-1 (Cooper strain). Three titers (10(5.7), 10(6.7) and 10(7.7) TCID(50)/ml) of the Cooper strain were used for incubation of hatched blastocysts (9 dpi). Bovine embryonic lung cells (BEL) on microcarriers were inoculated following the same protocol as for the embryos. At 0, 12, 24, 36 and 48 h post inoculation (hpi), groups of embryos and BEL cells were collected for virus titration and for the determination of the percentage of viral antigen positive cells by immunofluorescence. For the 3 developmental stages in ZP-free embryos, similar maximal intracellular virus progeny titers were obtained at 24 to 48 hpi ranging from 10(1.32) to 10(1.43) TCID(50)/ 100 embryonic cells. The intracellular virus titer in the BEL cells peaked at 10(3.08) TCID(50)/ 100 BEL cells. The percentage of cells which expressed viral antigens was 13% in ZP-free hatched blastocysts, 17% in ZP-free morulae and 100% in BEL cells. In ZP-intact embryos, no replication of BHV-1 was detected. These results clearly show that only after removal of the zona pellucida, BHV-1 is able to replicate within the in vitro produced embryos, with only a subset of embryonic cells being fully susceptible.

  19. Characterization of the finch embryo supports evolutionary conservation of the naive stage of development in amniotes

    PubMed Central

    Mak, Siu-Shan; Alev, Cantas; Nagai, Hiroki; Wrabel, Anna; Matsuoka, Yoko; Honda, Akira; Sheng, Guojun; Ladher, Raj K

    2015-01-01

    Innate pluripotency of mouse embryos transits from naive to primed state as the inner cell mass differentiates into epiblast. In vitro, their counterparts are embryonic (ESCs) and epiblast stem cells (EpiSCs), respectively. Activation of the FGF signaling cascade results in mouse ESCs differentiating into mEpiSCs, indicative of its requirement in the shift between these states. However, only mouse ESCs correspond to the naive state; ESCs from other mammals and from chick show primed state characteristics. Thus, the significance of the naive state is unclear. In this study, we use zebra finch as a model for comparative ESC studies. The finch blastoderm has mESC-like properties, while chick blastoderm exhibits EpiSC features. In the absence of FGF signaling, finch cells retained expression of pluripotent markers, which were lost in cells from chick or aged finch epiblasts. Our data suggest that the naive state of pluripotency is evolutionarily conserved among amniotes. DOI: http://dx.doi.org/10.7554/eLife.07178.001 PMID:26359635

  20. Two different concentrations of oxygen for culturing precompaction stage embryos on human embryo development competence: a prospective randomized sibling-oocyte study

    PubMed Central

    Guo, Na; Li, Yufeng; Ai, Jihui; Gu, Longjie; Chen, Wen; Liu, Qun

    2014-01-01

    The study was to investigate the effects of oxygen concentration at different levels for culturing pre-compaction embryos on human embryo development competence. A total of 1254 oocytes from 92 patients treated with conventional in vitro fertilization (IVF) were harvested in this study. Oocytes were randomly assigned to the atmospheric (~20%) or low (~5%) oxygen concentration groups on the retrieval day (day 0). Groups were compared with respect to fertilization rates, embryo development, and reproductive outcome. We failed to detect a significant difference on fertilization rate between two groups. However, the low oxygen group yielded more optimal embryos on day 3 when compared with the atmospheric group (72.4% vs. 64.2%). The low oxygen group had a significantly higher blastocyst formation rate than the atmospheric oxygen group (64.5% vs. 52.9%). It is seemly that the optimal blastocyst and frozen blastocyst rates was higher in the low oxygen group, but the data did not reach a statistical significance. Although the use of low oxygen will not affect the clinical outcome in the fresh cleavage-transfer cycles, but it will result in more favorable clinical outcomes in the subsequent warming blastocyst-transfer cycles, with statistically significantly higher clinical pregnancy rate (CPR) and implantation rate (IR) compared with atmospheric oxygen. In conclusion, a low oxygen concentration may significantly improve the developmental potential of pre-compaction embryos, thus resulting in a positive effect on subsequent blastocyst cultivation and optimizing the treatment cycle. PMID:25337269

  1. Development of the Superaltricial Monk Parakeet (Aves, Psittaciformes): Embryo Staging, Growth, and Heterochronies.

    PubMed

    Carril, Julieta; Tambussi, Claudia P

    2015-11-01

    Knowledge about the embryonic stages of birds is important in answering many questions about development and evolution. We give the first description of 41 embryological stages of the monk parakeet (Myiopsitta monachus) on the basis of external morphology and comparison with the chicken. We also provide measurements of some external morphological characters (i.e. body mass, crown-rump, beak, forelimb, and third toe lengths) and perform comparisons with other precocial and altricial birds with the aim of identifying heterochronous developmental features. The following differences in the development of characters in the monk parakeet when compared with other birds were found: (1) delay of the feathers primordia, (2) wing buds initially greater than leg buds, (3) forelimbs and hindlimbs with similar relative size, (4) retroversion of the toe IV, (5) ventral curvature of the upper jaw, (6) positive regressions between stages and beak length with acceleration and higher values and III toe lengths with deceleration and lower values in the monk parakeet compared to the chicken. The growth pattern of the monk paraket Myiopsitta monachus could be influenced by some heterochronic processes like post-displacement, acceleration and/or deceleration. Results of this research allow the standard identification of stages in different species of parrots, recognize similarities and differences between precocial (the chicken) and altricial species (Myiopsitta), and provide planning data for future studies.

  2. Stage-dependent susceptibility to copper in Rhinella arenarum embryos and larvae.

    PubMed

    Aronzon, Carolina M; Sandoval, Maria Teresa; Herkovits, Jorge; Pérezcoll, Cristina S

    2011-12-01

    Copper toxicity in different embryonic and larval stages of the common South American toad Rhinella arenarum was evaluated by means of continuous and 24-h pulse treatments in 12 different developmental stages. Lethal concentrations (LC) of 10, 50, and 90% of continuous treatment with Cu from early blastula (S.4), complete operculum (S.25), and hind limb bud (S.28) stages were plotted from 24 to 168 h, resulting from S.4 in a 24-h LC50 of 137 µg Cu(2+) /L and a 168-h LC50 of 19.5 µg Cu(2+) /L. This result was in agreement with pulse treatments that showed a high resistance to Cu at blastula and gastrula stages, whereas the organogenic period, between muscular response (S.18) and open mouth (S.21), was very susceptible to this metal. Continuous treatments from S.25 showed no significant differences along exposure time (168-h LC50 = 51 µg Cu(2+) /L), but in the case of S.28 toxicity increased slightly from a 24-h LC50 of 138.6 µg Cu(2+) /L to a 168-h LC50 of 104 µg Cu(2+) /L, pointing out that, although the larval period was significantly more resistant to Cu, there was also a remarkable stage-dependent susceptibility to this metal. Copper teratogenic potential was approximately two, and main adverse effects were reduced body size, axial flexure, microcephaly, acephaly, mouth malformations, agenesis of or underdeveloped gills, agenesis of or underdeveloped tail, and hydropsy. The results are discussed considering Cu toxicity mechanisms, an evolutionary perspective, and environmental protection.

  3. Isolation and characterization of a molecule stimulatory to growth of somatic embryos from early stage female gametophyte tissue of loblolly pine.

    PubMed

    De Silva, Veronica; Bostwick, David; Burns, Kristi L; Oldham, Charlie D; Skryabina, Anna; Sullards, M Cameron; Wu, Di; Zhang, Yalin; May, Sheldon W; Pullman, Gerald S

    2008-04-01

    Loblolly pine (LP, Pinus taeda) is the primary commercial species in southern forests of the US. Somatic embryogenesis (SE) is an effective technique to implement clonal tree production of high-value genotypes from breeding and genetic engineering programs. Unlike angiosperm embryos with attached cotyledons as seed storage organs, the diploid conifer embryo is surrounded by the unattached haploid female gametophyte (FG). The FG is not present in culture. This presents a dilemma if the FG produces necessary or regulatory compounds for embryo growth, since in culture these important compounds would be missing and would have to be added as supplements. We report here the direct evidence that extracts from early-stage FG indeed stimulate early-stage somatic embryo (SME) growth and multiplication, whereas extracts from late-stage FG inhibit early-stage SME growth. Furthermore, we have now isolated this stimulatory substance from early-stage FG tissue, and identified this substance as citric acid on the basis of NMR and mass spectrometry. We then demonstrated that topical application of citric acid to SMEs stimulates embryo colony growth at P = 0.05. Moreover, we find that there is a good correlation between the amount of citric acid isolated from FG tissue (65 nmoles per stage 2-3 FG) and the amount of citric acid that stimulates colony growth (25-50 nmoles) when applied topically to SMEs. This approach of isolating and characterizing a molecule from plant tissue, and investigating its role on SE processes can provide valuable information leading to further applications of these molecules to improve LP SE protocols.

  4. AMELIORATION OF ETHANOL-INDUCED DYSMORPHOGENESIS BY ADENOVIRAL-MEDIATED CU,ZN-SOD AND MN-SOD EXPRESSION IN NEURULATION STAGED MOUSE EMBRYOS IN VITRO

    EPA Science Inventory

    AMELIORATION OF ETHANOL-INDUCED DYSMORPHOGENESIS BY ADENOVIRAL-MEDIATED Cu,Zn-SOD AND Mn-SOD EXPRESSION IN NEURULATION STAGED MOUSE EMBRYOS IN VITRO. JB Smith1, PC Hartig3, MR Blanton3, KK Sulik1,2, and ES Hunter3. 1Department of Cell and Developmental Biology and 2Bowles Cente...

  5. The Relationship between Cell Number, Division Behavior and Developmental Potential of Cleavage Stage Human Embryos: A Time-Lapse Study

    PubMed Central

    Gong, Fei; Lu, Changfu; Zhang, Shuoping; Lu, Guangxiu; Lin, Ge

    2016-01-01

    Day 3 cleavage embryo transfer is routine in many assisted reproductive technology centers today. Embryos are usually selected according to cell number, cell symmetry and fragmentation for transfer. Many studies have showed the relationship between cell number and embryo developmental potential. However, there is limited understanding of embryo division behavior and their association with embryo cell number and developmental potential. A retrospective and observational study was conducted to investigate how different division behaviors affect cell number and developmental potential of day 3 embryos by time-lapse imaging. Based on cell number at day 3, the embryos (from 104 IVF/intracytoplasmic sperm injection (ICSI) treatment cycles, n = 799) were classified as follows: less than 5 cells (< 5C; n = 111); 5–6 cells (5–6C; n = 97); 7–8 cells (7–8C; n = 442), 9–10 cells (9–10C; n = 107) and more than 10 cells (>10C; n = 42). Division behavior, morphokinetic parameters and blastocyst formation rate were analyzed in 5 groups of day 3 embryos with different cell numbers. In <5C and 5–6C embryos, fragmentation (FR; 62.2% and 30.9%, respectively) was the main cause for low cell number. The majority of 7–8C embryos exhibited obvious normal behaviors (NB; 85.7%) during development. However, the incidence of DC in 9–10C and >10C embryos increased compared to 7–8C embryos (45.8%, 33.3% vs. 11.1%, respectively). In ≥5C embryos, FR and DC significantly reduced developmental potential, whereas <5C embryos showed little potential irrespective of division behaviors. In NB embryos, the blastocyst formation rate increased with cell number from 7.4% (<5C) to 89.3% (>10C). In NB embryos, the cell cycle elongation or shortening was the main cause for abnormally low or high cell number, respectively. After excluding embryos with abnormal division behaviors, the developmental potential, implantation rate and live birth rate of day 3 embryos increased with cell number

  6. The Relationship between Cell Number, Division Behavior and Developmental Potential of Cleavage Stage Human Embryos: A Time-Lapse Study.

    PubMed

    Kong, Xiangyi; Yang, Shuting; Gong, Fei; Lu, Changfu; Zhang, Shuoping; Lu, Guangxiu; Lin, Ge

    2016-01-01

    Day 3 cleavage embryo transfer is routine in many assisted reproductive technology centers today. Embryos are usually selected according to cell number, cell symmetry and fragmentation for transfer. Many studies have showed the relationship between cell number and embryo developmental potential. However, there is limited understanding of embryo division behavior and their association with embryo cell number and developmental potential. A retrospective and observational study was conducted to investigate how different division behaviors affect cell number and developmental potential of day 3 embryos by time-lapse imaging. Based on cell number at day 3, the embryos (from 104 IVF/intracytoplasmic sperm injection (ICSI) treatment cycles, n = 799) were classified as follows: less than 5 cells (< 5C; n = 111); 5-6 cells (5-6C; n = 97); 7-8 cells (7-8C; n = 442), 9-10 cells (9-10C; n = 107) and more than 10 cells (>10C; n = 42). Division behavior, morphokinetic parameters and blastocyst formation rate were analyzed in 5 groups of day 3 embryos with different cell numbers. In <5C and 5-6C embryos, fragmentation (FR; 62.2% and 30.9%, respectively) was the main cause for low cell number. The majority of 7-8C embryos exhibited obvious normal behaviors (NB; 85.7%) during development. However, the incidence of DC in 9-10C and >10C embryos increased compared to 7-8C embryos (45.8%, 33.3% vs. 11.1%, respectively). In ≥5C embryos, FR and DC significantly reduced developmental potential, whereas <5C embryos showed little potential irrespective of division behaviors. In NB embryos, the blastocyst formation rate increased with cell number from 7.4% (<5C) to 89.3% (>10C). In NB embryos, the cell cycle elongation or shortening was the main cause for abnormally low or high cell number, respectively. After excluding embryos with abnormal division behaviors, the developmental potential, implantation rate and live birth rate of day 3 embryos increased with cell number.

  7. The histone demethylase JMJD2C is stage-specifically expressed in preimplantation mouse embryos and is required for embryonic development.

    PubMed

    Wang, Jianle; Zhang, Miao; Zhang, Yu; Kou, Zhaohui; Han, Zhiming; Chen, Da-Yuan; Sun, Qing-Yuan; Gao, Shaorong

    2010-01-01

    Epigenetic modifications play a pivotal role in embryonic development by dynamically regulating DNA methylation and chromatin modifications. Although recent studies have shown that core histone methylation is reversible, very few studies have investigated the functions of the newly discovered histone demethylases during embryonic development. In the present study, we investigated the expression characteristics and function of JMJD2C, a histone demethylase that belongs to the JmjC-domain-containing histone demethylases, during preimplantation embryonic development of the mouse. We found that JMJD2C is stage-specifically expressed during preimplantation development, with the highest activity being observed from the two-cell to the eight-cell stage. Depletion of JMJD2C in metaphase II oocytes followed by parthenogenetic activation causes a developmental arrest before the blastocyst stage. Moreover, consistent with a previous finding in embryonic stem (ES) cells, depletion of JMJD2C causes a significant down-regulation of the pluripotency gene Nanog in embryos. However, contrary to a previous report in ES cells, we observed that other pluripotency genes, Pou5f1 and Sox2, are also significantly down-regulated in JMJD2C-depleted embryos. Furthermore, the depletion of JMJD2C in early embryos also caused significant down-regulation of the Myc and Klf4 genes, which are associated with cell proliferation. Our data suggest that the deregulation of these critical genes synergistically causes the developmental defects observed in JMJD2C-depleted embryos. PMID:19696013

  8. Outcomes of vitrified-warmed cleavage-stage embryo hatching after in vitro laser-assisted zona pellucida thinning in patients

    PubMed Central

    Wang, En-Hua; Wang, An-Cong; Wang, Bao-Song; Li, Bin

    2016-01-01

    The aim of the present study was to determine whether the size of the zona pellucida (ZP) thinning area by laser-assisted hatching affected the potential development of vitrified-warmed embryos. A total of 196 vitrified-warmed cleavage-stage embryos (from 49 patients, four sister embryos per patient) were used in the study, i.e., four sister embryos from each patient were randomly assigned to four groups: a control group of embryos that were not zona-manipulated (zona intact, group A); one experimental group of embryos in which a quarter of the zona pellucida was thinned using laser-assisted ZP thinning (group B); a second experimental group of embryos in which half of ZP was thinned (group C); and a third group in which two-thirds of the ZP was thinned (group D). Subsequent blastocyst development was assessed. Microscopy was performed to study the hatching process of the embryos after zona thinning. The blastocyst formation rates were 71.43% in group A, 67.35% in group B, 65.31% in group C, and 51.02% in group D (groups B-D vs. group A, P=0.661, P=0.515, P=0.038, respectively). The rates of complete hatching were 30.61% in group A, 38.78% in group B, 61.22% in group C, and 48.98% in group D (groups B-D vs. group A, P=0.396, P=0.002, P=0.063, respectively). For a subgroup of patients, there was a significant difference in the complete hatching in all the groups for women aged <35 years (P=0.011), and there was a significant difference in the complete hatching in all the groups for secondary infertility women (P=0.022). There was no significant difference in the blastocyst formation rates in the different groups of women aged ≥35 years (P=0.340). In addition, there was no significant difference in the complete hatching in the different groups among women aged ≥35 years (P=0.492). The results of the present study showed that in vitrified-warmed embryo transfers at the cleavage-stage, and the two-thirds zona pellucida thinning group demonstrated a significantly

  9. Outcomes of vitrified-warmed cleavage-stage embryo hatching after in vitro laser-assisted zona pellucida thinning in patients

    PubMed Central

    Wang, En-Hua; Wang, An-Cong; Wang, Bao-Song; Li, Bin

    2016-01-01

    The aim of the present study was to determine whether the size of the zona pellucida (ZP) thinning area by laser-assisted hatching affected the potential development of vitrified-warmed embryos. A total of 196 vitrified-warmed cleavage-stage embryos (from 49 patients, four sister embryos per patient) were used in the study, i.e., four sister embryos from each patient were randomly assigned to four groups: a control group of embryos that were not zona-manipulated (zona intact, group A); one experimental group of embryos in which a quarter of the zona pellucida was thinned using laser-assisted ZP thinning (group B); a second experimental group of embryos in which half of ZP was thinned (group C); and a third group in which two-thirds of the ZP was thinned (group D). Subsequent blastocyst development was assessed. Microscopy was performed to study the hatching process of the embryos after zona thinning. The blastocyst formation rates were 71.43% in group A, 67.35% in group B, 65.31% in group C, and 51.02% in group D (groups B-D vs. group A, P=0.661, P=0.515, P=0.038, respectively). The rates of complete hatching were 30.61% in group A, 38.78% in group B, 61.22% in group C, and 48.98% in group D (groups B-D vs. group A, P=0.396, P=0.002, P=0.063, respectively). For a subgroup of patients, there was a significant difference in the complete hatching in all the groups for women aged <35 years (P=0.011), and there was a significant difference in the complete hatching in all the groups for secondary infertility women (P=0.022). There was no significant difference in the blastocyst formation rates in the different groups of women aged ≥35 years (P=0.340). In addition, there was no significant difference in the complete hatching in the different groups among women aged ≥35 years (P=0.492). The results of the present study showed that in vitrified-warmed embryo transfers at the cleavage-stage, and the two-thirds zona pellucida thinning group demonstrated a significantly

  10. Effects of copper exposure on the hatching status and antioxidant defense at different developmental stages of embryos and larvae of goldfish Carassius auratus.

    PubMed

    Kong, Xianghui; Jiang, Hongxia; Wang, Shuping; Wu, Xiangmin; Fei, Wei; Li, Li; Nie, Guoxing; Li, Xuejun

    2013-09-01

    This study aims to assess the effects of copper exposure on hatching status and antioxidant defense at different stages of embryos and larvae of goldfish Carassius auratus. In this study, day-old embryos were randomly grouped after fertilization and then exposed to copper concentrations of 0, 0.1, 0.4, 0.7, and 1.0mgL(-1). Copper-exposed fish embryos were sampled every 24h to determine superoxide dismutase (SOD), and catalase (CAT) activities, as well as malondialdehyde (MDA) content. In addition, cumulative mortality and larval deformity were also investigated. The findings showed that cumulative mortality and larval deformity rate increased gradually with copper concentration increase. SOD and CAT activities were inhibited at higher copper concentrations. At a lower concentration (0.1mgL(-1)), SOD activity increased in larvae, whereas CAT activity showed no significant change (p>0.05). MDA, as the lipid peroxidation product, gradually accumulated in embryos and larvae with increasing copper concentration and the extension of exposure time. At 0.4mgL(-1) and more, copper toxicity was shown in embryos and larvae. In conclusion, copper-exposed effects on hatching status and antioxidant defense in C. auratus embryos and larvae showed concentration- and time-dependent patterns. The biochemical parameters in this study can be used as effective indicators for evaluating the responses of copper-exposed fish embryos. In addition, this study demonstrates that 0.4mgL(-1) copper (corresponding to 1mgL(-1) copper sulfate), used to kill parasites in aquaculture, is not safe concentration, because it can result in toxicity to larvae. Therefore, the copper concentration to kill pathogen should be less than 0.4mgL(-1) for C. auratus.

  11. Biphasic Aire expression in early embryos and in medullary thymic epithelial cells before end-stage terminal differentiation.

    PubMed

    Nishikawa, Yumiko; Hirota, Fumiko; Yano, Masashi; Kitajima, Hiroyuki; Miyazaki, Jun-ichi; Kawamoto, Hiroshi; Mouri, Yasuhiro; Matsumoto, Mitsuru

    2010-05-10

    The roles of autoimmune regulator (Aire)-expressing medullary thymic epithelial cells (mTECs) in the organization of the thymic microenvironment for establishing self-tolerance are enigmatic. We sought to monitor the production and maintenance of Aire-expressing mTECs by a fate-mapping strategy in which bacterial artificial chromosome transgenic (Tg) mice expressing Cre recombinase under the control of the Aire regulatory element were crossed with a GFP reporter strain. We found that, in addition to its well recognized expression within mature mTECs, Aire was expressed in the early embryo before emergence of the three germ cell layers. This observation may help to explain the development of ectodermal dystrophy often seen in patients with AIRE deficiency. With the use of one Tg line in which Cre recombinase expression was confined to mTECs, we found that Aire(+)CD80(high) mTECs further progressed to an Aire(-)CD80(intermediate) stage, suggesting that Aire expression is not constitutive from after its induction until cell death but instead is down-regulated at the beginning of terminal differentiation. We also demonstrated that many mTECs of Aire-expressing lineage are in close contact with thymic dendritic cells. This close proximity may contribute to transfer of tissue-restricted self-antigens expressed by mTECs to professional antigen-presenting cells.

  12. The p66Shc Adaptor Protein Controls Oxidative Stress Response in Early Bovine Embryos

    PubMed Central

    Betts, Dean H.; Bain, Nathan T.; Madan, Pavneesh

    2014-01-01

    The in vitro production of mammalian embryos suffers from high frequencies of developmental failure due to excessive levels of permanent embryo arrest and apoptosis caused by oxidative stress. The p66Shc stress adaptor protein controls oxidative stress response of somatic cells by regulating intracellular ROS levels through multiple pathways, including mitochondrial ROS generation and the repression of antioxidant gene expression. We have previously demonstrated a strong relationship with elevated p66Shc levels, reduced antioxidant levels and greater intracellular ROS generation with the high incidence of permanent cell cycle arrest of 2–4 cell embryos cultured under high oxygen tensions or after oxidant treatment. The main objective of this study was to establish a functional role for p66Shc in regulating the oxidative stress response during early embryo development. Using RNA interference in bovine zygotes we show that p66Shc knockdown embryos exhibited increased MnSOD levels, reduced intracellular ROS and DNA damage that resulted in a greater propensity for development to the blastocyst stage. P66Shc knockdown embryos were stress resistant exhibiting significantly reduced intracellular ROS levels, DNA damage, permanent 2–4 cell embryo arrest and diminished apoptosis frequencies after oxidant treatment. The results of this study demonstrate that p66Shc controls the oxidative stress response in early mammalian embryos. Small molecule inhibition of p66Shc may be a viable clinical therapy to increase the developmental potential of in vitro produced mammalian embryos. PMID:24475205

  13. Mitochondria-targeted DsRed2 protein expression during the early stage of bovine somatic cell nuclear transfer embryo development.

    PubMed

    Park, Hyo-Jin; Min, Sung-Hun; Choi, Hoonsung; Park, Junghyung; Kim, Sun-Uk; Lee, Seunghoon; Lee, Sang-Rae; Kong, Il-Keun; Chang, Kyu-Tae; Koo, Deog-Bon; Lee, Dong-Seok

    2016-09-01

    Somatic cell nuclear transfer (SCNT) has been widely used as an efficient tool in biomedical research for the generation of transgenic animals from somatic cells with genetic modifications. Although remarkable advances in SCNT techniques have been reported in a variety of mammals, the cloning efficiency in domestic animals is still low due to the developmental defects of SCNT embryos. In particular, recent evidence has revealed that mitochondrial dysfunction is detected during the early development of SCNT embryos. However, there have been relatively few or no studies regarding the development of a system for evaluating mitochondrial behavior or dynamics. For the first time, in mitochondria of bovine SCNT embryos, we developed a method for the visualization of mitochondria and expression of fluorescence proteins. To express red fluorescence in mitochondria of cloned embryos, bovine ear skin fibroblasts, nuclear donor, were stably transfected with a vector carrying mitochondria-targeting DsRed2 gene tagged with V5 epitope (mito-DsRed2-V5 tag) using lentivirus-mediated gene transfer because of its ability to integrate in the cell genome and the potential for long-term transgene expression in the transduced cells and their dividing cells. From western blotting analysis of V5 tag protein using mitochondrial fraction and confocal microscopy of red fluorescence using SCNT embryos, we found that the mitochondrial expression of the mito-DsRed2 protein was detected until the blastocyst stage. In addition, according to image analysis, it may be suggested possible use of the system for visualization of mitochondrial localization and evaluation of mitochondrial behaviors or dynamics in early development of bovine SCNT embryos. PMID:27287919

  14. Development of interspecies cloned embryos reconstructed with rabbit (Oryctolagus cuniculus) oocytes and cynomolgus monkey (Macaca fascicularis) fibroblast cell nuclei.

    PubMed

    Yamochi, Takayuki; Kida, Yuta; Oh, Noriyoshi; Ohta, Sei; Amano, Tomoko; Anzai, Masayuki; Kato, Hiromi; Kishigami, Satoshi; Mitani, Tasuku; Matsumoto, Kazuya; Saeki, Kazuhiro; Takenoshita, Makoto; Iritani, Akira; Hosoi, Yoshihiko

    2013-11-01

    Interspecies somatic cell nuclear transfer (ISCNT) has been proposed as a technique to produce cloned offspring of endangered species as well as to investigate nucleus-cytoplasm interactions in mammalian embryo. However, it is still not known which embryo culture medium is optimal for ISCNT embryos for the nuclear donor or the oocyte recipient. We assessed the effects of the culture medium on the developmental competence of the ISCNT embryos by introducing cynomolgus monkey (Macaca fascicularis) fibroblast nuclei into enucleated rabbit (Oryctolagus cuniculus) oocytes (monkey-rabbit embryo). The monkey-rabbit ISCNT embryos that were cultured in mCMRL-1066 developed to the blastocyst stage, although all monkey-rabbit ISCNT embryos cultured in M199 were arrested by the 4-cell stage. When monkey-rabbit ISCNT and rabbit-rabbit somatic cell nuclear transfer (SCNT) embryos were cultured in mCMRL-1066, the blastocyst cell numbers of the monkey-rabbit ISCNT embryos corresponded to the cell numbers of the control rabbit-rabbit SCNT embryos, which were produced from a rabbit fibroblast nucleus and an enucleated rabbit oocyte. In addition, the presence of mitochondria, which were introduced with monkey fibroblasts into rabbit recipient cytoplasm, was confirmed up to the blastocyst stage by polymerase chain reaction (PCR). This study demonstrated that: (1) rabbit oocytes can reprogramme cynomolgus monkey somatic cell nuclei, and support preimplantation development; (2) monkey-rabbit ISCNT embryos developed well in monkey culture medium at early embryonic developmental stages; (3) the cell number of monkey-rabbit ISCNT embryos is similar to that of rabbit-rabbit SCNT embryos; and (4) the mitochondrial fate of monkey-rabbit ISCNT embryos is heteroplasmic from the time just after injection to the blastocyst stage that has roots in both rabbit oocytes and monkey fibroblasts.

  15. Embryos, microscopes, and society.

    PubMed

    Maienschein, Jane

    2016-06-01

    Embryos have different meanings for different people and in different contexts. Seen under the microscope, the biological embryo starts out as one cell and then becomes a bunch of cells. Gradually these divide and differentiate to make up the embryo, which in humans becomes a fetus at eight weeks, and then eventually a baby. At least, that happens in those cases that carry through normally and successfully. Yet a popular public perception imagines the embryo as already a little person in the very earliest stages of development, as if it were predictably to become an adult. In actuality, cells can combine, pull apart, and recombine in a variety of ways and still produce embryos, whereas most embryos never develop into adults at all. Biological embryos and popular imaginations of embryos diverge. This paper looks at some of the historical reasons for and social implications of that divergence.

  16. Porcine Induced Pluripotent Stem Cells Require LIF and Maintain Their Developmental Potential in Early Stage of Embryos

    PubMed Central

    Cheng, De; Guo, Yanjie; Li, Zhenzhen; Liu, Yajun; Gao, Xing; Gao, Yi; Cheng, Xiang; Hu, Junhe; Wang, Huayan

    2012-01-01

    Porcine induced pluripotent stem (piPS) cell lines have been generated recently by using a cocktail of defined transcription factors, however, the features of authentic piPS cells have not been agreed upon and most of published iPS clones did not meet the stringent requirements of pluripotency. Here, we report the generation of piPS cells from fibroblasts using retrovirus carrying four mouse transcription factors (mOct4, mSox2, mKlf4 and mc-Myc, 4F). Multiple LIF-dependent piPS cell lines were generated and these cells showed the morphology similar to mouse embryonic stem cells and other pluripotent stem cells. In addition to the routine characterization, piPS cells were injected into porcine pre-compacted embryos to generate chimera embryos and nuclear transfer (NT) embryos. The results showed that piPS cells retain the ability to integrate into inner and outer layers of the blastocysts, and support the NT embryos development to blastocysts. The generations of chimera embryos and NT embryos derived from piPS clones are a practical means to determine the quality of iPS cells ex vivo. PMID:23251622

  17. Effect of mitotic inducers and retinoic acid blocker on expression of pluripotent genes in ES cells derived from early stage in vitro-produced embryos in buffalo.

    PubMed

    Kumar, Ashok; Kumar, Kuldeep; Singh, Renu; Puri, Gopal; Ranjan, R; Yasotha, T; Singh, R K; Sarkar, M; Bag, Sadhan

    2012-12-01

    So far, it has been difficult to generate embryonic stem (ES) cell from early stage preimplantation embryos of buffalo. These ES cells will be more helpful for efficient embryo cloning and generation of body cells as they are more primitive than inner cell mass (ICM)-derived ES cells. The present study was conducted to find the effect of lipopolysaccharide (LPS), melatonin (N-acetyl-5-methoxytryptamine, a pineal gland product), and citral (3,7-dimethyl-2,6-octadienal and a retinoic acid synthesis blocker) on establishment of primary ES cell colonies, the comparative size of the ES cell colonies, and expression of pluripotent genes during extended period of culture in buffalo. Zona-free eight-cell stage in vitro fertilization (IVF) embryos were cultured in ES cell medium supplemented with none (media I as control), LPS (media II), citral melatonin (media III), or melatonin (media IV). The multiplication of blastomere leading to ES cell colony formation and expression of pluripotent genes were assessed up to day 20 of culture. The primary colony formation, the comparative size of the ES cell colonies, and expression of pluripotent genes in these colonies were better in the medium supplemented with melatonin in all days of culture. Within melatonin supplementation, the colony size was comparatively larger on day 8 and day 12 of culture. Further, with this supplementation, the Oct-4 and Nanog expression was comparatively higher on all days of culture. The results indicated that supplementation of melatonin helped in the formation of better primary ES cell colony as well as in the maintenance of pluripotency. The results also indicated that primary colonies developed on day 8 to day 12 of culture may be better for passaging them for establishment of ES cell line from early stage preimplantation IVF embryos of in buffalo.

  18. Effect of mitotic inducers and retinoic acid blocker on expression of pluripotent genes in ES cells derived from early stage in vitro-produced embryos in buffalo.

    PubMed

    Kumar, Ashok; Kumar, Kuldeep; Singh, Renu; Puri, Gopal; Ranjan, R; Yasotha, T; Singh, R K; Sarkar, M; Bag, Sadhan

    2012-12-01

    So far, it has been difficult to generate embryonic stem (ES) cell from early stage preimplantation embryos of buffalo. These ES cells will be more helpful for efficient embryo cloning and generation of body cells as they are more primitive than inner cell mass (ICM)-derived ES cells. The present study was conducted to find the effect of lipopolysaccharide (LPS), melatonin (N-acetyl-5-methoxytryptamine, a pineal gland product), and citral (3,7-dimethyl-2,6-octadienal and a retinoic acid synthesis blocker) on establishment of primary ES cell colonies, the comparative size of the ES cell colonies, and expression of pluripotent genes during extended period of culture in buffalo. Zona-free eight-cell stage in vitro fertilization (IVF) embryos were cultured in ES cell medium supplemented with none (media I as control), LPS (media II), citral melatonin (media III), or melatonin (media IV). The multiplication of blastomere leading to ES cell colony formation and expression of pluripotent genes were assessed up to day 20 of culture. The primary colony formation, the comparative size of the ES cell colonies, and expression of pluripotent genes in these colonies were better in the medium supplemented with melatonin in all days of culture. Within melatonin supplementation, the colony size was comparatively larger on day 8 and day 12 of culture. Further, with this supplementation, the Oct-4 and Nanog expression was comparatively higher on all days of culture. The results indicated that supplementation of melatonin helped in the formation of better primary ES cell colony as well as in the maintenance of pluripotency. The results also indicated that primary colonies developed on day 8 to day 12 of culture may be better for passaging them for establishment of ES cell line from early stage preimplantation IVF embryos of in buffalo. PMID:23093464

  19. Zebrafish embryos as an alternative to animal experiments--a commentary on the definition of the onset of protected life stages in animal welfare regulations.

    PubMed

    Strähle, Uwe; Scholz, Stefan; Geisler, Robert; Greiner, Petra; Hollert, Henner; Rastegar, Sepand; Schumacher, Axel; Selderslaghs, Ingrid; Weiss, Carsten; Witters, Hilda; Braunbeck, Thomas

    2012-04-01

    Worldwide, the zebrafish has become a popular model for biomedical research and (eco)toxicology. Particularly the use of embryos is receiving increasing attention, since they are considered as replacement method for animal experiments. Zebrafish embryos allow the analysis of multiple endpoints ranging from acute and developmental toxicity determination to complex functional genetic and physiological analysis. Particularly the more complex endpoints require the use of post-hatched eleutheroembryo stages. According to the new EU Directive 2010/63/EU on the protection of animals used for scientific purposes, the earliest life-stages of animals are not defined as protected and, therefore, do not fall into the regulatory frameworks dealing with animal experimentation. Independent feeding is considered as the stage from which free-living larvae are subject to regulations for animal experimentation. However, despite this seemingly clear definition, large variations exist in the interpretation of this criterion by national and regional authorities. Since some assays require the use of post-hatched stages up to 120 h post fertilization, the literature and available data are reviewed in order to evaluate if this stage could still be considered as non-protected according to the regulatory criterion of independent feeding. Based on our analysis and by including criteria such as yolk consumption, feeding and swimming behavior, we conclude that zebrafish larvae can indeed be regarded as independently feeding from 120 h after fertilization. Experiments with zebrafish should thus be subject to regulations for animal experiments from 120 h after fertilization onwards.

  20. 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

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

    2014-06-01

    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. PMID:24659575

  1. 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

  2. Immunolocalization and expression of Na(+)/K(+) -ATPase in embryos, early larval stages and adults of the freshwater shrimp Palaemonetes argentinus (Decapoda, Caridea, Palaemonidae).

    PubMed

    Ituarte, Romina Belén; Lignot, Jehan-Hervé; Charmantier, Guy; Spivak, Eduardo; Lorin-Nebel, Catherine

    2016-06-01

    The euryhaline shrimp Palaemonetes argentinus exemplifies an evolutionary transition from brackish to freshwater habitats that requires adequate osmoregulatory capacities. Hyperosmoregulation is functional at hatching and it likely begins during the embryonic phase allowing this species to develop entirely in fresh water. Here, we investigated the Na(+)/K(+)-ATPase α-subunit gene (nka-α) expression using quantitative real-time PCR and localized Na(+)/K(+)-ATPase (NKA) in ion-transporting epithelia through immunofluorescence microscopy. We reared shrimps from spawning to juvenile stages at two salinities (1, 15 ‰) and maintained adults for 3 weeks at three salinity treatments (1, 15, 25 ‰). nka-α gene expression was measured in: (1) embryos at an early (SI), intermediate (SII) and late (SIII) stage of embryonic development; (2) newly hatched larvae (Zoea I, ZI); and (3) isolated gill tissue of adults. The nka-α expression was low in SI and SII embryos and reached maximum levels prior to hatching (SIII), which were similar to expression levels detected in the ZI. The nka-α expression in SIII and ZI was highest at 15 ‰, whereas salinity did not affect expression in earlier embryos. In SIII, in ZI and in a later zoeal stage ZIV, NKA was localized in epithelial cells of pleurae, in the inner-side epithelium of branchiostegite and in the antennal glands. Gills appeared in the ZIV but NKA immunolabeling of the cells of the gill shaft occurred in a subsequent developmental larval stage, the decapodid. Extrabranchial organs constitute the main site of osmoregulation in early ontogenetic stages of this freshwater shrimp.

  3. A comparative study between cleavage stage embryo transfer at day 3 and blastocyst stage transfer at day 5 in in-vitro fertilization/intra-cytoplasmic sperm injection on clinical pregnancy rates

    PubMed Central

    Kaur, Prabhleen; Swarankar, M. L.; Maheshwari, Manju; Acharya, Veena

    2014-01-01

    OBJECTIVE: To evaluate the efficacy of blastocyst transfer in comparison with cleavage stage transfer. STUDY DESIGN: A randomized, prospective study was conducted in Infertility clinic, Department of Obstetrics and Gynecology, Mahatma Gandhi Hospital, Jaipur on 300 patients aged 25-40 years undergoing in-vitro fertilization (IVF)/intra-cytoplasmic sperm injection (ICSI) cycle from May 2010-April 2011. When three or more Grade-I embryos were observed on day 2 of culture, patients were divided randomly into two study groups, cleavage stage transfer and blastocyst transfer group having 150 patients each. Primary outcomes evaluated were, Clinical pregnancy rate and Implantation rate. The results were analyzed using proportions, standard deviation and Chi-square test. RESULTS: Both the groups were similar for age, indication and number of embryos transferred. Clinical pregnancies after blastocyst transfer were significantly higher 66 (44.0%) compared to cleavage stage embryo transfer 44 (29.33%) (P < 0.01). Implantation rate for blastocyst transfer group was also significantly higher (P < 0.001). CONCLUSION: Blastocyst transfer having higher implantation rate and clinical pregnancy rate lead to reduction in multiple pregnancies. PMID:25395745

  4. The First Human Cloned Embryo.

    ERIC Educational Resources Information Center

    Cibelli, Jose B.; Lanza, Robert P.; West, Michael D.; Ezzell, Carol

    2002-01-01

    Describes a process known as parthenogenesis which produces cloned, early-stage embryos and human embryos generated only from eggs. Speculates that this technology puts therapeutic cloning within reach. (DDR)

  5. Endoscopic embryo collection and embryo transfer into the oviduct and the uterus of pigs.

    PubMed

    Besenfelder, U; Mödl, J; Müller, M; Brem, G

    1997-04-01

    We describe the first complete embryo transfer program, including flushing of embryos from the oviducts via the uterine horns, transfer of embryos into the Fallopian tubes or the uterine horns and recording of the number of piglets born live. The described procedure is minimally invasive and allows the use of pigs simultaneously for embryo collection and production of normal pregnancies. A 30 degrees forward oblique endoscope provided optimal visualization of the reproductive organs and free access to the organs for embryo flushing and transfer. In contrast to surgical and nonsurgical methods, endoscopy allows to pre-examine the genital tract for reproductive abnormalities and successful ovulation. A total of 95 prepuberal gilts or cyclic sows were used in this trial. Embryos or oocytes were collected from hormonally treated pigs via endoscopy(n = 17) on Day 3 and via laparotomy or post mortem after slaughter (control group, n = 38) on Day 3 and 6 after insemination. One (unilateral collection, n = 7) or both oviducts (bilateral collection, n = 10) were flushed endoscopically. We recovered 114 (average 16/pig) and 279 (average 28/pig) oocytes or embryos with fertilization rates of 89% and 72%, respectively. In the control group 834 oocytes or embryos were collected at Day 3 and 6 after insemination (fertilization rate 64%, total 534 embryos, 33 at 2-, 367 at 4-, 2 at 8-cell stage, 24 morulae and 108 blastocysts). Of 836 embryos recovered by endoscopy, surgery or slaughter 528 Day 3 embryos at 2- to 4-cell stage were transferred into (one) oviducts (n = 27 pigs, about 20/pig) resulting in 9 pregnant pigs diagnosed at Day 28 by sonography. Of the 9, 8 carried a total of 49 piglets to term. A total of 195 Day 6 embryos were transferred into uterine horns (n = 12 pigs, about 16/pig), resulting in 5 pregnant pigs carrying a total of 38 offspring to term. The use of endoscopy in assisted reproduction of pigs has the advantages of allowing easy access to the ovary

  6. Temperature and ration effects on components of the IGF system and growth performance of rainbow trout (Oncorhynchus mykiss) during the transition from late stage embryos to early stage juveniles.

    PubMed

    Li, Mao; Leatherland, John

    2008-02-01

    The study investigated the effects of incubation temperature, and the size of ration fed to the transitional embryo/juvenile stage of rainbow trout (Oncorhynchus mykiss) on growth, liver and gastrointestinal (GI) tract IGF-1 content, and the expression of insulin-like growth factor-related genes (IGF-1, IGF-2, IGF-RIa, and IGF-RIb) by the liver and GI tract. Embryos were reared from zygote to "swim-up" at either 8.5 degrees C (E(8.5)) or 6.0 degrees C (E(6.0)); at "swim-up" (51-days post-fertilization [dpf] and 72-dpf for the E(8.5) and E(6.0) groups, respectively), the embryos were transferred to grow-up tanks supplied with water at 8.5 degrees C. Late stage embryos (LSEs) at the same developmental stage from the two temperature treatment groups (64-dpf and 86-dpf for the E(8.5) and E(6.0) groups, respectively) were fed with salmonid starter diet at levels of 5.0%, 2.0%, and 0.5% of live body mass per day. Embryos were sampled just prior to first feeding (PFEs), and before complete absorption of the yolk [late stage embryos (LSEs)], and early stage juveniles (ESJs) were sampled after yolk sac absorption when they were fully reliant on exogenous sources of food. The early incubation temperature and ration levels had significant affects on mortality (with lower mortalities in the E(6.0) group) and growth performance of the fish; dry body mass values for fish fed the 5.0% ration were significantly lower in the E(6.0) group of LSEs and ESJs compared with the respective treatment in the E(8.5) group; a similar pattern was seen for total body length, although this was only significant for the LSEs. Whole embryo IGF-1 content was significantly lower in the E(6.0) group compared with the E(8.5) group of PFEs, and hepatic IGF-1 content was significantly lower in the E(6.0) group fed the maintenance ration (0.5%) compared with the E(8.5) fed a similar ration; restricted ration significantly elevated hepatic IGF-1 content in the LSE stage for both temperature treatment

  7. Developmental dynamics of IMSI-derived embryos: a time-lapse prospective study.

    PubMed

    Knez, Katja; Tomazevic, Tomaz; Vrtacnik-Bokal, Eda; Virant-Klun, Irma

    2013-08-01

    Because sperm vacuoles were marked as zones without chromatin in the sperm nucleus, which may reflect underlying chromosomal or DNA defects, this study considered whether they influence the morphology and dynamics of early developmental events in preimplantation embryos. Oocytes were injected with spermatozoa of four classes, according to the number and size of vacuoles at ×6000 magnification, and derived embryos were observed under time-lapse microscopy. For each embryo, the times of pronuclei appearance and disappearance and the first, second and third divisions were determined and related to its respective class of injected spermatozoa and its developmental stage. Embryos arising from normal class-I spermatozoa (without vacuoles) reached the 4-cell stage significantly earlier than embryos developed from class-IV spermatozoa (with large vacuoles and other abnormalities) (P=0.012). Blastocysts from class-I spermatozoa required the shortest mean time for all developmental events in comparison with blastocysts from spermatozoa of other classes (with vacuoles). Blastocysts also showed significantly earlier first division than arrested embryos in embryos arising from class-I spermatozoa (P=0.033). An insight into the developmental dynamics of embryo development according to morphology and head vacuoles of injected spermatozoa in morphologically selected sperm-derived embryos was observed for the first time.

  8. Co-culture of early cattle embryos to the blastocyst stage with oviducal tissue or in conditioned medium.

    PubMed

    Eyestone, W H; First, N L

    1989-03-01

    In Exp. 1, 5-8-cell embryos from superovulated cattle were co-cultured with oviducal tissue suspended in Ham's F10 + 10% fetal calf serum (F10FCS) or in F10FCS alone. After 4 days, the proportion of embryos developing into compact morulae or blastocysts was greater (P less than 0.005) in co-culture (38/82; 46%) than in F10FCS (1/27; 4%). In Exp. 2, a solution of collagenase, trypsin, DNAse and EDTA was used to disperse oviducal tissue, which was then cultured in TCM199 + 10% fetal calf serum (M199FCS) to obtain monolayers. Embryos (1-8 cells) were then co-cultured with monolayers or in M199FCS alone. The proportion of embryos developing into compact morulae and blastocysts after 4-5 days was higher (P less than 0.005) in co-culture (15/34; 43%) than in M199FCS (1/37; 3%); mean numbers of cells/embryo were also higher (P less than 0.001) (27.70; range 2-82 in co-culture; 8.83; range 2-18 in M199FCS). In Exp. 3, embryos obtained from in-vitro maturation and fertilization were used to compare development between co-culture and medium conditioned by oviducal tissue. Initial cleavage rate (no. embryos greater than 1 cell/total) was 76% (611/807) and did not differ among treatments. After 5 days, the proportion cleaving to greater than 16 cells was higher (P less than 0.005) in co-culture (71/203; 35%) and conditioned medium (48/205; 23%) compared to M199FCS (14/203; 7%). Similarly, the proportion developing into compact morulae and blastocysts was greater (P less than 0.005) in co-culture (44/203; 22%) and conditioned medium (46/205; 22%) than in M199FCS (7/203; 3%).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2704004

  9. Co-culture of early cattle embryos to the blastocyst stage with oviducal tissue or in conditioned medium.

    PubMed

    Eyestone, W H; First, N L

    1989-03-01

    In Exp. 1, 5-8-cell embryos from superovulated cattle were co-cultured with oviducal tissue suspended in Ham's F10 + 10% fetal calf serum (F10FCS) or in F10FCS alone. After 4 days, the proportion of embryos developing into compact morulae or blastocysts was greater (P less than 0.005) in co-culture (38/82; 46%) than in F10FCS (1/27; 4%). In Exp. 2, a solution of collagenase, trypsin, DNAse and EDTA was used to disperse oviducal tissue, which was then cultured in TCM199 + 10% fetal calf serum (M199FCS) to obtain monolayers. Embryos (1-8 cells) were then co-cultured with monolayers or in M199FCS alone. The proportion of embryos developing into compact morulae and blastocysts after 4-5 days was higher (P less than 0.005) in co-culture (15/34; 43%) than in M199FCS (1/37; 3%); mean numbers of cells/embryo were also higher (P less than 0.001) (27.70; range 2-82 in co-culture; 8.83; range 2-18 in M199FCS). In Exp. 3, embryos obtained from in-vitro maturation and fertilization were used to compare development between co-culture and medium conditioned by oviducal tissue. Initial cleavage rate (no. embryos greater than 1 cell/total) was 76% (611/807) and did not differ among treatments. After 5 days, the proportion cleaving to greater than 16 cells was higher (P less than 0.005) in co-culture (71/203; 35%) and conditioned medium (48/205; 23%) compared to M199FCS (14/203; 7%). Similarly, the proportion developing into compact morulae and blastocysts was greater (P less than 0.005) in co-culture (44/203; 22%) and conditioned medium (46/205; 22%) than in M199FCS (7/203; 3%).(ABSTRACT TRUNCATED AT 250 WORDS)

  10. Nervous necrosis virus replicates following the embryo development and dual infection with iridovirus at juvenile stage in grouper.

    PubMed

    Kuo, Hsiao-Che; Wang, Ting-Yu; Hsu, Hao-Hsuan; Chen, Peng-Peng; Lee, Szu-Hsien; Chen, Young-Mao; Tsai, Tieh-Jung; Wang, Chien-Kai; Ku, Hsiao-Tung; Lee, Gwo-Bin; Chen, Tzong-Yueh

    2012-01-01

    Infection of virus (such as nodavirus and iridovirus) and bacteria (such as Vibrio anguillarum) in farmed grouper has been widely reported and caused large economic losses to Taiwanese fish aquaculture industry since 1979. The multiplex assay was used to detect dual viral infection and showed that only nervous necrosis virus (NNV) can be detected till the end of experiments (100% mortality) once it appeared. In addition, iridovirus can be detected in a certain period of rearing. The results of real-time PCR and in situ PCR indicated that NNV, in fact, was not on the surface of the eggs but present in the embryo, which can continue to replicate during the embryo development. The virus may be vertically transmitted by packing into eggs during egg development (formation) or delivering into eggs by sperm during fertilization. The ozone treatment of eggs may fail to remove the virus, so a new strategy to prevent NNV is needed. PMID:22563447

  11. Blastomere removal from cleavage-stage mouse embryos alters placental function, which is associated with placental oxidative stress and inflammation

    PubMed Central

    Yao, Qi; Chen, Li; Liang, Yuanjiao; Sui, Liucai; Guo, Li; Zhou, Jingwei; Fan, Kai; Jing, Jun; Zhang, Yunhai; Yao, Bing

    2016-01-01

    Blastomere biopsy is an essential technique in preimplantation genetic diagnosis (PGD), a screening test that can detect genetic abnormalities of embryos before their transfer into uterus. Our results showed that the weights of fetuses derived from biopsied embryos were lower than that of non-biopsied counterparts at E12.5, E15.5, and E18.5. The ratio of fetal/placental (F/P) weights in the biopsied group was significantly lower than that in the non-biopsied group at E18.5. At E18.5, the mRNAs for selected glucose transporters, system A amino acid transporters, system L amino acid transporters, and imprinted genes were downregulated in the placentae of biopsied group, and the GLUT1 and CAT3 protein levels were decreased too. More apoptotic cells were detected by TUNEL in the placentae of biopsied group. Placentae from biopsied embryos exhibited lower levels of SOD and GSH. Furthermore, the concentration of MDA increased in the placentae from biopsied group. The levels of IL1B, IL6, and TNFA also significantly increased in the placentae of biopsied group. This study suggested that placental function may be sensitive to blastomere biopsy procedures, and placental oxidative stress and inflammation associated with blastomere biopsy may be critical factors of abnormal placental function and further influence the fetal development. PMID:27109212

  12. Roscovitine treatment improves synchronization of donor cell cycle in G0/G1 stage and in vitro development of handmade cloned buffalo (Bubalus bubalis) embryos.

    PubMed

    Selokar, Naresh L; Saini, Monika; Muzaffer, Mushariffa; Krishnakanth, G; Saha, Ambika P; Chauhan, Manmohan S; Manik, Radheysham; Palta, Prabhat; Madan, Pavneesh; Singla, Suresh K

    2012-04-01

    This study investigated the effects of serum-starvation, total confluence, and roscovitine treatment on cell-cycle synchronization of buffalo ear skin fibroblasts to the G0/G1 stage and on the developmental competence of cloned embryos. Serum starvation of total confluence cultures for 24 h had a higher (p<0.05) proportion of cells at G0/G1 stage (94.4%) compared with serum starved cyclic and nonstarved confluent cultures (76.8 and 86.0%, respectively), whereas differences between cyclic cells with or without serum starvation were not significant. The proportion of cells at G0/G1 was higher (p<0.05) with 20 and 30 μM roscovitine treatment than that with 10 μM (94.4, 96.4, and 86.6%, respectively), which was similar to that for total confluence (86.0%). MTT assay showed that cell viability decreased as dose of roscovitine increased. The blastocyst rate was significantly higher (p<0.05) when nuclear transfer embryos were reconstructed using donors cells from total confluence, confluence serum starved, and roscovitine-treated (20 and 30 μM) groups (48.8, 48.9, 57.9, and 62.9%, respectively) compared to nontreated cyclic cells (20.2%). However, the cleavage rate and total cell number of cloned embryos were similar for all the groups. The number of ICM cells was improved by 30 μM roscovitine treatment (45.25 ± 2.34). The cryosurvival rate of blastocysts derived from cells synchronized with 20 or 30 μM roscovitine was higher compared to that for total confluence group (33.6, 37.8 vs. 23.8%). In conclusion, treatment with 30 μM roscovitine is optimal for harvesting G0/G1 stage cells for producing high quality cloned buffalo embryos, and that it is better than serum-starvation or total confluence for cell synchronization.

  13. Urochordate Ascidians Possess a Single Isoform of Aurora Kinase That Localizes to the Midbody via TPX2 in Eggs and Cleavage Stage Embryos

    PubMed Central

    Hebras, Celine; McDougall, Alex

    2012-01-01

    Aurora kinases are key proteins found throughout the eukaryotes that control mitotic progression. Vertebrate Aurora-A and B kinases are thought to have evolved from a single Aurora-kinase isoform closest to that found in present day urochordates. In urochordate ascidians Aurora binds both TPX2 (a vertebrate AURKA partner) and INCENP (a vertebrate AURKB partner) and localizes to centrosomes and spindle microtubules as well as chromosomes and midbody during both meiosis and mitosis. Ascidian Aurora also displays this localization pattern during mitosis in echinoderms, strengthening the idea that non-vertebrate deuterostomes such as the urochordates and echinoderms possess a single form of Aurora kinase that has properties of vertebrate Aurora-kinase A and B. In the ascidian, TPX2 localizes to the centrosome and the spindle poles also as in vertebrates. However, we were surprised to find that TPX2 also localized strongly to the midbody in ascidian eggs and embryos. We thus examined more closely Aurora localization to the midbody by creating two separate point mutations of ascidian Aurora predicted to perturb binding to TPX2. Both forms of mutated Aurora behaved as predicted: neither localized to spindle poles where TPX2 is enriched. Interestingly, neither form of mutated Aurora localized to the midbody where TPX2 is also enriched, suggesting that ascidian Aurora midbody localization required TPX2 binding in ascidians. Functional analysis revealed that inhibition of Aurora kinase with a pharmacological inhibitor or with a dominant negative kinase dead form of Aurora caused cytokinesis failure and perturbed midbody formation during polar body extrusion. Our data support the view that vertebrate Aurora-A and B kinases evolved from a single non-vertebrate deuterostome ancestor. Moreover, since TPX2 localizes to the midbody in ascidian eggs and cleavage stage embryos it may be worthwhile re-assessing whether Aurora A kinase or TPX2 localize to the midbody in eggs and

  14. Accumulation and embryotoxicity of polystyrene nanoparticles at early stage of development of sea urchin embryos Paracentrotus lividus.

    PubMed

    Della Torre, C; Bergami, E; Salvati, A; Faleri, C; Cirino, P; Dawson, K A; Corsi, I

    2014-10-21

    Nanoplastic debris, resulted from runoff and weathering breakdown of macro- and microplastics, represents an emerging concern for marine ecosystems. The aim of the present study was to investigate disposition and toxicity of polystyrene nanoparticles (NPs) in early development of sea urchin embryos (Paracentrotus lividus). NPs with two different surface charges where chosen, carboxylated (PS-COOH) and amine (PS-NH2) polystyrene, the latter being a less common variant, known to induce cell death in several in vitro cell systems. NPs stability in natural seawater (NSW) was measured while disposition and embryotoxicity were monitored within 48 h of postfertilization (hpf). Modulation of genes involved in cellular stress response (cas8, 14-3-3ε, p-38 MAPK, Abcb1, Abcc5) was investigated. PS-COOH forms microaggregates (PDI > 0.4) in NSW, whereas PS-NH2 results are better dispersed (89 ± 2 nm) initially, though they also aggregated partially with time. Their respectively anionic and cationic nature was confirmed by ζ-potential measurements. No embryotoxicity was observed for PS-COOH up to 50 μg mL(-1) whereas PS-NH2 caused severe developmental defects (EC50 3.85 μg mL(-1) 24 hpf and EC50 2.61 μg mL(-1) 48 hpf). PS-COOH accumulated inside embryo's digestive tract while PS-NH2 were more dispersed. Abcb1 gene resulted up-regulated at 48 hpf by PS-COOH whereas PS-NH2 induced cas8 gene at 24 hpf, suggesting an apoptotic pathway. In line with the results obtained with the same PS NPs in several human cell lines, also in sea urchin embryos, differences in surface charges and aggregation in seawater strongly affect their embryotoxicity. PMID:25260196

  15. Laser microbeam-induced DNA damage inhibits cell division in fertilized eggs and early embryos.

    PubMed

    Wang, Zhong-Wei; Ma, Xue-Shan; Ma, Jun-Yu; Luo, Yi-Bo; Lin, Fei; Wang, Zhen-Bo; Fan, Heng-Yu; Schatten, Heide; Sun, Qing-Yuan

    2013-10-15

    DNA double-strand breaks are caused by both intracellular physiological processes and environmental stress. In this study, we used laser microbeam cut (abbreviated microcut or cut), which allows specific DNA damage in the pronucleus of a fertilized egg and in individual blastomere(s) of an early embryo, to investigate the response of early embryos to DNA double-strand breaks. Line type γH2AX foci were detected in the cut region, while Chk2 phosphorylation staining was observed in the whole nuclear region of the cut pronuclei or blastomeres. Zygotes with cut male or female pronucleus showed poor developmental capability: the percentage of cleavage embryos was significantly decreased, and the embryos failed to complete further development to blastocysts. The cut blastomeres in 2-cell, 4-cell, and 8-cell embryos ceased cleavage, and they failed to incorporate into compacted morulae, but instead underwent apoptosis and cell death at the blastocyst stage; the uncut part of embryos could develop to blastocysts, with a reduced percentage or decreased cell number. When both blastomeres of the 2-cell embryos were cut by laser microbeam, cell death occurred 24 h earlier, suggesting important functions of the uncut blastomere in delaying cell death of the cut blastomere. Taken together, we conclude that microbeam-induced DNA damage in early embryos causes compromised development, and that embryos may have their own mechanisms to exclude DNA-damaged blastomeres from participating in further development. PMID:24036543

  16. RNA-Seq Profiling of Intact and Enucleated Oocyte SCNT Embryos Reveals the Role of Pig Oocyte Nucleus in Somatic Reprogramming

    PubMed Central

    Bai, Lin; Li, Mengqi; Sun, Junli; Yang, Xiaogan; Lu, Yangqing; Lu, Shengsheng; Lu, Kehuan

    2016-01-01

    The specific molecular mechanisms involved in somatic reprogramming remain unidentified. Removal of the oocyte genome is one of the primary causes of developmental failure in cloned embryos, whereas intact oocyte shows stronger reprogramming capability than enucleated oocyte. To identify the reason for the low efficiency of cloning and elucidate the mechanisms involved in somatic reprogramming by the oocyte nucleus, we injected pig cumulus cells into 539 intact MII oocytes and 461 enucleated MII oocytes. Following activation, 260 polyploidy embryos developed to the blastocyst stage whereas only 93 traditionally cloned embryos (48.2% vs. 20.2%, P < 0.01) reached blastocyst stage. Blastocysts generated from intact oocytes also had more cells than those generated from enucleated oocytes (60.70 vs. 46.65, P < 0.01). To identify the genes that contribute to this phenomenon, two early embryos in 2-cell and 4-cell stages were collected for single-cell RNA sequencing. The two kinds of embryos were found to have dramatically different transcriptome profiles. Intact oocyte nuclear transfer embryos showed 1,738 transcripts that were up-regulated relative to enucleated cloned embryos at the 2-cell stage and 728 transcripts that were down-regulated (|log2Ratio| ≥ 5). They showed 2,941 transcripts that were up-regulated during the 4-cell stage and 1,682 that were down-regulated (|log2Ratio| ≥ 5). The most significantly enriched gene ontology categories were those involved in the regulation of binding, catalytic activity, and molecular transducer activity. Other genes that were notably up-regulated and expressed in intact oocyte nuclear transfer embryos were metabolic process. This study provides a comprehensive profile of the differences in gene expression between intact oocyte nuclear transfer embryos and traditional nuclear transfer embryos. This work thus paves the way for further research on the mechanisms underlying somatic reprogramming by oocytes. PMID:27070804

  17. RNA-Seq Profiling of Intact and Enucleated Oocyte SCNT Embryos Reveals the Role of Pig Oocyte Nucleus in Somatic Reprogramming.

    PubMed

    Bai, Lin; Li, Mengqi; Sun, Junli; Yang, Xiaogan; Lu, Yangqing; Lu, Shengsheng; Lu, Kehuan

    2016-01-01

    The specific molecular mechanisms involved in somatic reprogramming remain unidentified. Removal of the oocyte genome is one of the primary causes of developmental failure in cloned embryos, whereas intact oocyte shows stronger reprogramming capability than enucleated oocyte. To identify the reason for the low efficiency of cloning and elucidate the mechanisms involved in somatic reprogramming by the oocyte nucleus, we injected pig cumulus cells into 539 intact MII oocytes and 461 enucleated MII oocytes. Following activation, 260 polyploidy embryos developed to the blastocyst stage whereas only 93 traditionally cloned embryos (48.2% vs. 20.2%, P < 0.01) reached blastocyst stage. Blastocysts generated from intact oocytes also had more cells than those generated from enucleated oocytes (60.70 vs. 46.65, P < 0.01). To identify the genes that contribute to this phenomenon, two early embryos in 2-cell and 4-cell stages were collected for single-cell RNA sequencing. The two kinds of embryos were found to have dramatically different transcriptome profiles. Intact oocyte nuclear transfer embryos showed 1,738 transcripts that were up-regulated relative to enucleated cloned embryos at the 2-cell stage and 728 transcripts that were down-regulated (|log2Ratio| ≥ 5). They showed 2,941 transcripts that were up-regulated during the 4-cell stage and 1,682 that were down-regulated (|log2Ratio| ≥ 5). The most significantly enriched gene ontology categories were those involved in the regulation of binding, catalytic activity, and molecular transducer activity. Other genes that were notably up-regulated and expressed in intact oocyte nuclear transfer embryos were metabolic process. This study provides a comprehensive profile of the differences in gene expression between intact oocyte nuclear transfer embryos and traditional nuclear transfer embryos. This work thus paves the way for further research on the mechanisms underlying somatic reprogramming by oocytes. PMID:27070804

  18. Comparative Proteomic Analysis of Embryos between a Maize Hybrid and Its Parental Lines during Early Stages of Seed Germination

    PubMed Central

    Zhang, Guiping; Xing, Jiewen; Hu, Zhaorong; Feng, Wanjun; Yao, Yingyin; Peng, Huiru; Du, Jinkun; Zhang, Yirong; Ni, Zhongfu; Sun, Qixin

    2013-01-01

    In spite of commercial use of heterosis in agriculture, the molecular basis of heterosis is poorly understood. It was observed that maize hybrid Zong3/87-1 exhibited an earlier onset or heterosis in radicle emergence. To get insights into the underlying mechanism of heterosis in radicle emergence, differential proteomic analysis between hybrid and its parental lines was performed. In total, the number of differentially expressed protein spots between hybrid and its parental lines in dry and 24 h imbibed seed embryos were 134 and 191, respectively, among which 47.01% (63/134) and 34.55% (66/191) protein spots displayed nonadditively expressed pattern. Remarkably, 54.55% of nonadditively accumulated proteins in 24 h imbibed seed embryos displayed above or equal to the level of the higher parent patterns. Moreover, 155 differentially expressed protein spots were identified, which were grouped into eight functional classes, including transcription & translation, energy & metabolism, signal transduction, disease & defense, storage protein, transposable element, cell growth & division and unclassified proteins. In addition, one of the upregulated proteins in F1 hybrids was ZmACT2, a homolog of Arabidopsis thaliana ACT7 (AtACT7). Expressing ZmACT2 driven by the AtACT7 promoter partially complemented the low germination phenotype in the Atact7 mutant. These results indicated that hybridization between two parental lines can cause changes in the expression of a variety of proteins, and it is concluded that the altered pattern of gene expression at translational level in the hybrid may be responsible for the observed heterosis. PMID:23776561

  19. Transcriptome Analysis of Pig In Vivo, In Vitro–Fertilized, and Nuclear Transfer Blastocyst-Stage Embryos Treated with Histone Deacetylase Inhibitors Postfusion and Activation Reveals Changes in the Lysosomal Pathway

    PubMed Central

    Whitworth, Kristin M.; Mao, Jiude; Lee, Kiho; Spollen, William G.; Samuel, Melissa S.; Walters, Eric M.; Spate, Lee D.

    2015-01-01

    Abstract Genetically modified pigs are commonly created via somatic cell nuclear transfer (SCNT). Treatment of reconstructed embryos with histone deacetylase inhibitors (HDACi) immediately after activation improves cloning efficiency. The objective of this experiment was to evaluate the transcriptome of SCNT embryos treated with suberoylanilide hydroxamic acid (SAHA), 4-iodo-SAHA (ISAHA), or Scriptaid as compared to untreated SCNT, in vitro–fertilized (IVF), and in vivo (IVV) blastocyst-stage embryos. SAHA (10 μM) had the highest level of blastocyst development at 43.9%, and all treatments except 10 μM ISAHA had the same percentage of blastocyst development as Scriptaid (p<0.05). Two treatments, 1.0 μM ISAHA and 1.0 μM SAHA, had higher mean cell number than No HDACi treatment (p<0.021). Embryo transfers performed with 10 μM SAHA- and 1 μM ISAHA-treated embryos resulted in the birth of healthy piglets. GenBank accession numbers from up- and downregulated transcripts were loaded into the Database for Annotation, Visualization and Integrated Discovery to identify enriched biological themes. HDACi treatment yielded the highest enrichment for transcripts within the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway, lysosome. The mean intensity of LysoTracker was lower in IVV embryos compared to IVF and SCNT embryos (p<0.0001). SAHA and ISAHA can successfully be used to create healthy piglets from SCNT. PMID:26731590

  20. Utilization of endogenous fatty acid stores for energy production in bovine preimplantation embryos.

    PubMed

    Sutton-McDowall, Melanie L; Feil, Deanne; Robker, Rebecca L; Thompson, Jeremy G; Dunning, Kylie R

    2012-05-01

    Although current embryo culture media are based on carbohydrate metabolism of embryos, little is known about metabolism of endogenous lipids. L-carnitine is a β-oxidation cofactor absent in most culture media. The objective was to investigate the influence of L-carnitine supplementation on bovine embryo development. Abattoir-derived bovine cumulus oocyte complexes were cultured and fertilized. Post-fertilization, presumptive zygotes were transferred into a basic cleavage medium ± carbohydrates (glucose, lactate and pyruvate) ± 5 mm L-carnitine and cultured for 4 days in vitro. In the absence of carbohydrates during culture, embryos arrested at the 2- and 4-cell stages. Remarkably, +L-carnitine increased development to the morula stage compared to +carbohydrates alone (P < 0.001). The beneficial effects of L-carnitine were further demonstrated by inclusion of carbohydrates, with 14-fold more embryos reaching the morula stage after culture in the +carbohydrates +L-carnitine group compared to the +carbohydrates group (P < 0.05). Whereas there was a trend for +L-carnitine to increase ATP (P = 0.09), ADP levels were higher and ATP: ADP ratio were 1.9-fold lower (main effect, P < 0.05) compared to embryos cultured in -L-carnitine. Therefore, we inferred that +L-carnitine embryos were more metabolically active, with higher rates of ATP-ADP conversion. In conclusion, L-carnitine supplementation supported precompaction embryo development and there was an additive effect of +L-carnitine +carbohydrates on early embryo development, most likely through increased β-oxidation within embryos.

  1. Characterization of developmental arrest in early bovine embryos cultured in vitro.

    PubMed

    Eyestone, W H; First, N L

    1991-03-01

    The susceptibility of early bovine embryos to developmental arrest ("blocking") in vitro was examined. Embryos, obtained from superovulated donors, were cultured in vitro in Ham's F10 culture medium or in vivo in sheep oviducts. Treatments were terminated on Day 7 post-donor estrus (estrus = day 0), and the embryos were evaluated for development. Experiment 1 tested whether the 8- to 16-cell block was reversible. One- to two-cell embryos were cultured in vitro to the 8-cell stage (2 d), then in vivo for 3 d; controls were cultured in vitro or in vivo for 5 d. Forty-two percent (19/45) of in vivo controls developed normally; none (0/55; 0%) of the in vitro controls cleaved past the 9- to 16-cell stage. Only 4% (2/48) of the embryos cultured to eight cells in vitro developed normally after culture in sheep oviducts, indicating that the block was irreversible. Irreversibility was not caused by overt cell death, since 33/33 (100%) of blocked embryos responded positively to fluorescein diacetate vital staining. Experiment 2 tested the effect of in vitro exposure at specific cell stages on subsequent in vivo development. Embryos at the 1- to 2-, 3- to 4-, 5- to 8- and 9- to 16-cell stages were assigned randomly to one of the following treatments: in vivo culture; in vitro culture; or 24 h in vitro culture, followed by in vivo culture. Subsequent in vivo development was affected by 24 h of in vitro culture (P<0.05) only in 3- to 4-cell embryos (11/41, 27% vs 22/41, 54% for in vivo controls). We conclude that 1) the block is a manifestation of in vitro exposure during the four- to eight-cell stage, and 2) the block, while irreversible, is not the result of overt embryonic death. PMID:16726930

  2. Characterization of developmental arrest in early bovine embryos cultured in vitro.

    PubMed

    Eyestone, W H; First, N L

    1991-03-01

    The susceptibility of early bovine embryos to developmental arrest ("blocking") in vitro was examined. Embryos, obtained from superovulated donors, were cultured in vitro in Ham's F10 culture medium or in vivo in sheep oviducts. Treatments were terminated on Day 7 post-donor estrus (estrus = day 0), and the embryos were evaluated for development. Experiment 1 tested whether the 8- to 16-cell block was reversible. One- to two-cell embryos were cultured in vitro to the 8-cell stage (2 d), then in vivo for 3 d; controls were cultured in vitro or in vivo for 5 d. Forty-two percent (19/45) of in vivo controls developed normally; none (0/55; 0%) of the in vitro controls cleaved past the 9- to 16-cell stage. Only 4% (2/48) of the embryos cultured to eight cells in vitro developed normally after culture in sheep oviducts, indicating that the block was irreversible. Irreversibility was not caused by overt cell death, since 33/33 (100%) of blocked embryos responded positively to fluorescein diacetate vital staining. Experiment 2 tested the effect of in vitro exposure at specific cell stages on subsequent in vivo development. Embryos at the 1- to 2-, 3- to 4-, 5- to 8- and 9- to 16-cell stages were assigned randomly to one of the following treatments: in vivo culture; in vitro culture; or 24 h in vitro culture, followed by in vivo culture. Subsequent in vivo development was affected by 24 h of in vitro culture (P<0.05) only in 3- to 4-cell embryos (11/41, 27% vs 22/41, 54% for in vivo controls). We conclude that 1) the block is a manifestation of in vitro exposure during the four- to eight-cell stage, and 2) the block, while irreversible, is not the result of overt embryonic death.

  3. Label free cell-tracking and division detection based on 2D time-lapse images for lineage analysis of early embryo development.

    PubMed

    Cicconet, Marcelo; Gutwein, Michelle; Gunsalus, Kristin C; Geiger, Davi

    2014-08-01

    In this paper we report a database and a series of techniques related to the problem of tracking cells, and detecting their divisions, in time-lapse movies of mammalian embryos. Our contributions are (1) a method for counting embryos in a well, and cropping each individual embryo across frames, to create individual movies for cell tracking; (2) a semi-automated method for cell tracking that works up to the 8-cell stage, along with a software implementation available to the public (this software was used to build the reported database); (3) an algorithm for automatic tracking up to the 4-cell stage, based on histograms of mirror symmetry coefficients captured using wavelets; (4) a cell-tracking database containing 100 annotated examples of mammalian embryos up to the 8-cell stage; and (5) statistical analysis of various timing distributions obtained from those examples. PMID:24873887

  4. Label Free Cell-Tracking and Division Detection Based on 2D Time-Lapse Images For Lineage Analysis of Early Embryo Development

    PubMed Central

    Cicconet, Marcelo; Gutwein, Michelle; Gunsalus, Kristin C; Geiger, Davi

    2014-01-01

    In this paper we report a database and a series of techniques related to the problem of tracking cells, and detecting their divisions, in time-lapse movies of mammalian embryos. Our contributions are: (1) a method for counting embryos in a well, and cropping each individual embryo across frames, to create individual movies for cell tracking; (2) a semi-automated method for cell tracking that works up to the 8-cell stage, along with a software implementation available to the public (this software was used to build the reported database); (3) an algorithm for automatic tracking up to the 4-cell stage, based on histograms of mirror symmetry coefficients captured using wavelets; (4) a cell-tracking database containing 100 annotated examples of mammalian embryos up to the 8-cell stage; (5) statistical analysis of various timing distributions obtained from those examples. PMID:24873887

  5. Gene Coexpression and Evolutionary Conservation Analysis of the Human Preimplantation Embryos

    PubMed Central

    Liu, Tiancheng; Yu, Lin; Ding, Guohui; Wang, Zhen; Liu, Lei; Li, Hong; Li, Yixue

    2015-01-01

    Evolutionary developmental biology (EVO-DEVO) tries to decode evolutionary constraints on the stages of embryonic development. Two models—the “funnel-like” model and the “hourglass” model—have been proposed by investigators to illustrate the fluctuation of selective pressure on these stages. However, selective indices of stages corresponding to mammalian preimplantation embryonic development (PED) were undetected in previous studies. Based on single cell RNA sequencing of stages during human PED, we used coexpression method to identify gene modules activated in each of these stages. Through measuring the evolutionary indices of gene modules belonging to each stage, we observed change pattern of selective constraints on PED for the first time. The selective pressure decreases from the zygote stage to the 4-cell stage and increases at the 8-cell stage and then decreases again from 8-cell stage to the late blastocyst stages. Previous EVO-DEVO studies concerning the whole embryo development neglected the fluctuation of selective pressure in these earlier stages, and the fluctuation was potentially correlated with events of earlier stages, such as zygote genome activation (ZGA). Such oscillation in an earlier stage would further affect models of the evolutionary constraints on whole embryo development. Therefore, these earlier stages should be measured intensively in future EVO-DEVO studies. PMID:26273607

  6. Gene Coexpression and Evolutionary Conservation Analysis of the Human Preimplantation Embryos.

    PubMed

    Liu, Tiancheng; Yu, Lin; Ding, Guohui; Wang, Zhen; Liu, Lei; Li, Hong; Li, Yixue

    2015-01-01

    Evolutionary developmental biology (EVO-DEVO) tries to decode evolutionary constraints on the stages of embryonic development. Two models--the "funnel-like" model and the "hourglass" model--have been proposed by investigators to illustrate the fluctuation of selective pressure on these stages. However, selective indices of stages corresponding to mammalian preimplantation embryonic development (PED) were undetected in previous studies. Based on single cell RNA sequencing of stages during human PED, we used coexpression method to identify gene modules activated in each of these stages. Through measuring the evolutionary indices of gene modules belonging to each stage, we observed change pattern of selective constraints on PED for the first time. The selective pressure decreases from the zygote stage to the 4-cell stage and increases at the 8-cell stage and then decreases again from 8-cell stage to the late blastocyst stages. Previous EVO-DEVO studies concerning the whole embryo development neglected the fluctuation of selective pressure in these earlier stages, and the fluctuation was potentially correlated with events of earlier stages, such as zygote genome activation (ZGA). Such oscillation in an earlier stage would further affect models of the evolutionary constraints on whole embryo development. Therefore, these earlier stages should be measured intensively in future EVO-DEVO studies.

  7. Global Gene Expression Profiling of Individual Human Oocytes and Embryos Demonstrates Heterogeneity in Early Development

    PubMed Central

    Zeef, Leo; Kimber, Susan J.; Brison, Daniel R.

    2013-01-01

    Early development in humans is characterised by low and variable embryonic viability, reflected in low fecundity and high rates of miscarriage, relative to other mammals. Data from assisted reproduction programmes provides additional evidence that this is largely mediated at the level of embryonic competence and is highly heterogeneous among embryos. Understanding the basis of this heterogeneity has important implications in a number of areas including: the regulation of early human development, disorders of pregnancy, assisted reproduction programmes, the long term health of children which may be programmed in early development, and the molecular basis of pluripotency in human stem cell populations. We have therefore investigated global gene expression profiles using polyAPCR amplification and microarray technology applied to individual human oocytes and 4-cell and blastocyst stage embryos. In order to explore the basis of any variability in detail, each developmental stage is replicated in triplicate. Our data show that although transcript profiles are highly stage-specific, within each stage they are relatively variable. We describe expression of a number of gene families and pathways including apoptosis, cell cycle and amino acid metabolism, which are variably expressed and may be reflective of embryonic developmental competence. Overall, our data suggest that heterogeneity in human embryo developmental competence is reflected in global transcript profiles, and that the vast majority of existing human embryo gene expression data based on pooled oocytes and embryos need to be reinterpreted. PMID:23717564

  8. Observations of turkey eggs stored up to 27 days and incubated for 8 days: embryo developmental stage and weight differences and the differentiation of fertilized from unfertilized germinal discs.

    PubMed

    Bakst, M R; Welch, G R; Camp, M J

    2016-05-01

    For logistical reasons, egg storage prior to incubation is a growing practice in the commercial turkey industry. Yet the consequence of increasing egg storage over 7 d is a progressive increase in embryo mortality. The objective of this study was to provide the information necessary to differentiate an early dead embryo from an unfertilized egg after 8 days of incubation (DOI). Five groups of eggs each from inseminated and virgin hens were stored for progressively increasing periods of time (5-d or less, 6 to 10 d, 11 to 15 d, 16 to 20 d, and 21 to 27 d) and incubated. At 8 DOI, eggs were examined and the stage of development (Hamburger and Hamilton, 1951) and embryo weights in normally developed eggs were determined. There was a significant negative correlation between the stage of development and embryo weight with increasing storage periods. All remaining eggs from the inseminated and virgin hens were broken-out and the appearance of the yolk and the fertilized and unfertilized germinal discs examined. The yolks of both hen groups with unfertilized ova maintained a homogeneous uniform yellow-orange color. In contrast, yolks of ova that had been fertilized, with or without early-dead embryos, and yolks from virgin hens that showed evidence of parthenogenetic development (3%) had a heterogeneous appearance. Using fluorescence microscopy, the heterogeneous appearance was due to sheets of aberrant cells and less frequently dispersed cells and folds of the perivitelline layer. It was concluded that clear egg breakouts need to be performed to more accurately assess the impact of egg storage on embryonic mortality. Furthermore, such breakouts should be performed with a high intensity light directed across the surface of the germinal disc to clearly differentiate the subtle differences between an early-dead embryo and an unfertilized germinal disc. PMID:26957633

  9. Observations of turkey eggs stored up to 27 days and incubated for 8 days: embryo developmental stage and weight differences and the differentiation of fertilized from unfertilized germinal discs.

    PubMed

    Bakst, M R; Welch, G R; Camp, M J

    2016-05-01

    For logistical reasons, egg storage prior to incubation is a growing practice in the commercial turkey industry. Yet the consequence of increasing egg storage over 7 d is a progressive increase in embryo mortality. The objective of this study was to provide the information necessary to differentiate an early dead embryo from an unfertilized egg after 8 days of incubation (DOI). Five groups of eggs each from inseminated and virgin hens were stored for progressively increasing periods of time (5-d or less, 6 to 10 d, 11 to 15 d, 16 to 20 d, and 21 to 27 d) and incubated. At 8 DOI, eggs were examined and the stage of development (Hamburger and Hamilton, 1951) and embryo weights in normally developed eggs were determined. There was a significant negative correlation between the stage of development and embryo weight with increasing storage periods. All remaining eggs from the inseminated and virgin hens were broken-out and the appearance of the yolk and the fertilized and unfertilized germinal discs examined. The yolks of both hen groups with unfertilized ova maintained a homogeneous uniform yellow-orange color. In contrast, yolks of ova that had been fertilized, with or without early-dead embryos, and yolks from virgin hens that showed evidence of parthenogenetic development (3%) had a heterogeneous appearance. Using fluorescence microscopy, the heterogeneous appearance was due to sheets of aberrant cells and less frequently dispersed cells and folds of the perivitelline layer. It was concluded that clear egg breakouts need to be performed to more accurately assess the impact of egg storage on embryonic mortality. Furthermore, such breakouts should be performed with a high intensity light directed across the surface of the germinal disc to clearly differentiate the subtle differences between an early-dead embryo and an unfertilized germinal disc.

  10. Correlative scanning electron, transmission electron, and light microscopic studies of the in vitro development of mouse embryos on a plastic substrate at the implantation stage.

    PubMed

    Gonda, M A; Hsu, Y C

    1980-04-01

    Correlative scanning electron, transmission electron, and light microscopy were utilized to study the morphogenic events occurring during mouse blastocyst outgrowth and early-egg-cylinder development in vitro. After hatching and attachment of blastocysts on the plastic surface, the blastocoelic cavity collapses as the mural trophoblasts spread and migrate outward. The inner cell mass is covered with a differentiated endoderm on the blastocoelic cavity side and by the polar trophoblasts on the medium side at this stage. As the endoderm-covered inner cell mass proliferates, being physically restricted from further downward expansion by the plastic coverslip and by lack of space in the collapsed blastocoelic cavity, it migrates upward and protrudes into the culture medium in a break between the polar and mural trophoblast cells. Polar trophoblast cells apposed to the base of the egg cylinder continue to proliferate forming the ectoplacental cone. Thus, the early egg cylinder lacking a trophoblast barrier begins inverting its growth pattern from towards the culture dish surface to a more upright position. Egg-cylinder development in vitro from the inner cell mass and polar trophoblast cells closely paralleled in vivo. The functional nature of various embryonic cell types observed in these embryos was revealed by scanning electron microscopy. These studies as well as those of Wiley and Pedersen (1977) suggest that blastocysts can serve as a source of in vitro developing early mouse egg cylinders that appear to resemble their in vivo counterparts and can be used in experimental studies of mouse embryogenesis.

  11. Combined progesterone (IM + V) versus vaginal progesterone for luteal support in cleavage-stage embryo transfer cycles of good prognosis patients.

    PubMed

    Pabuccu, E G; Pabuccu, R; Evliyaoglu Ozdegirmenci, O; Bostancı Durmus, A; Keskin, M

    2016-01-01

    Many reports led to the consensus on the use of progesterone (P) for luteal-phase support. Vaginal P application is the method of choice due to its simplicity and high patient convenience but is hampered by application difficulties and personal or cultural aversions. Inappropriate vaginal P use may alter successful implantation, leading physicians to consider alternate P application routes. A worldwide survey revealed that intramuscular plus vaginal P (combined P) is the method used in nearly one-third of in vitro fertilization (IVF) cycles, particularly in Asia and North America; unfortunately, the outcomes of this approach have not been clearly elucidated. In the current analysis, we evaluated any additional benefit of short course parenteral P in addition to vaginal P capsules during a specific period in terms of implantation, pregnancy rates, miscarriages and ectopic pregnancies in cleavage stage embryo transfer (ET) cycles of good-prognosis patients. Despite significantly higher implantation rates in the combined arm, clinical and ongoing pregnancies were comparable in both groups, whereas a trend toward increased pregnancy rates was observed with combined support. The available data are too limited to draw conclusions.

  12. A semi-dominant mutation in the general splicing factor SF3a66 causes anterior-posterior axis reversal in one-cell stage C. elegans embryos.

    PubMed

    Keikhaee, Mohammad R; Nash, Eric B; O'Rourke, Sean M; Bowerman, Bruce

    2014-01-01

    Establishment of anterior-posterior polarity in one-cell stage Caenorhabditis elegans embryos depends in part on astral microtubules. As the zygote enters mitosis, these microtubules promote the establishment of a posterior pole by binding to and protecting a cytoplasmic pool of the posterior polarity protein PAR-2 from phosphorylation by the cortically localized anterior polarity protein PKC-3. Prior to activation of the sperm aster, the oocyte Meiosis I and II spindles assemble and function, usually at the future anterior pole, but these meiotic spindle microtubules fail to establish posterior polarity through PAR-2. Here we show that a semi-dominant mutation in the general splicing factor SF3a66 can lead to a reversed axis of AP polarity that depends on PAR-2 and possibly on close proximity of oocyte meiotic spindles with the cell cortex. One possible explanation is that reduced levels of PKC-3, due to a general splicing defect, can result in axis reversal due to a failure to prevent oocyte meiotic spindle microtubules from interfering with AP axis formation. PMID:25188372

  13. 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. PMID:27064112

  14. 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.

  15. Absence of nucleolus formation in raccoon dog-porcine interspecies somatic cell nuclear transfer embryos results in embryonic developmental failure

    PubMed Central

    JEON, Yubyeol; NAM, Yeong-Hee; CHEONG, Seung-A; KWAK, Seong-Sung; LEE, Eunsong; HYUN, Sang-Hwan

    2016-01-01

    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. PMID:27064112

  16. New techniques on embryo manipulation.

    PubMed

    Escribá, M J; Valbuena, D; Remohí, J; Pellicer, A; Simón, C

    2002-01-01

    For many years, experience has been accumulated on embryo and gamete manipulation in livestock animals. The present work is a review of these techniques and their possible application in human embryology in specific cases. It is possible to manipulate gametes at different levels, producing paternal or maternal haploid embryos (hemicloning), using different techniques including nuclear transfer. At the embryonic stage, considering practical, ethical and legal issues, techniques will be reviewed that include cloning and embryo splitting at the cleavage stage, morula, or blastocyst stage.

  17. Sterilising embryos for transgenic chimaeras.

    PubMed

    Aige-Gil, V; Simkiss, K

    1991-07-01

    1. Experiments were undertaken to attempt to sterilise fowl embryos with ultraviolet light. Such sterilised embryos would be useful as recipients of genetically manipulated germ cells. 2. The germinal crescents of embryos were exposed to a calibrated UV source at stages 4 and 8 to 10 of incubation for 30 s, 3 min and 10 min. Teratological and sterility effects were studied at periods up to 6 d of incubation. 3. Simply exposing embryos by opening the shell produced a number of abnormalities and mortalities. These decreased with the age of the embryo but increased with the dosage of irradiation. 4. Although there was abundant evidence for UV-induced cell damage, the sterility of the embryos was usually less than 75%. PMID:1893258

  18. Exposure time to caffeine affects heartbeat and cell damage-related gene expression of zebrafish Danio rerio embryos at early developmental stages.

    PubMed

    Abdelkader, Tamer Said; Chang, Seo-Na; Kim, Tae-Hyun; Song, Juha; Kim, Dong Su; Park, Jae-Hak

    2013-11-01

    Caffeine is white crystalline xanthine alkaloid that is naturally found in some plants and can be produced synthetically. It has various biological effects, especially during pregnancy and lactation. We studied the effect of caffeine on heartbeat, survival and the expression of cell damage related genes, including oxidative stress (HSP70), mitochondrial metabolism (Cyclin G1) and apoptosis (Bax and Bcl2), at early developmental stages of zebrafish embryos. We used 100 µm concentration based on the absence of locomotor effects. Neither significant mortality nor morphological changes were detected. We monitored hatching at 48 h post-fertilization (hpf) to 96 hpf. At 60 and 72 hpf, hatching decreased significantly (P < 0.05); however, the overall hatching rate at 96 hpf was 94% in control and 93% in caffeine treatment with no significant difference (P > 0.05). Heartbeats per minute were 110, 110 and 112 in control at 48, 72 and 96 hpf, respectively. Caffeine significantly increased heartbeat - 122 and 136 at 72 and 96 hpf, respectively. Quantitative RT-PCR showed significant up-regulation after caffeine exposure in HSP70 at 72 hpf; in Cyclin G1 at 24, 48 and 72 hpf; and in Bax at 48 and 72 hpf. Significant down-regulation was found in Bcl2 at 48 and 72 hpf. The Bax/Bcl2 ratio increased significantly at 48 and 72 hpf. We conclude that increasing exposure time to caffeine stimulates oxidative stress and may trigger apoptosis via a mitochondrial-dependent pathway. Also caffeine increases heartbeat from early phases of development without affecting the morphology and survival but delays hatching. Use of caffeine during pregnancy and lactation may harm the fetus by affecting the expression of cell-damage related genes.

  19. Prion Protein and Stage Specific Embryo Antigen 1 as Selection Markers to Enrich the Fraction of Murine Embryonic Stem Cell-Derived Cardiomyocytes

    PubMed Central

    Ikeda, Nobuhito; Nakayama, Yuji; Nakazawa, Natsumi; Yoshida, Akio; Ninomiya, Haruaki; Shirayoshi, Yasuaki

    2016-01-01

    Background The prion protein (PrP) might be useful as a tool to collect cardiac progenitor cells derived from embryonic stem (ES) cells. It is also possible that PrP+ cells include undifferentiated cells with a capacity to develop into tumors. Methods PrP+ cells isolated from embryoid bodies (EB) formed by mouse AB1 ES cells were examined using RT–PCR analysis and clonogeneic cell assay. To assess their potential to differentiate into cardiomyocytes, Nkx2.5GFP/+ (hcgp7) cells, another ES cell line that carries the GFP reporter gene in the Nkx2.5 loci, were used. Results PrP+ cells isolated from EB of day 7 and 14 did not express pluripotency markers, but expressed cardiac cell markers, while PrP+ cells isolated from EB of day 21 expressed pluripotency markers. Cultured PrP+ cells isolated from EB of day 21 expressed pluripotency markers to form colonies, whereas those isolated from EB of day 7 and 14 did not. To exclude proliferating cells from PrP+ cells, stage specific embryo antigen 1 (SSEA1) was employed as a second marker. PrP+/SSEA1– cells did not proliferate and expressed cardiac cell markers, while PrP+/SSEA1+ did proliferate. Conclusion PrP+ cells isolated from EB included undifferentiated cells in day 21. PrP+/SSEA1– cells included cardiomyoctes, suggesting PrP and SSEA1 may be useful as markers to enrich the fraction of cardiomyocytes. PMID:27493483

  20. Immature embryo rescue and culture.

    PubMed

    Shen, Xiuli; Gmitter, Fred G; Grosser, Jude W

    2011-01-01

    Embryo culture techniques have many significant applications in plant breeding, as well as basic studies in physiology and biochemistry. Immature embryo rescue and culture is a particularly attractive technique for recovering plants from sexual crosses where the majority of embryos cannot survive in vivo or become dormant for long periods of time. Overcoming embryo inviability is the most common reason for the application of embryo rescue techniques. Recently, fruit breeding programs have greatly increased the interest in exploiting interploid hybridization to combine desirable genetic traits of complementary parents at the triploid level for the purpose of developing improved seedless fruits. However, the success of this approach has only been reported in limited number of species due to various crossing barriers and embryo abortion at very early stages. Thus, immature embryo rescue provides an alternative means to recover triploid hybrids, which usually fail to completely develop in vivo. This chapter will provide a brief discussion of the utilization of interploid crosses between a monoembryonic diploid female with an allotetraploid male in a citrus cultivar improvement program, featuring a clear and comprehensive illustration of successful protocols for immature embryo rescue and culture. The protocols will cover the complete process from embryo excision to recovered plant in the greenhouse and can easily be adapted to other plant commodities. Factors affecting the success and failure of immature embryo rescue to recover triploid progeny from interploid crosses will be discussed.

  1. Effect of microinjection of a single IVF-derived blastomere on the development of cloned embryos at the eight-cell stage in bovine.

    PubMed

    Chen, Xiuping; Wang, Jianwu; Li, Rong; Ding, Fangrong; Li, Song; Zhang, Lei; Dai, Yunping; Li, Ning

    2010-12-01

    This study was conducted to determine the effect of microinjection of a single blastomere from in vitro fertilization (IVF)-derived eight-cell embryo into eight-cell cloned embryos harboring the gene encoding recombinant human lactoferrin (rhLF), GFP, and NEO markers in bovine. The reconstructed chimeric embryos were assessed for their development to blastocyst, or to term after transfer, and tissues of offspring were evaluated by polymerase chain reaction (PCR) for the presence of nuclear transfer (NT)-derived transgenic cells, and the cloned embryos without microinjection were used as controls. The chimeric embryos showed slightly higher blastocyst rate than that for controls. The single IVF-derived blastomere appeared to preferential contribute to inner cell mass (ICM) in the chimeric blastocysts. After transfer, the rates of development of chimeric embryos to day 60, to term, and to weaning were significantly higher than those of controls. Sixty-three chimeric blastocysts were transferred and 11 calves were born: 7 calves of them were dead, and the remaining 4 calves are apparently normal and healthy. Most of the tissues collected from dead fetus were transgenic, whereas NT-derived transgenic cells were not detected in some tissues of the living calves. Our results indicated that a single blastomere from IVF-derived eight-cell embryo improves the in vivo developmental potential of transgenic cloned eight-cell embryos in bovine; however, the single IVF-derived blastomere appears to be better able to populate the ICM and many tissues of offspring than NT-derived blastomeres.

  2. A role for histamine in cardiovascular regulation in late stage embryos of the red-footed tortoise, Chelonoidis carbonaria Spix, 1824.

    PubMed

    Crossley, Dane A; Sartori, Marina R; Abe, Augusto S; Taylor, Edwin W

    2013-08-01

    A chorioallantoic membrane artery in embryos of the red-footed tortoise, Chelonoidis carbonaria was occlusively cannulated for measurement of blood pressure and injection of drugs. Two age groups of embryos in the final 10 % of incubation were categorized by the ratio of embryonic body to yolk mass. All embryos first received cholinergic and β-adrenergic blockade. This revealed that β-adrenergic control was established in both groups whereas cholinergic control was only established in the older group immediately prior to hatching. The study then progressed as two series. Series one was conducted in a subset of embryos treated with histamine before or after injection of ranitidine, the antagonist of H2 receptors. Injection of histamine caused an initial phasic hypertension which recovered, followed by a longer lasting hypertensive response accompanied by a tachycardia. Injection of the H2 receptor antagonist ranitidine itself caused a hypotensive tachycardia with subsequent recovery of heart rate. Ranitidine also abolished the cardiac effects of histamine injection while leaving the initial hypertensive response intact. In series, two embryos were injected with histamine after injection of diphenhydramine, the antagonist to H1 receptors. This abolished the whole of the pressor response to histamine injection but left the tachycardic response intact. These data indicate that histamine acts as a non-adrenergic, non-cholinergic factor, regulating the cardiovascular system of developing reptilian embryos and that its overall effects are mediated via both H1 and H2 receptor types.

  3. Antisense inhibition of cyclin D1 expression is equivalent to flavopiridol for radiosensitization of zebrafish embryos

    SciTech Connect

    McAleer, Mary Frances; Duffy, Kevin T.; Davidson, William R.; Kari, Gabor; Dicker, Adam P.; Rodeck, Ulrich; Wickstrom, Eric . E-mail: eric@tesla.jci.tju.edu

    2006-10-01

    Purpose: Flavopiridol, a small molecule pan-cyclin inhibitor, has been shown to enhance Radiation response of tumor cells both in vitro and in vivo. The clinical utility of flavopiridol, however, is limited by toxicity, previously attributed to pleiotropic inhibitory effects on several targets affecting multiple signal transduction pathways. Here we used zebrafish embryos to investigate radiosensitizing effects of flavopiridol in normal tissues. Methods and Materials: Zebrafish embryos at the 1- to 4-cell stage were treated with 500 nM flavopiridol or injected with 0.5 pmol antisense hydroxylprolyl-phosphono nucleic acid oligomers to reduce cyclin D1 expression, then subjected to ionizing radiation (IR) or no radiation. Results: Flavopiridol-treated embryos demonstrated a twofold increase in mortality after exposure to 40 Gy by 96 hpf and developed distinct radiation-induced defects in midline development (designated as the 'curly up' phenotype) at higher rates when compared with embryos receiving IR only. Cyclin D1-deficient embryos had virtually identical IR sensitivity profiles when compared with embryos treated with flavopiridol. This was particularly evident for the IR-induced curly up phenotype, which was greatly exacerbated by both flavopriridol and cyclin D1 downregulation. Conclusions: Treatment of zebrafish embryos with flavopiridol enhanced radiation sensitivity of zebrafish embryos to a degree that was very similar to that associated with downregulation of cyclin D1 expression. These results are consistent with the hypothesis that inhibition of cyclin D1 is sufficient to account for the radiosensitizing action of flavopiridol in the zebrafish embryo vertebrate model.

  4. Improving efficiencies of locus-specific DNA methylation assessment for bovine in vitro produced embryos.

    PubMed

    Wroclawska, Ewa; Brant, Jason O; Yang, Thomas P; Moore, Karen

    2010-02-01

    Characterization of DNA methylation is one assessment of chromatin remodeling in early embryos. Unfortunately, evaluation at specific loci is hindered by their small cell numbers. Our objective was to determine if bisulfite sequencing could be optimized for preimplantation embryos, comparing conversion times, primer design, and DNA amplification methods. Methylation at three loci, SATI, OCT4, and IGF2, was investigated in bovine in vitro produced (IVP) embryos, somatic cells, and no template controls. Bisulfite treatment for 15-16 h gave higher quality DNA than treatment for 18 h. Three step primer design improved bisulfite primer specificity, yielding more PCR product than primers previously reported. Following optimization, methylation data were obtained from as few as 4 cell equivalents. Finally, DNA amplification efficiencies were evaluated using miniprep, TempliPhi, or 96-well glycerol stocks with automated TempliPhi. While TempliPhi was better than standard minipreps, the 96-well format proved most efficient. Preliminary methylation profiles of bovine IVP 2-cell, 8-cell, blastocyst stage embryos and somatic cells were 25, 10, 22, and 74% for SATI and 88, 88, 79, and 88% for OCT4, respectively, suggesting that SATI is demethylated during early embryonic reprogramming, while OCT4 remains hypermethylated. IGF2 methylation was 84, 28, and 84% for bovine IVP 8-cell, blastocyst stage embryos and somatic cells; blastocyst stage embryos exhibited more variability, ranging from 0 to 80%. This new assay will enhance assessment of chromatin remodeling in embryos, and be especially useful for evaluating those produced by assisted reproductive technologies. PMID:20170282

  5. Differences between Brahman and Holstein cows in response to estrus synchronization, superovulation and resistance of embryos to heat shock.

    PubMed

    Krininger, C E; Block, J; Al-Katanani, Y M; Rivera, R M; Chase, C C; Hansen, P J

    2003-09-15

    Embryos from Bos indicus are more resistant to elevated culture temperature (i.e. heat shock) than embryos from some Bos taurus breeds. The present experiment was designed to determine if Brahman embryos have greater resistance to heat shock than Holstein embryos at a stage in development before the embryonic genome was fully activated. A second objective was to test breed effects on estrus synchronization and superovulation responses. A total of 29 Brahman and 24 Holstein cows were subjected to estrus synchronization using gonadotropin releasing hormone (GnRH) and prostaglandin F2alpha (PGF2alpha) superovulation. Embryos were collected at 48 h and day 5 after insemination. There was a tendency for a lower proportion of Brahmans to be detected in standing estrus than Holsteins. There were no differences between breeds in the proportion of cows detected in estrus using both tailpaint and standing estrus as criteria or in interval from PGF2alpha to estrus. The degree of synchrony in estrus was greater for Brahmans. Superovulation response was generally similar between breeds. At 48 h after insemination, there was a tendency for a greater proportion of Brahman oocytes to have undergone cleavage. Uncleaved oocytes were cultured for an additional 24 h-at this time, cleavage rate was similar between breeds. When embryos reached the 2-4-cell stage, they were heat-shocked for 4.5 h at 41 degrees C. This heat shock reduced the proportion of embryos that developed to the blastocyst stage but there was no breedxtreatment interaction. At day 5 after insemination, the number of embryos recovered was too low to allow comparison of breed effects. In conclusion, genetic effects on cellular thermotolerance that make Brahman embryos more resistant to heat shock are not expressed at the 2-4-cell stage. There were few differences between Brahman and Holstein in response to estrus synchronization and superovulation. The fact that cleavage tended to occur earlier in Brahman than

  6. Differences in developmental competence and gene expression profiles between buffalo (Bubalus bubalis) preimplantation embryos cultured in three different embryo culture media.

    PubMed

    Sadeesh, E M; Selokar, N L; Balhara, A K; Yadav, P S

    2016-10-01

    The objective of this study was to compare effects of in vitro culture systems on embryonic development and expression patterns of developmentally important genes in preimplantation buffalo embryos. After IVM/IVF presumptive zygotes were cultured in one of three systems: undefined TCM-199, mCR2aa medium supplemented with 10 % FBS and defined PVA-myo-inositol-phosphate-EGF medium. No (P > 0.05) differences at 2-cell, 4-cell and 8-cell to 16- cell stages were observed among the three cultured media used, however, increased (P < 0.05) blastocyst yield, cell number and hatching rate were found in defined medium compared to undefined media. The expression patterns of genes implicated in embryo metabolism (GLUT-1), anti-apoptosis (BCL-2), imprinting (IGF-2R), DNA methylation (DNMT-3A) and maternal recognition of pregnancy (IFNT) were increased (P < 0.05) in hatched blastocysts derived from defined medium compared to undefined media. In conclusion, serum-free, defined medium improved developmental competence of in vitro cultured buffalo embryos. Whether these differences in morphological development and gene expression have long-term effects on buffalo calves born after embryo transfer remains unknown. However, it is possible that early adaptations of the preimplantation embryo to its environment persist during fetal and post-natal development. PMID:27481470

  7. Dam line and sire line effects on turkey embryo survival and thyroid hormone concentrations at the plateau stage in oxygen consumption

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Inheritance of embryo thyroid function was measured in lines of turkeys. Two lines that had been selected for either increased egg production (E) or increased 16-wk BW (F) and their respective randombred controls (i.e., RBC1 and RBC2) were examined. Reciprocal crosses of dams and sires from each sel...

  8. OCT-4 expression is essential for the segregation of trophectoderm lineages in porcine preimplantation embryos

    PubMed Central

    EMURA, Natsuko; SAKURAI, Nobuyuki; TAKAHASHI, Kazuki; HASHIZUME, Tsutomu; SAWAI, Ken

    2016-01-01

    Oct-4, a member of the POU family of transcription factors, is a key factor that regulates the segregation of the inner cell mass (ICM) and the trophectoderm (TE) during the transition from morula to blastocyst in mice. However, little is known about its role in porcine early embryogenesis. To determine the function of OCT-4 in the ICM and TE segregation of porcine embryos, we studied the developmental morphology of porcine embryos using RNA interference technology. Our experiments demonstrated that when 1-cell stage embryos were co-injected with the small interfering RNA (siRNA)for targeted knockdown of OCT-4 (OCT-4-siRNA) and tetramethylrhodamine isothiocyanate (TRITC)-dextran conjugate (Dx), they failed to form blastocysts. Therefore, in this study, we constructed chimeric embryos comprising blastomeres that either expressed OCT-4 normally or showed downregulated OCT-4 expression by co-injection of OCT-4-siRNA and Dx into one blastomere in 2- to 4-cell stage embryos. In control embryos, which were co-injected with control siRNA and Dx, Dx-positive cells contributed to the TE lineage in almost all the blastocysts examined. In contrast, Dx-positive cells derived from a blastomere co-injected with OCT-4-siRNA and Dx were degenerated in almost half the blastocysts. This was probably due to the inability of these cells to differentiate into the TE lineage. Real-time RT-PCR analysis revealed no difference in the levels of SOX2, TEAD4, FGF4 and FGFR1-IIIc, all of which are known to be regulated by OCT-4, between the OCT-4-siRNA-injected morulae and the control ones. However, the level of CDX2, a molecule specifically expressed in the TE lineage, was significantly higher in the former than in the latter. Our results indicate that continuous expression of OCT-4 in blastomeres is essential for TE formation of porcine embryos. PMID:27210587

  9. Ion currents in embryo development.

    PubMed

    Tosti, Elisabetta; Boni, Raffaele; Gallo, Alessandra

    2016-03-01

    Ion channels are proteins expressed in the plasma membrane of electrogenic cells. In the zygote and blastomeres of the developing embryo, electrical modifications result from ion currents that flow through these channels. This phenomenon implies that ion current activity exerts a specific developmental function, and plays a crucial role in signal transduction and the control of embryogenesis, from the early cleavage stages and during growth and development of the embryo. This review describes the involvement of ion currents in early embryo development, from marine invertebrates to human, focusing on the occurrence, modulation, and dynamic role of ion fluxes taking place on the zygote and blastomere plasma membrane, and at the intercellular communication between embryo cell stages.

  10. Cryobiological preservation of Drosophila embryos

    SciTech Connect

    Mazur, P.; Schreuders, P.D.; Cole, K.W.; Hall, J.W. ); Mahowald, A.P. )

    1992-12-18

    The inability to cryobiologically preserve the fruit fly Drosophila melanogaster has required that fly stocks be maintained by frequent transfer of adults. This method is costly in terms of time and can lead to loss of stocks. Traditional slow freezing methods do not succeed because the embryos are highly sensitive to chilling. With the procedures described here, 68 percent of precisely staged 15-hour Oregon R (wild-type) embryos hatch after vitrification at -205[degree]C, and 40 percent of the resulting larvae develop into normal adult flies. These embryos are among the most complex organisms successfully preserved by cryobiology.

  11. Embryo Development in Phaseolus vulgaris

    PubMed Central

    Smith, Jerry G.

    1973-01-01

    Endosperm liquid bathes the embryo of Phaseolus vulgaris from the heart stage through the late cotyledon state. This liquid was aspirated from many ovules of the same stage, pooled, and analyzed for the following constituents and parameters: [List: see text] PMID:16658350

  12. Intrauterine embryo transfer with canine embryos cryopreserved by the slow freezing and the Cryotop method

    PubMed Central

    HORI, Tatsuya; USHIJIMA, Hitoshi; KIMURA, Taku; KOBAYASHI, Masanori; KAWAKAMI, Eiichi; TSUTSUI, Toshihiko

    2016-01-01

    Canine embryos (8-cell to blastocyst stages) frozen-thawed using the slow-freezing method with glycerol (four recipients) or dimethyl sulfoxide (three recipients) as a cryoprotectant and vitrified-warmed using the Cryotop method (five recipients) were surgically transferred into the unilateral uterine horn of recipient bitches. As a result, the morphology of embryos frozen-thawed using the slow-freezing method was judged to be normal, but no conception occurred in any of the recipient bitches. Two of the five bitches that received transferred embryos (morula to early blastocyst stages) vitrified-warmed using the Cryotop method became pregnant and produced normal pups (1/9 embryos, 11.1% and 1/6 embryos, 17.0%). It was concluded that the Cryotop method was more appropriate for canine embryo cryopreservation than the slow-freezing method, which is used for the cryopreservation of embryos of other mammalian species. PMID:27041356

  13. Radioactive labeling of proteins in cultured postimplantation mouse embryos. I. Influence of the embryo preparation method

    SciTech Connect

    Nowak, J.; Klose, J. )

    1989-07-01

    Conditions for optimum incorporation of radioactive amino acids into proteins of cultured postimplantation mouse embryos were investigated under the aspect of using these proteins for two-dimensional electrophoretic separations followed by fluorography. The aim was to obtain highly radioactive proteins under conditions as physiological as possible. Embryos at Days 10, 11, and 12 of gestation were prepared in different ways and incubated for 4 h in Tyrode's solution containing ({sup 3}H)amino acids (mixture) at a concentration of 27 microCi/ml medium. The preparations were: (a) yolk sac opened, placenta and blood circulation intact; (b) yolk sac and amnion opened, placenta and blood circulation intact (Day 10 embryos only); (c) placenta, yolk sac, and amnion removed (embryo naked); (d) naked embryos cut randomly into pieces (Day 10 embryos only). After incubation whole embryos or certain parts (tail, liver, rest body) were investigated by determining the radioactivity taken up by the protein. The results are given in dpm per mg protein per embryo. Radioactivity of proteins was about 3 times higher in naked embryos than in embryos left in their yolk sacs. This was true for all three stages investigated. However, the degree of radioactivity in the various parts of naked embryos differed by a factor of 15, whereas radioactivity was evenly distributed in embryos incubated in their yolk sacs. Therefore, embryos prepared according to the first method (see above) fulfilled the conditions required at the best.

  14. Efficient blastomere biopsy for mouse embryo splitting for future applications in human assisted reproduction.

    PubMed

    Illmensee, K; Kaskar, K; Zavos, P M

    2005-12-01

    The objective of the current study was to establish a safe, efficient biopsy procedure for embryo splitting using the mouse model for future applications in human assisted reproduction. From mouse embryos at the 2-, 4-, 6- and 8-cell stage, half the number of blastomeres were microsurgically biopsied and transferred into empty mouse zonae pellucidae. Twin embryonic development was monitored during in-vitro culture. Blastocyst developmental rate using 2-, 4-, 6-, and 8-cell splitting was 74.4, 75.0, 66.7 and 38.4 respectively, with corresponding hatching rates of 94.9, 97.5, 92.7 and 83.8%. Blastocysts from 2-, 4-, and 6-cell splitting resulted in elevated hatching rates compared with non-operated blastocysts (87.5%), due to the Tyrode-assisted hatching effect. Blastocyst morphology was superior from 2- and 4-cell splitting when compared with 6- and 8-cell splitting. Furthermore, outgrowth of twin blastocysts from 2- and 4-cell splitting showed well-developed colonies with trophoblast cells and clusters of ICM cells, whereas those obtained from 6- and 8-cell splitting frequently formed small-sized colonies. Due to the high twinning success rate obtained under the experimental conditions employed in this study, it appears that with further modifications and proper safeguards, such embryo splitting efforts could have potential applications in humans.

  15. 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.

  16. The ART of studying early embryo development: progress and challenges in ruminant embryo culture.

    PubMed

    Lonergan, Pat; Fair, Trudee

    2014-01-01

    The study of preimplantation mammalian embryo development is challenging due to difficulties in accessing in vivo-derived embryos in large numbers at the early stages and the inability to culture embryos in vitro much beyond the blastocyst stage. Nonetheless, embryos exhibit an amazing plasticity and tolerance when it comes to adapting to the environment in which they are cultured. They are capable of developing in media ranging in composition from simple balanced salt solutions to complex systems involving serum and somatic cells. At least a proportion of the blastocysts that develop in culture are developmentally competent as evidenced by the fact that live offspring have resulted following transfer. However, several studies using animal models have shown that such embryos are sensitive to environmental conditions that can affect future pre- and post-natal growth and developmental potential. This review summarises some key aspects of early embryo development and the approaches taken to study this important window in early life.

  17. The MAPK cascade in equally cleaving spiralian embryos.

    PubMed

    Lambert, J David; Nagy, Lisa M

    2003-11-15

    Spiralian development is shared by several protostome phyla and characterized by regularities in early cleavage, fate map, and larva. Experimental evidence from multiple spiralian species implicates cells in the D quadrant lineage as the organizer of future axial development of the embryo. However, the mechanisms by which the D quadrant is specified differ between species with equal and unequal spiral cleavage. Equally cleaving mollusc embryos establish the D quadrant via cell-cell interactions between the micromeres and macromeres at the 24- to 36-cell stage. In unequally cleaving embryos, the D quadrant is established at the 4-cell stage via asymmetries in the first 2 cell divisions. We have begun to explore the molecular mechanisms of D quadrant patterning in spiralians. Previously, we showed that, in the unequally cleaving embryo of the mollusc Ilyanassa obsoleta, the MAPK pathway is activated and functionally required in 3D and also in the micromeres known to require a signal from 3D. Here, we examine the role of MAPK signaling in 4 spiralians with equal cleavage. In 3 equally cleaving molluscs, the chiton Chaetopleura, the limpet Tectura, and the snail Lymnaea, the MAPK pathway is activated in the 3D cell but not in the overlying micromeres. In the equally cleaving embryo of the polychaete annelid Hydroides, MAPK activation was not detected in the 3D macromere but was observed in one of its daughter cells, 4d. In addition, inhibiting Tectura MAPK activation disrupts differentiation of 3D and cells induced by it, supporting a functional role for MAPK in axis specification in equally cleaving spiralians. Thus, MAPK signaling may have a conserved role in the D quadrant organizer cell 3D in molluscs. However, there have been at least 2 evolutionary changes in the activation of the MAPK pathway during spiralian evolution. MAPK function in the Ilyanassa micromeres is a recent cooption and, since the divergence of annelids and molluscs, there has been a shift in

  18. Assay using embryo aggregation chimeras for the detection of nonlethal changes in X-irradiated mouse preimplantation embryos

    SciTech Connect

    Obasaju, M.F.; Wiley, L.M.; Oudiz, D.J.; Miller, L.; Samuels, S.J.; Chang, R.J.; Overstreet, J.W.

    1988-02-01

    We have developed a short-term in vitro assay for the detection of sublethal effects produced by very low levels of ionizing radiation. The assay utilizes mouse embryo aggregation chimeras consisting of one irradiated embryo paired with an unirradiated embryo whose blastomeres have been labeled with fluorescein isothiocyanate (FITC). X irradiation (from 0.05 to 2 Gy) and chimera construction were performed with four-cell stage embryos, and the chimeras were cultured for 40 h to the morula stage. The morulae were partially dissociated with calcium-free culture medium and viewed under phase contrast and epifluorescence microscopy to obtain total embryo cell number and the cellular contribution of irradiated (unlabeled) and control (FITC labeled) embryos per chimera. In chimeras where neither embryo was irradiated, the ratio of the unlabeled blastomeres to the total number of blastomeres per chimera embryo was 0.50 (17.8 +/- 5.6 cells per unlabeled embryo and 17.4 +/- 5.5 cells per FITC-labeled partner embryo). However, in chimeras formed after the unlabeled embryos were irradiated with as little as 0.05 Gy, the ratio of unlabeled blastomeres to the total number of blastomeres per chimera embryo was 0.43 (P less than 0.01). The apparent decreases in cell proliferation were not observed in irradiated embryos that were merely cocultured with control embryos, regardless of whether the embryos were zona enclosed or zona free. We conclude that very low levels of radiation induce sublethal changes in cleaving embryos that are expressed as a proliferative disadvantage within two cell cycles when irradiated embryos are in direct cell-to-cell contact with unirradiated embryos.

  19. Effects of Fluoxetine on Human Embryo Development.

    PubMed

    Kaihola, Helena; Yaldir, Fatma G; Hreinsson, Julius; Hörnaeus, Katarina; Bergquist, Jonas; Olivier, Jocelien D A; Åkerud, Helena; Sundström-Poromaa, Inger

    2016-01-01

    The use of antidepressant treatment during pregnancy is increasing, and selective serotonin reuptake inhibitors (SSRIs) are the most widely prescribed antidepressants in pregnant women. Serotonin plays a role in embryogenesis, and serotonin transporters are expressed in two-cell mouse embryos. Thus, the aim of the present study was to evaluate whether fluoxetine, one of the most prescribed SSRI antidepressant world-wide, exposure influences the timing of different embryo developmental stages, and furthermore, to analyze what protein, and protein networks, are affected by fluoxetine in the early embryo development. Human embryos (n = 48) were randomly assigned to treatment with 0.25 or 0.5 μM fluoxetine in culture medium. Embryo development was evaluated by time-lapse monitoring. The fluoxetine-induced human embryo proteome was analyzed by shotgun mass spectrometry. Protein secretion from fluoxetine-exposed human embryos was analyzed by use of high-multiplex immunoassay. The lower dose of fluoxetine had no influence on embryo development. A trend toward reduced time between thawing and start of cavitation was noted in embryos treated with 0.5 μM fluoxetine (p = 0.065). Protein analysis by shotgun mass spectrometry detected 45 proteins that were uniquely expressed in fluoxetine-treated embryos. These proteins are involved in cell growth, survival, proliferation, and inflammatory response. Culturing with 0.5 μM, but not 0.25 μM fluoxetine, caused a significant increase in urokinase-type plasminogen activator (uPA) in the culture medium. In conclusion, fluoxetine has marginal effects on the timing of developmental stages in embryos, but induces expression and secretion of several proteins in a manner that depends on dose. For these reasons, and in line with current guidelines, the lowest possible dose of SSRI should be used in pregnant women who need to continue treatment. PMID:27378857

  20. Effects of Fluoxetine on Human Embryo Development

    PubMed Central

    Kaihola, Helena; Yaldir, Fatma G.; Hreinsson, Julius; Hörnaeus, Katarina; Bergquist, Jonas; Olivier, Jocelien D. A.; Åkerud, Helena; Sundström-Poromaa, Inger

    2016-01-01

    The use of antidepressant treatment during pregnancy is increasing, and selective serotonin reuptake inhibitors (SSRIs) are the most widely prescribed antidepressants in pregnant women. Serotonin plays a role in embryogenesis, and serotonin transporters are expressed in two-cell mouse embryos. Thus, the aim of the present study was to evaluate whether fluoxetine, one of the most prescribed SSRI antidepressant world-wide, exposure influences the timing of different embryo developmental stages, and furthermore, to analyze what protein, and protein networks, are affected by fluoxetine in the early embryo development. Human embryos (n = 48) were randomly assigned to treatment with 0.25 or 0.5 μM fluoxetine in culture medium. Embryo development was evaluated by time-lapse monitoring. The fluoxetine-induced human embryo proteome was analyzed by shotgun mass spectrometry. Protein secretion from fluoxetine-exposed human embryos was analyzed by use of high-multiplex immunoassay. The lower dose of fluoxetine had no influence on embryo development. A trend toward reduced time between thawing and start of cavitation was noted in embryos treated with 0.5 μM fluoxetine (p = 0.065). Protein analysis by shotgun mass spectrometry detected 45 proteins that were uniquely expressed in fluoxetine-treated embryos. These proteins are involved in cell growth, survival, proliferation, and inflammatory response. Culturing with 0.5 μM, but not 0.25 μM fluoxetine, caused a significant increase in urokinase-type plasminogen activator (uPA) in the culture medium. In conclusion, fluoxetine has marginal effects on the timing of developmental stages in embryos, but induces expression and secretion of several proteins in a manner that depends on dose. For these reasons, and in line with current guidelines, the lowest possible dose of SSRI should be used in pregnant women who need to continue treatment. PMID:27378857

  1. Effects of Fluoxetine on Human Embryo Development.

    PubMed

    Kaihola, Helena; Yaldir, Fatma G; Hreinsson, Julius; Hörnaeus, Katarina; Bergquist, Jonas; Olivier, Jocelien D A; Åkerud, Helena; Sundström-Poromaa, Inger

    2016-01-01

    The use of antidepressant treatment during pregnancy is increasing, and selective serotonin reuptake inhibitors (SSRIs) are the most widely prescribed antidepressants in pregnant women. Serotonin plays a role in embryogenesis, and serotonin transporters are expressed in two-cell mouse embryos. Thus, the aim of the present study was to evaluate whether fluoxetine, one of the most prescribed SSRI antidepressant world-wide, exposure influences the timing of different embryo developmental stages, and furthermore, to analyze what protein, and protein networks, are affected by fluoxetine in the early embryo development. Human embryos (n = 48) were randomly assigned to treatment with 0.25 or 0.5 μM fluoxetine in culture medium. Embryo development was evaluated by time-lapse monitoring. The fluoxetine-induced human embryo proteome was analyzed by shotgun mass spectrometry. Protein secretion from fluoxetine-exposed human embryos was analyzed by use of high-multiplex immunoassay. The lower dose of fluoxetine had no influence on embryo development. A trend toward reduced time between thawing and start of cavitation was noted in embryos treated with 0.5 μM fluoxetine (p = 0.065). Protein analysis by shotgun mass spectrometry detected 45 proteins that were uniquely expressed in fluoxetine-treated embryos. These proteins are involved in cell growth, survival, proliferation, and inflammatory response. Culturing with 0.5 μM, but not 0.25 μM fluoxetine, caused a significant increase in urokinase-type plasminogen activator (uPA) in the culture medium. In conclusion, fluoxetine has marginal effects on the timing of developmental stages in embryos, but induces expression and secretion of several proteins in a manner that depends on dose. For these reasons, and in line with current guidelines, the lowest possible dose of SSRI should be used in pregnant women who need to continue treatment.

  2. Assessment of human embryos by time-lapse videography: A comparison of quantitative and qualitative measures between two independent laboratories.

    PubMed

    Liu, Yanhe; Copeland, Christopher; Stevens, Adam; Feenan, Katie; Chapple, Vincent; Myssonski, Kim; Roberts, Peter; Matson, Phillip

    2015-12-01

    A total of 488 Day 3 human embryos with known implantation data from two independent in vitro fertilization laboratories were included for analysis, with 270 from Fertility North (FN) and 218 from Canberra Fertility Centre (CFC). Implanting embryos grew at different rates between FN and CFC as indicated in hours of the time intervals between pronuclear fading and the 4- (13.9 ± 1.1 vs. 14.9 ± 1.8), 5- (25.7 ± 1.9 vs. 28.4 ± 3.7) and 8-cell stages (29.0 ± 3.2 vs. 32.2 ± 4.6), as well as the durations of 2- (10.8 ± 0.8 vs. 11.6 ± 1.1), 3- (0.4 ± 0.5 vs. 0.9 ± 1.2), and 4-cell stages (11.8 ± 1.4 vs. 13.6 ± 2.9), all p<0.05. The application of a previously published time-lapse algorithm on ICSI embryos from the two participating laboratories failed to reproduce a predictive pattern of implantation outcomes (FN: AUC=0.565, p=0.250; CFC: AUC=0.614, p=0.224). However, for the qualitative measures including poor conventional morphology, direct cleavage, reverse cleavage and <6 intercellular contact points at the end of the 4-cell stage, there were similar proportions of embryos showing at least one of these biological events in either implanting (3.1% vs. 3.3%, p>0.05) or non-implanting embryos (30.4% vs. 38.3%, p>0.05) between FN and CFC. Furthermore, implanting embryos favored lower proportions of the above biological events compared to the non-implanting ones in both laboratories (both p<0.01). To conclude, human embryo morphokinetics may vary between laboratories, therefore time-lapse algorithms emphasizing quantitative timing parameters may have reduced inter-laboratory transferability; qualitative measures are independent of cell division timings, with potentially improved inter-laboratory reproducibility.

  3. 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

  4. Efficacy of in vitro embryo transfer in lactating dairy cows using fresh or vitrified embryos produced in a novel embryo culture medium.

    PubMed

    Block, J; Bonilla, L; Hansen, P J

    2010-11-01

    Objectives were to determine whether pregnancy success could be improved in lactating cows with timed embryo transfer when embryos were produced in vitro using a medium designed to enhance embryo development and survival after cryopreservation. In experiment 1, embryos (n=569 to 922) were cultured in either modified synthetic oviduct fluid or a serum-free medium, Block-Bonilla-Hansen-7 (BBH7). Development to the blastocyst stage was recorded at d 7, and selected blastocysts (n=79 to 114) were vitrified using open pulled straws. Culture of embryos in BBH7 increased development to the blastocyst stage (41.9±2.0 vs. 14.7±2.0%) and advanced blastocyst stages (expanded, hatching, hatched; 31.1±1.3 vs. 6.4±1.3%) at d 7 and resulted in higher hatching rates at 24h postwarming compared with embryos cultured in modified synthetic oviduct fluid (59.0±0.5 vs. 26.7±0.5%). In experiment 2, embryos were produced using X-sorted semen and cultured in BBH7. At d 7 after insemination, embryos were transferred fresh or following vitrification. Lactating Holstein cows were either subjected to timed artificial insemination (TAI) on the day of presumptive ovulation or used as embryo recipients 7 d later. Embryo recipients received an embryo if a corpus luteum was present. The percentage of cows pregnant at d 32, 46, and 76 of gestation was higher among cows that received fresh embryos compared with TAI cows or cows that received vitrified embryos. At d 76, for example, the proportion and percentage pregnant was 47/150 (31.3%) for cows subjected to TAI, 48/95 (50.5%) for cows receiving fresh embryos, and 39/141 (27.7%) for cows receiving a vitrified embryo. No difference was observed in the percentage of cows pregnant among TAI cows and those that received vitrified embryos. There was a service or transfer number × treatment interaction because differences in pregnancy rate between embryo transfer recipients and cows bred by TAI were greater for cows with more than 3 services or

  5. Arabidopsis mitochondrial protein slow embryo development1 is essential for embryo development.

    PubMed

    Ju, Yan; Liu, Chunying; Lu, Wenwen; Zhang, Quan; Sodmergen

    2016-05-27

    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 analysis 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.

  6. Arabidopsis mitochondrial protein slow embryo development1 is essential for embryo development.

    PubMed

    Ju, Yan; Liu, Chunying; Lu, Wenwen; Zhang, Quan; Sodmergen

    2016-05-27

    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 analysis 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. PMID:27109472

  7. Effects of sperm pretreatment and embryo activation methods on the development of bovine embryos produced by intracytoplasmic sperm injection.

    PubMed

    Lee, Jai-Wei; Chang, Hsun-Chung; Wu, Hung-Yi; Liu, Shyh-Shyan; Wang, Chih-Hua; Chu, Chun-Yen; Shen, Perng-Chih

    2015-09-01

    The aim of the study was to examine the effects of different embryo activation methods and sperm pretreatments on the activation and development of bovine embryos produced by intracytoplasmic sperm injection (ICSI). Four activation agents, i.e., calcium ionophore (A23187), ionomycin (Ion), electric pulse (EP) and ethanol (Eth) were used in various combinations to activate bovine ICSI embryos. The normal fertilization rate was similar in bovine ICSI embryos activated by A23187+Eth, Ion+Eth, Ion+EP+Eth, and 2-Ion (Ion administered two times)+Eth. Increasing the frequency of ionomycin stimulation from two (2-Ion+Eth) to three times (3-Ion+Eth) significantly (p<0.05) increased the cell number per embryo at the blastocyst stage. In addition, spermatozoa were pretreated with dithiothreitol (DTT), glutathione (GSH) or GSH+lysolecithin (LL) and used for producing bovine ICSI embryos. The blastocyst rate of bovine ICSI embryos produced from sperm pretreated with GSH was significantly (p<0.05) higher than those of embryos produced from sperm pretreated with DTT and GSH+LL. In conclusion, the embryo activation methods and sperm pretreatments examined in the present study did not affect the normal fertilization rate of bovine ICSI embryos. However, activation with 3-Ion+Eth and sperm pretreatment with GSH increased the cell number per embryo at blastocyst stage and the blastocyst rate, respectively, in bovine ICSI embryos.

  8. Clonal propagation of primate offspring by embryo splitting.

    PubMed

    Chan, A W; Dominko, T; Luetjens, C M; Neuber, E; Martinovich, C; Hewitson, L; Simerly, C R; Schatten, G P

    2000-01-14

    Primates that are identical in both nuclear and cytoplasmic components have not been produced by current cloning strategies, yet such identicals represent the ideal model for investigations of human diseases. Here, genetically identical nonhuman embryos were produced as twin and larger sets by separation and reaggregation of blastomeres of cleavage-stage embryos. A total of 368 multiples were created by the splitting of 107 rhesus embryos with four pregnancies established after 13 embryo transfers (31% versus 53% in vitro fertilization controls). The birth of Tetra, a healthy female cloned from a quarter of an embryo, proves that this approach can result in live offspring.

  9. Development of porcine embryos and offspring after intracytoplasmic sperm injection with liposome transfected or non-transfected sperm into in vitro matured oocytes.

    PubMed

    Lai, L; Sun, Q; Wu, G; Murphy, C N; Kühholzer, B; Park, K W; Bonk, A J; Day, B N; Prather, R S

    2001-11-01

    The objective of this study was to evaluate in vitro and in vivo development of porcine in vitro matured (IVM) porcine oocytes fertilised by intracytoplasmic sperm injection (ICSI) and the possibility of producing transgenic embryos and offspring with this procedure. Activated ICSI oocytes had a higher pronuclear formation than non-activated ICSI oocytes (mean 64.8+/-17.3% vs 28.5+/-3.4%, p<0.05). When the zygotes with two pronuclei were cultured to day 2, there was no difference (p<0.05) in the cleavage rate (mean 60.0+/-7.0% vs 63.3+/-12.7%) between the two groups. The blastocyst rate in the activation group was significantly higher than that in the non-activation group (mean 30.0+/-11.6% vs 4.6+/-4.2%, p<0.05). After injection of the sperm transfected with DNA/liposome complex, destabilised enhanced green fluorescent protein (d2EGFP) expression was not observed on day 2 in either cleaved or uncleaved embryos. But from day 3, some of the embryos at the 2-cell to 4-cell stage started to express d2EGFP. On day 7, about 30% of cleaved embryos, which were in the range of 2-cell to blastocyst stage, expressed d2EGFP. However, for the IVF oocytes inseminated with sperm transfected with DNA/liposome complex, and for oocytes injected with sperm transfected with DNA/liposome complex, and for oocytes injected with DNA/liposome complex following insemination with sperm not treated with DNA/liposome complex, none of the embryos expressed d2EGFP. Sixteen day 4 ICSI embryos derived from sperm not treated with DNA/liposome complex were transferred into a day 3 recipient. One recipient delivered a female piglet with normal birthweight. After transfer of the ICSI embryos derived from sperm transfected with DNA/liposome complex, none of the four recipients maintained pregnancy. PMID:11771901

  10. Response of Mouse Zygotes Treated with Mild Hydrogen Peroxide as a Model to Reveal Novel Mechanisms of Oxidative Stress-Induced Injury in Early Embryos

    PubMed Central

    2016-01-01

    Our study aimed to develop embryo models to evaluate the impact of oxidative stress on embryo development. Mouse zygotes, which stayed at G1 phase, were treated with prepared culture medium (containing 0.00, 0.01, 0.02, 0.03, 0.04, 0.05, or 0.1 mM hydrogen peroxide (H2O2)) for 30 min in experiment 1. The dose-effects of H2O2 on embryo development were investigated via comparisons of the formation rate at each stage (2- and 4-cell embryos and blastocysts). Experiment 2 was carried out to compare behaviors of embryos in a mild oxidative-stressed status (0.03 mM H2O2) with those in a control (0 mM H2O2). Reactive oxygen species (ROS) levels, variation of mitochondrial membrane potential (MMP), expression of γH2AX, and cell apoptosis rate of blastocyst were detected. We observed a dose-dependent decrease on cleavage and blastocyst rates. Besides, higher level of ROS, rapid reduction of MMP, and the appearance of γH2AX revealed that embryos are injured early in mild oxidative stress. Additionally, γH2AX may involve during DNA damage response in early embryos. And the apoptotic rate of blastocyst may significantly increase when DNA damage repair is inadequate. Most importantly, our research provides embryo models to study cell cycle regulation and DNA damage response under condition of different levels of oxidative stress. PMID:27738489

  11. Toxicity of chlorine to zebrafish embryos.

    PubMed

    Kent, Michael L; Buchner, Cari; Barton, Carrie; Tanguay, Robert L

    2014-01-16

    Surface disinfection of fertilized fish eggs is widely used in aquaculture to reduce extraovum pathogens that may be released from brood fish during spawning, and this is routinely used in zebrafish Danio rerio research laboratories. Most laboratories use approximately 25 to 50 ppm unbuffered chlorine solution for 5 to 10 min. Treatment of embryos with chlorine has significant germicidal effects for many Gram-negative bacteria, viruses, and trophozoite stages of protozoa, but is less effective against cyst or spore stages of protozoa and certain Mycobacterium spp. Therefore, we evaluated the toxicity of unbuffered and buffered chlorine solutions to embryos exposed at 6 or 24 h post-fertilization (hpf) to determine whether higher concentrations can be used for treating zebrafish embryos. Most of our experiments entailed using an outbred line (5D), with both mortality and malformations as endpoints. We found that 6 hpf embryos consistently were more resistant than 24 hpf embryos to the toxic effects of chlorine. Chlorine is more toxic and germicidal at lower pH, and chlorine causes elevated pH. Consistent with this, we found that unbuffered chlorine solutions (pH ca. 8-9) were less toxic at corresponding concentrations than solutions buffered to pH 7. Based on our findings here, we recommend treating 6 hpf embryos for 10 min and 24 hpf embryos for 5 min with unbuffered chlorine solution at 100 ppm.

  12. Genetic Analysis of Human Preimplantation Embryos.

    PubMed

    Garcia-Herrero, S; Cervero, A; Mateu, E; Mir, P; Póo, M E; Rodrigo, L; Vera, M; Rubio, C

    2016-01-01

    Preimplantation development comprises the initial stages of mammalian development, before the embryo implants into the mother's uterus. In normal conditions, after fertilization the embryo grows until reaching blastocyst stage. The blastocyst grows as the cells divide and the cavity expands, until it arrives at the uterus, where it "hatches" from the zona pellucida to implant into the uterine wall. Nevertheless, embryo quality and viability can be affected by chromosomal abnormalities, most of which occur during gametogenesis and early embryo development; human embryos produced in vitro are especially vulnerable. Therefore, the selection of chromosomally normal embryos for transfer in assisted reproduction can improve outcomes in poor-prognosis patients. Additionally, in couples with an inherited disorder, early diagnosis could prevent pregnancy with an affected child and would, thereby, avoid the therapeutic interruption of pregnancy. These concerns have prompted advancements in the use of preimplantation genetic diagnosis (PGD). Genetic testing is applied in two different scenarios: in couples with an inherited genetic disorder or carriers of a structural chromosomal abnormality, it is termed PGD; in infertile couples with increased risk of generating embryos with de novo chromosome abnormalities, it is termed preimplantation genetic screening, or PGS. PMID:27475859

  13. 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.

  14. Embryo aggregation does not improve the development of interspecies somatic cell nuclear transfer embryos in the horse.

    PubMed

    Gambini, Andrés; De Stéfano, Adrián; Jarazo, Javier; Buemo, Carla; Karlanian, Florencia; Salamone, Daniel Felipe

    2016-09-01

    The low efficiency of interspecies somatic cell nuclear transfer (iSCNT) makes it necessary to investigate new strategies to improve embryonic developmental competence. Embryo aggregation has been successfully applied to improve cloning efficiency in mammals, but it remains unclear whether it could also be beneficial for iSCNT. In this study, we first compared the effect of embryo aggregation over in vitro development and blastocyst quality of porcine, bovine, and feline zona-free (ZF) parthenogenetic (PA) embryos to test the effects of embryo aggregation on species that were later used as enucleated oocytes donors in our iSCNT study. We then assessed whether embryo aggregation could improve the in vitro development of ZF equine iSCNT embryos after reconstruction with porcine, bovine, and feline ooplasm. Bovine- and porcine-aggregated PA blastocysts had significantly larger diameters compared with nonaggregated embryos. On the other hand, feline- and bovine-aggregated PA embryos had higher blastocyst cell number. Embryo aggregation of equine-equine SCNT was found to be beneficial for embryo development as we have previously reported, but the aggregation of three ZF reconstructed embryos did not improve embryo developmental rates on iSCNT. In vitro embryo development of nonaggregated iSCNT was predominantly arrested around the stage when transcriptional activation of the embryonic genome is reported to start on the embryo of the donor species. Nevertheless, independent of embryo aggregation, equine blastocyst-like structures could be obtained in our study using domestic feline-enucleated oocytes. Taken together, these results reported that embryo aggregation enhance in vitro PA embryo development and embryo quality but effects vary depending on the species. Embryo aggregation also improves, as expected, the in vitro embryo development of equine-equine SCNT embryos; however, we did not observe positive effects on equine iSCNT embryo development. Among oocytes

  15. Effect of epigenetic modification with trichostatin A and S-adenosylhomocysteine on developmental competence and POU5F1-EGFP expression of interspecies cloned embryos in dog.

    PubMed

    Mousai, M; Hosseini, S M; Hajian, M; Jafarpour, F; Asgari, V; Forouzanfar, M; Nasr-Esfahani, M H

    2015-10-01

    Adult canine fibroblasts stably transfected with either cytomegalovirus (CMV) or POU5F1 promoter-driven enhanced green fluorescent protein (EGFP) were used to investigate if pre-treatment of these donor cells with two epigenetic drugs [trichostatin A (TSA), or S-adenosylhomocysteine (SAH)] can improve the efficiency of interspecies somatic cell nuclear transfer (iSCNT). Fluorescence-activated cell sorting (FACS), analyses revealed that TSA, but not SAH, treatment of both transgenic and non-transgenic fibroblasts significantly increased acetylation levels compared with untreated relatives. The expression levels of Bcl2 and P53 were significantly affected in TSA-treated cells compared with untreated cells, whereas SAH treatment had no significant effect on cell apoptosis. Irrespective of epigenetic modification, dog/bovine iSCNT embryos had overall similar rates of cleavage and development to 8-16-cell and morula stages in non-transgenic groups. For transgenic reconstructed embryos, however, TSA and SAH could significantly improve development to 8-16-cell and morula stages compared with control. Even though, irrespective of cell transgenesis and epigenetic modification, none of the iSCNT embryos developed to the blastocyst stage. The iSCNT embryos carrying CMV-EGFP expressed EGFP at all developmental stages (2-cell, 4-cell, 8-16-cell, and morula) without mosaicism, while no POU5F1-EGFP signal was observed in any stage of developing iSCNT embryos irrespective of TSA/SAH epigenetic modifications. These results indicated that bovine oocytes partially remodel canine fibroblasts and that TSA and SAH have marginal beneficial effects on this process.

  16. Effect of epigenetic modification with trichostatin A and S-adenosylhomocysteine on developmental competence and POU5F1-EGFP expression of interspecies cloned embryos in dog.

    PubMed

    Mousai, M; Hosseini, S M; Hajian, M; Jafarpour, F; Asgari, V; Forouzanfar, M; Nasr-Esfahani, M H

    2015-10-01

    Adult canine fibroblasts stably transfected with either cytomegalovirus (CMV) or POU5F1 promoter-driven enhanced green fluorescent protein (EGFP) were used to investigate if pre-treatment of these donor cells with two epigenetic drugs [trichostatin A (TSA), or S-adenosylhomocysteine (SAH)] can improve the efficiency of interspecies somatic cell nuclear transfer (iSCNT). Fluorescence-activated cell sorting (FACS), analyses revealed that TSA, but not SAH, treatment of both transgenic and non-transgenic fibroblasts significantly increased acetylation levels compared with untreated relatives. The expression levels of Bcl2 and P53 were significantly affected in TSA-treated cells compared with untreated cells, whereas SAH treatment had no significant effect on cell apoptosis. Irrespective of epigenetic modification, dog/bovine iSCNT embryos had overall similar rates of cleavage and development to 8-16-cell and morula stages in non-transgenic groups. For transgenic reconstructed embryos, however, TSA and SAH could significantly improve development to 8-16-cell and morula stages compared with control. Even though, irrespective of cell transgenesis and epigenetic modification, none of the iSCNT embryos developed to the blastocyst stage. The iSCNT embryos carrying CMV-EGFP expressed EGFP at all developmental stages (2-cell, 4-cell, 8-16-cell, and morula) without mosaicism, while no POU5F1-EGFP signal was observed in any stage of developing iSCNT embryos irrespective of TSA/SAH epigenetic modifications. These results indicated that bovine oocytes partially remodel canine fibroblasts and that TSA and SAH have marginal beneficial effects on this process. PMID:25314965

  17. 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.

  18. Vitrification-based cryopreservation of Drosophila embryos

    SciTech Connect

    Schreuders, P.D.; Mazur, P.

    1994-12-31

    Currently, over 30,000 strains of Drosophila melanogaster are maintained by geneticists through regular transfer of breeding stocks. A more cost effective solution is to cryopreserve their embryos. Cooling and warming rates >10,000{degrees}C/min. are required to prevent chilling injury. To avoid the lethal intracellular ice normally produced at such high cooling rates, it is necessary to use {ge}50% (w/w) concentrations of glass-inducing solutes to vitrify the embryos. Differential scanning calorimetry (DSC) is used to develop and evaluate ethylene glycol and polyvinyl pyrrolidone based vitrification solutions. The resulting solution consists of 8.5M ethylene glycol + 10% polyvinylpyrrolidone in D-20 Drosophila culture medium. A two stage method is used for the introduction and concentration of these solutes within the embryo. The method reduces the exposure time to the solution and, consequently, reduces toxicity. Both DSC and freezing experiments suggest that, while twelve-hour embryos will vitrify using cooling rates >200{degrees}C/min., they will devitrify and be killed with even moderately rapid warming rates of {approximately}1,900{degrees}C/min. Very rapid warming ({approximately}100,000{degrees}C/min.) results in variable numbers of successfully cryopreserved embryos. This sensitivity to warming rite is typical of devitrification. The variability in survival is reduced using embryos of a precisely determined embryonic stage. The vitrification of the older, fifteen-hour, embryos yields an optimized hatching rate of 68%, with 35 - 40% of the resulting larvae developing to normal adults. This Success rite in embryos of this age may reflect a reduced sensitivity to limited devitrification or a more even distribution of the ethylene glycol within the embryo.

  19. Culture systems: embryo density.

    PubMed

    Reed, Michael L

    2012-01-01

    Embryo density is defined as the embryo-to-volume ratio achieved during in vitro culture; in other words, it is the number of embryos in a defined volume of culture medium. The same density can be achieved by manipulating either the number of embryos in a given volume of medium, or manipulating the volume of the medium for a given number of embryos: for example, a microdrop with five embryos in a 50 μl volume under oil has the same embryo-to-volume ratio (1:10 μl) as a microdrop with one embryo in a 10 μl volume under oil (1:10 μl). Increased embryo density can improve mammalian embryo development in vitro; however, the mechanism(s) responsible for this effect may be different with respect to which method is used to increase embryo density.Standard, flat sterile plastic petri dishes are the most common, traditional platform for embryo culture. Microdrops under a mineral oil overlay can be prepared to control embryo density, but it is critical that dish preparation is consistent, where appropriate techniques are applied to prevent microdrop dehydration during preparation, and results of any data collection are reliable, and repeatable. There are newer dishes available from several manufacturers that are specifically designed for embryo culture; most are readily available for use with human embryos. The concept behind these newer dishes relies on fabrication of conical and smaller volume wells into the dish design, so that embryos rest at the lowest point in the wells, and where putative embryotrophic factors may concentrate.Embryo density is not usually considered by the embryologist as a technique in and of itself; rather, the decision to culture embryos in groups or individually is protocol-driven, and is based more on convenience or the need to collect data on individual embryos. Embryo density can be controlled, and as such, it can be utilized as a simple, yet effective tool to improve in vitro development of human embryos. PMID:22829380

  20. Cryopreservation of embryos and oocytes: an update.

    PubMed

    Gelety, T; Surrey, E

    1993-10-01

    Widespread incorporation of human embryo cryopreservation into IVF programs may reduce the risk of multiple gestation and severe ovarian hyperstimulation syndrome as well as contributing to an overall increase in pregnancy rates. Slow cooling techniques appropriate to the stage of embryo development are most commonly employed. Growing experience with ultrarapid freezing suggests that this technique may offer similar success rates and has the advantages of simplicity and economy. The developmental stage at cryopreservation has not been conclusively shown to influence successful pregnancy outcome, although several retrospective studies suggest improved embryo survival and pregnancy rate following freezing at the pronuclear stage. Endometrial preparation using a number of different regimens before thawed-embryo transfer appears to confer no advantage over the natural cycle, but may be necessary with ovulatory dysfunction. The use of gonadotropin-releasing hormone (GnRH) analogs during ovarian stimulation for IVF has been suggested to decrease subsequent embryo freeze-thaw survival rates. However, increased pregnancy rates have been reported following transfer of previously cryopreserved embryos originating in cycles using GnRH analog (GnRHa), possibly reflecting increased numbers of embryos available for freezing. Cryopreservation of embryos originating from fertile donor oocytes has been very successful and may be used to facilitate synchronization between a donor and one or more recipients. Recent work in oocyte cryopreservation has addressed the problems of meiotic spindle disruption, the risks of aneuploidy, and decreased fertilization associated with zona hardening and polyspermy. Further refinements in technique will be required before widespread oocyte banking becomes feasible. PMID:8241436

  1. Effect of thermal injury on embryos of banded coral shrimp (Stenopus hispidus) under hypothermal conditions.

    PubMed

    Lin, Chiahsin; Han, Chiao-Chuan; Tsai, Sujune

    2013-02-01

    Cryopreservation technology regarding banded coral shrimp (Stenopus hispidus) embryos is important as it could improve cultivation and preservation of the species. The development of this technology is to reduce collections of this species from the wild, thus preventing damage to coral reefs. This study investigated the tolerance of different developmental stages of S. hispidus embryos in response to low temperature in the presence or absence of cryoprotectant. Embryos undergoing three stages of embryonic development (eye-formation, heart beat and pre-hatch stage) were selected and exposed to 5, 0 and -5°C in cryoprotectant solutions of 1 or 2M methanol for 1, 2, 4, 6, 8, 16 and 32h. Embryo survival was evaluated based on their hatching percentage. In experiments on the effect of different concentrations of methanol on chilling sensitivity of embryos, it was determined that methanol at 1M methanol reduced the chilling sensitivities of embryos most effectively when compared to the other tested concentrations. Experiments regarding the chilling sensitivity of embryos in different developmental stages indicated that pre-hatch stage embryos were more resistant to subzero temperatures than early stage embryos; they tolerated the 32h exposure at 5 and 0°C without a loss in survival. The study also indicated that late stage embryos are considered to be resistant to chilling, and that pre-late stage embryos are better candidate for cryopreservation.

  2. Effect of thermal injury on embryos of banded coral shrimp (Stenopus hispidus) under hypothermal conditions.

    PubMed

    Lin, Chiahsin; Han, Chiao-Chuan; Tsai, Sujune

    2013-02-01

    Cryopreservation technology regarding banded coral shrimp (Stenopus hispidus) embryos is important as it could improve cultivation and preservation of the species. The development of this technology is to reduce collections of this species from the wild, thus preventing damage to coral reefs. This study investigated the tolerance of different developmental stages of S. hispidus embryos in response to low temperature in the presence or absence of cryoprotectant. Embryos undergoing three stages of embryonic development (eye-formation, heart beat and pre-hatch stage) were selected and exposed to 5, 0 and -5°C in cryoprotectant solutions of 1 or 2M methanol for 1, 2, 4, 6, 8, 16 and 32h. Embryo survival was evaluated based on their hatching percentage. In experiments on the effect of different concentrations of methanol on chilling sensitivity of embryos, it was determined that methanol at 1M methanol reduced the chilling sensitivities of embryos most effectively when compared to the other tested concentrations. Experiments regarding the chilling sensitivity of embryos in different developmental stages indicated that pre-hatch stage embryos were more resistant to subzero temperatures than early stage embryos; they tolerated the 32h exposure at 5 and 0°C without a loss in survival. The study also indicated that late stage embryos are considered to be resistant to chilling, and that pre-late stage embryos are better candidate for cryopreservation. PMID:22634114

  3. Live embryo imaging to follow cell cycle and chromosomes stability after nuclear transfer.

    PubMed

    Balbach, Sebastian T; Boiani, Michele

    2015-01-01

    Nuclear transfer (NT) into mouse oocytes yields a transcriptionally and functionally heterogeneous population of cloned embryos. Most studies of NT embryos consider only embryos at predefined key stages (e.g., morula or blastocyst), that is, after the bulk of reprogramming has taken place. These retrospective approaches are of limited use to elucidate mechanisms of reprogramming and to predict developmental success. Observing cloned embryo development using live embryo cinematography has the potential to reveal otherwise undetectable embryo features. However, light exposure necessary for live cell cinematography is highly toxic to cloned embryos. Here we describe a protocol for combined bright-field and fluorescence live-cell imaging of histone H2b-GFP expressing mouse embryos, to record cell divisions up to the blastocyst stage. This protocol, which can be adapted to observe other reporters such as Oct4-GFP or Nanog-GFP, allowed us to quantitatively analyze cleavage kinetics of cloned embryos. PMID:25287344

  4. [Development of Brassica rapa L. embryos under conditions of microgravity].

    PubMed

    Popova, A F; Musgrave, M; Kuang, A

    2009-01-01

    Results of comparative studies of the embryos of identical age formed under microgravity and ground laboratory control are presented. Significant similarity of a rate of embryo development and degree of their differentiation in both variants has been shown. The single cases of the disturbances in embryo formation, and also a certain acceleration of endosperm development at the early stages of seed formation in microgravity are revealed.

  5. Fatty acid breakdown in developing embryos of Brassica napus L.

    PubMed

    Chia, T; Rawsthorne, S

    2000-12-01

    Developing Brassica napus embryos are primarily concerned with the accumulation of storage products, namely oil, starch and protein. The presence of fatty acid catabolic pathways in the background of this biosynthetic activity was investigated. Enzymes involved in the process of lipid mobilization, such as malate synthase and isocitrate lyase, are detectable towards the late stages of embryo development. [(14)C]Acetate feeding experiments also reveal that fatty acid catabolism becomes increasingly functional as the embryo matures.

  6. Embryo technologies in the horse.

    PubMed

    Squires, E L; Carnevale, E M; McCue, P M; Bruemmer, J E

    2003-01-01

    Recent studies demonstrated that zwitterionic buffers could be used for satisfactory storage of equine embryos at 5 degrees C. The success of freezing embryos is dependent upon size and stage of development. Morulae and blastocysts <300 microm can be slowly cooled or vitrified with acceptable pregnancy rates after transfer. The majority of equine embryos are collected from single ovulating mares, as there is no commercially available product for superovulation in equine. However, pituitary extract, rich in FSH, can be used to increase embryo recovery three- to four-fold. Similar to human medicine, assisted reproductive techniques have been developed for the older, subfertile mare. Transfer of in vivo-matured oocytes from young, healthy mares into a recipient's oviduct results in a 70-80% pregnancy rate compared with a 30-40% pregnancy rate when the oocytes are from older, subfertile mares. This procedure can also be used to evaluate in vitro maturation systems. In vitro production of embryos is still quite difficult in the horse. However, intracytoplasmic sperm injection (ICSI) has been used to produce several foals. Cleavage rates of 60% and blastocyst rates of 30% have been reported after ICSI of in vitro-matured oocytes. Gamete intrafallopian tube transfer (GIFT) is a possible treatment for subfertile stallions. Transfer of in vivo-matured oocytes with 200,000 sperm into the oviduct of normal mares resulted in a pregnancy rate of 55-82%. Oocyte freezing is a technique that has proven difficult in most species. However, equine oocytes vitrified in a solution of ethylene glycol, DMSO, and Ficoll and loaded onto a cryoloop resulted in three pregnancies of 26 transfers and two live foals produced. Production of a cloned horse appears to be likely, as several cloned pregnancies have recently been produced. PMID:12499026

  7. Electroporation into Cultured Mammalian Embryos

    NASA Astrophysics Data System (ADS)

    Nomura, Tadashi; Takahashi, Masanori; Osumi, Noriko

    Over the last century, mammalian embryos have been used extensively as a common animal model to investigate fundamental questions in the field of developmental biology. More recently, the establishment of transgenic and gene-targeting systems in laboratory mice has enabled researchers to unveil the genetic mechanisms under lying complex developmental processes (Mak, 2007). However, our understanding of cell—cell interactions and their molecular basis in the early stages of mammalian embryogenesis is still very fragmentary. One of the major problems is the difficulty of precise manipulation and limited accessibility to mammalian embryos via uterus wall. Unfortunately, existing tissue and organotypic culture systems per se do not fully recapitulate three-dimensional, dynamic processes of organogenesis observed in vivo. Although transgenic animal technology and virus-mediated gene delivery are useful to manipulate gene expression, these techniques take much time and financial costs, which limit their use.

  8. Deep cytoplasmic rearrangements in ventralized Xenopus embryos

    NASA Technical Reports Server (NTRS)

    Brown, E. E.; Denegre, J. M.; Danilchik, M. V.

    1993-01-01

    Following fertilization in Xenopus, dramatic rearrangements of the egg cytoplasm relocalize maternally synthesized egg components. During the first cell cycle the vegetal yolk mass rotates relative to the egg surface, toward the sperm entry point (SEP) (J. P. Vincent, G. F. Oster, and J. C. Gerhart, 1986, Dev. Biol. 113, 484-500), while concomitant deep cytoplasmic rearrangements occur in the animal hemisphere (M. V. Danilchik and J. M. Denegre, 1991, Development 111, 845-856). In this paper we examine the role of vegetal yolk mass rotation in producing the animal cytoplasmic rearrangements. We inhibited rotation by uv-irradiating embryos during the first cell cycle, a treatment that yields an extremely ventralized phenotype. Both uv-irradiated embryos and unirradiated control embryos show cytoplasmic rearrangements in the animal hemisphere during the first cell cycle. Cytoplasmic rearrangements on the SEP side of the embryo associated with the path of the sperm pronucleus, plus a swirl on the anti-SEP (dorsal) side, are seen, whether or not yolk mass rotation has occurred. This result suggests a role for the expanding sperm aster in directing animal hemisphere cytoplasmic movements. In unirradiated control embryos the anti-SEP (dorsal) swirl is larger than that in uv-irradiated embryos and often extends into the vegetal hemisphere, consistent with the animal cytoplasm having been pulled dorsally and vegetally by the sliding vegetal yolk mass. Thus the yolk mass rotation may normally enhance the dorsalward cytoplasmic movement, begun by the sperm aster, enough to induce normal axis formation. We extended our observations of unirradiated control and uv-irradiated embryos through early cleavages. The vegetal extent of the anti-SEP (dorsal) swirl pattern seen in control embryos persists through the early cleavage period, such that labeled animal cytoplasm extends deep into dorsal third-tier blastomeres at the 32-cell stage. Significantly, in uv-irradiated embryos

  9. Effect of embryo density on in vitro development and gene expression in bovine in vitro-fertilized embryos cultured in a microwell system.

    PubMed

    Sugimura, Satoshi; Akai, Tomonori; Hashiyada, Yutaka; Aikawa, Yoshio; Ohtake, Masaki; Matsuda, Hideo; Kobayashi, Shuji; Kobayashi, Eiji; Konishi, Kazuyuki; Imai, Kei

    2013-01-01

    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

  10. 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

  11. Transcriptional profiling of the Arabidopsis embryo.

    PubMed

    Spencer, Matthew W B; Casson, Stuart A; Lindsey, Keith

    2007-02-01

    We have used laser-capture microdissection to isolate RNA from discrete tissues of globular, heart, and torpedo stage embryos of Arabidopsis (Arabidopsis thaliana). This was amplified and analyzed by DNA microarray using the Affymetrix ATH1 GeneChip, representing approximately 22,800 Arabidopsis genes. Cluster analysis showed that spatial differences in gene expression were less significant than temporal differences. Time course analysis reveals the dynamics and complexity of gene expression in both apical and basal domains of the developing embryo, with several classes of synexpressed genes identifiable. The transition from globular to heart stage is associated in particular with an up-regulation of genes involved in cell cycle control, transcriptional regulation, and energetics and metabolism. The transition from heart to torpedo stage is associated with a repression of cell cycle genes and an up-regulation of genes encoding storage proteins, and pathways of cell growth, energy, and metabolism. The torpedo stage embryo shows strong functional differentiation in the root and cotyledon, as inferred from the classes of genes expressed in these tissues. The time course of expression of the essential EMBRYO-DEFECTIVE genes shows that most are expressed at unchanging levels across all stages of embryogenesis. We show how identified genes can be used to generate cell type-specific markers and promoter activities for future application in cell biology.

  12. Cryopreservation of embryos of Lucilia sericata (Diptera: Calliphoridae)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Embryos of Lucilia (Phaenicia) sericata (Meigen) (Diptera: Calliphoridae), the green blowfly, were successfully cryopreserved by vitrification in liquid nitrogen and stored for 8 yr. Embryos incubated at 19 deg. C for 17 h after oviposition were found to be the most appropriate stage to cryopreserve...

  13. 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

  14. Metabolite profiling of somatic embryos of Cyclamen persicum in comparison to zygotic embryos, endosperm, and testa.

    PubMed

    Winkelmann, Traud; Ratjens, Svenja; Bartsch, Melanie; Rode, Christina; Niehaus, Karsten; Bednarz, Hanna

    2015-01-01

    Somatic embryogenesis has been shown to be an efficient in vitro plant regeneration system for many crops such as the important ornamental plant Cyclamen persicum, for which this regeneration pathway of somatic embryogenesis is of interest for the vegetative propagation of parental lines as well as elite plants. However, somatic embryogenesis is not commercially used in many crops due to several unsolved problems, such as malformations, asynchronous development, deficiencies in maturation and germination of somatic embryos. In contrast, zygotic embryos in seeds develop and germinate without abnormalities in most cases. Instead of time-consuming and labor-intensive experiments involving tests of different in vitro culture conditions and plant growth regulator supplements, we follow a more directed approach. Zygotic embryos served as a reference and were compared to somatic embryos in metabolomic analyses allowing the future optimization of the in vitro system. The aims of this study were to detect differences in the metabolite profiles of torpedo stage somatic and zygotic embryos of C. persicum. Moreover, major metabolites in endosperm and testa were identified and quantified. Two sets of extracts of two to four biological replicates each were analyzed. In total 52 metabolites were identified and quantified in the different tissues. One of the most significant differences between somatic and zygotic embryos was that the proline concentration in the zygotic embryos was about 40 times higher than that found in somatic embryos. Epicatechin, a scavenger for reactive oxygen species, was found in highest abundance in the testa. Sucrose, the most abundant metabolite was detected in significantly higher concentrations in zygotic embryos. Also, a yet unknown trisaccharide, was significantly enriched in zygotic embryos. PMID:26300898

  15. Electrical polarity in embryos of wild carrot precedes cotyledon differentiation.

    PubMed

    Brawley, S H; Wetherell, D F; Robinson, K R

    1984-10-01

    Endogenous electrical currents traverse embryos of a higher plant, the wild carrot Daucus carota L. Current enters the apical pole and leaves the region near the presumptive radicle in the radially symmetric globular embryo. Current also enters the exposed surfaces of incipient globular embryos. This electrical polarity precedes differentiation of vascular tissue and cotyledon development. Localized current is observed at both growing ends of the embryos in subsequent stages of embryogenesis. Inward current is found at the cotyledons; outward current is found at the radicle/root. Exogenous indole-3-acetic acid (3 muM) reversibly inhibits these currents. Little current traverses the surface of intermediate regions of the embryo. The ionic gradients generated by these currents may be important in accumulation of metabolites and in other developmental processes within the embryo.

  16. Cell communication compartments in molluscan embryos.

    PubMed

    Serras, F; Kühtreiber, W M; Krul, M R; van den Biggelaar, J A

    1985-08-01

    Early embryos of Patella vulgata have been injected with Lucifer Yellow. No restriction of dye spread was found. We show that later in the development, the larval trochophore stage present evidence of compartments of cell communication. These dye compartments coincide with different presumptive regions. PMID:4028198

  17. Developmental kinetics of bovine nuclear transfer and parthenogenetic embryos.

    PubMed

    Holm, P; Booth, P J; Callesen, H

    2003-01-01

    Early developmental kinetics of nuclear transfer (NT) embryos reconstituted with blastomeres and parthenogenones produced by ionophore activation followed by either dimethylaminopurine (DMAP) or cycloheximide (CHX) treatment was studied. In vitro produced (IVP) embryos served as controls. Embryos were cultured to the hatched blastocyst stage, and images were recorded every 0.5 h throughout the culture period. The longest cell cycle shifted from 4th to 5th cycle (26 +/- 4 and 44 +/- 5 h) in NT-embryos compared to IVP-embryos (41 +/- 2 and 20 +/- 3 h) and showed greater asynchrony between blastomeres than any other embryo category. Compared to DMAP, CHX prolonged the 1(st) (23 +/- 1 vs. 33 +/- 1 h) and shortened the 3(rd) cell cycle (17 +/- 2 vs. 13 +/- 1 h). Moreover, though cytoskeleton activity was initialised, a larger proportion of CHX embryos was unable to accomplish first cleavage. The parthegenones differed from IVP embryos with respect to the lengths of the 1st, 3rd, and 4th cell cycles and time of hatching. The findings are discussed in relation to known ultrastructural, chromosomal and genomic aberrations found in NT embryos and parthenogenones. We hypothesize that the shift of the longest cell cycle in NT embryos is associated with a shift in the time of major genomic transition.

  18. Micromanipulation of mammalian embryos.

    PubMed

    Sugawara, S

    1988-11-01

    Micromanipulation of embryos provide a new and valuable biological tool for animal agriculture and medical fields. Current techniques for embryo manipulation are in practice but some of techniques is still way off for application. The present status in new biotechnology applying to animal science and medicine is reviewed and its future is also discussed. PMID:3072425

  19. Mouse Embryo Compaction.

    PubMed

    White, M D; Bissiere, S; Alvarez, Y D; Plachta, N

    2016-01-01

    Compaction is a critical first morphological event in the preimplantation development of the mammalian embryo. Characterized by the transformation of the embryo from a loose cluster of spherical cells into a tightly packed mass, compaction is a key step in the establishment of the first tissue-like structures of the embryo. Although early investigation of the mechanisms driving compaction implicated changes in cell-cell adhesion, recent work has identified essential roles for cortical tension and a compaction-specific class of filopodia. During the transition from 8 to 16 cells, as the embryo is compacting, it must also make fundamental decisions regarding cell position, polarity, and fate. Understanding how these and other processes are integrated with compaction requires further investigation. Emerging imaging-based techniques that enable quantitative analysis from the level of cell-cell interactions down to the level of individual regulatory molecules will provide a greater understanding of how compaction shapes the early mammalian embryo. PMID:27475854

  20. Algal-CAMs: isoforms of a cell adhesion molecule in embryos of the alga Volvox with homology to Drosophila fasciclin I.

    PubMed

    Huber, O; Sumper, M

    1994-09-15

    Proof that plants possess homologs of animal adhesion proteins is lacking. In this paper we describe the generation of monoclonal antibodies that interfere with cell-cell contacts in the 4-cell embryo of the multicellular alga Volvox carteri, resulting in a hole between the cells. The number of following cell divisions is reduced and the cell division pattern is altered drastically. Antibodies given at a later stage of embryogenesis specifically inhibit inversion of the embryo, a morphogenetic movement that turns the embryo inside out. Immunofluorescence microscopy localizes the antigen (Algal-CAM) at cell contact sites of the developing embryo. Algal-CAM is a protein with a three-domain structure: an N-terminal extensin-like domain characteristic for plant cell walls and two repeats with homology to fasciclin I, a cell adhesion molecule involved in the neuronal development of Drosophila. Alternatively spliced variants of Algal-CAM mRNA were detected that are produced under developmental control. Thus, Algal-CAM is the first plant homolog of animal adhesion proteins. PMID:7925267

  1. Retarded Embryo Development 1 (RED1) regulates embryo development, seed maturation and plant growth in Arabidopsis.

    PubMed

    Du, Qian; Wang, Huanzhong

    2016-07-20

    Plant seeds accumulate large amounts of protein and carbohydrate as storage reserves during maturation. Thus, understanding the genetic control of embryo and seed development may provide bioengineering tools for yield improvement. In this study, we report the identification of Retarded Embryo Development 1 (RED1) gene in Arabidopsis, whose two independent T-DNA insertion mutant lines, SALK_085642 (red1-1) and SALK_022583 (red1-2), show a retarded embryo development phenotype. The embryogenesis process ceases at the late heart stage in red1-1 and at the bent-cotyledon stage in red1-2, respectively, resulting in seed abortion in both lines. The retarded embryo development and seed abortion phenotypes reverted to normal when RED1 complementation constructs were introduced into mutant plants. Small red1-2 homozygous plants can be successfully rescued by culturing immature seeds, indicating that seed abortion likely results from compromised tolerance to the desiccation process associated with seed maturation. Consistent with this observation, red1-2 seeds accumulate less protein, and the expression of two late embryo development reporter transgenes, LEA::GUS and β-conglycinin::GUS, was significantly weak and started relatively late in the red1-2 mutant lines compared to the wild type. The RED1 gene encodes a plant specific novel protein that is localized in the nucleus. These results indicate that RED1 plays important roles in embryo development, seed maturation and plant growth. PMID:27477025

  2. Expression of the gap junction gene connexin43 (Cx43) in preimplantation bovine embryos derived in vitro or in vivo.

    PubMed

    Wrenzycki, C; Herrmann, D; Carnwath, J W; Niemann, H

    1996-09-01

    In this study we have examined the presence of mRNA encoding connexin 43 (Cx43) in bovine embryos derived in vivo and in vitro. Cumulus-oocyte complexes, immature and matured oocytes liberated from cumulus cells, zygotes, 2-4-cell and 8-16-cell embryos, morulae, blastocysts and hatched blastocysts were produced in vitro from ovaries obtained from an abattoir using TCM 199 supplemented with hormones and 10% oestrous cow serum for maturation. Cumulus-oocyte complexes matured for 24 h were exposed to bull spermatozoa for 19 h and then cultured in TCM 199 supplemented with 10% oestrous cow serum to the desired developmental stage. Morulae and blastocysts derived in vivo were collected from superovulated donor cows. Total RNA was extracted from pools of 60-200 bovine oocytes or embryos using a modified phenol-chloroform extraction method and analysed by reverse transcriptase polymerase chain reaction. Before reverse transcription, aliquots of DNase-digested embryonic RNA were tested by polymerase chain reaction using bovine-specific primers to control for residual genomic DNA contamination. DNA-free, total RNA was reverse transcribed after preincubation with the Cx43 specific 3'primer. The resultant cDNA was amplified by polymerase chain reaction using Cx43 specific primers that define a 516 bp fragment of Cx43. The reverse transcriptase polymerase chain reaction product was verified by restriction enzyme analysis with Alu I and sequencing. Assays were repeated at least twice for each developmental stage and provided identical results between replicates. Cx43 transcripts were detected in bovine morulae and blastocysts grown in vivo. In contrast, whereas the early in vitro stages from cumulus-oocyte complexes to morulae expressed Cx43, blastocysts and hatched blastocysts did not have detectable concentrations of mRNA from this gene. Restriction enzyme cutting revealed three fragments of the predicted size (139, 177, 200 bp). The amplified product showed 100% identity

  3. An Improved System for Generation of Diploid Cloned Porcine Embryos Using Induced Pluripotent Stem Cells Synchronized to Metaphase.

    PubMed

    Kim, Eunhye; Zheng, Zhong; Jeon, Yubyeol; Jin, Yong-Xun; Hwang, Seon-Ung; Cai, Lian; Lee, Chang-Kyu; Kim, Nam-Hyung; Hyun, Sang-Hwan

    2016-01-01

    Pigs provide outstanding models of human genetic diseases due to their striking similarities with human anatomy, physiology and genetics. Although transgenic pigs have been produced using genetically modified somatic cells and nuclear transfer (SCNT), the cloning efficiency was extremely low. Here, we report an improved method to produce diploid cloned embryos from porcine induced pluripotent stem cells (piPSCs), which were synchronized to the G2/M stage using a double blocking method with aphidicolin and nocodazole. The efficiency of this synchronization method on our piPSC lines was first tested. Then, we modified our traditional SCNT protocol to find a workable protocol. In particular, the removal of a 6DMAP treatment post-activation enhanced the extrusion rate of pseudo-second-polar bodies (p2PB) (81.3% vs. 15.8%, based on peak time, 4hpa). Moreover, an immediate activation method yielded significantly more blastocysts than delayed activation (31.3% vs. 16.0%, based on fused embryos). The immunofluorescent results confirmed the effect of the 6DMAP treatment removal, showing remarkable p2PB extrusion during a series of nuclear transfer procedures. The reconstructed embryos from metaphase piPSCs with our modified protocol demonstrated normal morphology at 2-cell, 4-cell and blastocyst stages and a high rate of normal karyotype. This study demonstrated a new and efficient way to produce viable cloned embryos from piPSCs when synchronized to the G2/M phase of the cell cycle, which may lead to opportunities to produce cloned pigs from piPSCs more efficiently. PMID:27472781

  4. An Improved System for Generation of Diploid Cloned Porcine Embryos Using Induced Pluripotent Stem Cells Synchronized to Metaphase

    PubMed Central

    Jeon, Yubyeol; Jin, Yong-Xun; Hwang, Seon-Ung; Cai, Lian; Lee, Chang-Kyu; Kim, Nam-Hyung; Hyun, Sang-Hwan

    2016-01-01

    Pigs provide outstanding models of human genetic diseases due to their striking similarities with human anatomy, physiology and genetics. Although transgenic pigs have been produced using genetically modified somatic cells and nuclear transfer (SCNT), the cloning efficiency was extremely low. Here, we report an improved method to produce diploid cloned embryos from porcine induced pluripotent stem cells (piPSCs), which were synchronized to the G2/M stage using a double blocking method with aphidicolin and nocodazole. The efficiency of this synchronization method on our piPSC lines was first tested. Then, we modified our traditional SCNT protocol to find a workable protocol. In particular, the removal of a 6DMAP treatment post-activation enhanced the extrusion rate of pseudo-second-polar bodies (p2PB) (81.3% vs. 15.8%, based on peak time, 4hpa). Moreover, an immediate activation method yielded significantly more blastocysts than delayed activation (31.3% vs. 16.0%, based on fused embryos). The immunofluorescent results confirmed the effect of the 6DMAP treatment removal, showing remarkable p2PB extrusion during a series of nuclear transfer procedures. The reconstructed embryos from metaphase piPSCs with our modified protocol demonstrated normal morphology at 2-cell, 4-cell and blastocyst stages and a high rate of normal karyotype. This study demonstrated a new and efficient way to produce viable cloned embryos from piPSCs when synchronized to the G2/M phase of the cell cycle, which may lead to opportunities to produce cloned pigs from piPSCs more efficiently. PMID:27472781

  5. Use of DNA strand damage (Comet assay) and embryo hatching effects to assess contaminant exposure in blue crab (Callinectes sapidus) embryos

    SciTech Connect

    Lee, R.F.; Steinert, S.A.; Nakayama, K.; Oshima, Y.

    1999-07-01

    After fertilization, blue crab eggs are embedded in a sponge which is attached to the female abdomen during embryo development. Embryos after 9 stages in the egg sac hatch into a swimming zoea stage (stage 10). The authors have developed a bioassay where embryo development is monitored in culture plates with and without toxicants in the water. Toxicant effects are based on determining the percentage of embryos which hatch to zoea. Hatching EC{sub 50} (toxicant concentration at which 50% of the embryos fail to hatch) for a number of pesticides, organometallics and metals were determined. The test takes from 2 to 6 days depending on the embryo stage selected for the study. In addition to embryo development effects the prevalence of DNA single-strand breaks in individual embryo cells were determined using the single cell gel electrophoresis method (Comet assay). A good correlation between DNA strand breakage and embryo defects was found after exposure to genotoxic contaminants. Thus, the bioassay linking DNA damage to embryo hatching effects is rapid, sensitive and mechanistically relevant.

  6. T-Cell Development in Early Partially Decapitated Chicken Embryos

    PubMed Central

    Moreno, Javier; Vicente, Angeles; Varas, Alberto

    1995-01-01

    We have evaluated the immunohistological and cytofluorometric changes that occur in the thymus of chicken embryos partially decapitated at 33-38 hr of incubation (DCx embryos) in an attempt to analyze possible neuroendocrinological influences on T-cell differentiation and, indirectly, the ontogeny of the so-called neuroendocrine-immune network. The thymus of DCx embryos shows important variations that profoundly and selectively affect different T-cell subsets, but not the nonlymphoid cell components of thymic stroma. These modifications include the accumulation of cell precursors, mainly DN (CD4- CD8-) cells and immature CD8high CD4- cells, which expand but do not differentiate, resulting in an extreme decline of both DP (CD4+ CD8+) cells and TcR c-expressing cells. Accordingly, both subcapsulary and outer cortex increase in size, whereas the deep cortex and principally the thymic medulla almost disappear in DCx embryos. In contrast, other T-cell subsets of DCx embryos, largely CDgglowCD4- cells and TcR γδ-expressing cells do not undergo significant variations throughout thymic ontogeny. PMID:8770560

  7. From Stress to Embryos: Some of the Problems for Induction and Maturation of Somatic Embryos.

    PubMed

    Ochatt, Sergio J; Revilla, Maria Angeles

    2016-01-01

    Although somatic embryogenesis has been successfully achieved in numerous plant species, little is known about the mechanism(s) underlying this process. Changes in the balance of growth regulators of the culture medium, osmolarity, or amino acids as well as the genotype and developmental stage of the tissue used as initial explant may have a pivotal influence on the induction of somatic embryogenic cultures. Moreover, different stress agents (ethylene, activated charcoal, cold or heat or electrical shocks), as well as abscisic acid, can also foster the induction or further development of somatic embryos. In the process, cells first return to a stem cell-like status and then either enter their new program or dye when the stress level exceeds cell tolerance. Recalcitrance to differentiation of somatic cells into embryos is frequently observed, and problems such as secondary or recurrent embryogenesis, embryo growth arrest (at the globular stage or during the transition from torpedo to cotyledonary stage), and development of only the aerial part of somatic embryos can appear, interfering with normal germination and conversion of embryos to plants. Some solutions to solve these problems associated to embryogenesis are proposed and two very efficient somatic embryogenesis protocols for two model plant species are detailed. PMID:26619886

  8. Characterization of somatic embryo attached structures in Feijoa sellowiana Berg. (Myrtaceae).

    PubMed

    Correia, Sandra M; Canhoto, Jorge M

    2010-06-01

    The presence of an attached organ to somatic embryos of angiosperms connecting the embryo to the supporting tissue has been a subject of controversy. This study shows that 67% of the morphologically normal somatic embryos of Feijoa sellowiana possess this type of organ and that its formation was not affected by culture media composition. Histological and ultrastructural analysis indicated that the attached structures of somatic embryos displayed a great morphological diversity ranging from a few cells to massive and columnar structures. This contrast with the simple suspensors observed in zygotic embryos which were only formed by five cells. As well as the suspensor of zygotic embryos, somatic embryo attached structures undergo a process of degeneration in later stages of embryo development. Other characteristic shared by zygotic suspensors and somatic embryo attached structures was the presence of thick cell walls surrounding the cells. Elongated thin filaments were often associated with the structures attached to somatic embryos, whereas in other cases, tubular cells containing starch grains connected the embryo to the supporting tissue. These characteristics associated with the presence of plasmodesmata in the cells of the attached structures seem to indicate a role on embryo nutrition. However, cell proliferation in the attached structures resulting into new somatic embryos may also suggest a more complex relationship between the embryo and the structures connecting it to the supporting tissue.

  9. Inducible gene expression in transgenic Xenopus embryos.

    PubMed

    Wheeler, G N; Hamilton, F S; Hoppler, S

    2000-07-13

    The amphibian Xenopus laevis has been successfully used for many years as a model system for studying vertebrate development. Because of technical limitations, however, molecular investigations have mainly concentrated on early stages. We have developed a straightforward method for stage-specific induction of gene expression in transgenic Xenopus embryos [1] [2]. This method is based on the Xenopus heat shock protein 70 (Xhsp70 [3]) promoter driving the expression of desired gene products. We found that ubiquitous expression of the transgene is induced upon relatively mild heat treatment. Green fluorescent protein (GFP) was used as a marker to monitor successful induction of gene expression in transgenic embryos. We used this method to study the stage specificity of Wnt signalling function. Transient ectopic Wnt-8 expression during early neurulation was sufficient to repress anterior head development and this capacity was restricted to early stages of neurulation. By transient over-expression at different stages of development, we show that frizzled-7 disrupted morphogenesis sequentially from anterior to posterior along the dorsal axis as development proceeds. These results demonstrate that this method for inducible gene expression in transgenic Xenopus embryos will be a very powerful tool for temporal analysis of gene function and for studying molecular mechanisms of vertebrate organogenesis.

  10. Human embryos in the original position?

    PubMed

    DiSilvestro, Russell

    2005-06-01

    Two different discussions in John Rawls' A Theory of Justice lead naturally to a rather conservative position on the moral status of the human embryo. When discussing paternalism, he claims that the parties in the original position would seek to protect themselves in case they end up as incapacitated or undeveloped human beings when the veil of ignorance is lifted. Since human embryos are examples of such beings, the parties in the original position would seek to protect themselves from their embryonic stages onward. When discussing the basis of equality, Rawls claims that the parties in the original position would guarantee basic rights for all those with the capacity to take part in this original position. To guarantee the basic rights of infants and young children, he goes on to interpret this capacity as a "potentiality that is ordinarily realized in due course." Since human embryos have this potentiality, they too should have basic rights.

  11. 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.

  12. Embryo fossilization is a biological process mediated by microbial biofilms

    PubMed Central

    Raff, Elizabeth C.; Schollaert, Kaila L.; Nelson, David E.; Donoghue, Philip C. J.; Thomas, Ceri-Wyn; Turner, F. Rudolf; Stein, Barry D.; Dong, Xiping; Bengtson, Stefan; Huldtgren, Therese; Stampanoni, Marco; Chongyu, Yin; Raff, Rudolf A.

    2008-01-01

    Fossilized embryos with extraordinary cellular preservation appear in the Late Neoproterozoic and Cambrian, coincident with the appearance of animal body fossils. It has been hypothesized that microbial processes are responsible for preservation and mineralization of organic tissues. However, the actions of microbes in preservation of embryos have not been demonstrated experimentally. Here, we show that bacterial biofilms assemble rapidly in dead marine embryos and form remarkable pseudomorphs in which the bacterial biofilm replaces and exquisitely models details of cellular organization and structure. The experimental model was the decay of cleavage stage embryos similar in size and morphology to fossil embryos. The data show that embryo preservation takes place in 3 distinct steps: (i) blockage of autolysis by reducing or anaerobic conditions, (ii) rapid formation of microbial biofilms that consume the embryo but form a replica that retains cell organization and morphology, and (iii) bacterially catalyzed mineralization. Major bacterial taxa in embryo decay biofilms were identified by using 16S rDNA sequencing. Decay processes were similar in different taphonomic conditions, but the composition of bacterial populations depended on specific conditions. Experimental taphonomy generates preservation states similar to those in fossil embryos. The data show how fossilization of soft tissues in sediments can be mediated by bacterial replacement and mineralization, providing a foundation for experimentally creating biofilms from defined microbial species to model fossilization as a biological process. PMID:19047625

  13. 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

  14. Preimplantation death of xenomitochondrial mouse embryo harbouring bovine mitochondria.

    PubMed

    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

  15. Embryo cryopreservation and in vitro culture of preimplantation embryos in Campbell's hamster (Phodopus campbelli).

    PubMed

    Amstislavsky, Sergei; Brusentsev, Eugeny; Kizilova, Elena; Igonina, Tatyana; Abramova, Tatyana; Rozhkova, Irina

    2015-04-01

    The aims of this study were to compare different protocols of Campbell's hamster (Phodopus campbelli) embryos freezing-thawing and to explore the possibilities of their in vitro culture. First, the embryos were flushed from the reproductive ducts 2 days post coitum at the two-cell stage and cultured in rat one-cell embryo culture medium (R1ECM) for 48 hours. Most (86.7%) of the two-cell embryos developed to blastocysts in R1ECM. Second, the embryos at the two- to eight-cell stages were flushed on the third day post coitum. The eight-cell embryos were frozen in 0.25 mL straws according to standard procedures of slow cooling. Ethylene glycol (EG) was used either as a single cryoprotectant or in a mixture with sucrose. The survival of frozen-thawed embryos was assessed by double staining with fluorescein diacetate and propidium iodide. The use of EG as a single cryoprotectant resulted in fewer alive embryos when compared with control (fresh embryos), but combined use of EG and sucrose improved the survival rate after thawing. Furthermore, granulocyte-macrophage colony-stimulating factor rat (2 ng/mL) improved the rate of the hamster frozen-thawed embryo development in vitro by increasing the final cell number and alleviating nuclear fragmentation. Our data show the first attempt in freezing and thawing Campbell's hamster embryos and report the possibility of successful in vitro culture for this species in R1ECM supplemented with granulocyte-macrophage colony-stimulating factor.

  16. Genetic diagnosis of human embryos.

    PubMed

    Bonnicksen, Andrea

    1992-01-01

    For all the worried talk about genetic engineering over the last two decades, it is surprising how quietly plans for the genetic diagnosis of human embryos have developed. The issues raised warrant careful examination: what needs are met through embryo diagnosis? Who bears responsibility for monitoring this technique? Under what overarching ethic should embryo diagnosis and, eventually, embryo therapy, be applied? What are the broader social implications raised by the genetic diagnosis of human embryos?

  17. Conflict over moral status of embryos continues.

    PubMed

    Callahan, S

    1988-04-01

    Ethical questions are addressed that have arisen due to the rapid development of new medical technology involving the fertilized human ovum. Moral issues have arisen with the new progesterone antagonist, RU-486, and in vitro fertilization (IVF). 3 different ethical views of the embryo are presented: A permissive pro-choice position states either that the moral value of the fertilized egg depends on each pregnant woman's decision to humanize the embryo in her body or that the moral value of the fertilized egg increases as human development progresses. Some pro-life advocates argue that after implantation an irreversibly developing human being exists who has all the rights of a human being. The author suggests that some medical technological interventions and experimentation on embryos may be morally permissible to these pro-life advocates. The Vatican statement that the human being must be respected--as a person--from the very 1st instant of his existence sums up the 3d, view of an embryo's status. New knowledge of the very complex earliest stages of development, combined with other concepts and worldviews, appear to create honest doubt about the fertilized egg's status. For example, approximately 50% of fertilized ova are naturally lost, and the combination of 1 or more embryos into a mosaic casts doubt on the existence of an irreversible human. Relevant worldviews of postmodern understandings of science, the universe, and a new theology of creation must be considered to solve these ethical dilemmas.

  18. Precambrian animal diversity: putative phosphatized embryos from the Doushantuo Formation of China.

    PubMed

    Chen, J Y; Oliveri, P; Li, C W; Zhou, G Q; Gao, F; Hagadorn, J W; Peterson, K J; Davidson, E H

    2000-04-25

    Putative fossil embryos and larvae from the Precambrian phosphorite rocks of the Doushantuo Formation in Southwest China have been examined in thin section by bright field and polarized light microscopy. Although we cannot completely exclude a nonbiological or nonmetazoan origin, we identified what appear to be modern cnidarian developmental stages, including both anthozoan planula larvae and hydrozoan embryos. Most importantly, the sections contain a variety of small (stage embryos of modern bilaterian forms.

  19. Precambrian animal diversity: putative phosphatized embryos from the Doushantuo Formation of China

    NASA Technical Reports Server (NTRS)

    Chen, J. Y.; Oliveri, P.; Li, C. W.; Zhou, G. Q.; Gao, F.; Hagadorn, J. W.; Peterson, K. J.; Davidson, E. H.

    2000-01-01

    Putative fossil embryos and larvae from the Precambrian phosphorite rocks of the Doushantuo Formation in Southwest China have been examined in thin section by bright field and polarized light microscopy. Although we cannot completely exclude a nonbiological or nonmetazoan origin, we identified what appear to be modern cnidarian developmental stages, including both anthozoan planula larvae and hydrozoan embryos. Most importantly, the sections contain a variety of small (stage embryos of modern bilaterian forms.

  20. Avian Egg Odour Encodes Information on Embryo Sex, Fertility and Development

    PubMed Central

    Webster, Ben; Hayes, William; Pike, Thomas W.

    2015-01-01

    Avian chemical communication is a rapidly emerging field, but has been hampered by a critical lack of information on volatile chemicals that communicate ecologically relevant information (semiochemicals). A possible, but as yet unexplored, function of olfaction and chemical communication in birds is in parent-embryo and embryo-embryo communication. Communication between parents and developing embryos may act to mediate parental behaviour, while communication between embryos can control the synchronicity of hatching. Embryonic vocalisations and vibrations have been implicated as a means of communication during the later stages of development but in the early stages, before embryos are capable of independent movement and vocalisation, this is not possible. Here we show that volatiles emitted from developing eggs of Japanese quail (Coturnix japonica) convey information on egg fertility, along with the sex and developmental status of the embryo. Specifically, egg volatiles changed over the course of incubation, differed between fertile and infertile eggs, and were predictive of embryo sex as early as day 1 of incubation. Egg odours therefore have the potential to facilitate parent-embryo and embryo-embryo interactions by allowing the assessment of key measures of embryonic development long before this is possible through other modalities. It also opens up the intriguing possibility that parents may be able to glean further relevant information from egg volatiles, such as the health, viability and heritage of embryos. By determining information conveyed by egg-derived volatiles, we hope to stimulate further investigation into the ecological role of egg odours. PMID:25629413

  1. Lack of embryotoxicity of homocysteine thiolactone in mouse embryos in vitro.

    PubMed

    Hansen, D K; Grafton, T F; Melnyk, S; James, S J

    2001-01-01

    Recent work from humans and chick embryos has suggested that homocysteine may play a role in producing neural tube defects (NTDs). In an effort to determine if homocysteine is able to produce NTDs in mammalian embryos, mouse embryos were explanted on GD 8 and cultured for 44 h. When either homocysteine or homocysteine thiolactone was added to the culture medium, treated embryos developed as well as controls and had closed neural tubes. Homocysteine thiolactone was also microinjected into the amniotic sac of mouse embryos. Again, development proceeded normally with no significant increase in the number of embryos with open neural tubes at the end of the culture period. HPLC analysis of embryonic thiols 24 h after microinjection revealed a significant increase in embryonic cystathionine levels. These data suggest that homocysteine does not produce NTDs in mouse embryos cultured in vitro and that early organogenesis-stage embryos are able to metabolize homocysteine.

  2. Lipidome signatures in early bovine embryo development.

    PubMed

    Sudano, Mateus J; Rascado, Tatiana D S; Tata, Alessandra; Belaz, Katia R A; Santos, Vanessa G; Valente, Roniele S; Mesquita, Fernando S; Ferreira, Christina R; Araújo, João P; Eberlin, Marcos N; Landim-Alvarenga, Fernanda D C

    2016-07-15

    Mammalian preimplantation embryonic development is a complex, conserved, and well-orchestrated process involving dynamic molecular and structural changes. Understanding membrane lipid profile fluctuation during this crucial period is fundamental to address mechanisms governing embryogenesis. Therefore, the aim of the present work was to perform a comprehensive assessment of stage-specific lipid profiles during early bovine embryonic development and associate with the mRNA abundance of lipid metabolism-related genes (ACSL3, ELOVL5, and ELOVL6) and with the amount of cytoplasmic lipid droplets. Immature oocytes were recovered from slaughterhouse-derived ovaries, two-cell embryos, and eight- to 16-cell embryos, morula, and blastocysts that were in vitro produced under different environmental conditions. Lipid droplets content and mRNA transcript levels for ACSL3, ELOVL5, and ELOVL6, monitored by lipid staining and quantitative polymerase chain reaction, respectively, increased at morula followed by a decrease at blastocyst stage. Relative mRNA abundance changes of ACSL3 were closely related to cytoplasmic lipid droplet accumulation. Characteristic dynamic changes of phospholipid profiles were observed during early embryo development and related to unsaturation level, acyl chain length, and class composition. ELOVL5 and ELOVL6 mRNA levels were suggestive of overexpression of membrane phospholipids containing elongated fatty acids with 16, 18, and 20 carbons. In addition, putative biomarkers of key events of embryogenesis, embryo lipid accumulation, and elongation were identified. This study provides a comprehensive description of stage-specific lipidome signatures and proposes a mechanism to explain its potential relationship with the fluctuation of both cytoplasmic lipid droplets content and mRNA levels of lipid metabolism-related genes during early bovine embryo development. PMID:27107972

  3. Facial Transplants in Xenopus laevis Embryos

    PubMed Central

    Sive, Hazel

    2014-01-01

    Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. To identify where signaling pathways and tissues act during patterning of the developing face, a 'face transplant' technique has been developed in embryos of the frog Xenopus laevis. A region of presumptive facial tissue (the "Extreme Anterior Domain" (EAD)) is removed from a donor embryo at tailbud stage, and transplanted to a host embryo of the same stage, from which the equivalent region has been removed. This can be used to generate a chimeric face where the host or donor tissue has a loss or gain of function in a gene, and/or includes a lineage label. After healing, the outcome of development is monitored, and indicates roles of the signaling pathway within the donor or surrounding host tissues. Xenopus is a valuable model for face development, as the facial region is large and readily accessible for micromanipulation. Many embryos can be assayed, over a short time period since development occurs rapidly. Findings in the frog are relevant to human development, since craniofacial processes appear conserved between Xenopus and mammals. PMID:24748020

  4. Inhibition of Endoplasmic Reticulum Stress Improves Mouse Embryo Development

    PubMed Central

    Zhang, Jin Yu; Diao, Yun Fei; Kim, Hong Rye; Jin, Dong Il

    2012-01-01

    X-box binding protein-1 (XBP-1) is an important regulator of a subset of genes during endoplasmic reticulum (ER) stress. In the current study, we analyzed endogenous XBP-1 expression and localization, with a view to determining the effects of ER stress on the developmental competency of preimplantation embryos in mice. Fluorescence staining revealed that functional XBP-1 is localized on mature oocyte spindles and abundant in the nucleus at the germinal vesicle (GV) stage. However, in preimplantation embryos, XBP-1 was solely detected in the cytoplasm at the one-cell stage. The density of XBP-1 was higher in the nucleus than the cytoplasm at the two-cell, four-cell, eight-cell, morula, and blastocyst stages. Furthermore, RT-PCR analysis confirmed active XBP-1 mRNA splicing at all preimplantation embryo stages, except the one-cell stage. Tunicamycin (TM), an ER stress inducer used as a positive control, promoted an increase in the density of nuclear XBP-1 at the one-cell and two-cell stages. Similarly, culture medium supplemented with 25 mM sorbitol displayed a remarkable increase active XBP-1 expression in the nuclei of 1-cell and 2-cell embryos. Conversely, high concentrations of TM or sorbitol led to reduced nuclear XBP-1 density and significant ER stress-induced apoptosis. Tauroursodeoxycholic acid (TUDCA), a known inhibitor of ER stress, improved the rate of two-cell embryo development to blastocysts by attenuating the expression of active XBP-1 protein in the nucleus at the two-cell stage. Our data collectively suggest that endogenous XBP-1 plays a role in normal preimplantation embryonic development. Moreover, XBP-1 splicing is activated to generate a functional form in mouse preimplantation embryos during culture stress. TUDCA inhibits hyperosmolar-induced ER stress as well as ER stress-induced apoptosis during mouse preimplantation embryo development. PMID:22808162

  5. Preliminary studies on cryopreservation of snakehead (Channa striata) embryos.

    PubMed

    Mohd Sharifuddin, M; Siti Azizah, M N

    2014-08-01

    This paper reports the findings of the ongoing studies on cryopreservation of the snakehead, Channa striata embryos. The specific objective of this study was to collect data on the sensitivity of C. striata embryo hatching rate to low temperatures at two different developmental stages in the presence of four different cryoprotectants. Embryos at morula and heartbeat stages were selected and incubated in 1M dimethyl sulfoxide (Me2SO), 1M ethylene glycol (EG), 1M methanol (MeOH) and 0.1M sucrose solutions at different temperatures for a period of time. Embryos were kept at 24 °C (control), 15 °C, 4 °C and -2 °C for 5 min, 1h and 3h. Following these treatments, the embryos were then transferred into a 24 °C water bath until hatch to evaluate the hatching rate. The results showed that there was a significant decrease of hatching rate in both developmental stages following exposure to 4 °C and -2 °C at 1h and 3h exposure in each treatment. Heartbeat stage was more tolerant against chilling at -2 °C for 3h exposure in Me2SO followed by MeOH, sucrose and EG. Further studies will be conducted to find the best method to preserve embryos for long term storage.

  6. Preliminary studies on cryopreservation of snakehead (Channa striata) embryos.

    PubMed

    Mohd Sharifuddin, M; Siti Azizah, M N

    2014-08-01

    This paper reports the findings of the ongoing studies on cryopreservation of the snakehead, Channa striata embryos. The specific objective of this study was to collect data on the sensitivity of C. striata embryo hatching rate to low temperatures at two different developmental stages in the presence of four different cryoprotectants. Embryos at morula and heartbeat stages were selected and incubated in 1M dimethyl sulfoxide (Me2SO), 1M ethylene glycol (EG), 1M methanol (MeOH) and 0.1M sucrose solutions at different temperatures for a period of time. Embryos were kept at 24 °C (control), 15 °C, 4 °C and -2 °C for 5 min, 1h and 3h. Following these treatments, the embryos were then transferred into a 24 °C water bath until hatch to evaluate the hatching rate. The results showed that there was a significant decrease of hatching rate in both developmental stages following exposure to 4 °C and -2 °C at 1h and 3h exposure in each treatment. Heartbeat stage was more tolerant against chilling at -2 °C for 3h exposure in Me2SO followed by MeOH, sucrose and EG. Further studies will be conducted to find the best method to preserve embryos for long term storage. PMID:24726775

  7. Phosphorylated H2AX in parthenogenetically activated, in vitro fertilized and cloned bovine embryos.

    PubMed

    Pereira, A F; Melo, L M; Freitas, V J F; Salamone, D F

    2015-08-01

    In vitro embryo production methods induce DNA damage in the embryos. In response to these injuries, histone H2AX is phosphorylated (γH2AX) and forms foci at the sites of DNA breaks to recruit repair proteins. In this work, we quantified the DNA damage in bovine embryos undergoing parthenogenetic activation (PA), in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT) by measuring γH2AX accumulation at different developmental stages: 1-cell, 2-cell and blastocyst. At the 1-cell stage, IVF embryos exhibited a greater number of γH2AX foci (606.1 ± 103.2) and greater area of γH2AX staining (12923.6 ± 3214.1) than did PA and SCNT embryos. No differences at the 2-cell stage were observed among embryo types. Although PA, IVF and SCNT were associated with different blastocyst formation rates (31.1%, 19.7% and 8.3%, P < 0.05), no differences in the number of γH2AX foci or area were detected among the treatments. γH2AX is detected in bovine preimplantation embryos produced by PA, IVF and SCNT; the amount of DNA damage was comparable among those embryos developing to the blastocyst stage among different methods for in vitro embryo production. While IVF resulted in increased damage at the 1-cell embryo stage, no difference was observed between PA and SCNT embryos at any developmental stage. The decrease in the number of double-stranded breaks at the blastocyst stage seems to indicate that DNA repair mechanisms are functional during embryo development.

  8. Phosphorylated H2AX in parthenogenetically activated, in vitro fertilized and cloned bovine embryos.

    PubMed

    Pereira, A F; Melo, L M; Freitas, V J F; Salamone, D F

    2015-08-01

    In vitro embryo production methods induce DNA damage in the embryos. In response to these injuries, histone H2AX is phosphorylated (γH2AX) and forms foci at the sites of DNA breaks to recruit repair proteins. In this work, we quantified the DNA damage in bovine embryos undergoing parthenogenetic activation (PA), in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT) by measuring γH2AX accumulation at different developmental stages: 1-cell, 2-cell and blastocyst. At the 1-cell stage, IVF embryos exhibited a greater number of γH2AX foci (606.1 ± 103.2) and greater area of γH2AX staining (12923.6 ± 3214.1) than did PA and SCNT embryos. No differences at the 2-cell stage were observed among embryo types. Although PA, IVF and SCNT were associated with different blastocyst formation rates (31.1%, 19.7% and 8.3%, P < 0.05), no differences in the number of γH2AX foci or area were detected among the treatments. γH2AX is detected in bovine preimplantation embryos produced by PA, IVF and SCNT; the amount of DNA damage was comparable among those embryos developing to the blastocyst stage among different methods for in vitro embryo production. While IVF resulted in increased damage at the 1-cell embryo stage, no difference was observed between PA and SCNT embryos at any developmental stage. The decrease in the number of double-stranded breaks at the blastocyst stage seems to indicate that DNA repair mechanisms are functional during embryo development. PMID:24735637

  9. In vitro production of cattle-water buffalo (Bos taurus--Bubalus bubalis) hybrid embryos.

    PubMed

    Kochhar, H P S; Rao, K B C Appa; Luciano, A M; Totey, S M; Gandolfi, F; Basrur, P K; King, W A

    2002-05-01

    Interspecific hybrid embryos are useful models for the study of maternal-fetal interactions, transmission pattern of species-specific markers and parental contributions to growth and developmental potential of pre-attachment embryos. In an attempt to investigate the possibility of producing hybrid embryos of domestic cattle (Bos taurus) and water buffalo (Bubalus bubalis), cattle oocytes were exposed to buffalo sperm and buffalo oocytes were exposed to cattle sperm and the cleavage rate and the post-fertilisation features of hybrid embryos up to the blastocyst stage were compared with those of buffalo and cattle embryos. The cleavage rate in buffalo oocytes exposed to cattle sperm was low (40.8%), with only 8.8% of these hybrid embryos reaching the blastocyst stage. Cattle oocytes exposed to buffalo sperm showed 86.3% cleavage, while 25.9% of these attained the blastocyst stage. The speed of development of both types of hybrids was intermediate between that of cattle and buffalo embryos, with hatching occurring on day 7.5 in hybrid embryos, day 8-9 in cattle and day 7 in buffalo. The proportions of cells contributing to the trophectoderm and the inner cell mass were closer to those of the maternal species in both types of hybrid embryos. Our results indicate that cattle-water buffalo hybrid embryos produced using inter species gametes are capable of developing to advanced blastocyst stages and that their in vitro fate, and developmental potential, are influenced by the origin of the oocyte.

  10. Global gene transcription patterns in in vitro-cultured fertilized embryos and diploid and haploid murine parthenotes

    SciTech Connect

    Cui Xiangshun; Li Xingyu; Kim, Nam-Hyung . E-mail: nhkim@chungbuk.ac.kr

    2007-01-19

    To gain insights into the roles the paternal genome and chromosome number play in pre-implantation development, we cultured fertilized embryos and diploid and haploid parthenotes (DPs and HPs, respectively), and compared their development and gene expression patterns. The DPs and fertilized embryos did not differ in developmental ability but HPs development was slower and characterized by impaired compaction and blastocoel formation. Microarray analysis revealed that fertilized blastocysts expressed several genes at higher levels than DP blastocysts; these included the Y-chromosome-specific gene eukaryotic translation initiation factor 2, subunit 3, structural gene Y-linked (Eif2s3y) and the imprinting gene U2 small nuclear ribonucleoprotein auxiliary factor 1, related sequence 1 (U2af1-rs1). We also found that when DPs and HPs were both harvested at 44 and 58 h of culture, they differed in the expression of 38 and 665 genes, respectively. However, when DPs and HPs were harvested at the midpoints of 4-cell stage (44 and 49 h, respectively), no differences in expression was observed. Similarly, when the DPs and HPs were harvested when they became blastocysts (102 and 138 h, respectively), only 15 genes showed disparate expression. These results suggest that while transcripts needed for early development are delayed in HPs, it does progress sufficiently for the generation of the various developmental stages despite the lack of genetic components.

  11. Acute toxicity and synergism of binary mixtures of antifouling biocides with heavy metals to embryos of sea urchin Glyptocidaris crenularis.

    PubMed

    Xu, Xue; Wang, Xia; Li, Yan; Wang, Yonghua; Wang, Yuan

    2011-08-01

    Acute toxicity and synergism of four antifouling biocides (Irgarol 1051, dichlofluanid, tolylfluanid and Sea-Nine 211) and five heavy metals (Ni, Pb, Zn, Cd and Cu) are investigated using the sea urchin embryos of Glyptocidaris crenularis (G. crenularis) at six typical developmental stages, that is, 2-cell, 4-cell, 8-cell, blastula, gastrula and 4-arm pluteus. Our results show that the toxicity of the four biocides is in an order of Sea-Nine 211 > tolylfluanid > dichlofluanid > Irgarol 1051 and their -log EC(50) values at all stages are strongly linearly correlated with the 1-octanol/water partition coefficient (log P) values (correlation coefficients R > 0.72) indicating the importance of hydrophobicity for the embryonic toxicity. For the five heavy metals, the EC(50) ranges from 0.36 to 30.78 μM and the toxicity follows an order of Cu > Pb > Zn > Cd >Ni. The significant correlation (R > 0.79) between the -log EC50 and the bioconcentration factor (log BCF) values of metals also indicate that the bioaccumulation property of metals contributes to their aquatic toxicity. In addition, the joint effects of the biocides with the heavy metals in embryonic development are assessed by using a concentration addition model. Synergistic effects are observed in almost all 25 mixtures, showing that Cu yields the strongest while Ni the weakest synergistic toxic effects on the embryos development. PMID:20930027

  12. Dual Positive Regulation of Embryo Implantation by Endocrine and Immune Systems--Step-by-Step Maternal Recognition of the Developing Embryo.

    PubMed

    Fujiwara, Hiroshi; Araki, Yoshihiko; Imakawa, Kazuhiko; Saito, Shigeru; Daikoku, Takiko; Shigeta, Minoru; Kanzaki, Hideharu; Mori, Takahide

    2016-03-01

    In humans, HCG secreted from the implanting embryo stimulates progesterone production of the corpus luteum to maintain embryo implantation. Along with this endocrine system, current evidence suggests that the maternal immune system positively contributes to the embryo implantation. In mice, immune cells that have been sensitized with seminal fluid and then the developing embryo induce endometrial differentiation and promote embryo implantation. After hatching, HCG activates regulatory T and B cells through LH/HCG receptors and then stimulates uterine NK cells and monocytes through sugar chain receptors, to promote and maintain pregnancy. In accordance with the above, the intrauterine administration of HCG-treated PBMC was demonstrated to improve implantation rates in women with repeated implantation failures. These findings suggest that the maternal immune system undergoes functional changes by recognizing the developing embryos in a stepwise manner even from a pre-fertilization stage and facilitates embryo implantation in cooperation with the endocrine system. PMID:26755274

  13. Phosphatized polar lobe-forming embryos from the Precambrian of southwest China.

    PubMed

    Chen, Jun-Yuan; Bottjer, David J; Davidson, Eric H; Dornbos, Stephen Q; Gao, Xiang; Yang, Yong-Hua; Li, Chia-Wei; Li, Gang; Wang, Xiu-Qiang; Xian, Ding-Chang; Wu, Hung-Jen; Hwu, Yeu-Kuang; Tafforeau, Paul

    2006-06-16

    In developing embryos of some extant spiralian animals, polar lobe formation is one of the symmetry-breaking mechanisms for segregation of maternal cytoplasmic substances to certain blastomeres and not others. Polar lobe formation leads to unique early cleavage morphologies that include trilobed, J-shaped, and five-lobed structures. Fossil embryos similar to modern lobeforming embryos are recognized from the Precambrian Doushantuo Formation phosphates, Weng'an, Guizhou Province, China. These embryos are abundant and form a developmental sequence comparable to different developing stages observed in lobe-forming embryos of extant spiralians. These data imply that lobe formation is an evolutionarily ancient process of embryonic specification.

  14. Culture of bovine embryos on a polydimethylsiloxane (PDMS) microwell plate.

    PubMed

    Akagi, Satoshi; Hosoe, Misa; Matsukawa, Kazutsugu; Ichikawa, Akihiko; Tanikawa, Tamio; Takahashi, Seiya

    2010-08-01

    We fabricated a polydimethylsiloxane (PDMS)-based microwell plate (PDMS-MP) containing 100 microwells with a rounded bottom and examined whether it can be used for culture of individual in vitro fertilized (IVF) embryos or parthenogenetically activated zona-free embryos in cattle. In Experiment 1, we examined the in vitro developmental ability of IVF embryos cultured individually on PDMS-MP. After IVF, 20 embryos were transferred into 100 microl drops on PDMS-MP and cultured individually in each well of PDMS-MP (PDMS group). After 7 days of culture, the embryos in the PDMS group developed to the blastocyst stage at the same rate of those in the control group cultured in a group of 20 embryos without PDMS-MP. There were no differences in total number of cells and the ratio of inner cell mass to total cells between the PDMS and control groups. In Experiment 2, we examined the in vitro developmental ability of parthenogenetically activated zona-free bovine embryos cultured individually on PDMS-MP. The zona-free embryos were cultured individually in each well of a PDMS-MP or in each well produced by pressing a darning needle onto the bottom of a culture dish (WOW group). After 7 days of culture, the blastocyst formation rate and cell number of blastocysts in the PDMS group did not differ from those of the zona-intact embryos in the control group. Also, there were no differences in the blastocyst formation rate and cell number of blastocysts between the WOW and PDMS groups. These results suggest that the culture system using PDMS-MP is useful for individual embryos or zona-free embryos in cattle. PMID:20484872

  15. Human pre-implantation embryo development

    PubMed Central

    Niakan, Kathy K.; Han, Jinnuo; Pedersen, Roger A.; Simon, Carlos; Pera, Renee A. Reijo

    2012-01-01

    Understanding human pre-implantation development has important implications for assisted reproductive technology (ART) and for human embryonic stem cell (hESC)-based therapies. Owing to limited resources, the cellular and molecular mechanisms governing this early stage of human development are poorly understood. Nonetheless, recent advances in non-invasive imaging techniques and molecular and genomic technologies have helped to increase our understanding of this fascinating stage of human development. Here, we summarize what is currently known about human pre-implantation embryo development and highlight how further studies of human pre-implantation embryos can be used to improve ART and to fully harness the potential of hESCs for therapeutic goals. PMID:22318624

  16. A formula for scoring human embryo growth rates in in vitro fertilization: its value in predicting pregnancy and in comparison with visual estimates of embryo quality.

    PubMed

    Cummins, J M; Breen, T M; Harrison, K L; Shaw, J M; Wilson, L M; Hennessey, J F

    1986-10-01

    Two systems for measuring embryo development in vitro were evaluated. One was a 1-4 scale based on a subjective evaluation of embryo quality (EQ) from microscopic appearance. In addition, a formula for scoring embryo growth rate in vitro was developed. The embryo development rating (EDR) was based on the ratio between the time at which embryos were observed at a particular stage after insemination and the time at which they would be expected to reach that stage in a hypothetical "ideal" growth rate with a cell cycle length of 11.9 hr. Using this scoring system, "normally" growing embryos scored 100. This approach was aimed at partially normalizing the data and allowed all embryos to be analyzed similarly regardless of the time of observation. Analysis of 1539 embryo replacements resulting in 232 clinical pregnancies showed that both EDR and embryo-quality scores were of value in predicting success, with clinical pregnancy most likely to eventuate from a combination of moderate to good EQ scores (2-4) coupled with average or above-average growth rates (EDR scores from 90 to 129). Poor-quality and very slowly or very rapidly growing embryos were underrepresented in cycles that proceeded to pregnancy. These inferences were based on all embryos transferred (mean, 2.73 per transfer cycle), and they were substantiated by an analysis of 33 pregnancies resulting from replacement of a single embryo and from 18 pregnancies in which all embryos scored the same with both systems. EQ and EDR were significantly associated with each other and together provide a valuable guide in predicting pregnancy, in selecting embryos for freezing, and in monitoring day-to-day performance in the in vitro fertilization (IVF) program.

  17. Efficient reproduction of cynomolgus monkey using pronuclear embryo transfer technique.

    PubMed

    Sun, Qiang; Dong, Juan; Yang, Wenting; Jin, Yujuan; Yang, Mingying; Wang, Yan; Wang, Philip L; Hu, Yinghe; Tsien, Joe Z

    2008-09-01

    One of the technical bottlenecks in producing nonhuman primate models is that current assisted reproductive techniques, such as in vitro culture and frozen conservation of multicell-stage embryos, often result in poor embryo quality and subsequently lead to low birth rates. We investigated whether pronuclear embryo transfer can be used as an effective means for improving pregnancy and live birth rates of nonhuman primates. We collected 174 metaphase II oocytes by laparoscopy from 22 superovulated mature females and then fertilized these eggs using either in vitro fertilization or intracytoplasmic sperm injection, resulting in a 33.3% and a 50% fertilization rate, respectively. These 66 fertilized pronuclear-stage embryos were then tubally transferred to 30 recipients and led to 7 births and 1 abortion. Importantly, we observed that the highest live birth rate of approximately 64% was obtained when the transfer of pronuclear embryos was performed in the presence of new corpus luteum in the ovary of recipients between 24 h and 36 h after estradiol peak. Therefore, our experiments demonstrate that by matching the critical time window in the recipient's reproductive cycle for achieving optimal embryo-uterine synchrony, pronuclear embryo transfer technology can significantly improve the pregnancy rate and live birth of healthy baby monkeys. This efficient method should be valuable to the systematic efforts in construction of various transgenic primate disease models. PMID:18725640

  18. Efficient reproduction of cynomolgus monkey using pronuclear embryo transfer technique

    PubMed Central

    Sun, Qiang; Dong, Juan; Yang, Wenting; Jin, Yujuan; Yang, Mingying; Wang, Yan; Wang, Philip L.; Hu, Yinghe; Tsien, Joe Z.

    2008-01-01

    One of the technical bottlenecks in producing nonhuman primate models is that current assisted reproductive techniques, such as in vitro culture and frozen conservation of multicell-stage embryos, often result in poor embryo quality and subsequently lead to low birth rates. We investigated whether pronuclear embryo transfer can be used as an effective means for improving pregnancy and live birth rates of nonhuman primates. We collected 174 metaphase II oocytes by laparoscopy from 22 superovulated mature females and then fertilized these eggs using either in vitro fertilization or intracytoplasmic sperm injection, resulting in a 33.3% and a 50% fertilization rate, respectively. These 66 fertilized pronuclear-stage embryos were then tubally transferred to 30 recipients and led to 7 births and 1 abortion. Importantly, we observed that the highest live birth rate of ≈64% was obtained when the transfer of pronuclear embryos was performed in the presence of new corpus luteum in the ovary of recipients between 24 h and 36 h after estradiol peak. Therefore, our experiments demonstrate that by matching the critical time window in the recipient's reproductive cycle for achieving optimal embryo-uterine synchrony, pronuclear embryo transfer technology can significantly improve the pregnancy rate and live birth of healthy baby monkeys. This efficient method should be valuable to the systematic efforts in construction of various transgenic primate disease models. PMID:18725640

  19. 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.

  20. Storage oil breakdown during embryo development of Brassica napus (L.).

    PubMed

    Chia, Tansy Y P; Pike, Marilyn J; Rawsthorne, Stephen

    2005-05-01

    In this study it is shown that at least 10% of the major storage product of developing embryos of Brassica napus (L.), triacylglycerol, is lost during the desiccation phase of seed development. The metabolism of this lipid was studied by measurements of the fate of label from [1-(14)C]decanoate supplied to isolated embryos, and by measurements of the activities of enzymes of fatty acid catabolism. Measurements on desiccating embryos have been compared with those made on embryos during lipid accumulation and on germinating seedlings. Enzymes of beta-oxidation and the glyoxylate cycle, and phosphoenolpyruvate carboxykinase were present in embryos during oil accumulation, and increased in activity and abundance as the seeds matured and became desiccated. Although the activities were less than those measured during germination, they were at least comparable to the in vivo rate of fatty acid synthesis in the embryo during development. The pattern of labelling, following metabolism of decanoate by isolated embryos, indicated a much greater involvement of the glyoxylate cycle during desiccation than earlier in oil accumulation, and showed that much of the (14)C-label from decanoate was released as CO(2) at both stages. Sucrose was not a product of decanoate metabolism during embryo development, and therefore lipid degradation was not associated with net gluconeogenic activity. These observations are discussed in the context of seed development, oil yield, and the synthesis of novel fatty acids in plants.

  1. 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

  2. Equine cloning: in vitro and in vivo development of aggregated embryos.

    PubMed

    Gambini, Andrés; Jarazo, Javier; Olivera, Ramiro; Salamone, Daniel F

    2012-07-01

    The production of cloned equine embryos remains highly inefficient. Embryo aggregation has not yet been tested in the equine, and it might represent an interesting strategy to improve embryo development. This study evaluated the effect of cloned embryo aggregation on in vitro and in vivo equine embryo development. Zona-free reconstructed embryos were individually cultured in microwells (nonaggregated group) or as 2- or 3-embryo aggregates (aggregated groups). For in vitro development, they were cultured until blastocyst stage and then either fixed for Oct-4 immunocytochemical staining or maintained in in vitro culture where blastocyst expansion was measured daily until Day 17 or the day on which they collapsed. For in vivo assays, Day 7-8 blastocysts were transferred to synchronized mares and resultant vesicles, and cloned embryos were measured by ultrasonography. Embryo aggregation improved blastocyst rates on a per well basis, and aggregation did not imply additional oocytes to obtain blastocysts. Embryo aggregation improved embryo quality, nevertheless it did not affect Day 8 and Day 16 blastocyst Oct-4 expression patterns. Equine cloned blastocysts expanded and increased their cell numbers when they were maintained in in vitro culture, describing a particular pattern of embryo growth that was unexpectedly independent of embryo aggregation, as all embryos reached similar size after Day 7. Early pregnancy rates were higher using blastocysts derived from aggregated embryos, and advanced pregnancies as live healthy foals also resulted from aggregated embryos. These results indicate that the strategy of aggregating embryos can improve their development, supporting the establishment of equine cloned pregnancies.

  3. 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

  4. Embryo loss in cattle between Days 7 and 16 of pregnancy.

    PubMed

    Berg, D K; van Leeuwen, J; Beaumont, S; Berg, M; Pfeffer, P L

    2010-01-15

    Embryo loss between embryonic Days 7 and 16 (Day 0=day of IVF) in nonlactating cattle, Bos taurus, was analyzed using transfer of 2449 (in groups of 3 to 30) in vitro-produced (IVP) blastocysts. In 152 transfers, pregnancy losses attributable solely to recipient failings amounted to between 6% (beef heifers) and 16% (parous dairy cows), of which 3% were caused by uterine infections. Neither season, year, nor the age of the embryos on retrieval affected pregnancy rates. The latter observation indicated that the reason that a recipient failed to retain embryos was already present at the time of transfer. Notably, the proportion of embryos recovered decreased (P=0.03) as more embryos were transferred, particularly at later stages (Day 14, P<0.01). The average length of embryos decreased by approximately 5% for every additional embryo transferred (P<0.0001). These effects may be linked to embryonic migration. Embryo mortality inherent to the embryo during the second week of pregnancy was 24%. Additionally, 9% of Day 14 embryos were of inferior quality, as they did not contain an epiblast. Combining embryo and recipient causes but excluding infection effects, embryonic loss of IVP embryos during the second week of pregnancy amounted to 26% (heifers) or 34% (parous dairy cows). The length of embryos doubled every day between Days 9 and 16, with a 4.4-fold range in sizes representing two thirds of the variation in length. Embryos retrieved from heifers were twice the size of those incubated in parous cows (P<0.0001), indicating faster embryonic development/trophoblast proliferation in heifers. Whereas season did not affect embryo recoveries, length was lower (50%) in winter (winter-autumn, P<0.05; winter-spring, P<0.001). Lastly, transuterine migration in cattle, when transferring multiple embryos, commenced at Day 14 (4%) and had occurred in all recipients by Day 16 (38% of embryos found contralaterally).

  5. Cooling of pirapitinga (Piaractus brachypomus) embryos stored at -10ºC.

    PubMed

    Pessoa, Nathalie Ommundsen; Galvão, José Agenor Soares; de Souza Filho, Francisco Gerson Mendes; de Sousa, Míriam Luiza Nogueira Martins; Sampaio, Célia Maria Souza

    2015-06-01

    Cryopreservation has not been used successfully to preserve fish embryos, although chilling techniques have been used with good results. The aim of this study was to chill Piaractus brachypomus embryos at - 10°C for various storage times. Embryos at the following ontogenetic stages were used: blastoderm - 1.2 hours post-fertilization (hpf); epiboly - 5 hpf; blastopore closure - 8 hpf; and appearance of the optic vesicle - 13 hpf. One hundred embryos were selected from each ontogenetic stage and chilled at - 10°C for 6 or 10 h. The results were analysed using analysis of variance (ANOVA) and Tukey's test at a 5% significance level. A significantly greater number of completely developed live larvae were observed following embryonic treatment with a cryoprotectant solution that contained 17.5% sucrose and 10% methanol. There was no survival for embryos cooled at - 10°C in initial developmental stages (1, 2 and 5 h hpf). Furthermore, higher survival rates were observed when embryos were treated at more advanced developmental stages (8 and 13 hpf). Therefore, P. brachypomus embryos at the blastopore-closure (8 hpf) or appearance-of-optic-vesicle (13 hpf) stages should be used for embryo chilling protocols and chilling should be performed using a 17.5% sucrose with a 10% methanol solution at - 10°C for up to 6 h. The best results were obtained with 13-hpf and 8-hpf embryos and cooling at 6 h of storage. PMID:24666580

  6. The Metabolomic Profile of Spent Culture Media from Day-3 Human Embryos Cultured under Low Oxygen Tension.

    PubMed

    de Los Santos, Maria José; Gámiz, Pilar; de Los Santos, José María; Romero, Josep Lluís; Prados, Nicolás; Alonso, Cristina; Remohí, José; Dominguez, Francisco

    2015-01-01

    Despite efforts made to improve the in vitro embryo culture conditions used during assisted reproduction procedures, human embryos must adapt to different in vitro oxygen concentrations and the new metabolic milieu provided by the diverse culture media used for such protocols. It has been shown that the embryo culture environment can affect not only cellular metabolism, but also gene expression in different species of mammalian embryos. Therefore we wanted to compare the metabolic footprint left by human cleavage-stage embryos under two types of oxygen atmospheric culture conditions (6% and 20% O2). The spent culture media from 39 transferred and implanted embryos from a total of 22 patients undergoing egg donation treatment was analyzed; 23 embryos came from 13 patients in the 6% oxygen concentration group, and 16 embryos from 9 patients were used in the 20% oxygen concentration group. The multivariate statistics we used in our analysis showed that human cleavage-stage embryos grown under both types of oxygen concentration left a similar metabolic fingerprint. We failed to observe any change in the net depletion or release of relevant analytes, such as glucose and especially fatty acids, by human cleavage-stage embryos under either type of culture condition. Therefore it seems that low oxygen tension during embryo culture does not alter the global metabolism of human cleavage-stage embryos. PMID:26562014

  7. The avian embryo responding to microgravity of space flight

    NASA Technical Reports Server (NTRS)

    Hullinger, Ronald L.

    1993-01-01

    Of all the many potential and real microenvironmental influences, only gravity would appear to have remained relatively constant and ubiquitous for developing organisms. Histo- and organogenesis as well as differential growth of the embryo and fetus may have evolved with a constant environmental factor of gravity. Chick embryos of 2-day and 9-day stages of incubation were flown in an incubator on the Space Shuttle during a 9-day mission. Significant differences in embryo response to this microgravity environment were observed. This paper offers an analysis and suggests mechanisms which may contribute to these results.

  8. Ethics for embryos.

    PubMed

    Parker, C

    2007-10-01

    This paper responds to DW Brock's technically strong case for the use of human embryonic stem cells in medical research. His main issue in this context is the question of whether it is moral to destroy viable human embryos. He offers a number of reasons to support his view that it is moral to destroy them, but his use of conceptual arguments is not adequate to secure his position. The purpose and scope of this paper is wholly concerned with his arguments rather than with the conclusion that it is justifiable to destroy human embryos. The author proceeds through his variety of arguments and offers reasons for rejecting them. The author concludes that Brock has not shown that it is moral to destroy viable human embryos.

  9. Cryopreservation and In Vitro culture of Preimplantation Embryos in Djungarian Hamster (Phodopus sungorus).

    PubMed

    Brusentsev, E Yu; Abramova, T O; Rozhkova, I N; Igonina, T N; Naprimerov, V A; Feoktistova, N Yu; Amstislavsky, S Ya

    2015-08-01

    Although embryo cryobanking was applied to Syrian golden and to Campbell's hamsters, no attempt has been made at freezing embryos in Djungarian hamsters. Four-cell stage embryos were flushed from the reproductive ducts of pregnant females before noon of the third-day post coitum and frozen in 0.25-ml straws according to standard procedures of slow cooling. A mixture of permeating (ethylene glycol) and non-permeating (sucrose) cryoprotectants was used. The thawing was performed by incubating at RT for 40 s followed by 40 s in a water bath at 30.0°C. Most (66.7%) of the non-frozen four-cell embryos developed up to the morula stage in rat one-cell embryo culture medium (R1ECM). The use of hamster embryo culture medium (HECM) yielded fewer morulas (18.2%) during the same 24-h period of culture. The rate of embryo's surviving the freezing-thawing procedures, as estimated by light microscopy, was 60.7-68.8%. After 24-h culturing in R1ECM, 64.7% of frozen-thawed four-cell embryos developed and all of them reached the morula stage. Supplementation of R1ECM with GM-CSF (2 ng/ml) improved the rate of Djungarian hamster frozen-thawed embryo development: 100% of the four-cell stage embryos developed, 50% of them achieved the morula stage, and 50% developed even further and reached the blastocyst stage within 24 h of culturing. This study reports the world's first successful transfer of frozen-thawed Djungarian hamster embryos yielding term pups. Taken together, the results of this study demonstrate the possibility of applying some key reproductive technologies, that is, embryo freezing/cryopreservation and in vitro culture, to Djungarian hamsters.

  10. Somitomeres: mesodermal segments of vertebrate embryos.

    PubMed

    Jacobson, A G

    1988-01-01

    Well before the somites form, the paraxial mesoderm of vertebrate embryos is segmented into somitomeres. When newly formed, somitomeres are patterned arrays of mesenchymal cells, arranged into squat, bilaminar discs. The dorsal and ventral faces of these discs are composed of concentric rings of cells. Somitomeres are formed along the length of the embryo during gastrulation, and in the segmental plate and tail bud at later stages. They form in strict cranial to caudal order. They appear in bilateral pairs, just lateral to Hensen's node in the chick embryo. When the nervous system begins to form, the brain parts and neuromeres are in a consistent relationship to the somitomeres. Somitomeres first appear in the head, and the cranial somitomeres do not become somites, but disperse to contribute to the head the same cell types contributed by somites in the trunk region. In the trunk and tail, somitomeres gradually condense and epithelialize to become somites. Models of vertebrate segmentation must now take into account the early presence of these new morphological units, the somitomeres. Somitomeres were discovered in the head of the chick embryo (Meier, 1979), with the use of stereo scanning electron microscopy. The old question of whether the heads of the craniates are segmented is now settled, at least for the paraxial mesoderm. Somitomeres have now been identified in the embryos of a chick, quail, mouse, snapping turtle, newt, anuran (Xenopus) and a teleost (the medaka). In all forms studied, the first pair of somitomeres abut the prosencephalon, but caudal to that, for each tandem pair of somitomeres in the amniote and teleost, there is but one somitomere in the amphibia. The mesodermal segments of the shark embryo are arranged like those of the amphibia.

  11. Identification of key factors conquering developmental arrest of somatic cell cloned embryos by combining embryo biopsy and single-cell sequencing

    PubMed Central

    Liu, Wenqiang; Liu, Xiaoyu; Wang, Chenfei; Gao, Yawei; Gao, Rui; Kou, Xiaochen; Zhao, Yanhong; Li, Jingyi; Wu, You; Xiu, Wenchao; Wang, Su; Yin, Jiqing; Liu, Wei; Cai, Tao; Wang, Hong; Zhang, Yong; Gao, Shaorong

    2016-01-01

    Differentiated somatic cells can be reprogrammed into totipotent embryos through somatic cell nuclear transfer. However, most cloned embryos arrest at early stages and the underlying molecular mechanism remains largely unexplored. Here, we first developed a somatic cell nuclear transfer embryo biopsy system at two- or four-cell stage, which allows us to trace the developmental fate of the biopsied embryos precisely. Then, through single-cell transcriptome sequencing of somatic cell nuclear transfer embryos with different developmental fates, we identified that inactivation of Kdm4b, a histone H3 lysine 9 trimethylation demethylase, functions as a barrier for two-cell arrest of cloned embryos. Moreover, we discovered that inactivation of another histone demethylase Kdm5b accounts for the arrest of cloned embryos at the four-cell stage through single-cell analysis. Co-injection of Kdm4b and Kdm5b can restore transcriptional profiles of somatic cell nuclear transfer embryos and greatly improve the blastocyst development (over 95%) as well as the production of cloned mice. Our study therefore provides an effective approach to identify key factors responsible for the developmental arrest of somatic cell cloned embryos. PMID:27462457

  12. Identification of key factors conquering developmental arrest of somatic cell cloned embryos by combining embryo biopsy and single-cell sequencing.

    PubMed

    Liu, Wenqiang; Liu, Xiaoyu; Wang, Chenfei; Gao, Yawei; Gao, Rui; Kou, Xiaochen; Zhao, Yanhong; Li, Jingyi; Wu, You; Xiu, Wenchao; Wang, Su; Yin, Jiqing; Liu, Wei; Cai, Tao; Wang, Hong; Zhang, Yong; Gao, Shaorong

    2016-01-01

    Differentiated somatic cells can be reprogrammed into totipotent embryos through somatic cell nuclear transfer. However, most cloned embryos arrest at early stages and the underlying molecular mechanism remains largely unexplored. Here, we first developed a somatic cell nuclear transfer embryo biopsy system at two- or four-cell stage, which allows us to trace the developmental fate of the biopsied embryos precisely. Then, through single-cell transcriptome sequencing of somatic cell nuclear transfer embryos with different developmental fates, we identified that inactivation of Kdm4b, a histone H3 lysine 9 trimethylation demethylase, functions as a barrier for two-cell arrest of cloned embryos. Moreover, we discovered that inactivation of another histone demethylase Kdm5b accounts for the arrest of cloned embryos at the four-cell stage through single-cell analysis. Co-injection of Kdm4b and Kdm5b can restore transcriptional profiles of somatic cell nuclear transfer embryos and greatly improve the blastocyst development (over 95%) as well as the production of cloned mice. Our study therefore provides an effective approach to identify key factors responsible for the developmental arrest of somatic cell cloned embryos. PMID:27462457

  13. 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 (embryo configuration, connected by a very thin cell bridge (figure eight in shape). To separate the parts of the embryo, the cell bridge was cut using a glass microneedle. During the separation only a few cells were damaged. As a result of the procedure 4 20 (20.0%), 48 144 (33.3%) and 3 40 (7.5%) middle, late and expanded blastocysts hatched according to the pattern described. The developed procedure could be considered as a non-invasive alternative to conventional embryo splitting.

  14. Production of embryos by oocyte cytoplast-blastomere fusion in domestic animals.

    PubMed

    First, N L; Prather, R S

    1991-01-01

    Embryos of amphibians, sheep, cattle, pigs and rabbits have been multiplied by nuclear transfer. Successful nuclear transfer in these species has been accomplished by transfer of a blastomere from a late-stage embryo into an enucleated oocyte with large scale multiplication by repeating the procedure using blastomeres from the embryos produced from nuclear transfer. This allows the production of clonal lines which, when appropriately selected for performance in a given trait, can be reproduced to capture in the offspring expression of additive and non-additive inheritance. The efficiency of these procedures is high only for amphibian embryos for which as many as 1000 offspring can be made from a blastula- or gastrula-stage embryo and the process repeated 60-100 times with descendant embryos. In domestic animals the largest number of offspring from one embryo has been 8 calves. Embryos as late as the 64-cell stage in cattle and 120-cell blastocyst in sheep have been used successfully as donors of blastomeres. Recloning has also been done in cattle. Nuclear transfer potentially provides a mechanism for multiplication and production testing of clonal lines, a method for rapid genetic improvement and rapid propagation of a selected genotype. Unfortunately the present efficiencies of subsequent embryo development, pregnancy and embryo survival are less than normal. This paper reviews variables contributing to reduced efficiency and research to improve nuclear transfer.

  15. Influence of Delipation on the Energy Metabolism in Pig Parthenogenetically Activated Embryos.

    PubMed

    Wang, C; Niu, Y; Chi, D; Zeng, Y; Liu, H; Dai, Y; Li, J

    2015-10-01

    This study was designed not only to measure the effect of delipation on the developmental viability of pig parthenogenetically activated (PA) embryos, but also to evaluate the changes of mitochondria DNA (mtDNA), reactive oxygen species (ROS) level, adenosine triphosphate (ATP) content and gene (Acsl3, Acadsb, Acaa2, Glut1) expression level at different stages after delipation. Results showed that no effect was observed on the cleavage ability, but significant lower blastocyst rate was obtained in delipated embryos. Copy number of mtDNA decreased gradually from MII to four-cell stages and subsequently kept consistent with blastocyst stage both in delipated and control embryos, but the copy number of mtDNA in delipated embryos was similar to that in the control groups no matter at which developmental stage was observed. Both in delipated and control embryos, ATP content progressive decreased from one-cell to blastocyst stages, while just at one-cell stage, a significant decrease of ATP level was observed in delipated embryos compared with that of control. The level of ROS increased obviously after delipation at cleavage stage, but no difference was seen at blastocyst stage. Finally, the expression level of genes related to fatty acids beta-oxidation (Acadsb and Acaa2) was decreased, while the expression level of genes related to glucose metabolism (Glut 1) was upregulated after delipation. In conclusion, the reduction of lipids in pig oocytes will affect the developmental competence of pig PA embryos by disturbed energy metabolism and ROS stress. PMID:26303295

  16. The Virtual Embryo Project

    EPA Science Inventory

    The v-Embryo™ is a far reaching new research program at the US EPA to develop a working computer model of a mammalian embryo that can be used to better understand the prenatal risks posed by environmental chemicals and to eventually predict a chemical’s potential developmental to...

  17. Alternation of apoptotic and implanting genes expression of mouse embryos after re-vitrification

    PubMed Central

    Majidi Gharenaz, Nasrin; Movahedin, Mansoureh; Mazaheri, Zohreh; Pour beiranvand, Shahram

    2016-01-01

    Background: Nowadays, oocytes and embryos vitrification has become a routine technique. Based on clinical judgment, re-vitrification maybe required. But little is known about re-vitrification impact on genes expression. Objective: The impact of re-vitrification on apoptotic and implanting genes, Bax, Bcl-2 and ErbB4, at compaction stage embryos were evaluated in this study. Materials and Methods: In this experimental study, 8 cell embryos (n=240) were collected from female mature mice, 60-62 hr post HCG injection. The embryos were divided randomly to 3 groups included: fresh (n=80), vitrified at 8 cell stage (n=80), vitrified at 8 cell stage thawed and re-vitrified at compaction stage (n=80). Embryos were vitrified by using cryolock, (open system) described by Kuwayama. Q-PCR was used to examine the expression of Bax, Bcl2 ErbB4 genes in derived blastocysts. Results: Our result showed that expanded blastocyst rate was similar between vitrified and re-vitrified groups, while re-vitrified embryos showed significant decrease in expanded blastocyst rate comparing with fresh embryos (p=0.03). In addition, significant difference was observed on apoptotic gene expression when comparing re-vitrified and fresh embryos (p=0.004), however expression of Bax and Bcl-2 (apoptotic) genes didn't demonstrate a significant difference between re-vitrified and vitrified groups. The expression rate of ErbB4, an implantation gene was decreased in re-vitrified embryos comparing with fresh embryos (p=0.003), but it was similar between re-vitrified and vitrified embryos. Conclusion: Re-vitrification can alter the expression of Bax, Bcl-2 and ErbB4 genes and developmental rate of mouse embryos in compaction stage. PMID:27679826

  18. Alternation of apoptotic and implanting genes expression of mouse embryos after re-vitrification

    PubMed Central

    Majidi Gharenaz, Nasrin; Movahedin, Mansoureh; Mazaheri, Zohreh; Pour beiranvand, Shahram

    2016-01-01

    Background: Nowadays, oocytes and embryos vitrification has become a routine technique. Based on clinical judgment, re-vitrification maybe required. But little is known about re-vitrification impact on genes expression. Objective: The impact of re-vitrification on apoptotic and implanting genes, Bax, Bcl-2 and ErbB4, at compaction stage embryos were evaluated in this study. Materials and Methods: In this experimental study, 8 cell embryos (n=240) were collected from female mature mice, 60-62 hr post HCG injection. The embryos were divided randomly to 3 groups included: fresh (n=80), vitrified at 8 cell stage (n=80), vitrified at 8 cell stage thawed and re-vitrified at compaction stage (n=80). Embryos were vitrified by using cryolock, (open system) described by Kuwayama. Q-PCR was used to examine the expression of Bax, Bcl2 ErbB4 genes in derived blastocysts. Results: Our result showed that expanded blastocyst rate was similar between vitrified and re-vitrified groups, while re-vitrified embryos showed significant decrease in expanded blastocyst rate comparing with fresh embryos (p=0.03). In addition, significant difference was observed on apoptotic gene expression when comparing re-vitrified and fresh embryos (p=0.004), however expression of Bax and Bcl-2 (apoptotic) genes didn't demonstrate a significant difference between re-vitrified and vitrified groups. The expression rate of ErbB4, an implantation gene was decreased in re-vitrified embryos comparing with fresh embryos (p=0.003), but it was similar between re-vitrified and vitrified embryos. Conclusion: Re-vitrification can alter the expression of Bax, Bcl-2 and ErbB4 genes and developmental rate of mouse embryos in compaction stage.

  19. Embryo Aggregation in Pig Improves Cloning Efficiency and Embryo Quality.

    PubMed

    Buemo, Carla Paola; Gambini, Andrés; Moro, Lucia Natalia; Hiriart, María Inés; Fernández-Martín, Rafael; Collas, Philippe; Salamone, Daniel Felipe

    2016-01-01

    In this study, we analyzed the effects of the cloned embryo aggregation on in vitro embryo development and embryo quality by measuring blastocyst diameter and cell number, DNA fragmentation levels and the expression of genes associated with pluripotency, apoptosis, trophoblast and DNA methylation in the porcine. Zona-free reconstructed cloned embryos were cultured in the well of the well system, placing one (1x non aggregated group) or three (3x group) embryos per microwell. Our results showed that aggregation of three embryos increased blastocyst formation rate and blastocyst diameter of cloned pig embryos. DNA fragmentation levels in 3x aggregated cloned blastocysts were significantly decreased compared to 1x blastocysts. Levels of Oct4, Klf4, Igf2, Bax and Dnmt 1 transcripts were significantly higher in aggregated embryos, whereas Nanog levels were not affected. Transcripts of Cdx2 and Bcl-xl were essentially non-detectable. Our study suggests that embryo aggregation in the porcine may be beneficial for cloned embryo development and embryo quality, through a reduction in apoptotic levels and an improvement in cell reprogramming.

  20. Embryo Aggregation in Pig Improves Cloning Efficiency and Embryo Quality

    PubMed Central

    Buemo, Carla Paola; Gambini, Andrés; Moro, Lucia Natalia; Hiriart, María Inés; Fernández-Martín, Rafael; Collas, Philippe; Salamone, Daniel Felipe

    2016-01-01

    In this study, we analyzed the effects of the cloned embryo aggregation on in vitro embryo development and embryo quality by measuring blastocyst diameter and cell number, DNA fragmentation levels and the expression of genes associated with pluripotency, apoptosis, trophoblast and DNA methylation in the porcine. Zona-free reconstructed cloned embryos were cultured in the well of the well system, placing one (1x non aggregated group) or three (3x group) embryos per microwell. Our results showed that aggregation of three embryos increased blastocyst formation rate and blastocyst diameter of cloned pig embryos. DNA fragmentation levels in 3x aggregated cloned blastocysts were significantly decreased compared to 1x blastocysts. Levels of Oct4, Klf4, Igf2, Bax and Dnmt 1 transcripts were significantly higher in aggregated embryos, whereas Nanog levels were not affected. Transcripts of Cdx2 and Bcl-xl were essentially non-detectable. Our study suggests that embryo aggregation in the porcine may be beneficial for cloned embryo development and embryo quality, through a reduction in apoptotic levels and an improvement in cell reprogramming. PMID:26894831

  1. Embryo Aggregation in Pig Improves Cloning Efficiency and Embryo Quality.

    PubMed

    Buemo, Carla Paola; Gambini, Andrés; Moro, Lucia Natalia; Hiriart, María Inés; Fernández-Martín, Rafael; Collas, Philippe; Salamone, Daniel Felipe

    2016-01-01

    In this study, we analyzed the effects of the cloned embryo aggregation on in vitro embryo development and embryo quality by measuring blastocyst diameter and cell number, DNA fragmentation levels and the expression of genes associated with pluripotency, apoptosis, trophoblast and DNA methylation in the porcine. Zona-free reconstructed cloned embryos were cultured in the well of the well system, placing one (1x non aggregated group) or three (3x group) embryos per microwell. Our results showed that aggregation of three embryos increased blastocyst formation rate and blastocyst diameter of cloned pig embryos. DNA fragmentation levels in 3x aggregated cloned blastocysts were significantly decreased compared to 1x blastocysts. Levels of Oct4, Klf4, Igf2, Bax and Dnmt 1 transcripts were significantly higher in aggregated embryos, whereas Nanog levels were not affected. Transcripts of Cdx2 and Bcl-xl were essentially non-detectable. Our study suggests that embryo aggregation in the porcine may be beneficial for cloned embryo development and embryo quality, through a reduction in apoptotic levels and an improvement in cell reprogramming. PMID:26894831

  2. State-of-the-art embryo technologies in cattle.

    PubMed

    Lonergan, P

    2007-01-01

    Over the past 30 years, basic and applied studies on classical and advanced embryo technologies have generated a vast literature on factors regulating oocyte and embryo development and quality. In addition, over this period, commercial bovine embryo transfer has become a large international business. It is well recognised that bovine embryos derived in vivo are of superior quality to those derived from in vitro maturation, fertilization and culture. Relatively little has changed in the techniques of producing embryos in vivo although there is increasing evidence of the importance of, for example, peripheral and follicular endocrine profiles for the subsequent developmental competence of the embryo. The in vitro production of ruminant embryos is a three-step process involving oocyte maturation, oocyte fertilization and in vitro culture. Only 30-40% of such oocytes reach the blastocyst stage, at which they can be transferred to a recipient or frozen for future use. We know now that the quality of the oocyte is crucial in determining the proportion of immature oocytes that form blastocysts while the post-fertilization culture environment has a major influence on the quality of the blastocyst. Use of sexed-sorted sperm in conjunction with in vitro embryo production is a potentially efficient means of obtaining offspring of the desired sex. Concerns regarding the use of sexed semen technology include the apparent lower fertility of sorted sperm, the lower survival of sorted sperm after cryopreservation and the reduced number of sperm that could be separated in a specified time period. Assessment of embryo quality is a challenge. Morphological assessment is at present the most popular method for embryo selection prior to transfer. Other non-invasive assessment methods include the timing of the first cleavage division which has been linked to developmental ability. Quantitative examination of gene expression is an additional valuable tool to assess the viability of

  3. Aneuploidy analysis of non-pronuclear embryos from IVF with use of array CGH: a case report.

    PubMed

    Lixin, Deng; Zhifeng, Xiang; Cong, He; Jinzhou, Zhang; Hongbin, Xie

    2014-06-01

    By using array comparative genomic hybridization (array CGH), to analyze the aneuploidy of the single blastomeres from non-pronuclear embryos on cleavage-stage in IVF cycle. Four non-pronuclear embryos were got from an IVF cycle, and the each single cell was biopsied from the four cleavage-stage embryos on the third day after the insemination which was investigated by using array CGH. After the biopsy, all the embryos continued to cleave, and lately entered the morula stage on the fifth day, just one embryo 3 was developed to early blastocyst stage on the sixth day. The four blastomere 24 chromosomes showed one X monomer and three normal XY diploids; the autosome chromosomes of blastomeres were abnormally gained or lost at different chromosome from four embryos, such as Embryo 1 : 49,X (-1, -5, -11, -19, -20, -21, -Y, +3, +6, +7, +8, +10, +13, +14, +16, +17, +18); Embryo 2 : 44,XY (-12, -15); Embryo 3: 47,XY (-3, -8, -9, -21, +7, +17, +18, +19, +20); Embryo 4 : 54,XY (+4, +7, +10, +12, +13, +16, +17, +22). With the use of the array CGH, the aneuploidy analysis could review the abnormal chromosomes of single blastomere from the non-pronuclear embryos, which can harbor the risk of abnormal sex chromosome and autosome chromosomes.

  4. HSPC117 deficiency in cloned embryos causes placental abnormality and fetal death

    SciTech Connect

    Wang, Yingying; Hai, Tang; Liu, Zichuan; Zhou, Shuya; Lv, Zhuo; Ding, Chenhui; Liu, Lei; Niu, Yuyu; Zhao, Xiaoyang; Tong, Man; Wang, Liu; Jouneau, Alice; Zhang, Xun; Ji, Weizhi; Zhou, Qi

    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 fertilized 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.

  5. Microwells support high-resolution time-lapse imaging and development of preimplanted mouse embryos

    PubMed Central

    Chung, Yu-Hsiang; Hsiao, Yi-Hsing; Kao, Wei-Lun; Hsu, Chia-Hsien; Chen, Chihchen

    2015-01-01

    A vital aspect affecting the success rate of in vitro fertilization is the culture environment of the embryo. However, what is not yet comprehensively understood is the affect the biochemical, physical, and genetic requirements have over the dynamic development of human or mouse preimplantation embryos. The conventional microdrop technique often cultures embryos in groups, which limits the investigation of the microenvironment of embryos. We report an open microwell platform, which enables micropipette manipulation and culture of embryos in defined sub-microliter volumes without valves. The fluidic environment of each microwell is secluded from others by layering oil on top, allowing for non-invasive, high-resolution time-lapse microscopy, and data collection from each individual embryo without confounding factors. We have successfully cultured mouse embryos from the two-cell stage to completely hatched blastocysts inside microwells with an 89% success rate (n = 64), which is comparable to the success rate of the contemporary practice. Development timings of mouse embryos that developed into blastocysts are statistically different to those of embryos that failed to form blastocysts (p–value < 10−10, two-tailed Student's t-test) and are robust indicators of the competence of the embryo to form a blastocyst in vitro with 94% sensitivity and 100% specificity. Embryos at the cleavage- or blastocyst-stage following the normal development timings were selected and transferred to the uteri of surrogate female mice. Fifteen of twenty-two (68%) blastocysts and four of ten (40%) embryos successfully developed into normal baby mice following embryo transfer. This microwell platform, which supports the development of preimplanted embryos and is low-cost, easy to fabricate and operate, we believe, opens opportunities for a wide range of applications in reproductive medicine and cell biology. PMID:26015830

  6. Early Aberrations in Chromatin Dynamics in Embryos Produced Under In Vitro Conditions

    PubMed Central

    Deshmukh, Rahul S.; Strejcek, Frantisek; Vejlsted, Morten; Lucas-Hahn, Andrea; Petersen, Bjorn; Li, Juan; Callesen, Henrik; Niemann, Heiner; Hyttel, Poul

    2012-01-01

    Abstract In vitro production of porcine embryos by means of in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT) is limited by great inefficienciy. The present study investigated chromatin and nucleolar dynamics in porcine embryos developed in vivo (IV) and compared this physiological standard to that of embryos produced by IVF, parthenogenetic activation (PA), or SCNT. In contrast to IV embryos, chromatin spatial and temporal dynamics in PA, IVF, and SCNT embryos were altered; starting with aberrant chromatin–nuclear envelope interactions at the two-cell stage, delayed chromatin decondensation and nucleolar development at the four-cell stage, and ultimately culminating in failure of proper first lineage segregation at the blastocyst stage, demonstrated by poorly defined inner cell mass. Interestingly, in vitro produced (IVP) embryos also lacked a heterochromatin halo around nucleolar precursors, indicating imperfections in global chromatin remodeling after fertilization/activation. Porcine IV-produced zygotes and embryos display a well-synchronized pattern of chromatin dynamics compatible with genome activation and regular nucleolar formation at the four-cell stage. Production of porcine embryos under in vitro conditions by IVF, PA, or SCNT is associated with altered chromatin remodeling, delayed nucleolar formation, and poorly defined lineage segregation at the blastocyst stage, which in turn may impair their developmental capacity. PMID:22468997

  7. Radial extracorporeal shock wave treatment harms developing chicken embryos.

    PubMed

    Kiessling, Maren C; Milz, Stefan; Frank, Hans-Georg; Korbel, Rüdiger; Schmitz, Christoph

    2015-02-06

    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.

  8. 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

  9. Constant transmission of mitochondrial DNA in intergeneric cloned embryos reconstructed from swamp buffalo fibroblasts and bovine ooplasm.

    PubMed

    Srirattana, Kanokwan; Matsukawa, Kazutsugu; Akagi, Satoshi; Tasai, Mariko; Tagami, Takahiro; Nirasawa, Keijiro; Nagai, Takashi; Kanai, Yukio; Parnpai, Rangsun; Takeda, Kumiko

    2011-04-01

    Although interspecies/intergeneric somatic cell nuclear transfer (iSCNT) has been proposed as a tool to produce offspring of endangered species, conflict between donor nucleus and recipient cytoplasm in iSCNT embryos has been identified as an impediment to implementation for agricultural production. To investigate the nuclear-mitochondrial interactions on the developmental potential of iSCNT embryos, we analyzed the mtDNA copy numbers in iSCNT embryos reconstructed with water buffalo (swamp type) fibroblasts and bovine enucleated oocytes (buffalo iSCNT). As controls, SCNT embryos were derived from bovine fibroblasts (bovine SCNT). Buffalo iSCNT and bovine SCNT embryos showed similar rates of cleavage and development to the 8-cell stage (P>0.05). However, buffalo iSCNT embryos did not develop beyond the 16-cell stage. Both bovine and buffalo mtDNA content in buffalo iSCNT embryos was stable throughout the nuclear transfer process, and arrested at the 8- to 16-cell stage (P>0.05). In bovine SCNT embryos that developed to the blastocyst stage, mtDNA copy number was increased (P<0.05). In conclusion, both the donor cell and recipient cytoplast mtDNAs of buffalo iSCNT embryos were identified and maintained through the iSCNT process until the 8-16-cell stage. In addition, the copy number of mtDNA per embryo was a useful monitor to investigate nuclear-mitochondrial interactions.

  10. Comparison of gender-specific human embryo development characteristics by time-lapse technology.

    PubMed

    Serdarogullari, Munevver; Findikli, Necati; Goktas, Cihan; Sahin, Oya; Ulug, Ulun; Yagmur, Erbil; Bahceci, Mustafa

    2014-08-01

    Numerous studies indicate that there might be differences in embryo growth dynamics between male and female embryos. However, current data in humans are scarce and the results are inconclusive or conflicting. This study asks whether there exist gender-specific embryo development kinetics or parameters between human male and female embryos that can be observed by time-lapse technology. Study included data from 139 consecutive cycles (177 embryos transferred, 179 sacs analysed) with positive pregnancy that resulted in 100% implantation. Single- or double-embryo transfers were performed. Cases were analysed for parameters including cleavage time points and duration in each cleavage from two cells to hatching blastocyst stages and time interval between cleavages. Morphokinetic parameters of 78 female and 60 male embryos from a total of 119 cycles (139 sacs were examined after transfer of 138 embryos) were processed for data analysis according to the gender group. A detailed analysis of the data regarding each time point or interval between consecutive events according to these groups showed them to be similar in cell division kinetics, from the early cleavage through their development to blastocyst stage. However, female embryos showed earlier cavitation than male embryos, but the results did not reach statistical significance.

  11. Treatments affecting maturation and germination of American chestnut somatic embryos.

    PubMed

    Robichaud, Rodney L; Lessard, Veronica C; Merkle, Scott A

    2004-08-01

    The effects of amino acids, abscisic acid (ABA), polyethylene glycol (PEG), and elevated sucrose were tested on the maturation and germination of American chestnut (Castanea dentata) somatic embryos. Somatic embryos from three lines were matured over an eight week period through a two-stage process. After maturation, somatic embryos were randomly divided into three groups to measure dry weight/ fresh weight ratios, starch levels, and germination rates. Prior to transfer to germination medium, somatic embryos received a four week cold treatment. While some treatments with amino acids, elevated sucrose, PEG or ABA increased either dry weight/fresh weight ratios, starch content or both, only addition of 25mM L-asparagine significantly increased germination rate and taproot length, and this response was only obtained with one of the three lines tested. Six plants survived the transfer to potting mix, acclimatization to greenhouse conditions and field planting. PMID:15384407

  12. Treatments affecting maturation and germination of American chestnut somatic embryos.

    PubMed

    Robichaud, Rodney L; Lessard, Veronica C; Merkle, Scott A

    2004-08-01

    The effects of amino acids, abscisic acid (ABA), polyethylene glycol (PEG), and elevated sucrose were tested on the maturation and germination of American chestnut (Castanea dentata) somatic embryos. Somatic embryos from three lines were matured over an eight week period through a two-stage process. After maturation, somatic embryos were randomly divided into three groups to measure dry weight/ fresh weight ratios, starch levels, and germination rates. Prior to transfer to germination medium, somatic embryos received a four week cold treatment. While some treatments with amino acids, elevated sucrose, PEG or ABA increased either dry weight/fresh weight ratios, starch content or both, only addition of 25mM L-asparagine significantly increased germination rate and taproot length, and this response was only obtained with one of the three lines tested. Six plants survived the transfer to potting mix, acclimatization to greenhouse conditions and field planting.

  13. Cells, embryos and development in space

    NASA Technical Reports Server (NTRS)

    Krikorian, A. D.

    1984-01-01

    Work continues to focus on the demonstrable totipotency of cultured somatic cells of various higher plants and has examined the conditions which regulate this propensity to be controllably released. This was done with special reference to cells obtained from cultured explants of daylily and carrot. For purposes of identifying the variables in question, work was carried out almost exclusively in liquid media. The events that intervene between the aseptic isolation of tissue explants, the culture of small derived units and free cells and the propagation in large numbers of adventive or somatic embryos to plantlets were traced and certain definitive stages at which control is exercised were identified. In daylily, morphologically competent units are now propagated with a high degree of precision in rotated liquid cultures in bulk, and under the conditions of continuous neutralized gravity, the development progresses so that embryo-plantlets are obtained.

  14. The Impact of Biopsy on Human Embryo Developmental Potential during Preimplantation Genetic Diagnosis

    PubMed Central

    Cimadomo, Danilo; Capalbo, Antonio; Ubaldi, Filippo Maria; Scarica, Catello; Palagiano, Antonio; Canipari, Rita; Rienzi, Laura

    2016-01-01

    Preimplantation Genetic Diagnosis and Screening (PGD/PGS) for monogenic diseases and/or numerical/structural chromosomal abnormalities is a tool for embryo testing aimed at identifying nonaffected and/or euploid embryos in a cohort produced during an IVF cycle. A critical aspect of this technology is the potential detrimental effect that the biopsy itself can have upon the embryo. Different embryo biopsy strategies have been proposed. Cleavage stage blastomere biopsy still represents the most commonly used method in Europe nowadays, although this approach has been shown to have a negative impact on embryo viability and implantation potential. Polar body biopsy has been proposed as an alternative to embryo biopsy especially for aneuploidy testing. However, to date no sufficiently powered study has clarified the impact of this procedure on embryo reproductive competence. Blastocyst stage biopsy represents nowadays the safest approach not to impact embryo implantation potential. For this reason, as well as for the evidences of a higher consistency of the molecular analysis when performed on trophectoderm cells, blastocyst biopsy implementation is gradually increasing worldwide. The aim of this review is to present the evidences published to date on the impact of the biopsy at different stages of preimplantation development upon human embryos reproductive potential. PMID:26942198

  15. An evaluation of background levels and sources of polycyclic aromatic hydrocarbons in naturally spawned embryos of Pacific herring (Clupea pallasii) from Puget Sound, Washington, USA.

    PubMed

    West, James E; O'Neill, Sandra M; Ylitalo, Gina M; Incardona, John P; Doty, Daniel C; Dutch, Margaret E

    2014-11-15

    Pacific herring embryos spawned in nearshore habitats may be exposed to toxic contaminants as they develop, from exogenous sources in spawning habitats and from maternal transfer. Determining baseline concentrations of these toxic contaminants is important for evaluating the health of this species, especially during this sensitive life stage. In this study we compared concentrations of polycyclic aromatic hydrocarbons, or PAHs, in naturally spawned herring embryos from five spawning areas across Puget Sound. The summed values of 31 PAH analytes (Σ31PAH) in early- to late-stage development embryos ranged from 1.1 to 140 ng/g, wet weight. Σ31PAH concentrations increased with development time in embryos from one spawning area where the greatest concentrations were observed, and the relative abundance of PAH chemicals in late-stage embryos was similar to those in nearby sediments, suggesting accumulation from local environmental sources. PAHs in both sediments and late-stage embryos appeared to exhibit a pyrogenic pattern. Although maternal transfer of PAHs appeared to be a negligible source to embryos in spawning areas with the greatest embryo PAH concentrations, maternal transfer may have been the dominant source in embryos from spawning areas where the lowest levels of embryo-PAHs occurred. Chronic embryo mortality has been reported in spawning habitats where we observed the greatest concentration of PAHs in embryos, and necrotic tissue in herring embryos from one such location was similar in description to phototoxic PAH necrosis reported elsewhere for embryonic zebrafish.

  16. Single-Cell XIST Expression in Human Preimplantation Embryos and Newly Reprogrammed Female Induced Pluripotent Stem Cells.

    PubMed

    Briggs, Sharon F; Dominguez, Antonia A; Chavez, Shawn L; Reijo Pera, Renee A

    2015-06-01

    The process of X chromosome inactivation (XCI) during reprogramming to produce human induced pluripotent stem cells (iPSCs), as well as during the extensive programming that occurs in human preimplantation development, is not well-understood. Indeed, studies of XCI during reprogramming to iPSCs report cells with two active X chromosomes and/or cells with one inactive X chromosome. Here, we examine expression of the long noncoding RNA, XIST, in single cells of human embryos through the oocyte-to-embryo transition and in new mRNA reprogrammed iPSCs. We show that XIST is first expressed beginning at the 4-cell stage, coincident with the onset of embryonic genome activation in an asynchronous manner. Additionally, we report that mRNA reprogramming produces iPSCs that initially express XIST transcript; however, expression is rapidly lost with culture. Loss of XIST and H3K27me3 enrichment at the inactive X chromosome at late passage results in X chromosome expression changes. Our data may contribute to applications in disease modeling and potential translational applications of female stem cells.

  17. 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.

  18. 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

  19. 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.

  20. Comparative Transcriptomic Analysis of Developing Cotton Cotyledons and Embryo Axis

    PubMed Central

    Jiao, Xiaoming; Zhao, Xiaochun; Zhou, Xue-Rong; Green, Allan G.; Fan, Yunliu; Wang, Lei; Singh, Surinder P.; Liu, Qing

    2013-01-01

    Background As a by product of higher value cotton fibre, cotton seed has been increasingly recognised to have excellent potential as a source of additional food, feed, biofuel stock and even a renewable platform for the production of many diverse biological molecules for agriculture and industrial enterprises. The large size difference between cotyledon and embryo axis that make up a cotton seed results in the under-representation of embryo axis gene transcript levels in whole seed embryo samples. Therefore, the determination of gene transcript levels in the cotyledons and embryo axes separately should lead to a better understanding of metabolism in these two developmentally diverse tissues. Results A comparative study of transcriptome changes between cotton developing cotyledon and embryo axis has been carried out. 17,384 unigenes (20.74% of all the unigenes) were differentially expressed in the two adjacent embryo tissues, and among them, 7,727 unigenes (44.45%) were down-regulated and 9,657 unigenes (55.55%) were up-regulated in cotyledon. Conclusions Our study has provided a comprehensive dataset that documents the dynamics of the transcriptome at the mid-maturity of cotton seed development and in discrete seed tissues, including embryo axis and cotyledon tissues. The results showed that cotton seed is subject to many transcriptome variations in these two tissue types and the differential gene expression between cotton embryo axis and cotyledon uncovered in our study should provide an important starting point for understanding how gene activity is coordinated during seed development to make a seed. Further, the identification of genes involved in rapid metabolite accumulation stage of seed development will extend our understanding of the complex molecular and cellular events in these developmental processes and provide a foundation for future studies on the metabolism, embryo differentiation of cotton and other dicot oilseed crops. PMID:23977137

  1. Real protection for the embryo.

    PubMed

    Mazzoni, Cosimo Marco

    2005-01-01

    This article fundamentally analyses the current protection that the Italian law offers to the embryo. Likewise, the author, contrary to those who are of the opinion that the embryo is a person subject to law, exposes his polemic theory in which he places the embryo within the scope of things. Specifically, he argues that the embryo has a quasi-personal category. In order to justify this, he analyses the moral and legal history of the statute of the embryo, he makes a difference between the biological life and the legal life. The author establishes that the concept of the person has been and will continue to be a very controversial concept, concluding with a study on the Italian legislation in respect to the protection of the embryo.

  2. Gender determination of avian embryo

    DOEpatents

    Daum, Keith A.; Atkinson, David A.

    2002-01-01

    Disclosed is a method for gender determination of avian embryos. During the embryo incubation process, the outer hard shells of eggs are drilled and samples of allantoic fluid are removed. The allantoic fluids are directly introduced into an ion mobility spectrometer (IMS) for analysis. The resulting spectra contain the relevant marker peaks in the positive or negative mode which correlate with unique mobilities which are sex-specific. This way, the gender of the embryo can be determined.

  3. Electrothermal branding for embryo labeling.

    PubMed

    Wang, L; Beebe, D J; Williams, A R; Easley, K D

    1997-11-01

    A novel embryo labeling technique based on electrothermal branding is developed. Two types of micro branding irons are fabricated and tested. One utilizes 25 microns tungsten wire as the heating element. The other utilizes surface micromachining techniques to fabricate polysilicon branding irons. The thermal behavior of the branding irons and the heat distributions in the embryos are analytically modeled. Micron-scale labels on unfertilized bovine embryos are achieved.

  4. Determination of pentose phosphate and Embden-Meyerhof pathway activities in bovine embryos.

    PubMed

    Javed, M H; Wright, R W

    1991-05-01

    Quantitative determination was made of the activity of pentose phosphate pathway (PPP) and Embden-Meyerhof pathway (EMP) in individual bovine embryos from the six-cell to the hatched blastocyst stage. Embryos were collected from superovulated cross-bred heifers and classified into good and poor categories. A single embryo in 1 microl of medium was mixed with 2 microl of medium containing 3 to 30 nCi radiolabeled glucose previously placed on a detached lid of the 1.5-ml polypropylene microcentrifugé vial. The lid was then fitted to its vial which had been loaded in advance with 1.5 ml of 0.1 N NaOH. Vials were then incubated at 37 degrees C for 3 h. At the end of the incubation period, a 1.5-ml NaOH trap was inverted and placed into a 20-ml scintillation vial containing 10 ml of aqueous counting solution and counted in a liquid scintillation spectrophotometer. The PPP activity in good-quality embryos was greatest at the six-cell stage and decreased with increasing embryo development. The EMP activity showed the reverse trend. Poor- quality embryos had a lower glucose metabolism and higher PPP activity. Similar measurements were made on embryos following 24 h of culture, and total glucose metabolism and percentage of PPP activity were increased. In conclusion, these data suggest that in good quality bovine embryos total glucose utilization is low until 16-cell stage, with PPP being the predominant pathway. Total glucose utilization increases significantly at the morula stage; EMP activity increases with increasing embryo development; and PPP activity increases significantly in poor quality embryos and in embryos 24 h in culture.

  5. Contour extraction of Drosophila embryos.

    PubMed

    Li, Qi; Kambhamettu, Chandra

    2011-01-01

    Contour extraction of Drosophila (fruit fly) embryos is an important step to build a computational system for matching expression pattern of embryonic images to assist the discovery of the nature of genes. Automatic contour extraction of embryos is challenging due to severe image variations, including 1) the size, orientation, shape, and appearance of an embryo of interest; 2) the neighboring context of an embryo of interest (such as nontouching and touching neighboring embryos); and 3) illumination circumstance. In this paper, we propose an automatic framework for contour extraction of the embryo of interest in an embryonic image. The proposed framework contains three components. Its first component applies a mixture model of quadratic curves, with statistical features, to initialize the contour of the embryo of interest. An efficient method based on imbalanced image points is proposed to compute model parameters. The second component applies active contour model to refine embryo contours. The third component applies eigen-shape modeling to smooth jaggy contours caused by blurred embryo boundaries. We test the proposed framework on a data set of 8,000 embryonic images, and achieve promising accuracy (88 percent), that is, substantially higher than the-state-of-the-art results.

  6. Determination of gene expression patterns using high-throughput RNA in situ hybridizaion to whole-mount Drosophila embryos

    SciTech Connect

    Weiszmann, R.; Hammonds, A.S.; Celniker, S.E.

    2009-04-09

    We describe a high-throughput protocol for RNA in situ hybridization (ISH) to Drosophila embryos in a 96-well format. cDNA or genomic DNA templates are amplified by PCR and then digoxigenin-labeled ribonucleotides are incorporated into antisense RNA probes by in vitro transcription. The quality of each probe is evaluated before ISH using a RNA probe quantification (dot blot) assay. RNA probes are hybridized to fixed, mixed-staged Drosophila embryos in 96-well plates. The resulting stained embryos can be examined and photographed immediately or stored at 4oC for later analysis. Starting with fixed, staged embryos, the protocol takes 6 d from probe template production through hybridization. Preparation of fixed embryos requires a minimum of 2 weeks to collect embryos representing all stages. The method has been used to determine the expression patterns of over 6,000 genes throughout embryogenesis.

  7. The biological basis of non-invasive strategies for selection of human oocytes and embryos.

    PubMed

    Scott, Lynette

    2003-01-01

    There is a need for more accurate embryo selection in human assisted reproduction, if the goal of reducing the number of embryos used in embryo transfer is to be realized. Furthermore, any selection strategy should be non-invasive if the embryos are to be used in embryo transfer. Currently, the strategy is selection by one to three parameters in the cleaving- and blastocyst-stage embryo, sometimes with additional pronuclear selection. It is clear that no one system is ideal, as the vast majority of transferred embryos do not implant. As the health of the embryo is largely dictated by the originating gametes, the very early events in oocyte development should be considered. This review will point to the early biological events in the unfertilized and fertilized oocyte that can be scored non-invasively and which can have a profound effect on the later developmental stages. Using a sequential scoring system, with emphasis on the oocyte, a system for selecting the most viable single embryo for transfer may hopefully be achieved.

  8. Role of melatonin in embryo fetal development

    PubMed Central

    Voiculescu, SE; Zygouropoulos, N; Zahiu, CD; Zagrean, AM

    2014-01-01

    Melatonin is an indoleamine produced by the pineal gland and secreted in a circadian manner. In the past few decades, research over this topic has been enhanced. Melatonin has many important roles in the human physiology: regulator of the circadian rhythms, sleep inducer, antioxidant, anticarcinogenic. This paper reviews the involvement of melatonin in embryo fetal development. The pineal gland develops completely postpartum, so both the embryo and the fetus are dependent on the maternal melatonin provided transplacentally. Melatonin appears to be involved in the normal outcome of pregnancy beginning with the oocyte quality and finishing with the parturition. Its pregnancy night-time concentrations increase after 24 weeks of gestation, with significantly high levels after 32 weeks. Melatonin receptors are widespread in the embryo and fetus since early stages. There is solid evidence that melatonin is neuroprotective and has a positive effect on the outcome of the compromised pregnancies. In addition, chronodisruption leads to a reproductive dysfunction. Thus, the influence of melatonin on the developing human fetus may not be limited to the entertaining of circadian rhythmicity, but further studies are needed. PMID:25713608

  9. Role of melatonin in embryo fetal development.

    PubMed

    Voiculescu, S E; Zygouropoulos, N; Zahiu, C D; Zagrean, A M

    2014-01-01

    Melatonin is an indoleamine produced by the pineal gland and secreted in a circadian manner. In the past few decades, research over this topic has been enhanced. Melatonin has many important roles in the human physiology: regulator of the circadian rhythms, sleep inducer, antioxidant, anticarcinogenic. This paper reviews the involvement of melatonin in embryo fetal development. The pineal gland develops completely postpartum, so both the embryo and the fetus are dependent on the maternal melatonin provided transplacentally. Melatonin appears to be involved in the normal outcome of pregnancy beginning with the oocyte quality and finishing with the parturition. Its pregnancy night-time concentrations increase after 24 weeks of gestation, with significantly high levels after 32 weeks. Melatonin receptors are widespread in the embryo and fetus since early stages. There is solid evidence that melatonin is neuroprotective and has a positive effect on the outcome of the compromised pregnancies. In addition, chronodisruption leads to a reproductive dysfunction. Thus, the influence of melatonin on the developing human fetus may not be limited to the entertaining of circadian rhythmicity, but further studies are needed.

  10. The influence of growth factors on the development of preimplantation mammalian embryos.

    PubMed

    Díaz-Cueto, L; Gerton, G L

    2001-01-01

    The development of the preimplantation mammalian embryo from a fertilized egg to a blastocyst capable of implanting in the uterus is a complex process. Cell division must be carefully programmed. The embryonic genome must be activated at the appropriate stage of development, and the pattern of gene expression must be carefully coordinated for the initiation of the correct program of differentiation. Cell fates must be chosen to establish specific cell types such as the inner cell mass and the trophectoderm, which give rise to the embryo proper and the placenta, respectively. This review summarizes recent findings concerning the influence of growth factors on the development of preimplantation mammalian embryos. Maternal factors secreted into the lumen of the female reproductive tract as well as substances synthesized by the developing embryo itself help to regulate this process. Studies of embryos in culture and investigations using homologous recombination to create embryos and animals null for specific genes have enabled the identification of several growth factors that appear essential for preimplantation mammalian embryo development. Some of the factors are required maternal factors; others are embryo-derived autocrine and paracrine factors. Studies using molecular biology are beginning to identify differences in the patterns of genes expressed by naturally derived embryos and those developing in culture. The knowledge gained from studies on growth factors, media, embryonic development, and gene expression should help improve culture conditions for embryos and will provide for safer outcomes from assisted reproductive procedures in human and animal clinics. PMID:11750739

  11. Radioactive labeling of proteins in cultured postimplantation mouse embryos. II. Dose and time dependency

    SciTech Connect

    Nowak, J.; Klose, J. )

    1989-07-01

    The conditions for optimum incorporation of radioactive amino acids into proteins of cultured postimplantation mouse embryos were investigated under the aspect of using these proteins for two-dimensional electrophoretic separations and fluorography. The aim was to obtain highly radioactively labeled proteins under conditions as physiological as possible. Mouse embryos of Days 8, 10, and 11 of gestation were cultured in Tyrode's solution. Incubation time and concentration of ({sup 3}H (or {sup 14}C))amino acids in the culture medium were varied over a broad range. Embryos were prepared with placenta and yolk sac or without any embryonic envelopes. After culturing, the physiologic-morphologic state of the embryos was registered on the basis of several criteria. The radioactivity taken up by the total protein of each embryo was determined and calculated in disintegrations per minute per milligram protein per embryo. To approach our aim, embryos of different developmental stages had to be cultured under different conditions. A good compromise for Day-8, Day-10, and Day-11 embryos was: embryos prepared with yolk sac (opened) and placenta, 150 microCi radioactive amino acids added per milliliter medium, incubation for 4 to 5 h. For maximum labeling of proteins it is advisable to culture Day-10 embryos without embryonic envelopes under particular conditions.

  12. Functional Analysis of Lysosomes During Mouse Preimplantation Embryo Development

    PubMed Central

    TSUKAMOTO, Satoshi; HARA, Taichi; YAMAMOTO, Atsushi; OHTA, Yuki; WADA, Ayako; ISHIDA, Yuka; KITO, Seiji; NISHIKAWA, Tetsu; MINAMI, Naojiro; SATO, Ken; KOKUBO, Toshiaki

    2012-01-01

    Abstract Lysosomes are acidic and highly dynamic organelles that are essential for macromolecule degradation and many other cellular functions. However, little is known about lysosomal function during early embryogenesis. Here, we found that the number of lysosomes increased after fertilization. Lysosomes were abundant during mouse preimplantation development until the morula stage, but their numbers decreased slightly in blastocysts. Consistently, the protein expression level of mature cathepsins B and D was high from the one-cell to morula stages but low in the blastocyst stage. One-cell embryos injected with siRNAs targeted to both lysosome-associated membrane protein 1 and 2 (LAMP1 and LAMP2) were developmentally arrested at the two-cell stage. Pharmacological inhibition of lysosomes also caused developmental retardation, resulting in accumulation of lipofuscin. Our findings highlight the functional changes in lysosomes in mouse preimplantation embryos. PMID:23080372

  13. Rhesus monkey embryos produced by nuclear transfer from embryonic blastomeres or somatic cells.

    PubMed

    Mitalipov, Shoukhrat M; Yeoman, Richard R; Nusser, Kevin D; Wolf, Don P

    2002-05-01

    Production of genetically identical nonhuman primates would reduce the number of animals required for biomedical research and dramatically impact studies pertaining to immune system function, such as development of the human-immunodeficiency-virus vaccine. Our long-term goal is to develop robust somatic cell cloning and/or twinning protocols in the rhesus macaque. The objective of this study was to determine the developmental competence of nuclear transfer (NT) embryos derived from embryonic blastomeres (embryonic cell NT) or fetal fibroblasts (somatic cell NT) as a first step in the production of rhesus monkeys by somatic cell cloning. Development of cleaved embryos up to the 8-cell stage was similar among embryonic and somatic cell NT embryos and comparable to controls created by intracytoplasmic sperm injection (ICSI; mean +/- SEM, 81 +/- 5%, 88 +/- 7%, and 87 +/- 4%, respectively). However, significantly lower rates of development to the blastocyst stage were observed with somatic cell NT embryos (1%) in contrast to embryonic cell NT (34 +/- 15%) or ICSI control embryos (46 +/- 6%). Development of somatic cell NT embryos was not markedly affected by donor cell treatment, timing of activation, or chemical activation protocol. Transfer of embryonic, but not of somatic cell NT embryos, into recipients resulted in term pregnancy. Future efforts will focus on optimizing the production of somatic cell NT embryos that develop in high efficiency to the blastocyst stage in vitro.

  14. Metabolomic assessment of embryo viability.

    PubMed

    Uyar, Asli; Seli, Emre

    2014-03-01

    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

  15. Effect of Substrate Stiffness on Early Mouse Embryo Development

    PubMed Central

    Kolahi, Kevin S.; Donjacour, Annemarie; Liu, Xiaowei; Lin, Wingka; Simbulan, Rhodel K.; Bloise, Enrrico; Maltepe, Emin; Rinaudo, Paolo

    2012-01-01

    It is becoming increasingly clear that cells are remarkably sensitive to the biophysical cues of their microenvironment and that these cues play a significant role in influencing their behaviors. In this study, we investigated whether the early pre-implantation embryo is sensitive to mechanical cues, i.e. the elasticity of the culture environment. To test this, we have developed a new embryo culture system where the mechanical properties of the embryonic environment can be precisely defined. The contemporary standard environment for embryo culture is the polystyrene petri dish (PD), which has a stiffness (1 GPa) that is six orders of magnitude greater than the uterine epithelium (1 kPa). To approximate more closely the mechanical aspects of the in vivo uterine environment we used polydimethyl-siloxane (PDMS) or fabricated 3D type I collagen gels (1 kPa stiffness, Col-1k group). Mouse embryo development on alternate substrates was compared to that seen on the petri dish; percent development, hatching frequency, and cell number were observed. Our results indicated that embryos are sensitive to the mechanical environment on which they are cultured. Embryos cultured on Col-1k showed a significantly greater frequency of development to 2-cell (68±15% vs. 59±18%), blastocyst (64±9.1% vs. 50±18%) and hatching blastocyst stages (54±25% vs. 21±16%) and an increase in the number of trophectodermal cell (TE,65±13 vs. 49±12 cells) compared to control embryos cultured in PD (mean±S.D.; p<.01). Embryos cultured on Col-1k and PD were transferred to recipient females and observed on embryonic day 12.5. Both groups had the same number of fetuses, however the placentas of the Col-1k fetuses were larger than controls, suggesting a continued effect of the preimplantation environment. In summary, characteristics of the preimplantation microenvironment affect pre- and post-implantation growth. PMID:22860009

  16. Patients with polycystic ovary syndrome have successful embryo arrest

    PubMed Central

    Yin, Baoli; Hao, Haoying; Wei, Duo; Song, Xiaobing; Xie, Juanke; Zhang, Cuilian

    2015-01-01

    In this retrospective study, we investigate the relationship between embryo arrest and polycystic ovary syndrome (PCOS) during in vitro fertilization-embryo transfer (IVF-ET). In this study, 667 subjects were enrolled, including 330 patients with PCOS and 337 subjects without PCOS. The subjects underwent in vitro fertilization/intracytoplasmic sperm injection and embryo transfer (IVF/ICSI-ET) cycles at the Reproductive Medical Centre of Henan Provincial Hospital from January 2009 to December 2012. Four protocols were used to stimulate the ovaries, including long protocol, super-long down-regulation protocol, short protocol and antagonist protocol. Oocytes were retrieved using transvaginal ultrasound guidance. Pronuclei were checked on the next morning after IVF/ICSI. Cleavage stage embryo was assessed after 62-66 hours. Women with PCOS had significantly elevated body mass index, basal luteinizing hormone, estradiol and testosterone compared with normal women. Basal Follicle stimulating hormone level in PCOS patients was lower compared with that in control group. After IVF-ET, PCOS patients had more available oocytes than subjects in control group. PCOS patients had slightly lower fertilization rate than the controls in IVF cycles, but in ICSI cycles, fertilization rate in PCOS patients was significantly higher than that in controls. For either IVF or ICSI, the embryo arrest rate was not changed by PCOS. Moreover, there was no significant difference in embryo arrest rate between both groups adopting different stimulation protocols. Interestingly, embryo arrest rate was not correlated with testosterone for patients in PCOS group. The data indicated that patients with PCOS had successful early embryo arrest during IVF-ET. PMID:26131233

  17. Inhibition of histone deacetylases enhances DNA damage repair in SCNT embryos.

    PubMed

    Bohrer, Rodrigo Camponogara; Duggavathi, Raj; Bordignon, Vilceu

    2014-01-01

    Recent studies have shown that DNA damage affects embryo development and also somatic cell reprogramming into induced pluripotent stem (iPS) cells. It has been also shown that treatment with histone deacetylase inhibitors (HDACi) improves development of embryos produced by somatic cell nuclear transfer (SCNT) and enhances somatic cell reprogramming. There is evidence that increasing histone acetylation at the sites of DNA double-strand breaks (DSBs) is critical for DNA damage repair. Therefore, we hypothesized that HDACi treatment enhances cell programming and embryo development by facilitating DNA damage repair. To test this hypothesis, we first established a DNA damage model wherein exposure of nuclear donor cells to ultraviolet (UV) light prior to nuclear transfer reduced the development of SCNT embryos proportional to the length of UV exposure. Detection of phosphorylated histone H2A.x (H2AX139ph) foci confirmed that exposure of nuclear donor cells to UV light for 10 s was sufficient to increase DSBs in SCNT embryos. Treatment with HDACi during embryo culture increased development and reduced DSBs in SCNT embryos produced from UV-treated cells. Transcript abundance of genes involved in either the homologous recombination (HR) or nonhomologous end-joining (NHEJ) pathways for DSBs repair was reduced by HDACi treatment in developing embryos at day 5 after SCNT. Interestingly, expression of HR and NHEJ genes was similar between HDACi-treated and control SCNT embryos that developed to the blastocyst stage. This suggested that the increased number of embryos that could achieve the blastocyst stage in response to HDACi treatment have repaired DNA damage. These results demonstrate that DNA damage in nuclear donor cells is an important component affecting development of SCNT embryos, and that HDACi treatment after nuclear transfer enhances DSBs repair and development of SCNT embryos. PMID:24841373

  18. Expression of microRNAs in bovine and human pre-implantation embryo culture media.

    PubMed

    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.

  19. Ultrastructural analyses of somatic embryo initiation, development and polarity establishment from mesophyll cells of Dactylis glomerata

    NASA Technical Reports Server (NTRS)

    Vasilenko, A.; McDaniel, J. K.; Conger, B. V.

    2000-01-01

    Somatic embryos initiate and develop directly from single mesophyll cells in in vitro-cultured leaf segments of orchardgrass (Dactylis glomerata L.). Embryogenic cells establish themselves in the predivision stage by formation of thicker cell walls and dense cytoplasm. Electron microscopy observations for embryos ranging from the pre-cell-division stage to 20-cell proembryos confirm previous light microscopy studies showing a single cell origin. They also confirm that the first division is predominantly periclinal and that this division plane is important in establishing embryo polarity and in determining the embryo axis. If the first division is anticlinal or if divisions are in random planes after the first division, divisions may not continue to produce an embryo. This result may produce an embryogenic cell mass, callus formation, or no structure at all. Grant numbers: NAGW-3141, NAG10-0221.

  20. Silver Nanoparticles Incite Size and Dose-Dependent Developmental Phenotypes and Nanotoxicity in Zebrafish Embryos

    PubMed Central

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

    2013-01-01

    Nanomaterials possess distinctive physicochemical properties and promise a wide range of applications, from advanced technology to leading-edge medicine. However, their effects on living organisms remain largely unknown. Here we report that the purified silver nanoparticles (Ag NPs, 97 ± 13 nm) incite specific developmental stage embryonic phenotypes and nanotoxicity in a dose-dependent manner, upon acute exposure of given-stage embryos to the NPs (0–24 pM) for only 2 h. The critical concentrations of the NPs that cause 50% of embryos develop normally for cleavage, early-gastrula, early-segmentation, late-segmentation, and hatching stage zebrafish embryos are 3.5, 4, 6, 6, and 8 pM, respectively, showing that the earlier developmental stage embryos are much more sensitive to the effects of the NPs than the later stage. Interestingly, distinctive phenotypes (head abnormality and no eyes) are observed only in cleavage and early-gastrula stage embryos treated with the NPs, showing the stage-specific effects of the NPs. By comparing with our study of the smaller Ag NPs (13.1 ± 2.5 nm), we found that the embryonic phenotypes strikingly depend upon the sizes of Ag NPs and embryonic developmental stages. These notable findings suggest that the Ag NPs are unlike any conventional chemicals or ions. They can potentially enable target specific study and therapy for early embryonic development in size, stage, dose, and exposure-duration dependent manners. PMID:24024906

  1. Intra- and interspecific embryo transfer.

    PubMed

    Kraemer, D C

    1983-11-01

    The procedures that are collectively referred to as embryo transfer (ET) have many uses. They were first used as research tools to study fetal-maternal physiology. Since the first successful mammalian embryo transfer in 1890, ET has been utilized for enhancement of genetic selection; diagnosis and treatment of infertility; control of infectious disease transmission; screening for genetic defects; propagation of rare and endangered species; and the study of developmental biology. Most of the embryo transfers have been intraspecific. A listing of the species includes rabbit, rat, sheep, mouse, goat, cattle, pig, hamster, ferret, mink, horse, baboon, cat, dog, water buffalo. In two species, rhesus monkey and humans, the successful embryo transfers have been limited to within-animal, homologous replacement of the embryos. There have been a few successful interspecific embryo transfers. The most common were between Bos taurus and B. indicus cattle. Other interspecific transfers involved Bos gaurus and B. taurus, cattle; Ovis musimon and O. aries, sheep; Equus asinus and E. caballus, horses. There are several examples of intergeneric embryo transfers in which embryos implanted but did not develop to term: sheep and goat, mouse and rat. The factors that determine the degree of compatibility between embryos and uteri of various species and genera are not clearly understood. The ability to hybridize successfully is probably a dependable indication of compatibility for embryo transfer. The limits of tolerance for differences between the donor and recipient in such factors as placental structure and gestation length have not been defined, but the recently developed technique of inner cell mass transfer will be very useful in such studies.

  2. 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. PMID:25115559

  3. Effect of vitrification using the Cryotop method on the gene expression profile of in vitro-produced bovine embryos.

    PubMed

    de Oliveira Leme, Ligiane; Dufort, Isabelle; Spricigo, José Felipe Warmling; Braga, Thiago Felipe; Sirard, Marc-André; Franco, Maurício Machaim; Dode, Margot Alves Nunes

    2016-03-01

    The present study analyzed the changes in gene expression induced by the Cryotop vitrification technique in bovine blastocyst-stage embryos, using Agilent EmbryoGENE microarray slides. Bovine in vitro-produced embryos were vitrified and compared with nonvitrified (control) embryos. After vitrification, embryos were warmed and cultured for an additional 4 hours. Survived embryos were used for microarray analysis and quantitative polymerase chain reaction (qPCR) quantification. Survival rates were higher (P < 0.05) in the control embryos (100%) than in the vitrified embryos (87%). Global gene expression analysis revealed that only 43 out of 21,139 genes exhibited significantly altered expression in the vitrified embryos compared to the control embryos, with a very limited fold change (P < 0.05). A total of 10 genes were assessed by qPCR. Only the FOS-like antigen 1 (FOSL1) gene presented differential expression (P < 0.05) according to both the array and qPCR methods, and it was overexpressed in vitrified embryos. Although, the major consequence of vitrification seems to be the activation of the apoptosis pathway in some cells. Indeed, FOSL1 is part of the activating protein 1 transcription factor complex and is implicated in a variety of cellular processes, including proliferation, differentiation, and apoptosis. Therefore, our results suggest that a limited increase in the rate of apoptosis was the only detectable response of the embryos to vitrification stress. PMID:26553569

  4. High levels of mitochondrial heteroplasmy modify the development of ovine-bovine interspecies nuclear transferred embryos.

    PubMed

    Hua, Song; Lu, Chenglong; Song, Yakun; Li, Ruizhe; Liu, Xu; Quan, Fusheng; Wang, Yongsheng; Liu, Jun; Su, Feng; Zhang, Yong

    2012-01-01

    To investigate the effect of mitochondrial heteroplasmy on embryo development, cloned embryos produced using bovine oocytes as the recipient cytoplasm and ovine granulosa cells as the donor nuclei were complemented with 2pL mitochondrial suspension isolated from ovine (BOOMT embryos) or bovine (BOBMT embryos) granulosa cells; cloned embryos without mitochondrial injection served as the control group (BO embryos). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and sodium bisulfite genomic sequencing were used to analyse mRNA and methylation levels of pluripotency genes (OCT4, SOX2) and mitochondrial genes (TFAM, POLRMT) in the early developmental stages of cloned embryos. The number of mitochondrial DNA copies in 2pL ovine-derived and bovine-derived mitochondrial suspensions was 960±110 and 1000±120, respectively. The blastocyst formation rates were similar in BOBMT and BO embryos (P>0.05), but significantly higher than in BOOMT embryos (P<0.01). Expression of OCT4 and SOX2, as detected by RT-qPCR, decreased significantly in BOOMT embryos (P<0.05), whereas the expression of TFAM and POLRMT increased significantly, compared with expression in BOOMT and BO embryos (P<0.05). In addition, methylation levels of OCT4 and SOX2 were significantly greater (P<0.05), whereas those of TFAM and POLRMT were significantly lower (P<0.01), in BOOMT embryos compared with BOBMT and BO embryos. Together, the results of the present study suggest that the degree of mitochondrial heteroplasmy may affect embryonic development.

  5. Preimplantation development of cloned canine embryos recovered by hysterectomy or surgical uterine flushing and subsequent pregnancy outcomes.

    PubMed

    Jeong, Yeon Woo; Kim, Joung Joo; Kim, Hyun Duk; Hwang, Kyu Chan; Hyun, Sang Hwan; Kim, Nam-Hyung; Jeung, Eui-Bae; Hwang, Woo Suk

    2016-11-01

    Dog cloning offers a substantial potential because of the advancements in assisted reproductive technology and development of the human disease model in line with the transgenic technique. However, little is known about the development of the canine cloned embryo during the preimplantation period. The aim of this study was to investigate the most efficient method and time for collecting cloned canine preimplantation embryos and to ascertain the developmental timeline of cloned canine embryos. Two hundred cloned embryos were created and transferred into 11 surrogates. The preimplantation stage cloned embryos were then collected on Days 7, 8, and 9 using an ovariohysterectomy or the Foley balloon catheter method. The recovery rate of reconstructed embryos was 63.6% and 60.6% for the ovariohysterectomy and Foley balloon catheter methods, respectively. Although significant differences were observed in the early developmental stages (one-cell and 16-cell stages), no significant difference was observed in the blastocyst stage. Significantly higher blastocyst rate was observed when the embryos were collected on Day 8 (11.4%) than on Day 7 (0.0%; P < 0.05). At the proximal uterine horn on Day 7, no embryos at any stage were found, whereas on Days 8 and 9, blastocysts were found. We have observed a 63% initial pregnancy rate at 25 to 30 days after embryo transfer and a 50% full-term pregnancy rate, whereas 6.3% of the puppies were born, and 5.5% were born live among the total transferred embryos. Our results suggest that cloned embryos can develop to blastocysts by Day 8, and full-term pregnancy can be achieved after embryo transfer in canine. PMID:27587271

  6. Preimplantation development of cloned canine embryos recovered by hysterectomy or surgical uterine flushing and subsequent pregnancy outcomes.

    PubMed

    Jeong, Yeon Woo; Kim, Joung Joo; Kim, Hyun Duk; Hwang, Kyu Chan; Hyun, Sang Hwan; Kim, Nam-Hyung; Jeung, Eui-Bae; Hwang, Woo Suk

    2016-11-01

    Dog cloning offers a substantial potential because of the advancements in assisted reproductive technology and development of the human disease model in line with the transgenic technique. However, little is known about the development of the canine cloned embryo during the preimplantation period. The aim of this study was to investigate the most efficient method and time for collecting cloned canine preimplantation embryos and to ascertain the developmental timeline of cloned canine embryos. Two hundred cloned embryos were created and transferred into 11 surrogates. The preimplantation stage cloned embryos were then collected on Days 7, 8, and 9 using an ovariohysterectomy or the Foley balloon catheter method. The recovery rate of reconstructed embryos was 63.6% and 60.6% for the ovariohysterectomy and Foley balloon catheter methods, respectively. Although significant differences were observed in the early developmental stages (one-cell and 16-cell stages), no significant difference was observed in the blastocyst stage. Significantly higher blastocyst rate was observed when the embryos were collected on Day 8 (11.4%) than on Day 7 (0.0%; P < 0.05). At the proximal uterine horn on Day 7, no embryos at any stage were found, whereas on Days 8 and 9, blastocysts were found. We have observed a 63% initial pregnancy rate at 25 to 30 days after embryo transfer and a 50% full-term pregnancy rate, whereas 6.3% of the puppies were born, and 5.5% were born live among the total transferred embryos. Our results suggest that cloned embryos can develop to blastocysts by Day 8, and full-term pregnancy can be achieved after embryo transfer in canine.

  7. Circus movements in dissociated cells in normal and hybrid frog embryos.

    PubMed

    Johnson, K E; Adelman, M R

    1981-06-01

    Circus movements, involving circumferential rotation of a hyaline cytoplasmic blister and endoplasmic flow, occur in EDTA-dissociated gastrula stage Rana pipiens embryos. Such cell movements occur in very few cells taken from pre-gastrula stage embryos. During gastrulation, there is a progressive increase in the proportion of a population of cells that is engaged in circus movements. Circus movements do not occur in dividing cells. Individual cells in culture, as well as small clusters of cells in vitro, are jostled about in an apparently aimless fashion over short distances by circus movements, although the translocation of masses of cells over long distances is substantially greater than the translocation of isolated cells. In an early gastrula stage normal embryo, cells from around the site of blastopore invagination are most active in circus movements. Cells taken from different stages of arrested hybrid embryos show variable depression in the formation of rotating hyaline blebs. Aggregates of cells from arrested hybrid embryos are also relatively immobile in culture. The morphogenetic significance of circus movements in normal embryos and gastrula-arrest hybrid embryos is discussed.

  8. Metabolite profiling reveals clear metabolic changes during somatic embryo development of Norway spruce (Picea abies).

    PubMed

    Businge, Edward; Brackmann, Klaus; Moritz, Thomas; Egertsdotter, Ulrika

    2012-02-01

    Progress on industrial-scale propagation of conifers by somatic embryogenesis has been hampered by the differences in developmental capabilities between cell lines, which are limiting the capture of genetic gains from breeding programs. In this study, we investigated the metabolic events occurring during somatic embryo development in Norway spruce to establish a better understanding of the fundamental metabolic events required for somatic embryo development. Three embryogenic cell lines of Norway spruce (Picea abies (L.) Karst) with different developmental capabilities were studied during somatic embryo development from proliferation of proembryogenic masses to mature somatic embryos. The three different cell lines displayed normal, aberrant and blocked somatic embryo development. Metabolite profiles from four development stages in each of the cell lines were obtained using combined gas chromatography-mass spectrometry. Multivariate discriminant analyses of the metabolic data revealed significant metabolites (P  ≤  0.05) for each development stage and transition. The results suggest that endogenous auxin and sugar signaling affects initial stages of somatic embryo development. Furthermore, the results highlight the importance of a timed stress response and the presence of stimulatory metabolites during late stages of embryo development.

  9. Ethics and embryos.

    PubMed Central

    Poplawski, N; Gillett, G

    1991-01-01

    In this paper we argue that the human form should be seen to exist, in a longitudinal way, throughout the continuum of human growth and development. This entails that the moral value of that form, which we link analytically to the adult, interacting, social and rational being, attaches to all phases of human life to some extent. Having established this we discuss the consequences it has for the moral status of the human embryo. We then apply this argument, and the resulting moral status, to the area of reproductive technology. In doing this we show that there are certain regulations and controls which ought to apply to the use of these infertility treatments. PMID:1870084

  10. 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.

  11. Use of dual section mRNA in situ hybridisation/immunohistochemistry to clarify gene expression patterns during the early stages of nephron development in the embryo and in the mature nephron of the adult mouse kidney.

    PubMed

    Georgas, Kylie; Rumballe, Bree; Wilkinson, Lorine; Chiu, Han Sheng; Lesieur, Emmanuelle; Gilbert, Thierry; Little, Melissa H

    2008-11-01

    The kidney is the most complex organ within the urogenital system. The adult mouse kidney contains in excess of 8,000 mature nephrons, each of which can be subdivided into a renal corpuscle and 14 distinct tubular segments. The histological complexity of this organ can make the clarification of the site of gene expression by in situ hybridisation difficult. We have defined a panel of seven antibodies capable of identifying the six stages of early nephron development, the tubular nephron segments and the components of the renal corpuscle within the embryonic and adult mouse kidney. We have analysed in detail the protein expression of Wt1, Calb1 Aqp1, Aqp2 and Umod using these antibodies. We have then coupled immunohistochemistry with RNA in situ hybridisation in order to precisely identify the expression pattern of different genes, including Wnt4, Umod and Spp1. This technique will be invaluable for examining at high resolution, the structure of both the developing and mature nephron where standard in situ hybridisation and histological techniques are insufficient. The use of this technique will enhance the expression analyses of genes which may be involved in nephron formation and the function of the mature nephron in the mouse.

  12. Assessment of ‘one-step’ versus ‘sequential’ embryo culture conditions through embryonic genome methylation and hydroxymethylation changes

    PubMed Central

    Salvaing, J.; Peynot, N.; Bedhane, M. N.; Veniel, S.; Pellier, E.; Boulesteix, C.; Beaujean, N.; Daniel, N.; Duranthon, V.

    2016-01-01

    STUDY QUESTION In comparison to in vivo development, how do different conditions of in vitro culture (‘one step’ versus ‘sequential medium’) impact DNA methylation and hydroxymethylation in preimplantation embryos? SUMMARY ANSWER Using rabbit as a model, we show that DNA methylation and hydroxymethylation are both affected by in vitro culture of preimplantation embryos and the effect observed depends on the culture medium used. WHAT IS KNOWN ALREADY Correct regulation of DNA methylation is essential for embryonic development and DNA hydroxymethylation appears more and more to be a key player. Modifications of the environment of early embryos are known to have long term effects on adult phenotypes and health; these probably rely on epigenetic alterations. STUDY DESIGN SIZE, DURATION The study design we used is both cross sectional (control versus treatment) and longitudinal (time-course). Each individual in vivo experiment used embryos flushed from the donor at the 2-, 4-, 8-, 16- or morula stage. Each stage was analyzed in at least two independent experiments. Each individual in vitro experiment used embryos flushed from donors at the 1-cell stage (19 h post-coïtum) which were then cultured in parallel in the two tested media until the 2-, 4-, 8- 16-cell or morula stages. Each stage was analyzed in at least three independent experiments. In both the in vivo and in vitro experiments, 4-cell stage embryos were always included as an internal reference. PARTICIPANTS/MATERIALS, SETTING, METHODS Immunofluorescence with antibodies specific for 5-methylcytosine (5meC) and 5-hydroxymethylcytosine (5hmeC) was used to quantify DNA methylation and hydroxymethylation levels in preimplantation embryos. We assessed the expression of DNA methyltransferases (DNMT), of ten eleven translocation (TET) dioxigenases and of two endogenous retroviral sequences (ERV) using RT-qPCR, since the expression of endogenous retroviral sequences is known to be regulated by DNA methylation

  13. HIV exceptionalism, CD4+ cell testing, and conscientious subversion

    PubMed Central

    Jansen, L

    2005-01-01

    In recent years, many states in the United States have passed legislation requiring laboratories to report the names of patients with low CD4 cell counts to their state Departments of Health. This name reporting is an integral part of the growing number of "HIV Reporting and Partner Notification Laws" which have emerged in response to recently revised guidelines suggested by the National Centers for Disease Control (CDC). Name reporting for patients with low CD4 cell counts allows for a more accurate tracking of the natural history of HIV disease. However, given that this test is now considered to be an "indicator" of HIV, should it be subject to the same strict consent required for HIV testing? While the CDC has recommended that each state develop its own consent requirements for CD4 cell testing, most states have continued to rely on the presumed consent standards for CD4 cell testing that were in place before the passage of name reporting statutes. This allows physicians who treat patients who refuse HIV testing to order a CD4 cell blood analysis to gather information that is indicative of their patient's HIV status. This paper examines the ethical and legal issues associated with the practice of "conscientious subversion" as it arises when clinicians use CD4 cell counts as a surrogate for HIV testing. PMID:15923478

  14. MLL2 is essential for porcine embryo development in vitro.

    PubMed

    Zhao, Ming-Hui; Liang, Shuang; Kim, Nam-Hyung; Cui, Xiang-Shun

    2016-06-01

    Several germ cell-specific transcription factors essential for ovarian formation and folliculogenesis have been identified and studied. However, their function during early embryo development has been poorly explored. In this study, we investigated the role of mixed-lineage leukemia protein 2 (MLL2) in the development of porcine preimplantation embryos. To explore the function of MLL2 in porcine embryo development, expression and localization of MLL2 were assessed by qRT-PCR and immunofluorescence assays. Results showed that expression of MLL2 was significantly reduced after the four-cell stage. However, immunofluorescence results showed that MLL2 only localized in the nucleus of blastocysts, revealing a potential role of MLL2 in regulating the gene expression in the blastocyst stage. Knockdown of Mll2 by double-stranded RNA (dsRNA) caused a reduction in MLL2 signal in porcine blastocyst cells and in blastocyst formation. No significant differences in the cleavage and morula stages were observed. The mechanism of MLL2 regulation in blastocysts was assessed by assaying the trimethylation of histone 3 at lysine 4 (H3K4m3). MLL2 knockdown significantly reduced H3K4m3 in the nucleus and further reduced expression of Sox2 and Magoh genes. In conclusion, MLL2 is essential for porcine embryo development by the regulation of methylation of H3K4 in vitro. PMID:27059328

  15. Optimization protocol for storage of goldfish (Carassius auratus) embryos in chilled state.

    PubMed

    Shaluei, F; Imanpoor, M R; Shabani, A; Nasr-Esfahani, M H

    2014-04-01

    A series of five experiments were conducted to explore suitable conditions for storing of goldfish embryos in a chilled state. The factors studied were embryo stage, storage temperature, physiological saline solutions and goldfish artificial coelomic fluid (GFACF) medium, antibiotics (penicillin and streptomycin), antioxidants (vitamin E, vitamin C), buffer (Hepes, Tris) and BSA (bovine serum albumin). First, goldfish embryos at eight developmental stages were incubated in aerated and dechlorinated tap water at 0 °C for 24 h. Result shows that early developmental stages were most sensitive to chilling. Heartbeat-stage goldfish embryos were chilled at 0, 4 or 8 °C for up to 72 h in water, and chilled storage was possible only for up to 18, 24 and 48 h at 0, 4 and 8 °C, respectively, without a decrease in viability. Chilling of goldfish embryos at 8 °C in GFACF medium and Dettlaff's solution instead of water and other physiological saline solutions prolonged their viability (p < 0.01). Nevertheless, viability of chilled embryos in GFACF medium was slightly, but non-significantly, higher than in Dettlaff's solution. Supplementation of the GFACF medium with antibiotics, Hepes or BSA increased the viability of chilled embryos, but the tested vitamin E analogue Trolox, vitamin C or Tris concentration had no effect on embryo viability. The outcome of this series of experiments shows that heartbeat-stage goldfish embryos could be chilled for 60 h in GFACF supplemented with 25 mm Hepes, 100 U/ml penicillin, 10 μg/l streptomycin and 1 g/l BSA in such a way that embryonic development does not proceed, and viability is not lost.

  16. Characterization of the Two Maize Embryo-Lethal Defective Kernel Mutants Rgh*-1210 and Fl*-1253b: Effects on Embryo and Gametophyte Development

    PubMed Central

    Clark, J. K.; Sheridan, W. F.

    1988-01-01

    We have examined the effects on embryonic and gametophytic development of two nonallelic defective-kernel mutants of maize. Earlier studies indicated that both mutants are abnormal in embryonic morphogenesis as well as in the formation of their endosperm. Mutant rgh*-1210 embryos depart from the normal embryogenic pathway at the proembryo and transition stage, by developing meristematic lobes and losing bilateral symmetry. They continue growth as irregular cell masses that enlarge and become necrotic. Somatic embryos arising in rgh*-1210 callus cultures display the rgh*-1210 mutant phenotype. Mutant fl*-1253B embryos are variably blocked from the coleoptilar stage through stage 2. Following formation of the shoot apex in the mutant embryos the leaf primordia and tissues surrounding the embryonic axis continue growth and cell division, while the scutellum ceases development and becomes hypertrophied. Mutant fl*-1253B embryos are unable to germinate, either in mutant kernels or as immature embryos in culture, and the mutant scutellar tissue does not produce regenerable callus. Expression of the fl*-1253B locus during male gametophytic development is revealed by a marked reduction in pollen transmission as a result of mutant expression during the interval between meiosis and the initiation of pollen tube growth. In both mutants, there is considerable proliferation of the aleurone cells of the endosperm. Mutant expression of rgh*-1210 in the female gametophyte is revealed by the abnormal antipodal cells of the embryo sac. These results show that these two gene loci play unique and crucial roles in normal morphogenesis of the embryo. In addition, it is evident that both mutants are pleiotropic in affecting the development of the endosperm and gametophyte as well as the embryo. These pleiotropisms suggest some commonality in the gene regulation of development in these three tissues. PMID:17246478

  17. Differential regulation of receptivity in two uterine horns of a recipient mouse following asynchronous embryo transfer

    PubMed Central

    Li, Shi-Jie; Wang, Tong-Song; Qin, Fu-Niu; Huang, Zhu; Liang, Xiao-Huan; Gao, Fei; Song, Zhuo; Yang, Zeng-Ming

    2015-01-01

    Receptivity is a limited time in which uterine endometrium can establish a successful dialogue with blastocyst. This study was to investigate the effect of asynchronous embryo transfer on uterine receptivity in mice. Embryos under different stages were transferred into two oviduct sides of a recipient mouse on day 1 of pseudopregnancy. Our results showed the asynchronously transferred embryos can implant in all groups. Compared to zygote-transfer group, the length of implanted embryos is longer in 8-cell embryo- or blastocyst-transfer group. The levels of Snail and COX-2 immunostaining in blastocyst-transfer group are significantly stronger than that in zygote-transfer group. Embryos in blastocyst-transfer group migrate faster than that in zygote-transfer group within uterus. Blastocysts are in a state of developmental delay after they are transferred into oviducts, and they are reactivated and implanted rapidly in uterus. The developmental rate to newborn in zygote-transfer group is obviously higher than that in blastocyst-transfer group, suggesting that a delay in embryo development and implantation will lead to a decrease of litter size. These results indicated that the window of implantation is differentially regulated in two uterine horns of a recipient by embryos at different stages. PMID:26531680

  18. Automating fruit fly Drosophila embryo injection for high throughput transgenic studies

    NASA Astrophysics Data System (ADS)

    Cornell, E.; Fisher, W. W.; Nordmeyer, R.; Yegian, D.; Dong, M.; Biggin, M. D.; Celniker, S. E.; Jin, J.

    2008-01-01

    To decipher and manipulate the 14 000 identified Drosophila genes, there is a need to inject a large number of embryos with transgenes. We have developed an automated instrument for high throughput injection of Drosophila embryos. It was built on an inverted microscope, equipped with a motorized xy stage, autofocus, a charge coupled device camera, and an injection needle mounted on a high speed vertical stage. A novel, micromachined embryo alignment device was developed to facilitate the arrangement of a large number of eggs. The control system included intelligent and dynamic imaging and analysis software and an embryo injection algorithm imitating a human operator. Once the injection needle and embryo slide are loaded, the software automatically images and characterizes each embryo and subsequently injects DNA into all suitable embryos. The ability to program needle flushing and monitor needle status after each injection ensures reliable delivery of biomaterials. Using this instrument, we performed a set of transformation injection experiments. The robot achieved injection speeds and transformation efficiencies comparable to those of a skilled human injector. Because it can be programed to allow injection at various locations in the embryo, such as the anterior pole or along the dorsal or ventral axes, this system is also suitable for injection of general biochemicals, including drugs and RNAi.

  19. PXD101 significantly improves nuclear reprogramming and the in vitro developmental competence of porcine SCNT embryos

    SciTech Connect

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

    2015-01-02

    Highlights: • First explored that the effects of PXD101 on the development of SCNT embryos in vitro. • 0.5 μM PXD101 treated for 24 h improved the development of porcine SCNT embryos. • Level of AcH3K9 was significantly higher than control group at early stages. - Abstract: In this study, we investigated the effects of the histone deacetylase inhibitor PXD101 (belinostat) on the preimplantation development of porcine somatic cell nuclear transfer (SCNT) embryos and their expression of the epigenetic markers histone H3 acetylated at lysine 9 (AcH3K9). We compared the in vitro developmental competence of SCNT embryos treated with various concentrations of PXD101 for 24 h. Treatment with 0.5 μM PXD101 significantly increased the proportion of SCNT embryos that reached the blastocyst stage, in comparison to the control group (23.3% vs. 11.5%, P < 0.05). We tested the in vitro developmental competence of SCNT embryos treated with 0.5 μM PXD101 for various amounts of times following activation. Treatment for 24 h significantly improved the development of porcine SCNT embryos, with a significantly higher proportion of embryos reaching the blastocyst stage in comparison to the control group (25.7% vs. 10.6%, P < 0.05). PXD101-treated SCNT embryos were transferred into two surrogate sows, one of whom became pregnant and four fetuses developed. PXD101 treatment significantly increased the fluorescence intensity of immunostaining for AcH3K9 in embryos at the pseudo-pronuclear and 2-cell stages. At these stages, the fluorescence intensities of immunostaining for AcH3K9 were significantly higher in PXD101-treated embryos than in control untreated embryos. In conclusion, this study demonstrates that PXD101 can significantly improve the in vitro and in vivo developmental competence of porcine SCNT embryos and can enhance their nuclear reprogramming.

  20. Atomic force microscopy of living and fixed Xenopus laevis embryos.

    PubMed

    Efremov, Yu M; Pukhlyakova, E A; Bagrov, D V; Shaitan, K V

    2011-12-01

    Xenopus laevis embryos are a rather simple and at the same time a very interesting animal model, which is widely used for research in developmental biology. Intensive coordinated cell movements take place during the multi-cellular organism development. Little is known of the cellular, molecular and biomechanical mechanisms of these movements. The conceptual framework for analysis of cell interactions within integrated populations is poorly developed. We have used atomic force microscopy (AFM) to observe the surface of fixed X. laevis embryos at different stages of their development. We have developed a new sample preparation protocol for these observations. The obtained images were compared with scanning electronic microscopy (SEM) data. Cell rearrangement during morphogenesis in vivo was also visualized by AFM. In the current paper we discuss facilities and challenges of using this technique for further embryo researching.

  1. Clinical outcomes of vitrified-thawed embryo transfer using a pull and cut straw method

    PubMed Central

    Lim, Joon Gyo; Heo, Young Tae; Min, Seung Gi; Min, Byeong Yeol; Uhm, Sang Jun

    2013-01-01

    Objective To compare the clinical outcomes of patients with vitrified-thawed embryos transferred using either the 0.25 mL straw method and the pull and cut straw (PNC) method. To evaluate the clinical outcomes of patients with transferred embryos that underwent assisted hatching at the cleaved embryo (day 3) or the blastocyst (day 5) stage. Methods The study population consisted of women who underwent vitrified-warmed embryo transfer between May 2000 and December 2011 and assisted hatching was performed after warming of embryos. Cycles of thawing between assisted hatching treated and non treated groups were compared for survival and pregnancy rates. Results The PNC vitrification method improved survival and pregnancy rates in partial lysed embryos. While assisted hatching did not affect the developmental and clinical pregnancy rates of the vitrified-warmed blastocyst group, it did increase the pregnancy rate of poor quality vitrified-warmed cleaved embryos. Conclusion These results suggest that PNC may increase the number of clinical pregnancies via the vitrification of both cleaved embryos and blastocysts. In addition, selective assisted hatching treatment of embryos that show a poor prognosis after warming may increase the rate of clinical pregnancy. PMID:24327999

  2. Abnormalities in centrosome number in human embryos and embryonic stem cells.

    PubMed

    Gu, Yi-Fan; OuYang, Qi; Dai, Can; Lu, Chang-Fu; Lin, Ge; Gong, Fei; Lu, Guang-Xiu

    2016-05-01

    Chromosomal abnormalities are common in human embryos. Previous studies have suggested links between centrosome number and chromosome abnormalities, but information regarding abnormalities in centrosome number in human embryos is limited. We analyzed abnormalities in centrosome number in human embryos and embryonic stem cells (hESCs). Following normal fertilization, supernumerary centrosomes were present at rates of 7.3% in two-pronucleus (2PN)-stage zygotes and 6.5% in first-cleavage zygotes. Supernumerary centrosomes were also detected in 24.4% of blastomeres from 60% of embryos derived from 2PN zygotes. Conversely, in mono- (1PN) and tri-pronucleus (3PN) zygotes, the frequency of abnormal centrosome number increased substantially at first cleavage. Rates in blastomeres of Day-3 embryos, however, were about the same between embryos derived from 1PN and 2PN zygotes, whereas abnormalities in centrosome number were higher in those from 3PN zygotes. By comparison, the rate of abnormal centrosome numbers in hESCs was 1.5-11.2%. Thus, abnormalities in centrosome number existed in human zygotes and cleaved embryos-especially those resulting from aberrant fertilization-but the frequency of such abnormalities was lower in hESCs derived from these embryos. These findings identify a source of the chromosomal instability in human embryos and hESCs, and highlight new safety issues for human assisted reproductive technology. Mol. Reprod. Dev. 83: 392-404, 2016. © 2016 Wiley Periodicals, Inc.

  3. The effect of hypoxia on facial shape variation and disease phenotypes in chicken embryos

    PubMed Central

    Smith, Francis; Hu, Diane; Young, Nathan M.; Lainoff, Alexis J.; Jamniczky, Heather A.; Maltepe, Emin; Hallgrimsson, Benedikt; Marcucio, Ralph S.

    2013-01-01

    SUMMARY Craniofacial anomalies can arise from both genetic and environmental factors, including prenatal hypoxia. Recent clinical evidence correlates hypoxia to craniofacial malformations. However, the mechanisms by which hypoxia mediates these defects are not yet understood. We examined the cellular mechanisms underlying malformations induced by hypoxia using a chicken (Gallus gallus) embryo model. Eggs were incubated in either hypoxic (7, 9, 11, 13, 15, 17 or 19% O2) or normoxic (21% O2) conditions. Embryos were photographed for morphological analysis at days 3–6. For analysis of skeletal development, 13-day embryos were cleared and stained with alcian blue and alizarin red for cartilage and bone, respectively. Quantitative analysis of facial shape variation was performed on images of embryos via geometric morphometrics. Early-stage embryos (day 2) were analyzed for apoptosis via whole-mount and section TUNEL staining and immunostaining for cleaved caspase-3, whereas later-stage embryos (days 4–6) were sectioned in paraffin for analysis of cell proliferation (BrdU), apoptosis (TUNEL) and metabolic stress (phospho-AMPK). Results demonstrate that survival is reduced in a dose-dependent manner. Hypoxic embryos displayed a spectrum of craniofacial anomalies, from mild asymmetry and eye defects to more severe frontonasal and cephalic anomalies. Skull bone development was delayed in hypoxic embryos, with some skeletal defects observed. Morphometric analysis showed facial shape variation relative to centroid size and age in hypoxic groups. Hypoxia disrupted cell proliferation and, in early-stage embryos, caused apoptosis of neural crest progenitor cells. Hypoxic embryos also displayed an increased metabolic stress response. These results indicate that hypoxia during early embryonic craniofacial development might induce cellular oxidative stress, leading to apoptosis of the neural crest progenitor cells that are crucial to normal craniofacial morphogenesis. PMID

  4. Development of water buffalo (Bubalus bubalis) embryos from in vitro matured oocytes reconstructed with fetal skin fibroblast cells as donor nuclei.

    PubMed

    Meena, C R; Das, S K

    2006-07-01

    The present study was carried out to explore the feasibility of using buffalo fetal skin fibroblasts as donor nuclei and to find out the developmental competence of embryos following transfer of these nuclei to in vitro matured enucleated buffalo oocytes. Skin cells were isolated from 1 to 2-month-old fetuses obtained from slaughterhouse, by enzymatic digestion (0.5% w/v trypsin +0.05% w/v collagenase in Dulbecco's PBS) for 15-20 min. The cells were washed 4 times with Dulbecco's PBS and then once with RPMI-1640+10% FBS by centrifugation at 600 x g. The cells were then cultured in the same medium in a CO2 incubator (5% CO2 in air) at 38.5 degrees C for 2-3 days. Cumulus-oocyte complexes (COCs) collected from slaughterhouse buffalo ovaries were subjected to IVM in the IVM medium (TCM-199 + 5 microg/ml FSH-P + 10 microg/ml LH+10% FBS) for 20-22 h in a CO2 incubator (5% CO2 in air) at 38.5 degrees C. Oocytes were denuded with 0.1% trypsin followed by repeated pipetting and then enucleated by aspirating the first polar body with 10-15% of nearby cytoplasm with a micromanipulator. Two different types of donor cells (growing cells and those arrested with cytochalasin-B) were used for reconstruction of oocytes. The reconstructs were electro fused and incubated in the activation medium (TCM-199 + 8 microg/ml cytochalasin-B+10% FBS) for 4 h. These were then cultured in IVC medium (TCM-199+10% FBS) in a CO2 incubator (5% CO2 in air) at 38.5 degrees C for 48 h. The cleaved embryos were then co-cultured with buffalo oviduct cells in embryo development media (EDM). Out of 119 denuded matured oocytes which were enucleated and reconstructed with growing cells, 78 (65.5%) were electro fused, activated and cultured, out of which 4 (5.1%) reconstructs cleaved and developed to 2-cell stage, 3 (3.8%) reached to 4-cell stage and 3 (3.8%) reached to 8-cell stage. In the synchronized group, out of 62 denuded matured oocytes which were reconstructed with cytochalasin-B blocked cells, 40

  5. Effects of perfluorinated compounds on development of zebrafish embryos.

    PubMed

    Zheng, Xin-Mei; Liu, Hong-Ling; Shi, Wei; Wei, Si; Giesy, John P; Yu, Hong-Xia

    2011-08-01

    Perfluorinated compounds (PFCs) have been widely used in industrial and consumer products and frequently detected in many environmental media. Potential reproductive effects of perfluorooctanesulfonate (PFOS), perfluorooctanoic acid (PFOA) and perfluorononanoic acid (PFNA) have been reported in mice, rats and water birds. PFOS and PFOA were also confirmed developing toxicants towards zebrafish embryos; however, the reported effect concentrations were contradictory. Polyfluorinated alkylated phosphate ester surfactants (including FC807) are precursor of PFOS and PFOA; however, there is no published information about the effects of FC807 and PFNA on zebrafish embryos. Therefore, this study was conducted to determine the effects of these four PFCs on zebrafish embryos. Normal fertilized zebrafish embryos were selected to be exposed to several concentrations of PFOA, PFNA, PFOS or FC807 in 24-well cell culture plates. A digital camera was used to image morphological anomalies of embryos with a stereomicroscope. Embryos were observed through matching up to 96-h post-fertilization (hpf) and rates of survival and abnormalities recorded. PFCs caused lethality in a concentration-dependent manner with potential toxicity in the order of PFOS > FC807 > PFNA > PFOA based on 72-h LC(50). Forty-eight-hour post-fertilization pericardial edema and 72- or 96-hpf spine crooked malformation were all observed. PFOA, PFNA, PFOS and FC807 all caused structural abnormalities using early stages of development of zebrafish. The PFCs all retarded the development of zebrafish embryos. The toxicity of the PFCs was related to the length of the PFC chain and functional groups. PMID:22828880

  6. [How can we nowadays select the best embryo to transfer?].

    PubMed

    Alter, L; Boitrelle, F; Sifer, C

    2014-01-01

    Multiple pregnancies stand as the most common adverse outcome of assisted reproduction technologies (ART) and the dangers associated with those pregnancies have been reduced by doing elective single embryo transfers (e-SET). Many studies have shown that e-SET is compatible with a continuously high pregnancy rate per embryo transfer. Yet, it still becomes necessary to improve the selection process in order to define the quality of individual embryos - so that the ones we choose for transfer are more likely to implant. First, analysis of embryo morphology has greatly helped in this identification and remains the most relevant criterion for choosing the embryo. The introduction of time-lapse imaging provides new criteria predictive of implantation potential, but the real contribution of this system - including the benefit/cost ratio - seems to be not yet properly established. In this context, extended culture until blastocyst stage is an essential practice but it appears wise to keep it for a population showing a good prognosis. Then, the failure of aneuploid embryos to implant properly led to achieve preimplantation genetic screening (PGS) in order to increase pregnancy and delivery rates after ART. However, PGS by fluorescence in situ hybridization (FISH) at day 3 is a useless process - and may even be harmful. Another solution involves using comparative genomic hybridisation (CGH) and moving to blastocyst biopsy. Finally, it is envisaged that morphology will also be significantly aided by non-invasive analysis of biomarkers in the culture media that give a better reflection of whole-embryo physiology and function. PMID:24951187

  7. Lipid droplet analysis using in vitro bovine oocytes and embryos.

    PubMed

    Ordoñez-Leon, E A; Merchant, H; Medrano, A; Kjelland, M; Romo, S

    2014-04-01

    The aim of this study was to quantify the content of lipid droplets in bovine oocytes and embryos from Bos indicus (Bi), Bos taurus (Bt) and Bos indicus × Bos taurus (Bi × Bt). Oocytes were aspirated post-mortem and subjected to in vitro maturation, in vitro fertilization and in vitro development; the medium employed at each stage (TCM-199, TALP, SOF) was supplemented with (i) serum replacement (SR), (ii) foetal calf serum (FCS) or (iii) oestrous cow serum (ECS). The structure and distribution of the lipid droplets were established using electron microscopy, but were quantified using an optical microscope on semi-fine toluidine blue-stained sections. The highest percentage of embryos corresponded to those produced with FCS and ECS, which differed from embryos generated with SR (p < 0.05). The highest percentage of morulae and the lowest percentage of blastocysts were obtained with the SR supplement (p < 0.05). The oocytes cultured in FCS demonstrated a higher number of lipid droplets compared to those cultured in SR and ECS (p < 0.05). Less accumulation of lipids was observed in embryos supplemented with SR. The lowest and highest numbers of lipid droplets in oocytes corresponded to the Bi and Bt strain, respectively. The lowest amount of lipid droplets in embryos was observed in Bi (p < 0.05). In conclusion, supplementation of the in vitro development culture medium (synthetic oviduct fluid) with a synthetic substitute serum produced similar results in terms of embryo development compared to those obtained with FCS, but a decreased degree of lipid droplet accumulation was observed in the in vitro-cultured embryos. PMID:24467659

  8. Direct evidence that suspensor cells have embryogenic potential that is suppressed by the embryo proper during normal embryogenesis

    PubMed Central

    Liu, Yuan; Li, Xinbo; Zhao, Jing; Tang, Xingchun; Tian, Shujuan; Chen, Junyi; Shi, Ce; Wang, Wei; Zhang, Liyao; Feng, Xianzhong; Sun, Meng-Xiang

    2015-01-01

    The suspensor is a temporary supporting structure of proembryos. It has been proposed that suspensor cells also possess embryogenic potential, which is suppressed by the embryo as an effect of the embryo–suspensor interaction. However, data to support this hypothesis are not yet available. In this report, using an in vivo living cell laser ablation technique, we show that Arabidopsis suspensor cells can develop into embryos after removing the embryo proper. The embryo proper plays a critical role in maintaining suspensor cell identity. However, this depends on the developmental stage; after the globular embryo stage, the suspensors no longer possess the potential to develop into embryos. We also reveal that hypophysis formation may be essential for embryo differentiation. Furthermore, we show that, after removing the embryo, auxin gradually accumulates in the top suspensor cell where cell division occurs to produce an embryo. Auxin redistribution likely reprograms the fate of the suspensor cell and triggers embryogenesis in suspensor cells. Thus, we provide direct evidence that the embryo suppresses the embryogenic potential of suspensor cells. PMID:26396256

  9. Live birth in a woman with recurrent implantation failure and adenomyosis following transfer of refrozen-warmed embryos

    PubMed Central

    Safari, Somayyeh; Faramarzi, Azita; Khalili, Mohammad Ali

    2016-01-01

    The aim was to report a healthy live birth using re-vitrified-warmed cleavage-stage embryos derived from supernumerary warmed embryos after frozen embryo transfer (ET) in a patient with recurrent implantation failure (RIF). The case was a 39-year-old female with a history of polycystic ovarian syndrome and adenomyosis, along with RIF. After ovarian hyperstimulation, 33 cumulus-oocyte complexes were retrieved and fertilized with conventional in vitro fertilization and intracytoplasmic sperm injection. Because of the risk of ovarian hyperstimulation syndrome, 16 grade B and C embryos were vitrified. After 3 and 6 months, 3 and 4 B–C warmed embryos were transferred to the uterus, respectively. However, implantation did not take place. Ten months later, four embryos were warmed, two grade B 8-cell embryos were transferred, and two embryos were re-vitrified. One year later, the two re-vitrified cleavage-stage embryos were warmed, which resulted in a successful live birth. This finding showed that following first warming, it is feasible to refreeze supernumerary warmed embryos for subsequent ET in patients with a history of RIF. PMID:27689042

  10. Live birth in a woman with recurrent implantation failure and adenomyosis following transfer of refrozen-warmed embryos

    PubMed Central

    Safari, Somayyeh; Faramarzi, Azita; Khalili, Mohammad Ali

    2016-01-01

    The aim was to report a healthy live birth using re-vitrified-warmed cleavage-stage embryos derived from supernumerary warmed embryos after frozen embryo transfer (ET) in a patient with recurrent implantation failure (RIF). The case was a 39-year-old female with a history of polycystic ovarian syndrome and adenomyosis, along with RIF. After ovarian hyperstimulation, 33 cumulus-oocyte complexes were retrieved and fertilized with conventional in vitro fertilization and intracytoplasmic sperm injection. Because of the risk of ovarian hyperstimulation syndrome, 16 grade B and C embryos were vitrified. After 3 and 6 months, 3 and 4 B–C warmed embryos were transferred to the uterus, respectively. However, implantation did not take place. Ten months later, four embryos were warmed, two grade B 8-cell embryos were transferred, and two embryos were re-vitrified. One year later, the two re-vitrified cleavage-stage embryos were warmed, which resulted in a successful live birth. This finding showed that following first warming, it is feasible to refreeze supernumerary warmed embryos for subsequent ET in patients with a history of RIF.

  11. Live birth in a woman with recurrent implantation failure and adenomyosis following transfer of refrozen-warmed embryos.

    PubMed

    Safari, Somayyeh; Faramarzi, Azita; Agha-Rahimi, Azam; Khalili, Mohammad Ali

    2016-09-01

    The aim was to report a healthy live birth using re-vitrified-warmed cleavage-stage embryos derived from supernumerary warmed embryos after frozen embryo transfer (ET) in a patient with recurrent implantation failure (RIF). The case was a 39-year-old female with a history of polycystic ovarian syndrome and adenomyosis, along with RIF. After ovarian hyperstimulation, 33 cumulus-oocyte complexes were retrieved and fertilized with conventional in vitro fertilization and intracytoplasmic sperm injection. Because of the risk of ovarian hyperstimulation syndrome, 16 grade B and C embryos were vitrified. After 3 and 6 months, 3 and 4 B-C warmed embryos were transferred to the uterus, respectively. However, implantation did not take place. Ten months later, four embryos were warmed, two grade B 8-cell embryos were transferred, and two embryos were re-vitrified. One year later, the two re-vitrified cleavage-stage embryos were warmed, which resulted in a successful live birth. This finding showed that following first warming, it is feasible to refreeze supernumerary warmed embryos for subsequent ET in patients with a history of RIF. PMID:27689042

  12. Deficiency in the response to DNA double-strand breaks in mouse early preimplantation embryos

    SciTech Connect

    Yukawa, Masashi; Oda, Shoji; Mitani, Hiroshi; Nagata, Masao; Aoki, Fugaku . E-mail: aokif@k.u-tokyo.ac.jp

    2007-06-29

    DNA double-strand breaks (DSBs) are caused by various environmental stresses, such as ionizing radiation and DNA-damaging agents. When DSBs occur, cell cycle checkpoint mechanisms function to stop the cell cycle until all DSBs are repaired; the phosphorylation of H2AX plays an important role in this process. Mouse preimplantation-stage embryos are hypersensitive to ionizing radiation, and X-irradiated mouse zygotes are arrested at the G2 phase of the first cell cycle. To investigate the mechanisms responding to DNA damage at G2 in mouse preimplantation embryos, we examined G2/M checkpoint and DNA repair mechanisms in these embryos. Most of the one- and two-cell embryos in which DSBs had been induced by {gamma}-irradiation underwent a delay in cleavage and ceased development before the blastocyst stage. In these embryos, phosphorylated H2AX ({gamma}-H2AX) was not detected in the one- or two-cell stages by immunocytochemistry, although it was detected after the two-cell stage during preimplantation development. These results suggest that the G2/M checkpoint and DNA repair mechanisms have insufficient function in one- and two-cell embryos, causing hypersensitivity to {gamma}-irradiation. In addition, phosphorylated ataxia telangiectasia mutated protein and DNA protein kinase catalytic subunits, which phosphorylate H2AX, were detected in the embryos at one- and two-cell stages, as well as at other preimplantation stages, suggesting that the absence of {gamma}-H2AX in one- and two-cell embryos depends on some factor(s) other than these kinases.

  13. Dynamic regulation of DNA methyltransferases in human oocytes and preimplantation embryos after assisted reproductive technologies.

    PubMed

    Petrussa, Laetitia; Van de Velde, Hilde; De Rycke, Martine

    2014-09-01

    DNA methylation is a key epigenetic modification which is essential for normal embryonic development. Major epigenetic reprogramming takes place during gametogenesis and in the early embryo; the complex DNA methylation patterns are established and maintained by DNA methyltransferases (DNMTs). However, the influence of assisted reproductive technologies (ART) on DNA methylation reprogramming enzymes has predominantly been studied in mice and less so in human oocytes and embryos. The expression and localization patterns of the four known DNMTs were analysed in human oocytes and IVF/ICSI embryos by immunocytochemistry and compared between a reference group of good quality fresh embryos and groups of abnormally developing embryos or embryo groups after cryopreservation. In humans, DNMT1o rather than DNMT1s seems to be the key player for maintaining methylation in early embryos. DNMT3b, rather than DNMT3a and DNMT3L, appears to ensure global DNA remethylation in the blastocysts before implantation. DNMT3L, an important regulator of maternal imprint methylation in mouse, was not detected in human oocytes (GV, MI and MII stage). Our study confirms the existence of species differences for mammalian DNA methylation enzymes. In poor quality fresh embryos, the switch towards nuclear DNMT3b expression was delayed and nuclear DNMT1, DNMT1s and DNMT3b expression was less common. Compared with the reference embryos, a smaller number of cryopreserved embryos showed nuclear DNMT1, while a delayed switch to nuclear DNMT3b and an extended DNMT1s temporal expression pattern were also observed. The spatial and temporal expression patterns of DNMTs seem to be disturbed in abnormally developing embryos and in embryos that have been cryopreserved. Further research must be performed in order to understand whether the potentially disturbed embryonic DNMT expression after cryopreservation has any long-term developmental consequences. PMID:24994815

  14. Standardization of grading embryo morphology.

    PubMed

    Racowsky, Catherine; Vernon, Michael; Mayer, Jacob; Ball, G David; Behr, Barry; Pomeroy, Kimball O; Wininger, David; Gibbons, William; Conaghan, Joseph; Stern, Judy E

    2010-08-01

    Standardization of morphologic assessment for an embryo grading system was developed and is being implemented by the Society for Assisted Reproductive Technology (SART). A recent European consensus conference of embryologists from Europe and America is working toward adopting an embryo classification system modeled similarly to that of SART that, if adopted, would produce a de facto international standard to aid cross-border collaboration. PMID:20580357

  15. Estrogen receptor and progesterone receptor genes are expressed differentially in mouse embryos during preimplantation development.

    PubMed Central

    Hou, Q; Gorski, J

    1993-01-01

    Estrogen and progesterone play an important role in the development and implantation of preimplantation embryos. However, it is controversial whether these hormones act directly on the embryos. The effects of these hormones depend on the existence of their specific receptors. To determine whether estrogen receptor (ER) and progesterone receptor genes are expressed in mouse preimplantation embryos, we examined RNA from embryos at different stages of preimplantation development by reverse transcription-polymerase chain reaction techniques. ER mRNA was found in oocytes and fertilized eggs. The message level began to decline at the two-cell stage and reached its lowest level at the five- to eight-cell stage. ER mRNA was not detectable at the morula stage but reappeared at the blastocyst stage. Progesterone receptor mRNA was not detectable until the blastocyst stage. The embryonic expression of ER and progesterone receptor genes in the blastocyst suggests a possible functional requirement for ER and progesterone receptor at this stage of development. These results provide a basis for determining the direct role of estrogen and progesterone in preimplantation embryos. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8415723

  16. Developmental defects in pelagic fish embryos from the western Baltic

    NASA Astrophysics Data System (ADS)

    v. Westernhagen, H.; Dethlefsen, V.; Cameron, P.; Berg, J.; Fürstenberg, G.

    1988-03-01

    In February/March 1983 and 1984 a survey of pelagic fish eggs was conducted in the western Baltic (Kiel Bight), employing a horizontally towed plankton net (1 m Ø and 300 μm mesh). Maximum egg numbers in the upper meter of the S=21×10-3 salinity layer were 200·100 m-3. The most abundant eggs were cod (up to 142 eggs·100 m-3), followed by plaice (up to 74 eggs·100 m-3) and flounder (20 eggs·100 m-3). A considerable percentage of embryos of all species displayed aberrant development. In 1983 18% of cod, 22% of flounder and 24% of plaice eggs caught contained defective embryos; in 1984 this number was larger, ranging from 28% in plaice over 32% in cod to 44% in flounder. Early developmental stages showed the highest malformation rates (up to 51% in the case of early flounder embryos). With progressive development, malformations decreased in numbers, being lowest prior to hatching. Highest rates of malformations were recorded in the Mecklenburg Bight in 1983. A second area with high incidence of malformation rates was located south and east of the island of Langeland. Several reasons, including environmental and anthropogenic factors, for the occurrence of malformed embryos in pelagic fish eggs are discussed. The potential of malformation rates in embryos of pelagic fish eggs as a tool for monitoring is considered.

  17. Filial cannibalism improves survival and development of beaugregory damselfish embryos.

    PubMed

    Payne, Adam G; Smith, Carl; Campbell, Andrew C

    2002-10-22

    Cannibalism of small numbers of offspring by a parent has been proposed as an adaptive parental strategy, by providing energy to support parental care. However, there are few empirical studies to support this hypothesis. We conducted field and laboratory experiments to investigate partial filial cannibalism in Stegastes leucostictus, a coral reef fish with paternal care. Partial cannibalism was shown to be common, and males were found to remove developing embryos from throughout a clutch in a random pattern, rather than in the more aggregated pattern seen during embryo predation. Males that received a diet supplement grew faster than control males, but did not engage in less cannibalism. Also, males did not concentrate cannibalism on early embryonic stages with the highest energetic value. Experimental reduction of embryo densities was found to significantly increase embryo development rate and survival from egg deposition to hatching, and experimental reduction of oxygen levels significantly increased rates of partial filial cannibalism by males. Artificial spawning sites with low oxygen levels were avoided by spawning females, and cannibalism rates by males were higher. We propose that partial filial cannibalism serves as an adaptive parental strategy to low oxygen levels in S. leucostictus by increasing the hatching success of embryos. PMID:12396483

  18. Sensitivity of early mouse embryos to (/sup 3/H)thymidine

    SciTech Connect

    Spindle, A.; Wu, K.; Pedersen, R.A.

    1982-12-01

    Effects of intranuclear radiation on the developmental capacity of early mouse embryos were studied by exposing embryos to (/sup 3/H)thymidine and counting the number of embryos forming blastocysts, trophoblast outgrowths, inner cell masses (ICMs), and two-layer ICMs (differentiated into primary endoderm and ectoderm). When embryos were cultured from the 2-cell stage for 8 days in the continuous presence of (/sup 3/H)thymidine, concentrations as low as 0.2 nCi/ml reduced the number of embryos forming two-layer ICMs. At 1 nCi/ml, the number of both ICMs and two-layer ICMs were reduced, and at 10 nCi/ml the number of embryos developing to all three post-blastocyst endpoints was reduced. Blastocyst formation was not affected even at the highst concentration (/sup 3/H)thymidine and then cultured further in unlabelled medium, the effects were similar to those of 8-day exposure. When embryos were exposed to (/sup 3/H)thymidine for 24 h at various developmental stages, effects were less severe than when they were exposed continuously for 3 or 8 days, and the sensitivity of embryos differed between stages. The 24-h exposure of immunosurgically isolated ICMS to (/sup 3/H)thymidine revealed that the high sensitivity of the ICM to (/sup 3/H)thymidine persists through the late blastocyst stage and declines progressively thereafter. Autoradiography indicated that the change in radiosensitivity of embryos or ICMs is generally related to their ability to incorporate (/sup 3/H)thymidine into the DNA.

  19. Alternative developmental pathways associated with diapause regulated by temperature and maternal influences in embryos of the annual killifish Austrofundulus limnaeus

    PubMed Central

    Podrabsky, Jason E.; Garrett, Ian D. F.; Kohl, Zachary F.

    2010-01-01

    Embryos of the annual killifish Austrofundulus limnaeus enter a state of developmental arrest termed diapause as part of their normal developmental program. Diapause can occur at two distinct developmental stages in this species, termed diapause II and III. When incubated at 25°C, most embryos enter diapause II, whereas a small percentage of ‘escape’ embryos develop continuously past diapause II and enter diapause III. Control of entry into diapause II can be altered by maternal influences and the incubation environment experienced by the embryos. Young females produce a higher proportion of escape embryos than do older females. In addition, increasing the incubation temperature from 25 to 30°C induces all embryos to escape from diapause. Surprisingly, escape embryos follow a different morphological and physiological developmental trajectory than do embryos that enter diapause II. Development of anterior structures is advanced compared with that of posterior structures in escape embryos when compared with embryos that will enter diapause II. The difference in timing of development for these two trajectories is consistent with changes observed between two species but is very atypical of variation observed within a species. Importantly, the two developmental pathways diverge early in development, during the segmentation period, when, according to evolutionary theory, constraint on developmental pathways should be relatively high. The possession of alternative developmental pathways in a vertebrate embryo is a novel finding, the ecological and evolutionary importance of which is still unknown, but potentially significant in terms of life-history evolution. PMID:20833920

  20. Production of rhesus monkey cloned embryos expressing monomeric red fluorescent protein by interspecies somatic cell nuclear transfer

    SciTech Connect

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

    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 rhesus 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.

  1. Morphokinetic behavior of euploid and aneuploid embryos analyzed by time-lapse in embryoscope

    PubMed Central

    Patel, Deven V.; Shah, Preeti B.; Kotdawala, Aditi P.; Herrero, Javier; Rubio, Irene; Banker, Manish R.

    2016-01-01

    BACKGROUND: Embryonic aneuploidy may result in miscarriage, implantation failure, or birth defects. Thus, it is clinically necessary to avoid the selection of aneuploid embryos during in vitro fertilization treatment. AIM: The aim of this study was to identify the morphokinetic differences by analyzing the development of euploid and aneuploid embryos using a time-lapse technology. We also checked the accuracy of a previously described model for selection of euploid embryos based on morphokinetics in our study population. MATERIALS AND METHODS: It is a retrospective study of 29 cycles undergoing preimplantation genetic screening from October 2013 to April 2015 at our center. Of 253 embryos, 167 suitable for biopsy embryos were analyzed for their chromosomal status using array-comparative genome hybridization (CGH). The morphokinetic behavior of these embryos was further analyzed in embryoscope using time-lapse technology. RESULTS: Among the analyzed embryos, 41 had normal and 126 had abnormal chromosome content. No significant difference in morphokinetics was found between euploid and aneuploid embryos. The percentage of embryos with blastulation was similar in the euploid (65.85%, 27/41) and aneuploid (60.31%, 76/126) embryos (P = 0.76). Although hard to define, majority of the chromosomal defects might be due to meiotic errors. On applying embryo selection model from Basile et al., embryos falling within optimal ranges for time to division to 5 cells (t5), time period of the third cell cycle (CC3), and time from 2 cell division to 5 cell division (t5-t2) exhibited greater proportion of normal embryos than those falling outside the optimal ranges (28.6%, 25.9%, and 26.7% vs. 17.5%, 20.8%, and 14.3%). CONCLUSION: Keeping a track of time interval between two stages can help us recognize aneuploid embryos at an earlier stage and prevent their selection of transfer. However, it cannot be used as a substitute for array CGH to select euploid embryos for transfer. PMID

  2. Lessons from Embryos: Haeckel's Embryo Drawings, Evolution, and Secondary Biology Textbooks

    ERIC Educational Resources Information Center

    Wellner, Karen L.

    2014-01-01

    In 1997, developmental biologist Michael Richardson compared his research team's embryo photographs to Ernst Haeckel's 1874 embryo drawings and called Haeckel's work "noncredible". "Science" soon published "Haeckel's Embryos: Fraud Rediscovered," and Richardson's comments further reinvigorated criticism of Haeckel by…

  3. Investigation into developmental potential and nuclear/mitochondrial function in early wood and plains bison hybrid embryos.

    PubMed

    Seaby, R P; Mackie, P; King, W A; Mastromonaco, G F

    2012-08-01

    Studies to date have shown that bison embryo development in vitro is compromised with few embryos developing to the blastocyst stage. The aim of this study was to use bison-cattle hybrid embryos, an interspecific cross that is known to result in live offspring in vivo, as a model for assessing species-specific differences in embryo development in vitro. Cattle oocytes fertilized with cattle, plains bison and wood bison sperm were assessed for various developmental parameters associated with embryo quality, including cell number, apoptosis and ATP content. Decreased development to the blastocyst stage was observed in hybrid wood bison embryos compared with the other treatment groups. Although both wood bison and plains bison hybrid blastocysts had significantly lower cell numbers than cattle blastocysts, only wood bison hybrid blastocysts had a greater incidence of apoptosis than cattle blastocysts. Among the treatment groups, ATP levels and expression profiles of NRF1, TFAM, MT-CYB, BAX and BCL2 were not significantly different in both 8- to 16-cell stage and blastocyst stage embryos. These data provide evidence of decreased developmental competence in the wood bison hybrid embryos, owing to inadequate culture conditions that have increased apoptotic events.

  4. Transcriptomic Analysis of the Porcine Endometrium during Embryo Implantation.

    PubMed

    Lin, Haichao; Wang, Huaizhong; Wang, Yanping; Liu, Chang; Wang, Cheng; Guo, Jianfeng

    2015-01-01

    In pigs, successful embryo implantation is an important guarantee for producing litter size, and early embryonic loss occurring on day 12-30 of gestation critically affects the potential litter size. The implantation process is regulated by the expression of numerous genes, so comprehensive analysis of the endometrium is necessary. In this study, RNA sequencing (RNA-Seq) technology is used to analyze endometrial tissues during early pregnancy. We investigated the changes of gene expression between three stages (day 12, 18, and 25) by multiple comparisons. There were 1557, 8951, and 2345 differentially expressed genes (DEGs) revealed between the different periods of implantation. We selected several genes for validation by the use of quantitative real-time RT-PCR. Bioinformatic analysis of differentially expressed genes in the endometrium revealed a number of biological processes and pathways potentially involved in embryo implantation in the pig, most noticeably cell proliferation, regulation of immune response, interaction of cytokine-cytokine receptors, and cell adhesion. These results showed that specific gene expression patterns reflect the different functions of the endometrium in three stages (maternal recognition, conceptus attachment, and embryo implantation). This study identified comprehensive transcriptomic profile in the porcine endometrium and thus could be a foundation for targeted studies of genes and pathways potentially involved in abnormal endometrial receptivity and embryo loss in early pregnancy. PMID:26703736

  5. Transcriptomic Analysis of the Porcine Endometrium during Embryo Implantation

    PubMed Central

    Lin, Haichao; Wang, Huaizhong; Wang, Yanping; Liu, Chang; Wang, Cheng; Guo, Jianfeng

    2015-01-01

    In pigs, successful embryo implantation is an important guarantee for producing litter size, and early embryonic loss occurring on day 12–30 of gestation critically affects the potential litter size. The implantation process is regulated by the expression of numerous genes, so comprehensive analysis of the endometrium is necessary. In this study, RNA sequencing (RNA-Seq) technology is used to analyze endometrial tissues during early pregnancy. We investigated the changes of gene expression between three stages (day 12, 18, and 25) by multiple comparisons. There were 1557, 8951, and 2345 differentially expressed genes (DEGs) revealed between the different periods of implantation. We selected several genes for validation by the use of quantitative real-time RT-PCR. Bioinformatic analysis of differentially expressed genes in the endometrium revealed a number of biological processes and pathways potentially involved in embryo implantation in the pig, most noticeably cell proliferation, regulation of immune response, interaction of cytokine-cytokine receptors, and cell adhesion. These results showed that specific gene expression patterns reflect the different functions of the endometrium in three stages (maternal recognition, conceptus attachment, and embryo implantation). This study identified comprehensive transcriptomic profile in the porcine endometrium and thus could be a foundation for targeted studies of genes and pathways potentially involved in abnormal endometrial receptivity and embryo loss in early pregnancy. PMID:26703736

  6. [Cyto B dependent and ouabain insensitive regulatory volume decrease in bicellular mouse embryo].

    PubMed

    Pogorelova, M A; Golichenkov, V A; Tarasov, A V; Pogorelova, V N; Panait, A I; Pogorelov, A G

    2012-01-01

    Mouse single-cell embryos exhibit robust Regulatory Volume Decrease (RVD). In what manner the very early mammalian embryo following zygote stage is appreciably altered by the anisotonic extracellular solution is, as yet, totally unclear. Little attention was paid to this direction since there was no way to determine the blastomere volume. This work has served to quantitatively investigate the osmotic response of bicellular mouse embryos employing Laser Scanning Microtomography (LSM) followed with three-dimensional reconstruction (3 DR). We have shown that bicellular mouse embryos in hypotonic Dulbecco's experience RVD. Embryonic cells subjected to hyposmolar exhibit rapid osmotic swelling followed by gradual shrinking back toward their original volume. The van't Hoff law defines swelling phase with the effective hydraulic conductivity of 0.3 micron x min(-1) x atm(-1). Water release during RVD in bicellular mouse embryos is abolished by Cytochalasin B (Cyto B) and the volume recovery is insensitive to ouabain treatment. PMID:22650075

  7. Post-implantation embryo culture: validation with selected compounds for teratogenicity testing.

    PubMed

    Cicurel, L; Schmid, B P

    1988-06-01

    1. Some chemical compounds selected by experts for the validation of in vitro teratogenicity testing were investigated in whole rat embryos cultured during the early stages of organogenesis. All sixteen known in vivo teratogens tested also induced specific malformations in embryos grown in culture. 2. Of the nine compounds which were negative in in vivo rat teratogenicity studies, none provoked dysmorphogenic effects in cultured embryos. Abnormal development of the embryos was only observed with these compounds at concentrations also high enough to affect significantly overall growth and/or differentiation. 3. The results showed a high predictability of this system for the compounds tested and suggest that the post-implantation embryo culture system may also be useful in the prospective testing of new drugs and environmental chemicals.

  8. Tablet-based two-dimensional measurement for estimating the embryo area of brown rice

    NASA Astrophysics Data System (ADS)

    Intaravanne, Yuttana; Sumriddetchkajorn, Sarun; Chaitavon, Kosom; Chanhorm, Sataporn; Pongsoon, Prasit; Prasertsak, Anchalee

    2014-10-01

    The embryo or germ of a rice seed is growing to the shoot and the root parts of a seedling. In the early stage, the germinated embryo directly receives food from the endosperm. How healthy of the seedling can be physically predicted by measuring the areas of the embryo and endosperm. In this work, we show for the first time how the embryo and endosperm areas of a brown rice can be spatially measured. Our key design is based on the utilization of a tablet equipped with our lens module for capturing the rice seed image under white light illumination. Our Windows-based program is developed to analyze and separate the image of the whole brown rice into the embryo and endosperm parts within 2 seconds per seed. Our tablet-based system is just 30×30×6 cm3 with 1 kilogram in weight, capable to easily carry to perform in the field.

  9. 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.

  10. Telomere elongation facilitated by trichostatin a in cloned embryos and pigs by somatic cell nuclear transfer.

    PubMed

    Kong, Qingran; Ji, Guangzhen; Xie, Bingteng; Li, Jingyu; Mao, Jian; Wang, Juan; Liu, Shichao; Liu, Lin; Liu, Zhonghua

    2014-06-01

    Telomere attrition and genomic instability are associated with organism aging. Concerns still exist regarding telomere length resetting in cloned embryos and ntES cells, and possibilities of premature aging of cloned animals achieved by somatic cell nuclear transfer (SCNT). Trichostatin A (TSA), a histone deacetylase inhibitor, effectively improves the developmental competence of cloned embryos and animals, and recently contributes to successful generation of human ntES cells by SCNT. To test the function of TSA on resetting telomere length, we analyzed telomeres in cloned blastocysts and pigs following treatment of SCNT embryos with TSA. Here, we show that telomeres of cloned pigs generated by standard SCNT methods are not effectively restored, compared with those of donor cells, however TSA significantly increases telomere lengths in cloned pigs. Telomeres elongate in cloned porcine embryos during early cleavage from one-cell to four-cell stages. Notably, TSA facilitates telomere lengthening of cloned embryos mainly at morula-blastocyst stages. Knockdown of pTert by shRNA in donor cells reduces telomerase activity in cloned blastocysts but does not abrogate telomere elongation in the TSA-treated embryos (p > 0.05). However, genes associated with recombination or telomerase-independent mechanism of alternative lengthening of telomeres (ALT) Rad50 and BLM show increased expression in TSA-treated embryos. These data suggest that TSA may promote telomere elongation of cloned porcine embryos by ALT. Together, TSA can elongate telomeres in cloned embryos and piglets, and this could be one of the mechanisms underlying improved development of cloned embryos and animals treated with TSA. PMID:24510582

  11. A chimera embryo assay reveals a decrease in embryonic cellular proliferation induced by sperm from X-irradiated male mice

    SciTech Connect

    Obasaju, M.F.; Wiley, L.M.; Oudiz, D.J.; Raabe, O.; Overstreet, J.W.

    1989-05-01

    Male mice were divided into three experimental groups and a control group. Mice in the experimental groups received one of three doses of acute X irradiation (1.73, 0.29, and 0.05 Gy) and together with the control unirradiated mice were then mated weekly to unirradiated female mice for a 9-week experimental period. Embryos were recovered from the weekly matings at the four-cell stage and examined by the chimera assay for proliferative disadvantage. Aggregation chimeras were constructed of embryos from female mice mated to irradiated males (experimental embryos) and embryos from females mated to unexposed males (control embryos) and contained either one experimental embryo and one control embryo (heterologous chimera) or two control embryos (control chimera). The control embryo in heterologous chimeras and either embryo in control chimeras were prelabeled with the vital dye fluorescein isothiocyanate (FITC), and the chimeras were cultured for 40 h and viewed under phase-contrast and epifluorescence microscopy to obtain total embryo cell number and the cellular contribution from the FITC-labeled embryo. Experimental and control embryos that were cultured singly were also examined for embryo cell number at the end of the 40-h culture period. In control chimeras, the mean ratio of the unlabeled cells:total chimera cell number (henceforth referred to as ''mean ratio'') was 0.50 with little or no weekly variation over the 9-week experimental period. During Weeks 4-7, the mean ratios of heterologous chimeras differed significantly from the mean ratio of control chimeras with the greatest differences occurring during Week 7 (0.41 for chimeras of 0.05 Gy dose group, 0.40 for chimeras of the 0.29 Gy dose group, and 0.17 for chimeras of the 1.73 Gy dose group).

  12. Histone deacetylase inhibitor improves the development and acetylation levels of cat-cow interspecies cloned embryos.

    PubMed

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

    2013-08-01

    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

  13. Histone deacetylase inhibitor improves the development and acetylation levels of cat-cow interspecies cloned embryos.

    PubMed

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

    2013-08-01

    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.

  14. Change of embryotoxic susceptibility to di-n-butyltin dichloride in cultured rat embryos.

    PubMed

    Ema, M; Iwase, T; Iwase, Y; Ohyama, N; Ogawa, Y

    1996-01-01

    Di-n-butylin dichloride (DBTCl), which is commonly used as heat and light stabilizer for polyvinyl chloride (PVC) plastics, is a teratogen in vivo. In the present study, the toxic effects were investigated of DBTCl on cultured rat embryos during three different stages of organogenesis. Rat embryos explanted on gestational day (GD) 8.5, GD 9.5, and GD 11.5 were cultured for 68, 46, and 48 h and were exposed to a range of DBTCl concentrations for the first 24, 46, and the last 46 h of culture, respectively. Significant decreases in the placental diameter at > or = 10 ng/ml and in the number of somite pairs and the morphological score at 30 ng/ml were noted in embryos cultured from GD 8.5. Significant decreases in the yolk sac diameter and the crown-rump length at 100 ng/ml, in the number of somite pairs at > or = 50 ng/ml, and in the morphological score at > or = 30 ng/ml were found in embryos cultured from GD 9.5. No adverse effects on these parameters were detected in embryos cultured from GD 11.5 even at 300 ng/ml. Dysmorphogenesis in embryos cultured from GD 8.5, GD 9.5, and GD 11.5 was observed at > or = 10, > or = 50, and 300 ng/ml, respectively. Incomplete turning and craniofacial defects in embryos cultured from GD 8.5 and GD 9.5 and defects of the forelimb buds and tail in embryos cultured from GD 11.5 were frequently observed. These results show that in vitro exposure to DBTCl interferes with normal development of embryos during three different stages of organogenesis and that susceptibility to the embryotoxicity, including the dysmorphogenic potential of DBTCl, varies with developmental stage. PMID:8896720

  15. External gills and adaptive embryo behavior facilitate synchronous development and hatching plasticity under respiratory constraint.

    PubMed

    Rogge, Jessica R; Warkentin, Karen M

    2008-11-01

    Plasticity in hatching timing allows embryos to balance egg- and larval-stage risks, and depends on the ability of hatching-competent embryos to continue developing in the egg. Hypoxia can slow development, kill embryos and induce premature hatching. For terrestrial eggs of red-eyed treefrogs, the embryonic period can extend approximately 50% longer than development to hatching competence, and development is synchronous across perivitelline oxygen levels (PO2) ranging from 0.5-16.5 kPa. Embryos maintain large external gills until hatching, then gills regress rapidly. We assessed the respiratory value of external gills using gill manipulations and closed-system respirometry. Embryos without external gills were oxygen limited in air and hatched at an external PO2 of 17 kPa, whereas embryos with gills regulated their metabolism and remained in the egg at substantially lower PO2. By contrast, tadpoles gained no respiratory benefit from external gills. We videotaped behavior and manipulated embryos to test if they position gills near the air-exposed portion of the egg surface, where PO2 is highest. Active embryos remained stationary for minutes in gills-at-surface positions. After manipulations and spontaneous movements that positioned gills in the O2-poor region of the egg, however, they returned their gills to the air-exposed surface within seconds. Even neural tube stage embryos, capable only of ciliary rotation, positioned their developing head in the region of highest PO2. Such behavior may be critical both to delay hatching after hatching competence and to obtain sufficient oxygen for normal, synchronous development at earlier stages.

  16. Factors affecting the success of a large embryo transfer program in Holstein cattle in a commercial herd in the southeast region of the United States.

    PubMed

    Ferraz, P A; Burnley, C; Karanja, J; Viera-Neto, A; Santos, J E P; Chebel, R C; Galvão, K N

    2016-10-15

    The objectives of this study were to evaluate factors affecting in vivo embryo production and pregnancy per embryo transfer (P/ET) in Holstein cattle in the southeast region of the United States. Data from a total of 516 embryo collections and 10,297 ETs performed from 2011 to 2014 were available. For embryo production, the effects of donor parity (nulliparous [N], primiparous [P], multiparous [M]), average temperature-humidity index (THI) at embryo collection, days in milk at embryo collection, occurrence of calving problems, and occurrence of metritis postpartum were evaluated. For P/ET, the effects of donor parity (N or parous), recipient parity (N, P, and M), embryo type (fresh, frozen, IVF, and IVF-frozen), embryo developmental stage (4-7), embryo quality (1-3), recipient estrous cycle day at ET (6-9), average THI at ET, days in milk at ET, milk yield at ET, occurrence of calving problems (abortion, dystocia, twins, fetal death, or retained placenta), and occurrence of metritis postpartum were evaluated. Pregnancy was diagnosed at 41 ± 3 days of gestation. Continuous and binary data were analyzed using the MIXED and GLIMMIX procedures of SAS, respectively. Parity affected embryo production; M had greater number and percentage of unfertilized embryos and lesser percentage of viable embryos than P and N. Recipient parity, embryo type, embryo stage, embryo quality, estrous cycle day at ET, and THI at ET affected P/ET. There was an interaction between recipient parity and THI at ET. P/ET was greater for N than P and greater for P than M, greater for fresh embryos than others, greater for stage 7 than others, greater for quality 1 than 2 and greater for quality 2 than 3, and greater for ET on estrous cycle Day 7 and 8 than 6. P/ET was decreased for THI ≥80 in N and THI ≥72 in P and M. Calving problems and metritis also affected P/ET in P and M and was lesser for cows that had calving problems and metritis. In conclusion, embryo production was affected by

  17. Avian embryos in hypoxic environments.

    PubMed

    León-Velarde, F; Monge-C, C

    2004-08-12

    Avian embryos at high altitude do not benefit of the maternal protection against hypoxia as in mammals. Nevertheless, avian embryos are known to hatch successfully at altitudes between 4,000 and 6,500 m. This review considers some of the processes that bring about the outstanding modifications in the pressure differences between the environment and mitochondria of avian embryos in hypoxic environments. Among species, some maintain normal levels of oxygen consumption ( VO2) have a high oxygen carrying capacity, lower the air cell-arterial pressure difference ( PAO2 - PaO2 ) with a constant pH. Other species decrease VO2, increase only slightly the oxygen carrying capacity, have a higher PAO2 - PaO2 difference than sea-level embryos and lower the PCO2 and pH. High altitude embryos, and those exposed to hypoxia have an accelerated decline of erythrocyte ATP levels during development and an earlier stimulation of 2,3-BPG synthesis. A higher Bohr effect may ensure high tissue PO2 in the presence of the high-affinity hemoglobin. Independently of the strategy used, they serve together to promote suitable rates of development and successful hatching of high altitude birds in hypoxic environments.

  18. Transgenic chicken, mice, cattle, and pig embryos by somatic cell nuclear transfer into pig oocytes.

    PubMed

    Gupta, Mukesh Kumar; Das, Ziban Chandra; Heo, Young Tae; Joo, Jin Young; Chung, Hak-Jae; Song, Hyuk; Kim, Jae-Hwan; Kim, Nam-Hyung; Lee, Hoon Taek; Ko, Dae Hwan; Uhm, Sang Jun

    2013-08-01

    This study explored the possibility of producing transgenic cloned embryos by interspecies somatic cell nuclear transfer (iSCNT) of cattle, mice, and chicken donor cells into enucleated pig oocytes. Enhanced green florescent protein (EGFP)-expressing donor cells were used for the nuclear transfer. Results showed that the occurrence of first cleavage did not differ significantly when pig, cattle, mice, or chicken cells were used as donor nuclei (p>0.05). However, the rate of blastocyst formation was significantly higher in pig (14.9±2.1%; p<0.05) SCNT embryos than in cattle (6.3±2.5%), mice (4.2±1.4%), or chicken (5.1±2.4%) iSCNT embryos. The iSCNT embryos also contained a significantly less number of cells per blastocyst than those of SCNT pig embryos (p<0.05). All (100%) iSCNT embryos expressed the EGFP gene, as evidenced by the green florescence under ultraviolet (UV) illumination. Microinjection of purified mitochondria from cattle somatic cells into pig oocytes did not have any adverse effect on their postfertilization in vitro development and embryo quality (p>0.05). Moreover, NCSU23 medium, which was designed for in vitro culture of pig embryos, was able to support the in vitro development of cattle, mice, and chicken iSCNT embryos up to the blastocyst stage. Taken together, these data suggest that enucleated pig oocytes may be used as a universal cytoplast for production of transgenic cattle, mice, and chicken embryos by iSCNT. Furthermore, xenogenic transfer of mitochondria to the recipient cytoplast may not be the cause for poor embryonic development of cattle-pig iSCNT embryos.

  19. Differences in heat tolerance between preimplantation embryos from Brahman, Romosinuano, and Angus breeds.

    PubMed

    Hernández-Cerón, J; Chase, C C; Hansen, P J

    2004-01-01

    Exposure to 41 degrees C reduces development of embryos of heat-sensitive breeds (Holstein and Angus) more than for embryos of the heat-tolerant Brahman breed. Here it was tested whether embryonic resistance to heat shock occurs for a thermotolerant breed of different genetic origin than the Brahman. In particular, the thermal sensitivity of in vitro produced embryos of the Romosinuano, a Bos taurus, Criollo-derived breed, was compared to that for in vitro produced Brahman and Angus embryos. At d 4 after insemination, embryos > or = 8 cells were randomly assigned to control (38.5 degrees C) or heat shock (41 degrees C for 6 h) treatments. Heat shock reduced the proportion of embryos that developed to the blastocyst stage on d 8 after insemination. At 38.5 degrees C, there were no significant differences in development between breeds. Among embryos exposed to 41 degrees C, however, development was lower for Angus embryos than for Brahman and Romosinuano embryos. Furthermore, an Angus vs. (Brahman + Romosinuano) x temperature interaction occurred because heat shock reduced development more in Angus (30.3 +/- 4.6% at 38.5 degrees C vs. 4.9 +/- 4.6% at 41 degrees C) than in Brahman (25.1 +/- 4.6% vs. 13.6 +/- 4.6%) and Romosinuano (28.3 +/- 4.1% vs. 17.5 +/- 4.1%). Results demonstrate that embryos from Brahman and Romosinuano breeds are more resistant to elevated temperature than embryos from Angus. Thus, the process of adaptation of Brahman and Romosinuano breeds to hot environments resulted in both cases in selection of genes controlling thermotolerance at the cellular level. PMID:14765810

  20. A Simple Method for Transportation of Mouse Embryos Using Microtubes and a Warm Box.

    PubMed

    Tokoro, Mikiko; Fukunaga, Noritaka; Yamanaka, Kaori; Itoi, Fumiaki; Terashita, Yukari; Kamada, Yuko; Wakayama, Sayaka; Asada, Yoshimasa; Wakayama, Teruhiko

    2015-01-01

    Generally, transportation of preimplantation embryos without freezing requires incubators that can maintain an optimal culture environment with a suitable gas phase, temperature, and humidity. Such incubators are expensive to transport. We reported previously that normal offspring were obtained when the gas phase and temperature could be maintained during transportation. However, that system used plastic dishes for embryo culture and is unsuitable for long-distance transport of live embryos. Here, we developed a simple low-cost embryo transportation system. Instead of plastic dishes, several types of microtubes-usually used for molecular analysis-were tested for embryo culture. When they were washed and attached to a gas-permeable film, the rate of embryo development from the 1-cell to blastocyst stage was more than 90%. The quality of these blastocysts and the rate of full-term development after embryo transfer to recipient female mice were similar to those of a dish-cultured control group. Next, we developed a small warm box powered by a battery instead of mains power, which could maintain an optimal temperature for embryo development during transport. When 1-cell embryos derived from BDF1, C57BL/6, C3H/He and ICR mouse strains were transported by a parcel-delivery service over 3 days using microtubes and the box, they developed to blastocysts with rates similar to controls. After the embryos had been transferred into recipient female mice, healthy offspring were obtained without any losses except for the C3H/He strain. Thus, transport of mouse embryos is possible using this very simple method, which might prove useful in the field of reproductive medicine.

  1. Aberrant epigenetic reprogramming of imprinted microRNA-127 and Rtl1 in cloned mouse embryos

    SciTech Connect

    Cui Xiangshun; Zhang Dingxiao; Ko, Yoeung-Gyu; Kim, Nam-Hyung

    2009-02-06

    The microRNA (miRNA) genes mir-127 and mir-136 are located near two CpG islands in the imprinted mouse retrotransposon-like gene Rtl1, a key gene involved in placenta formation. These miRNAs appear to be involved in regulating the imprinting of Rtl1. To obtain insights into the epigenetic reprogramming of cloned embryos, we compared the expression levels of mir-127 and mir-136 in fertilized mouse embryos, parthenotes, androgenotes and cloned embryos developing in vitro. We also examined the DNA methylation status of the promoter regions of Rtl1 and mir-127 in these embryos. Our data showed that mir-127 and mir-136 were highly expressed in parthenotes, but rarely expressed in androgenotes. Interestingly, the expression levels of mir-127 and mir-136 in parthenotes were almost twice that seen in the fertilized embryos, but were much lower in the cloned embryos. The Rtl1 promoter region was hyper-methylated in blastocyst stage parthenotes (75.0%), moderately methylated (32.4%) in the fertilized embryos and methylated to a much lower extent ({approx}10%) in the cloned embryos. Conversely, the promoter region of mir-127 was hypo-methylated in parthenogenetically activated embryos (0.4%), moderately methylated (30.0%) in fertilized embryos and heavily methylated in cloned blastocysts (63-70%). These data support a role for mir-127 and mir-136 in the epigenetic reprogramming of the Rtl1 imprinting process. Analysis of the aberrant epigenetic reprogramming of mir-127 and Rtl1 in cloned embryos may help to explain the nuclear reprogramming procedures that occur in donor cells following somatic cell nuclear transfer (SCNT)

  2. Feminists on the inalienability of human embryos.

    PubMed

    McLeod, Carolyn; Baylis, Francoise

    2006-01-01

    The feminist literature against the commodification of embryos in human embryo research includes an argument to the effect that embryos are "intimately connected" to persons, or morally inalienable from them. We explore why embryos might be inalienable to persons and why feminists might find this view appealing. But, ultimately, as feminists, we reject this view because it is inconsistent with full respect for women's reproductive autonomy and with a feminist conception of persons as relational, embodied beings. Overall, feminists should avoid claims about embryos' being inalienable to persons in arguments for or against the commodification of human embryos. PMID:17111554

  3. Cadence of procreation: orchestrating embryo-uterine interactions.

    PubMed

    Cha, Jeeyeon; Dey, Sudhansu K

    2014-10-01

    Embryo implantation in eutherian mammals is a highly complex process and requires reciprocal communication between different cell types of the embryo at the blastocyst stage and receptive uterus. The events of implantation are dynamic and highly orchestrated over a species-specific period of time with distinctive and overlapping expression of many genes. Delayed implantation in different species has helped elucidate some of the intricacies of implantation timing and different modes of the implantation process. How these events are coordinated in time and space are not clearly understood. We discuss potential regulators of the precise timing of these events with respect to central and local clock mechanisms. This review focuses on the timing and synchronization of early pregnancy events in mouse and consequences of their aberrations at later stages of pregnancy. PMID:24862857

  4. Improved Method for Ex Ovo-Cultivation of Developing Chicken Embryos for Human Stem Cell Xenografts

    PubMed Central

    Schomann, Timo; Qunneis, Firas; Widera, Darius; Kaltschmidt, Christian; Kaltschmidt, Barbara

    2013-01-01

    The characterization of human stem cells for the usability in regenerative medicine is particularly based on investigations regarding their differentiation potential in vivo. In this regard, the chicken embryo model represents an ideal model organism. However, the access to the chicken embryo is only achievable by windowing the eggshell resulting in limited visibility and accessibility in subsequent experiments. On the contrary, ex ovo-culture systems avoid such negative side effects. Here, we present an improved ex ovo-cultivation method enabling the embryos to survive 13 days in vitro. Optimized cultivation of chicken embryos resulted in a normal development regarding their size and weight. Our ex ovo-approach closely resembles the development of chicken embryos in ovo, as demonstrated by properly developed nervous system, bones, and cartilage at expected time points. Finally, we investigated the usability of our method for trans-species transplantation of adult stem cells by injecting human neural crest-derived stem cells into late Hamburger and Hamilton stages (HH26–HH28/E5—E6) of ex ovo-incubated embryos. We demonstrated the integration of human cells allowing experimentally easy investigation of the differentiation potential in the proper developmental context. Taken together, this ex ovo-method supports the prolonged cultivation of properly developing chicken embryos enabling integration studies of xenografted mammalian stem cells at late developmental stages. PMID:23554818

  5. 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.

  6. Development of fetuses from in vitro-produced and cloned bovine embryos.

    PubMed

    Farin, C E; Farin, P W; Piedrahita, J A

    2004-01-01

    The establishment of in vitro fertilization and culture systems for mammalian embryos has facilitated the application of embryo technologies in research, industry, and clinical settings. Furthermore, the advent of cloning by nuclear transfer has significantly enhanced the potential for genetic modification of livestock. Based on studies in cattle, sheep, and mice, it has become apparent that embryos produced using these systems can differ in morphology and developmental potential compared with embryos produced in vivo. Referred to as "large offspring syndrome," these abnormalities in the development of fetuses, placentas, and offspring are particularly evident following transfer of cloned embryos, but they also occur in pregnancies from embryos produced using in vitro culture alone. The objective of this review is to examine the effects of in vitro production and cloning on bovine embryo and fetal development. Literature pertaining to preimplantation embryo, conceptus, and fetal development, as well as gene expression occurring at each of these three stages, is reviewed. Physiologic and genetic mechanisms that contribute to large offspring syndrome also are discussed.

  7. Ribonuclease J is required for chloroplast and embryo development in Arabidopsis

    PubMed Central

    Chen, Hongyu; Zou, Wenxuan; Zhao, Jie

    2015-01-01

    Chloroplasts perform many essential metabolic functions and their proper development is critically important in embryogenesis. However, little is known about how chloroplasts function in embryogenesis and more relevant components need to be characterized. In this study, we show that Arabidopsis Ribonuclease J (RNase J) is required for chloroplast and embryo development. Mutation of AtRNJ led to albino ovules containing aborted embryos; the morphological development of rnj embryos was disturbed after the globular stage. Observation of ultrastructures indicated that these aborted embryos may result from impaired chloroplast development. Furthermore, by analyzing the molecular markers of cell fate decisions (STM, FIL, ML1, SCR, and WOX5) in rnj embryos, we found that this impairment of chloroplast development may lead to aberrant embryo patterning along the apical-basal axis, indicating that AtRNJ is important in initiating and maintaining the organization of shoot apical meristems (SAMs), cotyledons, and hypocotyls. Moreover, the transport and response of auxin in rnj embryos was found to be disrupted, suggesting that AtRNJ may be involved in auxin-mediated pathways during embryogenesis. Therefore, we speculate that RNJ plays a vital role in embryo morphogenesis and apical-basal pattern formation by regulating chloroplast development. PMID:25871650

  8. Automated microinjection of recombinant BCL-X into mouse zygotes enhances embryo development.

    PubMed

    Liu, Xinyu; Fernandes, Roxanne; Gertsenstein, Marina; Perumalsamy, Alagammal; Lai, Ingrid; Chi, Maggie; Moley, Kelle H; Greenblatt, Ellen; Jurisica, Igor; Casper, Robert F; Sun, Yu; Jurisicova, Andrea

    2011-01-01

    Progression of fertilized mammalian oocytes through cleavage, blastocyst formation and implantation depends on successful implementation of the developmental program, which becomes established during oogenesis. The identification of ooplasmic factors, which are responsible for successful embryo development, is thus crucial in designing possible molecular therapies for infertility intervention. However, systematic evaluation of molecular targets has been hampered by the lack of techniques for efficient delivery of molecules into embryos. We have developed an automated robotic microinjection system for delivering cell impermeable compounds into preimplantation embryos with a high post-injection survival rate. In this paper, we report the performance of the system on microinjection of mouse embryos. Furthermore, using this system we provide the first evidence that recombinant BCL-XL (recBCL-XL) protein is effective in preventing early embryo arrest imposed by suboptimal culture environment. We demonstrate that microinjection of recBCL-XL protein into early-stage embryos repairs mitochondrial bioenergetics, prevents reactive oxygen species (ROS) accumulation, and enhances preimplantation embryo development. This approach may lead to a possible treatment option for patients with repeated in vitro fertilization (IVF) failure due to poor embryo quality.

  9. Epigenetic and hormonal profile during maturation of Quercus Suber L. somatic embryos.

    PubMed

    Pérez, Marta; Viejo, Marcos; LaCuesta, Maite; Toorop, Peter; Cañal, María Jesús

    2015-01-15

    Somatic embryogenesis is a powerful alternative to conventional mass propagation of Quercus suber L. However, poor quality and incomplete maturation of somatic embryos restrict any application. Given that epigenetic and hormonal control govern many developmental stages, including maturation of zygotic embryos, global DNA methylation and abscisic acid (ABA) were analyzed during development and maturation of cork oak somatic embryos. Our results indicated that development of somatic embryos concurred with a decrease in 5-mdC. In contrast, endogenous ABA content showed a transient increase with a peak in immature E2 embryos denoting the onset of the maturation phase. A cold stratification phase was necessary for embryos to acquire germination ability, which coincided with a significant decrease in 5-mdC and ABA content. Immunohistochemical analyses showed that there was a specific spatial-temporal regulation during embryogenesis, particularly after the cold treatment. The acquisition of germination capacity concurred with a general low 5-mdC signal in the root meristem, while retention of the 5-mdC signal was mainly located in the shoot meristem and provascular tissues. Conversely, ABA immunolocalization was mainly located in the root and shoot apical meristems. Furthermore, a strong decrease in the ABA signal was observed in the root cap after the stratification treatment suggesting a role for the root cap during development of somatic embryos. These results suggest that, in addition to ABA, epigenetic control appears to play an important role for the correct maturation and subsequent germination of cork oak somatic embryos.

  10. Altering Intracellular pH Disrupts Development and Cellular Organization in Preimplantation Hamster Embryos

    PubMed Central

    Squirrell, Jayne M.; Lane, Michelle; Bavister, Barry D.

    2016-01-01

    In early cleavage stage hamster embryos, the inability to regulate intracellular pH (pHi) properly is associated with reduced developmental competence in vitro. The disruption of mitochondrial organization is also correlated with reduced development in vitro. To determine the relationship between pHi and the disruption of cytoplasmic organization, we examined the effects of altering pHi on hamster embryo development, mitochondrial distribution, and cytoskeletal organization. The weak base trimethylamine was used to increase pHi and was found to reduce embryo development and disrupt the perinuclear organization of mitochondria. The weak acid 5,5-dimethyl-2,4-oxazolinedione was used to decrease pHi and was also found to reduce development and disrupt the perinuclear organization of mitochondria. With either treatment, the microfilament organization was perturbed, but the microtubule cytoskeleton was not. However, the temporal progression of the disruption of mitochondrial distribution was more rapid in alkalinized embryos than acidified embryos, as revealed by two-photon imaging of living embryos. Additionally, the disruption of the microfilament network by the two treatments was not identical. The cytoplasmic disruptions observed were not due to acute toxicity of the compounds because embryos recovered developmentally when the treatment compounds were removed. These observations link ionic homeostasis, structural integrity and developmental competence in preimplantation hamster embryos. PMID:11369617

  11. Quick freezing of one-cell mouse embryos using ethylene glycol with sucrose.

    PubMed

    Rayos, A A; Takahashi, Y; Hishinuma, M; Kanagawa, H

    1992-03-01

    One-cell mouse embryos were frozen by direct plunging into liquid nitrogen (LN(2)) vapor after equilibration in 3 M ethylene glycol with 0.25 M sucrose (freezing medium) for 5 to 40 minutes. After thawing, the embryos were cultured in vitro and the effects of the equilibration period and dilution method were examined. No significant difference was observed in the in vitro survival of embryos when 0.5 or 1.0 M sucrose was used for the dilution of the cryoprotectant for each equilibration period. The highest survival rate (67.2%) was obtained when the embryos were equilibrated for 10 minutes, and the cryoprotectant diluted with either 0.5 or 1.0 M sucrose after thawing. Shorter (5 minutes) or prolonged (40 minutes) equilibration of embryos in the freezing medium yielded significantly lower survival rates. Dilution by direct transfer of the frozen-thawed embryos into PB1 resulted in lower survival rates than when 0.5 or 1.0 M sucrose was used. The in vitro development to the blastocyst stage of one-cell mouse embryos frozen after 10 minutes equilibration in the freezing medium and diluted after thawing in 0.5 M sucrose was significantly lower than the control (68.0 vs 92.7%). However, transfer of the blastocysts developing from frozen-thawed one-cell mouse embryos into the uterine horns of the recipients resulted in fetal development and implantation rates similar to the control.

  12. Buffalo and cattle hybrid embryo development is decreased by caffeine treatment during in vitro fertilization.

    PubMed

    Tatham, B G; Feehan, T; Pashen, R

    2003-02-01

    Water buffalo are renowned for difficulties in the implementation of assisted reproductive technologies, with both males and females being problematic. In this study, we used cattle oocytes to assess the effect of treatments with heparin and caffeine on buffalo spermatozoa and subsequent fertilization and embryo development in vitro. There was no significant difference between buffalo and bovine spermatozoa in the events associated with fertilization. Fertilization of cattle oocytes with buffalo spermatozoa resulted in 7.8% of oocytes developing into hybrid embryos. A difference in the developmental capability of hybrid embryos compared with the cattle control was observed. This has not been previously reported. The subsequent transfer of a limited number of hybrid embryos did not produce a viable pregnancy. However, control treatments in this experiment also failed to achieve pregnancy, so objective data is not available to provide conclusions about the developmental competence of the buffalo and cattle hybrid embryos. Optimal spermatozoa capacitation treatments achieved 61% fertilization and 21% zygote cleavage into two cell embryos. There was no significant difference in fertilization or development due to heparin or spermatozoa concentrations. However, treatment of buffalo and cattle spermatozoa with caffeine significantly decreased embryo cleavage but also tended to decrease embryo development to the blastocyst stage. These studies suggest that problems with reproduction in buffalo may reside with biological mechanisms associated with the oocyte that are often complicated by poor male reproductive performance. Selection for bull fertility would prevent some of these complications.

  13. 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.

  14. Proteomics of early zebrafish embryos

    PubMed Central

    Link, Vinzenz; Shevchenko, Andrej; Heisenberg, Carl-Philipp

    2006-01-01

    Background Zebrafish (D. rerio) has become a powerful and widely used model system for the analysis of vertebrate embryogenesis and organ development. While genetic methods are readily available in zebrafish, protocols for two dimensional (2D) gel electrophoresis and proteomics have yet to be developed. Results As a prerequisite to carry out proteomic experiments with early zebrafish embryos, we developed a method to efficiently remove the yolk from large batches of embryos. This method enabled high resolution 2D gel electrophoresis and improved Western blotting considerably. Here, we provide detailed protocols for proteomics in zebrafish from sample preparation to mass spectrometry (MS), including a comparison of databases for MS identification of zebrafish proteins. Conclusion The provided protocols for proteomic analysis of early embryos enable research to be taken in novel directions in embryogenesis. PMID:16412219

  15. Pine somatic embryogenesis using zygotic embryos as explants.

    PubMed

    Pullman, Gerald S; Bucalo, Kylie

    2011-01-01

    Somatic embryogenesis (SE) has the potential to be the lowest-cost method to rapidly produce large numbers of high-value somatic seedlings with desired characteristics for plantation forestry. At least 24 of the 115-120 known Pinus species can undergo SE. Initiation for most species works best with immature megagametophytes as starting material, although a few pines can initiate SE cultures from isolated mature seed embryos. Successful initiation depends heavily on explant type, embryo developmental stage, and medium salt base. Most first reports of initiation used 2,4-D and BAP or a combination of cytokinins. More recent reports have optimized initiation for many Pinus spp., but still use mostly the combinations of auxin and cytokinins. Initiation can be stimulated with medium supplements including abscisic acid (ABA), brassinosteroids, ethylene inhibitors, gibberellin inhibitors, organic acids, putrescine, specific sugar types (maltose, galactose, D-chiro-inositol, and D-xylose), triacontanol, vitamins (B12, biotin, vitamin E, and folic acid), or manipulation of environmental factors including pH, water potential, cone cold storage, gelling agent concentration, and liquid medium. Embryo development and maturation usually occur best on medium containing ABA along with water potential reduction (with sugars and polyethylene glycol) or water availability reduction (with raised gelling agent increasing gel-strength). Activated carbon and maltose may also improve embryo maturation. The main issues holding SE technology back are related to the high cost of producing a somatic seedling, incurred from low initiation percentages for recalcitrant species, culture loss, and decline after initiation and poor embryo maturation resulting in no or poor germination. Although vast progress has been made in pine SE technology over the past 24 years, fundamental studies on seed and embryo physiology, biochemistry, and gene expression are still needed to help improve the technology

  16. Osmoregulatory response to low salinities in the European sea bass embryos: a multi-site approach.

    PubMed

    Sucré, Elliott; Bossus, Maryline; Bodinier, Charlotte; Boulo, Viviane; Charmantier, Guy; Charmantier-Daures, Mireille; Cucchi, Patricia

    2013-01-01

    Embryonic osmoregulation effected by embryonic ionocytes in the European sea bass Dicentrarchus labrax has been studied at several sites, including the yolk sac membrane, the first gill slits and the gut ionocytes. D. labrax embryos, spawned in seawater (SW) (39 ‰), were exposed to dilute seawater (DSW) (5 ‰) during 48 h, from stage 10 pairs of somites (10S) to hatching time (HT). Control embryos originating from the same spawn were maintained in SW. Both SW and DSW embryos were examined after 24- and 48-h exposure. Nanoosmometric measurements of the embryonic fluids osmolality suggest that late embryos are confronted with the variations in external salinity and that they were able to slightly regulate their osmolality. Immunolocalization of Na⁺/K⁺ ATPase, NKCC and CFTR has shown that DSW-exposed embryos can limit ion losses due to compensatory physiological mechanisms. CFTR and NKCC were not observed in DSW embryos in the yolk sac ionocytes and in the tegumentary ionocytes of the gill slits. The quantification of mRNA indicated that NKA, NKCC1 and CFTR transcript levels increased from stage 10S to stage HT. At stage HT, following 48 h of DSW- or SW-exposure, different responses were observed according to salinity. These results, when compared to those obtained in D. labrax juveniles and adults long-term exposed to fresh water (FW), show that in embryos the physiological response following a short-term DSW exposure is different. The mechanisms of hyper-osmoregulation observed in D. labrax embryos, although not fully efficient, allow their survival for several days in DSW.

  17. Locomotor behavior of fish hatched from embryos exposed to flight conditions

    NASA Technical Reports Server (NTRS)

    Kleerekoper, H.

    1978-01-01

    Embryos of Fundulus heteroclitus in various stages of development were exposed to space flight conditions aboard Apollo spacecraft and Cosmos satellites. The objective of the study was to ascertain whether fish hatched from these embryos displayed locomotor behavior different from that of control fish of the same age. An electronic monitoring technique was used to record behavior. Results indicate no change in locomotor behavior in fish on Apollo Spacecraft, but inexplicable significant changes were noted in fish aboard Cosmos Satellites.

  18. Spermatozoa telomeres determine telomere length in early embryos and offspring.

    PubMed

    de Frutos, C; López-Cardona, A P; Fonseca Balvís, N; Laguna-Barraza, R; Rizos, D; Gutierrez-Adán, A; Bermejo-Álvarez, P

    2016-01-01

    Offspring telomere length (TL) has been correlated with paternal TL, but the mechanism for this parent of origin-specific inheritance remains unclear. The objective of this study has been to determine the role of spermatozoa TL in embryonic telomere lengthening by using two mouse models showing dimorphism in their spermatozoa TL: Mus musculus vs Mus spretus and old vs young Mus musculus. Mus spretus spermatozoa displayed a shorter TL than Mus musculus. Hybrid offspring exhibited lower TL compared with Mus musculus starting at the two-cell stage, before the onset of telomerase expression. To analyze the role of spermatozoa telomeres in early telomere lengthening, we compared the TL in oocytes, zygotes, two-cell embryos and blastocysts produced by parthenogenesis or by fertilization with Mus musculus or Mus spretus spermatozoa. TL was significantly higher in spermatozoa compared with oocytes, and it increased significantly from the oocyte to the zygote stage in those embryos fertilized with Mus musculus spermatozoa, but not in those fertilized with Mus spretus spermatozoa or produced by parthenogenesis. A further increase was noted from the zygote to the two-cell stage in fertilized Mus musculus embryos, whereas hybrid embryos maintained the oocyte TL. Spermatozoa TL shortened with age in Mus musculus and the offspring from young males showed a significantly higher TL compared with that fathered by old males. These significant differences were already noticeable at the two-cell stage. These results suggest that spermatozoa telomeres act as a guide for telomerase-independent telomere lengthening resulting in differences in TL that persist after birth.

  19. The DNA methylation landscape of human early embryos.

    PubMed

    Guo, Hongshan; Zhu, Ping; Yan, Liying; Li, Rong; Hu, Boqiang; Lian, Ying; Yan, Jie; Ren, Xiulian; Lin, Shengli; Li, Junsheng; Jin, Xiaohu; Shi, Xiaodan; Liu, Ping; Wang, Xiaoye; Wang, Wei; Wei, Yuan; Li, Xianlong; Guo, Fan; Wu, Xinglong; Fan, Xiaoying; Yong, Jun; Wen, Lu; Xie, Sunney X; Tang, Fuchou; Qiao, Jie

    2014-07-31

    DNA methylation is a crucial element in the epigenetic regulation of mammalian embryonic development. However, its dynamic patterns have not been analysed at the genome scale in human pre-implantation embryos due to technical difficulties and the scarcity of required materials. Here we systematically profile the methylome of human early embryos from the zygotic stage through to post-implantation by reduced representation bisulphite sequencing and whole-genome bisulphite sequencing. We show that the major wave of genome-wide demethylation is complete at the 2-cell stage, contrary to previous observations in mice. Moreover, the demethylation of the paternal genome is much faster than that of the maternal genome, and by the end of the zygotic stage the genome-wide methylation level in male pronuclei is already lower than that in female pronuclei. The inverse correlation between promoter methylation and gene expression gradually strengthens during early embryonic development, reaching its peak at the post-implantation stage. Furthermore, we show that active genes, with the trimethylation of histone H3 at lysine 4 (H3K4me3) mark at the promoter regions in pluripotent human embryonic stem cells, are essentially devoid of DNA methylation in both mature gametes and throughout pre-implantation development. Finally, we also show that long interspersed nuclear elements or short interspersed nuclear elements that are evolutionarily young are demethylated to a milder extent compared to older elements in the same family and have higher abundance of transcripts, indicating that early embryos tend to retain higher residual methylation at the evolutionarily younger and more active transposable elements. Our work provides insights into the critical features of the methylome of human early embryos, as well as its functional relation to the regulation of gene expression and the repression of transposable elements.

  20. Spermatozoa telomeres determine telomere length in early embryos and offspring.

    PubMed

    de Frutos, C; López-Cardona, A P; Fonseca Balvís, N; Laguna-Barraza, R; Rizos, D; Gutierrez-Adán, A; Bermejo-Álvarez, P

    2016-01-01

    Offspring telomere length (TL) has been correlated with paternal TL, but the mechanism for this parent of origin-specific inheritance remains unclear. The objective of this study has been to determine the role of spermatozoa TL in embryonic telomere lengthening by using two mouse models showing dimorphism in their spermatozoa TL: Mus musculus vs Mus spretus and old vs young Mus musculus. Mus spretus spermatozoa displayed a shorter TL than Mus musculus. Hybrid offspring exhibited lower TL compared with Mus musculus starting at the two-cell stage, before the onset of telomerase expression. To analyze the role of spermatozoa telomeres in early telomere lengthening, we compared the TL in oocytes, zygotes, two-cell embryos and blastocysts produced by parthenogenesis or by fertilization with Mus musculus or Mus spretus spermatozoa. TL was significantly higher in spermatozoa compared with oocytes, and it increased significantly from the oocyte to the zygote stage in those embryos fertilized with Mus musculus spermatozoa, but not in those fertilized with Mus spretus spermatozoa or produced by parthenogenesis. A further increase was noted from the zygote to the two-cell stage in fertilized Mus musculus embryos, whereas hybrid embryos maintained the oocyte TL. Spermatozoa TL shortened with age in Mus musculus and the offspring from young males showed a significantly higher TL compared with that fathered by old males. These significant differences were already noticeable at the two-cell stage. These results suggest that spermatozoa telomeres act as a guide for telomerase-independent telomere lengthening resulting in differences in TL that persist after birth. PMID:26475708

  1. The moral status of the human embryo: a tradition recalled.

    PubMed

    Dunstan, G R

    1984-03-01

    An Anglican theologian contends that the claim to absolute protection for the human embryo from the moment of conception is a recent one in the Roman Catholic moral tradition. Citing pre-classical, classical, biblical, and post-biblical Roman Catholic sources, Dunstan argues that these traditions attempted to grade the protection provided to the nascent human being according to the stages of its development.

  2. A chick embryo with a yet unclassified type of cephalothoracopagus malformation and a hypothesis for explaining its genesis.

    PubMed

    Maurer, B; Geyer, S H; Weninger, W J

    2013-06-01

    Cephalothoracopagus embryos are conjoined twins, who share parts of their heads, necks and bodies. Our study aims at presenting a detailed morphological analysis of a cephalothoracopagus chick embryo of developmental stage 31. Because none of the existing theories can explain the genesis of the phenotype of this embryo, we also suggest a hypothesis, which explains it. Beside the cephalothoracopagus embryo, we investigated five control embryos. With the aid of the high-resolution episcopic microscopy (HREM) technique, we created digital volume data and three-dimensional (3D) computer models of the organs and arteries of the embryos. We used the 3D models for topological analysis and for measuring the diameters of the great intrathoracic arteries. The malformed embryo showed two body backs, each containing a notochord, spinal cord and dorsal aorta. The body backs continued into separated lower bodies. The embryo had a single, four-chambered heart, single respiratory tract and single upper alimentary tract. The topology of the pharyngeal arch arteries was normal, and the diameters of these arteries were similar to that of the control embryos. We classified the embryo we investigated as a yet unknown malformation and suggest a hypothesis explaining its genesis. PMID:22971166

  3. A chick embryo with a yet unclassified type of cephalothoracopagus malformation and a hypothesis for explaining its genesis.

    PubMed

    Maurer, B; Geyer, S H; Weninger, W J

    2013-06-01

    Cephalothoracopagus embryos are conjoined twins, who share parts of their heads, necks and bodies. Our study aims at presenting a detailed morphological analysis of a cephalothoracopagus chick embryo of developmental stage 31. Because none of the existing theories can explain the genesis of the phenotype of this embryo, we also suggest a hypothesis, which explains it. Beside the cephalothoracopagus embryo, we investigated five control embryos. With the aid of the high-resolution episcopic microscopy (HREM) technique, we created digital volume data and three-dimensional (3D) computer models of the organs and arteries of the embryos. We used the 3D models for topological analysis and for measuring the diameters of the great intrathoracic arteries. The malformed embryo showed two body backs, each containing a notochord, spinal cord and dorsal aorta. The body backs continued into separated lower bodies. The embryo had a single, four-chambered heart, single respiratory tract and single upper alimentary tract. The topology of the pharyngeal arch arteries was normal, and the diameters of these arteries were similar to that of the control embryos. We classified the embryo we investigated as a yet unknown malformation and suggest a hypothesis explaining its genesis.

  4. Integrin beta 8 (ITGB8) regulates embryo implantation potentially via controlling the activity of TGF-B1 in mice.

    PubMed

    Kumar, Vijay; Maurya, Vineet Kumar; Joshi, Anubha; Meeran, Syed Musthapa; Jha, Rajesh Kumar

    2015-04-01

    Integrins (ITGs) are mediators of cell-cell and cell-matrix interactions, which are also associated with embryo implantation processes by controlling the interaction of blastocyst with endometrium. During early pregnancy, ITGbeta8 (ITGB8) has been shown to interact with latent transforming growth factor (TGF) beta 1 (TGFB1) at the fetomaternal interface. However, the precise role of ITGB8 in the uterus and its association with embryo implantation has not been elucidated. Therefore, we attempted to ascertain the role of ITGB8 during the window of embryo implantation process by inhibiting its function or protein expression. Uterine plasma membrane-anchored ITGB8 was augmented at peri-implantation and postimplantation stages. A similar pattern of mRNA expression was also found during the embryo implantation period. An immunolocalization study revealed the presence of ITGB8 on luminal epithelial cells along with mild expression on the stromal cells throughout the implantation period studied; however, an intense fluorescence was noted only during the peri- and postimplantation stages. Bioneutralization and mRNA silencing of the uterine Itgb8 at preimplantation stage reduced the rate/frequency of embryo implantation and subsequent pregnancy, suggesting its indispensable role during the embryo implantation period. ITGB8 can also regulate the liberation of active TGFB1 from its latent complex, which, in turn, acts on SMAD2/3 phosphorylation (activation) in the uterus during embryo implantation. This indicates involvement of ITGB8 in the embryo implantation process through regulation of activation of TGFB1. PMID:25788663

  5. Pollination and embryo development in Brassica rapa L. in microgravity.

    PubMed

    Kuang, A; Popova, A; Xiao, Y; Musgrave, M E

    2000-03-01

    Plant reproduction under spaceflight conditions has been problematic in the past. In order to determine what aspect of reproductive development is affected by microgravity, we studied pollination and embryo development in Brassica rapa L. during 16 d in microgravity on the space shuttle (STS-87). Brassica is self-incompatible and requires mechanical transfer of pollen. Short-duration access to microgravity during parabolic flights on the KC-135A aircraft was used initially to confirm that equal numbers of pollen grains could be collected and transferred in the absence of gravity. Brassica was grown in the Plant Growth Facility flight hardware as follows. Three chambers each contained six plants that were 13 d old at launch. As these plants flowered, thin colored tape was used to indicate the date of hand pollination, resulting in silique populations aged 8-15 d postpollination at the end of the 16-d mission. The remaining three chambers contained dry seeds that germinated on orbit to produce 14-d-old plants just beginning to flower at the time of landing. Pollen produced by these plants had comparable viability (93%) with that produced in the 2-d-delayed ground control. Matched-age siliques yielded embryos of equivalent developmental stage in the spaceflight and ground control treatments. Carbohydrate and protein storage reserves in the embryos, assessed by cytochemical localization, were also comparable. In the spaceflight material, growth and development by embryos rescued from siliques 15 d after pollination lagged behind the ground controls by 12 d; however, in the subsequent generation, no differences between the two treatments were found. The results demonstrate that while no stage of reproductive development in Brassica is absolutely dependent upon gravity, lower embryo quality may result following development in microgravity.

  6. Pollination and embryo development in Brassica rapa L. in microgravity

    NASA Technical Reports Server (NTRS)

    Kuang, A.; Popova, A.; Xiao, Y.; Musgrave, M. E.

    2000-01-01

    Plant reproduction under spaceflight conditions has been problematic in the past. In order to determine what aspect of reproductive development is affected by microgravity, we studied pollination and embryo development in Brassica rapa L. during 16 d in microgravity on the space shuttle (STS-87). Brassica is self-incompatible and requires mechanical transfer of pollen. Short-duration access to microgravity during parabolic flights on the KC-135A aircraft was used initially to confirm that equal numbers of pollen grains could be collected and transferred in the absence of gravity. Brassica was grown in the Plant Growth Facility flight hardware as follows. Three chambers each contained six plants that were 13 d old at launch. As these plants flowered, thin colored tape was used to indicate the date of hand pollination, resulting in silique populations aged 8-15 d postpollination at the end of the 16-d mission. The remaining three chambers contained dry seeds that germinated on orbit to produce 14-d-old plants just beginning to flower at the time of landing. Pollen produced by these plants had comparable viability (93%) with that produced in the 2-d-delayed ground control. Matched-age siliques yielded embryos of equivalent developmental stage in the spaceflight and ground control treatments. Carbohydrate and protein storage reserves in the embryos, assessed by cytochemical localization, were also comparable. In the spaceflight material, growth and development by embryos rescued from siliques 15 d after pollination lagged behind the ground controls by 12 d; however, in the subsequent generation, no differences between the two treatments were found. The results demonstrate that while no stage of reproductive development in Brassica is absolutely dependent upon gravity, lower embryo quality may result following development in microgravity.

  7. Single-embryo transfer versus multiple-embryo transfer.

    PubMed

    Gerris, Jan

    2009-01-01

    Despite the progress made in assisted reproductive technology, live birth rates remain disappointingly low. Multiple-embryo transfer has been an accepted practice with which to increase the success rate. This has led to a higher incidence of multiple-order births compared with natural conception, which not only increase the risk of mortality and morbidity to both mother and children but are also associated with social and economic consequences. Elective single-embryo transfer (eSET) was developed in an effort to increase singleton pregnancies in assisted reproduction. Studies comparing eSET with multiple-embryo transfer highlight the benefit of this approach and suggest that, with careful patient selection and the transfer of good-quality embryos, the risk of a multiple-order pregnancy can be reduced without significantly decreasing live birth rates. Although the use of eSET has gradually increased in clinical practice, its acceptance has been limited by factors such as availability of funding and awareness of the procedure. An open discussion of eSET is warranted in an effort to enable a broader understanding by physicians and patients of the merits of this approach. Ultimately, eSET may provide a more cost-effective, potentially safer approach to patients undergoing assisted reproduction technology. PMID:19406034

  8. Single-embryo transfer versus multiple-embryo transfer.

    PubMed

    Gerris, Jan

    2009-01-01

    Despite the progress made in assisted reproductive technology, live birth rates remain disappointingly low. Multiple-embryo transfer has been an accepted practice with which to increase the success rate. This has led to a higher incidence of multiple-order births compared with natural conception, which not only increase the risk of mortality and morbidity to both mother and children but are also associated with social and economic consequences. Elective single-embryo transfer (eSET) was developed in an effort to increase singleton pregnancies in assisted reproduction. Studies comparing eSET with multiple-embryo transfer highlight the benefit of this approach and suggest that, with careful patient selection and the transfer of good-quality embryos, the risk of a multiple-order pregnancy can be reduced without significantly decreasing live birth rates. Although the use of eSET has gradually increased in clinical practice, its acceptance has been limited by factors such as availability of funding and awareness of the procedure. An open discussion of eSET is warranted in an effort to enable a broader understanding by physicians and patients of the merits of this approach. Ultimately, eSET may provide a more cost-effective, potentially safer approach to patients undergoing assisted reproduction technology.

  9. 9 CFR 98.16 - The embryo collection unit.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... CERTAIN ANIMAL EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth Disease Exists § 98.16 The embryo collection unit. Ruminant and swine embryos may...

  10. 9 CFR 98.16 - The embryo collection unit.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... CERTAIN ANIMAL EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth Disease Exists § 98.16 The embryo collection unit. Ruminant and swine embryos may...

  11. 9 CFR 98.16 - The embryo collection unit.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... CERTAIN ANIMAL EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth Disease Exists § 98.16 The embryo collection unit. Ruminant and swine embryos may...

  12. 9 CFR 98.16 - The embryo collection unit.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... CERTAIN ANIMAL EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth Disease Exists § 98.16 The embryo collection unit. Ruminant and swine embryos may...

  13. 9 CFR 98.16 - The embryo collection unit.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... CERTAIN ANIMAL EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth Disease Exists § 98.16 The embryo collection unit. Ruminant and swine embryos may...

  14. The transcriptomic architecture of mouse Sertoli cell clone embryos reveals temporal–spatial-specific reprogramming.

    PubMed

    Cao, Feng; Fukuda, Atsushi; Watanabe, Hiroshi; Kono, Tomohiro

    2013-03-01

    Somatic cell nuclear transfer, a technique used to generate clone embryos by transferring the nucleus of a somatic cell into an enucleated oocyte, is an excellent approach to study the reprogramming of the nuclei of differentiated cells. Here, we conducted a transcriptomic study by performing microarray analysis on single Sertoli cell nuclear transfer (SeCNT) embryos throughout preimplantation development. The extensive data collected from the oocyte to the blastocyst stage helped to identify specific genes that were incorrectly reprogrammed at each stage, thereby providing a novel perspective for understanding reprogramming progression in SeCNT embryos.This attempt provided an opportunity to discuss the possibility that ectopic gene expression could be involved in the developmental failure of SeCNT embryos. Network analysis at each stage suggested that in total, 127 networks were involved in developmental and functional disorders in SeCNT embryos. Furthermore, chromosome mapping using our time-lapse expression data highlighted temporal–spatial changes of the abnormal expression, showing the characteristic distribution of the genes on each chromosome.Thus, the present study revealed that the preimplantation development of SeCNT embryos appears normal; however, the progression of incorrect reprogramming is concealed throughout development.

  15. De novo DNA methylation of the paternal genome in 2-cell mouse embryos.

    PubMed

    Ma, X S; Wang, X G; Qin, L; Song, C L; Lin, F; Song, J M; Zhu, C C; Liu, H L

    2014-10-27

    The developmental dynamics of DNA methylation events have been well studied. Active demethylation of the paternal genome occurs in the zygote, passive demethylation occurs during cleavage stages, and de novo methylation occurs by the blastocyst stage. It is believed that the paternal genome has lower levels of methylation during early development than the maternal genome. However, in this study, we provide direct and indirect evidence of genome-wide de novo DNA methylation of the paternal genome after the first cell cycle in mouse embryos. Although very little methylation was detected within the male pronucleus in zygotes, an intense methylation signal was clearly visible within the androgenetic 2-cell embryos. Moreover, the DNA methylation level of the paternal genome in the post-zygotic metaphase embryos was similar to that of the maternal genome. Using indirect immunofluorescence with an antibody to methylated lysine 9 in histone H3, we provided new evidence to support the concept of spatial compartmentalization of parental genomes in 2-cell mouse embryos. Nevertheless, the transient segregation of parental genomes was not observed by determining the DNA methylation distribution in the 2-cell embryos even though DNA methylation asymmetry between the maternal and paternal pronucleus existed in the 1-cell stage. The disappearance of separate immunofluorescence signals of 5-methyl cytosine in the 2-cell embryos might be attributed to the de novo methylation of the paternal genome during the first mitotic cycle.

  16. Telomere-to-centromere ratio of bovine clones, embryos, gametes, fetal cells, and adult cells.

    PubMed

    Meerdo, Lora N; Reed, William A; White, Kenneth L

    2005-01-01

    In 1997, Dolly, the first animal cloned from an adult cell, was born. It was announced in 1999 that Dolly might be aging faster than normal because her telomeres were shorter than age-matched control sheep. Telomeres, a repeated DNA sequence located at the ends of linear chromosomes, allow for base pair loss during DNA replication. Telomere shortening acts as a "mitotic clock," leading to replicative senescence. By using whole cell lysate and slot-blot analysis, we determined the telomere-to-centromere ratio (T/C) for bovine gametes, embryos, fetal tissues (brain, heart, lung, kidney, uterus, ovary, and skin), adult donor cells, and cloned embryos. Our data indicates a consistency in T/C among the various fetal tissues. The T/C of sperm is significantly lower than in oocytes. The T/C decreases from the oocyte to the 2-8-cell stage embryo, increases dramatically at the morula stage, and decreases at the blastocyst stage. Our data shows no significant difference in T/C between cloned embryos and in vitro fertilized (IVF) embryos, but there is a significant difference between cloned embryos and adult donor cells. In conclusion, the enucleated bovine oocyte has the ability to reestablish the telomere length of adult somatic cell donor nuclei. PMID:15996118

  17. Embryo splitting: a role in infertility?

    PubMed

    Wood, C

    2001-01-01

    Embryo splitting may be used to increase the potential fertility of couples requiring IVF. Using cattle as a model, it is possible to increase pregnancy rates from 70% per transfer of good quality in-vivo-produced embryos, to 110% by transferring the two demi-embryos resulting from the bisection of one embryo. The 30-40% greater chance of conception would reduce costs for the government, health authorities and patients, and reduce stress, time and complications for women having IVF treatment. Embryo splitting may also provide donor embryos for infertile couples that cannot conceive naturally or with IVF. The shortage of children for adoption and donor embryos may be overcome by the production of demi-embryos.

  18. Combined transcriptome and proteome analysis identifies pathways and markers associated with the establishment of rapeseed microspore-derived embryo development.

    PubMed

    Joosen, Ronny; Cordewener, Jan; Supena, Ence Darmo Jaya; Vorst, Oscar; Lammers, Michiel; Maliepaard, Chris; Zeilmaker, Tieme; Miki, Brian; America, Twan; Custers, Jan; Boutilier, Kim

    2007-05-01

    Microspore-derived embryo (MDE) cultures are used as a model system to study plant cell totipotency and as an in vitro system to study embryo development. We characterized and compared the transcriptome and proteome of rapeseed (Brassica napus) MDEs from the few-celled stage to the globular/heart stage using two MDE culture systems: conventional cultures in which MDEs initially develop as unorganized clusters that usually lack a suspensor, and a novel suspensor-bearing embryo culture system in which the embryo proper originates from the distal cell of a suspensor-like structure and undergoes the same ordered cell divisions as the zygotic embryo. Improved histodifferentiation of suspensor-bearing MDEs suggests a new role for the suspensor in driving embryo cell identity and patterning. An MDE culture cDNA array and two-dimensional gel electrophoresis and protein sequencing were used to compile global and specific expression profiles for the two types of MDE cultures. Analysis of the identities of 220 candidate embryo markers, as well as the identities of 32 sequenced embryo up-regulated protein spots, indicate general roles for protein synthesis, glycolysis, and ascorbate metabolism in the establishment of MDE development. A collection of 135 robust markers for the transition to MDE development was identified, a number of which may be coregulated at the gene and protein expression level. Comparison of the expression profiles of preglobular-stage conventional MDEs and suspensor-bearing MDEs identified genes whose differential expression may reflect improved histodifferentiation of suspensor-bearing embryos. This collection of early embryo-expressed genes and proteins serves as a starting point for future marker development and gene function studies aimed at understanding the molecular regulation of cell totipotency and early embryo development in plants.

  19. Combined transcriptome and proteome analysis identifies pathways and markers associated with the establishment of rapeseed microspore-derived embryo development.

    PubMed

    Joosen, Ronny; Cordewener, Jan; Supena, Ence Darmo Jaya; Vorst, Oscar; Lammers, Michiel; Maliepaard, Chris; Zeilmaker, Tieme; Miki, Brian; America, Twan; Custers, Jan; Boutilier, Kim

    2007-05-01

    Microspore-derived embryo (MDE) cultures are used as a model system to study plant cell totipotency and as an in vitro system to study embryo development. We characterized and compared the transcriptome and proteome of rapeseed (Brassica napus) MDEs from the few-celled stage to the globular/heart stage using two MDE culture systems: conventional cultures in which MDEs initially develop as unorganized clusters that usually lack a suspensor, and a novel suspensor-bearing embryo culture system in which the embryo proper originates from the distal cell of a suspensor-like structure and undergoes the same ordered cell divisions as the zygotic embryo. Improved histodifferentiation of suspensor-bearing MDEs suggests a new role for the suspensor in driving embryo cell identity and patterning. An MDE culture cDNA array and two-dimensional gel electrophoresis and protein sequencing were used to compile global and specific expression profiles for the two types of MDE cultures. Analysis of the identities of 220 candidate embryo markers, as well as the identities of 32 sequenced embryo up-regulated protein spots, indicate general roles for protein synthesis, glycolysis, and ascorbate metabolism in the establishment of MDE development. A collection of 135 robust markers for the transition to MDE development was identified, a number of which may be coregulated at the gene and protein expression level. Comparison of the expression profiles of preglobular-stage conventional MDEs and suspensor-bearing MDEs identified genes whose differential expression may reflect improved histodifferentiation of suspensor-bearing embryos. This collection of early embryo-expressed genes and proteins serves as a starting point for future marker development and gene function studies aimed at understanding the molecular regulation of cell totipotency and early embryo development in plants. PMID:17384159

  20. Studying bovine early embryo transcriptome by microarray.

    PubMed

    Dufort, Isabelle; Robert, Claude; Sirard, Marc-André

    2015-01-01

    Microarrays represent a significant advantage when studying gene expression in early embryo because they allow for a speedy study of a large number of genes even if the sample of interest contains small quantities of genetic material. Here we describe the protocols developed by the EmbryoGENE Network to study the bovine transcriptome in early embryo using a microarray experimental design.

  1. Gray level Co-occurrence Matrices (GLCM) to assess microstructural and textural changes in pre-implantation embryos.

    PubMed

    Tan, Tiffany C Y; Ritter, Lesley J; Whitty, Annie; Fernandez, Renae C; Moran, Lisa J; Robertson, Sarah A; Thompson, Jeremy G; Brown, Hannah M

    2016-08-01

    The preimplantation embryo is extraordinarily sensitive to environmental signals and events such that perturbations can alter embryo metabolism and program an altered developmental trajectory, ultimately affecting the phenotype of the adult individual; indeed, the physical environment associated with in vitro embryo culture can attenuate development. Defining the underlying metabolic changes and mechanisms, however, has been limited by the imaging technology used to evaluate metabolites and structural features in the embryo. Here, we assessed the impact of in vitro fertilization and culture on mouse embryos using three metabolic markers: peroxyfluor 1 (a reporter of hydrogen peroxide), monochlorobimane (a reporter of glutathione), and Mitotracker Deep Red (a marker of mitochondria). We also evaluated the distribution pattern of histone 2AX gamma (γH2AX) in the nuclei of 2- and 8-cell embryos and blastocysts to investigate the degree of DNA damage caused by in vitro embryo culture. In vitro-fertilized embryos, in vivo-developed embryos, and in vivo-fertilized embryos recovered and cultured in vitro were compared at the 2-, 8-cell, and blastocyst stages. In addition to assessments based on fluorescence intensity, textural analysis using Gray Level Co-occurrence Matrix (GLCM), a statistical approach that assesses texture within an image, was used to evaluate peroxyfluor 1, monochlorobimane, and Mitotracker Deep Red staining in an effort to develop a robust metric of embryo quality. Our data provide strong evidence of modified metabolic parameters identifiable as altered fluorescence texture in embryos developed in vitro. Thus, texture-analysis approach may provide a means of gaining additional insight into embryo programming beyond conventional measurements of staining intensity for metabolic markers. Mol. Reprod. Dev. 83: 701-713, 2016 © 2016 Wiley Periodicals, Inc. PMID:27409576

  2. Embryo adoption: Some further considerations.

    PubMed

    Patterson, Colin

    2015-02-01

    Recent discussions of embryo adoption have sought to make sense of the teaching of the Congregation for the Doctrine of the Faith (CDF) document Dignitas personae which appeared to provide a negative judgment on such a practice. This article aims to provide a personalist account of the process of fertilization and implantation that might serve as the basis for the negative judgment of the CDF document. In doing so, it relies upon the idea that a person, including an embryo, is not to be considered in isolation, but always in relation to God and to others. This approach extends the substantialist conceptualizations commonly employed in discussions of this issue. More generally, the article seeks to highlight the value of a personalist re-framing for an understanding of the moral questions surrounding the beginning of life. Lay summary: This article seeks to make sense of what appears to be a clear-cut rejection, set out in the Congregation for the Doctrine of the Faith (CDF) document Dignitas personae, of the proposal for women to "adopt" surplus frozen embryos. It draws upon more recently developed modes of philosophical/theological reasoning to argue that, in human procreation, both fertilization and implantation represent constitutive dimensions of divine creative activity and so must be protected from manipulative technological intervention. Since embryo adoption requires this kind of technology, it makes sense for the Church document not to approve it. PMID:25698841

  3. Embryo adoption: Some further considerations

    PubMed Central

    Patterson, Colin

    2015-01-01

    Recent discussions of embryo adoption have sought to make sense of the teaching of the Congregation for the Doctrine of the Faith (CDF) document Dignitas personae which appeared to provide a negative judgment on such a practice. This article aims to provide a personalist account of the process of fertilization and implantation that might serve as the basis for the negative judgment of the CDF document. In doing so, it relies upon the idea that a person, including an embryo, is not to be considered in isolation, but always in relation to God and to others. This approach extends the substantialist conceptualizations commonly employed in discussions of this issue. More generally, the article seeks to highlight the value of a personalist re-framing for an understanding of the moral questions surrounding the beginning of life. Lay summary: This article seeks to make sense of what appears to be a clear-cut rejection, set out in the Congregation for the Doctrine of the Faith (CDF) document Dignitas personae, of the proposal for women to “adopt” surplus frozen embryos. It draws upon more recently developed modes of philosophical/theological reasoning to argue that, in human procreation, both fertilization and implantation represent constitutive dimensions of divine creative activity and so must be protected from manipulative technological intervention. Since embryo adoption requires this kind of technology, it makes sense for the Church document not to approve it. PMID:25698841

  4. Untwisting the Caenorhabditis elegans embryo

    PubMed Central

    Christensen, Ryan Patrick; Bokinsky, Alexandra; Santella, Anthony; Wu, Yicong; Marquina-Solis, Javier; Guo, Min; Kovacevic, Ismar; Kumar, Abhishek; Winter, Peter W; Tashakkori, Nicole; McCreedy, Evan; Liu, Huafeng; McAuliffe, Matthew; Mohler, William; Colón-Ramos, Daniel A; Bao, Zhirong; Shroff, Hari

    2015-01-01

    The nematode Caenorhabditis elegans possesses a simple embryonic nervous system with few enough neurons that the growth of each cell could be followed to provide a systems-level view of development. However, studies of single cell development have largely been conducted in fixed or pre-twitching live embryos, because of technical difficulties associated with embryo movement in late embryogenesis. We present open-source untwisting and annotation software (http://mipav.cit.nih.gov/plugin_jws/mipav_worm_plugin.php) that allows the investigation of neurodevelopmental events in late embryogenesis and apply it to track the 3D positions of seam cell nuclei, neurons, and neurites in multiple elongating embryos. We also provide a tutorial describing how to use the software (Supplementary file 1) and a detailed description of the untwisting algorithm (Appendix). The detailed positional information we obtained enabled us to develop a composite model showing movement of these cells and neurites in an 'average' worm embryo. The untwisting and cell tracking capabilities of our method provide a foundation on which to catalog C. elegans neurodevelopment, allowing interrogation of developmental events in previously inaccessible periods of embryogenesis. DOI: http://dx.doi.org/10.7554/eLife.10070.001 PMID:26633880

  5. III. Gamete and embryo donation.

    PubMed

    2002-05-01

    Ethical considerations concerning gametes and embryo donation are discussed. Basic principles are outlined, focusing on the issues raised by the meaning of genetic links, regulation and the necessity for taking into account the welfare of the child. Relevant specific aspects concern anonymity, compensation for donation and the consent, screening and assessment of donors and recipients. PMID:11980773

  6. 9 CFR 98.9 - Embryos refused entry.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Embryos refused entry. 98.9 Section 98... EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos from Regions Free of Rinderpest and Foot-and-Mouth Disease; and Embryos of Horses and Asses § 98.9 Embryos refused entry. Any embryo refused entry into...

  7. 9 CFR 98.9 - Embryos refused entry.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Embryos refused entry. 98.9 Section 98... EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos from Regions Free of Rinderpest and Foot-and-Mouth Disease; and Embryos of Horses and Asses § 98.9 Embryos refused entry. Any embryo refused entry into...

  8. 9 CFR 98.9 - Embryos refused entry.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Embryos refused entry. 98.9 Section 98... EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos from Regions Free of Rinderpest and Foot-and-Mouth Disease; and Embryos of Horses and Asses § 98.9 Embryos refused entry. Any embryo refused entry into...

  9. 9 CFR 98.9 - Embryos refused entry.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Embryos refused entry. 98.9 Section 98... EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos from Regions Free of Rinderpest and Foot-and-Mouth Disease; and Embryos of Horses and Asses § 98.9 Embryos refused entry. Any embryo refused entry into...

  10. 9 CFR 98.9 - Embryos refused entry.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Embryos refused entry. 98.9 Section 98... EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos from Regions Free of Rinderpest and Foot-and-Mouth Disease; and Embryos of Horses and Asses § 98.9 Embryos refused entry. Any embryo refused entry into...

  11. Effects of High-Butterfat Diet on Embryo Implantation in Female Rats Exposed to Bisphenol A.

    PubMed

    Martinez, Alan M; Cheong, Ana; Ying, Jun; Xue, Jingchuan; Kannan, Kurunthachalam; Leung, Yuet-Kin; Thomas, Michael A; Ho, Shuk-Mei

    2015-12-01

    Bisphenol A (BPA) is an endocrine disruptor associated with poor pregnancy outcomes in human and rodents. The effects of butterfat diets on embryo implantation and whether it modifies BPA's actions are currently unknown. We aimed to determine the effects of butterfat diet on embryo implantation success in female rats exposed to an environmentally relevant dose of BPA. Female Sprague-Dawley rats were exposed to dietary butterfat (10% or 39% kcal/kg body weight [BW]) in the presence or absence of BPA (250 μg/kg BW) or ethinylestradiol (0.1 μg/kg BW) shortly before and during pregnancy to assess embryo implantation potentials by preimplantation development and transport, in vitro blastulation, outgrowth, and implantation. On gestational day (GD) 4.5, rats treated with BPA alone had higher serum total BPA level (2.3-3.7 ng/ml). They had more late-stage preimplantation embryos, whereas those receiving high butterfat (HBF) diet had the most advanced-stage embryos; dams cotreated with HBF and BPA had the most number of advanced embryos. BPA markedly delayed embryo transport to the uterus, but neither amount of butterfat had modifying effects. An in vitro implantation assay showed HBF doubled the outgrowth area, with BPA having no effect. In vivo, BPA reduced the number of implanted embryos on GD8, and cotreatment with HBF eliminated this adverse effect. HBF diet overall resulted in more and larger GD8 embryos. This study reveals the implantation disruptive effects of maternal exposure to an environmentally relevant dose of BPA and identifies HBF diet as a modifier of BPA in promoting early embryonic health.

  12. Protein degradation in preimplantation mouse embryos and the lethality of tritiated amino acids

    SciTech Connect

    Wielbold, J.L.

    1982-01-01

    The role of protein degradation in preimplantation development in the mouse was studied. Proteins of morulae and blastocysts (M and B) cultured in vitro after labeling for 1 hour (h) in /sup 3/H-leucine exhibit a mean half-life (t/sub 1///sub 2/) of 8.1 h. The t/sub 1///sub 2/ tends to increase (9.5 h) when 10% fetal calf serum is added to the chase medium. This decrease in protein degradation in the presence of serum is associated with an increase in the percentage of B that are hatching (P<0.02). This rate of protein degradation in vivo was affected by the stage of pseudopregnancy (PSP) of the recipient. Day 4 embryos in a Day 4 uterus (Day 1=vaginal plug) retained more of the /sup 3/H-leucine in their proteins than did Day 4 embryos remaining in culture (P<0.02), while Day 4 embryos in a Day 3 uterus retained the same amount of radioactivity as did Day 4 embryos in culture. This differential effect of uterine environment was also seen when Day 4 embryos were transferred to recipients. More fetuses developed to term when the recipient was in Day 3 of PSP (50.8%) than when the recipient was in Day 4 PSP (25.9%, P<0.001), regardless of the age of the recipient. Age of the recipient does affect the percentage of transferred embryos developing to term. Thus, protein degradation may vary with the stage of embryo development and the conditions to which the embryos are exposed. However, even low levels of incorporated tritiated leucine can have lethal effects on the embryos and compromise the validity of the protein half-lives determined.

  13. Influence of the embryonal-suspensor mass (ESM) sampling on development and proliferation of maritime pine somatic embryos.

    PubMed

    Ramarosandratana, A; Harvengt, L; Bouvet, A; Calvayrac, R; Pâques, M

    2001-02-01

    Two maturation media with high and low concentration of gellan gum were used to evaluate the maturation performances of four maritime pine ESM (embryonal-suspensor mass) lines. The maturation performance is influenced by sampling modalities; the outer part of the ESM yielded more cotyledonary embryos than the inner part or the whole colony. ESM lines showing several stage 1 embryos at the periphery (spiky) were more productive than those for which stage 1 embryos were rarely visible (smooth). This latter group develop preferably stage 3 embryos on the maturation medium containing high concentration of gellan gum. Biomass production is higher on a medium containing low concentration of gellan gum. However, sampling modalities did not affect the biomass production, and no relation was found between the biomass production and the maturation performance of each line. Stage 3 embryos developed on the medium with low concentration of gellan gum (0.45%, w/v) were shorter than those developed on medium with 0.9% of gellan gum. These short embryos were not able to germinate whereas about 48% of germination was reached with the longest embryos. The ability to develop primary root is dependent on the genotype while epicotyl elongation was observed among all lines.

  14. Self-organization of the in vitro attached human embryo.

    PubMed

    Deglincerti, Alessia; Croft, Gist F; Pietila, Lauren N; Zernicka-Goetz, Magdalena; Siggia, Eric D; Brivanlou, Ali H

    2016-05-12

    Implantation of the blastocyst is a developmental milestone in mammalian embryonic development. At this time, a coordinated program of lineage diversification, cell-fate specification, and morphogenetic movements establishes the generation of extra-embryonic tissues and the embryo proper, and determines the conditions for successful pregnancy and gastrulation. Despite its basic and clinical importance, this process remains mysterious in humans. Here we report the use of a novel in vitro system to study the post-implantation development of the human embryo. We unveil the self-organizing abilities and autonomy of in vitro attached human embryos. We find human-specific molecular signatures of early cell lineage, timing, and architecture. Embryos display key landmarks of normal development, including epiblast expansion, lineage segregation, bi-laminar disc formation, amniotic and yolk sac cavitation, and trophoblast diversification. Our findings highlight the species-specificity of these developmental events and provide a new understanding of early human embryonic development beyond the blastocyst stage. In addition, our study establishes a new model system relevant to early human pregnancy loss. Finally, our work will also assist in the rational design of differentiation protocols of human embryonic stem cells to specific cell types for disease modelling and cell replacement therapy. PMID:27144363

  15. Self-organization of the in vitro attached human embryo.

    PubMed

    Deglincerti, Alessia; Croft, Gist F; Pietila, Lauren N; Zernicka-Goetz, Magdalena; Siggia, Eric D; Brivanlou, Ali H

    2016-05-04

    Implantation of the blastocyst is a developmental milestone in mammalian embryonic development. At this time, a coordinated program of lineage diversification, cell-fate specification, and morphogenetic movements establishes the generation of extra-embryonic tissues and the embryo proper, and determines the conditions for successful pregnancy and gastrulation. Despite its basic and clinical importance, this process remains mysterious in humans. Here we report the use of a novel in vitro system to study the post-implantation development of the human embryo. We unveil the self-organizing abilities and autonomy of in vitro attached human embryos. We find human-specific molecular signatures of early cell lineage, timing, and architecture. Embryos display key landmarks of normal development, including epiblast expansion, lineage segregation, bi-laminar disc formation, amniotic and yolk sac cavitation, and trophoblast diversification. Our findings highlight the species-specificity of these developmental events and provide a new understanding of early human embryonic development beyond the blastocyst stage. In addition, our study establishes a new model system relevant to early human pregnancy loss. Finally, our work will also assist in the rational design of differentiation protocols of human embryonic stem cells to specific cell types for disease modelling and cell replacement therapy.

  16. Isolation of herpes simplex viruses by chick embryo culture.

    PubMed

    Akter, T; Tabassum, S; Jahan, M; Nessa, A; Islam, M N; Giasuddin, M

    2013-04-01

    The chick embryo is a versatile host system in diagnostic virology, especially for isolation of herpes simplex viruses. In this study, samples obtained from 57 clinically diagnosed patients with active herpetic lesions (35 genital & 22 non-genital) were cultured by chick embryo method for isolation of herpes simplex virus. After inoculation onto the chorioallantoic membrane (CAM) of 10-11 days old chick embryo, typical CAM reactions (pocks) appeared in 23(40.3%) samples after 3 days. CAM reactions were identified and typed by direct fluorescence antibody test and 22(95.6%) of 23 isolates gave positive results. Of this, 9(40.9%) were HSV-1 & 13(59.1%) were HSV-2. HSV-1 was isolated from 8(36.4%) of non-genital samples and from 1(7.1%) genital sample. HSV-2 was isolated from 13(92.8%) of genital samples, but none were isolated from non-genital samples. High isolation rate was obtained from vesicular stage of both non-genital (71.5%) and genital (57.1%) samples and from early lesions (sampled within 72 hours) of non-genital (50%) and genital (52.9%) specimen. The chorioallantoic membrane of chick embryo it is a simple, cheap and efficient method of cultivation of some viruses, including HSV. Thus, in settings where cell culture facilities are not available, it can be used for the isolation of herpes simplex viruses from clinical samples.

  17. Dignity, marriage and embryo adoption: a look at Dignitas Personae.

    PubMed

    Murphy, Timothy F

    2011-12-01

    The Catholic Church's 2008 Dignitas Personae discusses the moral implications of respecting the dignity of all human beings, regardless of the stage of development. In that text, the Vatican's Congregation for the Doctrine of the Faith argues that respect for this dignity is incompatible with the conception of embryos outside marriage as well as assisted reproduction treatments and certain kinds of human embryonic research. Not only that, but the Congregation also rejects efforts at embryo adoption. As a matter of secular moral philosophy, this view of dignity is disputable and this article shows how an alternate view of dignity--one that depends on interests as against status--serves as a better foundation for decisions about ways in which to help people have children. This view of dignity is entirely compatible with a wide array of assisted reproduction treatments and research and is compatible with the conception of embryos for single parents or opposite-sex couples looking to have children. Using its notion of human dignity, the Congregation makes a case against embryo adoption, but that case is unconvincing given the permissible exercise of individual conscience and the presumptive importance of rescuing human lives where they can be rescued.

  18. Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula.

    PubMed

    Kurdyukov, Sergey; Song, Youhong; Tiew, Terence W-Y; Wang, Xin-Ding; Nolan, Kim E; Rose, Ray J

    2015-01-01

    Early embryogenesis starting from a single cell zygote goes through rapid cell division and morphogenesis, and is morphologically characterized by pre-globular, globular, heart, torpedo and cotyledon stages. This progressive development is under the tight regulation of a complex molecular network. Harvesting sufficient early embryos at a similar stage of development is essential for investigating the cellular and molecular regulation of early embryogenesis. This is not straightforward since early embryogenesis undergoes rapid morphogenesis in a short while e.g. 8 days for Medicago truncatula to reach the early cotyledon stage. Here, we address the issue by two approaches. The first one establishes a linkage between embryo development and pod morphology in helping indicate the stage of the zygotic embryo. This is particularly based on the number of pod spirals and development of the spines. An alternative way to complement the in vivo studies is via culturing leaf explants to produce somatic embryos. The medium includes an unusual hormone combination - an auxin (1-naphthaleneacetic acid), a cytokinin (6-benzylaminopurine), abscisic acid and gibberellic acid. The different stages can be discerned growing out of the callus without dissection. PMID:26131626

  19. Blastocele fluid from in vitro- and in vivo-produced equine embryos contains nuclear DNA.

    PubMed

    Herrera, C; Morikawa, M I; Castex, C Baca; Pinto, M R; Ortega, N; Fanti, T; Garaguso, R; Franco, M J; Castañares, M; Castañeira, C; Losinno, L; Miragaya, M H; Mutto, A A

    2015-02-01

    Normal mammalian early embryonic development involves apoptosis of blastomeres as a remodeling process during differentiation, starting at the blastocyst stage. Genomic DNA has been recently detected in the blastocele fluid of human embryos and has been amplified by real-time polymerase chain reaction (PCR) to diagnose the sex of in vitro-produced human embryos. This new approach varies from conventional preimplantation genetic diagnosis in that no cells are extracted from the embryo and only the blastocele fluid is aspirated and used as a DNA sample for diagnosis. In the present work, we investigated whether the blastocele fluid of equine preimplantation embryos contains nuclear DNA and whether this DNA could be used to diagnose the sex of the embryos by conventional PCR, using specific primers that target the TSPY and AMEL equine genes. The sex of 11 of 13 in vivo-produced embryos and of four of five in vitro-produced embryos was successfully diagnosed. The PCR amplification product was analyzed using genetic sequencing reporting that the DNA present in blastocele fluid was genomic. Additionally, after polyacrylamide gel electrophoresis and silver staining, the blastocele fluid from three different embryos produced a ladder pattern characteristic of DNA fragmented during apoptosis. Therefore, the results presented in this work report that blastocele fluid from in vivo- and in vitro-produced equine embryos contains nuclear DNA which is probably originated by apoptosis of embryonic cells, and this DNA could be used to diagnose the sex of preimlpantation embryos by conventional PCR.

  20. Effects of brief hypoxia and hyperoxia on tissue element levels in the development chick embryo

    SciTech Connect

    Richards, M.P.; Stock, M.K.; Metcalfe, J. Oregon Health Sciences Univ., Portland )

    1991-03-15

    Brief hypoxia or hyperoxia has been shown to affect growth and metabolism of chick embryos during the later stages of development. The objective of this experiment was to alter the availability of oxygen to chick embryos developing in ovo and to determine the effects on tissue levels of Zn, Cu, Fe and Mn. Hypoxia reduced embryo, heart, brain and liver wts (wet wt), whereas, hyperoxia increased embryo, heart, lung and liver wts compared to normoxic controls. Chorioallantoic membrane (CAM) wt was increased by hypoxia and reduced by hyperoxia. Livers from hyperoxic embryos contained more Zn, Fe and Mn and less Cu than livers from hypoxic or normoxic embryos. Tissue levels of Zn, Cu, Fe and Mn were reduced in brains from hypoxic compared to hyperoxic or normoxic embryos. Hyperoxia increased the concentrations of Zn and Cu in CAM; whereas, hypoxia reduced the levels of Zn and Fe. The amounts of Zn and Cu were increased in hyperoxic compared to normoxic lungs. Hearts from hyperoxic embryos had more Zn, Cu and Mn than hypoxic or normoxic hearts. Hypoxic yolk sac contained more Zn, Cu and Mn than hyperoxic or normoxic yolk sac. Except for yolk sac, the amounts of Zn, Cu, Fe and Mn in tissues from normoxic embryos increased from day 15 to day 18 of incubation in concert with tissue growth. The authors conclude that the availability of O{sub 2} to the developing chick embryo affects tissue trace element levels either through its effects on tissue growth or via effects on the regulation of trace element uptake and assimilation by the tissues.

  1. Aquatic toxicity assessment of single-walled carbon nanotubes using zebrafish embryos

    NASA Astrophysics Data System (ADS)

    Pan, Huichin; Lin, Yu-Jun; Li, Meng-Wei; Chuang, Han-Ni; Chou, Cheng-Chung

    2011-07-01

    Zebrafish embryos selected at the 64-cell stage were exposed to various concentrations of amide functionalized single-walled carbon nanotubes (SWCNTs) ranging from 1 to 10 μg/ml dissolved in 1% Pluronic F-68 (a cell culture grade surfactant), and the development of embryos was examined from 24 to 120 hours post fertilization (hpf). Incubation of embryos in 1% F-68 did not induce overt abnormal phenotype as compared to the wild-type; neither did it cause significant mortality during the exposure period. Generally, there was a slight developmental delay in larvae treated with SWCNTs of 5 μg/ml or above. Only larvae exposed to >= 5 μg/ml SWCNTs showed significantly reduced survival rates. About 50% of the embryos exposed to 5 μg/ml showed abnormal phenotypes at 24 hpf as compared to the control group. As development proceeds to 120 hpf, more embryos displayed defective morphology. A slight hatching delay was observed in embryos exposed to concentrations above 5 μg/ml. There was a general reduction of body axes, including narrowed somite and shortened yolk stalk. In addition, pigmentation in the ventral trunk area was less than that observed in control group. The body lengths of the exposed embryos were decreased significantly at 48 hpf (3.11 mm in control vs. 3.00 mm in SWCNTs-exposed embryos). However, exposure to SWCNTs did not affect the number of somites. Other features that were noticed in the SWCNTs-exposed embryos included edema and shrinkage and blebbling of the epidermal lining. Most of these observed phenotypes persisted from 48 hpf through 120 hpf. Overall, the aforementioned results indicate that soluble amide-functionalized SWCNTs are toxic to zebrafish embryos at a minimum concentration of 5 μg/ml.

  2. Astroviruses associated with stunting and pre-hatching mortality in duck and goose embryos.

    PubMed

    Biđin, M; Lojkić, I; Tišljar, M; Biđin, Z; Majnarić, D

    2012-01-01

    The first detection of avian nephritis virus (ANV) in goose embryos and of turkey astrovirus-1 (TAstV-1) in duck embryos is described. Intestinal samples from duck and goose embryos from five duck and four goose flocks in Croatia were tested by polymerase chain reaction for the presence of ANV, TAstV-1, turkey astrovirus-2, chicken astrovirus, duck astrovirus and also for the presence of avian reovirus, Derzsy's disease virus and duck enteritis virus. The kidneys from duck and goose embryos were also tested for ANV, while liver samples were tested for duck astrovirus. Duck embryos were also tested to detect duck circovirus and goose embryos for the presence of goose circovirus and goose haemorrhagic polyomavirus. All embryos were in the final stage of incubation and were characterized by moderate to markedly retarded growth. ANV was confirmed in the intestines and kidneys of embryos from two duck and two goose flocks and TAstV-1 was found in embryos from two duck flocks. One duck flock was positive for both ANV and TAstV-1. No other viruses were found in tested flocks. Phylogenetic analysis based on the ANV polymerase gene fragment of ANV sequences detected in duck and goose embryos revealed greatest similarity (88.1 to 97.2%) with ANV isolates from chickens. Further, the existence of at least two types of ANV circulating in Croatian duck and goose flocks was confirmed. Based on the phylogenetic analysis of the portion of TAstV-1 polymerase gene, two detected TAstV-1 nucleotide sequences were 99.5% similar. Compared with six TAstV-1 sequences, Croatian sequences showed one unique nucleotide change. In addition to other possible causes of stunted growth and late embryonic death, these findings suggest that ANV and/or TAstV-1 infection may be a contributing factor in the pre-hatching mortality of ducklings and goslings.

  3. Triphasic low-dose response in zebrafish embryos irradiated by microbeam protons.

    PubMed

    Choi, Viann Wing Yan; Yum, Emily Hoi Wa; Konishi, Teruaki; Oikawa, Masakazu; Cheng, Shuk Han; Yu, Kwan Ngok

    2012-01-01

    The microbeam irradiation system (Single-Particle Irradiation System to Cell, acronym as SPICE) at the National Institute of Radiological Sciences (NIRS), Japan, was employed to irradiate dechorionated zebrafish embryos at the 2-cell stage at 0.75 h post fertilization (hpf) by microbeam protons. Either one or both of the cells of the embryos were irradiated with 10, 20, 40, 50, 80, 100, 160, 200, 300 and 2000 protons each with an energy of 3.37 MeV. The embryos were then returned back to the incubator until 24 hpf for analyses. The levels of apoptosis in zebrafish embryos at 25 hpf were quantified through terminal dUTP transferase-mediated nick end-labeling (TUNEL) assay, with the apoptotic signals captured by a confocal microscope. The results revealed a triphasic dose-response for zebrafish embryos with both cells irradiated at the 2-cell stage, namely, (1) increase in apoptotic signals for < 200 protons (< 30 mGy), (2) hormesis to reduce the apoptotic signals below the spontaneous number for 200-400 protons (at doses of 30-60 mGy), and (3) increase in apoptotic signals again for > 600 protons (at doses > 90 mGy). The dose response for zebrafish embryos with only one cell irradiated at the 2-cell stage was also likely a triphasic one, but the apoptotic signals in the first zone (< 200 protons or < 30 mGy) did not have significant differences from those of the background. At the same time, the experimental data were in line with induction of radiation-induced bystander effect as well as rescue effect in the zebrafish embryos, particular in those embryos with unirradiated cells.

  4. Assessment of developmental retardation and abnormality of in vivo produced preimplantation embryos in rat.

    PubMed

    Rupasri, A; Shivakumar, K R; Sreenath, B R; Seshagiri, P B

    1995-12-01

    In most mammals studied, a substantial numbers of preimplantation embryos are believed to be lost in vivo. In vitro, embryos develop slowly and lose viability. Hence, there is a need to assess the extent and cause of embryonic loss both in vivo and in vitro. In this study, we assessed the quality of in vivo produced ovulation products/embryos, recovered on days 1-5 pregnancy, from naturally bred wistar rats. From day 1 pregnant rats (n = 24), 226 ovulation products were recovered which included 52% (117) unfertilized oocytes and empty zonae with/without cell debris (UFO-EZ:CD) and 48% (109) 1-cells. Flushings of day 2 rats (n = 27) contained 229 ovulation products, consisting of 70% (160) 2-cells and 30% (69) UFO-EZ:CD. Flushings of day 3 rats (n = 27) had 23% (56) 2-cells, 6% (15) 3-cells, 23% (57) 4-cells, 1% (2) 5-7 cells, 2% (5) 8-cells and 45% (112) UFO-EZ:CD, total being 247. Flushings of day 4 rats (n = 28) had 193 ovulation products comprising of one morula, 45% (86) 8-cells, 5% (9) 5-7-cells and the rest w