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Sample records for 4-cell stage embryos

  1. Presence and distribution of E-cadherin in the 4-cell golden hamster embryo. Effect of maternal age and parity.

    PubMed

    Trejo, A; Ambriz, D; Navarro-Maldonado, M C; Mercado, E; Rosado, A

    2008-08-01

    Maternal age dependency of gestation time in hamster and in other mammals is a well demonstrated fact. We have recently shown that adult nulliparous and multiparous hamster females show significant asynchrony and retard on early embryo development (from two blastomeres to morula stages) when compared with nulliparous young females. The number of cell-cell adhesions between blastomeres in early embryo development has been reported to be a good indication of the ability of embryos to cleave and develop. In this work we studied, by indirect immunofluorescence, the presence and distribution of E-cadherin in 4-cell embryos obtained from nulliparous young (NYF), nulliparous adult (NAF) and multiparous adult (MAF) hamster females. Distribution and intensity of fluorescence was observed and registered using confocal microscopy. Staining intensities for E-cadherin were quantified by computed densitometry in the free membrane regions, in the cytoplasm region and in the cell-cell adhesion zones of each embryo. E-Cadherin in all the studied zones was significantly higher (p<0.01) in NYF. Cadherin concentration in the intercellular membranes was always statistically higher (p<0.05) than in the free membrane regions. An appreciable concentration of E-cadherin was found in the cytoplasm of the 4-cell embryos obtained from the three groups of females, but was significantly higher in NYF. No statistical differences were observed in any of the parameters studied between NAF and MAF. Our results seem to indicate that changes in the reproductive behavior related to age and/or multiparity may be correlated with changes in the processes related to intercellular adhesions during early cleavage.

  2. Survival of frozen-thawed sheep embryos cryopreserved at cleavage stages.

    PubMed

    Garcia-Garcia, R M; Gonzalez-Bulnes, A; Dominguez, V; Veiga-Lopez, A; Cocero, M J

    2006-02-01

    This study evaluated the effect of freezing-thawing procedures on the viability of sheep embryos cryopreserved at various developmental stages. The survival rates of frozen-thawed embryos were compared with non-frozen counterparts. Embryos were recovered from the oviduct and uterus, at different days of the early luteal phase, and were classified at six different developmental stages: 2- to 4-cell (n = 72), 5- to 8-cell (n = 73), 9- to 12-cell (n = 70), early morulae (n = 42), morulae (n = 41), and blastocyst (n = 70). For each early cleavage stage and blastocysts, approximately half of the embryos, were frozen immediately by slow freezing with an ethylene glycol-based solution. The remaining embryos were cultured to the hatched blastocyst stage. All morulae and compact morulae were frozen after recovery with the same protocol. Cryoprotectants were removed using 1M sucrose solution, and then warmed the embryos were cultured to the hatched stage in a standardized in vitro culture. Embryo developmental stage had a significant effect on the ability to hatch following freezing (P<0.0001). The cryotolerance of the embryos fitted a regression (r2 = 0.908), increasing linearly from 2- to 4-cell embryos (17.1%) to morula stage (46.3%) and in a quadratic regression from the morula to the blastocyst stage (83.7%). Frozen early cleavage stage embryos had a significantly lower viability than their fresh counterparts (23.1 vs 83.1%; P<0.0001), with a similar rate of viability between fresh or frozen blastocysts (92.5 vs 83.7%). In conclusion, early sheep embryos are very sensitive to freezing per se and the survival rates following conventional freezing improve as embryo developmental stage progresses.

  3. 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.

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

    PubMed

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

    2016-01-01

    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.

  5. Developmentally delayed cleavage-stage embryos maintain comparable implantation rates in frozen embryo transfers.

    PubMed

    Burks, Heather; Buckbinder, Jennifer; Francis-Hernandez, Mary; Chung, Karine; Jabara, Sami; Bendikson, Kristin; Paulson, Richard

    2015-10-01

    In fresh IVF cycles, embryos reaching the eight-cell stage on day 3 of development are thought to have a higher chance of implantation than those reaching this stage on day 4. To determine whether this difference persists after cryopreservation, we compared pregnancy and implantation rates between frozen embryo transfer (FET) cycles using delayed cleavage-stage embryos (cryopreserved day 4) and normal cleavage-stage embryos (cryopreserved day 3). Participants underwent FET between 2008 and 2012 using embryos cryopreserved on either day 3 (n = 76) or day 4 (n = 48), depending on the length of time needed to achieve the eight-cell stage. All embryos, regardless of day of cryopreservation, were thawed and transferred on the 4th day of vaginal progesterone following endometrial preparation with oral estradiol. Chi-square and Mann-Whitney U tests were used to compare patient demographics and cycle outcomes. More women in the day 4 group had diminished ovarian reserve (44 vs 16 %, p = 0.003). Pregnancy outcomes in preceding fresh cycles were not different between the two groups. Pregnancy, implantation, and live birth rates following FET did not differ between the day 3 and day 4 groups. This is the first study to address outcomes using day 3 versus day 4 cryopreserved embryos. Despite a higher prevalence of diminished ovarian reserve (DOR) in the day 4 group, delayed cleavage-stage embryos utilized in FET cycles performed as well as embryos growing at the normal rate, suggesting delayed embryo development does not affect embryo implantation as long as endometrial synchrony is maintained.

  6. Morphological and cytogenetic assessment of cleavage and blastocyst stage embryos.

    PubMed

    Fragouli, E; Alfarawati, S; Spath, K; Wells, D

    2014-02-01

    Morphological assessments are the main way in which fertility clinics select in vitro generated embryo(s) for transfer to the uterus. However, it is widely acknowledged that the microscopic appearance of an embryo is only weakly correlated with its viability. Furthermore, the extent to which morphology is affected by aneuploidy, a genetic defect common in human preimplantation embryos, remains unclear. Aneuploidy is of great relevance to embryo selection as it represents one of the most important causes of implantation failure and miscarriage. The current study aimed to examine whether morphological appearance can assist in identifying embryos at risk of aneuploidy. Additionally, the data produced sheds light on how chromosomal anomalies impact development from the cleavage to the blastocyst stage. A total of 1213 embryos were examined. Comprehensive chromosome analysis was combined with well-established criteria for the assessment of embryo morphology. At the cleavage stage, chromosome abnormalities were common even amongst embryos assigned the best morphological scores, indicating that aneuploidy has little effect on microscopic appearance at fixed time points up until Day 3 of development. However, at the blastocyst stage aneuploidies were found to be significantly less common among embryos of optimal morphological quality, while such abnormalities were overrepresented amongst embryos considered to be of poor morphology. Despite the link between aneuploidy and blastocyst appearance, many chromosomally abnormal embryos were able to achieve the highest morphological scores. In particular, blastocysts affected by forms of aneuploidy with the greatest capacity to produce clinical pregnancies (e.g. trisomy 21) were indistinguishable from euploid embryos. The sex ratio was seen to be equal throughout preimplantation development. Interestingly, however, females were overrepresented amongst the fastest growing cleavage-stage embryos, whereas a sex-related skew in the

  7. Should we be promoting embryo transfer at blastocyst stage?

    PubMed

    Maheshwari, Abha; Hamilton, Mark; Bhattacharya, Siladitya

    2016-02-01

    Improved laboratory standards and better culture media have made extended culture to blastocyst stage a reality to identify embryos with maximum implantation potential. The strategy of extended culture has become more popular across the world at a time when regulatory bodies have emphasized the need to increase the uptake of elective single embryo transfer, minimize complications associated with multiple births and aim for a healthy singleton live-birth as the preferred outcome in IVF. New data on perinatal outcomes suggest that pregnancies after embryo transfer at blastocyst stage are associated with a higher risk of preterm delivery, large for gestational age babies, monozygotic twins and altered sex ratio compared with those following embryo transfers at cleavage stage. In addition, concerns have been raised of increased congenital anomalies and epigenetic modifications with embryo transfer at blastocyst stage. Twenty-four years on from the first embryo transfer at blastocyst stage, we examine the reasons for extended embryo culture, evaluate the risks and benefits of this strategy and suggest the need to reconsider this policy in the interests of fetal safety.

  8. 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

  9. Establishment of rock bream Oplegnathus fasciatus embryo (RoBE-4) cells with cytolytic infection of red seabream iridovirus (RSIV).

    PubMed

    Oh, So-Young; Nishizawa, Toyohiko

    2016-12-01

    Red seabream iridovirus (RSIV) is a member of genus Megalocytivirus in the family Iridoviridae. RSIV infection causes significant economic losses of marine-fishes in East Asian countries. Grunt fin (GF) cell line has been commonly used for culturing RSIV. However, it is not suitable for definite evaluation of infectivity titer of RSIV because cells infected with RSIV are not completely cytolysed. Thus, we established a new cell line, RoBE-4, from rock bream (Oplegnathus fasciatus) eyed-egg embryos in this study. Morphologically, RoBE-4 cells were fibroblastic-like. They have been stably grown over two-years with 60 passages using Leibovitz's L-15 medium containing 10% (v/v) fetal bovine serum. RoBE-4 cells infected with RSIV exhibited cytopathic effects (CPE) with cell rounding. They were cytolysed completely after ≥2 weeks of culture. Numerous RSIV particles with icosahedral morphology of approximately 122nm in diameter were observed in cytoplasmic area of infected RoBE-4 cells. The RSIV-suceptibility and amount of extracellular RSIV released by RoBE-4 cells were 100-fold higher than those by GF cells. RSIV cultured with RoBE-4 cells was highly virulent to rock bream in infection experiments. Therefore, using RoBE-4 cells instead of GF cells will enable accurate and sensitive measurement of RSIV infectivity. In addition, RoBE-4 cells might be used to produce RSIV vaccine in the future with significant reduction in cost.

  10. Valproic acid treatment from the 4-cell stage improves Oct4 expression and nuclear distribution of histone H3K27me3 in mouse cloned blastocysts.

    PubMed

    Isaji, Yuuki; Murata, Moeko; Takaguchi, Naoya; Mukai, Toshita; Tajima, Yosuke; Imai, Hiroshi; Yamada, Masayasu

    2013-01-01

    We examined effects of treatment with valproic acid (0, 0.2, 1 or 2 mM, VPA), an inhibitor of class I and IIa histone deacetylases (HDACs), of mouse somatic cell nuclear transfer (SCNT) embryos for 24 h from 48 h (4-cell stage), 24 h (2-cell stage) or immediately after oocyte activation on blastocyst formation rates and qualities of the resultant blastocysts. Blastocyst formation rates (33.4-37.0%) were not improved by VPA treatments compared with the untreated control (35.1-36.4%). However, immunofluorescence staining revealed that Oct4 expression levels, evaluated from percentages of embryos expressing Oct4 strongly and having more than 10 Oct4-positive cells, in blastocysts from SCNT embryos treated with 1 mM VPA for 24 h from the 4-cell stage (VPA-4C) were highest among all the groups and that the proportion of cells with a normal nuclear distribution of histone H3 trimethylated at lysine 27 (H3K27me3), a marker of the state of X-chromosome inactivation, significantly increased in the VPA-4C group (36.6%) compared with the control group (12.4%, P<0.05). Treatments with scriptaid and sodium butyrate, inhibitors of class I and IIa/b HDACs, for 24 h from the 4-cell stage also had beneficial effects on SCNT blastocysts. These findings indicate that treatment with 1 mM VPA from the 4-cell stage improves the Oct4 expression and nuclear distribution of H3K27me3 in mouse SCNT blastocysts and suggest that the inhibition of class I and IIa HDACs from the 4-cell stage plays an important role in these effects.

  11. Telomere lengths in human oocytes, cleavage stage embryos and blastocysts

    PubMed Central

    Turner, S.; Wong, H.P.; Rai, J.; Hartshorne, G.M.

    2010-01-01

    Telomeres are repeated sequences that protect the ends of chromosomes and harbour DNA repair proteins. Telomeres shorten during each cell division in the absence of telomerase. When telomere length becomes critically short, cell senescence occurs. Telomere length therefore reflects both cellular ageing and capacity for division. We have measured telomere length in human germinal vesicle (GV) oocytes and preimplantation embryos, by quantitative fluorescence in situ hybridization (Q-FISH), providing baseline data towards our hypothesis that telomere length is a marker of embryo quality. The numbers of fluorescent foci suggest that extensive clustering of telomeres occurs in mature GV stage oocytes, and in preimplantation embryos. When calculating average telomere length by assuming that each signal presents one telomere, the calculated telomere length decreased from the oocyte to the cleavage stages, and increased between the cleavage stages and the blastocyst (11.12 versus 8.43 versus 12.22 kb, respectively, P < 0.001). Other methods of calculation, based upon expected maximum and minimum numbers of telomeres, confirm that telomere length in blastocysts is significantly longer than cleavage stages. Individual blastomeres within an embryo showed substantial variation in calculated average telomere length. This study implies that telomere length changes according to the stage of preimplantation embryo development. PMID:20573647

  12. [Analysis of sex chromosome mosaicisms in early cleavage-stage human embryos and blastocysts with poor embryo quality scores].

    PubMed

    Ou, Jian; Wang, Wei; Ding, Jie; Gu, Bin; Zheng, Ai-yan; Wang, Fu-xin; Li, Hong

    2011-12-01

    To analyze sex chromosome mosaicisms in early cleavage-stage human embryos and blastocysts with poor embryo quality score based on the numbers of pronucleus(PN) zygotes using X,Y dual color fluorescence in situ hybridization (FISH), and to discuss the possible mechanisms. Fresh or frozen-thawed early cleavage-stage human embryos and blastocysts with poor embryo quality score not suitable for embryo transfer were studied with dual color FISH. Double signal rate of 2PN among early cleavage-stage embryos was 66.67%, which was significantly higher than 1PN and 3PN embryos. Single signal rate of 1PN early cleavage-stage embryos was 90.41%, which was significantly higher than 2PN and 3PN ones. Three signal rate of 3PN early cleavage-stage embryos was 28.00%, which was significantly higher than 1PN and 2PN ones. Double signal rate of 3PN ones was 46.00%, which was significantly higher than 1PN ones. The polyploid rate of frozen-thawed early cleavage-stage embryos was 23.53%, which was slightly higher than that of fresh embryos, but with no statistical significance. The mosaicism rate of 24 blastocysts was 100.00% and the double signal dominant (≥ 50%) rate was 62.50%, which was significantly higher than the rate of early cleavage-stage embryos. Using 2PN as the criterion for embryo quality score cannot guarantee the selection of normal fertilized embryo for transplantation. Frozen-thawed embryos may harbor more polyploid cells. To avoid the selection of embryos with abnormal chromosomes, combinations of pre-implantation genetic diagnosis (PGD) and prenatal diagnosis are necessary. Meanwhile, blastocysts with poor quality scores may provide an important source for embryo stem cells.

  13. Stage dependent susceptibility to lead in Bufo arenarum embryos.

    PubMed

    Pérez-Coll, C S; Herkovits, J

    1990-01-01

    The stage dependent susceptibility to lead in amphibian development was studied by exposing Bufo arenarum embryos during neurulae, neuromuscular activity and gill circulation stages for twenty hours to 1 ppm Pb(2+). Survival, malformations and behavioral disorders were evaluated. The embryonic susceptibility to lead was markedly stage dependent. The survival at the neuromuscular activity stage was approximately half that of the other two periods; concerning malformations, the gill circulation stage was the least sensitive. The observed malformations consisted of failed closure of neural tube, hydropsy, small and cylindrical tails, reduced body size and incurvations in the body axis. Some alterations occurred in all experimental groups and therefore were considered non-dependent on the period of treatment. In all experimental embryos, neurological disorders such as trembles and loss of equilibrium were observed.

  14. Developmental stages in human embryos: revised and new measurements.

    PubMed

    O'Rahilly, Ronan; Müller, Fabiola

    2010-01-01

    The staging of human embryos, as distinct from seriation, depends on a morphological scheme devised by Streeter and completed by O'Rahilly, who proposed the term Carnegie stages. To avoid misconceptions and errors, and to place new findings in perspective, it is necessary to summarize the essentials of the Carnegie system: (1) Twenty-three stages cover the embryonic period, i. e. the first 8 postfertilizational weeks of development. (2) The system is based on internal as well as external features, and the use of only external criteria is subject to serious limitations. For example, precise delineation of stages 19-23 and of the embryonic-fetal transition depends on histological examination. (3) Prenatal measurements are not an integral component of the staging system, and hence a stage should never be assigned merely on the basis of embryonic length. A 20-mm embryo, for example, could belong to any of three stages. Measurements, however, are important for the assessment of age, and very few measurements are available for staged embryos. Presented here and based on accurate staging are the maximum diameter of the chorionic sac, the crown-heel length, the greatest length exclusive of the lower limbs, the biparietal diameter, the head circumference, the length of the hindbrain, the total length of the brain, and the lengths of the limbs as well as of their segments, including the foot length. (4) Prenatal ages are also not an integral part of the staging system and hence a stage should never be assigned merely on the basis of prenatal age. Ages, however, are of clinical importance and their estimate has been rendered more precise by accurate timing of fertilization followed by ultrasonography. Prenatal age is postfertilizational and hence some 2 weeks less than the postmenstrual interval. The term gestational age is ambiguous and should be discarded. Presented here is a new graph showing proposed estimates of age in relation to stages and based on current information.

  15. A set of stage-specific gene transcripts identified in EK stage X and HH stage 3 chick embryos.

    PubMed

    Lee, Bo Ram; Kim, Heebal; Park, Tae Sub; Moon, Sunjin; Cho, Seoae; Park, Taesung; Lim, Jeong Mook; Han, Jae Yong

    2007-06-01

    The embryonic developmental process in avian species is quite different from that in mammals. The first cleavage begins 4 h after fertilization, but the first differentiation does not occur until laying of the egg (Eyal-Giladi and Kochav (EK) stage X). After 12 to 13 h of incubation (Hamburger and Hamilton (HH) stage 3), the three germ layers form and germ cell segregation in the early chick embryo are completed. Thus, to identify genes associated with early embryonic development, we compared transcript expression patterns between undifferentiated (stage X) and differentiated (HH stage 3) embryos. Microarray analysis primarily showed 40 genes indicating the significant changes in expression levels between stage X and HH stage 3, and 80% of the genes (32/40) were differentially expressed with more than a twofold change. Among those, 72% (23/32) were relatively up-regulated at stage X compared to HH stage 3, while 28% (9/32) were relatively up-regulated at HH stage 3 compared to stage X. Verification and gene expression profiling of these GeneChip expression data were performed using quantitative RT-PCR for 32 genes at developmental four points; stage X (0 h), HH stage 3 (12 h), HH stage 6 (24 h), and HH stage 9 (30 h). Additionally, we further analyzed four genes with less than twofold expression increase at HH stage 3. As a result, we identified a set of stage-specific genes during the early chick embryo development; 21 genes were relatively up-regulated in the stage X embryo and 12 genes were relatively up-regulated in the HH stage 3 embryo based on both results of microarray and quantitative RT-PCR. We identified a set of genes with stage-specific expression from microarray Genechip and quantitative RT-PCR. Discovering stage-specific genes will aid in uncovering the molecular mechanisms involved the formation of the three germ layers and germ cell segregation in the early chick embryos.

  16. Using the amniotic cavity of the developing chick embryo for the in vivo culture of early-stage mammalian embryos.

    PubMed

    Blakewood, E G; Jaynes, J M; Johnson, W A; Godke, R A

    1989-12-01

    The fertile chicken egg may provide an effective, inexpensive method for promoting the development of early-stage embryos from other species. Presently, the loss of viability associated with the in vitro culture of mammalian embryos is hindering the use of in vitro fertilization with farm animals. Consequently, alternative in vitro laboratory methods are needed for the culture of mammalian embryos. A new method has been developed that involves the culture of mammalian embryos in the amniotic cavity of a developing chick embryo. Chick embryos were placed into shell-less incubation (37 C) at the 72-h developmental stage. After 24 h of shell-less incubation, agarose-embedded mammalian embryos were injected into the amniotic cavity of the chick embryo. The mammalian embryos were first placed into a drop of liquid agarose. One to four embryos were then aspirated into a beveled injection pipette and cooled, allowing the agarose to harden. Following penetration of the amnion with the beveled pipette, the agarose cylinder containing the embryos was expelled into the amniotic cavity. The shell-less culture system was then returned to incubation at 37 C for an additional 72 to 96 h. Following incubation, the amniotic cavity containing both chick and mammalian embryos was isolated and the agarose-embedded mammalian embryos were harvested. Significantly more embryos developed in the chick embryo amnion than in the control medium alone. Results obtained using this method on laboratory animals (mice) and on domestic mammals (goats and cattle) indicate that the chick-embryo amnion can support the development of early-stage, mammalian embryos to the blastocyst stage of development.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. The polycomb group protein EED varies in its ability to access the nucleus in porcine oocytes and cleavage stage embryos.

    PubMed

    Foust, Kallie B; Li, Yanfang; Park, Kieun; Wang, Xin; Liu, Shihong; Cabot, Ryan A

    2012-08-01

    Chromatin-modifying complexes serve essential functions during mammalian embryonic development. Polycomb group proteins EED, SUZ12, and EZH2 have been shown to mediate methylation of the lysine 27 residue of histone protein H3 (H3K27), an epigenetic mark that is linked with transcriptional repression. H3K27 trimethylation has been shown to be present on chromatin in mature porcine oocytes, pronuclear and 2-cell stage embryos, with H3K27 trimethylation decreasing at the 4-cell stage and not detectable in blastocyst stage embryos. The goals of this study were to determine the intracellular localization of the polycomb group protein EED in porcine oocytes and cleavage stage porcine embryos produced by in vitro fertilization and to determine the binding abilities of karyopherin α subtypes toward EED. Our results revealed that EED had a strong nuclear localization in 4-cell and blastocyst stage embryos and a strong perinuclear staining in GV-stage oocytes; EED was not detectable in the nuclei of pronuclear or 2-cell stage embryos. An in vitro binding assay was performed to assess the ability of EED to interact with a series of karyopherin α subtypes; results from this experiment revealed that EED can interact with several karyopherin α subtypes, but with varying degrees of affinity. Together these data indicate that EED displays a dynamic change in intracellular localization in progression from immature oocyte to cleavage stage embryo and that EED possess differing in vitro binding affinities toward individual karyopherin α subtypes, which may in part regulate the nuclear access of EED during this window of development. Copyright © 2012. Published by Elsevier B.V.

  18. 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.

  19. Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

    PubMed Central

    Sicaeros, Beatriz; O'Dowd, Diane K.

    2007-01-01

    This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24°C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function. PMID:18979024

  20. Pregnancy rate following transfer of in vitro produced lamb derived embryos in two embryonic stages.

    PubMed

    Shirazi, A; Shams-Esfandabadi, N; Ahmadi, E; Jadidi, M; Heidari, B

    2008-03-15

    Ovine embryos were produced by maturation, fertilization and in vitro culture (IVM/IVF/IVC) of oocytes collected from slaughtered prepubertal ewes. At 24 h post IVM, oocytes were fertilized with fresh semen collected from Lori-Bakhtiari breed at a concentration of 1.0 x l0(6) sperm mL(-1). The presumptive ova/embryos were transferred into the embryo culture medium at 22-24 h post IVF. Following 4 to 7 day in culture, embryos (at morula and blastocyst stage, respectively) were transferred surgically to the uterine horn of synchronized recipients. Pregnancy was diagnosed at day 30 by hormonal assay and at days 55 and 140 of gestation by ultrasonography and pregnancies were allowed to go to term. A total of nine ewes received 27 embryos (3 embryos/ewe). Five ewes received 15 embryos at morula stage and four ewes received 12 embryos at blastocyst stage. From those received morula stage embryos one was pregnant on day 30 (20%), though no pregnancy was diagnosed on each of days 55 and 140. While from those received blastocyst stage embryos, three ewes were pregnant on day 30 (75%) and two ewes (50%) remained pregnant on each of days 55 and 140. In conclusion, day 4 IVM-IVF morula stage embryos had a lower survival rate than did day 7 IVM-IVF blastocysts embryos, following transfer to the synchronized recipient ewes.

  1. [Comparison of clinical outcomes of blastocysts derived from non-top quality embryos and cleavage-stage high-quality embryos in frozen-thawed embryo transfer cycles].

    PubMed

    Xu, Li-Juan; Chen, Xin; Tian, Xiao-Long; Liu, Yu-Dong; Wang, Nan; Ye, De-Sheng; Guo, Ping-Ping; Chen, Shi-Ling

    2015-04-01

    To explore the developmental potential of embryos at different developmental days and provide evidence for blastocyst culture of non-top quality cleavage stage embryos in frozen-thawed embryo transfer (FET) cycles. The clinical data of 687 FET cycles were retrospectively analyzed. According to the embryo freezing time, the patients were divided into day 5 (D5) blastocyst group (n=87), day 6 (D6) blastocyst group (n=111) and day 3 cleavage-stage embryo (D3) group (n=489) with hormone replacement cycles or natural cycles for endometrial preparation. The clinical pregnancy rates, miscarriage rates, and implantation rates were compared between the 3 groups. The clinical pregnancy rate, miscarriage rate and implantation rate per transfer were 58.6%, 9.8%, and 42.9% in D5 group, 32.4%, 19.4%, and 23.3% in D6 group, and 44.9%, 16.4%, and 26.9% in D3 group, respectively. The clinical pregnancy rate and implantation rate were significantly higher in D5 group than in the other two groups (P<0.05). The D5 blastocysts derived from non-top quality D3 embryos after cryopreservation can have better clinical outcomes than those derived from D3 cleavage-stage embryos and D6 blastocysts, and are therefore a better option than D3 cleavage-stage embryos in FET cycles.

  2. Efficient harvesting methods for early-stage snake and turtle embryos.

    PubMed

    Matsubara, Yoshiyuki; Kuroiwa, Atsushi; Suzuki, Takayuki

    2016-04-01

    Reptile development is an intriguing research target for understating the unique morphogenesis of reptiles as well as the evolution of vertebrates. However, there are numerous difficulties associated with studying development in reptiles. The number of available reptile eggs is usually quite limited. In addition, the reptile embryo is tightly adhered to the eggshell, making it a challenge to isolate reptile embryos intact. Furthermore, there have been few reports describing efficient procedures for isolating intact embryos especially prior to pharyngula stage. Thus, the aim of this review is to present efficient procedures for obtaining early-stage reptilian embryos intact. We first describe the method for isolating early-stage embryos of the Japanese striped snake. This is the first detailed method for obtaining embryos prior to oviposition in oviparous snake species. Second, we describe an efficient strategy for isolating early-stage embryos of the soft-shelled turtle. © 2016 Japanese Society of Developmental Biologists.

  3. Axial differentiation and early gastrulation stages of the pig embryo.

    PubMed

    Hassoun, Romia; Schwartz, Peter; Feistel, Kerstin; Blum, Martin; Viebahn, Christoph

    2009-12-01

    Differentiation of the principal body axes in the early vertebrate embryo is based on a specific blueprint of gene expression and a series of transient axial structures such as Hensen's node and the notochord of the late gastrulation phase. Prior to gastrulation, the anterior visceral endoderm (AVE) of the mouse egg-cylinder or the anterior marginal crescent (AMC) of the rabbit embryonic disc marks the anterior pole of the embryo. For phylogenetic and functional reasons both these entities are addressed here as the mammalian anterior pregastrulation differentiation (APD). However, mouse and rabbit show distinct structural differences in APD and the molecular blueprint, making the search of general rules for axial differentiation in mammals difficult. Therefore, the pig was analysed here as a further species with a mammotypical flat embryonic disc. Using light and electron microscopy and in situ hybridisation for three key genes involved in early development (sox17, nodal and brachyury), two axial structures of early gastrulation in the pig were identified: (1) the anterior hypoblast (AHB) characterised by increased cellular height and density and by sox17 expression, and (2) the early primitive streak characterised by a high pseudostratified epithelium with an almost continuous but unusually thick basement membrane, by localised epithelial-mesenchymal transition, and by brachyury expression in the epiblast. The stepwise appearance of these two axial structures was used to define three stages typical for mammals at the start of gastrulation. Intriguingly, the round shape and gradual posterior displacement of the APD in the pig appear to be species-specific (differing from all other mammals studied in detail to date) but correlate with ensuing specific primitive streak and extraembryonic mesoderm development. APD and, hence, the earliest axial structure presently known in the mammalian embryo may thus be functionally involved in shaping extraembryonic membranes and

  4. Vitrification can modify embryo cleavage stage after warming. Should we change endometrial preparation?

    PubMed

    Cercas, R; Villas, C; Pons, I; Braña, C; Fernandez-Shaw, S

    2012-12-01

    Studies have shown that embryo metabolism and cell cleavage after warming vitrified embryos is faster than after thawing frozen embryos. We study vitrified embryo transfer (VET) results depending on the developmental stage of warmed embryos and the duration of progesterone treatment before embryo transfer. We designed a prospective study, patients were randomized in two groups, starting progesterone three (D + 3) or four days (D + 4) before embryo transfer. We recruited 88 patients with embryos vitrified on day 3. We didn't find statitistical differences in pregnancy rate when we transferred embryos in D + 3 vs D + 4 (38.2 % vs 40.5 % p ≥ 0.05). The day after warming, 54.6 % of embryos had developed to morula or early blastocyst, 32.4 % to cleavage stage and 13 % didn't cleave. Transfers were with morula/blastocysts stage embryos (52.1 %; n:37), cleavage stage embryos (18.3 %; n:13) or mixed (29.6 %; n:21). Implantation rate was significantly higher in morula/blastocyst stage than in cleavage stage or mixed transfers (44 %, 22 % and 16.3 %; p = 0.011). Pregnancy and implantation rates were significantly higher in morula/blastocyst transfers on D + 4 than on D + 3 (68.7 % and 64.7 % vs 33.3 %, and 33.3 %, p = 0.033 and p = 0.034). Our findings suggest that a majority of embryos will develop to morula/blastocyst stage after warming. VET results with morula/blastocysts, and after four days of progesterone supplementation, are better than with cleavage stage embryos.

  5. 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

  6. Assessment of early cleaving in vitro fertilized human embryos at the 2-cell stage before transfer improves embryo selection.

    PubMed

    Sakkas, D; Percival, G; D'Arcy, Y; Sharif, K; Afnan, M

    2001-12-01

    To determine the most viable embryos for transfer. Study 1: Preselection of early-cleaving 2-cell embryos for transfer. Study 2: Alternating weeks during which preselection was performed and not performed. ART program, Birmingham Women's Hospital, Birmingham, United Kingdom. Patients undergoing IVF or ICSI cycles with transfer on day 2. Culture of all fertilized embryos. Number of fertilized embryos cleaving to the 2-cell stage on day 1, embryo quality, implantation rates, and pregnancy rates. Patients with early-cleaving 2-cell embryos had significantly higher pregnancy and implantation rates (45 of 100 [45.0%] and 58 of 219 [25.5%], respectively) than did patients without early-cleaving 2-cell embryos (31 of 130 [23.8%] and 43 of 290 [14.8%], respectively). In weeks during which preselection was used, the overall pregnancy and implantation rates of the clinic improved. The presence of early-cleaving 2-cell embryos improves a patient's chance of achieving pregnancy. Use of more stringent embryo selection criteria can improve overall pregnancy rates.

  7. Cleavage stage versus blastocyst stage embryo transfer in assisted reproductive technology.

    PubMed

    Glujovsky, Demián; Farquhar, Cindy; Quinteiro Retamar, Andrea Marta; Alvarez Sedo, Cristian Roberto; Blake, Deborah

    2016-06-30

    Advances in cell culture media have led to a shift in in vitro fertilisation (IVF) practice from cleavage stage embryo transfer to blastocyst stage transfer. The rationale for blastocyst transfer is to improve both uterine and embryonic synchronicity and enable self selection of viable embryos, thus resulting in better live birth rates. To determine whether blastocyst stage (day 5 to 6) embryo transfers improve the live birth rate, and other associated outcomes, compared with cleavage stage (day 2 to 3) embryo transfers. We searched the Cochrane Gynaecology and Fertility Group Specialised Register of controlled trials, Cochrane Central Register of Controlled Trials (CENTRAL; the Cochrane Library; 2016, Issue 4), MEDLINE, EMBASE, PsycINFO, CINAHL, and Bio extracts from inception to 4th April 2016. We also searched registers of ongoing trials and the reference lists of studies retrieved. We included randomised controlled trials (RCTs) which compared the effectiveness of blastocyst versus cleavage stage transfers. We used standard methodological procedures recommended by Cochrane. Our primary outcomes were live birth and cumulative clinical pregnancy rates. Secondary outcomes were clinical pregnancy, multiple pregnancy, high order pregnancy, miscarriage, failure to transfer embryos, and embryo freezing. We assessed the overall quality of the evidence for the main comparisons using GRADE methods. We included 27 RCTs (4031 couples or women).The live birth rate following fresh transfer was higher in the blastocyst transfer group (odds ratio (OR) 1.48, 95% confidence interval (CI) 1.20 to 1.82; 13 RCTs, 1630 women, I(2) = 45%, low quality evidence) following fresh transfer. This suggests that if 29% of women achieve live birth after fresh cleavage stage transfer, between 32% and 42% would do so after fresh blastocyst stage transfer.There was no evidence of a difference between the groups in rates per couple of cumulative pregnancy following fresh and frozen

  8. Cleavage stage versus blastocyst stage embryo transfer in assisted reproductive technology.

    PubMed

    Glujovsky, Demián; Blake, Debbie; Farquhar, Cindy; Bardach, Ariel

    2012-07-11

    Advances in cell culture media have led to a shift in in vitro fertilization (IVF) practice from early cleavage embryo transfer to blastocyst stage transfer. The rationale for blastocyst culture is to improve both uterine and embryonic synchronicity and enable self selection of viable embryos thus resulting in higher implantation rates. To determine if blastocyst stage (Day 5 to 6) embryo transfers (ETs) improve live birth rate and other associated outcomes compared with cleavage stage (Day 2 to 3) ETs. Cochrane Menstrual Disorders and Subfertility Group Specialised Register of controlled trials, Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library), MEDLINE, EMBASE and Bio extracts. The last search date was 21 February 2012. Trials were included if they were randomised and compared the effectiveness of early cleavage versus blastocyst stage transfers. Of the 50 trials that were identified, 23 randomised controlled trials (RCTs) met the inclusion criteria and were reviewed (five new studies were added in this update). The primary outcome was rate of live birth. Secondary outcomes were rates per couple of clinical pregnancy, cumulative clinical pregnancy, multiple pregnancy, high order pregnancy, miscarriage, failure to transfer embryos and cryopreservation. Quality assessment, data extraction and meta-analysis were performed following Cochrane guidelines. Twelve RCTs reported live birth rates and there was evidence of a significant difference in live birth rate per couple favouring blastocyst culture (1510 women, Peto OR 1.40, 95% CI 1.13 to 1.74) (Day 2 to 3: 31%; Day 5 to 6: 38.8%, I(2) = 40%). This means that for a typical rate of 31% in clinics that use early cleavage stage cycles, the rate of live births would increase to 32% to 42% if clinics used blastocyst transfer.There was no difference in clinical pregnancy rate between early cleavage and blastocyst transfer in the 23 RCTs (Peto OR 1.14, 95% CI 0.99 to 1.32) (Day 2 to 3: 38

  9. Pregnancy outcome after blastocyst stage transfer comparing to early cleavage stage embryo transfer.

    PubMed

    Aziminekoo, Elham; Mohseni Salehi, Maryam Sadat; Kalantari, Vahid; Shahrokh Tehraninejad, Ensieh; Haghollahi, Fedyeh; Hossein Rashidi, Batool; Zandieh, Zahra

    2015-01-01

    Blastocyst transfer has been introduced as an alternative for improving the chance for in vitro fertilizations (IVF) implantation. The present study was to evaluate pregnancy rates when embryo transfer was performed either on day 2-3 (cleavage stage) or on day 4-5 (blastocyst stage). This randomized clinical trial included 118 infertile women. All the study subjects underwent controlled ovarian stimulation using a long protocol and randomized into two groups. BS group (n = 57), the culture was extended to day 5 (blastocyst stage) and in the CS-group (n = 61), embryo culture was continued to day 3 (cleavage stage). Ongoing pregnancies, abortion, implantation rate were evaluated. No significant differences were seen in the pregnancy rate between the two groups (33.3% in the BS group versus 27.9% in the CS group; p = 0.519). Abortion, implantation rate in two groups are not significant. Despite the lack of statistical difference between the two study groups, our data suggest that blastocyst transfer may be associated with a higher pregnancy and an overall better implantation rates. However, further studies with larger sample size are mandatory to confirm these findings.

  10. The effect of superovulation on the contributions of individual blastomeres from 2-cell stage CF1 mouse embryos to the blastocyst.

    PubMed

    Katayama, Mika; Roberts, R Michael

    2010-01-01

    It remains controversial whether blastomeres of 2-cell stage mouse embryos show bias in their contribution to the blastocyst and whether there is any effect of superovulation. Two-cell stage embryos from CF1 mice were derived by either natural breeding (N) or superovulation (S) and cultured in vitro. At blastocyst, inner cell mass and trophectoderm were distinguished by Cdx2 and Oct4 immunostaining. A fluorescent dye (CM-Dil) was also used to tag individual blastomeres at the 2-cell stage, and the descendant cells identified by their red fluorescence. S and N embryos developed to blastocyst at the same rate and contained a similar number of cells. However, with S embryos, the descendants of the blastomere labeled with CM-DiI contributed predominantly to either the embryonic or abembryonic pole about 70% of the time, whereas most N embryos displayed random patterning, with no restriction to one or other of the poles. In S-embryos, but not N-embryos, the leading blastomere at second cleavage contributed preferentially to the embryonic pole of the blastocyst and the lagging blastomere to the abembryonic pole and hence mural trophectoderm. In addition, a tetrahedral rather than a flat morphology at the 4-cell stage of S-embryos was strongly biased to displaying the embryonic/abembryonic pattern at blastocyst. In contrast, S-embryos lacking a zona pellucida resembled N embryos in their patterning. In CF1 mice, superovulation has little effect on development to blastocyst, but enforces a greater degree of lineage restriction than natural breeding, most likely through constraints imposed by the zona pellucida.

  11. Endoderm/mesoderm multiplication rates in stage 5-12 chick embryos

    SciTech Connect

    Rosenquist, G.C.

    1982-01-01

    Multiplication rates for the endoderm/mesoderm layer of the head-process to 17-somite-stage chick embryo were studied by implanting essentially identical transplants labeled with tritiated thymidine into paired recipient embryos. One recipient was fixed as soon as the transplant had healed (after 30 min) and the other was reincubated an additional 3.5 to 22.5 hr; the ratios of labeled cells in the paired embryos provided points on a graph that indicated that doubling of endoderm/mesoderm cells in head-process-stage chick embryos occurs at approximately 4.0 and 17.2 hr of reincubation.

  12. Does IVF cleavage stage embryo quality affect pregnancy complications and neonatal outcomes in singleton gestations after double embryo transfers?

    PubMed

    Zhu, Jinliang; Lian, Ying; Li, Ming; Chen, Lixue; Liu, Ping; Qiao, Jie

    2014-12-01

    Embryo quality is associated with successful implantation and live births. Our retrospective study was carried out to determine whether or not cleavage stage embryo quality affects the miscarriage rate, pregnancy complications and neonatal outcomes of singletons conceived with assisted reproduction technology. The current study included 11,721 In Vitro Fertilization-Embryo Transfer cycles (IVF-ET) between January 2009 (the date at which electronic medical records were implemented at our center) and March 2013. Only women < 40 years of age undergoing their first fresh embryo transfer cycle using non-donor oocytes were included. Our study indicated that the transfer of poor-quality embryos resulted in higher miscarriage (19.77% vs. 13.28%, p = 0.02) and lower ongoing pregnancy rates (15.33% vs. 48.06%, p < 0.001). Logistic regression analysis performed on data derived from 744 cycles culminating in miscarriages versus 4,333 cycles culminating in live births, suggested that embryo quality (p = 0.04) is significantly associated with miscarriage rate after adjusting for other confounding factors. Moreover, there were no differences in the mean birth weight, low birth weight (<2,500 g), very low birth weight (<1,500 g), gestational age, preterm delivery (<37 weeks), very preterm delivery (<32 weeks), congenital malformations, small-for-gestational-age singletons (SGA), and large-for-gestational-age singleton (LGA) rate (p > 0.05). Similarly, pregnancy complications resulting from poor-quality embryos were not different from good-quality embryos (4.04% vs. 2.57 %, p = 0.33). Finally, logistic regression suggested that embryo quality was not significantly associated with pregnancy complications after adjusting for other confounding factors (p = 0.40). Our study suggests that transfer of poor-quality embryos did not increase the risk of adverse outcomes; however, the quality of cleavage stage embryos significantly affected the miscarriage rate

  13. A simple breeding protocol for the procurement of accurately staged rat donor embryos for neural transplantation.

    PubMed

    Weyrauch, U M; Torres, E M; Baird, A L; Dunnett, S B

    2009-01-01

    Obtaining accurately staged rat embryos can be difficult because of the variety of breeding protocols employed and because precise staging cannot be confirmed until excision of the embryos from the dam. The detection of estrus, pairing of animals, and confirmation of pregnancies is generally left to commercial suppliers, as in-house breeding can be laborious and unpredictable. Here we describe a simple, reliable in-house breeding protocol for the generation of accurately staged embryos as assessed by measurements of average crown to rump length (CRL).

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

    USDA-ARS?s Scientific Manuscript database

    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...

  16. 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...

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

    PubMed

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

    2013-03-15

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

  18. Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

    PubMed Central

    Raissig, Michael T.; Gagliardini, Valeria; Jaenisch, Johan; Grossniklaus, Ueli; Baroux, Célia

    2013-01-01

    In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays. PMID:23770918

  19. Efficient and rapid isolation of early-stage embryos from Arabidopsis thaliana seeds.

    PubMed

    Raissig, Michael T; Gagliardini, Valeria; Jaenisch, Johan; Grossniklaus, Ueli; Baroux, Célia

    2013-06-07

    In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

  20. Mouse embryos stressed by physiological levels of osmolarity become arrested in the late 2-cell stage before entry into M phase.

    PubMed

    Wang, Fang; Kooistra, Megan; Lee, Martin; Liu, Lin; Baltz, Jay M

    2011-10-01

    Preimplantation mouse embryos of many strains become arrested at the 2-cell stage if the osmolarity of culture medium that normally supports development to blastocysts is raised to approximately that of their normal physiological environment in the oviduct. Arrest can be prevented if molecules that serve as "organic osmolytes" are present in the medium, because organic osmolytes, principally glycine, are accumulated by embryos to provide intracellular osmotic support and regulate cell volume. Medium with an osmolarity of 310 mOsM induced arrest of approximately 80% of CF1 mouse embryos at the 2-cell stage, in contrast to the approximately 100% that progressed beyond the 2-cell stage at 250 or 301 mOsM with glycine. The nature of this arrest induced by physiological levels of osmolarity is unknown. Arrest was reversible by transfer to lower-osmolarity medium at any point during the 2-cell stage, but not after embryos would normally have progressed to the 4-cell stage. Cessation of development likely was not due to apoptosis, as shown by lack of external annexin V binding, detectable cytochrome c release from mitochondria, or nuclear DNA fragmentation. Two-cell embryos cultured at 310 mOsM progressed through the S phase, and zygotic genome activation markers were expressed. However, most embryos failed to initiate the M phase, as evidenced by intact nuclei with decondensed chromosomes, low M-phase promoting factor activity, and an inactive form of CDK1, although a few blastomeres were arrested in metaphase. Thus, embryos become arrested late in the G(2) stage of the second embryonic cell cycle when stressed by physiological osmolarity in the absence of organic osmolytes.

  1. Mouse Embryos Stressed by Physiological Levels of Osmolarity Become Arrested in the Late 2-Cell Stage Before Entry into M Phase1

    PubMed Central

    Wang, Fang; Kooistra, Megan; Lee, Martin; Liu, Lin; Baltz, Jay M.

    2011-01-01

    Preimplantation mouse embryos of many strains become arrested at the 2-cell stage if the osmolarity of culture medium that normally supports development to blastocysts is raised to approximately that of their normal physiological environment in the oviduct. Arrest can be prevented if molecules that serve as “organic osmolytes” are present in the medium, because organic osmolytes, principally glycine, are accumulated by embryos to provide intracellular osmotic support and regulate cell volume. Medium with an osmolarity of 310 mOsM induced arrest of approximately 80% of CF1 mouse embryos at the 2-cell stage, in contrast to the approximately 100% that progressed beyond the 2-cell stage at 250 or 301 mOsM with glycine. The nature of this arrest induced by physiological levels of osmolarity is unknown. Arrest was reversible by transfer to lower-osmolarity medium at any point during the 2-cell stage, but not after embryos would normally have progressed to the 4-cell stage. Cessation of development likely was not due to apoptosis, as shown by lack of external annexin V binding, detectable cytochrome c release from mitochondria, or nuclear DNA fragmentation. Two-cell embryos cultured at 310 mOsM progressed through the S phase, and zygotic genome activation markers were expressed. However, most embryos failed to initiate the M phase, as evidenced by intact nuclei with decondensed chromosomes, low M-phase promoting factor activity, and an inactive form of CDK1, although a few blastomeres were arrested in metaphase. Thus, embryos become arrested late in the G2 stage of the second embryonic cell cycle when stressed by physiological osmolarity in the absence of organic osmolytes. PMID:21697513

  2. Transferring two grades I cleavage-stage embryo might not be a good protocol.

    PubMed

    Li, Mingzhao; Wang, Hui; Ma, Chun; Shi, Juanzi

    2017-07-01

    The aim of this study was to explore whether transferring two grades I cleavage-stage embryo was suitable for the patients in the first fresh transfer. This study included 202 single grades I cleavage-stage, 229 single grades III cleavage-stage, 743 single excellent blastocyst, 522 double grades I cleavage-stage, and 596 double grades III cleavage-stage embryo transfers. Main clinical outcomes: clinical pregnancy and twin-pregnancy rate. Among single excellent blastocyst, single grades I and single grades III group, the clinical pregnancy rate was significantly higher in single excellent blastocyst group than single grades I and grades III group (67.16% versus 42.08% versus 23.97%; p < 0.001). When transferred double grades I cleavage-stage embryos, the clinical pregnancy rate reached 68.20% which was no significant difference compared with the single excellent blastocyst group (67.16%). However, the twin-pregnancy rate was significantly higher in double grades I group than double grades III and single excellent blastocyst group (43.26% versus 26.70% versus 0.60%; p < 0.001). Because of higher twin-pregnancy incidence rate, transferring two grades I cleavage-stage embryo might not be a good protocol. Extended culture to blastocyst-stage could be considered for the patient with only two grades I cleavage-stage embryos.

  3. Early detection and staging of spontaneous embryo resorption by ultrasound biomicroscopy in murine pregnancy

    PubMed Central

    2014-01-01

    Background Embryo resorption is a major problem in human medicine, agricultural animal production and in conservation breeding programs. Underlying mechanisms have been investigated in the well characterised mouse model. However, post mortem studies are limited by the rapid disintegration of embryonic structures. A method to reliably identify embryo resorption in alive animals has not been established yet. In our study we aim to detect embryos undergoing resorption in vivo at the earliest possible stage by ultra-high frequency ultrasound. Methods In a longitudinal study, we monitored 30 pregnancies of wild type C57BI/6 mice using ultra-high frequency ultrasound (30-70 MHz), so called ultrasound biomicroscopy (UBM). We compared the sonoembryology of mouse conceptuses under spontaneous resorption and neighbouring healthy conceptuses and correlated the live ultrasound data with the respective histology. Results The process of embryo resorption comprised of four stages: first, the conceptus exhibited growth retardation, second, bradycardia and pericardial edema were observed, third, further development ceased and the embryo died, and finally embryo remnants were resorbed by maternal immune cells. In early gestation (day 7 and 8), growth retardation was characterized by a small embryonic cavity. The embryo and its membranes were ill defined or did not develop at all. The echodensity of the embryonic fluid increased and within one to two days, the embryo and its cavity disappeared and was transformed into echodense tissue surrounded by fluid filled caverns. In corresponding histologic preparations, fibrinoid material interspersed with maternal granulocytes and lacunae filled with maternal blood were observed. In later stages (day 9–11) resorption prone embryos were one day behind in their development compared to their normal siblings. The space between Reichert’s membrane and inner yolk sac membrane was enlarged The growth retarded embryos exhibited bradycardia and

  4. Early detection and staging of spontaneous embryo resorption by ultrasound biomicroscopy in murine pregnancy.

    PubMed

    Flores, Luis E; Hildebrandt, Thomas B; Kühl, Anja A; Drews, Barbara

    2014-05-10

    Embryo resorption is a major problem in human medicine, agricultural animal production and in conservation breeding programs. Underlying mechanisms have been investigated in the well characterised mouse model. However, post mortem studies are limited by the rapid disintegration of embryonic structures. A method to reliably identify embryo resorption in alive animals has not been established yet. In our study we aim to detect embryos undergoing resorption in vivo at the earliest possible stage by ultra-high frequency ultrasound. In a longitudinal study, we monitored 30 pregnancies of wild type C57BI/6 mice using ultra-high frequency ultrasound (30-70 MHz), so called ultrasound biomicroscopy (UBM). We compared the sonoembryology of mouse conceptuses under spontaneous resorption and neighbouring healthy conceptuses and correlated the live ultrasound data with the respective histology. The process of embryo resorption comprised of four stages: first, the conceptus exhibited growth retardation, second, bradycardia and pericardial edema were observed, third, further development ceased and the embryo died, and finally embryo remnants were resorbed by maternal immune cells. In early gestation (day 7 and 8), growth retardation was characterized by a small embryonic cavity. The embryo and its membranes were ill defined or did not develop at all. The echodensity of the embryonic fluid increased and within one to two days, the embryo and its cavity disappeared and was transformed into echodense tissue surrounded by fluid filled caverns. In corresponding histologic preparations, fibrinoid material interspersed with maternal granulocytes and lacunae filled with maternal blood were observed. In later stages (day 9-11) resorption prone embryos were one day behind in their development compared to their normal siblings. The space between Reichert's membrane and inner yolk sac membrane was enlarged The growth retarded embryos exhibited bradycardia and ultimately cessation of heart

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

    PubMed Central

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

    2016-01-01

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

  6. 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

  7. Optimized ex-ovo culturing of chick embryos to advanced stages of development.

    PubMed

    Cloney, Kellie; Franz-Odendaal, Tamara Anne

    2015-01-24

    Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos model organisms, such as the chicken, are often used. The chicken is an excellent model organism due to its low cost and minimal maintenance, however they present observational challenges because they are enclosed in an opaque eggshell. In order to properly view the embryo as it develops, the shell must be windowed or removed. Both windowing and ex ovo techniques have been developed to assist researchers in the study of embryonic development. However, each of the methods has limitations and challenges. Here, we present a simple, optimized ex ovo culture technique for chicken embryos that enables the observation of embryonic development from stage HH 19 into late stages of development (HH 40), when many organs have developed. This technique is easy to adopt in both undergraduate classes and more advanced research laboratories where embryo manipulations are conducted.

  8. 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.

  9. Distinct outcome of stage I lung adenocarcinoma with ACTN4 cell motility gene amplification.

    PubMed

    Noro, R; Honda, K; Tsuta, K; Ishii, G; Maeshima, A M; Miura, N; Furuta, K; Shibata, T; Tsuda, H; Ochiai, A; Sakuma, T; Nishijima, N; Gemma, A; Asamura, H; Nagai, K; Yamada, T

    2013-10-01

    Even if detected at an early stage, a substantial number of lung cancers relapse after curative surgery. However, no method for distinguishing such tumors has yet been established. The copy number of the actinin-4 (ACTN4) gene was determined by fluorescence in situ hybridization on tissue microarrays comprising 543 surgically resected adenocarcinomas of the lung. Amplification (an increase in the copy number by ≥ 2.0 fold) of the ACTN4 gene was detected in two of seven lung adenocarcinoma cell lines and 79 (15%) of 543 cases of pathological stage I-IV lung adenocarcinoma. Multivariate analysis revealed that ACTN4 gene amplification was the most significant independent factor associated with an extremely high risk of death (hazard ratio, 6.78; P = 9.48 × 10(-5), Cox regression analysis) among 290 patients with stage I lung adenocarcinoma. The prognostic significance of ACTN gene amplification was further validated in three independent cohorts totaling 1033 patients. Amplification of the ACTN4 gene defines a small but substantial subset of patients with stage I lung adenocarcinoma showing a distinct outcome. Such patients require intensive medical attention and might benefit from postoperative adjuvant chemotherapy.

  10. Clinical analysis of the patients with single fair cleavage-stage embryo on day 3.

    PubMed

    Li, Mingzhao; Wang, Hui; Xue, Xia; Shi, Juanzi

    2017-09-23

    This study aimed to explore an appropriate selection for the patients with single fair cleavage-stage embryo on day 3. This study included 469 fresh transfers and 220 frozen-thawed transfers from January 2014 to June 2016. Furthermore, in 72 patients who have only 4-6 fair embryos (4-5 blastomeres) on day 3, the blastocysts were cultured to day 5 for transfer. In the fresh transfers, the clinical pregnancy rate of 4-5 blastomeres group was significantly lower than 6-7 and 8-10 blastomeres group (5.88 vs. 30.13%, p<.001and 5.88 vs. 26.09%, p < .001). In the frozen-thawed transfers, the clinical pregnancy rate of 4-5 blastomeres group was also significantly lower than 6-7 and 8-10 blastomeres group (10.00 vs. 28.57%, p = .040 and 10.00 vs. 33.33%, p = .005). For the blastocyst transfers derived from fair embryos with 4-5 blastomeres, the clinical pregnancy rate was significantly higher than single and double fair embryo transfers of similar quality (44.44 vs. 7.04%, p < .001 and 44.44 vs. 28.09%, p = .013). For the patients with single fair embryo (6-7 blastomeres or 8-10 blastomeres), transfer at the cleavage stage is feasible. For the patients with single fair embryo (4-5 blastomeres), transfer of single fair embryo at the blastocyst stage or accumulating two fair embryos might be worthy of consideration.

  11. 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.

  12. Blastocyst vs cleavage-stage embryo transfer: systematic review and meta-analysis of reproductive outcomes.

    PubMed

    Martins, W P; Nastri, C O; Rienzi, L; van der Poel, S Z; Gracia, C; Racowsky, C

    2017-05-01

    Blastocyst transfer in assisted reproduction techniques could be advantageous because the timing of exposure of the embryo to the uterine environment is more analogous to a natural cycle and permits embryo self-selection after activation of the embryonic genome on day 3. Conversely, the in-vitro environment is likely to be inferior to that in vivo, and in-vitro culture beyond embryonic genomic activation could potentially harm the embryo. Our objective was to identify, appraise and summarize the available evidence comparing the effectiveness of blastocyst vs cleavage-stage embryo transfer. This was a systematic review and meta-analysis of randomized controlled trials (RCTs) comparing the transfer of blastocysts (days 5-6) with the transfer of cleavage-stage embryos (days 2-3) in women undergoing in-vitro fertilization or intracytoplasmic sperm injection. The last electronic searches were run on 1 August 2016. Abstracts and studies with a mean difference between the two study groups of > 0.5 for the number of embryos transferred were excluded. We screened 1187 records and assessed 33 potentially eligible studies. Twelve studies were included, comprising a total of 1200 women undergoing blastocyst transfer and 1218 undergoing cleavage-stage embryo transfer. We observed low-quality evidence of no significant difference of blastocyst transfer on live birth/ongoing pregnancy (relative risk (RR), 1.11 (95% CI, 0.92-1.35), 10 RCTs, 1940 women, I(2)  = 54%), clinical pregnancy (RR, 1.10 (95% CI, 0.93-1.31), 12 RCTs, 2418 women, I(2)  = 64%), cumulative pregnancy (RR, 0.89 (95% CI, 0.67-1.16), four RCTs, 524 women, I(2)  = 63%) and miscarriage (RR, 1.08 (95% CI, 0.74-1.56), 10 RCTs, 763 pregnancies, I(2)  = 0%). There was moderate-quality evidence of a decrease in the number of women with surplus embryos after the blastocyst-stage embryo transfer (RR, 0.78 (95% CI, 0.66-0.91)). Overall, the quality of the evidence was limited by the quality of the included

  13. Live births after polar body biopsy and frozen-thawed cleavage stage embryo transfer: case report

    PubMed Central

    Guimarães, Fernando; Roque, Matheus; Valle, Marcello; Kostolias, Alessandra; de Azevedo, Rodrigo A; Martinhago, Ciro D; Sampaio, Marcos; Geber, Selmo

    2016-01-01

    Pre-implantation genetic diagnosis (PGD) or screening (PGS) technology, has emerged and developed in the past few years, benefiting couples as it allows the selection and transfer of healthy embryos during IVF treatments. These techniques can be performed in oocytes (polar-body biopsy) or embryos (blastomere or trophectoderm biopsy). In this case report, we describe the first two live births to be published in Brazil after a polar-body (PB) biopsy. In case 1, a 42-year-old was submitted to PB biopsy with PGS due to advanced maternal age and poor ovarian reserve. Five MII oocytes underwent first and second polar body biopsy and four cleavage embryos were cryopreserved. The PGS analysis resulted in two euploid embryos (next generation sequence). A frozen-thawed embryo transfer (FET) was performed after endometrial priming and a healthy baby was delivered after a cesarean section (37 weeks, female, 3390g, 47.5 cm). In case 2, a 40-year old patient with balanced translocation and poor ovarian response was submitted to PB biopsy. Two MII oocytes underwent first and second polar body biopsy and two embryos were cryopreserved in cleavage stage. The analysis resulted in one euploid embryo that was transferred after endometrial priming. A preterm healthy baby (34 weeks, female, 2100g, 40 cm) was delivered via cesarean section. In conclusion, although the blastocyst biopsy is the norm when performing PGS/PGD during IVF treatments, other alternatives (as PB biopsy) should be considered in some specific situations. PMID:28050963

  14. Chromosome fragility at FRAXA in human cleavage stage embryos at risk for fragile X syndrome.

    PubMed

    Verdyck, Pieter; Berckmoes, Veerle; De Vos, Anick; Verpoest, Willem; Liebaers, Inge; Bonduelle, Maryse; De Rycke, Martine

    2015-10-01

    Fragile X syndrome (FXS), the most common inherited intellectual disability syndrome, is caused by expansion and hypermethylation of the CGG repeat in the 5' UTR of the FMR1 gene. This expanded repeat, also known as the rare fragile site FRAXA, causes X chromosome fragility in cultured cells from patients but only when induced by perturbing pyrimidine synthesis. We performed preimplantation genetic diagnosis (PGD) on 595 blastomeres biopsied from 442 cleavage stage embryos at risk for FXS using short tandem repeat (STR) markers. In six blastomeres, from five embryos an incomplete haplotype was observed with loss of all alleles telomeric to the CGG repeat. In all five embryos, the incomplete haplotype corresponded to the haplotype carrying the CGG repeat expansion. Subsequent analysis of additional blastomeres from three embryos by array comparative genomic hybridization (aCGH) confirmed the presence of a terminal deletion with a breakpoint close to the CGG repeat in two blastomeres from one embryo. A blastomere from another embryo showed the complementary duplication. We conclude that a CGG repeat expansion at FRAXA causes X chromosome fragility in early human IVF embryos at risk for FXS.

  15. Live births after polar body biopsy and frozen-thawed cleavage stage embryo transfer: case report.

    PubMed

    Guimarães, Fernando; Roque, Matheus; Valle, Marcello; Kostolias, Alessandra; Azevedo, Rodrigo A de; Martinhago, Ciro D; Sampaio, Marcos; Geber, Selmo

    2016-12-01

    Pre-implantation genetic diagnosis (PGD) or screening (PGS) technology, has emerged and developed in the past few years, benefiting couples as it allows the selection and transfer of healthy embryos during IVF treatments. These techniques can be performed in oocytes (polar-body biopsy) or embryos (blastomere or trophectoderm biopsy). In this case report, we describe the first two live births to be published in Brazil after a polar-body (PB) biopsy. In case 1, a 42-year-old was submitted to PB biopsy with PGS due to advanced maternal age and poor ovarian reserve. Five MII oocytes underwent first and second polar body biopsy and four cleavage embryos were cryopreserved. The PGS analysis resulted in two euploid embryos (next generation sequence). A frozen-thawed embryo transfer (FET) was performed after endometrial priming and a healthy baby was delivered after a cesarean section (37 weeks, female, 3390g, 47.5 cm). In case 2, a 40-year old patient with balanced translocation and poor ovarian response was submitted to PB biopsy. Two MII oocytes underwent first and second polar body biopsy and two embryos were cryopreserved in cleavage stage. The analysis resulted in one euploid embryo that was transferred after endometrial priming. A preterm healthy baby (34 weeks, female, 2100g, 40 cm) was delivered via cesarean section. In conclusion, although the blastocyst biopsy is the norm when performing PGS/PGD during IVF treatments, other alternatives (as PB biopsy) should be considered in some specific situations.

  16. 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

    2017-07-01

    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.

  17. 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.

  18. 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. Copyright © 2011 Wiley Periodicals, Inc., A Wiley Company.

  19. Blastocyst-stage versus cleavage-stage embryo transfer in the first frozen cycles of OHSS-risk patients who deferred from fresh embryo transfer.

    PubMed

    Chen, Hua; Lv, Jie-Qiang; Wu, Xin-Mei; Xiao, Yu; Xi, Hai-Tao; Zhu, Chun-Fang; Huang, Jian-Ying; Zhang, Fan; Ge, Hong-Shan

    2015-01-01

    Elective cryopreservation of all embryos has been the most effective means to avoid developing ovarian hyperstimulation syndrome (OHSS). However, it is still unknown which stage is optimal for freezing and transferring into uterus in OHSS-risk patients. This study was undertaken to evaluate whether OHSS-risk patients could benefit from transferring blastocysts. A total of 162 women were allocated to cleavage-stage embryo transfer (ET) (group A = 70) and blastocysts transfer (group B = 92) on the basis of patients' voluntary in their first frozen cycles. Although the mean number of transferred embryos in group A was significantly more than those in group B (2.37 ± 0.52 versus 2.11 ± 0.52, p < 0.05), the clinical pregnancy rates, implantation rates and live birth rates in group B were significantly higher than those in group A (47.83% versus 31.43%, p < 0.05; 31.44% versus 18.67%, p < 0.05; 40.21% versus 27.14%, p < 0.05), and the multiple pregnancy rates in both groups were comparable (34.09% versus 36.36%, p > 0.05). The observed results in OHSS-risk population allow us to take a position in favor of blastocyst transfer, thus pregnancy and live birth could be achieved with fewer ETs and in a shorter time frame.

  20. Culture system for embryos of blue-breasted quail from the blastoderm stage to hatching.

    PubMed

    Ono, Tamao; Nakane, Yoshifumi; Wadayama, Takahiro; Tsudzuki, Masaoki; Arisawa, Kenjiro; Ninomiya, Shoko; Suzuki, Toshihiko; Mizutani, Makoto; Kagami, Hiroshi

    2005-01-01

    The blue-breasted quail (Coturnix chinensis), the smallest species in the order Galliforms, is a candidate model animal for avian developmental engineering because it is precocious and prolific. This species requires 17 days to hatch and 8 to 9 weeks to mature to an adult body weight of about 50 g, whereas the Japanese quail (Coturnix japonica) requires 16 days to hatch and 6 to 8 weeks to mature to an adult body weight of 100 to 150 g. The early embryo is the most challenging embryonic stage in terms of culture and manipulation for avian biotechnology. We have evaluated various conditions for the culture of blue-breasted quail embryos from the blastoderm stage to hatching. A hatchability rate of 26% (10/39) is among the best of the various culture conditions examined in the present study and the embryo culture system should facilitate advances in avian biotechnology.

  1. (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 ...

  2. 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.

  3. (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 ...

  4. 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...

  5. 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...

  6. 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

  7. 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. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  8. Origin and destination of the median germ cells in late somite stage and early post-somite stage duck embryos.

    PubMed

    van Limborgh, J

    1975-11-01

    In continuation of preceding investigations, in 184 duck embryos of the developmental stages 18-27 according to Hamburger and Hamilton (about 85-140 hours of incubation) a study was done on the occurrence of median germ cells. These cells proved to be present in proportionally small numbers in the stages 19-26. Their numbers were independent of the sex of the embryos and did not show any relations whatever with changes in the numbers or distribution of the intragonadal germ cells. The temporary occurrence of the median germ cells could be shown to be due to the movement of the gonadal primordia from the splanchnopleure of the yolk sac to the ventro-medial surface of the mesonephros, and it was argued that ultimately all these germ cells reach the right of left gonad.

  9. Formation of embryos of the Earth-Moon system at the stage of rarefied condensations

    NASA Astrophysics Data System (ADS)

    Ipatov, S.

    2015-10-01

    The minimum value of the mass of the rarefied condensation that was a parent for the embryos of the Earth and the Moon could be about 0.02 of the Earth mass. There could be also another main collision of the parental condensation with another condensation, which changed the tilt of the Earth to its present value. Depending on eccentricities of planetesimals that collided with solid embryos of the Earth and the Moon, the Moon could acquire 0.04-0.3 of its mass at the stage of accumulation of solid bodies while the mass of the growing Earth increased by 10 times.

  10. Synchronization and superovulation of mature cycling gilts for the collection of pronuclear stage embryos

    PubMed Central

    Sommer, Jeffrey R.; Collins, E. Bruce; Estrada, Jose L.; Petters, Robert M.

    2007-01-01

    An efficient protocol was developed to synchronize and superovulate mature pigs for the collection of pronuclear stage embryos suitable for DNA microinjection. A timed and coordinated regimen of Lutalyse®, PG600® and Chorulon® along with daily checking for estrus allowed synchronization of groups of gilts having estrous cycles at regular intervals. Pigs 10 to 16 days after the beginning of standing estrus have been successfully synchronized into estrus using this protocol. A standard dose of each drug was used independent of size or age of the animal. One protocol averaged 38.9 ovulations and 31.1 1-cell embryos recovered per animal. PMID:17118586

  11. Nerve growth factor regulates axial rotation during early stages of chick embryo development.

    PubMed

    Manca, Annalisa; Capsoni, Simona; Di Luzio, Anna; Vignone, Domenico; Malerba, Francesca; Paoletti, Francesca; Brandi, Rossella; Arisi, Ivan; Cattaneo, Antonino; Levi-Montalcini, Rita

    2012-02-07

    Nerve growth factor (NGF) was discovered because of its neurotrophic actions on sympathetic and sensory neurons in the developing chicken embryo. NGF was subsequently found to influence and regulate the function of many neuronal and non neuronal cells in adult organisms. Little is known, however, about the possible actions of NGF during early embryonic stages. However, mRNAs encoding for NGF and its receptors TrkA and p75(NTR) are expressed at very early stages of avian embryo development, before the nervous system is formed. The question, therefore, arises as to what might be the functions of NGF in early chicken embryo development, before its well-established actions on the developing sympathetic and sensory neurons. To investigate possible roles of NGF in the earliest stages of development, stage HH 11-12 chicken embryos were injected with an anti-NGF antibody (mAb αD11) that binds mature NGF with high affinity. Treatment with anti-NGF, but not with a control antibody, led to a dose-dependent inversion of the direction of axial rotation. This effect of altered rotation after anti NGF injection was associated with an increased cell death in somites. Concurrently, a microarray mRNA expression analysis revealed that NGF neutralization affects the expression of genes linked to the regulation of development or cell proliferation. These results reveal a role for NGF in early chicken embryo development and, in particular, in the regulation of somite survival and axial rotation, a crucial developmental process linked to left-right asymmetry specification.

  12. Messenger RNAs in metaphase II oocytes correlate with successful embryo development to the blastocyst stage.

    PubMed

    Biase, Fernando Henrique; Everts, Robin Edward; Oliveira, Rosane; Santos-Biase, Weruska Karyna Freitas; Fonseca Merighe, Giovana Krempel; Smith, Lawrence Charles; Martelli, Lúcia; Lewin, Harris; Meirelles, Flávio Vieira

    2014-02-01

    The mRNAs accumulated in oocytes provide support for embryo development until embryo genomic activation. We hypothesized that the maternal mRNA stock present in bovine oocytes is associated with embryo development until the blastocyst stage. To test our hypothesis, we analyzed the transcriptome of the oocyte and correlated the results with the embryo development. Our goal was to identify genes expressed in the oocyte that correlate with its ability to develop to the blastocyst stage. A fraction of oocyte cytoplasm was biopsied using micro-aspiration and stored for further expression analysis. Oocytes were activated chemically, cultured individually and classified according to their capacity to develop in vitro to the blastocyst stage. Microarray analysis was performed on mRNA extracted from the oocyte cytoplasm fractions and correlated with its ability to develop to the blastocyst stage (good quality oocyte) or arrest at the 8-16-cell stage (bad quality oocyte). The expression of 4320 annotated genes was detected in the fractions of cytoplasm that had been collected from oocytes matured in vitro. Gene ontology classification revealed that enriched gene expression of genes was associated with certain biological processes: 'RNA processing', 'translation' and 'mRNA metabolic process'. Genes that are important to the molecular functions of 'RNA binding' and 'translation factor activity, RNA binding' were also enriched in oocytes. We identified 29 genes with differential expression between the two groups of oocytes compared (good versus bad quality). The content of mRNAs expressed in metaphase II oocytes influences the activation of the embryonic genome and enables further develop to the blastocyst stage.

  13. Quadrivalent asymmetry in reciprocal translocation carriers predicts meiotic segregation patterns in cleavage stage embryos.

    PubMed

    Zhang, Yueping; Zhu, Saijuan; Wu, Jialong; Liu, Suying; Sun, Xiaoxi

    2014-10-01

    The effect of quadrivalent geometry on meiotic behaviour was evaluated. Segregation patterns of 404 cleavage stage embryos from 40 reciprocal translocation carriers undergoing 75 PGD cycles were analysed according to the asymmetric degree of quadrivalent. The percentage of alternate products with severe asymmetric quadrivalents was significantly lower than patients with mild asymmetric quadrivalents (22.5% versus 38.7%, P = 0.001). The incidence of 3:1 products was significantly higher in patients with severe compared with mild asymmetric quadrivalents (23.1% versus 12.2%, P = 0.004). The incidence of adjacent 1 (25.8% versus 24.3%), 2 (11.5% versus 12.6%) and 4:0/other segregation products (17.0% versus 12.2%) were not statistically significantly different between embryos from patients with severe or mild asymmetric quadrivalents. After adjusting for the confounder of sex using a logistic regression model, the odds of alternate embryos is about one-half for carriers classified as severe (OR 0.456, 95% CI 0.291 to 0.705), and the odds of 3:1 embryos is 2.2 times higher for carriers with severe asymmetric quadrivalents (OR 2.235, 95% CI 1.318 to 3.846). Our results suggest that the meiotic segregation pattern is related to the degree of asymmetry of specific quadrivalents. Severe asymmetric quadrivalents increases the risk of abnormal embryos.

  14. The early development of the nervous system in staged insectivore and primate embryos.

    PubMed

    Müller, F; O'Rahilly, R

    1980-10-01

    The early development of the nervous system was studied in stage embryos of hemicentetes semispinosus, Microcebus murinus, Alouatta seniculus, Cebus appella, Cebus albifrons, macaca mulatta, and Homo sapiens. The specimens were assigned to Carnegie stages 11-13. Serial transverse sections were examined and graphic reconstructions were prepared. The early development of the neural tube is basically similar in all the species investigated but differences in detail are noticeable. The mesencephalic flexure serves in all cases as a landmark for malpighi's tripartite subdivision of the brain. The nonhuman embryos seem to show a little more variation than the human in the closure of the neuropores in relation to somitic count. With the exception of the later-appearing terminal-vomeronasal component, all major portions of the neural crest as classified by O'Rahilly ('65) are represented in both the nonhuman and the human embryos studied. No crest is present at the level of rhombomere 1, nor at rhombomere 3 except in the platyrrhines and some human embryos, nor at rhombomere 5 except in certain human specimens. An indication of the division of the trigeminal ganglion into its primary divisions is rare at stage 11 (C. apella), may be visible at stage 12 (Alouatta, macaca, Homo), and is definite (in Homo) at stage 13. Ganglionic contributions from head ectoderm (epipharyngeal placodes), as previously described in the human and some other vertebrate embryos, were sought and found in Cebus apella. In both nonhuman and human, a tendency is noted whereby the rostral limit of the occipitospinal crest, high at stage 11, seems to descend relatively at stage 12, and ascend again at stage 13 (at least in the human) to become associated with the appearance of the accessory and hypoglossal nerves. In general, the motor components of the nerves are identifiable before the sensory elements, and, in the present study, nerve fibers were first observed in the human at stage 13 in some of

  15. Timing of cell division in human cleavage-stage embryos is linked with blastocyst formation and quality.

    PubMed

    Cruz, María; Garrido, Nicolás; Herrero, Javier; Pérez-Cano, Inmaculada; Muñoz, Manuel; Meseguer, Marcos

    2012-10-01

    Noninvasive markers of embryo quality are being sought to improve IVF success. The present study aimed to discover possible associations between embryo division kinetics in the cleavage stage, the subsequent ability of human embryos to reach the blastocyst stage and the resulting blastocyst morphology. A retrospective cohort study analysed 834 embryos from 165 oocyte donation couples using a time-lapse monitoring system that allowed the recording of the exact timings for key events related to embryo development. Timing parameters were categorized into four quartiles. The probability of an embryo developing to a blastocyst was linked to a strict chronology of development. To further evaluate the relationships between these morphokinetic parameters and subsequent blastocyst formation, the ensuing blastocyst morphology was compared with a viability score based on a hierarchical classification of the cleavage-stage morphokinetic parameters. It is concluded that the kinetics of early embryo development and the potential for human embryos to develop to the blastocyst stage on day 5 are closely related and that time-lapse-based evaluation of the exact timing of early events in embryo development is a promising tool for the prediction of blastocyst formation and quality according to the proposed model.

  16. Producing fully ES cell-derived mice from eight-cell stage embryo injections.

    PubMed

    DeChiara, Thomas M; Poueymirou, William T; Auerbach, Wojtek; Frendewey, David; Yancopoulos, George D; Valenzuela, David M

    2010-01-01

    In conventional methods for the generation of genetically modified mice, gene-targeted embryonic stem (ES) cells are injected into blastocyst-stage embryos or are aggregated with morula-stage embryos, which are then transferred to the uterus of a surrogate mother. F0 generation mice born from the embryos are chimeras composed of genetic contributions from both the modified ES cells and the recipient embryos. Obtaining a mouse strain that carries the gene-targeted mutation requires breeding the chimeras to transmit the ES cell genetic component through the germ line to the next (F1) generation (germ line transmission, GLT). To skip the chimera stage, we developed the VelociMouse method, in which injection of genetically modified ES cells into eight-cell embryos followed by maturation to the blastocyst stage and transfer to a surrogate mother produces F0 generation mice that are fully derived from the injected ES cells and exhibit a 100% GLT efficiency. The method is simple and flexible. Both male and female ES cells can be introduced into the eight-cell embryo by any method of injection or aggregation and using all ES cell and host embryo combinations from inbred, hybrid, and outbred genetic backgrounds. The VelociMouse method provides several unique opportunities for shortening project timelines and reducing mouse husbandry costs. First, as VelociMice exhibit 100% GLT, there is no need to test cross chimeras to establish GLT. Second, because the VelociMouse method permits efficient production of ES cell-derived mice from female ES cells, XO ES cell subclones, identified by screening for spontaneous loss of the Y chromosome, can be used to generate F0 females that can be bred with isogenic F0 males derived from the original targeted ES cell clone to obtain homozygous mutant mice in the F1 generation. Third, as VelociMice are genetically identical to the ES cells from which they were derived, the VelociMouse method opens up myriad possibilities for creating mice with

  17. 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.

  18. Active loss of DNA methylation in two-cell stage goat embryos.

    PubMed

    Park, Jung S; Lee, Doosoo; Cho, Sunwha; Shin, Sang-Tae; Kang, Yong-Kook

    2010-01-01

    Early mammalian embryos are thought to gain nuclear totipotency through DNA methylation reprogramming (DMR). By this process, DNA methylation patterns acquired during gametogenesis that are unnecessary for zygotic development are erased. The DMR patterns of various mammalian species have been studied; however, they do not seem to have a conserved pattern. We examined early goat embryos to find conforming rules underlying mammalian DMR patterns. Immunocytochemical results showed that the overall level of DNA methylation was not greatly changed during the pronucleus stage. At the two-cell stage, active demethylation occurred and simultaneously affected both parental DNAs, resulting in a global loss of 5-methylcytosine. The level of DNA methylation was lowest in the four-cell stage, with increased de novo methylation during the eight-cell stage. Histone H3-lysine 9 was gradually trimethylated in the sperm-derived chromatin, continuing from the pronucleus stage through the two-cell stage. This goat DMR pattern is novel and distinct from the DMRs of other mammalian species. The more mammalian species we included for DMR analysis, the more multifarious patterns we obtained, adding an extra diversity each time to the known mammalian DMR patterns. Nevertheless, the evolutionary significance and developmental consequence of such diverse DMR patterns are currently unknown.

  19. Body movements during early stages of chick embryo under intermittent low oxygen environment

    NASA Astrophysics Data System (ADS)

    Moriya, Kenji; Chiba, Yuya; Shimouchi, Akito

    2017-07-01

    We have attempted to elucidate the characteristic pattern of body movements in early stages of chick embryos under intermittent low oxygen incubation environment. In order to achieve this aim, the oxygen control system that can be set arbitrary oxygen concentration was developed. We choose the 18% of O2 concentration and tried to measure the embryonic body movements. As a results, only one chick embryo in the early stages under intermittent 18% O2 environment (the cycle is 18%O2-10min and 21%O2-50min) was successfully recorded and its body movements were analyzed. The characteristic body movements, which are attributed to the instantaneous effect of low oxygen environment, compared with before and after normal O2 condition were not observed. Because the early stage embryos in which the significant organs aside from heart are not formed yet have a strong adaptation to environment changes, short hypoxic condition like a 10 min might not affect instantaneous embryonic physiological changes. Meanwhile, although the cyclic interval of the large body movements becomes short in the normal development, it became long in 18%O2 condition. This result might indicate that intermittent low oxygen condition accumulatively influenced physiological function. Further improvements of accuracy in the oxygen control system and the calculation system of body movements, and further experiments under low oxygen conditions are required in the next step.

  20. Mouse embryo motion and embryonic development from the 2-cell to blastocyst stage using mechanical vibration systems.

    PubMed

    Asano, Yuka; Matsuura, Koji

    2014-06-01

    We investigated the effect of mechanical stimuli on mouse embryonic development from the 2-cell to blastocyst stage to evaluate physical factors affecting embryonic development. Shear stress (SS) applied to embryos using two mechanical vibration systems (MVSs) was calculated by observing microscopic images of moving embryos during mechanical vibration (MV). The MVSs did not induce any motion of the medium and the diffusion rate using MVSs was the same as that under static conditions. Three days of culture using MVS did not improve embryonic development. MVS transmitted MV power more efficiently to embryos than other systems and resulted in a significant decrease in development to the morula or blastocyst stage after 2 days. Comparison of the results of embryo culture using dynamic culture systems demonstrated that macroscopic diffusion of secreted materials contributes to improved development of mouse embryos to the blastocyst stage. These results also suggest that the threshold of SS and MV to induce negative effects for mouse embryos at stages earlier than the blastocyst may be lower than that for the blastocyst, and that mouse embryos are more sensitive to physical and chemical stimuli than human or pig embryos because of their thinner zona pellucida.

  1. Exploring uptake and biodistribution of polystyrene (nano)particles in zebrafish embryos at different developmental stages.

    PubMed

    van Pomeren, M; Brun, N R; Peijnenburg, W J G M; Vijver, M G

    2017-09-01

    In ecotoxicology, it is continuously questioned whether (nano)particle exposure results in particle uptake and subsequent biodistribution or if particles adsorb to the epithelial layer only. To contribute to answering this question, we investigated different uptake routes in zebrafish embryos and how they affect particle uptake into organs and within whole organisms. This is addressed by exposing three different life stages of the zebrafish embryo in order to cover the following exposure routes: via chorion and dermal exposure; dermal exposure; oral and dermal exposure. How different nanoparticle sizes affect uptake routes was assessed by using polystyrene particles of 25, 50, 250 and 700nm. In our experimental study, we showed that particle uptake in biota is restricted to oral exposure, whereas the dermal route resulted in adsorption to the epidermis and gills only. Ingestion followed by biodistribution was observed for the tested particles of 25 and 50nm. The particles spread through the body and eventually accumulated in specific organs and tissues such as the eyes. Particles larger than 50nm were predominantly adsorbed onto the intestinal tract and outer epidermis of zebrafish embryos. Embryos exposed to particles via both epidermis and intestine showed highest uptake and eventually accumulated particles in the eye, whereas uptake of particles via the chorion and epidermis resulted in marginal uptake. Organ uptake and internal distribution should be monitored more closely to provide more in depth information of the toxicity of particles. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  2. 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.

  3. 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.

  4. Obstetric and neonatal outcomes in blastocyst-stage biopsy with frozen embryo transfer and cleavage-stage biopsy with fresh embryo transfer after preimplantation genetic diagnosis/screening.

    PubMed

    Jing, Shuang; Luo, Keli; He, Hui; Lu, Changfu; Zhang, Shuoping; Tan, Yueqiu; Gong, Fei; Lu, Guangxiu; Lin, Ge

    2016-07-01

    To study whether embryo biopsy for preimplantation genetic diagnosis/preimplantation genetic screening (PGD/PGS) can influence pregnancy complications and neonatal outcomes. Retrospective analysis. University-affiliated center. This study included data from women and their neonates born after PGD/PGS (n = 317). Questionnaires were designed to obtain information relating to pregnancy complications and neonatal outcomes. Two major strategies for PGD/PGS were evaluated. Blastocyst-stage biopsy and frozen embryo transfer (BB-FET) was carried out in 166 patients, and cleavage-stage biopsy and fresh embryo transfer (CB-ET) was carried out in 129 patients. The incidence of gestational hypertension was significantly higher in BB-FET compared with in CB-ET (9.0% vs. 2.3%, adjusted odds ratio [OR] and 95% confidence interval [CI], 4.85 [1.34, 17.56]). In twins, the birthweight (median [range], 2.70 kg [1.55-3.60 kg] vs. 2.50 kg [1.23-3.75 kg]) was higher in BB-FET than in CB-ET and the gestational age was longer in BB-FET than in CB-ET (median [range], 36.71 weeks [31.14-39.29 weeks] vs. 35.57 weeks [30.57-38.43 weeks]). There was no difference in the incidence of singleton births between the two groups except in the incidence of preterm births (28-37 weeks; 5.3% vs. 16.5% in CB-ET and BB-FET). No significant differences were detected in the incidence of perinatal deaths, birth defects, gender of neonates, and large for gestational age in both singletons and twins, although the numbers of some events were small. BB-FET is associated with a higher incidence of gestational hypertension but better neonatal outcomes compared with CB-ET, especially in twins. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  5. Culturing surplus poor-quality embryos to blastocyst stage have positive predictive value of clinical pregnancy rate.

    PubMed

    Zhu, Hai Bo; Zhang, Zhi Hong; Fadlalla, Elfateh; Wang, Rui Xue; Geng, Dong Feng; Liu, Rui Zhi

    2014-09-01

    Clinical reproductive centers produce large amounts of surplus poor-quality embryos annually, how to maximize the use of these embryos, and which of them have the potential to develop into blastocyst stage and influencing factors were lack of systematic research. To investigate the fate of surplus poor-quality embryos which were cultured to obtain blastocyst, determine the factors which may influence the blastulation, and discuss their application in predicting of the pregnancy outcomes. Day 3 (D3) after embryo transfer and freezing, surplus poor-quality embryos from IVF/ICSI cycles were cultured to blastocyst by the sequential method, then the blastulation outcomes were observed. Focusing on the blastulation rate of those embryos with different number cells and different embryonic grade; and last the relationship between the pregnancy outcomes of remained poor-quality embryos with successful blastulation or failed blastulation groups were studied. Of 127 patients with 569 poor-quality in vitro cultured embryos, there were formation of 248 blastocysts from 91 patients (43.59%), which lead to development of 138 high-quality blastocysts (24.25%). With the increase in cells number of D 3 blastomeres, the blastulation rate gradually increased, that, 7-cell blastomeres blastulation rate was the highest (70.59%), and 8-cell blastomeres is a little below (70.37%); while the embryonic levels and blastulation rate did not show this positive relationship. The clinical pregnancy rate and implantation rate of those who had successful blastulation (67.03% and 42.39%) were higher than of those who failed to develop to blastocyst (p=0.039). Day 3 poor-quality embryos with successful blastulation or with failed blastulation had predictive value on pregnancy outcomes. For embryo transfer 7-8 cells grade III-IV embryo is better than 4-5 cells grade I-II embryo, in case of lack good-quality embryos.

  6. 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

  7. Can repeated IVF-ICSI-cycles be avoided by using blastocysts developing from poor-quality cleavage stage embryos?

    PubMed

    Kaartinen, Noora; Das, Pia; Kananen, Kirsi; Huhtala, Heini; Tinkanen, Helena

    2015-03-01

    In many clinics, good-quality embryos are selected for embryo transfer and cryopreservation at the cleavage stage, and poor-quality embryos are discarded. The aim of this retrospective study was to examine how many repeated IVF cycles could be avoided by culturing the cleavage stage poor-quality embryos to blastocyst stage and transferring them after vitrification and warming (604 IVF and intracytoplasmic sperm injection [IVF-ICSI] cycles were included). Poor-quality cleavage stage embryos not eligible for transfer or cryopreservation were cultured until day 5 or 6, and those developing to the blastocyst stage were vitrified. The rate of vitrified blastocysts and clinical pregnancy and delivery rate of the warmed blastocysts was evaluated. The effect of the extended culture on the cumulative delivery rate, and the number of avoided new treatment cycles was calculated. The surplus blastocysts resulted in clinical pregnancy, spontaneous abortion and delivery rates of 24.6%, 27.3% and 17.2% respectively. The use of surplus blastocysts raised cumulative delivery rate from 43% to 47% and 53 repeated new cycles were avoided. This study shows that the cumulative delivery rate can be increased, and repeated IVF-ICSI treatments avoided by using blastocysts developing from poor-quality cleavage stage embryos, which otherwise would have been discarded.

  8. Development of lumbosacral spina bifida: three-dimensional computer graphic study of human embryos at Carnegie stage twelve.

    PubMed

    Haque, M; Ohata, K; Takami, T; Soares, S B; Aree, S N; Hakuba, A; Hara, M

    2001-11-01

    There is still some controversy as to whether sacral spina bifida in humans is the result of a defect of the primary or secondary neural tube. As somites are related to the development of vertebrae and the primary neural tube is related to the development of the spinal cord in embryos, it is very important to determine the number of somites in normal human embryos at the time of closure of the primary neural tube to understand the contribution of primary neural tube defects to the development of spina bifida. However, in the literature, the number of somites in stage 12 human embryos is still controversial. The aim of this study is to find the number of somites in human embryos at Carnegie stage 12. Four human embryos at Carnegie stage 12 were selected from the laboratory of the Congenital Anomaly Research Center in Japan. The neural tube and somites were reconstructed from their slices by a three-dimensional computer graphic reconstruction technique. The reconstructed embryos were examined from multidirectional magnified images. Thirty-three pairs of somites were present in all these reconstructed embryos. As the 33rd pair of somites corresponds to the fifth sacral segment, the presence of 33 pairs of somites at Carnegie stage 12 suggests that spina bifida develops from defects of the primary neural tube. Copyright 2001 S. Karger AG, Basel

  9. Correlation analysis of human embryo Le(Y) glycan antigen expression and embryo quality.

    PubMed

    Gu, Juan; Sui, Linlin; Ma, Yanni; Guo, Zhenzhen; Zhang, Man; Zhu, Chenyang; Cai, Zhu; Kong, Ying

    2017-07-01

    This study assessed the feasibility of using Le(Y) glycan secretion level in human embryos as a method of judging embryo quality. Embryo culture media from patients receiving in vitro fertilization-embryo transfer was collected, and quality scores of embryos were recorded. Secretions of Le(Y) in the culture media in different development stages (from 4-cell to 10-cell), embryos in the same development stage of the same patients (8-cell/I) and embryos in the same development stage of different patients (8-cell/I) were examined by dot-blot. Embryos were divided into a hypersecretion group and hyposecretion group, based on their Le(Y) secretion level. The embryo quality was evaluated by clinical observations, the number which developed to D3 cell stage and the number of successful embryo transplantations. Le(Y) secretion increased as embryos developed from 4-cell to 10-cell (P<0.05); secretion of Le(Y) of 8/I is not identical; development speed of embryos with different secretion level of Le(Y) was also different. The number of embryos which developed to 6-cell or higher was 82.2% in the Le(Y) hypersecretion group but only 60% in the hyposecretion group. The rate of successful transplantation was significantly higher in the hypersecretion group (71.1 vs. 40%). In conclusion, Le(Y) glycan secretion level in human embryos is closely related to embryo quality. Le(Y) may become a useful measure to evaluate embryo quality in the future.

  10. Efficient embryo transfer in the common marmoset monkey (Callithrix jacchus) with a reduced transfer volume: a non-surgical approach with cryopreserved late-stage embryos.

    PubMed

    Ishibashi, Hidetoshi; Motohashi, Hideyuki H; Kumon, Mami; Yamamoto, Kazuhiro; Okada, Hironori; Okada, Takashi; Seki, Kazuhiko

    2013-05-01

    Among primates, the common marmoset is suitable for primate embryology research. Its small body size, however, has delayed the technical development of efficient embryo transfer. Furthermore, three factors have been determined to adversely affect the performance of marmoset embryo transfer: nonsurgical approaches, the use of cryopreserved embryos, and the use of late-stage embryos. Here we performed embryo transfer under conditions that included the above three factors and using either a small (1 μl or less) or a large volume (2-3 μl) of medium. The pregnancy and birth rates were 50% (5/10) and 27% (3/11), respectively, when using the large volume, and 80% (8/10) and 75% (9/12), respectively, when using the small volume. The latter scores exceed those of previous reports using comparable conditions. Thus, it appears that these three previously considered factors could be overcome, and we propose that reducing the transfer volume to 1 μl or less is essential for successful marmoset embryo transfer.

  11. Low serum concentration in bovine embryo culture enhances early blastocyst rates on Day-6 with quality traits in the expanded blastocyst stage similar to BSA-cultured embryos.

    PubMed

    Murillo, A; Muñoz, M; Martín-González, D; Carrocera, S; Martínez-Nistal, A; Gómez, E

    2017-06-01

    In bovine, single in vitro embryo culture in protein-free medium from Day-6 to Day-7 leads to expanded blastocyst (XB) with improved pregnancy and birth rates after cryopreservation. Under these conditions, early blastocysts (EB) progress to the XB stage at higher rates than morulae (M). However, embryo production with BSA in culture prior to Day-6 leads to low EB rates. We investigated whether a very low FCS concentration (0.1%) in culture from Day-1 to Day-6 would improve EB rates and, subsequently, increase XB rates on Day-7 after single culture in protein-free medium. The quality of embryos produced was evaluated in terms of survival to cryopreservation, apoptosis percentage, lipid accumulation and transfer to recipients. On Day-6, EB rates from embryos cultured with FCS were higher than with BSA (P=0.022). On Day-7, XB rates were higher in embryos from Day-6 EB than from Day-6M, both with and without FCS (P<0.005). After vitrification/warming of Day-7 XB, 100% embryos survived at 24h in all treatments, and total cell number and apoptosis percentage were not affected by the presence of FCS or embryonic stage on Day-6. Cryopreserved and fresh embryos produced with FCS until Day-6, and then deprived of protein and cultured individually, led to pregnancies after ET. In conclusion, minute FCS concentration improves EB rates on Day-6 leading, after one-day single culture without protein, to more XBs. The quality of XB produced with FCS compares well with XB produced with BSA in terms of apoptosis, lipid accumulation and pregnancy. Copyright © 2017 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  12. Dissection and Downstream Analysis of Zebra Finch Embryos at Early Stages of Development

    PubMed Central

    Murray, Jessica R.; Stanciauskas, Monika E.; Aralere, Tejas S.; Saha, Margaret S.

    2014-01-01

    The zebra finch (Taeniopygiaguttata) has become an increasingly important model organism in many areas of research including toxicology1,2, behavior3, and memory and learning4,5,6. As the only songbird with a sequenced genome, the zebra finch has great potential for use in developmental studies; however, the early stages of zebra finch development have not been well studied. Lack of research in zebra finch development can be attributed to the difficulty of dissecting the small egg and embryo. The following dissection method minimizes embryonic tissue damage, which allows for investigation of morphology and gene expression at all stages of embryonic development. This permits both bright field and fluorescence quality imaging of embryos, use in molecular procedures such as in situ hybridization (ISH), cell proliferation assays, and RNA extraction for quantitative assays such as quantitative real-time PCR (qtRT-PCR). This technique allows investigators to study early stages of development that were previously difficult to access. PMID:24999108

  13. Maternal diabetes triggers DNA damage and DNA damage response in neurulation stage embryos through oxidative stress

    PubMed Central

    Dong, Daoyin; Yu, Jingwen; Wu, Yanqing; Fu, Noah; Villela, Natalia Arias; Yang, Peixin

    2015-01-01

    DNA damage and DNA damage response (DDR) in neurulation stage embryos under maternal diabetes conditions are not well understood. The purpose of this study was to investigate whether maternal diabetes and high glucose in vitro induce DNA damage and DDR in the developing embryo through oxidative stress. In vivo experiments were conducted by mating superoxide dismutase 1 (SOD1) transgenic male mice with wild-type (WT) female mice with or without diabetes. Embryonic day 8.75 (E8.75) embryos were tested for the DNA damage markers, phosphorylated histone H2A.X (p-H2A.X) and DDR signaling intermediates, including phosphorylated checkpoint 1 (p-Chk1), phosphorylated checkpoint 2 (p-Chk2), and p53. Levels of the same DNA damage markers and DDR signaling intermediates were also determined in the mouse C17.2 neural stem cell line. Maternal diabetes and high glucose in vitro significantly increased the levels of p-H2A.X. Levels of p-Chk1, p-Chk2, and p53, were elevated under both maternal diabetic and high glucose conditions. SOD1 overexpression blocked maternal diabetes-induced DNA damage and DDR in vivo. Tempol, a SOD1 mimetic, diminished high glucose-induced DNA damage and DDR in vitro. In conclusion, maternal diabetes and high glucose in vitro induce DNA damage and activates DDR through oxidative stress, which may contribute to the pathogenesis of diabetes-associated embryopathy. PMID:26427872

  14. Genome-Wide DNA Methylation Patterns of Bovine Blastocysts Developed In Vivo from Embryos Completed Different Stages of Development In Vitro

    PubMed Central

    Salilew-Wondim, Dessie; Fournier, Eric; Hoelker, Michael; Saeed-Zidane, Mohammed; Tholen, Ernst; Looft, Christian; Neuhoff, Christiane; Besenfelder, Urban; Havlicek, Vita; Rings, Franca; Gagné, Dominic; Sirard, Marc-André; Robert, Claude; A. Shojaei Saadi, Habib; Gad, Ahmed; Schellander, Karl; Tesfaye, Dawit

    2015-01-01

    Early embryonic loss and altered gene expression in in vitro produced blastocysts are believed to be partly caused by aberrant DNA methylation. However, specific embryonic stage which is sensitive to in vitro culture conditions to alter the DNA methylation profile of the resulting blastocysts remained unclear. Therefore, the aim of this study was to investigate the stage specific effect of in vitro culture environment on the DNA methylation response of the resulting blastocysts. For this, embryos cultured in vitro until zygote (ZY), 4-cell (4C) or 16-cell (16C) were transferred to recipients and the blastocysts were recovery at day 7 of the estrous cycle. Another embryo group was cultured in vitro until blastocyst stage (IVP). Genome-wide DNA methylation profiles of ZY, 4C, 16C and IVP blastocyst groups were then determined with reference to blastocysts developed completely under in vivo condition (VO) using EmbryoGENE DNA Methylation Array. To assess the contribution of methylation changes on gene expression patterns, the DNA methylation data was superimposed to the transcriptome profile data. The degree of DNA methylation dysregulation in the promoter and/or gene body regions of the resulting blastocysts was correlated with successive stages of development the embryos advanced under in vitro culture before transfer to the in vivo condition. Genomic enrichment analysis revealed that in 4C and 16C blastocyst groups, hypermethylated loci were outpacing the hypomethylated ones in intronic, exonic, promoter and proximal promoter regions, whereas the reverse was observed in ZY blastocyst group. However, in the IVP group, as much hypermethylated as hypomethylated probes were detected in gene body and promoter regions. In addition, gene ontology analysis indicated that differentially methylated regions were found to affected several biological functions including ATP binding in the ZY group, programmed cell death in the 4C, glycolysis in 16C and genetic imprinting and

  15. Genome-Wide DNA Methylation Patterns of Bovine Blastocysts Developed In Vivo from Embryos Completed Different Stages of Development In Vitro.

    PubMed

    Salilew-Wondim, Dessie; Fournier, Eric; Hoelker, Michael; Saeed-Zidane, Mohammed; Tholen, Ernst; Looft, Christian; Neuhoff, Christiane; Besenfelder, Urban; Havlicek, Vita; Rings, Franca; Gagné, Dominic; Sirard, Marc-André; Robert, Claude; Shojaei Saadi, Habib A; Gad, Ahmed; Schellander, Karl; Tesfaye, Dawit

    2015-01-01

    Early embryonic loss and altered gene expression in in vitro produced blastocysts are believed to be partly caused by aberrant DNA methylation. However, specific embryonic stage which is sensitive to in vitro culture conditions to alter the DNA methylation profile of the resulting blastocysts remained unclear. Therefore, the aim of this study was to investigate the stage specific effect of in vitro culture environment on the DNA methylation response of the resulting blastocysts. For this, embryos cultured in vitro until zygote (ZY), 4-cell (4C) or 16-cell (16C) were transferred to recipients and the blastocysts were recovery at day 7 of the estrous cycle. Another embryo group was cultured in vitro until blastocyst stage (IVP). Genome-wide DNA methylation profiles of ZY, 4C, 16C and IVP blastocyst groups were then determined with reference to blastocysts developed completely under in vivo condition (VO) using EmbryoGENE DNA Methylation Array. To assess the contribution of methylation changes on gene expression patterns, the DNA methylation data was superimposed to the transcriptome profile data. The degree of DNA methylation dysregulation in the promoter and/or gene body regions of the resulting blastocysts was correlated with successive stages of development the embryos advanced under in vitro culture before transfer to the in vivo condition. Genomic enrichment analysis revealed that in 4C and 16C blastocyst groups, hypermethylated loci were outpacing the hypomethylated ones in intronic, exonic, promoter and proximal promoter regions, whereas the reverse was observed in ZY blastocyst group. However, in the IVP group, as much hypermethylated as hypomethylated probes were detected in gene body and promoter regions. In addition, gene ontology analysis indicated that differentially methylated regions were found to affected several biological functions including ATP binding in the ZY group, programmed cell death in the 4C, glycolysis in 16C and genetic imprinting and

  16. 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.

  17. Evaluation of Stage-Dependent Genotoxic Effect of Roundup(®) (Glyphosate) on Caiman latirostris Embryos.

    PubMed

    Burella, Pamela Mariana; Simoniello, Maria Fernanda; Poletta, Gisela Laura

    2017-01-01

    The agricultural expansion over the past decades, along with the associated increase in the use of pesticides, represents a high risk for many wild species. Caiman latirostris is a South American caiman with many features that make it highly vulnerable to pesticide exposure. Considering previous finding on the genotoxicity of the glyphosate-based formulation Roundup(®) in this species, the aim of this study was to evaluate the possible stage-dependent effect of this compound on C. latirostris embryos through the Comet assay (CA), micronuclei (MN), and nuclear abnormalities (NA) tests. Caiman eggs were exposed to three effective concentrations of Roundup® (750, 1250, 1750 µg/egg) in three different stages of the incubation period (total duration 70 ± 3 days at 31 ± 2 °C) of approximately 23 days each. A statistically significant difference in DNA damage determined by the CA was found between groups exposed to different concentrations of RU (p < 0.05) and the negative control, but no difference was observed among the three stages of exposure within any treatment (p > 0.05). There was no differences in the MN or NA frequencies between the different groups and the negative control (p > 0.05), nor among the different stages within each treatment. The results obtained in this study indicate that RU produce DNA damage on C. latirostris embryos independently of the developmental stage where the exposure occurs, implying an important risk for the species during all its period of development, when pesticide application is at maximum rate.

  18. 50 SURVIVAL OF SEXED IVF-DERIVED BOVINE EMBRYOS FROZEN AT DIFFERENT PREIMPLANTATION STAGES OF DEVELOPMENT.

    PubMed

    Ferré, L; Fresno, C; Kjelland, M; Ross, P

    2016-01-01

    The ability to freeze in vitro-produced bovine embryos with a high post-thaw viability is still problematic and hampers logistics of on-farm embryo transfer. The objectives of this experiment were to compare different stages of development, freezing methods, and addition of cytoskeletal stabilisers (cytochalasin-B) before freezing. Ovaries were collected from an abattoir and oocytes aspirated from 2- to 6-mm follicles. Cumulus-oocyte complexes containing compact and complete cumulus cell layers were selected and matured in groups of 50 in 400µL of M199 medium supplemented with ALA-glutamine (0.1mM), Na pyruvate (0.2mM), gentamicin (5µgmL(-1)), EGF (50ngmL(-1)), ovine FSH (50ngmL(-1)), bLH (3µgmL(-1)), cysteamine (0.1mM), and 10% fetal bovine serum (FBS) for 22 to 24h. Fertilization (Day 0) was done using female sex-sorted semen selected with a discontinuous density gradient and diluted to a final concentration of 1×10(6) sperm/mL. Synthetic oviductal fluid (SOF)-FERT medium was supplemented with fructose (90µgmL(-1)), penicillamine (3µgmL(-1)), hypotaurine (11µgmL(-1)), and heparin (20µgmL(-1)). After 18h, presumptive zygotes were denuded and cultured in groups of 15 to 20 in 50-µL drops of SOF-BSA for 7 days. On Day 3.5 post-fertilization, 3% FBS was added. Low oxygen tension (5% O2) was used for culture. Morulae were selected at Day 5.5-6, blastocysts at Day 6-6.5, and expanded blastocysts at Day 6.5-7. Embryo harvesting for each stage was performed from a dedicated drop/dish and discarded in order to avoid further embryo stage collections. Grade 1 morulae, blastocysts, and expanded blastocysts were selected for freezing and placed randomly into 2 groups: slow-freezing and vitrification. Before freezing, half of the embryos from each stage were exposed to cytochalasin-B for 45min. The slow freezing protocol consisted of 1.5M ethylene glycol (EG)+20% FBS+0.4% BSA, and the cooling rate was 0.5°C/min. Slow-frozen embryo thawing was performed by exposing

  19. 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.

  20. Conversion of oat (Avena sativa L.) haploid embryos into plants in relation to embryo developmental stage and regeneration media.

    PubMed

    Noga, Angelika; Skrzypek, Edyta; Warchoł, Marzena; Czyczyło-Mysza, Ilona; Dziurka, Kinga; Marcińska, Izabela; Juzoń, Katarzyna; Warzecha, Tomasz; Sutkowska, Agnieszka; Nita, Zygmunt; Werwińska, Krystyna

    2016-01-01

    Obtaining oat DH lines is only effective via wide crossing with maize. Seven hundred haploid embryos from 21 single F1 progeny obtained from wide crosses with maize were isolated, divided into four groups according to their size (<0.5 mm, 0.5-0.9 mm, 1.0-1.4 mm, and ≥1.5 mm), and transferred into 190-2 regeneration medium with different growth regulators: 0.5 mg L(-1) kinetin (KIN) and 0.5 mg L(-1) 1-naphthaleneacetic acid (NAA); 1 mg L(-1) zeatin (ZEA) and 0.5 mg L(-1) NAA; or 1 mg L(-1) dicamba (DIC), 1 mg L(-1) picloram (PIC), and 0.5 mg L(-1) kinetin (KIN). Among all isolated embryos, approximately 46.1% were between 1.0-1.4 mm, while the smallest group of embryos (7.1%) were those <0.5 mm. The ability of haploid embryos to germinate varied depending on oat genotypes and the size of embryos. Haploid embryos <0.5 mm were globular and did not germinate, whereas embryos ≥1.5 mm had clearly visible coleoptiles, radicles, and scutella, and were able to germinate. Germination of oat haploid embryos varied depending on growth regulators in the regeneration medium. Most haploid embryos germinated on medium with 0.5 mg L(-1) NAA and 0.5 mg L(-1) KIN, while the fewest germinated on medium with 1 mg L(-1) DIC, 1 mg L(-1) PIC, and 0.5 mg L(-1) KIN. One hundred thirty germinated haploid embryos converted into haploid plants. Fifty oat DH lines were obtained after colchicine treatment.

  1. Correlative light and electron microscopy of intermediate stages of meiotic spindle assembly in the early Caenorhabditis elegans embryo.

    PubMed

    Woog, Ina; White, Silke; Büchner, Mandy; Srayko, Martin; Müller-Reichert, Thomas

    2012-01-01

    This chapter is an update of the previously published book chapter "Correlative Light and Electron Microscopy of Early C. elegans Embryos in Mitosis" (Müller-Reichert, Srayko, Hyman, O'Toole, & McDonald, 2007). Here, we have adapted and improved the protocol for the isolated meiotic embryos, which was necessary to meet the specific challenges a researcher faces while investigating the development of very early Caenorhabditis elegans embryos ex-utero. Due to the incompleteness of the eggshell assembly, the meiotic embryo is very fragile and much more susceptible to changes in the environmental conditions than the mitotic ones. To avoid phototoxicity associated with wide-field UV illumination, we stage the meiotic embryos primarily using transmitted visible light. Throughout the staging and high-pressure freezing, we incubate samples in an isotonic embryo buffer. The ex-utero approach allows precise tracking of the developmental events in isolated meiotic embryos, thus facilitating the comparison of structural features between wild-type and mutant or RNAi-treated samples.

  2. Production of mouse chimeras by injection of embryonic stem cells into the perivitelline space of one-cell stage embryos.

    PubMed

    De Repentigny, Yves; Kothary, Rashmi

    2010-12-01

    Generation of mouse chimeras is useful for the elucidation of gene function. In the present report, we describe a new technique for the production of chimeras by injection of R1 embryonic stem (ES) cells into the perivitelline space of one-cell stage mouse embryos. One-cell embryos are injected with 2-6 ES cells into the perivitelline space under the zona pellucida without laser-assistance. Our embryo culture experiments reveal that ES cells injected at the one-cell stage embryo start to be incorporated into the blastomeres beginning at the 8-cell stage and form a chimeric blastocyst after 4 days. We have used this approach to successfully produce a high rate of mouse chimeras in two different mouse genetic backgrounds permitting the establishment of germ line transmitters. This method allows for the earlier introduction of ES cells into mouse embryos, and should free up the possibility of using frozen one-cell embryos for this purpose.

  3. Improvement of the developmental ability of nuclear transfer embryos by using blastomeres from in vitro fertilized embryos selected according to the early developmental stage and cell division status as donor cells in cattle.

    PubMed

    Goto, Yuji; Matoba, Satoko; Imai, Kei; Geshi, Masaya

    2011-04-01

    This study was conducted to improve the developmental ability of nuclear transfer (NT) embryos by using blastomeres from in vitro fertilized (IVF) embryos with high quality as donor cells. The IVF embryos selected at the 2-cell stage at 24-h postinsemination (hpi) and again at the ≥8-cell stage at 48 hpi (Selected-IVF-embryos) showed the highest blastocyst formation rate among embryos. When blastomeres from the Selected-IVF-embryos (Selected-NT group) or Nonselected-IVF-embryos (Non-selected-NT group) were used as donor cells for NT, the blastocyst formation rate in the Selected-NT group (25.6%) was significantly higher than that in the Non-selected-NT group (13.5%). When blastomeres from the Selected-IVF-embryos at 108 (contained many cells before cell division) and 126 hpi (contained many cells immediately after cell division) were used as donor cells for NT (108- and 126-NT groups, respectively), the 126-NT group showed a significantly higher blastocyst formation rate (32.1%) than the 108-NT group (16.8%). Embryo transfer of blastocysts in the 126-NT group showed that 11 of 23 recipients became pregnant; nine calves were obtained. For the NT embryos reconstructed using in vivo derived embryos, 9 of 20 recipients became pregnant; seven calves were obtained. These results indicate that the blastocyst formation rate of NT embryos can be improved by using blastomeres from IVF embryos selected at the early developmental stage, especially immediately after cell division, and that the resultant NT embryos have a high developmental ability to progress to term that is comparable to NT embryos reconstructed using in vivo derived embryos.

  4. Maternal diabetes triggers DNA damage and DNA damage response in neurulation stage embryos through oxidative stress.

    PubMed

    Dong, Daoyin; Yu, Jingwen; Wu, Yanqing; Fu, Noah; Villela, Natalia Arias; Yang, Peixin

    2015-11-13

    DNA damage and DNA damage response (DDR) in neurulation stage embryos under maternal diabetes conditions are not well understood. The purpose of this study was to investigate whether maternal diabetes and high glucose in vitro induce DNA damage and DDR in the developing embryo through oxidative stress. In vivo experiments were conducted by mating superoxide dismutase 1 (SOD1) transgenic male mice with wild-type (WT) female mice with or without diabetes. Embryonic day 8.75 (E8.75) embryos were tested for the DNA damage markers, phosphorylated histone H2A.X (p-H2A.X) and DDR signaling intermediates, including phosphorylated checkpoint 1 (p-Chk1), phosphorylated checkpoint 2 (p-Chk2), and p53. Levels of the same DNA damage markers and DDR signaling intermediates were also determined in the mouse C17.2 neural stem cell line. Maternal diabetes and high glucose in vitro significantly increased the levels of p-H2A.X. Levels of p-Chk1, p-Chk2, and p53, were elevated under both maternal diabetic and high glucose conditions. SOD1 overexpression blocked maternal diabetes-induced DNA damage and DDR in vivo. Tempol, a SOD1 mimetic, diminished high glucose-induced DNA damage and DDR in vitro. In conclusion, maternal diabetes and high glucose in vitro induce DNA damage and activates DDR through oxidative stress, which may contribute to the pathogenesis of diabetes-associated embryopathy.

  5. Distribution of Mercury in Rainbow Trout Tissues at Embryo-Larval and Juvenile Stages

    PubMed Central

    Kenšová, Renáta; Kružíková, Kamila; Havránek, Jan; Haruštiaková, Danka; Svobodová, Zdeňka

    2012-01-01

    The aims of the study were to determine total mercury concentrations in “rainbow trout Oncorhynchus mykiss (Walbaum)” at their embryo-larval and juvenile stages and to assess mercury concentration dynamics in individual tissues. Samples of rainbow trout were collected at two-month intervals over a period of 18 months (one stock production cycle) at the Velká Losenice trout farm. Feedstuff samples were collected at the same time and analyzed for mercury concentrations. Tissue mercury concentrations were determined in muscle, liver, and kidneys. Analyses were performed using the AMA 254 atomic absorption spectrophotometer. The lowest mercury concentration was found in 14-day-old embryos (hard roe), and the highest concentrations in muscle tissue, liver, and kidneys at the end of monitoring, that is, in rainbow trout aged 18 months. The amount of mercury in feedstuffs showed an increasing trend and ranged between 0.0126 and 0.0859 mgkg−1. A significant effect (P < 0.001) of mercury intake on mercury concentrations in muscle tissue, liver, and kidneys was demonstrated. Muscle mercury concentrations in 18-month-old market-ready rainbow trout of 0.128 ± 0.048 mgkg−1 met the criteria for fish meat hygiene. PMID:22645443

  6. The amygdaloid complex and the medial and lateral ventricular eminences in staged human embryos.

    PubMed

    Müller, Fabiola; O'Rahilly, Ronan

    2006-05-01

    The amygdaloid complex was investigated in 36 serially sectioned staged human embryos, including 20 impregnated with silver. This is the first such account based on graphic reconstructions, 28 of which were prepared. Significant findings in the human include the following. (1) The medial (first) and (then) lateral ventricular eminences arise independently at stages 14 and 15, and unite only at stage 18 to form the floor of the lateral ventricle. (2) The future amygdaloid region is discernible at stage 14 and the amygdaloid primordium at stage 15. (3) The anterior amygdaloid area and the corticomedial and basolateral complexes appear at stage 16. (4) These three major divisions arise initially from the medial ventricular eminence, which is diencephalic. (5) Individual nuclei begin to be detectable at stages 17-21, the central nucleus at stage 23 and the lateral nucleus shortly thereafter. (6) The ontogenetic findings in the human embryonic period accord best with the classification used by Humphrey. (7) The lateral eminence, which is telencephalic, contributes to the cortical nucleus at stage 18. (8) The primordial plexiform layer develops independently of the cortical nucleus. (9) Spatial changes of the nuclei within the amygdaloid complex and of the complex as a whole begin in the embryonic period and continue during the fetal period, during the early part of which the definitive amygdaloid topography in relation to the corpus striatum is attained. (10) The developing amygdaloid nuclei are closely related to the medial forebrain bundle, which has already appeared in stage 15. (11) Fibre connections develop successively between the amygdaloid nuclei and the septal, hippocampal and diencephalic formations, constituting the beginning of the limbic system before the end of the embryonic period. Although the nucleus accumbens also appears relatively early (stage 19), connections between it and the amygdaloid complex are not evident during the embryonic period. (12

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

    PubMed

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

    2015-06-01

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

  8. From oocyte to 16-cell stage: cytoplasmic and cortical reorganizations that pattern the ascidian embryo.

    PubMed

    Sardet, Christian; Paix, Alexandre; Prodon, François; Dru, Philippe; Chenevert, Janet

    2007-07-01

    The dorsoventral and anteroposterior axes of the ascidian embryo are defined before first cleavage by means of a series of reorganizations that reposition cytoplasmic and cortical domains established during oogenesis. These domains situated in the periphery of the oocyte contain developmental determinants and a population of maternal postplasmic/PEM RNAs. One of these RNAs (macho-1) is a determinant for the muscle cells of the tadpole embryo. Oocytes acquire a primary animal-vegetal (a-v) axis during meiotic maturation, when a subcortical mitochondria-rich domain (myoplasm) and a domain rich in cortical endoplasmic reticulum (cER) and maternal postplasmic/PEM RNAs (cER-mRNA domain) become polarized and asymmetrically enriched in the vegetal hemisphere. Fertilization at metaphase of meiosis I initiates a series of dramatic cytoplasmic and cortical reorganizations of the zygote, which occur in two major phases. The first major phase depends on sperm entry which triggers a calcium wave leading in turn to an actomyosin-driven contraction wave. The contraction concentrates the cER-mRNA domain and myoplasm in and around a vegetal/contraction pole. The precise localization of the vegetal/contraction pole depends on both the a-v axis and the location of sperm entry and prefigures the future site of gastrulation and dorsal side of the embryo. The second major phase of reorganization occurs between meiosis completion and first cleavage. Sperm aster microtubules and then cortical microfilaments cause the cER-mRNA domain and myoplasm to reposition toward the posterior of the zygote. The location of the posterior pole depends on the localization of the sperm centrosome/aster attained during the first major phase of reorganization. Both cER-mRNA and myoplasm domains localized in the posterior region are partitioned equally between the first two blastomeres and then asymmetrically over the next two cleavages. At the eight-cell stage the cER-mRNA domain compacts and gives rise to

  9. Cumulative live birth rates after fresh and vitrified cleavage-stage versus blastocyst-stage embryo transfer in the first treatment cycle.

    PubMed

    De Vos, Anick; Van Landuyt, Lisbet; Santos-Ribeiro, Samuel; Camus, Michel; Van de Velde, Hilde; Tournaye, Herman; Verheyen, Greta

    2016-11-01

    Do cumulative live birth rates differ between single cleavage-stage Day 3 transfer and single blastocyst-stage Day 5 transfer? Cumulative live birth rates after Day 3 and 5 transfers were similar in young patients when the vitrified embryo transfers were also taken into account. Previous evidence has shown that the probability of live birth following IVF with a fresh embryo transfer is significantly higher after blastocyst-stage Day 5 transfer. However, because the introduction of vitrification has enhanced the survival of cryopreserved embryos and improved pregnancy rates, the optimal outcome measure for this comparison should now be cumulative live birth rates, as these include the eventual contribution of vitrified-warmed embryos. Our retrospective study included first IVF/ICSI cycles performed between January 2010 and December 2013 at a tertiary care centre. All patients were scheduled for fresh single embryo transfer, either on Day 3 (n = 377) or on Day 5 (n = 623). Both IVF and ICSI cycles were included and the sperm used were either fresh or frozen partner ejaculates, or frozen donor ejaculates. The primary outcome was cumulative live birth (after 24 weeks) rate per started cycle, including the eventual contribution of vitrification until the birth of a first child. Live birth rates per started cycle were significantly lower after transferring the fresh single cleavage-stage embryo, compared to a blastocyst (31.3% and 37.8%, respectively, P = 0.041). Furthermore, the number of embryo transfers necessary until the first live birth was significantly lower for blastocyst-stage embryos (P < 0.001). However, the cumulative live birth rates were 52.6% for cleavage-stage and 52.5% for blastocyst-stage transfers (P = 0.989). The extrapolation of the results is limited by the retrospective nature of the study. Furthermore, the analysis was restricted to patients under 36 years of age undergoing their first treatment cycle. These results deserve further

  10. Notch and Delta mRNAs in early-stage and mid-stage Drosophila embryos exhibit complementary patterns of protein producing potentials

    PubMed Central

    Shepherd, Andrew; Wesley, Uma; Wesley, Cedric

    2010-01-01

    Notch and Delta proteins generate Notch signaling that specifies cell fates during animal development. There is an intriguing phenomenon in Drosophila embryogenesis that has not received much attention and whose significance to embryogenesis is unknown. Notch and Delta mRNAs expressed in early-stage embryos are shorter than their counterparts in mid-stage embryos. We show here that the difference in sizes is due to mRNA 3′ processing at alternate polyadenylation sites. While the early-stage Notch mRNA has a lower protein-producing potential than the mid-stage Notch mRNA, the early-stage Delta mRNA has a higher protein-producing potential than the mid-stage Delta mRNA. Our data can explain the complementary patterns of Notch and Delta protein levels in early-stage and mid-stage embryos. Our data also raise the possibility that the manner and regulation of Notch signaling change in the course of embryogenesis and that this change is effected by 3′ UTR and mRNA 3′ processing factors. PMID:20201103

  11. Vitrification versus programmable rate freezing of late stage murine embryos: a randomized comparison prior to application in clinical IVF.

    PubMed

    Walker, David L; Tummon, Ian S; Hammitt, Diane G; Session, Donna R; Dumesic, Daniel A; Thornhill, Alan R

    2004-05-01

    A prospective randomized trial was performed to compare post-thaw development of murine blastocysts following programmable rate freezing and two methods of vitrification. Frozen 2-cell murine embryos (n = 429) thawed and cultured for 48 h, were randomly allocated by stage of development into four groups: control (not refrozen), programmable rate freezing (PR) in 0.25 ml straws, vitrification in flexible micropipettes by immersion in super-cooled (VSC) liquid nitrogen (LN2), and vitrification in flexible micropipettes by immersion in LN2 (VLN). Survival, developmental stage progression, presence or absence of an inner cell mass (ICM), and cell counts were recorded 24 h post-thaw. All measured outcomes were different between embryos from the control group and all freezing methods. Controlled-rate freezing resulted in the lowest total cell counts and fewest embryos with a distinct ICM. A higher percentage of embryos survived 24 h post-thaw, progressed to more advanced developmental stages and had higher total cell counts after VLN compared with PR. Moreover, fewer embryos, frozen by either PR or VSC, contained a detectable ICM compared with VLN. These data demonstrate that vitrification may be a better method for freezing murine blastocysts than PR, and may prove to be a superior method for freezing human blastocysts.

  12. 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.

  13. Differential pattern of Xist RNA accumulation in single blastomeres isolated from 8-cell stage mouse embryos following laser zona drilling.

    PubMed

    Hartshorn, Cristina; Rice, John E; Wangh, Lawrence J

    2003-01-01

    Xist gene expression begins at the late 2-cell stage in female mouse embryos and by the third division results in the accumulation of an average 100 copies of Xist RNA per cell, as measured by real-time reverse transcription-polymerase chain reaction (RT-PCR). In the blastocyst, the trophectoderm maintains the paternally imprinted pattern of Xist expression present during early development, while either the maternal or the paternal X chromosome can express Xist among cells of the inner mass. Fluorescent in situ hybridization (FISH) has previously established that Xist transcripts are localized on the silenced X chromosome, forming aggregates of variable dimensions in blastomeres of 8-cell embryos. This observation and the fact that Xist RNA accumulation per cell sharply decreases after morula stage raise the possibility that cells of cleaving embryos contain different levels of Xist RNA, perhaps linked to their subsequent developmental fates. We show here that Xist RNA is efficiently recovered from single blastomeres isolated from 8-cell embryos following laser zona drilling. Sexing of the samples and simultaneous quantification of Xist RNA in individual cells is achieved with a multiplex Xist/Sry real-time RT-PCR assay sensitive to the single-copy level. This analysis reveals that Xist RNA is indeed accumulated to substantially different levels in individual blastomeres of the same 8-cell embryo and that two blastomeres contain most of the molecules per embryo. These results support the conclusion that cells of the early mammalian embryo are not all functionally equivalent. Differential Xist gene expression could arise from differences in DNA methylation, or the order in which cells divide. Copyright 2003 Wiley-Liss, Inc.

  14. Timetable for upper eyelid development in staged human embryos and fetuses.

    PubMed

    Byun, Tae Ho; Kim, Jeong Tae; Park, Hyoung Woo; Kim, Won Kyu

    2011-05-01

    In this study, we examined the development of the upper eyelids to provide a basic understanding of gross anatomical structures and information relative to mechanisms of congenital anomalies in the upper eyelids. We studied the upper eyelids by external and histological observation in 48 human embryos and in fetuses from 5 to 36 weeks postfertilization. The upper eyelid fold began to develop at Stage 18. Upper and lower eyelids fused from the lateral cantus at Stage 22, and fusion was complete by 9 weeks of development. Mesenchymal condensations forming the orbital part of the orbicularis oculi (OO), tarsal plate, and the eyelashes and their appendages, were first seen at Week 9. Definite muscle structures of the upper eyelid, such as the orbital part of the OO and the levator palpebrae superioris and its aponeurosis, and the Müller's muscle were observed at 12 and 14 weeks, respectively. In addition, orbital septum, arterial arcade and orbital fat pad, and tarsal gland (TG) were apparent at 12, 14, and 18 weeks, respectively. Opening of the palpebral fissure was observed at Week 20. In addition, we defined the directional orientation between the levator aponeurosis and orbital septum and the growth pattern of the TG. Our results will be helpful in understanding the normal development of the upper eyelid and the origins of upper eyelid birth defects.

  15. Developmental Neurotoxicity of Methamidophos in the Embryo-Larval Stages of Zebrafish

    PubMed Central

    He, Xiaowei; Gao, Jiawei; Dong, Tianyu; Chen, Minjian; Zhou, Kun; Chang, Chunxin; Luo, Jia; Wang, Chao; Wang, Shoulin; Chen, Daozhen; Zhou, Zuomin; Tian, Ying; Xia, Yankai; Wang, Xinru

    2016-01-01

    Methamidophos is a representative organophosphate insecticide. The knowledge of its developmental neurotoxicity is limited, especially for zebrafish in the early stages of their life. Four hour post-fertilization (hpf) zebrafish embryos were exposed to several environmentally relevant concentrations of methamidophos (0, 25, and 500 μg/L) for up to 72 hpf. Locomotor behavior was then studied in the zebrafish larvae at this timepoint. Acridine orange (AO) staining was carried out in the zebrafish larvae, and the mRNA levels of genes associated with neural development (mbp and syn2a) were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). The number of escape responders for mechanical stimulation was significantly decreased in exposed groups. AO staining showed noticeable signs of apoptosis mainly in the brain. In addition, the mRNA levels of mbp and syn2a were both significantly down-regulated in exposed groups. Our study provides the first evidence that methamidophos exposure can cause developmental neurotoxicity in the early stages of zebrafish life, which may be caused by the effect of methamidophos on neurodevelopmental genes and the activation of cell apoptosis in the brain. PMID:28036051

  16. Vitrification of cleavage stage day 3 embryos results in higher live birth rates than conventional slow freezing: a RCT.

    PubMed

    Debrock, S; Peeraer, K; Fernandez Gallardo, E; De Neubourg, D; Spiessens, C; D'Hooghe, T M

    2015-08-01

    Is the live birth rate (LBR) per embryo thawed/warmed higher when Day 3 cleavage stage embryos are cryopreserved by vitrification compared with slow freezing? The LBR per embryo thawed/warmed was higher after vitrification than after slow freezing on Day 3, based on better embryo survival, quality and availability of embryos in the vitrification group. Post-thawing survival rate of cleavage-stage embryos has been reported to be higher after vitrification than after slow freezing. This RCT was performed in an academic tertiary center between September 2011 and March 2013. If supernumerary embryos were available on Day 3, patients were randomized at the time of cryopreservation using a computerized system to determine a simple allocation to the vitrification group or the slow freezing group and all embryos were frozen with the same technique. The primary outcome of this study was the LBR per embryo thawed/warmed. Power calculation revealed that 184 thawed embryos were needed in each group (β = 0.8, α < 0.05) to test the hypothesis that the LBR per embryo thawed/warmed was significantly higher (16%) after vitrification than after slow freezing (6%). Patients <40 years old undergoing their first oocyte retrieval (OR), with embryo transfer and with supernumerary embryos on Day 3, were randomized. Day 3 embryos with ≥6 cells, <25% fragmentation and morphologically equal blastomeres were cryopreserved by slow freezing (using 1,2-propanediol and 0.1 M sucrose as cryoprotectant) or by closed vitrification using commercially available freezing/vitrification media. Survival was defined as ≥50% cells were intact after thawing. Thawed embryos were further cultured overnight. In total, 307 patients were randomized to slow freezing (155 patients, 480 embryos) or vitrification (152 patients, 495 embryos). By March 2013, 200 embryos were thawed after slow freezing in 95 cycles for 79 patients and 217 embryos were warmed after vitrification in 121 cycles in 90 patients. The

  17. Identification of differentially expressed genes between cloned and zygote-developing zebrafish (Danio rerio) embryos at the dome stage using suppression subtractive hybridization.

    PubMed

    Luo, Daji; Hu, Wei; Chen, Shangping; Xiao, Yi; Sun, Yonghua; Zhu, Zuoyan

    2009-04-01

    Comparative analyses of differentially expressed genes between somatic cell nuclear transfer (SCNT) embryos and zygote-developing (ZD) embryos are important for understanding the molecular mechanism underlying the reprogramming processes. Herein, we used the suppression subtractive hybridization approach and from more than 2900 clones identified 96 differentially expressed genes between the SCNT and ZD embryos at the dome stage in zebrafish. We report the first database of differentially expressed genes in zebrafish SCNT embryos. Collectively, our findings demonstrate that zebrafish SCNT embryos undergo significant reprogramming processes during the dome stage. However, most differentially expressed genes are down-regulated in SCNT embryos, indicating failure of reprogramming. Based on Ensembl description and Gene Ontology Consortium annotation, the problems of reprogramming at the dome stage may occur during nuclear remodeling, translation initiation, and regulation of the cell cycle. The importance of regulation from recipient oocytes in cloning should not be underestimated in zebrafish.

  18. 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.

  19. Functional Genomics of 5- to 8-Cell Stage Human Embryos by Blastomere Single-Cell cDNA Analysis

    PubMed Central

    Galán, Amparo; Montaner, David; Póo, M. Eugenia; Valbuena, Diana; Ruiz, Verónica; Aguilar, Cristóbal; Dopazo, Joaquín; Simón, Carlos

    2010-01-01

    Blastomere fate and embryonic genome activation (EGA) during human embryonic development are unsolved areas of high scientific and clinical interest. Forty-nine blastomeres from 5- to 8-cell human embryos have been investigated following an efficient single-cell cDNA amplification protocol to provide a template for high-density microarray analysis. The previously described markers, characteristic of Inner Cell Mass (ICM) (n = 120), stemness (n = 190) and Trophectoderm (TE) (n = 45), were analyzed, and a housekeeping pattern of 46 genes was established. All the human blastomeres from the 5- to 8-cell stage embryo displayed a common gene expression pattern corresponding to ICM markers (e.g., DDX3, FOXD3, LEFTY1, MYC, NANOG, POU5F1), stemness (e.g., POU5F1, DNMT3B, GABRB3, SOX2, ZFP42, TERT), and TE markers (e.g., GATA6, EOMES, CDX2, LHCGR). The EGA profile was also investigated between the 5-6- and 8-cell stage embryos, and compared to the blastocyst stage. Known genes (n = 92) such as depleted maternal transcripts (e.g., CCNA1, CCNB1, DPPA2) and embryo-specific activation (e.g., POU5F1, CDH1, DPPA4), as well as novel genes, were confirmed. In summary, the global single-cell cDNA amplification microarray analysis of the 5- to 8-cell stage human embryos reveals that blastomere fate is not committed to ICM or TE. Finally, new EGA features in human embryogenesis are presented. PMID:21049019

  20. Human developmental anatomy: microscopic magnetic resonance imaging (μMRI) of four human embryos (from Carnegie Stage 10 to 20).

    PubMed

    Lhuaire, Martin; Martinez, Agathe; Kaplan, Hervé; Nuzillard, Jean-Marc; Renard, Yohann; Tonnelet, Romain; Braun, Marc; Avisse, Claude; Labrousse, Marc

    2014-12-01

    Technological advances in the field of biological imaging now allow multi-modal studies of human embryo anatomy. The aim of this study was to assess the high magnetic field μMRI feasibility in the study of small human embryos (less than 21mm crown-rump) as a new tool for the study of human descriptive embryology and to determine better sequence characteristics to obtain higher spatial resolution and higher signal/noise ratio. Morphological study of four human embryos belonging to the historical collection of the Department of Anatomy in the Faculty of Medicine of Reims was undertaken by μMRI. These embryos had, successively, crown-rump lengths of 3mm (Carnegie Stage, CS 10), 12mm (CS 16), 17mm (CS 18) and 21mm (CS 20). Acquisition of images was performed using a vertical nuclear magnetic resonance spectrometer, a Bruker Avance III, 500MHz, 11.7T equipped for imaging. All images were acquired using 2D (transverse, sagittal and coronal) and 3D sequences, either T1-weighted or T2-weighted. Spatial resolution between 24 and 70μm/pixel allowed clear visualization of all anatomical structures of the embryos. The study of human embryos μMRI has already been reported in the literature and a few atlases exist for educational purposes. However, to our knowledge, descriptive or morphological studies of human developmental anatomy based on data collected these few μMRI studies of human embryos are rare. This morphological noninvasive imaging method coupled with other techniques already reported seems to offer new perspectives to descriptive studies of human embryology.

  1. Inhibition of Apoptosis Overcomes Stage-Related Compatibility Barriers to Chimera Formation in Mouse Embryos.

    PubMed

    Masaki, Hideki; Kato-Itoh, Megumi; Takahashi, Yusuke; Umino, Ayumi; Sato, Hideyuki; Ito, Keiichi; Yanagida, Ayaka; Nishimura, Toshinobu; Yamaguchi, Tomoyuki; Hirabayashi, Masumi; Era, Takumi; Loh, Kyle M; Wu, Sean M; Weissman, Irving L; Nakauchi, Hiromitsu

    2016-11-03

    Cell types more advanced in development than embryonic stem cells, such as EpiSCs, fail to contribute to chimeras when injected into pre-implantation-stage blastocysts, apparently because the injected cells undergo apoptosis. Here we show that transient promotion of cell survival through expression of the anti-apoptotic gene BCL2 enables EpiSCs and Sox17(+) endoderm progenitors to integrate into blastocysts and contribute to chimeric embryos. Upon injection into blastocyst, BCL2-expressing EpiSCs contributed to all bodily tissues in chimeric animals while Sox17(+) endoderm progenitors specifically contributed in a region-specific fashion to endodermal tissues. In addition, BCL2 expression enabled rat EpiSCs to contribute to mouse embryonic chimeras, thereby forming interspecies chimeras that could survive to adulthood. Our system therefore provides a method to overcome cellular compatibility issues that typically restrict chimera formation. Application of this type of approach could broaden the use of embryonic chimeras, including region-specific chimeras, for basic developmental biology research and regenerative medicine. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Proteomic identification of abnormally expressed proteins in early-stage placenta derived from cloned cat embryos.

    PubMed

    Bang, Jae-Il; Lee, Hyo-Sang; Deb, Gautam Kumar; Ha, A-Na; Kwon, Young-Sang; Cho, Seong-Keun; Kim, Byeong-Woo; Cho, Kyu-Woan; Kong, Il-Keun

    2013-01-15

    It is unknown whether gene expression in cloned placenta during pre- and postimplantation is associated with early pregnancy failure in the cat. In this study, protein expression patterns were examined in early-stage (21-day-old) domestic cat placentas of fetuses derived from AI (CP; N = 4) and cloned embryo transfer (CEP; N = 2). Differentially expressed proteins were analyzed by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight (TOF) mass spectrometry (MS). A total of 21 proteins were aberrantly expressed (P < 0.05) by >1.5-fold in CEP compared with CP. Compared with CP, 12 proteins were upregulated in CEP (peptidyl-prolyl cis-trans isomerase A, annexin A2, protein DJ-1, adenylate kinase isoenzyme 1, protein disulfide-isomerase A3, actin cytoplasmic 1, serum albumin, protein disulfide-isomerase A6, and triosephosphate isomerase), and nine proteins were downregulated (triosephosphate isomerase; heterogeneous nuclear ribonucleoprotein H; tropomyosin alpha-4; triosephosphate isomerase 1; 60 kDa heat shock protein, mitochondrial; serum albumin; calumenin; keratin type 1; and prohibitin). The identities of the differentially expressed proteins were validated by peptide mass fingerprinting using matrix-assisted laser desorption/ionization-TOF/TOF MS/MS. The abnormally expressed proteins identified in this study might be associated with impaired development and dysfunction of CEP during early pregnancy. Abnormal protein expression might also induce fetal loss and contribute to failure to maintain pregnancy to term.

  3. 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.

  4. 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.

  5. Ongoing and cumulative pregnancy rate after cleavage-stage versus blastocyst-stage embryo transfer using vitrification for cryopreservation: impact of age on the results.

    PubMed

    Fernández-Shaw, S; Cercas, R; Braña, C; Villas, C; Pons, I

    2015-02-01

    To determine if blastocyst transfer increases the ongoing and cumulative pregnancy rates, compared with day 3 embryo transfer, in women of all ages when at least 4 zygotes are obtained. Prospective study including patients undergoing a first IVF/ICSI treatment and assigned to cleavage stage (n = 46) or blastocyst (n = 58) embryo transfer. Supernumerary embryos were vitrified and patients failing to achieve an ongoing pregnancy after fresh embryo transfer would go through cryopreserved cycles. The main outcome measure was the ongoing pregnancy rate after the fresh IVF/ICSI transfer and the cumulative ongoing pregnancy rate. Results were also analyzed according to age (under 35 and 35 or older). A majority of patients (96.6 %) had a blastocyst transfer when at least 4 zygotes were obtained. The ongoing pregnancy rate was significantly higher in the day-5 group compared with the day-3 group (43.1 % vs. 24 %, p = 0.041). The cumulative ongoing pregnancy rate was higher (but not significantly) with blastocyst than with cleavage stage embryos (56.8 % vs. 43.4 %, p = 0.174). When analysed by age, patients 35 or older showed significantly higher ongoing pregnancy rate (48.4 % vs. 19.3 %, p = 0.016) and cumulative ongoing pregnancy rate (58 % vs. 25.8 %, p = 0.01) in the day-5 group compared to the day-3 group, while no such differences were observed in women under 35. Blastocyst transfer can be suggested whenever there are at least 4 zygotes. While there are no differences in women under 35, the benefit of this option over cleavage stage transfer could be significant in women 35 or older.

  6. Porcine Cloned Embryos Reconstructed with the Cell Nuclei of Tetraploid M-phase Fibroblast Cells Can Restore Normal Diploidy at the Blastocyst Stage.

    PubMed

    Zhao, Q; Qiu, Y G; Tian, J T; Wang, C S; An, T Z

    2016-11-17

    The cell cycle of donor cells as a major factor that affects cloning efficiency remains debatable. G2/M phase cells as a donor can successfully produce cloned animals, but a minimal amount is known regarding nuclear remodeling events. In this study, porcine fetal fibroblasts (PFFs) were carefully synchronized at G1 or M phase as donor cells. Most of the cloned embryos reconstructed from PFFs at G1 (G1-embryos) or M (M-embryos) phase formed a pronucleus-like nucleus (PN) within 6-h post fusion (hpf), but the M-embryos formed PN earlier than the G1-embryos did. Moreover, 77.4% of the M-embryos formed two PNs, whereas the G1-embryos formed a single PN. The rate of extrusion of polar body-like structures by the M-embryos was significantly lower than that extruded by the G1-embryos (26.3% vs. 37.1%, P < 0.05), and DNA synthesis in most embryos in both groups was initiated at 9-12 hpf. Most of the M-embryos were octoploid before the first cleavage. Furthermore, 81.25% of the blastomeres of blastocysts developed from the M-embryos showed abnormal ploidy compared with those developed from the G1-embryos (22.55%). However, some of the blastomeres remained diploid in all the M-embryos tested. A portion of the blastomeres restored normal diploidy in some of the M-embryos at the blastocyst stage. This finding provides an explanation for M-embryos developing to term.

  7. 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.

  8. 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

  9. Neonatal outcomes among singleton births after blastocyst versus cleavage stage embryo transfer: a systematic review and meta-analysis.

    PubMed

    Dar, S; Lazer, T; Shah, P S; Librach, C L

    2014-01-01

    Several studies have evaluated outcomes of singleton pregnancies after blastocyst versus cleavage stage embryo transfer. Higher incidences of preterm birth (PTB), very preterm birth (VPTB), low birthweight (LBW) and congenital malformations were identified in a few of them. The objective of our study was to systematically review and meta-analyze pregnancy and neonatal outcomes among singleton births following blastocyst versus cleavage stage embryo transfer. METHODS EMBASE, MEDLINE, EBM Reviews and bibliographies of included studies were searched from their inception until March 2013. Observational studies or clinical trials comparing blastocyst with cleavage stage embryo transfer and reporting on outcomes of PTB (<37 weeks), VPTB (<32 weeks), LBW (<2500 g), very low birthweight (VLBW) (<1500 g) and/or congenital anomalies in singleton neonates were included. Data on the outcomes were extracted by two reviewers. Statistical heterogeneity among studies was evaluated by calculating I(2) values and χ(2) statistics. Meta-analyses were conducted to estimate the pooled unadjusted odds ratio (OR) and the adjusted OR (AOR) with a 95% confidence interval (CI) using the random effect model. RESULTS Six observational studies, of low to moderate risk of bias, were included in this review. There were significantly higher odds of PTB (four studies, 54 792 cleavage stage and 20 724 blastocyst stage births; AOR 1.32, 95% CI 1.19-1.46) and congenital anomalies (two studies, 22 068 cleavage stage and 4517 blastocyst stage births; AOR 1.29, 95% CI 1.03-1.62) among births after blastocyst transfer compared with cleavage stage transfer. There was no difference in the adjusted odds of VPTB (four studies, 54 792 cleavage stage and 20 724 blastocyst stage births; AOR 1.18, 95% CI 0.93-1.49), LBW (four studies, 54 109 cleavage stage and 20 392 blastocyst stage births; AOR 1.06, 95% CI 0.99-1.15) or VLBW (three studies, 22 088 cleavage stage and 5772 blastocyst stage births; AOR 1.01, 95

  10. 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

    2015-01-01

    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%.

  11. 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.

  12. Embryonic hematopoietic stem cells and interstitial Cajal cells in the hindgut of late stage human embryos: evidence and hypotheses.

    PubMed

    Ilie, C A; Rusu, M C; Didilescu, A C; Motoc, A G M; Mogoantă, L

    2015-07-01

    There have been few studies on human embryos describing a specific pattern of hindgut colonization by hematopoietic stem cells (HSCs) and interstitial Cajal cells (ICCs). We aimed to study CD34, CD45 and CD117/c-kit expression in late stage human embryos, to attain observational data that could be related to studies on the aorta-gonad-mesonephros (AGM)-derived HSCs, and data on hindgut ICCs. Antibodies were also applied to identify alpha-smooth muscle actin and neurofilaments. Six human embryos of 48-56 days were used. In the 48 day embryo, the hindgut was sporadically populated by c-kit+ ICCs, but, in all other embryos, a layer of myenteric ICCs had been established. Intraneural c-kit+ cells were found in pelvic nerves and vagal trunks, suggesting that the theory of Ramon y Cajal assuming that ICCs may be primitive neurons may not be so invalid. Also in the 48 day embryo, c-kit+/CD45+ perivascular cells were found along the pelvic neurovascular axes, suggesting that not only liver, but also other organs could be seeded with HSCs from the AGM region. CD45+ cells with dendritic morphologies were found in all hindgut layers, including the epithelium. This last evidence is suggestive of an AGM contribution to the tissue resident macrophages and could be related to processes of sprouting angiogenesis which, in turn, have been found to be guided by filopodia of endothelial tip cells. Further studies on human embryonic and fetal material should be performed to attempt to clarify whether the hindgut colonization with HSCs is a transitory or definitive process.

  13. A simplified technique for embryo biopsy for preimplantation genetic diagnosis.

    PubMed

    Wang, Wei-Hua; Kaskar, Khalied; Gill, Jimmy; DeSplinter, Traci

    2008-08-01

    To report a simplified embryo biopsy method for preimplantation genetic diagnosis (PGD). Technique and method. A regional hospital in vitro fertilization (IVF) laboratory and private reproductive medicine clinic. Women undergoing IVF and PGD. Blastomeres were successfully isolated from day-3 embryos at various stages. Blastomere integrity after biopsy, time of biopsy procedure, and subsequent blastocyst developmental rate. Twenty embryos derived from abnormally fertilized oocytes (one pronucleus or three pronuclei) were used for biopsy at four-cell to 10-cell stages (day 3) by a laser zona drilling and assisted hatching micropipette delivery of culture medium inside the zona to push one blastomere out. Biopsies of all embryos using this method were successful. In two cases for PGD, fourteen 6-9-cell and four 3-4-cell stage embryos were successfully biopsied by this method. Ten out of 14 embryos from the 6-9-cell stage developed to hatching or hatched blastocysts. When two hatched blastocysts were vitrified, warmed, and cultured, both reexpanded, showing normal morphologic features. This technique is easy to learn, less damaging to the embryos, and less time consuming. It can be used for all stages of embryos without damage to either embryos or isolated blastomeres. It is an alternative method for embryo biopsy in PGD.

  14. A novel application of motion analysis for detecting stress responses in embryos at different stages of development

    PubMed Central

    2013-01-01

    Background Motion analysis is one of the tools available to biologists to extract biologically relevant information from image datasets and has been applied to a diverse range of organisms. The application of motion analysis during early development presents a challenge, as embryos often exhibit complex, subtle and diverse movement patterns. A method of motion analysis able to holistically quantify complex embryonic movements could be a powerful tool for fields such as toxicology and developmental biology to investigate whole organism stress responses. Here we assessed whether motion analysis could be used to distinguish the effects of stressors on three early developmental stages of each of three species: (i) the zebrafish Danio rerio (stages 19 h, 21.5 h and 33 h exposed to 1.5% ethanol and a salinity of 5); (ii) the African clawed toad Xenopus laevis (stages 24, 32 and 34 exposed to a salinity of 20); and iii) the pond snail Radix balthica (stages E3, E4, E6, E9 and E11 exposed to salinities of 5, 10 and 15). Image sequences were analysed using Sparse Optic Flow and the resultant frame-to-frame motion parameters were analysed using Discrete Fourier Transform to quantify the distribution of energy at different frequencies. This spectral frequency dataset was then used to construct a Bray-Curtis similarity matrix and differences in movement patterns between embryos in this matrix were tested for using ANOSIM. Results Spectral frequency analysis of these motion parameters was able to distinguish stage-specific effects of environmental stressors in most cases, including Xenopus laevis at stages 24, 32 and 34 exposed to a salinity of 20, Danio rerio at 33 hpf exposed to 1.5% ethanol, and Radix balthica at stages E4, E9 and E11 exposed to salinities of 5, 10 and 15. This technique was better able to distinguish embryos exposed to stressors than analysis of manual quantification of movement and within species distinguished most of the developmental stages

  15. Developmental kinetics of in vitro-produced bovine embryos: An aid for making decisions.

    PubMed

    Carrocera, S; Caamaño, J N; Trigal, B; Martín, D; Díez, C

    2016-03-15

    Embryo developmental kinetics and embryo survival after cryopreservation have been correlated with embryo quality and viability. The main objectives of this work were to analyze developmental ability and quality of in vitro-produced bovine embryos in relation to their kinetics and to establish a criterion of quality to predict further viability. Embryos were classified and grouped by their specific stage of development (2, 3-4, or ≥ 5 cells) at 44 hours post insemination (hpi) and cultured separately up to Day 8. On Days 7 and 8, good quality expanded blastocysts were vitrified or frozen. Cryopreserved surviving hatched embryos were stained for cell counts. Embryos at a more advanced stage (3-4 cells, and ≥5 cells) developed to morulae (P < 0.001) and blastocysts (P < 0.01) at higher rates than those embryos that had cleaved once by 44 hpi. Vitrification improved the hatching rates of blastocysts at 48 hours (P < 0.001) when compared with slow-rate freezing within each group of embryos (3-4 cells and ≥5 cells). After vitrification/warming, blastocysts coming from 3- to 4-cell embryos had higher hatching rates at 48 hours than those that came from ≥5-cell embryos. With regard to differential cell counts, no effect of the initial developmental stage was observed after warming/thawing. However, trophectoderm and total cells were higher in vitrified/warmed than in the frozen/thawed embryos (P < 0.001). These data show that selecting IVF embryos at 44 hpi, after the evaluation of their in vitro embryo development, could be used as noninvasive markers of embryo developmental competence and may help to select IVF embryos that would be more suitable for cryopreservation.

  16. Beyond the American Society for Reproductive Medicine transfer guidelines: how many cleavage-stage embryos are safe to transfer in women ≥43 years old?

    PubMed

    Gunnala, Vinay; Reichman, David E; Meyer, Laura; Davis, Owen K; Rosenwaks, Zev

    2014-12-01

    To determine the number of cleavage-stage embryos that can be safely transferred in women ≥43 years old. Retrospective cohort. Academic medical center. All patients ≥43 years old undergoing transfer of five or more cleavage-stage embryos during the period from January 2004 through April 2012. In vitro fertilization. A total of 567 cycles in 464 patients aged 43-45 years, whose IVF cycles were characterized by transfer of five to eight cleavage-stage embryos were identified. Clinical outcomes and risk of multiples were analyzed, stratifying by age and number of embryos transferred. Live birth rates per transfer were 14.4%, 9.4%, and 1.3% for women aged 43, 44, and 45 years, respectively. In 43-year-old women, 2.9% (2/69) of pregnancies were triplet gestations (one selective reduction and one spontaneous reduction). Twin birth rate was 16.3%, 6.7%, and 0 (of all live births) for ages 43, 44, and 45 years, respectively. There was no higher order multiple births. Women aged 43 and 44 years having five or more embryos transferred experienced higher clinical pregnancy rates (PRs) than those patients receiving a transfer of three or four embryos. Clinical outcomes for patients undergoing transfer with six or more embryos were not better than those undergoing transfer with five embryos. Transferring five or more day 3 embryos may be a safe option for patients ≥43 years of age, as it is associated with an overall low rate of multiple gestations. Having more than five embryos available for transfer on day 5 is associated with improved IVF outcomes. Whether this benefit is from the additional embryo(s) for transfer or the inherently better prognosis of such patients remains to be determined. Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  17. Effects of high three pro-nuclei (3PN) proportion incidence on clinical outcomes in the fresh cleavage-stage and blastocyst-stage embryo transfer (ET) cycles.

    PubMed

    Li, Mingzhao; Xue, Xia; Zhao, Wanqiu; Li, Wei; Shi, Juanzi

    2017-01-01

    The aim of this study was to explore the effect of three pro-nuclei (3PN) incidence on clinical outcomes in the fresh cleavage-stage embryo transfer (CSET) and blastocyst-stage embryo transfer (BSET) cycles. This retrospective cohort study included 1427 CSET cycles, 632 BSET cycles, and 313 elective single BSET cycles from January 2013 to June 2015. The patients were divided into two groups as follows: Group 1 included patients with no 3PN zygotes and Group 2 included patients with >20% 3PN zygotes. We observed that the fertilization rate was significantly lower in 3PN = 0% than 3PN > 20% group (p < 0.05), but the day-3 grade I + II embryo and day-3 grade I + II + III embryo rates were not significantly different between 3PN = 0% and 3PN > 20% group (p > 0.05). Interestingly, in the CSET, the implantation (42.87% and 41.76%, p = 0.585) and clinical pregnancy (59.94% and 58.25%, p = 0.538) rates were not significantly different between two groups. In the BSET, the implantation (61.93% and 49.62%, p < 0.001) and clinical pregnancy rates (69.45% and 61.02%, p = 0.043) were significantly higher in 3PN = 0% than 3PN > 20% group. In the elective single BSET, the implantation (68.91% and 61.33%, p = 0.223) and clinical pregnancy rates (68.48% and 61.33%, p = 0.251) were higher in 3PN = 0% than 3PN > 20% group, but there was no significant difference. We concluded that a high 3PN incidence may predict poor outcomes in BSET but not CSET cleavage-stage.

  18. Differential Expression of Metallothionein Isoforms in Terrestrial Snail Embryos Reflects Early Life Stage Adaptation to Metal Stress

    PubMed Central

    Baurand, Pierre-Emmanuel; Pedrini-Martha, Veronika; de Vaufleury, Annette; Niederwanger, Michael; Capelli, Nicolas; Scheifler, Renaud; Dallinger, Reinhard

    2015-01-01

    The aim of this study was to analyze the expression of three metallothionein (MT) isoform genes (CdMT, CuMT and Cd/CuMT), already known from adults, in the Early Life Stage (ELS) of Cantareus aspersus. This was accomplished by detection of the MT isoform-specific transcription adopting Polymerase Chain Reaction (PCR) amplification and quantitative Real Time (qRT)-PCR of the three MT genes. Freshly laid eggs were kept for 24 hours under control conditions or exposed to three cadmium (Cd) solutions of increasing concentration (5, 10, and 15 mg Cd/L). The transcription of the three MT isoform genes was detected via PCR in 1, 6 and 12-day-old control or Cd-exposed embryos. Moreover, the transcription of this isoform genes during development was followed by qRT-PCR in 6 and 12-day-old embryos. Our results showed that the CdMT and Cd/CuMT genes, but not the CuMT gene, are expressed in embryos at the first day of development. The transcription of the 3 MT genes in control embryos increased with development time, suggesting that the capacities of metal regulation and detoxification may have gradually increased throughout embryogenesis. However in control embryos, the most highly expressed MT gene was that of the Cd/CuMT isoform, whose transcription levels greatly exceeded those of the other two MT genes. This contrasts with the minor significance of this gene in adult snails and suggests that in embryos, this isoform may play a comparatively more important role in metal physiology compared to adult individuals. This function in adult snails appears not to be related to Cd detoxification. Instead, snail embryos responded to Cd exposure by over-expression of the CdMT gene in a concentration-dependent manner, whereas the expression of the Cd/CuMT gene remained unaffected. Moreover, our study demonstrates the ability of snail embryos to respond very early to Cd exposure by up-regulation of the CdMT gene. PMID:25706953

  19. Differential expression of metallothionein isoforms in terrestrial snail embryos reflects early life stage adaptation to metal stress.

    PubMed

    Baurand, Pierre-Emmanuel; Pedrini-Martha, Veronika; de Vaufleury, Annette; Niederwanger, Michael; Capelli, Nicolas; Scheifler, Renaud; Dallinger, Reinhard

    2015-01-01

    The aim of this study was to analyze the expression of three metallothionein (MT) isoform genes (CdMT, CuMT and Cd/CuMT), already known from adults, in the Early Life Stage (ELS) of Cantareus aspersus. This was accomplished by detection of the MT isoform-specific transcription adopting Polymerase Chain Reaction (PCR) amplification and quantitative Real Time (qRT)-PCR of the three MT genes. Freshly laid eggs were kept for 24 hours under control conditions or exposed to three cadmium (Cd) solutions of increasing concentration (5, 10, and 15 mg Cd/L). The transcription of the three MT isoform genes was detected via PCR in 1, 6 and 12-day-old control or Cd-exposed embryos. Moreover, the transcription of this isoform genes during development was followed by qRT-PCR in 6 and 12-day-old embryos. Our results showed that the CdMT and Cd/CuMT genes, but not the CuMT gene, are expressed in embryos at the first day of development. The transcription of the 3 MT genes in control embryos increased with development time, suggesting that the capacities of metal regulation and detoxification may have gradually increased throughout embryogenesis. However in control embryos, the most highly expressed MT gene was that of the Cd/CuMT isoform, whose transcription levels greatly exceeded those of the other two MT genes. This contrasts with the minor significance of this gene in adult snails and suggests that in embryos, this isoform may play a comparatively more important role in metal physiology compared to adult individuals. This function in adult snails appears not to be related to Cd detoxification. Instead, snail embryos responded to Cd exposure by over-expression of the CdMT gene in a concentration-dependent manner, whereas the expression of the Cd/CuMT gene remained unaffected. Moreover, our study demonstrates the ability of snail embryos to respond very early to Cd exposure by up-regulation of the CdMT gene.

  20. 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

  1. Identification and quantitative analysis of stage-specific carbohydrates in loblolly pine (Pinus taeda) zygotic embryo and female gametophyte tissues.

    PubMed

    Pullman, Gerald S; Buchanan, Mike

    2008-07-01

    Stage-specific analyses of starch and 18 sugars, including pentoses, hexoses, disaccharides, trisaccharides, oligosaccharides and sugar alcohols, were made throughout seed development for zygotic embryo and female gametophyte (FG) tissues of loblolly pine (Pinus taeda L.). Tissue was most often analyzed in triplicate from two open-pollinated families grown in different locations and sampled in different years. Carbohydrates were analyzed by enzymatic assay, high performance liquid chromatography or gas chromatography/mass spectrometry. For all carbohydrates quantified, peak concentrations were higher in embryo tissue than in FG tissue. Significant changes in starch and sugar concentrations occurred over time, with both seed collections showing similar trends in temporal changes. Although concentrations were not always similar, embryo and FG tissues generally showed similar patterns of change in starch and sugar concentrations over time. Total starch concentration was highest during early seed development and decreased as development progressed. The major sugars contributing to osmotic potential during early seed development were D-pinitol, sucrose, fructose and glucose. During mid-seed development, D-pinitol, sucrose, fructose, glucose, melibiose and raffinose provided major contributions to the osmotic environment. During late seed development, sucrose, raffinose, melibiose, stachyose and fructose were the major contributors to osmotic potential. These data suggest stage-specific media composition for each step in the somatic embryogenesis protocol.

  2. Changes in the transcriptome of morula-stage bovine embryos caused by heat shock: relationship to developmental acquisition of thermotolerance

    PubMed Central

    2013-01-01

    Background While initially sensitive to heat shock, the bovine embryo gains thermal resistance as it progresses through development so that physiological heat shock has little effect on development to the blastocyst stage by Day 5 after insemination. Here, experiments using 3’ tag digital gene expression (3’DGE) and real-time PCR were conducted to determine changes in the transcriptome of morula-stage bovine embryos in response to heat shock (40 degrees C for 8 h) that could be associated with thermotolerance. Results Using 3’DGE, expression of 173 genes were modified by heat shock, with 94 genes upregulated by heat shock and 79 genes downregulated by heat shock. A total of 38 differentially-regulated genes were associated with the ubiquitin protein, UBC. Heat shock increased expression of one heat shock protein gene, HSPB11, and one heat shock protein binding protein, HSPBP1, tended to increase expression of HSPA1A and HSPB1, but did not affect expression of 64 other genes encoding heat shock proteins, heat shock transcription factors or proteins interacting with heat shock proteins. Moreover, heat shock increased expression of five genes associated with oxidative stress (AKR7A2, CBR1, GGH, GSTA4, and MAP2K5), decreased expression of HIF3A, but did not affect expression of 42 other genes related to free radical metabolism. Heat shock also had little effect on genes involved in embryonic development. Effects of heat shock for 2, 4 and 8 h on selected heat shock protein and antioxidant genes were also evaluated by real-time PCR. Heat shock increased steady-state amounts of mRNA for HSPA1A (P<0.05) and tended to increase expression of HSP90AA1 (P<0.07) but had no effect on expression of SOD1 or CAT. Conclusions Changes in the transcriptome of the heat-shocked bovine morula indicate that the embryo is largely resistant to effects of heat shock. As a result, transcription of genes involved in thermal protection is muted and there is little disruption of gene

  3. Effects of downregulating GLIS1 transcript on preimplantation development and gene expression of bovine embryos.

    PubMed

    Takahashi, Kazuki; Sakurai, Nobuyuki; Emura, Natsuko; Hashizume, Tsutomu; Sawai, Ken

    2015-01-01

    Krüppel-like protein Gli-similar 1 (GLIS1) is known as a direct reprogramming factor for the generation of induced pluripotent stem cells. The objective of this study was to investigate the role of GLIS1 in the preimplantation development of bovine embryos. GLIS1 transcripts in in vitro-matured oocytes and 1-cell to 4-cell stage embryos were detected, but they were either absent or at trace levels at the 8-cell to blastocyst stages. We attempted GLIS1 downregulation of bovine early embryos by RNA interference and evaluated developmental competency and gene transcripts, which are involved in zygotic gene activation (ZGA) in GLIS1-downregulated embryos. Injection of specific siRNA resulted in a distinct decrease in GLIS1 transcript in bovine embryos at the 4-cell stage. Although the bovine embryos injected with GLIS1-siRNA could develop to the 16-cell stage, these embryos had difficulty in developing beyond the 32-cell stage. Gene transcripts of PDHA1 and HSPA8, which are transcribed after ZGA, showed lower level in GLIS1 downregulated embryos. It is possible that GLIS1-downregulated embryos fail to initiate ZGA. Our results indicated that GLIS1 is an important factor for the preimplantation development of bovine embryos.

  4. Effects of downregulating GLIS1 transcript on preimplantation development and gene expression of bovine embryos

    PubMed Central

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

    2015-01-01

    Krüppel-like protein Gli-similar 1 (GLIS1) is known as a direct reprogramming factor for the generation of induced pluripotent stem cells. The objective of this study was to investigate the role of GLIS1 in the preimplantation development of bovine embryos. GLIS1 transcripts in in vitro-matured oocytes and 1-cell to 4-cell stage embryos were detected, but they were either absent or at trace levels at the 8-cell to blastocyst stages. We attempted GLIS1 downregulation of bovine early embryos by RNA interference and evaluated developmental competency and gene transcripts, which are involved in zygotic gene activation (ZGA) in GLIS1-downregulated embryos. Injection of specific siRNA resulted in a distinct decrease in GLIS1 transcript in bovine embryos at the 4-cell stage. Although the bovine embryos injected with GLIS1-siRNA could develop to the 16-cell stage, these embryos had difficulty in developing beyond the 32-cell stage. Gene transcripts of PDHA1 and HSPA8, which are transcribed after ZGA, showed lower level in GLIS1 downregulated embryos. It is possible that GLIS1-downregulated embryos fail to initiate ZGA. Our results indicated that GLIS1 is an important factor for the preimplantation development of bovine embryos. PMID:26074126

  5. Array comparative genomic hybridization analyses of all blastomeres of a cohort of embryos from young IVF patients revealed significant contribution of mitotic errors to embryo mosaicism at the cleavage stage.

    PubMed

    Chow, Judy F C; Yeung, William S B; Lau, Estella Y L; Lee, Vivian C Y; Ng, Ernest H Y; Ho, Pak-Chung

    2014-11-24

    Embryos produced by in vitro fertilization (IVF) have a high level of aneuploidy, which is believed to be a major factor affecting the success of human assisted reproduction treatment. The aneuploidy rate of cleavage stage embryos based on 1-2 biopsied blastomeres has been well-reported, however, the true aneuploidy rate of whole embryos remain unclear because of embryo mosaicism. To study the prevalence of mosaicism in top quality IVF embryos, surplus embryos donated from young patients (aged 28-32) in the assisted reproduction program at Queen Mary Hospital, Hong Kong were used. Thirty-six good quality day 2 embryos were thawed. Out of the 135 blastomeres in these embryos, 121 (89.6%) survived thawing. Twelve of these embryos without lysed blastomeres and which cleaved to at least seven cells after a 24-h culture were dissembled into individual blastomeres, which were analysed by array comparative genomic hybridization and microsatellite marker analysis by fluorescent PCR. Out of 12 day-3 embryos, 2 (16.7%) were normal, 3 (25%) were diploid/aneuploidy with <38% abnormality, 4 (33.3%) were diploid/aneuploidy mosaic with > =38% abnormality, and three (25%) were mosaic aneuploids. Conclusive chromosomal data were obtained from a high percentage of blastomeres (92.8%, 90/97). Microsatellite marker analysis performed on blastomeres in aneuploid embryos enabled us to reconstruct the chromosomal status of the blastomeres in each cleavage division. The results showed the occurrence of meiotic errors in 3 (25%) of the studied embryos. There were 16 mitotic errors (18.8%, 16/85) in the 85 mitotic divisions undertaken by the studied embryos. The observed mitotic errors were mainly contributed by endoreduplication (31.3%, 5/16), non-disjunction (25%, 4/16) and anaphase lagging (25%, 4/16). Chromosome breakages occurred in 6 divisions (7.1%, 6/85). Mosaicism occurs in a high percentage of good-quality cleavage stage embryos and mitotic errors contribute significantly to

  6. A Novel Sperm-Delivered Toxin Causes Late-Stage Embryo Lethality and Transmission Ratio Distortion in C. elegans

    PubMed Central

    Seidel, Hannah S.; Ailion, Michael; Li, Jialing; van Oudenaarden, Alexander; Rockman, Matthew V.; Kruglyak, Leonid

    2011-01-01

    The evolutionary fate of an allele ordinarily depends on its contribution to host fitness. Occasionally, however, genetic elements arise that are able to gain a transmission advantage while simultaneously imposing a fitness cost on their hosts. We previously discovered one such element in C. elegans that gains a transmission advantage through a combination of paternal-effect killing and zygotic self-rescue. Here we demonstrate that this element is composed of a sperm-delivered toxin, peel-1, and an embryo-expressed antidote, zeel-1. peel-1 and zeel-1 are located adjacent to one another in the genome and co-occur in an insertion/deletion polymorphism. peel-1 encodes a novel four-pass transmembrane protein that is expressed in sperm and delivered to the embryo via specialized, sperm-specific vesicles. In the absence of zeel-1, sperm-delivered PEEL-1 causes lethal defects in muscle and epidermal tissue at the 2-fold stage of embryogenesis. zeel-1 is expressed transiently in the embryo and encodes a novel six-pass transmembrane domain fused to a domain with sequence similarity to zyg-11, a substrate-recognition subunit of an E3 ubiquitin ligase. zeel-1 appears to have arisen recently, during an expansion of the zyg-11 family, and the transmembrane domain of zeel-1 is required and partially sufficient for antidote activity. Although PEEL-1 and ZEEL-1 normally function in embryos, these proteins can act at other stages as well. When expressed ectopically in adults, PEEL-1 kills a variety of cell types, and ectopic expression of ZEEL-1 rescues these effects. Our results demonstrate that the tight physical linkage between two novel transmembrane proteins has facilitated their co-evolution into an element capable of promoting its own transmission to the detriment of organisms carrying it. PMID:21814493

  7. Optimization and comparison of bottom-up proteomic sample preparation for early-stage Xenopus laevis embryos.

    PubMed

    Peuchen, Elizabeth H; Sun, Liangliang; Dovichi, Norman J

    2016-07-01

    Xenopus laevis is an important model organism in developmental biology. While there is a large literature on changes in the organism's transcriptome during development, the study of its proteome is at an embryonic state. Several papers have been published recently that characterize the proteome of X. laevis eggs and early-stage embryos; however, proteomic sample preparation optimizations have not been reported. Sample preparation is challenging because a large fraction (~90 % by weight) of the egg or early-stage embryo is yolk. We compared three common protein extraction buffer systems, mammalian Cell-PE LB(TM) lysing buffer (NP40), sodium dodecyl sulfate (SDS), and 8 M urea, in terms of protein extraction efficiency and protein identifications. SDS extracts contained the highest concentration of proteins, but this extract was dominated by a high concentration of yolk proteins. In contrast, NP40 extracts contained ~30 % of the protein concentration as SDS extracts, but excelled in discriminating against yolk proteins, which resulted in more protein and peptide identifications. We then compared digestion methods using both SDS and NP40 extraction methods with one-dimensional reverse-phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS). NP40 coupled to a filter-aided sample preparation (FASP) procedure produced nearly twice the number of protein and peptide identifications compared to alternatives. When NP40-FASP samples were subjected to two-dimensional RPLC-ESI-MS/MS, a total of 5171 proteins and 38,885 peptides were identified from a single stage of embryos (stage 2), increasing the number of protein identifications by 23 % in comparison to other traditional protein extraction methods.

  8. Acceptance of embryonic stem cells by a wide developmental range of mouse tetraploid embryos.

    PubMed

    Lin, Chih-Jen; Amano, Tomokazu; Zhang, Jifeng; Chen, Yuqing Eugene; Tian, X Cindy

    2010-08-01

    Tetraploid (4N) complementation assay is regard as the most stringent characterization test for the pluripotency of embryonic stem (ES) cells. The technology can generate mice fully derived from the injected ES cell (ES-4N) with 4N placentas. However, it remains a very inefficient procedure owing to a lack of information on the optimal conditions for ES incorporation into the 4N embryos. In the present study, we injected ES cells from embryos of natural fertilization (fES) and somatic cell nuclear transfer (ntES) into 4N embryos at various stages of development to determine the optimal stage of ES cells integration by comparing the efficiency of full-term ES-4N mouse generation. Our results demonstrate that fES/ntES cells can be incorporated into 4N embryos at 2-cell, 4-cell and blastocyst stages and full-term mice can be generated. Interestingly, ntES cells injected into the 4-cell group resulted in the lowest efficiency (5.6%) compared to the 2-cell (13.8%, P > 0.05) and blastocyst (16.7%, P < 0.05) stages. Because 4N embryos start to form compacted morulae at the 4-cell stage, we investigated whether the lower efficiency at this stage was due to early compaction by injecting ntES cells into artificially de-compacted embryos treated with calcium free medium. Although the treatment changed the embryonic morphology, it did not increase the efficiency of ES-4N mice generation. Immunochemistry of the cytoskeleton displayed microtubule and microfilament polarization at the late 4-cell stage in 4N embryos, which suggests that de-compaction treatment cannot reverse the polarization process. Taken together, we show here that a wide developmental range of 4N embryos can be used for 4N complementation and embryo polarization and compaction may restrict incorporation of ES cells into 4N embryos.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  10. Single stage incubators and Hypercapnia during incubation affect the vascularization of the chorioallantoic membrane in broiler embryos.

    PubMed

    Fernandes, J I M; Bortoluzzi, C; Schmidt, J M; Scapini, L B; Santos, T C; Murakami, A E

    2017-01-01

    Incubation management can have direct effects on neonate health and consequently affect post-hatching development. The effects of incubation in multiple and single stage incubators with different concentrations of CO2 were evaluated in terms of the vessel density in the chorioallantoic membrane, hatching, heart morphology, and body development of the neonate up to the tenth day. A total of 2,520 fertile eggs were used and distributed in a completely randomized design with 4 levels of CO2 in 4 single-stage incubators (4,000; 6,000; 8,000; and 10,000 ppm) and a control treatment based on multiple-stage incubation, totaling 5 treatments. The levels of CO2 were used during the first 10 d of the incubation period, and after this period, all eggs were submitted to the same level of CO2 (4,000 ppm). Eggs that were incubated in multiple-stage incubators presented a lower percentage of vessels in the chorioallantoic membrane, lower yolk absorption by the embryo, wall depth of the right ventricle, and greater humidity losses in the eggs when compared to eggs in the single-stage incubators. The eggs submitted to hypercapnia, between 5,000 and 6,000 ppm of CO2, had a higher percentage of vessels in the chorioallantoic membrane; the embryos originating from these eggs had higher weight, with higher relative weight of the liver. However, the same levels reduced the yolk absorption. Single-stage incubation with moderate levels of hypercapnia is an efficient tool to be adopted by the hatcheries when attempting to improve chick quality. © 2016 Poultry Science Association Inc.

  11. The Temperature and Type of Intracellular Ice Formation in Preimplantation Mouse Embryos as a Function of the Developmental Stage1

    PubMed Central

    Seki, Shinsuke; Mazur, Peter

    2010-01-01

    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°C/min to −70°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°C) vs. 2) those undergoing IIF within 1°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°C to −43°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°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. PMID:20164439

  12. Proteomic analysis of the Gallus gallus embryo at stage-29 of development.

    PubMed

    Agudo, David; Agudo Garcillán, David; Gómez-Esquer, Francisco; Díaz-Gil, Gema; Martínez-Arribas, Fernando; Delcán, José; Schneider, José; Palomar, María Angustias; Linares, Rafael

    2005-12-01

    The chicken (Gallus gallus) is one of the primary models for embryological and developmental studies. In order to begin to understand the molecular mechanisms underlying the normal and abnormal development of the chicken, we used 2-DE to construct a whole-embryo proteome map. Proteins were separated by IEF on IPG strips, and by 11% SDS-PAGE) gels. Protein identification was performed by means of PMF with MALDI-TOF-MS. In all, 105 protein spots were identified, 35 of them implicated in embryo development, 10 related with some diseases, and 16, finally, being proteins that have never been identified, purified or characterized in the chicken before. This map will be updated continuously and will serve as a reference database for investigators, studying changes at the protein level under different physiological conditions.

  13. 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.

  14. 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

  15. 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.

  16. 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. Copyright © 2013 Elsevier Inc. All rights reserved.

  17. A randomized clinical trial comparing recombinant hyaluronan/recombinant albumin versus human tubal fluid for cleavage stage embryo transfer in patients with multiple IVF-embryo transfer failure.

    PubMed

    Friedler, Shevach; Schachter, Morey; Strassburger, Devorah; Esther, Kasterstein; Ron El, Raphael; Raziel, Arieh

    2007-09-01

    We aimed to examine the efficacy of using an embryo transfer medium enriched with hyaluronan (HA) to improve implantation in a selected group of patients aged <43 years with repeated (>4) implantation failures after IVF-embryo transfer. About 101 patients, meeting our selection criteria, were randomly allocated to undergo embryo transfer either using our routine embryo transfer medium without HA (control group) or a HA enriched commercial embryo transfer medium (study group). The primary outcome was clinical pregnancy rate. After a similar treatment protocol, the ovarian hormonal response, the mean number of ova retrieved and injected per patient, fertilization and cleavage rates and mean embryo quality were comparable between the study and control groups. Although a similar number of embryos was transferred in both groups (3.1 +/- 0.7 versus 2.9 +/- 0.6, mean +/- SD), a significantly higher implantation rate (16.3% versus 4.8%, P = 0.002) and clinical pregnancy rate (35.2% versus 10.0%, P = 0.004) and delivered or ongoing pregnancy rate (31.3% versus 4.0%, P = 0.0005) were observed in the study group. When mean implantation rate per patient was calculated, the difference between the study (0.148 +/- 0.23) and control (0.04 +/- 0.13) group was significant (P = 0.003). In this selected group of patients after multiple IVF-embryo transfer failures, the use of HA enriched embryo transfer medium is beneficial.

  18. Single Cell Proteomics Using Frog (Xenopus laevis) Blastomeres Isolated from Early Stage Embryos, Which Form a Geometric Progression in Protein Content.

    PubMed

    Sun, Liangliang; Dubiak, Kyle M; Peuchen, Elizabeth H; Zhang, Zhenbin; Zhu, Guijie; Huber, Paul W; Dovichi, Norman J

    2016-07-05

    Single cell analysis is required to understand cellular heterogeneity in biological systems. We propose that single cells (blastomeres) isolated from early stage invertebrate, amphibian, or fish embryos are ideal model systems for the development of technologies for single cell analysis. For these embryos, although cell cleavage is not exactly symmetric, the content per blastomere decreases roughly by half with each cell division, creating a geometric progression in cellular content. This progression forms a ladder of single-cell targets for the development of successively higher sensitivity instruments. In this manuscript, we performed bottom-up proteomics on single blastomeres isolated by microdissection from 2-, 4-, 8-, 16-, 32-, and 50-cell Xenopus laevis (African clawed frog) embryos. Over 1 400 protein groups were identified in single-run reversed-phase liquid chromatography-electrospray ionization-tandem mass spectrometry from single balstomeres isolated from a 16-cell embryo. When the mass of yolk-free proteins in single blastomeres decreased from ∼0.8 μg (16-cell embryo) to ∼0.2 μg (50-cell embryo), the number of protein group identifications declined from 1 466 to 644. Around 800 protein groups were quantified across four blastomeres isolated from a 16-cell embryo. By comparing the protein expression among different blastomeres, we observed that the blastomere-to-blastomere heterogeneity in 8-, 16-, 32-, and 50-cell embryos increases with development stage, presumably due to cellular differentiation. These results suggest that comprehensive quantitative proteomics on single blastomeres isolated from these early stage embryos can provide valuable insights into cellular differentiation and organ development.

  19. A catalog of Xenopus tropicalis transcription factors and their regional expression in the early gastrula stage embryo.

    PubMed

    Blitz, Ira L; Paraiso, Kitt D; Patrushev, Ilya; Chiu, William T Y; Cho, Ken W Y; Gilchrist, Michael J

    2017-06-15

    Gene regulatory networks (GRNs) involve highly combinatorial interactions between transcription factors and short sequence motifs in cis-regulatory modules of target genes to control cellular phenotypes. The GRNs specifying most cell types are largely unknown and are the subject of wide interest. A catalog of transcription factors is a valuable tool toward obtaining a deeper understanding of the role of these critical effectors in any biological setting. Here we present a comprehensive catalog of the transcription factors for the diploid frog Xenopus tropicalis. We identify 1235 genes encoding DNA-binding transcription factors, comparable to the numbers found in typical mammalian species. In detail, the repertoire of X. tropicalis transcription factor genes is nearly identical to human and mouse, with the exception of zinc finger family members, and a small number of species/lineage-specific gene duplications and losses relative to the mammalian repertoires. We applied this resource to the identification of transcription factors differentially expressed in the early gastrula stage embryo. We find transcription factor enrichment in Spemann's organizer, the ventral mesoderm, ectoderm and endoderm, and report 218 TFs that show regionalized expression patterns at this stage. Many of these have not been previously reported as expressed in the early embryo, suggesting thus far unappreciated roles for many transcription factors in the GRNs regulating early development. We expect our transcription factor catalog will facilitate myriad studies using Xenopus as a model system to understand basic biology and human disease. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  20. Quantitation and characterization of a species-specific and embryo stage-dependent 55-kilodalton phosphoprotein also present in cells transformed by simian virus 40.

    PubMed Central

    Chandrasekaran, K; McFarland, V W; Simmons, D T; Dziadek, M; Gurney, E G; Mora, P T

    1981-01-01

    A 55-kilodalton (kDal) protein was detected recently in primary cultures of day 12 mouse embryos by immunoprecipitation with serum from simian virus 40 (SV40) tumor-bearing hamsters (T serum), Preliminary evidence suggested that this protein was similar to a cellular 55-kDal protein induced after SV40 transformation of mouse cells. We now show that specific approximately 55-kDal [35S]methionine-labeled proteins precipitate from primary cultures of midgestation mouse, rat, and hamster embryos on addition of T serum or monoclonal antiserum prepared against the SV40-induced mouse 55-kDal proteins. The two-dimensional maps of the [35S]methionine-labeled tryptic peptides of the mouse, hamster, and rat embryo proteins are similar to the maps of the corresponding proteins from SV40-transformed cells. Primary cells from midgestation mouse, hamster, or rat embryos contain one-third to one-half as much 55-kDal protein as a SV40-transformed mouse fibroblast cell and nearly the same amount as F9 mouse embryonal carcinoma cells. The amount of 55-kDal protein is greatly reduced on replating the mouse, rat, or hamster embryo primary cells. The amount of this protein in mouse embryos is dependent on the stage of the embryo. The embryo proteins are phosphoproteins. Images PMID:6273897

  1. The new Rapid-i carrier is an effective system for human embryo vitrification at both the blastocyst and cleavage stage.

    PubMed

    Desai, Nina N; Goldberg, Jeffrey M; Austin, Cynthia; Falcone, Tommaso

    2013-05-15

    The Rapid-i is a new FDA cleared closed carrier for embryo vitrification. The cooling rate of - 1220°C/min is far lower than that reported with open vitrification systems such as the cryoloop (-15,000°C/min). Little published data is currently available on this device. This study presents our initial clinical data, as well as live birth outcomes, with the Rapid-i. The efficacy of this device for the cryopreservation of cleavage, as well as blastocyst stage human embryos is also analyzed. We further compare outcomes to those achieved with the cryoloop, an "open" vitrification system routinely used in our laboratory. Human embryos were vitrified at either the 8-10 cell stage or else the blastocyst stage. The vitrification protocol was: 7.5% DMSO/7.5% ethylene glycol (EG) (2-3 min) followed by incubation in 15% DMSO /15% EG (45 sec) before loading on the vitrification carrier. Cryoprotectant was removed during warming by sequential washes in 0.25 M and 0.125 M sucrose in culture medium. Clinical outcome data for frozen cycles between January 2011 and August 2012 were stratified according to carrier and cell stage. The student t-test and chi square test were used to compare results. P value of < 0.05 was considered significant. A total of 486 vitrified-warmed embryos were assessed and 92% of them were transferred. The clinical pregnancy rate (CPR) and implantation rate (IR) with Rapid-i vitrified blastocysts were 59% and 49%, versus 47% and 37%, respectively for cleavage stage embryos. This was not statistically different from results with the cryoloop vitrified blastocysts (CPR 46%, IR 38%) nor the cleavage stage vitrified embryos (CPR 49%, IR 35%). To date, there have been 31 deliveries of 34 healthy infants from Rapid-i vitrified embryos, with another 12 pregnancies still on-going. The Rapid-i offers an excellent alternative to existing open vitrification devices for embryo cryopreservation at the 8-10 cell stage as well as the blastocyst stage. Use of this type

  2. Development of rat tetraploid and chimeric embryos aggregated with diploid cells.

    PubMed

    Shinozawa, T; Sugawara, A; Matsumoto, A; Han, Y-J; Tomioka, I; Inai, K; Sasada, H; Kobayashi, E; Matsumoto, H; Sato, E

    2006-11-01

    In the present study, we examined the preimplantation and postimplantation development of rat tetraploid embryos produced by electrofusion of 2-cell-stage embryos. Developmental rate of tetraploid embryos to morula or blastocyst stage was 93% (56/60) and similar to that found in diploid embryos (95%, 55/58). After embryo transfer, rat tetraploid embryos showed implantation and survived until day 8 of pregnancy, however the conceptuses were aberrant on day 9. In mouse, tetraploid embryos have the ability to support the development of blastomeres that cannot develop independently. As shown in the present study, a pair of diploid blastomeres from the rat 8-cell-stage embryo degenerated immediately after implantation. Therefore, we examined whether rat tetraploid embryos have the ability to support the development of 2/8 blastomeres. We produced chimeric rat embryos in which a pair of diploid blastomeres from an 8-cell-stage green fluorescent protein negative (GFP-) embryo was aggregated with three tetraploid blastomeres from 4-cell GFP-positive (GFP+) embryos. The developmental rate of rat 2n(GFP-) <--> 4n(GFP+) embryos to the morula or blastocyst stages was 93% (109/117) and was similar to that found for 2n(GFP-) <--> 2n(GFP+) embryos (100%, 51/51). After embryo transfer, 2n(GFP-) <--> 4n(GFP+) conceptuses were examined on day 14 of pregnancy, the developmental rate to fetus was quite low (4%, 4/109) and they were all aberrant and smaller than 2n(GFP-) <--> 2n(GFP+) conceptuses, whereas immunohistochemical analysis showed no staining for GFP in fetuses. Our results suggest that rat tetraploid embryos are able to prolong the development of diploid blastomeres that cannot develop independently, although postimplantation development was incomplete.

  3. Analysis of compaction initiation in human embryos by using time-lapse cinematography.

    PubMed

    Iwata, Kyoko; Yumoto, Keitaro; Sugishima, Minako; Mizoguchi, Chizuru; Kai, Yoshiteru; Iba, Yumiko; Mio, Yasuyuki

    2014-04-01

    To analyze the initiation of compaction in human embryos in vitro by using time-lapse cinematography (TLC), with the goal of determining the precise timing of compaction and clarifying the morphological changes underlying the compaction process. One hundred and fifteen embryos donated by couples with no further need for embryo-transfer were used in this study. Donated embryos were thawed and processed, and then their morphological behavior during the initiation of compaction was dynamically observed via time-lapse cinematography (TLC) for 5 days. Although the initiation of compaction occurred throughout the period from the 4-cell to 16-cell stage, 99 (86.1 %) embryos initiated compaction at the 8-cell stage or later, with initiation at the 8-cell stage being most frequent (22.6 %). Of these 99 embryos, 49.5 % developed into good-quality blastocysts. In contrast, of the 16 (13.9 %) embryos that initiated compaction prior to the 8-cell stage, only 18.8 % developed into good-quality blastocysts. Embryos that initiated compaction before the 8-cell stage showed significantly higher numbers of multinucleated blastomeres, due to asynchronism in nuclear division at the third mitotic division resulting from cytokinetic failure. The initiation of compaction primarily occurs at the third mitotic division or later in human embryos. Embryos that initiate compaction before the 8-cell stage are usually associated with aberrant embryonic development (i.e., cytokinetic failure accompanied by karyokinesis).

  4. The Dynamin 2 inhibitor Dynasore affects the actin filament distribution during mouse early embryo development.

    PubMed

    Wang, Qiao-Chu; Liu, Jun; Duan, Xing; Cui, Xiang-Shun; Kim, Nam-Hyung; Xiong, Bo; Sun, Shao-Chen

    2015-01-01

    Dynamin 2 is a large GTPase notably involved in clathrin-mediated endocytosis, cell migration and cytokinesis in mitosis. Our previous study identified that Dynamin 2 regulated polar body extrusion in mammalian oocytes, but its roles in early embryo development, remain elusive. Here, we report the critical roles of Dynamin 2 in mouse early embryo development. Dynamin 2 accumulated at the periphery of the blastomere during embryonic development. When Dynamin 2 activity was inhibited by Dynasore, embryos failed to cleave to the 2-cell or 4-cell stage. Moreover, the actin filament distribution and relative amount were aberrant in the treatment group. Similar results were observed when embryos were cultured with Dynasore at the 8-cell stage; the embryos failed to undergo compaction and develop to the morula stage, indicating a role of Dynamin 2 in embryo cytokinesis. Therefore, our data indicate that Dynamin 2 might participate in the early embryonic development through an actin-based cytokinesis.

  5. Expression pattern of oxygen and stress-responsive gene transcripts at various developmental stages of in vitro and in vivo preimplantation bovine embryos.

    PubMed

    Balasubramanian, S; Son, W J; Kumar, B Mohana; Ock, S A; Yoo, J G; Im, G S; Choe, S Y; Rho, G J

    2007-07-15

    The present study examined the expression pattern of oxygen (O(2)) and stress-responsive gene transcripts at various preimplantation developmental stages of in vitro produced (IVP) and in vivo derived (IVD) bovine embryos. Embryos were produced in vitro from oocytes matured, fertilized and cultured in synthetic oviductal fluid (SOF) medium under low (5%) and high (20%) O(2) concentrations. In vivo embryos were derived from 18 superovulated and artificially inseminated cows. In IVP and IVD groups, embryos were collected at 2-, 4-, 8-, 16-cell morula and blastocyst stages at specific time points for gene expression analysis. The cleavage rates (69.8+/-4.8%) did not differ significantly, but blastocyst rates were significantly higher (28.5+/-3.7%) in low O(2) than those in high O(2) group (18.7+/-3.9%). Mean cell number in low O(2) (145+/-12) and high O(2) (121+/-73) IVP blastocyst were lower (P<0.05) than those of IVD blastocyst (223+/-25). The ICM ratio of IVD blastocyst (26+/-4) was lower (P<0.05) than that of IVP embryos under 5% O(2) (33+/-5) and 20% O(2) (34+/-4) concentrations, respectively. Using real time PCR, for the set of target transcripts (Glut1, Glut5, Sox, G6PD, MnSOD, PRDX5, NADH and Hsp 70.1) analyzed, there were differences in the mRNA expression pattern at 2-, 4-, 8-, 16-cell morula and Day 7 blastocyst stages between the two embryo sources. It can be concluded that, although in vitro bovine embryo culture in SOF medium under low (5%) O(2) concentration provided a more conducive environment in terms of blastocyst formation; differences in the total cell number and gene expression pattern between the IVP and IVD embryos reflected the effect of O(2) concentration.

  6. 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.

  7. 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

  8. 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

  9. 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

  10. 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

  11. Absence of Sperm Factors as in the Parthenogenesis Does Not Interfere on Bovine Embryo Sensitiveness to Heat Shock at Pre-Implantation Stage.

    PubMed

    Camargo, L S A; Paludo, F; Pereira, M M; Wohlres-Viana, S; Gioso, M M; Carvalho, B C; Quintao, C C R; Viana, J H M

    2016-02-01

    Oocyte has been considered the major contributor for embryo thermo-tolerance. However, it was shown that sperm factors can be transferred to the oocyte during fertilization, raising the question of whether the absence of such factors could interfere on embryo thermo-tolerance. In this study, we used parthenogenesis to generate bovine embryos without spermatozoa in order to test whether the absence of sperm factors could influence their thermo-sensitiveness at early stages. In vitro fertilized (IVF) and parthenogenetic (PA) embryos at 44 h post-insemination/chemical activation were exposed to 38.5°C (control) or 41°C (heat shock) for 12 h and then developed for 48 h and up to blastocyst stage. Apoptosis index and expression of PRDX1, GLUT1, GLUT5 and IGF1r genes in blastocysts derived from heat-shocked embryos were also evaluated. The heat shock decreased the blastocyst rate at day seven (p < 0.05) for IVF embryos and at day eight (p < 0.01) for both IVF and PA embryos. Total cell number was not affected by heat shock in IVF and PA blastocysts, but there was an increased proportion (p < 0.05) of apoptotic cells in heat-shocked embryos when compared to controls. There was no interaction (p > 0.05) between method of activation (IVF and PA) and temperature (38.5°C or 41.5°C) for all developmental parameters evaluated. Expression of GLUT1 gene was downregulated (p < 0.05) by heat shock in both IVF and PA blastocyst whereas expression of GLUT5 and IGF1r genes was downregulated (p < 0.05) by heat shock in PA blastocysts. Those data show that the heat shock affects negatively the embryo development towards blastocysts stage, increases the apoptotic index and disturbed the expression of some genes in both IVF and PA embryos, indicating that the presence or absence of sperm factors does not influence the sensitivity of the bovine embryo to heat shock.

  12. Mercury (II) impairs nucleotide excision repair (NER) in zebrafish (Danio rerio) embryos by targeting primarily at the stage of DNA incision.

    PubMed

    Chang, Yung; Lee, Wei-Yuan; Lin, Yu-Jie; Hsu, Todd

    2017-09-14

    Mercuric ion (Hg(2+)) is the most prevalent form of inorganic Hg found in polluted aquatic environment. As inhibition of DNA damage repair has been proposed as one of the mechanisms of Hg(2+)-induced genotoxicity in aquatic animals and mammalian cells, this study explored the susceptibility of different stages of nucleotide excision repair (NER) in zebrafish (Danio rerio) embryos to Hg(2+) using UV-damaged DNA as the repair substrate. Exposure of embryos at 1h post fertilization (hpf) to HgCl2 at 0.1-2.5μM for 9h caused a concentration-dependent inhibition of NER capacity monitored by a transcription-based DNA repair assay. The extracts of embryos exposed to 2.5μM Hg(2+) almost failed to up-regulate UV-suppressed marker cDNA transcription. No inhibition of ATP production was observed in all Hg(2+)-exposed embryos. Hg(2+) exposure imposed either weak inhibitory or stimulating effects on the gene expression of NER factors, while band shift assay showed the inhibition of photolesion binding activities to about 40% of control in embryos treated with 1-2.5μM HgCl2. The damage incision stage of NER in zebrafish embryos was found to be more sensitive to Hg(2+) than photolesion binding capacity due to the complete loss of damage incision activity in the extracts of embryos exposed to 1-2.5μM Hg(2+). NER-related DNA incision was induced in UV-irradiated embryos based on the production of short DNA fragments matching the sizes of excision products generated by eukaryotic NER. Pre-exposure of embryos to Hg(2+) at 0.1-2.5μM all suppressed DNA incision/excision in UV-irradiated embryos, reflecting a high sensitivity of DNA damage incision/excision to Hg(2+). Our results showed the potential of Hg(2+) at environmental relevant levels to disturb NER in zebrafish embryos by targeting primarily at the stage of DNA incision/excision. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. 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

  14. Effect of freezing rate and exposure time to cryoprotectant on the development of mouse pronuclear stage embryos.

    PubMed

    Nowshari, M A; Brem, G

    2001-11-01

    The effects of exposure time (20 versus 45 s) to a high concentration of cryoprotectant (7.0 mol/l ethylene glycol with 0.5 mol/l sucrose) and freezing rates (1200-10 300 degrees C/min) during rapid freezing of mouse pronuclear stage embryos on survival and development to blastocysts were investigated. Different freezing rates were achieved by directly plunging the straws (rapid freezing) and open pulled straws (OPS) in liquid nitrogen (OPS freezing) and by plunging the straws (super rapid) and OPS (super OPS) in a super cooled liquid nitrogen chamber (at -212 degrees C) before storage in liquid nitrogen. Morphologically intact mouse zygotes (n = 891) pre-equilibrated in 1.5 mol/l ethylene glycol for 5 min were either loaded in 0.25 ml straws containing cryoprotectant or loaded in OPS with 2 microl cryoprotectant. After 20 or 45 s of loading the straws or mixing in cryoprotectant and loading in OPS, they were plunged either directly in to liquid nitrogen or were plunged first in to liquid nitrogen in a super cooled chamber and then stored in liquid nitrogen. Zygotes were thawed and intact embryos cultured in vitro. The rate of survival was higher (91%, P < 0.01) when zygotes were frozen with rapid freezing compared with super rapid, OPS and super OPS freezing rates with an exposure time of 20 s (70, 65, and 76% respectively). When zygotes were exposed to cryoprotectant for 45 s and frozen with rapid freezing rates, the survival was higher (86%, P < 0.01) compared with those frozen with OPS (62%) but was not different from those frozen with super rapid and super OPS freezing rates (81 and 75%). A higher rate of survival was observed when zygotes were exposed to cryoprotectant for 45 s and frozen with super OPS than with OPS freezing (75 versus 62%; P < 0.05). The rate of cleavage and development of intact zygotes to blastocysts was not different among the different groups. Exposure of zygotes to a high concentration of cryoprotectant (7.0 mol/l ethylene glycol with

  15. 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

  16. Intraovarian transplantation of stage I-II follicles results in viable zebrafish embryos.

    PubMed

    Csenki, Zsolt; Zaucker, Andreas; Kovács, Balázs; Hadzhiev, Yavor; Hegyi, Arpád; Lefler, Katalin-Kinga; Müller, Tamás; Kovács, Robert; Urbányi, Béla; Váradi, László; Müller, Ferenc

    2010-01-01

    Maternal gene products drive early embryogenesis almost exclusively until the mid blastula transition (MBT) in many animal models including fish. However, the maternal contribution to embryogenesis does not stop at MBT, but continues to be an essential regulator of key developmental processes. The extent to which maternal effects contribute to embryonic and larval development is hard to estimate due to the technical difficulty of interfering with maternal gene products by conventional forward and reverse genetic tools. Therefore, novel methods to manipulate maternal factors in oocytes need to be developed. Here, we provide a proof of principle protocol for transplanting stage I-II zebrafish follicles into recipient mothers where donor stage I oocytes can develop to stage IV in 2 weeks and in 3 weeks they develop into mature eggs and produce viable offspring. Moreover, we show that simple microinjection of stage I-II follicles with RNA results in reporter gene expression in oocytes and paves the way for developing tools for interfering with maternal gene activity. This early stage oocyte transplantation protocol provides a means to study cellular and molecular aspects of oocyte development in the zebrafish.

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

    PubMed

    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.

  18. 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. © 2015 Wiley Periodicals, Inc.

  19. Spontaneous locomotor activity in late-stage chicken embryos is modified by stretch of leg muscles

    PubMed Central

    Bradley, Nina S.; Ryu, Young U.; Yeseta, Marie C.

    2014-01-01

    Chicks initiate bilateral alternating steps several days before hatching and adaptively walk within hours of hatching, but emergence of precocious walking skills is not well understood. One of our aims was to determine whether interactions between environment and movement experience prior to hatching are instrumental in establishing precocious motor skills. However, physiological evidence of proprioceptor development in the chick has yet to be established; thus, one goal of this study was to determine when in embryogenesis proprioception circuits can code changes in muscle length. A second goal was to determine whether proprioception circuits can modulate leg muscle activity during repetitive limb movements for stepping (RLMs). We hypothesized that proprioception circuits code changes in muscle length and/or tension, and modulate locomotor circuits producing RLMs in anticipation of adaptive locomotion at hatching. To this end, leg muscle activity and kinematics were recorded in embryos during normal posture and after fitting one ankle with a restraint that supported the limb in an atypical posture. We tested the hypotheses by comparing leg muscle activity during spontaneous RLMs in control posture and ankle extension restraint. The results indicated that proprioceptors detect changes in muscle length and/or muscle tension 3 days before hatching. Ankle extension restraint produced autogenic excitation of the ankle flexor and reciprocal inhibition of the ankle extensor. Restraint also modified knee extensor activity during RLMs 1 day before hatching. We consider the strengths and limitations of these results and propose that proprioception contributes to precocious locomotor development during the final 3 days before hatching. PMID:24265423

  20. 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...

  1. 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...

  2. Improving the quality of miniature pig somatic cell nuclear transfer blastocysts: aggregation of SCNT embryos at the four-cell stage.

    PubMed

    Terashita, Y; Sugimura, S; Kudo, Y; Amano, R; Hiradate, Y; Sato, E

    2011-04-01

    Miniature pigs share many similar characteristics such as anatomy, physiology and body size with humans and are expected to become important animal models for therapeutic cloning using embryonic stem cells (ESCs) derived by somatic cell nuclear transfer (SCNT). In the present study, we observed that miniature pig SCNT blastocysts possessed a lower total number of nuclei and a lower percentage of POU5F1-positive cells than those possessed by in vitro fertilized (IVF) blastocysts. To overcome these problems, we evaluated the applicability of aggregating miniature pig SCNT embryos at the four-cell stage. We showed that (i) aggregation of two or three miniature pig SCNT embryos at the four-cell stage improves the total number of nuclei and the percentage of POU5F1-positive cells in blastocysts, and (ii) IVF blastocysts with low cell numbers induced by the removal of two blastomeres at the four-cell stage did not exhibit a decrease in the percentage of POU5F1-positive cells. These results suggest that the aggregation of miniature pig SCNT embryos at the four-cell stage can be a useful technique for improving the quality of miniature pig SCNT blastocysts and indicating that improvement in the percentage of POU5F1-positive cells in aggregated SCNT embryos is not simply the consequence of increased cell numbers. © 2010 Blackwell Verlag GmbH.

  3. Vitamin A deficiency in the late gastrula stage rat embryo results in a one to two vertebral anteriorization that extends throughout the axial skeleton.

    PubMed

    Kaiser, Mary E; Merrill, Ronald A; Stein, Adam C; Breburda, Edith; Clagett-Dame, Margaret

    2003-05-01

    Vitamin A and its metabolites are known to be involved in patterning the vertebrate embryo. Study of the effect of vitamin A on axial skeletal patterning has been hindered by the fact that deficient embryos do not survive past midgestation. In this study, pregnant vitamin A-deficient rats were maintained on a purified diet containing limiting amounts of all-trans retinoic acid (12 microg atRA/g diet) and given a daily oral bolus dose of retinol starting at embryonic day 0.5, 8.25, 8.5, 8.75, 9.25, 9.5, 9.75, or 10.5. Embryos were recovered at E21.5 for analysis of the skeleton and at earlier times for analysis of select mRNAs. Normal axial skeletal development and patterning were observed in embryos from pregnant animals receiving retinol starting on or before E8.75. Delay of retinol supplementation to E9.5 or later resulted in a marked increase in both occurrence and severity of skeletal malformations, extending from the craniocervical to sacral regions. Embryos from the groups receiving retinol starting at E9.5 and E9.75 had one-vertebral anterior transformations of the cervical, thoracic, lumbar, and sacral vertebrae. Few embryos survived in the E10.5 group, but these embryos yielded the most severe and extensive anteriorization events. The skeletal alterations seen in vitamin A deficiency are associated with posterior shifts in the mesodermal expression of Hoxa-4, Hoxb-3, Hoxd-3, Hoxd-4, and Hoxa-9 mRNAs, whereas the anterior domains of Hoxb-4 and Cdx2 expression are unaltered. This work defines a critical window of development in the late gastrula-stage embryo when vitamin A is essential for normal axial skeletal patterning and shows that vitamin A deficiency causes anterior homeotic transformations extending from the cervical to lumbosacral regions.

  4. 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.

  5. Comparative neonatal outcomes in singleton births from blastocyst transfers or cleavage-stage embryo transfers: a systematic review and meta-analysis.

    PubMed

    Wang, Xingling; Du, Mingze; Guan, Yichun; Wang, Bijun; Zhang, Junwei; Liu, Zihua

    2017-05-04

    Comparative neonatal outcomes with respect to singleton births from blastocyst transfers or cleavage-state embryo transfers are controversial with respect to which method is superior. Many studies have yielded contradictory results. We performed a systematic review and meta-analysis for the purpose of comparing neonatal outcomes in single births following IVF/ICSI. We searched the Medline, Embase and Cochrane Central Register of Clinical Trials (CCTR) databases until October 2016. Studies and trials that contained neonatal outcomes for singleton births were included. Data were extracted in 2 × 2 tables. The analysis was performed using Rev Man 5.1 software. Risk ratios (RRs) and risk differences, with 95% confidence intervals, were calculated to assess the results of each outcome. Subgroups were applied in all outcomes. Newcastle-Ottawa scale (NOS) checklists were used to assess the quality of the referenced studies. Twelve studies met the criteria in this meta-analysis. There was a high risk of preterm birth after blastocyst embryo transfer versus the risk after cleavage-stage transfer (RR: 1.11, 95% CI: 1.01-1.22). For the "only fresh" subgroup, the outcome was coincident (RR: 1.16, 95% CI: 1.06-1.27). For the "fresh and frozen" and "only frozen" subgroups, there were no differences. Patients who received fresh blastocyst embryo transfers had a high risk of very preterm births (RR: 1.16, 95% CI: 1.02-1.31). Finally, cleavage-stage embryo transfers were associated with a high risk of infants who were small for gestational age (0.83, 95% CI: 0.76-0.92) and a low risk of those who were large for gestation age (1.14, 95% CI: 1.04-1.25). The risks of preterm and very preterm births increased after fresh blastocyst transfers versus the risks after fresh cleavage-stage embryo transfers. However, in frozen embryo transfers, there were no differences. Blastocyst embryo transfers resulted in high risks of infants who were large for gestational age, and cleavage-stage

  6. A simplified table for staging embryos of the pipid frog Pipa arrabali.

    PubMed

    Araújo, Olívia G S; Haddad, Célio F B; Silva, Hélio R DA; Pugener, Lourdes A

    2016-01-01

    Pipa is a Neotropical genus of frogs that dwell in freshwater environments. It includes four species that lack free-swimming larvae (P. aspera, P. arrabali, P. pipa, and P. snethlageae) and three with tadpoles (P. carvalhoi, P. myersi, and P. parva). Developmental tables such as the one proposed by Nieuwkoop and Faber might be useful for Pipa species with tadpoles. However, for the other Pipa species, to determine stages by this table or by any of the tables already prepared for frogs without tadpoles (e.g., Crinia nimbus, Eleutherodactylus coqui, and Oreobates barituensis) is impossible. By using embryonic, juvenile, and subadult specimens, we generated a staging table for P. arrabali, from the moment limb buds were first observed until birth, based on diagnostic features such as snout-vent length; growth, morphology, and reabsorption of the external tail; growth and differentiation of fore and hind limbs; development of intestine and vent tube; position of the angle of the mouth relative to nostrils and eyes; and color of preserved individuals. Based on these observations, we discuss some noteworthy traits (e.g., posture of hands and feet). We also compare the pattern of development of P. arrabali with that of other anuran species (with and without tadpoles).

  7. Two culture systems showing a biphasic effect on ovine embryo development from the 1-2 cell stage to hatched blastocysts.

    PubMed

    Ledda, S; Bogliolo, L; Leoni, G; Loi, P; Cappai, P; Naitana, S

    1995-01-01

    This study compared the effect of using either CZB or TCM 199 media on both the development of 1-2 cell ovine embryos from superovulated ewes to the blastocyst stage (Experiment 1), and the hatching process of ovine blastocysts developed in vitro (Experiment 2). For the first 5 d, the CZB medium showed higher rates of embryo development than the TCM 199 medium (p < 0.001). The embryos reaching the > 16 cell stage were 79 vs 52% and 74 vs 20% with or without an oviductal monolayer, respectively, and those reaching the blastocyst stage were 71 vs 46% and 46 vs 13% with or without cells. The CZB medium was less able to support the hatching process of the blastocysts obtained in the first experiment than was the TCM-199 medium + 10% FCS (fetal calf serum) with cells (31 vs 92%; p < 0.001) or without cells (13 vs 66%; p < 0.001). No blastocysts completely escaped from the zona pellucida (ZP) in the CZB medium compared with 80 and 61% in the TCM 199 medium with or without cells, respectively. In Experiment 3, 47% of the blastocysts migrated through the artificial opening of the ZP and hatched completely. After 24 h of culture in the CZB medium, however, they showed blastocoelic cavity breakdown. During the preliminary cleavages, the ovine embryos developed better in CZB medium than in TCM 199, but the latter was more efficient in promoting the hatching process of the blastocysts.

  8. The innate immunity receptor TIR8/SIGIRR is expressed in the early developmental stages of chicken embryos.

    PubMed

    Turin, L; Manarolla, G; Rampin, T; Riva, F

    2014-01-01

    The orphan receptor TIR8, also known as SIGIRR (Single Immunoglobulin IL-1R-Related molecule), belongs to the IL-1R/TLR (TIR) superfamily and plays an important role in the inflammatory responses. The signaling pathways of the receptors belonging to the TIR family are tightly regulated by both extracellular and intracellular mechanisms. TIR8 does not activate the transcription factors NFkB (nuclear factor kB) and IRF3 (interferon regulatory factor 3), although it negatively modulates the inflammatory responses. It acts as an antagonist for the IL-1 receptor family and triggers a negative pathway of the Toll-like/IL-1 receptor system, crucial for dampening inflammation stimuli in the gastrointestinal (GI) tract and in other organs (e.g. lung and kidney). The recent findings of TLRs expression in ovary and embryos of different species (mammals and chickens) are very important for an understanding of reproductive physiology and transovarian pathogen transmission. TIR8 was well characterized in mouse, humans and in other mammalian species, but it is still poorly characterized in the chicken. When TIR8 expression was measured in selected organs of chicken embryos of both broiler and layer types at different time points a unique pattern of expression was observed. Interestingly, TIR8 was detected during the first stages of chicken development (day 1 of incubation), and reached a remarkable level of expression by day 10. We observed this receptor to be ubiquitously expressed in the kidney, GI tract, Bursa of Fabricius, with the highest expression levels in liver and kidney. This pattern was comparable to those observed in post-hatching chickens and in mammals examined to date. No expression differences were observed between the two different chicken breeds (layer- and broiler-type) in the first incubation period (8 days). Whereas in some organs starting from day 10, higher TIR8 expression was observed in broiler-type compared to layer-type. These are the first findings

  9. The early embryo response to intracellular reactive oxygen species is developmentally regulated.

    PubMed

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

    2011-01-01

    In vitro embryo production (IVP) suffers from excessive developmental failure. Its inefficiency is linked, in part, to reactive oxygen species (ROS) brought on by high ex vivo oxygen (O(2)) tensions. To further delineate the effects of ROS on IVP, the intracellular ROS levels of early bovine embryos were modulated by: (1) varying O(2) tension; (2) exogenous H(2)O(2) treatment; and (3) antioxidant supplementation. Although O(2) tension did not significantly affect blastocyst frequencies (P>0.05), 20% O(2) accelerated the rate of first cleavage division and significantly decreased and increased the proportion of permanently arrested 2- to 4-cell embryos and apoptotic 9- to 16-cell embryos, respectively, compared with embryos cultured in 5% O(2) tension. Treatment with H(2)O(2), when applied separately to oocytes, zygotes, 2- to 4-cell embryos or 9- to 16-cell embryos, resulted in a significant (P<0.05) dose-dependent decrease in blastocyst development in conjunction with a corresponding increase in the induction of either permanent embryo arrest or apoptosis in a stage-dependent manner. Polyethylene glycol-catalase supplementation reduced ROS-induced embryo arrest and/or death, resulting in a significant (P<0.05) increase in blastocyst frequencies under high O(2) culture conditions. Together, these results indicate that intracellular ROS may be signalling molecules that, outside an optimal range, result in various developmentally regulated modes of embryo demise.

  10. Chimeric honeybees (Apis mellifera) produced by transplantation of embryonic cells into pre-gastrula stage embryos and detection of chimerism by use of microsatellite markers.

    PubMed

    Bergem, M; Norberg, K; Roseth, A; Meuwissen, T; Lien, S; Aamodt, R H

    2006-04-01

    The production of chimeras, by use of cell transplantation, has proved to be highly valuable in studies of development by providing insights into cell fate, differentiation, and developmental potential. So far, chimeric honeybees have been created by nuclear transfer technologies. We have developed protocols to produce chimeric honeybees by use of cell transplantation. Embryonic cells were transplanted between pre-gastrula stage embryos (32-34 hr after oviposition) and hatched larvae were reared in vitro for 4 days. Chimeric individuals were detected by use of microsatellite analysis and a conservative estimation approach. 4.8% of embryos, posteriorly injected with embryonic cells, developed into chimeric honeybee larvae. By injection of cells pre-stained with fluorescent cell tracer dye, we studied the integration of transplanted cells in the developing embryos. Number of injected cells varied from 0 to 50 and cells remained and multiplied mainly in the area of injection.

  11. 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.

  12. Cervicovaginal cytological abnormalities in patients with human immunodeficiency virus infection, in relation to disease stage, CD4 cell count and viral load.

    PubMed

    Micheletti, Adilha Misson Rua; Dutra, Valéria de Freitas; Murta, Eddie Fernando Cândido; Paschoini, Marina Carvalho; Silva-Vergara, Mário Leon; Barbosa e Silva, Gisele; Adad, Sheila Jorge

    2009-03-01

    The objective of the present study was to assess infections and cytologic abnormalities in cervicovaginal smears from 153 HIV-positive women and 169 HIV-negative followed up at the UFTM School of Medicine between May 1999 and May 2002. The medical records and cervicovaginal smears were reviewed and the HIV-positive group was classified according to CD4 cell count, HIV viral load, antiretroviral therapy and HIV subgroups (with or without disease; with or without therapy) and compared to HIV-negative group. We conclude that the frequency of Candida sp, Trichomonas vaginalis and bacterial vaginosis in cervicovaginal smear, is not different between HIV-positive and HIV-negative women, even if the HIV-group is subdivided according to CD4 cell count, HIV viral load, antiretroviral therapy and HIV subgroups. The frequency of LSIL, in cervicovaginal smears, was greater in the HIV-group (17.6%) than in the HIV-negative (4.1%); there was no difference between the two groups according to frequency of HSIL (4.6% versus 1.8%), ASCUS/AGUS (7.8% versus 3.5%) and invasive carcinoma (1.3% versus 0.6%). The frequency of LSIL was greater in the HIV positive group with CD4 cell count < 350 cells/mm(3). The viral load, therapeutic regimen and HIV subgroups (HIV-positive without therapy, HIV-positive with therapy, AIDS by immunological criteria and AIDS by clinical criteria) have not shown relationship with LSIL frequency, until now.

  13. Vestigial Is Required during Late-Stage Muscle Differentiation in Drosophila melanogaster Embryos

    PubMed Central

    Deng, Hua; Bell, John B.

    2010-01-01

    The somatic muscles of Drosophila develop in a complex pattern that is repeated in each embryonic hemi-segment. During early development, progenitor cells fuse to form a syncytial muscle, which further differentiates via expression of muscle-specific factors that induce specific responses to external signals to regulate late-stage processes such as migration and attachment. Initial communication between somatic muscles and the epidermal tendon cells is critical for both of these processes. However, later establishment of attachments between longitudinal muscles at the segmental borders is largely independent of the muscle–epidermal attachment signals, and relatively little is known about how this event is regulated. Using a combination of null mutations and a truncated version of Sd that binds Vg but not DNA, we show that Vestigial (Vg) is required in ventral longitudinal muscles to induce formation of stable intermuscular attachments. In several muscles, this activity may be independent of Sd. Furthermore, the cell-specific differentiation events induced by Vg in two cells fated to form attachments are coordinated by Drosophila epidermal growth factor signaling. Thus, Vg is a key factor to induce specific changes in ventral longitudinal muscles 1–4 identity and is required for these cells to be competent to form stable intermuscular attachments with each other. PMID:20685961

  14. Vestigial is required during late-stage muscle differentiation in Drosophila melanogaster embryos.

    PubMed

    Deng, Hua; Bell, John B; Simmonds, Andrew J

    2010-10-01

    The somatic muscles of Drosophila develop in a complex pattern that is repeated in each embryonic hemi-segment. During early development, progenitor cells fuse to form a syncytial muscle, which further differentiates via expression of muscle-specific factors that induce specific responses to external signals to regulate late-stage processes such as migration and attachment. Initial communication between somatic muscles and the epidermal tendon cells is critical for both of these processes. However, later establishment of attachments between longitudinal muscles at the segmental borders is largely independent of the muscle-epidermal attachment signals, and relatively little is known about how this event is regulated. Using a combination of null mutations and a truncated version of Sd that binds Vg but not DNA, we show that Vestigial (Vg) is required in ventral longitudinal muscles to induce formation of stable intermuscular attachments. In several muscles, this activity may be independent of Sd. Furthermore, the cell-specific differentiation events induced by Vg in two cells fated to form attachments are coordinated by Drosophila epidermal growth factor signaling. Thus, Vg is a key factor to induce specific changes in ventral longitudinal muscles 1-4 identity and is required for these cells to be competent to form stable intermuscular attachments with each other.

  15. Is caprine arthritis encephalitis virus (CAEV) transmitted vertically to early embryo development stages (morulae or blastocyst) via in vitro infected frozen semen?

    PubMed

    Al Ahmad, M Z Ali; Chebloune, Y; Chatagnon, G; Pellerin, J L; Fieni, F

    2012-05-01

    The aim of this study was to determine, in vivo, whether in vitro infected cryopreserved caprine sperm is capable of transmitting caprine arthritis-encephalitis virus (CAEV) vertically to early embryo development stages via artificial insemination with in vitro infected semen. Sperm was collected from CAEV-free bucks by electroejaculation. Half of each ejaculate was inoculated with CAEV-pBSCA at a viral concentration of 10(4) TCID(50)/mL. The second half of each ejaculate was used as a negative control. The semen was then frozen. On Day 13 of superovulation treatment, 14 CAEV-free does were inseminated directly into the uterus under endoscopic control with thawed infected semen. Six CAEV-free does, used as a negative control, were inseminated intrauterine with thawed CAEV-free sperm, and eight CAEV-free does were mated with naturally infected bucks. Polymerase chain reaction (PCR) was used to detect CAEV proviral-DNA in the embryos at the D7 stage, in the embryo washing media, and in the uterine secretions of recipient does. At Day 7, all the harvested embryos were PCR-negative for CAEV proviral-DNA; however, CAEV proviral-DNA was detected in 8/14 uterine smears, and 9/14 flushing media taken from does inseminated with infected sperm, and in 1/8 uterine swabs taken from the does mated with infected bucks. The results of this study confirm that (i) artificial insemination with infected semen or mating with infected bucks may result in the transmission of CAEV to the does genital tack seven days after insemination, and (ii) irrespective of the medical status of the semen or the recipient doe, it is possible to obtain CAEV-free early embryos usable for embryo transfer.

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

    PubMed Central

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

    2010-01-01

    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+CD80high mTECs further progressed to an Aire−CD80intermediate 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. PMID:20404099

  17. 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.

  18. The nasal apparatus of the red squirrel (Sciurus vulgaris L.) embryo at the stage of the fully formed chondrocranium.

    PubMed

    Slabý, O

    1991-01-01

    The nasal apparatus of the squirrel embryo at the optimum stage of the chondrocranium displays simple (though not always primitive) features and individual structures developed largely as in other rodents and even in insectivores. Primitive features include the presence of a cartilago paraseptalis communis and probably the simplicity of the olfactory labyrinth, whose main support in the region as a whole is ethmoturbinale I, whose dorsal and ventral lamella divide off the basic recesses; anterior (frontalis), maxillaris and frontoturbinalis. Completely caudally we find ethmoturbinale I, the frontoturbinalia and the relevant secondary (greatly reduced) recesses. The zona annularis is interrupted by failure of the rostral processes of the lamina transversalis anterior (corresponding to the processes laterales ventrales) to fuse with the capsula nasi ant. or with the anlage of the septum nasi. We did find a fenestra nasi superior (lateralis). The atrioturbinale is well developed, the maxilloturbinale only as a trace, but we have a very striking nasoturbinale. In the rostral region of the olfactory segment, the cartilaginous capsule has a very conspicuous thickening, which in this part is formed of paranasal cartilage. The thickening presents outwardly as the prominentia anterior; cavity of the recessus anterior (frontalis) is formed inside it. The rest of the dorsal and caudal wall of this cavity forms the crista semicircularis, which further caudally circumscribes the foramen olfactorium. A foramen epiphaniale is present. The vestibular region of the epithelial nasal tube is interestingly formed. In cross section it is crescent-shaped; the nasal tube itself opens into the convexity, but the lamina transversalis ant. sends a turbinale into the concavity. A cartilago alaris superior, which develops independently in situ, is present; in our stage it is associated with the wall of the capsule, but ventrolaterally it terminates freely and is not joined to any other

  19. Piglets born after intrauterine laparoscopic embryo transfer.

    PubMed

    Wieczorek, J; Koseniuk, J; Mandryk, I; Poniedziałek-Kempny, K

    2015-01-01

    The aim of the study was the preliminary development of laparoscopic transfer of embryos to the uterus in the pig, which can become the alternative for more invasive surgical methods. We proposed the original method of embryo transfer. Donors (n = 40) and recipients (n = 15) of embryos were sows of age of 6-8 months. The estrus cycle of both recipients and donors was routinely synchronized. The experimental animals were divided into two groups. In the first group (10 donors and 3 recipients) embryos were transplanted according to the method described earlier and in the second group (30 donors and 12 recipients) embryos were transplanted according to our own proposed method. As the control group, we used 16 sows after insemination (AI). In animals from both experimental groups pregnancy was diagnosed between 28-31 day after transplantation and in the control group between 28-31 day after insemination. All animals were observed during pregnancy and weaning period in pig farm. Embryos at the development stage of 2-4 cell were obtained surgically and cultured in vitro for 4 days. Obtained blastocysts were transferred to donors. The original set of catheters for blastocysts transfer to pig uterus was constructed. Three trocars were placed in abdominal cavity for inserting endoscope and 2 grasps for uterus stabilization. After uterus stabilization, the slide was inserted into abdomen which was used for putting the needle to puncture uterus. Through this needle catheter with embryos was inserted into the uterus cavity. Embryos were placed by injection into lumen of the one uterine horn. From 12 recipients pregnancy was diagnosed in 6 recipients. From 6 litters, 57 piglets were born. We weaned 41 piglets (71.9%). In our study we obtained 50% efficacy, with the mean number of 9.5 alive piglets in litter and 6.8 weaned piglets. The efficacy of developed method of laparoscopic transfer of porcine embryos allows it to be used in routine practice.

  20. 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.

  1. The p66(Shc) adaptor protein controls oxidative stress response in early bovine embryos.

    PubMed

    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.

  2. Laser-assisted hatching improves clinical outcomes of vitrified-warmed blastocysts developed from low-grade cleavage-stage embryos: a prospective randomized study.

    PubMed

    Wan, Cai-Yun; Song, Cheng; Diao, Liang-Hui; Li, Guan-Gui; Bao, Zhong-Jian; Hu, Xiao-Dong; Zhang, Hong-Zhan; Zeng, Yong

    2014-05-01

    The aim of this study was to evaluate the effects of quarter zona-pellucida (ZP) opening by laser-assisted hatching (QLAH) on the clinical outcomes following transfer of vitrified-warmed blastocysts developed from low-grade cleavage-stage embryos in patients with all high-grade and fair-grade cleavage-stage embryos transferred without achieving pregnancy. Patients were randomized into two groups: QLAH (n=101) and control (n=102). The implantation and clinical pregnancy rates were significantly higher in the QLAH group compared with the control group (P=0.021 and P=0.034, respectively). The live birth rate of the QLAH group was also higher, although not significantly. When the clinical outcomes according to the day of blastocyst vitrification were compared between the groups, the implantation, clinical pregnancy and live birth rates of the QLAH group were significantly higher (P<0.05) than those of the control group for day 6 blastocysts, but not for day 5 or day 5/day 6 blastocysts. These results suggest that QLAH improves the clinical outcomes of vitrified-warmed blastocysts, especially of day 6 vitrified blastocysts, developed from low-grade cleavage-stage embryos. Copyright © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  3. 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.

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

    PubMed

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

    2017-09-01

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

  5. Early life stage and genetic toxicity of stannous chloride on zebrafish embryos and adults: toxic effects of tin on zebrafish.

    PubMed

    Şişman, Turgay

    2011-06-01

    Humans are exposed to stannous chloride (SnCl(2)), known as tin chloride, present in packaged food, soft drinks, biocides, dentifrices, etc. Health effects in children exposed to tin and tin compounds have not been investigated yet. Therefore, we evaluated the possible teratogenic effects and genotoxic of SnCl(2) in zebrafish (Danio rerio) adults and their embryos. In the embryo-larval study, SnCl(2) showed embryo toxicity and developmental delay after exposure to the various concentrations of 10-250 μM for 120 h. Teratogenic effects including morphological malformations of the embryos and larvae were observed. The embryos exposed to 100 μM displayed tail deformation at 28 hpf and the larvae exposed to 50 μM showed reduced body growth, smaller head and eyes, bent trunk, mild pericardial edema, and smaller caudal fin at 96 hpf. The results of the teratological study show that SnCl(2) induced a significant decrease in the number of living embryos and larvae. Regarding the chromosome analysis, SnCl(2) induced a dose-dependent increase in the micronucleus (MN) frequency in peripheral erythrocytes of adult zebrafish. In blood cells, the 25 μM dose of SnCl(2) caused a nonsignificant increase in the total chromosomal aberrations, but the high doses significantly increased the total number of chromosomal aberrations compared with the control groups. Overall, the results clearly indicate that SnCl(2) is teratogenic and genotoxic to zebrafish.

  6. 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.

  7. 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

  8. 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.

  9. Production of good-quality blastocyst embryos following IVF of ovine oocytes vitrified at the germinal vesicle stage using a cryoloop.

    PubMed

    Moawad, Adel R; Zhu, Jie; Choi, Inchul; Amarnath, Dasari; Chen, Wenchao; Campbell, Keith H S

    2013-01-01

    The cryopreservation of immature oocytes at the germinal vesicle (GV) stage would create an easily accessible, non-seasonal source of female gametes for research and reproduction. The present study investigated the ability of ovine oocytes vitrified at the GV stage using a cryoloop to be subsequently matured, fertilised and cultured in vitro to blastocyst-stage embryos. Selected cumulus-oocyte complexes obtained from mature ewes at the time of death were randomly divided into vitrified, toxicity and control groups. Following vitrification and warming, viable oocytes were matured in vitro for 24 h. Matured oocytes were either evaluated for nuclear maturation, spindle and chromosome configuration or fertilised and cultured in vitro for 7 days. No significant differences were observed in the frequencies of IVM (oocytes at the MII stage), oocytes with normal spindle and chromatin configuration and fertilised oocytes among the three groups. Cleavage at 24 and 48 h post insemination was significantly decreased (P<0.01) in vitrified oocytes. No significant differences were observed in the proportion of blastocyst development between vitrified and control groups (29.4% v. 45.1%, respectively). No significant differences were observed in total cell numbers, the number of apoptotic nuclei or the proportion of diploid embryos among the three groups. In conclusion, we report for the first time that ovine oocytes vitrified at the GV stage using a cryoloop have the ability to be matured, fertilised and subsequently developed in vitro to produce good-quality blastocyst embryos at frequencies comparable to those obtained using fresh oocytes.

  10. 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

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

    2014-07-01

    To evaluate the efficacy of blastocyst transfer in comparison with cleavage stage transfer. 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. 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). Blastocyst transfer having higher implantation rate and clinical pregnancy rate lead to reduction in multiple pregnancies.

  11. 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)

  12. 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)

  13. Developmental anatomy of the liver from computerized three-dimensional reconstructions of four human embryos (from Carnegie stage 14 to 23).

    PubMed

    Lhuaire, Martin; Tonnelet, Romain; Renard, Yohann; Piardi, Tullio; Sommacale, Daniele; Duparc, Fabrice; Braun, Marc; Labrousse, Marc

    2015-07-01

    Some aspects of human embryogenesis and organogenesis remain unclear, especially concerning the development of the liver and its vasculature. The purpose of this study was to investigate, from a descriptive standpoint, the evolutionary morphogenesis of the human liver and its vasculature by computerized three-dimensional reconstructions of human embryos. Serial histological sections of four human embryos at successive stages of development belonging to three prestigious French historical collections were digitized and reconstructed in 3D using software commonly used in medical radiology. Manual segmentation of the hepatic anatomical regions of interest was performed section by section. In this study, human liver organogenesis was examined at Carnegie stages 14, 18, 21 and 23. Using a descriptive and an analytical method, we showed that these stages correspond to the implementation of the large hepatic vascular patterns (the portal system, the hepatic artery and the hepatic venous system) and the biliary system. To our knowledge, our work is the first descriptive morphological study using 3D computerized reconstructions from serial histological sections of the embryonic development of the human liver between Carnegie stages 14 and 23. Copyright © 2015 Elsevier GmbH. All rights reserved.

  14. What is the optimal duration of progesterone administration before transferring a vitrified-warmed cleavage stage embryo? A randomized controlled trial.

    PubMed

    van de Vijver, A; Polyzos, N P; Van Landuyt, L; Mackens, S; Stoop, D; Camus, M; De Vos, M; Tournaye, H; Blockeel, C

    2016-05-01

    What is the impact on clinical pregnancy rates when vitrified cleavage stage Day 3 embryos, warmed and cultured overnight to Day 4, are transferred on the 3rd or 5th day of progesterone administration in an artificial cycle? Clinical pregnancy rates are similar when transferring a vitrified-warmed cleavage stage Day 3 embryo after overnight culture on the 3rd or 5th day of progesterone administration. In artificially prepared cycles, progesterone supplementation is generally started 3 days before embryo transfer, although the optimal length of exposure to progesterone before frozen embryo transfer (FET) has not been established. However, in a natural cycle, serum progesterone levels start to rise before ovulation, due to the LH-stimulated production by the peripheral granulosa cells. Hence, it could be postulated that progesterone supplementation before embryo transfer in an artificial cycle should start earlier or even later. Prospective, randomized controlled trial, encompassing 300 patients who had embryos frozen on Day 3 and who underwent FET in an artificial cycle. Between 1 November 2012 and 31 December 2014, 300 patients were allocated to one of two groups as soon as endometrial thickness reached ≥7 mm on ultrasound after estrogen supplementation. A computer-generated randomization list was used, not concealed to the physicians. Each patient was enrolled into the study only once. FET was performed on the fifth day of progesterone supplementation in Group A, whereas in Group B, FET was performed on the third day of vaginal micronized progesterone administration. Embryos were thawed the day before transfer and after overnight culture, one or two Day 4 embryos were transferred. One hundred and fifty patients in Group A received 5 days of vaginal micronized progesterone tablets and one hundred and fifty patients in Group B received 3 days of micronized progesterone vaginally before FET. In Group A, 13 patients did not have an embryo transfer, compared with 12

  15. 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.

  16. EXPOSURE TO A P13KINASE INHIBITOR PRODUCED DYSMORPHOGENESIS IN NEURULATION-STAGED MOUSE EMBRYOS IN CULTURE

    EPA Science Inventory

    The haloacetic acids (HAA) are a family of chemicals that are drinking water disinfection byproducts. We previously reported that bromo- and chloro-acetic acids alter embryonic development when mouse conceptuses are directly exposed to these xenobiotics in whole embryo culture. C...

  17. EXPOSURE TO A P13KINASE INHIBITOR PRODUCED DYSMORPHOGENESIS IN NEURULATION-STAGED MOUSE EMBRYOS IN CULTURE

    EPA Science Inventory

    The haloacetic acids (HAA) are a family of chemicals that are drinking water disinfection byproducts. We previously reported that bromo- and chloro-acetic acids alter embryonic development when mouse conceptuses are directly exposed to these xenobiotics in whole embryo culture. C...

  18. Synchronous regulation of the determinants of endometrial receptivity to interleukin 1 at key stages of early embryo implantation in vivo.

    PubMed

    Bourdiec, Amélie; Martel, Valéry; Akoum, Ali

    2014-04-01

    To investigate the expression kinetics of interleukin-1 receptors (IL-1R), receptor antagonist (IL-1RN), and monocyte chemotactic protein 1 (MCP-1) throughout early gestation in mice. Assessment of IL-1R, IL-1RN, and MCP-1 throughout early pregnancy. Reproduction laboratory. B6C3F1 female mice bred with fertile males of the same strain. Collection of endometrial tissue at necropsy from nonimplanted and implanted sites. IL-1R, IL-1RN, and MCP-1 mRNA expression by quantitative reverse-transcription polymerase chain reaction and protein expression by enzyme-linked immunosorbent assay and immunohistochemistry. The expression of the signaling IL-1R1 significantly increased in the first 2 days of gestation, which corresponded to the inflammatory-like period triggered by the seminal fluid, before increasing again at the implantation window and lasting throughout embryo implantation. The expression of inhibitory IL-1R2 and IL-1RN concomitantly increased during gestational days 1-2 but remained low, particularly within the embryo implantation sites and throughout the implantation period. The expression of MCP-1 significantly increased only at the embryo implantation sites and showed a significant positive correlation with IL-1R1 expression. Our data identified for the first time synchronous changes in endometrial IL-1R throughout early gestation in vivo and point to a deep modulation of endometrial receptivity to IL-1 by embryo-driven signals. This may play a key role in the creation of a receptive phenotype in the maternal endometrium and represent a key mechanism underlying embryo implantation. Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  19. Adaptive Transition of Aquaporin 5 Expression and Localization during Preimplantation Embryo Development by In Vitro Culture.

    PubMed

    Park, Jae-Won; Shin, Yun Kyung; Choen, Yong-Pil

    2014-09-01

    Adaptive development of early stage embryo is well established and recently it is explored that the mammalian embryos also have adaptive ability to the stressful environment. However, the mechanisms are largely unknown. In this study, to evaluate the possible role of aquaporin in early embryo developmental adaptation, the expression of aquaporin (AQP) 5 gene which is detected during early development were examined by the environmental condition. To compare expression patterns between in vivo and in vitro, we conducted quantitative RT-PCR and analyzed localization of the AQP5 by whole mount immunofluorescence. At in vivo condition, Aqp5 expressed in oocyte and in all the stages of preimplantation embryo. It showed peak at 2-cell stage and decreased continuously until morula stage. At in vitro condition, Aqp5 expression pattern was similar with in vivo embryos. It expressed both at embryonic genome activation phase and second midpreimplantation gene activation phase, but the fold changes were modified between in vivo embryos and in vitro embryos. During in vivo development, AQP5 was mainly localized in apical membrane of blastomeres of 4-cell and 8-cell stage embryos, and then it was localized in cytoplasm. However, the main localization area of AQP5 was dramatically shifted after 8-cell stage from cytoplasm to nucleus by in vitro development. Those results explore the modification of Aqp5 expression levels and location of its final products by in vitro culture. It suggests that expression of Aqp5 and the roles of AQP5 in homeostasis can be modulated by in vitro culture, and that early stage embryos can develop successfully by themselves adapting to their condition through modulation of the specific gene expression and localization.

  20. Adaptive Transition of Aquaporin 5 Expression and Localization during Preimplantation Embryo Development by In Vitro Culture

    PubMed Central

    Park, Jae-Won; Shin, Yun Kyung; Choen, Yong-Pil

    2014-01-01

    Adaptive development of early stage embryo is well established and recently it is explored that the mammalian embryos also have adaptive ability to the stressful environment. However, the mechanisms are largely unknown. In this study, to evaluate the possible role of aquaporin in early embryo developmental adaptation, the expression of aquaporin (AQP) 5 gene which is detected during early development were examined by the environmental condition. To compare expression patterns between in vivo and in vitro, we conducted quantitative RT-PCR and analyzed localization of the AQP5 by whole mount immunofluorescence. At in vivo condition, Aqp5 expressed in oocyte and in all the stages of preimplantation embryo. It showed peak at 2-cell stage and decreased continuously until morula stage. At in vitro condition, Aqp5 expression pattern was similar with in vivo embryos. It expressed both at embryonic genome activation phase and second midpreimplantation gene activation phase, but the fold changes were modified between in vivo embryos and in vitro embryos. During in vivo development, AQP5 was mainly localized in apical membrane of blastomeres of 4-cell and 8-cell stage embryos, and then it was localized in cytoplasm. However, the main localization area of AQP5 was dramatically shifted after 8-cell stage from cytoplasm to nucleus by in vitro development. Those results explore the modification of Aqp5 expression levels and location of its final products by in vitro culture. It suggests that expression of Aqp5 and the roles of AQP5 in homeostasis can be modulated by in vitro culture, and that early stage embryos can develop successfully by themselves adapting to their condition through modulation of the specific gene expression and localization. PMID:25949184

  1. Blood flow dynamics reflect degree of outflow tract banding in Hamburger–Hamilton stage 18 chicken embryos

    PubMed Central

    Midgett, Madeline; Goenezen, Sevan; Rugonyi, Sandra

    2014-01-01

    Altered blood flow during embryonic development has been shown to cause cardiac defects; however, the mechanisms by which the resulting haemodynamic forces trigger heart malformation are unclear. This study used heart outflow tract banding to alter normal haemodynamics in a chick embryo model at HH18 and characterized the immediate blood flow response versus the degree of band tightness. Optical coherence tomography was used to acquire two-dimensional longitudinal structure and Doppler velocity images from control (n = 16) and banded (n = 25, 6–64% measured band tightness) embryos, from which structural and velocity data were extracted to estimate haemodynamic measures. Peak blood flow velocity and wall shear rate (WSR) initially increased linearly with band tightness (p < 0.01), but then velocity plateaued between 40% and 50% band tightness and started to decrease with constriction greater than 50%, whereas WSR continued to increase up to 60% constriction before it began decreasing with increased band tightness. Time of flow decreased with constriction greater than 20% (p < 0.01), while stroke volume in banded embryos remained comparable to control levels over the entire range of constriction (p > 0.1). The haemodynamic dependence on the degree of banding reveals immediate adaptations of the early embryonic cardiovascular system and could help elucidate a range of cardiac adaptations to gradually increased load. PMID:25165602

  2. The developmental stage of chicken embryos modulates the impact of in ovo olfactory stimulation on food preferences.

    PubMed

    Bertin, Aline; Calandreau, Ludovic; Arnould, Cécile; Lévy, Frédéric

    2012-03-01

    Like mammals, bird embryos are capable of chemosensory learning, but the ontogeny of their feeding preferences has not been examined. We tested if the timing of stimulation in chicken embryos modulates the impact of in ovo olfactory stimulation on later food preferences. We exposed chicken embryos to an olfactory stimulus for a 4-day period in the middle or toward the end of the incubation period. The chicks were tested for their preference between foods with and without the olfactory stimulus in 3-min choice tests and on a 24-h time scale. Regardless of the type of food (familiar or novel) or the duration of the test, the control chicks not exposed to the olfactory stimulus consistently showed significant preferences for non-odorized foods. Chicks that were exposed in ovo to the olfactory stimulus did not show a preference for odorized or non-odorized foods. Only those chicks that were exposed to the olfactory stimulus toward the end of the incubation period differed from the controls and incorporated a higher proportion of odorized food into their diets on a 24-h time scale. This result indicates that olfactory stimulation at the end of embryonic development has a stronger impact on later feeding preferences. Our findings contribute to the growing pool of recent data appreciating the impact of olfactory signals on behavior regulation in avian species.

  3. Estrous cycle staging before mating led to increased efficiency in the production of pseudopregnant recipients without negatively affecting embryo transfer in mice.

    PubMed

    Heykants, Malte; Mahabir, Esther

    2016-03-15

    The goal was to increase pseudopregnant mice production by estrous cycle staging by visual examination before pairing and to determine the effect of such pseudopregnant recipients on embryo transfer. To compare methods of estrous cycle staging over 14 days, groups consisted of 10 females in proestrus-estrus and 10 vasectomized males; group 1: only daily visual observation; group 2: daily visual observation and cytological examination on day 1; group 3: daily visual observation and daily cytological examination. The average time to first vaginal plug was 1.8 days in group 1, 2.7 days in group 2, and 3.2 days in group 3, whereas the average time between consecutive vaginal plugs was 9.2 days (group 1), 10 days (group 2), and 9.25 days (group 3). The average time between consecutive estrous cycles was 9.7 days (group 1), 11.8 days (group 2), and 9.4 days (group 3). The congruence between visual and cytological examination in determining proestrus-estrus in group 2 was 100% and that for the four stages in group 3 was 79% with a range of 44% to 100%. From 162 plug-positive females originally selected in proestrus-estrus, 49%, 30%, 19%, and 2% were plug-positive on Day 1, Day 2, Day 3, and Day 4, respectively, showing that pseudopregnant mice production was significantly increased on the first 2 days. From 192 plug-positive females originally selected randomly, these values were 31%, 21%, 35%, 10%, and 3% on d1, d2, d3, d4, and d5, respectively. No significant differences were observed between groups with respect to embryo transfers with fresh or cryopreserved embryos although the number of pups born per litter was higher in group A with fresh (7.57 vs. 6.39) and cryopreserved-thawed embryos (5.0 vs. 4.38). Furthermore, the sex ratio and the genotype of the pups were not significantly affected.

  4. 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.

  5. Urochordate ascidians possess a single isoform of Aurora kinase that localizes to the midbody via TPX2 in eggs and cleavage stage embryos.

    PubMed

    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

  6. 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

  7. Peptidylarginine deiminase 1-catalyzed histone citrullination is essential for early embryo development

    PubMed Central

    Zhang, Xiaoqian; Liu, Xiaoqiu; Zhang, Mei; Li, Tingting; Muth, Aaron; Thompson, Paul R.; Coonrod, Scott A.; Zhang, Xuesen

    2016-01-01

    Peptidylarginine deiminase (PADI) enzymes are increasingly being associated with the regulation of chromatin structure and gene activity via histone citrullination. As one of the PADI family members, PADI1 has been mainly reported to be expressed in the epidermis and uterus, where the protein in keratinocytes is thought to promote differentiation by citrullinating filament proteins. However, the roles of PADI1 in preimplantation development have not been addressed. Using a PADI1-specific inhibitor and Padi1-morpholino knockdown, we found that citrullination of histone tails at H4R3 and H3R2/8/17 were markedly reduced in the 2- and 4-cell embryos. Consistent with this observation, early embryo development was also arrested at the 4-cell stage upon depletion of PADI1 or inhibition of PADI1 enzyme activity. Additionally, by employing 5-ethynyl uridine (EU) incorporation analysis, ablation of PADI1 function led to a dramatic decrease in overall transcriptional activity, correlating well with the reduced levels of phosphorylation of RNA Pol II at Ser2 observed at 2- or 4-cell stage of embryos under Padi1 knockdown or inhibiting PADI1. Thus, our data reveal a novel function of PADI1 during early embryo development transitions by catalyzing histone tail citrullination, which facilitates early embryo genome transactivation. PMID:27929094

  8. The X-linked imprinted gene family Fthl17 shows predominantly female expression following the two-cell stage in mouse embryos

    PubMed Central

    Kobayashi, Shin; Fujihara, Yoshitaka; Mise, Nathan; Kaseda, Kazuhiro; Abe, Kuniya; Ishino, Fumitoshi; Okabe, Masaru

    2010-01-01

    Differences between male and female mammals are initiated by embryonic differentiation of the gonad into either a testis or an ovary. However, this may not be the sole determinant. There are reports that embryonic sex differentiation might precede and be independent of gonadal differentiation, but there is little molecular biological evidence for this. To test for sex differences in early-stage embryos, we separated male and female blastocysts using newly developed non-invasive sexing methods for transgenic mice expressing green fluorescent protein and compared the gene-expression patterns. From this screening, we found that the Fthl17 (ferritin, heavy polypeptide-like 17) family of genes was predominantly expressed in female blastocysts. This comprises seven genes that cluster on the X chromosome. Expression analysis based on DNA polymorphisms revealed that these genes are imprinted and expressed from the paternal X chromosome as early as the two-cell stage. Thus, by the time zygotic genome activation starts there are already differences in gene expression between male and female mouse embryos. This discovery will be important for the study of early sex differentiation, as clearly these differences arise before gonadal differentiation. PMID:20185572

  9. Expression pattern of pluripotent markers in different embryonic developmental stages of buffalo (Bubalus bubalis) embryos and putative embryonic stem cells generated by parthenogenetic activation.

    PubMed

    Singh, Karn P; Kaushik, Ramakant; Garg, Veena; Sharma, Ruchi; George, Aman; Singh, Manoj K; Manik, Radhey S; Palta, Prabhat; Singla, Suresh K; Chauhan, Manmohan S

    2012-12-01

    In this study, we describe the production of buffalo parthenogenetic blastocysts and subsequent isolation of parthenogenetic embryonic stem cell (PGESC)-like cells. PGESC colonies exhibited dome-shaped morphology and were clearly distinguishable from the feeder layer cells. Different stages of development of parthenogenetic embryos and derived embryonic stem cell (ESC)-like cells expressed key ESC-specific markers, including OCT-4, NANOG, SOX-2, FOXD3, REX-1, STAT-3, TELOMERASE, NUCLEOSTEMIN, and cMYC. Immunofluorescence-based studies revealed that the PGESCs were positive for surface-based pluripotent markers, viz., SSEA-3, SSEA-4, TRA 1-80, TRA 1-60, CD-9, and CD-90 and exhibited high alkaline phosphatase (ALP) activity. PGEC cell-like cells formed embryoid body (EB)-like structures in hanging drop cultures and when cultured for extended period of time spontaneously differentiated into derivatives of three embryonic germ layers as confirmed by RT-PCR for ectodermal (CYTOKERATIN8, NF-68), mesodermal (MSX1, BMP-4, ASA), and endodermal markers (AFP, HNF-4, GATA-4). Differentiation of PGESCs toward the neuronal lineage was successfully directed by supplementation of serum-containing media with retinoic acid. Our results indicate that the isolated ESC-like cells from parthenogenetic blastocyst hold properties of ESCs and express markers of pluripotency. The pluripotency markers were also expressed by early cleavage-stage of buffalo embryos.

  10. Effects of superovulation with oFSH and norgestomet/GnRH-controlled release of the LH surge on hormone concentrations, and yield of oocytes and embryos at specific developmental stages.

    PubMed

    Knijn, H M; Fokker, W; van der Weijden, G C; Dieleman, S J; Vos, P L A M

    2012-04-01

    The objective of this study was to evaluate a new superovulation procedure with oFSH after temporary suppression of the endogenous LH surge by norgestomet followed by administration of GnRH, to collect bovine oocytes and embryos at specific developmental stages. Since 1999, our research group applies this superovulation procedure with controlled release of the endogenous LH surge. The objective of this study is to verify if this procedure is reliable for collection of oocytes and embryos at specific time points of development and if it produces a sufficient number of both oocytes and embryos of good quality. This procedure was validated regarding to hormonal characteristics, superovulatory response and both oocyte and embryo yield at different times of in vivo development. The results demonstrate that the procedure used to control the occurrence of the pre-ovulatory LH surge was effective in 92% of the animals (n = 238) and even in 99% of the animals the oocytes and embryos were collected at the intended stage of development. The superovulatory response and both oocyte, embryo yield and quality were similar to the average yield in Europe reported by Association Européenne de transfert embryonnaire (AETE). In conclusion, this superovulation procedure provides a valid tool to collect oocytes and embryos at specific time points of development. © 2008 Blackwell Verlag GmbH.

  11. Generation and developmental characteristics of porcine tetraploid embryos and tetraploid/diploid chimeric embryos.

    PubMed

    He, Wenteng; Kong, Qingran; Shi, Yongqian; Xie, Bingteng; Jiao, Mingxia; Huang, Tianqing; Guo, Shimeng; Hu, Kui; Liu, Zhonghua

    2013-10-01

    The aim of this study was to optimize electrofusion conditions for generating porcine tetraploid (4n) embryos and produce tetraploid/diploid (4n/2n) chimeric embryos. Different electric field intensities were tested and 2 direct current (DC) pulses of 0.9 kV/cm for 30 μs was selected as the optimum condition for electrofusion of 2-cell embryos to produce 4n embryos. The fusion rate of 2-cell embryos and the development rate to blastocyst of presumably 4n embryos, reached 85.4% and 28.5%, respectively. 68.18% of the fused embryos were found to be 4n as demonstrated by fluorescent in situ hybridization (FISH). Although the number of blastomeres in 4n blastocysts was significantly lower than in 2n blastocysts (P<0.05), there was no significant difference in developmental rates of blastocysts between 2n and 4n embryos (P>0.05), suggesting that the blastocyst forming capacity in 4n embryos is similar to those in 2n embryos. Moreover, 4n/2n chimeric embryos were obtained by aggregation of 4n and 2n embryos. We found that the developmental rate and cell number of blastocysts of 4-cell (4n)/4-cell (2n) chimeric embryos were significantly higher than those of 2-cell (4n)/4-cell (2n), 4-cell (4n)/8-cell (2n), 4-cell (4n)/2-cell (2n) chimeric embryos (P<0.05). Consistent with mouse chimeras, the majority of 4n cells contribute to the trophectoderm (TE), while the 2n cells are mainly present in the inner cell mass (ICM) of porcine 4n/2n chimeric embryos. Our study established a feasible and efficient approach to produce porcine 4n embryos and 4n/2n chimeric embryos.

  12. 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.

  13. 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.

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

    PubMed Central

    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

    2013-01-01

    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

  15. Lipid rafts enriched in monosialylGb5Cer carrying the stage-specific embryonic antigen-4 epitope are involved in development of mouse preimplantation embryos at cleavage stage

    PubMed Central

    2011-01-01

    Background Lipid rafts enriched in glycosphingolipids (GSLs), cholesterol and signaling molecules play an essential role not only for signal transduction started by ligand binding, but for intracellular events such as organization of actin, intracellular traffic and cell polarity, but their functions in cleavage division of preimplantation embryos are not well known. Results Here we show that monosialylGb5Cer (MSGb5Cer)-enriched raft domains are involved in development during the cleavage stage of mouse preimplantation embryos. MSGb5Cer preferentially localizes at the interfaces between blastomeres in mouse preimplantation embryos. Live-imaging analysis revealed that MSGb5Cer localizes in cleavage furrows during cytokinesis, and that by accumulating at the interfaces, it thickens them. Depletion of cholesterol from the cell membrane with methyl-beta-cyclodextrin (MbCD) reduced the expression of MSGb5Cer and stopped cleavage. Extensive accumulation of MSGb5Cer at the interfaces by cross-linking with anti-MSGb5Cer Mab (6E2) caused F-actin to aggregate at the interfaces and suppressed the localization of E-cadherin at the interfaces, which resulted in the cessation of cleavage. In addition, suppression of actin polymerization with cytochalasin D (CCD) decreased the accumulation of MSGb5Cer at the interfaces. In E-cadherin-targeted embryos, the MSGb5Cer-enriched raft membrane domains accumulated heterotopically. Conclusions These results indicate that MSGb5Cer-enriched raft membrane domains participate in cytokinesis in a close cooperation with the cortical actin network and the distribution of E-cadherin. PMID:21489308

  16. Epidermal growth factor improves developmental competence and embryonic quality of singly cultured domestic cat embryos

    PubMed Central

    THONGKITTIDILOK, Chommanart; THARASANIT, Theerawat; SONGSASEN, Nucharin; SANANMUANG, Thanida; BUARPUNG, Sirirak; TECHAKUMPHU, Mongkol

    2015-01-01

    This study examined the influence of EGF on the expression of EGF receptors (EGFR) and developmental competence of embryos cultured individually versus those cultured in groups. Cat oocytes were in vitro matured and fertilized (IVM/IVF), and cleaved embryos were randomly assigned to one of seven culture conditions: one group each in which embryos were subjected to group culture supplemented with or without 5 ng/ml EGF and five groups in which embryos were subjected to single-embryo culture supplemented with EGF (0, 5, 25, 50 or 100 ng/ml). Morulae, blastocysts and hatching blastocysts were assessed at days 5 and 7; post IVF, respectively, and total blastocyst cell numbers were assessed at day 7. Relative mRNA expressions of EGFR of 2–4-cell embryos, 8–16-cell embryos, morulae and blastocysts cultured in groups or singly with or without EGF supplementation were examined. OCT3/4 and Ki67 in blastocysts derived from the group or single-embryo culture systems with or without EGF supplementation were localized. A higher rate of embryos cultured in groups developed to blastocysts than individually incubated cohorts. Although EGF increased blastocyst formation in the single-embryo culture system, EGF did not affect embryo development in group culture. Expression levels of EGFR decreased in morulae and blastocysts cultured with EGF. An increased ratio of Ki67-positive cells to the total number of cells in the blastocyst was observed in singly cultured embryos in the presence of EGF. However, EGF did not affect the expression of OCT3/4. These findings indicate that EGF enhanced developmental competence of cat embryos cultured singly by stimulating cell proliferation and modulating the EGFR expression at various developmental stages. PMID:25985792

  17. Epidermal growth factor improves developmental competence and embryonic quality of singly cultured domestic cat embryos.

    PubMed

    Thongkittidilok, Chommanart; Tharasanit, Theerawat; Songsasen, Nucharin; Sananmuang, Thanida; Buarpung, Sirirak; Techakumphu, Mongkol

    2015-01-01

    This study examined the influence of EGF on the expression of EGF receptors (EGFR) and developmental competence of embryos cultured individually versus those cultured in groups. Cat oocytes were in vitro matured and fertilized (IVM/IVF), and cleaved embryos were randomly assigned to one of seven culture conditions: one group each in which embryos were subjected to group culture supplemented with or without 5 ng/ml EGF and five groups in which embryos were subjected to single-embryo culture supplemented with EGF (0, 5, 25, 50 or 100 ng/ml). Morulae, blastocysts and hatching blastocysts were assessed at days 5 and 7; post IVF, respectively, and total blastocyst cell numbers were assessed at day 7. Relative mRNA expressions of EGFR of 2-4-cell embryos, 8-16-cell embryos, morulae and blastocysts cultured in groups or singly with or without EGF supplementation were examined. OCT3/4 and Ki67 in blastocysts derived from the group or single-embryo culture systems with or without EGF supplementation were localized. A higher rate of embryos cultured in groups developed to blastocysts than individually incubated cohorts. Although EGF increased blastocyst formation in the single-embryo culture system, EGF did not affect embryo development in group culture. Expression levels of EGFR decreased in morulae and blastocysts cultured with EGF. An increased ratio of Ki67-positive cells to the total number of cells in the blastocyst was observed in singly cultured embryos in the presence of EGF. However, EGF did not affect the expression of OCT3/4. These findings indicate that EGF enhanced developmental competence of cat embryos cultured singly by stimulating cell proliferation and modulating the EGFR expression at various developmental stages.

  18. Allele-specific expression of the MAOA gene and X chromosome inactivation in in vitro produced bovine embryos.

    PubMed

    Ferreira, A R; Machado, G M; Diesel, T O; Carvalho, J O; Rumpf, R; Melo, E O; Dode, M A N; Franco, M M

    2010-07-01

    During embryogenesis, one of the two X chromosomes is inactivated in embryos. The production of embryos in vitro may affect epigenetic mechanisms that could alter the expression of genes related to embryo development and X chromosome inactivation (XCI). The aim of this study was to understand XCI during in vitro, pre-implantation bovine embryo development by characterizing the allele-specific expression pattern of the X chromosome-linked gene, monoamine oxidase A (MAOA). Two pools of ten embryos, comprised of the 4-, 8- to 16-cell, morula, blastocyst, and expanded blastocyst stages, were collected. Total RNA from embryos was isolated, and the RT-PCR-RFLP technique was used to observe expression of the MAOA gene. The DNA amplicons were also sequenced using the dideoxy sequencing method. MAOA mRNA was detected, and allele-specific expression was identified in each pool of embryos. We showed the presence of both the maternal and paternal alleles in the 4-, 8- to 16-cell, blastocyst and expanded blastocyst embryos, but only the maternal allele was present in the morula stage. Therefore, we can affirm that the paternal X chromosome is totally inactivated at the morula stage and reactivated at the blastocyst stage. To our knowledge, this is the first report of allele-specific expression of an X-linked gene that is subject to XCI in in vitro bovine embryos from the 4-cell to expanded blastocyst stages. We have established a pattern of XCI in our in vitro embryo production system that can be useful as a marker to assist the development of new protocols for in vitro embryo production. (c) 2010 Wiley-Liss, Inc.

  19. Stem Cell Differentiation Stage Factors from Zebrafish Embryo: A Novel Strategy to Modulate the Fate of Normal and Pathological Human (Stem) Cells

    PubMed Central

    Biava, Pier M.; Canaider, Silvia; Facchin, Federica; Bianconi, Eva; Ljungberg, Liza; Rotilio, Domenico; Burigana, Fabio; Ventura, Carlo

    2015-01-01

    In spite of the growing body of evidence on the biology of the Zebrafish embryo and stem cells, including the use of Stem Cell Differentiation Stage Factors (SCDSFs) taken from Zebrafish embryo to impact cancer cell dynamics, comparatively little is known about the possibility to use these factors to modulate the homeostasis of normal human stem cells or to modulate the behavior of cells involved in different pathological conditions. In the present review we recall in a synthetic way the most important researches about the use of SCDSFs in reprogramming cancer cells and in modulating the high speed of multiplication of keratinocytes which is characteristic of some pathological diseases like psoriasis. Moreover we add here the results about the capability of SCDSFs in modulating the homeostasis of human adipose-derived stem cells (hASCs) isolated from a fat tissue obtained with a novel-non enzymatic method and device. In addition we report the data not yet published about a first protein analysis of the SCDSFs and about their role in a pathological condition like neurodegeneration. PMID:26201607

  20. Stem Cell Differentiation Stage Factors from Zebrafish Embryo: A Novel Strategy to Modulate the Fate of Normal and Pathological Human (Stem) Cells.

    PubMed

    Biava, Pier M; Canaider, Silvia; Facchin, Federica; Bianconi, Eva; Ljungberg, Liza; Rotilio, Domenico; Burigana, Fabio; Ventura, Carlo

    2015-01-01

    In spite of the growing body of evidence on the biology of the Zebrafish embryo and stem cells, including the use of Stem Cell Differentiation Stage Factors (SCDSFs) taken from Zebrafish embryo to impact cancer cell dynamics, comparatively little is known about the possibility to use these factors to modulate the homeostasis of normal human stem cells or to modulate the behavior of cells involved in different pathological conditions. In the present review we recall in a synthetic way the most important researches about the use of SCDSFs in reprogramming cancer cells and in modulating the high speed of multiplication of keratinocytes which is characteristic of some pathological diseases like psoriasis. Moreover we add here the results about the capability of SCDSFs in modulating the homeostasis of human adiposederived stem cells (hASCs) isolated from a fat tissue obtained with a novel-non enzymatic method and device. In addition we report the data not yet published about a first protein analysis of the SCDSFs and about their role in a pathological condition like neurodegeneration.

  1. Comparative proteomic analysis of embryos between a maize hybrid and its parental lines during early stages of seed germination.

    PubMed

    Guo, Baojian; Chen, Yanhong; 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.

  2. 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

  3. 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

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

    2015-08-01

    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.

  4. 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. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

  5. Embryonic-stage-dependent changes in the level of eIF4E-binding proteins during early development of sea urchin embryos.

    PubMed

    Salaün, Patrick; Boulben, Sandrine; Mulner-Lorillon, Odile; Bellé, Robert; Sonenberg, Nahum; Morales, Julia; Cormier, Patrick

    2005-04-01

    The eukaryotic initiation factor 4E (eIF4E)-binding proteins (4E-BPs) inhibit translation initiation by binding eIF4E and preventing recruitment of the translation machinery to mRNA. We have previously shown that fertilization of sea urchin eggs triggers eIF4E-4E-BP complex dissociation and 4E-BP degradation. Here, we show that microinjection of eIF4E-binding motif peptide into unfertilized eggs delays the onset of the first mitosis triggered by fertilization, demonstrating that dissociation of the eIF4E-4E-BP complex is functionally important for the first mitotic division in sea urchin embryos. We also show by gel filtration analyses that eIF4E is present in unfertilized eggs as an 80 kDa molecular mass complex containing 4E-BP and a new 4E-BP of 40 kDa. Fertilization triggers the dissociation of eIF4E from these two 4E-BPs and triggers the rapid recruitment of eIF4E into a high-molecular-mass complex. Release of eIF4E from the two 4E-BPs is correlated with a decrease in the total level of both 4E-BPs following fertilization. Abundance of the two 4E-BPs has been monitored during embryonic development. The level of the two proteins remains very low during the rapid cleavage stage of early development and increases 8 hours after fertilization. These results demonstrate that these two 4E-BPs are down- and upregulated during the embryonic development of sea urchins. Consequently, these data suggest that eIF4E availability to other partners represents an important determinant of the early development of sea urchin embryos.

  6. Paternal exposure to testis cancer chemotherapeutics alters sperm fertilizing capacity and affects gene expression in the eight-cell stage rat embryo.

    PubMed

    Maselli, J; Hales, B F; Robaire, B

    2014-03-01

    Treatment of testicular cancer includes the coadministration of bleomycin, etoposide and cis-platinum (BEP); however, along with its therapeutic benefit, BEP exposure results in extensive reproductive chemotoxic effects, including alterations to sperm chromatin integrity. As an intact paternal genome is essential for successful fertilization and embryogenesis, we assessed the effect of paternal exposure to BEP on sperm fertilization capacity and the resulting consequences on early embryonic gene expression. Adult male Brown Norway rats received a 9-week treatment with BEP or saline and then were sacrificed immediately or subject to a 9-week recovery period. HSP90AA1, HSP90B1 and PDIA3, involved in spermatozoa-egg interactions, were overexpressed in BEP-exposed spermatozoa after the 9-week treatment period; overexpression was also observed in spermatozoa from BEP-treated rats after 9 weeks of recovery. These proteins were localized to the plasma membrane of the sperm head; this localization may facilitate their role in spermatozoa-egg interactions as the highest staining intensities were observed in capacitated spermatozoa. The fertilization potential of spermatozoa was determined by in vitro fertilization with oocytes from unexposed naturally cycling female rats. Interestingly, the fertilization potential of spermatozoa following a 9-week recovery period from BEP treatment was significantly enhanced compared with controls. Moreover, stem cell transcription factors, involved in the regulation of a plethora of early embryonic events, were upregulated by more than twofold in eight-cell stage embryos sired by BEP recovery males compared with controls; this suggests that there are potential deleterious effects on embryo development well after termination of BEP exposure.

  7. 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

  8. Nucleoli in a pronuclei-stage mouse embryo are represented by major satellite DNA of interconnecting chromosomes.

    PubMed

    Dozortsev, D; Coleman, A; Nagy, P; Diamond, M P; Ermilov, A; Weier, U; Liyanage, M; Reid, T

    2000-02-01

    To investigate the arrangement of chromosomes within pronuclei-stage mouse zygotes. In vitro study. Academic medical center. None. None. Location of major alpha-satellite DNA, centromeres, and telomeres, and relative location of chromosomes. Chromosomes appeared to be oriented inward by centromeres and to be interconnected by major alpha-satellite DNA, which appeared to be the sole DNA component of the nucleoli. This chromosomal arrangement persisted throughout interphase. Chromosomal painting failed to identify chromosomal ordering within pronuclei. Pronuclear nucleoli are represented by alpha-satellite sequences of interconnecting chromosomes that hold all chromosomes together during interphase. Chromosomes within the pronucleus are randomly positioned relative to each other.

  9. Kid depletion in mouse oocytes associated with multinucleated blastomere formation and inferior embryo development.

    PubMed

    Egashira, Akiyoshi; Yamauchi, Nobuhiko; Islam, Md Rashedul; Yamagami, Kazuki; Tanaka, Asami; Suyama, Hikaru; El-Sayed, El-Sharawy Mohamed; Tabata, Shoji; Kuramoto, Takashi

    2016-08-01

    This study investigated the knockdown (KD) of Kid on maturation developmental competence and multinucleation of mouse germinal vesicle (GV) oocytes after parthenogenetic activation. Data revealed that Kid messenger RNA (mRNA) was expressed in GV and MII stage oocyte and 1- and 2-cell embryos. Additionally, Kid mRNA expression in the Kid KD group decreased by nearly 46% compared to the control small interfering RNA (siRNA) groups. The rate of multinucleated embryos in the Kid KD group (52.4%) was significantly higher (P < 0.05) than the control siRNA group (4.7%). Finally, the developmental rates were significantly lower in the Kid siRNA group at > 4-cell stage (28.6% vs. 53.5%) and the blastocyst stage (2.4% vs. 23.3%) compared to the control siRNA groups. Suppression of Kid using siRNA caused multinucleation in early embryos with high frequency and it may increase 2- to 4-cell arrested embryos and reduce the developmental competence to blastocyst.

  10. Global gene expression profiling of individual human oocytes and embryos demonstrates heterogeneity in early development.

    PubMed

    Shaw, Lisa; Sneddon, Sharon F; 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.

  11. The effects of metals on embryo-larval and adult life stages of the sea urchin, Diadema antillarum.

    PubMed

    Bielmyer, G K; Brix, K V; Capo, T R; Grosell, M

    2005-09-10

    Since the massive population decline of the long-spined sea urchin, Diadema antillarum, in the early 1980s, the dynamics of coral reef ecosystems in the Caribbean have changed tremendously. The absence of D. antillarum, once a keystone herbivore, has led to macroalgal dominance in many of these reef communities. D. antillarum is not only important ecologically, but may also be a sensitive bioindicator species for toxicant exposure. Echinoderm larval development tests were conducted with D. antillarum exposed to elevated levels of aqueous copper (Cu), silver (Ag), nickel (Ni), or selenium (Se). All metals significantly affected larval development, based on normal development to the pluteus stage. The EC50s based on dissolved metal concentrations were 11 microg/L Cu, 6 microg/L Ag, 15 microg/L Ni, and 26 microg/L Se. Adult sea urchins were exposed to aqueous copper under flow through conditions for 96 h. The 96-h LC50 for this exposure was 25 microg/L dissolved Cu. Additionally, behavioral and physiological disturbance was observed. The physiological responses included both acid-base balance disturbance, as evidenced by reduced coelomic fluid pH and apparent ionoregulatory effects. In addition, behavioral effects included spatial orientation within the exposure tank, spine closure, and loss of spines. The high sensitivity of both adult and larval D. antillarum to these metals supports the use of this organism as an important biological indicator for metal exposure in marine environments.

  12. High sensitivity of embryo-larval stage of the Mediterranean mussel, Mytilus galloprovincialis to metal pollution in combination with temperature increase.

    PubMed

    Boukadida, Khouloud; Banni, Mohamed; Gourves, Pierre-Yves; Cachot, Jérôme

    2016-12-01

    The present work aimed to assess the effects of two widespread metallic pollutants, copper and silver, along with environmentally-realistic temperature increases, on embryo-larval development of the Mediterranean mussel Mytilus galloprovincialis. First, mussel embryos upon fertilization were exposed for 48 h to increasing concentrations of Cu (0.5-500 μg/L) and Ag (0.1-100 μg/L) at different temperatures (18, 20, 22 or 24 °C) in order to characterize toxicity of each toxicant at the different tested temperatures. Increasing concentrations of a Cu-Ag mixture were then tested in order to assess the mixture effect at different temperatures (18, 20 or 22 °C). Embryotoxicity was measured after 48 h of exposure (D-larvae stage) considering both the percentage of abnormalities and developmental arrest in D-larvae. The results suggest that the optimum temperature for mussel larvae development is 18 °C (12.65± 1.6% malformations) and beyond 20 °C a steep increase of abnormal larvae was observed up to 100% at 24 °C. Ag was more toxic than Cu with a 50% effective concentration (EC50) at 18 °C of 6.58 μg/L and 17.6 μg/L, respectively. Temperature increased the toxicity of both metals as proved with the EC50 at 20 °C at 3.86 μg/L and 16.28 μg/L for Ag and Cu respectively. Toxic unit calculation suggests additive effects of Cu and Ag in mixture at 18 and 20 °C. These results highlight a possible impairment of M. galloprovincialis reproduction in the Mediterranean Sea in relation to increase of both pollutants and water temperature due to global warming. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Effects of three pro-nuclei (3PN) proportion incidence on clinical outcomes of patients with lower retrieved oocytes in the fresh cleavage-stage embryo transfer (ET) cycles.

    PubMed

    Li, Mingzhao; Zhang, Silin; Shi, Wenhao; Ren, Wenjuan; Liu, Yanan; Tang, Qingqing; Shi, Juanzi

    2016-11-01

    To analyze the three pro-nuclei (3PN) incidence on clinical outcomes of patients with lower retrieved oocytes in the fresh cleavage-stage embryo transfer (ET) cycles. This study included 1200 fresh cleavage-stage ET cycles from January 2013 to June 2015. The patients were divided into 3PN = 0% (773 cycles) and 3PN > 0% (427 cycles) group. Main outcomes compared were fertilization, cleavage, normal fertilization, good quality embryo, implantation, clinical pregnancy, and early abortion rate. We observed that there was no significant difference in female's age, the number of retrieved oocytes, the number of transferred embryos, the number of good quality embryos, endometrial thickness, infertile time, basal serum follicle-stimulating hormone, and E2 value between two groups (p > 0.05). The fertilization (89.43 versus 83.90%, p < 0.001) and cleavage (98.34 versus 97.19%, p = 0.048) rates were significantly higher in 3PN > 0% than 3PN = 0% group. However, the normal fertilization (70.05 versus 50.67%, p < 0.001), good quality embryos (37.11 versus 26.47%, p < 0.001), and clinical pregnancy (49.81 versus 43.79%, p = 0.046) rates were significantly higher in 3PN = 0% than 3PN > 0% group. The implantation (35.88 versus 33.78%, p = 0.333) and early abortion (8.83 versus 10.70%, p = 0.474) rates were not significantly different between two groups. 3PN incidence might make a negative effect on clinical outcomes for patients with lower retrieved oocytes in the fresh cleavage-stage ET cycles.

  14. 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

  15. Evidence for stage-specific modulation of specific microRNAs (miRNAs) and miRNA processing components in zygotic embryo and female gametophyte of loblolly pine (Pinus taeda).

    PubMed

    Oh, Thomas J; Wartell, Roger M; Cairney, John; Pullman, Gerald S

    2008-01-01

    MicroRNAs (miRNAs) are known to regulate plant development, but have not been studied in gymnosperm seed tissues. The presence and characteristics of several miRNAs were examined in zygotic embryos (ZEs) and female gametophytes (FGs) of Pinus taeda (loblolly pine). Evidence for miRNAs was obtained using northern analyses and quantitative reverse transcription polymerase chain reaction (qRT-PCR) mediated with poly(A) polymerase. Partial sequences of two miRNAs were verified. Three regions of putative mRNA targets were analyzed by qRT-PCR to monitor the occurrence of stage-dependent miRNA-mediated cleavage. Five miRNAs were identified in ZEs and FGs along with partial sequences of Pta-miR166 and Pta-miR167. Both miRNAs showed differing degrees of tissue-specific and stage-specific modulation. Analysis of HB15L mRNA (a potential Pta-miR166 target) suggested miRNA-guided cleavage in ZEs and FGs. Analysis of ARF8L mRNA (a potential Pta-miR167 target) implied cleavage in ZEs but not in FGs. Argonaute9-like mRNA (ptAGO9L) showed stage-specific modulation of expression in ZEs that appeared to be inverted in the corresponding FGs. MicroRNAs and argonaute genes varied spatiotemporally during seed development. The peak levels of Pta-miR166 in FGs and ptAGO9L in embryos occurred at stage 9.1, a critical transition point during embryo development and a point where somatic embryo maturation often stops. MicroRNAs identified in FG tissue may play a role in embryogenesis.

  16. 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.

  17. Prevalence, consequence, and significance of reverse cleavage by human embryos viewed with the use of the Embryoscope time-lapse video system.

    PubMed

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

    2014-11-01

    To investigate the prevalence and potential causes of reverse cleavage (RC) by human early-cleavage embryos and its associations with embryonic development and implantation after transfer. Clinical retrospective cohort study. Private fertility treatment center. A total of 126 consecutive in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) treatment cycles, with 353 IVF and 436 ICSI embryos cultured in the Embryoscope until day 3. None. Embryo assessment on day 3, incidence of abnormal division, embryo morphokinetic parameters, and fetal heart beat. RC, referring to either blastomere fusion or failed cytokinesis, occurred up to three times per individual embryo in 27.4% of embryos during the first three cleavage cycles. A higher incidence was associated with GnRH antagonist cycles compared with agonist cycles (odds ratio [OR] 1.683), or with ICSI compared with IVF (OR 1.600). After ICSI, sperm progressive motility was associated with RC (area under the receiver operating characteristic curve: 0.573). Compared with RC-negative embryos, a lower proportion of RC-positive embryos reached 6-cell stage or beyond by day 3 (47.7% vs. 71.7%), and were more likely to have multinucleation at the 4-cell stage (10.1% vs. 5.0%). Embryos showing RC had significantly poorer performance in both conventional grading and morphokinetic parameters, and they implanted less (0/22 vs. 29/131) than those not showing RC. RC significantly compromised embryo development, culminating in poor implantation potential. For each embryo, it can occur on more than one occasion at any stage during the first 3 days of culture. It is associated with factors affecting both oocyte and sperm. Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  18. Genetic expression of hexokinase and glucose phosphate isomerase in late-stage mouse preimplantation embryos: transcription activities in glucose/phosphate-containing HTF and glucose/phosphate-free P1 media.

    PubMed

    Johnson, M D; Batey, D W; Behr, B; Barro, J

    1997-04-01

    In mouse and human preimplantation development, pyruvate is consumed preferentially during early embryogenesis; however, during the morula and blastocyst stages, glucose is the preferred energy substrate. Studies have suggested that the glycolytic enzymes, hexokinase and glucose phosphate isomerase, are important enzymes in glucose metabolism during these later stages of human and mouse preimplantation development. In order to investigate the genetic activities of these enzymes in late-stage mouse embryos developing in vitro, we analysed hexokinase and glucose phosphate isomerase transcription activities by qualitative RNA assays using reverse transcriptase-nested polymerase chain reaction amplification of individual mouse morulae and early blastocysts incubated in glucose/phosphate-free preimplantation stage one (P1) medium and glucose/phosphate-containing human tubal fluid (HTF) medium. We observed an increased incidence of hexokinase transcripts in the population of blastocysts compared with morulae, and differences in transcript incidence between early blastocysts developing in HTF medium and in P1 medium. In contrast, glucose phosphate isomerase transcripts were consistantly present in all embryos analysed, and appear to be constitutively expressed during late-stage mouse embryogenesis. The different activity patterns of the two glycolytic genes may reflect different mechanisms of gene regulation or differential transcript stability during the later stages of mouse preimplantation development.

  19. 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.

  20. 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.

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

    PubMed

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

    2016-04-01

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

  2. Successful embryo transfer following artificial insemination of superovulated fallow deer (Dama dama).

    PubMed

    Jabbour, H N; Marshall, V S; Argo, C M; Hooton, J; Loudon, A S

    1994-01-01

    Thirty-four European fallow deer (Dama dama dama) were randomly allocated into embryo donor (n = 12) or embryo recipient (n = 22) groups. All does were treated with controlled internal drug release (CIDR) devices for 14 days. Animals in the embryo donor group were further treated with 200 I.U. pregnant mare serum gonadotrophin (PMSG) and 0.5 units ovine follicle-stimulating hormone (FSH). PMSG was administered 72 h before withdrawal of CIDR devices and FSH was given in eight 0.063 unit injections at 12-hourly intervals starting at the time of PMSG administration. All embryo donor animals were inseminated, by laparoscopy in both uterine horns, 36 h after withdrawal of CIDR devices with 25 x 10(6) fresh spermatozoa collected from Mesopotamian fallow deer (Dama dama mesopotamica). Embryos were recovered by laparotomy on Day 3 (n = 6) or Day 6 (n = 6) after withdrawal of CIDR devices and the ovarian response was determined. In total, 22 embryos were transferred into the oviduct (2-4-cell stage, n = 14) or uterine horn (morula stage, n = 8) on Day 3 or Day 6 after withdrawal of CIDR devices respectively. The overall means (+/- s.e.m.) of total follicular response and corpora lutea were 24.2 +/- 3.5 and 14.1 +/- 3.6 respectively. The mean number of large unruptured follicles was higher on Day 6 than on Day 3 (13.5 +/- 2.9 v. 6.7 +/- 1.3, P < 0.05). The overall embryo recovery rate was 45.8%. There was no difference in pregnancy rate following transfer of embryos on either Day 3 or Day 6 (7/14 v. 5/8 respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

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

    PubMed

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

    2017-05-01

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

  5. Direct Unequal Cleavages: Embryo Developmental Competence, Genetic Constitution and Clinical Outcome

    PubMed Central

    Zhan, Qiansheng; Ye, Zhen; Clarke, Robert; Rosenwaks, Zev; Zaninovic, Nikica

    2016-01-01

    Objective To investigate the prevalence, developmental potential, chromosomal constitution and clinical outcome of embryos with direct unequal cleavages (DUC). Design A retrospective observational study. Setting Academic Institution. Participant 21,261 embryos from 3,155 cycles cultured in EmbryoScope®. Results The total incidence of DUCs per embryo occupying the first three cleavages were 26.1%. Depending of the cell stage, DUC rate was 9.8% at first cleavage (DUC-1), 9.1% at second cleavage (DUC-2), and 3.7% at third cleavage (DUC-3) with 3.6% of embryos exhibiting multiple DUCs (DUC-Plus). The occurrence of DUCs was not correlated with female gamete age or source. The incidence of DUC-1 was significantly higher in embryos fertilized by epididymal and testicular sperm (13.6% and 11.4%, respectively) compared to ejaculated sperm (9.1%, all p<0.05). The total incidences of DUCs were strongly correlated with the onset of blastomere multinucleation (MNB) during the first three divisions. In MNB embryos, DUCs incidence are two to three times more likely to develop when compared to non-MNB embryos (OR = 3.11, 95% CI [2.64, 3.67] at 1-cell stage, OR = 2.64, 95% CI [2.39, 2.91] at 2-cell stage and OR = 2.51, 95% CI [1.84, 3.43] at 4-cell stage). The blastocyst formation rates gradually decreased from 61.0% in non-DUC to 40.2% in DUC-3, 18.8% in DUC-2, 8.2% in DUC-1 and 5.6% in multiple DUC embryos (DUC-Plus). The known implantation rates (FH) for day 3 (D3) transfers were 12.42% (n = 3172) in Non-DUC embryos, 6.3% (n = 127) in DUC-3, and 2.7% (n = 260) in DUC-2 embryos. No live births resulted from either DUC-1 (n = 225) or DUC-Plus (n = 100) embryo transfers. For blastocyst transfers, lower implantation rates (33.3%) but similar live birth (LB) rates (40%) were observed if DUC blastocysts were transferred. Comparatively rates in Non-DUC blastocyst were 45.2% and 34.8%, respectively. The euploid rate gradually increased from DUC-1, -2, -3 to Non-DUC (13.3%, 19.5%, 33

  6. 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. Published by Oxford University Press on behalf of Poultry Science Association 2016. This work is written by (a) US

  7. Regulation of ATF1 and ATF2 transcripts by sequences in their 3' untranslated region in cleavage-stage cattle embryos.

    PubMed

    Orozco-Lucero, Ernesto; Dufort, Isabelle; Sirard, Marc-André

    2017-04-01

    The sequence of a 3' untranslated region (3'UTR) of mRNA governs the timing of its polyadenylation and translation in mammalian oocytes and early embryos. The objective of this study was to assess the influence of cis-elements in the 3'UTR of the developmentally important ATF1 and ATF2 transcripts on their timely translation during first cleavages in bovine embryos. Eight different reporter mRNAs (coding sequence of green fluorescent protein [GFP] fused to the 3'UTR of short or long isoforms of cattle ATF1 or -2, with or without polyadenylation) or a control GFP mRNA were microinjected separately into presumptive bovine zygotes at 18 hr post-insemination (hpi), followed by epifluorescence assessment for GFP translation between 24 and 80 hpi (expressed as percentage of GFP-positive embryos calculated from the total number of individuals). The presence of either polyadenine or 3'UTR sequence in deadenylated constructs is required for GFP translation (implying the need for polyadenylation), and all exogenous mRNAs that met either criteria were translated as soon as 24 hpi-except for long-deadenylated ATF2-UTR, whose translation began at 36 hpi. Overall, GFP was more visibly translated in competent (cleaving) embryos, particularly in long ATF1/2 constructs. The current data shows a timely GFP translation in bovine embryos depending on sequences in the 3'UTR of ATF1/2, and indicates a difference between short and long isoforms. In addition, cleaving embryos displayed increased translational capacity of the tested constructs. Functional confirmation of the identification cis-sequences in the 3'UTR of ATF1/2 will contribute to the understanding of maternal mRNA translation regulation during early cattle development. © 2017 Wiley Periodicals, Inc.

  8. Identical triplets and twins developed from isolated blastomeres of 8- and 16-cell mouse embryos supported with tetraploid blastomeres.

    PubMed

    Tarkowski, Andrzej K; Ozdzenski, Waclaw; Czolowska, Renata

    2005-01-01

    We studied the developmental potential of single blastomeres from early cleavage mouse embryos. Eight- and sixteen-cell diploid mouse embryos were disaggregated and single blastomeres from eight-cell embryos or pairs of sister blastomeres from sixteen-cell embryos were aggregated with 4, 5 or 6 tetraploid blastomeres from 4-cell embryos. Each diploid donor embryo gave eight sister aggregates, which later were manipulated together as one group (set). The aggregates were cultured in vitro until the blastocyst stage, when they were transferred (in sets) to the oviducts of pseudopregnant recipients. Eighteen live foetuses or pups were obtained from the transfer (11.0% of transferred blastocysts) and out of those, eleven developed into fertile adults (one triplet, one pair of twins and four singletons). In all surviving adults, pups and living foetuses, only diploid cells were detected in their organs and tissues as shown by analysis of coat pigmentation and distribution of glucose phosphate isomerase isoforms. In order to explain the observed high rate of mortality of transferred blastocysts, in an accompanying experiment, the diploid and tetraploid blastomeres were labelled with different fluorochromes and then aggregated. These experiments showed the diploid cells to be present not only in the inner cell mass (ICM) but also in the trophectoderm. The low number of diploid cells and the predominance of tetraploid cells in the ICM of chimaeric blastocysts might have been responsible for high postimplantation mortality of our experimental embryos.

  9. Messenger RNA expression of Pabpnl1 and Mbd3l2 genes in oocytes and cleavage embryos.

    PubMed

    Biase, Fernando Henrique; Martelli, Lúcia; Puga, Renato; Giuliatti, Silvana; Santos-Biase, Weruska Karyna Freitas; Fonseca Merighe, Giovana Krempel; Meirelles, Flávio Vieira

    2010-05-15

    To identify genes specifically expressed in mammalian oocytes using an in silico subtraction, and to characterize the mRNA patterns of selected genes in oocytes, embryos, and adult tissues. Comparison between oocyte groups and between early embryo stages. Laboratories of embryo manipulation and molecular biology from Departamento de Genética (FMRP) and Departamento de Ciências Básicas (FZEA)--University of São Paulo. Oocytes were collected from slaughtered cows for measurements, in vitro fertilization, and in vitro embryo culture. Somatic tissue, excluding gonad and uterus tissue, was collected from male and female cattle. Messenger RNA levels of poly(A)-binding protein nuclear-like 1 (Pabpnl1) and methyl-CpG-binding domain protein 3-like 2 (Mbd3l2). Pabpnl1 mRNA was found to be expressed in oocytes, and Mbd3l2 transcripts were present in embryos. Quantification of Pabpnl1 transcripts showed no difference in levels between good- and bad-quality oocytes before in vitro maturation (IVM) or between good-quality oocytes before and after IVM. However, Pabpnl1 transcripts were not detected in bad-quality oocytes after IVM. Transcripts of the Mbd3l2 gene were found in 4-cell, 8-cell, and morula-stage embryos, with the highest level observed in 8-cell embryos. Pabpnl1 gene expression is restricted to oocytes and Mbd3l2 to embryos. Different Pabpnl1 mRNA levels in oocytes of varying viability suggest an important role in fertility involving the oocyte potential for embryo development. Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  10. Reduction of Mitochondrial Function by FCCP During Mouse Cleavage Stage Embryo Culture Reduces Birth Weight and Impairs the Metabolic Health of Offspring.

    PubMed

    Zander-Fox, Deirdre L; Fullston, Tod; McPherson, Nicole O; Sandeman, Lauren; Kang, Wan Xian; Good, Suzanne B; Spillane, Marni; Lane, Michelle

    2015-05-01

    The periconceptual environment represents a critical window for programming fetal growth trajectories and susceptibility to disease; however, the underlying mechanism responsible for programming remains elusive. This study demonstrates a causal link between reduction of precompaction embryonic mitochondrial function and perturbed offspring growth trajectories and subsequent metabolic dysfunction. Incubation of embryos with carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), which uncouples mitochondrial oxidative phosphorylation, significantly reduced mitochondrial membrane potential and ATP production in 8-cell embryos and the number of inner cell mass cells within blastocysts; however, blastocyst development was unchanged. This perturbed embryonic mitochondrial function was concomitant with reduced birth weight in female offspring following embryo transfer, which persisted until weaning. FCCP-treated females also exhibited increased adiposity at 4 wk, increased adiposity gain between 4 and 14 wk, glucose intolerance at 8 wk, and insulin resistance at 14 wk. Although FCCP-treated males also exhibited reduced glucose tolerance, but their insulin sensitivity and adiposity gain between 4 and 14 wk was unchanged. To our knowledge, this is one of the first studies to demonstrate that reducing mitochondrial function and, thus, decreasing ATP output in the precompacting embryo can influence offspring phenotype. This is of great significance as a large proportion of patients requiring assisted reproductive technologies are of advanced maternal age or have a high body mass index, both of which have been independently linked with perturbed early embryonic mitochondrial function.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  12. 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.

  13. 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.

  14. Comparison of clinical outcomes between fresh embryo transfers and frozen-thawed embryo transfers.

    PubMed

    Shen, Chunjuan; Shu, Defeng; Zhao, Xiaojie; Gao, Ying

    2014-06-01

    Advances in embryo culture technology and cryopreservation have led to a shift in in vitro fertilization (IVF) from early fresh or frozen-thawed cleavage embryo transfer to fresh or frozen-thawed blastocyst stage transfer. To compare the clinical outcomes of fresh embryo transfers and frozen-thawed embryo transfers. In this retrospective case control study, patients undergoing IVF cycles from January 2012 to December 2012 were enrolled in Assisted Reproduction of Wuhan Union Hospital were enrolled. A total of 1891 cycle contains 1150 fresh embryo transfers and 741 frozen-thawed embryo transfers were studied. All data were transferred directly to SPSS 18 and analyzed. Clinical pregnancy rates of fresh cleavage-stage embryo transfers compared with fresh blastocyst transfers, frozen-thawed cleavage-stage embryo transfers, post thaw cleavage-stage extended blastocyst culture transfers and frozen-thawed blastocyst transfers were 52.7%, 35.88%, 35.29%, 47.75%, 59.8% in patients under 35 years of ages and 41.24%, 26.92%, 11.32%, 46.15%, 55.8% in patients older than 35 years old, respectively. The multiple pregnancy rates, abortion rates and ectopic pregnancy rates did not differ significantly among the five groups. The clinical pregnancy rates were not different significantly between fresh cleavage-stage embryo transfers and fresh blastocyst transfers. But the clinical pregnancy rate of frozen-thawed blastocyst transfer was the highest among fresh/frozen-thawed embryo transfers.

  15. Zygote arrest 1, nucleoplasmin 2, and developmentally associated protein 3 mRNA profiles throughout porcine embryo development in vitro.

    PubMed

    Wasielak, M; Więsak, T; Bogacka, I; Jalali, B Moza; Bogacki, M

    2016-12-01

    Maternal effect genes (MEGs) are expressed in oocytes and embryos and play an important role in activation of the embryonic genome. An abnormality in the expression of these genes may lead to arrest of embryonic cleavage or to altered transcription of factors responsible for further embryonic development. In vitro-produced porcine embryos have a lower developmental potential than embryos produced in vivo. We hypothesized that in vitro embryo culture conditions have an effect on the expression of MEGs at various developmental stages, which may affect their developmental potential. Here, using real-time polymerase chain reaction, we examined mRNA profiles of the MEGs, zygote arrest 1 (ZAR-1), nucleoplasmin 2 (NPM2), and developmentally associated pluripotency protein 3 (DPPA3), in porcine oocytes and embryos produced in vitro and in vivo. Further, we evaluated the effect of the combined addition of EGF, interleukin 1β, and leukemia inhibitory factor to the porcine in vitro embryo production system on mRNA profiles of selected MEGs. Finally, we studied localization of the MEG protein products in in vitro-obtained oocytes and embryos using confocal microscopy. We found that the ZAR-1 mRNA profile differed throughout in vitro and in vivo embryo development. In the embryos produced in vitro, the decrease in ZAR-1 mRNA levels was observed at the 2-cell stage, whereas in in vivo embryos, ZAR-1 mRNA levels declined significantly starting at the 4-cell stage (P < 0.05). In vitro culture conditions affected transiently also DPPA3 mRNA levels at the 4-cell stage (P < 0.05). There was no difference in the NPM2 mRNA profile during in vitro and in vivo embryo development. The ZAR-1 and DPPA3 proteins were localized in the cytoplasm of the oocytes and embryos, whereas the NPM2 protein was found both in the cytoplasm and in the nucleus. All proteins were expressed until blastocyst stage. The addition of EGF and cytokines to the culture medium decreased DPPA3 m

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

    PubMed

    Hori, Tatsuya; Ushijima, Hitoshi; Kimura, Taku; Kobayashi, Masanori; Kawakami, Eiichi; Tsutsui, Toshihiko

    2016-08-01

    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.

  17. 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

  18. Heterochromatin reprogramming in rabbit embryos after fertilization, intra-, and inter-species SCNT correlates with preimplantation development.

    PubMed

    Yang, Cai-Xia; Liu, Zichuan; Fleurot, Renaud; Adenot, Pierre; Duranthon, Véronique; Vignon, Xavier; Zhou, Qi; Renard, Jean-Paul; Beaujean, Nathalie

    2013-02-01

    To investigate the embryonic genome organization upon fertilization and somatic cell nuclear transfer (SCNT), we tracked HP1β and CENP, two well-characterized protein markers of pericentric and centromeric compartments respectively, in four types of embryos produced by rabbit in vivo fertilization, rabbit parthenogenesis, rabbit-to-rabbit, and bovine-to-rabbit SCNT. In the interphase nuclei of rabbit cultured fibroblasts, centromeres and associated pericentric heterochromatin are usually isolated. Clustering into higher-order chromatin structures, such as the chromocenters seen in mouse and bovine somatic cells, could not be observed in rabbit fibroblasts. After fertilization, centromeres and associated pericentric heterochromatin are quite dispersed in rabbit embryos. The somatic-like organization is progressively established and completed only by the 8/16-cell stage, a stage that corresponds to major embryonic genome activation in this species. In SCNT embryos, pericentric heterochromatin distribution typical for rabbit and bovine somatic cells was incompletely reverted into the 1-cell embryonic form with remnants of heterochromatin clusters in 100% of bovine-to-rabbit embryos. Subsequently, the donor cell nuclear organization was rapidly re-established by the 4-cell stage. Remarkably, the incomplete remodeling of bovine-to-rabbit 1-cell embryos was associated with delayed transcriptional activation compared with rabbit-to-rabbit embryos. Together, the results confirm that pericentric heterochromatin spatio-temporal reorganization is an important step of embryonic genome reprogramming. It also appears that genome reorganization in SCNT embryos is mainly dependent on the nuclear characteristics of the donor cells, not on the recipient cytoplasm.

  19. Ensoulment and IVF embryos.

    PubMed Central

    Shea, M C

    1987-01-01

    This paper examines the metaphysical question of 'ensoulment' in relation to the theory, put forward in an earlier paper, that human life begins when the newly formed body organs and systems of the embryo begin to function as an organised whole, at which stage there is evidence of a change of nature. Although Roman Catholic theology teaches that a human being is a union of physical body and spiritual soul, it is incorrect to interpret this in a dualistic sense. The meaning of 'soul' is considered and the conclusion reached that although both in the religious context and apart from it abortion is difficult to justify at any stage after conception, it does not follow that the use of 'spare' In Vitro Fertilisation (IVF) embryos should be rejected. If 'ensoulment' does not occur until the new organism functions as a whole then a decision not to make use of IVF embryos for medical purposes would be a heavy responsibility and not a 'safe' way out. PMID:3612702

  20. 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.

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

    PubMed

    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 [3H]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.

  2. Maternal hCG concentrations in early IVF pregnancies: associations with number of cells in the Day 2 embryo and oocytes retrieved.

    PubMed

    Tanbo, T G; Eskild, A

    2015-12-01

    Do number of cells in the transferred cleavage stage embryo and number of oocytes retrieved for IVF influence maternal hCG concentrations in early pregnancies? Compared with transfer of a 2-cell embryo, transfer of a 4-cell embryo results in higher hCG concentrations on Day 12 after transfer, and more than 20 oocytes retrieved were associated with low hCG concentrations. Maternal hCG concentration in very early pregnancy varies considerably among women, but is likely to be an indicator of time since implantation of the embryo into the endometrium, in addition to number and function of trophoblast cells. We followed 1047 pregnancies after IVF/ICSI from oocyte retrieval until Day 12 after embryo transfer. Women were recruited in Norway during the years 2005-2013. Successful pregnancies after transfer of one single embryo that had been cultured for 2 days were included. Maternal hCG was quantified on Day 12 after embryo transfer by chemiluminescence immunoassay, which measures intact hCG and the free β-hCG chain. Information on a successful pregnancy, defined as birth after >16 weeks, was obtained by linkage to the Medical Birth Registry of Norway. Transfer of a 4-cell embryo resulted in higher maternal hCG concentrations compared with transfer of a 2-cell embryo (134.8 versus 87.8 IU/l, P < 0.05). A high number of oocytes retrieved (>20) was associated with low hCG concentrations (P < 0.05). The factors studied explain a limited part of the total variation of hCG concentrations in early pregnancy. Although embryo transfer was performed at the same time after fertilization, we do not know the exact time of implantation. A further limitation to our study is that the number of pregnancies after transfer of a 2-cell embryo was small (27 cases). Number of cells in the transferred embryo and number of oocytes retrieved may influence the conditions and timing for embryo implantation in different ways and thereby influence maternal hCG concentrations. Such knowledge may be

  3. Autophagy and ubiquitin-mediated proteolysis may not be involved in the degradation of spermatozoon mitochondria in mouse and porcine early embryos.

    PubMed

    Jin, Yong-Xun; Zheng, Zhong; Yu, Xian-Feng; Zhang, Jia-Bao; Namgoong, Suk; Cui, Xiang-Shun; Hyun, Sang-Hwan; Kim, Nam-Hyung

    2016-02-01

    The mitochondrial genome is maternally inherited in animals, despite the fact that paternal mitochondria enter oocytes during fertilization. Autophagy and ubiquitin-mediated degradation are responsible for the elimination of paternal mitochondria in Caenorhabditis elegans; however, the involvement of these two processes in the degradation of paternal mitochondria in mammals is not well understood. We investigated the localization patterns of light chain 3 (LC3) and ubiquitin in mouse and porcine embryos during preimplantation development. We found that LC3 and ubiquitin localized to the spermatozoon midpiece at 3 h post-fertilization, and that both proteins were colocalized with paternal mitochondria and removed upon fertilization during the 4-cell stage in mouse and the zygote stage in porcine embryos. Sporadic paternal mitochondria were present beyond the morula stage in the mouse, and paternal mitochondria were restricted to one blastomere of 4-cell embryos. An autophagy inhibitor, 3-methyladenine (3-MA), did not affect the distribution of paternal mitochondria compared with the positive control, while an autophagy inducer, rapamycin, accelerated the removal of paternal mitochondria compared with the control. After the intracytoplasmic injection of intact spermatozoon into mouse oocytes, LC3 and ubiquitin localized to the spermatozoon midpiece, but remnants of undegraded paternal mitochondria were retained until the blastocyst stage. Our results show that paternal mitochondria colocalize with autophagy receptors and ubiquitin and are removed after in vitro fertilization, but some remnants of sperm mitochondrial sheath may persist up to morula stage after intracytoplasmic spermatozoon injection (ICSI).

  4. Renovation of a drop embryo cultures system by using refined mineral oil and the effect of glucose and/or hemoglobin added to a serum-free medium.

    PubMed

    Lee, Seungtae; Cho, Meeyoung; Kim, Eunjung; Kim, Taemin; Lee, Changkyu; Han, Jaeyong; Lim, Jeongmook

    2004-01-01

    This study was conducted to evaluate whether refining mineral oil and the addition of hemoglobin and/or glucose to a serum-free medium could improve in vitro-development of embryos cultured in a chemically semi-defined microdroplet culture system. Block strain, outbred (ICR) mouse 1- or 2-cell embryos were cultured in 5 microl droplets of Chatot, Ziomek and Bavister medium overlaid with mineral oil of different types, and preimplantation development to the blastocyst stage was subsequently monitored. In the experiment 1, either Sigma (M-8410) or BDH (GPR) mineral oil with or without washing was used for embryo culture and, distilled water (DW) or culture medium was used as a washing agent. As results, better (P<0.0001) development of 1-cell embryos was found in the Sigma than in the BDH; more blastocysts developed in Sigma oil washed with culture medium than in the others (37% vs. 0%). Subsequently, 1- (experiment 2) or 2-cell (experiment 3) embryos were cultured in the droplets overlaid with medium-washed Sigma oil, to which 0.001 mg/ml hemoglobin and/or 5.6 mM glucose were supplemented at the 1-cell and the 4-cell stages, respectively. Regardless of embryo stages, blastocyst formation was significantly improved by the addition of hemoglobin (54 to 48% vs. 42 to 31% in 1-cell and 83 to 78% vs. 65 to 68% in 2-cell embryos) and this effect was independent of glucose addition. In conclusion, the selection and washing of mineral oil, and the addition of hemoglobin is beneficial for improving the efficacy of a drop embryo culture system using a serum-free medium.

  5. Progestin priming before gonadotrophin stimulation and AI improves embryo development and normalises luteal function in the cat.

    PubMed

    Stewart, Rosemary A; Crosier, Adrienne E; Pelican, Katharine M; Pukazhenthi, Budhan S; Sitzmann, Brandon D; Porter, Tom E; Wildt, David E; Ottinger, Mary Ann; Howard, JoGayle

    2015-01-01

    Exogenous gonadotrophins administered before AI can adversely alter endocrine dynamics and inhibit embryo development in felids. In the present study, we tested the hypothesis that priming the domestic cat ovary with progestin mitigates the negative influence of gonadotrophin therapy by normalising early embryogenesis and luteal function. Queens were given either: (1) progestin pretreatment plus chorionic gonadotrophins (n=8; primed); or (2) gonadotrophins only (n=8; unprimed). Ovulatory response was assessed laparoscopically, and cats with fresh corpora lutea (CL) were inseminated in utero. Ovariohysterectomy was performed 3 days later to recover intra-oviductal embryos for in vitro culture; one ovary was prepared for histology, and CL from the remaining ovary were excised and assessed for progesterone content and targeted gene expression. Of the six primed and seven unprimed queens inseminated, embryo(s) were recovered from five individuals per group. Embryos from progestin-primed donors more closely simulated normal stage in vivo development (P<0.05). No 2- or 4-cell embryos from either group developed beyond 16-cells in vitro; however, 50% of unprimed and 66.7% of primed (P>0.05) 5-16-cell embryos progressed to morulae or blastocysts by Day 4 of culture. Although histological characteristics were unaffected by progestin priming (P>0.05), luteal progesterone was unusually high (P<0.05) in unprimed compared with primed cats (72.4±5.8 vs. 52.2±5.5 ng mg(-1), respectively). Two genes associated with progesterone biosynthesis (luteinising hormone receptor and 3β-hydroxysteroid dehydrogenase) were upregulated in unprimed versus primed individuals (P=0.05 and P<0.05, respectively), indicating potential mechanistic pathways for the protective influence of pre-emptive progestin treatment. Building on earlier findings that progestin priming prevents spontaneous ovulation, increases ovarian sensitivity to gonadotrophins and ensures a normative endocrine environment

  6. The human PRD-like homeobox gene LEUTX has a central role in embryo genome activation

    PubMed Central

    Jouhilahti, Eeva-Mari; Madissoon, Elo; Vesterlund, Liselotte; Töhönen, Virpi; Krjutškov, Kaarel; Plaza Reyes, Alvaro; Petropoulos, Sophie; Månsson, Robert; Linnarsson, Sten; Bürglin, Thomas; Lanner, Fredrik; Hovatta, Outi; Katayama, Shintaro

    2016-01-01

    Leucine twenty homeobox (LEUTX) is a paired (PRD)-like homeobox gene that is expressed almost exclusively in human embryos during preimplantation development. We previously identified a novel transcription start site for the predicted human LEUTX gene based on the transcriptional analysis of human preimplantation embryos. The novel variant encodes a protein with a complete homeodomain. Here, we provide a detailed description of the molecular cloning of the complete homeodomain-containing LEUTX. Using a human embryonic stem cell overexpression model we show that the complete homeodomain isoform is functional and sufficient to activate the transcription of a large proportion of the genes that are upregulated in human embryo genome activation (EGA), whereas the previously predicted partial homeodomain isoform is largely inactive. Another PRD-like transcription factor, DPRX, is then upregulated as a powerful repressor of transcription. We propose a two-stage model of human EGA in which LEUTX acts as a transcriptional activator at the 4-cell stage, and DPRX as a balancing repressor at the 8-cell stage. We conclude that LEUTX is a candidate regulator of human EGA. PMID:27578796

  7. Enhance beef cattle improvement by embryo biotechnologies.

    PubMed

    Wu, B; Zan, L

    2012-10-01

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

  8. 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.

  9. Generating chimeric zebrafish embryos by transplantation.

    PubMed

    Kemp, Hilary A; Carmany-Rampey, Amanda; Moens, Cecilia

    2009-07-17

    One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps.

  10. Gap junctional connexin messenger RNA expression in the ovine uterus and placenta: effects of estradiol-17β-treatment, early pregnancy stages, and embryo origin.

    PubMed

    Johnson, M L; Redmer, D A; Reynolds, L P; Grazul-Bilska, A T

    2017-01-01

    Gap junctions play a major role in direct, contact-dependent cell-cell communication, and they have been implicated in the regulation of cellular metabolism and the coordination of cellular functions during growth and differentiation of organs and tissues. Gap junctional channels, composed of connexin (Cx) proteins, have been detected and shown to be influenced by hormones (eg, estrogen and progesterone) in uterine and placental tissues in several species. We hypothesized that (1) the messenger RNA (mRNA) for Cx26, Cx32, Cx37, and Cx43 is expressed in the uterus of ovariectomized sheep treated with estradiol-17β (E2) and in ovine placenta during early pregnancy, (2) E2-treatment of ovariectomized ewes would cause time-specific changes in Cx26, Cx32, Cx37, and Cx43 mRNA expression (experiment 1), and (3) expression of these 4 Cx would vary across the days of early pregnancy (experiment 2) and will be affected by embryo origin (ie, after application of assisted reproductive technologies [ARTs]; experiment 3). Thus, we collected uterine tissues at 0 to 24 h after E2 treatments (experiment 1), and placental tissues during days 14 to 30 of early pregnancy after natural (NAT) breeding (experiment 2) and on day 22 of early pregnancy established after transfer of embryos generated through natural breeding (NAT-ET), in vitro fertilization (IVF), or in vitro activation (IVA, parthenotes; experiment 3). In experiment 1, the expression of Cx26, Cx37, and Cx43 mRNA increased (P < 0.05) and Cx32 mRNA decreased (P < 0.06) in both caruncular and intercaruncular tissues after E2 treatment. In experiment 2, during early pregnancy, there were significant changes (P < 0.01) across days in the expression of Cx26, Cx37, and Cx43 mRNA in the maternal placenta, accompanied by changes (P < 0.001) in Cx37 and Cx43 mRNA in the fetal placenta. In experiment 3, in maternal placenta, Cx32 mRNA expression was decreased (P < 0.001) in NAT-ET, IVF, and IVA groups compared to the NAT group; but

  11. DNA repair in mammalian embryos.

    PubMed

    Jaroudi, Souraya; SenGupta, Sioban

    2007-01-01

    Mammalian cells have developed complex mechanisms to identify DNA damage and activate the required response to maintain genome integrity. Those mechanisms include DNA damage detection, DNA repair, cell cycle arrest and apoptosis which operate together to protect the conceptus from DNA damage originating either in parental gametes or in the embryo's somatic cells. DNA repair in the newly fertilized preimplantation embryo is believed to rely entirely on the oocyte's machinery (mRNAs and proteins deposited and stored prior to ovulation). DNA repair genes have been shown to be expressed in the early stages of mammalian development. The survival of the embryo necessitates that the oocyte be sufficiently equipped with maternal stored products and that embryonic gene expression commences at the correct time. A Medline based literature search was performed using the keywords 'DNA repair' and 'embryo development' or 'gametogenesis' (publication dates between 1995 and 2006). Mammalian studies which investigated gene expression were selected. Further articles were acquired from the citations in the articles obtained from the preliminary Medline search. This paper reviews mammalian DNA repair from gametogenesis to preimplantation embryos to late gestational stages.

  12. Ultra rapid freezing and vitrification of human embryos derived from abnormally fertilised zygotes.

    PubMed

    Molina, I; Duque, C C; Alfonso, J; Cervera, R P; Romeu, A

    2006-01-01

    The present study was undertaken to compare the developmental capacity of human embryos derived from abnormally fertilised zygotes (1 PN, > 3 PN; 16-18 hours after ICSI) cryopreserved using two techniques: ultra rapid freezing and vitrification. At 2-4 cell stage, (48 hours after ICSI), these abnormally fertilised embryos were then distributed in three groups: a) embryos that were cryopreserved by ultra rapid freezing (URF Group), b) embryos cryopreserved by vitrification (V Group) and c) embryos that were not cryopreserved (Control group). Survival rates and embryo development after 24 hours of in vitro culture (72 hours after ICSI) were compared. 42 embryos were cryopreserved by ultra rapid freezing in 0.5 mL straws, using a mixture of dimethyl sulphoxide (3M) and sucrose (0.25M) in a base solution consisting of IVF medium plus 20 percent (v/v) of Human Serum Albumin (HSA), and 24 embryos were vitrified in 0.25 ml straws, using a two step protocol with an equilibration solution consisting of 10 percent ethylene glycol (1.79 M) and 10 percent dimethyl sulphoxide (1.41 M) in a base solution of modified phosphate buffered saline (PBS) with 20 percent of HSA and a vitrification solution consisting of 20 percent ethylene glycol (3.58 M), 20 percent dimethyl sulphoxide (2.82 M) and 0.5 M sucrose in base solution. The recovery rate after thawing/warming was lower for the vitrification group (75 percent V; 83 percent URF). The number of embryos with less than 50 percent of intact blastomeres after cryopreservation was significantly higher for the URF group (0 percent V; 34 percent URF). After in vitro culture, the rate of embryos not cryopreserved (Control group) that developed in vitro (72 hours after ICSI) was the highest (86 percent), followed by group V (50 percent), while group URF was the lowest (13 percent). These differences were statistically significant. This straw method of vitrification is successful and safe.

  13. Gene transfer into older chicken embryos by ex ovo electroporation.

    PubMed

    Luo, Jiankai; Yan, Xin; Lin, Juntang; Rolfs, Arndt

    2012-07-27

    The chicken embryo provides an excellent model system for studying gene function and regulation during embryonic development. In ovo electroporation is a powerful method to over-express exogenous genes or down-regulate endogenous genes in vivo in chicken embryos(1). Different structures such as DNA plasmids encoding genes(2-4), small interfering RNA (siRNA) plasmids(5), small synthetic RNA oligos(6), and morpholino antisense oligonucleotides(7) can be easily transfected into chicken embryos by electroporation. However, the application of in ovo electroporation is limited to embryos at early incubation stages (younger than stage HH20--according to Hamburg and Hamilton)(8) and there are some disadvantages for its application in embryos at later stages (older than stage HH22--approximately 3.5 days of development). For example, the vitelline membrane at later stages is usually stuck to the shall membrane and opening a window in the shell causes rupture of the vessels, resulting in death of the embryos; older embryos are covered by vitelline and allantoic vessels, where it is difficult to access and manipulate the embryos; older embryos move vigorously and is difficult to control the orientation through a relatively small window in the shell. In this protocol we demonstrate an ex ovo electroporation method for gene transfer into chicken embryos at late stages (older than stage HH22). For ex ovo electroporation, embryos are cultured in Petri dishes(9) and the vitelline and allantoic vessels are widely spread. Under these conditions, the older chicken embryos are easily accessed and manipulated. Therefore, this method overcomes the disadvantages of in ovo electroporation applied to the older chicken embryos. Using this method, plasmids can be easily transfected into different parts of the older chicken embryos(10-12).

  14. Impairment of preimplantation porcine embryo development by histone demethylase KDM5B knockdown through disturbance of bivalent H3K4me3-H3K27me3 modifications.

    PubMed

    Huang, Jiaojiao; Zhang, Hongyong; Wang, Xianlong; Dobbs, Kyle B; Yao, Jing; Qin, Guosong; Whitworth, Kristin; Walters, Eric M; Prather, Randall S; Zhao, Jianguo

    2015-03-01

    KDM5B (JARID1B/PLU1) is a H3K4me2/3 histone demethylase that is implicated in cancer development and proliferation and is also indispensable for embryonic stem cell self-renewal, cell fate, and murine embryonic development. However, little is known about the role of KDM5B during preimplantation embryo development. Here we show that KDM5B is critical to porcine preimplantation development. KDM5B was found to be expressed in a stage-specific manner, consistent with demethylation of H3K4me3, with the highest expression being observed from the 4-cell to the blastocyst stages. Knockdown of KDM5B by morpholino antisense oligonucleotides injection impaired porcine embryo development to the blastocyst stage. The impairment of embryo development might be caused by increased expression of H3K4me3 at the 4-cell and blastocyst stages, which disturbs the balance of bivalent H3K4me3-H3K27me3 modifications at the blastocyst stage. Decreased abundance of H3K27me3 at blastocyst stage activates multiple members of homeobox genes (HOX), which need to be silenced for faithful embryo development. Additionally, the histone demethylase KDM6A was found to be upregulated by knockdown of KDM5B, which indicated it was responsible for the decreased abundance of H3K27me3 at the blastocyst stage. The transcriptional levels of Ten-Eleven Translocation gene family members (TET1, TET2, and TET3) are found to be increased by knockdown of KDM5B, which indicates cross talk between histone modifications and DNA methylation. The studies above indicate that KDM5B is required for porcine embryo development through regulating the balance of bivalent H3K4me3-H3K27me3 modifications.

  15. Asymmetries in Cell Division, Cell Size, and Furrowing in the Xenopus laevis Embryo.

    PubMed

    Tassan, Jean-Pierre; Wühr, Martin; Hatte, Guillaume; Kubiak, Jacek

    2017-01-01

    Asymmetric cell divisions produce two daughter cells with distinct fate. During embryogenesis, this mechanism is fundamental to build tissues and organs because it generates cell diversity. In adults, it remains crucial to maintain stem cells. The enthusiasm for asymmetric cell division is not only motivated by the beauty of the mechanism and the fundamental questions it raises, but has also very pragmatic reasons. Indeed, misregulation of asymmetric cell divisions is believed to have dramatic consequences potentially leading to pathogenesis such as cancers. In diverse model organisms, asymmetric cell divisions result in two daughter cells, which differ not only by their fate but also in size. This is the case for the early Xenopus laevis embryo, in which the two first embryonic divisions are perpendicular to each other and generate two pairs of blastomeres, which usually differ in size: one pair of blastomeres is smaller than the other. Small blastomeres will produce embryonic dorsal structures, whereas the larger pair will evolve into ventral structures. Here, we present a speculative model on the origin of the asymmetry of this cell division in the Xenopus embryo. We also discuss the apparently coincident asymmetric distribution of cell fate determinants and cell-size asymmetry of the 4-cell stage embryo. Finally, we discuss the asymmetric furrowing during epithelial cell cytokinesis occurring later during Xenopus laevis embryo development.

  16. Amino acid starvation induced by protease inhibition produces differential alterations in redox status and the thiol proteome in organogenesis-stage rat embryos and visceral yolk sacs.

    PubMed

    Harris, Craig; Jilek, Joseph L; Sant, Karilyn E; Pohl, Jan; Reed, Matthew; Hansen, Jason M

    2015-12-01

    The process of embryonic nutrition in rodent conceptuses during organogenesis has been shown to involve a dominant histiotrophic mechanism where essential developmental substrates and micronutrients are supplied as whole maternal proteins or cargoes associated with proteins. The histiotrophic nutrition pathways (HNP) responsible for uptake and initial processing of proteins across maternal-conceptal interfaces involve uptake via receptor mediated endocytosis and protein degradation via lysosomal proteolysis. Chemical inhibition of either process can lead to growth deficits and malformation in the embryo (EMB), but selective inhibition of either HNP component will elicit a different subset of developmental perturbations. In vitro, whole embryo culture exposure of GD10 or GD11 rat conceptuses to the natural protease inhibitor, leupeptin, leads to significant reductions in all measured embryonic growth parameters as well as a myriad of other effects. Leupeptin doses of 10 μM or 20 μM over a 26-h period (GD10-GD11) and 50 μM over a 3 h pulse period produced significant decreases in the clearance of FITC-albumin from culture media. The near complete loss of acid soluble fluorescence and increased total visceral yolk sac (VYS) protein content confirmed the selective inhibition of proteolysis. Inhibition of lysosomal proteolysis thus deprives the developing EMB of essential nutrient amino acids producing conditions akin to amino acid starvation, but may also cause direct effects on pathways critical for normal growth and differentiation. Following leupeptin exposure for 26 or 6 h, total glutathione (GSH) concentrations dropped significantly in the VYS, but only slightly in yolk sac (YSF) and amniotic (AF) fluids. Cys concentrations increased in VYS and EMB, but dropped in YSF and AF fluids. Redox potentials (Eh) for the glutathione disulfide (GSSG)/glutathione (GSH) redox couple trended significantly toward the positive, confirming the net oxidation of conceptual

  17. Amino Acid Starvation Induced by Protease Inhibition Produces Differential Alterations in Redox Status and the Thiol Proteome in Organogenesis-Stage Rat Embryos and Visceral Yolk Sacs

    PubMed Central

    Harris, Craig; Jilek, Joseph L.; Sant, Karilyn E.; Pohl, Jan; Reed, Matthew; Hansen, Jason M.

    2015-01-01

    The process of embryonic nutrition in rodent conceptuses during organogenesis has been shown to involve a dominant histiotrophic mechanism where essential developmental substrates and micronutrients are supplied as whole maternal proteins or cargoes associated with proteins. The histiotrophic nutrition pathways (HNP) responsible for uptake and initial processing of proteins across maternal-conceptal interfaces involve uptake via receptor mediated endocytosis and protein degradation via lysosomal proteolysis. Chemical inhibition of either process can lead to growth deficits and malformation in the embryo (EMB), but selective inhibition of either HNP component will elicit a different subset of developmental perturbations. In vitro, whole embryo culture (WEC) exposure of GD10 or GD11 rat conceptuses to the natural protease inhibitor, leupeptin, leads to significant reductions in all measured embryonic growth parameters as well as a myriad of other effects. Leupeptin doses of 10 μM or 20 μM over a 26 hr period (GD10-GD11) and 50 μM over a 3 hr pulse period produced significant decreases in the clearance of FITC-albumin from culture media. The near complete loss of acid soluble fluorescence and increased total visceral yolk sac (VYS) protein content confirmed the selective inhibition of proteolysis. Inhibition of lysosomal proteolysis thus deprives the developing EMB of essential nutrient amino acids producing conditions akin to amino acid starvation, but may also cause direct effects on pathways critical for normal growth and differentiation. Following leupeptin exposure for 26 or 6 hr, total glutathione (GSH) concentrations dropped significantly in the VYS, but only slightly in yolk sac (YSF) and amniotic (AF) fluids. Cys concentrations increased in VYS and EMB, but dropped in YSF and AF fluids. Redox potentials (Eh) for the GSSG/GSH redox couple trended significantly toward the positive, confirming the net oxidation of conceptual tissues following leupeptin

  18. Factors associated with discordance between absolute CD4 cell count and CD4 cell percentage in patients coinfected with HIV and hepatitis C virus.

    PubMed

    Hull, Mark W; Rollet, Kathleen; Odueyungbo, Adefowope; Saeed, Sahar; Potter, Martin; Cox, Joseph; Cooper, Curtis; Gill, John; Klein, Marina B

    2012-06-01

    Liver cirrhosis has been associated with decreased absolute CD4 cell counts but preserved CD4 cell percentage in human immunodeficiency virus (HIV)-negative persons. We evaluated factors associated with discordance between the absolute CD4 cell count and the CD4 cell percentage in a cohort of patients coinfected with HIV and hepatitis C virus (HCV). Baseline data from 908 participants in a prospective, Canadian, multisite cohort of individuals with HIV-HCV coinfection were analyzed. Absolute CD4 cell count and CD4 cell percentage relationships were evaluated. We defined low and high discordance between absolute CD4 cell count/CD4 cell percentage relationships as CD4 cell percentages that differed from the expected CD4 cell percentage, given the observed absolute CD4 cell count, by ±7 percentage points; we defined very low and very high discordance as differences of ±14 percentage points. Factors associated with high or very high discordance, including either end-stage liver disease or aspartate transaminase to platelet ratio index (APRI) of >1.5, were analyzed using multivariate logistic regression models and compared to groups with concordant and low discordant results. High/very high discordance was seen in 31% (n = 286), while 35% (n = 321) had concordant values. Factors associated with very high discordance at baseline included history of end-stage liver disease (adjusted odds ratio [aOR], 6.52; 95% confidence interval [CI], 2.27-18.67) and APRI of >1.5 (aOR 4.69; 95% CI, 1.64-13.35). Compared with those with detectable HCV RNA, those who cleared HCV spontaneously were less likely to have very high discordance. Discordance between absolute CD4 cell count and CD4 cell percentage is common in an HIV/HCV-coinfected population and is associated with advanced liver disease and ongoing HCV replication.

  19. 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

  20. Polyethylene glycol-induced fusion of two-cell mouse embryo blastomeres

    SciTech Connect

    Spindle, A.

    1981-01-01

    Polyethylene glycol (PEG) was found to be an effective fusion-inducing agent for early mouse embryo blastomeres. A brief exposure of zona-intact 2-cell embryos to 40% PEG induced fusion of blastomeres in > 80% of embryos, and the treatment did not interfere with subsequent development of embryos to the blastocyst stage.

  1. Successful transfer of day 10 horse embryos: influence of donor-recipient asynchrony on embryo development.

    PubMed

    Wilsher, Sandra; Clutton-Brock, Amber; Allen, W R

    2010-03-01

    A total of 78 day 10 horse embryos were transferred non-surgically to recipient mares that had ovulated 9, 7, 6, 5, 4, 3, 2 or 1 day after (negative asynchrony), on the same day (synchronous), or 2 or 4 days before (positive asynchrony) the donor (n=6 or 8 mares per group). Pregnancy rates between 100% (6/6) and 63% (5/8) were seen in recipient mares that were between +2 and -6 days asynchronous. Embryo survival to the heartbeat stage declined in recipients that were -7 days asynchronous and no embryos survived in recipients that were -9 days asynchronous. Irrespective of uterine asynchrony, cessation of embryo mobility and fixation at the base of a uterine horn occurred when the conceptus was approximately 17 days old. Conceptus growth and development was slowed when embryos were placed in negatively asynchronous uteri. At the greatest degree of negative asynchrony at which embryos survived to the heartbeat stage, i.e. -7 and -6 days, development of the embryo proper and allantois was retarded. Luteostasis was achieved in recipient mares when day 10 embryos were transferred to recipient mares at any stage of asynchrony between -9 and +2 days with respect to the donor. These results indicate that in the horse, there is a wide window for establishment of pregnancy following embryo transfer to asynchronous recipients. Although progesterone priming of the uterus to a stage equivalent to that of the transferred embryo does not appear to be a prerequisite for embryo survival, it does nonetheless influence embryonic development.

  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. Copyright © 2015 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  3. Glassfrog embryos hatch early after parental desertion.

    PubMed

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

    2014-06-22

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

  4. 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

  5. Effect of zidovudine on preimplantation murine embryos.

    PubMed Central

    Toltzis, P; Mourton, T; Magnuson, T

    1993-01-01

    It previously has been demonstrated that zidovudine (AZT) is lethal to early murine embryos. The effect of the drug on pre- and postimplantation embryos was examined to delineate the timing of this toxicity and to investigate its possible mechanisms. Embryos exposed in the whole mouse during preblastocyst development were unable to proceed beyond the blastocyst stage. Similarly, when two-cell embryos harvested from unexposed females were exposed to low-concentration (1 microM) AZT in vitro over 24 h, development beyond the blastocyst stage was inhibited. In contrast, drug exposure during in vitro blastocyst and postblastocyst development resulted in little or no morphologic toxicity. Further investigation revealed that preblastocyst AZT exposure resulted in the development of blastocysts with significantly lower cell numbers than control embryos. While embryonic exposure to AZT at the blastocyst and postblastocyst stages also resulted in retarded cell division, the effects were milder than those recorded after preblastocyst exposure. These data demonstrate that the critical period of AZT toxicity toward murine embryos is between ovulation and implantation and indicate that AZT directly suppresses cell division in the preimplantation embryo. PMID:8215271

  6. Production of transgenic canine embryos using interspecies somatic cell nuclear transfer.

    PubMed

    Hong, So Gun; Oh, Hyun Ju; Park, Jung Eun; Kim, Min Jung; Kim, Geon A; Koo, Ok Jae; Jang, Goo; Lee, Byeong Chun

    2012-02-01

    Somatic cell nuclear transfer (SCNT) has emerged as an important tool for producing transgenic animals and deriving transgenic embryonic stem cells. The process of SCNT involves fusion of in vitro matured oocytes with somatic cells to make embryos that are transgenic when the nuclear donor somatic cells carry 'foreign' DNA and are clones when all the donor cells are genetically identical. However, in canines, it is difficult to obtain enough mature oocytes for successful SCNT due to the very low efficiency of in vitro oocyte maturation in this species that hinders canine transgenic cloning. One solution is to use oocytes from a different species or even a different genus, such as bovine oocytes, that can be matured easily in vitro. Accordingly, the aim of this study was: (1) to establish a canine fetal fibroblast line transfected with the green fluorescent protein (GFP) gene; and (2) to investigate in vitro embryonic development of canine cloned embryos derived from transgenic and non-transgenic cell lines using bovine in vitro matured oocytes. Canine fetal fibroblasts were transfected with constructs containing the GFP and puromycin resistance genes using FuGENE 6®. Viability levels of these cells were determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. Interspecies SCNT (iSCNT) embryos from normal or transfected cells were produced and cultured in vitro. The MTT measurement of GFP-transfected fetal fibroblasts (mean OD = 0.25) was not significantly different from non-transfected fetal fibroblasts (mean OD = 0.35). There was no difference between transgenic iSCNT versus non-transgenic iSCNT embryos in terms of fusion rates (73.1% and 75.7%, respectively), cleavage rates (69.7% vs. 73.8%) and development to the 8-16-cell stage (40.1% vs. 42.7%). Embryos derived from the transfected cells completely expressed GFP at the 2-cell, 4-cell, and 8-16-cell stages without mosaicism. In summary, our results demonstrated that

  7. Rho-kinase in sea urchin eggs and embryos.

    PubMed

    Aguirre-Armenta, Beatriz; López-Godínez, Juana; Martínez-Cadena, Guadalupe; García-Soto, Jesús

    2011-06-01

    The activation of sea urchin eggs at fertilization provides an ideal system for studying the molecular events involved in cellular activation. Rho GTPases, which are key signaling enzymes in eukaryotes, are involved in sustaining the activation of sea urchin eggs; however, their downstream effectors have not yet been characterized. In somatic cells, RhoA regulates a serine/threonine kinase known as Rho-kinase (ROCK). The activity of ROCK in early sea urchin development has been inferred, but not tested directly. A ROCK gene was identified in the sea urchin (Strongylocentrotus purpuratus) genome and the sequence of its cDNA determined. The sea urchin ROCK (SpROCK) sequence predicts a protein of 158 kDa with >72% and 45% identities with different protein orthologues of the kinase catalytic domain and the complete protein sequence, respectively. SpROCK mRNA levels are high in unfertilized eggs and decrease to 35% after 15 min postfertilization and remain low up to the 4 cell stage. Antibodies to the human ROCK-I kinase domain revealed SpROCK to be concentrated in the cortex of eggs and early embryos. Co-immunoprecipitation assays indicate that RhoA and SpROCK are physically associated. This association is destroyed by treatment with the C3 exoenzyme and with the ROCK antagonist H-1152. H-1152 also inhibited DNA synthesis in embryos. We conclude that the Rho-dependent signaling pathway, via SpROCK, is essential for early embryonic development.

  8. Refrigeration of rainbow trout gametes and embryos.

    PubMed

    Babiak, Igor; Dabrowski, Konrad

    2003-12-01

    Prolonged access to early embryos composed of undifferentiated, totipotent blastomeres is desirable in situations when multiple collections of gametes are not possible. The objective of the present study is to examine whether the refrigeration of rainbow trout Oncorhynchus mykiss gametes and early embryos would be a suitable, reliable, and efficient tool for prolonging the availability of early developmental stages up to the advanced blastula stage. The study was conducted continuously during fall, winter, and spring spawning seasons. In all, more than 500 experimental variants were performed involving individual samples from 26 females and 33 males derived from three strains. These strains represented three possible circumstances. In optimal one, gametes from good quality donors were obtained soon after ovulation. In the two non-optimal sources, either donors were of poor genetic quality or gametes were collected from a distant location and transported as unfertilized gametes. A highly significant effect of variability of individual sample quality on efficiency of gamete and embryo refrigeration was revealed. The source of gametes significantly affected viability of refrigerated oocytes and embryos, but not spermatozoa. On average, oocytes from optimal source retained full fertilization viability for seven days of chilled storage, significantly longer than from non-optimal sources. Spermatozoa, regardless of storage method, retained full fertilization ability for the first week of storage. Refrigeration of embryos at 1.4+/-0.4 degrees C significantly slowed the development. Two- week-old embryos were still in blastula stage. Average survival rate of embryos refrigerated for 10 days and then transferred to regular incubation temperatures of 9-14 degrees C was 92% in optimal and 51 and 71% in non-optimal source variants. No effect of gamete and embryo refrigeration on the occurrence of developmental abnormalities was observed. Cumulative refrigeration of oocytes and

  9. Development of interspecies cloned embryos in yak and dog.

    PubMed

    Murakami, Masao; Otoi, Takeshige; Wongsrikeao, Pimprapar; Agung, Budiyanto; Sambuu, Rentsenkhand; Suzuki, Tatsuyuki

    2005-01-01

    Interspecies nuclear transfer (NT) could be an alternative to replicate animals when supply of recipient oocytes is limited or in vitro embryo production systems are incomplete. In the present study, embryonic development was assessed following interspecies NT of donor cumulus cells derived from yak and dog into the recipient ooplasm of domestic cow. The percentages of fusion and subsequent embryo development to the eight-cell stage of interspecies NT embryos were comparable to those of intraspecies NT embryos (cow-cow NT embryos). The percentage of development to blastocysts was significantly lower (p < 0.05) in yak-cow NT embryos than that in cow-cow NT embryos (10.9% vs. 39.8%). In dog-cow NT embryos, only one embryo (0.4%) developed to the blastocyst stage. These results indicate that interspecies NT embryos possess equally developmental competence to the eight-cell stage as intraspecies NT embryos, but the development to blastocysts is very low when dog somatic cells are used as the donor nuclei.

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

    PubMed

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

    2014-01-01

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

  11. RNA Profiles of Porcine Embryos during Genome Activation Reveal Complex Metabolic Switch Sensitive to In Vitro Conditions

    PubMed Central

    Østrup, Olga; Olbricht, Gayla; Østrup, Esben; Hyttel, Poul; Collas, Philippe; Cabot, Ryan

    2013-01-01

    Fertilization is followed by complex changes in cytoplasmic composition and extensive chromatin reprogramming which results in the abundant activation of totipotent embryonic genome at embryonic genome activation (EGA). While chromatin reprogramming has been widely studied in several species, only a handful of reports characterize changing transcriptome profiles and resulting metabolic changes in cleavage stage embryos. The aims of the current study were to investigate RNA profiles of in vivo developed (ivv) and in vitro produced (ivt) porcine embryos before (2-cell stage) and after (late 4-cell stage) EGA and determine major metabolic changes that regulate totipotency. The period before EGA was dominated by transcripts responsible for cell cycle regulation, mitosis, RNA translation and processing (including ribosomal machinery), protein catabolism, and chromatin remodelling. Following EGA an increase in the abundance of transcripts involved in transcription, translation, DNA metabolism, histone and chromatin modification, as well as protein catabolism was detected. The further analysis of members of overlapping GO terms revealed that despite that comparable cellular processes are taking place before and after EGA (RNA splicing, protein catabolism), different metabolic pathways are involved. This strongly suggests that a complex metabolic switch accompanies EGA. In vitro conditions significantly altered RNA profiles before EGA, and the character of these changes indicates that they originate from oocyte and are imposed either before oocyte aspiration or during in vitro maturation. IVT embryos have altered content of apoptotic factors, cell cycle regulation factors and spindle components, and transcription factors, which all may contribute to reduced developmental competence of embryos produced in vitro. Overall, our data are in good accordance with previously published, genome-wide profiling data in other species. Moreover, comparison with mouse and human embryos

  12. 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.

  13. Expression of Apoptotic and Antioxidant Enzyme Genes in Sheep Oocytes and In Vitro Produced Embryos.

    PubMed

    Mishra, Ashish; Reddy, Ippala Janardhan; Gupta, Paluru Subramanyam Parameswara; Mondal, Sukanta

    2017-01-02

    The present study was to find out the expression pattern and relative expression level of apoptotic (Bcl2, Bax, Casp3, and PCNA) and antioxidant enzyme [(GPx, Cu/Zn-SOD (SOD1) and Mn-SOD (SOD2)] genes in sheep oocytes and developing embryos produced in vitro by conventional RT-PCR and real time qPCR, respectively. Different developmental stages of embryos were produced in vitro from oocytes collected from local slaughter house ovaries. RT-PCR amplicons showed expression of Bcl2 and PCNA in all stages except at morula. In contrast Bax and Casp3 were expressed in all stages. GPx and SOD1 were expressed in all stages but SOD2 was not expressed in 8-16 cells, although expressed in the remaining stages. The qPCR analysis reflected that Bcl2 expression was significantly (P < 0.05) downregulated in morula and maximum upregulated expression was observed in in vitro matured oocytes. Higher upregulated expression (P < 0.05) of Bax was in morula and downregulated expression was at 2-4 cells. Casp3 was significantly upregulated at 8-16 cells and downregulated in in vitro matured oocyte. PCNA expression was highest at blastocyst and least expression was at morula. GPx was expressed significantly highest in matured oocytes and least expression was at zygote. SOD1 was expressed significantly highest at 8-16 cells and least expression was at zygote. Expression of SOD2 was least among all the antioxidant enzymes but significantly higher expression of SOD2 was in immature oocyte; however, least expression was at 8-16 cells. It can be concluded from the study that the sheep embryos produced in vitro are highly sensitive to culture condition, which alters the expression level of apoptotic and antioxidant enzyme genes.

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

    PubMed

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

    2009-09-01

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

  15. 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

  16. Following the course of pre-implantation embryo patterning by non-linear microscopy.

    PubMed

    Kyvelidou, Christiana; Tserevelakis, George J; Filippidis, George; Ranella, Anthi; Kleovoulou, Anastasia; Fotakis, Costas; Athanassakis, Irene

    2011-12-01

    Embryo patterning is subject to intense investigation. So far only large, microscopically obvious structures like polar body, cleavage furrow, pro-nucleus shape can be evaluated in the intact embryo. Using non-linear microscopic techniques, the present work describes new methodologies to evaluate pre-implantation mouse embryo patterning. Third Harmonic Generation (THG) imaging, by detecting mitochondrial/lipid body structures, could provide valuable and complementary information as to the energetic status of pre-implantation embryos, time evolution of different developmental stages, embryo polarization prior to mitotic division and blastomere equivalence. Quantification of THG imaging detected highest signalling in the 2-cell stage embryos, while evaluating a 12-18% difference between blastomeres at the 8-cell stage embryos. Such a methodology provides novel, non-intrusive imaging assays to follow up intracellular structural patterning associated with the energetic status of a developing embryo, which could be successfully used for embryo selection during the in vitro fertilization process.

  17. Effect of culture medium volume and embryo density on early mouse embryonic development: tracking the development of the individual embryo.

    PubMed

    Dai, Shan-Jun; Xu, Chang-Long; Wang, Jeffrey; Sun, Ying-Pu; Chian, Ri-Cheng

    2012-07-01

    To determine the optimal volume or density of embryos for the well-of-the-well (WOW) system in order to track the development of individual embryos and to determine whether the WOW system can reverse the negative impact of culturing embryos singly. (1) Mouse embryos (groups of nine at the 2-cell stage) were cultured in 6.25 μl, 12.50 μl, 25.00 μl and 50.00 μl of droplets of culture medium under paraffin oil; (2) Groups of three, six, nine and twelve embryos at the 2-cell stage were cultured in 50 μl of droplet of culture medium under paraffin oil; (3) Groups of nine embryos at the 2-cell stage were cultured in 50 μl of droplet under paraffin oil with or without nine micro-wells made on the bottom of the Petri dish into each of which were placed one of the nine embryos (WOW system). Also single 2-cell stage embryos was cultured individually in 5.5 μl of droplet of culture medium under paraffin oil with or without a single micro-well made on the bottom of the Petri dish (WOW system for single culture). At the end of culture, the percentages of blastocyst development, hatching and hatched blastocysts were compared in each group. The blastocysts were fixed for differential staining. The blastocyst development was significantly higher (P < 0.05) when nine embryos were cultured in 50 μl of droplet of culture medium compared with other volumes. The blastocyst development was significantly reduced (P < 0.05) in single embryo culture compared to group embryo culture with or without the WOW system. The blastocyst development was not improved when single embryo cultured individually in a micro-well was compared to single embryo cultured individually without micro-well. The total cell numbers of blastocysts were significantly higher in group embryo culture than single embryo culture regardless of whether the WOW system was used. In addition, the total cell numbers of blastocysts were significantly higher (P < 0.05) in single embryo culture with the WOW

  18. Determination of the expression pattern of the dual promoter of zebrafish fushi tarazu factor-1a following microinjections into zebrafish one cell stage embryos.

    PubMed

    von Hofsten, J; Modig, C; Larsson, A; Karlsson, J; Olsson, P-E

    2005-05-15

    The zebrafish fushi tarazu factor-1a (ff1a) is a transcription factor belonging to the NR5A subgroup of nuclear receptors. The NR5A receptors bind DNA as monomers and are considered to be orphans due to their ability to promote transcription of downstream genes without ligands. In zebrafish, four ff1 homologues (Ff1a, Ff1b, Ff1c, and Ff1d) have been identified so far. The gene coding for Ff1a is driven by two separate promoters, and give rise to four splice variants. Ff1a is expressed in the somites and pronephric ducts during somitogenesis and in the brain, liver, and mandibular arch during later embryonic stages. In adults the gene is highly expressed in gonads, liver, and intestine, but can be detected in most tissues. The broad variety of embryonic expression domains indicates several important developmental features. One of the mammalian fushi tarazu factor-1 genes, steroidogenic factor-1 (SF-1), is essential for the development of gonads and adrenals. SF-1 is together with Sox9, WT1, and GATA4 a positive transcriptional regulator of human anti-mullerian hormone (AMH) and thereby linked to the male sex-determining pathway. The zebrafish ff1a dual promoter contains several GATA binding sites and E-boxes, a site for DR4, XFD2, MyoD, Snail, HNF3, S8, and an HMG-box recognition site for Sox9. In a first attempt to dissect the ff1a promoter in vivo we have produced first generation transgenes in order to determine the correlation between the expression of the endogenous ff1a gene and the microinjected ff1a dual promoter coupled to the pEGFP reporter vector. Our results show that the microinjected constructs are expressed in the correct tissues.

  19. 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

  20. 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.

  1. Miniaturized Embryo Array for Automated Trapping, Immobilization and Microperfusion of Zebrafish Embryos

    PubMed Central

    Akagi, Jin; Khoshmanesh, Khashayar; Evans, Barbara; Hall, Chris J.; Crosier, Kathryn E.; Cooper, Jonathan M.; Crosier, Philip S.; Wlodkowic, Donald

    2012-01-01

    Zebrafish (Danio rerio) has recently emerged as a powerful experimental model in drug discovery and environmental toxicology. Drug discovery screens performed on zebrafish embryos mirror with a high level of accuracy the tests usually performed on mammalian animal models, and fish embryo toxicity assay (FET) is one of the most promising alternative approaches to acute ecotoxicity testing with adult fish. Notwithstanding this, automated in-situ analysis of zebrafish embryos is still deeply in its infancy. This is mostly due to the inherent limitations of conventional techniques and the fact that metazoan organisms are not easily susceptible to laboratory automation. In this work, we describe the development of an innovative miniaturized chip-based device for the in-situ analysis of zebrafish embryos. We present evidence that automatic, hydrodynamic positioning, trapping and long-term immobilization of single embryos inside the microfluidic chips can be combined with time-lapse imaging to provide real-time developmental analysis. Our platform, fabricated using biocompatible polymer molding technology, enables rapid trapping of embryos in low shear stress zones, uniform drug microperfusion and high-resolution imaging without the need of manual embryo handling at various developmental stages. The device provides a highly controllable fluidic microenvironment and post-analysis eleuthero-embryo stage recovery. Throughout the incubation, the position of individual embryos is registered. Importantly, we also for first time show that microfluidic embryo array technology can be effectively used for the analysis of anti-angiogenic compounds using transgenic zebrafish line (fli1a:EGFP). The work provides a new rationale for rapid and automated manipulation and analysis of developing zebrafish embryos at a large scale. PMID:22606275

  2. Miniaturized embryo array for automated trapping, immobilization and microperfusion of zebrafish embryos.

    PubMed

    Akagi, Jin; Khoshmanesh, Khashayar; Evans, Barbara; Hall, Chris J; Crosier, Kathryn E; Cooper, Jonathan M; Crosier, Philip S; Wlodkowic, Donald

    2012-01-01

    Zebrafish (Danio rerio) has recently emerged as a powerful experimental model in drug discovery and environmental toxicology. Drug discovery screens performed on zebrafish embryos mirror with a high level of accuracy the tests usually performed on mammalian animal models, and fish embryo toxicity assay (FET) is one of the most promising alternative approaches to acute ecotoxicity testing with adult fish. Notwithstanding this, automated in-situ analysis of zebrafish embryos is still deeply in its infancy. This is mostly due to the inherent limitations of conventional techniques and the fact that metazoan organisms are not easily susceptible to laboratory automation. In this work, we describe the development of an innovative miniaturized chip-based device for the in-situ analysis of zebrafish embryos. We present evidence that automatic, hydrodynamic positioning, trapping and long-term immobilization of single embryos inside the microfluidic chips can be combined with time-lapse imaging to provide real-time developmental analysis. Our platform, fabricated using biocompatible polymer molding technology, enables rapid trapping of embryos in low shear stress zones, uniform drug microperfusion and high-resolution imaging without the need of manual embryo handling at various developmental stages. The device provides a highly controllable fluidic microenvironment and post-analysis eleuthero-embryo stage recovery. Throughout the incubation, the position of individual embryos is registered. Importantly, we also for first time show that microfluidic embryo array technology can be effectively used for the analysis of anti-angiogenic compounds using transgenic zebrafish line (fli1a:EGFP). The work provides a new rationale for rapid and automated manipulation and analysis of developing zebrafish embryos at a large scale.

  3. Multiple-embryo transfer for studying very early maternal-embryo interactions in cattle.

    PubMed

    Gómez, E; Muñoz, M

    2015-08-01

    In the present paper, we highlight the need to study very early maternal-embryo interactions and discuss how these interactions can be addressed. Bovine species normally carry one or, less frequently, two embryos to term; there are very rare cases of triplets or higher-order multiple pregnancies in which all the offspring are born alive. Multiple-embryo transfer (MET) in cattle allows for the detection of endometrial responses in scenarios where single-embryo transfer would not. Although MET is non-physiological, the present study shows that at the very early embryonic stages, a uterus carrying zona-enclosed embryos does not exhibit non-physiological reactions. On the contrary, MET should be considered the sum of multiple individual effects triggered by developing embryos. We provide arguments to support our hypothesis that describe a rationale for current work with MET, and we discuss alternative hypotheses. Using cattle as a model, we describe how technical approaches to analyzing zona-enclosed early embryo-maternal interactions (i.e., transcriptomics, proteomics, and endometrial cell culture) can help identify molecular changes that may be difficult to observe when only a single embryo is present. We conclude that MET can be used for studying very early maternal-embryo interactions in vivo in monotocous species. Free Spanish abstract: A Spanish translation of this abstract is freely available at http://www.reproduction-online.org/content/150/2/R35/suppl/DC1.

  4. Feasibility of cryopreservation of zebrafish (Danio rerio) primordial germ cells by whole embryo freezing.

    PubMed

    Higaki, Shogo; Mochizuki, Kentaro; Baba, Hiroko; Akashi, Yuichiro; Yamaha, Etsuro; Katagiri, Seiji; Takahashi, Yoshiyuki

    2009-08-01

    We investigated the feasibility of cryopreservation of zebrafish (Danio rerio) blastomeres and primordial germ cells (PGCs) by rapid freezing of dechorionated whole embryos at the blastula, gastrula and segmentation stages. Initially we examined the glass-forming properties and embryo toxicities of 5 cryoprotectants: methanol (MeOH), ethylene glycol (EG), dimethyl sulfoxide (DMSO), propylene glycol (PG), and 1,3-butylene glycol (1,3-BG). Embryos at the blastula and gastrula stages had high sensitivities to cryoprotectant toxicities and were fragile against mechanical damage. Thus the segmentation stage embryos, the PGCs of which were visualized by injecting green fluorescence protein-nos1 3'UTR mRNA, were frozen using solutions containing each cryoprotectant at 6 M (first trial) and 2 types of cryoprotectants at 3 M each (second trial). In the first trial, live PGCs were recovered from most of the embryos frozen with EG (about 2 cells/embryo); however, a few embryos had live PGCs when embryos were frozen with other cryoprotectants. In the second trial, a mixture of EG + PG better preserved the viability of PGCs in frozen embryos. Live PGCs were recovered from all embryos frozen with EG + PG (about 3 cells/embryo), and the survival rate of PGCs was estimated to be about 25% based on the number of live PGCs in fresh embryos (about 12 cells/embryo). The present study indicates that we can utilize rapid freezing of dechorionated whole embryos at the segmentation stage for the cryopreservation of PGCs.

  5. Association between Number of Formed Embryos, Embryo Morphology and Clinical Pregnancy Rate after Intracytoplasmic Sperm Injection.

    PubMed

    Luz, Caroline Mantovani da; Giorgi, Vanessa Silvestre Innocenti; Coelho Neto, Marcela Alencar; Martins, Wellington de Paula; Ferriani, Rui Alberto; Navarro, Paula Andrea

    2016-09-01

    Introduction Infertility has a high prevalence in the general population, affecting ∼ 5 to 15% of couples in reproductive age. The assisted reproduction techniques (ART) include in vitro manipulation of gametes and embryos and are an important treatment indicated to these couples. It is well accepted that the implantation rate is positively influenced by the morphology of transferred embryos. However, we question if, apart from the assessment of embryo morphology, the number of produced embryos per cycle is also related to pregnancy rates in the first fresh transfer cycle. Purpose To evaluate the clinical pregnancy rate according to the number of formed embryos and the transfer of top quality embryos (TQEs). Methods In a retrospective cohort study, between January 2011 and December 2012, we evaluated women who underwent intracytoplasmic sperm injection (ICSI), aged < 40 years, and with at least 1 formed embryo fresh transferred in cleavage stage. These women were stratified into 3 groups according to the number of formed embryos (1 embryo, 2-3 and ≥ 4 embryos). Each group was divided into 2 subgroups according to the presence or not of at least 1 transferred TQE (1 with TQE; 1 without TQE; 2-3 with TQE, 2-3 without TQE; ≥ 4 with TQE; ≥ 4 without TQE). The clinical pregnancy rates were compared in each subgroup based on the presence or absence of at least one transferred TQE. Results During the study period, 636 women had at least one embryo to be transferred in the first fresh cycle (17.8% had 1 formed embryo [32.7% with TQE versus 67.3% without TQE], 42.1% of women had 2-3 formed embryos [55.6% with TQE versus 44.4% without TQE], and 40.1% of patients had ≥ 4 formed embryos [73.7% with TQE versus 26.3% without TQE]). The clinical pregnancy rate was significantly higher in the subgroup with ≥ 4 formed embryos with at least 1 transfered TQE (45.2%) compared with the subgroup without TQE (28.4%). Conclusions Having at

  6. In vitro production of Sudanese camel (Camelus dromedarius) embryos from epididymal spermatozoa and follicular oocytes of slaughtered animals.

    PubMed

    Abdelkhalek, A E; Gabr, Sh A; Khalil, W A; Shamiah, Sh M; Pan, L; Qin, G; Farouk, M H

    2017-03-28

    Application of assisted reproductive technology in camelidea, such as artificial insemination (AI) and embryo transfer, has been slow in comparison to that for other livestock species. In Egypt, there are few attempts to establish in vitro maturation (IVM) and fertilization (IVF) techniques in dromedary camel. The present study was carried out to produce Sudanese camel embryos using in vitro matured oocytes and epididymal spermatozoa. Dromedary camel ovaries were collected from abattoirs and then, the oocytes were aspirated from all the visible follicles on the ovarian surface (~2-8 mm in a diameter). Meanwhile, Fetal Dromedary Camel Serum (FDCS) was obtained from camel fetuses after slaughtering. Thereafter, only Cumulus Oocyte Complexes (COCs) were matured in vitro in the Tissue Culture Medium (TCM-199) complemented with 10% FDCS. Spermatozoa required for in vitro fertilization were collected from testes (epididymal cauda) of the slaughtered camel bulls. The results clearly showed that the maturation rate of oocytes at metaphase II was about 59.5% while the fertilization rate was around 70.4%. Intriguingly, the embryo rates determined were 13.1%, in 2-cell; 0.0%, in 4-cell; 34.7%, in 8-16% cell; 39.1%, in morula and 13.1% in a blastocyst stage. This study represented a successful in vitro production of Sudanese dromedary camel embryos from epididymal sperm cells and in vitro matured oocytes recovered from slaughtered camels.

  7. Optimized CUBIC protocol for three-dimensional imaging of chicken embryos at single-cell resolution.

    PubMed

    Gómez-Gaviro, María Victoria; Balaban, Evan; Bocancea, Diana; Lorrio, María Teresa; Pompeiano, Maria; Desco, Manuel; Ripoll, Jorge; Vaquero, Juan José

    2017-06-01

    The CUBIC tissue-clearing protocol has been optimized to produce translucent immunostained whole chicken embryos and embryo brains. When combined with multispectral light-sheet microscopy, the validated protocol presented here provides a rapid, inexpensive and reliable method for acquiring accurate histological images that preserve three-dimensional structural relationships with single-cell resolution in whole early-stage chicken embryos and in the whole brains of late-stage embryos. © 2017. Published by The Company of Biologists Ltd.

  8. In vitro culture of embryos of the guppy, Poecilia reticulata.

    PubMed

    Martyn, Ulrike; Weigel, Detlef; Dreyer, Christine

    2006-03-01

    The rich variation in adult color patterns of male guppies (Poecilia reticulata) has attracted the attention of geneticists and ecologists for almost a century. Studies on their embryogenesis, however, have been limited by the fact that guppies are live bearers. We have observed normal development after explantation of guppy embryos from the ovary of pregnant females at various times after last parturition, and found that development of each batch of eggs is slightly asynchronous, most likely due to asynchronous fertilization. We have cultured explanted embryos in vitro and continuously observed their development. Although embryos explanted a few days after fertilization survived up to 4 weeks in culture, they did not complete their development. In contrast, embryos explanted at late stages of gestation could hatch and develop to fertile adults. Our embryo culture techniques overcome some of the limitations of using livebearers as study objects, and they allow continuous observation of and accessibility to live embryos at all stages.

  9. 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.

  10. 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.

  11. 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.

  12. Inoculation of somatic embryos of sweet potato with an arbuscular mycorrhizal fungus improves embryo survival and plantlet formation.

    PubMed

    Bressan, W; de Carvalho, C H; Sylvia, D M

    2000-08-01

    Responses of somatic embryos of sweet potato (Ipomoea batata (L.) Poir., cv. White Star) at different developmental stages to in vitro inoculation with Glomus etunicatum (Becker and Gerdemann) (isolate INVAM FL329) were evaluated. Somatic embryos were grown in glass tubes containing sterilized vermiculite and sand. A layer of natrosol plus White's medium was used as a carrier for arbuscular mycorrhizal (AM) fungal spores. Survival of embryos inoculated with AM fungi was significantly (P < 0.05) greater than that of noninoculated embryos at the rooted-cotyledonary-torpedo and rooted-elongated-torpedo developmental stages. Mycorrhizae significantly (P < 0.05) increased plantlet formation only when inoculation occurred at the rooted-elongated-torpedo developmental stage. The growth stage at which the embryos were inserted into the glass tubes exerted a significant influence upon plantlet formation, and plantlet formation was further enhanced by inoculation with G. etunicatum. Plantlet formation was greatest at the rooted-elongated-torpedo stage. These results demonstrate that inoculation of somatic embryos with AM fungi improves embryo survival and plantlet formation, and could enhance use of somatic embryos as synthetic seeds.

  13. Steroidal alkaloid toxicity to fish embryos.

    PubMed

    Crawford, L; Kocan, R M

    1993-02-01

    Embryos of two species of fish were evaluated for their suitability as model systems for steroidal alkaloid toxicity, the Japanese rice fish, medaka (Oryzius latipes) and the rainbow trout (Oncorhynchus mykiss). Additionally, the equine neurotoxic sesquiterpene lactone repin, was also tested. A PROBIT program was used to evaluate the EC1, EC50 and EC99 as well as the associated confidence limits. The steroidal alkaloids tested were the Solanum potato glycoalkaloids alpha-chaconine, alpha-solanine, the aglyclones solanidine and solasodine and the Veratrum alkaloid, jervine. Embryo mortality, likely due to structural or functional abnormalities in the early development stages of the embryo, were the only response observed in both species. The rainbow trout exhibited a toxic response to chaconine, solasidine, repin and solanine but the medaka embryos were only affected by the compounds, chaconine and solanine. Rainbow trout may indeed serve as a good lower vertebrate model for studying the toxicity of steroidal alkaloids.

  14. 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.

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

    SciTech Connect

    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. -- Highlights: •Arabidopsis SED1 is essential for embryo development. •The sed1 embryo accumulates less storage and has abnormal ultrastructure. •SED1 localizes to the mitochondrion.

  16. 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

  17. 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

  18. ROCK inhibition prevents early mouse embryo development.

    PubMed

    Duan, Xing; Chen, Kun-Lin; Zhang, Yu; Cui, Xiang-Shun; Kim, Nam-Hyung; Sun, Shao-Chen

    2014-08-01

    ROCK is a Rho-GTPase effector that is important for actin assembly and is involved in various cellular functions, including cell contraction, migration, motility, and tumor cell invasion. In this study, we investigated ROCK expression and function during early mouse embryo development. Inhibiting ROCK by Y-27632 treatment at the zygote stage resulted in first cleavage failure, and most embryos failed to develop to the 8-cell stage. When adding Y-27632 at the 8-cell stage, embryos failed to undergo compaction and could not develop into blastocysts. In addition, fluorescence staining intensity analysis indicated that actin expression at blastomere membranes was significantly reduced. After ROCK inhibition, two or more nuclei were observed in a cell, which indicated possible cytokinesis failure. Moreover, after ROCK inhibition with Y-27632, the phosphorylation levels of LIMK1/2, a downstream molecule of ROCK, were decreased at blastomere membranes. Thus, our results showed conserved roles for ROCK in this mammalian embryo model and indicated that a ROCK-LIMK1/2-actin pathway might regulate cleavage and blastocyst formation during early mouse embryo development.

  19. Microspore-derived embryos from Quercus suber anthers mimic zygotic embryos and maintain haploidy in long-term anther culture.

    PubMed

    Bueno, Maria A; Gomez, Arancha; Sepulveda, Federico; Seguí, José M; Testillano, Pilar S; Manzanera, José A; Risueño, Maria-Carmen

    2003-08-01

    Microspore-derived embryos produced from cork oak anther cultures after long-term incubations (up to 10-12 months) were analysed in order to determine the genetic variability and ploidy level stability, as well as morphology, developmental pattern and cellular organisation. Most of the embryos from long-term anther cultures were haploid (90.7%), corresponding to their microspore origin. The presence of a low percentage of diploid embryos (7.4%) was observed. Microsatellite analysis of haploid embryos, indicated different microspores origins of the same anther. In the diploid embryos, homozygosity for different alleles was detected from anther wall tissues, excluding the possibility of clonal origin. The maintenance of a high proportion of haploid embryos, in long-term anther cultures, is similar in percentage to that reported in embryos originating after 20 days of plating (Bueno et al. 1997). This suggests that no significant alterations in the ploidy level occurred during long incubations (up to 12 months). These results suggest that ploidy changes are rare in this in vitro system, and do not significantly increase during long-term cultures. Microscopical studies of the microspore embryos in various stages revealed a healthy and well developed anatomy with no aberrant or chimeric structures. The general morphology of embryos appearing at different times after plating, looked similar to that of earlier embryos, as well as the zygotic embryos, indicating that they represent high quality material for cork oak breeding.

  20. Assessment of 'one-step' versus 'sequential' embryo culture conditions through embryonic genome methylation and hydroxymethylation changes.

    PubMed

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

    2016-11-01

    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? 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. 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. 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. 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. Three repeats were first done for all stages; then three additional repetitions were performed for those stages showing

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

    USDA-ARS?s Scientific Manuscript database

    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...

  2. An Arabidopsis thaliana embryo arrest mutant exhibiting germination potential

    USDA-ARS?s Scientific Manuscript database

    The ability to initiate radicle elongation, or germination potential, occurs in developing embryos before the completion of seed maturation. Green embryos after walking-stick stage in developing Arabidopsis thaliana seeds germinate when excised from seeds and incubated in MS media containing 1 % suc...

  3. 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.

  4. 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

  5. Vitrification of zebrafish embryo blastomeres in microvolumes.

    PubMed

    Cardona-Costa, J; García-Ximénez, F

    2007-01-01

    Cryopreservation of fish embryos may play an important role in biodiversity preservation and in aquaculture, but it is very difficult. In addition, the cryopreservation of fish embryo blastomeres makes conservation strategies feasible when they are used in germ-line chimaerism, including interspecific chimaerism. Fish embryo blastomere cryopreservation has been achieved by equilibrium procedures, but to our knowledge, no data on vitrification procedures are available. In the present work, zebrafish embryo blastomeres were successfully vitrified in microvolumes: a number of 0.25 microl drops, sufficient to contain all the blastomeres of an embryo at blastula stage (from 1000-cell stage to oblong stage), were placed over a 2.5 cm loop of nylon filament. In this procedure, where intracellular cryoprotectant permeation is not required, blastomeres were exposed to cryoprotectants for a maximum of 25 sec prior vitrification. The assayed cryoprotectants (ethylene glycol, propylene glycol, dimethyl sulphoxide, glycerol and methanol) are all frequently used in fish embryo and blastomere cryopreservation. Methanol was finally rejected because of the excessive concentration required for the vitrification (15M). All other cryoprotectants were prepared (individually) at 5 M in Hanks' buffered salt solution (sigma) plus 20% FBS (vitrification solutions: vs). After direct thawing in Hanks' buffered salt solution plus 20% FBS, acceptable survival rates were obtained with ethylene glycol: 82.8%, propylene glycol: 87.7%, dimethyl sulphoxide: 93.4%, and glycerol: 73.9% (p < 0.05). Dimethyl sulphoxide showed the highest blastomere survival rate and allowed the rescue of as much as 20% of the total blastomeres from each zebrafish blastula embryo.

  6. Effects of deer velvet extract from Formosan sika deer on the embryonic development and anti-oxidative enzymes mRNA expression in mouse embryos.

    PubMed

    Cheng, Shih-Lin; Lai, Yi-Ling; Lee, Ming-Che; Shen, Perng-Chih; Liu, Shyh-Shyan; Liu, Bing-Tsan

    2014-07-03

    The deer velvet or its extracts has been widely used in clinic. It has been used in promoting reproductive performances and treating of oxidation and aging process. The aim of this study is to investigate the effects of velvet extract from Formosan sika deer (Formosan sika deer; Cervus nippon taiouanus, FSD) velvet on mouse embryonic development and anti-oxidant ability in vitro. Mouse 4-cells embryos were divided into 16 groups for 72 h in vitro incubation. The embryonic development stages and morphology were evaluated every 12h in experimental period. The quantitative real time PCR was used to measure the CuZn-SOD, GPx and CAT mRNA expression of the blastocysts. The 4-cells embryos of hydrogen peroxide (HP) groups did not continue developing after oxidant stress challenged. The blastocyst developmental rate (90.0-90.4%, P>0.05) and normal morphological rate (84.4-85.1%, P>0.05) of the 1% and 2% DV extract groups were similar to those in the control group (90.7% and 88.8%, respectively). The embryos challenged by HP (5, 10 and 25 μM) and subsequently incubated in mHTF medium with 1% and 2% of deer velvet (DV) extracts were able to continue development; the blastocyst developmental rate of these groups were similar to that in the control group. The relative mRNA expression of the focused anti-oxidative enzymes in the mouse embryos did not significantly differ among the designed DV treatment groups (P>0.05). The FSD velvet extract in adequate concentration could promote anti-oxidative enzymes mRNA expression followed the challenge of hydrogen peroxide, relieve the mouse embryo under oxidative stress, and maintain the blastocyst developmental ability in vitro. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  7. Potentiality and human embryos.

    PubMed

    Lizza, John P

    2007-09-01

    Consideration of the potentiality of human embryos to develop characteristics of personhood, such as intellect and will, has figured prominently in arguments against abortion and the use of human embryos for research. In particular, such consideration was the basis for the call of the US President's Council on Bioethics for a moratorium on stem cell research on human embryos. In this paper, I critique the concept of potentiality invoked by the Council and offer an alternative account. In contrast to the Council's view that an embryo's potentiality is determined by definition and is not affected by external conditions that may prevent certain possibilities from ever being realized, I propose an empirically grounded account of potentiality that involves an assessment of the physical and decisional conditions that may restrict an embryo's possibilities. In my view, some human embryos lack the potentiality to become a person that other human embryos have. Assuming for the sake of argument that the potential to become a person gives a being special moral status, it follows that some human embryos lack this status. This argument is then used to support Gene Outka's suggestion that it is morally permissible to experiment on 'spare' frozen embryos that are destined to be destroyed.

  8. Variable imprinting of the MEST gene in human preimplantation embryos

    PubMed Central

    Huntriss, John D; Hemmings, Karen E; Hinkins, Matthew; Rutherford, Anthony J; Sturmey, Roger G; Elder, Kay; Picton, Helen M

    2013-01-01

    There is evidence that expression and methylation of the imprinted paternally expressed gene 1/mesoderm-specific transcript homologue (PEG1/MEST) gene may be affected by assisted reproductive technologies (ARTs) and infertility. In this study, we sought to assess the imprinting status of the MEST gene in a large cohort of in vitro-derived human preimplantation embryos, in order to characterise potentially adverse effects of ART and infertility on this locus in early human development. Embryonic genomic DNA from morula or blastocyst stage embryos was screened for a transcribed AflIII polymorphism in MEST and imprinting analysis was then performed in cDNA libraries derived from these embryos. In 10 heterozygous embryos, MEST expression was monoallelic in seven embryos, predominantly monoallelic in two embryos, and biallelic in one embryo. Screening of cDNA derived from 61 additional human preimplantation embryos, for which DNA for genotyping was unavailable, identified eight embryos with expression originating from both alleles (biallelic or predominantly monoallelic). In some embryos, therefore, the onset of imprinted MEST expression occurs during late preimplantation development. Variability in MEST imprinting was observed in both in vitro fertilization and intracytoplasmic sperm injection-derived embryos. Biallelic or predominantly monoallelic MEST expression was not associated with any one cause of infertility. Characterisation of the main MEST isoforms revealed that isoform 2 was detected in early development and was itself variably imprinted between embryos. To our knowledge, this report constitutes the largest expression study to date of genomic imprinting in human preimplantation embryos and reveals that for some imprinted genes, contrasting imprinting states exist between embryos. PMID:22763377

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

    PubMed

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

    2013-06-01

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

  10. Measuring embryo metabolism to predict embryo quality.

    PubMed

    Thompson, Jeremy G; Brown, Hannah M; Sutton-McDowall, Melanie L

    2016-01-01

    Measuring the metabolism of early embryos has the potential to be used as a prospective marker for post-transfer development, either alone or in conjunction with other embryo quality assessment tools. This is necessary to maximise the opportunity of couples to have a healthy child from assisted reproduction technology (ART) and for livestock breeders to efficiently improve the genetics of their animals. Nevertheless, although many promising candidate substrates (e.g. glucose uptake) and methods (e.g. metabolomics using different spectroscopic techniques) have been promoted as viability markers, none has yet been widely used clinically or in livestock production. Herein we review the major techniques that have been reported; these are divided into indirect techniques, where measurements are made from the embryo's immediate microenvironment, or direct techniques that measure intracellular metabolic activity. Both have strengths and weaknesses, the latter ruling out some from contention for use in human ART, but not necessarily for use in livestock embryo assessment. We also introduce a new method, namely multi- (or hyper-) spectral analysis, which measures naturally occurring autofluorescence. Several metabolically important molecules have fluorescent properties, which we are pursuing in conjunction with improved image analysis as a viable embryo quality assessment methodology.

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

    PubMed Central

    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

  12. Fusion of blastomeres in mouse embryos under the action of femtosecond laser radiation. Efficiency of blastocyst formation and embryo development

    NASA Astrophysics Data System (ADS)

    Osychenko, A. A.; Zalesskii, A. D.; Krivokharchenko, A. S.; Zhakhbazyan, A. K.; Ryabova, A. V.; Nadtochenko, V. A.

    2015-05-01

    Using the method of femtosecond laser surgery we study the fusion of two-cell mouse embryos under the action of tightly focused femtosecond laser radiation with the fusion efficiency reaching 60%. The detailed statistical analysis of the efficiency of blastomere fusion and development of the embryo up to the blastocyst stage after exposure of the embryos from different mice to a femtosecond pulse is presented. It is shown that the efficiency of blastocyst formation essentially depends on the biological characteristics of the embryo, namely, the strain and age of the donor mouse. The possibility of obtaining hexaploid embryonal cells using the methods of femtosecond laser surgery is demonstrated.

  13. In vitro fertilization and embryo development in pigs.

    PubMed

    Abeydeera, L R

    2001-01-01

    Considerable progress has been made in the in vitro production of pig embryos using improved methods for in vitro maturation (IVM) and fertilization (IVF). Despite the progress, polyspermic penetration remains a problem for in vitro-matured oocytes. Variation among boars, ejaculates and IVF protocols used in different laboratories appears to influence the incidence of polyspermy. Recent studies indicate that oviduct cells and their secretions play a role in reducing polyspermy. Very early attempts to culture in vivo-derived pig embryos met with little success and most were arrested at the four-cell stage. At present, many culture media are available that can overcome the four-cell block and support development to the blastocyst stage. In contrast, blastocyst development of in vitro-produced (IVP) embryos in these culture media varies significantly. Significant differences in morphology and numbers of cells have been observed in in vitro-produced blastocysts compared with in vivo-derived blastocysts. Surgical transfer of in vitro-produced embryos to recipient animals has resulted in acceptable pregnancy rates with moderate litter sizes. Although several systems are available for the generation of in vitro-produced embryos, the problems of polyspermy and poor embryo survival prevent large-scale production of embryos. Further research should be directed to improve oocyte and embryo quality, and to develop methods to minimize polyspermy through development of better IVM, IVF and embryo culture techniques.

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

    Treesearch

    Rakesh Minocha; Subhash C. Minocha; Stephanie Long

    2004-01-01

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

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

    Treesearch

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

    1999-01-01

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

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

    PubMed

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

    2014-04-01

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

  17. Stimulus-triggered enhancement of chilling tolerance in zebrafish embryos

    PubMed Central

    Szabó, Katalin; Budai, Csilla; Losonczi, Eszter; Bernáth, Gergely; Csenki-Bakos, Zsolt; Urbányi, Béla; Pribenszky, Csaba; Horváth, Ákos; Cserepes, Judit

    2017-01-01

    Background Cryopreservation of zebrafish embryos is still an unsolved problem despite market demand and massive efforts to preserve genetic variation among numerous existing lines. Chilled storage of embryos might be a step towards developing successful cryopreservation, but no methods to date have worked. Methods In the present study, we applied a novel strategy to improve the chilling tolerance of zebrafish embryos by introducing a preconditioning hydrostatic pressure treatment to the embryos. In our experiments, 26-somites and Prim-5 stage zebrafish embryos were chilled at 0°C for 24 hours after preconditioning. Embryo survival rate, ability to reach maturation and fertilizing capacity were tested. Results Our results indicate that applied preconditioning technology made it possible for the chilled embryos to develop normally until maturity, and to produce healthy offspring as normal, thus passing on their genetic material successfully. Treated embryos had a significantly higher survival and better developmental rate, moreover the treated group had a higher ratio of normal morphology during continued development. While all controls from chilled embryos died by 30 day-post-fertilization, the treated group reached maturity (~90–120 days) and were able to reproduce, resulting in offspring in expected quantity and quality. Conclusions Based on our results, we conclude that the preconditioning technology represents a significant improvement in zebrafish embryo chilling tolerance, thus enabling a long-time survival. Furthermore, as embryonic development is arrested during chilled storage this technology also provides a solution to synchronize or delay the development. PMID:28166301

  18. Developmental table of the early mouse post-implantation embryo.

    PubMed

    Van Maele-Fabry, G; Delhaise, F; Gofflot, F; Picard, J J

    1993-11-01

    The developmental tables of early somite mouse embryos that are presently available from the literature give clear and useful descriptions of the differentiation at successive stages. However, they provide no easy access to the correlation between the growth of the embryo and its differentiation. In the present study, quantitative data concerning normal mouse embryonic development as well as the major developmental events occurring between 0 and 30 pairs of somites were established. Measurements of growth (crown-rump length, head length, absorbancy at 280 nm) and differentiation parameters (morphological score) of 168 to 310 explanted mouse embryos were recorded for each developmental stage (number of pairs of somites). A short description of the major events occurring at the corresponding stages is also presented. The table is more detailed than those presently available and provides a rapid and practical overview of the timing of the appearance of developmental events and differentiation in correlation to the progressive growth of the embryo. It could, therefore, be useful for embryologists and toxicologists. In addition, the development of 67 post-implantation mouse embryos cultured in vitro was compared with the reference table established from in vivo embryos. Our results confirm and extend previous reports showing that embryos cultured in vitro grow and differentiate at a pace very similar to that of embryos developed in vivo.

  19. Nucleolar development and allocation of key nucleolar proteins require de novo transcription in bovine embryos.

    PubMed

    Svarcova, Olga; Laurincik, Jozef; Avery, Birthe; Mlyncek, Milos; Niemann, Heiner; Maddox-Hyttel, Poul

    2007-11-01

    The goal of the present study was to investigate whether key nucleolar proteins involved in ribosomal RNA (rRNA) transcription and processing are transcribed de novo or from maternally inherited messenger RNAs (mRNA) in bovine embryos, and to which extent de novo transcription of these proteins mRNA is required for the development of functional nucleoli during the major activation of the embryonic genome. Immunofluorescence for localization of key nucleolar proteins, autoradiography for detection of transcriptional activity, and transmission electron microscopy were applied to in vitro produced bovine embryos cultured from the 2-cell stage with or without (control groups) alpha-amanitin, which blocks the RNA polymerases II and III transcription and, thus the synthesis of mRNA. In the control groups, weak autoradiographic labeling was initially observed in the periphery of few nuclei at the 4-cell and the early 8-cell stage, and the entire nucleoplasm as well as nucleolus precursor bodies (NBBs) were prominently labelled in all late 8-cell stages. The NPBs displayed initial transformation into fibrillo-granular nucleoli. In the alpha-amanitin group, lack of autoradiographic labeling was seen at all developmental stages and disintegrated NPBs stage were found at the late 8-cell. Our immunofluorescence data indicate that RNA polymerase I, UBF, topoisomerase I and fibrillarin are transcribed de novo whereas nucleolin and nucleophosmin are maternally inherited as demonstrated by alpha -amanitin inhibition. However, localization of these two proteins to the nucleolar compartments was negatively affected by the alpha-amanitin treatment. Consequently, functional nucleoli were not established.

  20. Replication and persistence of different strains of bovine viral diarrhea virus in an in vitro embryo production system.

    PubMed

    Givens, M D; Galik, P K; Riddell, K P; Brock, K V; Stringfellow, D A

    2000-10-15

    Recent studies have shown that exposed, in vitro-derived embryos remain contaminated with bovine viral diarrhea virus (BVDV) after washing. However, introduction of a Genotype II versus Genotype I strain of BVDV into an IVF system was reported to provide greater potential for transmission of disease. The primary objective of this study was to compare the potentials for different strains of noncytopathic BVDV to replicate in an IVF system, associate with IVF embryos and infect co-cultured cells via association with washed embryos. The secondary objective was to compare the effect of different strains of BVDV on embryonic development. Two Genotype I (SD-1 and NY-1) and 2 Genotype II (CD-87 and PA-131) strains of BVDV were evaluated. After IVM and IVF of oocytes, presumptive zygotes were washed and transferred into in vitro cultures containing uterine tubal cells (UTC) and medium that was free of BVDV-neutralizing activity. Immediately before addition of zygotes, the cultures were inoculated with 10(5) cell culture infective doses (50%, CCID50) of a strain of BVDV or maintained as a negative control. Cultures of zygotes were then incubated for 7 d. Embryonic development was observed on Days 3 and 7, and attempts were made to isolate BVDV from UTC and medium on Day 7. Also on Day 7, groups of intact, washed blastocysts were either transferred into virus-free secondary cultures containing UTC or sonicated with sonicate fluid assayed by both virus isolation and single-closed-tube reverse transcription nested polymerase chain reaction (RT-nPCR). After 3 d in secondary culture, hatched embryos were enumerated, and medium from the cultures, washed UTC and embryos were tested for BVDV by virus isolation. In addition, washed UTC and embryos were tested for BVDV using RT-nPCR. All strains of BVDV persisted and replicated in the embryo culture environment, but cleavage beyond the 4-cell stage, blastocyst development and hatching varied among cultures contaminated with different

  1. Studies of In Vitro Embryo Culture of Guppy (Poecilia reticulata).

    PubMed

    Liu, LiLi; Lee, Ki-Young

    2014-09-01

    Different with other fishes, the guppies (Poecilia reticulata) is ovoviviparity, which retain their fertilized eggs within the follicle throughout gestation. The synchronously growing diplotene oocytes store nutrients in droplets and yolk, before their maturation and fertilization. The lecithotrophic strategy of development entails the provisioning of embryos with resources from the maternal yolk deposit rather than from a placenta, it allows the extracorporeal culture of guppy embryo. Studies on their early development of live bearers like the guppy including lineage tracing and genetic manipulations, have been limited. Therefore, to optimize conditions of embryo in vitro culture, explanted embryos from pregnant females were incubated in embryo medium (L-15 medium, supplemented with 5, 10, 15, 20% fetal bovine serum, respectively). We investigated whether the contents of FBS in vitro culture medium impact the development of embryos, and whether they would hatch in vitro. Our study found that in 5% of FBS of the medium, although embryos developed significantly slower in vitro than in the ovary, it was impossible to exactly quantify the developmental delay in culture, due to the obvious spread in developmental stage within each batch of eggs, and embryos can only be maintained until the early-eyed. And although in culture with 20% FBS the embryos can sustain rapid development of early stage, but cannot be cultured for the entire period of their embryonic development and ultimately died. In the medium with 10% and 15% FBS, the embryos seems well developed, even some can continue to grow after follicle ruptures until it can be fed. We also observed that embryonic in these two culture conditions were significantly different in development speed, in 15% it is faster than 10%. But 10% FBS appears to be more optimizing condition than 15% one on development process of embryos and survival rate to larvae stage.

  2. Studies of In Vitro Embryo Culture of Guppy (Poecilia reticulata)

    PubMed Central

    Liu, LiLi; Lee, Ki-Young

    2014-01-01

    Different with other fishes, the guppies (Poecilia reticulata) is ovoviviparity, which retain their fertilized eggs within the follicle throughout gestation. The synchronously growing diplotene oocytes store nutrients in droplets and yolk, before their maturation and fertilization. The lecithotrophic strategy of development entails the provisioning of embryos with resources from the maternal yolk deposit rather than from a placenta, it allows the extracorporeal culture of guppy embryo. Studies on their early development of live bearers like the guppy including lineage tracing and genetic manipulations, have been limited. Therefore, to optimize conditions of embryo in vitro culture, explanted embryos from pregnant females were incubated in embryo medium (L-15 medium, supplemented with 5, 10, 15, 20% fetal bovine serum, respectively). We investigated whether the contents of FBS in vitro culture medium impact the development of embryos, and whether they would hatch in vitro. Our study found that in 5% of FBS of the medium, although embryos developed significantly slower in vitro than in the ovary, it was impossible to exactly quantify the developmental delay in culture, due to the obvious spread in developmental stage within each batch of eggs, and embryos can only be maintained until the early-eyed. And although in culture with 20% FBS the embryos can sustain rapid development of early stage, but cannot be cultured for the entire period of their embryonic development and ultimately died. In the medium with 10% and 15% FBS, the embryos seems well developed, even some can continue to grow after follicle ruptures until it can be fed. We also observed that embryonic in these two culture conditions were significantly different in development speed, in 15% it is faster than 10%. But 10% FBS appears to be more optimizing condition than 15% one on development process of embryos and survival rate to larvae stage. PMID:25949182

  3. Dynamics of maternal and paternal effects on embryo and seed development in wild radish (Raphanus sativus).

    PubMed

    Diggle, P K; Abrahamson, N J; Baker, R L; Barnes, M G; Koontz, T L; Lay, C R; Medeiros, J S; Murgel, J L; Shaner, M G M; Simpson, H L; Wu, C C; Marshall, D L

    2010-08-01

    Variability in embryo development can influence the rate of seed maturation and seed size, which may have an impact on offspring fitness. While it is expected that embryo development will be under maternal control, more controversial hypotheses suggest that the pollen donor and the embryo itself may influence development. These latter possibilities are, however, poorly studied. Characteristics of 10-d-old embryos and seeds of wild radish (Raphanus sativus) were examined to address: (a) the effects of maternal plant and pollen donor on development; (b) the effects of earlier reproductive events (pollen tube growth and fertilization) on embryos and seeds, and the influence of embryo size on mature seed mass; (c) the effect of water stress on embryos and seeds; (d) the effect of stress on correlations of embryo and seed characteristics with earlier and later reproductive events and stages; and (e) changes in maternal and paternal effects on embryo and seed characteristics during development. Eight maternal plants (two each from four families) and four pollen donors were crossed and developing gynoecia were collected at 10 d post-pollination. Half of the maternal plants experienced water stress. Characteristics of embryos and seeds were summarized and also compared with earlier and later developmental stages. In addition to the expected effects of the maternal plants, all embryo characters differed among pollen donors. Paternal effects varied over time, suggesting that there are windows of opportunity for pollen donors to influence embryo development. Water-stress treatment altered embryo characteristics; embryos were smaller and less developed. In addition, correlations of embryo characteristics with earlier and later stages changed dramatically with water stress. The expected maternal effects on embryo development were observed, but there was also evidence for an early paternal role. The relative effects of these controls may change over time. Thus, there may be

  4. Dynamics of maternal and paternal effects on embryo and seed development in wild radish (Raphanus sativus)

    PubMed Central

    Diggle, P. K.; Abrahamson, N. J.; Baker, R. L.; Barnes, M. G.; Koontz, T. L.; Lay, C. R.; Medeiros, J. S.; Murgel, J. L.; Shaner, M. G. M.; Simpson, H. L.; Wu, C. C.; Marshall, D. L.

    2010-01-01

    Background and Aims Variability in embryo development can influence the rate of seed maturation and seed size, which may have an impact on offspring fitness. While it is expected that embryo development will be under maternal control, more controversial hypotheses suggest that the pollen donor and the embryo itself may influence development. These latter possibilities are, however, poorly studied. Characteristics of 10-d-old embryos and seeds of wild radish (Raphanus sativus) were examined to address: (a) the effects of maternal plant and pollen donor on development; (b) the effects of earlier reproductive events (pollen tube growth and fertilization) on embryos and seeds, and the influence of embryo size on mature seed mass; (c) the effect of water stress on embryos and seeds; (d) the effect of stress on correlations of embryo and seed characteristics with earlier and later reproductive events and stages; and (e) changes in maternal and paternal effects on embryo and seed characteristics during development. Methods Eight maternal plants (two each from four families) and four pollen donors were crossed and developing gynoecia were collected at 10 d post-pollination. Half of the maternal plants experienced water stress. Characteristics of embryos and seeds were summarized and also compared with earlier and later developmental stages. Key Results In addition to the expected effects of the maternal plants, all embryo characters differed among pollen donors. Paternal effects varied over time, suggesting that there are windows of opportunity for pollen donors to influence embryo development. Water-stress treatment altered embryo characteristics; embryos were smaller and less developed. In addition, correlations of embryo characteristics with earlier and later stages changed dramatically with water stress. Conclusions The expected maternal effects on embryo development were observed, but there was also evidence for an early paternal role. The relative effects of these

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

    PubMed

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

    2016-10-01

    . Furthermore, the developed blastocysts were scored using the blastocyst quality score (BQS), and the correlation with oxygen consumption rate was also assessed. The device enabled the oxygen consumption rate of an embryo to be measured automatically within a minute. The oxygen concentration gradient profile showed excellent linearity in a distance-dependent change. A close correlation in the oxygen consumption rates of bovine embryos was observed between the SECM measuring system and CERMs, with a determination coefficient of 0.8203 (P = 0.0008). Oxygen consumption rates of human embryos that have reached the blastocyst stage were significantly higher than those of arrested embryos at 48, 72 and 96 h after thawing (P = 0.039, 0.004 and 0.049, respectively). Thus, in vitro development of frozen-thawed human embryos to the blastocyst stage would be predicted at 48 h after thawing (day 4) by measuring the oxygen consumption using CERMs. Although a positive linear relationship between BQS and the oxygen consumption rate was observed [the determination coefficient was R(2) = 0.6537 (P = 0.008)], two blastocysts exhibited low oxygen consumption rates considering their relatively high BQS. This suggests that morphology and metabolism in human embryos might not correlate consistently. Transfer of the embryo and pregnancy evaluation was not performed. Thus, a correlation between oxygen consumption and the in vivo viability of embryos remains unknown. Clinical trials, including embryo transfer, would be desirable to determine a threshold value to elect clinically relevant, quality embryos for transfer. We utilized frozen-thawed human embryos in this study. The effect of these manipulations on the respiratory activity of the embryo is also unknown. Selection of quality embryos, especially in a single embryo transfer cycle, by CERMs may have an impact on obtaining better clinical outcomes, albeit with clinical trials being required. Furthermore, the early determination of quality

  6. The development and expression of pluripotency genes in embryos derived from nuclear transfer and in vitro fertilization.

    PubMed

    Ma, Li-Bing; He, Xiao-Ying; Wang, Feng-Mei; Cao, Jun-Wei; Cheng, Teng

    2014-11-01

    Somatic cell nuclear transfer can be used to produce embryonic stem (ES) cells, cloned animals, and can even increase the population size of endangered animals. However, the application of this technique is limited by the low developmental rate of cloned embryos, a situation that may result from abnormal expression of some zygotic genes. In this study, sheep-sheep intra-species cloned embryos, goat-sheep inter-species cloned embryos, or sheep in vitro fertilized embryos were constructed and cultured in vitro and the developmental ability and expression of three pluripotency genes, SSEA-1, Nanog and Oct4, were examined. The results showed firstly that the developmental ability of in vitro fertilized embryos was significantly higher than that of cloned embryos. In addition, the percentage of intra-species cloned embryos that developed to morula or blastocyst stages was also significantly higher than that of the inter-species cloned embryos. Secondly, all three types of embryos expressed SSEA-1 at the 8-cell and morula stages. At the 8-cell stage, a higher percentage of in vitro fertilized embryos expressed SSEA-1 than occurred for cloned embryos. However, at the morula stage, all detected embryos could express SSEA-1. Thirdly, the three types of embryos expressed Oct4 mRNA at the morula and blastocyst stages, and embryos at the blastocyst stage expressed Nanog mRNA. The rate of expression of Oct4 and Nanog mRNA at these developmental stages was higher in in vitro fertilized embryos than in cloned embryos. These results indicated that, during early development, the failure to reactivate some pluripotency genes maybe is a reason for the low cloning efficiency found with cloned embryos.

  7. Embryo dignity: the status and juridical protection of the in vitro embryo.

    PubMed

    Raposo, Vera Lúcia; Osuna, Eduardo

    2007-12-01

    In the context of research and reproduction, the status of the human in vitro embryo ranges from being regarded as a person to being regarded as mere property. As regards the first view, one extreme of the spectrum for offering possible legal protection considers that the embryo constitutes a legal person from the moment of conception. For opponents of this view life is a continuum that runs from conception until death. In this process one of the most important stages is birth, the reason being that birth represents the transition between a potential person and a person. The term "embryo" is used to express the being that exists after fusion of the egg and a spermatozoon during the process of embryogenesis until it reaches eight weeks, after which time it is termed a foetus. The embryo's life is recognized as a constitutional value which deserves juridical protection, but not as a person. It only becomes a person with birth.

  8. 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.

  9. Comparison of gene expression in fresh and frozen-thawed human preimplantation embryos.

    PubMed

    Shaw, Lisa; Sneddon, Sharon F; Brison, Daniel R; Kimber, Susan J

    2012-11-01

    Identification and characterisation of differentially regulated genes in preimplantation human embryonic development are required to improve embryo quality and pregnancy rates in IVF. In this study, we examined expression of a number of genes known to be critical for early development and compared expression profiles in individual preimplantation human embryos to establish any differences in gene expression in fresh compared to frozen-thawed embryos used routinely in IVF. We analysed expression of 19 genes by cDNA amplification followed by quantitative real-time PCR in a panel of 44 fresh and frozen-thawed human preimplantation embryos. Fresh embryos were obtained from surplus early cleavage stage embryos and frozen-thawed embryos from cryopreserved 2PN embryos. Our aim was to determine differences in gene expression between fresh and frozen-thawed human embryos, but we also identified differences in developmental expression patterns for particular genes. We show that overall gene expression among embryos of the same stage is highly variable and our results indicate that expression levels between groups did differ and differences in expression of individual genes was detected. Our results show that gene expression from frozen-thawed embryos is more consistent when compared with fresh, suggesting that cryopreserved embryos may represent a reliable source for studying the molecular events underpinning early human embryo development.

  10. 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.

  11. 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.

  12. Relationship between pronuclear scoring and embryo quality and implantation potential in IVF-ET.

    PubMed

    Liu, Qun; Zhu, Guijin; Hu, Juan; Wei, Yulan; Ren, Xinling; Zhang, Hanwang; Li, Yufeng; Jin, Lei; Yue, Jing

    2008-04-01

    To assess the relationship between pronuclear scoring and day-3 embryo quality and pregnancy outcome and to determine the clinical value of pronuclear stage scoring system in human in vitro fertilization-embryo transfer (IVF-ET) program, a pronuclear scoring system was used to score zygotes 16-20 h after insemination during conventional IVF or intracytoplasmic sperm injection (ICSI). The embryos were classified into groups Z1, Z2, Z3 and Z4. Comparisons were made of the rates of arrested embryos and excellent embryos on day 3. Comparisons of pregnancy outcome were made only in those patients in whom cohorts of similarly Z-scored embryos were transferred. The results showed that there were less arrested embryos and more excellent embryos on day 3 in groups Z1 and Z2 than those in group Z3 and Z4. More embryos arrested and less excellent embryos developed in group Z4 than group Z3. The clinical pregnancy rates resulting from the transfer of single pronuclear score homologous embryo types were similar among groups Z1, Z2 and Z3. Implantation rates of group Z1 were higher (P<0.05) than that of group Z3. These findings suggests that pronuclear scoring can predict developmental ability on day 3 and implantation potential. A evaluation that combines the Z-score and day 3 embryo morphology is useful in the determination of the most viable embryos and the number of embryos for transfer.

  13. Freezing injuries in the embryos of Piaractus mesopotamicus.

    PubMed

    Fornari, Darci Carlos; Ribeiro, Ricardo Pereira; Streit, Danilo Pedro; Vargas, Lauro; Barrero, Nelson M Lopera; de Moraes, Gentil Vanini

    2011-11-01

    Cryopreservation of mammal embryos has been technically feasible for many years, but morphological injuries still persist in fish embryos during cryopreservation. Thus, the objective of the present study was to describe these freezing injuries in Piaractus mesopotamicus embryos. Two hundred and twenty-five embryos were collected at the post-gastrula stage and assigned into four treatments of sucrose at 8.5, 17.0, 25.0 or 34.0% plus 9.0% methanol. The control was prepared with distilled water only. The gradual decrease in the temperature was 0.5°C/min. After the seeding stage, the fish embryos were stored in liquid nitrogen at -33°C. Thereafter, they were thawed for evaluating per cent hatching, and the samples collected from every treatment were submitted to scanning electron microscopy for morphological analysis. The micrographic images showed that there was substantial alterations in embryo morphology under the highest concentrations of sucrose. These solutions did not prevent the formation of ice crystals, which lead to deformities and killed the embryos, but the observed reduced level of morphological structure in these embryos when treated with 17.0% sucrose plus 9.0% methanol is a compelling argument for additional studies.

  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

    2015-09-29

    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.

  15. 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

  16. Embryotoxic effects of chlorobutanol in cultured mouse embryos.

    PubMed

    Smoak, I W

    1993-03-01

    Chlorobutanol (CB) is a commonly used preservative which is added to numerous pharmaceutical preparations, and it is the active ingredient in certain oral sedatives and topical anesthetics. Chlorobutanol has demonstrated adverse effects in adult tissues, but CB has not been previously investigated for its effect on the developing whole embryo. The method of whole-embryo culture was used in this study to expose mouse embryos during two stages of organogenesis to CB at final concentrations of 0 (control), 10, 25, 50, 100, and 200 micrograms/ml. Embryos were evaluated for heart rate (HR), malformations, and somite number, and embryos and visceral yolk sacs (VYSs) were assayed for total protein content as a measure of overall growth. Neurulating (3-6 somite) embryos were malformed and growth retarded by exposure to CB concentrations > or = 25 micrograms/ml, with decreased VYS growth at > or = 50 micrograms/ml and decreased HR at > or = 100 micrograms/ml CB. Early limb-bud stage (20-25 somite) embryos were malformed at CB concentrations > or = 50 micrograms/ml and growth retarded at > or = 100 micrograms/ml, with decreased VYS growth at 200 micrograms/ml and decreased HR at > or = 100 micrograms/ml CB. Thus, CB produces dysmorphogenesis in mouse embryos in vitro, and neurulating embryos are somewhat less sensitive than early limb-bud stage embryos. The concentrations of CB that interfere with normal embryonic development are within the range of human blood levels measured following multiple doses of CB. Preparations containing CB should be used with caution during pregnancy, particularly when repeated dosing may allow accumulation of CB to potentially embryotoxic levels.

  17. Toxicity of chlorine to zebrafish embryos

    PubMed Central

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

    2014-01-01

    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 – 50 ppm unbuffered chlorine solution for 5 – 10 min. Treatment of embryos with chlorine has significant germicidal effects for many Gram-negative bacteria, viruses, and trophozoite stages of protozoa, it has reduced efficacy against cyst or spore stages of protozoa and certain Mycobacterium spp. Therefore, we evaluated the toxicity of unbufferred and buffered chlorine solution to embryos exposed at 6 or 24 hours post-fertilization (hpf) to determine if 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 pHs, and chlorine causes elevated pH. Consistent with this, we found that unbufferred 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 for 5 min with unbuffered chlorine solution at 100 ppm. One trial indicated that AB fish, a popular outbred line, are more susceptible to toxicity than 5Ds. This suggests that variability between zebrafish lines occurs, and researchers should evaluate each line or strain under their particular laboratory conditions for selection of the optimum chlorine treatment procedure. PMID:24429474

  18. Zscan4: a novel gene expressed exclusively in late 2-cell embryos and embryonic stem cells

    PubMed Central

    Falco, Geppino; Lee, Sung-Lim; Stanghellini, Ilaria; Bassey, Uwem C.; Hamatani, Toshio; Ko, Minoru S. H.

    2007-01-01

    The first wave of transcription, called zygotic genome activation (ZGA), begins during the 2-cell stage in mouse preimplantation development and marks a vital transition from the maternal genetic to the embryonic genetic program. Utilizing DNA microarray data, we looked for genes that are expressed only during ZGA and found Zscan4, whose expression is restricted to late 2-cell stage embryos. Sequence analysis of genomic DNA and cDNA clones revealed nine paralogous genes tightly clustered in 0.85 Mb on mouse Chromosome 7. Three genes are not transcribed and are thus considered pseudogenes. Among the six expressed genes named Zscan4a-Zscan4f, three -- Zscan4c, Zscan4d, and Zscan4f -- encode full-length ORFs with 506 amino acids. Zscan4d is a predominant transcript at the late 2-cell stage, whereas Zscan4c is a predominant transcript in embryonic stem (ES) cells. No transcripts of any Zscan4 genes are detected in any other cell types. Reduction of Zscan4 transcript levels by siRNAs delays the progression from the 2-cell to the 4-cell stage and produces blastocysts that fail to implant or proliferate in blastocyst outgrowth culture. Zscan4 thus seems to be essential for preimplantation development. PMID:17553482

  19. 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.

  20. Human embryos cultured in vitro to 14 days

    PubMed Central

    2017-01-01

    We know a great deal about the development of the mammalian embryo until the time that the blastocyst implants into the uterus. With model organisms such as the mouse, we have also developed a considerable understanding of development immediately around gastrulation as embryos can be recovered at this stage for short-term in vitro culture. However, the intervening period of development remained a ‘black box’ because it takes place as the blastocyst is implanting into the uterus. Over the past 6 years, techniques pioneered and developed in Magdalena Zernicka-Goetz's laboratory for the in vitro culture of embryos through these implantation stages have opened up this box, affording the first glimpse of embryonic development through these previously hidden stages. Remarkably, the techniques developed with mouse embryos are equally applicable to human embryos, ushering the very first opportunities for studying our own development throughout this time. Here, I outline how the culture methods were developed, paving the way to culture of the human embryo to the point of gastrulation, an accomplishment recognized as the People's Choice for the Scientific Breakthrough of 2016 in Science magazine. I also discuss the new ethical challenges raised by the possibility of extending the time limits for human embryo culture. PMID:28123056

  1. 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.

  2. 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.

  3. 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.

  4. A dysmorphology score system for assessing embryo abnormalities in rat whole embryo culture.

    PubMed

    Zhang, Cindy X; Danberry, Tracy; Jacobs, Mary Ann; Augustine-Rauch, Karen

    2010-12-01

    The rodent whole embryo culture (WEC) system is a well-established model for characterizing developmental toxicity of test compounds and conducting mechanistic studies. Laboratories have taken various approaches in describing type and severity of developmental findings of organogenesis-stage rodent embryos, but the Brown and Fabro morphological score system is commonly used as a quantitative approach. The associated score criteria is based upon developmental stage and growth parameters, where a series of embryonic structures are assessed and assigned respective scores relative to their gestational stage, with a Total Morphological Score (TMS) assigned to the embryo. This score system is beneficial because it assesses a series of stage-specific anatomical landmarks, facilitating harmonized evaluation across laboratories. Although the TMS provides a quantitative approach to assess growth and determine developmental delay, it is limited to its ability to identify and/or delineate subtle or structure-specific abnormalities. Because of this, the TMS may not be sufficiently sensitive for identifying compounds that induce structure or organ-selective effects. This study describes a distinct morphological score system called the "Dysmorphology Score System (DMS system)" that has been developed for assessing gestation day 11 (approximately 20-26 somite stage) rat embryos using numerical scores to differentiate normal from abnormal morphology and define the respective severity of dysmorphology of specific embryonic structures and organ systems. This method can also be used in scoring mouse embryos of the equivalent developmental stage. The DMS system enhances capabilities to rank-order compounds based upon teratogenic potency, conduct structure- relationships of chemicals, and develop statistical prediction models to support abbreviated developmental toxicity screens. © 2010 Wiley-Liss, Inc.

  5. Efficacy of fish embryo vitrification protocols in terms of embryo morphology - a systematic review.

    PubMed

    Souza de Carvalho, A F; Ramos, S E; Gonzaga de Carvalho, T S; Pomarico de Souza, Y C; Zangeronimo, M G; Pereira, L J; Solis Murgas, L D

    2014-01-01

    Embryo cryopreservation has been used for the creation of genetic banks with diploid resources, and among different techniques, vitrification is considered as the most promising method. The goal is to evaluate the major aspects of the existing vitrification techniques and to evaluate their efficacy in terms of embryo morphology. Electronic searches in the PubMed and ScienceDirect databases were performed with the keyword combination: fish, embryo and vitrification. Pubmed retrieved 26 articles and Science Direct resulted in 464 articles. For this review, only studies that developed and tested vitrification protocols in fish embryos were included. Research regarding cryoprotectant toxicity and permeability were excluded. There were no restrictions on publication date or language. With these criteria, a total of ten articles were evaluated. In these articles, the major aspects to be considered for the development of new vitrification protocols are: the cryoprotectants' toxicity, the embryos' development stage, the exposure to and the permeability of the cryoprotectants, vitrification devices and vitrification-warning cycle. The survival were limited, however, the preservation of embryonic morphology after thawing indicates the possibility of preserving fish embryos via the vitrification technique.

  6. Biopsy of embryos produced by in vitro fertilization affects development in C57BL/6 mouse strain

    PubMed Central

    Sugawara, Atsushi; Ward, Monika A.

    2012-01-01

    Preimplantation genetic diagnosis (PGD) is considered highly successful in respect to its accuracy in detecting genetic anomalies but the effects of embryo biopsy on embryonic/fetal growth and development are less known, particularly in conjunction with in vitro fertilization (IVF). Here, we compared biopsied (B) and non-biopsied (NB) mouse embryos for their developmental competence. Embryos C57BL/6 (B6) and B6D2F2 (F2) generated by IVF were subjected to single blastomere biopsy at the 4-cell stage, and were either cultured for 120 h and subjected to differential inner cell mass (ICM) and trophoblast (T) staining, or were transferred into the uterine tubes of surrogate mothers after 72 h of culture, to examine their pre- and post-implantation development, respectively. Non-biopsied embryos from the same IVF cohorts served as controls. Embryo biopsy negatively affected preimplantation development to blastocyst in C57BL/6 (69 vs 79%, P<0.01) but not in B6D2F1 mice (89 vs 91%, P=NS). Although B6 embryos had lower total cell number than F2 (B6: 47 and 61 vs. F1: 53 and 70; B and NB, respectively, P<0.05) there were no differences between B and NB blastocysts in %ICM (B6: 19.8 vs 19.8; F2: 20.9 vs 20.4, P=NS) and ICM:T ratio (B6: 4.7 vs 4.7; F2: 4.4 vs. 4.7) in both mouse strains. Post-implantation development to live fetuses of B embryos as compared to NB counterparts was impaired in C57BL/6 (6 vs 18%, P<0.001) but not in B6D2F1 mice (26 vs 35%, P=NS). We conclude that blastomere biopsy impairs embryonic/fetal development in mice known to be sensitive to in vitro culture and manipulations. Such mice model infertile couples with poor quality gametes seeking help in assisted reproduction technologies (ART) clinics. PMID:23174776

  7. Supplementation with small-extracellular vesicles from ovarian follicular fluid during in vitro production modulates bovine embryo development

    PubMed Central

    Andrade, Gabriella M.; del Collado, Maite; Sampaio, Rafael V.; Sangalli, Juliano R.; Silva, Luciano A.; Pinaffi, Fábio V. L.; Jardim, Izabelle B.; Cesar, Marcelo C.; Nogueira, Marcelo F. G.; Cesar, Aline S. M.; Coutinho, Luiz L.; Pereira, Rinaldo W.; Perecin, Felipe; Meirelles, Flávio V.

    2017-01-01

    Pregnancy success results from the interaction of multiple factors, among them are folliculogenesis and early embryonic development. Failure during these different processes can lead to difficulties in conception. Alternatives to overcome these problems are based on assisted reproductive techniques. Extracellular vesicles are cell-secreted vesicles present in different body fluids and contain bioactive materials, such as messenger RNA, microRNAs (miRNAs), and proteins. Thus, our hypothesis is that extracellular vesicles from follicular fluid from 3–6 mm ovarian follicles can modulate bovine embryo development in vitro. To test our hypothesis follicular fluid from bovine ovaries was aspirated and small-extracellular vesicles (<200 nm) were isolated for further analysis. Additionally, small-extracellular vesicles (EVs) were utilized for functional experiments investigating their role in modulating messenger RNA, microRNA as well as global DNA methylation and hydroxymethylation levels of bovine blastocysts. EVs from 3–6 mm follicles were used for RNA-seq and miRNA analysis. Functional annotation analysis of the EVs transcripts revealed messages related to chromatin remodeling and transcriptional regulation. EVs treatment during oocyte maturation and embryo development causes changes in blastocyst rates, as well as changes in the transcription levels of genes related to embryonic metabolism and development. Supplementation with EVs from 3–6 mm follicles during oocyte maturation and early embryo development (until the 4-cell stage) increased the levels of bta-miR-631 (enriched in EVs from 3–6 mm follicles) in embryos. Interestingly, the addition of EVs from 3–6 mm follicles induced changes in global DNA methylation and hydroxymethylation levels compared to embryos produced by the standard in vitro production system. Our results indicate that the supplementation of culture media with EVs isolated from the follicular fluid of 3–6 mm follicles during oocyte

  8. Avian egg odour encodes information on embryo sex, fertility and development.

    PubMed

    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.

  9. 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.

  10. 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. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. 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.

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

    PubMed

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

    2011-06-01

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

  13. Mitochondrial and DNA damage in bovine somatic cell nuclear transfer embryos.

    PubMed

    Hwang, In-Sun; Bae, Hyo-Kyung; Cheong, Hee-Tae

    2013-01-01

    The generation of reactive oxygen species (ROS) and subsequent mitochondrial and DNA damage in bovine somatic cell nuclear transfer (SCNT) embryos were examined. Bovine enucleated oocytes were electrofused with donor cells and then activated by a combination of Ca-ionophore and 6-dimethylaminopurine culture. The H2O2 and ˙OH radical levels, mitochondrial morphology and membrane potential (ΔΨ), and DNA fragmentation of SCNT and in vitro fertilized (IVF) embryos at the zygote stage were analyzed. The H2O2 (35.6 ± 1.1 pixels/embryo) and ˙OH radical levels (44.6 ± 1.2 pixels/embryo) of SCNT embryos were significantly higher than those of IVF embryos (19.2 ± 1.5 and 23.8 ± 1.8 pixels/embryo, respectively, p < 0.05). The mitochondria morphology of SCNT embryos was diffused within the cytoplasm. The ΔΨ of SCNT embryos was significantly lower (p < 0.05) than that of IVF embryos (0.95 ± 0.04 vs. 1.21 ± 0.06, red/green). Moreover, the comet tail length of SCNT embryos was longer than that of IVF embryos (515.5 ± 26.4 μm vs. 425.6 ± 25.0 μm, p < 0.05). These results indicate that mitochondrial and DNA damage increased in bovine SCNT embryos, which may have been induced by increased ROS levels.

  14. 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.

  15. A zygote is not an embryo: ethical and legal considerations.

    PubMed

    Tesarik, Jan; Greco, Ermanno

    2004-07-01

    In spite of several past attempts at defining the point at which conception can be considered completed, resulting in the formation of an embryo, the existing definitions are still contradictory. In the absence of clear terminology, the application of laws aimed at the protection of early human life may have inadequate consequences for the efficacy of the current techniques of human infertility treatment. In this paper biological arguments are revisited, suggesting that the only point at which a clear demarcation line between what is and what is still not an embryo can be drawn is the moment of nuclear syngamy at the outset of the first cleavage division. The term 'zygote' is suggested to denote entities composed of spermatozoon and oocyte components before nuclear syngamy. It is suggested that the current embryo protection laws should not concern the zygote stage: at this stage, the main features that are said, in documents issued by different ethical and legal authorities, to characterize the early human embryo, namely the inseparable union of the male and female contribution, cell division and an autonomous control over cell division, are still not present. This reasoning strictly applies to embryos of biparental (paternal and maternal contribution) origin and cannot be extrapolated to embryos created by cell nuclear transfer (cloning). The application of embryo protection laws from the nuclear syngamy stage onwards can regulate embryo and embryo-derived stem cell research while still preserving the current high standard and efficacy of infertility treatment, which is of immediate interest to millions of infertile couples throughout the world.

  16. 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. © 2016 Elsevier Inc. All rights reserved.

  17. 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.

  18. 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.

  19. 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.

  20. Human oocyte cryopreservation: a valid alternative to embryo cryopreservation?

    PubMed

    Tucker, Michael; Morton, Paula; Liebermann, Juergen

    2004-04-05

    Embryo cryopreservation has become an ethical necessity due to the way human in vitro fertilization (IVF) infertility therapy has developed. Limited embryonic implantation has by necessity driven IVF therapy to adopt ways to maximize the harvest of oocytes following ovarian hyperstimulation with its attendant risks. Collection of more oocytes has allowed more embryos to be generated to compensate for poor embryonic viability, often leading to transfer of multiple embryos to increase per transfer pregnancy rates. In an era of improving embryonic viability and prevailing trend toward single embryo transfers, production of excessive numbers of surplus embryos appears increasingly inappropriate. At which stage embryo cryopreservation can be undertaken most effectively remains controversial. Embryo cryopreservation nevertheless represents the current solution to the problem of excessive embryo production, but inherently raises ethical concerns for certain couples uncomfortable with what they might perceive to be "experimental" cryostorage, who in extreme circumstances may even choose to limit the number of oocytes inseminated to obviate the production of spare embryos. On a more practical level, cryostored embryos are co-owned by two people who may separate, and as such the embryos then face an uncertain fate, commonly decided in courts of law. Oocyte cryopreservation, if consistent and successful, offers a way to avoid the above complications of routine IVF therapy. Oocytes may need to be cryostored in the event of unforeseen non-production of sperm during IVF therapy, allowing a more measured consideration of donor sperm use or other means of sperm retrieval. Beyond IVF for infertility therapy using a couple's own gametes, oocyte cryopreservation provides a wonderful opportunity to optimize donor oocyte cryo-banking, reducing costs and improving convenience. Meanwhile, frozen embryo donation is an approach that many couples are uncomfortable with, and allows only for

  1. Heteroparental blastocyst production from microsurgically corrected tripronucleated human embryos.

    PubMed

    Escribá, María-José; Martín, Julio; Rubio, Carmen; Valbuena, Diana; Remohí, José; Pellicer, Antonio; Simón, Carlos

    2006-12-01

    To prove the efficiency of identification and removal of one of the surplus paternal pronuclei in dispermic IVF zygotes to obtain heteroparental blastocysts. Experimental. One hundred fourteen tripronucleated (3PN) embryos from conventional IVF. After informed and signed consent, the patients from Instituto Valenciano Infertilidad (IVI), Valencia, donated their abnormally fertilized embryos. Seventy-two embryos were diploidized by microsurgical removal of the pronucleus located at the farthest position to the second polar body. Forty-two 3PN embryos served as controls. Survival and correction rate; in vitro development up to the blastocyst stage; X, Y, and 18 chromosome determination by triple fluorescent in situ hybridization and, inheritance analysis for 10 polymorphic repeat regions using polymerase chain reaction (PCR) amplification and sequencing. Seventy-eight percent of 3PN zygotes (56/72) survived manipulation and eventually 51 zygotes had two pronuclei (71%). Forty-one percent of manipulated embryos progressed in vitro to the blastocyst stage (21/51). Fluorescent in situ hybridization analysis performed on eight manipulated embryos confirmed their diploid state; all four controls were triploid. Heteroparental inheritances were also confirmed in four of six manipulated embryos. Heteroparental blastocysts can be derived from corrected dispermic zygotes.

  2. 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.

  3. 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.

  4. A successful technique for the preservation of rabbit embryos.

    PubMed

    Prins, J B; Fox, R R

    1984-10-01

    A technique for successfully freezing, thawing and transferring rabbit embryos has been developed. Morula stage embryos were collected from super-ovulated female rabbits by flushing both oviducts and uterine horns with a tissue culture medium. Well developed, viable embryos were then transferred to freezing vials and a cryoprotectant, dimethyl sulfoxide (DMSO) was added in several steps to bring its final concentration to 1.6 molar. To freeze the embryos the temperature was lowered slowly (either 0.5 degrees C/min or 1.0 degrees C/min) to -80 degrees C at which point the vials were transferred directly to liquid nitrogen (-196 degrees C). Thawing was done at 8 degrees C/min. After thawing, phosphate buffered saline was added in a stepwise manner to dilute the DMSO. The thawed embryos were then cultured at 37 degrees C. Transfer of the embryos was accomplished by laparotomizing a pseudopregnant doe and introducing the embryos into the fimbriated ends of the oviducts. The 101 positively transferred embryos resulted in 45 implantations and 34 live born young.

  5. 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.

  6. Human embryo research and the 14-day rule.

    PubMed

    Pera, Martin F

    2017-06-01

    In many jurisdictions, restrictions prohibit the culture of human embryos beyond 14 days of development. However, recent reports describing the successful maintenance of embryos in vitro to this stage have prompted many in the field to question whether the rule is still appropriate. This Spotlight article looks at the original rationale behind the 14-day rule and its relevance today in light of advances in human embryo culture and in the derivation of embryonic-like structures from human pluripotent stem cells. © 2017. Published by The Company of Biologists Ltd.

  7. The avian embryo responding to microgravity of space flight

    NASA Techni