WorldWideScience

Sample records for two-cell stage embryos

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

    Science.gov (United States)

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

    2011-03-01

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

  2. Factors affecting the cryosurvival of mouse two-cell embryos.

    Science.gov (United States)

    Critser, J K; Arneson, B W; Aaker, D V; Huse-Benda, A R; Ball, G D

    1988-01-01

    A series of 4 experiments was conducted to examine factors affecting the survival of frozen-thawed 2-cell mouse embryos. Rapid addition of 1.5 M-DMSO (20 min equilibration at 25 degrees C) and immediate, rapid removal using 0.5 M-sucrose did not alter the frequency (mean +/- s.e.m.) of blastocyst development in vitro when compared to untreated controls (90.5 +/- 2.7% vs 95.3 +/- 2.8%). There was an interaction between the temperature at which slow cooling was terminated and thawing rate. Termination of slow cooling (-0.3 degrees C/min) at -40 degrees C with subsequent rapid thawing (approximately 1500 degrees C/min) resulted in a lower frequency of blastocyst development than did termination of slow cooling at -80 degrees C with subsequent slow thawing (+8 degrees C/min) (36.8 +/- 5.6% vs 63.9 +/- 5.7%). When slow cooling was terminated between -40 and -60 degrees C, higher survival rates were achieved with rapid thawing. When slow cooling was terminated below -60 degrees C, higher survival rates were obtained with slow thawing rates. In these comparisons absolute survival rates were highest among embryos cooled below -60 degrees C and thawed slowly. However, when slow cooling was terminated at -32 degrees C, with subsequent rapid warming, survival rates were not different from those obtained when embryos were cooled to -80 degrees C and thawed slowly (52.4 +/- 9.5%, 59.5 +/- 8.6%). These results suggest that optimal cryosurvival rates may be obtained from 2-cell mouse embryos by a rapid or slow thawing procedure, as has been found for mouse preimplantation embryos at later stages.(ABSTRACT TRUNCATED AT 250 WORDS)

  3. Nuclear reprogramming by interphase cytoplasm of two-cell mouse embryos.

    Science.gov (United States)

    Kang, Eunju; Wu, Guangming; Ma, Hong; Li, Ying; Tippner-Hedges, Rebecca; Tachibana, Masahito; Sparman, Michelle; Wolf, Don P; Schöler, Hans R; Mitalipov, Shoukhrat

    2014-05-01

    Successful mammalian cloning using somatic cell nuclear transfer (SCNT) into unfertilized, metaphase II (MII)-arrested oocytes attests to the cytoplasmic presence of reprogramming factors capable of inducing totipotency in somatic cell nuclei. However, these poorly defined maternal factors presumably decline sharply after fertilization, as the cytoplasm of pronuclear-stage zygotes is reportedly inactive. Recent evidence suggests that zygotic cytoplasm, if maintained at metaphase, can also support derivation of embryonic stem (ES) cells after SCNT, albeit at low efficiency. This led to the conclusion that critical oocyte reprogramming factors present in the metaphase but not in the interphase cytoplasm are 'trapped' inside the nucleus during interphase and effectively removed during enucleation. Here we investigated the presence of reprogramming activity in the cytoplasm of interphase two-cell mouse embryos (I2C). First, the presence of candidate reprogramming factors was documented in both intact and enucleated metaphase and interphase zygotes and two-cell embryos. Consequently, enucleation did not provide a likely explanation for the inability of interphase cytoplasm to induce reprogramming. Second, when we carefully synchronized the cell cycle stage between the transplanted nucleus (ES cell, fetal fibroblast or terminally differentiated cumulus cell) and the recipient I2C cytoplasm, the reconstructed SCNT embryos developed into blastocysts and ES cells capable of contributing to traditional germline and tetraploid chimaeras. Last, direct transfer of cloned embryos, reconstructed with ES cell nuclei, into recipients resulted in live offspring. Thus, the cytoplasm of I2C supports efficient reprogramming, with cell cycle synchronization between the donor nucleus and recipient cytoplasm as the most critical parameter determining success. The ability to use interphase cytoplasm in SCNT could aid efforts to generate autologous human ES cells for regenerative

  4. The Effects of Ethanol and Strontium on Growth and Development of Two-Cell Arrested Mouse Embryos

    Directory of Open Access Journals (Sweden)

    Mohammad Reza Darabi

    2012-01-01

    Full Text Available Background: Arresting at a certain stage of development like the two-cell stage could be one of the causes of infertility. The aim of this study is to evaluate the effects of ethanol and strontium on growth and development of mice embryos arrested at the two-cell stage.Materials and Methods: In this experimental study, female mice were coupled with a male following superovulation. Positive vaginal plug mice were sacrificed 48 hours after human chorionic gonadotropin (hCG injection. Two-cell embryos were transferred to M16 medium and divided to four groups. The first control group was incubated without any exposure to low temperatures. Groups 2, 3 and 4 were exposed to 4°C for 24 hours. The second control group was incubated immediately, while the third and fourth groups were exposed to 10 mM strontium for five minutes and 0.1% ethanol for a further five minutes. Growth rate and developmental parameters of embryos were analyzed by one-way ANOVA. The significant difference between the groups was determined by Post Hoc.Results: The data shows that developmental rate is decreased significantly by 4°C exposure. The mean percentage of degenerated embryo was significantly different between groups but the mean cleavage rate was not significantly different. The mean percent of morula, blastocyst and hatched blastocyst formation were significantly different between groups during a 120 hours study post hCG injection.Conclusion: The effect of strontium and ethanol on arrested two-cell embryos had no significant effect on the mean percentage of morula, but ethanol treatment significantly increased the percentage of blastocyst and hatched blastocyst formation compared to strontium.

  5. Chromosomal mosaicism in mouse two-cell embryos after paternal exposure to acrylamide

    Energy Technology Data Exchange (ETDEWEB)

    Marchetti, Francesco; Bishop, Jack; Lowe, Xiu; Wyrobek, Andrew J

    2008-10-14

    Chromosomal mosaicism in human preimplantation embryos is a common cause ofspontaneous abortions, however, our knowledge of its etiology is limited. We used multicolor fluorescence in situ hybridization (FISH) painting to investigate whether paternally-transmitted chromosomal aberrations result in mosaicism in mouse 2-cell embryos. Paternal exposure to acrylamide, an important industrial chemical also found in tobacco smoke and generated during the cooking process of starchy foods, produced significant increases in chromosomally defective 2-cell embryos, however, the effects were transient primarily affecting the postmeiotic stages of spermatogenesis. Comparisons with our previous study of zygotes demonstrated similar frequencies of chromosomally abnormal zygotes and 2-cell embryos suggesting that there was no apparent selection against numerical or structural chromosomal aberrations. However, the majority of affected 2-cell embryos were mosaics showing different chromosomal abnormalities in the two blastomeric metaphases. Analyses of chromosomal aberrations in zygotes and 2-cell embryos showed a tendency for loss of acentric fragments during the first mitotic division ofembryogenesis, while both dicentrics and translocations apparently underwent propersegregation. These results suggest that embryonic development can proceed up to the end of the second cell cycle of development in the presence of abnormal paternal chromosomes and that even dicentrics can persist through cell division. The high incidence of chromosomally mosaic 2-cell embryos suggests that the first mitotic division of embryogenesis is prone to missegregation errors and that paternally-transmitted chromosomal abnromalities increase the risk of missegregation leading to embryonic mosaicism.

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

    Science.gov (United States)

    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.

  7. Optimal developmental stage for vitrification of parthenogenetically activated porcine embryos

    DEFF Research Database (Denmark)

    Li, Rong; Li, Juan; Liu, Ying;

    2012-01-01

    The objective of this experiment was to determine the optimal developmental stage to vitrify in-vitro cultured porcine parthenogenetically activated (PA) embryos. Embryos were vitrified by Cryotop on Day 4, 5 or 6 after oocyte activation (Day 0), and immediately after warming they were either time......-lapse monitored for 24 h or analyzed by diffential staining. After warming, the embryos had to be cultured for at least 8 h before their survival rates were stabilized. Both the survival rate and 8 h and the hatching rate at 24 h of Day 4 embryos were significantly higher than those vitrified on Day5 or Day 6 (P...

  8. Effects of melatonin on in vitro development of mouse two-cell embryos cultured in HTF medium.

    Science.gov (United States)

    Tian, Xiu-Zhi; Wen, Qing; Shi, Jian-Min; Liang-Wang; Zeng, Shen-Ming; Tian, Jian-Hui; Zhou, Guang-Bin; Zhu, Shi-En; Liu, Guo-Shi

    2010-01-01

    Melatonin is capable of improving the developmental capacity of ovine, porcine and bovine embryos in vitro. However, whether melatonin possesses similar benefits to the in vitro mouse embryonic development has yet to be determined. In this study, we assessed the effects of various concentrations of melatonin (10-13 to 10-3 M) on the in-vitro development of mouse embryos cultured in HTF medium for 96 hr; embryos cultured without melatonin were used as control. The in vitro development of mouse two-cell embryos significantly benefited from treatment with melatonin in a concentration-dependent manner. The effects of melatonin on the rates of blastocyst formation, hatching/hatched blastocysts and cell number per blastocyst were bi-phasic; all significantly increased by melatonin at 10-13 to 10-5 M and decreased by melatonin at 10-3 M. Maximal benefit of melatonin on in vitro mouse 2-cell embryo development was achieved at a concentration of 10-9 M. In comparison to control, 10-9 M melatonin increased blastocyst formation rate from 48.08 +/- 5.25% to 82.08 +/- 2.34% (p HTF medium.

  9. Stage-specific proteome signatures in early bovine embryo development.

    Science.gov (United States)

    Deutsch, Daniela R; Fröhlich, Thomas; Otte, Kathrin A; Beck, Andrea; Habermann, Felix A; Wolf, Eckhard; Arnold, Georg J

    2014-10-03

    Development of early embryonic stages before activation of the embryonic genome depends on sufficiently stored products of the maternal genome, adequate recruitment and degradation of mRNAs, as well as activation, deactivation, and relocation of proteins. By application of an isobaric tagging for relative and absolute quantification (iTRAQ)-based approach, the proteomes of bovine embryos at the zygote and 2-cell and 4-cell stage with MII oocytes as a reference were quantitatively analyzed. Of 1072 quantified proteins, 87 differed significantly in abundance between the four stages. The proteomes of 2-cell and 4-cell embryos differed most from the reference MII oocyte, and a considerable fraction of proteins continuously increased in abundance during the stages analyzed, despite a strongly attenuated rate of translation reported for this period. Bioinformatic analysis revealed particularly interesting proteins involved in the p53 pathway, lipid metabolism, and mitosis. Verification of iTRAQ results by targeted SRM (selected reaction monitoring) analysis revealed excellent agreement for all five proteins analyzed. By principal component analysis, SRM quantifications comprising a panel of only five proteins were shown to discriminate between all four developmental stages analyzed here. For future experiments, an expanded SRM protein panel will provide the potential to detect developmental disturbances with high sensitivity and enable first insights into the underlying molecular pathways.

  10. Extended Culture of Early Stage Embryos in Frozen-thawed Cycles

    Institute of Scientific and Technical Information of China (English)

    Hong-bo WANG; Yan-hui LI

    2009-01-01

    Objective To investigate the impact of extended culture of early stage embryos on pregnancy outcome of frozen embryo transfer (FET).Methods The survival rates of embryos after thawing and pregnancy outcome following FET were compared retrospectively between zygote and cleavage embryos which cultured to cleavage stage or extended cultured to blastocysts. Results A total of 425 zygote embryos in 67 cycles were thawed. After thawing, the survival rate was 94.4% and with an average transfer of 2.8 embryos, the clinical pregnancy rate was 55.2% (37/67). In 222 FET cycles, totally 1 270 cleavage stage embryos were thawed and the overall survival rates were 80.3%. With an average transfer of 2.7 embryos, the clinical pregnancy rate was 55.4% (123/222). A significantly lower percentage of degenerated embryos were found for zygotes (5.6%) than that for cleavage stage embryos (19.7%) (P0.05). Conclusion Although the clinical pregnancy rate was not different between patients with freeze-thaw zygote and cleavage stage embryo transfer, higher survival rate for zygote was shown compared with that for cleavage stage embryo. However, the present studies did not demonstrate that extended culture thawing embryos to blastocyst could achieve favor clinical outcome.

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

    OpenAIRE

    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 ...

  12. The effects of MRI on mouse embryos during fetal stage

    Energy Technology Data Exchange (ETDEWEB)

    Nakamura, Takashi; Sakazaki, Takahiko; Itokawa, Yuka [Suzuka University of Medical Science, Koriyama (Japan)] (and others)

    2006-06-15

    The effects of Magnetic Resonance Imaging (MRI) on mouse embryos at the early stage of organogenesis were investigated. Pregnant ICR mice were exposed on day 8 of gestation to MRI at 0.5 T for 0.5 hour to 3 hours. The mortality rates of embryos or fetuses, the incidence of external malformations, fetal body weight and sex ratio were observed at day 18 of gestation. A significant increase in embryonic mortality was observed after exposure to either 0.5 T MRI for 0.5 hour or 2 hours. However, the exposure to MRI for 1 hour or 3 hours did not induce any significant increase in embryonic mortality when compared with control. External malformations such as exencephaly, cleft palate and anomalies of tail were observed in all experimental groups exposed to each MRI. A statistically significant increase of external malformations was observed in all groups treated with 0.5 T MRI for 0.5 hour and 3 hours. The incidence of external malformations in the mice group exposed to 0.5 T MRI for 0.5-hour was found to be higher than those of mice group exposed to 0.5 T MRI for 2 hours. The effects of MRI on the external malformations might not to be dose-dependent. There was no statistically significant difference in fetal body weight and sex ratio among each MRI exposure groups.

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

    Science.gov (United States)

    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.

  14. A quantification model for apoptosis in mouse embryos in the early stage of fetation

    Institute of Scientific and Technical Information of China (English)

    WANG PengFei; FU JianHua; MA WanYun; CHEN DieYan; Lü DanYu; BAI WenJia

    2009-01-01

    Apoptosis is the most important inducement and modulator for embryos in the early stage of fetation, i.e. after the 8-cell stage, mostly the morula and blastula stage, to proceed to the stage of nonlinear development. Using a two-photon laser scanning microscopy (TPLSM) system, we obtained 3-dimensional (3D) fluorescent images of preimplantation mouse embryos. A model for quantification was established. The statistical results for the spatial location of apoptosis bodies in embryos was obtained following image processing, as well as investigation of the kinetics of apoptosis. It was found that most (70%) apoptosis occurred in the trophectoderm, and the departure between the centroid and geometric center of embryos had a step transition when embryos developed into the 32-cell stage,which was consistent with the theoretical prediction that the blastocele would induce a symmetry break of the distribution of cells in embryos.

  15. A quantification model for apoptosis in mouse embryos in the early stage of fetation

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Apoptosis is the most important inducement and modulator for embryos in the early stage of fetation, i.e. after the 8-cell stage, mostly the morula and blastula stage, to proceed to the stage of nonlinear development. Using a two-photon laser scanning microscopy (TPLSM) system, we obtained 3-dimensional (3D) fluorescent images of preimplantation mouse embryos. A model for quantification was established. The statistical results for the spatial location of apoptosis bodies in embryos was obtained following image processing, as well as investigation of the kinetics of apoptosis. It was found that most (70%) apoptosis occurred in the trophectoderm, and the departure between the centroid and geometric center of embryos had a step transition when embryos developed into the 32-cell stage, which was consistent with the theoretical prediction that the blastocele would induce a symmetry break of the distribution of cells in embryos.

  16. Effect of Different Concentrations of Melatonin on Live Births Resulting from the Transfer of Two-Cell Embryos of NMRI Mice

    Directory of Open Access Journals (Sweden)

    Mahdi Saadati

    2014-12-01

    Full Text Available Background & objectives : Infertility is a global problem affecting millions of men and women in developed and developing countries. In this regard, in-vitro fertilization (IVF plays an important role in improving the quality of life in infertile patients. However, studies have shown that the implantation failure in IVF is the main challenge of this procedure. Melatonin can increase the survival rate of embryos and IVF success rate through eliminating free radicals and removing reactive oxygen species. So, this study is conducted to investigate the effects of different concentrations of melatonin on the rate of newborns of mice following transfer oftwo-cell embryos .   Methods : In this study, female mice with average age of six to eight weeks were superovulated by administering pregnant mares serum gonadotropin (PMSG intraperitoneally (7.5 IU. ip, and followed after 48h by human chorionic gonadotropin (hCG (7.5 IU. ip. Two-cell mouse embryos were obtained from female mice oviduct after 48 h. The embryos transferred bilaterally into pseudopregnant mice of the same strain through surgical procedure and 8-14 embryos were transferred to each tube. The study included 4 treatment groups and one control group (6 mice in each group. The treatment groups were exposed to subcutaneous injection of concentrations of 100 µm , 10 µm , 1 µm and 100 nm of melatonin. After the cesarean on 18th day of pregnancy, the percentage of live births was assessed. The outcomes of the live birth rate were as­sessed using the chi-square test and statistical analyses were carried out using SPSS version 16.0. Percentage of live birth was calculated and compared with the control group.   Results: A total of 701 two-cell mouse embryos were transferred into one control group and four experimental groups. The number and percentage of live births at concentrations of 100 µm and 10 µm of melatonin and the control groups were 21 (15.55%, 13 (9.15% and 9 (6

  17. Influence of recipient cytoplasm cell stage on transcription in bovine nucleus transfer embryos

    DEFF Research Database (Denmark)

    Smith, Steven D.; Soloy, Eva; Kanka, Jiri

    1996-01-01

    relies upon maternally derived RNA transcripts up to the 8-cell stage, at which time it begins to transcribe its own RNA. In this experiment, RNA synthesis was detected in nucleus transfer embryos (NTE) and control embryos by pulsing with 3H-uridine, fixation, and autoradiography on semithin sections...... of maturation. Control in-vitro-produced embryos were 3H-uridine-labelled and fixed at the 2-, 4-, early 8-, and late 8-cell stages. NTE were similarly prepared at 1, 3, and 20 hr postfusion and at the 2-, 4-, and 8-cell stages. In the control embryos, RNA synthesis was absent in the 2-, 4-, and early 8-cell...

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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).

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

    Science.gov (United States)

    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...

  1. Relationship Between Morphology, Euploidy and Implantation Potential of Cleavage and Blastocyst Stage Embryos

    Directory of Open Access Journals (Sweden)

    Gaurav Majumdar

    2017-01-01

    Full Text Available Aim: The aim of this study was to investigate the relationship between the morphology, euploidy and implantation rate of cleavage stage and blastocyst stage embryos. Setting: Institution-based, tertiary care in-vitro fertilization centre. Study Design: This study included a retrospective data analysis of 306 embryos: 154 cleavage stage embryos and 152 blastocysts that underwent biopsy on day 3 and day 5/6, respectively, which were subsequently screened for aneuploidy by array comparative genomic hybridization analysis. Materials and Methods: Both cleavage stage and blastocyst stage embryos were categorized according to their morphology into the following three groups: good, average and poor. In addition, blastocysts were categorized into day 5 and day 6 embryos on the basis of their developmental rate. Results: The euploidy rate was found to be significantly higher for blastocysts with good morphology as compared to those with poor morphology, with 73.2, 50 and 40.5% euploid embryos in the good, average and poor morphology groups, respectively (P = 0.001. No significant association was found between day 3 embryo morphology and euploidy rates with 40.6, 29.3 and 25.8% euploid embryos in the three groups, respectively (P = 0.254. The implantation rates, as per morphology, for the transferred euploid cleavage stage and blastocyst stage embryos were 43.8, 37.5 and 0% (P = 0.354 and 51.7, 71.4 and 66.7% (P = 0.562 in the good, average and poor morphology groups, respectively. The euploidy rate for day 5 blastocysts was significantly higher (70% vs. 34.1%, P < 0.001 than that of day 6 blastocysts, but the implantation rate was similar in both the groups (58.8 and 50%, respectively. The miscarriage rates for the euploid cleavage stage and the blastocysts stage embryos were 18.2 and 8.3% (P = 0.575, respectively. Conclusion: Blastocyst morphology and the rate of development were found to be significantly associated with euploidy

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

    Science.gov (United States)

    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.

  3. The Early Stages of Heart Development: Insights from Chicken Embryos

    Directory of Open Access Journals (Sweden)

    Johannes G. Wittig

    2016-04-01

    Full Text Available The heart is the first functioning organ in the developing embryo and a detailed understanding of the molecular and cellular mechanisms involved in its formation provides insights into congenital malformations affecting its function and therefore the survival of the organism. Because many developmental mechanisms are highly conserved, it is possible to extrapolate from observations made in invertebrate and vertebrate model organisms to humans. This review will highlight the contributions made through studying heart development in avian embryos, particularly the chicken. The major advantage of chick embryos is their accessibility for surgical manipulation and functional interference approaches, both gain- and loss-of-function. In addition to experiments performed in ovo, the dissection of tissues for ex vivo culture, genomic, or biochemical approaches is straightforward. Furthermore, embryos can be cultured for time-lapse imaging, which enables tracking of fluorescently labeled cells and detailed analysis of tissue morphogenesis. Owing to these features, investigations in chick embryos have led to important discoveries, often complementing genetic studies in mice and zebrafish. As well as including some historical aspects, we cover here some of the crucial advances made in understanding early heart development using the chicken model.

  4. Repression of global protein synthesis by Eif1a-like genes that are expressed specifically in the two-cell embryos and the transient Zscan4-positive state of embryonic stem cells.

    Science.gov (United States)

    Hung, Sandy S C; Wong, Raymond C B; Sharov, Alexei A; Nakatake, Yuhki; Yu, Hong; Ko, Minoru S H

    2013-08-01

    Mouse embryonic stem (ES) cells are prototypical stem cells that remain undifferentiated in culture for long periods, yet maintain the ability to differentiate into essentially all cell types. Previously, we have reported that ES cells oscillate between two distinct states, which can be distinguished by the transient expression of Zscan4 genes originally identified for its specific expression in mouse two-cell stage embryos. Here, we report that the nascent protein synthesis is globally repressed in the Zscan4-positive state of ES cells, which is mediated by the transient expression of newly identified eukaryotic translation initiation factor 1A (Eif1a)-like genes. Eif1a-like genes, clustered on Chromosome 12, show the high sequence similarity to the Eifa1 and consist of 10 genes (Eif1al1-Eif1al10) and 9 pseudogenes (Eif1al-ps1-Eif1al-ps9). The analysis of the expressed sequence tag database showed that Eif1a-like genes are expressed mostly in the two-cell stage mouse embryos. Microarray analyses and quantitative real-time polymerase chain reaction analyses show that Eif1a-like genes are expressed specifically in the Zscan4-positive state of ES cells. These results indicate a novel mechanism to repress protein synthesis by Eif1a-like genes and a unique mode of protein synthesis regulation in ES cells, which undergo a transient and reversible repression of global protein synthesis in the Zscan4-positive state.

  5. Repression of Global Protein Synthesis by Eif1a-Like Genes That Are Expressed Specifically in the Two-Cell Embryos and the Transient Zscan4-Positive State of Embryonic Stem Cells

    Science.gov (United States)

    Hung, Sandy S. C.; Wong, Raymond C. B.; Sharov, Alexei A.; Nakatake, Yuhki; Yu, Hong; Ko, Minoru S. H.

    2013-01-01

    Mouse embryonic stem (ES) cells are prototypical stem cells that remain undifferentiated in culture for long periods, yet maintain the ability to differentiate into essentially all cell types. Previously, we have reported that ES cells oscillate between two distinct states, which can be distinguished by the transient expression of Zscan4 genes originally identified for its specific expression in mouse two-cell stage embryos. Here, we report that the nascent protein synthesis is globally repressed in the Zscan4-positive state of ES cells, which is mediated by the transient expression of newly identified eukaryotic translation initiation factor 1A (Eif1a)-like genes. Eif1a-like genes, clustered on Chromosome 12, show the high sequence similarity to the Eifa1 and consist of 10 genes (Eif1al1–Eif1al10) and 9 pseudogenes (Eif1al-ps1–Eif1al-ps9). The analysis of the expressed sequence tag database showed that Eif1a-like genes are expressed mostly in the two-cell stage mouse embryos. Microarray analyses and quantitative real-time polymerase chain reaction analyses show that Eif1a-like genes are expressed specifically in the Zscan4-positive state of ES cells. These results indicate a novel mechanism to repress protein synthesis by Eif1a-like genes and a unique mode of protein synthesis regulation in ES cells, which undergo a transient and reversible repression of global protein synthesis in the Zscan4-positive state. PMID:23649898

  6. Monozygotic Triplets and Dizygotic Twins following Transfer of Three Poor-Quality Cleavage Stage Embryos

    Directory of Open Access Journals (Sweden)

    Reshef Tal

    2012-01-01

    Full Text Available Background. Assisted reproductive technology has been linked to the increased incidence of monozygotic twinning. It is of clinical importance due to the increased risk of complications in multiple pregnancies in general and in monozygotic twins in particular. Case. A 29-year-old female, nulligravida underwent her first IVF cycle. Three poor-quality cleavage stage embryos were transferred resulting in monochorionic triamniotic triplets and dichorionic diamniotic twins. Selective embryo reduction was performed at 12 weeks leaving dichorionic twins. The patient underwent emergency cesarean section due to preterm labor and nonreassuring fetal heart tracing at 30 weeks of gestation. Conclusion. Our case emphasizes that even embryos with significant morphological abnormalities should be considered viable and the possibility of simultaneous spontaneous embryo splitting must be factored into determining number of embryos to transfer.

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

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Ting; Zhao, Jing [Key Laboratory of Yangtze River Water Environment, Ministry of Education, College of Environmental Science and Technology, Tongji University, Shanghai 200092 (China); Hu, Ping [Key Laboratory of Model Animal for Disease Study, Ministry of Education, Model Animal Research Center, Nanjing University, Nanjing 210061 (China); State Key Laboratory of Reproductive Medicine, Department of Prenatal Diagnosis, Nanjing Maternity and Child Health Care Hospital Affiliated to Nanjing Medical University, Nanjing 210029 (China); Dong, Zhangji; Li, Jingyun [Key Laboratory of Model Animal for Disease Study, Ministry of Education, Model Animal Research Center, Nanjing University, Nanjing 210061 (China); Zhang, Hongchang [Key Laboratory of Yangtze River Water Environment, Ministry of Education, College of Environmental Science and Technology, Tongji University, Shanghai 200092 (China); Yin, Daqiang, E-mail: yindq@tongji.edu.cn [Key Laboratory of Yangtze River Water Environment, Ministry of Education, College of Environmental Science and Technology, Tongji University, Shanghai 200092 (China); Zhao, Qingshun, E-mail: qingshun@nju.edu.cn [Key Laboratory of Model Animal for Disease Study, Ministry of Education, Model Animal Research Center, Nanjing University, Nanjing 210061 (China)

    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

  8. Biopsy of human morula-stage embryos: outcome of 215 IVF/ICSI cycles with PGS.

    Directory of Open Access Journals (Sweden)

    Elena E Zakharova

    Full Text Available Preimplantation genetic diagnosis (PGD is commonly performed on biopsies from 6-8-cell-stage embryos or blastocyst trophectoderm obtained on day 3 or 5, respectively. Day 4 human embryos at the morula stage were successfully biopsied. Biopsy was performed on 709 morulae from 215 ICSI cycles with preimplantation genetic screening (PGS, and 3-7 cells were obtained from each embryo. The most common vital aneuploidies (chromosomes X/Y, 21 were screened by fluorescence in situ hybridization (FISH. No aneuploidy was observed in 72.7% of embryos, 91% of those developed to blastocysts. Embryos were transferred on days 5-6. Clinical pregnancy was obtained in 32.8% of cases, and 60 babies were born. Patients who underwent ICSI/PGS treatment were compared with those who underwent standard ICSI treatment by examining the percentage of blastocysts, pregnancy rate, gestational length, birth height and weight. No significant differences in these parameters were observed between the groups. Day 4 biopsy procedure does not adversely affect embryo development in vitro or in vivo. The increased number of cells obtained by biopsy of morulae might facilitate diagnostic screening. There is enough time after biopsy to obtain PGD results for embryo transfer on day 5-6 in the current IVF cycle.

  9. 4D atlas of the mouse embryo for precise morphological staging.

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Tachi, Norihide; Aoyama, Mitsuko (Nagoya City Univ. Medical School (Japan))

    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.

  11. Temporal and Developmental-Stage Variation in the Occurrence of Mitotic Errors in Tripronuclear Human Preimplantation Embryos

    NARCIS (Netherlands)

    Mantikou, Eleni; van Echten-Arends, Jannie; Sikkema-Raddatz, Birgit; van der Veen, Fulco; Repping, Sjoerd; Mastenbroek, Sebastiaan

    2013-01-01

    Mitotic errors during early development of human preimplantation embryos are common, rendering a large proportion of embryos chromosomally mosaic. It is also known that the percentage of diploid cells in human diploid-aneuploid mosaic embryos is higher at the blastocyst than at the cleavage stage. I

  12. Confinement and clearance of OCT4 in the porcine embryo at stereomicroscopically defined stages around gastrulation

    DEFF Research Database (Denmark)

    Vejlsted, Morten; Offenberg, Hanne Kjær; Thorup, Flemming;

    2006-01-01

    In the areas of developmental biology and embryonic stem cell research, reliable molecular markers of pluripotency and early lineage commitment are sparse in large animal species. In this study, we present morphological and immunohistochemical findings on the porcine embryo in the period around...... gastrulation, days 8-17 postinsemination, introducing a steromicroscopical staging system in this species. In embryos at the expanding hatched blastocyst stage, OCT4 is confined to the inner cell mass. Following detachment of the hypoblast, and formation of the embryonic disk, this marker of pluripotency...

  13. Regulation of somatic embryo development in Norway spruce (Picea abies). A molecular approach to the characterization of specific developmental stages

    Energy Technology Data Exchange (ETDEWEB)

    Sabala, I. [Swedish Univ. of Agricultural Sciences, Uppsala (Sweden). Dept. of Forest Genetics

    1998-12-31

    Embryo development is a complex process involving a set of strictly regulated events. The regulation of these events is poorly understood especially during the early stages of embryo development. Somatic embryos go through the same developmental stages as zygotic embryos making them an ideal model system for studying the regulation of embryo development. We have used embryogenic cultures of Picea abies to study some aspects of the regulation of embryo development in gymnosperms. The bottle neck during somatic embryogenesis is the switch from the proliferation stage to the maturation stage. This switch is initiated by giving somatic embryos a maturation treatment i.e. the embryos are treated with abscisic acid (ABA). Somatic embryos which respond to ABA by forming mature somatic embryos were stimulated to secret a 70 kDa protein, AF70. The af70 gene was isolated and characterised. The expression of the af70 gene was constitutive in embryos but was highly ABA-induced in seedlings. Moreover, expression of this gene was stimulated during cold acclimation of Picea abies seedlings. A full length Picea abies cDNA clone Pa18, encoding a protein with the characteristics of plant lipid transfer proteins (LTPs), was isolated and characterised. The Pa18 gene is constitutively expressed in embryogenic cultures of Picea abies representing different stages of development as well as in nonembryogenic callus and seedlings. In situ hybridization showed that Pa18 gene is expressed in all embryonic cells of proliferating somatic embryos but the expression of the gene in mature somatic and zygotic embryos is restricted to the outer cell layer. Southern blot analysis at different stringencies was consistent with a single gene. An alteration in expression of Pa18 causes disturbance in the formation of the proper outer cell layer in the maturing somatic embryos. In addition to its influence on embryo development the Pa18 gene product also inhibits growth of Agrobacterium tumefaciens 195

  14. Influence of recipient cytoplasm cell stage on transcription in bovine nucleus transfer embryos

    DEFF Research Database (Denmark)

    Smith, Steven D.; Soloy, Eva; Kanka, Jiri

    1996-01-01

    relies upon maternally derived RNA transcripts up to the 8-cell stage, at which time it begins to transcribe its own RNA. In this experiment, RNA synthesis was detected in nucleus transfer embryos (NTE) and control embryos by pulsing with 3H-uridine, fixation, and autoradiography on semithin sections...... of maturation. Control in-vitro-produced embryos were 3H-uridine-labelled and fixed at the 2-, 4-, early 8-, and late 8-cell stages. NTE were similarly prepared at 1, 3, and 20 hr postfusion and at the 2-, 4-, and 8-cell stages. In the control embryos, RNA synthesis was absent in the 2-, 4-, and early 8-cell...... stages, whereas in all late 8-cell stages, it was present. In NTE from nonactivated (MII phase) cytoplasts, there was a sharp decline in RNA synthesis at 1 hr and 3 hr after fusion and a total absence by 20 hr after fusion. In contrast, NTE from activated (S phase) cytoplasts exhibited continued high...

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

    Science.gov (United States)

    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.

  16. Cryotop vitrification for in vitro produced bovine and buffalo (Bubalus bubalis embryos at different stages of development

    Directory of Open Access Journals (Sweden)

    B. Gasparrini

    2010-02-01

    Full Text Available The aim of this study was to evaluate the possibility to vitrify in vitro produced (IVP buffalo and bovine embryos at different stages of development by an advanced version of the “minimal volume approaches”: the Cryotop method. In both experiments, the embryos were vitrified at the tight morula (TM, early blastocyst (eBl, blastocyst (Bl, expanded blastocyst (xBl and, only for buffalo, at the hatched blastocyst (hBl stage. After warming, the embryos were cultured in vitro for 24 hours. Stage of development affected the freezability of IVP embryos of both species with the highest embryo survival rates at advanced stages (xBl=76% and hBl=75% for buffalos and xBl=75% for bovine. These results suggest that Cryotop vitrification is an efficient method for buffalo and bovine IVP embryo cryopreservation.

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

    Science.gov (United States)

    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.

  18. Involvement of insulin in early development of mouse one-cell stage embryos

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Recent studies have suggested that growth factors and hormones play important roles in cell prolif-eration and differentiation during early embryonic development. In the present study, we examined the expression and localization of insulin in the mouse oocytes and one-cell stage embryos by quantitative ELISA, RT-PCR, Western blot and immunofluorescence. In the mouse oocytes and one-cell stage em-bryos, expression of insulin was uniformly distributed in the cytoplasm. We also examined the expres-sion, activity and localization of mTOR (mammalian target of rapamycin) and p70S6K. The expression of mTOR and p70S6K was not significantly different at the cell cycle of mouse one-cell stage embryos. mTOR and S6K were distributed evenly in the cytoplasm at G1, G2 and M phase phase, but at S phase, the distribution of mTOR and S6K was around the pronucleus. At different phases, the activity of mTOR fluctuated. We also used the PI3K specific inhibitor-Wortmannin to investigate the cleavage rate of eggs. The result showed that the rate obviously decreased. When the mTOR specific inhibitor Rapa-mycin was used, the first mitotic division of the mouse one-cell stage embryo was delayed. These re-sults suggested that insulin was expressed both in mouse oocytes and one-cell stage embryos, and may play functional roles in regulation of mouse early embryogenesis by activating the signal pathway of PI3K/PKB/mTOR/S6K.

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

    Science.gov (United States)

    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

  20. Nucleolar ultrastructure in bovine nuclear transfer embryos

    DEFF Research Database (Denmark)

    Kaňka, Jiří; Smith, Steven Dale; Soloy, Eva

    1999-01-01

    (nonactivated) or S phase (activated) cytoplasts. Control embryos were fixed at the two-, four-, early eight- and late eight-cell stages; nuclear transfer embryos were fixed at 1 and 3 hr post fusion and at the two-, four-, and eight-cell stages. Control embryos possessed a nucleolar precursor body throughout...... at 1 hr after fusion and, by 3 hr after fusion, it was restored again. At this time, the reticulated fibrillo-granular nucleolus had an almost round shape. The nucleolar precursor body seen in the two-cell stage nuclear transfer embryos consisted of intermingled filamentous components and secondary...... time intervals after fusion. In the two-cell stage nuclear transfer embryo, the originally reticulated nucleolus of the donor blastomere had changed into a typical nucleolar precursor body consisting of a homogeneous fibrillar structure. A primary vacuole appeared in the four-cell stage nuclear...

  1. Nucleolar ultrastructure in bovine nuclear transfer embryos

    DEFF Research Database (Denmark)

    Kaňka, Jiří; Smith, Steven Dale; Soloy, Eva

    1999-01-01

    (nonactivated) or S phase (activated) cytoplasts. Control embryos were fixed at the two-, four-, early eight- and late eight-cell stages; nuclear transfer embryos were fixed at 1 and 3 hr post fusion and at the two-, four-, and eight-cell stages. Control embryos possessed a nucleolar precursor body throughout...... at 1 hr after fusion and, by 3 hr after fusion, it was restored again. At this time, the reticulated fibrillo-granular nucleolus had an almost round shape. The nucleolar precursor body seen in the two-cell stage nuclear transfer embryos consisted of intermingled filamentous components and secondary...... time intervals after fusion. In the two-cell stage nuclear transfer embryo, the originally reticulated nucleolus of the donor blastomere had changed into a typical nucleolar precursor body consisting of a homogeneous fibrillar structure. A primary vacuole appeared in the four-cell stage nuclear...

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  6. Vitrification in sealed containers : Evaluation of a new technique (Rapid-i™) for cleavage stage embryos and blastocysts

    OpenAIRE

    Lannsjö, Christine

    2009-01-01

    Ovarian stimulation in assisted reproduction often leads to the production of a high number of oocytes. After fertilization of these oocytes, the resulting embryos can be cryopreserved for later use. Vitrification is a recently introduced method for cryostoring embryos, showing high survival rates for both cleavage stage embryos and blastocysts. Characteristic of vitrification are high concentrations of cryoprotectants and ultra fast freezing which makes the material glassily. A major concern...

  7. Dissection of Cell Division Processes in the One Cell Stage Caenorhabditis elegans Embryo by Mutational Analysis

    OpenAIRE

    Gönczy, Pierre; Schnabel, Heinke; Kaletta, Titus; Amores, Ana Duran; Hyman, Tony; Schnabel, Ralf

    1999-01-01

    To identify novel components required for cell division processes in complex eukaryotes, we have undertaken an extensive mutational analysis in the one cell stage Caenorhabditis elegans embryo. The large size and optical properties of this cell permit observation of cell division processes with great detail in live specimens by simple differential interference contrast (DIC) microscopy. We have screened an extensive collection of maternal-effect embryonic lethal mutations on chromosome III wi...

  8. Dissection of Cell Division Processes in the One Cell Stage Caenorhabditis elegans Embryo by Mutational Analysis

    Science.gov (United States)

    Gönczy, Pierre; Schnabel, Heinke; Kaletta, Titus; Amores, Ana Duran; Hyman, Tony; Schnabel, Ralf

    1999-01-01

    To identify novel components required for cell division processes in complex eukaryotes, we have undertaken an extensive mutational analysis in the one cell stage Caenorhabditis elegans embryo. The large size and optical properties of this cell permit observation of cell division processes with great detail in live specimens by simple differential interference contrast (DIC) microscopy. We have screened an extensive collection of maternal-effect embryonic lethal mutations on chromosome III with time-lapse DIC video microscopy. Using this assay, we have identified 48 mutations in 34 loci which are required for specific cell division processes in the one cell stage embryo. We show that mutations fall into distinct phenotypic classes which correspond, among others, to the processes of pronuclear migration, rotation of centrosomes and associated pronuclei, spindle assembly, chromosome segregation, anaphase spindle positioning, and cytokinesis. We have further analyzed pronuclear migration mutants by indirect immunofluorescence microscopy using antibodies against tubulin and ZYG-9, a centrosomal marker. This analysis revealed that two pronuclear migration loci are required for generating normal microtubule arrays and four for centrosome separation. All 34 loci have been mapped by deficiencies to distinct regions of chromosome III, thus paving the way for their rapid molecular characterization. Our work contributes to establishing the one cell stage C. elegans embryo as a powerful metazoan model system for dissecting cell division processes. PMID:10085292

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  11. AFSC/RACE/SAP/Foy: Effects of ocean acidification on embryo stages of Tanner crab: Kodiak Island, Alaska.

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — To study the effects of ocean acidification we examined the effects of ocean acidification on the embryo stages of the economically important southern Tanner crab,...

  12. Embryo splitting

    Directory of Open Access Journals (Sweden)

    Karl Illmensee

    2010-04-01

    Full Text Available Mammalian embryo splitting has successfully been established in farm animals. Embryo splitting is safely and efficiently used for assisted reproduction in several livestock species. In the mouse, efficient embryo splitting as well as single blastomere cloning have been developed in this animal system. In nonhuman primates embryo splitting has resulted in several pregnancies. Human embryo splitting has been reported recently. Microsurgical embryo splitting under Institutional Review Board approval has been carried out to determine its efficiency for blastocyst development. Embryo splitting at the 6–8 cell stage provided a much higher developmental efficiency compared to splitting at the 2–5 cell stage. Embryo splitting may be advantageous for providing additional embryos to be cryopreserved and for patients with low response to hormonal stimulation in assisted reproduction programs. Social and ethical issues concerning embryo splitting are included regarding ethics committee guidelines. Prognostic perspectives are presented for human embryo splitting in reproductive medicine.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  15. 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.

    Science.gov (United States)

    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 (Pcultured 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.

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

    Science.gov (United States)

    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.

  17. Developmental toxicity of cypermethrin in embryo-larval stages of zebrafish.

    Science.gov (United States)

    Shi, Xiangguo; Gu, Aihua; Ji, Guixiang; Li, Yuan; Di, Jing; Jin, Jing; Hu, Fan; Long, Yan; Xia, Yankai; Lu, Chuncheng; Song, Ling; Wang, Shoulin; Wang, Xinru

    2011-10-01

    Cypermethrin, a type II pyrethroid insecticide, is widely used throughout the world in agriculture, forestry, horticulture and homes. Though the neurotoxicity of cypermethrin has been thoroughly studied in adult rodents, little is so far available regarding the developmental toxicity of cypermethrin to fish in early life stages. To explore the potential developmental toxicity of cypermethrin, 4-h post-fertilization (hpf) zebrafish embryos were exposed to various concentrations of cypermethrin (0, 25, 50, 100, 200 and 400 μg L⁻¹) until 96 h. Among a suite of morphological abnormalities, the unique phenotype curvature was observed at concentrations as low as 25 μg L⁻¹. Studies revealed that 400 μg L⁻¹ cypermethrin significantly increased malondialdehyde production. In addition, activity of antioxidative enzymes including superoxide dismutase and catalase were significantly induced in zebrafish larvae in a concentration-dependent manner. To further investigate the toxic effects of cypermethrin on fish, acridine orange (AO) staining was performed at 400 μg L⁻¹ cypermethrin and the result showed notable signs of apoptosis mainly in the nervous system. Cypermethrin also down-regulated ogg1 and increased p53 gene expression as well as the caspase-3 activity. Our results demonstrate that cypermethrin was able to induce oxidative stress and produce apoptosis through the involvement of caspases in zebrafish embryos. In this study, we investigated the developmental toxicity of cypermethrin using zebrafish embryos, which could be helpful in fully understanding the potential mechanisms of cypermethrin exposure during embryogenesis and also suggested that zebrafish could serve as an ideal model for studying developmental toxicity of environmental contaminants.

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

    Science.gov (United States)

    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 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.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

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

    2002-01-01

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

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

    Science.gov (United States)

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

    2011-02-01

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

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Xiaowei He

    2016-12-01

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  5. Functional genomics of 5- to 8-cell stage human embryos by blastomere single-cell cDNA analysis.

    Directory of Open Access Journals (Sweden)

    Amparo Galán

    Full Text Available 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.

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

    Science.gov (United States)

    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

  7. Fast Cryopreservation of the Mammalian Embryos in Different Developmental Stages by 0.25 mL Straws Vitrification with One Equilibration Step

    Directory of Open Access Journals (Sweden)

    Ada Cean

    2010-05-01

    Full Text Available The aim of our study was to test the cryoprotective proprieties of 7 vitrification media, designed in our laboratory, using the 0.25 mL straws vitrification method, with one equilibration step. As biological material we used mouse females, age 2 months superovulated with 5UI PMSG (Pregnant Mare Serum Gonadotropine and 5 UI hCG (human Corionic Gonadotropine. For freezing we used embryos in three developmental stages: 2 cells, morula and blastocyst. After recovery, the embryos were placed in equilibration media, after 5 minutes, the embryos were introduced in straws, in vitrification media and plugged directly into liquid nitrogen. After vitrification the straws were thawed in water bath at 37°C, the embryos were rehydrated for 5 minutes and then in vitro cultured. The percent of embryos that rehydrated, resumed development and hatched were registered. The best results were obtained with embryos in morula stage that had a hatching rate of 20.83% when MV1 was used for vitrification. None of the embryos in 2 cells and blastocyst stage hatched after thawing and in vitro culture, regardless of the vitrification media used. From the vitrification media tested, the worst results were obtained with MV4 and MV6, none of the embryos reached hatching stage, regardless of the development stage. The vitrification method in 0.25 mL straws, with one equilibration step can be used for cryopreservation of the morula stage embryos, but is ineffective for vitrification of the 2 cells and blastocyst stage embryo. Media VM4 and VM6 are not suited for vitrification in 0.25 mL straws, with one equilibration step, of mouse embryos.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    Kyono, Koichi

    2013-08-01

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

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Eliana Beltrán-Pardo

    Full Text Available 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.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Jessica van Leeuwen

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

  14. Perkembangan Praimplantasi Embrio Mencit dengan Materi Genetik yang Berasal dari Parental, Maternal, dan Inti Sel Somatik (PRE-IMPLANTATION DEVELOPMENT OF MOUSE EMBRYO WITH GENETIC MATERIAL DERIVED FROM PARENTAL, MATERNAL AND SOMATIC CELL NUCLEUS

    Directory of Open Access Journals (Sweden)

    Harry Murti

    2014-05-01

    Full Text Available Cloned embryo and parthenogenetic embryo are a potential source of stem cells for regenerativemedicine. Stem cells derived from those embryos are expected to overcome the ethical issues to the use offertilization embryos for therapeutic purposes. The pre-implantation development is a critical step fordeveloping embryos reach the blastocyst stage. The objectives in vivo of this research are to produce mousecloned embryo, parthenogenetic embryo, and fertilized embryo and to study stages of  in vitro pre-implantation development culture. In vivo fertilized embryos, mouse oocytes, and cumulus cells were usedin this study. Treatment was performed on female mice superovulated with PMSG and hCG injections.Two-cell stage of in vivo fertilized embryos were collected on the second day post hCG injection. Clonedembryos were produced through Somatic Cell Nuclear Transfer (SCNT, which included enucleation, nucleartransfer and artificial activation. Parthenogenetic embryos were produced with artificial activationtechnique. The result of the research indicated that SCNT application was able to produce cloned embryos which could develop to blastocyst stage (3,2%. In addition, artificial activation of oocytes could produceparthenogenetic embryos which were able to develop up to the blastocyst stage (8,6%. In conclusion,efficiency level of parthenogenetic embryos that is able to reach the blastocyst stage was higher than in thecloned embryos. Fertilized embryos shows a better development and more efficient compared to in vitrocloned embryos and parthenogenetic embryos cultures.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Sakatani Miki

    2013-01-01

    Full Text Available Abstract 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 (PHSP90AA1 (PSOD1 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 networks involved in embryonic development. It is likely that

  17. A novel sperm-delivered toxin causes late-stage embryo lethality and transmission ratio distortion in C. elegans.

    Directory of Open Access Journals (Sweden)

    Hannah S Seidel

    2011-07-01

    Full Text Available 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.

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

    OpenAIRE

    Guimar?es, Fernando; Roque,Matheus; Valle, Marcello; Kostolias, Alessandra; Azevedo, Rodrigo A de; 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...

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    Schlisser, Ava E; Hales, Barbara F

    2013-08-01

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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2016-09-01

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

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

    Science.gov (United States)

    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 (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 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 division behaviors. In NB embryos, the blastocyst formation rate increased with cell number from 7.4% (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. [Rudimentary stages of the extremities of Scelotes gronovii (Daudin) embryos, a South African Scincidea reptile].

    Science.gov (United States)

    Raynaud, A; Van den Elzen, P

    1976-01-01

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

  6. Early aberrations in chromatin dynamics in embryos produced under In vitro conditions

    DEFF Research Database (Denmark)

    Deshmukh, Rahul Shahaji; Østrup, Olga; Strejcek, Frantisek;

    2012-01-01

    In vitro production of porcine embryos by means of in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT) is limited by great inefficienciy. The present study investigated chromatin and nucleolar dynamics in porcine embryos developed in vivo (IV) and compared this physiological...... standard to that of embryos produced by IVF, parthenogenetic activation (PA), or SCNT. In contrast to IV embryos, chromatin spatial and temporal dynamics in PA, IVF, and SCNT embryos were altered; starting with aberrant chromatin-nuclear envelope interactions at the two-cell stage, delayed chromatin...... decondensation and nucleolar development at the four-cell stage, and ultimately culminating in failure of proper first lineage segregation at the blastocyst stage, demonstrated by poorly defined inner cell mass. Interestingly, in vitro produced (IVP) embryos also lacked a heterochromatin halo around nucleolar...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    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

  8. Germination ecology of the endemic Iberian daffodil Narcissus radinganorum (Amaryllidaceae. Dormancy induction by cold stratification or desiccation in late stages of embryo growth

    Directory of Open Access Journals (Sweden)

    José María Herranz

    2015-04-01

    Full Text Available Aim of study: We studied the germination ecology of a threatened daffodil in order to develop a protocol to produce plants ex-situ from seeds, a key tool for population reinforcement. Area of study: Experiments were carried out both outdoors and in the laboratory in Albacete (Spain. Material and methods: Embryo length, radicle and shoot emergence were analyzed to determine the level of morphophysiological dormancy (MPD. Effects on germination of cold stratification or desiccation in late stages of embryo growth were also studied. Main results: Mean embryo length in fresh seeds was 1.36 mm, needing to grow up to 2.20 mm to be able to germinate. In the laboratory, embryo growth occurred during warm stratification (28/14, 25/10 ºC, and then radicle emerged when temperatures went down (15/4 ºC in darkness. Phenology study in outdoors conditions revealed that embryo grew during summer-early autumn, short time after seed dispersal in nature (i.e., May; radicle emerged in autumn. The shoot however did not emerge until late winter-early spring, because it was physiologically dormant and required a cold (5 ºC period of 30 days to break dormancy. Early cold temperatures interrupted the embryo growth and induced dormancy in seeds whose embryo had grown 30% with respect to the initial length. Desiccation in seeds whose embryo had grown 30% did not induce dormancy, but did it when the embryo growth reached 70%. Research highlights: Seeds of Narcissus radinganorum have deep simple epicotyl MPD.

  9. Effective cryopreservation of golden Syrian hamster embryos by open pulled straw vitrification.

    Science.gov (United States)

    Fan, Z; Meng, Q; Bunch, T D; White, K L; Wang, Z

    2016-02-01

    Golden Syrian hamster embryos are difficult to cryopreserve due to their high sensitivity to cryoprotectants and in vitro handling. The objective of this study is to develop a robust open pulled straw (OPS) vitrification technique for cryopreserving hamster embryos at various developmental stages. We first systematically tested the concentrations of cryoprotectants and the exposure times of two-cell embryos to various vitrification solutions. We identified pretreatment of two-cell embryos with 10% (v/v) ethylene glycol (EG) + 10% (v/v) dimethylsulfoxide (DMSO) for 30 s followed by exposure in the vitrification solution, EDFS30 (containing 15% EG + 15% DMSO), for 30 s before plunging into liquid nitrogen (two-step exposure method) as the optimal OPS vitrification protocol. We then investigated the resourcefulness of this protocol for vitrifying hamster embryos at different developmental stages. The results showed that high blastocyst rates from embryos vitrified at two-cell, four-cell, eight-cell, or morula stage (62%, 78%, 80%, or 72%, respectively), but not those verified at pronuclear (0%) or blastocyst stage (24%; P  0.05) from the 40% birth rate of the unvitrified controls. In conclusion, we have developed an effective two-step OPS vitrification protocol for hamster embryos.

  10. Runx expression is mitogenic and mutually linked to Wnt activity in blastula-stage sea urchin embryos.

    Directory of Open Access Journals (Sweden)

    Anthony J Robertson

    Full Text Available BACKGROUND: The Runt homology domain (Runx defines a metazoan family of sequence-specific transcriptional regulatory proteins that are critical for animal development and causally associated with a variety of mammalian cancers. The sea urchin Runx gene SpRunt-1 is expressed throughout the blastula stage embryo, and is required globally during embryogenesis for cell survival and differentiation. METHODOLOGY/PRINCIPAL FINDINGS: Depletion of SpRunt-1 by morpholino antisense-mediated knockdown causes a blastula stage deficit in cell proliferation, as shown by bromodeoxyuridine (BrdU incorporation and direct cell counts. Reverse transcription coupled polymerase chain reaction (RT-PCR studies show that the cell proliferation deficit is presaged by a deficit in the expression of several zygotic wnt genes, including wnt8, a key regulator of endomesoderm development. In addition, SpRunt-1-depleted blastulae underexpress cyclinD, an effector of mitogenic Wnt signaling. Blastula stage cell proliferation is also impeded by knockdown of either wnt8 or cyclinD. Chromatin immunoprecipitation (ChIP indicates that Runx target sites within 5' sequences flanking cyclinD, wnt6 and wnt8 are directly bound by SpRunt-1 protein at late blastula stage. Furthermore, experiments using a green fluorescent protein (GFP reporter transgene show that the blastula-stage operation of a cis-regulatory module previously shown to be required for wnt8 expression (Minokawa et al., Dev. Biol. 288: 545-558, 2005 is dependent on its direct sequence-specific interaction with SpRunt-1. Finally, inhibitor studies and immunoblot analysis show that SpRunt-1 protein levels are negatively regulated by glycogen synthase kinase (GSK-3. CONCLUSIONS/SIGNIFICANCE: These results suggest that Runx expression and Wnt signaling are mutually linked in a feedback circuit that controls cell proliferation during development.

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

    Science.gov (United States)

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

    2014-03-15

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  14. Aberrant DNA methylation patterns in cultured mouse embryos

    Institute of Scientific and Technical Information of China (English)

    HOU Jian; CUI Xiuhong; LEI Tinghua; LIU Lei; AN Xiaorong; CHEN Yongfu

    2005-01-01

    Mouse early embryos undergo genome-wide demethylation and remethylation events during pre-implantation development. Abnormal methylation reprogramming is thought to be associated with development arrest. Using immunofiuorescence staining with an antibody against 5-methylcytosine (MeC), we examined the genome methylation patterns of mouse embryos cultured in vitro. The results did not show the difference in staining patterns between development-blocked two-cell embryos that cultured in vitro and the two-cell embryos that were freshly collected from the donor mice. But in vitro-arrested morulae displayed a strong positive staining when compared to the morulae freshly collected from the donor mice. At the blastocyst stage, although most embryos showed the expected methylation patterns, with highly stained inner cell mass (ICM) and weekly stained trophectoderm (TE), a proportion of embryos were dimly stained in both ICM and TE. These results indicated that the methylation profile of the embryos could be changed by culturing in vitro when the embryos were in the transition from morulae to blastocyst.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    OLÍVIA G.S. ARAÚJO

    Full Text Available ABSTRACT 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.

  17. Embryonic death, dwarfism and fetal malformations after irradiation of embryos at the zygote stage. Studies on two mouse strains

    Energy Technology Data Exchange (ETDEWEB)

    Jacquet, P.; Saint-Georges, L. de; Baugnet-Mahieu, L. [Laboratory of Radiobiology, Department of Radioprotection, CEN/SCK, Mol (Belgium); Vankerkom, J. [Division of Environmental Research, VITO, Mol (Belgium)

    1995-11-01

    Female mice of the BALB/c and CF1 strains were mated and irradiated with various doses of X-rays 7 h after presumed fertilization. 18 days later, females were killed and their uteri examined for prenatal mortality at the different stages of development. Living fetuses were weighed and examined for the presence of external malformations. A number of them were also examined for skeletal anomalies. Radiation induced mainly a dose-dependent increase of the preimplantation loss in the BALB/c strain and of the early postimplantation loss in the CF1 strain. Embryos of the BALB/c strain were refractory to the induction of teratogenic effects after such preimplantation irradiation. In CF1 mice, the frequency of malformed fetuses increased regularly after irradiation, the difference with controls being significant for the doses of 10, 50 and 100 cGy. Dwarfism occurrence also appeared to be increased by irradiation in this strain, although the importance of this effect varied depending on the criterion chosen for the assessment of dwarfs. With the definition proposed in the present paper, the increase in the frequency of dwarfs paralleled that of malformed fetuses, being significant after doses of 50 and 100 cGy. Irradiation did not increase the frequency of skeletal anomalies. A careful examination of the various data obtained to date led us to conclude that radiation may possibly be teratogenic in several mouse strains, when administered as early as during the one-cell stage and, to a lesser extent, during the following preimplantation stages. However, early prenatal mortality will remain by far the greatest risk associated with an exposure to radiation during this period. Moreover, the relativity of the risk of abnormality due to such irradiation should be considered in the context of the high prevalence of developmental defects spontaneously occurring during human pregnancy.

  18. Promoter analysis of the rabbit POU5F1 gene and its expression in preimplantation stage embryos

    Directory of Open Access Journals (Sweden)

    Bock Istvan

    2009-09-01

    Full Text Available Abstract Background The POU5F1 gene encodes the octamer-binding transcription factor-4 (Oct4. It is crucial in the regulation of pluripotency during embryonic development and widely used as molecular marker of embryonic stem cells (ESCs. The objective of this study was to identify and to analyse the promoter region of rabbit POU5F1 gene; furthermore to examine its expression pattern in preimplantation stage rabbit embryos. Results The upstream region of rabbit POU5F1 was subcloned sequenced and four highly conserved promoter regions (CR1-4 were identified. The highest degree of similarity on sequence level was found among the conserved domains between rabbit and human. Among the enhancers the proximal enhancer region (PE-1A exhibited the highest degree of homology (96.4%. Furthermore, the CR4 regulator domain containing the distal enhancer (DE-2A was responsible for stem cell-specific expression. Also, BAC library screen revealed the existence of a processed pseudogene of rabbit POU5F1. The results of quantitative real-time PCR experiments showed that POU5F1 mRNA was abundantly present in oocytes and zygotes, but it was gradually reduced until the activation of the embryonic genome, thereafter a continuous increase in POU5F1 mRNA level was observed until blastocyst stage. By using the XYClone laser system the inner cell mass (ICM and trophoblast portions of embryos were microdissected and examined separately and POU5F1 mRNA was detected in both cell types. Conclusion In this study we provide a comparative sequence analysis of the regulatory region of rabbit POU5F1 gene. Our data suggest that the POU5F1 gene is strictly regulated during early mammalian development. We proposed that the well conserved CR4 region containing the DE-2A enhancer is responsible for the highly conserved ESC specific gene expression. Notably, we are the first to report that the rabbit POU5F1 is not restricted to ICM cells only, but it is expressed in trophoblast cells as

  19. Activation of ribosomal RNA genes in porcine embryos produced in vitro or by somatic cell nuclear transfer

    DEFF Research Database (Denmark)

    Bjerregaard, Bolette; Pedersen, Hanne Gervi; Jakobsen, Anne Sørig

    2007-01-01

    The onset of ribosomal RNA (rRNA) synthesis occurs during the second half of the third cell cycle, that is, at the four-cell stage, in porcine embryos developed in vivo. In the present study the onset of rRNA synthesis was investigated in porcine embryos produced in vitro (IVP) or by somatic cell...... nuclear transfer (SCNT) using fluorescence in situ hybridization (FISH) with an rDNA probe and subsequent visualization of the nucleolar proteins by silver staining. In the 205 IVP embryos investigated, all two-cell embryos (n = 34) were categorized as transcriptionally inactive. At the late four......-cell stage (n = 45), 38% of the embryos contained 1-3 nuclei with signs of rRNA transcription, indicating an asynchronous transcription initiation. This pattern continued in the following stages, as 78% (n = 47), 47% (n = 42) and 83% (n = 37) of the embryos revealed a mixture of transcriptionally inactive...

  20. Expression profiles of the pluripotency marker gene POU5F1 and validation of reference genes in rabbit oocytes and preimplantation stage embryos

    Directory of Open Access Journals (Sweden)

    Polgar Zsuzsanna

    2008-07-01

    Full Text Available Abstract Background The surge in the number of gene expression studies and tendencies to increase the quality of analysis have necessitated the identification of stable reference genes. Although rabbits are classical experimental model animals, stable reference genes have not been identified for normalization. The aims of this study were to compare the expression profiles of the widely used reference genes in rabbit oocytes and preimplantation stage embryos, and to select and validate stable ones to use as reference. Results Quantitative real time PCR method was used to evaluate 13 commonly used references (Actb, Gapdh, Hprt1, H2afz, Ubc, Ppia, Eef1e1, Polr2a, Tbp, G6pdx, B2m, Pgk1, and Ywhaz and POU5F1 (Oct4 genes. Expressions of these genes were examined in multiple individual embryos of seven different preimplantation developmental stages and embryo types (in vivo and in vitro. Initial analysis identified three genes (Ubc, Tbp, and B2m close to the detection limit with irregular expression between the different stages. As variability impedes the selection of stable genes, these were excluded from further analysis. The expression levels of the remaining ten genes, varied according to developmental stage and embryo types. These genes were ranked using the geNorm software and finally the three most stable references (H2afz, Hprt1, and Ywhaz were selected. Normalization factor was calculated (from the geometric averages of the three selected genes and used to normalize the expressions of POU5F1 gene. The results showed the expected expression patterns of the POU5F1 during development. Conclusion Compared to the earlier studies with similar objectives, the comparison of large number of genes, the use of multiple individual embryos as compared to pools, and simultaneous analyses of in vitro and in vivo derived embryo samples were unique approaches in our study. Based on quantification, pattern and geNorm analyses, we found the three genes (H2afz

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

    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.

  3. Altered development of Xenopus embryos in a hypogeomagnetic field.

    Science.gov (United States)

    Mo, Wei-Chuan; Liu, Ying; Cooper, Helen M; He, Rong-Qiao

    2012-04-01

    The hypogeomagnetic field (HGMF; magnetic fields HGMF exposure on living systems remains unclear. In this article, we examine the biological effects of HGMF on the embryonic development of Xenopus laevis (African clawed frog). A decrease in horizontal third cleavage furrows and abnormal morphogenesis were observed in Xenopus embryos growing in the HGMF. HGMF exposure at the two-cell stage, but no later than the four-cell stage, is enough to alter the third cleavage geometry pattern. Immunofluorescent staining for α-tubulin showed reorientation of the spindle of four-cell stage blastomeres. These results indicate that a brief (2-h) exposure to HGMF is sufficient to interfere with the development of Xenopus embryos at cleavage stages. Also, the mitotic spindle could be an early sensor to the deprivation of the geomagnetic field, which provides a clue to the molecular mechanism underlying the morphological and other changes observed in the developing and/or developed embryos.

  4. ECTOPIC PREGNANCY RATES WITH CLEAVAGE STAGE EMBRYO TRANSFER AT DAY 3 VERSUS BLASTOCYST STAGE TRANSFER AT DAY 5: A PROSPECTIVE ANALYSIS

    Directory of Open Access Journals (Sweden)

    Prabhleen

    2014-10-01

    Full Text Available OBJECTIVE: The purpose of our study was to compare the tubal pregnancy rates between day 3 and day 5 transfers. As theoretically blastocyst transfer is said to decrease the incidence of ectopic pregnancy following IVF-ET due to the decreased uterine contractility reported on day 5. METHODS: A prospective analysis of all clinical pregnancies conceived in our IVF program between May 2010 to April 2011 was performed. The ectopic pregnancy rates were compared for day 3 and day 5 transfers. RESULTS: There were 44 pregnancies resulting from day 3 transfers of which one was ectopic (2.27%. In day 5 transfers, there was also one ectopic pregnancies out of 66 clinical pregnancies (1.52%, difference between these rates was not statistically significant (P>0.05 CONCLUSION: This suggests that the ectopic pregnancy rate is not reduced following blastocyst transfer on day 5 compared to cleavage stage embryo on day 3. While there may be several benefits to extended culture in IVF, the decision to offer blastocyst transfer should be made independently from the issue of ectopic pregnancy risk.

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

    Science.gov (United States)

    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.

  6. Use of picosecond infrared laser for micromanipulation of early mammalian embryos.

    Science.gov (United States)

    Karmenyan, Artashes V; Shakhbazyan, Avetik K; Sviridova-Chailakhyan, Tatiana A; Krivokharchenko, Alexander S; Chiou, Arthur E; Chailakhyan, Levon M

    2009-10-01

    A high repetition rate (80 MHz) picosecond pulse (approximately 2 psec) infrared laser was used for the inactivation (functional enucleation) of oocytes and two-cell mouse embryos and also for the fusion of blastomeres of two-cell mouse embryos. The laser inactivation of both blastomeres of two-cell mouse embryos by irradiation of nucleoli completely blocked further development of the embryo. The inactivation of one blastomere, however, did not affect the ability of the second intact blastomere to develop into a blastocyst after treatment. Laser inactivation of oocytes at Metaphase II (MII) stage and parthenogenetically activated pronuclear oocytes also completely blocked their ability for further development. Suitable doses of irradiation in cytoplasm region did not affect the ability of embryos and activated oocytes to development. The efficiency of laser induced fusion for blastomeres of two-cell embryos was 66.7% and all the tetraploid embryos developed successfully into blastocysts in culture. Our results demonstrate unique opportunities of the applications of a suitable infrared periodic pulse laser as a universal microsurgery tool for individual living cells.

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

    Science.gov (United States)

    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

  8. Cumulus Cell Role on Mouse Germinal Vesicle Oocyte Maturation, Fertilization, and Subsequent Embryo Development to Blastocyst Stage In Vitro

    Directory of Open Access Journals (Sweden)

    Reza Mahmodi

    2009-01-01

    Full Text Available Objective: The purpose of this study is to investigate the effect of cumulus cells on maturation,fertilization and subsequent development of mouse germinal vesicle oocytes.Materials and Methods: A total of 470 germinal vesicle (GV oocytes were obtained from26 ovaries of 3- 4 week old ICR female mice 48 hours after injection of 5 IU pregnant mareserum gonadotropin (PMSG. Collected oocytes were divided into two groups; group I: GVoocytes without cumulus cells (denuded oocyte, group II: GV oocytes with cumulus cells(cumulus-oocyte complex. The oocytes in both groups were cultured in TCM-199 mediumsupplemented with 10% fetal bovine serum (FBS for 22- 24 hours in a humidified atmosphereof 5% CO2 in air at 37°C. Oocyte maturation was scored under inverted microscope.To do in vitro fertilization, matured oocytes from each group were placed in T6 mediumand capacitated spermatozoa were added. Then the fertilized oocytes were cultured andassessed for cleavage to the 2-cell stage 24 hours and production of blastocyst 120 hoursafter fertilization. Data was analyzed by chi-square test and differences in the values wereconsiderable significant when p<0.05.Results: Maturation, fertilization, cleavage and blastocyst rates in denuded oocytes were:76.32%, 57.49%, 51.15% and 19.14% respectively. In the cumulus-oocyte complex rateswere: 89.41%, 80.76%, 75.58% and 45.62% respectively; all in the cumulus-oocyte complexwere significantly higher than those of denuded oocytes (p<0.05.Conclusion: The present study indicates that cumulus cells have important role duringmaturation, fertilization and subsequent embryo development to the blastocyst stage.

  9. Frailty of two cell cycle checkpoints which prevent entry into mitosis and progression through early mitotic stages in higher plant cells.

    Science.gov (United States)

    del Campo, A; Giménez-Martín, G; López-Sáez, J F; de la Torre, C

    1997-11-01

    Allium cepa L. root meristems were given two short caffeine treatments spaced by 15 hours, the time which roughly corresponds to the duration of one cell cycle. In this way two subsequent cytokineses were prevented, and multinucleate cells with their in complement distributed into two, three or four nuclei were formed. Though all nuclei started to replicate synchronously in these cells, some of them (fast nuclei) completed their replication earlier than others (slow nuclei). The present report shows that two successive checkpoints operate before prometaphase in these cells. The first one prevents the entry of the fast nuclei into prophase until the slow ones have completed their replication. The second checkpoint ensures the synchronous entry into prometaphase after all nuclei have reached and finished prophase. By treating the multinucleate cells with an inhibitor of DNA synthesis at that time when fast but not slow nuclei had finished their replication, it was observed that both checkpoint mechanisms became leaky with time. Under these conditions the fast nuclei entered prophase in the presence of nuclei which were prevented from finishing the replication of their DNA. Subsequently, even prometaphase was triggered after a prolonged prophase. Finally, as expected from the presence of mitotic stages in these cells, nuclei with incompletely replicated DNA endured premature chromosome condensation. The prematurely condensed chromosomes either remained in a prometaphase-like stage until reconstitution nuclei formed or they followed the progression of the fast nuclei into metaphase and anaphase leading to the appearance of acentric chromosomal segments which after reconstitution gave rise to aneuploid nuclei containing unstable and broken DNA.

  10. Intra- and interobserver analysis in the morphological assessment of early stage embryos during an IVF procedure: a multicentre study

    Directory of Open Access Journals (Sweden)

    Devroe Johanna

    2011-09-01

    Full Text Available Abstract Background Quality control programs are necessary to maintain good clinical practice. Embryo grading has been described as one of the external quality assurance schemes. Although the evaluation of embryos is based on the assessment of morphological characteristics, considerable intra- and inter-observer variability has been described. In this multicentre study, the variability in the embryo evaluation has been evaluated using morphological characteristics on day 1, day 2 and day 3 of embryo development. Methods Five embryologists of four different IVF centers participated in this study. Multilevel images of embryos were presented on a website at different time points to evaluate intra-and inter-observer agreement in the assessment of embryo morphology. The embryos were evaluated on day 1, day 2 and day 3 of their development and each embryologist had to decide if the embryo had to be transferred, cryopreserved or discarded. Results Both intra-observer agreement and inter-observer agreement were good to excellent for the position of the pronuclei on day 1, the number of blastomeres on day 2 and day 3 and the clinical decision (transfer, cryopreservation, discard. For all other characteristics (size of pronuclei, presence of cytoplasomic halo, degree of fragmentation and size of blastomeres the intra- and inter-observer agreement was moderate to very poor. Conclusions Mono- or multicentre quality control on embryo scoring by morphological assessment can easily be performed through the design of a simple website. In the future the website design can be adapted to generate statistical feedback upon scoring and can even include a training module.

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

    Science.gov (United States)

    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.

  12. Effects of Single-embryo Vitrification on the Cleavage Stage Embryos Developmental Ability in vitro%单胚胎玻璃化冷冻对小鼠胚胎体外发育的影响

    Institute of Scientific and Technical Information of China (English)

    贾思思; 付锴; 张昌军; 彭海英

    2011-01-01

    Objective To compare the effect between single-embryo vitrification and slow-freezing on development of mice cleavage stage embryos in vitro,and to explore whether that vitrification is feasible to freeze preserve a great quantity of embryos. Methods Mice 8-cell embryos were cryopreserved by vitrification or slow-freezing randomly and cultured until the blastocyst hatched out after thawed. The embryo recovery rate,blastomere mortality,blastocyst formation rate,the number of cell clusters,hatched rate were counted and statistically analyzed. Results In the vitrification group,the recovery rate,blas-tomere mortality,blastocyst formation rate,hatched rate,the number of cell clusters were 100% , 12.7% ,75.7% ,66.4% , 112,respectively. While they were 87.1% ,31.0% ,35.5% ,58.7% ,78 in slow freezing group,respectively. All these indexes were significantly difference between two groups excluding the hatched rate. Conclusion The survival rate of mice cleavage stage embryos cryopreserved by single-embryo vitrification were high,the structure integrality and developmental abillity of embryos could be better maintained by this method.%目的:比较单胚胎玻璃化冷冻与慢速冷冻对小鼠分裂期胚胎损伤和体外发育的影响,探讨使用玻璃化冷冻保存大量胚胎的可行性。方法:选择8-细胞胚胎随机进行改良的玻璃化和慢速冷冻,复苏后培养至囊胚孵出,计数胚胎复苏率、卵裂球死亡率、囊胚形成率、细胞团数、孵出率,并进行组间比较分析。结果:玻璃化冷冻组复苏率、卵裂球死亡率、囊胚形成率、孵出率、细胞团数分别为100%、12.7%、75.7%、66.4%、112,慢速冷冻组分别为87.1%、31.0%、35.5%、58.7%、78,除囊胚孵出率其余各指标两组间差异均具有统计学意义。结论:单胚胎玻璃化法冻存小鼠分裂期胚胎复苏后细胞成活率高,结构完整,更好的保持了胚胎的发育潜能。

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

    Directory of Open Access Journals (Sweden)

    Dessie Salilew-Wondim

    Full Text Available 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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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 (proscovitine 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 (proscovitine-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.

  17. Developmental stage on day-5 and fragmentation rate on day-3 can influence the implantation potential of top-quality blastocysts in IVF cycles with single embryo transfer

    Directory of Open Access Journals (Sweden)

    Devroey Paul

    2007-01-01

    Full Text Available Abstract Background In IVF-ICSI cycles with single embryo transfer (SET, embryo selection for transfer is of crucial importance. The present study aimed to define which embryo parameters might be related to the implantation potential of advanced blastocysts. Methods Overall, in 203 cycles with SET, developmental characteristics of 93 implanted (group A and 110 non-implanted (group B advanced blastocysts of good quality were compared. The following developmental parameters were assessed in the two groups: normal fertilization, developmental stage on day 5, number of blastomeres on day 2 and on day 3, fragmentation rate on day 3, compaction on day 4 and cleavage pattern on day 2 and day 3. Results Expanded blastocysts compared to full blastocysts have higher implantation potential (56.5% vs. 29.3%, p 10–50% fragments on day 3 showed a significant lower implantation (29.7% than those with ≤ 10%fragments (49.4%, P = 0.03. All the other parameters analysed were comparable for the two groups. Conclusion Developmental stage on day 5 and fragmentation rate on day 3 were related to the implantation potential of advanced blastocysts and should also be taken into account in the selection of the best advanced blastocyst for transfer.

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

    Energy Technology Data Exchange (ETDEWEB)

    Osychenko, A A; Zalesskii, A D; Krivokharchenko, A S; Zhakhbazyan, A K; Nadtochenko, V A [N N Semenov Institute of Chemical Physics, Russian Academy of Sciences, Moscow (Russian Federation); Ryabova, A V [A M Prokhorov General Physics Institute, Russian Academy of Sciences, Moscow (Russian Federation)

    2015-05-31

    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. (extreme light fields and their applications)

  19. Electroactivation of Oocyte and Electrofusion of Two-cell Embryos of Mouse%小鼠卵母细胞的电激活及胚胎电融合

    Institute of Scientific and Technical Information of China (English)

    李莉; 赵浩斌; 魏庆信

    2001-01-01

    The electric impulse was applied in oocyte electroactivation and two-cell embryonic electrofusion of mouse. The results showed that the difference of the rate of oocyte activation was obvious(P<0.01) as a result of different electric field strength,while using 30μs ,two pulses and 0.1 kV/cm pulse could get the best result(76.9%). The difference existed among different electric field strength,pulse number and pulse duration,for embryonic fusion too.The best fusion result(84.6%) could be obtained by using 0.1kV/cm, 30μs and two pulses.

  20. 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.

    Science.gov (United States)

    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.

  1. 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

    Directory of Open Access Journals (Sweden)

    Prabhleen Kaur

    2014-01-01

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

  2. The First Human Cloned Embryo.

    Science.gov (United States)

    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)

  3. Superovulation and Development of Early-Stage Embryo in Rabbits%家兔超数排卵与早期胚胎发育研究

    Institute of Scientific and Technical Information of China (English)

    林峰; 孙克宁; 陈玉霞; 杨婷; 高汉婷; 高腾云

    2011-01-01

    为了提高家兔繁殖力,使用不同剂量的FSH对家兔进行了超数排卵研究,结果表明:采用高剂量FSH处理母兔的平均排卵点数与平均卵泡发育数显著(P<0.05)高于低剂量FSH组,两种处理方法的平均卵泡囊肿数与平均卵泡数则差异不显著(P>0.05).家兔早期胚胎发育观察结果表明:母兔交配后24 h采胚所获取的胚胎大多处于1细胞期,交配后30 h胚胎多处于2细胞期,交配后35 h胚胎多处于4细胞期.采用52IU FSH进行超数排卵处理,对家兔的胚胎发育无影响.%The method of superovulation in rabbits was studied by using different dosages of FSH in order to improve their reproductive ability. The results showed that the average number of the ovulation points and the developed follicles on the ovaries of the does used the high dosage of FSH was obviously higher (F0. 05). The observating results of early-stage embryos' development also indicated that most of the collected embryos were in 1-cell stage during 24 hours after the does were accepted mating, and in 2-cell stage during 30 hours after mating and in 4-cell stage during 35 hours after mating. The dosage of 52IU FSH didn't influence the embryos' development of the rabbits when it was applied to conduct their superovulation treatment.

  4. Blastocyst transfer ameliorates live birth rate compared with cleavage-stage embryos transfer in fresh in vitro fertilization or intracytoplasmic sperm injection cycles: reviews and meta-analysis.

    Science.gov (United States)

    Wang, Shan-Shan; Sun, Hai-Xiang

    2014-05-01

    Blastocyst transfer has been recommended to raise the implantation rate without affecting the pregnancy rate. The objective of this meta-analysis is to systematically evaluate whether the live birth rate and other pregnancy outcomes can be improved by blastocyst transfer compared with cleavage-stage embryos transfer. EMBASE and MEDLINE databases were searched for papers published between March 2004 and March 2013. An extensive range of the electronic databases yielded initially 317 studies from which seven trials met the inclusion criteria for further analysis. Our outcome measures were the live birth rate, clinical pregnancy rate, implantation rate, ongoing pregnancy rate, multiple pregnancy rate, first trimester miscarriage rate and ectopic pregnancy rate. Fixed effects models were chosen to calculate the odds ratio (OR). Seven trials (n=1446 cases) were finally analyzed. Compared with cleavage-stage embryos transfer, the blastocyst transfer was statistically significantly associated with an increase in clinical pregnancy rate [OR 1.43; 95% confidence interval (CI), 1.15-1.78], implantation rate (OR 1.38; 95% CI, 1.09-1.74) and ongoing pregnancy rate (OR 2.15; 95% CI, 1.57-2.94), and also a reduction in the probability of first trimester miscarriage rate (OR 0.51; 95% CI, 0.30-0.87). The improvement in the live birth rate was also observed (OR 1.77; 95% CI, 1.32-2.37). Moreover, there was no evidence of difference in multiple pregnancy and ectopic pregnancy rates. The available evidences suggest that live birth and other pregnancy outcomes after fresh in vitro fertilization or intracytoplasmic sperm injection (IVF/ICSI) are significantly improved following blastocyst transfer as compared to cleavage-stage embryo transfer.

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

    Directory of Open Access Journals (Sweden)

    Celine Hebras

    Full Text Available 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

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

    Science.gov (United States)

    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.

  7. 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.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Baojian Guo

    Full Text Available 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.

  10. Attenuation of N-glycosylation causes polarity and adhesion defects in the C. elegans embryo.

    Science.gov (United States)

    Stevens, Julia; Spang, Anne

    2017-04-01

    The Caenorhabditiselegans early embryo is highly polarized, requiring sequestration of cytoplasmic polarity factors at the plasma membrane. This compartmentalization aids asymmetric distribution of lipids and proteins, which is partially responsible for the fates of the daughter cells. Since most plasma membrane proteins are glycosylated, we determined the effect of attenuation of N-glycosylation on cell polarity. While polarity establishment was not perturbed, the size difference between the two cells formed in first cell division (AB and P1) was more variable in embryos with reduced N-glycosylation than in the mock-treated embryos. In addition, among other deficiencies, we observed spindle orientation defects in two-cell embryos. Moreover, cell-cell adhesion was specifically lost at the two-cell stage when N-glycosylation was reduced. This loss-of-adhesion phenotype was rescued by interfering with polarity establishment, indicating that polarity establishment enforces plasma membrane compartmentalization. Consistent with this idea, the decreased plasma membrane levels of the adhesion proteins E-cadherin and MAGI-1 in ribo-1(RNAi) embryos were restored in the absence of functional PAR-2. Our data suggest a general role for N-glycosylation in plasma membrane compartmentalization and cell polarity.

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

    Science.gov (United States)

    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.

  12. PAR-4/LKB1 regulates DNA replication during asynchronous division of the early C. elegans embryo

    Science.gov (United States)

    Descoteaux, Catherine; Chartier, Nicolas T.; Pintard, Lionel; Labbé, Jean-Claude

    2014-01-01

    Regulation of cell cycle duration is critical during development, yet the underlying molecular mechanisms are still poorly understood. The two-cell stage Caenorhabditis elegans embryo divides asynchronously and thus provides a powerful context in which to study regulation of cell cycle timing during development. Using genetic analysis and high-resolution imaging, we found that deoxyribonucleic acid (DNA) replication is asymmetrically regulated in the two-cell stage embryo and that the PAR-4 and PAR-1 polarity proteins dampen DNA replication dynamics specifically in the posterior blastomere, independently of regulators previously implicated in the control of cell cycle timing. Our results demonstrate that accurate control of DNA replication is crucial during C. elegans early embryonic development and further provide a novel mechanism by which PAR proteins control cell cycle progression during asynchronous cell division. PMID:24841566

  13. 非优良胚胎形成的囊胚与卵裂期优良胚胎的冻融胚胎移植的临床结局比较%Comparison of clinical outcomes of blastocysts derived from non-top quality embryos and cleavage-stage high-quality embryos in frozen-thawed embryo transfer cycles

    Institute of Scientific and Technical Information of China (English)

    许丽娟; 陈薪; 田小龙; 刘玉东; 王楠; 叶德盛; 郭萍萍; 陈士岭

    2015-01-01

    目的:探讨不同发育天数胚胎的发育潜能,为D3非优良胚胎进行囊胚培养及其冻融移植提供依据。方法回顾性分析687例复苏周期胚胎移植患者的资料,根据胚胎冷冻时间不同,分为3组:D5冷冻组(n=87)、D6冷冻组(n=111)和D3冷冻组(n=489),采用外源性雌孕激素或自然周期准备内膜,比较各组间的临床妊娠率、流产率、种植率等指标。结果每组移植周期临床妊娠率、流产率、种植率分别为:D5冷冻组58.6%、9.8%、42.9%;D6冷冻组32.4%、19.4%、23.3%;D3冷冻组44.9%、16.4%、26.9%。D5冷冻组的临床妊娠率和种植率均明显高于另外两组,且差异均具有统计学意义(P<0.05)。结论 D3的非优良胚胎继续培养,若能够形成囊胚,所形成的D5囊胚冻融移植的临床结局优于D3的优良胚胎和非优良胚胎形成的D6囊胚冻融移植的临床结局。因此D3的非优良胚胎的囊胚培养及其冻融移植具有临床应用价值,且在冻融胚胎移植周期若有不同冷冻时间的胚胎可供选择时,可以优先选择非优良胚胎来源的D5囊胚,其次选择D3的卵裂期胚胎。%Objective 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. Methods 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. Results The clinical pregnancy rate, miscarriage rate and implantation rate per transfer were 58.6%, 9.8%, and 42.9% in D

  14. Expression of growth factor ligand and receptor genes in the preimplantation bovine embryo.

    Science.gov (United States)

    Watson, A J; Hogan, A; Hahnel, A; Wiemer, K E; Schultz, G A

    1992-02-01

    The sensitive technique of mRNA phenotyping with the reverse transcription-polymerase chain reaction was employed to determine the patterns of gene expression for several growth factor ligand and receptor genes during bovine preimplantation development. Several thousand bovine embryos encompassing a developmental series from one-cell zygotes to hatched blastocysts were produced by the application of in vitro maturation, fertilization, and oviductal epithelial cell embryo coculture methods. Transcripts for transforming growth factor (TGF-alpha) and platelet-derived growth factor (PDGF-A) are detectable in all preimplantation bovine stages as observed in the mouse. Transcripts for TGF-beta 2 and insulin-like growth factor (IGF-II) and the receptors for PDGF-alpha, insulin, IGF-I, and IGF-II are also detectable throughout bovine preimplantation development, suggesting that these mRNAs are products of both the maternal and the embryonic genomes in the cow, whereas in the mouse they are present only following the activation of the embryonic genome at the two-cell stage. In contrast to the mouse embryo, IGF-I mRNA was detected within preimplantation bovine embryos. Basic fibroblast growth factor (bFGF) is a maternal message in the bovine embryo, since it is only detectable up until the eight-cell embryo stage. Bovine trophoblast protein (bTP) mRNA was detectable within day 8 bovine blastocysts. As was observed in the mouse, the transcripts for insulin, epidermal growth factor (EGF), or nerve growth factor (NGF) were not detectable in any bovine embryo stage. Analyses of this type should aid the development of a completely defined culture medium for the more efficient production of preimplantation bovine embryos.

  15. Radiation sensitivity of the gastrula-stage embryo: Chromosome aberrations and mutation induction in lacZ transgenic mice: The roles of DNA double-strand break repair systems.

    Science.gov (United States)

    Jacquet, Paul; van Buul, Paul; van Duijn-Goedhart, Annemarie; Reynaud, Karine; Buset, Jasmine; Neefs, Mieke; Michaux, Arlette; Monsieurs, Pieter; de Boer, Peter; Baatout, Sarah

    2015-10-01

    At the gastrula phase of development, just after the onset of implantation, the embryo proper is characterized by extremely rapid cell proliferation. The importance of DNA repair is illustrated by embryonic lethality at this stage after ablation of the genes involved. Insight into mutation induction is called for by the fact that women often do not realize they are pregnant, shortly after implantation, a circumstance which may have important consequences when women are subjected to medical imaging using ionizing radiation. We screened gastrula embryos for DNA synthesis, nuclear morphology, growth, and chromosome aberrations (CA) shortly after irradiation with doses up to 2.5Gy. In order to obtain an insight into the importance of DNA repair for CA induction, we included mutants for the non-homologous end joining (NHEJ) and homologous recombination repair (HRR) pathways, as well as Parp1-/- and p53+/- embryos. With the pUR288 shuttle vector assay, we determined the radiation sensitivity for point mutations and small deletions detected in young adults. We found increased numbers of abnormal nuclei 5h after irradiation; an indication of disturbed development was also observed around this time. Chromosome aberrations 7h after irradiation arose in all genotypes and were mainly of the chromatid type, in agreement with a cell cycle dominated by S-phase. Increased frequencies of CA were found for NHEJ and HR mutants. Gastrula embryos are unusual in that they are low in exchange induction, even after compromised HR. Gastrula embryos were radiation sensitive in the pUR288 shuttle vector assay, giving the highest mutation induction ever reported for this genetic toxicology model. On theoretical grounds, a delayed radiation response must be involved. The compromised developmental profile after doses up to 2.5Gy likely is caused by both apoptosis and later cell death due to large deletions. Our data indicate a distinct radiation-sensitive profile of gastrula embryos, including

  16. A simple method for producing tetraploid porcine parthenogenetic embryos.

    Science.gov (United States)

    Sembon, S; Fuchimoto, D; Iwamoto, M; Suzuki, S; Yoshioka, K; Onishi, A

    2011-09-01

    The objective was to produce porcine tetraploid parthenogenetic embryos using cytochalasin B, which inhibits polar body extrusion. Porcine cumulus-enclosed oocytes aspirated from antral follicles were cultured for 51 h, and treated with cytochalasin B from 35 h to 42 h after the start of culture. After maturation culture, 74.7% (2074/2775) of oocytes treated with cytochalasin B did not extrude a polar body (0PB oocytes). In contrast, 80.4% (1931/2403) of control oocytes extruded a polar body (1PB oocytes). The 0PB oocytes were electrically stimulated, treated with cytochalasin B again for 3 h, and then cultured without cytochalasin B. Six days after electrical stimulation, 49.8% (321/644) reached the blastocyst stage. The number of cells in these blastocysts derived from 0PB oocytes was significantly lower than that from 1PB oocytes (0PB: 24.9 ± 10.6; 1PB: 43.0 ± 17.1; mean ± SD). A porcine chromosome 1-specific sequence was detected in parthenogenetic 0PB embryos by fluorescence in situ hybridization (FISH) analysis. Typical pronucleus-stage samples derived from 0PB embryos had two pronuclei, each with two signals. In two-cell and blastocyst-stage embryos, four signals were detected in each nucleus derived from 0PB embryos. We inferred that 0PB oocytes, which had a tetraploid number of chromosomes, started to develop as tetraploid parthenotes after electrical stimulation, and that tetraploid status was stably maintained during early embryonic development, at least until the blastocyst stage.

  17. Embryo-endometrial expression of leukemia inhibitory factor in the golden hamster (Mesocricetus auratus): increased expression during proestrous and window of implantation stages.

    Science.gov (United States)

    Rao, Rajnish P; Fischer, Bernd; Seshagiri, Polani B

    2008-01-01

    Leukemia inhibitory factor (LIF) is a pleiotropic IL-6 family cytokine and its maternal uterine expression is critical for mouse blastocyst implantation. In the golden hamster (Mesocricetus auratus), although the blastocyst hatching phenomenon is quite interesting and LIF is shown to regulate hatching, information is not available on the embryonic and uterine expression of LIF and hormonal regulation of LIF expression during the peri-implantation period. The present investigation is aimed at studying embryonic and uterine expression of LIF during preimplantation hamster development. We observed embryonic expression of LIF mRNA and protein in the 8-cell, morula and blastocyst stages. In cycling females, uterine LIF mRNA expression was maximal during the oestrogen-dominant phase of the oestrous cycle, i.e. proestrous stage. Interestingly, during pregnancy, both LIF mRNA and protein were highly upregulated on Days 3.5 and 4 ('window of implantation'), implying a role for this cytokine in blastocyst hatching and implantation. Cell type-specific localisation of LIF mRNA and protein was observed predominantly in luminal epithelium and uterine glands with faint staining being detected in the stroma. The hamster uterus encoded a approximately 4.2 kb LIF transcript whose coding region, when cloned and sequenced, showed a high degree of identity to the murine cDNA counterpart. These data demonstrate that: (1) hamster preimplantation embryos show LIF mRNA and protein expression; (2) uterine expression of LIF mRNA and protein was dependent on elevated levels of circulating oestrogen, and (3) there is a possible functional association of LIF with the peri-implantation development in the golden hamster.

  18. New techniques on embryo manipulation.

    Science.gov (United States)

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

    2002-01-01

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

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

    Science.gov (United States)

    Yokoo, Masaki; Mori, Miho

    2017-05-27

    The aim of the present study was to assess the effects of near-infrared laser irradiation on the in vitro development of mouse embryos. Female ICR mice were superovulated with pregnant mare serum gonadotropin and human chorionic gonadotropin (hCG), and mated with male mice. Two-cell stage embryos were collected 40 h after administering hCG and cultured in M16 medium. Two-cell embryos (0 h after culture), 8-cell embryos (approx. 30 h after culture), morula (approx. 48 h after culture), and blastocysts (approx. 73 h after culture) were irradiated at 904 nm for 60 s. These embryos were cultured in a time-lapse monitoring system and the timing of blastocyst hatching was evaluated. Some of the irradiated blastocysts were transferred to the uterine horns of pseudopregnant recipients immediately after irradiation. Pregnancy rates, and offspring growth and fertility, were evaluated. Near-infrared laser irradiation increased the speed of in vitro mouse embryo development. In irradiated blastocysts, hatching was faster than in control (non-irradiated) blastocysts (18.4 vs. 28.2 h, P infrared laser irradiation improves the quality of mouse embryo development in vitro, and increases the live birth rate without affecting the normality of the offspring. Thus, the near-infrared laser method may enhance the quality of embryos and contribute to improvements in reproductive technologies in mammals. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. 人卵裂胚和囊胚的玻璃化冷冻及临床应用%Vitrified cryopreservation for human embryoes in cleavage or blastocyst stage and its preliminarily applications in clinic

    Institute of Scientific and Technical Information of China (English)

    孙迎利; 张敏; 常秀峰; 张建平; 朱爱萍; 马晓伟; 余裕炉

    2011-01-01

    Objective: To evaluate the efficacy of vitrified cryopreservation for human embryos in cleavage and blastocyst stages using cryoloop. Methods: 270 embryos ( 167 and 103 in cleavage and blastocyst stage respectively), from the hospital IVF center,were divided into two groups, as their stages, I. e. cleavage or blastocyst stage, for the later artifical shrinked must be done prior to be freezed, and then rates for thawing survival, implantation, clinic pregnancy and miscarrage were observed. Results: 97.0%,33. 1%, 42. 9%, 13. 3% and 96. 1%, 43.9%, 52%, 15.4%; in rates mentioned above, were observed in 70 and 75 transfer cycles, respectively for cleavage and blastocyst stage embryos. Conclusion: This protocol for vitrified cryopreservation, using cryoloop, can work well and suitable for cleavage stage embryos and blastocyst stage embryos as well.%目的 探索人卵裂胚及囊胚玻璃化冷冻的有效性.方法 来源于生殖中心IVF或ICSI助孕的共270个胚胎(卵裂胚167个.囊胚103个),用Cryoloop作为胚胎载体和相同的玻璃化方法进行冷冻、复苏和移植,观察其复苏存活率、临床妊娠率、植入率和流产率.结果 卵裂胚和囊胚的复苏率、植入率和妊娠率分别为97.0%、33.1%、42.9%和96.1%、43.9%、52%,流产率分别为13.3%和15.4%.结论 以Cryoloop为胚胎载体,使用相同的玻璃化冷冻方法对人类卵裂胚和囊胚均可获得同样理想的冷冻效果和临床结局.

  1. Comparion of the Outcomes Between Vitrification and Slow-Freezing Methods for Cryopreservation of Embryos in Cleavage Stage%快冻和慢冻法对胚胎复苏结局的比较

    Institute of Scientific and Technical Information of China (English)

    赵庆红; 杨菁; 尹太郎; 龙文; 李星; 方健叶; 李磊

    2011-01-01

    Objective: To compare the outcomes of vitrification and slow-freezing methods for cryopreservation of cleavage stage embryos in assisted reproductive technology. Methods: A retrospective statistical analysis was performed in our center on 577 vitrification cycles and 276 slow-freezing cycles. The embryo survival and intact survical rate after embryo recovery, embryo implantation rate and clinical pregnancy rate and other indicators were compared. Results: In rapid freezing group, the numbers of embryos thawed each cycle were less than in the slow-freezing group, but the numbers of survival embryos were more, and the period recurrence rate was higher. Vitrified embryos was superior to the procedural slowly-frozen embryos in embryo survival rate, intact embryo survival rate, embryo implantation rate, clinical pregnant rate, and transfer cancel rate (all P<0.05). Conclusion: Vitrification is better to preserve the potential development of frozed embryo after recovery, and is an ideal method for cryopreservation of the cleavage embryos.%目的:比较辅助生殖技术中快冻(玻璃化法)和慢冻(程序化法)两种冷冻方法对卵裂期胚胎的冻融效果.方法:将复苏后全部卵裂球存活的胚胎定义完整存活胚胎,同一病人由于有多余冻融胚胎而行2次及以上移植周期的称为重复周期.对577个快冻周期和276个慢冻周期的相关资料进行回顾性统计学分析,比较两组冻融胚胎的存活率、完整存活率、重复周期率、胚胎种植率和临床妊娠率等指标.结果:快冻组的每周期胚胎解冻数低于慢冻组,而每周期存活胚胎数和重复周期率均高于慢冻组;玻璃化冷冻后的胚胎存活率和完整性胚胎存活率均显著高于慢冻组,复苏周期移植取消率显著低于慢冻组,而胚胎种植率和临床妊娠率则明显高于慢冻组,均有统计学差异(P<0.05).结论:玻璃化法能够较好地保存冻融胚胎的发育潜能,得到较好的临床妊

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

    Science.gov (United States)

    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.

  3. 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.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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 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.

  5. A concentration gradient of retinoids in the early Xenopus laevis embryo.

    Science.gov (United States)

    Chen, Y; Huang, L; Solursh, M

    1994-01-01

    Previous studies have postulated that Xenopus embryos contain an endogenous retinoic acid (RA) concentration gradient from posterior to anterior during the process of primary axis formation, since RA is able to alter profoundly primary axis formation in Xenopus embryos, to increase the expression of some posterior markers, and to inhibit the expression of some anterior markers, including homeobox-containing genes. Here, we provide direct evidence for this hypothesis. By using a reporter cell system, we demonstrate that the endogenous biologically active retinoid concentration in whole Xenopus embryos increases 3-fold from the two-cell stage to the neurula stage, and that the active retinoid concentration in the dorsal marginal zone, a region wherein the Spemann's organizer is located, increases about 5-fold from the early gastrula to late gastrula stages, suggesting the developmental regulation of the retinoid levels. In the early neurula stage (stage 13-14), endogenous active retinoids are present in a concentration gradient with the highest level at the posterior end, about 10-fold higher than that at the anterior end, of the embryo. This concentration gradient may be established during gastrulation and may provide positional cues for primary axis formation.

  6. Comparation of multiple-pregnancy rate between cleavage stage embryos transfer and blastocyst transfer%胚胎卵裂期移植与囊胚移植多胎率的比较

    Institute of Scientific and Technical Information of China (English)

    施文浩; 张四林; 任文娟; 张鑫; 师娟子

    2016-01-01

    目的:通过胚胎卵裂期移植与囊胚移植的不同移植胚胎数目间比较,寻找降低多胎率的方法和策略。方法2014年1月1日至2015年6月30日在陕西省妇幼保健院生殖中心行常规体外受精/卵胞浆内单精子注射-胚胎移植( IVF/ICSI-ET)的患者为研究对象,其中受精后第三天新鲜移植或解冻胚胎第三天移植为卵裂期组,受精后第五天新鲜移植或解冻后囊胚移植为囊胚期组。根据移植胚胎数目(1~3枚)分别进行两组间临床妊娠及多胎率的比较。结果共计移植7454周期,其中新鲜移植4756周期,冻融胚胎移植2698周期。移植数目相同的情况下,囊胚移植的临床妊娠率比卵裂期胚胎明显增加(移植1枚、2枚、3枚时,P值分别为0.001,0.001,0.033);卵裂期胚胎移植由2枚增加到3枚时,临床妊娠率并未增加反而下降(P=0.002),同时多胎率无明显变化(P=0.252),因此移植3枚胚胎临床妊娠率下降且多胎率无明显变化;囊胚移植由2枚增加到3枚时临床妊娠率略升高(P=0.739),同时多胎率升高(P=0.595),但无统计学差异。结论卵裂期胚胎移植数目为1~3枚,可通过增加移植胚胎数目维持一定的临床妊娠率,多胎风险并无增加;囊胚移植由2枚增加至3枚时并未显著提高临床妊娠率,相反有增加多胎率的风险。故囊胚移植数目应该为1~2枚,并应禁止移植3枚囊胚。%Objective To compare the number of embryo between cleavage stage embryos transfer and blastocyst transfer so as to look for the strategies to reduce the rate of multiple-pregnancies.Methods The patients, undergoing in vitro fertilization /intracytoplasmic sperm injection-embryo transfer (IVF/ICSI-ET) in Reproductive Center of Northwest Women and Children's Hospital during January 1, 2014 to June 30, 2015 were enrolled as research objects.The cleavage stage group

  7. Relationship Between Development, Metabolism, and Mitochondrial Organization in 2-Cell Hamster Embryos in the Presence of Low Levels of Phosphate

    Science.gov (United States)

    Ludwig, Tenneille E.; Squirrell, Jayne M.; Palmenberg, Ann C.; Bavister, Barry D.

    2016-01-01

    The effect of low concentrations of inorganic phosphate (Pi) on development, metabolic activity, and mitochondrial organization in the same cohorts of cultured hamster embryos was evaluated. Two-cell embryos were collected from eCG-stimulated golden hamsters and cultured in HECM-10 with 0.0 (control), 1.25, 2.5, or 5.0 µM KH2PO4. Glucose utilization through the Embden-Meyerhof pathway (EMP) and tricarboxylic acid (TCA)-cycle activity were determined following 5 h of culture. Mitochondrial organization in living embryos was evaluated using multiphoton microscopy at 6 h of culture. Development was assessed at 27 h (on-time 8-cell stage) and 51 h (on-time blastocyst stage) of culture. Total cell numbers, as well as cell allocation to the trophectoderm and inner cell mass were determined for morula- and blastocyst-stage embryos. Culture with Pi did not alter TCA-cycle activity. However, culture with ≥2.5 µM Pi significantly increased (P organization was significantly (P culture medium dramatically alters embryo physiology. Additionally, although 2-cell embryos can tolerate some structural disruption without concomitant, detrimental effects on development or metabolic activity, metabolic disturbance is associated with decreased developmental competence. PMID:11717124

  8. Effects of developmental stages of tetraploid embryos on mouse chimeras from embryonic stem cells%四倍体胚胎发育阶段对胚胎干细胞嵌合体小鼠制备的影响

    Institute of Scientific and Technical Information of China (English)

    吴兴龙; 张鹏; 赵虎; 王鹏博; 胡春超; 李相运

    2011-01-01

    目的 探讨四倍体胚胎发育阶段对胚胎干细胞(ES)嵌合体小鼠制备的影响.方法 通过2-细胞胚胎电融合法制备四倍体胚胎,采用显微注射方法将ES细胞分别注入1-细胞、4-细胞、囊胚3个发育阶段的四倍体胚胎中.所用ES细胞分别为杂交系B6D2F1×129/Sv和近交系C57BL/6J,经胚胎移植和剖腹产以获得ES小鼠.结果 实验表明,2-细胞胚胎电融合率为92.45%,4-细胞胚胎发育率为93.51%,囊胚发育率为90.42%.杂交ES细胞注射四倍体囊胚获得22只ES小鼠,效率显著高于近交系ES细胞以及其他发育阶段的四倍体胚胎,ES小鼠与B6D2F1×129/Sv杂交小鼠毛色一致且具有正常生殖能力.结论 四倍体胚胎的发育阶段显著影响ES小鼠的制备.%Objective To investigate the effects of developmental stages of tetraploid embryos on mouse chimeras from embryonic stem cells ( ES ).Methods Tetraploid embryo complementation and microinjection were utilized to produce ES mice that derived completely from ES cells.Tetraploid embryos were firstly prepared by electrofusion of 2-cell mouse embryos, and then embryonic stem ( ES ) cells with different genetic background ( hybrid or inbred ) were injected into tetraploid 1-cell, 4-cell, and blastocyst stage embryos.The injected embryos were transferred into uterine horns of pseudopregnant 2.5days female CDl mice.The CD1 mice were suhjected into cesarean sections after 16 days.Results Our data showed that 92.45% of 2-cell embryos were electrofused, 93.51% and 90.42% of the electrofused embryos developed to 4-cell and blastocyst stages, respectively.Blastocysts were injected with hybrid ES cells and 22 ES mice were obtained by Cesarean section.Blastocyst was more effective than 1-cell and 4-cell.ES mice had normal germline transmission capahility and the same coat color as the hybrid mice which hybrid ES cells were drived from.Conclusion The tetraploid embryo developmental stage is able to affect the ES mouse

  9. Screening of biotechnical parameters for production of bovine inter-subspecies embryonic chimeras by the aggregation of tetraploid Bos indicus and diploid crossbred Bos taurus embryos.

    Science.gov (United States)

    Razza, Eduardo M; Satrapa, Rafael A; Emanuelli, Isabele P; Barros, Ciro M; Nogueira, Marcelo F G

    2016-03-01

    The aggregation of a tetraploid zebu embryo (Bos indicus, a thermotolerant breed) with a diploid taurine embryo (Bos taurus, a thermosensitive breed) should create a complete taurine fetus, whose extra-embryonic components, e.g., the chorion, is derived mainly from the zebu embryo. These zebu-derived extra-embryonic components may interact positively with the taurine embryo/fetus during pregnancy in a tropical environment. We tested different parameters for the production of tetraploid Nelore (Bos indicus) embryos to be combined via aggregation with crossbred Bos taurus (diploid) embryos in order to produce viable chimeric blastocysts. Bovine (Bos indicus or crossbred Bos taurus) embryos were produced in vitro according to standard procedures. Two-cell Bos indicus embryos were submitted to electrofusion with varying numbers of pulses (1 or 2), voltages (0.4, 0.5, 0.75, 1.0, 1.4 and 5.0 kV/cm) and time (20, 25, 50 and 60 μs) to produce tetraploid embryos. Electrofused embryos were cultured with crossbred non-fused embryos to form chimeras that developed until the blastocyst stage. The best fusion parameter was 0.75 kV/cm for 60 μs. Four chimeric blastocysts (tetraploid Nelore with diploid crossbred Holstein) were formed after 31 attempts in 4 replicates (13%). We established an optimal procedure for the production of tetraploid Bos indicus (4n) embryos and embryonic chimeras by aggregation of crossbred Bos taurus (2n) with Bos indicus (4n) embryos. This technique would be valid in applied research, by producing exclusively taurine calves, but with placental elements from the Bos indicus breed, following transfer of these chimeras into recipient cows.

  10. Chromosome remodeling and differentiation of tetraploid embryos during preimplantation development.

    Science.gov (United States)

    Park, Mi-Ryung; Lee, Ah-Reum; Bui, Hong-Thuy; Park, Chankyu; Park, Keun-Kyu; Cho, Ssang-Goo; Song, Hyuk; Kim, Jae-Hwan; Nguyen, Van Thuan; Kim, Jin-Hoi

    2011-07-01

    Although it is known that the tetraploid embryo contributes only to the placenta, the question of why tetraploid embryos differentiate into placenta remains unclear. To study the effect of electrofusion on the development of mouse tetraploid oocytes, mouse two-cell embryos were fused and cultured in vitro in Chatot-Ziomek-Bavister medium. After electrofusion, two chromosome sets from the tetraploid blastomere were individually duplicated before nuclear fusion. At 8-10 hr after electrofusion, each chromosome set was condensing and the nuclear membrane was breaking down. Around 12-14 hr after electrofusion, the two chromosome sets had combined together and had reached the second mitotic metaphase, at this point with 8n sets of chromosomes. Interestingly, we discovered that expression of OCT4, an inner cell mass cells biomarker, is lost by the tetraploid expanded blastocysts, but that CDX2, a trophectoderm cells biomarker, is strongly expressed at this stage. This observation provides evidence clarifying why tetraploid embryos contribute only to trophectoderm.

  11. Aberrant DNA methylation in cloned ovine embryos

    Institute of Scientific and Technical Information of China (English)

    LIU Lei; HOU Jian; LEI TingHua; BAI JiaHua; GUAN Hong; AN XiaoRong

    2008-01-01

    By using the approach of immunofluorescence staining with an antibody against 5-methylcytosine (5MeC), the present study detected the DNA methylation patterns of cloned ovine embryos. The em-bryos derived from in vitro fertilization were also examined for reference purpose. The results showed that: (1) during the pre-implantation development, cloned embryos displayed a similar demethylation profile to the fertilized embryos; that is, the methylation level decreased to the lowest at 8-cell stage, and then increased again at morulae stage. However, methylation level was obviously higher in cloned embryos than in stage-matched fertilized embryos, especially at 8-cell stage and afterwards; (2) at blastocyst stage, the methylation pattern in cloned embryos was different from that in fertilized em-bryos. In cloned blastocyst, inner cell mass (ICM) exhibited a comparable level to trophectoderm cells (TE), while in in-vitro fertilized blastocyst the methylation level of ICM was lower than that of TE, which is not consistent with that reported by other authors. These results indicate that DNA methylation is abnormally reprogrammed in cloned embryos, implying that aberrant DNA methylation reprogramming may be one of the factors causing cloned embryos developmental failure.

  12. Human embryo twinning with applications in reproductive medicine.

    Science.gov (United States)

    Illmensee, Karl; Levanduski, Mike; Vidali, Andrea; Husami, Nabil; Goudas, Vasilios T

    2010-02-01

    To assess the efficacy of human embryo twinning by blastomere biopsy at different early embryonic stages (splitting efficiency) and to determine the in vitro developmental capacity of twinned human embryos (developmental efficiency). Randomized comparative study. Private IVF centers. Couples undergoing IVF donating triploid embryos. Embryos at the 2- to 5- and 6- to 8-cell stage were split into twin embryos. Half the number of blastomeres from donor embryos were removed and inserted into recipient empty zonae pellucidae. After embryo splitting, donor and recipient embryos were cultured in vitro. Development of twinned embryos to the blastocyst stage. The number of developing embryos obtained after splitting could be increased in comparison with the number of embryos available before splitting at the 6- to 8-cell stage but not at the 2- to 5-cell stage (splitting efficiency). Splitting of 6- to 8-cell embryos yielded superior rates of twin embryos developing to blastocysts (developmental efficiency). Twinning success was related to the superior morphological quality of embryos used for splitting. This is the first report on twinned human embryos developing to blastocysts. This study exhibits the potential for novel applications in human assisted reproduction. Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  13. Embryo forming cells in carrot suspension cultures.

    OpenAIRE

    Toonen, M.A.J.

    1997-01-01

    Somatic cells of many plant species can be cultured in vitro and induced to form embryos that are able to develop into mature plants. This process, termed somatic embryogenesis, was originally described in carrot (Daucus carota L.). Somatic embryos develop through the same characteristic morphological stages, i.e. the globular-, heartand torpedo-stage respectively, as their zygotic counterparts. Due to the different cellular origin of somatic embryos, it is less clear to what extent the earli...

  14. RESEARCHES REGARDING THE INFLUENCE OF RECOVERY MEDIA ON THE IN VITRO DEVELOPMENT CAPACITY OF THE PREIMPLANTATIONAL MOUSE EMBRYO

    Directory of Open Access Journals (Sweden)

    ADA CEAN

    2009-05-01

    Full Text Available Phosphate Bufered Saline with 0.4% BSA and M2 medium are one of the most common media used in embryorecovery. The aim of our paper was to investigate if the recovery media used for the recovery of the mouseembryo is influencing in vitro developmental capacity. As biological material we used 10 used were mousefemales, age 2 months superovulated with 5UI PMSG (Pregnant Mare Serum Gonadotropine and 5 UI hCG(human Corionic Gonadotropine. The embryos used were recovered, by oviduct flushing, at 24 hours from theidentification of the vaginal plug. The majority of the embryos (78.3% were in two cells stage. A total of 123, 2cells embryos were cultivated in M16 medium. The evolution of the embryos was examined at 24, 48 and 72hours interval. The proportion of hatched blastocyst was higher at the embryos recovered with M2 (53.7%compared with the embryos recovered with PBS 0.4% BSA. The difference is statistically very significant(p<0.001. Embryos recovered in M2 media have a higher in vitro developmental capacity compared with theembryos recovered in PBS media supplemented with 0,4% BSA, possibly because of the sodium bicarbonate andlactate used in M2 media for pH regulation.

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

    Science.gov (United States)

    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.

  16. Factors influencing the outcome of embryo freezing and thawing program

    Institute of Scientific and Technical Information of China (English)

    叶英辉; 金帆; 徐晨明; 邢兰凤

    2002-01-01

    Objective: To investigate the factors that might influence the succ ess of an embryo freezing and thawing program. Method: The relationship betwee n the pregnancy rate in 73 cycles of embryo freezing and thawing program and the following factors was analyzed: maternal age, E2 level at the time of HCG trigg er, embryo storage time, number of thawed embryos transferred, presence of spons oring embryos and intact embryos. And the survival rate of thawed embryos with d ifferent morphology, cell stage and storage time was evaluated. Result: Tra nsfer with three or more than three thawed embryos resulted in pregnancy rates o f 38.5% and 35.7%, respectively, compared with 5.3% for transfer of fewer th an t hree embryos. The presence of sponsoring embryos and intact embryos significantl y increases pregnancy rate in embryo freezing and thawing program. No other fact or examined had any effect on pregnancy outcome. The survival rate of good morph ology embryos was higher than poor ones, but was not influenced by cell stage an d storage time. Conclusion: Embryo morphology before freezing, number of thaw ed embryos transferred and the presence of intact embryos are important to the o utcome of embryo freezing and thawing program.

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

    Science.gov (United States)

    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 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.

  18. 小鼠胚胎干细胞与四倍体胚胎的嵌合%Chimera of mouse ES cells and tetraploid embryos

    Institute of Scientific and Technical Information of China (English)

    李相运; 窦忠英; 李松

    2003-01-01

    The oviducts of superovulated Kunming white females were flushed 44-46 hours after treatment with human chorionic gonadotropin to collect 1074 late two-cell-stage embryos.The embryos were placed twenty at a time between two platinum electrodes laid 1 mm apart in 0.3M mannitol in the electrode chamber.The blastomeres were fused by a short electric pulse(80V for 50μsec) applied by a pulse generator.Fusion of blastomeres was usually completed in 20-60minutes.After 25 hours of culture,most of the tetraploid embryos developed to the four-cell stage.Zonae pellucidae of 387 four-cell-stage tetraploid embryos were removed by treatment with acid Tyrode's buffer.The embryos were plated on an ES cell layer,After 40 hours of coculture,248 embryos aggregated with ES cells were collected and transferred into the uteri of twenty four 2.5-day pseudopregnant recipinets.Ten recipients were pregnant.but no live fetuses were born.Three pregnant recipients were routinely subject to a Caesarean section on day 18 of pregnancy and seven abnormal fetuses were obtained.The results demonstrate that ES cells derived from C57BL/6 mice are pluripotential to a certain extent.

  19. [Influence of "zero" magnetic field on the growth of embryonic cells and primary embryos of mouse in vitro].

    Science.gov (United States)

    Osipenko, M A; Mezhevikina, L M; Krasts, I V; Iashin, V A; Novikov, V V; Fesenko, E E

    2008-01-01

    The present investigation reveals that a 250-fold screening of the geomagnetic field ("zero" geomagnetic fields, 200 nT) is a biologically active factor that adversely affects embryonic cells and the processes of early embryogenesis as a whole. In particular, the cultivation of primary embryonic fibroblasts in "zero" geomagnetic fields causes reduces the capacity for adhesion and proliferation, changes the monolayer morphology and increases cell death. In a more highly organized experimental model, two-celled mouse embryos, the exposure to the "zero" field results in an increase of plasma membrane permeability for dyes, a reorganization of the cytoskeleton because of alpha-actin redistribution, and the disturbance of the spatial orientation of blastomeres. As a result, the development of two-celled mouse embryos stops, and they do not reach the stage of blastocyst. These data show the significant role of geomagnetic fields in the normal growth of embryonic cells in vitro and the regulation of mammalian embryogenesis.

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

    Science.gov (United States)

    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.

  1. Factors influencing the outcome of embryo freezing and Ihawing program

    Institute of Scientific and Technical Information of China (English)

    叶英辉; 金帆; 徐晨明; 邢兰凤

    2002-01-01

    Objective: To investigate the factors that might influence the sucess of an ernbryo freezing and thawing program.Method: The relationship between the pregnancy rate in 73 cycles of embryo freezing and thewing program and the following factors was analyzed;matermal age,E2 level at the time of HCG trigger,embryo storage time,number of thawed embryos transferred,presence of sponsoring embryos and intact embryos.And the suvival rate of thawed embryos with different morphology,cell stage and storage time was evaluated.Result:Transfer with three of more than three thawed embryos resulted in pragnancy rates of 38.5% and 35.7%,respectively.compared with 5.3% for transfer of fewer than three embryos.The presence of sponsoring embryos and intact embryos significantly incresses pregnancy rate in embryo freezing and thawing program .No other factor examined had any effect on pregnancy outcome.The survival rate of good morphology embryos was higher than poor ones,but was not influenced by cell stage and storage time.Conclusion:Embryo morphology before freezing , number of thawed embryos transferred and the presence of intact embryos are important to the outcome of embryo freezing and thawing program.

  2. [Distribution of foreign mitochondrial DNA during the first splittings of the transmitochondrial mouse embryos].

    Science.gov (United States)

    Kustova, M E; Sokolova, V A; Bass, M G; Zakharova, F M; Sorokin, A V; Vasil'ev, V B

    2008-01-01

    Distribution of human mitochondrial DNA (mtDNA) among separate murine blastomeres was analyzed during the splitting of embryos in which the suspension of human mitochondria had been injected at the one- or two-cell stage. Human mtDNA was detected by PCR with species specific primers. The total amount of the two- and four-cell murine embryos analyzed in the study was 339. In all embryos examined the copies of human mitochondrial genome were revealed along with murine mtDNA, which indicated the phenomenon of an artificially modeled heteroplasmy. The foreign mtDNA was not ubiquitous among the blastomeres of transmitochondrial embryos. Mathematical analysis of the results showed that in the period between the injection of human mitochondria and the subsequent splitting no equal distribution of the human mtDNA occurred in the cytoplasm. These results also point at the presence of more than 2-3 segregation units of mtDNA in the entire pool of mitochondria (about 5 x 10(2)) introduced into an embryo by microinjection.

  3. Facial Transplants in Xenopus laevis Embryos

    OpenAIRE

    Jacox, Laura A.; Dickinson, Amanda J.; Sive, Hazel

    2014-01-01

    Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. To identify where signaling pathways and tissues act during patterning of the developing face, a 'face transplant' technique has been developed in embryos of the frog Xenopus laevis. A region of presumptive facial tissue (the "Extreme Anterior Domain" (EAD)) is removed from a donor embryo at tailbud stage, and transplanted to a host embryo ...

  4. Derivation and characterisation of hESC lines from supernumerary embryos, experience from Odense, Denmark

    DEFF Research Database (Denmark)

    Harkness, Linda; Rasmussen, Iben Anne; Erb, Karin

    2010-01-01

    . Analysis of clinical data showed that the majority of embryos (94.5%) failed to reach the blastocyst stage of development and of all embryos, regardless of developmental status, 248 embryos were needed to create one stem cell line. From the number of embryos (69) which developed to the blastocyst stage 8...

  5. Rape embryogenesis. IV. Appearance and disappearance of starch during embryo development

    Directory of Open Access Journals (Sweden)

    Teresa Tykarska

    2014-01-01

    Full Text Available Starch appears first in the suspensor of the proembryo with two-cell apical part. It is observed in the embryo proper from the octant stage. At first it is visible in all the embryo cells in the form of minute transient grains which disappear during cell divisions. But the columella mother cells and their derivatives have persistent large grains. When the embryo turns green in the heart stage a gradual accumulation of storage starch begins and lasts to the end of embryogenesis. Storage starch grains appear first in the auter cortex layers of the hypocotyl where the largest grains are to be found later, and afterwards in all the other tissues. Starch is usually absent in the frequently dividing cells, but even there it appears in the form of minute grains after the end of cell divisions. Disappearance of starch starts when the intensive green colour of the seed coat begins to fade. The first to disappear are the smallest granules in the regions where they were noted latest. In the embryo axis the starch grains remain deposited longest in dermatogen and cortex cells in the lower hypocotyl part. They are visible there, still when the seed turns brown. In black seeds starch may be only found in the columella the cells of which throughout embryogenesis contain amyloplasts filled with starch. These grains disappear completely at the time when the seeds become dry.

  6. In vitroembryo outgrowth is a bioassay ofin vivo embryo implantation and development

    Institute of Scientific and Technical Information of China (English)

    Natalie K. Binder; Natalie J. Hannan; David K. Gardner

    2015-01-01

    Objective:To determine the efficacy of embryo outgrowth on fibronectin as a low cost, high throughput alternative to embryo transfer to model embryo attachment and the initial stages of implantation.Methods:Following in vitro embryo culture, embryo quality was assessedviaembryo transfer or embryo outgrowth with metabolic assessment.Results:This study shows that blastocysts attach to fibronectin at the same rate that they implantin vivo, and that the carbohydrate utilisation of embryos that successfully outgrow is comparable to those that are able to develop into a fetus.Conclusions:Embryo outgrowth is a suitable alternative endpoint to embryo transfer.

  7. Evaluation of Embryos Derived from in vitro Fertilized Oocytes Reconstructed by Meiosis-II Chromosome Transplantation from Aged Mice to Ooplasms of Young Mice

    Directory of Open Access Journals (Sweden)

    Abdolhossein Shahverdi

    2010-01-01

    Full Text Available Background: To assess embryos derived by the transfer of meiosis-II chromosomes (M-II-t fromaged mice oocytes into ooplasms from younger mice to overcome the problem of age-relateddecline in female fertility.Materials and Methods: The developmental capacity, karyotype, and ultrastructure of reconstructedoocytes derived from meiosis-II chromosome transplantation from aged mice into the ooplasms ofyoung mice by piezo-micromanipulation were assessed.Results: The survival rate of enucleated young oocytes was 54% and the percent of fertilizedreconstructed oocytes was 23%. The rate of embryo development to the two-cell stage aftercultivation was 40%. Since 82.4% of the analyzed embryos derived from reconstructed oocyteshad condensed nuclei, it was not possible to analyze their chromosomal integrity. However, 17.6%of analyzable reconstructed old oocyte derived embryos (old-ODEs, had normal diploid sets ofchromosomes. Major structural differences were not observed between young, old, and M-II-tderived two-cell embryos.Conclusion: Our findings suggested that ooplasms from younger mice may overcome ageassociatedproblems in older mice.

  8. Enhance beef cattle improvement by embryo biotechnologies.

    Science.gov (United States)

    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.

  9. Generating chimeric zebrafish embryos by transplantation.

    Science.gov (United States)

    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. Oocytes selected using BCB staining enhance nuclear reprogramming and the in vivo development of SCNT embryos in cattle.

    Directory of Open Access Journals (Sweden)

    Jianmin Su

    Full Text Available The selection of good quality oocytes is crucial for in vitro fertilization and somatic cloning. Brilliant cresyl blue (BCB staining has been used for selection of oocytes from several mammalian species. However, the effects of differential oocyte selection by BCB staining on nuclear reprogramming and in vivo development of SCNT embryos are not well understood. Immature compact cumulus-oocyte complexes (COCs were divided into control (not exposed to BCB, BCB+ (blue cytoplasm and BCB- (colorless cytoplasm groups. We found that BCB+ oocytes yielded a significantly higher somatic cell nuclear transfer (SCNT blastocyst rate and full term development rate of bovine SCNT embryos than the BCB- and control oocytes. BCB+ embryos (embryos developed from BCB+ oocytes showed increased acetylation levels of histone H3 at K9 and K18 (AcH3K9, AcH3K18, and methylation levels of histone H3 at K4 (H3K4me2 than BCB- embryos (embryos developed from BCB- oocytes at the two-cell stage. Furthermore, BCB+ embryos generated more total cells, trophectoderm (TE cells, and inner cell mass (ICM cells, and fewer apoptotic cells than BCB- embryos. The expression of SOX2, CDX2, and anti-apoptotic microRNA-21 were up-regulated in the BCB+ blastocysts compared with BCB- blastocysts, whereas the expression of pro-apoptotic gene Bax was down-regulated in BCB+ blastocysts. These results strongly suggest that BCB+ oocytes have a higher nuclear reprogramming capacity, and that BCB staining can be used to select developmentally competent oocytes for nuclear transfer.

  11. Diploid oocyte formation and tetraploid embryo development induced by cytochalasin B in bovine.

    Science.gov (United States)

    Bai, Chunling; Liu, Hui; Liu, Ying; Wu, Xia; Cheng, Lei; Bou, Shorgan; Li, Guang-Peng

    2011-02-01

    Tetraploid embryos are a useful model for postimplantation development of polyploidy cells, and tetraploid cells are an advantage in studies for chimeras yielding offspring completely derived from embryo stem cells or induced pluripotent cells. This study was designed to investigate the effects of cytochalasin B (CB) on bovine oocyte meiosis, and to induce the formation of diploid oocytes and tetraploid embryos. The results showed that: (1) incubation of oocytes in CB at ≥2.0 μg/mL concentrations for 24 h significantly decreased oocyte maturation and the matured oocytes' haploid composition. Over 50% of the CB-treated oocytes did not expel PB1 (non-PB1), and most of the non-PB1 oocytes contained 2n (60) chromosomes. (2) Pretreatment of oocytes with CB at concentrations of 7.5 and 15 μg/mL for 10 h significantly decreased oocyte maturation. Posttreatment of oocytes with CB resulted in most of the oocytes containing 2n chromosomes. (3) The parthenogenetic blastocysts (25-28%) derived from the non-PB1 oocytes of posttreatment group was significantly higher than that from pretreatment, whole period treatment, and the control oocytes (12-16%). (4) Cytogenetic analysis of the embryos derived from CB-treated non-PB1 oocytes resulted in 74% of the one-cell stage embryos being 4n = 120 chromosomes, 82% of two-cell stage embryos contained 4n chromosomes in each blastomere, and 75% of the blastocysts were tetraploidy (4n = 120). (6) The stopped uncleaved one-cell embryos showed an amazing phenomenon of over 15% of them containing extra chromosomes, which suggested multiple DNA duplication occurred within 40 h after activation. In conclusion, CB inhibits PB1 extrusion, disfigures spindle structure, decreases oocyte maturation, and results in formation of diploid (2n or 4c) oocytes. The diploid oocytes resulted in a higher development of tetraploid embryos, which would be a unique approach for the production of tetraploid embryos in bovine.

  12. Developmental competence of porcine chimeric embryos produced by aggregation

    DEFF Research Database (Denmark)

    Li, Juan; Jakobsen, Jannik E.; Xiong, Qiang

    2015-01-01

    The purpose of our study was to compare the developmental competence and blastomere allocation of porcine chimeric embryos formed by micro-well aggregation. Chimeras were created by aggregating either two blastomeres originating from 2-cell embryos or two whole embryos, where embryos were produced...... either by parthenogenetic activation (PA) or handmade cloning (HMC). Results showed that the developmental competence of chimeric embryos, evaluated based on their blastocyst rate and total cell number per blastocyst, was increased when two whole 2-cell stage embryos (PA or HMC) were aggregated....... In comparison, when two blastomeres were aggregated, the developmental competence of the chimeric embryos decreased if the blastomeres were either from PA or from HMC embryos, but not if they were from different sources, i.e. one PA and one HMC blastomere. To evaluate the cell contribution in embryo formation...

  13. Developmental competence of porcine chimeric embryos produced by aggregation

    DEFF Research Database (Denmark)

    Li, Juan; Jakobsen, Jannik E.; Xiong, Qiang

    2015-01-01

    The purpose of our study was to compare the developmental competence and blastomere allocation of porcine chimeric embryos formed by micro-well aggregation. Chimeras were created by aggregating either two blastomeres originating from 2-cell embryos or two whole embryos, where embryos were produced...... either by parthenogenetic activation (PA) or handmade cloning (HMC). Results showed that the developmental competence of chimeric embryos, evaluated based on their blastocyst rate and total cell number per blastocyst, was increased when two whole 2-cell stage embryos (PA or HMC) were aggregated....... In comparison, when two blastomeres were aggregated, the developmental competence of the chimeric embryos decreased if the blastomeres were either from PA or from HMC embryos, but not if they were from different sources, i.e. one PA and one HMC blastomere. To evaluate the cell contribution in embryo formation...

  14. Cutin fluorescence in early embryos of Pinus and Tsuga

    OpenAIRE

    Ewa Szczuka; Irena Gielwanowska

    2014-01-01

    Embryos of Pinus nigra Arnold and Tsuga canadensis Carr. (Pinaceae) at different stages of development were dissected from fresh, unfixed seeds and examined in a fluorescence microscope with 400 nm excitation light. The embryos of the investigated species showed cutin fluorescence after auramine 0 staining. At first the fluorescing cutin layer was formed on the apical part of the embryo with a well developed secondary suspensor, then it extended over the lateral surface of the embryo; the sus...

  15. Research Progress on Technique of Frozen Embryo Transfer in Sheep

    Institute of Scientific and Technical Information of China (English)

    SHE Qiu-sheng; HU Jian-ye; LOU Peng-yan; TAO Jing; XIE Zhao-hui

    2011-01-01

    The paper introduced the research progress on the technique of frozen embryo transfer in sheep, illustrated selection of donors and receptors, superovulation, synchronization of estrus, embryo cryopreservation and embryo transplantation. Frozen embryo transfer in sheep is another breakthrough in the high-quality sheep raising, and this technique in China is in its infancy recommendation stage, but it will be comprehensively popularized in the future.

  16. Effect of zona pellucida on porcine parthenogenetically activated embryos

    DEFF Research Database (Denmark)

    Li, Rong; Liu, Ying; Li, Juan

    2011-01-01

    The need for zona pellucida (ZP) during pre-implantation embryo development is still debated. In porcine parthenogenetically activated (PA) embryos, we have previously shown a different distribution in cell numbers on Day 6 blastocysts cultured with or without ZP (Li et al. 2010 Reprod. Fertil. D...... embryos, especially at the timing of embryonic genome activation (5-cell stage). Furthermore, the zona pellucida can benefit the blastocyst formation and cryo-tolerance for PA embryos, perhaps by creating a more stable microenvironement....

  17. Expression of estrogen receptor alpha in preimplantation mice embryos

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective:To study the expression of estrogen receptor alpha (ERα) in preimplantation mice embryos.Methods:Mice zygotes were collected from superovulated Kunming mice and cultured in vitro.Embryos at different developmental stages were collected at 0,24,36,48,72 and 96hours after cultivation.The expression of ERα in early mice embryos was detected by reverse transcription-PCR (RT-PCR) and immunocytochemistry.Results:The expression of ERα mRNA was detected in all of the examined embryonic stages.The relative amount of ERα mRNA showed no significant difference between 1-cell stage embryos and 4-cell stage embryos (P>0.05).However,the relative level of ERα mRNA significantly decreased (P<0.05) at 2-cell stage and was the lowest at this stage.Over 2-cell stage,the ERα mRNA relative level would increase and achieve the peak level at blastocyst stage.The location of immunocytochemistry showed that ERα immunopositive cells could be firstly detected at 8-cell stage,after which they are consistently detected until blastocyst stage.In addition,the intensity of ERα positive staining was higher at blastocyst stage compared with that at 8-cell stage and morula stage.Conclusion:ERα is expressed in preimplantation mice embryos in a temporal and spatial pattern and may be involved in regulating the development of early mice embryos,which probably plays crucial roles in early embryonic development.

  18. 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.

    Science.gov (United States)

    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

  19. New perspectives on embryo biopsy: not how, but when and why?

    Science.gov (United States)

    Xu, Kangpu; Montag, Markus

    2012-08-01

    During the past 2 decades, biopsy for preimplantation genetic diagnosis at three stages, that is, before conception (the first polar body), after fertilization (the second polar body), and early cleavage (D3 blastomeres) or blastocyst stage (D5/D6 trophectoderm biopsy), have been optimized and performed clinically in hundreds of in vitro fertilization centers around the world. Although opening the zona pellucida by mechanical or chemical means is still effectively in use, noncontact laser has become the indispensable instrument. Overall, the invasive nature of biopsy at any given stage is recognized. It is believed that removal of the polar bodies from M-II oocytes and fertilized zygotes may have the least detrimental effects on subsequent embryonic development; hence increasing applications of polar body biopsy are anticipated. Although D3 biopsy is currently the most frequently used method, the effectiveness of D3 cleavage-stage biopsy is unsettling because of the mosaicism in early cleavage human embryos. Controversies exist in several areas; particularly, the efficacy of preimplantation genetic screening based on one cell removed from a D3 embryo remains to be confirmed. With new genetic testing technology, there may be no need to biopsy two cells because accuracy from one cell is high and the risk of misdiagnosis is very low when sufficient markers are used for chromosome copy number assessment or for mutation detection of single-gene disorders. And finally, it appears that limited harm is caused by biopsy at the blastocyst stage and mosaicism seems to be less common as compared with earlier stages. Therefore, use of the blastocyst-stage biopsy combined with cryopreservation protocol can be effectively used for several indications. Furthermore, faster genetic analytical methods that can be completed within several hours will make this strategy more practical and attractive as fresh embryo transfer is possible. Thieme Medical Publishers 333 Seventh Avenue, New

  20. Developing Xenopus embryos recover by compacting and expelling single wall carbon nanotubes.

    Science.gov (United States)

    Holt, Brian D; Shawky, Joseph H; Dahl, Kris Noel; Davidson, Lance A; Islam, Mohammad F

    2016-04-01

    Single wall carbon nanotubes are high aspect ratio nanomaterials being developed for use in materials, technological and biological applications due to their high mechanical stiffness, optical properties and chemical inertness. Because of their prevalence, it is inevitable that biological systems will be exposed to nanotubes, yet studies of the effects of nanotubes on developing embryos have been inconclusive and are lacking for single wall carbon nanotubes exposed to the widely studied model organism Xenopus laevis (African clawed frog). Microinjection of experimental substances into the Xenopus embryo is a standard technique for toxicology studies and cellular lineage tracing. Here we report the surprising finding that superficial (12.5 ± 7.5 µm below the membrane) microinjection of nanotubes dispersed with Pluronic F127 into one- to two-cell Xenopus embryos resulted in the formation and expulsion of compacted, nanotube-filled, punctate masses, at the blastula to mid-gastrula developmental stages, which we call "boluses." Such expulsion of microinjected materials by Xenopus embryos has not been reported before and is dramatically different from the typical distribution of the materials throughout the progeny of the microinjected cells. Previous studies of microinjections of nanomaterials such as nanodiamonds, quantum dots or spherical nanoparticles report that nanomaterials often induce toxicity and remain localized within the embryos. In contrast, our results demonstrate an active recovery pathway for embryos after exposure to Pluronic F127-coated nanotubes, which we speculate is due to a combined effect of the membrane activity of the dispersing agent, Pluronic F127, and the large aspect ratio of nanotubes.

  1. 冻融卵裂期胚胎移植中过夜培养对临床妊娠结局的影响%Impact of culture overnight during frozen-thawed cleavage-stage embryo transfers on preg-nancy outcome

    Institute of Scientific and Technical Information of China (English)

    李荣; 许常龙; 谭秀群; 邓志华; 李春苑; 丘映

    2015-01-01

    Objective To explore the impact of culture overnight during frozen -thawed cleavage-stage embryo transfers on pregnancy outcome.Methods Data of 237 cycles of vitrified cleavage-stage embryos from the Third Affiliated Hospital between De -cember 2011 and December 2012 was retrospectively analyzed.The embryo were divided into group A (embryo was thawed on the day of transplantation) and group B (embryo was culture overnight ).The embryo implantation rate,clinical pregnancy rate and abortion rate were compared between the two groups .Results The embryo implantation rate was significantly lower in the group A when compared with that in the group B (P <0.05).However,no significant difference in clinical pregnancy rate and abortion rate was found between the two groups.Conclusion It may provide a valuable reference for the selection of high quality of embryo when embryos are thawed in advance and cultured overnight ,and then the development of embryos can be observed .This method also facilitates the arrangement of laboratory working.%目的:探讨冻融卵裂期胚胎移植中过夜培养对临床妊娠结局的影响。方法2011年12月至2012年12月在我院生殖医疗中心接受玻璃化冷冻的卵裂期胚胎移植患者共237个周期,其中胚胎解冻时间为移植当日128个周期(A 组),过夜培养109个周期(B 组),比较两组的胚胎种植率、临床妊娠率、流产率。结果 A 组胚胎种植率低于 B 组(P <0.05),而临床妊娠率和流产率与 B 组,差异无统计学意义(P >0.05)。结论将胚胎提前解冻培养过夜,观察解冻后胚胎的进一步发育情况,可提高胚胎种植率,为临床选择移植胚胎提供有意义的参考。

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

    Science.gov (United States)

    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.

  3. Somatic Embryos in Catharanthus roseus: A Scanning Electron Microscopic Study

    Directory of Open Access Journals (Sweden)

    Junaid ASLAM

    2014-06-01

    Full Text Available Catharanthus roseus (L. G. Don is an important medicinal plant as it contains several anti-cancerous compounds, like vinblastine and vincristine. Plant tissue culture technology (organogenesis and embryogenesis has currently been used in fast mass propagating raw materials for secondary metabolite synthesis. In this present communication, scanning electron microscopic (SEM study of somatic embryos was conducted and discussed. The embryogenic callus was first induced from hypocotyls of in vitro germinated seeds on which somatic embryos, differentiated in numbers, particularly on 2,4-D (1.0 mg/L Murashige and Skoog (MS was medium. To understand more about the regeneration method and in vitro formed embryos SEM was performed. The SEM study revealed normal somatic embryo origin and development from globular to heart-, torpedo- and then into cotyledonary-stage of embryos. At early stage, the embryos were clustered together in a callus mass and could not easily be detached from the parental tissue. The embryos were often long cylindrical structure with or without typical notch at the tip. Secondary embryos were also formed on primary embryo structure. The advanced cotyledonary embryos showed prominent roots and shoot axis, which germinated into plantlets. The morphology, structure and other details of somatic embryos at various stages were presented.

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

    Science.gov (United States)

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

    2010-11-01

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

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

    Science.gov (United States)

    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.

  6. Study on cryopreservation of mouse embryos at various development stages with hemi-straw vitrification and slow-freezing%Hemi-straw玻璃化与程序化冷冻小鼠不同发育阶段胚胎的效果

    Institute of Scientific and Technical Information of China (English)

    吴志南; 华月琴; 陈瑞华

    2011-01-01

    目的:探讨半细管(Hemi-straw)玻璃化方法与程序化方法冷冻不同发育阶段小鼠胚胎的效果.方法:采用慢速程序化方法与玻璃化方法冷冻小鼠原核期胚胎、卵裂期及早期囊胚期胚胎.解冻后观察存活率、囊胚形成率.结果:采用玻璃化方法冷冻小鼠卵裂期与早期囊胚期胚胎的存活率为92.7%与95.5%,显著高于玻璃化冷冻原核期胚胎的存活率79.8%与程序化冷冻的各组(均P0.05),但均显著低于玻璃化冷冻组(均P<0.01).玻璃化冷冻卵裂期与早期囊胚组的囊胚形成率高达90.1%与92.2%,显著高于原核期胚胎玻璃化冷冻组69.5%(均P<0.01).结论:胚胎行冷冻保存时,玻璃化冷冻效果较程序化冷冻好,且选择早期囊胚可获最佳冷冻效果.%Objective: To compare cryopreservation of mouse embryos at various development stages with hemi -straw vitrification and slow- freezing Methods: The mouse embryos at pronuclear stage, cleavage stage and early blastocyst stage were cryopreservated with hemi-straw vitrification and slow -freezing.Then cryosurvival rate and blastocyst formation rate were observed after ice-out.Results:The cryosurvival rates of embryos at cleavage stage and early blastocyst stage cryopreservated with vitrification were 92.7% and 95.5%,which were significantly higher than that at pronuclear stage (79.8%) and with slow-freezing groups (all P<0.01).The cryosurvival rate of embryos at pronuclear stage cryopreservated with slow - freezing was 50.2%, which was significantly lower than others ( all P <0.01 ).The blastocyst formation rates of embryos cryopreservated with slow - freezing were significantly lower than with vitrification ( all P <0.01 ).The blastocyst formation rates of embryos at cleavage stage and early blaatocyst stage cryopreservated with vitrification were 90.1% and 92.2%, which were significantiy higher than at pronuclear stage (69.5% ) ( all P <0.01 ).Conclusion: The embryos can be

  7. Ultrastructural changes in goat interspecies and intraspecies reconstructed early embryos

    DEFF Research Database (Denmark)

    Tao, Yong; Gheng, Lizi; Zhang, Meiling;

    2008-01-01

    and dispered gradually from the 4-cell period. The nucleolus of GC and GG embryos changed from electron dense to a fibrillo-granular meshwork at the 16-cell stage, showing that nucleus function in the reconstructed embryos was activated. The broken nuclear envelope and multiple nucleoli in one blastomere......- and intraspecies reconstructed embryos have a similar pattern of developmental change to that of in vivo-produced embryos for ZP, rough ER, Gi and nucleolus, but differ for mitochondria, LD, vesicles, nucleus and gap junction development. In particular, the interspecies cloned embryos showed more severe...

  8. Clonal propagation of primate offspring by embryo splitting.

    Science.gov (United States)

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

    2000-01-14

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

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

    Science.gov (United States)

    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

  10. 囊胚培养及移植在体外受精周期中的应用%Application of Embryo Culture and Transplanting in Blastocyst Stage in the In Vitro Fertilization Cycle

    Institute of Scientific and Technical Information of China (English)

    杨世俊

    2016-01-01

    目的 探讨囊胚培养及移植在体外受精周期中的应用. 方法 整群选取2012年1月—2014年12月期间来该院接受体外受精-胚胎移植的64例患者作为该次研究对象,按照选择胚胎培养及移植时期的不同将64例体外受精-胚胎移植患者分成研究组和对照组,研究组为选择囊胚期胚胎培养及移植的患者,共33例,对照组为选择卵裂期胚胎培养及移植的患者,共31例. 受精成功后采用G1进行早期胚胎培养,采用G2进行囊胚培养. 结果 两组患者中各有1例消移植周期病例, 实际移植周期数分别为32个和30个, 研究组患者的胚胎植入率为37.1%, 妊娠率为53.1%,与对照组患者的19.4%和33.3%比较,差异有统计学意义(P0.05),但对照组患者的多胎率明显高于研究组患者(P0.05), however, the multiple pregnancy rate in the control group was obviously higher than that in the research group (P<0.05). Conclusion Embryo culture and transplanting in blastocyst stage can effectively improve the embryo implantation rate and the pregnancy rate, and can reduce the incidence rate of adverse pregnancy outcome and transfer cycle cancellation rate.

  11. The DNA methylation events in normal and cloned rabbit embryos

    Institute of Scientific and Technical Information of China (English)

    TaoChen; Yan-LingZhang; YanJiang; Shu-ZhenLiu; HeideSchatten; Da-YuanChen; Qing-YuanSun

    2005-01-01

    To study the DNA methylation events in normal and cloned rabbit embryos, we investigated the methylation status of a satellite seqnence and the promoter region of a single-copy gene using bisulfite-sequencing technology. During normal rabbit embryo development, both sequences maintained hypermethylation status until the 8- to 16-cell stage when progressive demethylation took place. In cloned embryos, the single-copy gene promoter sequence was rapidly demethylated and preco-ciously de novo methylated, while the satellite sequence mainrained the donor-type methylation status in all examined stages. Our results indicate that unique sequences as well as satellitesequences may have aberrant methylation patterns in cloned embryos.

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

    Directory of Open Access Journals (Sweden)

    Jin Akagi

    Full Text Available 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.

  13. Cryopreservation of Primordial Germ Cells by Rapid Cooling of Whole Zebrafish (Danio rerio) Embryos

    National Research Council Canada - National Science Library

    HIGAKI, Shogo; MOCHIZUKI, Kentaro; AKASHI, Yuichiro; YAMAHA, Etsuro; KATAGIRI, Seiji; TAKAHASHI, Yoshiyuki

    2010-01-01

    ...) of dechorionated whole embryos at the 14- to 20-somite stage was investigated. Initially, we examined the glass-forming properties and embryo toxicities of six cryoprotectants: methanol (MeOH), ethylene glycol (EG), glycerol (GC...

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  16. Culture systems: embryo density.

    Science.gov (United States)

    Reed, Michael L

    2012-01-01

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

  17. Acute toxicity of fenoxycarb to zebrafish(Brachydanio rerio)at different development stages and chronic toxicity to embryo and sac-fry stage%苯氧威对斑马鱼不同发育阶段急性毒性及胚胎卵黄囊仔鱼阶段慢性毒性作用

    Institute of Scientific and Technical Information of China (English)

    孙兴泽; 王成菊; 李学锋; 郑明奇; 邱立红

    2015-01-01

    为了解苯氧威对斑马鱼的毒性作用,采用半静态方法,研究了苯氧威对斑马鱼不同发育阶段的急性毒性。结果表明:苯氧威对斑马鱼胚胎、孵化12 h和72 h的仔鱼以及成鱼的96 h-LC50值分别为2.35、0.98、1.87和1.67 mg/L,其对斑马鱼成鱼具有中等毒性;苯氧威对斑马鱼胚胎的自主运动、心率、孵化率、仔鱼体长等亚致死效应指标均有影响,并使得胚胎出现了心包囊肿、脊柱弯曲及无黑色素附着等畸形现象。进一步研究了苯氧威对斑马鱼胚胎卵黄囊仔鱼阶段的慢性毒性作用。结果显示:苯氧威对斑马鱼胚胎24 h自主运动、48 h心率、72 h畸形率及11 d孵化仔鱼体长均有不同程度的抑制作用,其最低可观测效应浓度( LOEC )值分别为0.60、0.20、0.60和0.80 mg/L,而与之对应的无可观测效应浓度( NOEC)值分别为0.40、0.10、0.40和0.60 mg/L。试验中还发现:苯氧威对斑马鱼胚胎孵化有明显抑制作用;心率是斑马鱼胚胎卵黄囊仔鱼阶段毒性试验最敏感的观察指标。%In order to provide information for the safety and rational use of fenoxycarb,the acute toxicity of fenoxycarb to different development stages of zebrafish( Brachydanio rerio)was investigated by semi-static method. Results showed that the 96 h LC50 values of fenoxycarb were 2. 35,0. 98,1. 87 and 1. 67 mg/L for embryo,12 h-old larva,72 h-old larva and adult of zebrafish,respectively. There were also some sublethal and teratogenic effects observed. Fenoxycarb could affect the spontaneous movements,heart rate,hatching rate of embryo and the body length of hatched larvae,as well as cause pericardial edema,axial malformation and unnormal develpoment of melanocytes in the embryo of zebrafish. The chronic toxicity of fenoxycarb to zebrafish embryo and sac-fry stage was further studied. According to different endpoints tested,the lowest observed effect concentration( LOEC )

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

    Science.gov (United States)

    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.

  19. MiRNA-320 in the human follicular fluid is associated with embryo quality in vivo and affects mouse embryonic development in vitro.

    Science.gov (United States)

    Feng, Ruizhi; Sang, Qing; Zhu, Yan; Fu, Wei; Liu, Miao; Xu, Yan; Shi, Huijuan; Xu, Yao; Qu, Ronggui; Chai, Renjie; Shao, Ruijin; Jin, Li; He, Lin; Sun, Xiaoxi; Wang, Lei

    2015-03-03

    Previous work from our laboratory demonstrated the existence of miRNAs in human follicular fluid. In the current study, we have sought to identify miRNAs that might affect oocyte/embryo quality in patients undergoing intracytoplasmic sperm injection and to investigate their roles in in vitro fertilization outcomes in mouse oocytes. 53 samples were classified as Group 1 (high quality) if the day-3 embryos had seven and more cells or as Group 2 (low quality) if the embryos had six and fewer cells. TaqMan Human microRNAs cards and qRT-PCR were performed to verify differently expressed miRNAs. The function of the corresponding miRNA was investigated in mouse oocytes by injecting them with miRNA-inhibitor oligonucleotides. We found that hsa-miR-320a and hsa-miR-197 had significantly higher expression levels in the Group 1 follicular fluids than in Group 2 (p = 0.0073 and p = 0.008, respectively). Knockdown of mmu-miR-320 in mouse oocytes strongly decreased the proportions of MII oocytes that developed into two-cell and blastocyst stage embryos (p = 0.0048 and p = 0.0069, respectively). Wnt signaling pathway components had abnormal expression level in miR-320 inhibitor-injected oocytes. This study provides the first evidence that miRNAs in human follicular fluid are indicative of and can influence embryo quality.

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

    Science.gov (United States)

    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.

  1. Steroidal alkaloid toxicity to fish embryos.

    Science.gov (United States)

    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.

  2. Electroporation into Cultured Mammalian Embryos

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Ju, Yan; Liu, Chunying; Lu, Wenwen; Zhang, Quan; Sodmergen, E-mail: sodmergn@pku.edu.cn

    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.

  4. Effects of species, development periods and different experimental conditions on the outcomes of embryo freezing and thawing in cleavage stage%种属和发育期及不同实验条件对卵裂期胚胎冷冻复苏结局的影响

    Institute of Scientific and Technical Information of China (English)

    巩晓芸; 赵静; 胡泊; 王鹏; 蔡霞

    2011-01-01

    背景:人卵裂期胚胎冷冻复苏的研究中,不同的实验条件及实验动物模型是否与人类胚胎具有相同的敏感性从而反映出实验方案的优劣值得探讨.目的:观察胚胎种属和发育期及不同冷冻条件对卵裂期胚胎冷冻复苏结局的影响.方法:将人卵裂期胚胎作为对照组,将KM小鼠胚分为2细胞,4细胞,8细胞组.各组胚胎随机采用以下实验方案:①冷冻操作环境温度18~20 ℃、24~26 ℃和37 ℃.②慢速程序化方案、自制straw叶片玻璃化方案和CPS玻璃化方案.③与玻璃化液接触时间 0.05).③各组胚胎与冷冻保护剂接触不同时间胚胎复苏率差异无显著性意义(P > 0.05).表明卵裂期胚胎玻璃化冷冻复苏效果优于慢速程序化,24~26 ℃操作环境、减少冷冻保护剂剂量和缩短接触时间可改善玻璃化冷冻复苏结局;相同冷冻条件下,胚胎种属和发育阶段对冷冻复苏结局有影响,4细胞鼠胚更适合作为研究人类卵裂期胚胎冷冻复苏的实验模型.%BACKGROUND: In the studies of frozen-thawed human cleavage embryos, whether the experimental conditions and experimental animal model have the same sens ft h/ity with human to reflect the experiment quality e worth exploring. OBJECT P/E: To investigate the effect of embryo species, development periods and drffererrt frozen ccndrticns on the outcomes of embryo freezing and thawing in cleavage stage.METHODS: Human embryos in cleavage stage served as control group, KM mouse embryos were randomly divided into 2-cell-stage group. 4-cell-stage group and 8-cell-stage group. Experimentscheme: Operating environmenttemperaturewere 1S-2O "C, 24-26 "C and 37 "C. Slow programmed cryopreservation. self-made Straw leaf vitrification cryopreservation and close pulled straw vitrification ciyopreservation were used in the study. Contact durations of vitrified solution were less than 40 seconds. 40-60 seconds and 60-90 seconds. The embryo survival rates

  5. ROCK inhibition prevents early mouse embryo development.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  7. Preliminary studies on cryopreservation of common tench (Tinca tinca) embryos (work in progress).

    Science.gov (United States)

    El-Battawy, K A; Linhart, O

    2009-08-01

    Vitrification could provide a promising tool for the cryopreservation of fish embryos. However, to achieve cryopreservation using vitrification, chilling sensitivity and cryoprotectants toxicity were determined using tench embryos at four developmental stages (11, 17, 23 and 29 h). Embryos treated with alcalase (2 ml/998 ml, 2 min at 22 degrees C) were exposed to chilling with/without warming. Other embryos were exposed to methanol and glycerol at the concentration of 10% and 20% for periods of 20 min. At last, embryos were incubated at special incubator cages where hatching rates were counted. Regarding chilling sensitivity and exposure to chilling followed by warming, the hatching rates of embryos decreased significantly (p < 0.001) after exposure to 0 degrees C at all developmental stages except the 29-h stage compared with the controls. The embryo stage most sensitive to chilling was 11-h stage. The 29-h stage exhibited the least sensitivity to low temperature while 17-h and 23-h stages were intermediate in their sensitivity to chilling. The toxicity of methanol increased significantly (p < 0.001) with developmental stage for 11, 17 and 23-h stages. The highest hatching rates of tench embryos were obtained with 29-h embryos using various concentrations of methanol. The hatching rates of tench embryos exposed to glycerol concentrations were approximately similar to those embryos exposed to methanol concentrations except for 11-h embryos that showed no hatching. Unfortunately, we could not obtain living embryos in any of the conditions examined after vitrification. In conclusion, it was quite difficult to vitrify the tench embryos during this study using various vitrifying solutions and the method reported by Chen & Tian (2005) and further studies are needed to achieve successful cryopreservation.

  8. Contrasting pattern of somatic and zygotic embryo development in alfalfa (Medicago sativa L.) as revealed by scanning electron microscopy.

    Science.gov (United States)

    Xu, N; Bewley, J D

    1992-06-01

    Scanning electron microscopy has been used to investigate the morphological changes occurring during the development of alfalfa somatic embryos. Embryos were initiated from callus, transferred to suspension culture and matured on solid agar medium. This developmental pattern was compared to that of zygotic embryos developing in ovulo. Somatic embryos begin as distinct pro-embryos within the callus tissue pieces placed in suspension culture. They become globular and heart-shaped while on solid agar medium and then undergo cotyledon elongation and maturation. Somatic embryos develop comparatively slower at early stages of development and faster at the later stages than zygotic embryos. They lack a well-defined suspensor and have a very rough, poorly-differentiated epidermis, the first layer of which is lost after pro-embryo formation. The cotyledons of somatic embryos are multiple and poorlydeveloped; there appears to be a correlation between the amount of surface roughness of the developing embryo and the extent to which polycotyledony occurs.

  9. Bovine pretransfer endometrium and embryo transcriptome fingerprints as predictors of pregnancy success after embryo transfer.

    Science.gov (United States)

    Salilew-Wondim, Dessie; Hölker, Michael; Rings, Franca; Ghanem, Nasser; Ulas-Cinar, Mehmet; Peippo, Jaana; Tholen, Ernst; Looft, Christian; Schellander, Karl; Tesfaye, Dawit

    2010-07-07

    Aberrant gene expression in the uterine endometrium and embryo has been the major causes of pregnancy failure in cattle. However, selecting cows having adequate endometrial receptivity and embryos of better developmental competence based on the gene expression pattern has been a greater challenge. To investigate whether pretransfer endometrial and embryo gene expression pattern has a direct relation with upcoming pregnancy success, we performed a global endometrial and embryo transcriptome analysis using endometrial and embryo biopsy technology and the pregnancy outcome information. For this, endometrial samples were collected from Simmental heifers at day 7 and 14 of the estrous cycle, one cycle prior to embryo transfer. In the next cycle, blastocyst stage embryos were transferred to recipients at day 7 of the estrous cycle after taking 30-40% of the blastocyst as a biopsy for transcriptome analysis. The results revealed that at day 7 of the estrous cycle, the endometrial gene expression pattern of heifers whose pregnancy resulting in calf delivery was significantly different compared with those resulting in no pregnancy. These differences were accompanied by qualitative and quantitative alteration of major biological process and molecular pathways. However, the transcriptome difference was minimal between the two groups of animals at day 14 of the estrous cycle. Similarly, the transcriptome analysis between embryos biopsies that resulted in calf delivery and those resulted in no pregnancy revealed a total of 70 differentially expressed genes. Among these, the transcript levels of 32 genes including SPAG17, PF6, UBE2D3P, DFNB31, AMD1, DTNBP1, and ARL8B were higher in embryo biopsies resulting in calf delivery. Therefore, the present study highlights the potential of pretransfer endometrial and embryo gene expression patterns as predictors of pregnancy success in cattle.

  10. In vivo assay for the developmental competence of embryo-derived zebrafish cell lines

    NARCIS (Netherlands)

    Speksnijder, JE; Hage, WJ; Lanser, PH; Collodi, P; Zivkovic, D

    1997-01-01

    We have produced chimeric zebrafish embryos by transplanting permanent embryo-derived cell lines into blastula-stage embryos. Furthermore, we have established a fluorescent in vivo assay to monitor the developmental effects and fate of such transplanted cells using confocal laser scanning microscopy

  11. Cutin fluorescence in early embryos of Pinus and Tsuga

    Directory of Open Access Journals (Sweden)

    Ewa Szczuka

    2014-01-01

    Full Text Available Embryos of Pinus nigra Arnold and Tsuga canadensis Carr. (Pinaceae at different stages of development were dissected from fresh, unfixed seeds and examined in a fluorescence microscope with 400 nm excitation light. The embryos of the investigated species showed cutin fluorescence after auramine 0 staining. At first the fluorescing cutin layer was formed on the apical part of the embryo with a well developed secondary suspensor, then it extended over the lateral surface of the embryo; the suspensor remained nonfluorescent. The fluorescing cutin layer occurred on the apical and side surface of the embryo, undergoing differentiation into the shoot axis and root initials. It is assumed that polarization and nutrition of the embryo may be influenced by presence of the cuticle.

  12. Role of glucose in cloned mouse embryo development

    National Research Council Canada - National Science Library

    Zhiming Han; Rita Vassena; Maggie M. Y. Chi; Santhi Potireddy; Miriam Sutovsky; Kelle H. Moley; Peter Sutovsky; Keith E. Latham

    2008-01-01

    Cloned mouse embryos display a marked preference for glucose-containing culture medium, with enhanced development to the blastocyst stage in glucose-containing medium attributable mainly to an early...

  13. Impact of Zona Pellucida Thinning by Laser Assisted Hatching on Clinical Outcomes of Cleavage Stage Embryos Cryopreserved by Slow Freezing and Vitrification%透明带薄化法激光辅助孵化对慢速冷冻和玻璃化冷冻卵裂期胚胎临床结局的影响

    Institute of Scientific and Technical Information of China (English)

    陈曦; 梁蓉; 石程; 王筠; 田莉; 沈浣

    2011-01-01

    Objective: To examine the impact of zona pellucida thinning by laser assisted hatching (AH) on clinical outcomes of cleavage stage embryos cryopereserved by slow freezing and vitrification. Methods: A retrospective analysis was performed on 564 frozen embryo transfer (FET) cycles with at least one intactness survival embryo cryopreserved by slow freezing or vitrification. All the cycles were divided into AH group (experimental group) and non-AH group (the control) according to whether the AH was performed before the embryos transferred. The clinical pregnancy, implantation and miscarriage rates of the cleavage embryos were analyzed. Results: When the embryos thawed from slow freezing, the implantation rate increased in AH group compared with control group significantly (19.5% vs 13.5%, P0.05). Conclusion: Quarter zona thinning assisted hatching may increase the implantation rate when embryos cryopreserved by slow freezing, but it is not an effective strategy for improving the implantation and clinical pregnancy rates of vitrified-warmed embryos at the cleavage stage. Moreover, AH would not increase the risk of embryos miscarriage.%目的:探讨透明带薄化法激光辅助孵化技术对慢速冷冻和玻璃化冷冻卵裂期胚胎临床结局的影响.方法:回顾性分析复苏后至少有1个胚胎是完整存活的564例卵裂期冻融胚胎移植周期,根据胚胎移植前是否行激光辅助孵化,分为辅助孵化组(研究组)与非辅助孵化组(对照组),分别观察透明带薄化法激光辅助孵化技术对慢速冷冻胚胎及玻璃化冷冻胚胎的临床妊娠率、种植率及流产率的影响.结果:胚胎经慢速冷冻后,研究组胚胎种植率(19.5%)显著高于对照组(13.5%),差异有统计学意义(P<0.05),而临床妊娠率及流产率(37.9% vs 28.5%、15.5% vs 10.8%)无显著差异;胚胎经玻璃化冷冻后,研究组和对照组胚胎的临床妊娠率(38.0% vs 35.6%)、种植率(17.3% vs 15.9

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

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

    OpenAIRE

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

    2016-01-01

    Preimplantation Genetic Diagnosis and Screening (PGD/PGS) for monogenic diseases and/or numerical/structural chromosomal abnormalities is a tool for embryo testing aimed at identifying nonaffected and/or euploid embryos in a cohort produced during an IVF cycle. A critical aspect of this technology is the potential detrimental effect that the biopsy itself can have upon the embryo. Different embryo biopsy strategies have been proposed. Cleavage stage blastomere biopsy still represents the most...

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

    Science.gov (United States)

    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).

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  19. Enhancement of NMRI Mouse Embryo Development In vitro

    Directory of Open Access Journals (Sweden)

    Abedini, F.

    2013-12-01

    Full Text Available Most of the systematic studies used in the development of human embryo culture media have been done first on mouse embryos. The general use of NMRI outbred mice is a model for toxicology, teratology and pharmacology. NMRI mouse embryo exhibit the two-cell block in vitro. The objective of this study was to evaluate and compare the effects of four kinds of culture media on the development of zygotes (NMRI after embryo vitrification. One-cell mouse embryos were obtained from NMRI mice after superovulation and mating with adult male NMRI mice. And then randomly divided into 4 groups for culture in four different cultures media including: M16 (A, DMEM/Ham, F-12 (B, DMEM/Ham's F-12 co-culture with Vero cells(C and DMEM/Ham's F-12 co-culture with MEF cells (D. Afterward all of the embryos were vitrified in EFS40 solution and collected. Results of our study revealed, more blastocysts significantly were developed with co-culture with MEF cells in DMEM/Ham's F-12 medium. More research needed to understand the effect of other components of culture medium, and co-culture on NMRI embryo development.

  20. Relationship between Pronuclear Scoring and Embryo Quality and Implantation Potential in IVF-ET

    Institute of Scientific and Technical Information of China (English)

    Qun LIU; Guijin ZHU; Juan HU; Yulan WEI; Xinling REN; Hanwang ZHANG; Yufeng LI; Lei JIN; Jing YUE

    2008-01-01

    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 (WF-ET) program, a pronuclear scoring system was used to score zygotes 16-20 h after insemination during conventional WF or intracytoplasmic sperm injec- tion (ICS1). 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. Implanta- tion rates of group Z1 were higher (P<0.05) than that of group Z3. These findings suggests that pro- nuclear 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.

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

    Science.gov (United States)

    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.

  2. Human embryos in the original position?

    Science.gov (United States)

    DiSilvestro, Russell

    2005-06-01

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

  3. Wide crossing in lentil through embryo rescue.

    Science.gov (United States)

    Fratini, Richard; Ruiz, Maria L

    2011-01-01

    Lentil seeds have provided an appreciated source of protein, carbohydrates and fibre to the diet of humans since the dawn of agriculture. Low amounts of variation have been detected in the cultivated lentil germplasm collections. Interspecific crosses allow for the introgression of important alleles of agricultural interest from wild species, such as the resistance or tolerance to abiotic and biotic stresses. Interspecific crosses within the genus Lens generally abort and embryo rescue techniques are necessary to recover hybrids. The in vitro culture procedure to rescue interspecific hybrids of Lens consists of at least four different stages: (1) in ovulo embryo culture (2), embryo culture, (3) plantlet development and finally, (4) the gradual habituation to ex vitro conditions of the recovered interspecific hybrid plantlets. In this chapter, the approach to rescue interspecific hybrids in the genus Lens is outlined.

  4. Selection of Norway spruce somatic embryos by computer vision

    Science.gov (United States)

    Hamalainen, Jari J.; Jokinen, Kari J.

    1993-05-01

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

  5. EMBRYO RESCUE Dendrobium anosmum Lindl. USING IN VITRO CULTURE

    Directory of Open Access Journals (Sweden)

    Putu Yuni Astriani Dewi

    2016-09-01

    Full Text Available The aim of the research is to analyse germination percentage, effect of coconut water addition in the media to number of last stage embryos and development of final stage D. anosmum Lindl. embryo. Research was conducted at Plant Structure and Development of Laboratory, Department of Biology, Faculty of Mathematics and Natural Sciences University of Udayana and UPT. Balai Benih Induk Tanaman Pangan dan Hortikultura Provinsi Bali from February 2015 to July 2015. Three type of media were employed, i.e. PDA, Organic, MS. Three different concentration of coconut was added to media, i.e. 0 ml/L 50 ml/L, 100 ml/L. Results showed that seed D. anosmum Lindl. on PDA, Organic and MS has not germinated yet on all media, only swollen cells observed. Seed dormancy and maturity of seed were the main factors affecting seed germination. Addition of 100 ml / L coconut water in MS showed that media is capable to increase the formation of final stages embryos (> 32 cells. Embryo development on PDA and Organic largely showed cell stage embryos 1 to 8 cells, while MS media already showed embryo development stages of > 32 cells.

  6. Detection of teratogens in human serum using rat embryo culture: cancer and epilepsy treatments. [Detecting teratogenicity of anticonvulsant and antineoplastic drugs

    Energy Technology Data Exchange (ETDEWEB)

    Chatot, C. L.

    1979-01-01

    Growth (protein and DNA contents) of headfold stage rat embryos cultured for 48 hrs on human serum was enhanced by glucose supplementation. Embryo growth varied with the source of the serum. Sera from 3 of the 19 control subjects produced abnormal embryos. Sera from 5 subjects undergoing cancer chemotherapy and 6 subjects receiving anticonvulsants were either lethal or teratogenic to cultured rat embryos.

  7. Analysis of Related Factors Influencing the Cleavage Stage Outcome of Frozen Thawed Embryo Transfer%探析卵裂期胚胎解冻移植结局相关影响原因

    Institute of Scientific and Technical Information of China (English)

    王琦; 陈蔚清; 张程

    2015-01-01

    目的:探析人卵裂期胚胎解冻移植结局的主要影响因素。方法研究对象是2012年2月1日~2014年2月1日在我院接受冻融胚胎移植的患者,解冻周期205个。结果玻璃化冷冻组的可移植胚胎率、胚胎复苏卵裂球存活率、临床妊娠率显著高于程序化冷冻组,两组有显著差别(0.05). Conclusion The method of embryo freezingsuitable is the main factor af ecting the cleavage stagefrozen thawed embryo transfer outcomes.

  8. Label-free Quantification of Proteins in Single Embryonic Cells with Neural Fate in the Cleavage-Stage Frog (Xenopus laevis) Embryo using Capillary Electrophoresis Electrospray Ionization High-Resolution Mass Spectrometry (CE-ESI-HRMS).

    Science.gov (United States)

    Lombard-Banek, Camille; Reddy, Sushma; Moody, Sally A; Nemes, Peter

    2016-08-01

    Quantification of protein expression in single cells promises to advance a systems-level understanding of normal development. Using a bottom-up proteomic workflow and multiplexing quantification by tandem mass tags, we recently demonstrated relative quantification between single embryonic cells (blastomeres) in the frog (Xenopus laevis) embryo. In this study, we minimize derivatization steps to enhance analytical sensitivity and use label-free quantification (LFQ) for single Xenopus cells. The technology builds on a custom-designed capillary electrophoresis microflow-electrospray ionization high-resolution mass spectrometry platform and LFQ by MaxLFQ (MaxQuant). By judiciously tailoring performance to peptide separation, ionization, and data-dependent acquisition, we demonstrate an ∼75-amol (∼11 nm) lower limit of detection and quantification for proteins in complex cell digests. The platform enabled the identification of 438 nonredundant protein groups by measuring 16 ng of protein digest, or embryo. LFQ intensity was validated as a quantitative proxy for protein abundance. Correlation analysis was performed to compare protein quantities between the embryo and n = 3 different single D11 blastomeres, which are fated to develop into the nervous system. A total of 335 nonredundant protein groups were quantified in union between the single D11 cells spanning a 4 log-order concentration range. LFQ and correlation analysis detected expected proteomic differences between the whole embryo and blastomeres, and also found translational differences between individual D11 cells. LFQ on single cells raises exciting possibilities to study gene expression in other cells and models to help better understand cell processes on a systems biology level.

  9. Effects of different nuclear recipients on developmental potential of mouse somatic nuclear transfer embryos

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    In order to investigate the effects of different kinds of nuclear recipients from Kunming (KM) mouse on developmental potential of somatic nuclear transfer em- bryos, the enucleated MⅡ oocytes, enucleated zygotes and 2-cell blastomere were used to produce cloned mouse embryos. Using fibroblast deriving from C57/BL6 ear tissue as nuclear donor, we produced cloned embryos by transferring the fibroblast nuclei into enucleated KM mouse oocytes (single nuclear transfer, SNT), transferring pronuclei from the SNT embryos into enucleated KM zygotes (nuclear into zygote, NZ), and 2-cell blastomere nuclei from SNT embryos into enucleated KM mouse oocytes (nuclear into oocytes, NO); tetraploid embryos (tetraploid embryos, TE) were obtained by fusing two blastomeres, one is from the SNT cloned embryos, and the other from normal 2-cell KM mouse embryos. In group SNT, the cloned embryos could not develop beyond 8-cell stage and the rate of 8-cell stage is only 0.3%; in group NO, the reconstructed embryos could develop to morula stage, the rate of 8-cell stage was significantly greater than that of SNT group (P < 0.05); in group NZ, the development rate was further improved, and the reconstructed embryos could develop into blastocyst stage, the rate of blastocyst was 1.9%; in group TE, as high as 62.3% of the reconstructed embryos could develop into blastocyst. Results suggested that different nuclear recipients could significantly affect the developmental potential of cloned mouse embryos; KM MⅡ oocyte cytoplasm was not so effective as zygotes to reprogram the mouse somatic cell nuclei; serial nuclear transfer could improve the developmental potential of cloned mouse embryos.

  10. Calciumreleasing activity induced by nuclei of mouse fertilized early embryos

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    At fertilization, repetitive transient rises of intracellular calcium concentration occur in all mammals studied so far. It has been shown that calcium rises could be induced when mouse fertilized 1-, 2-cell nuclei were transplanted into unfertilized eggs and that the reconstituted embryo could be activated. However, whether the capability of inducing calcium rises occurs in all stages of mammalian embryos remains unknown. In this study, by using the nuclear transplantation technique and measurement of intracellular calcium rises in living cells, we showed that only the nuclei from mouse fertilized 1-cell and 2-cell embryos, neither the nuclei from 4-, 8-cell and ethanol activated parthenogenetic embryos nor 2 or 3 nuclei of electrofused 4-cell stage syncytium, have calcium-releasing activity when they were transferred into unfertilized mature oocytes. Our results indicate that the calcium-releasing activity in nuclei of 1-, 2-cell embryos is produced during fertilization and exists at the special stage of fertilized early embryos. These suggested that the capacity of inducing calcium release activity in fertilized early embryos is important for normal embryonic development.

  11. Embryo-maternal communication

    DEFF Research Database (Denmark)

    Østrup, Esben; Hyttel, Poul; Østrup, Olga

    2011-01-01

    Communication during early pregnancy is essential for successful reproduction. In this review we address the beginning of the communication between mother and developing embryo; including morphological and transcriptional changes in the endometrium as well as epigenetic regulation mechanisms...... directing the placentation. An increasing knowledge of the embryo-maternal communication might not only help to improve the fertility of our farm animals but also our understanding of human health and reproduction....

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

    OpenAIRE

    HIGAKI, SHOGO; Mochizuki, Kentaro; Baba, Hiroko; Akashi, Yuichiro; Yamaha, Etsuro; Katagiri, Seiji; Takahashi, Yoshiyuki

    2009-01-01

    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 se...

  13. 冻融胚胎移植周期中不同发育阶段胚胎妊娠结局比较%The comparison of clinical outcomes of frozen-thawed embryos transfer at different develop-mental stages

    Institute of Scientific and Technical Information of China (English)

    张小建; 李晓洁; 廖梅旭; 彭礼繁; 余鲲; 曹雪华; 贾彦全; 邓芳; 吕群

    2016-01-01

    Objective To compare the clinical outcomes of frozen-thawed embryos transfer at different developmental stages in order to provide the basis for further improvement of FET pregnancy rate .Methods A total of 830 frozen-thawed embryos from 627 pa-tients were divided into D3 frozen group (n =385),D5 frozen group (n =300)and D6 frozen group (n =145)according to frozen days.The frozen-thawed embryos were transferred when the endometrial thickness was around 8~12 mm and embryos incubation time over 18~20 h (D3 group)or 2~3 h (D5/D6 groups)after thawing.Results The number of transplanted embryos in the D3 group was significantly higher than that in the D 5 and D6 groups ( P<0.05 ) .The clinical pregnancy rates and implantation rate of the D 5 group were significantly higher than that of the D 6 group (61.67%vs.51.03%for clinic pregnancy rate;46.37%vs.37.94%for implanta-tion rate,P<0.01)and the D3 group (61.67%vs.40.26%for clinic pregnancy rate;46.37%vs.24.57%for implantation rate,P<0.01 ) .Multiple pregnancy rate in the D 5 group was higher than that in the D 6 groups which was higher than that in the D 3 group ( P<0.01 ) .Conclusion If the patients with more embryos ,freezing embryos at blastocyst stage can achieve better clinical outcomes .%目的:探讨冻融胚胎移植周期中不同发育天数胚胎的发育潜能,为进一步提高FET妊娠率提供依据。方法627例患者830个复苏周期胚胎,根据冷冻时间不同,分为D3冷冻组( n =385),D5冷冻组( n =300)和D6冷冻组( n =145),待患者内膜达到8~12 mm时,复苏18~20 h(D3胚胎)或2~3 h(D5/D6)后移植。结果移植胚胎数,D3组显著高于D5和D6组(P<0.05),D5冷冻组的临床妊娠率和着床率均显著高于 D6冷冻组(61.67% vs 51.03%,46.37% vs 37.94%,P<0.01)和D3冷冻组(61.67%vs 40.26%,46.37%vs 24.57%,P<0.01),多胎妊娠率,D5>D6>D3组(P<0.01)。结论在患者有较多胚

  14. 体外共培养条件下种植窗期子宫内膜与胚胎的相互作用%Interaction between endometrium and embryo under the condition of in vitro co-culture during the implantation window stage

    Institute of Scientific and Technical Information of China (English)

    王婧; 谭丽

    2016-01-01

    目的:探讨体外共培养条件下种植窗期子宫内膜与胚胎的相互作用。方法:选择2014年6月至2015年6月在郑州大学第二附属医院生殖中心取卵后因有卵巢过度刺激综合征倾向取消移植的20例患者,于取卵后第3天取其子宫内膜,和胚胎组成共培养组(A组),以单独内膜组(B组)和单独胚胎组(C组)作为对照组,采用RT-PCR法分别于(取卵后)第3、4、5、6、7天检测共培养组和单独内膜组LIF mRNA和IGF mRNA的表达水平,并在显微镜下观察共培养组和单独胚胎组的胚胎生长情况。结果:共培养组LIF mRNA和IGF mRNA表达要高于单独内膜组,且在种植窗期达到最高,差异有统计学意义(P <0.05)。共培养组胚胎生长发育要优于单独胚胎组。结论:共培养对胚胎发育有积极的支持作用,使子宫内膜容受性提高,同时提高胚胎质量,要优于单独培养。%Objective To investigate the interaction between endometrium and embryo under the co-culture condition during the planting window stage. Methods Twenty patients who cancelled transplant for OHSS in the Reproductive Center of the second affiliated hospital of Zhengzhou university were enrolled.The discarded embryos were used to build the embryos co-culture system (Group A), with a single membrane (group B) and a single embryo(group C)as the control group. Levels of LIF and IGF expression and embryo growth were checked on 3, 4,5,6,7 day post-oocyte retrieval. Results Expressions of LIF mRNA and IGF mRNA in Group A were significantly higher than those in Group B. Embryo growth in the Group A was better than that in Group C. Conclusion Co-culture of endometrium and embryo supports the development of embryo and improves the receptivity of endometrium.

  15. Somatic Embryos in Catharanthus roseus: A Scanning Electron Microscopic Study

    Directory of Open Access Journals (Sweden)

    Junaid ASLAM

    2014-06-01

    Full Text Available Catharanthus roseus (L. G. Don is an important medicinal plant as it contains several anti-cancerous compounds, like vinblastine and vincristine. Plant tissue culture technology (organogenesis and embryogenesis has currently been used in fast mass propagating raw materials for secondary metabolite synthesis. In this present communication, scanning electron microscopic (SEM study of somatic embryos was conducted and discussed. The embryogenic callus was first induced from hypocotyls of in vitro germinated seeds on which somatic embryos, differentiated in numbers, particularly on 2,4-D (1.0 mg/L Murashige and Skoog (MS was medium. To understand more about the regeneration method and in vitro formed embryos SEM was performed. The SEM study revealed normal somatic embryo origin and development from globular to heart-, torpedo- and then into cotyledonary-stage of embryos. At early stage, the embryos were clustered together in a callus mass and could not easily be detached from the parental tissue. The embryos were often long cylindrical structure with or without typical notch at the tip. Secondary embryos were also formed on primary embryo structure. The advanced cotyledonary embryos showed prominent roots and shoot axis, which germinated into plantlets. The morphology, structure and other details of somatic embryos at various stages were presented.

  16. A cutin fluorescence pattern in developing embryos of some angiosperms

    Directory of Open Access Journals (Sweden)

    Ewa Szczuka

    2014-01-01

    Full Text Available A cuticle visualized by auramine O fluorescence appears on the developing embryos of 9 species belonging to Cruciferae, Caryophyllaceae, Plantaginaceae, Linaceae and Papilionaceae. In the investigated species the formation and extent of fluorescing and non-fluorescing embryonic areas follow a similar pattern. At first the cutin fluorescing layer is formed on the apical part of the proembryo without delimited protoderm. This layer extends and at the late globular stage envelops the embryo proper, except for a cell adjoining the suspensor. Fluorescing cutin persists during the heart stage but disappears from the torpedo embryo. During these stages there is no cutine fluorescence on suspensorial cells. Continuous cutin fluorescence appears again on the surface of the whole embryo by the late torpedo stage. Then fluorescence disappears from the radicular part of U-shaped embryos, but persists on the shoot apex, cotyledons and at least on the upper part of hypocotyl. It is assumed that polarization and nutrition of the embryo may be influenced by cuticular changes.

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

    Science.gov (United States)

    Mohd Sharifuddin, M; Siti Azizah, M N

    2014-08-01

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

  18. Development in vitro and mitochondrial fate of interspecies cloned embryos.

    Science.gov (United States)

    Ma, L-B; Yang, L; Hua, S; Cao, J-W; Li, J-X; Zhang, Y

    2008-06-01

    Although the technique of interspecies somatic cell nuclear transfer can be used to increase the population size of endangered mammals, the mitochondrial heteroplasmy in cloned embryos and animals makes this idea doubtful. In present study, goat-sheep cloned embryos were constructed by fusing goat foetal fibroblasts (GFFs) into sheep oocytes and then cultured in vitro to investigate the capability of sheep oocyte dedifferentiating GFF nucleus. Moreover, at each stage of 1- (immediately after fused), 2-, 4-, 8-, 16-cell, morula and blastocyst, the copy number of mtDNA from GFF and sheep oocyte was examined using real-time PCR. The results showed that: 7.4% of the fused cloned embryos can develop to the blastocyst stage; in the process of one cell to the morula stage, the copy number of two kinds of mtDNA was stable relatively; however, in the process of morula to the blastocyst stage, the decreasing in the copy number of GFF-derived mtDNA, while the increasing in sheep oocyte-derived, resulted in their ratio of decreasing sharply from 2.0 +/- 1.0% to 0.012 +/- 0.004%. This study demonstrates that: (i) the goat-sheep cloned embryos have the ability to develop to blastocyst in vitro; (ii) from the morula stage to the blastocyst stage of goat-sheep cloned embryos, goat derived mitochondria can be gradually replaced with those from sheep oocyte.

  19. Children born after cryopreservation of embryos or oocytes: a systematic review of outcome data

    DEFF Research Database (Denmark)

    Wennerholm, U-B; Söderström-Anttila, V; Bergh, C;

    2009-01-01

    embryos, blastocysts and oocytes. METHODS: A systematic review was performed. We searched the PubMed, Cochrane and Embase databases from 1984 to September 2008. Inclusion criteria for slow freezing of early cleavage stage embryos were controlled studies reporting perinatal or child outcomes. For slow...... freezing and vitrification of blastocysts and oocytes, and vitrification of early cleavage stage embryos, case reports on perinatal or child outcomes were also included. Three reviewers independently read and evaluated all selected studies. RESULTS: For early cleavage embryos, data from controlled studies...... of blastocysts and for vitrification of early cleavage stage embryos, blastocysts and oocytes, limited neonatal data was reported. We found no long-term child follow-up data for any cryopreservation technique. CONCLUSION: Data concerning infant outcome after slow freezing of embryos was reassuring. Properly...

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  2. Embryo implantation: A time for recalling and forwarding

    Institute of Scientific and Technical Information of China (English)

    CHEN Qi; PENG HongYing; ZHANG Ying; LEI Li; CAO YuJing; Duan EnKui

    2009-01-01

    The success of embryo implantation is a critical step towards further embryo development and pregnancy outcome.The observations and investigations on embryo implantation have been over a century.A huge body of knowledge has been accumulated in anatomy,histology,ultrastructure and hormonal regulation; as well as recently in depth information about molecular signaling pathways got from studies of genomic wide gene screening and specific gene deletion.The knowledge from basic research has also substantially helped to initiate and improve the Artificial Reproductive Technology (ART) in clinical applications.Now we've known that the normal embryo implantation involves the embryo's development into an implantation-competent blastocyst and the synchronized transformation of uteri into a receptive stage.The interdependent relationship between the blastocyst and uterus involves complicated hormonal regulation and local paracrine,juxtacrine interactions.In this paper,we review some important historical findings regarding uterine receptivity and blastocyst activation,as well as some less discussed topics such as embryo spacing,embryo orientation.Further understandings on detailed mechanisms during the process of embryo implantation will help cure women infertility as well as develop new generation of non-steroids contraceptives.

  3. Early embryo development in Fucus distichus is auxin sensitive

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  5. Embryo sac haustorium in Dryas octopetala L. (Rosaceae)

    OpenAIRE

    Romana Czapik

    2014-01-01

    The embryo sac haustorium found in Dryas octopetala L. from the Tatra Mts is the first record of its occurrence in Rosaceae. At the eight-nucleate stage of the embryo sac, the antipodal end of the central cell began to grow into a narrow caecum filled with dense cytoplasm and elongated in the chalazal direction leaving the three antipodals in situ. The haustorium enlarged and lost its characteristic shape after the period of fertilization. Finally, the embryo sac occupied almost the whole len...

  6. The avian embryo responding to microgravity of space flight

    Science.gov (United States)

    Hullinger, Ronald L.

    1993-01-01

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

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

    Science.gov (United States)

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

    2015-08-01

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

  8. Endodcytic labelling of visceral endoderm of mouse perigastrulation embryos

    OpenAIRE

    sprotocols

    2015-01-01

    Authors: Yoh Wada, Minako Aoyama, Ge-Hong Sun-Wada, Nobuyuki Kawamura & Hiroyuki Tabata ### Abstract In this protocol we describe methods for observation endocytic activity in the mouse embryos. The methods are optimised for mouse embryos at E5.5~E7.2 pregastrulation/gastrulation stages. We optimise three different experimental schemes for tracing the embryonic endocytosis. In utero labelling scheme, an endocytic tracer is introduced into circulation of a pregnant mother to follow...

  9. Oxygen diffusion in fish embryos

    NARCIS (Netherlands)

    Kranenbarg, S.

    2002-01-01

    All vertebrate embryos pass through a developmental period of remarkably low morphological variability. This period has been called phylotypic period. During the phylotypic period, organogenesis takes place, including blood vessel development. Before the phylotypic period, the embryos

  10. Demi-embryo production from hatching of zona-drilled bovine and rabbit blastocysts.

    Science.gov (United States)

    Skrzyszowska, M; Smorag, Z; Katska, L

    1997-09-01

    It is known that the pregnancy rate resulting after transfer of bisected embryos is lower than after transfer of whole embryos. The main reason is the reduced cell number in the demi-embryo which is less than 1 2 of that in the intact embryo, since a number of blastomeres is damaged as a result of the procedure used in conventional embryo splitting. The aim of our experiment was to develop a non-invasive procedure which would limit cell losses during microsurgery. The experiment was carried out on bovine IVM-IVF embryos at middle, late and expanded blastocyst stage and rabbit embryos at late blastocyst stage cultured in vitro from in vivo produced zygotes. The zona pellucida of these embryos was drilled on the line between the inner cell mass and the trophoblast using a glass microneedle (embryo configuration, connected by a very thin cell bridge (figure eight in shape). To separate the parts of the embryo, the cell bridge was cut using a glass microneedle. During the separation only a few cells were damaged. As a result of the procedure 4 20 (20.0%), 48 144 (33.3%) and 3 40 (7.5%) middle, late and expanded blastocysts hatched according to the pattern described. The developed procedure could be considered as a non-invasive alternative to conventional embryo splitting.

  11. Introduction of DT40 cells into chick embryos

    Institute of Scientific and Technical Information of China (English)

    Mariko Toba; Fumio Ebara; Hiroki Furuta; Yuichi Matsushimal; Yasuo Kitagawa; Noboru Fujihara

    2001-01-01

    To examine the transfection of exogenous genes into chick embryos, applying the characteristics of avian leukosis virus (ALV)-induced chicken B cell line DT40 to the production of chimeric birds. Methods: The DT40cells incorporated with exogenous gene (lacZ constructs encoding Escherichia coli β-galactosidase: β-gal) were introduced into chick embryos by the injection of cells into stage X blastoderm. Manipulated eggs were incubated for 3 (trial 1 ) or 6 (trial 2) days, and the expression of lacZ DNA was detected by a histochemical staining method of β-galactosidase and polymerase chain reaction (PCR) analysis. Results: The survival rates of the manipulated embryos incubated for 3 days (stage 18-20: trial 1) and 6 days (stage 28, 30: trial 2) were about 42% and 38%, respectively.The expression rates of the lacZ gene in the embryos in the trials 1 and 2 were about 60% and 23%, respectively, for the survived embryos. Conclusio: The rate of embryonic viability and expression rate of introduced genes were not so high, but it suggested the possibility of utilizing the DT40 cells as a vector for carrying exogenous genes into chick embryos.

  12. 氟虫腈和铜对斑马鱼早期发育的联合毒性效应%The joint toxicity of fipronil and Cu2+ in the embryo-larval stages of Zebrafish

    Institute of Scientific and Technical Information of China (English)

    章晓凤; 张微; 周聪

    2012-01-01

    氟虫腈是新一代苯基吡唑类杀虫剂,近年来关于氟虫腈的生态安全和健康风险的研究已成为热点,但有关氟虫腈与环境中其他污染物的复合污染的报道甚少.实验以氟虫腈和铜为研究对象,以斑马鱼胚胎为实验模型,采用双因子方差分析法研究氟虫腈和铜对斑马鱼早期发育的联合毒性.单一毒性结果表明:氟虫腈能够引起胚胎、仔鱼的死亡,以及仔鱼脊柱弯曲.而铜离子对胚胎死亡及畸形没有显著影响,但能够推迟胚胎的孵化.联合毒性结果显示:氟虫腈和铜离子对胚胎孵化的联合作用显著(p<0.05),两者表现为协同作用;对胚胎死亡和脊柱弯曲的联合作用不明显(p>0.05),两者表现为相加作用.研究结果为准确评价氟虫腈在实际环境中的复合污染提供依据.%Fipronil, a widespread used phenylpyrazole insecticide, is highly concerned on the eco-safety and human healthy. However, few reports have been issued on the joint effects between fipronil and other contaminants. In this study, the joint effect of fipronil and Cu2+ on the development in zebrafish embryo and larvae was studied by two-factor analysis of variance methods. The results showed that embryos exposed to fipronil displayed mortality and curved body axis, whereas only hatching process inhibition was observed when exposed to Cu2+. Moreover, significant interaction on inhibiting the hatching of zebrafish embryo was observed when exposed to the mixture of fipronil and Cu2+ suggesting synergistic effect between them. No significant interaction existed on the mortality and curved body axis indicating additive effect between them. The results provide substantial knowledge towards identifying and evaluating the environmental risk of fipronil.

  13. Ovarian stimulation and embryo quality

    NARCIS (Netherlands)

    Baart, Esther; Macklon, Nick S.; Fauser, Bart J. C. M.

    2009-01-01

    To Study the effects of different ovarian stimulation approaches on oocyte and embryo quality, it is imperative to assess embryo quality with a reliable and objective method. Embryos rated as high quality by standardized morphological assessment are associated with higher implantation and pregnancy

  14. Co-Culture of Early Embryo with Human Decidual Stromal Cells in vitro by Improvement of Early Embryo Development

    Institute of Scientific and Technical Information of China (English)

    YAN Jie; ZHU Guijin; LIU Jianxin; AI Jihui

    2000-01-01

    An early embryo co-culture system with human decidual stromal cells was established to study its effect on early embryonic cleavage and growth in vitro. Three hundred and eight 2-cell mouse embryos were co-cultured with human decidual stromal cell monolayer in MEM+0.4%bovine serum albumin (BSA) and 163 embryos cultured in MEM+15 % FCS alone as control. Among the mouse 2-cell embryos co-cultured with human decidual stromal cells, 72.73% developed to the morula stage and 67.21% cavitated to blastocysts with 59.74 % hatching, as compared with 61.34% to morula stage, 48.47% to blastocysts and none hatching in the controls,respectively. Co-cultured embryos cleaved slightly faster than controls and showed no or less fragmentation than those in the control. These results suggested that human decidual stromal cells can support early embryonic development and yield a reasonable number of embryos with good quality up to blastocyst stage.

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

    Science.gov (United States)

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

    2016-01-01

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

  16. Description of Phaseolus vulgaris L. aborting embryos from ethyl methanesulfonate (EMS mutagenized plants

    Directory of Open Access Journals (Sweden)

    Silué, S.

    2013-01-01

    Full Text Available The aim of this study was to describe the embryos abortion process and the inheritance of the embryos abortion trait in Phaseolus vulgaris plants deficient in seed development. These plants were isolated within the second generation of an ethyl methanesulfonate (EMS TILLING population of P. vulgaris cv. 'BAT93'. Mutant embryos show abnormalities mainly in suspensors, shoot apical meristem (SAM and cotyledons from the globular to the cotyledon stages and abort before maturity compared to those observed in wild-type samples. Mutant embryos show also hyperhydricity and contain low amount of chlorophyll. Genetic analyses of F1, F2 and F3 populations from the crosses carried out between the mutagenized plants with aborting embryos and the wild-type plants indicated that the embryo abortion phenotype is maternally inherited and controlled by a single recessive gene. These Phaseolus mutant plants with aborting embryos constitute a valuable material for plant embryogenesis studies.

  17. Analysis of the expression of putatively imprinted genes in bovine peri-implantation embryos

    DEFF Research Database (Denmark)

    Tveden-Nyborg, Pernille Yde; Alexopoulos, N.I.; Cooney, M.A.

    2008-01-01

    imprinted genes (Ata3, Dlk1, Gnas, Grb10, Magel2, Mest-1, Ndn and Sgce) in bovine peri-implantation embryos. Two embryonic developmental stages were examined, Day 14 and Day 21. The gene expression pattern of single embryos was recorded for in vivo, in vitro produced (IVP) and parthenogenetic embryos...... (LOS) in bovine embryos resulting in increased embryonic morbidity and mortality. In the bovine, limited numbers of imprinted genes have been studied and studies have primarily been restricted to pre-implantation stages. This study reports original data on the expression pattern of 8 putatively...... procedures, either by in vitro maturation, fertilization or culture. In conclusion, effects of genomic imprinting and of in vitro procedures for embryo production may influence the success of bovine embryo implantation....

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

    Science.gov (United States)

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

    2014-06-01

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

  19. Beneficial effect of two culture systems with small groups of embryos on the development and quality of in vitro-produced bovine embryos.

    Science.gov (United States)

    Cebrian-Serrano, A; Salvador, I; Silvestre, M A

    2014-02-01

    Currently, in vitro-produced embryos derived by ovum pick up (OPU) and in vitro fertilization (IVF) technologies represent approximately one-third of the embryos worldwide in cattle. Nevertheless, the culture of small groups of embryos from an individual egg donor is an issue that OPU-IVF laboratories have to face. In this work, we tested whether the development and quality of the preimplantation embryos in vitro cultured in low numbers (five embryos) could be improved by the addition of epidermal growth factor, insulin, transferrin and selenium (EGF-ITS) or by the WOW system. With this aim, immature oocytes recovered from slaughtered heifers were in vitro matured and in vitro fertilized. Presumptive zygotes were then randomly cultured in four culture conditions: one large group (LG) (50 embryos/500 μl medium) and three smaller groups [five embryos/50 μl medium without (control) or with EGF-ITS (EGF-ITS) and five embryos per microwell in the WOW system (WOW)]. Embryos cultured in LG showed a greater ability to develop to blastocyst stage than embryos cultured in smaller groups, while the blastocyst rate of WOW group was significantly higher than in control. The number of cells/blastocyst in LG was higher than control or WOW, whereas the apoptosis rate per blastocyst was lower. On the other hand, the addition of EGF-ITS significantly improved both parameters compared to the control and resulted in similar embryo quality to LG. In conclusion, the WOW system improved embryo development, while the addition of EGF-ITS improved the embryo quality when smaller groups of embryos were cultured.

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

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yingying [Department of Reproduction and Development, Kunming Institute of Zoology and Kunming Primate Research Center, Chinese Academy of Sciences, Kunming 650223 (China); State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080 (China); Graduate University of Chinese Academy of Sciences, Beijing 100049 (China); Hai, Tang; Liu, Zichuan; Zhou, Shuya; Lv, Zhuo [State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080 (China); Graduate University of Chinese Academy of Sciences, Beijing 100049 (China); Ding, Chenhui; Liu, Lei [State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080 (China); Niu, Yuyu [Department of Reproduction and Development, Kunming Institute of Zoology and Kunming Primate Research Center, Chinese Academy of Sciences, Kunming 650223 (China); Zhao, Xiaoyang; Tong, Man [State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080 (China); Graduate University of Chinese Academy of Sciences, Beijing 100049 (China); Wang, Liu [State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080 (China); Jouneau, Alice [INRA, UMR 1198, ENVA, CNRS, FRE 2857, Biologie du Developpement et Reproduction, Jouy en Josas F-78350 (France); Zhang, Xun [Neuroendocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114 (United States); Ji, Weizhi, E-mail: wji@mail.kiz.ac.cn [Department of Reproduction and Development, Kunming Institute of Zoology and Kunming Primate Research Center, Chinese Academy of Sciences, Kunming 650223 (China); Zhou, Qi, E-mail: qzhou@ioz.ac.cn [State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080 (China)

    2010-07-02

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

  1. Radial extracorporeal shock wave treatment harms developing chicken embryos

    Science.gov (United States)

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

    2015-01-01

    Radial extracorporeal shock wave treatment (rESWT) has became one of the best investigated treatment modalities for cellulite, including the abdomen as a treatment site. Notably, pregnancy is considered a contraindication for rESWT, and concerns have been raised about possible harm to the embryo when a woman treated with rESWT for cellulite is not aware of her pregnancy. Here we tested the hypothesis that rESWT may cause serious physical harm to embryos. To this end, chicken embryos were exposed in ovo to various doses of radial shock waves on either day 3 or day 4 of development, resembling the developmental stage of four- to six-week-old human embryos. We found a dose-dependent increase in the number of embryos that died after radial shock wave exposure on either day 3 or day 4 of development. Among the embryos that survived the shock wave exposure a few showed severe congenital defects such as missing eyes. Evidently, our data cannot directly be used to draw conclusions about potential harm to the embryo of a pregnant woman treated for cellulite with rESWT. However, to avoid any risks we strongly recommend applying radial shock waves in the treatment of cellulite only if a pregnancy is ruled out. PMID:25655309

  2. Rape embryogenesis. II. Development of embryo proper

    Directory of Open Access Journals (Sweden)

    Teresa Tykarska

    2015-05-01

    Full Text Available It was found in the continued studies on rape embryogenesis, started by the description of the proembryo (Tykarska, 1976 that the development of embryo is extremely regular and based on differentiating divisions. It appeared that the transverse segmentation boundary and cell walls separating the mother cells of the histogens in the proembryo can be distinguished in all the later stages of the embryo. The border between the cytoledons and epicotyl part of the embryonal axis, and the hypocotyl corresponds to the segmentation boundary between layer l and layer l' at the octant stage. As border between the hypocotyl and radicle was assumed the upper boundary of the root cap reaching usually to the level of the boundary between segments II and III of dermatogen and periblem. The apical meristem of the shoot forms from dermatogen and the periaxial cells of the globular embryo subepidermis. The promeristem of the radicle constists of 3 layers of initial cells surrounding on all sides the inactive layer of central binding cells.

  3. Effect of Clenbuterol Hydrochloride on the in vitro Development of Mouse Embryo

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Objective To investigate the effect of clenbuterol hydrochloride on the in vitro devel-opment of both 1-cell and 2-cell mouse embryos.Methods The cultural systems of both 1-cell and 2-cell mouse embryo were used todetermine the effect of clenbuterol hydrochloride at doses of 1 ng/mL, 3 ng/mL, and10 ng/mL on developmental rates of mouse embryos.Results When 1-cell embryos cultured with 1 ng/mL of clenbuterol hydrochloride,developmental rates from the 4-cell stage to blastocyst stage were significantly lowerthan those in the control group (P< 0. 05), but on dosages of 3 ng/mL and 10ng/mL,the inhibiting effects on embryo development were significantly increased (P< 0. 01).When 2-cell embryos cultured with 1 ng/mL of clenbuterol hydrochloride, obvious dif-ferences in developmental rates were not found between the 2-cell embryo group and thecontrol (P> 0. 05). However, at levels of 3 ng/mL and 10 ng/mL, significant de-crease of developmental rates in 2-cell embryos was observed from the 4-cell and fromthe 8-cell stage, respectively (P< 0. 05). Embryos cultured with clenbuterol hydrochlo-ride appeared to have more granules, fragments and degeneration than those in thecontrol.Conclusion Clenbuterol hydrochloride has a toxic effect on the mouse embryos, and theeffect is in a dose-dependent. 1-cell mouse embryos cultured with clenbuterolhydrochloride could be easily inhibited at 2-cell stage, but the effect of clenbuterolhydrochloride on development of the late 2-cell embryos would be reduced.

  4. Relevance of LIF and EGF on Mouse Preimplantation Embryo Development

    Directory of Open Access Journals (Sweden)

    Iraj Amiri

    2008-01-01

    Full Text Available Objective: Recent evidence suggests that Leukemia Inhibitory Factor (LIF, a member ofinterleukin-6 family, has biological actions on preimplantation embryo development. Alsoit is established that Epidermal Growth Factor (EGF, a strong mitosis-promoting agent,improves the preimplantation embryo development by increasing the cell metabolism andproliferation. The purpose of the present study is to investigate the effects of these factors,alone and in combination together, on preimplantation and development of the embryo.Materials and Methods: Six to eight weeks old NMRI mice were super ovulated by injectionof 10IU PMSG and 10IU hCG, then the mated mice were killed 46 hours later. Theiroviducts were flushed, two-cell embryos collected and divided randomly to the four groupsas following: Control, treatment 1 (LIF, treatment 2 (EGF, treatment 3 (LIF+EGF. In eachgroup, the embryos were cultured in an incubator at 37°C with 5% CO2 and 90% humidityfor 72hrs. The state of embryo development was evaluated in 24,36,48,60 and 72hrsfollowing the embryos cultures. By the end of the cultures, cell apoptosis was studiedby the terminal deoxynucleotidyl transferas-mediated dUTP nick end-labeling (TUNELtechnique.Results: Significant difference was detected in the rate of hatching in the LIF and LIF+EGFgroups. This difference was also seen in the rate of blastocyst formation after 36hrs(p<0.05 and in the average of the total cell number (p<0.05 after 72hrs. In comparison tothe apoptotic index, there was no significant difference between the control and treatmentgroups.Conclusion: The findings in this study show a beneficial effect of LIF and EGF on theblastocyst formation, hatching and its total cell numbers in vitro.

  5. Photobiomodulation of early mouse embryo development

    Science.gov (United States)

    Sviridova-Chailakhyan, T. A.; Fakhranurova, L. I.; Simonova, N. B.; Khramov, R. N.; Manokhin, A. A.; Paskevich, S. I.; Chailakhyan, L. M.

    2008-04-01

    The effect of artificial sunlight (AS) from a xenon source and of converted AS with an additional orange-red luminescent (λ MAX=626 nm) component (AS+L) on the development of mouse zygotes was investigated. A plastic screen with a photoluminophore layer was used for production of this orange-red luminescent (L) component. A single short-term (15 min) exposure produced a long-term stable positive effect on early embryo development of mice, which persisted during several days. After exposure to AS+L, a stimulating influence on preimplantation development was observed, in comparison with the control group without AS exposure. The positive effects were as follows: increase in percent of embryos (P <= 0.05) developed to the blastocyst stage (96.2 %) with hatching from the zona pellucida (80.8 %) within 82-96 hours in vitro compared to the control (67.1 % and 28.8 %, respectively).

  6. Search for the genes involved in oocyte maturation and early embryo development in the hen

    Directory of Open Access Journals (Sweden)

    Blesbois Elisabeth

    2008-02-01

    Full Text Available Abstract Background The initial stages of development depend on mRNA and proteins accumulated in the oocyte, and during these stages, certain genes are essential for fertilization, first cleavage and embryonic genome activation. The aim of this study was first to search for avian oocyte-specific genes using an in silico and a microarray approaches, then to investigate the temporal and spatial dynamics of the expression of some of these genes during follicular maturation and early embryogenesis. Results The in silico approach allowed us to identify 18 chicken homologs of mouse potential oocyte genes found by digital differential display. Using the chicken Affymetrix microarray, we identified 461 genes overexpressed in granulosa cells (GCs and 250 genes overexpressed in the germinal disc (GD of the hen oocyte. Six genes were identified using both in silico and microarray approaches. Based on GO annotations, GC and GD genes were differentially involved in biological processes, reflecting different physiological destinations of these two cell layers. Finally we studied the spatial and temporal dynamics of the expression of 21 chicken genes. According to their expression patterns all these genes are involved in different stages of final follicular maturation and/or early embryogenesis in the chicken. Among them, 8 genes (btg4, chkmos, wee, zpA, dazL, cvh, zar1 and ktfn were preferentially expressed in the maturing occyte and cvh, zar1 and ktfn were also highly expressed in the early embryo. Conclusion We showed that in silico and Affymetrix microarray approaches were relevant and complementary in order to find new avian genes potentially involved in oocyte maturation and/or early embryo development, and allowed the discovery of new potential chicken mature oocyte and chicken granulosa cell markers for future studies. Moreover, detailed study of the expression of some of these genes revealed promising candidates for maternal effect genes in the

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

    Directory of Open Access Journals (Sweden)

    Danilo Cimadomo

    2016-01-01

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

  8. In vivo and in vitro development of Tibetan antelope (Pantholops hodgsonii interspecific cloned embryos

    Directory of Open Access Journals (Sweden)

    Guanghua SU,Lei CHENG,Yu GAO,Kun LIU,Zhuying WEI,Chunling BAI,Fengxia YIN,Li GAO,Guangpeng LI,Shorgan BOU

    2014-02-01

    Full Text Available The Tibetan antelope is endemic to the Tibetan Plateau, China, and is now considered an endangered species. As a possible rescue strategy, the development of embryos constructed by interspecies somatic cell nuclear transfer (iSCNT was examined. Tibetan antelope fibroblast cells were transferred into enucleated bovine, ovine and caprine oocytes. These cloned embryos were then cultured in vitro or in the oviducts of intermediate animals. Less than 0.5% of the reconstructed antelope-bovine embryos cultured in vitro developed to the blastocyst stage. However, when the cloned antelope-bovine embryos were transferred to caprine oviducts, about 1.6% of the embryos developed to the blastocyst stage. In contrast, only 0.7% of the antelope-ovine embryos developed to the morula stage and none developed to blastocysts in ovine oviducts. The treatment of donor cells and bovine oocytes with trichostatin A did not improve the embryo development even when cultured in the oviducts of ovine and caprine. When the antelope-bovine embryos, constructed from oocytes treated with roscovitine or trichostatin A, were cultured in rabbit oviducts 2.3% and 14.3% developed to blastocysts, respectively. It is concluded that although some success was achieved with the protocols used, interspecies cloning of Tibetan antelope presents difficulties still to be overcome. The mechanisms resulting in the low embryo development need investigation and progress might require a deeper understanding of cellular reprogramming.

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

    Science.gov (United States)

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

    2016-01-01

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

  10. Embryo development and corresponding factors affecting in vitro germination of Cymbidium faberi × C. sinense hybrid seeds

    OpenAIRE

    Li Fengtong; Bao Jianzhong; Sun Ye; Liu Chungui; Ma Hui; Zhang Tian; Chen Xiulan

    2016-01-01

    A better understanding of embryo development would provide insights into seed quality and subsequent germination events in the interspecific hybridization of Cymbidium faberi ‘Jiepeimei’ × C. sinense ‘Qijianheimo’. At the mature stage, 26.1% of the ovules were abnormal. Most of the hybrid embryos could develop normally. Abortions mainly occurred at the zygote (9.5%) and 2-4-celled embryo (15.1%) stages. No germination was observed at 90 and 105 days after p...

  11. The effect of unilateral ovariectomy on early embryonic survival and embryo development in rabbits

    Directory of Open Access Journals (Sweden)

    R. Peiró

    2014-06-01

    Full Text Available Unilateral ovariectomy can be used to study uterine capacity in rabbits because an overcrowding of the functional uterine horn is produced. Due to the uterus duplex, the rabbit is the ideal model for such studies. However, this technique may affect embryo survival. The aim of this work is to study the effect of unilateral ovariectomy on early embryo survival and development in rabbit. A total of 101 unilateral ovariectomised females and 52 intact females were compared after slaughter at 30 h post-mating. Early embryo survival was estimated as the ratio between number of embryo recovered and ovulation rate. No differences were found between intact and unilaterally ovariectomised females in this trait. Unilateral ovariectomy did not change embryo development, measured as the number of embryo cells. Variability of embryo development was not affected either. At 30 h post-mating, the majority of embryos (86.2% were 4-cell stage. Embryo quality was evaluated according to morphological criteria. No difference in embryo quality between intact and unilaterally ovariectomised females was found. Therefore, unilateral ovariectomy performed before puberty in rabbit does not modify early embryo survival and development.

  12. Utero-tubal embryo transfer and vasectomy in the mouse model.

    Science.gov (United States)

    Bermejo-Alvarez, Pablo; Park, Ki-Eun; Telugu, Bhanu P

    2014-02-28

    The transfer of preimplantation embryos to a surrogate female is a required step for the production of genetically modified mice or to study the effects of epigenetic alterations originated during preimplantation development on subsequent fetal development and adult health. The use of an effective and consistent embryo transfer technique is crucial to enhance the generation of genetically modified animals and to determine the effect of different treatments on implantation rates and survival to term. Embryos at the blastocyst stage are usually transferred by uterine transfer, performing a puncture in the uterine wall to introduce the embryo manipulation pipette. The orifice performed in the uterus does not close after the pipette has been withdrawn, and the embryos can outflow to the abdominal cavity due to the positive pressure of the uterus. The puncture can also produce a hemorrhage that impairs implantation, blocks the transfer pipette and may affect embryo development, especially when embryos without zona are transferred. Consequently, this technique often results in very variable and overall low embryo survival rates. Avoiding these negative effects, utero-tubal embryo transfer take advantage of the utero-tubal junction as a natural barrier that impedes embryo outflow and avoid the puncture of the uterine wall. Vasectomized males are required for obtaining pseudopregnant recipients. A technique to perform vasectomy is described as a complement to the utero-tubal embryo transfer.

  13. Developmental toxicity of cartap on zebrafish embryos.

    Science.gov (United States)

    Zhou, Shengli; Dong, Qiaoxiang; Li, Shaonan; Guo, Jiangfeng; Wang, Xingxing; Zhu, Guonian

    2009-12-13

    Cartap is a widely used insecticide which belongs to a member of nereistoxin derivatives and acts on nicotinic acetylcholine receptor site. Its effects on aquatic species are of grave concern. To explore the potential developmental toxicity of cartap, zebrafish embryos were continually exposed, from 0.5 to 144h post-fertilization, to a range of concentrations of 25-1000microg/l. Results of the experiment indicated that cartap concentrations of 100microg/l and above negatively affected embryo survival and hatching success. Morphological analysis uncovered a large suite of abnormalities such as less melanin pigmentation, wavy notochord, crooked trunk, fuzzy somites, neurogenesis defects and vasculature defects. The most sensitive organ was proved to be the notochord which displayed defects at concentrations as low as 25microg/l. Both sensitivity towards exposure and localization of the defect were stage specific. To elucidate mechanisms concerning notochord, pigmentation, and hatching defects, enzyme assay, RT Q-PCR, and different exposure strategies were performed. For embryos with hatching failure, chorion was verified not to be digested, while removing cartap from exposure at early pre-hatching stage could significantly increase the hatching success. However, cartap was proved, via vitro assay, to have no effect on proteolytic activity of hatching enzyme. These findings implied that the secretion of hatching enzyme might be blocked. We also revealed that cartap inhibited the activity of melanogenic enzyme tyrosinase and matrix enzyme lysyl oxidase and induced expression of their genes. These suggested that cartap could impaired melanin pigmentation of zebrafish embryos through inhibiting tyrosinase activity, while inhibition of lysyl oxidase activity was responsible for notochord undulation, which subsequently caused somite defect, and at least partially responsible for defects in vasculature and neurogenesis.

  14. Comparative transcriptomic analysis of developing cotton cotyledons and embryo axis.

    Directory of Open Access Journals (Sweden)

    Xiaoming Jiao

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

  15. The slow growing embryo and premature progesterone elevation: compounding factors for embryo-endometrial asynchrony.

    Science.gov (United States)

    Healy, Mae Wu; Yamasaki, Meghan; Patounakis, George; Richter, Kevin S; Devine, Kate; DeCherney, Alan H; Hill, Micah J

    2017-02-01

    combined with an advanced endometrium, ultimately leading to decreased live births. This suggests that premature elevated P4 may be a factor in the lower live birth rates in day 6 fresh embryo transfers. Further studies are needed to evaluate if a frozen embryo transfer cycle can ameliorate the effect of elevated P4 on the day of trigger among these slower growing embryos that reach blastocyst staging on day 6. No external funding was received for this study. There are no conflicts of interest to declare. Not applicable. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  16. Manifestation of embryogenic potential in culture of zygotic embryos of Quercus robur L.

    Directory of Open Access Journals (Sweden)

    Maria G. Ostrolucká

    2014-01-01

    Full Text Available For the initiation of somatic embryogenesis early cotyledonary stage of zygotic embryo explants (from 15th July until late August was suitable. The highest frequency of differentiation of somatic embryos was obtained on cotyledons of zygotic embryos cultured on basal modified medium MS (with 1/2 concentration macronutrients or WPM medium containing 500 mg•l-1 glutamine, proline and casein hydrolysate and supplemented with 2,4-D (1,0-2,0 mg•l-1 and BAP (0,5-1,0 mg•l-1. The development of somatic embryos was direct and indirect and the process was continuous over a long period. Primary somatic embryos were able to produce secondary embryos. Repetitive somatic embryogenesis led to the proliferation of a large number of new somatic embryos on their cotyledons, hypocotyl or radicula. The process of embryo differentation is asynchronous - various stages of somatic embryos could be observed in embryogenic culture. A somatic embryo conversion was rare on tested media. Embryo germination occured on medium containing BAP (0,1 mg•l-1 or on medium with ABA and GA3 (each 0,2 mg•l-1 after a previous culture on WPM medium without plant growth regulators supplemented with sorbitol (6%. The embryo germination occurred also on WPM medium with 0.2 mg•l-1 BAP when cultures were mantained at 2oC for 4 weeks. Only 8 somatic embryos developed into plantlets. Their transplantation to in vivo conditions was unsuccessful.

  17. DEVELOPMENT OF MICE AND HAMSTER EMBRYOS IN KSOMAA AND HECM-6 MEDIUM

    Directory of Open Access Journals (Sweden)

    Bayu Rosadi

    2008-12-01

    Full Text Available The purpose of the present study was to investigate the viability of mice and hamster embryos developed in Kalium Simplex Optimized Medium amino acid (KSOMaa and Hamster Embryo Culture Medium-6 (HECM-6 medium. Female DDY mice were superovulated by injection i.p. of 5 IU Pregnant Mare Serum Gonadotropine (PMSG and 5 IU Human Chorionic Gonadotropine (hCG in 48 h interval, hamster (Phodopus campbelli injected by 2.5 IU PMSG and 2.5 IU hCG 48 h later. Then females were mated with fertile males. Eight-cell embryos were recovered at day 3 after natural mating. The mice embryos were cultured in KSOMaa+5% NBCS (New Born Calf Serum (T1 and HECM-6+5% NBCS (T2, the hamster embryos were cultured in KSOMaa+5% NBCS (T3 and HECM-6 + 5% NBCS (T4 for further development at 37oC in a humidified atmosphere of 5% CO2 in air for 48 h. The examinations were replicated five times. The T1 embryos developed to compact morulla and early blastocyst 100% (140/140, 92.1% (129/140 to blastocyst and expanded blastocyst, and 22.9% (32/140 became hatching/hatched. The T3 reached 100% (60/60 to compact morulla and early blastocyst, 85.0% (51/60 blastocyst, and 48.3% (29/60 expanded blastocyst, no embryo observed hatching/hatced. The T2 embryos had more expanded blastocyst than T3 (P<0.05, hatching/hatched rate higher than T1 and T3 but lower than T4 (P<0.05. Shortly, KSOMaa enable to support 8-cell stage mice and hamster embryo, but the hamster embryo developed lower at expanded blastocyst stage. HECM-6 is more appropriate than KSOMaa to support 8-cell mice embryos development and suitable to develop 8-cell stage hamster embryos.

  18. Blastocyst Morphology Holds Clues Concerning The Chromosomal Status of The Embryo

    Directory of Open Access Journals (Sweden)

    Rita de Cassia Savio Figueira

    2015-07-01

    Full Text Available Background: Embryo morphology has been proposed as an alternative marker of chromosomal status. The objective of this retrospective cohort study was to investigate the association between the chromosomal status on day 3 of embryo development and blastocyst morphology. Materials and Methods: A total of 596 embryos obtained from 106 cycles of intracytoplasmic sperm injection (ICSI followed by preimplantation genetic aneuploidy screening (PGS were included in this retrospective study. We evaluated the relationship between blastocyst morphological features and embryonic chromosomal alteration. Results: Of the 564 embryos with fluorescent in situ hybridization (FISH results, 200 reached the blastocyst stage on day 5 of development. There was a significantly higher proportion of euploid embryos in those that achieved the blastocyst stage (59.0% compared to embryos that did not develop to blastocysts (41.2% on day 5 (P<0.001. Regarding blastocyst morphology, we observed that all embryos that had an abnormal inner cell mass (ICM were aneuploid. Embryos with morphologically normal ICM had a significantly higher euploidy rate (62.1%, P<0.001. As regards to the trophectoderm (TE morphology, an increased rate of euploidy was observed in embryos that had normal TE (65.8% compared to embryos with abnormal TE (37.5%, P<0.001. Finally, we observed a two-fold increase in the euploidy rate in high-quality blastocysts with both high-quality ICM and TE (70.4% compared to that found in low-quality blastocysts (31.0%, P<0.001. Conclusion: Chromosomal abnormalities do not impair embryo development as aneuploidy is frequently observed in embryos that reach the blastocyst stage. A high-quality blastocyst does not represent euploidy of chromosomes 13, 14, 15, 16, 18, 21, 22, X and Y. However, aneuploidy is associated with abnormalities in the ICM morphology. Further studies are necessary to confirm whether or not the transfer of blastocysts with low-quality ICM should be

  19. Gender determination of avian embryo

    Science.gov (United States)

    Daum, Keith A.; Atkinson, David A.

    2002-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Coulam Carolyn B

    2011-05-01

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

  1. Cryopreservation of primordial germ cells by rapid cooling of whole zebrafish (Danio rerio) embryos.

    Science.gov (United States)

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

    2010-04-01

    The feasibility of cryopreservation of zebrafish (Danio rerio) primordial germ cells (PGCs) by rapid cooling (i.e., vitrification) of dechorionated whole embryos at the 14- to 20-somite stage was investigated. Initially, we examined the glass-forming properties and embryo toxicities of six cryoprotectants: methanol (MeOH), ethylene glycol (EG), glycerol (GC), dimethyl sulfoxide (DMSO), propylene glycol (PG) and 1,3-butylene glycol (1,3-BG). According to the results of glass-forming and embryo toxicity tests, pretreatment solution (PS) containing 2 or 3 M cryoprotectant and vitrification solution (VS) containing 5 M cryoprotectant and 0.5 M sucrose were prepared using each cryoprotectant. Dechorionated embryos, the PGCs of which were visualized by injection of green fluorescence protein-nos1 3'UTR mRNA, were cooled rapidly by plunging into liquid nitrogen after serial exposure to PS and VS. All embryos cooled with MeOH, PG and 1,3-BG showed ice formation during cooling, and few embryos had live PGCs after warming. Most embryos cooled with GC did not show ice formation; however, few embryos had live PGCs. All embryos cooled with EG and most embryos cooled with DMSO had live PGCs when the embryos did not show ice formation during cooling. Based on the number of live PGCs in fresh embryos, the maximum survival rates of PGCs recovered from embryos cooled with EG and DMSO were estimated to be about 40 and 20%, respectively. The present study indicates that rapid cooling of dechorionated whole embryos, especially using EG-based solutions, could be utilized as a simple and promising tool for cryopreservation of PGCs.

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

    Science.gov (United States)

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

    2012-12-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-04-09

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

  4. Morphokinetic Characteristics and Developmental Potential of In Vitro Cultured Embryos from Natural Cycles in Patients with Poor Ovarian Response

    Directory of Open Access Journals (Sweden)

    N. Hojnik

    2016-01-01

    Full Text Available Background. Patients with poor ovarian response to ovarian hyperstimulation represent an interesting group for studying the impact of embryo cleavage irregularities on clinical outcome since all embryos, regardless of their quality, are usually transferred to the uterus. The aim of our study was to follow the morphokinetics of fertilized oocytes from natural cycles in poor responders. Methods. Zygotes from 53 cycles were cultured in vitro for 3 days. The morphokinetics of their development and transfer outcomes were retrospectively analyzed for the normally and irregularly cleaved embryos. Results. Of all embryos, 30.2% had single and 20.8% multiple cleavage irregularities with the following prevalence: developmental arrest 30.2%, direct cleavage to more than two cells 24.5%, chaotic cleavage 13.2%, and reverse cleavage 11.3%. These embryos had longer pronuclear phases, first cytokinesis, second embryo cell cycles, and less synchronized divisions. The transfer of normally developing embryos resulted in an implantation rate of 30.8% and a delivery rate of 23.1%, but irregularly cleaved embryos did not implant. Conclusions. The use of time-lapse microscopy in poor responder patients identified embryos with cleavage abnormalities that are related with no or extremely low implantation potential. Gained information about embryo quality is important for counselling patients about their expectations.

  5. Wound healing ability of Xenopus laevis embryos. I. Rapid wound closure achieved by bisectional half embryos.

    Science.gov (United States)

    Yoshii, Yasuko; Noda, Masahiro; Matsuzaki, Takashi; Ihara, Setsunosuke

    2005-10-01

    We examined wound closure in 'half embryos' produced by the transverse bisection of Xenopus laevis embryos at the primary eye vesicle stage. Both the anterior- and posterior-half embryos survived for more than 6 days, and grew into 'half tadpoles'. Histology and videomicroscopy revealed that the open wound in the half embryo was rapidly closed by an epithelial sheet movement in the wound marginal zone. The time-course of wound closure showed a downward convex curve: the wound area decreased to one-fifth of the original area within 30 min, and the wound continued to contract slowly thereafter. The rapidity of closure of the epidermis as well as the absence of inflammatory cells are typical features of an embryonic type of wound healing. There was a dorso-ventral polarity in the motility of the epidermis: the wound was predominantly closed by the ventral and lateral epidermis. The change in the contour of the wound edge with time suggested a complex mechanism involved in the wound closure that could not be explained only by the purse-string theory. The present experimental system would be a unique and useful model for analyses of cellular movements in the embryonic epithelia.

  6. Comparison between Quality of Cryopreserved Embryos Generated from Short and Long Gamete Incubation

    Directory of Open Access Journals (Sweden)

    Morteza Anvari

    2009-01-01

    Full Text Available Background: The purpose was to investigate the effect of the duration of gamete incubation onfertilization rate, embryo cleavage, and embryo quality before and after freezing in mice.Materials and Methods: Ovulated oocytes collected from superovulated mice after ip injectionof PMSG and hCG were divided randomly into control and experimental groups. Oocytes fromthe control group were inseminated for six hours and the experimental group were inseminated forone hour, respectively. The differences in fertilization rates, embryo cleavage and percent of goodquality embryos in four grades (A, B, C, D were analyzed. Finally, two cell embryos were frozen;and after thawing, the quality of embryos from the two groups were compared.Results: There was no difference between the two groups in regards to fertilization and cleavagerates. However, the proportion of grade A embryos was significantly higher among the experimentalgroup (41.7% when compared to the control group (19%. Also the proportion of grade D embryoswas significantly (p=0.04 lower in the experimental group (8.3% as compared to the controlgroup (23.8%. In addition, percentage of good quality embryos in the experimental group did notdecrease after freezing (p=0.3, however the percentage of good quality embryos were significantlydecreased after freezing in the control group (p=0.01.Conclusion: Insemination of oocytes for a short period produced embryos of superior quality thaninsemination for a longer period in the experimental group . Also, the effect of freezing on embryosproduced from short insemination was less than the long insemination period. After freezing, ahigher percentage of good quality embryos survived post thawing in mice.

  7. Who abandons embryos after IVF?

    LENUS (Irish Health Repository)

    Walsh, A P H

    2010-04-01

    This investigation describes features of in vitro fertilisation (IVF) patients who never returned to claim their embryos following cryopreservation. Frozen embryo data were reviewed to establish communication patterns between patient and clinic; embryos were considered abandoned when 1) an IVF patient with frozen embryo\\/s stored at our facility failed to make contact with our clinic for > 2 yrs and 2) the patient could not be located after a multi-modal outreach effort was undertaken. For these patients, telephone numbers had been disconnected and no forwarding address was available. Patient, spouse and emergency family contact\\/s all escaped detection efforts despite an exhaustive public database search including death records and Internet directory portals. From 3244 IVF cycles completed from 2000 to 2008, > or = 1 embryo was frozen in 1159 cases (35.7%). Those without correspondence for > 2 yrs accounted for 292 (25.2%) patients with frozen embryos; 281 were contacted by methods including registered (signature involving abandoned embryos did not differ substantially from other patients. The goal of having a baby was achieved by 10\\/11 patients either by spontaneous conception, adoption or IVF. One patient moved away with conception status unconfirmed. The overall rate of embryo abandonment was 11\\/1159 (< 1%) in this IVF population. Pre-IVF counselling minimises, but does not totally eliminate, the problem of abandoned embryos. As the number of abandoned embryos from IVF accumulates, their fate urgently requires clarification. We propose that clinicians develop a policy consistent with relevant Irish Constitutional provisions to address this medical dilemma.

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

    Science.gov (United States)

    Scott, Lynette

    2003-01-01

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

  9. Parthenogenetic activation of rhesus monkey oocytes and reconstructed embryos.

    Science.gov (United States)

    Mitalipov, S M; Nusser, K D; Wolf, D P

    2001-07-01

    This study determines the efficiency of sequential calcium treatments (electroporation or ionomycin) combined with protein synthesis (cycloheximide) or phosphorylation inhibitors (6-dimethylaminopurine) or the specific maturation promoting factor (MPF) inhibitor, roscovitine, in inducing artificial activation and development of rhesus macaque parthenotes or nuclear transfer embryos. Exposure of oocytes arrested at metaphase II (MII) to ionomycin followed by 6-dimethylaminopurine or to electroporation followed by cycloheximide and cytochalasin B induced pronuclear formation and development to the blastocyst stage at a rate similar to control embryos produced by intracytoplasmic sperm injection. Parthenotes did not complete meiosis or extrude a second polar body, consistent with their presumed diploid status. In contrast, oocytes treated sequentially with ionomycin and roscovitine extruded the second polar body and formed a pronucleus at a rate higher than that observed in controls. Following reconstruction by nuclear transfer, activation with ionomycin/6-dimethylaminopurine resulted in embryos that contained a single pronucleus and no polar bodies. All nuclear transfer embryos activated with ionomycin/roscovitine contained one large pronucleus. However, a third of these embryos emitted one or two polar bodies, clearly containing chromatin material. In summary, we have identified simple yet effective methods of oocyte or cytoplast activation in the monkey, ionomycin/6-dimethylaminopurine, electroporation/cycloheximide/cytochalasin B, and ionomycin/roscovitine, which are applicable to parthenote or nuclear transfer embryo production.

  10. Nucleolar re-activation is delayed in mouse embryos cloned from two different cell lines

    DEFF Research Database (Denmark)

    Svarcova, Olga; Dinnyes, A.; Polgar, Z.

    2009-01-01

    displayed early NPBs transformation. In conclusion, despite normal onset of EGA in cloned embryos, activation of functional nucleoli was one cell cycle delayed in NT embryos. NT-MEF embryos displayed normal targeting but delayed activation of nucleolar proteins. Contrary, in NT-HM1 embryos, both......Aim of this study was to evaluate and compare embryonic genome activation (EGA) in mouse embryos of different origin using nucleolus as a marker. Early and late 2-cell and late 4-cell stage embryos, prepared by in vitro fertilization (IVF), parthenogenetic activation (PG), and nuclear transfer...... ofmouse embryonic fibroblast (MEF) and mouse HM1 emryonic stem cells (HM1), were processed for autoradiography following 3H-uridine incubation (transcriptional activity), transmission electron microscopy (ultrastructure) and immunofluorescence (nucleolar proteins; upstream binding factor, UBF...

  11. Development and quality of porcine parthenogenetically activated embryos after removal of zona pellucida

    DEFF Research Database (Denmark)

    Li, Rong; Liu, Ying; Pedersen, Hanne Skovsgaard

    2013-01-01

    The need of the zona pellucida (ZP) for in vitro development is controversial because it might be an obstacle to hatching of the blastocyst. This study investigated the development and quality of porcine parthenogenetically activated (PA) embryos by observation of the developmental kinetics......, the developmental percentages, the frequency of apoptosis, and robustness after removal of the ZP by pronase. Three experiments were made between zona-free PA embryos and zona-intact embryos: (1) determination of the timing of developmental stages using time-lapse observations for 6 days; (2) determination...... of developmental percentages and occurrence of apoptosis on Day 6 and Day 7 (Time of PA, Day 0); and (3) investigation of the robustness of embryos using vitrification on Day 4. The developmental kinetics showed that there was a general trend for zona-free PA embryos to develop faster than zona intact PA embryos...

  12. The miR-125 family is an important regulator of the expression and maintenance of maternal effect genes during preimplantational embryo development.

    Science.gov (United States)

    Kim, Kyeoung-Hwa; Seo, You-Mi; Kim, Eun-Young; Lee, Su-Yeon; Kwon, Jini; Ko, Jung-Jae; Lee, Kyung-Ah

    2016-11-01

    Previously, we reported that Sebox is a new maternal effect gene (MEG) that is required for early embryo development beyond the two-cell (2C) stage because this gene orchestrates the expression of important genes for zygotic genome activation (ZGA). However, regulators of Sebox expression remain unknown. Therefore, the objectives of the present study were to use bioinformatics tools to identify such regulatory microRNAs (miRNAs) and to determine the effects of the identified miRNAs on Sebox expression. Using computational algorithms, we identified a motif within the 3'UTR of Sebox mRNA that is specific to the seed region of the miR-125 family, which includes miR-125a-5p, miR-125b-5p and miR-351-5p. During our search for miRNAs, we found that the Lin28a 3'UTR also contains the same binding motif for the seed region of the miR-125 family. In addition, we confirmed that Lin28a also plays a role as a MEG and affects ZGA at the 2C stage, without affecting oocyte maturation or fertilization. Thus, we provide the first report indicating that the miR-125 family plays a crucial role in regulating MEGs related to the 2C block and in regulating ZGA through methods such as affecting Sebox and Lin28a in oocytes and embryos. © 2016 The Authors.

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

    Science.gov (United States)

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

    2002-05-01

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

  14. Characterization of the proximal region of the goat NANOG promoter that is used for monitoring cell reprogramming and early embryo development.

    Science.gov (United States)

    Guo, Yanjie; Lei, Lei; Ma, Xiaoling; Wang, Huayan

    2014-01-01

    Nanog is a key transcription regulatory molecule that plays an important role in maintaining stem cell pluripotency. However, the molecular features and transcription regulation of the NANOG gene in domestic animals are not well investigated. In this study, the 751-base pairs (bp) fragment of the proximal region of the goat NANOG promoter (GNP), which has a 572-bp promoter sequence retaining multiple transcription binding sites and a 179-bp 5' untranslated region of the goat NANOG gene, was cloned and characterized. The recombinant construct of pGNP-EGFP (enhanced green fluorescent protein) was solely activated in pluripotent cells and could be upregulated by the Oct4/Sox2 complex. The construct was stably transfected into goat fetal fibroblast (GFF) cells that were then used as the recipient cells to generate the induced pluripotent stem (iPS) cells. GNP-directed EGFP expression could be used to monitor the progression of cell reprogramming and the formation of iPS cells. The pGNP-EGFP construct was also delivered into goat oocytes cultured in vitro by microinjection. Interestingly, NANOG expression pattern in early stage goat embryos matured in vitro was asymmetrical. In two-cell embryos, the expression level of NANOG was uneven with one blastomere expressing EGFP and the next blastomere with no expression of EGFP. This was also observed in four-cell embryos. This asymmetrical expression may be due to the heterozygous expression of NANOG because of the quality of embryos and the culture environment. In conclusion, the GNP-EGFP reporter system represents a useful tool to monitor endogenous NANOG activation and for research with goat pluripotent stem cells.

  15. A Review of the Teratogenic Factors Effect on Embryo

    Directory of Open Access Journals (Sweden)

    Manzarbanoo Shojaei fard

    2017-02-01

    Full Text Available Background & Objectives: Teratology is a branch of embryology science that studies causes, mechanisms and abnormal pattern development. Embryo growth traumatic factors during pregnancy are called teratogens that some teratogens pass the placental barrier and cause adverse effect during development stages and malformation, however a drug may improve general health of the mother, but it might be poisonous for embryo and cause diverse malformation. Since study of embryo health and risk factor in this stage is important, the aim of this review article was the investigation of some types of teratosgens (such as radiation, infectious agents, heat disorders, maternal conditions and particularly the effect of teratogenic drugs on embryo including some legal drugs (such as acetaminophen, thalidomide, acyclovir, sedatives and anticonvulsants and illegal drugs (such as nicotine, alcohol, cocaine and marijuana. Conclusion: In general, teratogens depending on the type and duration of exposure in pregnancyperiod, adversely affect embryo and cause various disorders. A better understanding of these teratogens can contribute to prevent these defects, since many other drugs with similar effects and lower teratogenicity can be used to improve mothers’ health.

  16. Female gametophyte and embryo development in Helleborus bocconei Ten. (Ranunculaceae).

    Science.gov (United States)

    Bartoli, Giacomo; Felici, Cristiana; Ruffini Castiglione, Monica

    2017-01-01

    In this study, we investigated cytohistochemistry, cycle progression, and relative DNA content of the female gametophyte cells of Helleborus bocconei Ten. before and after fertilization process. The early stages of embryo development were also investigated. H. bocconei possesses a monosporic seven-celled/eight-nucleate Polygonum type female gametophyte, characterized by a morpho-functional polarity. The cells of the embryo sac showed abundant reserves of polysaccharides, strongly increasing in the egg cell just before fertilization. With different timing in DNA replication during cell cycle progression, synergids, egg cells, and polar nuclei showed a haploid DNA content at the end of their differentiation, while antipodes underwent three DNA endoreduplication cycles. Programmed cell death symptoms were detectable in synergid and antipodal cells. After double fertilization, the central cell quickly underwent many mitotic cycles forming the endosperm, which exhibited a progressive increase in protein bodies and starch grains. Close to the developing embryo, the endosperm differentiated a well-defined region rich in a fibrillar carbohydrate matrix. The zygote, that does not start immediately to divide after double fertilization, developed in to an embryo that reached the heart stage at fruit maturation time. A weakly differentiated embryo at this time indicates a morpho-physiological dormancy of seeds, as a survival strategy imposed by the life cycle of this plant with seed dispersal in spring and their germination in the following winter.

  17. The Effects of Progesterone on Oocyte Maturation and Embryo Development

    Directory of Open Access Journals (Sweden)

    Saeed Zavareh

    2013-01-01

    Full Text Available Oocyte maturation and embryo development are controlled by intra-ovarian factors suchas steroid hormones. Progesterone (P4 exists in the follicular fluid that contributes tonormal mammalian ovarian function and has several critical functions during embryodevelopment and implantation, including endometrial receptivity, embryonic survivalduring gestation and transformation of the endometrial stromal cells to decidual cells.It is well known that the physiological effects of P4 during the pre-implantation stages ofsome mammal’s embryos are mediated by P4 receptors and their gene expression is determined.The effects of P4 on oocytes and embryo development have been assessed bysome investigations, with contradictory results. P4, a dominant steroid in follicular fluidat approximately 18 hours after the luteinizing hormone (LH surge may have a criticalrole in maturation of oocytes at the germinal stage. However, it has been shown that differentconcentrations of P4 could not improve in vitro maturation rates of germinal vesicles(GV in cumulus oocyte complexes (COCs and cumulus denuded oocytes (CDOs.Culture media supplemented with P4 significantly improved mouse embryo development.In addition, an in vivo experimental design has shown high blastocyst survival andimplantation rates in P4-treated mice.In this review we explain some of the findings that pertain to the effects of P4 onoocyte maturation and embryo development both in vitro and in vivo.

  18. Abnormalities occurring during female gametophyte development result in the diversity of abnormal embryo sacs and leads to abnormal fertilization in indica/japonica hybrids in rice.

    Science.gov (United States)

    Zeng, Yu-Xiang; Hu, Chao-Yue; Lu, Yong-Gen; Li, Jin-Quan; Liu, Xiang-Dong

    2009-01-01

    Embryo sac abortion is one of the major reasons for sterility in indica/japonica hybrids in rice. To clarify the causal mechanism of embryo sac abortion, we studied the female gametophyte development in two indica/japonica hybrids via an eosin B staining procedure for embryo sac scanning using confocal laser scanning microscope. Different types of abnormalities occurred during megasporogenesis and megagametogenesis were demonstrated. The earliest abnormality was observed in the megasporocyte. A lot of the chalazal-most megaspores were degenerated before the mono-nucleate embryo sac stage. Disordered positioning of nucleus and abnormal nucellus tissue were characteristics of the abnormal female gametes from the mono-nucleate to four-nucleate embryo sac stages. The abnormalities that occurred from the early stage of the eight-nucleate embryo sac development to the mature embryo sac stage were characterized by smaller sizes and wrinkled antipodals. Asynchronous nuclear migration, abnormal positioning of nucleus, and degeneration of egg apparatus were also found at the eight-nucleate embryo sac stage. The abnormalities that occurred during female gametophyte development resulted in five major types of abnormal embryo sacs. These abnormal embryo sacs led to abnormal fertilization. Hand pollination using normal pollens on the spikelets during anthesis showed that normal pollens could not exclude the effect of abnormal embryo sac on seed setting.

  19. Abnormalities Occurring during Female Gametophyte Development Result in the Diversity of Abnormal Embryo Sacs and Leads to Abnormal Fertilization in indicaljaponica Hybrids in Rice

    Institute of Scientific and Technical Information of China (English)

    Yu-Xiang Zeng; Chao-Yue Hu; Yong-Gen Lu; Jin-Quan Li; Xiang-Dong Liu

    2009-01-01

    Embryo sac abortion is one of the major masons for sterility in indicaljaponica hybrids In rice. To clarify the causal mechanism of embryo sac abortion, we studied the female gametophyte development in two indicaljaponica hybrids via an eosin B staining procedure for embryo sac scanning using confocal laser scanning microscope. Different types of abnormalities occurred during megasporogenesis and megagamatogenesis were demonstrated. The earliest abnormality was observed in the megasporocyte. A lot of the chalazal-most megaspores were degenerated before the mono-nucleate embryo sac stage. Disordered positioning of nucleus and abnormal nucallus tissue were characteristics of the abnormal female gametes from the mono-nucleate to four-nucleate embryo sac stages. The abnormalities that occurred from the early stage of the eight-nucleate embryo sac development to the mature embryo sac stage were characterized by smaller sizes and wrinkled antipodals. Asynchronous nuclear migration, abnormal positioning of nucleus, and degeneration of egg apparatus were also found at the eight-nucleate embryo sac stage. The abnormalities that occurred during female gametophyte development resulted in five major types of abnormal embryo sacs. These abnormal embryo sacs led to abnormal fertilization. Hand pollination using normal pollens on the spikelets during anthesis showed that normal pollens could not exclude the effect of abnormal embryo sac on seed setting.

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

    Directory of Open Access Journals (Sweden)

    Jenna eKropp

    2014-04-01

    Full Text Available MicroRNAs (miRNA are short non-coding RNAs which act to regulate expression of genes driving numerous cellular processes. These RNAs are secreted within exosomes from cells into the extracellular environment where they may act as signaling molecules. In addition, they are relatively stable and are specifically expressed in association to certain cancers making them strong candidates as biological markers. Moreover, miRNAs have been detected in body fluids including urine, milk, saliva, semen, and blood plasma. However, it is unknown whether they are secreted by embryonic cells into the culture media. Given that miRNAs are expressed throughout embryonic cellular divisions and embryonic genome activation, we hypothesized that they are secreted from the embryo into the extracellular environment and may play a role in the developmental competence of bovine embryos. To test this hypothesis, bovine embryos were cultured individually from day 5 to day 8 of development in an in vitro fertilization system and gene expression of 5 miRNAs was analyzed in both embryos and culture media. Differential miRNA gene expression was observed between embryos that developed to the blastocyst stage and those that failed to develop from the morula to blastocyst stage, deemed degenerate embryos. MiR-25, mir-302c, miR-196a2, and miR-181a expression was found to be higher in degenerate embryos compared to blastocyst embryos. Interestingly, these miRNAs were also found to be expressed in the culture media of both bovine and human pre-implantation embryos. Overall, our results show for the first time that miRNAs are secreted from pre-implantation embryos into culture media and that miRNA expression may correlate with developmental competence of the embryo. Expression of miRNAs in in vitro culture media could allow for the development of biological markers for selection of better quality embryos and for subsequent successful pregnancy.

  1. Self-correction in human embryos%胚胎自我修复

    Institute of Scientific and Technical Information of China (English)

    徐艳文

    2013-01-01

    Reanalysis of aneuploid embryos diagnosed by preimplantation genetic screening (PGS) using fluorescence in situ hybridization(FISH) showed that part or all cells in some human cleavage-stage embryos may undergo self-correction during preimplantation development. Putative embryo self-correction mechanisms include embryonic mosaicism, preferential segregation of chromosomal abnormalities to the trophectoderm and extrusion or duplication of aneuploid chromosomes resulting in uniparental disomy. However, embryo self-correction has not been proved in the study using a single nucleotide polymorphism (SNP)microarray-based 24 chromosome aneuploidy screening technology. Neither preferential segregation of aneuploidy to trophectoderm nor uniparental disomy was found. Further study to improve the accuracy of karyotyping on cleavage-stage embryos is definitely needed.

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

    Science.gov (United States)

    Pullman, Gerald S; Johnson, Shannon

    2009-06-01

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

  3. The effect of peritoneal fluid from patients with endometriosis on mitochondrial function and development of early mouse embryos.

    Directory of Open Access Journals (Sweden)

    Jing Shu

    Full Text Available BACKGROUND: Peritoneal fluid (PF from patients with endometriosis can inhibit early embryo development via probable functional changes of embryo mitochondria in the early stage of embryo development. The purpose of this study was to determine the effect of PF from patients with endometriosis on mitochondrial function and development of early mouse embryos. METHODOLOGY/PRINCIPAL FINDINGS: PF was collected from patients with infertility and endometriosis, infertility due to tubal factors, and normal control subjects, and the level of NO was measured. Early murine embryos were then cultured with PF from normal control subjects, those with endometriosis, and with human tubal fluid (HTF, respectively. Cleavage and blastulation rates, mitochondrial DNA (mtDNA copy numbers, adenosine triphosphate (ATP level, and mitochondrial membrane potential (ΔΨm of the different groups were compared. The NO level in the PF of patients with endometriosis was significantly greater than in those without endometriosis and control patients. The embryos cultures with PF from patients with endometriosis had a lower cleavage rate and blastulation rate, and higher ATP and ΔΨm level at the 2- and 4-cell stages. No significant difference was found in mtDNA copies among the 3 groups. CONCLUSIONS/SIGNIFICANCE: PF from patients with endometriosis can inhibit early embryo development via probable functional changes of embryo mitochondria in the early stage of embryo development. Understanding the effects of PF on embryo development may assist in developing new methods of treatment for infertility.

  4. From embryo sac to oil and protein bodies: embryo development in the model legume Medicago truncatula.

    Science.gov (United States)

    Wang, Xin-Ding; Song, Youhong; Sheahan, Michael B; Garg, Manohar L; Rose, Ray J

    2012-01-01

    • The cell and developmental biology of zygotic embryogenesis in the model legume Medicago truncatula has received little attention. We studied M. truncatula embryogenesis from embryo sac until cotyledon maturation, including oil and protein body biogenesis. • We characterized embryo development using light and electron microscopy, measurement of protein and lipid fatty acid accumulation and by profiling the expression of key seed storage genes. • Embryo sac development in M. truncatula is of the Polygonum type. A distinctive multicellular hypophysis and suspensor develops before the globular stage and by the early cotyledon stage, the procambium connects the developing apical meristems. In the storage parenchyma of cotyledons, ovoid oil bodies surround protein bodies and the plasma membrane. Four major lipid fatty acids accumulate as cotyledons develop, paralleling the expression of OLEOSIN and the storage protein genes, VICILIN and LEGUMIN. • Zygotic embryogenesis in M. truncatula features the development of a distinctive multicellular hypophysis and an endopolyploid suspensor with basal transfer cell. A clear procambial connection between the apical meristems is evident and there is a characteristic arrangement of oil bodies in the cotyledons and radicle. Our data help link embryogenesis to the genetic regulation of oil and protein body biogenesis in legume seed.

  5. Viability of bovine demi embryo after splitting of fresh and frozen thawed embryo derived from in vitro embryo production

    Directory of Open Access Journals (Sweden)

    M Imron

    2007-06-01

    Full Text Available In vivo embryo production was limited by number of donor, wide variability respond due to superovulation program and also immunoactifity of superovulation hormone (FSH. Splitting technology could be an alternative to increase the number of transferrable embryos into recipien cows. Splitting is done with cutting embryo becoming two equal pieces (called demi embrio base on ICM orientation. The objective of this research was to determine the viability of demi embryo obtained from embryo splitting of fresh and frozen thawed embryo. The results showed that demi embryos which performed blastocoel reexpansion 3 hours after embryo splitting using fresh and frozen thawed embryos were 76.9 and 76.2% respectively. Base on existention of inner cell mass (ICM, the number of demi embryos developed with ICM from fresh and frozen thawed embryos were not significantly different (90.6 and 85.7% respectively. The cell number of demi embryo from fresh embryos splitting was not different compared with those from frozen thawed embryos (36.1 and 35.9 respectively. These finding indicated that embryo splitting can be applied to frozen thawed embryos with certain condition as well as fresh embryos.

  6. Construction and Analysis of a Full-Length cDNA Library of Peanut Embryos at Different Developmental Stages%不同发育时期花生胚混合全长cDNA文库的构建与分析

    Institute of Scientific and Technical Information of China (English)

    陈华; 邓烨; 张冲; 蔡铁城; 郑奕雄; 庄伟建

    2014-01-01

    以及DREB转录因子等。%[Objective] The objective of this study is to understand the molecular mechanism of peanut embryo development and obtain important genes related to peanut embryo development. [Method] Using peanut variety Minhua 6 as the experimental material, embryos on 10, 20, 30, 40, 50, and 60th day after pegging were sampled. Total RNA was extracted by improved CTAB method. Double strand cDNA was synthesized based on SMART technique. The purified dscDNA was ligated to pDNR-LIB vector digested by SfiⅠ and transformed into DH5α by electroporation to construct a full-length cDNA library of peanut embryos at different developmental stages. Bioinformatics analysis was performed following small-scale EST sequencing.[Result]A successful full-length cDNA library of peanut embryos at different development stages was constructed. The titer of unamplified cDNA library was about 3.5×106cfu/mL. The average cDNA inserts were more than 1 000 bp with a recombination frequency of 95.8%. Small-scale plasmid extraction and subsequent sequencing resulted in 60 ESTs, which were used for further analysis. BLASTX analysis showed that 39 sequences (65% of total sequences) had high similarity with reported genes in Glycine max, Arachis hypogaea, Medicago truncatula, etc. on NCBI with 32 sequences having known or putative functions and functions of other 7 sequences were unclear. The other 21 (35%of total sequences) could not find similarity with known genes in NCBI, which may be novel genes for peanut. GO annotation was performed with BLAST2GO software and the results revealed that the ESTs generated in this study mainly included responsive to stresses and defenses, protein synthesis and transport, lipid synthesis and metabolism, transcription and regulation, seed germination, dormancy and embryo development related genes. Besides, some genes were involved in signal transduction and light morphogenesis process. KEGG pathway analysis showed that the ESTs generated by randomly sequencing in this study mainly

  7. Is there a link between blastomere contact surfaces of day 3 embryos and live birth rate?

    Directory of Open Access Journals (Sweden)

    Paternot Goedele

    2012-09-01

    Full Text Available Abstract Background Cell-cell communication and adhesion are essential for the compaction process of early stage embryos. The aim of this study was to develop a non-invasive objective calculation system of embryo compaction in order to test the hypothesis that embryos with a larger mean contact surface result in a higher live birth rate compared to embryos with a lower mean contact surface. Methods Multilevel images of 474 embryos transferred on day 3 were evaluated by the Cellify software. This software calculates the contact surfaces between the blastomeres. The primary outcome of this study was live birth. An ideal range of contact surface was determined and the positive and negative predictive value, the sensitivity, the specificity and the area under the curve for this new characteristic were calculated. Results In total, 115 (24% transferred embryos resulted in a live birth. Selection of an embryo for transfer on its mean contact surface could predict live birth with a high sensitivity (80% and high negative predicting value (83% but with a low positive predictive value (27%, a low specificity (31% and low area under the ROC curve (0.56. The mean contact surface of embryos cultured in a single medium was significantly higher compared to the mean contact surface of embryos cultured in a sequential medium (p = 0.0003. Conclusions Neither the mean contact surface nor the number of contact surfaces of a day 3 embryo had an additional value in the prediction of live birth. The type of culture medium, however, had an impact on the contact surface of an embryo. Embryos cultured in a single medium had a significant larger contact surface compared to embryos cultured in the sequential medium.

  8. The Fate of Mitochondria in Ibex-hirus Reconstructed Early Embryos

    Institute of Scientific and Technical Information of China (English)

    Yan JIANG; Shu-Zhen LIU; Yan-Ling ZHANG; Man-Xi JIANG; Qing-Yuan SUN; Da-Yuan CHEN

    2004-01-01

    Inter-species nuclear transfer could be used to preserve North Goat (Capra ibex), an endan-gered species. We established the culture conditions for ibex-hirus reconstructed embryos and optimized themethod for DNA extractions of a single cell and early cloned embryo. By using mitochondria-specific probesof ibex and hirus respectively we found that mitochondria of donor cells can co-exist with recipients in 1-celland 2-cell stages of the reconstructed embryos but not in the following developmental stages.

  9. Estagiamento de embriões de Macrobrachium olfersi (Wiegman (Crustacea, Palaemonidae através de critérios morfológicos nos dias embrionários Macrobrachium olfersi (Wiegman (Crustacea, Palaemonidae embryo staging through morphological landmarks identified in each embryonic day

    Directory of Open Access Journals (Sweden)

    Marcos S. Simões-Costa

    2005-06-01

    was characterized through daily staging system. Living and fixed embryos were analyzed (48x in intervals of 24 hours (embryonic day. The eye index was calculated in each embryonic day from the appearance of the eye pigmentation. The development of M. olfersi was described in 14 embryonic days (E, where the cleavage, gastrulation, germinal disk and egg nauplius are developed from E1 to E4. The subsequent days were characterized by the growth of the egg nauplius, as well by the formation and the bent of the post-nauplius. At E7, the eye pigmentation appeared and was followed by the beginning of heartbeats at E8. From E9 to E14, more intensive organogenesis processes occurred, mainly on the nervous, cardiac and digestive systems. The daily staging of development of M. olfersi development enabled the recognition of different embryonic forms, as well as growth and differentiation rhythms of embryo, which were fundamental to the gradual formation of the body plan.

  10. Reconstruction of human embryos derived from somatic cells

    Institute of Scientific and Technical Information of China (English)

    LU Changfu; LIN Ge; XIE Changqing; GONG Fei; ZHOU Hong; TAN Yueqiu; LU Guangxiu

    2003-01-01

    Reconstruction of human nuclear transfer embryos is a necessary step of therapeutic cloning. In this study we injected somatic cell nuclei into MⅡ oocytes and activated reconstructed oocytes with calcium ionophore A23187 (CaA) and 6-dimethylaminopurine (6-DMAP). After oocyte activation and 2PN formation, we removed the female PN. By using this method, we avoided the application of DNA fluorescent stain and ultraviolet light for oocyte enucleation, and over elimination of ooplasm was also mitigated. Some reconstructed embryos developed into theblastocyst stage in vitro.

  11. Cytological-cytogenetic analyses of winter flounder embryos collected from the Benthos at the barge North Cape oil spill

    Energy Technology Data Exchange (ETDEWEB)

    Hughes, J.B. [Milford Laboratory, CT (United States). Northeast Fisheries Science Center

    1999-01-01

    The oil barge North Cape ran aground on Moonstone Beach in Rhode Island on January 19, 1996, spilling 828 000 gallons (US) of a combination of diesel and home heating oils. Samples of winter flounder embryos were collected from salt ponds in the area of the spill using an epibenthic sled. An examination of the field-sampled embryos based on gross morphology, levels of moribund embryos, mitotic index, and chromosomal anomalies found that a significant number exhibited high levels of one or more of these conditions when compared with flounder embryos raised under laboratory-controlled conditions. No chorion damage was noted in embryos collected from the field nor were there any significant findings of lordosis or scoliosis. The cumulative impact on winter flounder embryos of North Cape oil exposure was an estimated 51% reduction in the number of embryos surviving to the larval stage. (author)

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

    Science.gov (United States)

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

    2008-11-01

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

  13. Contrasting Storage Protein Synthesis and Messenger RNA Accumulation during Development of Zygotic and Somatic Embryos of Alfalfa (Medicago sativa L.).

    Science.gov (United States)

    Krochko, J E; Pramanik, S K; Bewley, J D

    1992-05-01

    During development on hormone-free media, somatic embryos pass through distinct morphological stages that superficially resemble those of zygotic embryo development (globular, heart, torpedo, cotyledonary stages). Despite these similarities, they differ from zygotic embryos in the extent of cotyledonary development and the patterns of synthesis and quantitative expression of seed-specific storage proteins (7S, 11S, and 2S proteins). Alfin (7S) is the first storage protein synthesized in developing zygotic embryos (stage IV). The 11S (medicagin) and 2S (Low Molecular Weight, LMW) storage proteins are not detectable until the following stage of development (stage V), although all three are present before the completion of embryo enlargement. Likewise, the 7S storage protein is the first to be synthesized in developing somatic embryos (day 5). Medicagin is evident by day 7 and the LMW protein by day 10. In contrast to zygotic embryos, alfin remains the predominant storage protein in somatic embryos throughout development. Not only are the relative amounts of medicagin and the LMW protein reduced in somatic embryos but the LMW protein is accumulated much later than the other proteins. Quantification of the storage protein mRNAs (7S, 11S, and 2S) by northern blot analysis confirms that there are substantial differences in the patterns of message accumulation in zygotic and somatic embryos of alfalfa (Medicago sativa). In zygotic embryos, the 7S, 11S, and 2S storage protein mRNAs are abundant during maturation and, in particular, during the stages of maximum protein synthesis (alfin, stages VI and VII; medicagin, stage VII; LMW, stage VII). In somatic embryos, the predominance of the 7S storage protein is correlated with increased accumulation of its mRNA, whereas the limited synthesis of the 11S storage protein is associated with much lower steady-state levels of its message. The mRNA for the LMW protein is present already by 3 days after transfer to hormone-free media

  14. Effective embryo production from Holstein cows treated with gonadotropin-releasing hormone during early lactation.

    Science.gov (United States)

    Ogata, Yasuhiro; Yu, Guang-Min; Hidaka, Takemasa; Matzushige, Tadami; Maeda, Teruo

    2016-10-01

    The low efficiency of embryo production in Holstein cows during early lactation presents many challenges for animal production. To improve its efficiency, the outcomes of single GnRH injections 48 hours before each of three cycles of ovum pick up (OPU; weeks 2, 4, and 6) were compared with three cycles of unstimulated OPU (controls; weeks 1, 3, and 5) in 35 Holstein cows during 6 weeks of early lactation (40-80 days postpartum). More total follicle numbers (19.5 vs. 16.0; P controls (15.3 vs. 11.5; P controls (2.8 vs. 1.7 and 5.8 vs. 4.2, respectively; P control cycles (13.7 vs. 9.6; P controls (9.0 vs. 6.2 two-cell embryos; 4.7 vs. 3.0 four-cell embryos; 3.3 vs. 2.0 morulae; and 3.0 vs. 1.7 blastocysts, respectively). Moreover, there was no significant difference in pregnancy rate of the recipient cows after embryo transfer (57.1% vs. 42.1%; P > 0.05) no matter if the embryos came from the GnRH-treated cycles or not. Thus, GnRH-stimulated OPUs improved the efficiency of embryo production in Holstein cows during early lactation. This novel method for in vitro embryo production should benefit the dairy industry.

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

    Science.gov (United States)

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

    2014-04-01

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

  16. Developmental toxicity and oxidative stress induced by gamma irradiation in zebrafish embryos

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Miao; Hu, Nan; Ding, Dexin; Zhao, Weichao; Feng, Yongfu; Zhang, Hui; Li, Guangyue; Wang, Yongdong [University of South China, Key Discipline Laboratory for National Defense for Biotechnology in Uranium Mining and Hydrometallurgy, Hengyang, Hunan Province (China)

    2016-11-15

    This study aimed to evaluate the biological effects of gamma irradiation on zebrafish embryos. Different doses of gamma rays (0.01, 0.05, 0.1, 0.5 and 1 Gy) were used to irradiate zebrafish embryos at three developmental stages (stage 1, 6 h post-fertilization (hpf); stage 2, 12 hpf; stage three, 24 hpf), respectively. The survival, malformation and hatching rates of the zebrafish embryos were measured at the morphological endpoint of 96 hpf. The activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx) and glutathione S-transferase (GST) were assayed. Morphology analysis showed that gamma irradiation inhibited hatching and induced developmental toxicity in a dose-dependent manner. Interestingly, after irradiation the malformation rate changed not only in a dose-dependent manner but also in a developmental stage-dependent manner, indicating that the zebrafish embryos at stage 1 were more sensitive to gamma rays than those at other stages. Biochemical analysis showed that gamma irradiation modulated the activities of antioxidant enzymes in a dose-dependent manner. A linear relationship was found between GPx activity and irradiation dose in 0.1-1 Gy group, and GPx was a suitable biomarker for gamma irradiation in the dose range from 0.1 to 1 Gy. Furthermore, the activities of SOD, CAT, GR and GPx of the zebrafish embryos at stage 3 were found to be much higher than those at other stages, indicating that the zebrafish embryos at stage 3 had a greater ability to protect against gamma rays than those at other stages, and thus the activities of antioxidant enzymes changed in a developmental stage-dependent manner. (orig.)

  17. 不同激光辅助孵化对玻璃化冷冻和慢速程序冷冻卵裂期胚胎移植妊娠率的影响%Effects of two kinds of laser assisted hatching method on frozen thawed embryo transfer pregnancy rate during cleavage stage

    Institute of Scientific and Technical Information of China (English)

    王琦; 陈蔚清; 张程

    2016-01-01

    Objective:To study the influence of two kinds of laser assisted hatching method on frozen thawed embryo transfer pregnancy rate during cleavage stage by vitrification freezing and slow freezing program.Methods:In our hospital a clinical study was performed on 160 patients with frozen embryo used laser zona drilling assisted hatching,while the study group used laser zona pellucida thinning assisted hatching,clinical pregnancy outcome was compared between two groups.Results:Firstly,after cryopreservation,the embryo implantation rate in study group (19.1 %)was significantly higher than that of the control group (12.3%),the difference was statistically significant (P 0.05 );In addition,after vitrification freezing,the thawing cleavage survival rate (87.48% vs.87.41 %),the clinical pregnancy rate of embryo (37.5% vs.35%)and implantation rate(17.5% vs.15.1 %) had no statistically significant difference (P >0.05 );What is more,the thawing cleavage survival rate (87.48% vs.82.21 %),the clinical pregnancy rate of embryo (37.5% vs.37.5%)and the implantation rate (17.5% vs.19.1 %)in the study group from two freezing methods,had no significant difference (P >0.05).Conclusion:The laser zona pellucida thinning assisted hatching is superior to laser zona drilling assisted hatching,it doesn't affect the patient’s pregnancy rate and can reduce the damage for the embryo.%目的:研究分析两种激光辅助孵化方法对玻璃化冷冻和慢速程序冷冻卵裂期胚胎移植妊娠率的影响。方法:对2013年11月到2014年7月期间在我院接受冷冻胚胎移植的160例患者进行临床研究,随机分为两组,对照组为进行激光透明带打孔辅助孵化,研究组进行激光透明带薄化辅助孵化,比较组间患者的临床妊娠结局。结果:胚胎经程序冷冻后,研究组胚胎种植率(19.1%)显著高于对照组(12.3%),差异有统计学意义(P <0.05),而解冻后卵裂球存活率(82.21% vs

  18. Developmental sequence of Cambrian embryo Markuelia

    Institute of Scientific and Technical Information of China (English)

    DONG XiPing

    2007-01-01

    Based on more exquisitely preserved specimens of Markuelia hunanensis recently recovered from Middle and Upper Cambrian in western Hunan and in the light of Synchrotron radiation X-ray tomographic microscopy, the developmental sequence from cleavage through organogenesis to the pre-hatching of Cambrian embryo Markuelia, especially the developmental sequence during the pre-hatching stage, i.e. from the earliest period when the scalids and tail spines only took shape to the latest period (just about hatching), is established. This developmental sequence provides a pattern of embryonic development during the pre-hatching stage, which has not been established in the living scalidophorans (priapulids, Ioriciferans and kinorhynchs). Thus, it not only enriches our knowledge on the embryonic development of the extant descendants of Markuelia, but also opens a new window to the evolution and development of the animal.

  19. Physical influences on embryo development.

    Science.gov (United States)

    Deeming, D C; Rowlett, K; Simkiss, K

    1987-01-01

    There is a critical period between 3 and 7 days of incubation when the absence of turning in eggs of the domestic fowl leads to increased mortality and decreased embryo growth. This critical period coincides with the time of subembryonic fluid formation, and it is suggested that the absence of turning leads to the presence of unstirred layer effects in fluid secretion. This fluid deficiency persists throughout the subsequent development of the embryo. Experiments on shell-less culture systems support this interpretation in preference to other explanations of embryo death in unturned eggs, which usually refer to chorion adhesion to shell membranes.

  20. Studies on Nuclear Transplantation in Mammalian Embryos

    Institute of Scientific and Technical Information of China (English)

    TanJinghe; ZhouQi; 等

    1995-01-01

    A series of experiments were conducted to study the major procedures in nuclear transplanation such as oocyte enucleation and activation,electrofusion and developent of the nuclear transplant embryos in the mouse,rabbits and sheep.The important results are as follows:1.In the mouse,only 35% of the oocytes collected 15-16h after hCG had a notable first polar body(FPb) and those without FPb were enucleated by removing cytoplasm from the PVS-wider side and the enucleation rate was similar to that in the oocytes with FPb,and the enucleation rate of removing 1/3 cytoplasm was remarkably higher than that of removing 1/4 cytoplasm.2.Among the three fusion media tested;mannitol and sucrose solutions produced better results than M2 in electrofusion of mouse 2-cell embryos.Under favorable pulse conditions,the osmotic pressure of fusion medium had no motable effect on electrofusion,but as the conditions became so unfavorable that some embryos began to lyse,the fusion rates in hypertonic mannitol solution were significantly higher than those in isotonic or hypotonic solutions.A wide range of pulse strengths (0.31-2.04kv/cm) and durations(10-1280us) were used and 100% of fusion were obtained in many cases.Optimal pulse durations were plotted for field strengths to obtain high fusion rates(96%-100%)in mouse2-cell embryos.3.With one pulse of 0.45kv/cm,satisfactory results of mouse oocyte activation were obatined only when the duration increased to 160us or longer,The activation rate increased as the oocytes got older.Some of the oocytes ar.rested at metaphaseⅢ after electrical stimulation and their proportion to the number of oocytes not activated increased with egg age.4.10% and 31% of the nuclear transplant embryos developed to morula or blastocyst stage in sheep and rabbits,respectively,with Chinese-made hormones and chemicals.

  1. Genomic DNA methylation patterns in bovine preim-plantation embryos derived from in vitro fertilization

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    By using the approach of immunofluorescence staining with an antibody against 5-methylcytosine (5MeC), the present study detected the DNA methylation patterns of bovine zygotes and preimplanta-tion embryos derived from oocyte in vitro maturation (IVM), in vitro fertilization (IVF) and embryo in vitro culture (IVC). The results showed that: a) paternal-specific demethylation occurred in 61.5% of the examined zygotes, while 34.6% of them showed no demethylation; b) decreased methylation level was observed after the 8-cell stage and persisted through the morula stage, however methylation levels were different between blastomeres within the same embryos; c) at the blastocyst stage, the methyla-tion level was very low in inner cell mass, but high in trophectoderm cells. The present study suggests, at least partly, that IVM/IVF/IVC may have effects on DNA methylation reprogramming of bovine zygotes and early embryos.

  2. Lower Cambrian yolk-pyramid embryos from Southern Shaanxi,China

    Institute of Scientific and Technical Information of China (English)

    CHEN Junyuan; Andreas BRAUN; Dieter WALOSZEK; PENG Qingqing; Andreas MAAS

    2004-01-01

    Phosphatized globules with radially arranged pillars were recently recorded from the Lower Cambrian phosphate deposit, Ningqiang, Shaanxi by Yue and Bengtson. These authors interpreted the globules as blastula stage of embryos and the pillars as blastomeres. On the basis of new additional material, we reinterpret these structures as yolk-pyramid stages of possible arthropod eggs. The 20 embryos under present study range from 380 μm to 600 μm in diameter and contain about 120 pyramids. Some embryos having a higher number of pyramids are tentatively interpreted as slightly later developmental stages of the same animal. These 543-million-year-old embryos may push back the evolutionary history of the arthropods to a deeper time and also suggest that one important pattern of arthropod development was already present at the beginning of the Cambrian.

  3. Genomic DNA methylation patterns in bovine preimplantation embryos derived from in vitro fertilization

    Institute of Scientific and Technical Information of China (English)

    HOU Jian; LIU Lei; LEI TingHua; CUI XiuHong; AN XiaoRong; CHEN YongFu

    2007-01-01

    By using the approach of immunofluorescence staining with an antibody against 5-methylcytosine (5MeC), the present study detected the DNA methylation patterns of bovine zygotes and preimplantation embryos derived from oocyte in vitro maturation (IVM), in vitro fertilization (IVF) and embryo in vitro culture (IVC). The results showed that: a) paternal-specific demethylation occurred in 61.5% of the examined zygotes, while 34.6% of them showed no demethylation; b) decreased methylation level was observed after the 8-cell stage and persisted through the morula stage, however methylation levels were different between blastomeres within the same embryos; c) at the blastocyst stage, the methylation level was very low in inner cell mass, but high in trophectoderm cells. The present study suggests, at least partly, that IVM/IVF/IVC may have effects on DNA methylation reprogramming of bovine zygotes and early embryos.

  4. Effect of washing mineral oil on development of mouse embryos in vitro and in vivo after embryo transfer

    Institute of Scientific and Technical Information of China (English)

    Li Hui; Zhang Li-xuan; Zhong Yu; Zhu Kai; Zhang Tian; Wang Min-kang

    2008-01-01

    Objective:To establish a simple and effective washing procedure for both used and purchased mineral oil,that can be used for embryo culture.Methods:A complete test system has been used for this purpose.There are 3 steps in our new washing proto-col.First,the oil was mixed with 95% ethanol at 1:1,the bottle being shaken by hand for 10 minutes,then sepa-rated.Second,the oil was heated to boiling point with 0.31 mol/L NaCl for 30 minutes.Third,anhydrous Na:SO4 was put into the oil for further treatment.1-cell stage embryos of a KM strain mouse have been collected surgically and cultured.Cleavage and blastocyst stage development were recorded and some embryos were transferred into re-cipients.Results:The results show that recycled oil can promote the development from 2-cell to blastocyst stage(23.3%)when compared with that of control(16.9%).Offspring have been obtained at 44 %(7/16),16 %(3/19)from washed recycled oil and control oil respectively.Conclusion:This washing procedure is safe and effective for the used treatment and for other sources of mineral oil used for embryo culture.

  5. Relationship between Different Pronuclear Patterns and Potential of Embryo Development and Pregnancy

    Institute of Scientific and Technical Information of China (English)

    Zhong-mei LI; Yu-bao WANG; Dun-yu GU; Xiao-yun WU; De-qi JIANG; Zhen LU; Zuan-chong FENG

    2003-01-01

    Objective To explore the relationship between the patterns of pronucleus and embryo development and pregnancy potential in the pronuclear stageMethods According to the number and distribution of nucleolar precursor bodies, the embryos at pronuclear stage were classified into 6 pronuclear patterns from 0 to 5, 16~18 h after in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). For each pattern, the subsequent embryonic morphology and the pregnancy rate were analyzed. Results Embryos of Pattern 0 developed to significantly more embryos with good quality and higher pregnancy potential than the embryos developing from other patterns (83.14% and 76.11% respectively, P<0.05). The pregnancy rate was decreased as less embryos of Pattern 0 were transferred . The pregnancy rate of the groups of only Pattern 0, with Pattern 0, and without Pattern 0 were 48.08%, 32.14% and 21.28% respectively (P<0.05).Conclusions The pronuclear patterns are of the predictive value of embryo development and pregnancy potential, which can be used as a new tool for the selection of embryos in IVF and ICSI.

  6. Non-surgical embryo transfer in pigs

    NARCIS (Netherlands)

    Hazeleger, W.

    1999-01-01

    Embryo transfer in pigs has been performed surgically for a long time. However, a less invasive, non-surgical, procedure of embryo transfer could be a valuable tool for research (to study embryo survival and embryo-uterus interactions) and practical applications (export, prevention of

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

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Jun-Xue; Kang, Jin-Dan; Li, Suo; Jin, Long; Zhu, Hai-Ying; Guo, Qing; Gao, Qing-Shan; Yan, Chang-Guo; Yin, Xi-Jun, E-mail: yinxj33@msn.com

    2015-01-02

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

  8. Embryo sac haustorium in Dryas octopetala L. (Rosaceae

    Directory of Open Access Journals (Sweden)

    Romana Czapik

    2014-01-01

    Full Text Available The embryo sac haustorium found in Dryas octopetala L. from the Tatra Mts is the first record of its occurrence in Rosaceae. At the eight-nucleate stage of the embryo sac, the antipodal end of the central cell began to grow into a narrow caecum filled with dense cytoplasm and elongated in the chalazal direction leaving the three antipodals in situ. The haustorium enlarged and lost its characteristic shape after the period of fertilization. Finally, the embryo sac occupied almost the whole length of the ovule. Situated at its chalazal end there was either dense cytoplasm with a group of endosperm nuclei or dense, grainy cytoplasm only, if fertilization had not taken place.

  9. Abnormalities in centrosome number in human embryos and embryonic stem cells.

    Science.gov (United States)

    Gu, Yi-Fan; OuYang, Qi; Dai, Can; Lu, Chang-Fu; Lin, Ge; Gong, Fei; Lu, Guang-Xiu

    2016-05-01

    Chromosomal abnormalities are common in human embryos. Previous studies have suggested links between centrosome number and chromosome abnormalities, but information regarding abnormalities in centrosome number in human embryos is limited. We analyzed abnormalities in centrosome number in human embryos and embryonic stem cells (hESCs). Following normal fertilization, supernumerary centrosomes were present at rates of 7.3% in two-pronucleus (2PN)-stage zygotes and 6.5% in first-cleavage zygotes. Supernumerary centrosomes were also detected in 24.4% of blastomeres from 60% of embryos derived from 2PN zygotes. Conversely, in mono- (1PN) and tri-pronucleus (3PN) zygotes, the frequency of abnormal centrosome number increased substantially at first cleavage. Rates in blastomeres of Day-3 embryos, however, were about the same between embryos derived from 1PN and 2PN zygotes, whereas abnormalities in centrosome number were higher in those from 3PN zygotes. By comparison, the rate of abnormal centrosome numbers in hESCs was 1.5-11.2%. Thus, abnormalities in centrosome number existed in human zygotes and cleaved embryos-especially those resulting from aberrant fertilization-but the frequency of such abnormalities was lower in hESCs derived from these embryos. These findings identify a source of the chromosomal instability in human embryos and hESCs, and highlight new safety issues for human assisted reproductive technology. Mol. Reprod. Dev. 83: 392-404, 2016. © 2016 Wiley Periodicals, Inc.

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

    Directory of Open Access Journals (Sweden)

    Dean H Betts

    Full Text Available 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.

  11. Comparison Study On In Vitro morphogenesis of Mature and Immature Wheat (Triticum aestivum L. Embryos

    Directory of Open Access Journals (Sweden)

    Hala Al. A. Almobasher

    2016-09-01

    Full Text Available The present study comparedthe in vitro performance of two stages of wheat embryos;mature and immature embryos explantswere cultured in MS medium supplemented with different concentrations of 2,4-Dfor callus induction,mature embryosgenerally showed ostensibly preference to the immature embryo and achieved the highest fresh weight value (62.0±0.01a at 2.0 mg/l 2,4-D. Calluses derived were transferred to regeneration mediasupplemented with different levels of activated charcoal, sucrose and silver nitrate. Mature embryo derived calluses achieved the highestpercentages (50-100% of nodular callus at all types of regeneration mediatested compared to immature embryo derived callusespercentages (8-66%.Shootregenerationfrequencies obtained were better in the mature embryo derived callus cultures, where it achieved 25% regeneration percentage at 3.0mg/l activated charcoal;16% regeneration percentage at 60.0 g/l sucrose and 25% regeneration percentage at 10.0 ml/l silver nitrate, while immature embryo derived callus cultures achieved 8% regeneration percentage at all activated charcoal concentrations, failed to regenerate shoots in sucrose concentrations and the highest regeneration percentage obtained was 8% at silver nitrate treatments. The present study showed the mature embryo culturespreference to immature embryo culturesfor callus induction, embryogenesis and plantlet regeneration in Elnileen wheat cultivar.

  12. The effect of hypoxia on facial shape variation and disease phenotypes in chicken embryos

    Directory of Open Access Journals (Sweden)

    Francis Smith

    2013-07-01

    Craniofacial anomalies can arise from both genetic and environmental factors, including prenatal hypoxia. Recent clinical evidence correlates hypoxia to craniofacial malformations. However, the mechanisms by which hypoxia mediates these defects are not yet understood. We examined the cellular mechanisms underlying malformations induced by hypoxia using a chicken (Gallus gallus embryo model. Eggs were incubated in either hypoxic (7, 9, 11, 13, 15, 17 or 19% O2 or normoxic (21% O2 conditions. Embryos were photographed for morphological analysis at days 3–6. For analysis of skeletal development, 13-day embryos were cleared and stained with alcian blue and alizarin red for cartilage and bone, respectively. Quantitative analysis of facial shape variation was performed on images of embryos via geometric morphometrics. Early-stage embryos (day 2 were analyzed for apoptosis via whole-mount and section TUNEL staining and immunostaining for cleaved caspase-3, whereas later-stage embryos (days 4–6 were sectioned in paraffin for analysis of cell proliferation (BrdU, apoptosis (TUNEL and metabolic stress (phospho-AMPK. Results demonstrate that survival is reduced in a dose-dependent manner. Hypoxic embryos displayed a spectrum of craniofacial anomalies, from mild asymmetry and eye defects to more severe frontonasal and cephalic anomalies. Skull bone development was delayed in hypoxic embryos, with some skeletal defects observed. Morphometric analysis showed facial shape variation relative to centroid size and age in hypoxic groups. Hypoxia disrupted cell proliferation and, in early-stage embryos, caused apoptosis of neural crest progenitor cells. Hypoxic embryos also displayed an increased metabolic stress response. These results indicate that hypoxia during early embryonic craniofacial development might induce cellular oxidative stress, leading to apoptosis of the neural crest progenitor cells that are crucial to normal craniofacial morphogenesis.

  13. Live birth in a woman with recurrent implantation failure and adenomyosis following transfer of refrozen-warmed embryos.

    Science.gov (United States)

    Safari, Somayyeh; Faramarzi, Azita; Agha-Rahimi, Azam; Khalili, Mohammad Ali

    2016-09-01

    The aim was to report a healthy live birth using re-vitrified-warmed cleavage-stage embryos derived from supernumerary warmed embryos after frozen embryo transfer (ET) in a patient with recurrent implantation failure (RIF). The case was a 39-year-old female with a history of polycystic ovarian syndrome and adenomyosis, along with RIF. After ovarian hyperstimulation, 33 cumulus-oocyte complexes were retrieved and fertilized with conventional in vitro fertilization and intracytoplasmic sperm injection. Because of the risk of ovarian hyperstimulation syndrome, 16 grade B and C embryos were vitrified. After 3 and 6 months, 3 and 4 B-C warmed embryos were transferred to the uterus, respectively. However, implantation did not take place. Ten months later, four embryos were warmed, two grade B 8-cell embryos were transferred, and two embryos were re-vitrified. One year later, the two re-vitrified cleavage-stage embryos were warmed, which resulted in a successful live birth. This finding showed that following first warming, it is feasible to refreeze supernumerary warmed embryos for subsequent ET in patients with a history of RIF.

  14. Live birth in a woman with recurrent implantation failure and adenomyosis following transfer of refrozen-warmed embryos

    Science.gov (United States)

    Safari, Somayyeh; Faramarzi, Azita; Khalili, Mohammad Ali

    2016-01-01

    The aim was to report a healthy live birth using re-vitrified-warmed cleavage-stage embryos derived from supernumerary warmed embryos after frozen embryo transfer (ET) in a patient with recurrent implantation failure (RIF). The case was a 39-year-old female with a history of polycystic ovarian syndrome and adenomyosis, along with RIF. After ovarian hyperstimulation, 33 cumulus-oocyte complexes were retrieved and fertilized with conventional in vitro fertilization and intracytoplasmic sperm injection. Because of the risk of ovarian hyperstimulation syndrome, 16 grade B and C embryos were vitrified. After 3 and 6 months, 3 and 4 B–C warmed embryos were transferred to the uterus, respectively. However, implantation did not take place. Ten months later, four embryos were warmed, two grade B 8-cell embryos were transferred, and two embryos were re-vitrified. One year later, the two re-vitrified cleavage-stage embryos were warmed, which resulted in a successful live birth. This finding showed that following first warming, it is feasible to refreeze supernumerary warmed embryos for subsequent ET in patients with a history of RIF. PMID:27689042

  15. Delay of ZGA initiation occurred in 2-cell blocked mouse embryos

    Institute of Scientific and Technical Information of China (English)

    JIA JING QIU; WU WEN ZHANG; ZHI LI WU; YI HONG WANG; MIN QIAN; YI PING LI

    2003-01-01

    One-cell mouse embryos from KM strain and B6C3F1 strain were cultured in M16 medium, in which2-cell block generally occurs. Embryos of KM strain exhibited 2-cell block, whereas B6C3F1 embryos,which are regarded as a nonblocking strain, proceeded to the 4-cell stage in our culture condition. It is oftenassumed that the block of early development is due to the failure of zygotic gene activation (ZGA) in culturedembryos. In this study we examined protein synthesis patterns by two-dimensional gel electrophoresis of[35S] methionine radiolabeled 2-cell embryos. Embryos from the blocking strain and the nonblocking strainwere compared in their development both in vitro and in vivo. The detection of TRC expression, a markerof ZGA, at 42 h post hCG in KM embryos developed in vitro suggested that ZGA was also initiated even inthe 2-cell arrested embryos. Nevertheless, a significant delay of ZGA was observed in KM strain as comparedwith normally developed B6C3F1 embryos. At the very beginning of major ZGA as early as 36 h post hCG,TRC has already been expressed in B6C3F1 embryos developed in vitro and KM embryos developed in vivo.But for 2-cell blocked KM embryos, TRC was still not detectable even at 38 h post hCG. These evidencessuggest that 2-cell-blocked embryos do initiate ZGA, and that 2-cell block phenomenon is due not to thedisability in initiating ZGA, but to a delay of ZGA.

  16. Transmission electron microscopic evaluation of neuronal changes in methylmercury-exposed zebrafish embryos (Danio rerio).

    Science.gov (United States)

    Hassan, Said A; Farouk, Sameh M; Abbott, Louise C

    2016-01-01

    Our work aimed to elucidate the ultrastructural changes associated with brain neurons in wild-type zebrafish embryos exposed to different concentrations of methylmercury. Zebrafish embryos were exposed to one of five concentrations of methylmercury (0 [negative control], 5, 10, 50, and 80 parts per billion) starting at six hours post fertilization (hpf). At 96 hpf, cells in the zebrafish embryo brains were examined using transmission electron microscopy. The developing neurons of the control embryos sowed normal cellular ultrastructure. Few alterations were observed among the neurons of zebrafish embryos exposed to 5 ppb methylmercury. The cells of the embryos exposed to 10 ppb methylmercury showed slight cellular degeneration as demonstrated by the accumulation of electron dens bodies which were presumably lysosomes in different stages of formation. In embryos exposed to 50 ppb methylmercury, the neuronal cytoplasm conained large electron dense lysosomes and the rough endoplasmic reticulum appeared to be reduced and irregular in shape. Furthermore, the embryonic brain neurons exposed to 80 ppb methylmercury showed the most severe ultrastructural changes, including some that were consistent with different stages of the cell death process. Obvious cellular changes were observed in this highest exposure group included: disrupted or degenerating nuclei; fragmentation or vacuolization of mitochondrial cristae; and loss of mitochondrial matrix density. Based on these observations, we conclude that these different morphological patterns of cellular changes may reflect either different stages of the cell death process or different types of cell death due to 24 hours of exposure to 80 ppb methylmercury.

  17. DAPI Staining of Drosophila Embryos.

    Science.gov (United States)

    Rothwell, Wendy F; Sullivan, William

    2007-10-01

    INTRODUCTIONDrosophila embryos can be stained with specific fluorescent probes or antibodies through either direct or indirect immunofluorescence. In particular, several effective probes exist for visualizing DNA. 4',6-diamidino-2-phenylindole (DAPI) is a commonly used DNA-binding dye. Because it is specific for double-stranded DNA, no prior RNase treatment is required. While the embryo staining method described here uses DAPI, other fluorescent DNA probes can be processed similarly.

  18. Cadmium-induced ectopic apoptosis in zebrafish embryos

    Energy Technology Data Exchange (ETDEWEB)

    Chan, Po Kwok; Cheng, Shuk Han [Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon (Hong Kong)

    2003-02-01

    In this study, we tested the hypothesis that cadmium-induced developmental toxicity was mediated via ectopic occurrence of apoptosis during embryonic development. We employed confocal microscopy to acquire images of whole-mount staining of apoptotic cells in zebrafish embryo exposed to 100 {mu}M cadmium from 5 hours post fertilisation (hpf) to 28 hpf. Three-dimensional reconstruction of the images was performed and the spatial and temporal distributions of apoptotic cells in the embryos were compared. In cadmium-treated embryos with varying degrees of gross developmental malformations, significantly higher numbers of apoptotic cells were detected with this method. In order to detect the precise locations of apoptotic cells, we performed terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay in sectioned embryos. In the degenerating neural tube of cadmium-treated embryos apoptotic cells were detected, while in the healthy neural tube of the untreated controls no apoptotic cells were found. We then employed flow cytometry to investigate whether cadmium exposure would affect the dynamics of apoptosis or induce any abnormalities in cell-cycle progression. It appeared that cadmium did not induce cell-cycle arrest. The percentages of apoptotic cells did not differ in the two groups at 13, 16 or 19 hpf. At 28 hpf, however, a significantly higher percentage of apoptotic cells were found in the cadmium-treated group. Exposure to cadmium, therefore, induced ectopic apoptosis at 28 hpf without affecting the dynamics of apoptosis at earlier developmental stages. (orig.)

  19. Factors of importance when selecting sows as embryo donors.

    Science.gov (United States)

    Nohalez, A; Martinez, C A; Reixach, J; Diaz, M; Vila, J; Colina, I; Parrilla, I; Vazquez, J L; Roca, J; Gil, M A; Rodriguez-Martinez, H; Martinez, E A; Cuello, C

    2017-08-01

    The improvement in porcine embryo preservation and non-surgical embryo transfer (ET) procedures achieved in recent years represents essential progress for the practical use of ET in the pig industry. This study aimed to evaluate the effects of parity, weaning-to-estrus interval (WEI) and season on reproductive and embryonic parameters at day 6 after insemination of donor sows superovulated after weaning. The selection of donor sows was based on their reproductive history, body condition and parity. The effects of parity at weaning (2 to 3, 4 to 5 or 6 to 7 litters), season (fall, winter and spring), and WEI (estrus within 3 to 4 days), and their interactions on the number of corpus luteum, cysts in sows with cysts, number and quality of viable and transferable embryos, embryo developmental stage and recovery and fertilization rates were evaluated using linear mixed effects models. The analyses showed a lack of significant effects of parity, season, WEI or their interactions on any of the reproductive and embryonic parameters examined. In conclusion, these results demonstrate that fertilization rates and numbers of viable and transferable embryos collected at day 6 of the cycle from superovulated donor sows are not affected by their parity, regardless of the time of the year (from fall to spring) and WEI (3 or 4 days).

  20. Filial cannibalism improves survival and development of beaugregory damselfish embryos.

    Science.gov (United States)

    Payne, Adam G; Smith, Carl; Campbell, Andrew C

    2002-01-01

    Cannibalism of small numbers of offspring by a parent has been proposed as an adaptive parental strategy, by providing energy to support parental care. However, there are few empirical studies to support this hypothesis. We conducted field and laboratory experiments to investigate partial filial cannibalism in Stegastes leucostictus, a coral reef fish with paternal care. Partial cannibalism was shown to be common, and males were found to remove developing embryos from throughout a clutch in a random pattern, rather than in the more aggregated pattern seen during embryo predation. Males that received a diet supplement grew faster than control males, but did not engage in less cannibalism. Also, males did not concentrate cannibalism on early embryonic stages with the highest energetic value. Experimental reduction of embryo densities was found to significantly increase embryo development rate and survival from egg deposition to hatching, and experimental reduction of oxygen levels significantly increased rates of partial filial cannibalism by males. Artificial spawning sites with low oxygen levels were avoided by spawning females, and cannibalism rates by males were higher. We propose that partial filial cannibalism serves as an adaptive parental strategy to low oxygen levels in S. leucostictus by increasing the hatching success of embryos. PMID:12396483

  1. Effect of Semen Quality on the Embryo Development

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    To investigate the influences of sperm quality on the zygotes and embryos development,as the role of the paternal factor in early human embryogenesis is gaining more attention because of the application of techniques such as intracytoplasmic sperm injection (ICSI) for the treatment of men infertility, 136 infertility couples with men factors (Group Ⅰ ) were included from May 2002 to January 2004. One hundred and seventy two infertility couples with tube factors (Group Ⅱ ) served as controls. The sperm parameters, gemmates and embryos quality, implantation rate and pregnant rate in both groups were analyzed. It was found that there was no significant differences in the number of oocytes retrieved, the fertilization rate and number of embryos transferred between two groups. Sperm concentration, percentage of motile sperm and percentage of sperm with normal morphology were significantly lower in group Ⅰ than in group Ⅱ (P<0.01). The proportion of good quality zygotes and good quality embryos were significantly lower in the male infertility group than in the tubal disease group (P<0.05). Implantation rate and pregnancy rate were similar in two groups. It was concluded that spermatozoa is involved in the embryo quality, even in the early stages of development, which limited the treatment potency of IVF procedure.

  2. High frequency production of haploid embryos in asparagus anther culture.

    Science.gov (United States)

    Feng, X R; Wolyn, D J

    1991-12-01

    A method for obtaining a high frequency of haploid asparagus embryos through anther culture was developed. Flowers collected from plants in the field in July, August and September 1990, for the genotype G203, were stored at 5°C for 24 h. Anthers were placed on Murashige and Skoog medium (MS) containing 500 mg l (-1) casein hydrolysate, 800 mg l(-1) glutamine, 2 mg l (-1) NAA, 1 mg l (-1) BA and 5 % sucrose at 32 °C in the dark for three to four weeks to induce calli. Calli were then grown at 25 °C with a 16 h photoperiod for three to four weeks. Developing embryos and calli were transferred to embryo maturation medium, MS containing 6% sucrose, 0.1 mg l (-1) NAA, 0.1 mg l (-1) kinetin and 0.65 mg l (-1) ancymidol, for four weeks. More than 50% of the recovered mature embryos germinated on MS containing l mg l (-1) GA3. Anthers with microspores at the late-uninucleate stage had the highest frequency of total and embryogenic calli formation, 40% and 15%, respectively. Each embryogenic callus usually produced 10-15 embryos. Aproximately 75 plants per 100 anthers cultured were recovered: 76% haploid, 22% diploid and 2% triploid. High temperature was critical for the induction of embryogenic callus.

  3. Phaseolus immature embryo rescue technology.

    Science.gov (United States)

    Geerts, Pascal; Toussaint, André; Mergeai, Guy; Baudoin, Jean-Pierre

    2011-01-01

    Predominant among the production constraints of the common bean Phaseolus vulgaris are infestation of Ascochyta blight, Bean Golden Mosaic virus (BGMV), and Bean Fly. Interbreeding with Phaseolus -coccineus L. and/or Phaseolus polyanthus Greenm has been shown to provide P. vulgaris with greater resistance to these diseases. For interspecific crosses to be successful, it is important to use P. coccineus and P. polyanthus as female parents; this prevents rapid reversal to the recurrent parent P. vulgaris. Although incompatibility barriers are post-zygotic, early hybrid embryo abortion limits the success of F1 crosses. While rescue techniques for globular and early heart-shaped embryos have improved in recent years, -success in hybridization remains very low. In this study, we describe six steps that allowed us to rescue 2-day-old P. vulgaris embryos using a pod culture technique. Our methods consisted of (i) pod culture, (ii) extraction and culture of immature embryos, (iii) dehydration of embryos, (iv) germination of embryos, (v) rooting of developed shoots, and (vi) hardening of plantlets.

  4. Production of rhesus monkey cloned embryos expressing monomeric red fluorescent protein by interspecies somatic cell nuclear transfer

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Hai-Ying; Kang, Jin-Dan; Li, Suo; Jin, Jun-Xue; Hong, Yu; Jin, Long; Guo, Qing; Gao, Qing-Shan; Yan, Chang-Guo; Yin, Xi-Jun, E-mail: yinxj33@msn.com

    2014-02-21

    Highlights: • Rhesus monkey cells were electroporated with a plasmid containing mRFP1, and an mRFP1-expressing cell line was generated. • For the first time, mRFP1-expressing rhesus monkey cells were used as donor cells for iSCNT. • The effect of VPA on the development of embryos cloned using iSCNT was determined. - Abstract: Interspecies somatic cell nuclear transfer (iSCNT) is a promising method to clone endangered animals from which oocytes are difficult to obtain. Monomeric red fluorescent protein 1 (mRFP1) is an excellent selection marker for transgenically modified cloned embryos during somatic cell nuclear transfer (SCNT). In this study, mRFP-expressing rhesus monkey cells or porcine cells were transferred into enucleated porcine oocytes to generate iSCNT and SCNT embryos, respectively. The development of these embryos was studied in vitro. The percentage of embryos that underwent cleavage did not significantly differ between iSCNT and SCNT embryos (P > 0.05; 71.53% vs. 80.30%). However, significantly fewer iSCNT embryos than SCNT embryos reached the blastocyst stage (2.04% vs. 10.19%, P < 0.05). Valproic acid was used in an attempt to increase the percentage of iSCNT embryos that developed to the blastocyst stage. However, the percentages of embryos that underwent cleavage and reached the blastocyst stage were similar between untreated iSCNT embryos and iSCNT embryos treated with 2 mM valproic acid for 24 h (72.12% vs. 70.83% and 2.67% vs. 2.35%, respectively). These data suggest that porcine-rhesus monkey interspecies embryos can be generated that efficiently express mRFP1. However, a significantly lower proportion of iSCNT embryos than SCNT embryos reach the blastocyst stage. Valproic acid does not increase the percentage of porcine-rhesus monkey iSCNT embryos that reach the blastocyst stage. The mechanisms underling nuclear reprogramming and epigenetic modifications in iSCNT need to be investigated further.

  5. Embryo transfer day does not affect the initial maternal serum β-hCG levels: A retrospective cohort study.

    Science.gov (United States)

    Dahiya, Mona; Rupani, Karishma; Yu, Su Ling; Fook-Chong, Stephanie M C; Siew Fui, Diana Chia; Rajesh, Hemashree

    2017-05-01

    The aim of this study is to compare the serum β-hCG values post transfer of a cleavage stage embryo versus a blastocyst stage embryo at equal time intervals post oocyte retrieval (OR) in clinically pregnant patients, and to ascertain a β-hCG value to predict pregnancy outcomes. This is a retrospective cohort study of 560 women with clinical pregnancy who underwent an embryo transfer performed at either the cleavage stage or the blastocyst stage of embryo development between January 2003 and June 2014 at the Center for Assisted Reproduction (CARE), Singapore General Hospital. The serum β-hCG level was measured on day 17 post OR. The β-hCG values were not significantly different in the cleavage stage versus the blastocyst stage embryos (mean±SD: 387±486IU/L D3 vs. 352±268IU/L D5, p=0.96, median value 297 in both groups). Our study suggests that the initial maternal serum β-hCG values were not affected by the day of transfer of the embryos since assessing the β-hCG at equivalent points after transfer should not lead to a significant difference assuming the progress and development of the embryos occurred as expected. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Lessons from Embryos: Haeckel's Embryo Drawings, Evolution, and Secondary Biology Textbooks

    Science.gov (United States)

    Wellner, Karen L.

    2014-01-01

    In 1997, developmental biologist Michael Richardson compared his research team's embryo photographs to Ernst Haeckel's 1874 embryo drawings and called Haeckel's work "noncredible". "Science" soon published "Haeckel's Embryos: Fraud Rediscovered," and Richardson's comments further reinvigorated criticism of Haeckel by…

  7. Lessons from Embryos: Haeckel's Embryo Drawings, Evolution, and Secondary Biology Textbooks

    Science.gov (United States)

    Wellner, Karen L.

    2014-01-01

    In 1997, developmental biologist Michael Richardson compared his research team's embryo photographs to Ernst Haeckel's 1874 embryo drawings and called Haeckel's work "noncredible". "Science" soon published "Haeckel's Embryos: Fraud Rediscovered," and Richardson's comments further reinvigorated criticism of Haeckel by…

  8. Principles guiding embryo selection following genome-wide haplotyping of preimplantation embryos.

    Science.gov (United States)

    Dimitriadou, Eftychia; Melotte, Cindy; Debrock, Sophie; Esteki, Masoud Zamani; Dierickx, Kris; Voet, Thierry; Devriendt, Koen; de Ravel, Thomy; Legius, Eric; Peeraer, Karen; Meuleman, Christel; Vermeesch, Joris Robert

    2017-03-01

    How to select and prioritize embryos during PGD following genome-wide haplotyping? In addition to genetic disease-specific information, the embryo selected for transfer is based on ranking criteria including the existence of mitotic and/or meiotic aneuploidies, but not carriership of mutations causing recessive disorders. Embryo selection for monogenic diseases has been mainly performed using targeted disease-specific assays. Recently, these targeted approaches are being complemented by generic genome-wide genetic analysis methods such as karyomapping or haplarithmisis, which are based on genomic haplotype reconstruction of cell(s) biopsied from embryos. This provides not only information about the inheritance of Mendelian disease alleles but also about numerical and structural chromosome anomalies and haplotypes genome-wide. Reflections on how to use this information in the diagnostic laboratory are lacking. We present the results of the first 101 PGD cycles (373 embryos) using haplarithmisis, performed in the Centre for Human Genetics, UZ Leuven. The questions raised were addressed by a multidisciplinary team of clinical geneticist, fertility specialists and ethicists. Sixty-three couples enrolled in the genome-wide haplotyping-based PGD program. Families presented with either inherited genetic variants causing known disorders and/or chromosomal rearrangements that could lead to unbalanced translocations in the offspring. Embryos were selected based on the absence or presence of the disease allele, a trisomy or other chromosomal abnormality leading to known developmental disorders. In addition, morphologically normal Day 5 embryos were prioritized for transfer based on the presence of other chromosomal imbalances and/or carrier information. Some of the choices made and principles put forward are specific for cleavage-stage-based genetic testing. The proposed guidelines are subject to continuous update based on the accumulating knowledge from the implementation of

  9. Transcriptomic Analysis of the Porcine Endometrium during Embryo Implantation

    Directory of Open Access Journals (Sweden)

    Haichao Lin

    2015-12-01

    Full Text Available In pigs, successful embryo implantation is an important guarantee for producing litter size, and early embryonic loss occurring on day 12–30 of gestation critically affects the potential litter size. The implantation process is regulated by the expression of numerous genes, so comprehensive analysis of the endometrium is necessary. In this study, RNA sequencing (RNA-Seq technology is used to analyze endometrial tissues during early pregnancy. We investigated the changes of gene expression between three stages (day 12, 18, and 25 by multiple comparisons. There were 1557, 8951, and 2345 differentially expressed genes (DEGs revealed between the different periods of implantation. We selected several genes for validation by the use of quantitative real-time RT-PCR. Bioinformatic analysis of differentially expressed genes in the endometrium revealed a number of biological processes and pathways potentially involved in embryo implantation in the pig, most noticeably cell proliferation, regulation of immune response, interaction of cytokine-cytokine receptors, and cell adhesion. These results showed that specific gene expression patterns reflect the different functions of the endometrium in three stages (maternal recognition, conceptus attachment, and embryo implantation. This study identified comprehensive transcriptomic profile in the porcine endometrium and thus could be a foundation for targeted studies of genes and pathways potentially involved in abnormal endometrial receptivity and embryo loss in early pregnancy.

  10. Effect of Different High CO2 Concentrations on the Development of 2-cell Mouse Embryos in vitro

    Institute of Scientific and Technical Information of China (English)

    Li-hua LU; Wei-jie ZHU

    2003-01-01

    Objective To investigate effects of different high CO2 concentrations on the development of 2-cell mouse embryos in vitroMethods At levels of 5% CO2 (control group), 5.7% CO2, 6.0% CO2 and 15% CO2, embryos were incubated in drops with CZB medium, respectively, and the drops were covered by paraffin oil which was treated with three-distilled water. In addition, at the level of 15% CO2, there were another two groups, in which paraffin oil was treated with phosphate-buffered saline (PBS) solution or the drops were uncovered. The development of embryos in all stages was noted.Results The developmental rates of blastocysts in five experimental groups were significantly lower than that of the control group (P0.05). At the level of 15% CO2, 15% embryos developed in the 4-cell stage with irregular blastomere and degenerated quickly in the group which paraffin oil was treated with distilled water; 2.2% embryos developed in the 4-cell stage in the group which paraffin oil was treated with PBS and the rest stagnated in the 2-cell stage. Conclusions High CO2 concentrations had toxic effect on the in vitro development of 2-cell mouse embryos, and was responsible for the inhibition of the embryos. It is important for the development of embryos in vitro to detect strictly CO2 concentration.

  11. TERATOGENIC EFFECTS OF TRANSPLACENTAL TRANSFUSION OF HETEROLOGOUS ANTISERA SIMULATED IN AN EXPERIMENTAL-MODEL USING INVITRO WHOLE RAT EMBRYO CULTURE

    NARCIS (Netherlands)

    VANDERZEE, DC; POELMANN, RE; ZWIERSTRA, RP; MENTINK, MMT; VERMEIJKEERS, C

    1991-01-01

    The effects of the transplacental transfusion of heterologous rabbit-anti-rat antiserum (RAR antiserum) and subsequent immunological interaction on the development of 9-10 days old rat embryos (stages 8-10 somites) were studied using an in vitro whole rat embryo culture. Transplacental transfusion w

  12. Effects of EGF or bFGF on the development of porcine parthenogenetic embryos in vitro

    Institute of Scientific and Technical Information of China (English)

    Ji LIU; Shutang FENG; Dengke PAN; Liguo GONG; Li ZHANG; Yulian MU; Zirong WANG

    2008-01-01

    Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) were added into the cul-ture medium in different culturing stages. The effects of EGF or bFGF on the development of porcine partheno-genetic embryos were studied in vitro. The results were as follows: The addition of EGF significantly enhanced the cleavage rate of porcine parthenogenetic embryos (P<0.05). The addition of EGF or bFGF also signifi-cantly enhanced the rate of blastocysts formation of 2-4-cell porcine parthenogenetic embryos (P<0.05). Additionally, the group of bFGF had more numbers of blastocysts and higher rates of blastocysts formation than the groups of EGF and the control. In conclusion, EGF and bFGF were found propitious to the development of porcine parthenogenetic embryos in vitro, and bFGF increased the quality of blastocysts by increasing the total cell number in porcine parthenogenetic embryos.

  13. Post-implantation embryo culture: validation with selected compounds for teratogenicity testing.

    Science.gov (United States)

    Cicurel, L; Schmid, B P

    1988-06-01

    1. Some chemical compounds selected by experts for the validation of in vitro teratogenicity testing were investigated in whole rat embryos cultured during the early stages of organogenesis. All sixteen known in vivo teratogens tested also induced specific malformations in embryos grown in culture. 2. Of the nine compounds which were negative in in vivo rat teratogenicity studies, none provoked dysmorphogenic effects in cultured embryos. Abnormal development of the embryos was only observed with these compounds at concentrations also high enough to affect significantly overall growth and/or differentiation. 3. The results showed a high predictability of this system for the compounds tested and suggest that the post-implantation embryo culture system may also be useful in the prospective testing of new drugs and environmental chemicals.

  14. Somatic Embryogenesis and Plant Regeneration from Cultured Immature Embryos of Rye (Secale cereale L.).

    Science.gov (United States)

    Lu, C Y; Chandler, S F; Vasil, I K

    1984-07-01

    Somatic embryogenesis was initiated in immature embryos of rye (Secale cereale L.) cultured on Murashige and Skoog's (1962) medium supplemented with various concentrations of 2,4-D and sucrose. The developmental stage of the embryo (optimal embryo length 0.5-2.0 mm) and concentration of 2,4-D (optimal concentration 2.5 mg · l(-1)) were found to be critical in determining embryo response. The phenoxyacetic acids, 2,4-D, 2,4,5-T, and MCPA were more effective in initiating embryogenic callus and embryoid formation than other auxins (NAA, IBA, IAA, IPA). Embryogenic callus and embryoid production was greater in embryos cultured scutellum up, and more embryoids were formed in the dark than in the light. Embryoids germinated after transfer to basal medium with/without cytokinin or GA3. The regenerated plants were grown to maturity in soil and were shown to have the normal diploid chromosome number of 14.

  15. A genetic component of resistance to fungal infection in frog embryos.

    Science.gov (United States)

    Sagvik, Jörgen; Uller, Tobias; Olsson, Mats

    2008-06-22

    The embryo has traditionally been considered to completely rely upon parental strategies to prevent threats to survival posed by predators and pathogens, such as fungi. However, recent evidence suggests that embryos may have hitherto neglected abilities to counter pathogens. Using artificial fertilization, we show that among-family variation in the number of Saprolegnia-infected eggs and embryos in the moor frog, Rana arvalis, cannot be explained by maternal effects. However, analysed as a within-females effect, sire identity had an effect on the degree of infection. Furthermore, relatively more eggs and embryos were infected when eggs were fertilized by sperm from the same, compared with a different, population. These effects were independent of variation in fertilization success. Thus, there is likely to be a significant genetic component in embryonic resistance to fungal infection in frog embryos. Early developmental stages may show more diverse defences against pathogens than has previously been acknowledged.

  16. EGF increases expression and activity of PAs in preimplantation rat embryos and their implantation rate

    Directory of Open Access Journals (Sweden)

    Har-Vardi Iris

    2007-01-01

    Full Text Available Abstract Background Embryo implantation plays a major role in embryogenesis and the outcome of pregnancy. Plasminogen activators (PAs have been implicated in mammalian fertilization, early stages of development and embryo implantation. As in-vitro developing embryos resulted in lower implantation rate than those developed in-vivo we assume that a reduced PAs activity may be involved. In the present work we studied the effect of EGF on PAs activity, quantity and embryo implantation. Methods Zygotes were flushed from rat oviducts on day one of pregnancy and grown in-vitro in R1ECM supplemented with EGF (10 ng/ml and were grown up to the blastocyst stage. The control groups were grown in the same medium without EGF. The distribution and quantity of the PAs were examined using fluorescence immunohistochemistry followed by measurement of PAs activity using the chromogenic assay. Implantation rate was studied using the embryo donation model. Results PAs distribution in the embryos was the same in EGF treated and untreated embryos. Both PAs were localized in the blastocysts' trophectoderm, supporting the assumption that PAs play a role in the implantation process in rats. EGF increased the quantity of uPA at all stages studied but the 8-cell stage as compared with controls. The tissue type PA (tPA content was unaffected except the 8-cell stage, which was increased. The activity of uPA increased gradually towards the blastocyst stage and more so due to the presence of EGF. The activity of tPA did not vary with the advancing developmental stages although it was also increased by EGF. The presence of EGF during the preimplantation development doubled the rate of implantation of the treated group as compared with controls.

  17. Splitting of IVP bovine blastocyst affects morphology and gene expression of resulting demi-embryos during in vitro culture and in vivo elongation.

    Science.gov (United States)

    Velasquez, Alejandra E; Castro, Fidel O; Veraguas, Daniel; Cox, Jose F; Lara, Evelyn; Briones, Mario; Rodriguez-Alvarez, Lleretny

    2016-02-01

    Embryo splitting might be used to increase offspring yield and for molecular analysis of embryo competence. How splitting affects developmental potential of embryos is unknown. This research aimed to study the effect of bovine blastocyst splitting on morphological and gene expression homogeneity of demi-embryos and on embryo competence during elongation. Grade I bovine blastocyst produced in vitro were split into halves and distributed in nine groups (3 × 3 setting according to age and stage before splitting; age: days 7-9; stage: early, expanded and hatched blastocysts). Homogeneity and survival rate in vitro after splitting (12 h, days 10 and 13) and the effect of splitting on embryo development at elongation after embryo transfer (day 17) were assessed morphologically and by RT-qPCR. The genes analysed were OCT4, SOX2, NANOG, CDX2, TP1, TKDP1, EOMES, and BAX. Approximately 90% of split embryos had a well conserved defined inner cell mass (ICM), 70% of the halves had similar size with no differences in gene expression 12 h after splitting. Split embryos cultured further conserved normal and comparable morphology at day 10 of development; this situation changes at day 13 when embryo morphology and gene expression differed markedly among demi-embryos. Split and non-split blastocysts were transferred to recipient cows and were recovered at day 17. Fifty per cent of non-split embryos were larger than 100 mm (33% for split embryos). OCT4, SOX2, TP1 and EOMES levels were down-regulated in elongated embryos derived from split blastocysts. In conclusion, splitting day-8 blastocysts yields homogenous demi-embryos in terms of developmental capability and gene expression, but the initiation of the filamentous stage seems to be affected by the splitting.

  18. Embryo density may affect embryo quality during in vitro culture in a microwell group culture dish.

    Science.gov (United States)

    Lehner, Adam; Kaszas, Zita; Murber, Akos; Rigo, Janos; Urbancsek, Janos; Fancsovits, Peter

    2017-08-01

    Culturing embryos in groups is a common practice in mammalian embryology. Since the introduction of different microwell dishes, it is possible to identify oocytes or embryos individually. As embryo density (embryo-to-volume ratio) may affect the development and viability of the embryos, the purpose of this study was to assess the effect of different embryo densities on embryo quality. Data of 1337 embryos from 228 in vitro fertilization treatment cycles were retrospectively analyzed. Embryos were cultured in a 25 μl microdrop in a microwell group culture dish containing 9 microwells. Three density groups were defined: Group 1 with 2-4 (6.3-12.5 μl/embryo), Group 2 with 5-6 (4.2-5.0 μl/embryo), and Group 3 with 7-9 (2.8-3.6 μl/embryo) embryos. Proportion of good quality embryos was higher in Group 2 on both days (D2: 18.9 vs. 31.5 vs. 24.7%; p Culturing 5-6 embryos together in a culture volume of 25 μl may benefit embryo quality. As low egg number, position, and distance of the embryos may influence embryo quality, results should be interpreted with caution.

  19. In Vitro Embryo Production and Transfer of Bubaline Embryos using Oocytes Derived from Transvaginal Ultrasound-Guided Follicular Aspiration (TUFA

    Directory of Open Access Journals (Sweden)

    FP Aquino

    2014-06-01

    Full Text Available Transvaginal ultrasound-guided follicular aspiration (TUFA has become a popular tool for embryo production in vitro due to its high degree of repeatability in terms of recovering oocytes from live animals. In Study 1, the quantity and quality of oocytes from Bulgarian Murrah buffalo cows (n=10 of varying ages (Group 1, 8-12; and Group 2, 13-17 years were assessed. Group 1 buffalo donor cows yielded significantly higher (P<0.05 number of oocytes vs Group 2 buffalo donor cows (71 vs 29 oocytes, respectively, though in terms of oocyte quality, no difference was observed. In Study 2, oocytes collected (n=100 in Study 1 were matured, fertilized in vitro and the resulting zygotes were cultured which developed to blastocyst stage embryos. The maturation, fertilization and blastocyst development rates obtained were 53.0%, 40.0% and 32.5%, respectively. In Study 3, the viability of resulting blastocyst stage embryos was determined by transferring to recipient cows. Of 10 recipients 1 got pregnant and delivered a 35 kg male calf after 310 days gestation period. Overall, the results of the studies conducted demonstrated the potential of TUFA technology in the in vitro production of embryos which eventually could be used in the production of live offspring.

  20. Localization of parathyroid hormone-related protein in the preimplantation mouse embryo is associated with events of blastocyst hatching.

    Science.gov (United States)

    Erbach, Gregory T; Biggers, John D; Manning, Peter C; Nowak, Romana A

    2013-08-01

    To determine the pattern of expression of parathyroid hormone-related protein (PTHrP) and its receptor, parathyroid hormone receptor 1 (PTHR1), in mouse embryos in different stages of preimplantation development. Embryos were cultured from the pronuclear zygote stage and harvested as 2-cell, 4-cell and 8-cell embryos, morulae and blastocysts. RT-PCR was carried out on mRNAs of these and of trophoblast outgrowths for detection of PTHrP and PTHR1. Whole mounted embryos intact or stripped of zonae pellucidae were immunofluorescently stained for PTHrP and PTH receptor and observed with confocal microscopy. PTHrP mRNA was present in the pronuclear zygote, not present in 2-cell, 4-cell and uncompacted 8-cell embryos, present in the 8-cell compacting embryo, and not detected in 16-cell morulae or blastocysts. The mRNA was present in trophoblasts growing on fibronectin beds. mRNA for PTHR1 was detected in the pronuclear zygote, then undetected until the compacted 8-cell stage and thereafter. PTH receptor protein was observed in 2-cell embryos, morulae and in the inner cell mass and trophectoderm of blastocysts. PTHrP was observed dispersed in the cytoplasm of 2-cell, 4-cell and uncompacted 8-cell embryos, and in distinct foci near the nuclei of morulae. In blastocysts, PTHrP appeared on the apical surface of only trophoblast cells which had extruded from the zona pellucida. Fully hatched blastocysts expressed the protein on the apical side of all trophoblasts. When morulae were prematurely stripped of their zonae, PTHrP was observed on the embryos' outer surface. PTHrP protein is expressed throughout early embryo development, and its receptor PTHR1 is expressed from the morula stage. Embryo hatching is associated with translocation of PTHrP to the apical plasma membrane of trophoblasts. PTHrP may thus have autocrine effects on the developing blastocyst.

  1. Tracking individual nanodiamonds in Drosophila melanogaster embryos

    CERN Document Server

    Simpson, David A; Kowarsky, Mark; Zeeshan, Nida F; Barson, Michael S J; Hall, Liam; Yan, Yan; Kaufmann, Stefan; Johnson, Brett C; Ohshima, Takeshi; Caruso, Frank; Scholten, Robert; Saint, Robert B; Murray, Michael J; Hollenberg, Lloyd C L

    2013-01-01

    Tracking the dynamics of fluorescent nanoparticles during embryonic development allows insights into the physical state of the embryo and, potentially, molecular processes governing developmental mechanisms. In this work, we investigate the motion of individual fluorescent nanodiamonds micro-injected into Drosophila melanogaster embryos prior to cellularisation. Fluorescence correlation spectroscopy and wide-field imaging techniques are applied to individual fluorescent nanodiamonds in blastoderm cells during stage 5 of development to a depth of ~40 \\mu m. The majority of nanodiamonds in the blastoderm cells during cellularisation exhibit free diffusion with an average diffusion coefficient of (6 $\\pm$ 3) x 10$^{-3}$ \\mu m$^2$/s, (mean $\\pm$ SD). Driven motion in the blastoderm cells was also observed with an average velocity of 0.13 $\\pm$ 0.10 \\mu m/s (mean $\\pm$ SD) \\mu m/s and an average applied force of 0.07 $\\pm$ 0.05 pN (mean $\\pm$ SD). Nanodiamonds in the periplasm between the nuclei and yolk were also...

  2. Somatic embryogenesis and plant regeneration from immature embryo explant of papaya (Carica papaya L. cv. washington and honey dew).

    Science.gov (United States)

    Bhattacharya, J; Khuspe, S S; Renukdas, N N; Rawal, S K

    2002-05-01

    Immature zygotic embryo explants of Carica papaya were cultured on MS medium supplemented with 2,4-D (2.0 mg/l) and formed globular embryos on explants without callus formation in 4-6 weeks. Maturation and conversion of somatic embryos was also achieved on the same medium. Cotyledonary stage embryos germinated to 63.66 and 68.33% in cv. honey dew and washington respectively in MS basal medium supplemented ABA (0.5 microm/l). Robust development and proliferation of plantlet roots in vitro was obtained on MS basal medium. Hardened plantlets have 60% survival rate.

  3. A chimera embryo assay reveals a decrease in embryonic cellular proliferation induced by sperm from X-irradiated male mice

    Energy Technology Data Exchange (ETDEWEB)

    Obasaju, M.F.; Wiley, L.M.; Oudiz, D.J.; Raabe, O.; Overstreet, J.W.

    1989-05-01

    Male mice were divided into three experimental groups and a control group. Mice in the experimental groups received one of three doses of acute X irradiation (1.73, 0.29, and 0.05 Gy) and together with the control unirradiated mice were then mated weekly to unirradiated female mice for a 9-week experimental period. Embryos were recovered from the weekly matings at the four-cell stage and examined by the chimera assay for proliferative disadvantage. Aggregation chimeras were constructed of embryos from female mice mated to irradiated males (experimental embryos) and embryos from females mated to unexposed males (control embryos) and contained either one experimental embryo and one control embryo (heterologous chimera) or two control embryos (control chimera). The control embryo in heterologous chimeras and either embryo in control chimeras were prelabeled with the vital dye fluorescein isothiocyanate (FITC), and the chimeras were cultured for 40 h and viewed under phase-contrast and epifluorescence microscopy to obtain total embryo cell number and the cellular contribution from the FITC-labeled embryo. Experimental and control embryos that were cultured singly were also examined for embryo cell number at the end of the 40-h culture period. In control chimeras, the mean ratio of the unlabeled cells:total chimera cell number (henceforth referred to as ''mean ratio'') was 0.50 with little or no weekly variation over the 9-week experimental period. During Weeks 4-7, the mean ratios of heterologous chimeras differed significantly from the mean ratio of control chimeras with the greatest differences occurring during Week 7 (0.41 for chimeras of 0.05 Gy dose group, 0.40 for chimeras of the 0.29 Gy dose group, and 0.17 for chimeras of the 1.73 Gy dose group).

  4. What is the preimplantation embryo?

    Science.gov (United States)

    Krones, Tanja; Schlüter, Elmar; Neuwohner, Elke; El Ansari, Susan; Wissner, Thomas; Richter, Gerd

    2006-07-01

    We present results from our 'bioethical field studies', which explore and compare the views of experts, patients and the general public on the beginning of human life and the status of the preimplantation embryo in Germany. Using a qualitative and quantitative multi-method approach, we found crucial differences in the categorization of the beginning of human life within the expert group (representative samples of human geneticists n=104, ethicists n=168, midwives n=294, obstetricians n=147, paediatricians n=166), and between expert and lay samples (IVF couples n=108, high genetic risk couples n=324, general population n=1017). The majority of lay respondents as well as paediatricians and obstetricians chose nidation, the moment when the implantation of the fertilized egg into the uterus takes place, as the crucial boundary that marks the beginning of human life, whereas the majority of (female) human geneticists, ethicists and midwives voted for conception as the decisive point in time. The views of all groups on the status of the preimplantation embryo differed from the assumptions underlying German legislation (Embryo Protection Act). Religiousness and religious affiliation, gender, attitudes towards disabled people, post-material values and a present desire for a child were identified as independent factors influencing attitudes towards the preimplantation embryo in the population sample. The results are discussed within a broader philosophical and social science perspective of constructivism versus essentialism, proposing a truly interdisciplinary approach to such bioethical core issues as new reproductive technologies and the status of the preimplantation embryo.

  5. Differences in heat tolerance between preimplantation embryos from Brahman, Romosinuano, and Angus breeds.

    Science.gov (United States)

    Hernández-Cerón, J; Chase, C C; Hansen, P J

    2004-01-01

    Exposure to 41 degrees C reduces development of embryos of heat-sensitive breeds (Holstein and Angus) more than for embryos of the heat-tolerant Brahman breed. Here it was tested whether embryonic resistance to heat shock occurs for a thermotolerant breed of different genetic origin than the Brahman. In particular, the thermal sensitivity of in vitro produced embryos of the Romosinuano, a Bos taurus, Criollo-derived breed, was compared to that for in vitro produced Brahman and Angus embryos. At d 4 after insemination, embryos > or = 8 cells were randomly assigned to control (38.5 degrees C) or heat shock (41 degrees C for 6 h) treatments. Heat shock reduced the proportion of embryos that developed to the blastocyst stage on d 8 after insemination. At 38.5 degrees C, there were no significant differences in development between breeds. Among embryos exposed to 41 degrees C, however, development was lower for Angus embryos than for Brahman and Romosinuano embryos. Furthermore, an Angus vs. (Brahman + Romosinuano) x temperature interaction occurred because heat shock reduced development more in Angus (30.3 +/- 4.6% at 38.5 degrees C vs. 4.9 +/- 4.6% at 41 degrees C) than in Brahman (25.1 +/- 4.6% vs. 13.6 +/- 4.6%) and Romosinuano (28.3 +/- 4.1% vs. 17.5 +/- 4.1%). Results demonstrate that embryos from Brahman and Romosinuano breeds are more resistant to elevated temperature than embryos from Angus. Thus, the process of adaptation of Brahman and Romosinuano breeds to hot environments resulted in both cases in selection of genes controlling thermotolerance at the cellular level.

  6. Analysis of the sex-chromosome constitution of digynic triploid mouse embryos.

    Science.gov (United States)

    Speirs, S; Kaufman, M H

    1989-01-01

    LT/Sv strain mice regularly ovulate up to 50% of their eggs as primary oocytes, which are fertilisable and give rise to digynic triploid embryos. A similar number of eggs are ovulated as secondary oocytes and, following fertilisation, give rise to normal diploid embryos. Pregnant LT/Sv females were autopsied at about midday on day 10 of gestation, when normal diploid embryos would be expected to possess between 25 and 30 pairs of somites. While a few of the triploid embryos either consisted of disorganised embryonic masses or were resorbing, most were at readily recognisable embryonic stages. Just over half of the embryos recovered were "unturned," while the remainder had "turned" and possessed between 15 and 25 pairs of somites. The triploids were usually readily recognised, owing to their small size and because they often displayed neural tube and cardiac defects. All of the embryos recovered were analysed cytogenetically by G-banding to establish their ploidy and sex-chromosome constitution. The XY:XX sex ratio of the 105 diploid embryos recovered, all of which had "turned," was 1.06:1, while the overall XXY:XXX sex ratio of the 120 triploids was 1:1. Analysis of only the developmentally most advanced triploid embryos (i.e., the 49 that had "turned") revealed that the XXY:XXX sex ratio in this group was 1.13:1, which was not significantly different from the expected ratio of 1:1. The crown-rump lengths of the XY and XX "turned" embryos were almost identical, as were those of the XXY and XXX "turned" embryos, although the triploids were significantly smaller than the diploids. No obvious effect of sex-chromosome constitution on developmental potential was therefore observed in this study in relation to either the digynic triploid or the control diploid embryos.

  7. A Simple Method for Transportation of Mouse Embryos Using Microtubes and a Warm Box.

    Directory of Open Access Journals (Sweden)

    Mikiko Tokoro

    Full Text Available Generally, transportation of preimplantation embryos without freezing requires incubators that can maintain an optimal culture environment with a suitable gas phase, temperature, and humidity. Such incubators are expensive to transport. We reported previously that normal offspring were obtained when the gas phase and temperature could be maintained during transportation. However, that system used plastic dishes for embryo culture and is unsuitable for long-distance transport of live embryos. Here, we developed a simple low-cost embryo transportation system. Instead of plastic dishes, several types of microtubes-usually used for molecular analysis-were tested for embryo culture. When they were washed and attached to a gas-permeable film, the rate of embryo development from the 1-cell to blastocyst stage was more than 90%. The quality of these blastocysts and the rate of full-term development after embryo transfer to recipient female mice were similar to those of a dish-cultured control group. Next, we developed a small warm box powered by a battery instead of mains power, which could maintain an optimal temperature for embryo development during transport. When 1-cell embryos derived from BDF1, C57BL/6, C3H/He and ICR mouse strains were transported by a parcel-delivery service over 3 days using microtubes and the box, they developed to blastocysts with rates similar to controls. After the embryos had been transferred into recipient female mice, healthy offspring were obtained without any losses except for the C3H/He strain. Thus, transport of mouse embryos is possible using this very simple method, which might prove useful in the field of reproductive medicine.

  8. A Simple Method for Transportation of Mouse Embryos Using Microtubes and a Warm Box.

    Science.gov (United States)

    Tokoro, Mikiko; Fukunaga, Noritaka; Yamanaka, Kaori; Itoi, Fumiaki; Terashita, Yukari; Kamada, Yuko; Wakayama, Sayaka; Asada, Yoshimasa; Wakayama, Teruhiko

    2015-01-01

    Generally, transportation of preimplantation embryos without freezing requires incubators that can maintain an optimal culture environment with a suitable gas phase, temperature, and humidity. Such incubators are expensive to transport. We reported previously that normal offspring were obtained when the gas phase and temperature could be maintained during transportation. However, that system used plastic dishes for embryo culture and is unsuitable for long-distance transport of live embryos. Here, we developed a simple low-cost embryo transportation system. Instead of plastic dishes, several types of microtubes-usually used for molecular analysis-were tested for embryo culture. When they were washed and attached to a gas-permeable film, the rate of embryo development from the 1-cell to blastocyst stage was more than 90%. The quality of these blastocysts and the rate of full-term development after embryo transfer to recipient female mice were similar to those of a dish-cultured control group. Next, we developed a small warm box powered by a battery instead of mains power, which could maintain an optimal temperature for embryo development during transport. When 1-cell embryos derived from BDF1, C57BL/6, C3H/He and ICR mouse strains were transported by a parcel-delivery service over 3 days using microtubes and the box, they developed to blastocysts with rates similar to controls. After the embryos had been transferred into recipient female mice, healthy offspring were obtained without any losses except for the C3H/He strain. Thus, transport of mouse embryos is possible using this very simple method, which might prove useful in the field of reproductive medicine.

  9. Effect of the P1 Medium and the ECM Medium on Embryo Quality in IVF

    Institute of Scientific and Technical Information of China (English)

    Qian CHEN; Ai-jun ZHANG; Yun FENG; Xiao-wei LU; Dong-mei JI; Zhi-peng XU

    2009-01-01

    Objective To investigate the effect of the glucose-free reimplantation stage one(P1) medium and the ECM medium on embryo development quality in IVF.Methods The patients with ≥4 zygotes of 2PN were studied.A total of 201 retrieval cycles were included in a prospective randomized study.Each patient was herself control Half of zygotes of 2PN were transferred into ECM medium(group A)and half into P1 medium(group B)for further culture.Embryo development was evaluated on the day of embryo transfer.The efficacy of ECM was compared with P1 as culture medium for the development of preimplantation embryos. Results No statistically significant differences were noted between the two groups regarding embryo-cleavage rate(97.13% vs 97.55%)and rate of normal-cleaving embryos(58.29% and 58.37%).The rate of top-quality embryos was statistically higher in group A than in group B(27.59% vs 19.75%,P<0.05).Embryo quality,as assessed by morphological parameters(the amount of detached anuclear fragments>30%),was better in group A than in group B(19.86% vs 21.75%),however,there was no statistically significance.Both the rate of good-quality embryos(47.95% vs 46.17%)and available embryos(63.22% vs 61.,9%)were higher in group A than in group B,but there was also no statistically significance.Conclusion The ECM medium may be associated with a better embryo quality compared with the P1 medium.

  10. Dynamic Properties of Electrotonic Coupling between Cells of Early Xenopus Embryos.

    Science.gov (United States)

    Dicaprio, R A; French, A S; Sanders, E J

    1974-05-01

    Frequency response functions were measured between the cells of Xenopus laevis embryos during the first two cleavage stages. Linear systems theory was then used to produce electronic models which account for the electrical behavior of the systems. Coupling between the cells may be explained by models which have simple resistive elements joining each cell to its neighbors. The vitelline, or fertilization, membrane which surrounds the embryos has no detectable resistance to the passage of electric current. The electrical properties of the four-cell embryo can only be explained by the existence of individual junctions linking each pair of cells. This arrangement suggests that electrotonic coupling is important in the development of the embryos, at least until the four-cell stage.

  11. Altered cleavage patterns in human tripronuclear embryos and their association to fertilization method

    DEFF Research Database (Denmark)

    Joergensen, Mette Warming; Agerholm, Inge; Hindkjaer, Johnny

    2014-01-01

    PURPOSE: To analyze the cleavage patterns in dipronuclear (2PN) and tripronuclear (3PN) embryos in relation to fertilization method. METHOD: Time-lapse analysis. RESULTS: Compared to 2PN, more 3PN IVF embryos displayed early cleavage into 3 cells (p cell...... stage (p cell divisions within the cleavage cycles differed between the two groups. In contrast......, the completion of the 1st, 2nd, and 3rd cleavage cycle was delayed, but with a similar division pattern for 3PN ICSI compared with the 2PN ICSI embryos. 3PN, more often than 2PN ICSI embryos, displayed early cleavage into 3 cells (p = 0.03) and arrested development from the compaction stage and onwards (p = 0...

  12. Comparative study of Msx-1 expression in early normal and vitamin A-deficient avian embryos.

    Science.gov (United States)

    Chen, Y; Kostetskii, I; Zile, M H; Solursh, M

    1995-07-01

    Homeobox-containing genes may play an important role in establishing embryonic patterns during development of vertebrates. Retinoic acid is able to induce expression of Hox genes in cells in culture and to alter expression patterns in the developing vertebrate embryos. Using wholemount in situ hybridization, we have examined and compared the expression patterns of a homeobox-containing gene, Msx-1, in early normal and vitamin A-deficient quail embryos. At gastrulation stage, Msx-1 is primarily expressed in the posterior half of both normal and vitamin A-deficient embryos. However, the gene is expressed wider and stronger in the vitamin A-deficient embryos. At neurulation stages, Msx-1 is continuously expressed in the posterior region up to Hensen's node and in the edge of the neural fold in both normal and vitamin A-deficient embryos. Notably, in the vitamin A-deficient embryos, Msx-1 is expressed more strongly and is also expressed ectopically in the anterior and precardiac regions. These results provide evidence that endogenous retinoids are involved in the normal expression of Msx-1 in avian embryo and that the expression of Msx-1 is downregulated by endogenous and physiological retinoids in vivo during early avian embryogenesis.

  13. Improved Method for Ex Ovo-Cultivation of Developing Chicken Embryos for Human Stem Cell Xenografts

    Directory of Open Access Journals (Sweden)

    Timo Schomann

    2013-01-01

    Full Text Available The characterization of human stem cells for the usability in regenerative medicine is particularly based on investigations regarding their differentiation potential in vivo. In this regard, the chicken embryo model represents an ideal model organism. However, the access to the chicken embryo is only achievable by windowing the eggshell resulting in limited visibility and accessibility in subsequent experiments. On the contrary, ex ovo-culture systems avoid such negative side effects. Here, we present an improved ex ovo-cultivation method enabling the embryos to survive 13 days in vitro. Optimized cultivation of chicken embryos resulted in a normal development regarding their size and weight. Our ex ovo-approach closely resembles the development of chicken embryos in ovo, as demonstrated by properly developed nervous system, bones, and cartilage at expected time points. Finally, we investigated the usability of our method for trans-species transplantation of adult stem cells by injecting human neural crest-derived stem cells into late Hamburger and Hamilton stages (HH26–HH28/E5—E6 of ex ovo-incubated embryos. We demonstrated the integration of human cells allowing experimentally easy investigation of the differentiation potential in the proper developmental context. Taken together, this ex ovo-method supports the prolonged cultivation of properly developing chicken embryos enabling integration studies of xenografted mammalian stem cells at late developmental stages.

  14. Automated microinjection of recombinant BCL-X into mouse zygotes enhances embryo development.

    Directory of Open Access Journals (Sweden)

    Xinyu Liu

    Full Text Available Progression of fertilized mammalian oocytes through cleavage, blastocyst formation and implantation depends on successful implementation of the developmental program, which becomes established during oogenesis. The identification of ooplasmic factors, which are responsible for successful embryo development, is thus crucial in designing possible molecular therapies for infertility intervention. However, systematic evaluation of molecular targets has been hampered by the lack of techniques for efficient delivery of molecules into embryos. We have developed an automated robotic microinjection system for delivering cell impermeable compounds into preimplantation embryos with a high post-injection survival rate. In this paper, we report the performance of the system on microinjection of mouse embryos. Furthermore, using this system we provide the first evidence that recombinant BCL-XL (recBCL-XL protein is effective in preventing early embryo arrest imposed by suboptimal culture environment. We demonstrate that microinjection of recBCL-XL protein into early-stage embryos repairs mitochondrial bioenergetics, prevents reactive oxygen species (ROS accumulation, and enhances preimplantation embryo development. This approach may lead to a possible treatment option for patients with repeated in vitro fertilization (IVF failure due to poor embryo quality.

  15. Embryo yield in dairy cattle after superovulation with Folltropin or Pluset.

    Science.gov (United States)

    Mikkola, M; Taponen, J

    2017-01-15

    Two commercial FSH products were compared in a retrospective study on 3990 commercial superovulations and embryo recoveries in dairy heifers and cows. In addition, the 56-day nonreturn rate of 19,400 embryos produced with these two preparations was analyzed. Embryo collections were performed during a 16-year period from donors of Holstein and Ayrshire breeds. Folltropin (Vetoquinol S.A., Lure cedex, France) group (Group F) consisted of 2592 superovulations, of which 80% were performed on heifers and 20% on cows, and Pluset (Laboratorios Calier, S.A., Barcelona, Spain) group (Group P) of 1398 treatments, of which 66% and 34% were on heifers and cows, respectively. Total number of recovered structures, number of transferable embryos, and the proportion of unfertilized ova (UFO) and degenerated embryos were analyzed. Distribution of embryos into quality grades (1-3) and developmental stages (4-9) according to the IETS classification guidelines and means for each collection were evaluated. The proportion of low-responders having fewer than five corpora lutea and yielding fewer than five embryos or ova was investigated for each treatment. Group P yielded 1.1 recovered structures more than Group F (P superovulated with Folltropin or Pluset. It was concluded that equal numbers of transferable embryos and pregnancies can be achieved with Folltropin and Pluset. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Factors that affect the reproductive efficiency of the recipient within a bovine embryo transfer program

    Directory of Open Access Journals (Sweden)

    Arturo Duica A.

    2007-12-01

    Full Text Available The embryo transfer is a biotechnological technique that allows increasing the descendant of animals with high genetic value. The positive results, represented in pregnancy after the application of this technique, are affected by some factors that are inherent to the donor, the embryo, the technique, and the recipients which receive a strange embryo in the uterus allowing pregnancy. This review describes some factors affecting the reproductive efficiency of the recipients of bovine embryos within a program of embryo transfer. Its important to evaluate the parameters in this kind of recipients, as race, age, physiological status, health status, weight, reproductive tract integrity and management, and also too monitoring the ovarian structures while the estrus synchronization, and within previous and posterior stages in embryo transfer procedure. Therefore an optimum follicular development will be determinant to corpus luteum formation which generates enough serum progesterone concentrations to offer a right uterine environment allowing the optimum embryo development. Controlling the factors that affect the efficiency of the embryo transfer, it will obtain an increasing of positive results represented in pregnancies and births of individuals come from animals with high genetic value.

  17. Epigenetic and hormonal profile during maturation of Quercus Suber L. somatic embryos.

    Science.gov (United States)

    Pérez, Marta; Viejo, Marcos; LaCuesta, Maite; Toorop, Peter; Cañal, María Jesús

    2015-01-15

    Somatic embryogenesis is a powerful alternative to conventional mass propagation of Quercus suber L. However, poor quality and incomplete maturation of somatic embryos restrict any application. Given that epigenetic and hormonal control govern many developmental stages, including maturation of zygotic embryos, global DNA methylation and abscisic acid (ABA) were analyzed during development and maturation of cork oak somatic embryos. Our results indicated that development of somatic embryos concurred with a decrease in 5-mdC. In contrast, endogenous ABA content showed a transient increase with a peak in immature E2 embryos denoting the onset of the maturation phase. A cold stratification phase was necessary for embryos to acquire germination ability, which coincided with a significant decrease in 5-mdC and ABA content. Immunohistochemical analyses showed that there was a specific spatial-temporal regulation during embryogenesis, particularly after the cold treatment. The acquisition of germination capacity concurred with a general low 5-mdC signal in the root meristem, while retention of the 5-mdC signal was mainly located in the shoot meristem and provascular tissues. Conversely, ABA immunolocalization was mainly located in the root and shoot apical meristems. Furthermore, a strong decrease in the ABA signal was observed in the root cap after the stratification treatment suggesting a role for the root cap during development of somatic embryos. These results suggest that, in addition to ABA, epigenetic control appears to play an important role for the correct maturation and subsequent germination of cork oak somatic embryos.

  18. Metamizole Sodium Induces Neural Tube Defects in a Chick Embryo Model.

    Science.gov (United States)

    Guvenc, Yahya; Billur, Deniz; Aydin, Sevim; Ozeren, Ersin; Demirci, Adnan; Alagoz, Fatih; Dalgic, Ali; Belen, Deniz

    The aim of this study was to investigate the effects of metamizole sodium on neural tube development in the early stage chick embryo model that complies with the first month of embryonic development in mammals. A total of 40 fertilized chicken eggs were divided into 4 equal groups. The eggs were incubated in the incubator at a temperature of 37.8±2°C with 60±5% humidity. Group A was the control, Group B was administered physiological saline, Group C was administered 30 mg/kg metamizole sodium (based on the therapeutic index range of it used in humans) and Group D was administered 90 mg/kg metamizole sodium. All embryos were removed from the egg at the 48th hour and morphologically and histologically examined. Normal development was seen and the neural tube was closed in 17 embryos in Groups A and B. A neural tube defect was seen in 2 embryos in group A and in 1 embryo in group B. A neural tube closure defect was seen in all embryos in group C and 9 embryos in group D. There was 1 dead embryo in Group D. Metamizole sodium was seen to produce a neural tube defect in the chicken embyro model.

  19. Distinct roles of ROCK1 and ROCK2 during development of porcine preimplantation embryos.

    Science.gov (United States)

    Zhang, Jin Yu; Dong, Huan Sheng; Oqani, Reza K; Lin, Tao; Kang, Jung Won; Jin, Dong Il

    2014-07-01

    Cell-to-cell contact mediated by cell adhesion is fundamental to the compaction process that ensures blastocyst quality during embryonic development. In this study, we first showed that Rho-associated coiled-coil protein kinases (ROCK1 and ROCK2) were expressed both in porcine oocytes and IVF preimplantation embryos, playing different roles in oocytes maturation and embryo development. The amount of mRNA encoding ROCK1 and the protein concentration clearly increased between the eight-cell and morula stages, but decreased significantly when blastocysts were formed. Conversely, ROCK2 was more abundant in the blastocyst compared with other embryonic stages. Moreover, immunostaining showed that ROCK1 protein distribution changed as the embryo progressed through cleavage and compaction to the morula stage. Initially, the protein was predominantly associated with the plasma membrane but later became cytoplasmic. By contrast, ROCK2 protein was localized in both the cytoplasm and the spindle rotation region during oocyte meiosis, but in the cytoplasm and nucleus as the embryo developed. In addition, ROCK2 was present in the trophectoderm cells of the blastocyst. Treatment with 15 μM Y27632, a specific inhibitor of ROCKs, completely blocked further development of early four-cell stage embryos. Moreover, we did not detect the expression of ROCK1 but did detect ROCK2 expression in blastocysts. Moreover, lysophosphatidic acid an activator of ROCKs significantly improved the rates of blastocyst formation. These data demonstrate that ROCKs are required for embryo development to the blastocyst stage. Together, our results indicate that ROCK1 and ROCK2 may exert different biological functions during the regulation of compaction and in ensuring development of porcine preimplantation embryos to the blastocyst stage.

  20. Alterations of intraembryonic metabolites in preimplantation mouse embryos exposed to elevated concentrations of glucose: a metabolic explanation for the developmental retardation seen in preimplantation embryos from diabetic animals.

    Science.gov (United States)

    Moley, K H; Chi, M M; Manchester, J K; McDougal, D B; Lowry, O H

    1996-06-01

    Preimplantation mouse embryos exposed to hyperglycemia, whether in vivo or in vitro, experience delayed development from the 2-cell to blastocyst stage. By comparing metabolites from embryos exposed to high vs. normal glucose conditions, a metabolic explanation for the delayed growth pattern was sought. Fertilized 1-cell embryos obtained from superovulated B5 x CBA F1 mice were cultured for 96 h in medium containing 2.8 mM glucose (C) or in medium with added glucose to give 10 mM, 30 mM, or 52 mM glucose (HG). After incubation, each embryo was quick-frozen and freeze-dried. Metabolites were assayed by the ultramicrofluorometric technique and enzymatic cycling to obtain measurable levels in single embryos. Embryos cultured in HG exhibited 7-fold higher intracellular glucose levels than those cultured in C (C: 2.25 +/- 0.6 vs. HG: 16.61 +/- 2.4 mmol/kg wet weight; p Krebs cycle metabolites are elevated and correspond to the degree of developmental delay. These findings suggest that a metabolic abnormality may be responsible for retarded development experienced by embryos exposed to high glucose.

  1. The Well of the Well (WOW) system: an efficient approach to improve embryo development

    DEFF Research Database (Denmark)

    Vajta, G; Korösi, T; Du, Y

    2008-01-01

    Transfer of human embryos at the blastocyst stage may offer considerable benefits including the increased implantation rates and decreased risks of multiple pregnancies, however, it requires an efficient and reliable in vitro embryo culture system. In our study, the effect of the Well of the Well......-four blastocysts transferred to 45 patients resulted in 48.9% clinical pregnancy rates including 8 twin, 8 single, 5 miscarriages and 1 ectopic pregnancies. Our results indicate that the WOW system means a promising alternative of microdrops for culture of mammalian embryos including human...

  2. Proteomics of early zebrafish embryos

    Directory of Open Access Journals (Sweden)

    Heisenberg Carl-Philipp

    2006-01-01

    Full Text Available Abstract Background Zebrafish (D. rerio has become a powerful and widely used model system for the analysis of vertebrate embryogenesis and organ development. While genetic methods are readily available in zebrafish, protocols for two dimensional (2D gel electrophoresis and proteomics have yet to be developed. Results As a prerequisite to carry out proteomic experiments with early zebrafish embryos, we developed a method to efficiently remove the yolk from large batches of embryos. This method enabled high resolution 2D gel electrophoresis and improved Western blotting considerably. Here, we provide detailed protocols for proteomics in zebrafish from sample preparation to mass spectrometry (MS, including a comparison of databases for MS identification of zebrafish proteins. Conclusion The provided protocols for proteomic analysis of early embryos enable research to be taken in novel directions in embryogenesis.

  3. Cell adhesion in embryo morphogenesis.

    Science.gov (United States)

    Barone, Vanessa; Heisenberg, Carl-Philipp

    2012-02-01

    Visualizing and analyzing shape changes at various scales, ranging from single molecules to whole organisms, are essential for understanding complex morphogenetic processes, such as early embryonic development. Embryo morphogenesis relies on the interplay between different tissues, the properties of which are again determined by the interaction between their constituent cells. Cell interactions, on the other hand, are controlled by various molecules, such as signaling and adhesion molecules, which in order to exert their functions need to be spatiotemporally organized within and between the interacting cells. In this review, we will focus on the role of cell adhesion functioning at different scales to organize cell, tissue and embryo morphogenesis. We will specifically ask how the subcellular distribution of adhesion molecules controls the formation of cell-cell contacts, how cell-cell contacts determine tissue shape, and how tissue interactions regulate embryo morphogenesis.

  4. In amnio MRI of mouse embryos.

    Directory of Open Access Journals (Sweden)

    Thomas A Roberts

    Full Text Available Mouse embryo imaging is conventionally carried out on ex vivo embryos excised from the amniotic sac, omitting vital structures and abnormalities external to the body. Here, we present an in amnio MR imaging methodology in which the mouse embryo is retained in the amniotic sac and demonstrate how important embryonic structures can be visualised in 3D with high spatial resolution (100 µm/px. To illustrate the utility of in amnio imaging, we subsequently apply the technique to examine abnormal mouse embryos with abdominal wall defects. Mouse embryos at E17.5 were imaged and compared, including three normal phenotype embryos, an abnormal embryo with a clear exomphalos defect, and one with a suspected gastroschisis phenotype. Embryos were excised from the mother ensuring the amnion remained intact and stereo microscopy was performed. Embryos were next embedded in agarose for 3D, high resolution MRI on a 9.4T scanner. Identification of the abnormal embryo phenotypes was not possible using stereo microscopy or conventional ex vivo MRI. Using in amnio MRI, we determined that the abnormal embryos had an exomphalos phenotype with varying severities. In amnio MRI is ideally suited to investigate the complex relationship between embryo and amnion, together with screening for other abnormalities located outside of the mouse embryo, providing a valuable complement to histology and existing imaging methods available to the phenotyping community.

  5. Effect of progesterone supplementation in the first week post conception on embryo survival in beef heifers

    OpenAIRE

    2009-01-01

    Progesterone is essential for establishment and maintenance of pregnancy in mammals. The objective of this study was to examine the effect of elevating progesterone during the different physiological stages of early embryo development on embryo survival. Estrus was synchronized in cross-bred beef heifers (n = 197, ∼2-years old) and they were inseminated 12–18 h after estrus onset (=Day 0). Inseminated heifers were randomly assigned to 1 of 3 treatments: (1) Control, n = 69; (2) progesterone s...

  6. Further Development and Validation of the Frog Embryo Teratogenesis Assay-Xenopus (FETAX).

    Science.gov (United States)

    1992-11-23

    first used in studying the hollow blistula stage of a few hundred cells developmental toxicity of selenium (Browne and (Figure 1) to a free-swimming...ONTROLS pigmentation, locomotion and hatchability are described in the ASTM New Standard Guide It is necessary to use appropriate controls in (Bantle and...Dumnont IN: Toxicity of selenium to acety enic alcohols on embryos and larvae of developin Xeno usr kawvs embryos. J Xenous laevis. In: Aquatic

  7. A chick embryo with a yet unclassified type of cephalothoracopagus malformation and a hypothesis for explaining its genesis.

    Science.gov (United States)

    Maurer, B; Geyer, S H; Weninger, W J

    2013-06-01

    Cephalothoracopagus embryos are conjoined twins, who share parts of their heads, necks and bodies. Our study aims at presenting a detailed morphological analysis of a cephalothoracopagus chick embryo of developmental stage 31. Because none of the existing theories can explain the genesis of the phenotype of this embryo, we also suggest a hypothesis, which explains it. Beside the cephalothoracopagus embryo, we investigated five control embryos. With the aid of the high-resolution episcopic microscopy (HREM) technique, we created digital volume data and three-dimensional (3D) computer models of the organs and arteries of the embryos. We used the 3D models for topological analysis and for measuring the diameters of the great intrathoracic arteries. The malformed embryo showed two body backs, each containing a notochord, spinal cord and dorsal aorta. The body backs continued into separated lower bodies. The embryo had a single, four-chambered heart, single respiratory tract and single upper alimentary tract. The topology of the pharyngeal arch arteries was normal, and the diameters of these arteries were similar to that of the control embryos. We classified the embryo we investigated as a yet unknown malformation and suggest a hypothesis explaining its genesis.

  8. Anteroposterior patterning in Xenopus embryos: egg fragment assay system reveals a synergy of dorsalizing and posteriorizing embryonic domains.

    Science.gov (United States)

    Fujii, Hidefumi; Nagai, Takeharu; Shirasawa, Hiroki; Doi, Jun-ya; Yasui, Kinya; Nishimatsu, Shin-ichirou; Takeda, Hiroyuki; Sakai, Masao

    2002-12-01

    Two distinct types of axis lacking embryos resulted from partial deletion of the vegetal part of early one-cell-stage embryos. When the deleted volume was 20-40% (relative surface area), the embryos underwent ventral-type gastrulation and formed ventral mesodermal tissues. When the deleted volume was more than 60%, the embryo did not gastrulate nor make mesodermal structures (M. Sakai, 1996, Development 122, 2207-2214). We have designated these two types of embryos as "gastrulating nonaxial embryos (GNEs)" and "permanent blastula-type embryos (PBEs)," respectively. Using these embryos as recipients, a series of Einsteck transplantation experiments were carried out to investigate mechanisms controlling anteroposterior patterning during early Xenopus development. GNEs receiving dorsal marginal zone (DMZ) transplants (GNE/DMZs) elongated and formed posteriorized phenotypes, which had muscle cells, melanocytes, and tail fins. In contrast, PBE/DMZs did not elongate but formed cement glands and brain-like structures showing strong anteriorization. Simultaneous transplantation of the cells from various regions of normal embryos with the DMZ into PBEs revealed that the entire vegetal half of normal embryos, except for the DMZ, showed posteriorizing activity. These results strongly suggest that anteroposterior patterning in Xenopus is not achieved solely by the dorsal marginal zone (the Spemann organizer), but instead by a synergistic mechanism of the dorsalizing domain (DMZ) and the posteriorizing domain (the entire vegetal half except for the DMZ).

  9. Supplementation of culture medium with L-carnitine improves development and cryotolerance of bovine embryos produced in vitro.

    Science.gov (United States)

    Takahashi, Toshikiyo; Inaba, Yasushi; Somfai, Tamas; Kaneda, Masahiro; Geshi, Masaya; Nagai, Takashi; Manabe, Noboru

    2013-01-01

    High lipid content in embryos is associated with low freezing tolerance. This study assessed the effects of exogenous L-carnitine, an enhancer of lipid metabolism, on the in vitro development and freezing survival of bovine embryos. Also, effects on metabolic activity, reactive oxygen species (ROS) and apoptosis were investigated. Supplementation of embryo culture medium with 1.518 mM or 3.030 mM L-carnitine significantly increased the rates of zygote development to the blastocyst stage and blastocyst cell numbers whereas 6.072 mM of this compound did not improve embryo development. Survival rates after slow freezing of blastocysts were significantly higher when embryos were cultured in the presence of 1.518 mM or 3.030 mM L-carnitine compared with the control. A lower density of lipid droplets was detected in L-carnitine-treated blastocysts compared with the control. L-carnitine significantly reduced ROS levels in 2-cell embryos but did not reduce ROS levels at later stages. The apoptotic cell rate was not different between control and L-carnitine-treated blastocysts. L-carnitine significantly increased ATP levels in 2-cell embryos but not at the 8-cell or blastocyst stages. L-carnitine increased the expression of metabolism-related ATP6 and COX1 genes in blastocysts. In conclusion, L-carnitine supplementation enhanced lipid metabolism in embryos resulting in improved development and cryotolerance of bovine blastocysts produced in vitro.

  10. Nuclear reprogramming by interphase cytoplasm of 2-cell mouse embryos

    Science.gov (United States)

    Kang, Enugu; Wu, Guangming; Ma, Hong; Li, Ying; Tippner-Hedges, Rebecca; Tachibana, Masahito; Sparman, Michelle; Wolf, Don P.; Schöler, Hans; Mitalipov, Shoukhrat

    2014-01-01

    Summary Successful mammalian cloning employing somatic cell nuclear transfer (SCNT) into unfertilized, metaphase II-arrested (MII) oocytes attests to the cytoplasmic presence of reprogramming factors capable of inducing pluripotency in somatic cell nuclei1-3. However, these poorly defined maternal factors presumably decline sharply after fertilization since cytoplasm of pronuclear stage zygotes is reportedly inactive4, 5. Recent evidence suggests that zygotic cytoplasm, if maintained at metaphase (M-phase) can also support derivation of embryonic stem cells (ESCs) following SCNT6-8, albeit at low efficiency. This led to the conclusion that critical oocyte reprogramming factors present in M-phase but not in interphase cytoplasm are “trapped” inside the nucleus during interphase and effectively removed during enucleation9. Here, we investigated the presence of reprogramming activity in the interphase cytoplasm of 2-cell mouse embryos (I2C). First, the presence of candidate reprogramming factors was documented in both intact and enucleated M-phase and interphase zygotes and 2-cell embryos. Consequently, enucleation did not provide a likely explanation for the inability of interphase cytoplasm to induce reprogramming. Then, when we carefully synchronized the cell cycle stage between the transplanted nucleus (ESC, fetal fibroblast or terminally differentiated cumulus cell) and the recipient I2C cytoplasm, the reconstructed SCNT embryos developed into blastocysts and ESCs capable of contributing to traditional germline and tetraploid chimeras. In addition, direct transfer of cloned embryos, reconstructed with ESC nuclei, into recipients resulted in live offspring. Thus, the cytoplasm of I2C supports efficient reprogramming with cell cycle synchronization between the donor nucleus and recipient cytoplasm as the most critical parameter determining success. The ability to utilize interphase cytoplasm in SCNT could impact efforts to generate autologous human ESCs for

  11. SOMATIC EMBRYOGENESIS AND MORPHOANATOMY OF Ocotea porosa SOMATIC EMBRYOS

    Directory of Open Access Journals (Sweden)

    Luciana Luiza Pelegrini

    2013-01-01

    Full Text Available Ocotea porosa seeds have strong tegument dormancy, recalcitrant behavior, low and irregular germination and that makes its natural propagation difficult. The aim of this study was to establish a protocol of regeneration of Ocotea porosa from somatic embryogenesis. Immature embryonic axes were inoculated on WPM culture medium supplemented with 2.4-D (200 μM combined or not with hydrolyzed casein or glutamine (0.5 or 1 g l-1, during 90 days. The repetitive embryogenesis was induced on medium with 2.4-D (22.62 μM combined with 2-iP (2.46 μM followed by transfer to culture medium with hydrolyzed casein or glutamine (1 g l-1 during 90 days. The maturation of somatic embryos was tested in culture medium containing NAA (0.5 μM and 2-iP (5; 10 and 20 μM. The highest percentage of somatic embryos induction (8.3% was observed in WPM culture medium containing 200 μM 2.4-D and 1 g L-1 hydrolyzed casein and the development of somatic embryos occurred indirectly. Repetitive somatic embryogenesis was promoted in WPM medium containing hydrolyzed casein or glutamine. However, the culture medium containing hydrolyzed casein promoted the maintenance of embryogenic capacity for more than two years. During the maturity phase, there was a low progression of globular embryos to cordiform and torpedo stages. The different ontogenetic stages of somatic embryos of Ocotea porosa were characterized by histological studies.

  12. Pollination and embryo development in Brassica rapa L. in microgravity

    Science.gov (United States)

    Kuang, A.; Popova, A.; Xiao, Y.; Musgrave, M. E.

    2000-01-01

    Plant reproduction under spaceflight conditions has been problematic in the past. In order to determine what aspect of reproductive development is affected by microgravity, we studied pollination and embryo development in Brassica rapa L. during 16 d in microgravity on the space shuttle (STS-87). Brassica is self-incompatible and requires mechanical transfer of pollen. Short-duration access to microgravity during parabolic flights on the KC-135A aircraft was used initially to confirm that equal numbers of pollen grains could be collected and transferred in the absence of gravity. Brassica was grown in the Plant Growth Facility flight hardware as follows. Three chambers each contained six plants that were 13 d old at launch. As these plants flowered, thin colored tape was used to indicate the date of hand pollination, resulting in silique populations aged 8-15 d postpollination at the end of the 16-d mission. The remaining three chambers contained dry seeds that germinated on orbit to produce 14-d-old plants just beginning to flower at the time of landing. Pollen produced by these plants had comparable viability (93%) with that produced in the 2-d-delayed ground control. Matched-age siliques yielded embryos of equivalent developmental stage in the spaceflight and ground control treatments. Carbohydrate and protein storage reserves in the embryos, assessed by cytochemical localization, were also comparable. In the spaceflight material, growth and development by embryos rescued from siliques 15 d after pollination lagged behind the ground controls by 12 d; however, in the subsequent generation, no differences between the two treatments were found. The results demonstrate that while no stage of reproductive development in Brassica is absolutely dependent upon gravity, lower embryo quality may result following development in microgravity.

  13. SOMATIC EMBRYOGENESIS AND MORPHOANATOMY OF Ocotea porosa SOMATIC EMBRYOS

    Directory of Open Access Journals (Sweden)

    Luciana Luiza Pelegrini

    2013-12-01

    Full Text Available http://dx.doi.org/10.5902/1980509812343Ocotea porosa seeds have strong tegument dormancy, recalcitrant behavior, low and irregular germinationand that makes its natural propagation difficult. The aim of this study was to establish a protocol ofregeneration of Ocotea porosa from somatic embryogenesis. Immature embryonic axes were inoculatedon WPM culture medium supplemented with 2.4-D (200 μM combined or not with hydrolyzed casein orglutamine (0.5 or 1 g l-1, during 90 days. The repetitive embryogenesis was induced on medium with 2.4-D(22.62 μM combined with 2-iP (2.46 μM followed by transfer to culture medium with hydrolyzed caseinor glutamine (1 g l-1 during 90 days. The maturation of somatic embryos was tested in culture mediumcontaining NAA (0.5 μM and 2-iP (5; 10 and 20 μM. The highest percentage of somatic embryos induction(8.3% was observed in WPM culture medium containing 200 μM 2.4-D and 1 g L-1 hydrolyzed casein andthe development of somatic embryos occurred indirectly. Repetitive somatic embryogenesis was promotedin WPM medium containing hydrolyzed casein or glutamine. However, the culture medium containinghydrolyzed casein promoted the maintenance of embryogenic capacity for more than two years. Duringthe maturity phase, there was a low progression of globular embryos to cordiform and torpedo stages.The different ontogenetic stages of somatic embryos of Ocotea porosa were characterized by histologicalstudies.

  14. Phenotypically plastic responses of green frog embryos to conflicting predation risk.

    Science.gov (United States)

    Ireland, D H; Wirsing, A J; Murray, D L

    2007-05-01

    Predators have been shown to alter the timing of switch points between life history stages, but few studies have addressed switch point plasticity in prey exposed simultaneously to conflicting predation pressure. We tested hatching responses of green frog (Rana clamitans) embryos subject to perceived predation risk from chemical cues released by two stage-specific predators, predicting that these predators would elicit: (1) directional hatching responses when presented independently, and (2) intermediate phenotypic responses when presented simultaneously. R. clamitans embryos in outdoor exclosures were exposed to cues from an egg predator (freshwater leeches; Nephelopsis obscura), a larval predator (dragonfly nymphs, Aeschna canadensis), and both predators in a 2 x 2 factorial experiment, and changes in hatchling size, hatchling developmental stage, and hatching time were compared to those for control embryos. Leeches alone induced embryos to hatch at a smaller size and an earlier developmental stage than controls, while dragonfly nymphs elicited a delay in egg hatching time that was associated with larger size and later developmental stage at hatching. Embryos failed to respond to simultaneous exposure to both predators, implying that responses to each occurred concurrently and were therefore dampened. Our results indicate that prey under threat from conflicting predators may manifest intermediate defensive phenotypes. Such intermediate responses may result in elevated rates of prey mortality with possible consequences at the population level.

  15. Dielectrospectroscopic monitoring of early embryogenesis in single frog embryos

    Science.gov (United States)

    Asami, Koji; Irimajiri, Akihiko

    2000-11-01

    Dielectric spectroscopy has been used to monitor the early embryogenesis of frog (Xenopus laevis) eggs. The dielectric spectra of a single egg in suspension over the frequency range 10 Hz to 10 MHz were collected at various stages of its development. The uncleaved egg showed a dielectric dispersion with a narrow distribution of relaxation times. After the first cleavage, the dielectric spectra were mainly composed of two subdispersions. In the cleavage process, up to the morula stage, changes in the spectra were quantitatively simulated by the `cell-aggregate' model in which the embryo is regarded as a concentrated suspension of shell-spheres that correspond to the blastomeres (i.e. the cells within the embryo). In the stages from the morula to the blastula, the changes in the dielectric spectra were explained as due to a reduction in the size of the blastomere accompanied by an expansion of the blastocoel (i.e. the central cavity in the embryo) using the `vesicle-inclusion' model that is a cell aggregate covered with a less conducting shell corresponding to the outermost layer of tightly interconnected cells.

  16. Future aspects of micromanipualtion with embryos for

    African Journals Online (AJOL)

    Embryo micromanipulation techniques and their potential genetic impact in dairy cattle ... bovine embryos and the subsequent transfer of halfembryos has reached a .... (e) to provide synchronously developing pronuclear srage ova for nuclear ...

  17. Embryo splitting: a role in infertility?

    Science.gov (United States)

    Wood, C

    2001-01-01

    Embryo splitting may be used to increase the potential fertility of couples requiring IVF. Using cattle as a model, it is possible to increase pregnancy rates from 70% per transfer of good quality in-vivo-produced embryos, to 110% by transferring the two demi-embryos resulting from the bisection of one embryo. The 30-40% greater chance of conception would reduce costs for the government, health authorities and patients, and reduce stress, time and complications for women having IVF treatment. Embryo splitting may also provide donor embryos for infertile couples that cannot conceive naturally or with IVF. The shortage of children for adoption and donor embryos may be overcome by the production of demi-embryos.

  18. The fate of frozen human embryos when transferred either on the day of thawing or after overnight culture

    Institute of Scientific and Technical Information of China (English)

    Yanhe Liu; Kelli Peirce; Kailin Yap; Kate McKenzie; Jay Natalwala; Vince Chapple; Margo Norman; Phillip Matson

    2012-01-01

    Objective:To study the performance of thawed zygotes and cleavage stage embryos transferred either on the day of thaw or after overnight culture.Methods:A retrospective study of864 frozen embryo transfer cycles.Cryosurvival rates per thawed embryo and implantation rates were analysed for embryos frozen onDay1,Day2 orDay3 relative to oocyte collection(Day0) and transferred on the day of thaw or after overnight culture, together with clinical pregnancy rates and prevalence of multiple gestations.Results:Survival ofDay3 embryos was significantly lower than those frozen onDay1(P=0.017) orDay2(P=0.015).Following overnight culture, resumption of mitosis of zygotes was more frequent thanDay2(P=0.000) which are in turn higher thanDay3(P=0.000) embryos.The implantation rate forDay2 embryos dividing overnight was significantly higher than those that did not divide for women <35 yrs(P=0.001) but not those women≥35 yrs(P=0.055).There were no differences in the implantation rates for those dividing or not after culture, for embryos frozen onDay3 for women <35 yrs(P=0.254) or≥35 yrs(P=0.403). Conclusions:Later cleavage stage post-thaw embryos survive and resume mitosis less frequently compared to earlier stages.Embryos not resuming mitosis after culture overnight can implant, particularlyDay3 embryos, suggesting that they can further increase the cumulative pregnancy rate per oocyte collection and that discarding them is wasteful.Overnight culture is best used for logistical reasons rather than a strategy to improve pregnancy rates.

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

    Science.gov (United States)

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

    2017-10-01

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

  20. Embryo growth in mature celery seeds

    NARCIS (Netherlands)

    Toorn, van der P.

    1989-01-01

    Germination of celery seeds is slow, due to the need for embryo growth before radicle protrusion can occur. Germination rate was correlated with embryo growth rate. Celery seeds with different embryo growth rates were obtained with fluid density separation of a seed lot. Low density seeds g

  1. Embryo temperature during incubation: practice and theory

    NARCIS (Netherlands)

    Lourens, A.

    2008-01-01

    (Key words: incubation, embryo temperature, embryonic development, heat production, heat loss) Until recently, all incubator studies were performed using a constant machine temperature (MT). But it is embryo temperature (ET) that is of importance to the embryo, and not MT. In practice, MT is often

  2. Improving embryo quality in assisted reproduction

    NARCIS (Netherlands)

    Mantikou, E.

    2013-01-01

    The goal of this thesis was to improve embryo quality in assisted reproductive technologies by gaining more insight into human preimplantation embryo development and by improving in vitro culture conditions. To do so, we investigated an intriguing feature of the human preimplantation embryo, i.e. it

  3. Embryo growth in mature celery seeds

    NARCIS (Netherlands)

    Toorn, van der P.

    1989-01-01

    Germination of celery seeds is slow, due to the need for embryo growth before radicle protrusion can occur. Germination rate was correlated with embryo growth rate. Celery seeds with different embryo growth rates were obtained with fluid density separation of a seed lot. Low density seeds

  4. Gray level Co-occurrence Matrices (GLCM) to assess microstructural and textural changes in pre-implantation embryos.

    Science.gov (United States)

    Tan, Tiffany C Y; Ritter, Lesley J; Whitty, Annie; Fernandez, Renae C; Moran, Lisa J; Robertson, Sarah A; Thompson, Jeremy G; Brown, Hannah M

    2016-08-01

    The preimplantation embryo is extraordinarily sensitive to environmental signals and events such that perturbations can alter embryo metabolism and program an altered developmental trajectory, ultimately affecting the phenotype of the adult individual; indeed, the physical environment associated with in vitro embryo culture can attenuate development. Defining the underlying metabolic changes and mechanisms, however, has been limited by the imaging technology used to evaluate metabolites and structural features in the embryo. Here, we assessed the impact of in vitro fertilization and culture on mouse embryos using three metabolic markers: peroxyfluor 1 (a reporter of hydrogen peroxide), monochlorobimane (a reporter of glutathione), and Mitotracker Deep Red (a marker of mitochondria). We also evaluated the distribution pattern of histone 2AX gamma (γH2AX) in the nuclei of 2- and 8-cell embryos and blastocysts to investigate the degree of DNA damage caused by in vitro embryo culture. In vitro-fertilized embryos, in vivo-developed embryos, and in vivo-fertilized embryos recovered and cultured in vitro were compared at the 2-, 8-cell, and blastocyst stages. In addition to assessments based on fluorescence intensity, textural analysis using Gray Level Co-occurrence Matrix (GLCM), a statistical approach that assesses texture within an image, was used to evaluate peroxyfluor 1, monochlorobimane, and Mitotracker Deep Red staining in an effort to develop a robust metric of embryo quality. Our data provide strong evidence of modified metabolic parameters identifiable as altered fluorescence texture in embryos developed in vitro. Thus, texture-analysis approach may provide a means of gaining additional insight into embryo programming beyond conventional measurements of staining intensity for metabolic markers. Mol. Reprod. Dev. 83: 701-713, 2016 © 2016 Wiley Periodicals, Inc.

  5. Overexpression of S-adenosylmethionine decarboxylase (SAMDC) in Xenopus embryos activates maternal program of apoptosis as a "fail-safe" mechanism of early embryogenesis

    Institute of Scientific and Technical Information of China (English)

    MASATAKE KAI; CHIKARA KAITO; HIROSHI FUKAMACHI; TAKAYASU HIGO; EIJI TA-KAYAMA; HIROSHI HARA; YOSHIKAZU OHYA; KAZUEI IGARASHI; KOICHIRO SHIOKAWA

    2003-01-01

    In Xenopus, injection of S-adenosylmethionine decarboxylase (SAMDC) mRNA into fertilized eggs or2-cell stage embryos induces massive cell dissociation and embryo-lysis at the early gastrula stage due toactivation of the maternal program of apoptosis. We injected SAMDC mRNA into only one of the animalside blastomeres of embryos at different stages of cleavage, and examined the timing of the onset of theapoptotic reaction. In the injection at 4- and 8-cell stages, a considerable number of embryos developed intotadpoles and in the injection at 16- and 32-cell stages, all the embryos became tadpoles, although tadpolesobtained were sometimes abnormal. However, using GFP as a lineage tracer, we found that descendant cellsof the blastomere injected with SAMDC mRNA at 8- to 32-cell stages are confined within the blastocoel atthe early gastrula stage and undergo apoptotic cell death within the blastocoel, in spite of the continueddevelopment of the injected embryos. These results indicate that cells overexpressed with SAMDC undergoapoptotic cell death consistently at the early gastrula stage, irrespective of the timing of the mRNA injection.We assume that apoptosis is executed in Xenopus early gastrulae as a "fail-safe" mechanism to eliminatephysiologically-severely damaged cells to save the rest of the embryo.

  6. Effects of High-Butterfat Diet on Embryo Implantation in Female Rats Exposed to Bisphenol A.

    Science.gov (United States)

    Martinez, Alan M; Cheong, Ana; Ying, Jun; Xue, Jingchuan; Kannan, Kurunthachalam; Leung, Yuet-Kin; Thomas, Michael A; Ho, Shuk-Mei

    2015-12-01

    Bisphenol A (BPA) is an endocrine disruptor associated with poor pregnancy outcomes in human and rodents. The effects of butterfat diets on embryo implantation and whether it modifies BPA's actions are currently unknown. We aimed to determine the effects of butterfat diet on embryo implantation success in female rats exposed to an environmentally relevant dose of BPA. Female Sprague-Dawley rats were exposed to dietary butterfat (10% or 39% kcal/kg body weight [BW]) in the presence or absence of BPA (250 μg/kg BW) or ethinylestradiol (0.1 μg/kg BW) shortly before and during pregnancy to assess embryo implantation potentials by preimplantation development and transport, in vitro blastulation, outgrowth, and implantation. On gestational day (GD) 4.5, rats treated with BPA alone had higher serum total BPA level (2.3-3.7 ng/ml). They had more late-stage preimplantation embryos, whereas those receiving high butterfat (HBF) diet had the most advanced-stage embryos; dams cotreated with HBF and BPA had the most number of advanced embryos. BPA markedly delayed embryo transport to the uterus, but neither amount of butterfat had modifying effects. An in vitro implantation assay showed HBF doubled the outgrowth area, with BPA having no effect. In vivo, BPA reduced the number of implanted embryos on GD8, and cotreatment with HBF eliminated this adverse effect. HBF diet overall resulted in more and larger GD8 embryos. This study reveals the implantation disruptive effects of maternal exposure to an environmentally relevant dose of BPA and identifies HBF diet as a modifier of BPA in promoting early embryonic health.

  7. Proteomic analysis of zebrafish embryos exposed to simulated-microgravity

    Science.gov (United States)

    Hang, Xiaoming; Ma, Wenwen; Wang, Wei; Liu, Cong; Sun, Yeqing

    Microgravity can induce a serial of physiological and pathological changes in human body, such as cardiovascular functional disorder, bone loss, muscular atrophy and impaired immune system function, etc. In this research, we focus on the influence of microgravity to vertebrate embryo development. As a powerful model for studying vertebrate development, zebrafish embryos at 8 hpf (hour past fertilization) and 24 hpf were placed into a NASA developed bioreac-tor (RCCS) to simulate microgravity for 64 and 48 hours, respectively. The same number of control embryos from the same parents were placed in a tissue culture dish at the same temper-ature of 28° C. Each experiment was repeated 3 times and analyzed by two-dimensional (2-D) gel electrophoresis. Image analysis of silver stained 2-D gels revealed that 64 from total 292 protein spots showed quantitative and qualitative variations that were significantly (Pprotein spots with significant expression alteration (Pproteins, 3 down-regulated proteins were identified as bectin 2, centrosomal protein of 135kDa and tropomyosin 4, while the up-regulated protein was identified as creatine kinase muscle B. Other protein spots showed significant expression alteration will be identified successively and the corresponding genes expression will also be measured by Q-PCR method at different development stages. The data presented in this study illustrate that zebrafish embryo can be significantly induced by microgravity on the expression of proteins involved in bone and muscle formation. Key Words: Danio rerio; Simulated-microgravity; Proteomics

  8. Blastocele fluid from in vitro- and in vivo-produced equine embryos contains nuclear DNA.

    Science.gov (United States)

    Herrera, C; Morikawa, M I; Castex, C Baca; Pinto, M R; Ortega, N; Fanti, T; Garaguso, R; Franco, M J; Castañares, M; Castañeira, C; Losinno, L; Miragaya, M H; Mutto, A A

    2015-02-01

    Normal mammalian early embryonic development involves apoptosis of blastomeres as a remodeling process during differentiation, starting at the blastocyst stage. Genomic DNA has been recently detected in the blastocele fluid of human embryos and has been amplified by real-time polymerase chain reaction (PCR) to diagnose the sex of in vitro-produced human embryos. This new approach varies from conventional preimplantation genetic diagnosis in that no cells are extracted from the embryo and only the blastocele fluid is aspirated and used as a DNA sample for diagnosis. In the present work, we investigated whether the blastocele fluid of equine preimplantation embryos contains nuclear DNA and whether this DNA could be used to diagnose the sex of the embryos by conventional PCR, using specific primers that target the TSPY and AMEL equine genes. The sex of 11 of 13 in vivo-produced embryos and of four of five in vitro-produced embryos was successfully diagnosed. The PCR amplification product was analyzed using genetic sequencing reporting that the DNA present in blastocele fluid was genomic. Additionally, after polyacrylamide gel electrophoresis and silver staining, the blastocele fluid from three different embryos produced a ladder pattern characteristic of DNA fragmented during apoptosis. Therefore, the results presented in this work report that blastocele fluid from in vivo- and in vitro-produced equine embryos contains nuclear DNA which is probably originated by apoptosis of embryonic cells, and this DNA could be used to diagnose the sex of preimlpantation embryos by conventional PCR.

  9. Expression of renin–angiotensin system components in the early bovine embryo

    Science.gov (United States)

    Pijacka, Wioletta; Hunter, Morag G; Broughton Pipkin, Fiona; Luck, Martin R

    2012-01-01

    The renin–angiotensin system (RAS), mainly associated with the regulation of blood pressure, has been recently investigated in female reproductive organs and the developing foetus. Angiotensin II (Ang II) influences oviductal gamete movements and foetal development, but there is no information about RAS in the early embryo. The aim of this study was to determine whether RAS components are present in the pre-implantation embryo, to determine how early they are expressed and to investigate their putative role at this stage of development. Bovine embryos produced in vitro were used for analysis of RAS transcripts (RT-PCR) and localisation of the receptors AGTR1 and AGTR2 (immunofluorescent labelling). We also investigated the effects of Ang II, Olmesartan (AGTR1 antagonist) and PD123319 (AGTR2 antagonist) on oocyte cleavage, embryo expansion and hatching. Pre-implanted embryos possessed AGTR1 and AGTR2 but not the other RAS components. Both receptors were present in the trophectoderm and in the inner cell mass of the blastocyst. AGTR1 was mainly localised in granular-like structures in the cytoplasm, suggesting its internalisation into clathrin-coated vesicles, and AGTR2 was found mainly in the nuclear membrane and in the mitotic spindle of dividing trophoblastic cells. Treating embryos with PD123319 increased the proportion of hatched embryos compared with the control. These results, the first on RAS in the early embryo, suggest that the pre-implanted embryo responds to Ang II from the mother rather than from the embryo itself. This may be a route by which the maternal RAS influences blastocyst hatching and early embryonic development. PMID:23781300

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

    Directory of Open Access Journals (Sweden)

    Tiancheng Liu

    2015-01-01

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

  11. DNA delivery into anterior neural tube of zebrafish embryos by electroporation.

    Science.gov (United States)

    Teh, Cathleen; Chong, Shang Wei; Korzh, Vladimir

    2003-11-01

    The zebrafish is widely used for functional studies of vertebrate genes. It is accessible to manipulations during all stages of embryogenesis because the embryo develops externally and is optically transparent. However, functional studies conducted on the zebrafish have been generally limited to the earliest phase of activity of the gene of interest, which is a limitation in studies of genes that are expressed at various stages of embryonic development. It is therefore necessary to develop methods that allow for the modulation of gene activity during later stages of zebrafish development while leaving earlier functions intact. We have successfully electroporated the green fluorescent protein (GFP) reporter gene into the neural tube of the zebrafish embryo in a unidirectional or bilateral manner. This approach can be used for the functional analysis of the late role of developmental genes in the neural tube of zebrafish embryo and larvae.

  12. Lethal effect of dehydroleucodine (DhL) on amphibian Bufo arenarum embryos.

    Science.gov (United States)

    Moreno, Liliana Elizabeth; Juárez, Américo Osvaldo; Pelzer, Lilian Eugenia

    2012-03-01

    The dehydroleucodine is a sesquiterpene lactone isolated from Artemisia douglasiana Besser which is used in popular medicine. Toxicity tests using embryos of amphibian have been widely used in order to predict toxic effects of different compounds. However, to our knowledge, there are not studies focussed on the toxic effects of dehydroleucodine on Bufo arenarum, which is an anuran widely distributed in South America. The effect of dehydroleucodine on the survival of embryos was evaluated in an acute test during the early life stage of B. arenarum embryos. Lethality and the degree of adverse effects were dehydroleucodine dose-dependent. Overall, amphibian early life stages appeared to be more susceptible to the embryotoxicity associated with exposure to dehydroleucodine, especially at concentration greater that 3mM. This increased susceptibility may result from the relatively high rate of cellular differentiation and morphogenesis that occurs at this early stage of development. Copyright © 2012 Elsevier Ltd. All rights reserved.

  13. The onset of foreign gene transcription in nuclear-transferred embryos of fish

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The transcriptional onset of hGH-transgene in fish was studied in the following three cases: the first is in MThGH-transgenic F4 common carp (Cyprinus carpio) embryos, the second is in nuclear-transferred embryos supported by the transgenic F4 embryonic nuclei, and the third is in nuclear-transferred embryos supported by the transgenic F4 tail-fin nuclei. RT-PCR results show that the hGH-transgene initiates its transcriptional activity from early-gastrula stage, the early blas-tula stage and even 16-cell stage in the first, second and third cases, respectively. It looks like that fish egg cytoplasm could just offer a very restricted reprogramming on transcriptional activity of specific gene in differentiated cell nuclei by nuclear transplantation.

  14. The onset of foreign gene transcription in nuclear-transferred embryos of fish

    Institute of Scientific and Technical Information of China (English)

    孙永华; 陈尚萍; 汪亚平; 朱作言

    2000-01-01

    The transcriptional onset ot hGH-transgene in fish was studied in the following three cases: the first is in MThGH-transgenic F4 common carp (Cyprinus carpio) embryos, the second is in nuclear-transferred embryos supported by the transgenic F4 embryonic nuclei, and the third is in nuclear-transferred embryos supported by the transgenic F4 tail-fin nuclei. RT-PCR results show that the hGH-transgene initiates its transcriptional activity from early-gastrula stage, the early blastula stage and even 16-cell stage in the first, second and third cases, respectively. It looks like that fish egg cytoplasm could just offer a very restricted reprogramming on transcriptional activity of specific gene in differentiated cell nuclei by nuclear transplantation.

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

    Directory of Open Access Journals (Sweden)

    Lisa Shaw

    Full Text Available 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.

  16. Developmental kinetics of the first cell cycles of bovine in vitro PRODUCED EMBRYOS IN RELATION TO THEIR IN VITRO VIABILITY AND SEX

    DEFF Research Database (Denmark)

    Holm, P; Shukri, N.N; Vajta, Gabor

    1998-01-01

    The development of bovine IVP-embryos was observed in a time-lapse culture system to determine cell cycle lengths of 1) embryos that developed into compact morulae (CM) or blastocysts (BL) within 174 h after insemination (viable), 2) embryos that arrested during earlier stages (nonviable) and 3...... were included (n=392), and the times of cleavage events noted. After culture, 100 CM or BL were randomly selected for sexing by PCR. BL developed equally well in the time-lapse and control culture systems (36 vs 38. The respective lengths of the first 4 cell cycles of viable embryos were 32.0 + 3.9, g......) male and female embryos. In 4 replicates, inseminated oocytes were cultured on a microscope stage in 3 to 4 groups on a granulosa cell monolayer in supplemented TCM 199. Images were sequentially recorded and stored at 30-min intervals. All embryos that could be identified throughout the culture period...

  17. Use of cultured rat embryos to evaluate the teratogenic activity of serum: cadmium and cyclophosphamide. [Serum-based culture media for growing rat embryos is used to determine the teratogenicity of cadmium and cyclophosphamide

    Energy Technology Data Exchange (ETDEWEB)

    Klein, N. W.; Vogler, M. A.; Chatot, C. L.; Pierro, L. J.

    1979-01-01

    Head fold stage rat embryos were cultured for 48 hrs in vitro on serum taken at various intervals from rats that had been injected ip with either cadmium or cyclophosphamide. Their response was compared to that of embryos cultured for the same period on control serum to which these substances were added directly. One and 4 hr sera from cadmium injected rats (2.13 mg Cd/sup + +//kg) were lethal. Eight hr serum allowed survival but embryos were exencephalic and contained reduced amounts of protein and DNA. The response to direct cadmium was characteristically different and was related to dosage and the extent to which zero-time embryos had progressed through the head fold stage. At 1.6 ..mu..M, Cd/sup + +/-susceptible embryos were hemorrhagic but not exencephalic. One hr serum from rats given cyclophosphamide (180 mg/kg) was lethal. On 4 hr serum, embryos survived but were exencephalic and contained less protein and DNA than controls. Embryos were resistant to direct cyclophosphamide up to 800 ..mu..g per ml of medium. At this concentration, embryos appeared morphologically normal but contained reduced amounts of protein.

  18. MORPHOLOGICAL CHANGES DURING THE DEVELOPMENT OF SOMATIC EMBRYOS OF SAGO (Metroxylon sagu Rottb.

    Directory of Open Access Journals (Sweden)

    Pauline D. Kasi

    2016-10-01

    Full Text Available Development of somatic embryos of sago (Metroxylon sagu Rottb. on agar-solidified medium are highly varied producing heterogeneous seedlings. Understanding of this phenomenon may help in improving the cultural procedures and conditions of sagosomatic embryogenesis to obtain uniform seedlings in a large scale. This experiment was conducted at the laboratory for plant cell culture and micropropagation, Indonesian Biotechnology Research Institute for Estate Crops from January to March 2006 to examine morphological changes i.e. color and development stages of sago during their somatic embryo development on an agar-solidified medium. Twenty single globular somatic embryos of sago with specific color (yellowish, greenish, and reddish were cultured in a Petri dish supplemented with a solid medium. The medium was a micronutrients-modified MS (MMS with half strength of macronutrients containing 0.01 mg l-1 ABA, 2 mg l-1 kinetin, 20 g l-1 sucrose, 0.5 g l-1 activated charcoal, and 2 g l-1 gelrite. Parameter observed was the percentage of embryo’s number based on color and developmental stage. The result showed that at the end of 6-week culture passage, most originally greenish (80.8% and reddish (95.8% embryos remained unchanged in their colors, whereas almost half of the originally yellowish embryos turned to greenish and only 30%remained yellowish. At the same time, single globular embryos have changed gradually into the next developmental stages, although not all of the embryos were germinated. The initial color of embryo affected the rate of the developmental stage changes. Yellowish and greenish globular embryos developed more rapidly into cotyledon or germinant stages at 58% and 55% respectively, in 6 weeks than the reddish ones (41%. Therefore, the yellowish and greenish embryos are the best sources of material for in vitro mass propagation and synthetic seed production of sago.

  19. Re-analysis by fluorescence in situ hybridisation of spare embryos cultured until Day 5 after preimplantation genetic diagnosis for a 47, XYY infertile patient demonstrates a high incidence of diploid mosaic embryos: a case report.

    Science.gov (United States)

    Emiliani, S; Merino, E G; Van den Bergh, M; Abramowicz, M; Vassart, G; Englert, Y; Delneste, D

    2000-12-01

    Mosaicism in 4-8-cell human embryos analysed by fluorescence in situ hybridisation (FISH) has been widely reported, but few studies have addressed the incidence of mosaicism in more advanced embryonic stages. In the present study we analysed spare human embryos in a case of preimplantation genetic diagnosis (PGD) for increased risk of aneuploidy because of an infertile 47,XYY man. After replacement of two embryos typed as 1818XX at PGD, six spare embryos (not frozen because of their low quality) were re-analysed on Day 5 for PGD confirmation. Out of five embryos typed as 1818XY at PGD, four were diploid mosaic (DM) and one was normal in all cells. The sixth embryo, typed as 18XYY/1818181818X at PGD, was a DM. In spite of the bias of our small series of morphologically low-quality embryos, the surprisingly high proportion of mosaics (which confirms previous findings) questions the validity of PGD, but supports the strategy of transferring only the embryos where two blastomeres gave normal and concordant results at PGD. More data are required to understand the clinical significance of early diploid mosaicism (and its impact on implantation rate) and to determine whether some diploid mosaic embryos might be considered safe for transfer.

  20. Minimally invasive transabdominal collection of preimplantation embryos from the common marmoset monkey (Callithrix jacchus).

    Science.gov (United States)

    Hanazawa, K; Mueller, T; Becker, T; Heistermann, M; Behr, R; Sasaki, E

    2012-09-01

    A novel, minimally invasive, transabdominal embryo collection method (transabdominal method) was developed as an alternative to a standard abdominal incision for embryo collection in the common marmoset. The abdominal incision method was used for 304 flushes using 36 female animals, whereas the transabdominal method was used for 488 flushes using 48 females; successful embryo collection rates were 48.0% and 48.4% (P > 0.05), respectively. These techniques were successfully duplicated at another institute (German Primate Center, DPZ). At that institution, successful embryo collection rates were 88.9% and 77.8% for the abdominal incision and transabdominal methods, respectively (P > 0.05), whereas the average numbers of preimplantation embryos obtained per flush were (mean ± SD) 1.91 ± 0.35 and 1.71 ± 0.14 (P > 0.05). The transabdominal method reduced animal stress, did not require incisional wound healing, and enabled successive embryo recoveries to be done much sooner. More embryos in early developmental stages (zygotes/morulae) were recovered using the transabdominal method (76.1%) than the abdominal incision method (52.6%, P marmoset developmental biology and embryology.

  1. ADAM10 Is Involved in Cell Junction Assembly in Early Porcine Embryo Development.

    Directory of Open Access Journals (Sweden)

    Jeongwoo Kwon

    Full Text Available ADAM10 (A Disintegrin and Metalloprotease domain-containing protein 10 is a cell surface protein with a unique structure possessing both potential adhesion and protease domains. However, the role of ADAM10 in preimplantation stage embryos is not clear. In this study, we examined the expression patterns and functional roles of ADAM10 in porcine parthenotes during preimplantation development. The transcription level of ADAM10 dramatically increased from the morula stage onward. Immunostaining revealed that ADAM10 was present in both the nucleus and cytoplasm in early cleavage stage embryos, and localized to the apical region of the outer cells in morula and blastocyst embryos. Knockdown (KD of ADAM10 using double strand RNA did not alter preimplantation embryo development until morula stage, but resulted in significantly reduced development to blastocyst stage. Moreover, the KD blastocyst showed a decrease in gene expression of adherens and tight junction (AJ/TJ, and an increase in trophectoderm TJ permeability by disrupting TJ assembly. Treatment with an ADAM10 specific chemical inhibitor, GI254023X, at the morula stage also inhibited blastocyst development and led to disruption of TJ assembly. An in situ proximity ligation assay demonstrated direct interaction of ADAM10 with coxsackie virus and adenovirus receptor (CXADR, supporting the involvement of ADAM10 in TJ assembly. In conclusion, our findings strongly suggest that ADADM10 is important for blastocyst formation rather than compaction, particularly for TJ assembly and stabilization in preimplantation porcine parthenogenetic development.

  2. Myoinositol Improves Embryo Development in PCOS Patients Undergoing ICSI

    Science.gov (United States)

    2016-01-01

    The aim of this study was to investigate the activity of myoinositol, in a court of 217 PCOS women undergoing intracytoplasmic sperm injection (ICSI), on pregnancy rate, embryo development, estradiol, and progesterone concentration in blood serum, superoxide dismutase (SOD), and catalase (CAT) in follicular fluid. Concerning the court of patient, 112 (groups I and II) out of 217 were PCOS women, whereas group III consisted of healthy subjects (not PCOS). Group I patients were treated with 400 μg of folic acid per day for 3 months before ICSI, whereas group II patients received 4000 mg of myoinositol and 400 μg of folic acid per day for 3 months before ICSI. Group II revealed a shorter embryo/blastocyst development period between microinjection and 5-cell stage compared to group I. The difference in SOD concentration between groups I and II and between groups II and III was statistically significant. In group II, 34.62% of pregnancies were obtained, whereas in group I this number reached 20% (NS). Myoinositol increased embryo development dynamics and accelerated blastocyst stage reaching time; however, no effect was shown on clinical pregnancy. Furthermore, it restored SOD concentration, lowered in PCOS women, but did not exert any effect on CAT concentration. PMID:27777587

  3. Knee joint morphogenesis of the quail (Coturnix japonica) embryo.

    Science.gov (United States)

    Shojaei, Bahador; Talebhemat, Mahdokht; Hashemnia, Shadi; Shojaeepour, Saeedeh

    2015-01-01

    Knee joint development and its morphogenetic events have been studied in human, chicken and other animal models and differences have been found in the pattern of the knee joint morphogenesis among the studied species. According to the small number of studies which have focused on the chronology of knee morphogenesis, a "morphogenetic timely pattern" is hard to suggest. Quail is an animal model for which there is no information about knee joint morphogenesis. This study was planned to define the time table of the knee joint structures formation in this bird. For this purpose embryonated Japanese quail eggs were incubated for 3 to 12 days. Embryos were removed from their eggs every twelve hours and staged according to Ainsworth et al. The hind limbs of the embryos at the stages 17 to 41 were dissected and 6 μm thick slides were prepared from their knee region. The time of appearance of menisci, ligaments, articular cavity and other knee joint components were identified in the quail embryo. During quail knee morphogenesis we observed the appearance of a three layered interzone, femorotibial cavitation and long bone ossification earlier than in chicken. A hypothesis is presented on the differential role of the flexor and extensor muscles of the knee joint on embryonic knee development in birds as compared with humans.

  4. In vivo Characterization of the Effects of Abscisic Acid and Drying Protocols Associated with the Acquisition of Desiccation Tolerance in Alfalfa (Medicago sativa L.) Somatic Embryos

    OpenAIRE

    SREEDHAR, LEKHA; Wolkers, Willem F.; Hoekstra, Folkert A.; BEWLEY, J. DEREK

    2002-01-01

    Although somatic embryos of alfalfa (Medicago sativa L.) had acquired some tolerance to desiccation at the cotyledonary stage of development (22 d after plating), additional culturing in 20 μm abscisic acid (ABA) for 8 d induced greater desiccation tolerance, as determined by increased germination. Compared with fast drying, slow drying of the ABA‐treated embryos improved desiccation tolerance. However, slow drying of non‐ABA‐treated embryos led to the complete loss of germination capacity, w...

  5. Acid peptidase activity released from in vitro produced porcine embryos: a candidate marker to predict developmental competence.

    Science.gov (United States)

    Telugu, Bhanu Prakash V L; Spate, Lee; Prather, Randall S; Green, Jonathan A

    2009-04-01

    The ability to efficiently create high quality embryos, competent to produce normal viable offspring in vitro, facilitates diverse technological advancements in animal agriculture and assisted reproduction. Current methods for evaluation of embryos are predominantly based on morphological characteristics which are prone to potential bias of the scorer. Metabolic and genetic markers have also been explored for quality assessment, but they are cost prohibitive or require longer periods of time for evaluation. We hypothesized that secreted enzymes could provide another means of embryo quality assessment. In this report, we provide evidence that medium conditioned by porcine embryos often has proteolytic activity that operates in acidic conditions (acid peptidase activity or APA). The APA could be inhibited by pepstatin A, suggesting that the activity is derived from one or more aspartic peptidases. We also provide evidence that single embryos, incubated for as few as 24 hr, released enough APA that it was possible to measure it accurately at day 5 of culture. We also observed that such activity on day 6 could be positively correlated with advanced developmental stage and embryo quality. In addition, those embryos that were graded identically by morphological evaluations often differed in the amount of APA--with some being significantly higher than the experimental threshold value. Therefore, the APA of embryos might serve as an additional marker for evaluation of embryos.

  6. The development al potential of the embryos of wolf spider Xerolycosa nemoralis from areas variously burdened with metals

    Directory of Open Access Journals (Sweden)

    Babczyńska A.

    2013-04-01

    Full Text Available The aim of this study was to assess and describe the developmental potential of the embryos of the wolf spider X. nemoralis collected from the reference site (Pilica and a site heavily polluted with metals (Welnowiec (southern Poland. For the analyses of the progress of development the embryos the computer microtomography was used. Adenylate concentration was measured using luminometric method. The macroscopic observations of the development of individual embryos revealed significant differences in the duration of embryogenesis (14,5 ±0.5 days in Pilica vs 16.5±0.53 in Welnowiec. The computer microtomography analyses enabled us to describe the geometry of the eggs inside the cocoon as well as to see the morphology of embryos inside the cocoon and to recognize selected developmental stages of the embryos. The concentration of cadmium in embryos in Welnowiec was significantly lower than in the embryos from the reference site, while the concentration of copper in the embryos from Pilica was significantly, 4 times lower than in Wełnowiec. The energy status indices (ATP concentration and ADP/ATP ratio did not differ significantly in the embryos from the two sites.

  7. Production of fertile zebrafish (Danio rerio) possessing germ cells (gametes) originated from primordial germ cells recovered from vitrified embryos.

    Science.gov (United States)

    Higaki, Shogo; Eto, Yoshiki; Kawakami, Yutaka; Yamaha, Etsuro; Kagawa, Noriko; Kuwayama, Masashige; Nagano, Masashi; Katagiri, Seiji; Takahashi, Yoshiyuki

    2010-04-01

    This study aimed to produce fertile zebrafish (Danio rerio) possessing germ cells (gametes) that originated from cryopreserved primordial germ cells (PGCs). First, to improve the vitrification procedure of PGCs in segmentation stage embryos, dechorionated yolk-intact and yolk-removed embryos, the PGCs of which were labeled with green fluorescent protein, were cooled rapidly after serial exposures to equilibration solution (ES) and vitrification solution (VS), which contained ethylene glycol, DMSO, and sucrose. Yolk removal well prevented ice formation in the embryos during cooling and improved the viability of cryopreserved PGCs. The maximum recovery rate of live PGCs in the yolk-removed embryos vitrified after optimum exposure to ES and VS was estimated to be about 90%, and about 50% of the live PGCs showed pseudopodial movement. Next, to elucidate the ability of cryopreserved PGCs to differentiate into functional gametes, PGCs recovered from the yolk-removed embryos (striped-type) that were vitrified under the optimum exposure to ES and VS were transplanted individually into 218 sterilized recipient blastulae (golden-type). Two days after the transplantation, 7.5% (14/187) of morphologically normal embryos had PGC(s) in the genital ridges. Six (5 males and 1 female) of the 14 recipient embryos developed into mature fish and generated progeny with characteristics inherited from PGC donors. In conclusion, we demonstrated the successful cryopreservation of PGCs by vitrification of yolk-removed embryos and the production of fertile zebrafish possessing germ cells that originated from the PGCs in vitrified embryos.

  8. Identification of CD146 Expression in Human and Mouse Preimplantation Embryo

    Institute of Scientific and Technical Information of China (English)

    Hong-bo WANG; Xuan DU; Ya-hui XU; Ze-hua WANG

    2008-01-01

    Objective To investigate whether CD146, a cell adhesion molecule, is expressed in mouse and human preimplantation blastocysts and to localize CD146 in the layer of trophectoderm(TE) and/or inner cell mass(ICM). Methods Human and mouse embryos were collected. Using reverse transcription polymerase chain reaction(RT-PCR), the expression of CD146 mRNA in blastocyst was evaluated in human and mouse embryos. Single embryo immunohistochemical staining was applicated in the examination of the expression of CD146 in protein level. The statistical significance of the data was analyzed using t-test. Results CD146 transcript was detected in all human and mouse preimplantation morula and blastocyst. The expression of CD146 was found to localize in human and mouse compacted morula stage embryos and the TE and ICM of the expanded blastocysts. Conclusion mRNA and protein of CD146 was expressed in preimplantation embryos,which may have a profound influence on early preimplantation development for the differentiation of the trophectoderm and the morphogenesis of the blastocyst.Furthermore, the expression of CD146 in blastocyst stage may be implicated in the assistance of embryo implantation.

  9. Prediction model for aneuploidy in early human embryo development revealed by single-cell analysis

    Science.gov (United States)

    Vera-Rodriguez, Maria; Chavez, Shawn L.; Rubio, Carmen; Pera, Renee A. Reijo; Simon, Carlos

    2015-01-01

    Aneuploidies are prevalent in the human embryo and impair proper development, leading to cell cycle arrest. Recent advances in imaging and molecular and genetic analyses are postulated as promising strategies to unveil the mechanisms involved in aneuploidy generation. Here we combine time-lapse, complete chromosomal assessment and single-cell RT–qPCR to simultaneously obtain information from all cells that compose a human embryo until the approximately eight-cell stage (n=85). Our data indicate that the chromosomal status of aneuploid embryos (n=26), including those that are mosaic (n=3), correlates with significant differences in the duration of the first mitotic phase when compared with euploid embryos (n=28). Moreover, gene expression profiling suggests that a subset of genes is differentially expressed in aneuploid embryos during the first 30 h of development. Thus, we propose that the chromosomal fate of an embryo is likely determined as early as the pronuclear stage and may be predicted by a 12-gene transcriptomic signature. PMID:26151134

  10. Developmental Toxicity of Dextromethorphan in Zebrafish Embryos/Larvae

    Science.gov (United States)

    Xu, Zheng; Williams, Frederick E.; Liu, Ming-Cheh

    2012-01-01

    Dextromethorphan is widely used in over-the-counter cough and cold medications. Its efficacy and safety for infants and young children remains to be clarified. The present study was designed to use the zebrafish as a model to investigate the potential toxicity of dextromethorphan during the embryonic and larval development. Three sets of zebrafish embryos/larvae were exposed to dextromethorphan at 24 hours post fertilization (hpf), 48 hpf, and 72 hpf, respectively, during the embryonic/larval development. Compared with the 48 and 72 hpf exposure sets, the embryos/larvae in the 24 hpf exposure set showed much higher mortality rates which increased in a dose-dependent manner. Bradycardia and reduced blood flow were observed for the embryos/larvae treated with increasing concentrations of dextromethorphan. Morphological effects of dextromethorphan exposure, including yolk sac and cardiac edema, craniofacial malformation, lordosis, non-inflated swim bladder, and missing gill, were also more frequent and severe among zebrafish embryos/larvae exposed to dextromethorphan at 24 hpf. Whether the more frequent and severe developmental toxicity of dextromethorphan observed among the embryos/larvae in the 24 hpf exposure set, as compared with the 48 and 72 hpf exposure sets, is due to the developmental expression of the Phase I and Phase II enzymes involved in the metabolism of dextromethorphan remains to be clarified. A reverse transcription-polymerase chain reaction (RT-PCR) analysis, nevertheless, revealed developmental stage-dependent expression of mRNAs encoding SULT3 ST1 and SULT3 ST3, two enzymes previously shown to be capable of sulfating dextrorphan, an active metabolite of dextromethorphan. PMID:20737414

  11. Developmental kinetics of pig embryos by parthenogenetic activation or by handmade cloning.

    Science.gov (United States)

    Li, J; Li, R; Liu, Y; Villemoes, K; Purup, S; Callesen, H

    2013-10-01

    The developmental kinetics of pig embryos produced by parthenogenetic activation without (PAZF) or with (PAZI) zona pellucida or by handmade cloning (HMC) was compared by time-lapse videography. After cumulus cell removal, the matured oocytes were either left zona intact (PAZI) or were made zona free by pronase digestion (PAZF) before they were activated (PA). Other matured oocytes were used for HMC based on foetal fibroblast cells. On Day 0 (day of PA or reconstruction), the embryos were cultured for 7 days in vitro in our time-lapse system. Pictures were taken every 30 min, and afterwards, each cell cycle was identified for each embryo to be analysed. Results showed that the PA embryos (both PAZF and PAZI) had shorter first cell cycle compared with HMC (17.4. 17.8 vs 23.6 h), but had a longer time length from four cell to morula stages (57.9, 53.8 vs 44.9 h). However, at the second cell cycle, PAZF embryos needed shorter time, while PAZI embryos had similar time length as HMC embryos, and both were longer than PAZF (23.4, 24.8 vs 14.6 h). Both PAZF and PAZI embryos used similar time to reach the blastocyst stage, and this was later than HMC embryos. In addition, when all of these embryos were grouped into viable (developed to blastocysts) and non-viable (not developed to blastocysts), the only difference in the time length was observed on the first cell cycle (18.6 vs 24.5 h), but not on the later cell cycles. In conclusion, our results not only give detailed information regarding the time schedule of in vitro-handled pig embryos, but also indicate that the first cell cycle could be used as a selecting marker for embryo viability. However, to evaluate the effect of the produced techniques, the whole time schedule of the pre-implantation developmental kinetics should be observed.

  12. Cysteine modified polyaniline films improve biocompatibility for two cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Yslas, Edith I., E-mail: eyslas@exa.unrc.edu.ar [Departamento de Biología Molecular, Universidad Nacional de Río Cuarto, Agencia Postal Nro3, X580BYA Río Cuarto (Argentina); Cavallo, Pablo; Acevedo, Diego F.; Barbero, César A. [Departamento de Química, Universidad Nacional de Río Cuarto, Agencia Postal Nro3, X580BYA Río Cuarto (Argentina); Rivarola, Viviana A. [Departamento de Biología Molecular, Universidad Nacional de Río Cuarto, Agencia Postal Nro3, X580BYA Río Cuarto (Argentina)

    2015-06-01

    This work focuses on one of the most exciting application areas of conjugated conducting polymers, which is cell culture and tissue engineering. To improve the biocompatibility of conducting polymers we present an easy method that involves the modification of the polymer backbone using L-cysteine. In this publication, we show the synthesis of polyaniline (PANI) films supported onto Polyethylene terephthalate (PET) films, and modified using cysteine (PANI-Cys) in order to generate a biocompatible substrate for cell culture. The PANI-Cys films are characterized by Fourier Transform infrared and UV–visible spectroscopy. The changes in the hydrophilicity of the polymer films after and before the modification were tested using contact angle measurements. After modification the contact angle changes from 86° ± 1 to 90° ± 1, suggesting a more hydrophylic surface. The adhesion properties of LM2 and HaCaT cell lines on the surface of PANI-Cys films in comparison with tissue culture plastic (TCP) are studied. The PANI-Cys film shows better biocompatibility than PANI film for both cell lines. The cell morphologies on the TCP and PANI-Cys film were examined by florescence and Atomic Force Microscopy (AFM). Microscopic observations show normal cellular behavior when PANI-Cys is used as a substrate of both cell lines (HaCaT and LM2) as when they are cultured on TCP. The ability of these PANI-Cys films to support cell attachment and growth indicates their potential use as biocompatible surfaces and in tissue engineering. - Highlights: • A new surface PANI-Cys was produced on films of polyethylene terephthalate. • The relationship between surface characteristics and biocompatibility is analyzed. • The PANI-Cys film presents good biocompatibility for two cell lines.

  13. The effect of hepatocyte growth factor on mouse oocyte in vitro maturation and subsequent fertilization and embryo development

    Directory of Open Access Journals (Sweden)

    Mohammad H. Bahadori

    2011-05-01

    Full Text Available Background: Oocyte invitro maturation is an enormously promising technology for the treatment of infertility, yet its clinical application remains limited owing to poor success rates. Therefore, this study was devised to evaluate the effect of hepatocyte growth factor (HGF on in vitro maturation of immature mouse oocytes and resulting embryos development. Materials and Method: Cumulus – oocyte complex and germinal vesicle were obtained from eighteen 6-8 weeks-old female NMRI mice 46-48 hours after administration of an injection of 5 IU PMSG (Pregnant Mares’ Serum Gonadotrophin. Oocytes were culture in TCM199 (Tissue culture medium-199 supplemented with dosages of 0, 10, 20, 50 and 100 ng/ml of HGF. After 24 hours, metaphase ІІ oocytes were co-incubated with sperms for 4-6 hours in T6 medium. Following isolation of two pronucleus embryos, cleavage of embryos was assessed in the same medium till blastocyst stage. The number of oocytes and embryos was recorded under an invert microscope and the rate of oocyte maturation, fertilization and embryos cleavage until blastocyst stage compared using of student χ2 test. Results: In all compared groups, oocytes growth and embryos development rate in the 20 ng/ml of HGF treatment group was significantly higher (p<0.05 than the control group (p<0.05.Conclusion: 20 ng/ml of HGF improved the nuclear maturation and embryo development up to blastocyst stage during culture condition

  14. Role of reactive oxygen species in diabetes-induced embryotoxicity: studies on pre-implantation mouse embryos cultured in serum from diabetic pregnant women.

    Science.gov (United States)

    Ornoy, A; Kimyagarov, D; Yaffee, P; Abir, R; Raz, I; Kohen, R

    1996-11-01

    Sera from diabetic patients or sera with high levels of diabetic metabolic products, were found to affect mouse and rat blastocysts. In the present study we examined the earliest developmental stages at which human diabetic serum will be lethal to mouse pre-implantation embryos, and whether reactive oxygen species (ROS) are involved in these diabetes-induced injuries. We cultured 2-4 cell-stage embryos and blastocysts in a medium containing 30 or 50% serum obtained from pregnant women with diabetes Type I, Type II and gestational diabetes (GDM) for 72 h. The development of the 2-4 cell-stage embryos was delayed when cultured in 30% diabetic serum, but the viability was impaired to a lesser extent. Viability was reduced in blastocysts cultured in 50% diabetic serum, but the development of the living embryos was not delayed. Cyclic voltametry measures the oxidation potential of the tissue and the concentration of antioxidants, thus reflecting the total antioxidative activity of the embryos. Pre-implantation embryos cultured in diabetic serum had a lower concentration of antioxidants than embryos cultured in non-diabetic serum. It seems, therefore, that diabetic metabolic factors may induce embryotoxicity in pre-implantation embryos through derangement of the antioxidant defense mechanism. A similar mechanism is suggested for the diabetes-induced teratogenicity in post-implantation embryos.

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

    Science.gov (United States)

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

    2013-11-01

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

  16. Micronucleus formation causes perpetual unilateral chromosome inheritance in mouse embryos.

    Science.gov (United States)

    Vázquez-Diez, Cayetana; Yamagata, Kazuo; Trivedi, Shardul; Haverfield, Jenna; FitzHarris, Greg

    2016-01-19

    Chromosome segregation defects in cancer cells lead to encapsulation of chromosomes in micronuclei (MN), small nucleus-like structures within which dangerous DNA rearrangements termed chromothripsis can occur. Here we uncover a strikingly different consequence of MN formation in preimplantation development. We find that chromosomes from within MN become damaged and fail to support a functional kinetochore. MN are therefore not segregated, but are instead inherited by one of the two daughter cells. We find that the same MN can be inherited several times without rejoining the principal nucleus and without altering the kinetics of cell divisions. MN motion is passive, resulting in an even distribution of MN across the first two cell lineages. We propose that perpetual unilateral MN inheritance constitutes an unexpected mode of chromosome missegregation, which could contribute to the high frequency of aneuploid cells in mammalian embryos, but simultaneously may serve to insulate the early embryonic genome from chromothripsis.

  17. Embryo donation in Iran: an ethical review.

    Science.gov (United States)

    Afshar, Leila; Bagheri, Alireza

    2013-12-01

    Iran is the only Muslim country that has legislation on embryo donation, adopted in 2003. With an estimated 10-15% of couples in the country that are infertile, there are not any legal or religious barriers that prohibit an infertile couple from taking advantage of Assisted Reproductive Technologies (ARTs). Although all forms of ARTs available in Iran have been legitimized by religious authorities, there is a lack of legislation in all ARTs except embryo donation. By highlighting ethical issues in embryo donation, the paper presents a critical review of the Act of Embryo Donation in Iran. The paper argues that the Act does not provide enough safeguards for the future child and assurance for the safety of the donated embryos. It also does not restrict embryo donation to surplus embryos from infertile couples and is silent about the number of embryos that could be donated by each couple as well as the number of recipients for donated embryos by a couple. The Act is also silent about the issues of genetic linkage (nasab) and heritage which are challenging issues, especially in a conservative Islamic society. As a result, the future child may not inherit from their birth parents, as it is not required by the Act, or from the genetically related parents under the anonymity policy. Finally there is no standard national protocol or guidelines to evaluate the safety of the donated embryos. The paper concludes that despite its benefits, the Act lacks clarity, and it is subject to misunderstanding and confusion.

  18. Early developing pig embryos mediate their own environment in the maternal tract.

    Directory of Open Access Journals (Sweden)

    Carmen Almiñana

    Full Text Available The maternal tract plays a critical role in the success of early embryonic development providing an optimal environment for establishment and maintenance of pregnancy. Preparation of this environment requires an intimate dialogue between the embryo and her mother. However, many intriguing aspects remain unknown in this unique communication system. To advance our understanding of the process by which a blastocyst is accepted by the endometrium and better address the clinical challenges of infertility and pregnancy failure, it is imperative to decipher this complex molecular dialogue. The objective of the present work is to define the local response of the maternal tract towards the embryo during the earliest stages of pregnancy. We used a novel in vivo experimental model that eliminated genetic variability and individual differences, followed by Affymetrix microarray to identify the signals involved in this embryo-maternal dialogue. Using laparoscopic insemination one oviduct of a sow was inseminated with spermatozoa and the contralateral oviduct was injected with diluent. This model allowed us to obtain samples from the oviduct and the tip of the uterine horn containing either embryos or oocytes from the same sow. Microarray analysis showed that most of the transcripts differentially expressed were down-regulated in the uterine horn in response to blastocysts when compared to oocytes. Many of the transcripts altered in response to the embryo in the uterine horn were related to the immune system. We used an in silico mathematical model to demonstrate the role of the embryo as a modulator of the immune system. This model revealed that relatively modest changes induced by the presence of the embryo could modulate the maternal immune response. These findings suggested that the presence of the embryo might regulate the immune system in the maternal tract to allow the refractory uterus to tolerate the embryo and support its development.

  19. New technology for vitrification and field (microscope-free) warming and transfer of small ruminant embryos.

    Science.gov (United States)

    Isachenko, Vladimir; Alabart, Jose Luis; Dattena, Maria; Nawroth, Frank; Cappai, Pietro; Isachenko, Eugenia; Cocero, Maria Jesus; Olivera, Julio; Roche, Alberto; Accardo, Carla; Krivokharchenko, Alexander; Folch, Jose

    2003-03-01

    This study was designed to test the efficiency of recently developed vitrification technology followed by microscope-free thawing and transfer of sheep embryos. In a first set of experiments, in vivo derived embryos at the morula to blastocyst stage were frozen in an automated freezer in ethylene glycol, and after thawing and removal of cryoprotectants, were transferred to recipient ewes according to a standard protocol (control group). A second group of embryos were loaded into open-pulled straws (OPS) and plunged into liquid nitrogen after exposure at room temperature to the media: 10% glycerol (G) for 5 min, 10% G+20% ethylene glycol (EG) for 5 min, 25% G+25% EG for 30s; or 10% EG+10% DMSO for 3 min, 20% EG+20% DMSO+0.3M trehalose for 30s. The OPS were thawed by plunging into tubes containing 0.5M trehalose. After this rapid thawing, the embryos were directly transferred using OPS as the catheter for the transplantation process. In a second set of experiments, in vivo derived and in vitro produced expanded blastocysts were vitrified in OPS and then transferred as described above. The lambing rates recorded (59% for the conventionally cryopreserved in vivo derived embryos, 56% for the vitrified in vivo derived embryos, and 20% for the vitrified in vitro produced embryos), suggest the suitability of the vitrification technique for the transfer of embryos obtained both in vivo and in vitro. This simple technology gives rise to a high embryo survival rate and will no doubt have applications in rearing sheep or other small ruminants.

  20. Comparative effects of zinc oxide nanoparticles and dissolved zinc on zebrafish embryos and eleuthero-embryos: importance of zinc ions.

    Science.gov (United States)

    Brun, Nadja Rebecca; Lenz, Markus; Wehrli, Bernhard; Fent, Karl

    2014-04-01

    The increasing use of zinc oxide nanoparticles (nZnO) and their associated environmental occurrence make it necessary to assess their potential effects on aquatic organisms. Upon water contact, nZnO dissolve partially to zinc (Zn(II)). To date it is not yet completely understood, whether effects of nZnO are solely or partly due to dissolved Zn(II). Here we compare potential effects of 0.2, 1 and 5mg/L nZnO and corresponding concentrations of released Zn(II) by water soluble ZnCl2 to two development stages of zebrafish, embryos and eleuthero-embryos, by analysing expressional changes by RT-qPCR. Another objective was to assess uptake and tissue distribution of Zn(II). Laser ablation-ICP-MS analysis demonstrated that uptake and tissue distribution of Zn(II) were identical for nZnO and ZnCl2 in eleuthero-embryos. Zn(II) was found particularly in the retina/pigment layer of eyes and brain. Both nZnO and dissolved Zn(II) derived from ZnCl2 had similar inhibiting effects on hatching, and they induced similar expressional changes of target genes. At 72hours post fertilization (hpf), both nZnO and Zn(II) delayed hatching at all doses, and inhibited hatching at 1 and 5 mg/L at 96 hpf. Both nZnO and Zn(II) lead to induction of metallothionein (mt2) in both embryos and eleuthero-embryos at all concentrations. Transcripts of oxidative stress related genes cat and Cu/Zn sod were also altered. Moreover, we show for the first time that nZnO exposure results in transcriptional changes of pro-inflammatory cytokines IL-1β and TNFα. Overall, transcriptional alterations were higher in embryos than eleuthero-embryos. The similarities of the effects lead to the conclusion that effects of nZnO are mainly related to the release of Zn(II).

  1. The Chromosomal Constitution of Embryos Arising from Monopronuclear Oocytes in Programmes of Assisted Reproduction

    Directory of Open Access Journals (Sweden)

    Bernd Rosenbusch

    2014-01-01

    Full Text Available The assessment of oocytes showing only one pronucleus during assisted reproduction is associated with uncertainty. A compilation of data on the genetic constitution of different developmental stages shows that affected oocytes are able to develop into haploid, diploid, and mosaic embryos with more or less complex chromosomal compositions. In the majority of cases (~80%, haploidy appears to be caused by gynogenesis, whereas parthenogenesis or androgenesis is less common. Most of the diploid embryos result from a fertilization event involving asynchronous formation of the two pronuclei or pronuclear fusion at a very early