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Transcription factor AP2 gamma (TFAP2C) is a well-established regulator of the trophoblast lineage in mice and humans, but a handful of studies indicate that TFAP2C may play an important role in pluripotency. Here, we hypothesize and provide new evidence that TFAP2C functions as an activator of trophoblast and pluripotency genes during preimplantation embryo development. [ABSTRACT FROM AUTHOR]

For the intracytoplasmic sperm injection (ICSI) procedure in pigs, an electrical pulse (EP) has been used as an effective method for oocyte stimulation, but unlike sperm, EP is unable to induce Ca2+ oscillations. In this study, we investigated the effects of generating artificial Ca2+ oscillations with phospholipase Cζ (PLCZ) mRNA, a candidate sperm factor, on fertilization, embryonic development, and gene expression after ICSI. Firstly, the concentration of PLCζ mRNA of a fixed volume (1.0 pl) that would induce a pattern of Ca2+ rise similar to that of in vitro fertilized (IVF) sperm was examined and determined to be 300 ng/µl. Secondly, the effects of oocyte stimulation methods on fertilization and embryonic development were investigated. ICSI-oocytes were activated by EP (EP group) or by PLCζ mRNA (PLCζ group). Furthermore, IVF-oocytes (IVF group) and ICSI-oocytes with and without an injection of buffer (buffer and untreated groups, respectively) were used as controls. It was found that the rates of normal fertilization in the PLCζ and EP groups were significantly higher than those in the buffer and untreated groups. The blastocyst formation rates did not differ among the groups. The embryo quality in the EP group was inferior to those in the PLCζ and IVF groups. Additionally, the expression level of a proapoptosis-related gene (Caspase-3) in the EP group was significantly higher than those in the PLCζ and IVF groups. Our data suggest that oocyte activation by PLCζ mRNA has the effect of improving embryo quality. [ABSTRACT FROM AUTHOR]

Tead4, a critical transcription factor expressed during preimplantation development, is essential for the expression of trophectoderm-specific genes in mice. However, the functional mechanism of TEAD4 in mouse preimplantation development and its conservation across mammals remain unclear. Here, we report that Tead4 is a crucial transcription factor necessary for blastocyst formation in mice. Disruption of Tead4 through base editing results in developmental arrest at the morula stage. Additionally, RNA-seq analysis reveals dysregulation of 670 genes in Tead4 knockout embryos. As anticipated, Tead4 knockout led to a decrease in trophectoderm genes Cdx2 and Gata3. Intriguingly, we observed a reduction in Krt8, suggesting that Tead4 influences the integrity of the trophectoderm epithelium in mice. More importantly, we noted a dramatic decrease in nuclear Yap in outside cells for Tead4-deficient morula, indicating that Tead4 directly regulates Hippo signaling. In contrast, bovine embryos with TEAD4 depletion could still develop to blastocysts with normal expression of CDX2, GATA3, and SOX2, albeit with a decrease in total cell number and ICM cell number. In conclusion, we propose that Tead4 regulates mouse blastocyst formation via Krt8 and Yap, both of which are critical regulators of mouse preimplantation development. [ABSTRACT FROM AUTHOR]

Context. In vitro maturation is an important process in the production of embryos. It has been shown that three cytokines, fibroblast growth factor 2, leukemia inhibitory factor and insulin-like growth factor 1 (FLI), increased efficiency of in vitro maturation,somatic cell nucleartransfer(SCNT) blastocyst production, and in vivo development of genetically engineered piglets. Aims. Assess effects of FLI on oocyte maturation, quality of oocytes, and embryo development in bovine in vitro fertilisation (IVF) and SCNT. Key results. Cytokine supplementation resulted in significant increases in maturation rates and decreased levels of reactive oxygen species. Oocytes matured in FLI had increased blastocyst rates when used in IVF (35.6% vs 27.3%, P < 0.05) and SCNT (40.6% vs 25.7%, P < 0.05). SCNT blastocysts contained significantly more inner cell mass and trophectodermal cells when compared to the control group. Importantly, SCNT embryos derived from oocytes matured in FLI medium resulted in a four-fold increase in full-term development compared to control medium (23.3% vs 5.3%, P < 0.05). Relative mRNA expression analysis of 37 genes associated with embryonic and fetal development revealed one gene had differential transcript abundance in metaphase II oocytes, nine genes at the 8-cell stage, 10 genes at the blastocyst stage in IVF embryos and four genes at the blastocyst stage in SCNT embryos. Conclusions. Cytokine supplementation increased efficiency of in vitro production of IVF and SCNT embryos and in vivo development of SCNT embryos to term. Implications. Cytokine supplementation is beneficial to embryo culture systems, which may shed light on requirements of early embryo development. [ABSTRACT FROM AUTHOR]

STUDY QUESTION Can recurrent embryo developmental problems after ICSI be overcome by assisted oocyte activation (AOA)? SUMMARY ANSWER AOA did not improve blastocyst formation in our patient cohort with recurrent embryo developmental problems after ICSI. WHAT IS KNOWN ALREADY The use of AOA to artificially induce calcium (Ca2+) rises by using Ca2+ ionophores (mainly calcimycin and ionomycin) has been reported as very effective in overcoming fertilization failure after ICSI, especially in patients whose Ca2+ dynamics during fertilization are deficient. However, there is only scarce and contradictory literature on the use of AOA to overcome embryo developmental problems after ICSI, and it is not clear whether abnormal Ca2+ patterns during fertilization disturb human preimplantation embryo development. Moreover, poor embryo development after ICSI has also been linked to genetic defects in the subcortical maternal complex (SCMC) genes. STUDY DESIGN, SIZE, DURATION This prospective cohort single-center study compared ICSI-AOA cycles and previous ICSI cycles in couples with normal fertilization rates (≥60%) but impaired embryonic development (≤15% blastocyst formation) in at least two previous ICSI cycles. In total, 42 couples with embryo developmental problems were included in this study from January 2018 to January 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS Of the 42 couples included, 17 underwent an ICSI-AOA cycle consisting of CaCl2 injection and double ionomycin exposure. Fertilization, blastocyst development, pregnancy, and live birth rates after ICSI-AOA were compared to previous ICSI cycles. In addition, the calcium pattern induced by the male patient's sperm was investigated by mouse oocyte calcium analysis. Furthermore, all 42 couples underwent genetic screening. Female patients were screened for SCMC genes (TLE6 , PADI6 , NLRP2 , NLRP5 , NLRP7 , and KHDC3L) and male patients were screened for the sperm–oocyte-activating factor PLCZ1. MAIN RESULTS AND THE ROLE OF CHANCE We compared 17 AOA cycles to 44 previous ICSI cycles from the same patient cohort. After AOA, a total fertilization rate of 68.95% (131/190), a blastocyst development rate of 13.74% (18/131), a pregnancy rate of 29.41% (5/17), and a live birth rate of 23.53% (4/17) were achieved, which was not different from the previous ICSI cycles (76.25% (321/421, P -value = 0.06); 9.35% (30/321, P -value = 0.18), 25.00% (11/44, P -value = 0.75), and 15.91% (7/44, P -value = 0.48), respectively). Calcium analysis showed that patient's sperm induced calcium patterns similar to control sperm samples displaying normal embryo developmental potential. Genetic screening revealed 10 unique heterozygous variants (in NLRP2 , NLRP5 , NLRP7 , TLE6 , and PADI6) of uncertain significance (VUS) in 14 females. Variant NLRP5 c.623-12_623-11insTTC (p.?) was identified in two unrelated individuals and variant NLRP2 c.1572T>C (p.Asp524=) was identified in four females. Interestingly, we identified a previously reported homozygous mutation PLCZ1 , c.1499C>T (p.Ser500Leu), in a male patient displaying impaired embryonic development, but not showing typical fertilization failure. LIMITATIONS, REASONS FOR CAUTION Our strict inclusion criteria, requiring at least two ICSI cycles with impaired embryo development, reduced cycle-to-cycle variability, while the requirement of a lower blastocyst development not influenced by a poor fertilization excluded couples who otherwise would be selective cases for AOA; however, these criteria limited the sample size of this study. Targeted genetic screening might be too restricted to identify a genetic cause underlying the phenotype of poor embryo development for all patients. Moreover, causality of the identified VUS should be further determined. WIDER IMPLICATIONS OF THE FINDINGS Strong evidence for AOA overcoming impaired embryonic development is still lacking in the literature. Thus far, only one article has reported a beneficial effect of AOA (using calcimycin) compared to previous ICSI cycles in this patient population, whilst two more recent sibling-oocyte control studies (one using calcimycin and the other ionomycin) and our research (using ionomycin) could not corroborate these findings. Although no major abnormalities have been found in children born after AOA, this technique should be reserved for couples with a clear Ca2+-release deficiency. Finally, genetic screening by whole-exome sequencing may reveal novel genes and variants linked to embryo developmental problems and allow the design of more personalized treatment options, such as wild-type complementary RNA or recombinant protein injection. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by the Flemish Fund for Scientific Research (grant FWO.OPR.2015.0032.01 to B.H. and grant no. 1298722N to A.B.). A.C.B. D.B. A.B. V.T. R.P. F.M. I.D.C. L.L. D.S. P.D.S. P.C. and F.V.M. have nothing to disclose. B.H. reports a research grant from the Flemish Fund for Scientific Research and reports being a board member of the Belgian Society for Reproductive Medicine and the Belgian Ethical Committee on embryo research. TRIAL REGISTRATION NUMBER NCT03354013 [ABSTRACT FROM AUTHOR]

Current understandings of the initiation of the trophectoderm (TE) program during mammalian embryonic development lack evidence of how TE-associated factors such as GATA3 and CDX2 participate in bovine lineage specification. In this study, we describe the effects of TE-associated factors on the expression of lineage specification marker genes such as SOX2, OCT4, NANOG, GATA6, and SOX17, by using cytosine base editor system. We successfully knockout GATA3 or CDX2 in bovine embryos with a robust efficiency. However, GATA3 or CDX2 deletion does not affect the developmental potential of embryos to reach the blastocyst stage. Interestingly, GATA3 deletion downregulates the NANOG expression in bovine blastocysts. Further analysis of the mosaic embryos shows that GATA3 is required for NANOG in the TE of bovine blastocysts. Single blastocyst RNA-seq analysis reveals that GATA3 deletion disrupts the transcriptome in bovine blastocysts. Altogether, we propose that GATA3 plays an important role in maintaining TE lineage program in bovine embryos and the functional role of GATA3 is species-specific. [ABSTRACT FROM AUTHOR]

Protein synthesis is an essential step in gene expression during the development of mammalian preimplantation embryos. This is a complex and highly regulated process. The accuracy of the translation initiation codon is important in various gene expression programs. However, the mechanisms that regulate AUG and non-AUG codon initiation in early embryos remain poorly understood. BZW1 is a key factor in determining the mRNA translation start codon. Here, we show that BZW1 is essential for early embryonic development in mice. Bzw1-knockdown embryos fail to undergo compaction, and show decreased blastocyst formation rates. We also observe defects in the differentiation capacity and implantation potential after Bzw1 interference. Further investigation revealed that Bzw1 knockdown causes the levels of translation initiation with CUG as the start codon to increase. The decline in BZW1 levels result in a decrease in protein synthesis in preimplantation embryos, whereas the total mRNA levels are not altered. Therefore, we concluded that BZW1 contributes to protein synthesis during early embryonic development by restricting non-AUG translational initiation. It is unclear how translational efficiency and codon stringency affect zygotic genome activation and early embryonic development. Here they show that the zygotic gene BZW1 increases start codon stringency, which is important for preimplantation development. [ABSTRACT FROM AUTHOR]

Objective: The objective of the study was to evaluate the impact of artificial oocyte activation (AOA) with calcium ionophore (A23187) on in vitro fertilization outcomes of patients with previous history of cycle cancellations due to arrested embryos before formation of day 5 blastocysts. Material and Methods: This retrospective study was conducted by evaluating records of patients that admitted for in vitro fertilization between 2013 and 2021. Patients with the previous history of developmental arrest in whole embryo cohort without fertilization failure were included in the study. Cycle outcomes of women with and without AOA were compared. Results: A total of 347 women were found eligible and included in the study. One hundred and eighty-five of them were treated with AOA that constituted the study group and 162 women were included in the control group. Rate of good quality embryos (65.3% vs. 31.1%; p<0.001), implantation rates (0.33±0.41 vs. 0.22±0.39; p=0.003), clinical pregnancy rates (43.2% vs. 25.3%; p<0.001), and live birth delivery rates (40.5% vs. 21%; p<0.001) were higher in women treated with AOA with calcium ionophore in comparison to controls. Rate of multiple pregnancy was also found higher in calcium ionophore group (5.4% vs. 1.9%; p=0.01). Mean infant birth weights were similar among groups. Conclusion: AOA with A23187 appears to improve progression to blastocyst rates, embryo quality, clinical pregnancy rates, and live birth delivery rates in women with a previous history of complete embryo development arrest before formation of day 5 blastocysts. [ABSTRACT FROM AUTHOR]

Background: Artificial oocyte activation (AOA) is used to improve fertilization rate following fertilization failure after intracytoplasmic sperm injection (ICSI). Several studies have also shown that AOA may be involved in embryo development. Women with poor ovarian response are more likely to encounter in vitro fertilization (IVF) failure due to poor embryo quality. The aim of this study was to investigate whether AOA could improve embryo quality in older patients with diminished ovarian reserve undergoing IVF-ICSI cycles. Methods: The retrospective cohort study consisted of 308 patients who fulfilled the POSEIDON Group 4 criteria and received IVF-ICSI cycles. The study group included 91 patients receiving AOA with calcium ionophores following ICSI. A total of 168 patients in the control group underwent ICSI without AOA. The baseline and cycle characteristics and embryo quality were compared between the two groups. Results: At baseline, there were more IVF attempts, greater primary infertility, higher basal FSH levels and lower anti-Müllerian hormone (AMH) levels in the AOA group than in the non-AOA group. In terms of embryo quality, there were higher cleavage rates and top-quality Day 3 embryo (TQE) rates, as well as higher percentages of more than 1 TQE and TQE rates ≥50 in the AOA group than in the non-AOA group. The multivariate analysis revealed that AOA was positively associated with more than 1 TQE (adjusted OR 3.24, 95% CI 1.63–6.45, P = 0.001) and a TQE rate ≥ 50 (adjusted OR 2.14, 95% CI 1.20–3.80, P = 0.010). When the study population was divided into 2 subgroups based on the age of 40 years old, the beneficial effects of AOA on embryo quality were only observed in the subgroup of age ≥ 40 years old. Conclusions: Our data suggest that AOA with calcium ionophores may improve embryo quality in older patients with diminished ovarian reserve undergoing IVF-ICSI cycles, especially in women aged ≥40 years. [ABSTRACT FROM AUTHOR]

Context: Male exposure to environmental toxicants can disrupt spermatogenesis and sperm function. However, consequences of environmentally relevant organotin exposure to post-ejaculatory mammalian spermatozoa on fertility are poorly understood. Aims: Determine the consequences of tributyltin chloride (TBT) exposure on post-ejaculatory sperm function and subsequent embryo development. Methods: Frozen-thawed bovine sperm were exposed to TBT (0.1–100 nM) for 90 min (acute) and 6 h (short-term) followed by quantification of multiple sperm kinematics via computer aided sperm analysis. JC-1 dye was used to measure mitochondrial membrane potential. Sperm were then exposed to TBT for 90 min in non-capacitating conditions, washed several times by centrifugation and applied to gamete co-incubation for in vitro embryo production to the blastocyst stage. Key results: 100 nM TBT decreased total motility (88 vs 79%), progressive motility (80 vs 70%) curvilinear velocity and beat-cross frequency for 90 min with similar phenotypes at 6 h (P < 0.05). Sperm mitochondrial membrane potential was lower in 10 and 100 nM groups after 6 h (P ≤ 0.05). Embryos fertilised from TBT-exposed sperm had reduced cleavage rate (80 vs 62%) and 8–16 cell morula development (55 vs 24%) compared to development from unexposed sperm. Conclusions: Exposure of post-ejaculatory mammalian sperm to TBT alters sperm function through lowered motility and mitochondrial membrane potential. Fertilisation of oocytes with TBT-exposed sperm reduces embryo development through mechanisms of paternal origin. Implications: Acute and short-term environmental exposure of post-ejaculatory sperm to organotins and endocrine disrupting chemicals such as TBT contribute to idiopathic subfertility and early embryo loss. Environmental contaminant exposure to humans and animals is a global concern for negative effects on reproductive health and fertility. Consequences of the common and environmentally stable compound tributyltin chloride on post-ejaculatory sperm exposure were previously unknown and established herein to alter sperm function through reducing embryo development. These findings indicate that short-term exposure to environmental contaminants impact male fertility through a mechanism of paternal origin that negatively alters embryo development. [ABSTRACT FROM AUTHOR]

Context: Maternal-effect genes (MEGs) play a critical role in modulating both cellular and molecular biology events in preimplantation embryonic development. Damage-specific DNA binding protein 1 (DDB1) is a gene that participates in meiotic resumption, ovulation, and embryonic stem cell maintenance. Its function in preimplantation development is not well-studied. Aims: We aimed to explore the expression pattern, genomic heritage, and potential molecular mechanisms of DDB1 in preimplantation embryos in porcine. Methods: In this study, RNA interference, microinjection, RT-qPCR, immunofluorescence staining and single-cell RNA sequencing were used to explore the molecular function of DDB1 in porcine preimplantation embryos. Key results: DDB1 was found to be expressed in germinal vesicle (GV) and Meiosis II (MII) oocytes and in preimplantation embryos. We confirmed it is a MEG. DDB1 -deficient blastocysts had a significantly reduced number of trophectoderm cells, an increased apoptotic cell number and increased apoptosis index. According to a next-generation sequencing (NGS) analysis, 236 genes (131 upregulated and 105 downregulated) significantly changed in the DDB1 -deficient morula. The myeloid leukaemia factor 1 (MLF1) and yes-associated protein 1 (YAP1) expressions were significantly upregulated and downregulated respectively, in the DDB1 -deficient morula. In combination with the decreased expression of TEAD4 , CDX2 , GATA3 , OCT4 , and NANOG and the increased expression of SOX2 in the blastocyst, DDB1 may play a role in determining lineage differentiation and pluripotency maintenance. Conclusions: DDB1 is a MEG and it plays a crucial role in porcine preimplantation embryonic development. Implications: This study provides a theoretical basis for further understanding the molecular mechanisms of preimplantation embryo development. DDB1 is a gene that participates in meiotic resumption, ovulation, and embryonic stem cell maintenance. It is produced from maternal origin transcripts in early porcine embryos. DDB1 -deficiency enhanced cellular apoptosis in the blastocyst. Lack of DDB1 impaired trophectoderm formation and the pluripotency maintenance of blastocyst. DDB1 is a MEG, which plays a the crucial role of in porcine preimplantation embryonic development. [ABSTRACT FROM AUTHOR]

Cryopreservation is a process in which the intact living cells, tissues, or embryos are preserved at subzero temperatures for preservation. The cryopreservation process highly impacts the survival and quality of the in vitro-produced (IVP) embryos. Some studies have highlighted the use of oviduct extracellular vesicles (EVs) to improve the cryotolerance of IVP embryos but the mechanism has not been well studied. The present study unravels the role of in vitro cultured bovine oviduct epithelial cells-derived EVs in improving the re-expansion and hatching potential of thawed blastocysts (BLs). The comparison of cryotolerance between synthetic oviduct fluid (SOF) and SOF + EVs-supplemented day-7 cryopreserved BLs revealed that the embryo's ability to re-expand critically depends on the intact paracellular sealing which facilitates increased fluid accumulation during cavity expansion after shrinkage. Our results demonstrated that BLs cultured in the SOF + EVs group had remarkably higher re-expansion (67.5 ± 4.2%) and hatching rate (84.8 ± 1.4%) compared to the SOF group (53.4 ± 3.4% and 63.9 ± 0.9%, respectively). Interestingly, EVssupplemented BLs exhibited greater influence on the expression of core genes involved in trophectoderm (TE) maintenance, formation of tight junction (TJ) assembly, H2O channel proteins (aquaporins), and Na+/K+ ATPase alpha 1. The EVs improved the fluid flux and allowed the transport of H2O into an actively re-expanded cavity in EVs-cultured cryo-survived BLs relative to control BLs. Our findings explored the function of EVs in restoring the TE integrity, improved the cell junctional contacts and H2O movement which helps the blastocoel re-expansion after thawing the cryopreserved BLs. [ABSTRACT FROM AUTHOR]

Oocytes reconstructed by spindle transfer (ST) are prone to chromosome abnormality, which is speculated to be caused by mechanical interference or premature activation, the mechanism is controversial. In this study, C57BL/6N oocytes were used as the model, and electrofusion ST was performed under normal conditions, Ca2+ free, and at room temperature, respectively. The effect of enucleation and electrofusion stimulation on MPF activity, spindle morphology, γ-tubulin localization and chromosome arrangement was compared. We found that electrofusion stimulation could induce premature chromosome separation and abnormal spindle morphology and assembly by decreasing the MPF activity, leading to premature activation, and thus resulting in chromosome abnormality in oocytes reconstructed via ST. Electrofusion stimulation was an independent factor of chromosome abnormality in oocytes reconstructed via ST, and was not related to enucleation, fusion status, temperature, or Ca2+. The electrofusion stimulation number should be minimized, with no more than 2 times being appropriate. As the electrofusion stimulation number increased, several typical abnormalities in chromosome arrangement and spindle assembly occurred. Although blastocyst culture could eliminate embryos with chromosomal abnormalities, it would significantly decrease the number of normal embryos and reduce the availability of embryos. The optimum operating condition for electrofusion ST was the 37°C group without Ca2+. [ABSTRACT FROM AUTHOR]

Dehydroepiandrosterone sulphate (DHEAS) concentrations stringently regulate fertilisation, embryo development and IVF outcomes: are we looking at a potentially compelling "oocyte-related factor" in oocyte activation? Keywords: DHEAS; Oocyte activation; Poor responder; PCOS; IVF EN DHEAS Oocyte activation Poor responder PCOS IVF 193 202 10 02/01/21 20210101 NES 210101 Introduction Generation of calcium ions (Ca SP 2+ sp ) is a pre-requisite for the oocyte activation process. Although present in cells surrounding the oocyte (therefore our term "oocyte-related"), the negatively charged sulphate group (SO SB 4 sb SP 2- sp ) may help DHEAS exert its effect either by acting via putative oocyte receptors [[20]] or by transporting across oocyte membrane through sodium-dependent organic anion transporters (SOAT) [[22]]. Metformin treatment was offered to this subgroup women in agreement with a report suggesting that metformin significantly lowers DHEAS as well as insulin levels in PCOS women with raised baseline DHEAS whereas it apparently has no effect on DHEAS levels in women with normal baseline DHEAS concentrations [[27]]. [Extracted from the article]

Tris(1,3‐dichloro‐2‐propyl) phosphate (TDCPP) is a kind of additive flame retardants (FRs) and was found to affect early embryonic development in zebrafish; however, there are few studies to investigate whether TDCPP will disturb the development of early mouse embryos. In our studies, we used mouse embryos as models to study the toxicology of TDCPP on the early embryos. The results showed that TDCPP disturbed the development of early mouse embryos in a dose‐dependent manner. 10 μM TDCPP decreased the blastocyst formation and 100 μM TDCPP was a lethal concentration for the mouse embryos. We proved that TDCPP was detrimental to embryonic development potential by increasing the reactive oxygen species level and inducing early apoptosis. Furthermore, TDCPP changed the DNA methylation patterns of imprinted genes in treated blastocysts. The methylation of H19 and Snrpn promoter regions was increased from 37.67% to 46.00% and 31.56% to 44.38% in treated groups, respectively. In contrast, Peg3 promoter region methylation was declined from 86.55% to 73.27% in treated embryos. Taken together, our results demonstrated that TDCPP could adversely impair the early embryonic development in mouse. Environ. Mol. Mutagen. 2019. © 2019 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Summary: Much effort has been devoted to improving the efficiency of animal cloning. The aim of this study was to investigate the effect of BRG1 contained in Xenopus egg extracts on the development of cloned mouse embryos. The results showed that mouse NIH/3T3 cells were able to express pluripotent genes after treatment with egg extracts, indicating that the egg extracts contained reprogramming factors. After co-injection of Xenopus egg extracts and single mouse cumulus cells into enucleated mouse oocytes, statistically higher pronucleus formation and development rates were observed in the egg Extract− co-injected group compared with those in the no egg extract-injected (NT) group (38–66% vs 18–34%, P <0.001). Removal of BRG1 protein from Xenopus egg extracts was conducted, and the BRG1-depleted extracts were co-injected with single donor cells into recipient oocytes. The results showed that the percentages of pronucleus formation were significantly higher in both BRG1-depleted and BRG1-intact groups than that in the nuclear transfer (NT) group (94, 64% vs 50%, P <0.05). Furthermore, percentages in the BRG1-depleted group were even higher than in the BRG1-intact group (94% vs 64%). More confined expression of Oct4 in the inner cell mass (ICM) was observed in the blastocyst derived from the egg extract-injected groups. However, Nanog expression was more contracted in the ICM of cloned blastocysts in the BRG1-depleted group than in the BGR1-intact group. Based on the present study, BRG1 might not play an essential role in reprogramming, but the factors enhancing pronucleus formation and development of cloned mouse embryos are contained in Xenopus egg extracts. [ABSTRACT FROM AUTHOR]

Serum starvation is a routine protocol for synchronizing nuclear donor cells to G0/G1 phase during somatic cell nuclear transfer (SCNT). However, abrupt serum deprivation can cause serious stress to the cells cultured in vitro, which might result in endoplasmic reticulum (ER) stress, chromosome damage, and finally reduce the success rate of SCNT. In the present study, the effects of tauroursodeoxycholic acid (TUDCA), an effective ER stress-relieving drug, on the nuclear donor cells under serum deprivation condition as well as following SCNT procedures were first assessed in the bovine. The results showed that TUDCA significantly reduced ER stress and cell apoptosis in those nuclear donor cells. Moreover, it significantly decreased the expression of Hdac1 and Dnmt1, and increased the level of H3K9 acetylation in nuclear donor cells compared with control group. SCNT reconstructed embryos cloned from TUDCA-treated donor cells showed significantly higher fusion, cleavage, blastocyst formation rate, total cell number in day 7 blastocysts, and lower apoptotic index than that from control group. In addition, the expression of Hdac1, Dnmt1 and Bax was significantly lower in blastocysts derived from TUDCA-treated donor cells than that from control group. In conclusion, TUDCA significantly reduced the ER stress of nuclear donor cells under serum starvation condition, and significantly improved the developmental competence of following SCNT reconstructed embryos when these TUDCA-treated cells were used as the nuclear donors. [ABSTRACT FROM AUTHOR]

The mammalian embryo undergoes a dramatic amount of epigenetic remodeling during the first week of development. In this review, we discuss several epigenetic changes that happen over the course of cleavage development, focusing on covalent marks (e.g., histone methylation and acetylation) and non-covalent remodeling (chromatin remodeling via remodeling complexes; e.g., SWI/SNF-mediated chromatin remodeling). Comparisons are also drawn between remodeling events that occur in embryos from a variety of mammalian species. [ABSTRACT FROM AUTHOR]

Background: TGF-β signaling pathways regulate several crucial processes in female reproduction. AKT is a non-SMAD signaling pathway regulated by TGF-β ligands essential for oocyte maturation and early embryonic development in the mouse, but its regulatory role in bovine early embryonic development is not well established. Previously, we demonstrated a stimulatory role for follistatin (a binding protein for specific members of TGF-β superfamily) in early bovine embryonic development. The objectives of the present studies were to determine the functional role of AKT signaling in bovine early embryonic development and embryotrophic actions of follistatin. Methods: We used AKT inhibitors III and IV as pharmacological inhibitors of AKT signaling pathway during the first 72 h of in vitro embryo culture. Effects of AKT inhibition on early embryonic development and AKT phosphorylation were investigated in the presence or absence of exogenous follistatin. Results: Pharmacological inhibition of AKT signaling resulted in a significant reduction in early embryo cleavage, and development to the 8- to 16-cell and blastocyst stages (d7). Treatment with exogenous follistatin increased AKT phosphorylation and rescued the inhibitory effect of AKT inhibitors III and IV on AKT phosphorylation and early embryonic development. Conclusions: Collectively, results suggest a potential requirement of AKT for bovine early embryonic development, and suggest a potential role for follistatin in regulation of AKT signaling in early bovine embryos. [ABSTRACT FROM AUTHOR]

The oviductal microenvironment is a site for key events that involve gamete maturation, fertilization and early embryo development. Secretions into the oviductal lumen by either the lining epithelium or by transudation of plasma constituents are known to contain elements conducive for reproductive success. Although previous studies have identified some of these factors involved in reproduction, knowledge of secreted proteins in the oviductal fluid remains rudimentary with limited definition of function even in extensively studied species like cattle. In this study, we used a shotgun proteomics approach followed by bioinformatics sequence prediction to identify secreted proteins present in the bovine oviductal fluid (ex vivo) and secretions from the bovine oviductal epithelial cells (in vitro). From a total of 2087 proteins identified, 266 proteins could be classified as secreted, 109 (41%) of which were common for both in vivo and in vitro conditions. Pathway analysis indicated different classes of proteins that included growth factors, metabolic regulators, immune modulators, enzymes, and extracellular matrix components. Functional analysis revealed mechanisms in the oviductal lumen linked to immune homeostasis, gamete maturation, fertilization and early embryo development. These results point to several novel components that work together with known elements mediating functional homeostasis, and highlight the diversity of machinery associated with oviductal physiology and early events in cattle fertility. [ABSTRACT FROM AUTHOR]

The developmental competence of in vitro cultured (IVC) embryos is markedly lower than that of their in vivo counterparts, suggesting the need for optimization of IVC protocols. Embryo culture medium is routinely replaced three days after initial culture in bovine, however, whether this protocol is superior to continuous nonrenewal culture method under current conditions remains unclear. Using bovine somatic cell nuclear transfer (SCNT) embryos as the model, our results showed that compared with routine renewal treatment, nonrenewal culture system significantly improved blastocyst formation, blastocyst quality (increased total cell number, decreased stress and apoptosis, enhanced Oct-4 expression and ratio of ICM/TE), as well as following development to term. Existence and function of SCNT embryo-derived exosomes were then investigated to reveal the cause of impaired development induced by culture medium replacement. Exosomes were successfully isolated through differential centrifugation and identified by both electron microscopy and immunostaining against exosomal membrane marker CD9. Supplementation of extracted exosomes into freshly renewed medium significantly rescued not only blastocyst formation and quality (in vitro development), but also following growth to term (in vivo development). Notably, ratio of ICM/TE and calving rate were enhanced to a similar level as that in nonrenewal group. In conclusion, our results for the first time indicate that 1: bovine SCNT embryos can secrete exosomes into chemically defined culture medium during IVC; 2: secreted exosomes are essential for SCNT blastocyst formation, blastocyst quality, and following development to term; 3: removal of exosomes induced by culture medium replacement impairs SCNT embryo development, which can be avoided by nonrenewal culture procedure or markedly recovered by exosome supplementation. [ABSTRACT FROM AUTHOR]

Our previous studies demonstrated that maternal (oocyte derived) follistatin (FST) expression is positively associated with bovine oocyte competence and exogenous follistatin treatment during the pre-compaction period of development (d 1–3 post insemination) is stimulatory to bovine early embryogenesis in vitro [blastocyst rates and cell numbers/allocation to trophectoderm (TE)]. In the present study, bovine embryos were treated with exogenous follistatin during d 1–3, d 4–7 and d 1–7 post insemination to test the hypothesis that embryotropic effects of exogenous follistatin are specific to the pre-compaction period (d 1–3) of early embryogenesis. Follistatin treatment during d 4–7 (peri-/post-compaction period) of embryo culture increased proportion of embryos reaching blastocyst and expanded blastocyst stage and total cell numbers compared to controls, but blastocyst rates and total cell numbers were lower than observed following d 1–3 (pre-compaction) follistatin treatment. Follistatin supplementation during d 1–7 of embryo culture increased development to blastocyst and expanded blastocyst stages and blastocyst total cell numbers compared to d 1–3 and d 4–7 follistatin treatment and untreated controls. A similar increase in blastocyst CDX2 mRNA and protein (TE cell marker) was observed in response to d 1–3, d 4–7 and d 1–7 follistatin treatment. However, an elevation in blastocyst BMP4 protein (TE cell regulator) was observed in response to d 1–3 and d 1–7, but not d 4–7 (peri-/post-compaction) follistatin treatment. In summary, our study revealed the potential utility of follistatin treatment for increasing the success rate of in vitro embryo production in cattle. Such results also expand our understanding of the embryotropic actions of follistatin and demonstrate that follistatin actions on blastocyst development and cell allocation to the TE layer are not specific to the pre-compaction period. [ABSTRACT FROM AUTHOR]

Somatic cell nuclear transfer (SCNT) is a well-known laboratory technique. The principle of the SCNT involves the reprogramming a somatic nucleus by injecting a somatic cell into a recipient oocyte whose nucleus has been removed. Therefore, the nucleus donor cells are considered as a crucial factor in SCNT. Cell cycle synchronization of nucleus donor cells at G0/G1 stage can be induced by contact inhibition or serum starvation. In this study, acteoside, a phenylpropanoid glycoside compound, was investigated to determine whether it is applicable for inducing cell cycle synchronization, cytoprotection, and improving SCNT efficiency in canine fetal fibroblasts. Primary canine fetal fibroblasts were treated with acteoside (10, 30, 50 μM) for various time periods (24, 48 and 72 hours). Cell cycle synchronization at G0/G1 stage did not differ significantly with the method of induction: acteoside treatment, contact inhibition or serum starvation. However, of these three treatments, serum starvation resulted in significantly increased level of reactive oxygen species (ROS) (99.5 ± 0.3%) and apoptosis. The results also revealed that acteoside reduced ROS and apoptosis processes including necrosis in canine fetal fibroblasts, and improved the cell survival. Canine fetal fibroblasts treated with acteoside were successfully arrested at the G0/G1 stage. Moreover, the reconstructed embryos using nucleus donor cells treated with acteoside produced a healthy cloned dog, but not the embryos produced using nucleus donor cells subjected to contact inhibition. In conclusion, acteoside induced cell cycle synchronization of nucleus donor cells would be an alternative method to improve the efficiency of canine SCNT because of its cytoprotective effects. [ABSTRACT FROM AUTHOR]

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

The initiation of normal embryo development depends on the completion of all events of egg activation. In all species to date, egg activation requires an increase(s) in the intracellular concentration of calcium ([Ca2+]i), which is almost entirely mediated by inositol 1,4,5-trisphosphate receptor 1 (IP3R1). In mammalian eggs, fertilization-induced [Ca2+]i responses exhibit a periodic pattern that are called [Ca2+]i oscillations. These [Ca2+]i oscillations are robust at the beginning of fertilization, which occurs at the second metaphase of meiosis, but wane as zygotes approach the pronuclear stage, time after which in the mouse oscillations cease altogether. Underlying this change in frequency are cellular and biochemical changes associated with egg activation, including degradation of IP3R1, progression through the cell cycle, and reorganization of intracellular organelles. In this study, we investigated the system requirements for IP3R1 degradation and examined the impact of the IP3R1 levels on the pattern of [Ca2+]i oscillations. Using microinjection of IP3 and of its analogs and conditions that prevent the development of [Ca2+]i oscillations, we show that IP3R1 degradation requires uniform and persistently elevated levels of IP3. We also established that progressive degradation of the IP3R1 results in [Ca2+]i oscillations with diminished periodicity while a near complete depletion of IP3R1s precludes the initiation of [Ca2+]i oscillations. These results provide insights into the mechanism involved in the generation of [Ca2+]i oscillations in mouse eggs. J. Cell. Physiol. 222:238–247, 2010. © 2009 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Background: In humans, normal healthy children are regularly produced through fertilization of fresh oocytes with fresh spermatozoa. However, asynchrony between oocytes and spermatozoa, especially when aged oocytes are fertilized by fresh or senescent spermatozoa, will not only affect the rate of fertilization and pre- and post-implantation embryo development but also the life of the offspring. As many failures in assisted reproduction technologies (ART) are related to oocyte aging, new methods are needed to control oocyte aging to benefit modern ART. Methods: We review changes associated with decreased fertilization rates and developmental potential of aged oocytes, and we present methods and approaches that prevent or delay oocyte aging. Results: Cellular and molecular abnormalities occur during oocyte aging, but prevention, delay or reversal is possible to various extents. Modifying existing culture conditions, or treatment of oocytes with agents such as caffeine, DL-dithiothreitol, nitric oxide or trichostatin A may correct molecular pathways that are affected by aging, and thus benefit and improve success rates in modern ART. Conclusions: Aging of oocytes is characterized by a sequence of molecular processes that deteriorate during aging and negatively impact fertilization and development. However, oocyte aging can be delayed or reversed by various treatments to increase success rates and produce increased numbers of healthy embryos, preventing failures or abnormalities that are frequently associated with ART using aged oocytes. [ABSTRACT FROM AUTHOR]

Studies the evidence of oocyte donor cow effect over oocyte production and embryo development in vitro. Formation of normal embryos with the capacity to development to term; Reprogamming of the nuclei of somatic cells.