Pluripotency and embryonic lineage genes expression in the presence of small molecule inhibitors of FGF, TGFß and GSK3 during pre-implantation development of goat embryos.

Generating stable livestock pluripotent stem cells (PSCs) can be used for complex genome editing, cellular agriculture, gamete generation, regenerative medicine and in vitro breeding schemes. Over the past decade, significant progress has been made in characterizing pluripotency markers for livestock species. In this study, we investigated embryo development and gene expression of the core pluripotency triad (OCT4, NANOG, SOX2) and cell lineage commitment markers (REX1, CDX2, GATA4) in the presence of three small molecules and their combination [PD0325901 (FGF inhibitor), SB431542 (TGFß inhibitor), and CHIR99021 (GSK3B inhibitor)] from day 2-7 post-insemination in goat. Significant reduction in rate of blastocyst formation was observed when SB was used along with PD or CHIR and their three combinations had more sever effect. SB and CHIR decreased the expression of SOX2 while increasing the GATA4 expression. PD decrease the relative expression of NANOG, OCT4 and GATA4, while increased the expression of REX1. Among the combination of two molecules, only SB + CHIR combination significantly decreased the expression of GATA4, while the combination of the three molecules significantly decreases the expression of NANOG, SOX2 and CDX2. According to these results, the inhibition of the FGF signaling pathway, by PD may lead to blocking the hypoblast formation as observed by reduction of GATA4. OCT4 and NANOG expressions did not show signs of maintenance pluripotency. GATA4, NANOG and OCT4 in the PD group were downregulated and REX1 as EPI-marker was upregulated thus REX1 may be considered as a marker of EPI/ICM in goat.

Oct-4 activating compound 1 (OAC1) could improve the quality of somatic cell nuclear transfer embryos in the bovine.

Previous studies described aberrant nuclear reprogramming in somatic cell nuclear transfer (SCNT) embryos that is distinctly different from fertilized embryos. This abnormal nuclear reprogramming hampers the proper pre- and/or post-implantation development. It has been demonstrated that SCNT blastocysts aberrantly expressed POU5F1 and POU5F1-related genes. With regard to this, it has been postulated that promoting the expression of POU5F1 in SCNT embryos may enhance reprogramming in SCNT embryos. In this study, we treated either fibroblast donor cells or SCNT embryos with OAC1 as a novel small molecule that has been reported to induce POU5F1 expression. Quantitative results from the MTS assay revealed that lower concentrations of OAC1 (1, 1.5, and 3 µM) are non-toxic after 2, 4, and 6 days, but higher concentrations (6, 8, 10, and 12 µM) are toxic and reduced the proliferation of cells after 6 days. No enhancement in the expression of endogenous POU5F1 was observed when both mouse and bovine fibroblast cells were treated with 1.5 and 3 µM OAC1 for up to 6 consecutive days. Subsequently, we treated either fibroblast as donor cells in the SCNT procedure (BFF-OAC1 group) or SCNT embryos [for 4 days (IVC-OAC1: D4-D7 group) or 7 days (IVC-OAC1: D0-D7 group)] with 1.5 µM OAC1. We observed that neither treatment of fibroblast donor cells nor SCNT embryos improved the cleavage and blastocyst rates. Interestingly, we observed that treatment of SCNT embryos all throughout the in vitro culture (IVC) (IVC-OAC1: D0-D7) with 1.5 µM OAC1 improves the quality of derived blastocyst which was indexed by morphological grading, blastomere allocation, epigenetic marks and mRNA expression of target genes. In conclusion, our results showed that supplementation of IVC medium with 1.5 µM OAC1 (D0-D7) accelerates SCNT reprogramming in bovine species.

Effect of rosiglitazone on developmental competence of mouse embryos treated with lipopolysaccharide.

Lipopolysaccharide (LPS) significantly reduces pre- and post-implantation developmental competence of embryos. One of the reason of this effect could be a consequence of TLR4-mediated inflammation. In this study, we assessed the anti-inflammatory effect of peroxisome proliferator activated receptor γ (PPAR γ) agonist, rosiglitazone (RGZ), in LPS-treated mouse embryos. Initially, the optimal doses of LPS, RGZ and GW9662 (a potent and selective PPARγ antagonist) were determined by treating the mouse zygotes up to blastocyst stage and assessment of compaction and blastocyst rates. Quantitative PCR was used to assess the mRNA expression of inflammatory cytokines. Immunostaining was used to study the translocation of PPARγ in blastocysts. Finally, the blastocysts were transferred to surrogate mouse to determine the post-implantation developmental competence. 0.0625 mg/mL of LPS significantly reduced the developmental competency by around 50% compared to control group. 10 µM of RGZ significantly ameliorated the toxic effect of LPS, which was also significantly reversed by 1.25 µM GW9662. Through immunostaining, it was shown that LPS could prevent the translocation of PPARγ to nucleus; and translocation was facilitated by RGZ and this effect was reversed by GW9662. A similar effect was also observed for the mRNA expression of inflammatory cytokines (Il-1ß and Il-6). LPS significantly increased the expression of these cytokines, while RGZ significantly reduced their expression, which was also significantly reversed by GW9662. It was also shown that embryos exposed to LPS had significantly reduced post implantation developmental competence which was considerably improved by treatment with RGZ. In conclusion, these data may have clinical implications for ameliorating the adverse effects of LPS in dairy farming and infertility treatment.

Granulosa secreted factors improve the developmental competence of cumulus oocyte complexes from small antral follicles in sheep.

Oocyte in vitro maturation can be improved by mimicking the intra-follicular environment. Oocyte, cumulus cells, granulosa cells, and circulating factors act as meiotic regulators in follicles and maintain oocyte in the meiotic phase until oocyte becomes competent and ready to be ovulated. In a randomized experimental design, an ovine model was used to optimize the standard in vitro maturation media by Granulosa secreted factors. At first, the development capacity of oocyte derived from medium (>4 to 6 mm) and small (2 to ≤4 mm) size follicles was determined. Differential gene expression of granulosa secreted factors and their receptors were compared between the cumulus cells of the two groups. Then, the best time and concentration for arresting oocytes at the germinal vesicle stage by natriuretic peptide type C (CNP) were determined by nuclear staining in both groups. Oocyte quality was further confirmed by calcein uptake and gene expression. The developmental competence of cumulus oocyte complexes derived from small size follicles that were cultured in the presence of CNP in combination with amphiregulin (AREG) and prostaglandin E2 (PGE2) for 24 h was determined. Finally, embryo quality was specified by assessing expressions of NANOG, SOX2, CDX2, OCT4, and TET1. The cumulus oocyte complexes derived from small size follicles had a lower capacity to form blastocyst in comparison with cumulus oocyte complexes derived from medium size follicles. Prostaglandin E receptor 2 and prostaglandin-endoperoxide synthase 2 had significantly lower expression in cumulus cells derived from small size follicles in comparison with cumulus cells derived from medium size follicles. Natriuretic peptide type C increased the percentage of cumulus oocyte complexes arresting at the germinal vesicle stage in both oocytes derived from medium and small follicles. Gap junction communication was also improved in the presence of natriuretic peptide type C. In oocytes derived from small size follicles; best blastocyst rates were achieved by sequential exposure of cumulus oocyte complexes in [TCM+CNP (6 h), then cultured in TCM+AREG+PGE2 (18h)] and [TCM+CNP (6 h), then cultured in conventional IVM supplements+AREG+PGE2 (18h)]. Increased SOX2 expression was observed in [TCM+CNP (6 h), then cultured in TCM+AREG+PGE2 (18h)], while decreased OCT4 expression was observed in [TCM+CNP (6 h), then cultured in conventional IVM supplements+AREG+PGE2 (18h)]. It seems that the natriuretic peptide type C modulates meiotic progression, and oocyte development is probably mediated by amphiregulin and prostaglandin E2. These results may provide an alternative IVM method to optimize in vitro embryo production in sheep and subsequently for humans.

Effects of ovary storage temperature and embryo vitrification on somatic cell nuclear transfer outcomes in goats.

Improving the genetic potential of farm animals is one of the primary aims in the field of assisted reproduction. In this regard, somatic cell nuclear transfer (SCNT) can be used to produce a large number of embryos from genetically elite animals. The aims of the present study were to assess the effects of: (1) ovary storage conditions on preimplantation development of recovered oocytes and the freezability of the derived blastocysts; and (2) vitrification of goat SCNT-derived blastocysts on postimplantation development. Goat oocytes were recovered from ovaries and stored under warm (25°C-27°C) or cold (11°C-12°C) conditions before being used to produce SCNT embryos. There were no differences in oocytes recovered from ovaries kept under cold versus warm storage conditions in terms of cleavage (mean (±s.d.) 95.68±1.67% vs 95.91±2.93% respectively) and blastocyst formation (10.69±1.17% vs 10.94±0.9% respectively) rates. The re-expansion rate of vitrified blastocysts was significantly lower for cold- than warm-stored ovaries (66.3±8.7% vs 90±11% respectively). To assess the effects of vitrification on postimplantation development, blastocysts from cold-stored ovaries only were transferred from fresh and vitrified-warmed groups. The pregnancy rate was comparable between the fresh and vitrified-warmed groups (41.65% and 45.45% respectively). In addition, established pregnancy in Day 28-38 and full-term pregnancy rates were similar between the two groups. In conclusion, this study shows similar invitro preimplantation developmental potential of warm- and cold-stored ovaries. This study introduces the vitrification technique as an appropriate approach to preserve embryos produced by SCNT for transfer to recipient goats at a suitable time.