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Despite evidence of the presence of prostaglandin (PG) I 2 in mammalian oviducts, its role in early development of in vitro-produced (IVP) embryos is largely unknown. Thus, in the present study we examined the effects of iloprost, a PGI 2 analogue, on the in vitro developmental competence of early porcine embryos and the underlying mechanism(s). To examine the effects of iloprost on the development rate of IVF embryos, iloprost was added to the in vitro culture (IVC) medium and cultured for 6 days. Supplementation of the IVC medium with iloprost significantly improved developmental parameters, such as blastocyst formation rate, the trophectoderm:inner cell mass ratio and cell survival in IVF and parthenogenetically activated (PA) embryos. In addition, post-blastulation development into the expanded blastocyst stage was improved in iloprost-treated groups compared with controls. Interestingly, the phosphatidylinositol 3-kinase (PI3K)/AKT signalling pathway was significantly activated by iloprost supplementation in a concentration-dependent manner (10-1000nM), and the beneficial effects of iloprost on the early development of porcine IVF and PA embryos was completely ablated by treatment with 2.5μM wortmannin, a PI3K/AKT signalling inhibitor. Importantly, expression of the PI3K/AKT signalling pathway was significantly reduced in somatic cell nuclear transfer (SCNT) compared with IVF embryos, and iloprost supported the early development of SCNT embryos, as was the case for IVF and PA embryos, suggesting a consistent effect of iloprost on the IVC of IVP porcine embryos. Together, these results indicate that iloprost can be a useful IVC supplement for production of IVP early porcine embryos with high developmental competence.

While a critical role of autophagy in mammalian early embryogenesis has been demonstrated, few studies have been conducted regarding the role of autophagy in in vitro maturation (IVM) of immature oocytes. In the present study we investigated the effect of rapamycin, a chemical autophagy inducer, on the nuclear and cytoplasmic maturation of porcine oocytes. Rapamycin treatment led to increased expression of LC3-II, an autophagy marker. Compared with the control group, as well as the 5 and 10nM rapamycin treatment groups, the rate of MII oocyte production was higher in the 1nM rapamycin treatment group, indicating improvement in nuclear maturation. In the analyses of cytoplasmic maturation, we found that the level of p34(cdc2), a cytoplasmic maturation marker, and the monospermic fertilisation rate were higher in the 1nM rapamycin treatment group than in the other groups. Moreover, the beneficial effect of 1nM rapamycin on cytoplasmic maturation of MII oocytes was further evidenced by increases in blastocyst formation rate, total cell number and cell survival. In the blastocyst embryos, anti-apoptotic Bcl-xL transcript levels were elevated in the 1nM rapamycin-treated group, whereas pro-apoptotic Bax transcript levels were decreased. Collectively, these results suggest that induction of autophagy during IVM contributes to enhancement of the nuclear and cytoplasmic maturation of porcine oocytes.

Despite the positive roles of histone deacetylase inhibitors in somatic cell nuclear transfer (SCNT), few studies have evaluated valproic acid (VPA) and its associated developmental events. Thus, the present study was conducted to elucidate the effect of VPA on the early development of bovine SCNT embryos and the underlying mechanisms of action. The histone acetylation level of SCNT embryos was successfully restored by VPA, with optimal results obtained by treatment with 3mM VPA for 24h. Importantly, the increases in blastocyst formation rate and inner cell mass and trophectoderm cell numbers were not different between the VPA and trichostatin A treatment groups, whereas cell survival was notably improved by VPA, indicating the improvement of developmental competence of SCNT embryos by VPA. Interestingly, VPA markedly reduced the transcript levels of endoplasmic reticulum (ER) stress markers, including sXBP-1 and CHOP. In contrast, the levels of GRP78/BiP, an ER stress-alleviating transcript, were significantly increased by VPA. Furthermore, VPA greatly reduced cell apoptosis in SCNT blastocysts, which was further evidenced by the increased levels of the anti-apoptotic transcript Bcl-xL and decreased level of the pro-apoptotic transcript Bax. Collectively, these results suggest that VPA enhances the developmental competence of bovine SCNT embryos by alleviating ER stress and its associated developmental damage.

Somatic cell nuclear transfer (SCNT) is a powerful tool, not only for producing cloned animals, but also in revealing various early developmental events. However, relatively little is known regarding the biological events and underlying mechanism(s) directly associated with early development of SCNT embryos. Here, we show that production of high-quality bovine SCNT blastocysts is dependent on the method used for fusion and the associated reduction in endoplasmic reticulum (ER) stress. During fusion between the donor cell and the enucleated oocyte, electrofusion triggers spontaneous oocyte activation, accompanied by an increase in intracellular Ca(2+) and improper nuclear remodelling. These events can be greatly reduced by the use of Sendai virus (SV)-mediated fusion. Moreover, SV-SCNT improves the blastulation rate and blastocyst quality, defined by the number and ratio of inner cell mass and trophectoderm cells in each blastocyst, in comparison with electrofusion-mediated SCNT (E-SCNT). Interestingly, expression of ER-stress-associated genes and blastomere apoptosis were significantly increased in E-SCNT embryos. These increases could be reversed by inhibition of ER stress or by using the SV-mediated fusion method. Collectively, these results indicate that SV-mediated fusion improves the developmental competence and quality of SCNT blastocysts, by reducing ER-stress-associated apoptosis., (© CSIRO 2011 Open Access)