Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2/B1) is an N6-methyladenosine (m6A) RNA modification regulator and a key determinant of premRNA processing, mRNA metabolism and transportation in cells. Currently, m6A reader proteins such as hnRNPA2/B1 and YTHDF2 has functional roles in mice embryo. However, the role of hnRNPA2/B1 in porcine embryogenic development are unclear. Here, we investigated the developmental competence and mRNA expression levels in porcine parthenogenetic embryos after hnRNPA2/B1 knock-down. HhnRNPA2/B1 was localized in the nucleus during subsequent embryonic development since zygote stage. After hnRNPA2/B1 knock-down using double stranded RNA injection, blastocyst formation rate decreased than that in the control group. Moreover, hnRNPA2/B1 knock-down embryos show developmental delay after compaction. In blastocyste stage, total cell number was decreased. Interestingly, gene expression patterns revealed that transcription of Pou5f1, Sox2, TRFP2C, Cdx2 and PARD6B decreased without changing the junction protein, ZO1, OCLN, and CDH1. Thus, hnRNPA2/B1 is necessary for porcine early embryo development by regulating gene expression through epigenetic RNA modification.

The coupling of autophagy and endoplasmic reticulum (ER) stress has been implicated in a variety of biological processes. However, little is known regarding the involvement of the autophagy/ER stress pathway in early embryogenesis or the underlying mechanism (s). Here, we showed that the developmental competence of in vitro-produced (IVP) bovine embryos was highly dependent on the autophagy/ER stress balance. Although relative abundances of autophagy-associated gene transcripts, including LC3, Atg5, and Atg7 transcripts, were high in oocytes and throughout the early stages of preattachment development, extensive autophagosome formation was only detected in fertilized embryos. Using inducer and inhibitor of autophagy, we showed that transient elevation of autophagic activity during early preattachment development greatly increased the blastocyst development rate, trophectoderm cell numbers, and blastomere survival; these same parameters were reduced by both inhibition and prolonged induction of autophagy. Interestingly, the induction of autophagy reduced ER stress and associated damage, while the developmental defects in autophagy-inhibited embryos were significantly alleviated by ER stress inhibitor treatment, indicating that autophagy is a negative regulator of ER stress inearly embryos. Collectively, these results suggest that early embryo genesis of IVP bovine embryos depends on an appropriate balance between autophagy and ER stress. These findings may increase our understanding of important early developmental events by providing compelling evidence concerning the tight association between autophagy and ER stress, and may contribute to the development of strategies for the production of IVP bovine blastocysts with high developmental competence.

Autophagy is known to be involved in a variety of biological processes. However, relatively a little is known regarding oocyte maturation and preimplantation development in mammals. Thus, the current study was conducted to investigate the role of autophagy in oocyte maturation and subsequent preimplantation development in pigs. Porcine oocytes were matured in the presence or absence of 1 μM rapamycin, an autophagy inducing agent, fertilized in vitro, and cultured to blastocyst stage. From Western blotting analysis, we found that active form LC3 was detected during in vitro maturation (IVM) period, suggesting the possible role of autophagy in oocyte maturation. Interestingly, treatment of rapamycin during IVM significantly increased nuclear maturation compared to control group. Importantly, rapamycin-assisted IVM greatly improved monospermic fertilization and blastocyst development rates compared to control embryos. In addition, we also found that cell number and blastomere survival in blastocysts were markedly increased in rapamycin treatment group, which was further evidenced by both elevation of anti-apoptotic transcript Bcl-XL and decrease of pro-apoptotic transcript Bax. Collectively, these results strongly suggest that induction of autophagy may contribute to the completion of nuclear and cytoplasmic maturation of porcine oocytes.

Despite of the presence of estradiol-17β (E2) in ovarian follicles, its role(s) in in vitro maturation (IVM) is still largely unknown, especially in pigs. Thus, the current study was conducted to investigate the effect of E2 on in vitro maturation (IVM) of porcine oocytes and subsequent preimplantation development using in vitro fertilization (IVF)- or somatic cell nuclear transfer (SCNT)-derived embryos. To define the effects of E2 on IVM and early embryogenesis, porcine oocytes were matured in the presence or absence of E2, fertilized in vitro and cultivated to blastocyst stage. Compared to control group, the production of MII oocytes was significantly increased by treatment with E2, accompanying with the increase in MPF content and ERK phosphorylation, and monospermic fertilization and blastocyst development rates were also greatly elevated in the E2-treated oocytes. In addition, the advantageous role of E2 was also found in blastomere survival, which was further evidenced by both elevation of anti-apoptotic transcript Bcl-XL and decrease of pro-apoptotic transcript Bax. Furthremore, these positive effects of E2 were highly reproducible in early development of SCNT embryos. Collectively, the current study strongly suggests that E2 can be used as a efficient IVM supplement leading to successful nuclear/cytoplasmic maturatioin in pigs.

Successful early embryogenesis of somatic cell nuclear transfer (SCNT) embryos is very important to produce cloned animals. However, poor preimplantation development of SCNT embryos has been a major obstacle to the generation of cloned animals due to a lack of understanding of developmental events and underlying mechanism(s). In the current study, we show that production of SCNT embryos with high developmental competence is dependent on the fusion method. Electrofusion causes spontaneous egg activation, accompanied by an increase in intracellular Ca2+ and improper nuclear remodeling, whereas Sendai virus (SV)-mediated fusion greatly reduces these events. In addition, SV-SCNT increased the blastocyst development rate and trophectoderm cell number compared to electrofusion-mediated SCNT (E-SCNT). In particular, expression of ER stress-associated genes and blastomere apoptosis were significantly increased in E-SCNT embryos, which could be alleviated by inhibition of ER stress or by using the SV-mediated fusion method. Taken together, these results strongly suggest that SV is a useful fusion material for improvement of preimplantation development of SCNT embryos through reduction of ER stress-associated apoptosis.