Circular RNAs (circRNAs) are thought to play essential roles in multiple biological processes, including apoptosis, an important process in antral follicle atresia. We aimed to investigate the potential involvement of circRNAs in granulosa cell apoptosis and thus antral follicle atresia. CircRNA expression profiles were generated from porcine granulosa cells isolated from healthy antral (HA) and atretic antral (AA) follicles. Over 9632 circRNAs were identified, of which 62 circRNAs were differentially expressed (DE-circRNAs). Back-splicing, RNase R resistance, and stability of DE-circRNAs were validated, and miRNA binding sites and related target genes were predicted. Two exonic circRNAs with low false discovery rate (FDR) high fold change, miRNA binding sites, and relevant biological functions—circ_CBFA2T2 and circ_KIF16B—were selected for further characterization. qRT-PCR and linear regression analysis confirmed expression and correlation of the targeted genes—the antioxidant gene GCLC (potential target of circ_CBFA2T2) and the apoptotic gene TP53 (potential target of circ_KIF16B). Increased mRNA content of TP53 in granulosa cells of AA follicles was further confirmed by strong immunostaining of both p53 and its downstream target pleckstrin homology like domain family a member 3 (PHLDA3) in AA follicles compared to negligible staining in granulosa cells of HA follicles. Therefore, we concluded that aberrantly expressed circRNAs presumably play a potential role in antral follicular atresia.

Purpose: Epigallocatechin-3-gallate (EGCG) is a major ingredient of catechin polyphenols and exerts protective effects because of its strong antioxidant properties. As far as we know, there is still a lack of systematic research on the effects of EGCG on the in vitro maturation (IVM) and in vitro fertilization (IVF) of porcine oocytes. The present study aimed to determine the effects of EGCG on the IVM and IVF of porcine oocytes. Methods: Porcine oocytes were treated with different concentrations of EGCG (5, 10 and 20 μM), and the cumulus cell expansion, oocyte maturation rate, reactive oxygen species (ROS), glutathione (GSH) and malondialdehyde (MDA) levels, total antioxidant capacity were determined. The mRNA expression levels of oxidative stress- and apoptosis-associated genes were determined by quantitative real-time PCR. The cleavage rate and blastocyst rate of oocytes after 10 μM EGCG treatment during IVM and IVF were also evaluated. Results: EGCG at 5, 10 and 20 μM significantly promoted cumulus cell expansion, and EGCG at 10 μM increased the oocyte maturation rate. EGCG (10 μM) treatment reduced the ROS and MDA levels, while increased the antioxidant capacity and GSH concentrations in the mature oocytes. The qRT-PCR results showed that EGCG treatment up-regulated the mRNA expression of catalase, glutathione peroxidase and superoxide dismutase in the mature oocytes. In addition, EGCG treatment also decreased the mRNA expression levels of Bax and caspase-3 and increased the Bcl-2 mRNA expression level in the mature oocytes. In addition, the cleavage rate and blastocyst rate of oocytes treated with 10 μM EGCG during IVM and IVF were significantly higher than those of the control group. Conclusion: Our results suggest that EGCG promotes the in vitro maturation and embryo development following IVF of porcine oocytes. The protective effects of EGCG on the oocytes may be associated with its antioxidant and anti-apoptosis properties.

Accumulating evidence suggests that leptin may play important roles in preimplantation embryonic development, although this remain controversial, and little is known about whether leptin has a stage-dependent regulatory effect on development of porcine embryos derived by parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT). The objective of this study was to investigate the effects of addition of leptin to in vitro culture (IVC) medium on development of porcine embryos derived by PA and SCNT. We found that addition of 50 ng/ml human recombinant leptin improved the rate of PA embryos reaching the blastocyst stage and increased the total cell number of blastocysts compared with the control group. The maximal blastocyst rate of SCNT embryos was achieved at 50 ng/ml, and the total cell number of blasocysts was increased significantly at 500 ng/ml leptin concentration. However, the ratio of the inner cell mass (ICM) to total cell number was not affected in any of the groups. Supplementation of leptin (50 ng/ml) from day 3, approximately the 4-8-cell stage, as in the case of the positive control, significantly increased the blatocyst rate of PA embryos compared with the negative control and inhibited cell apoptosis. There were no beneficial effects on embryonic development when 50 ng/ml leptin was added to the culture medium from day1 to day 3 or from day 4 to day 6. These results indicate that leptin could improve the development and the quality of PA and SCNT embryos; and 50 ng/ml leptin performs its primary stimulatory effect at 4-8-cell stage and that leptin may have no effect on the maternal-zygote transition (MZT) of porcine PA and SCNT embryos.