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The specific expression profile and function of circular RNAs (circRNAs) in mammalian ovarian follicles, especially during the atresia process, are unclear. In this study, we verified and explored the expression and function of circ-ANKHD1 in granulosa cells. Our results showed that abundance of circ-ANKHD1 was significantly lower in the granulosa cells than that of ANKHD1. The expression of ANKHD1 was highest in the granulosa cells from follicles with a diameter of 5-6 mm and lowest in that with a diameter of 3-4 mm. Furthermore, the expression level of circ-ANKHD1 in the ovarian tissue of 1-day-old piglets was significantly higher than that of 17-month-old multiparous sows. The luciferase reporter assay showed the potential interaction between circ-ANKHD1 and miR-27a-3p/miR-142-5p. Furthermore, circ-ANKHD1 overexpression up-regulated SFRP1 expression, while miR-27a-3p overexpression suppressed SFRP1 expression in granulosa cells. Circ-ANKHD1 overexpression significantly decreased the cell apoptotic rates of the granulosa cells and repressed the cell population at G0/G1 and S phases but increased cell population at G2/M phase. Finally, circ-ANKHD1 overexpression increased the mRNA expression levels of Bcl-2 and cyclin D1 in the granulosa cells, while there are no effects on the mRNA expression levels of caspase-3, p53, Bax, and proliferating cell nuclear antigen. In conclusion, our study for the first time identified a novel circRNA, circ-ANKHD1 that may be associated with the biological functions of granulosa cells. Circ-ANKHD1 may promote the granulosa cell proliferation, but attenuate apoptosis, and these effects may be associated with modulation of miR-27a-3p/SFRP1.

Kangfa Huang,1 Chengde Li,1 Fenglei Gao,2 Yushan Fan,1 Fanwen Zeng,1 Li Meng,1 Li Li,1 Shouquan Zhang,1 Hengxi Wei1 1National Engineering Research Center for Breeding Swine Industry, Guangdong Provincial Key Laboratory of Agro-Animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, 510642, People’s Republic of China; 2Department of Tropical Agriculture and Forestry, College of Guangdong Agriculture Industry Business Polytechnic, Guangzhou, Guangdong, 510507, People’s Republic of ChinaCorrespondence: Hengxi WeiNational Engineering Research Center for Breeding Swine Industry, Guangdong Provincial Key Laboratory of Agro-Animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, 510642, People’s Republic of ChinaTel +86-20-85284869Email weihengxi@scau.edu.cnPurpose: 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.Keywords: EGCG, porcine oocytes, IVM, antioxidant, anti-apoptosis, IVF

The proteins in the seminal plasma and on the sperm surface play important roles in sperm function and numerous reproductive processes. The cysteine-rich secretory proteins (CRISPs) are enriched biasedly in the male reproductive tract of mammals, and CRISP2 is the sole member of CRISPs produced during spermatogenesis; whereas the role of CRISP2 in fertilization and its association with fertility of boars are still unclear. This study aimed to investigate the relationship between the sperm CRISP2 and boar fertility, and explore its impact sperm fertilizing ability. The levels of CRISP2 protein in sperm were quantified by ELISA; correlation analysis was performed to evaluate the association between CRISP2 protein levels and boar reproductive parameters. Meanwhile, the expression of CRISP2 in boar reproductive organs and sperm, and the effects of CRISP2 on in vitro fertilization (IVF) were examined. The results showed that boars with high sperm levels of CRISP2 had high fertility. The protein levels of CRISP2 in sperm were positively correlated with the litter size (r = 0.412, p = 0.026), the number of live-born piglets (r = 0.421, p = 0.023) and the qualified piglets per litter (r = 0.381, p = 0.042). CRISP2 is specifically expressed in the testis and sperm of adult boars, and its location on sperm changed mainly from the post-acrosomal region to the apical segment of acrosome during capacitation. The cleavage rate was significantly decreased by adding the anti-CRISP2 antibody to the IVF medium, which indicates CRISP2 plays a critical role in fertilization. In conclusion, CRISP2 protein is specifically expressed in the adult testis and sperm and is associated with sperm fertilizing ability and boar fertility. Further mechanistic studies are warranted, in order to fully decipher the role of CRISP2 in the boar reproduction.