VD₃ Promotes Autophagy Of Porcine Ovarian Granulosa Cells Through The ROS-BNIP3-PINK1 Pathway

Vitamin D（VD）is one of the important essential nutrients in livestock,and early studies have shown that VD has a potential role in reproductive physiological activities.1,25-dihydroxyvitamin D₃（1α,25（OH）₂D₃,VD₃）is one of the effective active forms of VD,which plays a biological role in reproduction through vitamin D receptor（VDR）.However,scientists’understanding of the mechanism by which VD affects reproductive physiological activities is still limited.Follicular atresia is a common phenomenon during female reproduction.In livestock production,closed follicles lead to waste of genetic resources,reduce the reproductive efficiency of female animals and affect the development of the industry.Autophagy and apoptosis are involved in the regulation of follicular atresia.Granulosa cell autophagy is one of the key factors leading to follicular atresia.VD can regulate cell function and autophagy of various cells.VD may play a role in follicular atresia by regulating granulosa cell autophagy.However,its effect on autophagy of porcine ovarian granulosa cells and its mechanism of action have not been reported.Therefore,this study took PGCs as the research object to explore the effect of VD₃on porcine ovarian granulosa cells and investigate the mechanism of action of VD₃in porcine ovarian granulosa cells autophagy.The main research results are as follows:VD₃promotes porcine ovarian granulosa cell survival.After treatment with 10,100 and200 nM VD₃for 12,24 and 36 h,the viability of porcine ovarian granulosa cells was significantly increased.In porcine ovarian granulosa cells treated with 10 nM VD₃for 24 h,the expressions of VDR,CDK1 and CCNB1 genes were increased,while the expressions of P21 genes were decreased.In the follow-up experiment,10 nM VD₃was used to treat porcine ovarian granulosa cells for 24 h.VD₃significantly down-regulates the expressions of SOD1 and GSH-Px-1 genes in porcine ovarian granulosa cells,reduces the enzyme activities of SOD and GPX in porcine ovarian granulosa cells,and induces the increase of ROS levels in porcine ovarian granulosa cells.VD₃significantly increased the mitochondrial abundance and the relative expression of ND1 gene in porcine ovarian granulosa cells,but had no significant effect on mitochondrial membrane potential.In order to study the effect of VD₃on the autophagy of porcine ovarian granulosa cells,the experiment treated porcine ovarian granulosa cells with VD₃and autophagy inhibitor Chloroquine（CQ）respectively.The results showed that compared with the control group,VD₃significantly up-regulated the expression of autophagy related genes ATG7,Beclin1 and LC3,significantly increased the level of LC3Ⅱ/LC3Ⅰ in porcine ovarian granulosa cells,significantly down-regulated the expression of P62 gene and protein,and significantly increased the number of autophagy vesicles in porcine ovarian granulosa cells.Compared with the VD₃treatment group,the expression of ATG7,Beclin1 and LC3 genes was significantly up-regulated,LC3Ⅱ/LC3Ⅰ levels were significantly reduced,the expression of P62 gene and protein were significantly up-regulated,and the number of autophagy vesicles in porcine ovarian granulocyte cells was significantly increased after the co-treatment with VD₃.Compared with the control group,the concentrations of E₂and P₄in cell medium were significantly increased after VD₃treatment.Compared with the VD₃group,the concentrations of E₂and P₄in porcine ovarian granulosa cell medium treated with VD₃and CQ were significantly decreased.Compared with the control group,VD₃treatment significantly up-regulated the expressions of ESR1,CYP19A1,PGR and STAR genes in porcine ovarian granulosa cells.Compared with the VD₃group,the combination of VD₃and CQ significantly down-regulated the expression of these genes.Compared with control group,VD₃significantly increased the expression of STAR protein.However,compared with the VD₃treatment group,the combined treatment of VD₃and CQ significantly down-regulated the protein expression of STAR,and the autophagy induced by VD₃affected the secretion of estrogen and progesterone in porcine ovarian granulosa cells.To investigate the mechanism of autophagy induced by VD₃,porcine ovarian granulosa cells were treated with 4 mM n-acetylcysteine（NAC,ROS scavger）,10 nM VD₃,and 10μM CQ for 24 h.The results showed that VD₃significantly improved the viability and ROS content of porcine ovarian granulosa cells,and NAC could significantly reverse the effects of VD₃on the viability and ROS content of porcine ovarian granulosa cells.Compared with the VD₃treatment group,the gene expressions of ATG7,Beclin1 and LC3 in porcine ovarian granulosa cells were significantly down-regulated,the gene expression of P62 was up-regulated,LC3Ⅱ/LC3Ⅰ levels were decreased,the protein expression of P62 was increased,and the number of autophagy vesicles in porcine ovarian granulosa cells treated with VD₃was significantly decreased by NAC.VD₃induced autophagy in porcine ovarian granulosa cells by ROS.Compared with the control group,VD₃treatment significantly up-regulated the expression of mitochondria autophagy receptor BNIP3 and PINK1 gene and protein.Compared with VD₃alone,CQ co-treated with VD₃significantly up-regulated the expressions of BNIP3 and PINK1 genes and proteins.In addition,NAC co-treated with VD₃significantly decreased the expression of BNIP3 and PINK1 genes and proteins in porcine ovarian granulosa cells,compared with VD₃,which induced autophagy in porcine ovarian granulosa cells through the ROS-BNIP3-PINK1 pathway.In conclusion,in porcine ovarian granulosa cells,10 nM VD₃treatment for 24 h can significantly promote the survival of porcine ovarian granulosa cells and the secretion of estrogen and progesterone,increase the abundance of mitochondria in porcine ovarian granulosa cells,affect the intracellular antioxidant enzyme system,increase the intracellular ROS level,and promote autophagy.VD₃regulates autophagy through ROS-BNIP3-PINK1,thus affecting steroid secretion in porcine ovarian granulosa cells.The results provide theoretical support for the study of the mechanism of VD₃in porcine follicle development.