Regulation Mechanism Of ER? On Autophagy Of Porcine Ovarian Granulosa Cells

The fecundity of sows has an important influence on the economic benefits of pigs.The growth and development of follicles determine the litter rate.Most of the follicles in the ovary cannot ovulate because of atresia and degeneration,which is the main reason for the waste of genetic resources.The development and atresia of ovarian follicles are affected by many factors,but the specific regulatory process and mechanism have not been clarified.Estrogen Receptor ?(ER?),as one of the important transcription factors in follicular development,regulates the survival,proliferation and differentiation of granulosa cells(GCs)mainly through the realization of estrogen function.In view of the expression characteristics of ER? in ovary,combined with the regulation of ER? on autophagy function of different human cells,it is suggested that ER? may induce autophagy and lead to follicular atresia.PHB2 was initially considered as an estrogen receptor activity inhibitor(REA),binding to Estrogen Receptor ?(ER?),and inhibiting ER? specific regulatory gene transcription in the presence of E2.In recent years,PHB2 has been found to have ER? binding sites,but the specific regulatory mechanism is not clear.Therefore,in this study,the effect of ER? and PHB2 on the autophagy of GCs was studied,and the role of AKT/m TOR signaling pathway in this process was explored.1.In order to investigate the occurrence of autophagy and apoptosis of GCs during follicular development,GCs were isolated from three types of follicles: small(<2mm),medium(2-6mm)and large(>6mm).The results showed that compared with small follicles,the proliferation rate of GCs in medium follicles was higher and that of large follicles was lower(P<0.05).Bax and Caspase 3 m RNA levels in GCs of large follicles increased significantly(P<0.05),but the Bcl-2 / Bax ratio was low.The levels of ATG3,ATG7 and LC3 m RNA were higher in the middle follicle(P<0.05).The expression level of apoptosis related protein and autophagy related protein was similar to that of m RNA.Our results showed that the ERK level in GCs of large follicles was significantly higher than that in GCs of middle follicles and small follicles(P<0.05),while the AKT and m TOR phosphorylation levels in GCs of middle follicles were significantly higher than that in GCs of large follicles and small follicles(P<0.05).The results of gene transcription and protein expression in GCs with different follicle sizes were confirmed by DAPI and MDC.We conclude that autophagy and apoptosis occur in different sizes of follicles during follicular development,in which autophagy mainly occurs in GCs of middle follicles and apoptosis mainly occurs in GCs of large follicles.2.ER? can induce autophagy of GCs.The overexpression vector of ER? was transfected into cells,and the expression levels of genes and proteins were detected by q RT-PCR and Western Blot.The results showed that the expression of GCs autophagy related genes ATG3,BECN,LC3 and their proteins were significantly higher than that of the control group(P<0.05),while the expression of GCs autophagy related genes ATG3,BECN and LC3 in ER? silenced group was significantly lower than that of the control group(P<0.05),and the expression of GCs autophagy related proteins ATG3 and BECN in ER? silenced group was significantly lower than that of the control group(P<0.05).The above results showed that ER? could regulate the autophagy activity of GCs.The high expression of ER? in ovarian atresia follicles and late growth follicles suggests that ER? may play an important role in inducing follicular atresia.3.PHB2 can induce autophagy of GCs.The overexpression vector of ER? was transfected into cells,and the expression levels of genes and proteins were detected by q RT-PCR and Western Blot.The expression levels of ATG3,BECN and LC3 m RNA and protein were measured.The results showed that the expression levels of ATG3,BECN and LC3 m RNA and protein in PHB2 overexpression GCs were significantly higher than those in the control group(P<0.05).Meanwhile,the m RNA and protein levels of ATG3,BECN and LC3 in PHB2 silenced GCs were significantly lower than those in the control group(P<0.05).The above results show that PHB2 can regulate the activity of autophagy,overexpression of PHB2 can induce autophagy,and silencing PHB2 can inhibit the occurrence of autophagy.4.In order to investigate the interaction between ER? and PHB2 in GCs,this experiment verified the ability of ER? and PHB2 to bind to each other to form protein complexes by CO-IP.At the same time,laser confocal microscopy was used to demonstrate the co-localization of ER? and PHB2 in cells.In addition,in order to further confirm whether the protein complex formed by ER? and PHB2 can induce GCs autophagy,the experiment silenced PHB2 while overexpressing ER?.The results showed that the m RNA levels and protein levels of ATG3,BECN and LC3 in the co-transfected ER? and si-PHB2 GCs were not significantly different from those of the blank group(P>0.05).In the GCs of co-transfected PHB2 and si-ER?,the m RNA levels and protein levels of ATG3,BECN and LC3 were not significantly different from those of the blank group(P>0.05).The experimental results confirmed that silencing PHB2 in GCs can rescue autophagy caused by over-expression of ER?.Similarly,silencing ER? in GCs can rescue autophagy caused by over-expression of PHB2.Therefore,ER? cells and PHB2 induce autophagy through protein complexes in cells,and without ER? or PHB2,GCs autophagy cannot be induced.5.In this study,ER? and PHB2 overexpression vectors were co-transfected.The results showed that overexpression of ER? and PHB2 could cause a decrease in m TOR phosphorylation level(P<0.05)and induce autophagy.Silencing ER?,or PHB2 will increase the phosphorylation level of m TOR(P<0.05),thus inhibiting autophagy.In order to verify whether the protein complex formed by ER? and PHB2 induces autophagy through the m TOR signaling pathway,this experiment silenced PHB2 while overexpressing ER?,and the results showed that there was no significant difference in the phosphorylation level of m TOR compared with the blank group(P>0.05).Similarly,in the GCs of the co-transfected PHB2 overexpression vector and si-ER?,the phosphorylation level of m TOR was not significantly different from that of the blank group(P>0.05).The results showed that silencing PHB2 could effectively inhibit the reduction of m TOR phosphorylation caused by overexpression of ER?,and similarly,silencing ER? could effectively inhibit the autophagy induced by overexpression of PHB2.In conclusion,during the development of follicles,GCs in medium follicles are more likely to autophagy,and GCs in large follicles are more likely to apoptosis.ER? can induce granulosa cell autophagy through m TOR signal pathway through the protein complex formed by binding with PHB2.Because of the high expression of ER? in ovarian atresia follicle and late growth follicle,it indicates that ER? may play an important role in the process of inducing follicular atresia.