Regulation Mechanism Of ERβ 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 β(ERβ),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 ERβ in ovary,combined with the regulation of ERβ on autophagy function of different human cells,it is suggested that ERβ may induce autophagy and lead to follicular atresia.PHB2 was initially considered as an estrogen receptor activity inhibitor(REA),binding to Estrogen Receptor α(ERα),and inhibiting ERα specific regulatory gene transcription in the presence of E2.In recent years,PHB2 has been found to have ERβ binding sites,but the specific regulatory mechanism is not clear.Therefore,in this study,the effect of ERβ 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.ERβ can induce autophagy of GCs.The overexpression vector of ERβ 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 ERβ 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 ERβ silenced group was significantly lower than that of the control group(P<0.05).The above results showed that ERβ could regulate the autophagy activity of GCs.The high expression of ERβ in ovarian atresia follicles and late growth follicles suggests that ERβ may play an important role in inducing follicular atresia.3.PHB2 can induce autophagy of GCs.The overexpression vector of ERβ 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 ERβ and PHB2 in GCs,this experiment verified the ability of ERβ 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 ERβ and PHB2 in cells.In addition,in order to further confirm whether the protein complex formed by ERβ and PHB2 can induce GCs autophagy,the experiment silenced PHB2 while overexpressing ERβ.The results showed that the m RNA levels and protein levels of ATG3,BECN and LC3 in the co-transfected ERβ 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-ERβ,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 ERβ.Similarly,silencing ERβ in GCs can rescue autophagy caused by over-expression of PHB2.Therefore,ERβ cells and PHB2 induce autophagy through protein complexes in cells,and without ERβ or PHB2,GCs autophagy cannot be induced.5.In this study,ERβ and PHB2 overexpression vectors were co-transfected.The results showed that overexpression of ERβ and PHB2 could cause a decrease in m TOR phosphorylation level(P<0.05)and induce autophagy.Silencing ERβ,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 ERβ and PHB2 induces autophagy through the m TOR signaling pathway,this experiment silenced PHB2 while overexpressing ERβ,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-ERβ,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 ERβ,and similarly,silencing ERβ 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.ERβ 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 ERβ in ovarian atresia follicle and late growth follicle,it indicates that ERβ may play an important role in the process of inducing follicular atresia.