Expression Of Inhibin In Bovine Antral Follicles And Its Effect On The Proliferation Of Bovine Follicular Granulosa Cells In Vitro

Inhibin A produced by granulosa cells(GCs)is well known for its inhibitory properties against follicle-stimulating hormone(FSH),released through a pituitary-gonadal negative feedback loop to regulate follicular development.Ovarian folliculogenesis,hormonal biosynthesis,and gametogenesis are dependent on inhibins,playing vital roles in promoting or inhibiting cell proliferation.The previous research mainly focused on the effect of inhibin on FSH secretion,but the effect of exogenous FSH on inhibin synthesis and secretion is not clear.In our current experiment,effects of FSH on expressions of inhibin subunits genes were studied in bovine antral follicles cultured in vitro.It has been reported that inhibin also plays an important role in regulating cell proliferation.Still,there is a lack of knowledge about the effect of inhibin on the proliferation of bovine follicular GCs.Therefore,GCs were treated with different concentrations of inhibin A in vitro to study its effect on the proliferation of GCs.The contents and results of the study are as follows:The antral follicles isolated from the bovine ovaries were divided into three groups: small,medium,and large follicles based on their sizes ? 4,5-8 and 9-11 mm,respectively.Granulosa cells and follicular fluid were collected in vitro.The expression of inhibin gene and the concentration of gonadotropin hormones(FSH and LH)in follicular fluid were detected by qRT-PCR and ELISA,respectively.The results of qRT-PCR showed that the expression of inhibin ?(INH?)and inhibin ?A(INH?A)in GCs of large follicles were significantly(P < 0.05)higher than those in small follicles,while the expression of INH? in theca cells of large follicles was significantly lower than that in small follicles(P < 0.05).Results of the hormonal assay showed no significant difference in the concentration of FSH and LH in follicular fluid of large,medium,and small follicles(P > 0.05).Through the model of in vitro cultured antral follicle,the effects of different concentrations of exogenous FSH(0,1,10,25 and 50 ng/mL)on the expression of inhibin genes in GCs isolated from large follicles(9-11 mm)were also studied.The results showed that FSH treatment significantly(P < 0.05)down-regulated the expression of INH?A and INH?B in GCs.Aim to investigate the effect of inhibin A(INHA)on GCs functions,especially on the proliferation of GCs and its molecular mechanism,we treated GCs with different concentrations of exogenous INHA(0,20,50 and 100 ng/mL)in vitro.The results showed that high concentrations of INHA(50 and 100 ng/mL)could significantly(P < 0.05)increase the viability of cultured GCs,while 100 ng/mL INHA could promote the proliferation of bovine GCs by increasing the number of cells in S phase which was further manifested by significantly up-regulating the expression of PCNA and CyclinB1 genes.Likewise,100 ng/mL INHA could also inhibit GCs apoptosis by down-regulating the expression of Bax and Caspase-3 genes.All together,exogenous FSH can directly regulate the expression of inhibin gene in bovine GCs,while the addition of high concentration inhibin A in vitro can promote the proliferation of GCs and reduce its apoptosis rate.