The Effect And Mechanism Of FRBI On Ovarian And Oocyte Development

Binding of FSH to the cognate receptor FSHR on the surface of granulosa cells activates adenylate cyclase?AC?and phospholipase C?PLC?to produce cAMP,IP3and diacylglycerol?DAG?.These second messengers further contribute to the activation of the downstream signaling cascade through several mechanisms,and the cAMP-guanine exchange factor?cAMP-GEF?,in turn,activates some protein kinases,such as the GTP-binding protein?RAP1/RAP2/RAS?,these proteins in turn can further activate ERK1/2 or p38MAPK.These protein kinases phosphorylate these factors by activating different transcription factors,ultimately leading to steroid production in granule cells and promoting granulosa cell proliferation or survival.Follicle stimulating hormone receptor binding inhibitor?FRBI?is an 8-peptide compound extracted from human and sheep follicular fluid and further purified by reversed-phase high performance liquid chromatography.By inhibiting the binding of FSH to FSHR,signal transduction is inhibited,which in turn affects the proliferation of granulosa cells.Most of the current research is to use FSH and FRBI to conduct experiments on animals.However,whether FRBI alone can effectively inhibit the function of FSH and the best dose effect is not reported.In this study,firstly mice injected with FRBI were divided into three groups:M-20,M-30 and M-40,and intramuscularly injected with 20,30 and 40 mg/kg FRBI;positive control group?M-FSH group?was injected with 10 IU/FSH;control group?M-CG group?was injected with 0.2 mL saline for 5 days.The weight of bilateral ovaries in each mouse was accurately weighed.The thickness of cortical thickness?OCT?and secondary follicle morphology in ovarian tissue sections were determined by HE staining.Serum FSH and E₂ concentrations were determined by ELISA.FSHR and ER?was detected by RT-PCR.Western Blot analysis of ovarian FSHR and ER?expression.Secondly,Under the condition of 5%CO₂ concentration and saturated humidity,the number of polar bodies first polar bodies appeared at 22³0h at36³9°C;the oocytes were cultured at 0 according to the above conditions.In the medium add 5,10,15,20 IU/mL FSH and 10,20,30,40?g/mL FRBI,the number of polar bodies first polar bodies was observed in the oocytes.At last,the sheep cumulus oocyte complex bodies were randomLy divided into five experimental groups?O-CG group,O-10 group,O-20 group,O-30 group,O-40 group?and one positive?O-FSH group?.The experimental group cultured genes were added with 0,10,20,30 and 40?g/mL FRBI respectively.The medium of the positive control group was added with10 IU/mL FSH,and the cell culture medium was collected at 20h,22h and 24h for ELISA to analysis the change of cAMP and IP3 signaling pathways.The expression levels of FSHR,ER?and PKA protein in oocytes were detected by Western blot.FSHR and ER?gene expression in oocytes were detected by RT-PCR.Research indicates,FSH treatment can increase the number of SF and MF and increase follicular development;while treatment with FRBI reduces the number of SF and MF and inhibits follicular development in mice.In addition,FRBI reduced serum FSH and E2 concentrations and down-regulated ovarian FSHR and ER?protein expression and mRNA expression levels.?2?The mature rate of sheep oocytes cultured at 10 IU/mL FSH and 38.5°C for 26 h was the highest.With the increase of FRBI concentration,the oocytes were cultured at 38.5°C for 26 h,and the number of first polar bodies gradually decreased.?3?FRBI can reduce the expression levels of FSHR gene and ER?gene.Besides,FRBI can reduce the expression levels of FSHR,ER?and PKA protein in sheep oocytes.