Molecular And Cellular Mechanisms Of Inhibin βA And βB Subunits On Folliculogenesis In Mice

Inhibins,members of the TGF-β superfamily,are suppressors of follicle stimulating hormone(FSH)secretion through pituitary gonad negative feedback to regulate folliculogenesis and spermatogenesis.In the present study,we studied the role of inhibin Inhba,and Inhbb subunits genes during folliculogenesis in mice.We designed and constructed 3 vectors for each subunit by RNAi-Ready p SIREN-Retro Q-Zs Green vector mediated recombinant psh RNA plasmids which transfected into micegranulosa cells(GCs),to identify the mechanism of inhibin Beta subunits on the development of GCs in the mouse.The main results are as follows:1.Action mechanism of inhibin βB-subunit on the development of granulosa cellsIn this study,the expression of Inhbb gene was partially silenced in mice GCs with 3Inhbb RNAi recombinant plasmids,we studied action mechanism of inhibin bb on development of GCs in the mouse by Real Time PCR,Western blot,cell cycle,apoptosis and Elisa.The results were as follows:(1)The relative m RNA and protein expression of Inhbb in mice GCs can be significantly reduced by RNAi with psh RNA-B1,psh RNA-B2 and psh RNA-B3 recombined plasmids.The suppression rate of psh RNA-B3 was 58%,which was the highest among these 3 plasmids.(2)CYP11A1and CYP19A1 m RNA level were significantly downregulated after Inhbb gene silencing by Real Time PCR.(3)The cell cycle was arrested at G1 phase after inhibin βB knock down by flow cytometry(4)BAX protein level was upregulated,BCL-2,Cyclin D1 and Cyclin E protein level were downregulated after Inhbb silencing by Western blot analysis.(5)Granulosa cell apoptosis increased after Inhbb silencing by flow cytometry.(6)The concentration of estradiol and progesterone was reduced in the culture medium INHBB silencing.In conclusion,these findings suggest that inhibin βB is important in regulation of apoptosis and cell cycle progression in granulosa cells.Furthermore,inhibin βB-subunit has a role in the regulation of steroid hormone biosynthesis.Evidence is accumulating to support the concept that inhibin βBis physiologically essential for early folliculogenesis in mouse.2.Mechanism of action of Inhibin βA on the development of ovarian granulosa cells in the mouseIn this study,the expression of Inhba gene was partially silenced in the mouse granulosa cells with 3 Inhba RNAi recombinant plasmids,we studied action mechanism of inhibin on development of GCs in the mouse and role of follicular development related genes by Real Time PCR,Western blot,flow cytometry,cell cycle and cell apoptosis.The results were as follows:(1)The relative m RNA and protein expression of Inhba in mice GCs can be significantly reduced by RNAi with psh RNA-A1,psh RNA-A2 and psh RNA-A3 recombined plasmids.The suppression rate of psh RNA-A3 was 42%,which was the highest among these 3 plasmids.(2)CYP11A1and CYP19A1 m RNA level were significantly downregulated after Inhba gene silencing by Real Time PCR.(3)Cyclin D1 and cyclin E protein level were downregulated,Capspase 3,BAX and P21 protein level were upregulated after Inhba silencing by Western blot analysis.(4)The proportion of apoptotic cells increased significantly after Inhba silencing by flow cytometry.(5)Estradiol and progesterone concentration was reduced in the culture medium INHBB silencing by Elisa.(6)The cell cycle was arrested at G1 phase after inhibin βB knock down by flow cytometryIn conclusion,these findings indicate that Inhba may play an important role in the regulation of apoptosis,growth and proliferation and steroidogenesis in mouse granulosa cells.