Molecular And Cellular Action Mechanisms Of Inhibin On Folliculogenesis And Spermatogenesis In Mice

Inhibin, a member of the TGFβ superfamily, is a suppresser 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 during spermatogenesis by analysis of the temporal expression of Inha, Inhba, and Inhbb mRNA and the INHA protein and we designed and constructed3Inhibin a-subunit (Inha) gene RNAi vectors by RNAi-Ready pSIREN-RetroQ-ZsGreen vector mediated recombinant pshRNA plasmids which transfected into mice granulosa cells (GCs) and Setoli cells (SCs), identify the mechanism of inhibin on development of GCs and SCs in the mouse and the role of spermatogenesis and folliculogenesis related genes by means of RNA interference of Inha gene. Then, we studied the recombinant plasmids expreesion in the mouse ovary by direct intraovarian injection. The main results are as follows:1. Temporal expression of inhibin subunit mRNA and proteinTestes tissue total RNA and total protein was extracted from male neonates at days1,7,10,14,21,28,35, and56of age. Developmental expression of transcript of Inha, inhba and inhbb in postnatal testes by Real Time PCR and developmental expression of INHA protein in postnatal testes by Western blot. The results were as follows:(1) Inha relative expression general trend were decreased from day1to day10, increased from day10to day21, decreased from day21to day28. There was no significant difference from day28to day56. Inha relative expression at day1were significantly higher than day7, day10, day14, day21, day35, day56, respectively. Both Inhba and inhbb expression were significantly lower compared with Inha during different days except day28.(2) INHA protein were higher at1,7,10,14,21days of age than28,35,56days of age and showed little expression level after28days of age.In conclusion, these findings indicate that inhibin plays an important role in the formation of round spermatid during the first wave of spermatogenesis in mice.2. Action mechanism of inhibin a-subunit on the development of Sertoli cellsIn this study, the expression of Inha gene was partially silenced in mice SCs with3Inha RNAi recombinant plasmids, we studied action mechanism of inhibin on development of SCs in the mouse and role of spermatogenesis related genes by Real Time PCR, Western blot, cell cycle. The results were as follows: (1) The relative mRNA and protein expression of Inha in mice SCs can be significantly reduced by RNAi with pshRNA-1, psiRNA-2and psiRNA-3recombined plasmids. The suppression rate of pshRNA-2was58%, which was the highest among these3plasmids.(2) Inhba, Inhbb, Dhh and Tip1mRNA level were significantly up regulated, Pdgfa and Kitl mRNA level were significantly down regulated after Inha gene silencing by Real Time PCR.(3) INHBA, INHBB and P21protein level were up regulated and CyclinDl and CyclinE were down regulated after Inha silencing by Western blot analysis.(4) S phase cell significantly reduced and G1phase cell apoptosis increased after Inha silencing by flow cytometry.In conclusion, these findings indicate that Inha has the potential to influence the availability of the ligand inhibin in the SC in an autocrine manner and inhibit the progression of SC from G1to S. It may also participate in the development of the blood-testis barrier, Leydig cells, and spermatogenesis through its effect on Dhh, Tjp1, Kitl, and Pdgfa.3. Mechanism of action of Inhibin on development of ovarian granulosa cells in the mouseIn this study, the expression of Inha gene was partially silenced in the mouse granulosa cells with3Inha 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 mRNA and protein expression of Inha in mice GCs can be significantly reduced by RNAi with pshRNA-1, pshRNA-2and pshRNA-3recombined plasmids. The suppression rate of pshRNA-2was60%, which was the highest among these3plasmids.(2) Inhba, Inhbb, P450scc, P450arom, Bax, Ar and Caspase-3mRNA level were significantly up regulated, Bcl2and Tgfbr3mRNA level were significantly down regulated after Inha gene silencing by Real Time PCR.(3) INHBA, INHBB and P21protein level were up regulated after Inha silencing by Western blot analysis.(4) S phase cell significantly reduced and apoptosis increased after Inha silencing by flow cytometry. In conclusion, these findings indicate that Inha has the potential to regulate expression of the Bcl2gene family's induction of the Caspase-3dependent apoptosis pathway to affect GCs development. The influenced activities of a number of TGF superfamily members and the changed cell cycle by Inha were involved in this regulation.4. Inha RNAi plasmid expression in mice ovary by direct intraovarian injectionIn this study, pshRNA-2was expressed in the mouse ovary using direct intraovarian injection by detection of GFP with immunohistochemical. The ovary development and litter sizes were not affected with this method. It provided a foundation for studying the mechanisms of inhibin in mouse ovary.