Effects Of Bcl-2 And Inhibin α Gene On Apoptosis And Proliferation Of Goose Granulose Cells

To study the genetic effects of Bcl-2 and inhibin a on apoptosis and proliferation of F₁ and atretic follicular granulosa cells in vitro culture,(psiRNAb₁,psiRNAb₂,psiRNAb₃) were constructed to knock down Bcl-2 gene expression,(psiRNA-INHα1 and psiRNA-INHα2) were constructed to knock down Bcl-2 gene expression.After 48 h of transfection, the efficiency of these RNAi expression vectors were examined by fluorescence microscopy.Meanwhile,Bcl-2 and inhibin protein expression levels,apoptosis indexes (AI) and proliferation indexes(PI) of granulosa cells were analyzed by flow cytometry.In addition,the supernatants were collected to assay the concentrations of progesterone(P) and estradiol(E₂) by radioimmunoassay.The main results are as follows:1.Bcl-2(GenBank,D11381) and Inhibinα(GenBank,NM-001031257) siRNAs were designed according to Ambion web-based criteria and BLAST searching showed no significant homology with other genes.Three RNAi expression vectors,psiRNAb₁, psiRNAb₂ and psiRNAb₃ were constructed to interfere Bcl-2 mRNA expression, psiRNA-INHα1 and psiRNA-INHα2,were constructed to interfere InhibinαmRNA expression.2.The harvested granulosa cells were cultured in Dulbecco's modified Eagle's medium supplemented without serum,the RNAi plasmids were contained with transfection reagents,After 48 h of transfection,the efficiency of these RNAi expression vectors were over 60%examined by fluorescence microscop.3.Bcl-2 protein expression levels were analyzed by flow cytometry.Bcl-2 protein levels of test groups were significant lower than that of control(p＜0.05),the psiRNAb3 group were lowest than other RNAi groups(p＜0.05).4.As the Bcl-2 protein levels in granulose cells were reduced,the apoptosis indexes and proliferation indexes of granulose cells in the RNAi groups were significantly higher than those in the control groups,the correlation coefficient between Bcl-2 level and apoptosis index was -0.951(p＜0.05),and the correlation coefficient between the Bcl-2 level and proliferation index was -0.872(p＜0.05).The concentrations of E₂ secreted by granulosa cells were reduced and the P levels were increased with Bcl-2 gene silencing.5.The Inhibin levels in F₁ follicle granulosa cells were significantly higher than atretic follicular granulosa cells.The inhibin levels in interference groups of F₁ and atretic follicular granulosa cells were decreased copared with those in the control groups.6.The concentrations of E₂ secreted by granulosa cells were reduced and the P levels were increased with Inhibinαgene silencing,the inhibin has a direct effect on E₂ and P secretion by granulosa cells.The results showed that:(1) The expression content of Inhibinαof RNAi group were decreased 30～40%than those of control groups(p＜0.05),the apoptosis indexes and proliferation indexes of RNAi groups were significantly higher than those of the control groups(p＜0.05),the E₂ levels of RNAi groups were lower than those of control groups (p＜0.05).(2) The Bcl-2 protein level of test groups were significant lower than that of control,the apoptosis index of test group were higher than that of control,the proliferation index of test group were higher than that of control,the E₂ secretion of test group were lower than that of control,while the P secretion of test group were higher than that of control.These results indicate that Bcl-2 and Inhibinαhas antagonistic effect on granulosa cell apoptosis.