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Background  The important role of  WT1  in early folliculogenesis was evident from its restricted expression pattern in immature follicles and from its involvement in transcriptional control of  inhibin-α  and FSH receptor. There is also considerable evidence that  WT1  is a potent inhibitor of apoptotic cell death in the developing kidney and male germ cells, suggesting that it could play a role in the regulation of follicle survival. Therefore, we evaluated if  WT1  involves in cell survival of granulosa cells (GCs) during the FSH-independent stage.    Methods  GCs were obtained from small preantral follicles of immature rat ovary.  Bax  and  bcl-2  mRNA and protein levels in GCs transfected with  WT1  (−KTS) or  WT1  (+KTS) were analyzed by Real-time RT-PCR and immune-blotting analysis. Cell viability was measured with MTT assays and apoptosis was analyzed with  caspase  3/7 activity and TUNEL assay. The mechanism by which  WT1  regulates  Bax  expression was investigated using Bax promoter-luciferase reporter assay and ChIP assays from GCs.    Results  Here, we showed that  WT1  (−KTS) suppressed endogenous  Bax  transcript and protein expression, and this inhibition resulted from direct binding of  WT1  in the  Bax  promoter region in vivo. In addition, anti-apoptotic effects of  WT1  (−KTS) were demonstrated based on MTT assays, a sensitive bioluminescence  caspase  3/7 assay and TUNEL assays. On the other hand,  WT1  has no role on  bcl-2  expression in GCs.    Conclusion  These findings suggest that activation of  WT1  is necessary for maintenance of GC survival during early stage of follicles and  WT1  can play a role in protecting apoptosis through the regulation of upstream activator ( Bax ), as well as through regulation of downstream effecter ( caspase s 3 and 7).

Wilms’ tumor suppressor gene (WT1) suppresses apoptosis by transcriptionally downregulating BAX expression in immature rat granulosa cells