Abstract
BackgroundThe 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.MethodsGCs 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.ResultsHere, 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.ConclusionThese 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 (caspases 3 and 7).
Highlights
The fate of the follicles depends on the balance between anti- and pro-apoptotic factors that determine the rate of granulosa cell (GC) apoptosis
There is considerable evidence that WT1 is a potent inhibitor of apoptotic cell death in the developing kidney [5] and male germ cells [6], suggesting that it could play a role in the regulation of follicle survival
The percentage of apoptotic cells significantly decreased at 36? 48 h of culture in WT1 transfected cells as compared with the control (CT vs. WT1; 41.9 ? 15.7% vs. 19.7 ? 10.3%)(P < 0.01) (Figure 4B). These results showed that apoptosis was suppressed in GCs transfected with WT1 compared with the control, suggesting that WT1 is involved in GCs survival by regulating apoptosis
Summary
The fate of the follicles depends on the balance between anti- and pro-apoptotic factors that determine the rate of granulosa cell (GC) apoptosis. The important role of WT1 in early folliculogenesis is evident from the fact that its expression is restricted to immature follicles [2] and from its involvement in the transcriptional control of ovarian marker genes that encode inhibin-α [3] and the follicle stimulating hormone (FSH) receptor [4]. 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 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. We evaluated if WT1 involves in cell survival of granulosa cells (GCs) during the FSH-independent stage
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