Regulation of WT1, FSH, and FSH receptor in the immature rat granulosa cells

Abstract

Objectives: FSH receptor is essential for integrating FSH signal to gonadal response, while WT1 is essential transcriptional regulator in the gonad. The inverse relationship of FSHR & WT1 mRNA levels in the developing rat ovary, suggest that gonadotropin may also regulate WT1 expression in the ovary. Here we studied the regulatory relationships between WT1, FSH, & FSHR in order to better understand the balance of factors that contribute to preantral follicle selection.

Methods: Granulosa cells were obtained from small antral follicles of immature rat ovary. WT1 & FSHR expression were analyzed by IHC in the ovary. GCs were cultured in the presence of FSH & then endogenous WT1 protein levels were determined by immunoblot analysis. For evaluating promoter activity induced by WT1 overexpression or FSH, GCs were transfected with FSHR promoter construct and increasing amounts of WT1 expression vector [WT1 (-KTS)], or WT1 promoter construct. After 16 hr transfection, cells were lysed and then assayed for luciferase activity.

Results: IHC analysis showed that FSHR was strongly expressed in GCs of larger follicles, whereas WT1 was strongly expressed in small follicles. FSH dose-dependently decreased endogenous WT1 protein levels as well as suppressed WT1 promoter activity in GCs transiently transfected with the WT1 promoter construct. On the other hand, Basal activity of the FSHR promoter was stimulated with increasing amounts of WT1 (-KTS).

Conclusion: The present data demonstrate that WT1 regulates FSHR expression & could be controlled by FSH in GCs. The regulation of FSHR by WT1 could play an important role in the initiation of follicle growing until GCs attained enough numbers of FSHR, because inhibition of WT1 by FSH most likely depends on GCs responsiveness to FSH as follicles grow. This will further substantiate the hypothesis that WT1 represents an endogenous key factor for follicle transition.