Endocrine Abstracts

Background: The Wilms’ Tumour-1 (WT1) transcription factor is a critical regulator of embryonic gonadogenesis, but is also expressed by granulosa cells (GCs) in preantral follicles in the ovary after birth. Evidence from animal models suggests an important role for WT1 in regulating GC steroidogenesis and apoptosis. However, the role of WT1 in human GC biology has not been extensively explored.

Aim: To investigate the role of WT1 in regulating human GC function, proliferation and apoptosis.

Methods: Human KGN granulosa tumour cells were transfected with anti-WT1 or scrambled (control) siRNAs. Gene expression was measured using RT-qPCR. Proliferation was assessed using phosphorylated histone H3 immunohistochemistry, and apoptosis by cleaved caspase-3 and cleaved PARP immunoblotting. FSH treatments (10nM, 24h) were performed on serum-starved KGNs.

Results: Expression of the proliferation marker CCND1 was reduced ~64% in WT1-KD cells vs controls (P = 0.016), concomitant with a reduction in the proportion of proliferating cells from 8.08% ± 0.97% (controls) to 5.21% ± 0.46% (WT1-KD; P < 0.026). Expression of pro-apoptotic gene BAX1 was increased ~80% in WT1-KD cells vs controls (P = 0.032), and sensitivity to apoptotic stimuli was enhanced, demonstrated by 2.4-fold increases in cleaved caspase-3 and cleaved PARP in WT1-KD KGNs (both P = 0.029). In contrast to data from rodent and bovine models, WT1-depletion in KGNs was associated with repression of CYP11A1 (~30% reduction vs controls, P = 0.032) while other steroidogenic genes remained unchanged. Depletion of WT1 also dysregulated FSHR expression (3.6-fold increase in WT1-KD cells vs control, P = 0.016) and enhanced FSH-mediated upregulation of CYP19A1 by ~2.2-fold vs controls (P = 0.029).

Conclusion: These data identify WT1 as a potential regulator of human GC proliferation, survival and function, and reveal possible species-specific differences in the regulation of steroidogenic genes by WT1. As WT1 mutations have been associated with premature ovarian failure, this work may provide insight into the molecular basis of female infertility.