Regulated expression and potential roles of p53 and Wilms' tumor suppressor gene (WT1) during follicular development in the human ovary

It has been previously demonstrated that the gonadotropin-mediated inhibiti on of apoptosis in rat ovarian granulosa cells is associated with changes i n the expression of several cell death-regulatory genes, including p53. In addition, it has been shown that the actions of p53 may be amplified throug h a cooperative interaction with the Wilms' tumor suppressor gene product ( WT1). Based on these findings, the present studies were conducted to determ ine whether p53 and WT1 are expressed and gonadotropin regulated in the hum an ovary and to study the relationship between tumor suppressor gene expres sion and apoptosis in human granulosa/lutein cells (GCs). Analysis of total RNA prepared from human GCs using the RT-PCR demonstrated the presence of p53 messenger RNA (mRNA) and four WT1 mRNA splice variants. These observati ons were supported by Northern blot analysis of total RNA prepared from hum an GCs, which revealed the presence of a single (similar to 2.8 kb) p53 mRN A transcript and two primary (similar to 1.8 and similar to 3.5 kb) WT1 mRN A transcripts. Western blot analysis of nuclear protein extracts from human GCs yielded one immunoreactive protein of the expected size (similar to 53 kDa) recognized by a p53 antibody and one immunoreactive protein of the ex pected size (similar to 52-54 kDa)recognized by the WT1 antibody. Immunohis tochemical staining showed that both molecules were localized to nuclei of human GCs and were coordinately regulated during follicular development. Im munofluorescence analysis showed that p53 protein was localized exclusively to nuclei of GCs undergoing apoptosis during in vitro culture and was simi larly localized to nuclei and cytoplasm of apoptotic granulosa cells in atr etic follicles in vivo. To further evaluate whether human GC apoptosis is l inked to increased expression of tumor suppressor genes, we analyzed levels of p53 and WT1 mRNA and protein in GCs induced to undergo apoptosis in vit ro. Healthy (nonapoptotic) GCs snap-frozen immediately after isolation from patients undergoing in vitro fertilization-embryo transfer possessed relat ively low, but detectable, levels of p53 and WT1 mRNA and protein. However, following serum-free culture to induce apoptosis, p53 mRNA and protein lev els increased significantly after 24 h, paralleling the increase in the num ber of apoptotic GCs. The induction of both p53 mRNA and protein in GCs was inhibited by the addition of human CG to the culture medium. In contrast, WT1 mRNA and protein levels remained constitutive in GCs incubated for 24 h compared with GCs snap-frozen immediately after isolation. We conclude tha t the p53 and WT1 genes are expressed at the mRNA and protein levels in hum an GCs and that expression of p53 is regulated during follicular maturation . Nuclear accumulation of p53 protein occurs in human GCs during apoptosis in vitro and in vivo, and p53 mRNA and protein are up-regulated in GCs star ved of hormonal support but down-regulated by the presence of human CG. We propose that the products of these two principal tumor suppressor genes ser ve as important regulators of human follicular development and corpus luteu m function.