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Regulated Expression and Potential Roles of p53 and Wilms’ Tumor Suppressor Gene (WT1) during Follicular Development in the Human Ovary¹

Division of Human Reproduction and the Center for Research on Reproduction and Women’s Health, Department of Obstetrics and Gynecology (A.M., G.C., C.C.) and Pulmonary Division, Department of Medicine (K.A.), University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104; and Vincent Center for Reproductive Biology, Department of Obstetrics and Gynecology (J.L.T.), Massachusetts General Hospital/Harvard Medical School, Boston, Massachusetts 02114

Address correspondence and requests for reprints to: Christos Coutifaris M.D., Ph.D., Division of Human Reproduction, Department of Obstetrics and Gynecology, University of Pennsylvania, 106 Dulles, 3400 Spruce Street, Philadelphia, Pennsylvania 19104. E-mail: ccoutifaris{at}obgyn.upenn.edu

It has been previously demonstrated that the gonadotropin-mediated inhibition of apoptosis in rat ovarian granulosa cells is associated with changes in the expression of several cell death-regulatory genes, including p53. In addition, it has been shown that the actions of p53 may be amplified through a cooperative interaction with the Wilms’ tumor suppressor gene product (WT1). Based on these findings, the present studies were conducted to determine whether p53 and WT1 are expressed and gonadotropin regulated in the human ovary and to study the relationship between tumor suppressor gene expression 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 observations were supported by Northern blot analysis of total RNA prepared from human GCs, which revealed the presence of a single (2.8 kb) p53 mRNA transcript and two primary (1.8 and 3.5 kb) WT1 mRNA transcripts. Western blot analysis of nuclear protein extracts from human GCs yielded one immunoreactive protein of the expected size (53 kDa) recognized by a p53 antibody and one immunoreactive protein of the expected size (52–54 kDa) recognized by the WT1 antibody. Immunohistochemical staining showed that both molecules were localized to nuclei of human GCs and were coordinately regulated during follicular development. Immunofluorescence analysis showed that p53 protein was localized exclusively to nuclei of GCs undergoing apoptosis during in vitro culture and was similarly localized to nuclei and cytoplasm of apoptotic granulosa cells in atretic follicles in vivo. To further evaluate whether human GC apoptosis is linked to increased expression of tumor suppressor genes, we analyzed levels of p53 and WT1 mRNA and protein in GCs induced to undergo apoptosis in vitro. Healthy (nonapoptotic) GCs snap-frozen immediately after isolation from patients undergoing in vitro fertilization-embryo transfer possessed relatively low, but detectable, levels of p53 and WT1 mRNA and protein. However, following serum-free culture to induce apoptosis, p53 mRNA and protein levels increased significantly after 24 h, paralleling the increase in the number 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 that the p53 and WT1 genes are expressed at the mRNA and protein levels in human 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 starved of hormonal support but down-regulated by the presence of human CG. We propose that the products of these two principal tumor suppressor genes serve as important regulators of human follicular development and corpus luteum function.

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