The majority of ovarian follicles undergo atresia. We have demonstrated apoptosis as the underlying mechanism of atresia and identified multiple follicle survival and atretogenic hormones. Transgenic mice with ovarian over-expression of the anti-apoptosis protein Bcl-2 were generated and found to have decreased follicle cell apoptosis and increased tumor formation. We then prepared an ovarian fusion cDNA library to identify ovarian Bcl-2-interacting proteins using the yeast 2-hybrid system. BAD was found as a Bcl-2-binding protein whereas 14-3-3, a molecule capable of mediating cross talks between different signaling pathways, attenuated BAD-induced cell killing. The use of a mutant BAD incapable of binding 14- 3-3 as bait allowed the identification of P11, an early responses and survival gene induced by NGF in neuronal cells. Because P11 also attenuated BAD killing, it may mediate the actions of upstream survival factors in the ovary. Further screening using mutant BAD led to the isolation of Mcl-1, a key anti-apoptotic protein important in the """"""""decision"""""""" step of apoptosis in the ovary. Additional screening using Mcl- 1 allowed isolation of two novel pro-apoptotic Bcl-2-related proteins. Bok and BOD. Bok is highly expressed in the ovary and induces apoptosis in transfected mammalian cells whereas the function of BOD remains to be elucidated. We propose to analyze the expression pattern of P11, Mcl-1, Bok and BOD in ovarian follicles during development and under regulation by gonadotropins, estrogens and growth factors in vivo and in vitro. We will establish the role of BAD as a key bridging molecule between upstream signals and diverse Bcl-2 members. We will study the structure-function relationship of Bok. The full-length sequence of BOD, its role in ovarian cell apoptosis and its structural requirement will also be studied. Attempts will be made to characterize additional Mcl-1 binding proteins as signaling molecules in the ovary. Studies on the hormonal control of Bcl- 2-interacting proteins during follicle atresia provide a unique paradigm to elucidate intracellular apoptosis mechanisms. Because ovarian pathologies such as pre-mature ovarian failure and tumor formation are associated with aberrant apoptosis regulation, the proposed studies may provide new treatments for infertility and tumorigenesis.
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