Selective destruction of germ cells (oocytes) within the mammalian ovary following exposure to the environmental toxin, 4-vinyl-l-cyclohexene diepoxide (VCD), leads to permanent sterility in females. The mechanisms by which VCD selectively kills oocytes enclosed in follicles at the earliest stages of development are unknown. Recent data have indicated that changes in expression of a conserved subset of genes, most notably members of the bcl-2 gene family, may play a conserved role in triggering cell death. The product of the bcl-2 gene, when elevated in cells, promotes survival under conditions which normally trigger death. Recent cloning of a related gene product, Bax, has provided evidence that Bax antagonizes the actions of Bcl-2 via heterodimerization and inactivation of the Bcl-2 protein. Thus, it is believed that the ratio of bcl-2 to bax gene expression forms a final common event which directs either cell survival (high Bcl-2, low Bax) or death (low Bcl-2, high Bax). Previous genetic mutation analyses as well as more recent in vitro studies have revealed that the primary trophic hormone which supports oocyte survival is the Steel factor (SCF) synthesized by the somatic granulosa cells which surround the oocyte. The receptor for SCF in the ovary, expressed almost exclusively by the oocyte, has been identified as the c-kit protein. Based on these data, we have.hypothesized that VCD-induced oocyte destruction is mediated at multiple levels within the developing follicle, including a suppression of SCF gene expression in granulosa cells which support the oocyte, a loss of c-kit receptor expression required for binding and transduction of the SCF signal, as well as a direct alteration of bcl-2 (decrease) and bax (increase) gene expression in oocytes. To elucidate how VCD leads to sterility in females, the following aims are proposed: 1) to characterize the type and time-course of cell death induced in oocytes following exposure of follicles to VCD in vivo and in vitro through biochemical and in situ analysis of DNA integrity (apoptotic cell death: internucleosomal DNA breakdown; necrotic cell death: random DNA cleavage; programmed cell death: DNA integrity maintained) and/or polyubiquitin gene expression (a marker for programmed cell death); 2) to assess changes in mRNA levels for SCF and c-kit relative to the initiation and progression of oocyte death following exposure of follicles to VCD in vivo and in vitro, and determine if exogenous SCF can protect oocytes from the toxic effects of VCD in vitro; and, 3) to determine the effects of VCD, in vivo and in vitro, on bcl-2 and bax mRNA levels in cellular components of follicles, and assess the requirement for elevated bax expression in VCD-mediated oocyte death in vitro.
