OVARY SELECTIVE GENES: CRITICAL OVARIAN REGULATORS

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Ovary-selective genes constitute a subset of genes characterized by preferential or exclusive ovarian expression. The critical importance of the ovary-selective genes c-mos, ZP-3, GDF-9, and alpha-inhibin to murine ovarian function was established with null mutants which displayed markedly aberrant ovarian phenotypes and consequent infertility. A loss of function mutation in women established the critical role of the FSH receptor in ovarian function. These observations underlie the hypothesis that ovary- selective genes constitute critical molecular determinants of ovarian function. Thus far, the isolation, identification, and characterization of such ovary-selective genes has proceeded on a case-by-case basis. In this application, we propose a systematic approach. In this context, we report preliminary studies on the use of Suppression Subtractive Hybridization to construct an ovary-selective cDNA library. Ongoing screening of 327 clones has yielded 35 putative novel cDNAs lacking homology to any sequence entry deposited in publicly-accessible databases. We also report preliminary studies on the utility of conditional Cre/LoxP technology with an eye to effect ovary-selective gene deletion. Using the igf-1 gene as a testing paradigm, we document its successful "floxing" and selective deletion in the liver following crossing with a Cre transgenic driven by the albumin promoter. Preliminary studies are reported as to the generation of a Cre transgenic driven by the alpha-inhibin promoter in the hope of effecting granulosa cell-selective deletion of any "floxed" gene of interest. To complete the identification and begin the characterization of novel ovary- selective cDNAs, this proposal outlines a series of complementary in vitro and in vivo experiments. Specifically, we propose to: I) complete the identification of ovary-selective cDNAs using high-throughput robotics and microarrayed DNA chip technology, II) isolate and sequence the full-length of novel ovary-selective cDNAs, III) study the hormonal dependence, (cycle) phase-specific expression, and cellular localization of novel ovary-selective genes and IV) assess the functional role of three novel ovary- selective genes by effecting conditional ovarian gene deletion. Insight derived from this investigation may contribute to novel strategies for the promotion of fertility or its control.