MOLECULAR BIOLOGY OF HORMONES THAT REGULATE REPRODUCTION

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The goal of this project is to examine the expression and regulation of genes encoding brain and gonadal hormones that are involved in controlling mammalian reproduction. Four polypeptide hormones that modulate the synthesis and secretion of the pituitary gonadotropins have been described. These are the hypothalamic peptide gonadotropin-releasing hormone (GnRH), the related gonadal hormones inhibin and activin, and the recently described ovarian hormone follistatin. Inhibin, activin, and follistatin regulate the secretion of follicle-stimulating hormone (FSH) and GnRH regulates the secretion of FSH and lutienizing hormone (LH) from the pituitary. Because FSH and LH play a crucial role in modulating the gametogenic and steroidogenic functions of the gonads, these regulatory hormones are likely to be important for homeostasis in the reproductive system. We have isolated cDNA clones from the rat for all of these peptide hormones: GnRH, follistatin, and the alpha- and beta-chains of inhibin/activin. We have used these cDNAs to examine expression of the corresponding mRNAs in reproductive tissues using in situ hybridization histochemistry, and have begun to explore the regulation of these genes during the rat estrous cycle and during pregnancy. We now propose to extend these studies in two directions. Firstly, we plan to explore the molecular mechanisms by which these genes are regulated. We will complete in vivo experiments to examine changes in GnRH, inhibin, and activin mRNA levels during the estrous cycle and in response to specific hormones. We will develop in vitro assays, including ovarian organ cultures and primary rat granulosa cell cultures, to directly examine the effects of steroid and polypeptide hormones on inhibin gene expression. The granulosa cells will also be utilized for transfection studies in which inhibin and activin gene regulatory elements will be characterized. Secondly, we plan to examine the biological significance of expression of these genes in several novel tissues. The inhibin/activin beta-chain mRNA has been found in diverse tissues including the brain, pituitary, hematopoietic system, and the placenta. This is particularly intriguing in light of its similarity to the transforming growth factor-beta family of growth and differentiation regulators. We will investigate when in development the inhibin/activin beta-chain gene is expressed in these tissues, and determine whether the gene is regulated in a manner similar to that observed in the gonads. We will also examine extra-gonadal expression of the rat follistatin gene, pursuing our recent observation that this gene is highly expressed in decidual tissue early in pregnancy. To aid in these studies, we will generate immunological reagents specific for the activin and follistatin proteins. We expect these experiments to enhance our understanding of the roles these hormones play in regulating normal reproduction, and to provide a framework for examining how alterations in their activities might lead to reproductive disorders or dysfunction.