Normal ovarian follicular development culminates in a biosynthetically competent corpus luteum, which is essential for normal reproductive function in the female. Extensive experimental work in various mammalian species has demonstrated significant endocrine regulation of certain key steps in gonadal steroidogenesis and intracellular sterol metabolism. We have used a well-defined monolayer cell culture system consisting of hormonally responsive untransformed swine ovarian (granulosa) cells to examine the specific enzymes by which estradiol, FSH, and IGF-I act singly and in combination to regulate specific sterol-metabolizing enzymes under experimentally controlled conditions in vitro. Among the major regulated sites we have studied, and propose to study further are: (i) the LDL receptor; (ii) the cholesterol side-chain cleavage enzyme; (iii) the HMG CoA reductase enzyme; and (iv) the sterol-carrier protein (SCP-2). To date, most of our and other studies have evaluated hormonal regulation of sterol processing at the level of LDL receptor number, enzyme biochemical activity and/or the amount of specific protein synthesized. Our renewal proposal continues logically from these earlier investigations by focusing on the specific hormonal regulation of genomic expression. This emphasis is strongly supported by methods and progress achieved under my parallel RCDA, for example in which we have been able to demonstrate prominent (3-10 fold) stimulation of specific CSCC mRNA accumulation not only by FSH or IGF-I acting alone, but also by estradiol FSH and IGF-I acting synergistically. In contrast, gene expression of a constitutively active enzyme, phosphoglyceraldehyde dehydrogenase, is not induced by these hormones. Accordingly, we are now in an excellent position to use this primary granulosa-cell culture system further to examine the coordinate regulation of gonadal-cell sterol metabolism at the mRNA level. Notably, the swine granulosa cell in vitro system is almost unique in that it manifests prominent steroidogenic responses to three major classes of hormones acting-alone or in concert: viz., the steroid hormone, estradiol; the gonadotropin, FSH; and the insulin-like growth factor, IGF-I. Thus, three significant and distinct """"""""pathways of hormone action can be in, and jointly-in this experimental model.
Our aims encompass an investigation of steady-state mRNA concentrations, gene transcription rates, and in situ molecular hybridization. Studies of the individual and coordinate regulation of sterol-metabolizing enzymes and genes as proposed here should contribute significantly to our better understanding of the essential mechanisms by which relevant hormones act both singly and in concert to promote the remarkable increases in steroidogenic differentiation required during follicular maturation. The preceding studies should contribute important new insights into and further developments in fertility regulation in man, domestic animals, and endangered wild species.
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