This is an amended competing renewal application to investigate the mechanisms controlling steroidogenesis within developing Graafian follicles. The P.I. has discovered synergistic hormonal regulation of two critical genes responsible for sterol metabolism, namely, low density lipoprotein (LDL) receptor and steroidogenic acute regulatory protein (StAR). These two functionally complementary genes are critical to regulated ovarian steroidogenesis, since the LDL receptor serves as the primary gateway for delivering extracellular cholesterol into the granulosa-luteal cell, and StAR acts as the rate-limiting transporter protein to convey sterol substrate to the inner mitochondrial cholesterol side-chain cleavage enzyme complex. The P.I. has found that the gonadotropin, follicle stimulating hormone (FSH), and the intrafollicular growth factor, insulin like growth factor 1 (IGF-1) together produce a synergistic stimulation of 8-20 fold in the enhancement of expression of LDL receptor and StAR mRNA. This proposal concerns the mechanisms of this synergistic action. The specific hypotheses to be investigated are: 1) LDL-receptor and StAR genes are expressed coordinately over time throughout the pig estrous cycle; 2) FSH and IGF-1 synergistically stimulate increased LDL receptor and StAR mRNA expression via amplification of the protein kinase A intracellular signaling pathway; 3) FSH and IGF-1 induce expression of LDL receptor and StAR genes at the single granulosa cell level by amplifying the amount of each gene transcript expressed per granulosa cell and/or by increasing the number of granulosa cells expressing measurable sterol-regulatory gene transcripts; 4) FSH and IGF-1 increase specific LDL-receptor and StAR gene transcriptional initiation rates or decrease specific mRNA degradation (or both). These studies are expected to reveal fundamental mechanisms by which a pituitary hormone (FSH) and the growth factor (IGF-1) act either alone or in concert on ovarian cell populations and on single steroidogenic granulosa cells to control the coordinated expression and synergistic amplification of pivotal genes that direct overall steroidogenesis.
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