Induction of FSH-beta by TGF-Beta Family Members

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Follicle-stimulating homrone (FSH) is an alpha/beta heterodimer produced only in pituitary gonadotropes that orchestrates ovarian follicular development leading to egg production in mammals. Its synthesis and secretion depend primarily on transcription of its beta subunit (FSHB) which is tightly regulated, but can vary >20-fold in vivo. The central hypothesis of this proposal is that activin-like induction is the most important transcriptional event regulating FSHB, and its corollary is that this induction depends on discrete response elements in the FSHB proximal promoter. AIM 1 will use in vitro methods to locate these elements on ovine and mouse FSHB promoters. Transient expression of mutated FSHB-promoter-luciferase (FSHBLuc) constructs will be used in LbetaT2 cells (transformed mouse gonadotropes) to detect the presence (or absence) of critical response elements. Already deletion mutagenesis has located two regions in the ovine FSHB promoter required for activin action. AIM 2 will determine the physiological relevance of these two regions and other elements identified in aim 1. FSHBLuc (5 to 7 constructs) with mutated response elements will be expressed in transgenic mice, and their regulation in vivo will be examined for deviations caused by the dysfunctional response elements(s) to determine their physiological importance. Regulation will be monitored in vivo during the normal estrous cycle, after gonadectomy or following treatment with GnRH agonists or antagonists. The effects of activin, inhibin or follistatin will be tested in primary transgenic pituitary cultures. AIM 3 will characterize 5' and 3' FSHB sequences that allow the FSHB proximal promoter to function properly in vivo in gonadotropes. DNA transfer methods, as in aim 1, and DNAse hypersensitivity assays will be used to locate key DNA sequences. Then transgenic mice will carry FSHBLuc with mutations in these sequences (1 to 3 constructs) to determine the importance of the 5' and 3' regions in vivo. AIMS 1-3 will locate DNA elements that ultimately affect egg (and sperm) production in mammals. Deleterious mutations of these elements (or their binding proteins) will alter fertility. Our studies will show the importance of each element in vivo and predict how destruction of each element will be evident clinically. Moreover, AIM 3 will begin to characterize a cell-targeting mechanism on the FSHB gene that controls gonadotrope-specific express on and it likely to be prototypical of mechanisms that express most endocrine hormones.