The hypothalamic hormone gonadotropinreleasing hormone (GnRH) is released in a pulsatile fashion, with its frequency varying throughout the reproductive cycle. Varying pulse frequencies and amplitudes differentially regulate the biosynthesis and secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) The hypothalamic decapeptide gonadotropin-releasing hormone (GnRH) plays a critical role in reproductive development and function by regulating the biosynthesis and secretion of the pituitary gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH). GnRH is released into the hypophysial portal circulation and transported to the anterior pituitary, where it binds to specific, high-affinity cell surface receptors on gonadotropes, the pituitary cell type that produces gonadotropins. LH and FSH are heterodimers, each composed of a common a subunit associated noncovalently with a unique 1B subunit (1).The stimulation of gonadotropin secretion by GnRH is dependent on the pulsatile nature of GnRH delivery to the anterior pituitary. Administration of exogenous GnRH in a continuous fashion results in the down-regulation of LH and FSH secretion, whereas pulsatile GnRH stimulates LH and FSH secretion, in several species, including primates and rat (2-4). Furthermore, the frequency and amplitude of GnRH pulses secreted by the hypothalamus, which vary during different phases of the menstrual or estrous cycle, regulate differentially . Similarly, the levels of gonadotropin subunit gene expression in the rat pituitary vary by 2-to 4-fold, depending on the GnRH pulse frequency and amplitude (8)(9)(10)(11) GnRHR activation include inositol phospholipid turnover, which leads to calcium mobilization and protein kinase C activation (19,20).The mechanism by which GnRH is able to regulate differentially LH and FSH biosynthesis and secretion is unknown. Previous studies have been limited by the lack of available cell lines that express the LH and FSH subunit genes and respond to GnRH. We have taken advantage of the availability of the GnRHR cDNA to facilitate studies of the mechanisms of action of GnRH. GH3 cells are a well-characterized pituitary cell line generated from a rat pituitary adenoma that express prolactin (PRL) and growth hormone in a regulated fashion (21). These cells express thyrotropin-releasing hormone (TRH) receptors endogenously and respond to TRH with an increase in PRL gene expression, biosynthesis, and secretion (22). Like the GnRHR, the TRH receptor is a member of the family of G protein-coupled receptors and is coupled to pertussis toxin-insensitive G proteins of the Gq/11 family (23).Furthermore, like GnRH, TRH activates its receptor to stimulate inositol phospholipid turnover, leading to calcium mobilization and protein kinase C activation (24). Thus, the effects of TRH and GnRH appear to be mediated through similar intracellular signal transduction pathways.We hypothesized that GH3 cells may present a useful model for the study of the mechanisms of GnRH action...

The hypothalamic hormone gonadotropin-releasing hormone (GnRH) plays a critical role in reproductive function by regulating the biosynthesis and secretion of the pituitary gonadotropins. Although it is known that GnRH induces luteinizing hormone ␤ (LH␤) gene transcription, the mechanisms by which this occurs remain to be elucidated. We have shown previously that GH 3 cells transfected with the rat GnRH receptor cDNA (GGH 3 -1 cells) support the expression of a cotransfected fusion gene composed of 797 base pairs of rat LH␤ gene 5-flanking sequence and the first 5 base pairs of the 5-untranslated region fused to a luciferase reporter (؊797/؉5LH␤LUC) and respond to a GnRH agonist with a 10-fold stimulation of activity. Furthermore, we have shown that DNA sequences at ؊490/؊352 confer GnRH responsiveness to the rat LH␤ gene. We have now identified two putative binding sites for Sp1, a three-zincfinger transcription factor, within this region. Using electrophoretic mobility shift assay, DNase I footprinting, and methylation interference assays, we demonstrate that Sp1 can bind to these sites and that Sp1 is responsible for DNA-protein complexes formed using GGH 3 -1 and ␣T3-1 nuclear extracts. Mutations of the Sp1 binding sites, which block binding of Sp1, blunt the stimulation of the LH␤ gene promoter by GnRH. These data define GnRH-responsive elements in the LH␤ 5-flanking sequence and suggest that Sp1 plays an important role in conferring GnRH responsiveness to the LH␤ subunit gene. The pituitary gonadotropins luteinizing hormone (LH)1 and follicle-stimulating hormone (FSH) play integral roles in the regulation of normal reproductive development and function. The biosynthesis and secretion of these pituitary glycoproteins are controlled by the complex interaction of multiple factors, among the most important of which is gonadotropin-releasing hormone (GnRH). Pulsatile GnRH stimulates the secretion of LH and FSH as well as transcription, steady-state mRNA levels, and biosynthesis of the gonadotropin subunits ␣, LH␤, and FSH␤ (1-4). This regulation is dependent on GnRH pulse amplitude and frequency, which varies with physiologic state, during puberty, during the rat estrous and human menstrual cycles, and during menopause (5, 6). An understanding of the mechanisms of regulation of LH␤ gene expression is an important first step in elucidating the mechanisms of physiologic differential regulation of LH and FSH by GnRH.Studies of the gonadotropin ␣-subunit gene have identified a number of DNA elements in the 5Ј-flanking region that mediate tissue-specific and regulated expression and their cognate binding factors. Recently, two transcription factors, steroidogenic factor-1 (SF-1) and early growth response-1 (Egr-1), have been recognized to be involved in expression of the LH␤ gene (7-9). Nevertheless, relatively little is known about transcription factors that direct gonadotrope-specific or hormonally regulated expression of the LH␤ and FSH␤ subunit genes. A systematic approach to identifying mechanisms of hormonal re...

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