Abstract
BackgroundRegulation of gonadotropin-releasing hormone (GnRH) receptor (GnRHR) numbers on gonadotropes within the anterior pituitary gland represents a critical point for control of reproductive function. Binding of GnRH to its receptor regulates follicle stimulating hormone (FSH) and luteinizing hormone (LH) release and levels of this G-protein coupled receptor on the surface of gonadotropes determines their sensitivity to GnRH pulses. While transcriptional regulation of this gene has been studied in mice, rats, humans and sheep, little is known about its regulation in the pig, an important agricultural species and human research model.MethodsWe isolated 5118 bp of 5′ flanking sequence for the porcine GnRHR gene and generated luciferase reporter vectors. Deletion and mutation constructs were evaluated in gonadotrope-derived alphaT3-1 cells to determine regions important for gene transcription. Additionally, electrophoretic mobility shift assays (EMSAs) were performed to identify transcription factors binding to the GnRHR promoter.ResultsTransient transfections revealed that the GnRHR promoter was functional in alphaT3-1 cells but not in cells of non-gonadotrope origin. Mutation of the highly conserved gonadotrope specific element (GSE) located at -179/-171 of proximal promoter completely ablated luciferase activity, whereas mutation of another GSE at -315/-310 reduced activity by 34%. Consistent with this, EMSAs using alphaT3-1 nuclear extracts and a steroidogenic factor (SF)1 antibody confirmed SF1 binding to both GSEs. EMSAs also demonstrated that a retinoid X receptor (RXR) binding site at -279/-274 binds RXRalpha and RXRbeta and mutation of this site eliminated promoter activity. Transient transfection of alphaT3-1 cells with reporter vectors containing selective removal of 5′ flanking region for the porcine GnRHR gene indicated that the -1915/-1431 segment was important for promoter activity. Definition of this region via transfection assays and EMSAs revealed an upstream enhancing region located at -1779/-1667 that increases porcine GnRHR gene expression in alphaT3-1 cells and includes a SF1 binding site at -1760/-1753.ConclusionsPorcine GnRHR promoter activity in alphaT3-1 cells is partially conferred by a distal GSE, two proximal GSEs and a RXR binding site. Basal gonadotrope expression of the porcine GnRHR gene uniquely involves three GSEs and RXR is newly identified as a regulator of GnRHR promoter activity.
Highlights
Regulation of gonadotropin-releasing hormone (GnRH) receptor (GnRHR) numbers on gonadotropes within the anterior pituitary gland represents a critical point for control of reproductive function
Porcine GnRH receptors (GnRHR) promoter activity in alphaT3-1 cells is partially conferred by a distal gonadotrope specific element (GSE), two proximal GSEs and a retinoid X receptor (RXR) binding site
Basal gonadotrope expression of the porcine GnRHR gene uniquely involves three GSEs and RXR is newly identified as a regulator of GnRHR promoter activity
Summary
Regulation of gonadotropin-releasing hormone (GnRH) receptor (GnRHR) numbers on gonadotropes within the anterior pituitary gland represents a critical point for control of reproductive function. Basal expression of the mouse GnRHR gene in the gonadotrope-derived αT3-1 cell line is mediated by 600 bp of proximal promoter, comprised of a gonadotrope specific element (GSE), an activator protein (AP)-1 binding site and an element termed GnRH receptor activating sequence or GRAS [11,12,13,14]. Basal activity of the mouse GnRHR promoter is dependent upon: LHX3, a member of the LIM homeodomain family [15]; two octamer transcription factor (OCT1) binding sites [16]; a pituitary homeobox (Pitx)-1 site that acts synergistically with an AP-1 element [17]; the sequence underlying responsiveness to GnRH (SURG)-1 that binds OCT1 and nuclear factor (NF)-Y [18]; and E-boxes that bind CLOCK protein [19]. An SF1 binding site has been implicated in basal expression of the ovine GnRHR gene and 2700 bp of promoter confers tissue-specific expression in transgenic mice [26]
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