Identification and Analysis of Two Novel Sites of Rat GnRH Receptor Gene Promoter Activity: The Pineal Gland and Retina

Univ Paris Diderot Paris 7, Sorbonne Paris Cité, Biologie Fonctionnelle et Adaptative, EAC CNRS 4413, Physiologie de l'Axe Gonadotrope, Paris, France.
Neuroendocrinology (Impact Factor: 4.37). 03/2012; 97(2). DOI: 10.1159/000337661
Source: PubMed


Background and Aims: In mammals, activation of pituitary GnRH receptor (GnRHR) by hypothalamic GnRH increases the synthesis and secretion of LH and FSH, which, in turn, regulate gonadal functions. However, GnRHR gene (Gnrhr) expression is not restricted to the pituitary. Methods: To gain insight into the extrapituitary expression of Gnrhr, a transgenic mouse model that expresses the human placental alkaline phosphatase reporter gene driven by the rat Gnrhr promoter was created. Results: This study shows that the rat Gnrhr promoter is operative in two functionally related organs, the pineal gland, as early as embryonic day (E) 13.5, and the retina where activity was only detected at E17.5. Accordingly, Gnrhr mRNA were present in both tissues. Transcription factors known to regulate Gnrhr promoter activity such as the LIM homeodomain factors LHX3 and ISL1 were also detected in the retina. Furthermore, transient transfection studies in CHO and gonadotrope cells revealed that OTX2, a major transcription factor in both pineal and retina cell differentiation, is able to activate the Gnrhr promoter together with either CREB or PROP1, depending on the cell context. Conclusion: Rather than using alternate promoters, Gnrhr expression is directed to diverse cell lineages through specific associations of transcription factors acting on distinct response elements along the same promoter. These data open new avenues regarding GnRH-mediated control of seasonal and circadian rhythms in reproductive physiology.

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    • "Cell culture and transient transfection assays LbT2 cells kindly provided by Dr Pamela Mellon (University of California, San Diego, CA, USA) were maintained in monolayer cultures using high-glucose DMEM supplemented with 10% foetal bovine serum and penicillin/streptomycin at 37 8C in humidified 5% CO 2 , 95% air. They were transiently transfected using Lipofectamine 2000 (Invitrogen) following a reverse transfection protocol in 96-well plates as described previously (Schang et al. 2013). After 5 h transfection, cells were treated for 20 h with or without 10 ng/ml activin A (R&D, Minneapolis, MN, USA) or 150 ng/ml recombinant mouse follistatin (Biovision, Milpitas, CA, USA) followed by a 4 h treatment with an increasing concentration of the GnRH agonist triptorelin (Ipsen Biotech, Paris, France). "
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    ABSTRACT: The GnRH receptor (GnRHR) is expressed in several non-pituitary tissues, notably in gonads. However, mechanisms underlying gonad-specific expression of the Gnrhr are not well understood. Here, Gnrhr expression was analysed in developing testes and pituitaries of rats and transgenic mice bearing the human placental alkaline phosphatase reporter gene (ALPP) under the control of rat Gnrhr promoter. We showed that 3.3 kb, but not the pituitary-specific 1.1 kb promoter, directs ALPP expression exclusively to testis Leydig cells from embryonic day 12 onward. Real-time PCR analysis revealed that promoter activity displayed the same biphasic profile as marker genes in Leydig cells, i.e. abrupt declines after birth followed by progressive rises after a latency phase, in coherence with differentiation and evolution of foetal and adult Leydig cell lineages. Interestingly, the developmental profile of transgene expression showed high similarity with the endogenous Gnrhr profile in rat testis while mouse Gnrhr was only poorly expressed in mouse testis. In the pituitary, both transgene and Gnrhr were co-expressed at measurable levels with similar ontogenetic profiles, which were markedly distinct from those in testis. Castration that induced pituitary Gnrhr up-regulation in rat did not affect mouse Gnrhr. However, it duly up-regulated the transgene. In addition, in LβT2 cells, rat but not mouse Gnrhr promoter was sensitive to GnRH agonist stimulation. Collectively, our data highlight inter-species variations in expression and regulation of the Gnrhr in two different organs and reveal that the rat promoter sequence contains relevant genetic information that dictates rat-specific gene expression in the mouse context.
    Full-text · Article · Mar 2013 · Journal of Molecular Endocrinology
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    • "In a neutral cell context, OTX2 acted in synergy with CREB and formed a regulatory unit that involved CRE, an AP1 response element together with a new OTX2 binding site containing a TAAT core motif located at −163/−160. This motif is thus located 50 bp upstream of the CRE and 180 bp downstream of the AP1 element establishing a regulatory unit extending over 200 bp within the proximal promoter, markedly larger than the PROP1/OTX2 regulatory unit (Schang et al., 2012a). Whether such difference in size of these regulatory units as well as the other cited above is functionally relevant remains to be determined. "
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    ABSTRACT: The GnRH receptor (GnRHR) plays a central role in the development and maintenance of reproductive function in mammals. Following stimulation by GnRH originating from the hypothalamus, GnRHR triggers multiple signaling events that ultimately stimulate the synthesis and the periodic release of the gonadotropins, luteinizing-stimulating hormone (LH) and follicle-stimulating hormones (FSH) which, in turn, regulate gonadal functions including steroidogenesis and gametogenesis. The concentration of GnRHR at the cell surface is essential for the amplitude and the specificity of gonadotrope responsiveness. The number of GnRHR is submitted to strong regulatory control during pituitary development, estrous cycle, pregnancy, lactation, or after gonadectomy. These modulations take place, at least in part, at the transcriptional level. To analyze this facet of the reproductive function, the 5' regulatory sequences of the gene encoding the GnRHR have been isolated and characterized through in vitro and in vivo approaches. This review summarizes results obtained with the mouse, rat, human, and ovine promoters either by transient transfection assays or by means of transgenic mice.
    Full-text · Article · Dec 2012 · Frontiers in Endocrinology
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    ABSTRACT: Extra-gonadal role of gonadotropins has been re-evaluated over the last 20 years. In addition to pituitary secretion of luteinizing hormone (LH) and follicle stimulating hormone (FSH), the CNS has been clearly identified as a source of hCG acting locally to influence behaviour. Here we demonstrated that human retina is producing this gonadotropin that acts as a neuroactive molecule. Müller glial and retinal pigmented epithelial (RPE) cells are producing hCG that may affects neighbour cells expressing its receptor, namely cone photoreceptors. It was previously described that amacrine and retinal ganglion (RGC) cells are targets of the gonadotropin releasing hormone that control the secretion of all gonadotropins. Therefore our findings suggest that a complex neuroendocrine circuit exists in the retina, involving hCG secreting cells (glial and RPE), hCG targets (photoreceptors) and hCG-release controlling cells (amacrine and RGC). The exact physiological functions of this circuit have still to be identified, but the proliferation of photoreceptor-derived tumor induced by hCG demonstrated the need to control this neuroendocrine loop.
    Full-text · Article · Dec 2012 · PLoS ONE