Kisspeptin increases GnRH mRNA expression and secretion in GnRH secreting neuronal cell lines
Introduction
The hormonal network responsible for the control of reproduction is composed of three major hierarchical elements: the hypothalamic gonadotropin-releasing hormone (GnRH), the pituitary gonadotropins (LH and FSH) and the products of the gonads, principally, sex steroids. Reproductive capacity is attained at puberty as the end-point of a complex series of developmental and neuroendocrine events that lead to full activation of the GnRH pulse generator, enhanced gonadotropin secretion and complete gonadal maturation and function (Terasawa and Fernandez, 2001, Plant and Witchel, 2006). Since GnRH neurons play a critical central role in the regulation of pubertal development and reproduction, the central and peripheral signals that regulate these cells are important to elucidate.
Gonadal sex steroids are major regulators of GnRH secretion through negative and positive feedback loops. In addition, most recently, the physiological control of the reproductive axis was advanced by the identification of the essential role of kisspeptin, the peptide product of the KiSS-1 gene, and its receptor, G-protein coupled receptor 54 (GPR54), in the neuroendocrine regulation of reproduction (de Roux et al., 2003, Funes et al., 2003, Seminara et al., 2003, Castellano et al., 2005). Investigations by many laboratories over the last 5 years have led to the general concept that kisspeptin neurons activate GnRH neurons (Tena-Sempere, 2006, Kauffman et al., 2007, Caraty and Franceschini, 2008, Roa et al., 2008a, Roa et al., 2009, Seminara and Crowley, 2008).
Navarro et al. (2004) and Shahab et al. (2005) observed an increase of KiSS-1 and GPR54 mRNAs during pubertal development in experiments performed in intact rats and monkeys, suggesting that, these developmental changes in the expression of KiSS-1 mRNA and GPR54 may play a role in the onset of puberty, and could be considered proximal signaling events for pubertal maturation (Han et al., 2005, Gottsch et al., 2006). Central or systemic administration of kisspeptin leads to increased GnRH and gonadotropin secretion in both prepubertal and adult animals (Plant and Barker-Gibb, 2004, Navarro et al., 2005a, Gottsch et al., 2006, Roa et al., 2006, Pielecka-Fortuna et al., 2008, Roa et al., 2008b). Furthermore, GPR54 mutations in humans or targeted deletions in mice produce isolated hypogonadotropic hypogonadism and infertility, thereby demonstrating a required regulatory function in both sexes (Messager et al., 2005, Smith et al., 2006a, Tena-Sempere, 2006, Gottsch et al., 2006, d’Anglemont de Tassigny et al., 2007, Dungan et al., 2007, Clarkson et al., 2008).
Estradiol is one of the most important regulators of GnRH neuronal activity (Herbison, 1998, Wintermantel et al., 2006, Herbison, 2008). Irwig et al. (2004) and Navarro et al. (2004) have provided evidence in rats that kisspeptin-expressing neurons are targets for regulation by sex steroids, furthermore, these neurons are directly regulated by the negative and positive feedback actions of sex steroids in distinct regions of the forebrain (Gottsch et al., 2006, Smith et al., 2006a). These observations suggest that kisspeptin/GPR54 signaling provides tonic stimulatory input to GnRH neurons, which could be governed by the feedback effects of sex steroids acting on kisspeptin secreting neurons (Gottsch et al., 2006). In the female, the estradiol-dependent induction of KiSS-1 mRNA in the anteroventral periventricular nucleus (AVPV) may play a role in mediating the preovulatory GnRH/LH surge, that drives ovulation or the regulation of sexual behavior (Kinoshita et al., 2005, Smith et al., 2005a, Smith et al., 2005b, Smith et al., 2006b, Clarkson et al., 2008, Herbison, 2008). Unlike in the AVPV, kisspeptin neurons in the arcuate nucleus (Arc) play the same role in both sexes (negative feedback regulation of gonadotropin secretion by gonadal steroids). Recently, studies in GPR54 KO mice reported that GPR54 signaling is critical for the maintenance of tonic LH secretion, reflecting a lack of normal follicular development and resulting in infertility (Dungan et al., 2007). In addition to receiving input from estradiol sensitive presynaptic afferents, GnRH neurons have been shown to express estrogen receptors, although the studies have been difficult and controversial. In vivo evidence for the presence of ERβ has been demonstrated, suggesting that the effects of estrogen may be directly transmitted (Skynner et al., 1999, Hrabovszky et al., 2000, Herbison and Pape, 2001, Petersen et al., 2003, Skinner and Dufourny, 2005, Wintermantel et al., 2006). The model GnRH neuronal cell lines, GT1–7 and GN11 have been shown to also express functional ERα and ERβ (Radovick et al., 1991a, Roy et al., 1999, Ng et al., 2009).
The KiSS-1/GPR54 system revealed a fundamental role in the control of puberty and/or maintenance of reproductive function; although the direct targets of kisspeptin, its specific pharmacokinetics and the cellular signaling mechanism remained obscure (Tena-Sempere, 2006). In the present studies both the GN11 and GT1–7 GnRH expressing cell lines were used as models to explore the direct regulation of the GnRH neuron by kisspeptin. The aim of this work is to determine whether the GnRH neuron can be directly regulated by kisspeptin; the potential mechanism of kisspeptin signaling in GnRH neurons, and whether GnRH neurons may be integrators of sex steroid feedback with kisspeptin regulation.
Section snippets
Cell culture
GN11 cells were grown in Dulbecco's modified Eagle's medium (DMEM; Mediatech Inc., Herndon, VA, USA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA) and 25 mM glucose, 5 mM l-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco, Grand Island, NY, USA) in an atmosphere with 5% CO2 at 37 °C. GT1–7 cells were grown in a similar manner, except supplemented with 10% heat-inactivated fetal bovine serum. Cells were placed in media supplemented with 10% dextran and charcoal
GnRH mRNA and protein expression in GT1–7 and GN11 cells
Initial studies using RT-PCR revealed the presence of GPR54 mRNA in both GT1–7 and GN11 neuronal cell lines (Fig. 1A). The detection of a 175 bp band corresponds to amplification of a region between exons 2 and 3 from murine genomic DNA. No product was detected in the lane containing no cDNA (H2O) or performed without RT (RT(−)). Furthermore, studies using immunoprecipitation and Western blot assay revealed the presence of GPR54 protein as a 43 kDa product (Prentice et al., 2007) in these cells (
Discussion
The onset of puberty is heralded by activation of neurons in the forebrain that produce GnRH. Although the central reproductive axis has been studied in many mammalian species, precise identification of the molecular and cellular events in the forebrain that initiate pubertal processes and maintain reproductive competence remains elusive (Plant, 2008).
The goal of our study was to define whether kisspeptin had a direct role in GnRH neuronal function, and if so, the pharmacology of its effect on
Acknowledgements
This research was supported by NICHD/NIH through cooperative agreement [U54 HD 933067 (The Baltimore-Chicago Center for Reproductive Research)] as part of the Specialized Cooperative Centers Program in Reproduction and Infertility Research (SCCPIR) and R01 as NIH HD 370246. The authors would like to thank Dr Pamela Mellon for kindly providing the GT1–7 cell line and Dr. Jennifer Mammen for the assistance in the writing of the manuscript.
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