Ontogeny and mechanisms of action for the stimulatory effect of kisspeptin on gonadotropin-releasing hormone system of the rat

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Abstract

Kisspeptins have recently emerged as essential regulators of gonadotropin secretion and puberty onset. These functions are primarily conducted by stimulation of hypothalamic gonadotropin-releasing hormone (GnRH) secretion. However, relevant aspects of KiSS-1 physiology, including the ontogeny and major signaling systems of its stimulatory action, remain to be fully elucidated. To cover these issues, the effects of kisspeptin-10 on GnRH and LH secretion were monitored at early stages of postnatal maturation, and potential changes in the sensitivity to kisspeptin were assessed along the pubertal transition in the rat. In addition, the signaling cascades involved in kisspeptin-induced GnRH secretion were explored by means of pharmacological blockade using rat hypothalamic explants. Despite sexual immaturity, kisspeptin-10 potently elicited GnRH release ex vivo and LH secretion in vivo at early stages (neonatal to juvenile) of postnatal development. Yet, LH responsiveness to low doses of kisspeptin was enhanced in peri-pubertal animals. Concerning GnRH secretion, the stimulatory action of kisspeptin-10 required activation of phospholipase-C, mobilization of intracellular Ca2+ and recruitment of ERK1/2 and p38 kinases, but was preserved after blockade of type 2 cyclo-oxygenase and prostaglandin synthesis. In summary, our present data document the ontogeny, sensitivity and intracellular signals for the stimulatory action of kisspeptin on the GnRH/LH axis in the rat. Although LH responses to low doses of kisspeptin appeared to be enhanced at puberty, kisspeptin was able to readily activate the GnRH system at early stages of postnatal maturation. These observations further stress the essential role of kisspeptin in normal, and eventually pathological, timing of puberty.

Introduction

KiSS-1 was originally identified as a metastasis suppressor gene encoding a number of structurally related peptides, with a common RF-amide signature at C-terminus, which include metastin and other kisspeptins (Kotani et al., 2001, Muir et al., 2001, Ohtaki et al., 2001). The known biological actions of KiSS-1 peptides, which are exerted through interaction with the previously orphan G protein-coupled receptor GPR54 (Kotani et al., 2001, Muir et al., 2001, Ohtaki et al., 2001), were originally restricted to the inhibition of tumor progression and, later on, control of trophoblast invasion (Ohtaki et al., 2001, Bilban et al., 2004). However, in late 2003, an unsuspected, fundamental role for KiSS-1 peptides in the control of the gonadotropic axis was suggested on the basis of the reproductive phenotypes of human and mouse models carrying null mutations of GPR54. Thus, a number of point mutations and deletions of the GPR54 gene were found in patients suffering idiopathic hypogonadotropic hypogonadism (de Roux et al., 2003, Seminara et al., 2003); a syndrome that was reproduced in mice where the GPR54 gene had been knocked out (Seminara et al., 2003, Funes et al., 2003). Such observations boosted an extraordinary interest among reproductive biologists, who aimed to characterize the role of this novel system in the control of development and/or function of the reproductive axis.

Indeed, in the last 2 years and a half, a number of studies on the reproductive physiology of the KiSS-1 system have been published. These reports have now demonstrated that hypothalamic expression of KiSS-1 and GPR54 genes is developmentally (maximum at puberty) and hormonally (by sex steroids) regulated (Navarro et al., 2004a, Shahab et al., 2005, Smith et al., 2005a, Smith et al., 2005b), and have documented the extraordinary potency of kisspeptins in inducing gonadotropin release in vivo (Gottsch et al., 2004, Irwig et al., 2004, Matsui et al., 2004, Navarro et al., 2004a, Navarro et al., 2004b, Navarro et al., 2005a, Navarro et al., 2005b, Thompson et al., 2004, Dhillo et al., 2005, Messager et al., 2005, Shahab et al., 2005), which is likely the highest among the different elicitors of LH secretion known so far (see Tena-Sempere, 2006; and references therein). Moreover, functional studies have provided solid evidence for a relevant role of KiSS-1 signaling in timing of puberty onset in rodent and primate species (Navarro et al., 2004b, Shahab et al., 2005, Han et al., 2005). From a mechanistic standpoint, such effects primarily involve direct stimulatory actions upon the hypothalamic GnRH system, as activation of GnRH neurons (Irwig et al., 2004, Han et al., 2005) or induction of GnRH release (Thompson et al., 2004, Castellano et al., 2005, Messager et al., 2005) by kisspeptins have been reported. Yet, the possibility of additional sites of action at other levels of the gonadotropic axis (e.g. the pituitary) cannot be ruled out (Navarro et al., 2005a, Navarro et al., 2005b).

Despite the extensive progress in the field, some critical aspects of KiSS-1 physiology in the context of puberty onset and the control of reproductive function remain less well defined. These include the ontogeny for the activational effect of kisspeptins on GnRH and gonadotropin secretion (a phenomenon that may pose interesting implications in terms of timing of puberty) and the potential changes in responsiveness and sensitivity to kisspeptin along the pubertal transition. Of note, during the final stage of preparation of this study, Han and co-workers reported an increase in the sensitivity to kisspeptin and the efficiency of GPR54 signaling during mouse postnatal maturation (Han et al., 2005), which is yet to be confirmed in other species. In addition, the signaling systems recruited following GPR54 activation to induce GnRH release at the hypothalamus have not been directly evaluated. On the latter, in vitro assays had previously demonstrated that binding of kisspeptin to GPR54 activates phospholipase-C and increases PIP2 hydrolysis, which is followed by accumulation of inositol-(1,4,5)-triphosphate, Ca2+ mobilization, arachidonic acid release, and phosphorylation of ERK1/2 and p38 MAP kinases (Kotani et al., 2001). However, such signaling studies were conducted using heterologous systems (i.e., Chinese Hamster Ovary K1 cells stably expressing GPR54). Indeed, although metastin has been proven to activate ERK1/2 and/or p38 kinases in thyroid and pancreatic cancer cells (Ringel et al., 2002, Masui et al., 2004), the signaling cascades used by GPR54 in normal tissues (such as the hypothalamus), where this receptor is expressed and where major biological effects of kisspeptins have been reported, remain totally unexplored. Given the proposed role of KiSS-1 system as master regulator of puberty onset (Tena-Sempere, 2006), dissection of such intracellular pathways appears especially appropriate during maturational stages (such as the juvenile period) preceding the onset puberty.

In this context, the present experimental work was undertaken to cover the above uncertainties pertaining the neuroendocrine actions of kisspeptin in the control of the GnRH/LH axis, using the rat as an animal model. To this end, the effects of kisspeptin-10 on GnRH and LH secretion were monitored by combination of in vivo and ex vivo settings at early stages of rat postnatal maturation, i.e. the neonatal, late infantile and juvenile periods. Moreover, potential changes in the sensitivity to kisspeptin-induced LH responses during the pubertal transition were monitored in vivo. Finally, the involvement of different signaling cascades in kisspeptin-induced GnRH secretion was evaluated ex vivo, by means of pharmacological blockade of key routes in static incubations of hypothalamic explants from pre-pubertal (juvenile) female rats.

Section snippets

Animals and drugs

Wistar rats bred in the vivarium of the University of Córdoba were used. The day the litters were born was considered as d-1 of age. The animals were maintained under constant conditions of light (14 h of light, from 07:00) and temperature (22 °C), and were weaned at 21-d of age in groups of five rats per cage with free access to pelleted food and tap water. Experimental procedures were approved by the Córdoba University Ethical Committee for animal experimentation and conducted in accordance

Effects of kisspeptin on LH and GnRH secretion at early stages of postnatal development

To characterize the ontogeny of the stimulatory action of KiSS-1 system upon the gonadotropic axis, the effects of kisspeptin-10 upon LH and GnRH secretion were monitored by a combination of in vivo and ex vivo settings. Thus, LH secretory responses to a single bolus of kisspeptin-10 (1 μg/10 g BW) were assessed at 15-min after systemic (i.p.) injection of the peptide to neonatal (5-d-old), late infantile (15-d-old), and juvenile (25-d-old) male and female rats. In addition, the ability of 10−8 M

Discussion

In the last 2 years, the pivotal role of kisspeptin and its receptor, GPR54, in the central control of the reproductive axis has been substantiated on the basis of genetic, molecular and pharmacological studies. These analyses have now firmly established that the KiSS-1 system operates as a major gatekeeper of GnRH secretion (Seminara and Kaiser, 2005, Dungan et al., 2006, Tena-Sempere, 2006), thus serving an essential function in the activation of gonadotropic axis at puberty (Colledge, 2004),

Acknowledgments

Radioimmunoassay kits for hormone determinations were kindly supplied by Dr. A.F. Parlow, NIDDK National Hormone and Peptide Program; Torrance, CA, USA. M. T.-S. is indebted to Dr. David J. Handelsman (ANZAC Research Institute, Sydney, Australia) for helpful comments during preparation of this manuscript.

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    This work was supported by grants BFI 2002-00176 and BFI 2005-07446 from Ministerio de Educación y Ciencia (Spain), funds from Instituto de Salud Carlos III (Red de Centros RCMN C03/08 and Project PI042082; Ministerio de Sanidad, Spain), and EU research contract EDEN QLK4-CT-2002-00603.

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