The awakening of the gonadotrophic axis at puberty is the end-point of a complex cascade of sex developmental events that leads to the attainment of reproductive capacity. Recently, loss-of-function mutations of the gene encoding GPR54, the putative receptor for the KiSS-1-derived peptide metastin, have been linked to hypogonadotrophic hypogonadism, both in rodents and humans. However, the actual role of the KiSS-1/GPR54 system in the timing of puberty onset remains unexplored. We report herein that chronic central administration of KiSS-1 peptide to immature female rats induced the precocious activation of the gonadotrophic axis, as estimated by advanced vaginal opening, elevated uterus weight, and increased serum levels of luteinizing hormone (LH) and oestrogen. The central effect of KiSS-1 upon LH release appeared to be mediated via the hypothalamic LH-releasing hormone. In contrast, despite the well-documented permissive role of body fat stores and the adipocyte-derived hormone leptin in puberty maturation, acute activation of the gonadotrophic axis by KiSS-1 was persistently observed in pubertal animals under food deprivation, after central immunoneutralization of leptin, and in a model of leptin resistance. Overall, the present results, together with our recent data on maximum expression of KiSS-1 and GPR54 genes in the hypothalamus at puberty, provide novel evidence for a role of the KiSS-1 system as a downstream element in the hypothalamic network triggering the onset of puberty.

Loss-of-function mutations of the gene encoding GPR54, the putative receptor for the KiSS-1-derived peptide metastin, have been recently associated with hypogonadotropic hypogonadism, in both rodents and humans. Yet the actual role of the KiSS-1/GPR54 system in the neuroendocrine control of gonadotropin secretion remains largely unexplored. To initiate such analysis, the effects of KiSS-1 peptide on LH secretion were monitored using in vivo and in vitro settings under different experimental conditions. Central intracerebroventricular administration of KiSS-1 peptide potently elicited LH secretion in vivo over a range of doses from 10 pmol to 1 nmol. The effect of centrally injected KiSS-1 appeared to be mediated via the hypothalamic LHRH. However, no effect of central administration of KiSS-1 was detected on relative LHRH mRNA levels. Likewise, systemic (i.p. and i.v.) injection of KiSS-1 markedly stimulated LH secretion. This effect was similar in terms of maximum response to that of central administration of KiSS-1 and might be partially attributed to its ability to stimulate LH secretion directly at the pituitary. Finally, the LH-releasing activity of KiSS-1 was persistently observed after blockade of endogenous excitatory amino acid and nitric oxide pathways, i.e. relevant neurotransmitters in the neuroendocrine control of LH secretion. In summary, our results provide solid evidence for a potent stimulatory effect of KiSS-1 on LH release, acting at central levels (likely the hypothalamus) and eventually at the pituitary, and further document a novel role of the KiSS-1/GPR54 system as a relevant downstream element in the neuroendocrine network governing LH secretion.

KiSS-1 was originally identified as a metastasis suppressor gene encoding an array of structurally related peptides, namely kisspeptins, which acting through the G protein-coupled receptor GPR54 are able to inhibit tumor progression. Unexpectedly, a reproductive facet of this newly discovered system has recently arisen, and characterization of the role of the KiSS-1/GPR54 system in the neuroendocrine control of gonadotropin secretion has been initiated. However, such studies have been so far mostly restricted to LH, and very little is known about the actual contribution of this system in the regulation of FSH release. To address this issue, the effects of KiSS-1 peptide on FSH secretion were monitored in vivo and in vitro under different experimental conditions. Intracerebroventricular administration of KiSS-1 peptide significantly stimulated FSH secretion in prepubertal and adult rats. Yet, dose-response analyses in vivo demonstrated an ED(50) value for the FSH-releasing effects of KiSS-1 of 400 pmol, i.e. approximately 100-fold higher than that of LH. In addition, systemic (ip and iv) injection of KiSS-1 significantly stimulated FSH secretion in vivo. However, KiSS-1 failed to elicit basal FSH release directly at the pituitary level, although it moderately enhanced GnRH-stimulated FSH secretion in vitro. Finally, mechanistic studies revealed that the ability of KiSS-1 to elicit FSH secretion was abolished by the blockade of endogenous GnRH actions, but it was persistently observed in different models of leptin insufficiency and after blockade of endogenous excitatory amino acid and nitric oxide pathways, i.e. relevant signals in the neuroendocrine control of gonadotropin secretion. In summary, our results extend previous recent observations on the role of KiSS-1 in the control of LH secretion and provide solid evidence for a stimulatory effect of KiSS-1 on FSH release, acting at central level. Overall, it is proposed that the KiSS-1/GPR54 system is a novel, pivotal downstream element in the neuroendocrine network governing gonadotropin secretion.

Compelling evidence has now demonstrated direct biological actions of sex steroids at the cerebellum. Likewise, the expression of key steroidogenic factors, such as the steroidogenic acute regulatory protein (StAR), cytochrome P450 side chain cleavage (P450scc), and aromatase, at this neural site has been reported. Little is known, however, about the regulation of their genes in the cerebellum. Assessment of StAR, P450scc, and aromatase mRNAs in the cerebellum of male and female rats revealed that the expression of these genes is developmentally regulated, with the highest levels at early postnatal ages in both sexes and with significantly higher mRNA levels in postnatal males. Expression of these genes in the female remained unaltered after perinatal androgenization and along the estrous cycle. In contrast, damage of cerebellar afferent neurons of the inferior olivary nucleus evoked a significant increase in StAR, P450scc, and aromatase mRNA levels at this site, as well as a transient elevation in StAR mRNA at the cerebellum. Finally, enhancement of cAMP levels in cultured cerebellar neurons induced a significant increase in StAR and aromatase mRNA levels. In summary, we present herein novel evidence for the developmentally regulated and partially sexually dimorphic pattern of expression of StAR, P450scc, and aromatase genes in the rat cerebellum. These observations, together with the finding that the mRNA levels of these steroidogenic molecules are sensitive to injury and are regulated by intracellular cAMP, strongly suggest that local steroidogenesis is likely to play an important role during development and adaptation to neurodegenerative processes in the olivocerebellar system.

Net estrogen sensitivity in target tissues critically depends on the regulated expression of full-length and alternately processed estrogen receptor (ER) isoforms. However, the molecular mechanisms for the control of pituitary responsiveness to estrogen remain partially unknown. In the present communication, we report the ability of different ligands, with distinct agonistic or antagonistic properties at the ER, to modulate the expression of the transcripts encoding ERalpha and ERbeta isoforms, as well as those for the truncated ERalpha product (TERP), and the variant ERbeta2, in pituitaries from ovariectomized rats, i.e., a background devoid of endogenous estrogen. Compared with expression levels at the morning of proestrus, ovariectomy (OVX) resulted in increased pituitary expression of ERbeta and ERbeta2 mRNAs, whereas it decreased TERP-1 and -2 levels without affecting those of ERalpha. Administration of estradiol benzoate (as potent agonist for alpha and beta forms of ER) or the selective ERalpha agonist, propyl pyrazole triol, fully reversed the responses to OVX, while the ERbeta ligand, diarylpropionitrile, failed to induce any significant effect except for a partial stimulation of TERP-1 and -2 mRNA expression levels. To note, the ERbeta agonist was also ineffective in altering pituitary expression of progesterone receptor-B mRNA, i.e., a major estrogen-responsive target. In all parameters tested, tamoxifen, a selective ER modulator with mixed agonist/antagonist activity, behaved as ERalpha agonist, although the magnitude of tamoxifen effects was significantly lower than those of the ERalpha ligand, except for TERP induction. In contrast, the pure antiestrogen RU-58668 did not modify the expression of any of the targets under analysis. Overall, our results indicate that endogenous estrogen differentially regulates pituitary expression of the mRNAs encoding several ER isoforms with distinct functional properties, by a mechanism that is mostly conducted through ERalpha. Differential regulation of ER isoforms may represent a relevant system for the self-tuning of estrogen responsiveness in female pituitary.