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In seasonal mammals, a distinct photoneuroendocrine circuit that involves the pineal hormone melatonin tightly synchronizes reproduction with seasons. In the Syrian hamster, a seasonal model in which sexual activity is inhibited by short days, we have previously shown that the potent GnRH stimulator, kisspeptin, is crucial to convey melatonin's message; however, the precise mechanisms through which melatonin affects kisspeptin remain unclear. Interestingly, rfrp gene expression in the neurons of the dorsomedial hypothalamic nucleus, a brain region in which melatonin receptors are present in the Syrian hamster, is strongly down-regulated by melatonin in short days. Because a large body of evidence now indicates that RFamide-related peptide (RFRP)-3, the product of the rfrp gene, is an inhibitor of gonadotropin secretion in various mammalian species, we sought to investigate its effect on the gonadotrophic axis in the Syrian hamster. We show that acute central injection of RFRP-3 induces c-Fos expression in GnRH neurons and increases LH, FSH, and testosterone secretion. Moreover, chronic central administration of RFRP-3 restores testicular activity and Kiss1 levels in the arcuate nucleus of hamsters despite persisting photoinhibitory conditions. By contrast RFRP-3 does not have a hypophysiotrophic effect. Overall, these findings demonstrate that, in the male Syrian hamster, RFRP-3 exerts a stimulatory effect on the reproductive axis, most likely via hypothalamic targets. This places RFRP-3 in a decisive position between the melatonergic message and Kiss1 seasonal regulation. Additionally, our data suggest for the first time that the function of this peptide depends on the species and the physiological status of the animal model.

Kiss1 neurons have recently emerged as a putative conduit for the metabolic gating of reproduction, with leptin being a regulator of hypothalamic Kiss1 expression. Early perturbations of the nutritional status are known to predispose to different metabolic disorders later in life and to alter the timing of puberty; however, the potential underlying mechanisms remain poorly defined. Here we report how changes in the pattern of postnatal feeding affect the onset of puberty and evaluate key hormonal and neuropeptide [Kiss1/kisspeptin (Kp)] alterations linked to these early nutritional manipulations. Female rats were raised in litters of different sizes: small (four pups per dam: overfeeding), normal (12 pups per dam), and large litters (20 pups per litter: underfeeding). Postnatal overfeeding resulted in persistently increased body weight and earlier age of vaginal opening, as an external sign of puberty, together with higher levels of leptin and hypothalamic Kiss1 mRNA. Conversely, postnatal underfeeding caused a persistent reduction in body weight, lower ovarian and uterus weights, and delayed vaginal opening, changes that were paralleled by a decrease in leptin and Kiss1 mRNA levels. Kisspeptin-52 immunoreactivity (Kp-IR) in the hypothalamus displayed similar patterns, with lower numbers of Kp-IR neurons in the arcuate nucleus of postnatally underfed animals, and a trend for increased Kp-positive fibers in the periventricular area of early overfed rats. Yet, gonadotropin responses to Kp at puberty were similar in all groups, except for enhanced responsiveness to low doses of Kp-10 in postnatally underfed rats. In conclusion, our data document that the timing of puberty is sensitive to both overfeeding and subnutrition during early (postnatal) periods and suggest that alterations in hypothalamic expression of Kiss1/kisspeptin may underlie at least part of such programming phenomenon.

Severe inflammatory challenges are frequently coupled to decreased food intake and disruption of reproductive function, the latter via deregulation of different signaling pathways that impinge onto GnRH neurons. Recently, the hypothalamic Kiss1 system, a major gatekeeper of GnRH function, was suggested as potential target for transmitting immune-mediated repression of the gonadotropic axis during acute inflammation, and yet key facets of such a phenomenon remain ill defined. Using lipopolysaccharide S (LPS)-treated male rats as model of inflammation, we document herein the pattern of hypothalamic kisspeptin immunoreactivity (IR) and hormonal responses to kisspeptin during the acute inflammatory phase. LPS injections induced a dramatic but transient drop of serum LH and testosterone levels. Suppression of gonadotropic function was associated with a significant decrease in kisspeptin-IR in the arcuate nucleus (ARC) that was not observed under conditions of metabolic stress induced by 48-h fasting. In addition, absolute responses to kisspeptin-10 (Kp-10), in terms of LH and testosterone secretion, were significantly attenuated in LPS-treated males that also displayed a decrease in food intake and body weight. Yet pair-fed males did not show similar alterations in LH and testosterone secretory responses to Kp-10, whose magnitude was preserved, if not augmented, during food restriction. In summary, our data document the impact of acute inflammation on kisspeptin content at the ARC as key center for the neuroendocrine control of reproduction. Our results also suggest that suppressed gonadotropic function following inflammatory challenges might involve a reduction in absolute responsiveness to kisspeptin that is independent of the anorectic effects of inflammation.

Neurokinin B (NKB), encoded by Tac2 in rodents, and its receptor, NK3R, have recently emerged as important regulators of reproduction; NKB has been proposed to stimulate kisspeptin output onto GnRH neurons. Accordingly, NKB has been shown to induce gonadotropin release in several species; yet, null or even inhibitory effects of NKB have been also reported. The basis for these discrepant findings, as well as other key aspects of NKB function, remains unknown. We report here that in the rat, LH responses to the NK3R agonist, senktide, display a salient sexual dimorphism, with persistent stimulation in females, regardless of the stage of postnatal development, and lack of LH responses in males from puberty onward. Such dimorphism was independent of the predominant sex steroid after puberty, because testosterone administration to adult females failed to prevent LH responses to senktide, and LH responsiveness was not restored in adult males treated with estradiol or the nonaromatizable androgen, dihydrotestosterone. Yet, removal of sex steroids by gonadectomy switched senktide effects to inhibitory, both in adult male and female rats. Sexual dimorphism was also evident in the numbers of NKB-positive neurons in the arcuate nucleus (ARC), which were higher in adult female rats. This is likely the result of differences in sex steroid milieu during early periods of brain differentiation, because neonatal exposures to high doses of estrogen decreased ARC NKB neurons at later developmental stages. Likewise, neonatal estrogenization resulted in lower serum LH levels that were normalized by senktide administration. Finally, we document that the ability of estrogen to inhibit hypothalamic Tac2 expression seems region specific, because estrogen administration decreased Tac2 levels in the ARC but increased them in the lateral hypothalamus. Altogether, our data provide a deeper insight into relevant aspects of NKB function as major regulator of the gonadotropic axis in the rat, including maturational changes, sexual dimorphism, and differential regulation by sex steroids.

In seasonal breeders, reproduction is synchronised by day length via the pineal hormone melatonin. In short winter days (short day, SD), the Syrian hamster displays a complete gonadal atrophy together with a marked reduction in expression of kisspeptins (Kp), a family of potent hypothalamic stimulators of GNRH neurons. Both central and peripheral acute injections of Kp have been reported to activate the gonadotropic axis in mammals. The aim of this study was to determine if and how peripheral administration of Kp54 could restore gonadal function in photo-inhibited hamsters. Testicular activity of hamsters kept in SD was reactivated by two daily i.p. injections of Kp54 but not by chronic subcutaneous delivery of the same peptide via mini-pumps. Acute i.p. injection of Kp54-induced FOS (c-Fos) expression in a large number of GNRH neurons and pituitary gonadotrophs together with a strong increase in circulating testosterone. The activation of pituitary cells by Kp was inhibited by preadministration of the GNRH receptor antagonist acyline. Altogether, our results demonstrate that peripheral Kp54 activates the gonadotropic axis by stimulating GNRH release and indicate that an appropriate protocol of long-term systemic Kp administration can recrudesce a photo-inhibited reproductive axis.