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.

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In seasonal breeders, reproduction is synchronized to seasons by day length via the pineal hormone melatonin. Recently, we have demonstrated that Kiss1, a key activator of the reproductive function, is down-regulated in sexually inactive hamsters maintained in inhibitory short days (SDs). In rodents, Kiss1 is expressed in the anteroventral periventricular nucleus (AVPV) and in the arcuate nucleus (ARC). Because both the duration of the nocturnal peak of melatonin and circulating sex steroid levels vary with photoperiod, the aim of this study was to determine whether melatonin and sex steroids differentially regulate Kiss1 expression in the ARC and the AVPV. Kiss1 expression was examined by in situ hybridization in both male and female hamsters kept in various experimental conditions, and we observed that 1) SD exposure markedly reduced Kiss1 expression in the ARC and AVPV of male and female hamsters as compared to LD animals, 2) sex steroid treatment in SD-adapted male and female hamsters increased the number of Kiss1 neurons in the AVPV but decreased it in the ARC, 3) melatonin administration to LD-adapted hamsters decreased Kiss1 mRNA level in both the AVPV and the ARC in intact animals, whereas in castrated hamsters, melatonin rapidly inhibited Kiss1 expression in the ARC but not in the AVPV, and 4) pinealectomy of male or female SD-adapted hamsters increased the number of Kiss1 neurons in the ARC but not in the AVPV. In conclusion, our data demonstrate that Kiss1 expression in the Syrian hamster hypothalamus is down-regulated in SD via different mechanisms. In the ARC, melatonin inhibits Kiss1 via a direct effect on the hypothalamus, and this effect is probably sex steroid dependent, whereas in the AVPV, the decrease in Kiss1 expression appears to be secondary to the melatonin-driven reduction of sex steroid levels. Taken together, our data support the hypothesis that ARC Kiss1 neurons mediate melatonin effects on the gonadotropic axis of the Syrian hamster.

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.

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Kisspeptins, encoded by the Kiss1 gene, play a key role in the regulation of reproductive function, although very little is known about the ontogenesis of this system. The present study aimed to determine the period of arcuate nucleus (ARC) kisspeptin cell birth and the embryonic stage and neuroanatomical sites of onset of kisspeptin immunoreactivity. Bromodeoxyuridine (BrdU) was administered to female rats at various gestational stages and double immunohistochemistry against kisspeptin and BrdU was performed on brain sections from their offspring. The period of neurogenesis of ARC kisspeptin neurones begun between embryonic day (E) 12.5 and E13.5, reached its peak at E15.5 and was not completely over at E17.5. Kiss1 mRNA was detected in mediobasal hypothalamic punches of embryos aged E14.5, E16.5, E18.5 and E22.5 by real-time reverse transcriptase-polymerase chain reaction. Accordingly, kisspeptin-immunoreactive (-IR) cells were consistently detected in the embryonic ARC from E14.5 and their number increased until E18.5 to reach approximately half the level observed in adults. Between E18.5 and E22.5, the number of kisspeptin-IR cells and hypothalamic Kiss1 expression significantly decreased, regardless of sex, and this decrease persisted until birth. Taken together, these results demonstrate that rat ARC kisspeptin neurones are born locally during an extended embryonic period and are able to synthesise kisspeptins rapidly after their birth, consistent with the hypothesis of a role during embryonic activation of the hypothalamic-hypophyseal-gonadal axis. A sex-independent decrease of kisspeptin-IR cell numbers was observed during the perinatal period, suggestive of important regulations of kisspeptin neurones around birth.