Leptin, but not Estradiol, Signaling in PACAP Neurons Modulates Puberty Onset.

The adipose-derived hormone leptin critically modulates reproductive function, such that its absence results in hypothalamic hypogonadism. Pituitary adenylate cyclase-activating polypeptide (PACAP)-expressing neurons are potential mediators of leptin's action on the neuroendocrine reproductive axis because they are leptin-sensitive and involved in both feeding behavior and reproductive function. In the complete absence of PACAP, male and female mice exhibit metabolic and reproductive abnormalities, yet there is some sexual dimorphism in the reproductive impairments. We tested whether PACAP neurons play a critical and/or sufficient role in mediating leptin's effects on reproductive function by generating PACAP-specific leptin receptor (LepR) knockout and rescue mice, respectively. We also generated PACAP-specific estrogen receptor alpha knockout mice to determine whether estradiol-dependent regulation of PACAP was critically involved in the control of reproductive function and whether it contributed to the sexually dimorphic effects of PACAP. We showed that LepR signaling in PACAP neurons is critically involved in the timing of female, but not male, puberty onset, but not fertility. Rescuing LepR-PACAP signaling in otherwise LepR-deficient mice was unable to rescue the reproductive deficits observed in LepR null mice but led to a marginal improvement in body weight and adiposity in females. Finally, PACAP-specific estrogen receptor alpha knockout did not lead to any changes in body weight or puberty onset compared with control mice. These data highlight that PACAP is a critical mediator of some of leptin's, but not estradiol's, influence on puberty onset in females, but is not critically involved in relaying leptin's effects in males or in adult females.

Agouti-related peptide neuronal silencing overcomes delayed puberty in neonatally underfed male mice.

Agouti-related peptide (AgRP) neurons are thought to indirectly regulate the activity of hypothalamic gonadotrophin-releasing hormone neurons which control fertility. AgRP neurons also drive caloric intake and are modulated by metabolically-relevant hormones, providing a link to the hypothalamic-pituitary-gonadal axis. In mice expressing Cre-dependant designer receptors (DREADDs) in AgRP neurons, we activated or silenced these neurons in vivo using the synthetic ligand clozapine-N-oxide (CNO) to observe the effect of AgRP neuron activity on timing of puberty. To validate these animals, we chronically treated both stimulatory (hM3Dq) and inhibitory (hM4Di) DREADD × AgRP-Cre mice with CNO, observing a pronounced increase and decrease of food intake, respectively, consistent with the known orexigenic effects of these neurons. RNAscope was performed to visually confirm the activation of AgRP neurons. Puberty onset was assessed in males and females. There was no effect on preputial separation in males or vaginal opening and first oestrus in females after CNO treatment from day 26 to 30 to chronically modulate AgRP neurons. Next, to determine whether the delay in puberty onset occurring in response to neonatal underfeeding could be overcome by inhibiting AgRP neuronal activity, mice were raised in large (neonatally underfed) or normal litter sizes. The delay in puberty from underfeeding was completely reversed in CNO-treated AgRP-hM4Di male mice. These data highlight the inhibitory role of AgRP neurons to delay puberty onset when undernutrition occurs during the neonatal period, at least in male mice. TRAIL REGISTRATION NUMBER: JNE-22-0081-OA.R2.

Deletion of PTP1B From Brain Neurons Partly Protects Mice From Diet-Induced Obesity and Minimally Improves Fertility.

Reproductive dysfunction in women has been linked to high caloric diet (HCD)-feeding and obesity. Central resistance to leptin and insulin have been shown to accompany diet-induced infertility in rodent studies, and we have previously shown that deleting suppressor of cytokine signaling 3, which is a negative regulator of leptin signaling, from all forebrain neurons partially protects mice from HCD-induced infertility. In this study, we were interested in exploring the role of protein tyrosine phosphatase 1B (PTP1B), which is a negative regulator of both leptin and insulin signaling, in the pathophysiology of HCD-induced obesity and infertility. To this end, we generated male and female neuron-specific PTP1B knockout mice and compared their body weight gain, food intake, glucose tolerance, and fertility relative to control littermates under both normal calorie diet and HCD feeding conditions. Both male and female mice with neuronal PTP1B deletion exhibited slower body weight gain in response to HCD feeding, yet only male knockout mice exhibited improved glucose tolerance compared with controls. Neuronal PTP1B deletion improved the time to first litter in HCD-fed mice but did not protect female mice from eventual HCD-induced infertility. While the mice fed a normal caloric diet remained fertile throughout the 150-day period of assessment, HCD-fed females became infertile after producing only a single litter, regardless of their genotype. These data show that neuronal PTP1B deletion is able to partially protect mice from HCD-induced obesity but is not a critical mediator of HCD-induced infertility.

RFamide-Related Peptide Neurons Modulate Reproductive Function and Stress Responses.

RF-amide related peptide 3 (RFRP-3) is a neuropeptide thought to inhibit central regulation of fertility. We investigated whether alterations in RFRP neuronal activity led to changes in puberty onset, fertility, and stress responses, including stress and glucocorticoid-induced suppression of pulsatile luteinizing hormone secretion. We first validated a novel RFRP-Cre mouse line, which we then used in combination with Cre-dependent neuronal ablation and DREADD technology to selectively ablate, stimulate, and inhibit RFRP neurons to interrogate their physiological roles in the regulation of fertility and stress responses. Chronic RFRP neuronal activation delayed male puberty onset and female reproductive cycle progression, but RFRP-activated and ablated mice exhibited apparently normal fertility. When subjected to either restraint- or glucocorticoid-induced stress paradigms. However, we observed a critical sex-specific role for RFRP neurons in mediating acute and chronic stress-induced reproductive suppression. Female mice exhibiting RFRP neuron ablation or silencing did not exhibit the stress-induced suppression in pulsatile luteinizing hormone secretion observed in control mice. Furthermore, RFRP neuronal activation markedly stimulated glucocorticoid secretion, demonstrating a feedback loop whereby stressful stimuli activate RFRP neurons, which in turn further activate the stress axis. These data provide evidence for a neuronal link between the stress and reproductive axes.

Central RFRP-3 Stimulates LH Secretion in Male Mice and Has Cycle Stage-Dependent Inhibitory Effects in Females.

RFamide-related peptide (RFRP)-3 is a neuropeptide thought to play an inhibitory role in the regulation of reproduction in various mammalian species, although some stimulatory effects have been reported. To date, the effects of RFRP-3 on gonadotropin secretion have been scarcely studied in mice. The aim of the current study was to characterize the effect of RFRP-3 administration on gonadotropin secretion in male and female mice. Adult intact and castrated male mice received acute central injections of 0.5 to 5 nmol of RFRP-3, and luteinizing hormone (LH) concentration was assayed in tail-tip blood samples. RFRP-3 had a dose-dependent stimulatory effect on LH secretion when administered centrally to both intact and castrated mice, and this effect was diminished when RFRP-3 was administered to kisspeptin receptor knockout mice. In female mice, central RFRP-3 had an inhibitory effect on LH secretion when administered at the time of the preovulatory LH surge in intact mice, or of an estradiol-induced LH surge in ovariectomized mice. Conversely, central RFRP-3 administration had no effect on LH levels in intact diestrus or ovariectomized, low-dose estradiol-implanted mice. Finally, peripheral administration of RFRP-3 to intact males and to females at the time of the preovulatory LH surge or during diestrus had no effect on LH secretion. Taken together, these results provide a detailed description of sex- and cycle stage-dependent effects of RFRP-3 on gonadotrophin secretion in mice. Moreover, it appears that the stimulatory effects are mediated in part by the receptor for kisspeptin, a potent stimulator of the reproductive axis.

RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes effects of RF-amide-related peptide-3 and opioid-induced hyperalgesia in rodents.

Although opiates represent the most effective analgesics, their use in chronic treatments is associated with numerous side effects including the development of pain hypersensitivity and analgesic tolerance. We recently identified a novel orally active neuropeptide FF (NPFF) receptor antagonist, RF313, which efficiently prevents the development of fentanyl-induced hyperalgesia in rats. In this study, we investigated the properties of this compound into more details. We show that RF313 exhibited a pronounced selectivity for NPFF receptors, antagonist activity at NPFF1 receptor (NPFF1R) subtype both in vitro and in vivo and no major side effects when administered in mice up to 30 mg/kg. When co-administered with opiates in rats and mice, it improved their analgesic efficacy and prevented the development of long lasting opioid-induced hyperalgesia. Moreover, and in marked contrast with the dipeptidic NPFF receptor antagonist RF9, RF313 displayed negligible affinity and no agonist activity (up to 100 µM) toward the kisspeptin receptor. Finally, in male hamster, RF313 had no effect when administered alone but fully blocked the increase in LH induced by RFRP-3, while RF9 per se induced a significant increase in LH levels which is consistent with its ability to activate kisspeptin receptors. Altogether, our data indicate that RF313 represents an interesting compound for the development of therapeutic tools aiming at improving analgesic action of opiates and reducing adverse side effects associated with their chronic administration. Moreover, its lack of agonist activity at the kisspeptin receptor indicates that RF313 might be considered a better pharmacological tool, when compared to RF9, to examine the regulatory roles of RF-amide-related peptides and NPFF1R in reproduction.

Roles of RFRP-3 in the Daily and Seasonal Regulation of Reproductive Activity in Female Syrian Hamsters.

In females, reproductive activity relies on proper integration of daily and environmental changes as well as cyclic sex-steroid feedback. This study sought to investigate the role of the hypothalamic Arg-Phe amide-related peptide (RFRP)-3 in the daily and seasonal control of reproductive activity in female Syrian hamsters by analyzing the RFRP system and investigating the effects of central administration of RFRP-3 at different reproductive stages. In long day-adapted sexually active female hamsters, the number of c-Fos-activated RFRP immunoreactive neurons was reduced in the afternoon of diestrus and proestrus; the latter was correlated with increased kisspeptin activity and the luteinizing hormone (LH) surge. Moreover, acute RFRP-3 administration decreased LH secretion when given midafternoon, before the LH surge, and had no effect at other time points of proestrus or diestrus. These data indicate that RFRP-3 exerts a tonic inhibition on LH secretion, which is lifted at the time of the preovulatory surge on the afternoon of proestrus. In short day-adapted sexually inactive female hamsters, Rfrp expression is strongly inhibited in a sex steroid-independent manner, and prolonged central infusion of RFRP-3 completely reactivated the reproductive axis through increased kisspeptin expression, gonadotropin and estradiol secretion, and gonadal weight. These findings reveal a critical role of RFRP-3 in the control of reproductive activity in female rodents and suggest that RFRP neurons, acting alongside kisspeptin neurons, are essential for proper synchronization of reproductive activity with the time of the day, the stage of the estrous cycle, and the seasonal changes in photoperiod.

Development of Dipeptidic hGPR54 Agonists.

A series of dipeptides were designed as potential agonists of the human KiSS1-derived peptide receptor (hGPR54). While the sequence Arg-Trp-NH2 was the most efficient in terms of affinity, we established a convergent synthetic strategy to optimize the N terminus. Using two successive Sonogashira cross-coupling reactions on a solid-supported peptide, we were able to introduce various alkynes at the N terminus to afford compounds with sub-micromolar affinities for hGPR54. However, functional assays indicated the benzoylated dipeptide Bz-Arg-Trp-NH2 as the most promising compound in terms of agonistic properties. Interestingly, this compound appeared much more stable than the endogenous neuropeptide kisspeptin, both in serum and in liver microsomes of rats. This compound was also found to be able to induce a significant in vivo increase in testosterone levels in male rats.

Kisspeptins and RFRP-3 Act in Concert to Synchronize Rodent Reproduction with Seasons.

Seasonal mammals use the photoperiodic variation in the nocturnal production of the pineal hormone melatonin to synchronize their reproductive activity with seasons. In rodents, the (SD) short day profile of melatonin secretion has long been proven to inhibit reproductive activity. Lately, we demonstrated that melatonin regulates the expression of the hypothalamic peptides kisspeptins (Kp) and RFamide-related peptide-3 (RFRP-3), recently discovered as potent regulators of gonadotropin-releasing hormone (GnRH) neuron activity. In the male Syrian hamster, Kp expression in the arcuate nucleus is down-regulated by melatonin independently of the inhibitory feedback of testosterone. A central or peripheral administration of Kp induces an increase in pituitary gonadotropins and gonadal hormone secretion, but most importantly a chronic infusion of the peptide reactivates the photo-inhibited reproductive axis of Syrian hamsters kept in SD conditions. RFRP-3 expression in the dorsomedial hypothalamus is also strongly inhibited by melatonin in a SD photoperiod. Although RFRP-3 is usually considered as an inhibitory component of the gonadotropic axis, a central acute administration of RFRP-3 in the male Syrian hamster induces a marked increase in gonadotropin secretion and testosterone production. Furthermore, a chronic central infusion of RFRP-3 in SD-adapted hamsters reactivates the reproductive axis, in the same manner as Kp. Both Kp and RFRP-3 neurons project onto GnRH neurons and both neuropeptides regulate GnRH neuron activity. In addition, central RFRP-3 infusion was associated with a significant increase in arcuate Kp expression. However, the actual sites of action of both peptides in the Syrian hamster brain are still unknown. Altogether our findings indicate that Kp and RFRP neurons are pivotal relays for the seasonal regulation of reproduction, and also suggest that RFRP neurons might be the primary target of the melatoninergic message.

RFRP neurons are critical gatekeepers for the photoperiodic control of reproduction.

Seasonally breeding mammals rely on the photoperiodic signal to restrict their fertility to a certain time of the year. The photoperiodic information is translated in the brain via the pineal hormone melatonin, and it is now well-established that it is the variation in the duration of the nocturnal peak of melatonin which synchronizes reproduction with the seasons. The Syrian hamster is a long day breeder, and sexual activity is therefore promoted by exposure to a long day photoperiod and inhibited by exposure to a short day photoperiod. Interestingly, in this species electrolytic lesion of the mediobasal hypothalamus abolishes the short day-induced gonadal regression. We have shown that the expression of a recently discovered neuronal population, namely RFamide-related peptide (rfrp) neurons, present in the mediobasal hypothalamus, is strongly down-regulated by melatonin in short day conditions, but not altered by circulating levels of sex steroids. The role of rfrp and its product RFRP-3 in the regulation of reproductive activity has been extensively studied in mammals, and our recent findings indicate that this peptide is a potent stimulator of the reproductive axis in the Syrian hamster. It induces a marked increase in GnRH neuron activity and gonadotropin secretion, and it is able to rescue reproductive activity in short day sexually inactive hamsters. Little is known about the localization of the RFRP-3 receptor, GPR147, in the rodent brain. Accumulating evidence suggests that RFRP-3 could be acting via two intermediates, the GnRH neurons in the preoptic area and the Kiss1 neurons in the arcuate nucleus, but future studies should aim at describing the localization of Gpr147 in the Syrian hamster brain. Altogether our data indicate that the rfrp neuronal population within the mediobasal hypothalamus might be a serious candidate in mediating the photoperiodic effects of melatonin on the regulation of the reproductive axis.

A kiss for daily and seasonal reproduction.

Reproduction is a fundamental biological function ensuring individual descendant survival and species perpetuity. It is an energy-consuming process, and therefore, all underlying mechanisms have to work in synchrony to ensure reproductive success. Synchronization of reproductive activity with the best time of the day or the year is part of such adaptive processes. Recently, a neuropeptide named kisspeptin, synthesized in two discrete hypothalamic nuclei, the anteroventral periventricular nucleus and the arcuate nucleus, has been demonstrated to be a potent stimulator operating upstream of the gonadotropic axis. In this review, we show how kisspeptinergic neurons integrate daily and seasonal time cues to synchronize reproductive activity with the cycling environment.

Stimulatory effect of RFRP-3 on the gonadotrophic axis in the male Syrian hamster: the exception proves the rule.

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.