Systemic metabolic benefits of 17α-estradiol are not exclusively mediated by ERα in glutamatergic or GABAergic neurons.

17α-Estradiol (17αE2), a less-feminising enantiomer of 17ß-estradiol, has been shown to prolong lifespan and improve metabolic health in a sex-specific manner in male, but not in female mice. Recent studies have demonstrated the pivotal role of estrogen receptor α (ERα) in mediating the effects of 17αE2 on metabolic health. However, the specific tissues and/or neuronal signalling pathways that 17αE2 acts through remain to be elucidated. ERα expression in glutamatergic and GABAergic neurons (principal excitatory and inhibitory neurons respectively) in the hypothalamus is essential for estradiol signalling. Therefore, we hypothesised that knocking out ERα from one of these neuronal populations would attenuate the established beneficial metabolic effects of 17αE2 in male mice exposed to a high fat diet. To test this hypothesis we used two established brain specific ERα KO models, targeting either glutamatergic or GABAergic neurons (Vglut2/Vgat-ERαKO). We show that both of these ERα KO models exhibit a strong reduction in ERα expression in the arcuate nucleus of the hypothalamus, a control centre for metabolic regulation. Deletion of ERα from GABAergic neurons significantly diminished the effect of 17αE2 on body weight relative to controls, although these animals still show metabolic benefits with 17αE2 treatment. The response to 17αE2 was unaffected by ERα deletion in glutamatergic neurons. Our results support a benefit of 17αE2 treatment in protection against metabolic dysfunction, but these effects do not depend on exclusive ERα expression in glutamatergic and GABAergic neurons and persist when ERα expression is strongly reduced in the arcuate nucleus of the hypothalamus.

Androgen receptor actions on AgRP neurons are not a major cause of reproductive and metabolic impairments in peripubertally androgenized mice.

Excess levels of circulating androgens during prenatal or peripubertal development are an important cause of polycystic ovary syndrome (PCOS), with the brain being a key target. Approximately half of the women diagnosed with PCOS also experience metabolic syndrome; common features including obesity, insulin resistance and hyperinsulinemia. Although a large amount of clinical and preclinical evidence has confirmed this relationship between androgens and the reproductive and metabolic features of PCOS, the mechanisms by which androgens cause this dysregulation are unknown. Neuron-specific androgen receptor knockout alleviates some PCOS-like features in a peripubertal dihydrotestosterone (DHT) mouse model, but the specific neuronal populations mediating these effects are undefined. A candidate population is the agouti-related peptide (AgRP)-expressing neurons, which are important for both reproductive and metabolic function. We used a well-characterised peripubertal androgenized mouse model and Cre-loxP transgenics to investigate whether deleting androgen receptors specifically from AgRP neurons can alleviate the induced reproductive and metabolic dysregulation. Androgen receptors were co-expressed in 66% of AgRP neurons in control mice, but only in <2% of AgRP neurons in knockout mice. The number of AgRP neurons was not altered by the treatments. Only 20% of androgen receptor knockout mice showed rescue of DHT-induced androgen-induced anovulation and acyclicity. Furthermore, androgen receptor knockout did not rescue metabolic dysfunction (body weight, adiposity or glucose and insulin tolerance). While we cannot rule out developmental compensation in our model, these results suggest peripubertal androgen excess does not markedly influence Agrp expression and does not dysregulate reproductive and metabolic function through direct actions of androgens onto AgRP neurons.

Deletion of Androgen Receptors From Kisspeptin Neurons Prevents PCOS Features in a Letrozole Mouse Model.

Polycystic ovarian syndrome (PCOS) is the leading cause of anovulatory infertility and is a heterogenous condition associated with a range of reproductive and metabolic impairments. While its etiology remains unclear, hyperandrogenism and impaired steroid negative feedback have been identified as key factors underpinning the development of PCOS-like features both clinically and in animal models. We tested the hypothesis that androgen signaling in kisspeptin-expressing neurons, which are key drivers of the neuroendocrine reproductive axis, is critically involved in PCOS pathogenesis. To this end, we used a previously validated letrozole (LET)-induced hyperandrogenic mouse model of PCOS in conjunction with Cre-lox technology to generate female mice exhibiting kisspeptin-specific deletion of androgen receptor (KARKO mice) to test whether LET-treated KARKO females are protected from the development of reproductive and metabolic PCOS-like features. LET-treated mice exhibited hyperandrogenism, and KARKO mice exhibited a significant reduction in the coexpression of kisspeptin and androgen receptor mRNA compared to controls. In support of our hypothesis, LET-treated KARKO mice exhibited improved estrous cyclicity, ovarian morphology, and insulin sensitivity in comparison to LET-treated control females. However, KARKO mice were not fully protected from the effects of LET-induced hyperandrogenism and still exhibited reduced corpora lutea numbers and increased body weight gain. These data indicate that increased androgen signaling in kisspeptin-expressing neurons plays a critical role in PCOS pathogenesis but highlight that other mechanisms are also involved.

Central Irisin Signaling Is Required for Normal Timing of Puberty in Female Mice.

Timing of puberty requires exquisite coordination of genes, hormones, and brain circuitry. An increasing level of body adiposity, signaled to the brain via the fat-derived hormone leptin, is recognized as a major factor controlling puberty onset. However, it is clear that leptin is not the only metabolic cue regulating puberty, and that developmental regulation of this process also involves tissues other than adipose, with muscle development potentially playing a role in the timing of puberty. The proteolytic processing of fibronectin type 3 domain-containing protein 5 (FNDC5) releases a hormone, irisin. Irisin is primarily produced by muscle and is released into circulation, where levels increase dramatically as puberty approaches. We investigated the effects of a global deletion of the Fndc5 gene on pubertal timing. The absence of irisin induced a delay in puberty onset in female knockout mice compared with controls, without affecting body weight or gonadotropin-releasing hormone (GnRH) neuronal density. We next treated pre-pubertal wild-type male and female mice with an irisin receptor antagonist, cilengitide, for 7 days and observed a delay in first estrus occurrence compared to vehicle-treated control mice. Male puberty timing was unaffected. Next, we deleted the irisin receptor (integrin subunit alpha V) in all forebrain neurons and found a delay in the occurrence of first estrus in knockout females compared to controls. Taken together, these data suggest irisin plays a role in the timing of puberty onset in female mice via a centrally mediated mechanism.

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.

A customized long acting formulation of the kisspeptin analog C6 triggers ovulation in anestrus ewe.

The modulation of the kisspeptin system holds promise as a treatment for human reproductive disorders and for managing livestock breeding. The design of analogs has overcome some unfavorable properties of the endogenous ligands. However, for applications requiring a prolongation of drug activity, such as ovulation induction in the ewe during the non-breeding season, additional improvement is required. To this aim, we designed and tested three formulations containing the kisspeptin analog C6. Two were based on polymeric nanoparticles (NP1 and NP2) and the third was based on hydrogels composed of a mixture of cyclodextrin polymers and dextran grafted with alkyl side chains (MD/pCD). Only the MD/pCD formulation prolonged C6 activity, as shown by monitoring luteinizing hormone (LH) plasma concentration (elevation duration 23.4 ± 6.1, 13.7 ± 4.7 and 12.0 ± 2.4 h for MD/pCD, NP1 and NP2, respectively). When compared with the free C6 (15 nmol/ewe), the formulated (MD/pCD) doses of 10, 15 and 30 nmol/ewe, but not the 90 nmol/ewe dose, provided a more gradual release of C6 as shown by an attenuated LH release during the first 6 h post-treatment. When tested during the non-breeding season without progestogen priming, only, the formulated 30 nmol/ewe dose triggered ovulation (50% of ewes). Hence, we showed that a formulation with an adapted action time would improve the efficacy of C6 with respect to inducing ovulation during the non-breeding season. This result suggests that formulations containing a kisspeptin analog might find applications in the management of livestock reproduction but also point to the possibility of their use for the treatment of some human reproductive pathologies.

The kisspeptin analog C6 is a possible alternative to PMSG (pregnant mare serum gonadotropin) for triggering synchronized and fertile ovulations in the Alpine goat.

In temperate regions goat's reproduction is seasonal. To obtain year-round breeding, hormonal treatments are currently applied. These treatments usually combine a progesterone analog with the pregnant mare serum gonadotropin (PMSG). However, their use has significant ethical and environmental drawbacks. Therefore, alternative methods to manage reproduction are needed. The discovery that in mammals the neuropeptide kisspeptin is a major positive regulator of hypothalamo-pituitary gonadal axis offered an attractive alternative strategy to control reproduction. We have previously designed a kisspeptin analog, called C6, which offers pharmacological advantages over endogenous kisspeptin. These include a longer lasting effect and enhanced activity following intramuscular injection. In the present work, we evaluated C6 effect on LH and FSH plasma concentrations in the Alpine goat breed and tested whether C6 could replace PMSG to trigger ovulation. An intramuscular injection of C6 (15 nmol/doe) given 24 hours after the end of progestogen treatment induced a surge-like peak of both LH and FSH. This was followed by an increase of progesterone, a hallmark of ovulation induction and corpus luteus formation. These results were obtained at three different time of the year: during the breeding season, the non-breeding season and at the onset of the breeding season. Furthermore, we compared the efficacy of C6 and PMSG to induce fertile ovulations when these treatments are given at the onset of the breeding season and are followed by artificial insemination. The results of this first attempt were extremely promising with gestation rates of 45% and 64% for C6 and PMSG respectively. Pending optimization of the treatment procedure in order to improve efficacy, kisspeptin analogs could be the long sought-after alternative to PMSG.

Neuroanatomical connections between kisspeptin neurones and somatostatin neurones in female and male rat hypothalamus: a possible involvement of SSTR1 in kisspeptin release.

Somatostatin (SST) a neuropeptide involved in the central modulation of several physiological functions, is co-distributed in the same hypothalamic areas as kisspeptin (KP), the most potent secretagogue of the gonadotropin-releasing hormone (GnRH) secretion known to date. As SST infused intracerebroventricularly (icv) evoked a potent inhibition of GnRH release, we explored neuroanatomical relationships between KP and SST populations in male and female rats. For that, intact males and ovariectomised oestradiol-replaced females were killed and their brains processed in order to simultaneously detect KP, SST and synapsin, a marker for synapses. We observed numerous appositions of KP on SST neurones both in female and male arcuate nucleus (ARC) and ventromedial hypothalamus. A large association between SST terminals and KP neurones at the level of the pre-optic area (POA) was also observed in female rats and in a more limited frame in males. Finally, most KP neurones from the ARC showed SST appositions in both sexes. To determine whether SST could affect KP cell activity, we assessed whether SST receptors (SSTR) were present on KP neurones in the ARC. We also looked for the presence of SSTR1 and SSTR2A in the brain of male rats. Brains were processed through a sequential double immunocytochemistry in order to detect KP and SSTR1 or KP and SSTR2A. We observed overlapping distributions of immunoreactive neurones for SSTR1 and KP and counted approximately one third of KP neurones with SSTR1. In contrast, neurones labelled for SSTR2A or KP were often juxtaposed in the ARC and the occurrence of double-labelled neurones was sporadic (<5%). These results suggest that SST action on KP neurones would pass mainly through SSTR1 at the level of the ARC. This article is protected by copyright. All rights reserved.

New insights on the neuroendocrine control of puberty and seasonal breeding in female sheep.

Timing of puberty has a great influence on animal productivity. For example, reproduction in sheep can be affected by seasonality, leading to fluctuations in availability of animal products. Therefore, optimization of birth dates would improve reproductive success in sheep. Since the discovery of the major role of kisspeptin and Kiss1R, its cognate receptor, in reproductive function, there are new opportunities for interventions. Repeated or continuous administration of native kisspeptin are able to hasten puberty and induce ovulation during breeding and non-breeding seasons of sheep. However, due to the short half-life of kisspeptin, protocols involving native kisspeptin are usually proof of concept, but not practical under field conditions. Consequently, there are efforts to develop kisspeptin analogues capable of replicating effects of repeated/continuous administration of native kisspeptin. In this review, we intended to provide a comprehensive summary of the neuroendocrine requirements for puberty onset and ovulation in adult ewes, focusing on kisspeptin, its physiological effects and responses to its analogues on reproductive function in ewes.

Seasonal breeding in mammals: From basic science to applications and back.

Seasonal breeding is a remarkable adaptive feature, which allows animals to coordinate physiological functions throughout the year. However, in the context of animal production, it becomes an undesirable complication, which needs to be circumvented. Therefore, eco-friendly methods based on photoperiodic treatments and the use of the male effect have been developed to control seasonal reproduction in small ruminants. In practice, such treatments are hardly used and hormonal treatments constitute the benchmark, but practicality of hormonal treatments comes at a high cost for human health and the environment. Here, we summarize our current understanding of the molecular and neuroendocrine mechanisms underlying seasonal breeding in small ruminants. We then move on to describe current methods to control reproduction and detail why such methods are not sustainable. Finally, using the neuropeptide kisspeptin as an example, we show that an improved understanding of the molecular and neuroendocrine mechanisms that underlie photoperiodism might help design novel strategies for the development of improved and sustainable breeding schemes.

Rational design of triazololipopeptides analogs of kisspeptin inducing a long-lasting increase of gonadotropins.

New potent and selective KISS1R agonists were designed using a combination of rational chemical modifications of the endogenous neuropeptide kisspeptin 10 (KP10). Improved resistance to degradation and presumably reduced renal clearance were obtained by introducing a 1,4-disubstituted 1,2,3-triazole as a proteolysis-resistant amide mimic and a serum albumin-binding motif, respectively. These triazololipopeptides are highly potent full agonists of KISS1R and are >100 selective over the closely related NPFF1R. When injected in ewes with a quiescent reproductive system, the best compound of our series induced a much prolonged increase of luteinizing hormone release compared to KP10 and increased follicle-stimulating hormone plasma concentration. Hence, this KISS1R agonist is a new valuable pharmacological tool to explore the potential of KP system in reproduction control. Furthermore, it represents the first step to develop drugs treating reproductive system disorders due to a reduced activity of the hypothalamo-pituitary-gonadal axis such as delayed puberty, hypothalamic amenorrhea, and hypogonadotropic hypogonadism.

Acute injection and chronic perfusion of kisspeptin elicit gonadotropins release but fail to trigger ovulation in the mare.

Kisspeptin has emerged as the most potent gonadotropin-releasing hormone (GnRH) secretagogue and appears to represent the penultimate step in the central control of reproduction. In the sheep, we showed that kisspeptin could be used to manipulate gonadotropin secretion and control ovulation. Prompted by these results, we decided to investigate whether kisspeptin could be used as an ovulation-inducing agent in another photoperiodic domestic mammal, the horse. Equine kisspeptin-10 (eKp10) was administered intravenously as bolus injections or short- to long-term perfusions to Welsh pony mares, either during the anestrus season or at various stages of the cycle during the breeding season. In all the experimental conditions, eKp10 reliably increased peripheral concentrations of both luteinizing hormone and follicle-stimulating hormone. The nature of the response to eKp10 was consistent across experimental conditions and physiological states: the increase in gonadotropins was always rapid and essentially transient even when eKp10 was perfused for prolonged periods. Furthermore, eKp10 consistently failed to induce ovulation in the mare. To gain insights into the underlying mechanisms, we used acute injections or perfusions of GnRH. We also cloned the equine orthologues of the kisspeptin precursor and Kiss1r; this was justified by the facts that the current equine genome assembly predicted an amino acid difference between eKp10 and Kp10 in other species while an equine orthologue for Kiss1r was missing altogether. In light of these findings, potential reasons for the divergence in the response to kisspeptin between ewe and mare are discussed. Our data highlight that kisspeptin is not a universal ovulation-inducing agent.