Massimiliano Beltramo

RF9 Powerfully Stimulates Gonadotrophin Secretion in the Ewe: Evidence for a Seasonal Threshold of Sensitivity

GPR147 and its endogenous ligands, RFRPs, are emerging as important actors in hypothalamic‐pituitary axis control. The role of this system would be to inhibit gonadotrophin secretion. However, data on the subject are contradictory. The discovery of RF9 (adamantanecarbonyl‐RF‐2‐NH2), a GPR147 antagonist, prompted us to use this new tool to further investigate this system in the ewe. Accordingly, we tested the effect of i.c.v. administration of RF9 on gonadotrophin secretion in the ewe during anoestrous and the breeding season. Intracerebroventricular injections of RF9 (from 50–450 nmol) caused a clear elevation in peripheral blood plasma luteinising hormone (LH) concentrations. The effect of RF9 on LH was more pronounced during the anoestrous season. Furthermore, peripheral administration of RF9 as a bolus (2.1, 6.2 and 12.4 μmol per ewe) or as a constant i.v. infusion (2.1, 6.2, 12.4 and 18.6 μmol/h per ewe) to anoestrous acyclic ewes induced a sustained increase in LH plasma concentrations. A pharmacokinetic study showed that RF9 (12.4 μmol bolus i.v.) has an effective half life of 5.5 h in the plasma. Conversely, RF9 is not detectable in the cerebrospinal fluid, suggesting that it does not cross the blood–brain barrier. The increase in LH plasma concentrations induced by RF9 was blocked by previous administration of 1.3 μmol per ewe of gondotrophin‐releasing hormone (GnRH) antagonist Teverelix. This suggests that GnRH is involved in the stimulatory effect of RF9 on gonadotrophin secretion. Finally, no variation in LH plasma concentrations could be detected in ovariectomised ewes injected either i.c.v. or i.v. with RFRP3 (VPNLPQRF‐NH2). The lack of effect of RFRP3 in our experimental setting suggests that the mechanisms involved in RF9 action are probably more complex than previously assumed. Our results indicate that delivery of RF9 in the ewe greatly increases gondadotrophin secretion in both the oestrus and anoestrus season, suggesting a potential new way of controlling reproduction in mammals.

Cellular mechanisms and integrative timing of neuroendocrine control of GnRH secretion by kisspeptin

The hypothalamus integrates endogenous and exogenous inputs to control the pituitary-gonadal axis. The ultimate hypothalamic influence on reproductive activity is mediated through timely secretion of GnRH in the portal blood, which modulates the release of gonadotropins from the pituitary. In this context neurons expressing the RF-amide neuropeptide kisspeptin present required features to fulfill the role of the long sought-after hypothalamic integrative centre governing the stimulation of GnRH neurons. Here we focus on the intracellular signaling pathways triggered by kisspeptin through its cognate receptor KISS1R and on the potential role of proteins interacting with this receptor. We then review evidence implicating both kisspeptin and RFRP3--another RF-amide neuropeptide--in the temporal orchestration of both the pre-ovulatory LH surge in female rodents and the organization of seasonal breeding in photoperiodic species.

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.

Gonadotrophin-Releasing Hormone Release into the Hypophyseal Portal Blood of the Ewe Mirrors Both Pulsatile and Continuous Intravenous Infusion of Kisspeptin: An Insight into Kisspeptin's Mechanism of Action

Recent studies have demonstrated that kisspeptin (Kp) administration, given as a slow constant infusion of Kp10 (the shortest endogenous form of the Kp molecules which carries biological activity), is able to stimulate gonadotrophin secretion and induce ovulation in anoestrus acyclic ewes. Detailed analysis of peripheral luteinising hormone (LH) concentrations, obtained at 10‐min intervals, suggested that this Kp10 treatment induced the continuous release of gonadotrophins. Whether this apparent constant secretion of LH resulted from a continuous elevation of GnRH or discrete high‐frequency pulses could not be determined. In the present study, we monitored the patterns of gonadotrophin‐releasing homrone (GnRH) secreted into hypophyseal portal blood (HPB) and LH in the peripheral circulation when Kp10 was administered either as discrete pulses or by means of a continuous infusion. Samples of HPB and peripheral blood were obtained at 2 and 10‐min intervals, respectively, over a 6‐h period, from anoestrous acyclic ewes that received an i.v. bolus injection of Kp10 at 1 h and an infusion of Kp10 between hours 2 and 6. GnRH release following Kp10 administration appeared to be dose‐dependent, with larger responses being seen to the 20 μg bolus and 20 μg/h infusion than to the 10 μg bolus and 10 μg/h infusion, with the latter being marginally effective in inducing LH release. Bolus injections of Kp10 (either 20 or 10 μg) induced a sharp GnRH pulse in HPB and a discrete LH pulse in peripheral blood. By contrast, constant infusion of Kp10 (either 20 or 10 μg/h for 4 h) induced a sustained increase in baseline GnRH secretion with no convincing evidence of strictly episodic release. Values remained continuously elevated in HPB. No sign of pituitary desensitisation was observed at either concentration. Finally, i.v. injection of a large bolus (500 μg) of Kp10 produced immediate pharmacological concentrations of Kp10 in the peripheral circulation but were not associated with detectable levels of the peptide in the cerebrospinal fluid. In summary, our results demonstrate that the mode of Kp10 administration (pulsatile versus continuous) is important in shaping the pattern of GnRH secretion and suggests that this regulatory effect is most likely exerted at the level of the terminals of GnRH neurones. Moreover our data also suggest that Kp is involved in, rather than having a permissive role in, the control of endogenous GnRH pulsatility.

Immunohistochemical Evidence for the Presence of Various Kisspeptin Isoforms in the Mammalian Brain

Kisspeptins are small peptides encoded by the Kiss1 gene that have been the focus of intense neuroendocrine research during the last decade. Kisspeptin is now considered to have important roles in the regulation of puberty onset and adult oestrogen‐dependent feedback mechanisms on gonadotrophin‐releasing hormone secretion. Several kisspeptin antibodies have been generated that have enabled an overall view of kisspeptin peptide distribution in the brain of many mammalian species. However, it remains that the distribution of the different kisspeptin isoforms is unclear in the mammalian brain. In the present study, we report on two new N‐terminal‐directed kisspeptin antibodies, one against the mouse kisspeptin‐52 sequence (AC053) and one against the rat kisspeptin‐52 sequence (AC067), and use them to specifically map these long isoforms in the brains of mouse and rat, respectively. Kisspeptin‐52 immunoreactivity was detected in the two main kisspeptin neuronal populations of the rostral periventricular area and arcuate nucleus but not in the dorsomedial hypothahamus. A large number of fibres throughout the ventral forebrain were also labelled with these two antibodies. Finally, a comparison with the most commonly used C‐terminal‐directed kisspeptin antibodies further suggests the presence of shorter kisspeptin fragments in the brain with specific inter‐ and intracellular expression patterns.

Kisspeptins and the reproductive axis: potential applications to manage reproduction in farm animals.

Kisspeptins (Kp) are a family of neuropeptides produced mainly by two hypothalamic neuronal cell populations. They have recently emerged as a major regulator of the gonadotropin axis and their action is located upstream of the gonadotropin-releasing hormone (GnRH) cell population. In less than 10 yr a growing body of literature has demonstrated the involvement of these peptides in most, if not all, aspects of reproductive axis maturation and function. In contrast to these abundant basic research studies, few experiments have evaluated the potential application of Kp as tools to manipulate reproduction in domestic animals. In mammals, exogenous Kp administration potently stimulates gonadotropin secretion. This action is exerted mainly, if not exclusively, through the stimulation of GnRH release. Intravenous, intraperitoneal, or subcutaneous administration of Kp induced a robust and rapid increase in plasma gonadotropins (luteinizing hormone [LH] and follicle-stimulating hormone [FSH]). However, this stimulatory effect is of short duration. Prolonged LH and FSH release over several hours can be achieved only when Kp are given as repeated multiple bolus or as an infusion. Kp administration was used in two experimental models, ewe and pony mare, with the aim of inducing well-timed and synchronized ovulations. During the breeding season, progesterone-synchronized ewes were given an intravenous infusion of Kp starting 30 h after the removal of progesterone implants. An LH surge was induced in all Kp-treated animals within 2 h of infusion onset. In contrast, in pony mares a constant infusion of Kp for 3 d in the the late follicular phase was unable to induce synchronized ovulation. Another set of studies showed that Kp could be used to activate reproductive function in acyclic animals. Pulsatile administration of Kp in prepubertal ewe lambs was shown to activate ovarian function, leading to enhanced ovarian steroidogenesis, stimulation of LH preovulatory surge, and ovulation. In anestrous ewes, an intravenous infusion of a low dose of Kp induced an immediate and sustained release of gonadotropins, followed a few hours later by an LH surge. This hormonal pattern mimicked hormonal changes normally observed during the estrous cycle follicular phase and was associated with a high percentage of ovulating animals (80%). In summary, exogenous administration of Kp appears to be a new tool to manipulate reproduction. However, optimal doses and periods of treatment should be defined for each species, and the development of powerful analogs or long-term release formulations is necessary before large-scale applications in domestic animals could be envisaged.

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.

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

ABSTRACT 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.

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

ABSTRACT 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 &mgr;M) 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. HIGHLIGHTSRF313 displays antagonist activity at NPFF1 receptor both in vitro and in vivo.RF313 potentiates fentanyl and morphine analgesia by subcutaneous and oral routes.RF313 blocks fentanyl hyperalgesia and attenuates morphine hyperalgesia and tolerance.RF313 prevents the release of LH induced by RFRP‐3 in hamster.

Sexually active bucks are a critical social cue that activates the gonadotrope axis and early puberty onset in does

Abstract In rodents, early exposure to adult male is well known to induce an early puberty in females (Vandenbergh effect). This phenomenon has been less studied in other mammals. In goats, despite our extensive knowledge about the “male‐effect” phenomenon in adults (i.e. ovulation induced by the introduction of the male during the anestrous), there are few data on the consequences of an early exposure of females to males. Here, we evaluated the puberty onset of young alpine goats when raised since weaning with intact bucks (INT), with castrated bucks (CAS) or isolated from bucks (ISOL). The INT group had the first ovulation 1.5 month before the two other groups. Despite the earlier puberty the INT group of females had normal and regular ovarian cycles. Morphological study of the genital tract showed that at 6 months, uterus of INT goats was 40% heavier than CAS and ISOL goats. Moreover, INT females had a myometrium significantly thicker and INT was the only group having corpora lutea. In our study, INT females were pubescent in the month following the entry of bucks into the breeding season, suggesting that only sexually active bucks provide the signal responsible for puberty acceleration. By removing direct contact with the bucks, we showed that somatosensory interactions were dispensable for an early puberty induction. Finally, no difference in the GnRH network (fiber density and number of synaptic appositions) can be detected between pubescent and non‐pubescent females, suggesting that the male stimulations triggering puberty onset act probably on upstream neuronal networks, potentially on kisspeptin neurons. HighlightsIn Goats, the social environment is a key regulator of reproductive functions.Female goats exposed to intact bucks since weaning exhibit a precocious pubertyFemales with an early puberty remain cycling regularly and have a heavier uterus.Exposure to castrated bucks has no effect on the sexual maturation of young females.Somatosensory interactions with males are not needed to induce a precocious puberty.