Photoperiod, but not progesterone, has a strong impact upon the transcriptome of the medio-basal hypothalamus in female goats and ewes.

Photoperiod is the main environmental driver of seasonal responses in organisms living at temperate and polar latitudes. Other external cues such as food and temperature, and internal cues including hormones, intervene to fine-tune phasing of physiological functions to the solar year. In mammals, the medio-basal hypothalamus (MBH) is the key integrator of these cues, which orchestrates a wide array of seasonal functions, including breeding. Here, using RNAseq and RT-qPCR, we demonstrate that molecular components of the photoperiodic response previously identified in ewes are broadly conserved in does (female goats, Capra hircus), with a common core of ∼50 genes. This core group can be defined as the "MBH seasonal trancriptome", which includes key players of the pars tuberalis-tanycytes neuroendocrine retrograde pathway that governs intra-MBH photoperiodic switches of triiodothyronine (T3) production (Tshb, Eya3, Dio2 and SlcO1c1), the two histone methyltransferases Suv39H2 and Ezh2 and the secreted protein Vmo1. Prior data in ewes revealed that T3 and estradiol (E2), both key hormones for the proper timing of seasonal breeding, differentially impact the MBH seasonal transcriptome, and identified cellular and molecular targets through which these hormones might act. In contrast, information regarding the potential impact of progesterone (P4) upon the MBH transcriptome was nonexistent. Here, we demonstrate that P4 has no discernible transcriptional impact in either does or ewes. Taken together, our data show that does and ewes possess a common core set of photoperiod-responsive genes in the MBH and conclusively demonstrate that P4 is not a key regulator of the MBH transcriptome.

Male-induced early puberty correlates with the maturation of arcuate nucleus kisspeptin neurons in does.

In goats, early exposure of spring-born females to sexually active bucks induces an early puberty onset assessed by the first ovulation. This effect is found when females are continuously exposed well before the male breeding season starting in September. The first aim of this study was to evaluate whether a shortened exposure of females to males could also lead to early puberty. We assessed the onset of puberty in Alpine does isolated from bucks (ISOL), exposed to wethers (CAS), exposed to intact bucks from the end of June (INT1), or mid-August (INT2). Intact bucks became sexually active in mid-September. At the beginning of October, 100% of INT1 and 90% of INT2 exposed does ovulated, in contrast to the ISOL (0%) and CAS (20%) groups. This demonstrated that contact with males that become sexually active is the main factor prompting precocious puberty in females. Furthermore, a reduced male exposure during a short window before the breeding season is sufficient to induce this phenomenon. The second aim was to investigate the neuroendocrine changes induced by male exposure. We found a significant increase in kisspeptin immunoreactivity (fiber density and number of cell bodies) in the caudal part of the arcuate nucleus of INT1 and INT2 exposed females. Thus, our results suggest that sensory stimuli from sexually active bucks (e.g., chemosignals) may trigger an early maturation of the ARC kisspeptin neuronal network leading to gonadotropin-releasing hormone secretion and first ovulation.

Acid-Resistant BODIPY Amino Acids for Peptide-Based Fluorescence Imaging of GPR54 Receptors in Pancreatic Islets.

The G protein-coupled kisspeptin receptor (GPR54 or KISS1R) is an important mediator in reproduction, metabolism and cancer biology; however, there are limited fluorescent probes or antibodies for direct imaging of these receptors in cells and intact tissues, which can help to interrogate their multiple biological roles. Herein, we describe the rational design and characterization of a new acid-resistant BODIPY-based amino acid (Trp-BODIPY PLUS), and its implementation for solid-phase synthesis of fluorescent bioactive peptides. Trp-BODIPY PLUS retains the binding capabilities of both short linear and cyclic peptides and displays notable turn-on fluorescence emission upon target binding for wash-free imaging. Finally, we employed Trp-BODIPY PLUS to prepare some of the first fluorogenic kisspeptin-based probes and visualized the expression and localization of GPR54 receptors in human cells and in whole mouse pancreatic islets by fluorescence imaging.

Short photoperiod modulates behavior, cognition and hippocampal neurogenesis in male Japanese quail.

The mechanisms underlying the photoperiodic control of reproduction in mammals and birds have been recently clarified. In contrast, the potential impact of photoperiod on more complex, integrative processes, such as cognitive behaviors, remains poorly characterized. Here, we investigated the impact of contrasted long and short photoperiods (LP, 16 h light/day and SP, 8 h light/day, respectively) on learning, spatial orientation abilities, and emotional reactivity in male Japanese quail. In addition, we quantified cell proliferation and young cell maturation/migration within the hippocampus, a brain region involved in spatial orientation. Our study reveals that, in male quail, SP increases emotional responses and spatial orientation abilities, compared to LP. Behaviorally, SP birds were found to be more fearful than LP birds, exhibiting more freezing in the open field and taking longer to exit the dark compartment in the emergence test. Furthermore, SP birds were significantly less aggressive than LP birds in a mirror test. Cognitively, SP birds were slower to habituate and learn a spatial orientation task compared to LP birds. However, during a recall test, SP birds performed better than LP birds. From a neuroanatomical standpoint, SP birds had a significantly lower density of young neurons, and also tended to have a lower density of mature neurons within the hippocampus, compared to LP birds. In conclusion, our data reveal that, beyond breeding control, photoperiod also exerts a profound influence on behavior, cognition, and brain plasticity, which comprise the seasonal program of this species.

Acid-Resistant BODIPY Amino Acids for Peptide-Based Fluorescence Imaging of GPR54 Receptors in Pancreatic Islets.

The G protein-coupled kisspeptin receptor (GPR54 or KISS1R) is an important mediator in reproduction, metabolism and cancer biology; however, there are limited fluorescent probes or antibodies for direct imaging of these receptors in cells and intact tissues, which can help to interrogate their multiple biological roles. Herein, we describe the rational design and characterization of a new acid-resistant BODIPY-based amino acid (Trp-BODIPY PLUS), and its implementation for solid-phase synthesis of fluorescent bioactive peptides. Trp-BODIPY PLUS retains the binding capabilities of both short linear and cyclic peptides and displays notable turn-on fluorescence emission upon target binding for wash-free imaging. Finally, we employed Trp-BODIPY PLUS to prepare some of the first fluorogenic kisspeptin-based probes and visualized the expression and localization of GPR54 receptors in human cells and in whole mouse pancreatic islets by fluorescence imaging.

Impact of food restriction on the medio-basal hypothalamus of intact ewes as revealed by a large-scale transcriptomics study.

In mammals, the medio-basal hypothalamus (MBH) integrates photoperiodic and food-related cues to ensure timely phasing of physiological functions, including seasonal reproduction. The current human epidemics of obesity and associated reproductive disorders exemplifies the tight link between metabolism and reproduction. Yet, how food-related cues impact breeding at the level of the MBH remains unclear. In this respect, the sheep, which is a large diurnal mammal with a marked dual photoperiodic/metabolic control of seasonal breeding, is a relevant model. Here, we present a large-scale study in ewes (n = 120), which investigated the impact of food restriction (FRes) on the MBH transcriptome using unbiased RNAseq, followed by RT-qPCR. Few genes (~100) were impacted by FRes and the transcriptional impact was very modest (<2-fold increase or < 50% decrease for most genes). As anticipated, FRes increased expression of Npy/AgRP/LepR and decreased expression of Pomc/Cartpt, while Kiss1 expression was not impacted. Of particular interest, Eya3, Nmu and Dio2, genes involved in photoperiodic decoding within the MBH, were also affected by FRes. Finally, we also identified a handful of genes not known to be regulated by food-related cues (e.g., RNase6, HspA6, Arrdc2). In conclusion, our transcriptomics study provides insights into the impact of metabolism on the MBH in sheep, which may be relevant to human, and identifies possible molecular links between metabolism and (seasonal) reproduction.

The Kisspeptin analogue C6 induces ovulation in jennies.

Kisspeptins (KPs) are the most potent stimulating neurotransmitters of GnRH release, and consequently KP administration triggers LH and/or FSH release. In small ruminants, KP or its analogs induced an LH surge followed by ovulation in both cyclic and acyclic animals, while in the mare KP only increased LH plasma levels but failed to induce ovulation. This study in jennies compares the endocrinological effects, ovulatory and pregnancy rates of the KP analog C6 and the GnRH analog buserelin acetate. The ovarian activity of nine Amiata jennies was monitored daily by transrectal ultrasound for three complete estrous cycles. Jennies in estrus were assigned, to one of three treatment groups: 50 nmol of the KP analog C6 (injected twice, 24 h apart, C6 group); 0.4 mg buserelin acetate (injected once, Bu group); and 2 mL of saline (injected once, CTRL group). Blood samples were collected at Day-1 (-24 h) Day0 (h0, before treatment), h2, h4, h6, h8, h10, h24 (before second treatment with C6), h26, h28, h30, h32, h34, h48 and every 24 h until ovulation. Jennies were inseminated once at h24 with fresh extended semen from a donkey stallion. Pregnancy diagnoses were performed 14 days after ovulation. On days 5, 10, and 14 after ovulation, for every CL the cross-sectional area (CSA) and the vascularized area (VA) were recorded by color doppler ultrasound and measured. Significantly higher plasma LH levels were found after induction between the Bu and CTRL groups at h6 and h8 (P < 0.05), while tendentially higher differences were found between the Bu/C6 groups and CTRL at h10. Five/9, 4/9, and 2/9 jennies ovulated between 24 and 48 h after induction from the Bu, C6, and CTRL groups respectively, (P > 0.05). Correlations between corpora lutea CSA and VA with serum progesterone concentration were r = 0.31, P = 0.01, r = 0.38, P = 0.01, respectively. Pregnancy rates after artificial insemination did not differ among groups (CTRL: 6/9, 66.7%; C6: 7/9, 77.8%; Bu: 6/9, 66.7%; P > 0.05). Ovulation rates after C6 treatment were comparable to that of Bu, although not different from the CTRL. Pregnancy rates were comparable to the literature in terms of fresh extended donkey semen in every group. This study suggests that stimulation of the Kp system in jennies, in contrast to findings observed in mares, induces ovulation. Further studies using higher doses and/or more animals are needed to better characterize the efficacy of C6 in jennies.

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.

hCG is more effective than the GnRH agonist buserelin for inducing the first ovulation of the breeding season in mares.

BACKGROUND: Human Chorionic Gonadotropin (hCG) and Gonadotropin Releasing Hormone agonists (GnRHa) are routinely used to induce ovulation in mares. However, GnRHa efficacy in transitional mares has been suggested to be low. OBJECTIVES: The aims of this study were as follows: (a) to compare the efficacy of hCG and GnRHa in inducing the first ovulation of the breeding season and (b) to evaluate the correlation between ovulatory response, uterine oedema and teasing score at the time of treatment during the early or late transitional phase. STUDY DESIGN: Randomised controlled superiority trial. METHODS: Mares in winter anoestrus were treated with sulpiride when at least two follicles reached a diameter of 25 mm. The day after the follicle reached 35 mm in diameter, mares in oestrus were treated with GnRHa buserelin (N = 29) or hCG (N = 33) and checked daily for ovulation. RESULTS: More mares (30/33, 90.1%) ovulated when the first ovulation after winter anoestrus was induced with hCG, than with GnRHa, (11/29, 38.0%) (P = .0001). Ovulation rate was lower in mares that did not show uterine oedema and full acceptance of the teaser stallion for at least three days before the treatment (32/41, 78% vs 9/21, 42.9%) P = .01. MAIN LIMITATIONS: Plasma LH and oestrogen concentrations were not performed. CONCLUSIONS: These results demonstrate that hCG was more effective than GnRHa for inducing ovulation in the first cycle after winter anoestrus. Uterine oedema and behavioural signs of oestrus, for at least three days before the treatment, were predictors for a positive response to ovulation induction.

No evidence that Spexin impacts LH release and seasonal breeding in the ewe.

Spexin (SPX) is a recently identified peptide hormone of 14 amino acids. Interestingly, Spx and Kiss1 genes share a common ancestor gene. Considering that KISS1 peptides are key controllers of breeding in mammals and circumstantial evidence that SPX regulates gonadotropins in some fish species, we hypothesized that SPX may play a KISS1-related role in sheep. Here, we cloned the ovine Spx cDNA, performed in vivo injection and infusion of SPX (i.c.v. route, with or without concomittant KISS1 presence) and assessed a potential regulation of Spx expression by season, thyroid hormone and estradiol in the medio-basal hypothalamus of the ewe. Our data do not provide support for a role of SPX in the control of the gonadotropic axis in the ewe.

Circuit-level analysis identifies target genes of sex steroids in ewe seasonal breeding.

Thyroid hormone (TH) and estradiol (E2) direct seasonal switches in ovine reproductive physiology. In sheep, as in other mammals and birds, control of thyrotropin (TSH) production by the pars tuberalis (PT) links photoperiod responsiveness to seasonal breeding. PT-derived TSH governs opposite seasonal patterns of the TH deiodinases Dio2/Dio3 expression in tanycytes of the neighboring medio-basal hypothalamus (MBH), which explain the key role of TH. We recently used RNA-Seq to identify seasonal markers in the MBH and define the impact of TH. This impact was found to be quite limited, in terms of number of target genes, and very restricted with regards to neuroanatomical location, as TH specifically impacts genes expressed in tanycytes and hypothalamus, not in the PT. Here we address the impact of E2 on these seasonal markers, which are specifically expressed in either PT, tanycytes or hypothalamus. We also investigate if progesterone (P4) may be involved in timing the seasonal transition to anestrus. Our analysis provides circuit-level insights into the impact of sex steroids on the ewe seasonal breeding cycle. First, seasonal gene expression in the PT is independent of the sex steroid status. The fact that seasonal gene expression in the PT is also TH-independent strengthens the view that the PT is a circannual timer. Second, select tanycytic markers display some level of responsiveness to E2 and P4, which indicates another potential level of feedback control by sex steroids. Third, Kiss1 neurons of the arcuate nucleus are responsive to both TH and E2, which places them at the crossroads of photoperiodic transduction pathway and sex steroid feedback. This provides strong support to the concept that these Kiss1 neurons are pivotal to the long-recognized "seasonal switch in the ability of E2 to exert negative feedback", which drives seasonal breeding.

In Silico Peptide Ligation: Iterative Residue Docking and Linking as a New Approach to Predict Protein-Peptide Interactions.

Peptide⁻protein interactions are corner-stones of living functions involved in essential mechanisms, such as cell signaling. Given the difficulty of obtaining direct experimental structural biology data, prediction of those interactions is of crucial interest for the rational development of new drugs, notably to fight diseases, such as cancer or Alzheimer's disease. Because of the high flexibility of natural unconstrained linear peptides, prediction of their binding mode in a protein cavity remains challenging. Several theoretical approaches have been developed in the last decade to address this issue. Nevertheless, improvements are needed, such as the conformation prediction of peptide side-chains, which are dependent on peptide length and flexibility. Here, we present a novel in silico method, Iterative Residue Docking and Linking (IRDL), to efficiently predict peptide⁻protein interactions. In order to reduce the conformational space, this innovative method splits peptides into several short segments. Then, it uses the performance of intramolecular covalent docking to rebuild, sequentially, the complete peptide in the active site of its protein target. Once the peptide is constructed, a rescoring step is applied in order to correctly rank all IRDL solutions. Applied on a set of 11 crystallized peptide⁻protein complexes, the IRDL method shows promising results, since it is able to retrieve experimental binding conformations with a Root Mean Square Deviation (RMSD) below 2 Šin the top five ranked solutions. For some complexes, IRDL method outperforms two other docking protocols evaluated in this study. Hence, IRDL is a new tool that could be used in drug design projects to predict peptide⁻protein interactions.

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.

Brain mapping of the gonadotropin-inhibitory hormone-related peptide 2 with a novel antibody suggests a connection with emotional reactivity in the Japanese quail (Coturnix japonica, Temminck & Schlegel, 1849).

Gonadotropin-inhibitory hormone (GnIH) is a neuropeptide first discovered in the quail brain that is involved in the control of reproductive physiology and behaviors, and stress response. GnIH gene encodes a second peptide, GnIH-related peptide-2 (RP2), the distribution and function of which remain unknown. We therefore studied GnIH-RP2 distribution by immunohistochemistry using a novel antibody capable of discriminating between GnIH and GnIH-RP2. The overall distribution of GnIH-RP2 is similar to that of GnIH. The vast majority of labeled neurons is located in the paraventricular nucleus (PVN) of the hypothalamus. Labeling of fibers is conspicuous in the diencephalon, but present also in the mesencephalon and telencephalon. Several regions involved in the control of reproduction and stress response (the PVN, septum, bed nucleus of the stria terminalis and nucleus commissura pallii) showed a dense network of immunolabeled fibers. To investigate the potential function of GnIH-RP2 we compared its expression in two quail lines genetically selected for divergence in their emotional reactivity. A quantitative analysis in the above-mentioned brain regions showed that the density of fibers was similar in the two lines. However, the number of GnIH-RP2 labeled neurons was higher in the median portion of the PVN in birds with higher emotional reactivity. These results point to a possible involvement of GnRH-RP2 in modulating stress response and/or emotional reactivity.

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

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.

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.

Synthesis of aryl-thioglycopeptides through chemoselective Pd-mediated conjugation.

We describe herein a Pd-catalyzed methodology for the thioglycoconjugation of iodoaryl peptides and aminoacids. This operationally simple process occurs under semi-aqueous conditions and displays wide substrate scope. The strategy has been successfully applied to both the thioglycosylation of unprotected peptides and the generation of thioglyco-aminoacid building blocks, including those suitable for solid phase peptide synthesis. To demonstrate the broad potential of this technique for late stage functionalization, we successfully incorporated challenging unprotected ß-S-GlcNAc- and α-S-GalNAc-derivatives into very long unprotected peptides. This study opens the way to new applications in chemical biology, considering the well-recognized advantages of S-glycosides over O-glycosides in terms of resistance towards both enzymatic and chemical degradation.

Cross-Linking Furan-Modified Kisspeptin-10 to the KISS Receptor.

Chemical cross-linking is well-established for investigating protein-protein interactions. Traditionally, photo cross-linking is used but is associated with problems of selectivity and UV toxicity in a biological context. We here describe, with live cells and under normal growth conditions, selective cross-linking of a furan-modified peptide ligand to its membrane-presented receptor with zero toxicity, high efficiency, and spatio-specificity. Furan-modified kisspeptin-10 is covalently coupled to its glycosylated membrane receptor, GPR54(KISS1R). This newly expands the applicability of furan-mediated cross-linking not only to protein-protein cross-linking but also to cross-linking in situ. Moreover, in our earlier reports on nucleic acid interstrand cross-linking, furan activation required external triggers of oxidation (via addition of N-bromo succinimide or singlet oxygen). In contrast, we here show, for multiple cell lines, the spontaneous endogenous oxidation of the furan moiety with concurrent selective cross-link formation. We propose that reactive oxygen species produced by NADPH oxidase (NOX) enzymes form the cellular source establishing furan oxidation.

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.