Characterizing the relationship between gonadotropin releasing hormone (GnRH), kisspeptin, and RFamide related peptide 3 (RFRP-3) neurons in the equine hypothalamus across the estrous cycle and in the anovulatory seasons.

To understand better the role that kisspeptin plays in regulating seasonal and estrous cycle changes in the mare, this study investigated the number, location and interactions between GnRH, kisspeptin and RFRP-3 neurons in the equine hypothalamus. Hypothalami were collected from mares during the non-breeding season, vernal transition and various stages of the breeding season. Fluorescent immunohistochemistry was used to label the neuropeptides of interest. GnRH cells were observed primarily in the arcuate nucleus (ARC), while very few labeled cells were identified in the pre-optic area (POA). Kisspeptin cells were identified primarily in the ARC, with a small number of cells observed dorsal to the ARC, surrounding the third ventricle (3V). The mean number of kisspeptin cells varied between animals and typically showed no pattern associated with season or stage of estrous cycle, but a seasonal difference was identified in the ARC population. Small numbers of RFRP-3 cells were observed in the ARC, ventromedial hypothalamus (VMH) and dorsomedial hypothalamus (DMH). The mean number of RFRP-3 cells appeared higher in pre-ovulatory animals compared to all other stages. The percentage of GnRH cell bodies with kisspeptin appositions did not change with season or stage of estrous cycle. The percentage of kisspeptin cells receiving inputs from RFRP-3 fibers did not vary with season or stage of estrous cycle. These interactions suggest the possibility of the presence of an ultra-short loop feedback system between these three peptides. The changes in RFRP-3 neurons suggest the possibility of a role in the regulation of reproduction in the horse, but it is unlikely to be as a gonadotropin inhibitory factor.

Effect of nourishing and purging fire Chinese herbal mixture on delaying light-induced premature puberty in rats.

OBJECTIVE: To elucidate the mechanism of the nourishing Yin and purging fire Chinese herbal mixture (NYPF) in delaying light-induced premature puberty in rats. METHODS: Twenty-one days old female Sprague-Dawley rats were randomly assigned to normal group (N), long light exposure group (L), NYPF and normal saline group (NS). Rats in the L, NYPF and NS groups were exposed to 16 h: 350 lux light/8 h: dark, while rats in the N group were exposed to 12 h: 50 lux light/12 h: dark. NYPF and normal saline was administered to the rats in the NYPF group or NS group, respectively, from day 21. Five rats in every group were sacrificed at 9 p.m. on day 28 (P28), on the day when rat's vulva opened in the L group (L-VO), on the day when the first estrous interphase occurred in rats of L group (L-E1), and on the day when the second estrous interphase occurred in rats of L group (L-E2), respectively. RESULITS: On day 34, all rats in the L group, 80% of rats in the NS group, 40% of rats in the N group, and 20% of rats in the NYPF group showed complete opening of the vulva. At P28, mRNA level of hypothalamic kisspeptin (Kiss-1) in the L group was significantly higher than that in the N group (P < 0.05). The rats in the L and NS groups had significantly lower hypothalamic arginine-phenylalanine-amide (RFamide)-related peptide 3 (RFRP-3) mRNA levels than those in the N group (P < 0.05), whereas RFRP-3 mRNA level was significantly higher in the NYPF group than that in the L group (P < 0.05). At L-VO, the ovarian index of the L and NS groups was significantly higher than that of the N group (P < 0.05) and estradiol (E2) level of the NYPF group was significantly lower than that of the N and NS groups (P < 0.05); hypothalamic Kiss-1 mRNA level in the L and NS groups was significantly higher than that in the N and NYPF groups (P < 0.05), whereas hypothalamic RFRP-3 mRNA level in the L, NYPF, and NS groups was significantly lower than that in the N group (P < 0.05). At L-E1, E2 level of the L and NS groups was significantly higher than that of the N group (P < 0.01), whereas it was significantly lower in the NYPF group than that of the N, L, and NS groups (P < 0.01), and serum luteinizing hormone level of the L and NS groups was significantly higher than that of the N group (P < 0.05); levels of serum melatonin and ovarian melatonin receptor 1 (MT-1) mRNA in the L, NYPF, and NS groups were significantly lower than those in the N group (P < 0.05). At L-E2, the uterine organ index of the NYPF group was significantly lower than that of the L group (P < 0.05); and ovarian MT-1 mRNA level of the L and NS groups was significantly lower than that in the N group (P < 0.05). CONCLUSIONS: NYPF can delay puberty onset in rats exposed to strong light for a prolonged duration, and regulation of the gene expression of Kiss-1 and RFRP-3 in the hypothalamus has been suggested as one of the mechanisms.

Rfamide-related peptide-3(RFRP-3) receptor gene is expressed in mouse ovarian granulosa cells: Potential role of RFRP-3 in steroidogenesis and apoptosis.

RFRP-3 is a functional ortholog of avian GnIH and regulates reproductive activities in the gonads of animals. However, the role of RFRP-3 in the function of ovarian granulosa cells in mice remains unclear. First, we detected the expression of the RFRP-3 receptor (GPR147) in the ovarian granulosa cells of mice. Second, the effect of RFRP-3 treatment on estradiol and progesterone secretions from granulosa cells was tested by ELISA. Meanwhile, the expression of genes and proteins regulating steroid hormone synthesis was respectively examined by qPCR and western blot. Furthermore, the effect of RFRP-3 treatment on the apoptosis of granulosa cells was analyzed. The results revealed that the GPR147 protein (a RFRP-3 receptor) was expressed in the ovarian granulosa cells of mice. Low and medium doses RFRP-3 treatment significantly reduced progesterone secretion in the granulosa cells (P < 0.05), while RFRP-3 suppressed p450scc, 3ß-HSD, StAR, and FSHR expression in a non-dose-dependent manner. Moreover, RFRP-3 treatment might induce the apoptosis of granulosa cells. Additionally, low doses RFRP-3 significantly reduced p-ERK1/2 protein expression (P < 0.05) in the ovarian granulosa cells. We here, for the first time, confirmed that GPR147 was expressed in the ovarian granulosa cells of mice. Our findings suggested that and RFRP-3 regulates the granulosa cell function through the ERK signaling pathway, which will lay the foundation for uncovering molecular mechanisms by which RFRP-3 regulates follicle development in future.

Global cerebral ischemia in adult female rats interrupts estrous cyclicity and induces lasting changes in hypothalamic-pituitary-gonadal axis signaling peptides.

Persistent post-ischemic alterations to the hypothalamic-pituitary-adrenal (HPA) axis occur following global cerebral ischemia (GCI) in rodents. However, similar effects on hypothalamic-pituitary-gonadal (HPG) axis activation remain to be determined. Therefore, this study evaluated the effects of GCI in adult female rats (via four-vessel occlusion) on the regularity of the estrous cycle for 24-days post ischemia. A second objective aimed to assess persistent alterations of HPG axis activation through determination of the expression of estrogen receptor alpha (ERα), kisspeptin (Kiss1), and gonadotropin-inhibitory hormone (GnIH/RFamide-related peptide; RFRP3) in the medial preoptic area (POA), arcuate nucleus (ARC), dorsomedial nucleus (DMH) of the hypothalamus, and CA1 of the hippocampus 25 days post ischemia. Expression of glucocorticoid receptors (GR) in the paraventricular nucleus of the hypothalamus (PVN) and CA1 served as a proxy of altered HPA axis activation. Our findings demonstrated interruption of the estrous cycle in 87.5 % of ischemic rats, marked by persistent diestrus, lasting on average 11.86 days. Moreover, compared to sham-operated controls, ischemic female rats showed reduced Kiss1 expression in the hypothalamic ARC and POA, concomitant with elevated ERα in the ARC and increased GnIH in the DMH and CA1. Reduced GR expression in the CA1 was associated with increased GR-immunoreactivity in the PVN, indicative of lasting dysregulation of HPA axis activation. Together, these findings demonstrate GCI disruption of female rats' estrous cycle over multiple days, with a lasting impact on HPG axis regulators within the reproductive axis.

The Role of RFRP Neurons in the Allostatic Control of Reproductive Function.

Reproductive function is critical for species survival; however, it is energetically costly and physically demanding. Reproductive suppression is therefore a physiologically appropriate adaptation to certain ecological, environmental, and/or temporal conditions. This 'allostatic' suppression of fertility enables individuals to accommodate unfavorable reproductive circumstances and safeguard survival. The mechanisms underpinning this reproductive suppression are complex, yet culminate with the reduced secretion of gonadotropin-releasing hormone (GnRH) from the hypothalamus, which in turn suppresses gonadotropin release from the pituitary, thereby impairing gonadal function. The focus of this review will be on the role of RFamide-related peptide (RFRP) neurons in different examples of allostatic reproductive suppression. RFRP neurons release the RFRP-3 peptide, which negatively regulates GnRH neurons and thus appears to act as a 'brake' on the neuroendocrine reproductive axis. In a multitude of predictable (e.g., pre-puberty, reproductive senescence, and seasonal or lactational reproductive quiescence) and unpredictable (e.g., metabolic, immune and/or psychosocial stress) situations in which GnRH secretion is suppressed, the RFRP neurons have been suggested to act as modulators. This review examines evidence for and against these roles.

RFRP-3 Influences Apoptosis and Steroidogenesis of Yak Cumulus Cells and Compromises Oocyte Meiotic Maturation and Subsequent Developmental Competence.

RF amide-related peptide 3 (RFRP-3), a mammalian ortholog of gonadotropin-inhibitory hormone (GnIH), is identified to be a novel inhibitory endogenous neurohormonal peptide that regulates mammalian reproduction by binding with specific G protein-coupled receptors (GPRs) in various species. Herein, our objectives were to explore the biological functions of exogenous RFRP-3 on the apoptosis and steroidogenesis of yak cumulus cells (CCs) and the developmental potential of yak oocytes. The spatiotemporal expression pattern and localization of GnIH/RFRP-3 and its receptor GPR147 were determined in follicles and CCs. The effects of RFRP-3 on the proliferation and apoptosis of yak CCs were initially estimated by EdU assay and TUNEL staining. We confirmed that high-dose (10-6 mol/L) RFRP-3 suppressed viability and increased the apoptotic rates, implying that RFRP-3 could repress proliferation and induce apoptosis. Subsequently, the concentrations of E2 and P4 were significantly lower with 10-6 mol/L RFRP-3 treatment than that of the control counterparts, which indicated that the steroidogenesis of CCs was impaired after RFRP-3 treatment. Compared with the control group, 10-6 mol/L RFRP-3 treatment decreased the maturation of yak oocytes efficiently and subsequent developmental potential. We sought to explore the potential mechanism of RFRP-3-induced apoptosis and steroidogenesis, so we observed the levels of apoptotic regulatory factors and hormone synthesis-related factors in yak CCs after RFRP-3 treatment. Our results indicated that RFRP-3 dose-dependently elevated the expression of apoptosis markers (Caspase and Bax), whereas the expression levels of steroidogenesis-related factors (LHR, StAR, 3ß-HSD) were downregulated in a dose-dependent manner. However, all these effects were moderated by cotreatment with inhibitory RF9 of GPR147. These results demonstrated that RFRP-3 adjusted the expression of apoptotic and steroidogenic regulatory factors to induce apoptosis of CCs, probably through binding with its receptor GPR147, as well as compromised oocyte maturation and developmental potential. This research revealed the expression profiles of GnIH/RFRP-3 and GPR147 in yak CCs and supported a conserved inhibitory action on oocyte developmental competence.

Recombinant HBsAg-S and RFRP-3 DNA vaccine promotes reproduction hormone secretion in sheep.

RF-amide related peptides (RFRP) have been proposed as critical regulators of gonadotropin secretion in mammals. This study was designed to construct a DNA vaccine and investigate the effect of vaccine encoding RFRP-3 on reproduction physiology in ewe. A recombinant vaccine was constructed using two copies of the RFRP-3 gene and HBsAg-S that generate a fusion protein to induce an immunology response. Results showed this recombinant vaccine could produce a significant antibody titer in the treated animals (P < 0.05). The specific RFRP-3 antibody response induced by the vaccine was detected at week 2 with a peak at week 6 after the initial immunization. Furthermore, we found that ewes inoculated with pVAX-tPA-HBsAg-S-2RFRP-asd vaccine significantly raised the concentration of GnRH, LH and E2 in serum compared to the control group. LH and E2 concentration in the treated ewes (Group T) was significantly higher than that in control ewes (Group C) at weeks 10, 12 and 14 after the initial immunization, respectively (P < 0.05). Therefore, RFRP-3 can be used as a target for DNA immunization to promote reproductive hormone secretion in ewes and RFRP-3 gene immunization might be a candidate tool to regulate mammal reproduction.

A mammalian gonadotropin-inhibitory hormone homolog RFamide-related peptide 3 regulates pain and anxiety in mice.

RFamide-related peptide (RFRP) is a homologous neuropeptide to gonadotropin-inhibitory hormone (GnIH), which is a hypothalamic neuropeptide that negatively regulates the hypothalamic-pituitary-gonadal axis. RFRP/GnIH is thought to be the mediator of stress because various stressors increase RFRP/GnIH mRNA expression and/or RFRP/GnIH neuronal activities. RFRP/GnIH may also directly regulate behavior, because RFRP/GnIH neuronal fibers and RFRP/GnIH receptor are widely expressed in the brain. Here, we create a RFRP/GnIH knockout (GnIH-KO) mice and conduct various behavioral tests. Dense RFRP/GnIH neuronal fibers are located in the limbic system and broad areas in the thalamus, hypothalamus, and midbrain in wild-type mice but not in RFRP/GnIH-KO mice. Spatial working memory is not improved in GnIH-KO mice as shown by Y-maze test. GnIH-KO mice perform intensive wheel running exercise for several hours after light-off. Hot plate test shows that GnIH-KO mice have decreased sensitivity to pain and central administration of RFRP3 to GnIH-KO mice recovers pain sensitivity. Elevated plus maze test shows that GnIH-KO mice have decreased level of anxiety and central administration of RFRP3 to GnIH-KO mice recovers anxiety level. These results indicate that RFRP3 regulates pain and anxiety in mice. RFRP3 may be involved in the negative regulation of spontaneous activity in addition to negatively regulating the reproductive neuroendocrine axis in stressful conditions.

Induced-ovulation in female dromedary camel involves kisspeptin neuron activation by ß nerve growth factor†.

The dromedary camel (Camelus dromedarius) is a short-day desert breeder in which female ovulation is induced by mating. Current data indicate that male-induced ovulation is triggered by its seminal plasma nerve growth factor beta (ß-NGF), but the exact mechanisms involved in the induction of ovulation are still unknown. In this study, we report that an intramuscular injection of ß-NGF in sexually active short-day-adapted female camels induces an ovulation attested by a surge of circulating LH (2-6 h after treatment) followed by an oocyte release with its cumulus oophorus (confirmed by ultrasonography 72 h after treatment) and a large and progressive increase in circulating progesterone (significant from the 2nd to the 10th days after ß-NGF injection). In addition, this ß-NGF treatment induces a broad nuclear c-FOS activation in cells located in various hypothalamic areas, notably the preoptic area, the arcuate nucleus, the dorso- and ventromedial hypothalamus, the paraventricular nucleus, and the supraoptic nucleus. A double immunostaining with neuropeptides known to be involved in the central control of reproduction indicates that ~28% kisspeptin neurons and 43% GnRH neurons in the proptic area, and ~10% RFRP-3 neurons in the dorso- and ventromedial hypothalamus are activated following ß-NGF injection. In conclusion, our study demonstrates that systemic ß-NGF induces ovulation in the female dromedary camel and indicates that this effect involves the central activation of hypothalamic neurons, notably the kisspeptin neurons.

Expression and co-localization of RFRP-3 and kisspeptin during breeding and non-breeding season in the hypothalamus of male rhesus monkey (Macaca mulatta).

Propose: The mechanism that underpins how RFRP-3 and kisspeptin interacts are not fully understood in higher primates. This study therefore set out to assess RFRP-3 and kisspeptin expression and their morphological interactions in the breeding, and in the non-breeding period in monkey hypothalamus. Methods: Eight mature male macaques (Macaca mulatta) in the breeding season (February; n = 4) and non-breeding season (June; n = 4) were used. To reveal the expression and co-localization of RFRP-3 and kisspeptin, double-labeled immunohistochemistry was performed. Testicular volume, sperm count, and plasma testosterone level were also measured to validate the breeding and non-breeding paradigms. Results: Testicular volume, plasma testosterone level, and sperm count showed a significant reduction during non-breeding season. The number of kisspeptin-positive cells was significantly increased during the breeding season (p < 0.05), whereas more RFRP-3-positive cell bodies were seen in the non-breeding season (p < 0.01). Close contacts of RFRP-3 fibers with kisspeptin cells showed no significant difference (p > 0.05) across seasons. However, co-localization of RFRP-3-ir cell bodies onto kisspeptin IR cell bodies showed a statistical increase (p < 0.01) in non-breeding season. Conclusion: In higher primates, RFRP-3 decreases kisspeptin drives from the same cells to GnRH neurons in an autocrine manner causing suppression of the reproductive axis during the non-breeding period.

Impact of Chronic Prenatal Stress on Maternal Neuroendocrine Function and Embryo and Placenta Development During Early-to-Mid-Pregnancy in Mice.

Psychological stress, both leading up to and during pregnancy, is associated with increased risk for negative pregnancy outcomes. Although the neuroendocrine circuits that link the stress response to reduced sexual motivation and mating are well-described, the specific pathways by which stress negatively impacts gestational outcomes remain unclear. Using a mouse model of chronic psychological stress during pregnancy, we investigated 1) how chronic exposure to stress during gestation impacts maternal reproductive neuroendocrine circuitry, and 2) whether stress alters developmental outcomes for the fetus or placenta by mid-pregnancy. Focusing on the stress-responsive neuropeptide RFRP-3, we identified novel contacts between RFRP-3-immunoreactive (RFRP-3-ir) cells and tuberoinfundibular dopaminergic neurons in the arcuate nucleus, thus providing a potential pathway linking the neuroendocrine stress response directly to pituitary prolactin production and release. However, neither of these cell populations nor circulating levels of pituitary hormones were affected by chronic stress. Conversely, circulating levels of steroid hormones relevant to gestational outcomes (progesterone and corticosterone) were altered in chronically-stressed dams across gestation, and those dams were qualitatively more likely to experience delays in fetal development. Together, these findings suggest that, up until at least mid-pregnancy, mothers appear to be relatively resilient to the effects of elevated glucocorticoids on reproductive neuroendocrine system function. We conclude that understanding how chronic psychological stress impacts reproductive outcomes will require understanding individual susceptibility and identifying reliable neuroendocrine changes resulting from gestational stress.

Exposure to Cadmium Alters the Population of Glial Cell Types and Disrupts the Regulatory Mechanisms of the HPG Axis in Prepubertal Female Rats.

Despite the fact that the brain is susceptible to neurotoxicity induced by cadmium (Cd), the effects of Cd on the neuroanatomical development in the hypothalamus and regulatory mechanisms of the hypothalamic-pituitary-gonadal (HPG) axis are not fully understood. To clarify this issue, we investigated the effects of 25 mg/kg BW/day cadmium chloride (CdCl2) on neuroanatomical alterations in the hypothalamus of prepubertal female rats. Twenty-four Sprague-Dawley rats were randomly assigned to two groups (n = 12), and CdCl2 was administered via gavage from postnatal days (PND) 21 to PND35. The results of the stereological analysis demonstrated that prepubertal exposure to Cd reduced the number of neurons and oligodendrocytes in the arcuate (ARC) and dorsomedial hypothalamus nucleus (DMH) nuclei. In contrast, Cd exposure increased the number of microglial cells in the ARC and DMH nuclei. Cd exposure decreased the mRNA levels of gonadotropin-releasing hormone (GnRH) and increased the mRNA levels of RFamide-related peptide (RFRP-3), but not kisspeptin (Kiss1) in the hypothalamus. Moreover, hormonal assay showed that Cd exposure caused a reduction in the concentration of gonadotropins: luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in serum. Immunohistochemical expression of RFRP-3 in neuronal cell bodies demonstrated that the mean number of RFRP-3 expressing neurons in the DMH nucleus of cadmium-treated rats was dramatically higher than the vehicle group. Overall, exposure to Cd during the prepubertal period alters the population of neurons and glial cell types in the hypothalamus. Additionally, Cd exposure disrupts the regulatory mechanisms of the HPG axis.

Developmental sex differences in the peri-pubertal pattern of hypothalamic reproductive gene expression, including Kiss1 and Tac2, may contribute to sex differences in puberty onset.

The mechanisms regulating puberty still remain elusive, as do the underlying causes for sex differences in puberty onset (girls before boys) and pubertal disorders. Neuroendocrine puberty onset is signified by increased pulsatile GnRH secretion, yet how and when various upstream reproductive neural circuits change developmentally to govern this process is poorly understood. We previously reported day-by-day peri-pubertal increases (Kiss1, Tac2) or decreases (Rfrp) in hypothalamic gene expression of female mice, with several brain mRNA changes preceding external pubertal markers. However, similar pubertal measures in males were not previously reported. Here, to identify possible neural sex differences underlying sex differences in puberty onset, we analyzed peri-pubertal males and directly compared them with female littermates. Kiss1 expression in male mice increased over the peri-pubertal period in both the AVPV and ARC nuclei but with lower levels than in females at several ages. Likewise, Tac2 expression in the male ARC increased between juvenile and older peri-pubertal stages but with levels lower than females at most ages. By contrast, both DMN Rfrp expressionand Rfrp neuronal activation strongly decreased in males between juvenile and peri-pubertal stages, but with similar levels as females. Neither ARC KNDy neuronal activation nor Kiss1r expression in GnRH neurons differed between males and females or changed with age. These findings delineate several peri-pubertal changes in neural populations in developing males, with notable sex differences in kisspeptin and NKB neuron developmental patterns. Whether these peri-pubertal hypothalamic sex differences underlie sex differences in puberty onset deserves future investigation.

Environmental disruption of reproductive rhythms.

Reproduction is a key biological function requiring a precise synchronization with annual and daily cues to cope with environmental fluctuations. Therefore, humans and animals have developed well-conserved photoneuroendocrine pathways to integrate and process daily and seasonal light signals within the hypothalamic-pituitary-gonadal axis. However, in the past century, industrialization and the modern 24/7 human lifestyle have imposed detrimental changes in natural habitats and rhythms of life. Indeed, exposure to an excessive amount of artificial light at inappropriate timing because of shift work and nocturnal urban lighting, as well as the ubiquitous environmental contamination by endocrine-disrupting chemicals, threaten the integrity of the daily and seasonal timing of biological functions. Here, we review recent epidemiological, field and experimental studies to discuss how light and chemical pollution of the environment can disrupt reproductive rhythms by interfering with the photoneuroendocrine timing system.

Regulation of the gonadotropin-releasing hormone neuron during stress.

The effect of stress on reproduction and gonadal function has captivated investigators for almost 100 years. Following the identification of gonadotropin-releasing hormone (GnRH) 50 years ago, a niche research field emerged fixated on how stress impairs this central node controlling downstream pituitary and gonadal function. It is now clear that both episodic GnRH secretion in males and females and surge GnRH secretion in females are inhibited during a variety of stress types. There has been considerable advancement in our understanding of numerous stress-related signaling molecules and their ability to impair reproductive neuroendocrine activity during stress. Recently, much attention has turned to the effects of stress on two populations of kisspeptin neurons: the stimulatory afferents to GnRH neurons that regulate pulsatile and surge-type gonadotropin secretion. Indeed, future work is still required to fully construct the neuroanatomical framework underlying stress effects, directly or indirectly, on GnRH neuron function. The present review evaluates and synthesizes evidence related to stress-related signaling molecules acting directly on GnRH neurons. Here, we review the evidence for and against the action of a handful of signaling molecules as inhibitors of GnRH neuron function, including corticotropin-releasing hormone, urocortins, norepinephrine, cortisol/corticosterone, calcitonin gene-related peptide and arginine-phenylalanine-amide-related peptide-3.

Central and peripheral neuropeptide RFRP-3: A bridge linking reproduction, nutrition, and stress response.

This article is an amalgamation of the current status of RFRP-3 (GnIH) in reproduction and its association with the nutrition and stress-mediated changes in the reproductive activities. GnIH has been demonstrated in the hypothalamus of all the vertebrates studied so far and is a well-known inhibitor of GnRH mediated reproduction. The RFRP-3 neurons interact with the other hypothalamic neurons and the hormonal signals from peripheral organs for coordinating the nutritional, stress, and environmental associated changes to regulate reproduction. RFRP-3 has also been shown to regulate puberty, reproductive cyclicity and senescence depending upon the nutritional status. A favourable nutritional status and the environmental cues which are permissive for the successful breeding and pregnancy outcome keep RFRP-3 level low, whereas unfavourable nutritional status and stressful conditions increase the expression of RFRP-3 which impairs the reproduction. Still our knowledge about RFRP-3 is incomplete regarding its therapeutic application for nutritional or stress-related reproductive disorders.

Effect of RFRP-3, the mammalian ortholog of GnIH, on apoptosis and autophagy in porcine ovarian granulosa cells via the p38MAPK pathway.

RFamide-related peptide-3 (RFRP-3) has been proposed as a key inhibitory regulator of mammalian reproduction. Our previous studies demonstrated that RFRP-3 mediated apoptosis and autophagy of the epididymis in rats and inhibited porcine granulosa cell (GC) proliferation. However, the molecular mechanisms of the RFRP-3 effect on porcine GC apoptosis and autophagy have not been studied before. Herein, we first investigated the role of RFRP-3 in apoptosis and autophagy in cultured porcine GCs in vitro. Our results showed that different doses of RFRP-3 dose-dependently elevated the expression of autophagy markers at both the mRNA and protein levels, whereas the expression of apoptosis markers exhibited a bidirectional, dose-dependent effect. Because the p38MAPK signaling pathway plays essential roles in apoptosis and autophagy, we subsequently evaluated the effect of RFRP-3 on p38MAPK activation. The results showed that 10-6 M RFRP-3 treatment not only significantly decreased p38MAPK phosphorylation but also inhibited the p38MAPK activator U-46619 to promote p38MAPK activation in porcine GCs. Finally, we applied U-46619 to investigate the role of the p38MAPK signaling pathway in apoptosis and autophagy in RFRP-3-treated porcine GCs. The results showed that all doses of RFRP-3 significantly inhibited the U-46619-induced increase in apoptosis in a dose-dependent manner. However, except for the U-46619-induced Beclin-1 expression increase, which was significantly suppressed in high-dose RFRP-3-treated porcine GCs, other doses of RFRP-3 treatment strengthened the U-46619-induced increase in other autophagy markers. In summary, our data demonstrate a critical role for the p38MAPK signaling pathway in the porcine GC cellular response to RFRP-3 by controlling the balance between apoptosis and autophagy.

Age-dependent change of RFRP-3 neuron numbers and innervation in female mice.

In female mammals, reproductive senescence is a complex process involving progressive ovarian dysfunction, associated with altered central control of the hypothalamic-pituitary-gonadal axis and desynchronization of the circadian system. The objective of this study was to investigate age-dependent changes in the daily regulation of Arg-Phe amide-related peptide-3 (RFRP-3), a hypothalamic peptide involved in reproduction, in female C57BL/6 J mice of different age groups (4, 13, and 19 months old) sampled at their diestrus stage. We found an age-dependent decrease in the total number of RFRP-3 neurons and in the relative number of activated (i.e. c-Fos-positive) RFRP-3 neurons. RFRP-3 neuronal activation exhibited a daily variation in young and middle-aged mice, which was abolished in 19-month-old mice. We also found a daily variation in the number of RFRP-3 neurons receiving close vasopressin (AVP)- and vasoactive intestinal peptide (VIP)-ergic fiber appositions in mice aged 4 and 13 months, but not in 19-month-old mice. However, we found no daily or age-dependent changes in the AVP and VIP fiber density in the dorsomedial hypothalamus. Plasma LH levels were similar in mice aged 4 and 13 months, but were markedly increased in 19-month-old mice. The present findings indicate that the number of RFRP-3 positive neurons is downregulated during old age and that the daily changes in their innervation by the circadian peptides AVP and VIP are abolished. This age-associated reduced (rhythmic) activity of the inhibitory RFRP-3 system could be implicated in the elevated LH secretion observed during reproductive senescence.

Distribution of the Novel RFRP-3/receptors system in the epididymis of the seasonal desert rodent, Gerbillus tarabuli, during sexual activity.

RFamide-related peptide (RFRP-3), the Mammalian ortholog of the Avian gonadotropin-inhibitory hormone (GnIH), is a novel neuropeptide known for its inhibitory regulatory effect on reproduction in various mammalian species. However, a stimulatory action has been reported. This paper aims to: i) study the histology of the epididymis (caput) of Gerbillus tarabuli during the breeding period; and ii) to determine the distribution of the "RFRP-3/receptors system" in the epididymis (caput) of this desert rodent during the active season, and thus, to inspect its potential local interfering in sperm maturation. For that, immunohistochemistry was performed to detect the epididymal immunolocalizations of the three molecules, RFRP-3, GPR147, and GPR74. This is the first report of the epididymis histology in Gerbillus tarabuli, as it is the first evidence of the existence of the RFRP-3/Receptor system in the same organ of the same species. During the breeding season, moderate immunostaining of the RFRP-3/receptors system was present in the caput epididymis' epithelial parts (basal and principal cells) and spermatozoa. In contrast, these three molecules were absent in the peritubular and muscle coat's myoid cells and of the interstitial part of the caput epididymis. The results suggest that the epididymis is a potential source of RFRP-3 in the desert Rodent, Gerbillus tarabuli, which may function as a paracrine and/or autocrine factor affecting the main epididymis' function: sperm maturation.

Photoperiod is involved in the regulation of seasonal breeding in male water voles (Arvicola terrestris).

Mammals living at temperate latitudes typically display annual cyclicity in their reproductive activity: births are synchronized when environmental conditions are most favorable. In a majority of these species, day length is the main proximate factor used to anticipate seasonal changes and to adapt physiology. The brain integrates this photoperiodic signal through key hypothalamic structures, which regulate the reproductive axis. In this context, our study aimed to characterize regulations that occur along the hypothalamo-pituitary-gonadal (HPG) axis in male fossorial water voles (Arvicola terrestris, also known as Arvicola amphibius) throughout the year and to further probe the implication of photoperiod in these seasonal regulations. Our monthly field monitoring showed dramatic seasonal changes in the morphology and activity of reproductive organs, as well as in the androgen-dependent lateral scent glands. Moreover, our data uncovered seasonal variations at the hypothalamic level. During the breeding season, kisspeptin expression in the arcuate nucleus (ARC) decreases, while RFRP3 expression in the dorsomedial hypothalamic nucleus (DMH) increases. Our follow-up laboratory study revealed activation of the reproductive axis and confirmed a decrease in kisspeptin expression in males exposed to a long photoperiod (summer condition) compared with those maintained under a short photoperiod (winter condition) that retain all features reminiscent of sexual inhibition. Altogether, our study characterizes neuroendocrine and anatomical markers of seasonal reproductive rhythmicity in male water voles and further suggests that these seasonal changes are strongly impacted by photoperiod.