Transcriptome profiling of kisspeptin neurons from the mouse arcuate nucleus reveals new mechanisms in estrogenic control of fertility.

Kisspeptin neurons in the mediobasal hypothalamus (MBH) are critical targets of ovarian estrogen feedback regulating mammalian fertility. To reveal molecular mechanisms underlying this signaling, we thoroughly characterized the estrogen-regulated transcriptome of kisspeptin cells from ovariectomized transgenic mice substituted with 17ß-estradiol or vehicle. MBH kisspeptin neurons were harvested using laser-capture microdissection, pooled, and subjected to RNA sequencing. Estrogen treatment significantly (p.adj. < 0.05) up-regulated 1,190 and down-regulated 1,139 transcripts, including transcription factors, neuropeptides, ribosomal and mitochondrial proteins, ion channels, transporters, receptors, and regulatory RNAs. Reduced expression of the excitatory serotonin receptor-4 transcript (Htr4) diminished kisspeptin neuron responsiveness to serotonergic stimulation. Many estrogen-regulated transcripts have been implicated in puberty/fertility disorders. Patients (n = 337) with congenital hypogonadotropic hypogonadism (CHH) showed enrichment of rare variants in putative CHH-candidate genes (e.g., LRP1B, CACNA1G, FNDC3A). Comprehensive characterization of the estrogen-dependent kisspeptin neuron transcriptome sheds light on the molecular mechanisms of ovary-brain communication and informs genetic research on human fertility disorders.

Nitric oxide mediated kisspeptin regulation of steroidogenesis and gametogenesis in the catfish, Clarias batrachus.

Nitric oxide (NO) is a gaseous molecule that regulates various reproductive functions. It is a well-recognized regulator of GnRH-FSH/LH-sex steroid secretion in vertebrates including fish. Kisspeptin is a recently discovered neuropeptide which also regulates GnRH secretion. Nitrergic and kisspeptin neurons are reported in close physical contact in the mammalian brain suggesting their interactive role in the release of GnRH. The existence of kisspeptin and NOS is also demonstrated in vertebrate gonads, but information on their reciprocal relation in gonads, if any, is obscure. Therefore, attempts were made to evaluate the functional reciprocal relation between nitric oxide and kisspeptin in the catfish gonads, if any, by administering the nitric oxide synthase (NOS) inhibitor, L-NAME {N(G)-nitro-L-arginine methyl ester}, which reduces NO production, and kisspeptin agonist (KP-10) and assessing their impacts on the expressions of kisspeptin1, different NOS isoforms, NO and steroid production in the gonadal tissue. The results revealed that L-NAME suppressed the expression of kiss1 in gonads of the catfish establishing the role of NO in kisspeptin expression. However, KP-10 increased the expression of all the isoforms of NOSs (iNOS, eNOS, nNOS) and concurrently NO and steroids in the ovary and testis. In vitro studies also indicate that kisspeptin stimulates the production of NO and estradiol and testosterone levels in the gonadal explants and medium. Thus, in vivo results clearly suggest a reciprocal interaction between kisspeptin and NO to regulate the gonadal activity of the catfish. The in vitro findings further substantiate our contention regarding the interactive role of kisspeptin and NO in gonadal steroidogenesis.

Role of Neurokinin B in gametogenesis and steroidogenesis of freshwater catfish, Clarias batrachus.

Neurokinin B (NKB), a recently discovered neuropeptide, plays a crucial role in regulating the kiss-GnRH neurons in vertebrate's brain. NKB is also characterized in gonadal tissues; however, its role in gonads is poorly understood. Therefore, in the present study, the effects of NKB on gonadal steroidogenesis and gametogenesis through in vivo and in vitro approaches using NKB antagonist MRK-08 were evaluated. The results suggest that the NKB antagonist decreases the development of advanced ovarian follicles and germ cells in the testis. In addition, MRK-08 further reduces the production of 17ß-estradiol in the ovary and testosterone in the testis under both in vivo and in vitro conditions in a dose-dependent manner. Furthermore, the in vitro MRK-08 treatment of gonadal explants attenuated the expression of steroidogenic marker proteins, i.e., StAR, 3ß-HSD, and 17ß-HSD dose-dependently. Moreover, the MAP kinase proteins, pERK1/2 & ERK1/2 and pAkt & Akt were also downregulated by MRK-08. Thus, the study suggests that NKB downregulates steroidogenesis by modulating the expressions of steroidogenic marker proteins involving ERK1/2 & pERK1/2 and Akt/pAkt signalling pathways. NKB also appears to regulate gametogenesis by regulating gonadal steroidogenesis in the catfish.

The HGR motif is the antiangiogenic determinant of vasoinhibin: implications for a therapeutic orally active oligopeptide.

The hormone prolactin acquires antiangiogenic and antivasopermeability properties after undergoing proteolytic cleavage to vasoinhibin, an endogenous prolactin fragment of 123 or more amino acids that inhibits the action of multiple proangiogenic factors. Preclinical and clinical evidence supports the therapeutic potential of vasoinhibin against angiogenesis-related diseases including diabetic retinopathy, peripartum cardiomyopathy, rheumatoid arthritis, and cancer. However, the use of vasoinhibin in the clinic has been limited by difficulties in its production. Here, we removed this barrier to using vasoinhibin as a therapeutic agent by showing that a short linear motif of just three residues (His46-Gly47-Arg48) (HGR) is the functional determinant of vasoinhibin. The HGR motif is conserved throughout evolution, its mutation led to vasoinhibin loss of function, and oligopeptides containing this sequence inhibited angiogenesis and vasopermeability with the same potency as whole vasoinhibin. Furthermore, the oral administration of an optimized cyclic retro-inverse vasoinhibin heptapeptide containing HGR inhibited melanoma tumor growth and vascularization in mice and exhibited equal or higher antiangiogenic potency than other antiangiogenic molecules currently used as anti-cancer drugs in the clinic. Finally, by unveiling the mechanism that obscures the HGR motif in prolactin, we anticipate the development of vasoinhibin-specific antibodies to solve the on-going challenge of measuring endogenous vasoinhibin levels for diagnostic and interventional purposes, the design of vasoinhibin antagonists for managing insufficient angiogenesis, and the identification of putative therapeutic proteins containing HGR.

Provocative tests with Kisspeptin-10 and GnRH set the scene for determining social status and environmental impacts on reproductive capacity in male African lions (Panthera leo).

Understanding the hypothalamic factors regulating reproduction facilitates maximising the reproductive success of breeding programmes and in the management and conservation of threatened species, including African lions. To provide insight into the physiology and pathophysiology of the hypothalamic-pituitary-gonadal reproductive axis in lions, we studied the luteinising hormone (LH) and steroid hormone responses to gonadotropin-releasing hormone (GnRH) and its upstream regulator, kisspeptin. Six young (13.3 ± 1.7 months, 56.2 ± 4.3 kg) and four adult (40.2 ± 1.4 months, 174 ± 6 kg) male lions (Ukutula Conservation Centre, South Africa) were used in this study. Lions were immobilised with a combination of medetomidine and ketamine and an intravenous catheter was placed in a jugular, cephalic or medial saphenous vein for blood sampling at 10-min intervals for 220 min. The ten-amino acid kisspeptin which has full intrinsic activity (KP-10, 1 µg/kg) and GnRH (1 µg/kg) were administered intravenously to study their effects on LH and steroid hormone plasma concentrations, measured subsequently by ELISA and liquid chromatography tandem mass spectrometry (LC-MS/MS), respectively. Basal LH levels were similarly low between the age groups, but testosterone and its precursor levels were higher in the adult animals. Adult lions showed a significant LH response to KP-10 (10-fold) and GnRH (11-fold) administration (p < 0.05 and P < 0.001, respectively) whereas in young lions LH increased significantly only in response to GnRH. In adults alone, testosterone and its precursors steadily increased in response to KP-10, with no significant further increase in response to GnRH. Plasma levels of glucocorticoids in response to KP-10 remained unchanged. We suggest that provocative testing of LH and steroid stimulation with kisspeptin provides a new and sensitive tool for determining reproductive status and possibly an index of exposure to stress, environmental insults such as disease, endocrine disruptors and nutritional status. 272 words.

Functional Rescue of Inactivating Mutations of the Human Neurokinin 3 Receptor Using Pharmacological Chaperones.

G protein-coupled receptors (GPCRs) facilitate the majority of signal transductions across cell membranes in humans, with numerous diseases attributed to inactivating GPCR mutations. Many of these mutations result in misfolding during nascent receptor synthesis in the endoplasmic reticulum (ER), resulting in intracellular retention and degradation. Pharmacological chaperones (PCs) are cell-permeant small molecules that can interact with misfolded receptors in the ER and stabilise/rescue their folding to promote ER exit and trafficking to the cell membrane. The neurokinin 3 receptor (NK3R) plays a pivotal role in the hypothalamic-pituitary-gonadal reproductive axis. We sought to determine whether NK3R missense mutations result in a loss of cell surface receptor expression and, if so, whether a cell-permeant small molecule NK3R antagonist could be repurposed as a PC to restore function to these mutants. Quantitation of cell surface expression levels of seven mutant NK3Rs identified in hypogonadal patients indicated that five had severely impaired cell surface expression. A small molecule NK3R antagonist, M8, increased cell surface expression in four of these five and resulted in post-translational receptor processing in a manner analogous to the wild type. Importantly, there was a significant improvement in receptor activation in response to neurokinin B (NKB) for all four receptors following their rescue with M8. This demonstrates that M8 may have potential for therapeutic development in the treatment of hypogonadal patients harbouring NK3R mutations. The repurposing of existing small molecule GPCR modulators as PCs represents a novel and therapeutically viable option for the treatment of disorders attributed to mutations in GPCRs that cause intracellular retention.

Sex Disparities in COVID-19 Severity and Outcome: Are Men Weaker or Women Stronger?

The coronavirus disease 2019 (COVID-19) outbreak, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global public health issue which has profound effects on most aspects of societal well-being, including physical and mental health. A plethora of studies globally have suggested the existence of a sex disparity in the severity and outcome of COVID-19 patients, mainly due to mechanisms of virus infection, immune response to the virus, development of systemic inflammation, and consequent systemic complications, particularly thromboembolism. Epidemiological data report a sex difference in the severity of COVID-19, with a more favorable course of the disease in women compared to men regardless of age, although the rate of SARS-CoV-2 infection seems to be similar in both sexes. Sex hormones, including androgens and estrogens, may not only impact virus entry and load, but also shape the clinical manifestations, complications, and ultimately the outcome of the disease. The current review comprehensively summarizes the current literature on sex disparities in susceptibility and outcome of COVID-19 as well as the literature underpinning the pathophysiological and molecular mechanisms, which may provide a rationale to a sex disparity. These mechanisms include sex hormone influence on factors that facilitate virus entry and priming, immune and inflammatory response, as well as coagulation and thrombosis diathesis. Based on present evidence, women appear to be relatively protected from COVID-19 because of a more effective immune response and a less pronounced systemic inflammation, with consequent moderate clinical manifestations of the disease, together with a lesser predisposition to thromboembolism. Conversely, men appear to be particularly susceptible to COVID-19 because of a less effective immune response with consequent severe clinical manifestations of the disease, together with a greater predisposition to thromboembolism. In the elderly, generally characterized by the phenomenon of inflammaging, sex disparities in overall mortality following SARS-CoV-2 infection are even more palpable as elderly men appear to be more prone to severe COVID-19 because of a greater predisposition to infections, a weaker immune defense, and an enhanced thrombotic state compared to women. The information revealed from the review highlights potential novel therapeutic approaches employing the administration of hormonal or antihormonal therapy in combination with antiviral drugs in COVID-19 patients.

Congenital Hypogonadotropic Hypogonadism with Anosmia and Gorlin Features Caused by a PTCH1 Mutation Reveals a New Candidate Gene for Kallmann Syndrome.

BACKGROUND: Two loci (CHD7 and SOX10) underlying Kallmann syndrome (KS) were discovered through clinical and genetic analysis of CHARGE and Waardenburg syndromes, conditions that include congenital anosmia caused by olfactory bulb (CA/OBs) defects and congenital hypogonadotropic hypogonadism (CHH). We hypothesized that other candidate genes for KS could be discovered by analyzing rare syndromes presenting with these signs. Study Design, Size, Duration: We first investigated a family with Gorlin-Goltz syndrome (GGS) in which affected members exhibited clinical signs suggesting KS. Participants/Materials, Methods: Proband and family members underwent detailed clinical assessment. The proband received detailed neuroendocrine evaluation. Genetic analyses included sequencing the PTCH1 gene at diagnosis, followed by exome analyses of causative or candidate KS/CHH genes, in order to exclude contribution to the phenotypes of additional mutations. Exome analyses in additional 124 patients with KS/CHH probands with no additional GGS signs. RESULTS: The proband exhibited CA, absent OBs on magnetic resonance imaging, and had CHH with unilateral cryptorchidism, consistent with KS. Pulsatile Gonadotropin-releasing hormone (GnRH) therapy normalized serum gonadotropins and increased testosterone levels, supporting GnRH deficiency. Genetic studies revealed 3 affected family members harbor a novel mutation of PTCH1 (c.838G> T; p.Glu280*). This unreported nonsense deleterious mutation results in either a putative truncated Ptch1 protein or in an absence of translated Ptch1 protein related to nonsense mediated messenger RNA decay. This heterozygous mutation cosegregates in the pedigree with GGS and CA with OBs aplasia/hypoplasia and with CHH in the proband suggesting a genetic linkage and an autosomal dominant mode of inheritance. No pathogenic rare variants in other KS/CHH genes cosegregated with these phenotypes. In additional 124 KS/CHH patients, 3 additional heterozygous, rare missense variants were found and predicted in silico to be damaging: p.Ser1203Arg, p.Arg1192Ser, and p.Ile108Met. CONCLUSION: This family suggests that the 2 main signs of KS can be included in GGS associated with PTCH1 mutations. Our data combined with mice models suggest that PTCH1 could be a novel candidate gene for KS/CHH and reinforce the role of the Hedgehog signaling pathway in pathophysiology of KS and GnRH neuron migration.

Kisspeptin and neurokinin B interactions in modulating gonadotropin secretion in women with polycystic ovary syndrome.

STUDY QUESTION: What is the role of the hypothalamic neuropeptide neurokinin B (NKB) and its interaction with kisspeptin on GnRH/LH secretion in women with polycystic ovary syndrome (PCOS)? SUMMARY ANSWER: Administration of neurokinin 3 receptor antagonist (NK3Ra) for 7 days reduced LH and FSH secretion and LH pulse frequency in women with PCOS, whilst the stimulatory LH response to kisspeptin-10 was maintained. WHAT IS KNOWN ALREADY: PCOS is characterized by abnormal GnRH/LH secretion. NKB and kisspeptin are master regulators of GnRH/LH secretion, but their role in PCOS is unclear. STUDY DESIGN, SIZE, DURATION: The NK3Ra MLE4901, 40 mg orally twice a day, was administered to women with PCOS for 7 days (n = 8) (vs no treatment, n = 7). On the last day of NK3Ra administration or the equivalent day in those not treated, women were randomized to 7-h kisspeptin-10 (4 µg/kg/h i.v.) or vehicle infusion. This was repeated with the alternate infusion in a subsequent cycle. PARTICIPANTS/MATERIALS, SETTING, METHODS: Subjects were women with PCOS, studied in a Clinical Research Facility. Reproductive hormones were measured before and after NK3Ra administration. On the last day of NK3Ra administration (or the equivalent cycle day in untreated women), all women attended for an 8-h frequent blood sampling to allow analysis of the pulsatile LH secretion. MAIN RESULTS AND THE ROLE OF CHANCE: NK3Ra reduced LH secretion (4.0 ± 0.4 vs 6.5 ± 0.8 IU/l, P < 0.05) and pulse frequency (0.5 ± 0.1 vs 0.8 ± 0.1 pulses/h, P < 0.05); FSH secretion was also reduced (2.0 ± 0.3 vs 2.5 ± 0.4 IU/l, P < 0.05). Without NK3Ra pre-treatment, kisspeptin-10 increased LH secretion (5.2 ± 0.5 to 7.8 ± 1.0 IU/L, P < 0.05), with a positive relationship to oestradiol concentrations (r2 = 0.59, P < 0.05). After NK3Ra administration, the LH response to kisspeptin-10 was preserved (vehicle 3.5 ± 0.3 vs 9.0 ± 2.2 IU/l with kisspeptin-10, P < 0.05), but the positive correlation with oestradiol concentrations was abolished (r2 = 0.07, ns. after NK3Ra). FSH secretion was increased by kisspeptin-10 after NK3Ra treatment, but not without NK3Ra treatment. LIMITATIONS, REASONS FOR CAUTION: The study did not explore the dose relationship of the effect of NK3R antagonism. The impact of obesity or other aspects of the variability of the PCOS phenotype was not studied due to the small number of subjects. WIDER IMPLICATIONS OF THE FINDINGS: These data demonstrate the interactive regulation of GnRH/LH secretion by NKB and kisspeptin in PCOS, and that the NKB system mediates aspects of oestrogenic feedback. STUDY FUNDING/COMPETING INTEREST(S): Wellcome Trust through Scottish Translational Medicine and Therapeutics Initiative (102419/Z/13/A) and MRC grants (G0701682 to R.P.M. and R.A.A.) and MR/N022556/1 to the MRC Centre for Reproductive Health. This work was performed within the Edinburgh Clinical Research Facility. J.T.G. has undertaken consultancy work for AstraZeneca and Takeda Pharmaceuticals and is an employee of Boehringer Ingelheim. R.P.M. has consulted for Ogeda and was CEO of Peptocrine. R.A.A. has undertaken consultancy work for Merck, Ferring, NeRRe Therapeutics and Sojournix Inc. J.D.V. and K.S. have nothing to disclose. TRIAL REGISTRATION NUMBER: N/A.

Fasting affects amino acid nitrogen isotope values: a new tool for identifying nitrogen balance of free-ranging mammals.

Changes in the nutritional status of free-ranging animals have a strong influence on individual fitness, yet it remains challenging to monitor longitudinally. Nitrogen (δ15N) and carbon (δ13C) isotope values measured chronologically along the length of metabolically inert keratinous tissues can be used as a nutritional biomarker to retrospectively reconstruct the foraging ecology and eco-physiology of consumers. We quantitatively describe the physiological effects of fasting on amino acid metabolism using sequentially measured bulk tissue and amino acid δ15N values along the length of whiskers sampled from free-ranging juvenile, subadults, adult female, and male southern elephant seals (SES; Mirounga leonina) on Marion Island in the Southern Ocean. For both juveniles and adult females, whisker segments representing fasting had significantly higher bulk tissue δ15N values of 0.6 ± 0.5‰ and 1.3-1.8‰, respectively, in comparison to segments unaffected by fasting. We also found a large increase (2-6‰) in δ15N values for most glucogenic amino acids and a simultaneous depletion (2-3‰) of alanine in segments reflecting fasting, which enabled us to accurately predict (74%) the nutritional status of our model species. We hypothesize that the glucose-alanine cycle is the mechanism driving the observed depletion of alanine δ15N values during fasting. We demonstrated that keratinaceous tissues can be used as a longitudinal nutritional biomarker to detect changes in the nitrogen balance of an individual. Moreover, it is evident that physiological factors have an important influence on tissue δ15N values and can lead to erroneous bulk tissue or amino acid isotope-based reconstructions of foraging habits.

Kisspeptins and reproduction: physiological roles and regulatory mechanisms.

Procreation is essential for survival of species. Not surprisingly, complex neuronal networks have evolved to mediate the diverse internal and external environmental inputs that regulate reproduction in vertebrates. Ultimately, these regulatory factors impinge, directly or indirectly, on a final common pathway, the neurons producing the gonadotropin-releasing hormone (GnRH), which stimulates pituitary gonadotropin secretion and thereby gonadal function. Compelling evidence, accumulated in the last few years, has revealed that kisspeptins, a family of neuropeptides encoded by the Kiss1 gene and produced mainly by neuronal clusters at discrete hypothalamic nuclei, are pivotal upstream regulators of GnRH neurons. As such, kisspeptins have emerged as important gatekeepers of key aspects of reproductive maturation and function, from sexual differentiation of the brain and puberty onset to adult regulation of gonadotropin secretion and the metabolic control of fertility. This review aims to provide a comprehensive account of the state-of-the-art in the field of kisspeptin physiology by covering in-depth the consensus knowledge on the major molecular features, biological effects, and mechanisms of action of kisspeptins in mammals and, to a lesser extent, in nonmammalian vertebrates. This review will also address unsolved and contentious issues to set the scene for future research challenges in the area. By doing so, we aim to endow the reader with a critical and updated view of the physiological roles and potential translational relevance of kisspeptins in the integral control of reproductive function.

Similarities and differences in the reproductive phenotypes of women with congenital hypogonadotrophic hypogonadism caused by GNRHR mutations and women with polycystic ovary syndrome.

STUDY QUESTION: Does the phenotype of women with normosmic congenital hypogonadotrophic hypogonadism (nCHH) and pituitary resistance to GnRH caused by biallelic mutations in the GnRH receptor (GNRHR) (nCHH/bi-GNRHR) differ from that of women with polycystic ovary syndrome (PCOS)? SUMMARY ANSWER: Women with nCHH/bi-GNRHR have variable pubertal development but nearly all have primary amenorrhea and an exaggerated LH response to GnRH stimulation, similar to that seen in women with PCOS. WHAT IS KNOWN ALREADY: Women with nCHH/bi-GNRHR are very rare and their phenotype at diagnosis is not always adequately documented. The results of gonadotrophin stimulation by acute GnRH challenge test and ovarian features have not been directly compared between these patients and women with PCOS. STUDY DESIGN, SIZE, DURATION: We describe the phenotypic spectrum at nCHH/bi-GNRHR diagnosis in a series of 12 women. Their reproductive characteristics and acute responses to GnRH were compared to those of 70 women with PCOS. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients and controls (healthy female volunteers aged over 18 years) were enrolled in a single French referral centre. Evaluation included clinical and hormonal studies, pelvic ultrasonography and GnRH challenge test. We also functionally characterized two missense GNRHR mutations found in two new consanguineous families. MAIN RESULTS AND THE ROLE OF CHANCE: Breast development was highly variable at nCHH/bi-GNRHR diagnosis, but only one patient had undeveloped breasts. Primary amenorrhea was present in all but two cases. In untreated nCHH/bi-GNRHR patients, uterine height (UH) correlated (P = 0.01) with the circulating estradiol level and was shorter than in 23 nulliparous post-pubertal age-matched controls (P < 0.0001) and than in 15 teenagers with PCOS under 20-years-old (P < 0.0001) in which PCOS was revealed by primary amenorrhea or primary-secondary amenorrhea. Unexpectedly, the stimulated LH peak response in nCHH/bi-GNRHR patients was variable, and often normal or exaggerated. Interestingly, the LH peak response was similar to that seen in the PCOS patients, but the latter women had significantly larger mean ovarian volume (P < 0.001) and uterine length (P < 0.001) and higher mean estradiol (P < 0.001), anti-Müllerian hormone (AMH) (P = 0.02) and inhibin-B (P < 0.001) levels. In the two new consaguineous families, the affected nCHH/bi-GNRHR women carried the T269M or Y290F GNRHR missense mutation in the homozygous state. In vitro analysis of GnRHR showed complete or partial loss-of-function of the T269M and Y290F mutants compared to their wildtype counterpart. LIMITATIONS, REASONS FOR CAUTION: The number of nCHH/bi-GNRHR patients reported here is small. As this disorder is very rare, an international study would be necessary to recruit a larger cohort and consolidate the phenotypic spectrum observed here. WIDER IMPLICATIONS OF THE FINDINGS: In teenagers and young women with primary amenorrhea, significant breast and uterine development does not rule out CHH caused by biallelic GNRHR mutations. In rare patients with PCOS presenting with primary amenorrhea and a mild phenotype, the similar exaggerated pituitary LH responses to GnRH in PCOS and nCHH/bi-GNRHR patients could lead to diagnostic errors. This challenge test should therefore not be recommended. As indicated by consensus and guidelines, careful analysis of clinical presentation and measurements of testosterone circulating levels remain the basis of PCOS diagnosis. Also, analysis of ovarian volume, UH and of inhibin-B, AMH, estradiol and androgen circulating levels could help to distinguish between mild PCOS and nCHH/bi-GNRHR. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the French National Research Agency (ANR) grant ANR-09-GENO-017 KALGENOPATH, France; and by the Italian Ministry of Education, University and Research (MIUR) grant PRIN 2012227FLF_004, Italy. The authors declare no conflict of interest.

Medial Amygdala Kiss1 Neurons Mediate Female Pheromone Stimulation of Luteinizing Hormone in Male Mice.

BACKGROUND/AIMS: The medial amygdala (MeA) responds to olfactory stimuli and alters reproductive physiology. However, the neuronal circuit that relays signals from the MeA to the reproductive axis remains poorly defined. This study aimed to test whether MeA kisspeptin (MeAKiss) neurons in male mice are sensitive to sexually relevant olfactory stimuli and transmit signals to alter reproductive physiology. We also investigated whether MeAKiss neurons have the capacity to elaborate glutamate and GABA neurotransmitters and potentially contribute to reproductive axis regulation. METHODS: Using female urine as a pheromone stimulus, MeAKiss neuronal activity was analysed and serum luteinizing hormone (LH) was measured in male mice. Next, using a chemogenetic approach, MeAKiss neurons were bi-directionally modulated to measure the effect on serum LH and evaluate the activation of the preoptic area. Lastly, using in situ hybridization, we identified the proportion of MeAKiss neurons that express markers for GABAergic (Vgat) and glutamatergic (Vglut2) neurotransmission. RESULTS: Male mice exposed to female urine showed a two-fold increase in the number of c-Fos-positive MeAKiss neurons concomitant with raised LH. Chemogenetic activation of MeAKiss neurons significantly increased LH in the absence of urine exposure, whereas inhibition of MeAKiss neurons did not alter LH. In situ hybridization revealed that MeAKiss neurons are a mixed neuronal population in which 71% express Vgat mRNA, 29% express Vglut2 mRNA, and 6% express both. CONCLUSIONS: Our results uncover, for the first time, that MeAKiss neurons process sexually relevant olfactory signals to influence reproductive hormone levels in male mice, likely through a complex interplay of neuropeptide and neurotransmitter signalling.

Social rank does not affect sperm quality in male African wild dogs (Lycaon pictus).

Sperm banking and AI could benefit endangered African wild dog conservation. However, it is unclear whether their dominance hierarchy causes a decrease in reproductive and sperm quality parameters in subordinate males that typically do not breed. In this study, we investigated the effect of social rank on male reproductive parameters, including faecal androgen and glucocorticoid metabolite concentrations, prostate and testes volume, preputial gland size, semen collection success and sperm quality. Samples were obtained from captive males (prebreeding season: n=12 from four packs; breeding season: n=24 from seven packs) that were classified as alpha (dominant), beta or gamma (subordinates) based on the frequency of dominant versus submissive behaviours. In the prebreeding season, semen was successfully collected from all alpha but only half the subordinate males, with urine contamination (associated with lower rank) significantly reducing total and progressive motility, sperm motility index, normal sperm morphology and acrosome integrity. The breeding season was associated with a significant increase in faecal androgens, prostate and testis volume, as well as progressive motility and the total number of spermatozoa ejaculated. However, with the exception of prostate volume (mean±s.e.m: 12.5±4.5, 7.1±1.0 and 7.3±1.0cm3 in alpha, beta and gamma males respectively; P=0.035), all other reproductive and sperm quality parameters did not differ between males of each social rank. In conclusion, reproductive suppression of subordinate males appears to be behaviourally mediated, because males of all social ranks produce semen of similar quality, making them suitable candidates for sperm banking, particularly during the breeding season when sperm quality improves.

GnRH Antagonists Produce Differential Modulation of the Signaling Pathways Mediated by GnRH Receptors.

Commercial gonadotropin-releasing hormone (GnRH) antagonists differ by 1-2 amino acids and are used to inhibit gonadotropin production during assisted reproduction technologies (ART). In this study, potencies of three GnRH antagonists, Cetrorelix, Ganirelix and Teverelix, in inhibiting GnRH-mediated intracellular signaling, were compared in vitro. GnRH receptor (GnRHR)-transfected HEK293 and neuroblastoma-derived SH-SY5Y cell lines, as well as mouse pituitary LßT2 cells endogenously expressing the murine GnRHR, were treated with GnRH in the presence or absence of the antagonist. We evaluated intracellular calcium (Ca2+) and cAMP increases, cAMP-responsive element binding-protein (CREB) and extracellular-regulated kinase 1 and 2 (ERK1/2) phosphorylation, ß-catenin activation and mouse luteinizing-hormone ß-encoding gene (Lhb) transcription by bioluminescence resonance energy transfer (BRET), Western blotting, immunostaining and real-time PCR as appropriate. The kinetics of GnRH-induced Ca2+ rapid increase revealed dose-response accumulation with potency (EC50) of 23 nM in transfected HEK293 cells, transfected SH-SY5Y and LßT2 cells. Cetrorelix inhibited the 3 × EC50 GnRH-activated calcium signaling at concentrations of 1 nM-1 µM, demonstrating higher potency than Ganirelix and Teverelix, whose inhibitory doses fell within the 100 nM-1 µM range in both transfected HEK293 and SH-SY5Y cells in vitro. In transfected SH-SY5Y, Cetrorelix was also significantly more potent than other antagonists in reducing GnRH-mediated cAMP accumulation. All antagonists inhibited pERK1/2 and pCREB activation at similar doses, in LßT2 and transfected HEK293 cells treated with 100 nM GnRH. Although immunostainings suggested that Teverelix could be less effective than Cetrorelix and Ganirelix in inhibiting 1 µM GnRH-induced ß-catenin activation, Lhb gene expression increase occurring upon LßT2 cell treatment by 1 µM GnRH was similarly inhibited by all antagonists. To conclude, this study has demonstrated Cetrorelix-, Ganirelix- and Teverelix-specific biased effects at the intracellular level, not affecting the efficacy of antagonists in inhibiting Lhb gene transcription.

Neurokinin 3 Receptor Antagonism Reveals Roles for Neurokinin B in the Regulation of Gonadotropin Secretion and Hot Flashes in Postmenopausal Women.

OBJECTIVES: Neurokinin B (NKB) and kisspeptin are obligate for normal gonadotropin secretion, and links between gonadotropin-releasing hormone (GnRH) pulsatility and vasomotor symptoms have been proposed. Using a selective NKB receptor (NK3R) antagonist, the role of NKB in the hypergonadotropic state in menopausal women was explored. METHODS: Eleven postmenopausal women were administered the NK3R antagonist MLE4901 at 40 mg twice daily orally for 7 days. Ten-minute blood sampling for 8 h was performed before and on the last day of NK3R antagonist treatment for luteinising hormone (LH) pulsatility analysis with kisspeptin-10 (0.3 µg/kg i.v. bolus) administered at 6 h on both days. Hot flash frequency and severity were self-reported for 7 days before and during NK3R antagonist administration. RESULTS: LH fell from 29.3 ± 4.1 to 24.4 ± 3.8 IU/L (p < 0.05) after 7 days of NK3R antagonist treatment, with no change in follicle-stimulating hormone (FSH). Basal (non-pulsatile) LH secretion was reduced (549.0 ± 70.8 vs. 366.1 ± 92.1 IU/L/6 h, p = 0.006), and while the LH pulse frequency did not change in the group as a whole (from 0.8 ± 0.1 to 0.7 ± 0.1 pulses/h, ns), it did fall in the 8 women with hot flashes (from 1.0 ± 0.1 to 0.7 ± 0.1 pulses/h, p < 0.05). These women also reported a reduction in hot flash frequency (from 3.4 ± 1.2 to 1.0 ± 0.6 hot flashes/day, p = 0.008) whilst taking the NK3R antagonist. Kisspeptin-10 did not affect LH secretion with or without the NK3R antagonist. CONCLUSIONS: The administration of an NK3R antagonist indicates a role for NKB in the regulation of LH/GnRH in postmenopausal women, whereas the lack of response to kisspeptin may reflect the hypo-oestrogenic state. These data support a link of LH/GnRH pulsatility and vasomotor symptoms with NK3R antagonism as a potential therapeutic approach.

Tight Coupling of Astrocyte pH Dynamics to Epileptiform Activity Revealed by Genetically Encoded pH Sensors.

UNLABELLED: Astrocytes can both sense and shape the evolution of neuronal network activity and are known to possess unique ion regulatory mechanisms. Here we explore the relationship between astrocytic intracellular pH dynamics and the synchronous network activity that occurs during seizure-like activity. By combining confocal and two-photon imaging of genetically encoded pH reporters with simultaneous electrophysiological recordings, we perform pH measurements in defined cell populations and relate these to ongoing network activity. This approach reveals marked differences in the intracellular pH dynamics between hippocampal astrocytes and neighboring pyramidal neurons in rodent in vitro models of epilepsy. With three different genetically encoded pH reporters, astrocytes are observed to alkalinize during epileptiform activity, whereas neurons are observed to acidify. In addition to the direction of pH change, the kinetics of epileptiform-associated intracellular pH transients are found to differ between the two cell types, with astrocytes displaying significantly more rapid changes in pH. The astrocytic alkalinization is shown to be highly correlated with astrocytic membrane potential changes during seizure-like events and mediated by an electrogenic Na(+)/HCO3 (-) cotransporter. Finally, comparisons across different cell-pair combinations reveal that astrocytic pH dynamics are more closely related to network activity than are neuronal pH dynamics. This work demonstrates that astrocytes exhibit distinct pH dynamics during periods of epileptiform activity, which has relevance to multiple processes including neurometabolic coupling and the control of network excitability. SIGNIFICANCE STATEMENT: Dynamic changes in intracellular ion concentrations are central to the initiation and progression of epileptic seizures. However, it is not known how changes in intracellular H(+) concentration (ie, pH) differ between different cell types during seizures. Using recently developed pH-sensitive proteins, we demonstrate that astrocytes undergo rapid alkalinization during periods of seizure-like activity, which is in stark contrast to the acidification that occurs in neighboring neurons. Rapid astrocytic pH changes are highly temporally correlated with seizure activity, are mediated by an electrogenic Na(+)/HCO3- cotransporter, and are more tightly coupled to network activity than are neuronal pH changes. As pH has profound effects on signaling in the nervous system, this work has implications for our understanding of seizure dynamics.

Neurokinin 3 receptor antagonism decreases gonadotropin and testosterone secretion in healthy men.

OBJECTIVE: Patients with mutations of neurokinin B (NKB) and its receptor show hypogonadotrophic hypogonadism, but there is little evidence for the importance of this pathway in reproductive function in normal men, or its functional hierarchy with kisspeptin. DESIGN: An open label study wherein men (n = 6) were administered the NK3R antagonist MLE4901 40 mg orally twice a day for 7 days. Kisspeptin-10 (0.3 µg/kg iv bolus) was given before and on day 7 of NK3R antagonist treatment. PATIENTS: Subjects were healthy men. MEASUREMENTS: Reproductive hormones were measured before and during the NK3R antagonist administration, including frequent sampling on two occasions for analysis of pulsatile LH secretion. RESULTS: LH, FSH and testosterone secretion were decreased during NK3R antagonist administration. LH showed a biphasic response, being reduced after 24 hours of treatment (4.5 ± 0.6 IU/L pretreatment to 1.7 ± 0.2 IU/L, P < .05), with partial recovery thereafter, but it was again decreased on day 7 (2.5 ± 0.6 IU/L, P < .05 vs pretreatment). FSH secretion was also suppressed, with a similar temporal pattern to that of LH. Testosterone secretion was decreased from 24 hours (18.4 ± 1.6 pretreatment vs 5.6 ± 1.5 nmol/L, P < .01) and remained suppressed throughout the treatment period. Analysis of LH pulsatility showed that both basal and pulsatile LH secretion were markedly suppressed but there was no detected change in LH pulse frequency. Kisspeptin-10 stimulated LH secretion, with similar responses before and during NK3R antagonist administration. CONCLUSIONS: These data demonstrate a central role for NKB/NK3R in the physiological regulation of reproductive function in men, and that this is functionally upstream of kisspeptin-mediated GnRH secretion.

Amygdala Kisspeptin Neurons: Putative Mediators of Olfactory Control of the Gonadotropic Axis.

Kisspeptins and their receptors are potent regulators of the gonadotropic axis. Kisspeptin neurons are found mainly in the hypothalamic arcuate nucleus and the anteroventral periventricular nucleus. However, there is also a third population of kisspeptin neurons, located in the amygdala. We used fluorescence immunohistochemistry to quantify and localize the amygdala kisspeptin neurons and to reveal close apposition and putative innervations by vasopressinergic and tyrosine hydroxylase-positive dopaminergic neurons. Using microinjections of retro- and anterograde tracers, and viral transfection systems in rats and transgenic mice, we showed reciprocal connectivity between the accessory olfactory bulb and the amygdala kisspeptin neurons. In vitro recordings indicate an inhibitory action of kisspeptin on mitral cells in the accessory olfactory bulb. Using viral specific-cell gene expression in transgenic mice in combination with double immunofluorescence histochemistry, we found that the amygdala kisspeptin neurons also project to gonadotropin-releasing hormone (GnRH) neurons in the preoptic area. Our neuroanatomical and electrophysiological data suggest that amygdala kisspeptin neurons integrate social behaviour and odour information into GnRH neurons in the preoptic area to coordinate the gonadotropic axis and the appropriate output behaviour to odour cues.