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.

The iron-sulfur cluster assembly (ISC) protein Iba57 executes a tetrahydrofolate-independent function in mitochondrial [4Fe-4S] protein maturation.

Mitochondria harbor the bacteria-inherited iron-sulfur cluster assembly (ISC) machinery to generate [2Fe-2S; iron-sulfur (Fe-S)] and [4Fe-4S] proteins. In yeast, assembly of [4Fe-4S] proteins specifically involves the ISC proteins Isa1, Isa2, Iba57, Bol3, and Nfu1. Functional defects in their human equivalents cause the multiple mitochondrial dysfunction syndromes, severe disorders with a broad clinical spectrum. The bacterial Iba57 ancestor YgfZ was described to require tetrahydrofolate (THF) for its function in the maturation of selected [4Fe-4S] proteins. Both YgfZ and Iba57 are structurally related to an enzyme family catalyzing THF-dependent one-carbon transfer reactions including GcvT of the glycine cleavage system. On this basis, a universally conserved folate requirement in ISC-dependent [4Fe-4S] protein biogenesis was proposed. To test this idea for mitochondrial Iba57, we performed genetic and biochemical studies in Saccharomyces cerevisiae, and we solved the crystal structure of Iba57 from the thermophilic fungus Chaetomium thermophilum. We provide three lines of evidence for the THF independence of the Iba57-catalyzed [4Fe-4S] protein assembly pathway. First, yeast mutants lacking folate show no defect in mitochondrial [4Fe-4S] protein maturation. Second, the 3D structure of Iba57 lacks many of the side-chain contacts to THF as defined in GcvT, and the THF-binding pocket is constricted. Third, mutations in conserved Iba57 residues that are essential for THF-dependent catalysis in GcvT do not impair Iba57 function in vivo, in contrast to an exchange of the invariant, surface-exposed cysteine residue. We conclude that mitochondrial Iba57, despite structural similarities to both YgfZ and THF-binding proteins, does not utilize folate for its function.

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.

Aerosol generation during paediatric procedural sedation with continuous-flow nitrous oxide suggests a low risk of airborne viral transmission to health-care workers.

AIM: Inhaled nitrous oxide is a common form of procedural sedation in paediatric care. During the COVID-19 pandemic, concerns about potential aerosol generation and associated viral transmission to health-care workers have led to controversy regarding its use. We aimed to measure the degree of aerosol generation during continuous flow nitrous oxide sedation to inform future guidelines. METHODS: Aerosol numbers in the respirable range were measured using a particle counter during 30 procedures undertaken in children under nitrous oxide sedation in the Emergency Department. RESULTS: Changes from baseline measurements were greatest in particles in the 0.3 µm range. The mean increase from baseline in 0.3 µm particles per cubic metre was 18 022 (95% confidence interval (CI) 5949-30 096) after the child entered the room, and 2931 (95% CI -4407 to 10 269) during nitrous oxide administration. CONCLUSION: Variation of respirable particle numbers from baseline levels was no greater during nitrous oxide administration than for breathing and talking asymptomatic children. These results suggest the additional risk of airborne viral transmission to staff during inhaled nitrous oxide sedation is low.

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.

Starvation Ketosis in the Pediatric Emergency Department: A Prospective Estimate of Prevalence.

OBJECTIVES: Starvation ketosis may occur in children during intercurrent illnesses due to metabolic adaptation to fasting, resulting in significant ketonemia and sometimes ketoacidosis. Also known as accelerated starvation, common symptoms are vomiting, lethargy, and seizures. Previous studies found the prevalence of ketotic hypoglycemia to be 4 per 100,000 presentations to the emergency department (ED). We hypothesized that the prevalence had been underestimated due to the retrospective nature and restricted definitions of previous studies. Our aim was to determine if a prospective study would confirm a higher prevalence of starvation ketosis in pediatric patients presenting to the ED. METHODS: A prospective observational study of consecutive patients was performed in an urban pediatric ED utilizing point-of-care testing of capillary blood glucose and ketones in symptomatic children. RESULTS: This study found the prevalence of clinically significant ketosis (defined as beta-hydroxybutyrate 2.5 mmol/L or more) to be approximately 1800 per 100,000 ED presentations, with more than 170 per 100,000 also being hypoglycemic. Affected patients were 3 months to 9 years of age. Fifty-five percent of the presentations were boys, and 56% were under the 50th centile for weight. CONCLUSIONS: This prospective observational study using a targeted testing regimen found the prevalence of both starvation ketosis and hypoglycemia to be much greater than previous studies which used retrospective analyses. A simple capillary test for ketones should be considered in unwell children younger than 10 years who present with vomiting or lethargy, as this may identify the need for specific therapy to resolve ketosis.

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.

Rescue of Function of Mutant Luteinising Hormone Receptors with Deficiencies in Cell Surface Expression, Hormone Binding, and Hormone Signalling.

INTRODUCTION: G protein-coupled receptor (GPCR) mutations are implicated in many diseases. Most inactivating mutations cause receptor misfolding and prevent trafficking to the plasma membrane. Pharmacological chaperones can "rescue" cell surface expression of such mutants, presumably by stabilising correct folding of the nascent protein. OBJECTIVE: Here we examine the scope of intracellularly retained luteinising hormone receptor (LHR) mutants that can be "rescued" by the pharmacological chaperone LHR-Chap, and whether this allosteric agonist can also restore the function of mutant LHRs with deficiencies in hormone binding or hormone-induced signalling. METHODS: Mutant LHRs were expressed in HEK 293-T cells. Cell surface expression/localisation, hormone binding, and hCG/LHR-Chap signalling were determined by ELISA, radioligand binding, and inositol phosphate accumulation assays, respectively. Molecular modelling predicted LHR-Chap interactions. RESULTS: LHR-Chap increased cell surface expression of a subset of retained mutants located in transmembrane helices predicted to be stabilised by LHR-Chap binding. For 3 (T4613.47I, L5024.61P, and S6167.46Y) hCG-responsiveness was increased following treatment. LHRs with mutations in the hormone-binding site (C131ECDR and I152ECDT) or in the hinge region (E354HingeK) had good cell surface expression but poor response to hormone stimulation, yet were responsive to allosteric activation by LHR-Chap. CONCLUSIONS: LHR-Chap, in addition to rescuing cell surface expression of intracellularly retained LHR mutants, can rescue function in mutant receptors with binding and signalling deficiencies that have normal cell surface expression. This demonstration of rescue of multiple elements of LHR dysfunction arising from inactivating mutations offers exceptional potential for treating patients with diseases arising from GPCR mutations in general.

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.

Too far to fall: Exploring the relationship between playground equipment and paediatric upper limb fractures.

AIM: Playground equipment, most notably monkey bars, appears to be involved in a high proportion of upper limb fractures (ULFs) in the Australian paediatric population. Despite this, in 2014, Australian playground standards increased the maximum acceptable 'free height of fall' playground equipment from 2.5 to 3.0 m, and maintained monkey bars at 2.2 m. An updated snapshot regarding playground ULFs since these changes is important. This study aimed to determine the number and severity of playground ULFs in a paediatric population, compared to other common activities involved in ULFs. METHODS: Records of all patients aged 0-17 presenting to the Austin Hospital Paediatric Emergency Department with ULFs over a 12-month period were analysed retrospectively. ULFs included fractures to the carpal bones, radius, ulna or humerus. Variables documented included the activity involved with the fracture, and fracture management. Results were analysed using descriptive statistics. RESULTS: A total of 725 ULF cases were collected. Playground equipment was involved in 23% (n = 162/697, missing n = 28) of fractures. Monkey bars were involved in 14% of fractures (n = 100/697, missing n = 28), 1.64 times more than the next most common activity. Monkey bars were involved in 62% (n = 100/162) of all playground fractures. In children aged 5-9, monkey bars were involved in 27% (n = 83/304) of ULFs. Monkey bars were furthermore involved in most cases of 'severe' fractures (requiring reduction/operation) (n = 33), with one-third of monkey bar fractures being severe. CONCLUSION: Given these findings, the authors recommend a renewed focus on measures that reduce the frequency and severity of falls from monkey bars.

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.