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.

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.

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.

Vasopressin acts as a synapse organizer in limbic regions by boosting PSD95 and GluA1 expression.

Hypothalamic arginine vasopressin (AVP)-containing magnocellular neurosecretory neurons (AVPMNN) emit collaterals to synaptically innervate limbic regions influencing learning, motivational behaviour, and fear responses. Here, we characterize the dynamics of expression changes of two key determinants for synaptic strength, the postsynaptic density (PSD) proteins AMPAR subunit GluA1 and PSD scaffolding protein 95 (PSD95), in response to in vivo manipulations of AVPMNN neuronal activation state, or exposure to exogenous AVP ex vivo. Both long-term water deprivation in vivo, which powerfully upregulates AVPMNN metabolic activity, and exogenous AVP application ex vivo, in brain slices, significantly increased GluA1 and PSD95 expression as measured by western blotting, in brain regions reportedly receiving direct ascending innervations from AVPMNN (i.e., ventral hippocampus, amygdala and lateral habenula). By contrast, the visual cortex, a region not observed to receive AVPMNN projections, showed no such changes. Ex vivo application of V1a and V1b antagonists to ventral hippocampal slices ablated the AVP stimulated increase in postsynaptic protein expression measured by western blotting. Using a modified expansion microscopy technique, we were able to quantitatively assess the significant augmentation of PSD95 and GLUA1 densities in subcellular compartments in locus coeruleus tyrosine hydroxylase immunopositive fibres, adjacent to AVP axon terminals. Our data strongly suggest that the AVPMNN ascending system plays a role in the regulation of the excitability of targeted neuronal circuits through upregulation of key postsynaptic density proteins corresponding to excitatory synapses.

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.

Onset of normal cycles in postpartum anovulatory dairy cattle treated with kisspeptin.

The efficacy of a long-acting synthetic derivative of kisspeptin (Kp) to initiate normal oestrous cycles was tested in 24 mixed-aged, Holstein-Friesian cows that were 18-25 days postpartum on the day of treatment (D0). Groups of eight cows received saline (Sal) vehicle by intramuscular injection at 8:00 and 16:00 h (Sal-Sal), Kp at 8:00 h and vehicle at 16:00 h (Kp-Sal) or Kp on both occasions (Kp-Kp). The Kp dose was 15 nmol per 60 kg body weight. The ovaries of the cows were examined daily by ultrasonography between D4 and D14. Blood samples were collected from a tail vessel at 0, 2, 4, 8, 10 and 12 h relative to the time of the first injection for luteinizing hormone (LH) and follicle-stimulating hormone assay. Additional samples were collected daily from D4 until D14 and D19, 22, 26 and 29 for progesterone assay. LH surge-like responses were observed in cows treated with Kp at 8:00 h. Ovulation was consistently induced by Kp within 48 h when a dominant ovarian follicle of at least 10 mm in diameter was observed (8/14) but in no cases (6/14) during a new wave of ovarian follicular development comprising follicles <10 mm in diameter. The subsequent ovulatory cycle was of normal length in most cases as compared with short 8- to 12-day cycles observed in spontaneously ovulating cows. We conclude that Kp treatment can induce ovulation in postpartum dairy cows, with ensuing oestrous cycles of normal length, if administered when a mature dominant follicle is present in the ovaries. LAY SUMMARY: Cow fertility is important for efficient, profitable dairy farming. Cows that take too long after calving to become fertile are problematic. We tested a synthetically made, long-acting hormone called kisspeptin (Kp) to advance the time that cows become fertile after calving. Twenty-four dairy cows that had been calved for 3-4 weeks were used. One group of eight cows received an injection of Kp at the morning milking, another eight cows received Kp at both the morning and afternoon milking, while the last group of eight cows served as untreated controls. Kp treatment caused a desirable hormone response from the cows' brain. Normal oestrous cycles resulted, but only when a mature follicle was present in the ovary. Further study is required to analyse whether the use of a long-acting Kp drug could be used as an effective treatment for stimulating dairy cows to become more fertile after calving.

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.

The roles of kisspeptin and neurokinin B in GnRH pulse generation in humans, and their potential clinical application.

The delivery of gonadotropin-releasing hormone (GnRH) in a pulsatile mode to the gonadotropes has long been known to be essential for normal reproductive function. There have been numerous studies aimed at dissecting out the mechanisms underlying GnRH pulse generation. The discovery of kisspeptin as an upstream regulator of GnRH attracted the possibility that pulsatile kisspeptin governed the pulsatile secretion of GnRH. Subsequent studies have shown the importance of the neurokinin B (NKB) system in modulating kisspeptin secretion and this GnRH. A number of studies in laboratory rodents have supported this notion. By contrast, we present data from clinical studies in men and women, in a range of contexts, showing that continuous infusion of kisspeptin 10 at receptor-saturating levels gives rise to an increase in luteinizing hormone (LH) (GnRH) pulse frequency. This has been demonstrated in normal healthy and hypogonadal men, in normal women during the mid-cycle LH surge, in men and women with mutations in the genes encoding NKB or its receptor, neurokinin 3 receptor (NK3R), in women with polycystic ovary syndrome treated with NK3R antagonist, and in women treated with NK3R antagonist during the LH surge. These finds indicate that pulsatile secretion and action of kisspeptin on GnRH neurons is not required for the generation of LH (GnRH) pulses in humans. We also report that there is an absence of desensitization in humans exposed to continuous infusion of kisspeptin-10 at receptor-saturating concentrations over 22 h and briefly review GnRH, kisspeptin and NKB analogs and their clinical application.

A dual kisspeptin-GnRH immunogen for reproductive immunosterilization.

GnRH immunogens have been extensively employed in immunocontraception of animals. While they are effective, they are not 100% efficacious and of limited duration. GnRH secretion is dependent on upstream stimulation by kisspeptin. We therefore hypothesised that a dual immunogen combining GnRH and kisspeptin may be more efficacious through targeting two levels of the axis. We have previously shown GnRH immunogen elicits permanent sterilisation when sheep are vaccinated neonatally suggesting that the efficacy of GnRH immunisation may be dependent on the stage of reproductive development. We have now studied over 300 days the efficacy of immunisation with a dual immunogen comprising GnRH linked to kisspeptin via a hepatitis B T helper peptide sequence (GKT) administered to male and female rats prepubertally, pubertally and as adults. At all stages of development all immunised animals produced antibodies to GnRH, kisspeptin and GKT but differentially in titre with respect to sex and stage of development. In immunised adult, prepubertal and pubertal males testosterone and testes length was markedly reduced by 60 days and remained at low levels until day 150. Thereafter, testosterone recovered to pre immunisation levels and testes length increased to a maximum of about 40% of controls. 80% of males were infertile in three matings over 250 days. In prepubertal and pubertal female rats a single immunisation at day 0 reduced estradiol to low levels by day 60 which remained low until termination of the experiment on day 300. In matings of these females with fertile males on days 90, 120 and 250, 74% of prepubertal females were infertile and impressively, 100% (10/10) of pubertal females were infertile after a single immunisation on day 0. These findings set the scene for exploration of immunosterilisation of wild and domestic animals after a single immunisation.

Seasonal expression and distribution of kisspeptin1 (kiss1) in the ovary and testis of freshwater catfish, Clarias batrachus: A putative role in steroidogenesis.

The central role of kisspeptin (kiss) in mammalian reproduction is well established; however, its intra-gonadal role is poorly addressed. Moreover, studies investigating intra-gonadal role of kiss in fish reproduction are scanty, contradictory and inconclusive. The expression of kiss1 mRNA has been detected in the fish brain, and functionally attributed to the regulation of reproduction, feeding and behavior. The kiss1 mRNA has also been demonstrated in tissues other than the brain in some studies, but its cellular distribution and role at the tissue level have not been adequately addressed in fish. Therefore, an attempt was made in the present study to localize kiss1 in gonadal cells of the freshwater catfish, Clarias batrachus. This study reports the presence of kiss1 in the theca cells and granulosa cells of the ovarian oocytes and interstitial cells in the testis of the catfish. The role of kiss1 in the ovary and testis of the catfish was also investigated using kiss1 receptor (kiss1r) antagonist (p234). The p234 treatment decreased the production of 17ß-estradiol in ovary and testosterone in the testis by lowering the activities of 3ß-hydroxysteroid dehydrogenase and 17ß-hydroxysteroid dehydrogenase under both, in vivo as well as in vitro conditions. The p234 treatment also arrested the progression of oogenesis, as evident from the low number of advancing/advanced oocytes in the treated ovary in comparison to the control ovary. It also reduced the area and perimeter of the seminiferous tubules in the treated catfish testis. Thus, our findings suggest that kiss is involved in the regulation of gonadal steroidogenesis, independent of known endocrine/ autocrine/ paracine regulators, and thereby it accelerates gametogenic processes in the freshwater catfish.

Gametogenic and steroidogenic action of kisspeptin-10 in the Asian catfish, Clarias batrachus: Putative underlying mechanistic cascade.

Unlike mammals, two kisspeptins genes encoding, kiss1 and kiss2 are detected in fishes with highly varied and contradictory difference in their reproductive activities. The present study was undertaken to examine the direct action of kisspeptin-10 and its role in gonadal activities in the gonadally quiescent Asian catfish using native mammalian kisspeptin decapeptide (KP-10) involving in vivo and in vitro approaches. The in vivo KP-10 treatment caused precocious onset of gametogenesis and its rapid progression, as was evident from the appearance of advanced stages of ovarian follicles in ovary, and advanced germ cells (spermatocytes/ spermatids) in the testis of the treated Clarias batrachus in comparison to the control gonads. It also elevated the steroid levels in gonads of the catfish in vivo and in vitro conditions. Simultaneously, it increased the expressions of key steroidogenic enzymes like 3ß-HSD, 17ß-HSD, and StAR protein, responsible for transfer of cholesterol from outer to inner membrane of the mitochondria of steroidogenic cells. Concurrently, it augmented the activities of 3ß-HSD and 17ß-HSD in the ovarian explants. The expressions of MAPK component (pERK1/2 and ERK1/2) were also up-regulated by KP-10 in gonadal explants. Thus, the data suggest that kisspeptin-10 stimulates gametogenesis by enhancing gonadal steroid production. The study also describes the putative mechanistic cascade of steroidogenic actions of kisspeptin-10 in the catfish so much so in teleost fish. The study also suggests that, kisspeptin may act locally to regulate gonadal activities in an autocrine/paracine manner, independent of known extra-gonadal factors in the catfish.

Rescue of Cell Surface Expression and Signaling of Mutant Follicle-Stimulating Hormone Receptors.

Mutations in G protein-coupled receptors (GPCRs) underlie numerous diseases. Many cause receptor misfolding and failure to reach the cell surface. Pharmacological chaperones are cell-permeant small molecules that engage nascent mutant GPCRs in the endoplasmic reticulum, stabilizing folding and "rescuing" cell surface expression. We previously demonstrated rescue of cell surface expression of luteinizing hormone receptor mutants by an allosteric agonist. Here we demonstrate that a similar approach can be employed to rescue mutant follicle-stimulating hormone receptors (FSHRs) with poor cell surface expression using a small-molecule FSHR agonist, CAN1404. Seventeen FSHR mutations described in patients with reproductive dysfunction were expressed in HEK 293T cells, and cell surface expression was determined by enzyme-linked immunosorbent assay of epitope-tagged FSHRs before/after treatment with CAN1404. Cell surface expression was severely reduced to ≤18% of wild-type (WT) for 11, modestly reduced to 66% to 84% of WT for 4, and not reduced for 2. Of the 11 with severely reduced cell surface expression, restoration to ≥57% of WT levels was achieved for 6 by treatment with 1 µM CAN1404 for 24 h, and a corresponding increase in FSH-induced signaling was observed for 4 of these, indicating restored functionality. Therefore, CAN1404 acts as a pharmacological chaperone and can rescue cell surface expression and function of certain mutant FSHRs with severely reduced cell surface expression. These findings aid in advancing the understanding of the effects of genetic mutations on GPCR function and provide a proof of therapeutic principle for FSHR pharmacological chaperones.

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.

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.

Can the carbon and nitrogen isotope values of offspring be used as a proxy for their mother's diet? Using foetal physiology to interpret bulk tissue and amino acid δ15N values.

The measurement of bulk tissue nitrogen (δ15N) and carbon isotope values (δ13C) chronologically along biologically inert tissues sampled from offspring can provide a longitudinal record of their mothers' foraging habits. This study tested the important assumption that mother-offspring stable isotope values are positively and linearly correlated. In addition, any change in the mother-offspring bulk tissues and individual amino acids that occurred during gestation was investigated. Whiskers sampled from southern elephant seal pups (Mirounga leonina) and temporally overlapping whiskers from their mothers were analyzed. This included n = 1895 chronologically subsampled whisker segments for bulk tissue δ15N and δ13C in total and n = 20 whisker segments for amino acid δ15N values, sampled from recently weaned pups (n = 17), juvenile southern elephant seals (SES) < 2 years old (n = 23) and adult female SES (n = 17), which included nine mother-offspring pairs. In contrast to previous studies, the mother-offspring pairs were not in isotopic equilibrium or linearly correlated during gestation: the Δ15N and Δ13C mother-offspring offsets increased by 0.8 and 1.2‰, respectively, during gestation. The foetal bulk δ15N values were 1.7 ± 0.5‰ (0.9-2.7‰) higher than mothers' δ15N values before birth, while the foetal δ13C increased by ~1.7‰ during gestation and were 1.0 ± 0.5‰ (0.0-1.9‰) higher than their mothers' δ13C at the end of pregnancy. The mother-offspring serine and glycine Δ15N differed by ~4.3‰, while the foetal alanine δ15N values were 1.4‰ lower than that of their mothers during the third trimester of pregnancy. The observed mother-offspring δ15N differences are likely explained by shuttling of glutamate-glutamine and glycine-serine amongst skeletal muscle, liver, placenta and foetal tissue. Foetal development relies primarily on remobilized endogenous maternal proteinaceous sources. Researchers should consider foetal physiology when using offspring bulk tissue isotope values as biomarkers for the mother's isotopic composition as part of monitoring programmes.

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.