Pulsatile GnRH Release Research Articles

Neuroendocrine Blockade of the Reproductive Axis in Female Athletes

This review aims at defining the neuroendocrine mechanisms underlying the sport-induced restrictions of the reproductive axis in female athletes. Episodic gonadotropin release was found to be compromised, presumably a result of impaired hypothalamic pulsatile GnRH release. Any deviation from optimal gonadotropin release may result in a suboptimal function of the ovaries, leading to disorders of the menstrual cycle and ovulation. A whole spectrum of menstrual dysfunctions ranging from ovulatory eumenorrhea to luteal phase defects and amenorrhea has been reported in sportive women. As essential neuroendocrine factors underlying these observations, activation of the adrenal axis and altered central nervous neurotransmitter activity have been identified to transfer metabolic, nutritional, and stress signals into the hypothalamic GnRH release. The degree by which the neuroendocrine axis governing reproduction is impaired critically depends on the intensity and duration of exercise and the state of training. Other decisive factors may be energy expenditure and availability, nutritional components, and the maturity of the hypothalamic-pituitary-ovarian (HPO) axis when sport activity was initiated. In conclusion, the gradual cessation of reproductive function observed in female athletes may be interpreted as an adaptive mechanism in response to physical and psychological endurance during sport. This sport-induced restriction of reproductive capacity may serve as protection (endogenous contraception) to preserve a woman’s health.

Lactational Amenorrhea: Neuroendocrine Pathways Controlling Fertility and Bone Turnover.

Lactation is a physiological state of hyperprolactinemia and associated amenorrhea. Despite the fact that exact mechanisms standing behind the hypothalamus–pituitary–ovarian axis during lactation are still not clear, a general overview of events leading to amenorrhea may be suggested. Suckling remains the most important stimulus maintaining suppressive effect on ovaries after pregnancy. Breastfeeding is accompanied by high levels of prolactin, which remain higher than normal until the frequency and duration of daily suckling decreases and allows normal menstrual function resumption. Hyperprolactinemia induces the suppression of hypothalamic Kiss1 neurons that directly control the pulsatile release of GnRH. Disruption in the pulsatile manner of GnRH secretion results in a strongly decreased frequency of corresponding LH pulses. Inadequate LH secretion and lack of pre-ovulatory surge inhibit the progression of the follicular phase of a menstrual cycle and result in anovulation and amenorrhea. The main consequences of lactational amenorrhea are connected with fertility issues and increased bone turnover. Provided the fulfillment of all the established conditions of its use, the lactational amenorrhea method (LAM) efficiently protects against pregnancy. Because of its accessibility and lack of additional associated costs, LAM might be especially beneficial in low-income, developing countries, where modern contraception is hard to obtain. Breastfeeding alone is not equal to the LAM method, and therefore, it is not enough to successfully protect against conception. That is why LAM promotion should primarily focus on conditions under which its use is safe and effective. More studies on larger study groups should be conducted to determine and confirm the impact of behavioral factors, like suckling parameters, on the LAM efficacy. Lactational bone loss is a physiologic mechanism that enables providing a sufficient amount of calcium to the newborn. Despite the decline in bone mass during breastfeeding, it rebuilds after weaning and is not associated with a postmenopausal decrease in BMD and osteoporosis risk. Therefore, it should be a matter of concern only for lactating women with additional risk factors or with low BMD before pregnancy. The review summarizes the effect that breastfeeding exerts on the hypothalamus–pituitary axis as well as fertility and bone turnover aspects of lactational amenorrhea. We discuss the possibility of the use of lactation as contraception, along with this method’s prevalence, efficacy, and influencing factors. We also review the literature on the topic of lactational bone loss: its mechanism, severity, and persistence throughout life.

Modulation of pulsatile GnRH dynamics across the ovarian cycle via changes in the network excitability and basal activity of the arcuate kisspeptin network.

Pulsatile GnRH release is essential for normal reproductive function. Kisspeptin secreting neurons found in the arcuate nucleus, known as KNDy neurons for co-expressing neurokinin B, and dynorphin, drive pulsatile GnRH release. Furthermore, gonadal steroids regulate GnRH pulsatile dynamics across the ovarian cycle by altering KNDy neurons' signalling properties. However, the precise mechanism of regulation remains mostly unknown. To better understand these mechanisms, we start by perturbing the KNDy system at different stages of the estrous cycle using optogenetics. We find that optogenetic stimulation of KNDy neurons stimulates pulsatile GnRH/LH secretion in estrous mice but inhibits it in diestrous mice. These in vivo results in combination with mathematical modelling suggest that the transition between estrus and diestrus is underpinned by well-orchestrated changes in neuropeptide signalling and in the excitability of the KNDy population controlled via glutamate signalling. Guided by model predictions, we show that blocking glutamate signalling in diestrous animals inhibits LH pulses, and that optic stimulation of the KNDy population mitigates this inhibition. In estrous mice, disruption of glutamate signalling inhibits pulses generated via sustained low-frequency optic stimulation of the KNDy population, supporting the idea that the level of network excitability is critical for pulse generation. Our results reconcile previous puzzling findings regarding the estradiol-dependent effect that several neuromodulators have on the GnRH pulse generator dynamics. Therefore, we anticipate our model to be a cornerstone for a more quantitative understanding of the pathways via which gonadal steroids regulate GnRH pulse generator dynamics. Finally, our results could inform useful repurposing of drugs targeting the glutamate system in reproductive therapy.

Exposure to Iodoacetic Acid, a Water Disinfection Byproduct, Leads to Abnormal Expression of Key Reproductive Axis Genes in the Hypothalamus and Pituitary

Iodoacetic acid (IAA) – a water disinfection byproduct (DBP) formed from the reaction between an oxidizing disinfectant, i.e. chlorine, and iodide – is an understudied, yet potentially dangerous environmental toxicant. DBPs have been epidemiologically associated with reproductive dysfunction. In vitro studies have indicated that IAA is one of the most cyto- and genotoxic DBPs. Further, murine ovarian research has shown that IAA exposure significantly inhibits antral follicle growth and reduces estradiol levels. Despite this evidence, little is known about the other components of the reproductive axis: the hypothalamus and pituitary. To address this, we tested the hypothesis that IAA exposure would lead to disrupted expression of key hypothalamic and pituitary genes related to reproductive function. We exposed adult female CD1 mice to 0.5, 10, 100, or 500 mg/L IAA in their drinking water from postnatal day 40 (P40) to their first day in diestrus after P75. From this experiment, we collected whole pituitaries and hypothalamic punches containing the arcuate nucleus (ARC), anteroventral periventricular zone (AVPV), and medial preoptic nucleus (mPOA), and processed them for mRNA analysis. We also exposed pituitary explant cultures to IAA to observe direct effects on gene expression. In vivo, we found that mRNA levels of kisspeptin (Kiss1) are significantly increased in the ARC, the region that controls pulsatile GnRH release, at 0.5 and 10 mg/L IAA concentrations. Kiss1 is unchanged in the AVPV, the neuron population responsible for generating the LH surge. We also measured ARC expression of neurokinin B (Tac2) and dynorphin (Pdyn), neuropeptides secreted by kisspeptin co-expressing neurons to autosynaptically stimulate Kiss1 release. We saw no difference in either. GnRH (Gnrh1) expression was also unchanged. Both in vivo at 10 mg/L IAA and in culture, we found IAA exposure significantly reduced Fshb mRNA. Preliminary immunohistochemistry (IHC) data suggests it also leads to an apparent reduction in FSH-positive cells in vitro (N=2). Lhb and the α-subunit (Cga) were unaltered in vivo, though were significantly reduced with in vitro exposure. In neither context was mRNA expression of the GnRH receptor (Gnrhr) changed. Noting apparent direct effects of IAA on the pituitary, we assessed expression of the cell-cycle inhibitor p21 (Cdkn1a), which has been shown to increase with toxicant exposure. We found Cdkn1a increased in vivo at 500 mg/L IAA, trending at 100 mg/L (p=.070), and in vitro. IHC data in vitro suggests a marked increase in P21-positivity following IAA exposure. These data, together with prior ovarian findings, implicate IAA as a potential reproductive axis disruptor at each major level – through ARC Kiss1 expression, Fshb expression in vivo and in vitro, FSH expression in vitro, and Lhb and Cga in vitro. Further, Cdkn1a/P21 induction indicates IAA toxicity at the level of the pituitary.

Innervation of GnRH Neuron Distal Projections and Activation by Kisspeptin in a New GnRH-Cre Rat Model.

The neural mechanisms generating pulsatile GnRH release from the median eminence (ME) remain unclear. Studies undertaken in the mouse demonstrate that GnRH neurons extend projections to the ME that have properties of both dendrites and axons, termed "dendrons," and that the kisspeptin neuron pulse generator targets these distal dendrons to drive pulsatile GnRH secretion. It presently remains unknown whether the GnRH neuron dendron exists in other species. We report here the generation of a knock-in Gnrh1-Ires-Cre rat line with near-perfect targeting of Cre recombinase to the GnRH neuronal phenotype. More than 90% of adult male and female GnRH neurons express Cre with no ectopic expression. Adeno-associated viruses were used in adult female Gnrh1-Ires-Cre rats to target mCherry or GCAMP6 to rostral preoptic area GnRH neurons. The mCherry tracer revealed the known unipolar and bipolar morphology of GnRH neurons and their principal projection pathways to the external zone of the ME. Synaptophysin-labeling of presynaptic nerve terminals revealed that GnRH neuron distal projections received numerous close appositions as they passed through the arcuate nucleus and into the median eminence. Confocal GCaMP6 imaging in acute horizontal brain slices demonstrated that GnRH neuron distal projections lateral to the median eminence were activated by kisspeptin. These studies indicate the presence of a dendron-like arrangement in the rat with GnRH neuron distal projections receiving synaptic input and responding to kisspeptin.

SUN-243 Deletion of KNDy Neuron-Specific KISS1 Disrupts Estrous Cyclicity and LH Pulsatility in Female Mice

Kisspeptin (encoded by Kiss1), a neuropeptide critically involved in neuroendocrine regulation of reproduction, is primarily synthesized in two discrete hypothalamic nuclei: the anteroventral periventricular area (AVPV) and arcuate nucleus (ARC). AVPV Kiss1 is important for the pre-ovulatory luteinizing hormone (LH) surge unique to females as well as estrogen-induced positive feedback control of GnRH and LH. In contrast, ARC Kiss1 neurons, which largely co-express the neuropeptides NKB and dynorphin (collectively known as KNDy neurons), are major regulators of pulsatile release of GnRH and LH, and mediate estrogen-induced negative feedback control of both GnRH and LH. Previous studies have not fully separated the specific roles for Kiss1 in the AVPV versus KNDy-ARC neurons in the downstream control of GnRH and LH release. Therefore, we generated a Pdyn-Cre/Kiss1fl/fl (KO) mouse model to target Kiss1 in the KNDy neurons to differentiate KNDy neuron-specific function from AVPV Kiss1 function in the maturation and maintenance of the reproductive axis. qRT-PCR data documented a significant reduction of Kiss1 expression in the mediobasal hypothalamus (containing ARC) compared to controls, whereas Kiss1 in the preoptic area (containing AVPV) was similar in both KO and controls. Immunofluorescent IHC confirmed a loss of kisspeptin immunoreactivity in the ARC of KO animals while expression in the AVPV remained intact. Markers of pubertal onset (day of vaginal opening and first estrus in females; day of preputial separation in males) were normal in KO mice, suggesting that AVPV Kiss1 and/or other neural signals may be sufficient for pubertal onset. In addition, body weight throughout pubertal growth was comparable between KO and control animals of both sexes. Interestingly, KO female mice had disrupted estrous cycles presenting with persistent diestrus and a small vaginal opening. In order to test our hypothesis that conditional deletion of Kiss1 in KNDy neurons disrupts or ablates episodic GnRH/LH pulsatile release, we collected serial tail blood samples from mice at diestrus and measured LH. KO female mice exhibited significantly fewer LH pulses in a 3-hour timespan compared to controls, suggesting that KNDy neurons were functionally compromised. These observations indicate the central role of KNDy neurons in the regulation of GnRH/LH pulsatility and estrous cyclicity. The functional effects of disrupted estrous cyclicity and slower LH pulses observed in KO females are currently under study to assess potential abnormalities in ovarian folliculogenesis and fertility. Future experiments will determine whether ARC Kiss1 deletion disrupts the KNDy-driven negative feedback response of LH to gonadectomy, as well as address potential sex differences in ARC Kiss1-mediated negative feedback control of LH release.

SUN-241 Adult Exposure to Iodoacetic Acid Leads to Abnormal Expression of Key Genes Related to Hypothalamic and Pituitary Control of Reproductive Function

Water disinfection byproducts (DBPs) are formed when chemicals used to decontaminate water come into contact with natural or synthetic organic material. DBPs have been linked to a range of health concerns including reproductive disfunction. One such DBP, the monohalogenated iodoacetic acid (IAA), is formed when iodide reacts with a disinfectant, for example, chlorine. IAA is of particular health concern; not only is iodide widely present in the water supply, especially in coastal communities and those near fracking sites, but IAA has been found to be one of the most cyto- and genotoxic DBPs. Further, a previous study has indicated that in vitro IAA exposure significantly inhibits antral follicle growth and reduces estradiol levels in ovaries. However, little is known about how IAA affects the other major components of the reproductive axis: the hypothalamus and pituitary. The reproductive axis relies on homeostatic release of hormones to communicate from one organ to another and alterations at any level may impact reproduction. So, we set out to test the hypothesis that exposure to IAA would lead to disrupted expression of key hypothalamic and pituitary genes related to reproductive function. We continually exposed female adult CD1 mice to 0.5, 10, 100, or 500 mg/L IAA in their drinking water for approximately 35 days (postnatal day 40 (P40) to their first day in diestrus following P75.) Whole pituitaries and hypothalamic punches containing the arcuate nucleus (ARC), anteroventral periventricular zone (AVPV), and medial preoptic nucleus (mPOA) were collected and processed for qPCR analysis. We find that while kisspeptin (Kiss1) expression in the AVPV - the population responsible for generating the LH surge - is unchanged, 0.5 mg/L IAA exposure significantly increases Kiss1 in the ARC, which controls pulsatile GnRH release, and there is a trending increase (p=.056) at 10mg/L. We also measured ARC expression of Neurokinin B (NKB; Tac2), a neuropeptide secreted by kisspeptin co-expressing neurons to autosynaptically stimulate Kiss1 release. We found no change in mRNA levels of Tac2. We also saw no significant changes in GnRH (Gnrh1) mRNA expression. At the level of the pituitary, there is no change in Lhb mRNA levels. Exposure to 10 mg/L IAA leads to significantly reduced Fshb expression, however FSH serum levels are not significantly changed. These data, taken together with previous findings in the ovary, indicate that IAA has the potential to disrupt each major level of the reproductive axis: ovarian follicle development and steroid synthesis, hypothalamic arcuate Kiss1 synthesis, and Fshb synthesis from the pituitary. Further research is necessary to elucidate at which levels IAA acts directly and at which it acts through action on another component of the axis. Additionally, future studies can clarify the mechanism through which IAA has these effects.

Acute suppression of LH secretion by prolactin in female mice is mediated by kisspeptin neurons in the arcuate nucleus

Elevated prolactin levels suppress pulsatile release of GnRH from the hypothalamus, with a consequent reduction in pulsatile LH secretion from the pituitary. In this paper, the authors showed in mice that kisspeptin neurons located in the arcuate nucleus (ARC) respond to prolactin and conditional knockout of the prolactin receptor specifically in ARC kisspeptin neurons prevented prolactin-induced suppression of LH secretion.

SAT-420 Gonadotropin-Releasing Hormone Stimulates Luteinizing Hormone Exocytosis without Elevating Lhb Expression

Continuous but not pulsatile delivery of GnRH leads to marked down regulation of luteinizing hormone (LH) secretion and blockade of reproductive functions. However, the relationship between LH beta subunit gene (Lhb) expression and LH secretion has not been studied in detail. Here we studied the relationship between Lhb expression, LH release and intracellular LH content under different experimental paradigms. Experiments were performed in vivo and in vitro using female and male rats and in cultured rat pituitary cells. In vitro experiments included evaluation of Lhb expression during the prolonged absence of GnRH, influence of culture medium on gene expression, continuous vs repetitive GnRH application, and ELISA vs Western blot analysis of LH content. Experiments showed that Lhb expression declined progressively after pituitary removal and cell dispersion, indicating the loss of regulated gene expression. Addition of GnRH receptor agonists did not rescue Lhb expression, suggesting that other factor(s) account(s) for regulated gene expression. On the other hand, GnRH stimulated LH secretion in vitro, leading to gradual depletion of the secretory pool during continuous receptor activation. Inhibition of transcription by actinomycin D had only a minor effect on in vitro LH secretion, in contrast to cycloheximide, a protein synthesis inhibitor. In vivo administration of a GnRH receptor agonist was accompanied with a rapid increase in serum LH levels and a progressive depletion of the intrapituitary LH levels, without affecting Lhb expression. These results indicate that GnRH controls LH synthesis and exocytosis post-transcriptionally and that pulsatile GnRH release protects gonadotrophs from depletion of the LH secretory pool.

Acute Suppression of LH Secretion by Prolactin in Female Mice Is Mediated by Kisspeptin Neurons in the Arcuate Nucleus.

Hyperprolactinemia causes infertility, but the specific mechanism is unknown. It is clear that elevated prolactin levels suppress pulsatile release of GnRH from the hypothalamus, with a consequent reduction in pulsatile LH secretion from the pituitary. Only a few GnRH neurons express prolactin receptors (Prlrs), however, and thus prolactin must act indirectly in the underlying neural circuitry. Here, we have tested the hypothesis that prolactin-induced inhibition of LH secretion is mediated by kisspeptin neurons, which provide major excitatory inputs to GnRH neurons. To evaluate pulsatile LH secretion, we collected serial blood samples from diestrous mice and measured LH levels by ultrasensitive ELISA. Acute prolactin administration decreased LH pulses in wild-type mice. Kisspeptin neurons in the arcuate nucleus and in the rostral periventricular area of the third ventricle (RP3V) acutely responded to prolactin, but prolactin-induced signaling in kisspeptin neurons was up to fourfold higher in the arcuate nucleus when compared with the RP3V. Consistent with this, conditional knockout of Prlr specifically in arcuate nucleus kisspeptin neurons prevented prolactin-induced suppression of LH secretion. Our data establish that during hyperprolactinemia, suppression of pulsatile LH secretion is mediated by Prlr on arcuate kisspeptin neurons.

Potential roles for the kisspeptin/kisspeptin receptor system in implantation and placentation.

BACKGROUNDInitially identified as suppressors of metastasis in various types of cancer, kisspeptins are a family of neuropeptides that are key regulators of the mammalian reproductive axis. Accumulating evidence has shown that kisspeptin is able to control both the pulsatile and surge GnRH release, playing fundamental roles in female reproduction, which include the secretion of gonadotropins, puberty onset, brain sex differentiation, ovulation and the metabolic regulation of fertility. Furthermore, recent studies have demonstrated the involvement of the kisspeptin system in the processes of implantation and placentation. This review summarizes the current knowledge of the pathophysiological role and utility of these local placental regulatory factors as potential biomarkers during the early human gestation.OBJECTIVE AND RATIONALEA successful pregnancy, from the initiation of embryo implantation to parturition, is a complex process that requires the orchestration of a series of events. This review aims to concisely summarize what is known about the role of the kisspeptin system in implantation, placentation, early human pregnancy and pregnancy-related disorders, and to develop strategies for predicting, diagnosing and treating these abnormalities.SEARCH METHODSUsing the PubMed and Google Scholar databases, we performed comprehensive literature searches in the English language describing the advancement of kisspeptins and the kisspeptin receptor (KISS1R) in implantation, placentation and early pregnancy in humans, since its initial identification in 1996 and ending in July 2018.OUTCOMESRecent studies have shown the coordinated spatial and temporal expression patterns of kisspeptins and KISS1R during human pregnancy. The experimental data gathered recently suggest putative roles of kisspeptin signaling in the regulation of trophoblast invasion, embryo implantation, placentation and early pregnancy. Dysregulation of the kisspeptin system may negatively affect the processes of implantation as well as placentation. Clinical studies indicate that the circulating levels of kisspeptins or the expression levels of kisspeptin/KISS1R in the placental tissues may be used as potential diagnostic markers for women with miscarriage and gestational trophoblastic neoplasia.WIDER IMPLICATIONSComprehensive research on the pathophysiological role of the kisspeptin/KISS1R system in implantation and placentation will provide a dynamic and powerful approach to understanding the processes of early pregnancy, with potential applications in observational and analytic screening as well as the diagnosis, prognosis and treatment of implantation failure and early pregnancy-related disorders.

The Gonadotropin-Releasing Hormone Pulse Generator.

The pulsatile release of GnRH and LH secretion is essential for fertility in all mammals. Pulses of LH occur approximately every hour in follicular-phase females and every 2 to 3 hours in luteal-phase females and males. Many studies over the last 50 years have sought to identify the nature and mechanism of the "GnRH pulse generator" responsible for pulsatile LH release. This review examines the characteristics of pulsatile hormone release and summarizes investigations that have led to our present understanding of the GnRH pulse generator. There is presently little compelling evidence for an intrinsic mechanism of pulse generation involving interactions between GnRH neuron cell bodies. Rather, data support the presence of an extrinsic pulse generator located within the arcuate nucleus, and attention has focused on the kisspeptin neurons and their projections to GnRH neuron dendrons concentrated around the median eminence. Sufficient evidence has been gathered in rodents to conclude that a subpopulation of arcuate kisspeptin neurons is, indeed, the GnRH pulse generator. Findings in other species are generally compatible with this view and suggest that arcuate/infundibular kisspeptin neurons represent the mammalian GnRH pulse generator. With hindsight, it is likely that past arcuate nucleus multiunit activity recordings have been from kisspeptin neurons. Despite advances in identifying the cells forming the pulse generator, almost nothing is known about their mechanisms of synchronicity and the afferent hormonal and transmitter modulation required to establish the normal patterns of LH pulsatility in mammals.

Estrogenic-dependent glutamatergic neurotransmission from kisspeptin neurons governs feeding circuits in females.

The neuropeptides tachykinin2 (Tac2) and kisspeptin (Kiss1) in hypothalamic arcuate nucleus Kiss1 (Kiss1ARH) neurons are essential for pulsatile release of GnRH and reproduction. Since 17β-estradiol (E2) decreases Kiss1 and Tac2 mRNA expression in Kiss1ARH neurons, the role of Kiss1ARH neurons during E2-driven anorexigenic states and their coordination of POMC and NPY/AgRP feeding circuits have been largely ignored. Presently, we show that E2 augmented the excitability of Kiss1ARH neurons by amplifying Cacna1g, Hcn1 and Hcn2 mRNA expression and T-type calcium and h-currents. E2 increased Slc17a6 mRNA expression and glutamatergic synaptic input to arcuate neurons, which excited POMC and inhibited NPY/AgRP neurons via metabotropic receptors. Deleting Slc17a6 in Kiss1 neurons eliminated glutamate release and led to conditioned place preference for sucrose in E2-treated KO female mice. Therefore, the E2-driven increase in Kiss1 neuronal excitability and glutamate neurotransmission may play a key role in governing the motivational drive for palatable food in females.

Role of Kisspeptin and Neurokinin B Signaling in Male Rhesus Monkey Puberty.

Despite the well-established concept that an increase in pulsatile GnRH release triggers puberty, the precise signaling mechanism responsible for the pubertal increase in GnRH release remains unclear. A recent study indicates that developmental changes in the network formation between kisspeptin and neurokinin B (NKB) signaling greatly contribute to the pubertal increase in GnRH release in female monkeys. It is, however, unknown whether similar developmental changes in the kisspeptin and NKB network are involved in male puberty. In the current study, we first characterized the pubertal stages in male rhesus monkeys by assessing physiological and hormonal changes during sexual development. Subsequently, we examined the role of the kisspeptin and NKB signaling network in the pubertal increase in GnRH release. Results suggest that while collaborative kisspeptin and NKB signaling to GnRH neurons was active before puberty onset, after initiation of puberty the role of NKB signaling in GnRH neurons diminished and kisspeptin signaling assumed the primary stimulatory role in the regulation of GnRH release in male monkeys. These findings in males differ from those seen in females.

Investigating the KNDy Hypothesis in Humans by Coadministration of Kisspeptin, Neurokinin B, and Naltrexone in Men.

Context:A subpopulation of hypothalamic neurons colocalize three neuropeptides, namely kisspeptin, neurokinin B (NKB), and dynorphin, collectively termed KNDy neurons. Animal studies suggest they interact to affect pulsatile GnRH release (KNDy hypothesis); kisspeptin stimulates, NKB modulates, and dynorphin (an opioid) inhibits.Objective:To investigate the KNDy hypothesis in humans, we assessed for the first time the effects of the coadministration of kisspeptin-54, NKB, and an opioid receptor antagonist, naltrexone, on LH pulsatility (surrogate marker for GnRH pulsatility) and gonadotropin release.Design, Setting, and Participants:This was an ethically approved prospective, single-blinded, placebo-controlled study. Healthy male volunteers (n = 5/group) attended our research facility for eight study visits.Intervention and Main Outcome Measure:After 1 hour of baseline blood sampling, participants received a different intervention at each visit: oral 50 mg naltrexone, 8-hour iv infusions of vehicle, 2.56 nmol/kg · h NKB, 0.1 nmol/kg · h kissspeptin-54 (KP) alone and in combination. Frequent blood sampling to measure plasma gonadotropins and sex steroids was conducted and LH pulsatility was determined using blinded deconvolution analysis.Results:All kisspeptin and naltrexone containing groups potently increased LH and LH pulsatility (P < .001 vs vehicle). NKB alone did not affect gonadotropins. NKB+KP had significantly lower increases in gonadotropins compared with kisspeptin alone (P < .01). Naltrexone+KP was the only group to significantly increase LH pulse amplitude (P < .001 vs vehicle).Conclusions:Our results suggest significant interactions between the KNDy neuropeptides on LH pulsatility and gonadotropin release in humans. This has important implications for improving our understanding of GnRH pulse generation in humans.

GnRH Pulse Frequency Control of Fshb Gene Expression Is Mediated via ERK1/2 Regulation of ICER.

The pulsatile release of GnRH regulates the synthesis and secretion of pituitary FSH and LH. Two transcription factors, cAMP-response element-binding protein (CREB) and inducible cAMP early repressor (ICER), have been implicated in the regulation of rat Fshb gene expression. We previously showed that the protein kinase A pathway mediates GnRH-stimulated CREB activation. We hypothesized that CREB and ICER are activated by distinct signaling pathways in response to pulsatile GnRH to modulate Fshb gene expression, which is preferentially stimulated at low vs high pulse frequencies. In the LβT2 gonadotrope-derived cell line, GnRH stimulation increased ICER mRNA and protein. Blockade of ERK activation with mitogen-activated protein kinase kinase I/II (MEKI/II) inhibitors significantly attenuated GnRH induction of ICER mRNA and protein, whereas protein kinase C, calcium/calmodulin-dependent protein kinase II, and protein kinase A inhibitors had minimal effects. GnRH also stimulated ICER in primary mouse pituitary cultures, attenuated similarly by a MEKI/II inhibitor. In a perifusion paradigm, MEKI/II inhibition in LβT2 cells stimulated with pulsatile GnRH abrogated ICER induction at high GnRH pulse frequencies, with minimal effect at low frequencies. MEKI/II inhibition reduced GnRH stimulation of Fshb at high and low pulse frequencies, suggesting that the ERK pathway has additional effects on GnRH regulation of Fshb, beyond those mediated by ICER. Indeed, induction of the activating protein 1 proteins, cFos and cJun, positive modulators of Fshb transcription, by pulsatile GnRH was also abrogated by inhibition of the MEK/ERK signaling pathway. Collectively, these studies indicate that the signaling pathways mediating GnRH activation of CREB and ICER are distinct, contributing to the decoding of the pulsatile GnRH to regulate FSHβ expression.

The Distribution of Substance P and Kisspeptin in the Mediobasal Hypothalamus of the Male Rhesus Monkey and a Comparison of Intravenous Administration of These Peptides to Release GnRH as Reflected by LH Secretion

Substance P (SP) was recently reported to be expressed in human kisspeptin/neurokinin B/dynorphin (KNDy) neurons and to enhance KNDy neuron excitability in the mouse hypothalamus. We therefore examined (1) interactions of SP and kisspeptin in the mediobasal hypothalamus of adult male rhesus monkeys using immunofluorescence, and (2) the ability of SP to induce LH release in GnRH-primed, agonadal juvenile male monkeys. SP cell bodies were observed only occasionally in the arcuate nucleus (Arc), but more frequently dorsal to the Arc in the region of the premammillary nucleus. Castration resulted in an increase in the number of SP cell bodies in the Arc but not in the other regions. SP fibers innervated the Arc, where they were found in close apposition with kisspeptin perikarya in the periphery of this nucleus. Beaded SP axons projected to the median eminence, where they terminated in the external layer and intermingled with beaded kisspeptin axons. Colocalization of the two peptides, however, was not observed. Although close apposition between SP fibers and kisspeptin neurons suggest a role for SP in modulating GnRH pulse generator activity, i.v. injections of SP failed to elicit release of GnRH (as reflected by LH) in the juvenile monkey. Although the finding of structural interactions between SP and kisspeptin neurons is consistent with the notion that this tachykinin may be involved in regulating pulsatile GnRH release, the apparent absence of expression of SP in KNDy neurons suggests that this peptide is unlikely to be a fundamental component of the primate GnRH pulse generator.

A Reevaluation of the Question: Is the Pubertal Resurgence in Pulsatile GnRH Release in the Male Rhesus Monkey (Macaca mulatta) Associated With a Gonad-Independent Augmentation of GH Secretion?

A somatic signal has been posited to trigger the pubertal resurgence in pulsatile GnRH secretion that initiates puberty in highly evolved primates. That GH might provide such a signal emerged in 2000 as a result of a study reporting that circulating nocturnal GH concentrations in castrated juvenile male monkeys increased in a 3-week period immediately preceding the pubertal resurgence of LH secretion. The present study was conducted to reexamine this intriguing relationship, again in an agonadal model. Four castrated juvenile male monkeys were implanted with indwelling jugular catheters, housed in remote sampling cages, and subjected to 24 hours of sequential blood sampling (every 30 min) every 2 weeks from 19.5 to 22 months of age. Twenty-four-hour profiles of circulating GH concentrations were analyzed using the pulse detection algorithm, PULSAR, and developmental changes in pulsatile GH release with respect to the initiation of the pubertal rise of LH secretion (week 0; observed between 22.5 and 32 mo of age) were examined for significance by a repeated-measures ANOVA. Changes in the parameters of pulsatile GH secretion, including mean 24-hour GH concentration and GH pulse frequency and pulse amplitude for 3 (n = 4) and 6 (n = 3) months before week 0 were unremarkable and nonsignificant. These findings fail to confirm those of the earlier study and lead us to conclude that the timing of the pubertal resurgence of GnRH release in the male monkey is not dictated by GH. Reasons for the discrepancy between the two studies are unclear.

Expression of Vesicular Glutamate Transporter 2 (vGluT2) on Large Dense-Core Vesicles within GnRH Neuroterminals of Aging Female Rats.

The pulsatile release of GnRH is crucial for normal reproductive physiology across the life cycle, a process that is regulated by hypothalamic neurotransmitters. GnRH terminals co-express the vesicular glutamate transporter 2 (vGluT2) as a marker of a glutamatergic phenotype. The current study sought to elucidate the relationship between glutamate and GnRH nerve terminals in the median eminence—the site of GnRH release into the portal capillary vasculature. We also determined whether this co-expression may change during reproductive senescence, and if steroid hormones, which affect responsiveness of GnRH neurons to glutamate, may alter the co-expression pattern. Female Sprague-Dawley rats were ovariectomized at young adult, middle-aged and old ages (~4, 11, and 22 months, respectively) and treated four weeks later with sequential vehicle + vehicle (VEH + VEH), estradiol + vehicle (E2 + VEH), or estradiol + progesterone (E2+P4). Rats were perfused 24 hours after the second hormone treatment. Confocal microscopy was used to determine colocalization of GnRH and vGluT2 immunofluorescence in the median eminence. Post-embedding immunogold labeling of GnRH and vGluT2, and a serial electron microscopy (EM) technique were used to determine the cellular interaction between GnRH terminals and glutamate signaling. Confocal analysis showed that GnRH and vGluT2 immunofluorescent puncta were extensively colocalized in the median eminence and that their density declined with age but was unaffected by short-term hormone treatment. EM results showed that vGluT2 immunoreactivity was extensively associated with large dense-core vesicles, suggesting a unique glutamatergic signaling pathway in GnRH terminals. Our results provide novel subcellular information about the intimate relationship between GnRH terminals and glutamate in the median eminence.

Stress-induced inhibition of LH pulses in female rats: role of GABA in arcuate nucleus.

Stress exerts profound inhibitory effects on reproductive function by suppression of the pulsatile release of GnRH and therefore LH. Besides the corticotrophin-releasing factor (CRF), this effect also might be mediated via GABAergic signaling within the arcuate nucleus (ARC) since its inhibitory effects on LH pulses and increased activity during stress. In the present study, we investigated the role of endogenous GABAergic signaling within the ARC in stress-induced suppression of LH pulses. Ovariectomised oestradiol-replaced rats were implanted with bilateral and unilateral cannulae targeting toward the ARC and lateral cerebral ventricle respectively. Blood samples (25 μl) were taken via chronically implanted cardiac catheters every 5 min for 6 h for measurement of LH pulses. Intra-ARC infusion of GABAA receptor antagonist, bicuculline (0.2 pmol in 200 nl artificial cerebrospinal fluid (aCSF) each side, three times at 20-min intervals) markedly attenuated the inhibitory effect of lipopolysaccharide (LPS; 25 μg/kg i.v.) but not restraint (1 h) stress on pulsatile LH secretion. In contrast, restraint but not LPS stress-induced suppression of LH pulse frequency was reversed by intra-ARC administration of GABABR antagonist, CGP-35348 (1.5 nmol in 200 nl aCSF each side, three times at 20-min intervals). Moreover, intra-ARC application of either bicuculline or CGP-35348 attenuated the inhibitory effect of CRF (1 nmol in 4 μl aCSF, i.c.v.) on the LH pulses. These data indicate a pivotal and differential role of endogenous GABAA and GABAB signaling mechanisms in the ARC with respect to mediating immunological and psychological stress-induced suppression of the GnRH pulse generator respectively.