Gonadotropin-releasing Hormone Release Research Articles

Revolutionizing Infertility Management through Novel Peptide-based Targets.

Around 48 million couples and 186 million people worldwide have infertility; of these, approximately 85% have an identifiable cause, the most common being ovulatory dysfunctions, male infertility, polycystic ovary syndrome, and tubule disease. The remaining 15% have infertility for unknown reasons, including lifestyle and environmental factors. The regulation of the hypothalamic- pituitary-adrenal axis (HPA) is crucial for the secretion of gonadotropin-releasing hormone (GnRH), luteinizing hormone (LH), and follicle-stimulating hormone (FSH), which are essential for female reproductive functions. GnRH is the primary reproductive axis regulator. The pattern of GnRH, FSH, and LH release is determined by its pulsatile secretion, which in turn controls endocrine function and gamete maturation in the gonads. Peptides called Kisspeptin (KP), Neurokinin-B (NKB), and Orexin influence both positive and negative feedback modulation of GnRH, FSH, and LH secretion in reproduction. This review article mainly focuses on the historical perspective, isoform, and signaling pathways of KP, NKB, and Orexin novel peptide-based targets including clinical and preclinical studies and having a promising effect in the management of infertility.

The role of neuroestrogens in the estrogen-induced gonadotropin surge in male monkeys.

Neuroestrogens locally synthesized in the brain are known to play a role in sexual behaviors. However, the question of whether neuroestrogens are involved in the regulation of the gonadotropin-releasing hormone (GnRH) release is just emerging. Because previous studies in this lab indicate that neuroestradiol is also important for the pulsatile release as well as the surge release of GnRH in female rhesus monkeys, in the present study, we examined whether neuroestradiol plays a role in the estrogen-induced LH surge in orchidectomized (ORX) male rhesus monkeys. Unlike in rodents, it is known that a high dose of estrogen treatment can result in the LH surge in ORX male rhesus monkeys. Results that the administration of the aromatase inhibitor, letrozole, failed to attenuate the estrogen-induced LH surge, suggest that unlike in ovariectomized females, neuroestrogens do not play a role in the LH surge experimentally induced by the exogenous estrogen treatment in ORX male monkeys.

Novel insights into minipuberty and GnRH: Implications on neurodevelopment, cognition, and COVID-19 therapeutics.

In humans, the first 1000 days of life are pivotal for brain and organism development. Shortly after birth, gonadotropin-releasing hormone (GnRH) neurons in the hypothalamus are activated, a phenomenon known as minipuberty. This phenomenon, observed in all mammals studied, influences the postnatal development of the hypothalamic-pituitary-gonadal (HPG) axis and reproductive function. This review will put into perspective the results of recent studies showing that the impact of minipuberty extends beyond reproductive function, influencing sensory and cognitive maturation. Studies in mice have revealed the role of nitric oxide (NO) in regulating minipuberty amplitude, with NO deficiency linked to cognitive and olfactory deficits. Additionally, findings indicate that cognitive and sensory defects in adulthood in a mouse model of Down syndrome are associated with an age-dependent decline of GnRH production, whose origin can be traced back to minipuberty, and point to the potential therapeutic role of pulsatile GnRH administration in cognitive disorders. Furthermore, this review delves into the repercussions of COVID-19 on GnRH production, emphasizing potential consequences for neurodevelopment and cognitive function in infected individuals. Notably, GnRH neurons appear susceptible to SARS-CoV-2 infection, raising concerns about potential long-term effects on brain development and function. In conclusion, the intricate interplay between GnRH neurons, GnRH release, and the activity of various extrahypothalamic brain circuits reveals an unexpected role for these neuroendocrine neurons in the development and maintenance of sensory and cognitive functions, supplementing their established function in reproduction. Therapeutic interventions targeting the HPG axis, such as inhaled NO therapy in infancy and pulsatile GnRH administration in adults, emerge as promising approaches for addressing neurodevelopmental cognitive disorders and pathological aging.

Development and prenatal exposure to androgens alter potassium currents in gonadotropin-releasing hormone neurons from female mice.

Pulsatile gonadotropin-releasing hormone (GnRH) release is critical for reproduction. Disruptions to GnRH secretion patterns may contribute to polycystic ovary syndrome (PCOS). Prenatally androgenized (PNA) female mice recapitulate many neuroendocrine abnormalities observed in PCOS patients. PNA and development induce changes in spontaneous GnRH neuron firing rate, response to synaptic input, and the afterhyperpolarization potential of the action potential. We hypothesized potassium currents are altered by PNA treatment and/or development. Whole-cell patch-clamp recordings were made of transient and residual potassium currents of GnRH neurons in brain slices from 3-week-old and adult control and PNA females. At 3 weeks of age, PNA treatment increased transient current density versus controls. Development and PNA altered voltage-dependent activation and inactivation of the transient current. In controls, transient current activation and inactivation were depolarized at 3 weeks of age versus in adulthood. In GnRH neurons from 3-week-old mice, transient current activation and inactivation were more depolarized in control than PNA mice. Development and PNA treatment interacted to shift the time-dependence of inactivation and recovery from inactivation. Notably, in cells from adult PNA females, recovery was prolonged compared to all other groups. Activation of the residual current occurred at more depolarized membrane potentials in 3-week-old than adult controls. PNA depolarized activation of the residual current in adults. These findings demonstrate the properties of GnRH neuron potassium currents change during typical development, potentially contributing to puberty, and further suggest PNA treatment may both alter some typical developmental changes and induce additional modifications, which together may underlie aspects of the PNA phenotype. There was not any clinical trial involved in this work.

Effect of Caffeine on the Inflammatory-Dependent Changes in the GnRH/LH Secretion in a Female Sheep Model.

Caffeine is one of the most widely consumed psychoactive drugs in the world. It easily crosses the blood-brain barrier, and caffeine-interacting adenosine and ryanodine receptors are distributed in various areas of the brain, including the hypothalamus and pituitary. Caffeine intake may have an impact on reproductive and immune function. Therefore, in the present study performed on the ewe model, we decided to investigate the effect of peripheral administration of caffeine (30 mg/kg) on the secretory activity of the hypothalamic-pituitary unit which regulates the reproductive function in females during both a physiological state and an immune/inflammatory challenge induced by lipopolysaccharide (LPS; 400 ng/kg) injection. It was found that caffeine stimulated (p < 0.01) the biosynthesis of gonadotropin-releasing hormone (GnRH) in the hypothalamus of ewe under both physiological and inflammatory conditions. Caffeine also increased (p < 0.05) luteinizing hormone (LH) secretion in ewes in a physiological state; however, a single administration of caffeine failed to completely release the LH secretion from the inhibitory influence of inflammation. This could result from the decreased expression of GnRHR in the pituitary and it may also be associated with the changes in the concentration of neurotransmitters in the median eminence (ME) where GnRH neuron terminals are located. Caffeine and LPS increased (p < 0.05) dopamine in the ME which may explain the inhibition of GnRH release. Caffeine treatment also increased (p < 0.01) cortisol release, and this stimulatory effect was particularly evident in sheep under immunological stress. Our studies suggest that caffeine affects the secretory activity of the hypothalamic-pituitary unit, although its effect appears to be partially dependent on the animal's immune status.

Modulatory Effects of Ethinyl Estradiol Plus Drospirenone Contraceptive Pill on Spontaneous and GnRH-Induced LH Secretion

Background: Combined oral contraceptives (COCs) work mostly by preventing the pre-ovulatory gonadotropin surge, but the action of COCs on spontaneous episodic and GnRH (gonadotropin-releasing hormone)-induced LH (luteinizing hormone) release has been poorly evaluated. Oral contraceptives are known to act on the spontaneous hypothalamic–pituitary functions reducing both GnRH and gonadotropin release and blocking ovulation. Aim: To evaluate spontaneous and GnRH-induced LH release during both phases of the menstrual cycle or under the use of the contraceptive pill. Methods: A group of 12 women, subdivided into two groups, volunteered for the study. Group A (n = 6, controls) received no treatments, while Group B (n = 6) received a 21 + 7 combination of ethinyl-estradiol (EE) 30 µg + drospirenone (DRSP) 3 mg. Both groups were evaluated twice: Group A during follicular and luteal phases, Group B during pill assumption and during the suspension interval, performing a pulsatility test, GnRH stimulation test, and hormonal parameters evaluation. Spontaneous and GnRH-induced secretory pulses were evaluated, as well as the instantaneous secretory rate (ISR). Results: COC treatment lowered LH and FSH (follicle stimulating hormone) levels significantly if compared to the follicular phase of spontaneous cycles. During the suspension interval, hormone levels rapidly rose and became comparable to those of the follicular phase of the control group. The LH pulse frequency under COC administration during the suspension interval was similar to that observed during the follicular phase (2.6 ± 0.3 pulses/180 min and 2.3 ± 0.2 pulses/180 min, respectively). The GnRH-induced LH peaks were greater in amplitude and duration than those observed after ISR computation in both groups. The GnRH-induced LH release during the luteal phase of the control subjects was higher than in the follicular phase (51.2 ± 12.3 mIU/mL and 14.9 ± 1.8 mIU/mL, respectively). Conversely, subjects under COC showed a GnRH-induced LH response similar during COC and during the suspension interval. Conclusions: Our data support that the EE + DRSP preparation acts on both spontaneous pulsatile release and GnRH-induced LH release during the withdrawal period of the treatment, and that after 5–7 days from the treatment suspension, steroidal secretion from the ovary is resumed, such as that of androgens. This suggests that in hyperandrogenic patients, a suspension interval as short as 4 days might be clinically better.

Genetic and epigenetic aspects of the KISS1 and KISS1R genes in pubertal development and central precocious puberty: A review

The onset of puberty is a pivotal developmental milestone, and the release of gonadotropin-releasing hormone (GnRH) and luteinizing hormone is a key factor in the initiation of puberty. Both kisspeptin and its receptor (KISS1) and KISS1 receptor (KISS1R) play significant roles in regulating GnRH release, and consequently, the initiation of puberty. Central precocious puberty (CPP) is a condition in which the development of puberty is driven by the premature activation of the hypothalamic-pituitary-gonadal axis. In girls, CPP is primarily idiopathic, and genetic and epigenetic aspects of &lt;i&gt;KISS1&lt;/i&gt; and &lt;i&gt;KISS1R&lt;/i&gt; have been implicated in its etiology. This review aimed to provide an overview of the current knowledge regarding mutations and polymorphisms in &lt;i&gt;KISS1&lt;/i&gt; and &lt;i&gt;KISS1R&lt;/i&gt; associated with CPP. Additionally, this study provides a comprehensive review of the epigenetic regulation of the &lt;i&gt;KISS1&lt;/i&gt; gene in the context of puberty onset and CPP.

Troxerutin dampened hypothalamic neuroinflammation via microglial IL-22/IL-22R1/IRF3 activation in dihydrotestosterone-induced polycystic ovary syndrome rats

BackgroundPolycystic ovary syndrome (PCOS) is the most common reproductive-endocrine condition in premenopausal women. Troxerutin, a common clinical anti-coagulant agent, was shown to work as a strong IL-22 boosting agent counteracting the hyperactivated gonadotrophin releasing hormone (GnRH) neurons and heightened GnRH release, the neuroendocrine origin of PCOS with unknown mechanism in rats. Exploring the off-label use of troxerutin medication for PCOS is thus sorely needed. MethodsSerum IL-22 content and hypothalamic IL-22 protein were detected. Inflammatory factor levels in hypothalamo-pituitary were evaluated. Immunofluorescence staining was employed to determine the activation and M1/M2-prone polarization of microglia in arcuate hypothalamus and median eminence. RNA-sequencing and transcriptome analysis were applied to explore the potential driver of microglia M2-polarization in response to IL-22 bolstering effect. The function of microglial IL-22/IL-22R1/IRF3 system was further verified using in vivo knockdown of IL-22R1 and a potent IRF3 inhibitor in BV2 microglial cell lines in vitro. ResultsTroxerutin augmented serum IL-22 content, and its consequent spillover into the hypothalamus led to the direct activation of IL-22R1/IRF3 system on microglia, thereby promoted microglia M2 polarization in arcuate hypothalamus and median eminence, dampened hypothalamic neuroinflammation, inhibited hyperactive GnRH and rescued a breadth of PCOS-like traits in dihydrotestosterone (DHT) rats. The salutary effects of troxerutin treatment on hypothalamic neuroinflammation, microglial M1/2 polarization, GnRH secretion and numerous PCOS-like features were blocked by in vivo knockdown of IL-22R1. Moreover, evidence in vitro illustrated that IL-22 supplement to BV-2 microglia cell lines promoted M2 polarization, overproduction of anti-inflammatory marker and limitation of pro-inflammatory factors, whereas these IL-22 effects were blunted by geldanamycin, a potent IRF3 inhibitor. ConclusionHere, the present study reported the potential off-label use of troxerutin medication, a common clinical anti-coagulant agent and an endogenous IL-22 enhancer, for multiple purposes in PCOS. The rational underlying the application of troxerutin as a therapeutic choice in PCOS derived from its activity as an IL-22 memetic agent targeting the neuro-endocrine origin of PCOS, and its promotive impact on microglia M2 polarization via activating microglial IL-22R1/IRF3 system in the arcuate hypothalamus and median eminence of DHT female rats.

The functional study of a novel MKRN3 missense mutation associated with familial central precocious puberty.

Central precocious puberty (CPP) refers to a syndrome of early puberty initiation with a characteristic increase in the release of gonadotropin-releasing hormone (GnRH); therefore, it is also called GnRH-related precocious puberty. About a quarter of idiopathic central precocious puberty (ICPP) may be familial. Studies suggest that mutations of makorin ring finger protein 3 (MKRN3) can cause familial central precocious puberty (FCPP). In this report, we describe a Chinese female patient carrying a novel MKRN3 variant (c.980G>A/p.Arg327His) and presenting the CPP phenotype. This novel variant attenuated its own ubiquitination, degradation, and inhibition on the transcriptional and translational activity of GNRH1, which was verified through functional tests. We can consider this variant as a loss-of-function mutation, which subsides the inhibition of GnRH1-related signaling and gives rise to GnRH-related precocious puberty.

24 Effects of Gestational and Postweaning Nutrition on Brain Development and Puberty in Beef Heifers

Abstract Approximately 5 million beef heifers enter the U.S. cow herd annually, and their lifetime productivity is largely dependent upon their ability to attain puberty and produce a calf by 24 mo of age. However, a considerable proportion of heifers within existing U.S. production systems fail to achieve these goals, particularly in southern regions where Bos indicus-influenced cattle predominate. Consequently, managerial approaches are needed that employ nutritional programming of the reproductive neuroendocrine axis, while minimizing feeding costs and optimizing the consistent attainment of puberty by approximately 14 mo of age. Our previous research has clearly demonstrated that increased BW gain between 4 and 8 mo of age facilitates pubertal development by programming hypothalamic centers that regulate gonadotropin releasing-hormone (GnRH) secretion. Among the different metabolic hormones, leptin has a critical role in conveying nutritional information to the hypothalamus and controlling puberty. Two hypothalamic neuronal populations that express the orexigenic peptide neuropeptide Y (NPY) and the anorexigenic peptide alpha melanocyte-stimulating hormone (αMSH; a product of the POMC gene) are key components of afferent pathways that convey inhibitory (NPY) and excitatory (αMSH) inputs to GnRH and kisspeptin neurons. Our studies have demonstrated that short-term increases in dietary energy intake after early weaning at 4 mo of age result in epigenetic, structural, and functional modifications in these hypothalamic pathways to promote high-frequency, episodic release of GnRH and luteinizing hormone. However, integrating the foundational knowledge of metabolic imprinting of the brain for early puberty with issues related to lifetime performance is complex. One approach has been to employ a novel stair-step nutritional regimen involving alternating periods of dietary energy-restriction and re-feeding during juvenile development. This approach is designed to support early onset of puberty by imprinting functional alterations in the hypothalamus during key periods of brain development while optimizing other aspects of growth and performance. Finally, while several key processes of fetal brain development occur during late gestation, neither maternal undernutrition (BCS 3.0-3.5) nor maternal obesity (BCS 7.5-8.0) during the last two trimesters of gestation impacted the organization of hypothalamic neurocircuitries controlling GnRH/LH secretion or age at puberty in the heifer offspring. Collectively, these results indicate that the adult reproductive phenotype in Bos indicus-influenced females is resilient to significant degrees of nutritional stress imposed during prenatal development, particularly if early gestation (first trimester) is avoided. These findings are in contrast with reports in Bos taurus females in which late gestation nutritional extremes result in delayed puberty in heifer offspring, suggesting that potential genotype-specific differences may exist. Research support: USDA-NIFA-AFRI (2018-67015-27595 and 2021-67015-33676).

Endocrine profile of the kisspeptin receptor agonist MVT-602 in healthy premenopausal women with and without ovarian stimulation: results from 2 randomized, placebo-controlled clinical tricals

Kisspeptin is an essential regulator of hypothalamic gonadotropin-releasing hormone release and is required for physiological ovulation. Native kisspeptin-54 (KP54) can induce oocyte maturation during in vitro fertilization treatment, including in women at high risk of ovarian hyperstimulation syndrome. MVT-602 is a potent kisspeptin receptor agonist with prospective utility to treat anovulatory disorders by triggering oocyte maturation and ovulation during medically assisted reproduction (MAR). Currently, the endocrine profile of MVT-602 during ovarian stimulation is unreported. ObjectiveTo determine the endocrine profile of MVT-602 in the follicular phase of healthy premenopausal women (Phase-1 trial), and after minimal ovarian stimulation to more closely reflect the endocrine milieu encountered during MAR (Phase-2a trial). DesignTwo randomized, placebo-controlled, parallel group, dose-finding trials. SettingClinical trials unit, Netherlands. ParticipantsHealthy women aged 18-35 years, either without (Phase-1; n=24), or with ovarian stimulation (Phase-2a; n=75). InterventionsPhase-1: Single subcutaneous dose of MVT-602 (0.3, 1.0, or 3.0 μg) or placebo, (n=6 per dose).Phase-2a: Single subcutaneous dose of MVT-602 (0.1, 0.3, 1.0, or 3.0 μg; n=16-17 per dose), triptorelin 0.2 mg (n=5; active comparator), or placebo (n=5). Main Objectives and Outcome MeasuresPhase-1: Safety/tolerability; pharmacokinetics; pharmacodynamics (LH and other reproductive hormones).Phase-2a: Safety/tolerability; pharmacokinetics; pharmacodynamics (LH and other reproductive hormones); time to ovulation assessed by transvaginal ultrasound. ResultsIn both trials, MVT-602 was safe and well-tolerated across the entire dose-range. It was rapidly absorbed and eliminated, with a mean elimination half-life of 1.3-2.2 hours.In the Phase-2a trial, LH concentrations increased dose-dependently; mean maximum change from baseline of 82.4 IU/L at 24.8 hours was observed after administration of 3μg MVT-602 and remained above 15 IU/L for 33 hours. Time to ovulation following drug administration was 3.3-3.9 days (MVT-602), 3.4 days (triptorelin), and 5.5 days (placebo). Ovulation occurred within 5 days of administration in 100% (3 μg), 88% (1μg), 82% (0.3μg), and 75% (0.1μg), of women after MVT-602, 100% after triptorelin, and 60% after placebo. ConclusionsMVT-602 induces LH concentrations of similar amplitude and duration as the physiological mid-cycle LH surge with potential utility for induction of oocyte maturation and ovulation during MAR. Trial RegistrationsEUDRA-CT: 2017-003812-38, 2018-001379-20

The effect of NK3-Saporin injection within the arcuate nucleus on puberty, the LH surge, and the response to Senktide in female sheep†.

The timing of puberty onset is reliant on increased gonadotropin-releasing hormone (GnRH). This elicits a corresponding increase in luteinizing hormone (LH) due to a lessening of sensitivity to the inhibitory actions of estradiol (E2). The mechanisms underlying the increase in GnRH release likely involve a subset of neurons within the arcuate (ARC) nucleus of the hypothalamus that contain kisspeptin, neurokinin B (NKB), and dynorphin (KNDy neurons). We aimed to determine if KNDy neurons in female sheep are critical for: timely puberty onset; the LH surge; and the response to an intravenous injection of the neurokinin-3 receptor (NK3R) agonist, senktide. Prepubertal ewes received injections aimed at the ARC containing blank-saporin (control, n = 5) or NK3-saporin (NK3-SAP, n = 6) to ablate neurons expressing NK3R. Blood samples taken 3/week for 65days following surgery were assessed for progesterone to determine onset of puberty. Control ewes exhibited onset of puberty at 33.2 ± 3.9days post sampling initiation, whereas 5/6 NK3-SAP treated ewes didn't display an increase in progesterone. After an artificial LH surge protocol, surge amplitude was lower in NK3-SAP ewes. Finally, ewes were treated with senktide to determine if an LH response was elicited. LH pulses were evident in both groups in the absence of injections, but the response to senktide vs saline was similar between groups. These results show that KNDy cells are necessary for timely puberty onset and for full expresson of the LH surge. The occurrence of LH pulses in NK3-SAP treated ewes may indicate a recovery from an apulsatile state.

Integrative proteomic and phosphoproteomic analysis in the female goat hypothalamus to study the onset of puberty

BackgroundPuberty marks the end of childhood and achieve sexual maturation and fertility. The role of hypothalamic proteins in regulating puberty onset is unclear. We performed a comprehensive differential proteomics and phosphoproteomics analysis in prepubertal and pubertal goats to determine the roles of hypothalamic proteins and phosphoproteins during the onset of puberty.ResultsWe used peptide and posttranslational modifications peptide quantification and statistical analyses, and identified 69 differentially expressed proteins from 5,057 proteins and 576 differentially expressed phosphopeptides from 1574 phosphorylated proteins. Combined proteomic and phosphoproteomics, 759 correlated proteins were identified, of which 5 were differentially expressed only at the protein level, and 201 were only differentially expressed at the phosphoprotein level. Pathway enrichment analyses revealed that the majority of correlated proteins were associated with glycolysis/gluconeogenesis, Fc gamma R-mediated phagocytosis, focal adhesion, GABAergic synapse, and Rap1 signaling pathway. These pathways are related to cell proliferation, neurocyte migration, and promoting the release of gonadotropin-releasing hormone in the hypothalamus. CTNNB1 occupied important locations in the protein-protein interaction network and is involved in focal adhesion.ConclusionThe results demonstrate that the proteins differentially expression only at the protein level or only differentially expressed at the phosphoprotein level and their related signalling pathways are crucial in regulating puberty in goats. These differentially expressed proteins and phosphorylated proteins may constitute the proteomic backgrounds between the two different stages.

Serotonin stimulates female preoptic area kisspeptin neurons via activation of type 2 serotonin receptors in mice.

The neuroendocrine control of ovulation is orchestrated by neuronal circuits that ultimately drive the release of gonadotropin-releasing hormone (GnRH) from the hypothalamus to trigger the preovulatory surge in luteinizing hormone (LH) secretion. While estrogen feedback signals are determinant in triggering activation of GnRH neurons, through stimulation of afferent kisspeptin neurons in the rostral periventricular area of the third ventricle (RP3VKISS1 neurons), many neuropeptidergic and classical neurotransmitter systems have been shown to regulate the LH surge. Among these, several lines of evidence indicate that the monoamine neurotransmitter serotonin (5-HT) has an excitatory, permissive, influence over the generation of the surge, via activation of type 2 5-HT (5-HT2) receptors. The mechanisms through which this occurs, however, are not well understood. We hypothesized that 5-HT exerts its influence on the surge by stimulating RP3VKISS1 neurons in a 5-HT2 receptor-dependent manner. We tested this using kisspeptin neuron-specific calcium imaging and electrophysiology in brain slices obtained from male and female mice. We show that exogenous 5-HT reversibly increases the activity of the majority of RP3VKISS1 neurons. This effect is more prominent in females than in males, is likely mediated directly at RP3VKISS1 neurons and requires activation of 5-HT2 receptors. The functional impact of 5-HT on RP3VKISS1 neurons, however, does not significantly vary during the estrous cycle. Taken together, these data suggest that 5-HT2 receptor-mediated stimulation of RP3VKISS1 neuron activity might be involved in mediating the influence of 5-HT on the preovulatory LH surge.

Chronic estradiol exposure suppresses luteinizing hormone surge without affecting kisspeptin neurons and estrogen receptor alpha in anteroventral periventricular nucleus†.

Mammalian ovulation is induced by a luteinizing hormone surge, which is triggered by elevated plasma estrogen levels; however, chronic exposure to high levels of estradiol is known to inhibit luteinizing hormone secretion. In the present study, we hypothesized that the inhibition of the luteinizing hormone surge by chronic estradiol exposure is due to the downregulation of the estrogen receptor alpha in kisspeptin neurons at hypothalamic anteroventral periventricular nucleus, which is known as the gonadotropin-releasing hormone/luteinizing hormone surge generator. Animals exposed to estradiol for 2days showed an luteinizing hormone surge, whereas those exposed for 14days showed a significant suppression of luteinizing hormone. Chronic estradiol exposure did not affect the number of kisspeptin neurons and the percentage of kisspeptin neurons with estrogen receptor alpha or c-Fos in anteroventral periventricular nucleus, but it did affect the number of kisspeptin neurons in arcuate nucleus. Furthermore, chronic estradiol exposure did not affect gonadotropin-releasing hormone neurons. In the pituitary, 14-day estradiol exposure significantly reduced the expression of Lhb mRNA and LHβ-immunoreactive areas. Gonadotropin-releasing hormone-induced luteinizing hormone release was also reduced significantly by 14-day estradiol exposure. We revealed that the suppression of an luteinizing hormone surge by chronic estradiol exposure was induced in association with the significant reduction in kisspeptin neurons in arcuate nucleus, luteinizing hormone expression in the pituitary, and pituitary responsiveness to gonadotropin-releasing hormone, and this was not caused by changes in the estrogen receptor alpha-expressing kisspeptin neurons in anteroventral periventricular nucleus and gonadotropin-releasing hormone neurons, which are responsible for estradiol positive feedback.

Gonadal Feedback Alters the Relationship between Action Potentials and Hormone Release in Gonadotropin-Releasing Hormone Neurons in Male Mice.

In vertebrates, the pulsatile release of gonadotropin-releasing hormone (GnRH) from neurons in the hypothalamus triggers secretion of anterior pituitary gonadotropins, which activate steroidogenesis, and steroids in turn exert typically homeostatic negative feedback on GnRH release. Although long-term episodic firing patterns of GnRH neurons in brain slices resemble the pulsatile release of GnRH and LH in vivo, neither the relationship between GnRH neuron firing and release nor whether this relationship is influenced by gonadal feedback are known. We combined fast-scan cyclic voltammetry and patch-clamp to perform simultaneous measurements of neuropeptide release with either spontaneous action potential firing or in response to neuromodulator or action-potential-spike templates in brain slice preparations from male mice. GnRH release increased with higher frequency spontaneous firing to a point; release reached a plateau after which further increases in firing rate did not elicit further increased release. Kisspeptin, a potent GnRH neuron activator via a Gq-coupled signaling pathway, triggered GnRH release before increasing firing rate, whether globally perfused or locally applied. Increasing the number of spikes in an applied burst template increased release; orchidectomized mice had higher sensitivity to the increased action potential number than sham-operated mice. Similarly, Ca2+ currents triggered by these burst templates were increased in GnRH neurons of orchidectomized mice. These results suggest removal of gonadal feedback increases the efficacy of the stimulus-secretion coupling mechanisms, a phenomenon that may extend to other steroid-sensitive regions of the brain.SIGNIFICANCE STATEMENT Pulsatile secretion of GnRH plays a critical role in fertility. The temporal relationship between GnRH neuron action potential firing and GnRH release remains unknown as does whether this relationship is influenced by gonadal feedback. By combining techniques of fast-scan cyclic voltammetry and patch-clamp we, for the first time, monitored GnRH concentration changes during spontaneous and neuromodulator-induced GnRH neuron firing. We also made the novel observation that gonadal factors exert negative feedback on excitation-secretion coupling to reduce release in response to the same stimulus. This has implications for the control of normal fertility, central causes of infertility, and more broadly for the effects of sex steroids in the brain.

The opioid peptide leucine-enkephalin disrupts seasonal and gonadotropin-induced ovarian recrudescence in the gecko Hemidactylus frenatus

The enkephalins are known to regulate many physiological functions, including reproduction in vertebrates. However, the role of leucine-enkephalin (L-ENK) in the ovarian recrudescence activity of reptiles is not known. In the present study, we studied the influence of L-ENK on seasonal and FSH–induced ovarian recrudescence during the breeding and non-breeding phases of the cycle in the tropical and subtropical gecko Hemidactylus frenatus. In the first experiment, treatment with 5 and 25 μg L-ENK resulted in a dose-dependent inhibitory effect on the hypothalamic gonadotropin-releasing hormone (GnRH) neurons and ovary, as indicated by a significantly decreased percent area of GnRH-immunoreactive (GnRH-ir) fibres in the median eminence and pars distalis of the pituitary gland, concomitant with complete absence of stage V (late vitellogenic) follicles in the ovary compared to those of experimental controls. In the second experiment, administration of FSH to lizards in the regression phase stimulated the recruitment of stage IV and V (vitellogenic) follicles in contrast to their absence in initial controls or treatment controls. However, similar treatment of FSH in combination with 25 μg L-ENK did not result in the development of stage IV or V follicles. Together, these results suggest for the first time that treatment with 5 and 25 μg L-ENK exerts a dose-dependent inhibitory effect on the hypothalamic GnRH release into the median eminence and pituitary gland, leading to the blockade of ovarian recrudescence. These results also suggest a possible direct inhibitory effect of L-ENK at the level of the ovary in the gecko.

Dynamics of pulsatile activities of arcuate kisspeptin neurons in aging female mice.

Reproductive senescence is broadly observed across mammalian females, including humans, eventually leading to a loss of fertility. The pulsatile secretion of gonadotropin-releasing hormone (GnRH), which is essential for gonad function, is primarily controlled by kisspeptin neurons in the hypothalamic arcuate nucleus (ARCkiss), the pulse generator of GnRH. The pulsatility of GnRH release, as assessed by the amount of circulating gonadotropin, is markedly reduced in aged animals, suggesting that the malfunctions of ARCkiss may be responsible for reproductive aging and menopause-related disorders. However, the activity dynamics of ARCkiss during the natural transition to reproductive senescence remain unclear. Herein, we introduce chronic in vivo Ca2+ imaging of ARCkiss in female mice by fiber photometry to monitor the synchronous episodes of ARCkiss (SEskiss), a known hallmark of GnRH pulse generator activity, from the fully reproductive to acyclic phase over 1 year. During the reproductive phase, we find that not only the frequency, but also the intensities and waveforms of individual SEskiss, vary depending on the stage of the estrus cycle. During the transition to reproductive senescence, the integrity of SEskiss patterns, including the frequency and waveforms, remains mostly unchanged, whereas the intensities tend to decline. These data illuminate the temporal dynamics of ARCkiss activities in aging female mice. More generally, our findings demonstrate the utility of fiber-photometry-based chronic imaging of neuroendocrine regulators in the brain to characterize aging-associated malfunction.

Review: Unveiling the effect of beta-nerve growth factor on the reproductive function in llamas and cows.

The actions of the beta-nerve growth factor (β-NGF) on the neuroendocrine and reproductive system have challenged classical views on the control of reproductive function. After endometrial absorption, β-NGF triggers ovulation and promotes the development of functional corpora lutea in camelids. In this article, we review evidence showing that, in camelids, β-NGF exerts its actions by acting in both the hypothalamus and the ovary. In the hypothalamus, β-NGF may induce gonadotropin-releasing hormone (GnRH) release by interacting with neurons or glial cells expressing receptors for β-NGF. The LH surge occurs under the influence of ovarian estradiol and requires the release of GnRH into the portal vessels to reach the pituitary gland. In the ovary, β-NGF may be promoting the differentiation of follicular to luteal cells by modifying the steroidogenic profile of ovarian follicular cells in both camelids and ruminants. Although the mechanisms for these actions are largely undetermined, we aim to offer an update on the current understanding of the effects of β-NGF controlling reproductive function in camelids and ruminants.

Review: Gestational and postnatal nutritional effects on the neuroendocrine control of puberty and subsequent reproductive performance in heifers.

Pubertal attainment is an intricate biological process that involves maturation of the reproductive neuroendocrine axis and increased pulsatile release of gonadotropin-releasing hormone (GnRH) and luteinizing hormone. Nutrition is a critical environmental factor controlling the timing of puberty attainment. Nutrient restriction during early postnatal development delays puberty, whereas increased feed intake and adiposity during this period hasten pubertal maturation by imprinting the hypothalamus. Moreover, the dam's nutrition during gestation can program the neuroendocrine system in the developing fetus and has the potential to advance or delay puberty in the offspring. Leptin, a hormone produced primarily by adipose cells, plays an important role in communicating energy status to the brain and regulating sexual maturation. Leptin's regulation of GnRH release is mediated by an upstream neuronal network since GnRH neurons do not contain the leptin receptor. Two groups of neurons located in the arcuate nucleus of the hypothalamus that express neuropeptide Y (NPY), an orexigenic peptide, and alpha melanocyte-stimulating hormone (αMSH), an anorexigenic peptide, are central elements of the neural circuitry that relay inhibitory (NPY) and excitatory (αMSH) inputs to GnRH neurons. Moreover, KNDy neurons, neurons in the arcuate nucleus that co-express kisspeptin, neurokinin B (NKB), and dynorphin, also play a role in the metabolic regulation of puberty. Our studies in beef heifers demonstrate that increased rates of BW gain during early postweaning (4-9 mo of age) result in reduced expression of NPY mRNA, increased expression of proopiomelanocortin and kisspeptin receptor mRNA, reduced NPY inhibitory inputs to GnRH neurons, and increased excitatory αMSH inputs to KNDy neurons. Finally, our most recent data demonstrate that nutrition of the cow during the last two trimesters of gestation can also induce transcriptional and structural changes in hypothalamic neurocircuitries in the heifer progeny that likely persist long-term after birth. Managerial approaches, such as supplementation of the dam during gestation (fetal programming), creep feeding, early weaning, and stair-step nutritional regimens have been developed to exploit brain plasticity and advance pubertal maturation in heifers.