Kisspeptin Cells Research Articles

7869 Conditional deletion of androgen receptors from kisspeptin neurons only partially rescues the reproductive phenotype of a prenatal androgen-induced mouse model of polycystic ovary syndrome

Abstract Disclosure: A.G. Novak: None. A.V. Petriv: None. D.B. Lohr: None. K.M. Dillon: None. N.T. Neifert: None. A.M. Moore: None. Polycystic ovary syndrome (PCOS) is a prevalent cause of infertility in people with ovaries of reproductive age. Although the etiology of PCOS remains unclear, research in the clinic and with animal models suggests that hyperandrogenism (the presence of elevated circulating androgens) plays a significant role in the development of reproductive dysfunction, including irregular menstrual/estrous cycles and altered ovarian morphology. Although the site at which androgens disrupt the reproductive axis is unclear, neurons expressing kisspeptin are crucial regulators of the hypothalamic-pituitary-gonadal axis and express androgen receptors. Therefore, we hypothesize that androgen signaling at kisspeptin cells is a critical mediator of reproductive dysfunction in PCOS. To test this, we utilized Cre-lox technology to conditionally delete androgen receptors from kisspeptin cells (KARKO) in a commonly used mouse model of PCOS induced by using prenatal dihydrotestosterone treatment (prenatal androgen-treated (PNA) mice). To assess estrous cyclicity, vaginal cytology was examined daily for three weeks beginning at 50 days of age. Prenatal vehicle-treated (PNV) wild-type (WT) and PNV KARKO mice (n=6/group) exhibited regular estrous cycles with no significant differences in the percentage of time spent in proestrus, estrus, or metestrus/diestrus, indicating androgen receptors are not required to maintain fertility. As expected, PNA WT mice (n=6) rarely entered proestrus, spent significantly less time in estrus (p<0.05), and spent significantly more time in metestrus and diestrus (p<0.001) compared to the PNV groups (n=6/group, two-way ANOVA), replicating the previously reported acyclic phenotype of PNA mice. No significant differences in the percentage of time spent in cycle stages were detected between PNA WT and PNA KARKO (n=5) mice in this analysis, suggesting androgen signaling at kisspeptin cells is not required to induce acyclicity in PNA mice. Despite this acyclic phenotype, preliminary data using ovarian sections (5μm thickness) stained with hematoxylin and eosin revealed that the percentage of ovarian area composed of corpora lutea was significantly elevated in PNA KARKO mice (n=3) compared to PNA WT mice (n=4) (p<0.05, one-way ANOVA). Further, in this analysis, the area composed of corpora lutea in PNA KARKO mice was not significantly different from PNV WT and KARKO controls (n=6/group), suggesting PNA KARKO mice exhibit a comparable ovulation rate to fertile controls. Together, these data show that androgen signaling at kisspeptin cells partially contributes to the development of anovulation in PNA mice. However, additional mechanisms likely contribute to developing the full PCOS-like reproductive phenotype. Presentation: 6/1/2024

8602 Classical progesterone signaling in kisspeptin neurons is not required for negative feedback regulation of the GnRH pulse generator in mice

Abstract Disclosure: K.M. Dillon: None. D.B. Lohr: None. A.G. Novak: None. A.V. Petriv: None. N.T. Neifert: None. A.M. Moore: None. Mammalian reproductive function depends on the ability of progesterone to suppress pulsatile gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion in a homeostatic negative feedback loop. Impairments in progesterone negative feedback are associated with the development of polycystic ovary syndrome (PCOS), the leading cause of infertility for people with ovaries. Although previous research indicates cells upstream from GnRH neurons expressing the classical progesterone receptor (PR) are required for negative feedback, the identity of these cells and the mechanism by which they reduce GnRH/LH pulsatile secretion remain unclear. In this study, we aimed to address the hypothesis that PR expressed by a neuronal population in the arcuate nucleus expressing Kisspeptin, Neurokinin B, and Dynorphin, recently defined as the GnRH pulse generator, is required for negative feedback regulation of LH pulses. To achieve this, we utilized Cre-lox technology in mice to conditionally delete PR from kisspeptin cells (KPRKO mice) and assessed fecundity, LH pulsatility, and the mRNA expression of KNDy neuropeptides. Female KPRKO mice paired with B6 stud males for 3 months produced significantly fewer litters (p<0.05, n=5) and pups per litter (p<0.05) compared to WT controls (n=5) (students t-tests), recapitulating a previously reported subfertile phenotype in this model. In a separate cohort of animals, serial blood samples were collected every 6 minutes for 2 hours from the tail-tip of habituated WT and KPRKO mice in diestrus (n=6/group) and estrus (n=6/group), and an ultra-sensitive ELISA was used to detect LH pulses. However, compared to WT controls, KPRKO mice did not exhibit any significant changes in total and baseline LH release, pulse frequency or pulse amplitude in either cycle stages (p>0.05, students t-tests). At the completion of serial blood sampling, brains were collected from WT and KPRKO mice and RNAscope in situ hybridization was used to quantify mRNA for kisspeptin (KISS1) and dynorphin (PDYN), which are excitatory and inhibitory to GnRH/LH secretion, respectively. No differences in the number of KISS1- and PDYN-expressing cells or the number of KISS1 and PDYN mRNA transcripts were observed between WT and KPRKO mice (n=4/group, p>0.05, students t-tests). These data suggest that progesterone signaling in kisspeptin cells through PR is not essential for negative feedback regulation of LH pulses and is unlikely to contribute to the infertile phenotype of KPRKO mice. These results suggest that negative feedback may be maintained via non-classical mechanisms or an unidentified PR-positive neuronal population. Presentation: 6/1/2024

The deletion of nuclear progesterone receptors from kisspeptin cells does not impair negative feedback in female mice.

Reproductive function in mammals depends on the ability of progesterone to suppress pulsatile gonadotrophin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion in a homeostatic negative feedback loop. Previous research identified that cells upstream from GnRH neurons expressing the nuclear progesterone receptor (PGR) are required for progesterone-negative feedback. However, the identity of these cells and the mechanism by which they reduce GnRH/LH pulsatile secretion is unknown. We aimed to address the hypothesis that PGR expressed by a neural population in the arcuate nucleus recently identified as the GnRH pulse generator, cells expressing Kisspeptin, Neurokinin B, and Dynorphin (KNDy cells), mediate progesterone negative feedback. To achieve this, we utilized female mice with the PGR gene conditionally deleted from kisspeptin cells (KPRKO mice) and observed a substantial decrease in the percentage of KNDy neurons co-expressing PGR mRNA (11% in KPRKO mice versus 86% in wildtype mice). However, KPRKO mice did not display changes in the frequency or amplitude of LH pulses in diestrus or estrus, nor in the ability of exogenous progesterone to blunt a post-castration rise in LH. Further, mRNA expression of arcuate kisspeptin and dynorphin, which are excitatory and inhibitory to GnRH secretion, respectively, remained unaltered in KPRKO mice compared to wildtype controls. Together, these findings show that the near-complete loss of PGR signaling from KNDy cells does not impact negative feedback regulation of GnRH pulse generation in mice, suggesting that feedback through this receptor can occur via a small number of KNDy cells or a yet unidentified cell population.

Targeted inhibition of kisspeptin neurons reverses hyperandrogenemia and abnormal hyperactive LH secretion in a preclinical mouse model of polycystic ovary syndrome.

Do hyperactive kisspeptin neurons contribute to abnormally high LH secretion and downstream hyperandrogenemia in polycystic ovary syndrome (PCOS)-like conditions and can inhibition of kisspeptin neurons rescue such endocrine impairments? Targeted inhibition of endogenous kisspeptin neuron activity in a mouse model of PCOS reduced the abnormally hyperactive LH pulse secretion and hyperandrogenemia to healthy control levels. PCOS is a reproductive disorder characterized by hyperandrogenemia, anovulation, and/or polycystic ovaries, along with a hallmark feature of abnormal LH hyper-pulsatility, but the mechanisms underlying the endocrine impairments remain unclear. A chronic letrozole (LET; aromatase inhibitor) mouse model recapitulates PCOS phenotypes, including polycystic ovaries, anovulation, high testosterone, and hyperactive LH pulses. LET PCOS-like females also have increased hypothalamic kisspeptin neuronal activation which may drive their hyperactive LH secretion and hyperandrogenemia, but this has not been tested. Transgenic KissCRE+/hM4Di female mice or littermates Cre- controls were treated with placebo, or chronic LET (50 µg/day) to induce a PCOS-like phenotype, followed by acute (once) or chronic (2 weeks) clozapine-N-oxide (CNO) exposure to chemogenetically inhibit kisspeptin cells (n = 6 to 10 mice/group). Key endocrine measures, including in vivo LH pulse secretion patterns and circulating testosterone levels, were assessed before and after selective kisspeptin neuron inhibition and compared between PCOS groups and healthy controls. Alterations in body weights were measured and pituitary and ovarian gene expression was determined by qRT-PCR. Acute targeted inhibition of kisspeptin neurons in PCOS mice successfully lowered the abnormally hyperactive LH pulse secretion (P < 0.05). Likewise, chronic selective suppression of kisspeptin neuron activity reversed the previously high LH and testosterone levels (P < 0.05) down to healthy control levels and rescued reproductive gene expression (P < 0. 05). N/A. Ovarian morphology was not assessed in this study. Additionally, mouse models can offer mechanistic insights into neuroendocrine processes in PCOS-like conditions but may not perfectly mirror PCOS in women. These data support the hypothesis that overactive kisspeptin neurons can drive neuroendocrine PCOS-like impairments, and this may occur in PCOS women. Our findings complement recent clinical investigations using NKB receptor antagonists to lower LH in PCOS women and suggest that pharmacological dose-dependent modulation of kisspeptin neuron activity may be a valuable future therapeutic target to clinically treat hyperandrogenism and lower elevated LH in PCOS women. This research was supported by NIH grants R01 HD111650, R01 HD090161, R01 HD100580, P50 HD012303, R01 AG078185, and NIH R24 HD102061, and a pilot project award from the British Society for Neuroendocrinology. There are no competing interests.

SAT592 Sexually Dimorphic Modulation Of Arcuate Kisspeptin Neurons Activity In Response To NPY

Abstract Disclosure: N.D. Mansano: None. R. Frazao: None. Kisspeptin neurons are part of an intricate brain circuit that receives neuronal inputs related tonutritional status from at least three neuronal populations, including NPY/AgRP and POMC neurons of the arcuate nucleus of the hypothalamus (ARH) and cells in the ventral premammillary nucleus. Its well-known action on the HPG axis, hypothalamic kisspeptin neurons also modulate the activity of specific neurons of the paraventricular and the dorsomedial nuclei of the hypothalamus, regions involved in the regulation of appetite and energy expenditure. However, it is not completely elucidated if NPY can influence the HPG axis through the modulation of kisspeptin neuron activity. To determine the NPY effect in the hypothalamic kisspeptin neurons we performed whole-cell patch-clamp recordings. The hypothalamic slices were obtained from the adult Kiss1/hrGFP female (diestrus-stage) or male mice. Kisspeptin cells located in the arcuate nucleus of the hypothalamus (ARHkisspeptin) and at the anteroventral periventricular and rostral periventricular nuclei (AVPV/PeNkisspeptin) were recorded. On female mice, ARHkisspeptin neurons exhibited a rest membrane potential (RMP) average of -52.5±2.5 mV (-40 to -62 mV) and 1.3±0.2 GΩ input resistance (IR). NPY induced the hyperpolarization of 45% of the ARHkisspeptin neurons (n= 9 of 20 cells, out of 9 mice). ARHkisspeptin neuron’s RMP hyperpolarization was followed by a significant decrease in IR and frequency of action potentials (fAPS). In the AVPV/PeNkisspeptin neurons showed RMP average of -69.6±1.9 mV (-51 to - 88 mV) and IR of 0.8±0.09 GΩ (n=18 cells out of 9 mice). No significant NPY-induced effect was noted when recordings were performed on AVPV/PeNkisspeptin cells from female mice. To elucidate if the ARHkisspeptin neuron’s hyperpolarization depends on action potentials (APs) mediated by synaptic transmission we further evaluated NPY effects in the presence of TTX and amino acid receptor antagonists (6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10μM), DL-2-amino-5-phosphonopentanoic acid (AP5, 50μM), and picrotoxin (50μM). NPY induced no effect on ARHkisspeptin neuron’s RMP, and IR in the presence of TTX and amino acid receptor antagonists. Suggesting that NPY-induced hyperpolarization of ARHkisspeptin neurons is an indirect effect. To further verify whether sex must be considered as a biological variable to study the NPY effect, we recorded ARHkisspeptin cells from male mice. ARHkisspeptin neurons recorded from male mice exhibited RPM average of -55.6±1.5 (-45 to -66 mV) and IR of 1.5±0.15 GΩ (n= 13 cells out of 10 mice). Interestingly, NPY induced no effect on RMP, IR, or fAPs in male animals. In conclusion, our results suggest that NPY-induced hyperpolarization of ARHkisspeptin cells depends on AP’s mediate synaptic transmission, as a sex-specific effect. Presentation: Saturday, June 17, 2023

OR17-3 Immortalized AVPV Kisspeptin Neurons Transiently Express GnRH Receptors under Specific Sex Steroid Conditions in vitro

Abstract In female animals, ovarian estradiol (E2) acts as negative feedback inhibition of GnRH secretion and positive feedback stimulation at ovulation. Both E2-regulated mechanisms work via stimulation or repression of distinct neuronal populations of Kisspeptin (KP)-synthesizing neurons. While E2 clearly stimulates AVPV KP neurons to increase kiss1 expression during the preovulatory surge, mechanisms required for optimal potentiation of maximal GnRH surge release remain incompletely characterized. We used two immortalized KP cell lines demonstrating increased kiss1 expression under high E2 (KTaV-3), or kiss1 suppression under low E2 (KTaR-1), to probe GnRH receptor (GnRHR) expression levels under E2 conditions of varying doses and times of exposure. Additionally, we have begun to investigate transcriptional mechanisms underlying gnrhr expression in KP neurons in vitro. AVPV-derived KTaV-3 cells were treated with either E2 alone (5-100pM) or E2 with progesterone (4-40nM) for varying durations (4-96h), either constitutively or under modulating levels approximating changes found during the murine estrous cycle. All treatments were undertaken in serum-free (SF) media (DMEM) following a 50% serum synchronization. After total RNA isolation, cDNAs were probed with mouse gnrhr primers. Results reveal that gnrhr expression, not observed in typical growth media conditions, is induced in KTaV-3 cells to varying degrees dependent on prior E2/P4 exposure. Following shorter treatment times (12-24h), proestrus levels (100pM E2; 4nM P4) of sex steroids noticeably induced gnrhr, while exposure of KTaV-3 cells to a 96h estrous cycle revealed maximal gnrhr expression at the diestrus II→proestrus transition, with exposure cycles concluding with declining E2/rising P4 exhibiting no to minimal gnrhr expression. These results suggest that maximal KP neuronal expression of gnrhr may occur prior to the E2 peak on proestrus, potentially preparing AVPV KP neurons for GnRH surge release 12-16h later. Similar steroid conditions also increased dlx3 expression, a transcription factor required for GnRHR in pituitary, only in KTaV-3 cells. In KTaR-1 cells, gnrhr expression was observed only following E2 decreases, while dlx3 was constitutively elevated. Since cFos/JunB complex formation on the gnrhr promoter has been implicated in gonadotrope GnRHR levels, we explored how transfection of these transcription factors might affect gnrhr expression in KTaV-3 cells. Preliminary results reveal that fos/jun co-transfection stimulates gnrhr expression maximally at intermediate (DII) E2 levels, while higher E2 levels are required with fos or jun expression alone. While reciprocal GnRH-Kisspeptin connections have not yet been observed in vivo, these results suggest KP neurons may possess the capacity to respond to intrahypothalamic GnRH only in certain estrous cycle stages, by differentially modulating GnRH receptivity in the AVPV. These findings supplement the prevailing model of feed-forward KP regulation of GnRH with potential short-loop KP-GnRH positive feedback regulation, providing an increased understanding of feedback mechanisms required for normal neuroendocrine regulation of reproduction. Presentation: Sunday, June 12, 2022 11:30 a.m. - 11:45 a.m.

Conditional Deletion of KOR (Oprk1) in Kisspeptin Cells Does Not Alter LH Pulses, Puberty, or Fertility in Mice.

Classic pharmacological studies suggested that endogenous dynorphin-KOR signaling is important for reproductive neuroendocrine regulation. With the seminal discovery of an interconnected network of hypothalamic arcuate neurons co-expressing kisspeptin, neurokinin B, and dynorphin (KNDy neurons), the KNDy hypothesis was developed to explain how gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) pulses are generated. Key to this hypothesis is dynorphin released from KNDy neurons acting in a paracrine manner on other KNDy neurons via kappa opioid receptor (KOR) signaling to terminate neural "pulse" events. While in vitro evidence supports this aspect of the KNDy hypothesis, a direct in vivo test of the necessity of KOR signaling in kisspeptin neurons for proper LH secretion has been lacking. We therefore conditionally knocked out KOR selectively from kisspeptin neurons of male and female mice and tested numerous reproductive measures, including in vivo LH pulse secretion. Surprisingly, despite validating successful knockout of KOR in kisspeptin neurons, we found no significant effect of kisspeptin cell-specific deletion of KOR on any measure of puberty, LH pulse parameters, LH surges, follicle-stimulating hormone (FSH) levels, estrous cycles, or fertility. These outcomes suggest that the KNDy hypothesis, while sufficient normally, may not be the only neural mechanism for sculpting GnRH and LH pulses, supported by recent findings in humans and mice. Thus, besides normally acting via KOR in KNDy neurons, endogenous dynorphin and other opioids may, under some conditions, regulate LH and FSH secretion via KOR in non-kisspeptin cells or perhaps via non-KOR pathways. The current models for GnRH and LH pulse generation should be expanded to consider such alternate mechanisms.

Crosstalk between kisspeptin and gonadotropin-inhibitory hormone in the silence of puberty: preclinical evidence from a calcium signaling study

Kisspeptin and gonadotropin-inhibitory hormone (GnIH) are among suggested neuroendocrine modulators of reproductive function. Intracellular calcium signaling is a critical component in the regulation of a variety of physiological and pathological processes including neurotransmitter release, and, therefore, can be used as signaling indicator for investigating the involvement of kisspeptin, GnIH, and gonadotropin-releasing hormone (GnRH) release. Hence, this study investigated the effects of kisspeptin and GnIH on calcium signaling using immortalized hypothalamic cells (rHypoE-8) as a model. Kisspeptin neurons were loaded with the ratiometric calcium dye (Fura-2 AM, 1 μmol) and intracellular free calcium ([Ca2+]i) responses were quantified using digital fluorescence imaging system. Kisspeptin-10 (100, 300, and 1000 nM) caused a significant increase in [Ca2+]i in rHypoE-8 cells (n = 58, n = 64, and n = 49, respectively, p < 0.001). The kisspeptin receptor antagonist, P234, inhibited the calcium responses to kisspeptin (p < 0.001, n = 32). GnIH (100 and 1000 nM), alone, did not cause any significant change in the mean basal [Ca2+]i levels in kisspeptin cells, but GnIH attenuated the kisspeptin-evoked [Ca2+]i transients (n = 47, p < 0.001). This novel findings of [Ca2+]i signaling in in vitro setting implicate that kisspeptin and GnIH may exert their effects on hypothalamus–pituitary–gonadal (HPG) axis by modulating kisspeptin neurons. These results also implicate that kisspeptin neurons may have an autocrine regulation.

Socs3 ablation in kisspeptin cells partially prevents lipopolysaccharide-induced body weight loss

Many cytokines have been proposed to regulate reproduction due to their actions on hypothalamic kisspeptin cells, the main modulators of gonadotropin-releasing hormone (GnRH) neurons. Hormones such as leptin, prolactin and growth hormone are good examples of cytokines that lead to Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway activation, consequently exerting effects in kisspeptin neurons. Different studies have investigated how specific components of the JAK/STAT signaling pathway affect the functions of kisspeptin cells, but the role of the suppressor of cytokine signaling 3 (SOCS3) in mediating cytokine actions in kisspeptin cells remains unknown. Cre-Loxp technology was used in the present study to ablate Socs3 expression in kisspeptin cells (Kiss1/Socs3-KO). Then, male and female control and Kiss1/Socs3-KO mice were evaluated for sexual maturation, energy homeostasis features, and fertility. It was found that hypothalamic Kiss1 mRNA expression is significantly downregulated in Kiss1/Socs3-KO mice. Despite reduced hypothalamic Kiss1 mRNA content, these mice did not present any sexual maturation or fertility impairments. Additionally, body weight gain, leptin sensitivity and glucose homeostasis were similar to control mice. Interestingly, Kiss1/Socs3-KO mice were partially protected against lipopolysaccharide (LPS)-induced body weight loss. Our results suggest that Socs3 ablation in kisspeptin cells partially prevents the sickness behavior induced by LPS, suggesting that kisspeptin cells can modulate energy metabolism in mice in certain situations.

Norepinephrine modulation of heat dissipation in female rats lacking estrogen.

Postmenopausal hot flushes are caused by lack of estradiol (E2) but their neuroendocrine basis is still poorly understood. Here, we investigated the interrelationship between norepinephrine and hypothalamic neurons, with emphasis on kisspeptin neurons in the arcuate nucleus (ARC), as a regulatory pathway in the vasomotor effects of E2. Ovariectomized (OVX) rats displayed increased tail skin temperature (TST), and this increase was prevented in OVX rats treated with E2 (OVX + E2). Expression of Fos in the hypothalamus and the number of ARC kisspeptin neurons coexpressing Fos were increased in OVX rats. Likewise, brainstem norepinephrine neurons of OVX rats displayed higher Fos immunoreactivity associated with the increase in TST. In the ARC, the density of dopamine-ß-hydroxylase (DBH)-immunoreactive (ir) fibers was not altered by E2 but, importantly, DBH-ir terminals were found in close apposition to kisspeptin cells, revealing norepinephrine inputs to ARC kisspeptin neurons. Intracerebroventricular injection of the α2-adrenergic agonist clonidine (CLO) was used to reduce central norepinephrine release, confirmed by the decreased 3-methoxy-4-hydroxyphenylglycol/norepinephrine ratio in the preoptic area and ARC. Accordingly, CLO treatment in OVX rats reduced ARC Kiss1 mRNA levels and TST to the values of OVX + E2 rats. Conversely, CLO stimulated Kiss1 expression in the anteroventral periventricular nucleus (AVPV) and increased luteinizing hormone secretion. These findings provide evidence that augmented heat dissipation in OVX rats involves the increase in central norepinephrine that modulates hypothalamic areas related to thermoregulation, including ARC kisspeptin neurons. This neuronal network is suppressed by E2 and its imbalance may be implicated in the vasomotor symptoms of postmenopausal hot flushes.

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 17β-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.

Expression and co-localization of RFRP-3 and kisspeptin during breeding and non-breeding season in the hypothalamus of male rhesus monkey (Macaca mulatta).

ProposeThe mechanism that underpins how RFRP‐3 and kisspeptin interacts are not fully understood in higher primates. This study therefore set out to assess RFRP‐3 and kisspeptin expression and their morphological interactions in the breeding, and in the non‐breeding period in monkey hypothalamus.MethodsEight mature male macaques (Macaca mulatta) in the breeding season (February; n = 4) and non‐breeding season (June; n = 4) were used. To reveal the expression and co‐localization of RFRP‐3 and kisspeptin, double‐labeled immunohistochemistry was performed. Testicular volume, sperm count, and plasma testosterone level were also measured to validate the breeding and non‐breeding paradigms.ResultsTesticular volume, plasma testosterone level, and sperm count showed a significant reduction during non‐breeding season. The number of kisspeptin‐positive cells was significantly increased during the breeding season (p < 0.05), whereas more RFRP‐3‐positive cell bodies were seen in the non‐breeding season (p < 0.01). Close contacts of RFRP‐3 fibers with kisspeptin cells showed no significant difference (p > 0.05) across seasons. However, co‐localization of RFRP‐3‐ir cell bodies onto kisspeptin IR cell bodies showed a statistical increase (p < 0.01) in non‐breeding season.ConclusionIn higher primates, RFRP‐3 decreases kisspeptin drives from the same cells to GnRH neurons in an autocrine manner causing suppression of the reproductive axis during the non‐breeding period.

Reproductive Deficits Induced by Prenatal Antimüllerian Hormone Exposure Require Androgen Receptor in Kisspeptin Cells.

Polycystic ovary syndrome (PCOS) is a common reproductive disorder characterized by elevated androgens and antimüllerian hormone (AMH). These hormones remain elevated throughout pregnancy, and potential effects of hormone exposure on offspring from women with PCOS remain largely unexplored. Expanding on recent reports of prenatal AMH exposure in mice, we have fully characterized the reproductive consequences of prenatal AMH (pAMH) exposure throughout the lifespan of first- and second-generation offspring of both sexes. We also sought to elucidate mechanisms underlying pAMH-induced reproductive effects. There is a known reciprocal relationship between AMH and androgens, and in PCOS and PCOS-like animal models, androgen feedback is dysregulated at the level of the hypothalamus. Kisspeptin neurons express androgen receptors and play a critical role in sexual development and function. We therefore hypothesized that pAMH-induced reproductive phenotypes would be mediated by androgen signaling at the level of kisspeptin cells. We tested the pAMH model in kisspeptin-specific androgen receptor knockout (KARKO) mice and found that virtually all pAMH-induced phenotypes assayed are eliminated in KARKO offspring compared to littermate controls. By demonstrating the necessity of androgen receptor in kisspeptin cells to induce pAMH phenotypes, we have advanced understanding of the interactions between AMH and androgens in the context of prenatal exposure, which could have significant implications for children of women with PCOS.

Progesterone Receptors in AVPV Kisspeptin Neurons Are Sufficient for Positive Feedback Induction of the LH Surge.

Kisspeptin, encoded by Kiss1, stimulates gonadotropin-releasing hormone neurons to govern reproduction. In female rodents, estrogen-sensitive kisspeptin neurons in the rostral anteroventral periventricular (AVPV) hypothalamus are thought to mediate estradiol (E2)-induced positive feedback induction of the preovulatory luteinizing hormone (LH) surge. AVPV kisspeptin neurons coexpress estrogen and progesterone receptors (PGRs) and are activated during the LH surge. While E2 effects on kisspeptin neurons have been well studied, progesterone's regulation of kisspeptin neurons is less understood. Using transgenic mice lacking PGR exclusively in kisspeptin cells (termed KissPRKOs), we previously demonstrated that progesterone action specifically in kisspeptin cells is essential for ovulation and normal fertility. Unlike control females, KissPRKO females did not generate proper LH surges, indicating that PGR signaling in kisspeptin cells is required for positive feedback. However, because PGR was knocked out from all kisspeptin neurons in the brain, that study was unable to determine the specific kisspeptin population mediating PGR action on the LH surge. Here, we used targeted Cre-mediated adeno-associated virus (AAV) technology to reintroduce PGR selectively into AVPV kisspeptin neurons of adult KissPRKO females, and tested whether this rescues occurrence of the LH surge. We found that targeted upregulation of PGR in kisspeptin neurons exclusively in the AVPV is sufficient to restore proper E2-induced LH surges in KissPRKO females, suggesting that this specific kisspeptin population is a key target of the necessary progesterone action for the surge. These findings further highlight the critical importance of progesterone signaling, along with E2 signaling, in the positive feedback induction of LH surges and ovulation.

Kisspeptin Neurons Excitability Is Not Modulate by Fasting

Abstract Kisspeptin is the most important neuromodulator of gonadotrophin releasing hormone neurons. Hypothalamic Kiss1-expressing neurons can be found in the anteroventral periventricular and rostral periventricular nuclei (AVPV/PeN) and in the arcuate nucleus of the hypothalamus (ARH). Food intake and energy balance are modulated by leptin, which acts primarily in the ARH. Leptin acts inhibiting orexigen neurotransmitters and stimulating anorectic neurotransmitters. Few ARH kisspeptin cells (10-15%) coexpress the leptin receptor and have their resting membrane potential (RMP) depolarized by leptin. Since the reproductive axis is sensitive to metabolic disorders, in the present study we used immunohistochemical and electrophysiological approach to understand the effects of fasting on kisspeptin neurons activity. To determine whether AVPV/PeN and ARH kisspeptin neurons activity are modulated by fasting, Kiss1/hrGFP female mice were fed regular chow ad libitum or fasted for 24 hours. Mice were euthanized after 24h. Fasting induced a significant increase of Fos protein on the ARH nucleus, as expected (rostral level of ARH, control: 19.3 ± 3.5 vs fasted: 81.3 ± 3.5 cells; caudal level, control: 15.7 ± 1.3 vs fasted: 103.3 ± 6.2 cells, P &amp;lt;0.0001, n=3/6 mice per group). Despite the significant increase of Fos-immunoreactive in the ARH nucleus, kisspeptin neurons did not co-express this neuronal marker. Next, we determined the RMP of kisspeptin cells obtained from control or fasted mice. Compared to the control group (AVPV/PeN, -56.5 ± 2.0 mV, n=13 cells from 4 animals; ARH, - 49.8 ± 2.4 mV, n= 9 cells from 6 animals), fasting was not sufficient to induce changes in RMP (AVPV/PeN, -56.1 ± 4.6 mV, n=6 cells from 3 animals, P=0.95; ARH, - 50.9 ± 2.6 mV, n=9 cells from 3 animals, P=0.11). The frequency (freq) and amplitude (amp) of the excitatory postsynaptic currents acting on kisspeptin neurons was further investigated. No significant difference was observed by comparing data obtained from control (AVPV/PeN: freq 0.76 ± 0.1 Hz, n=19; amp, 28.6 ± 1.8 pA, n=16; ARH: freq 0.36 ± 0.1 Hz, n=15; amp, 28.5 ± 2 pA, n=15) and fasted mice (AVPV/PeN: freq 0.7 ± 0.1 Hz, n=18; amp, 28.5 ± 1.1 pA; n=18; ARH: freq 0.5 ± 0.2 Hz, n=24; amp, 27.9 ± 1.3 pA; n=24; P &amp;gt; 0.5, 7/9 animals per group). Considering that 24hr fasting is not enough to inhibit estrous cyclicity, even though it is sufficient to induce a significant reduction of hypothalamic Kiss1 mRNA expression (unpublished data), our results suggest that prolonged periods of food restriction may be required to disturb excitatory inputs into kisspeptin cells.

Estrogen Regulation of the Molecular Phenotype and Active Translatome of AVPV Kisspeptin Neurons.

In females, ovarian estradiol (E2) exerts both negative and positive feedback regulation on the neural circuits governing reproductive hormone secretion, but the cellular and molecular mechanisms underlying this remain poorly understood. In rodents, estrogen receptor α-expressing kisspeptin neurons in the hypothalamic anteroventral periventricular region (AVPV) are prime candidates to mediate E2 positive feedback induction of preovulatory gonadotropin-releasing hormone and luteinizing hormone (LH) surges. E2 stimulates AVPV Kiss1 expression, but the full extent of estrogen effects in these neurons is unknown; whether E2 stimulates or inhibits other genes in AVPV Kiss1 cells has not been determined. Indeed, understanding of the function(s) of AVPV kisspeptin cells is limited, in part, by minimal knowledge of their overall molecular phenotype, as only a few genes are currently known to be co-expressed in AVPV Kiss1 cells. To provide a more detailed profiling of co-expressed genes in AVPV Kiss1 cells, including receptors and other signaling factors, and test how these genes respond to E2, we selectively isolated actively translated mRNAs from AVPV Kiss1 cells of female mice and performed RNA sequencing (RNA-seq). This identified >13 000 mRNAs co-expressed in AVPV Kiss1 cells, including multiple receptor and ligand transcripts positively or negatively regulated by E2. We also performed RNAscope to validate co-expression of several transcripts identified by RNA-seq, including Pdyn (prodynorphin), Penk (proenkephalin), Vgf (VGF), and Cartpt (CART), in female AVPV Kiss1 cells. Given the important role of AVPV kisspeptin cells in positive feedback, E2 effects on identified genes may relate to the LH surge mechanism and/or other physiological processes involving these cells.

Distribution of GnRH and Kisspeptin Immunoreactivity in the Female Llama Hypothalamus.

Llamas are induced non-reflex ovulators, which ovulate in response to the hormonal stimulus of the male protein beta-nerve growth factor (β-NGF) that is present in the seminal plasma; this response is dependent on the preovulatory gonadotrophin-releasing hormone (GnRH) release from the hypothalamus. GnRH neurones are vital for reproduction, as these provide the input that controls the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the pituitary gland. However, in spontaneous ovulators, the activity of GnRH cells is regulated by kisspeptin neurones that relay the oestrogen signal arising from the periphery. Here, we investigated the organisation of GnRH and kisspeptin systems in the hypothalamus of receptive adult female llamas. We found that GnRH cells exhibiting different shapes were distributed throughout the ventral forebrain and some of these were located in proximity to blood vessels; sections of the mediobasal hypothalamus (MBH) displayed the highest number of cells. GnRH fibres were observed in both the organum vasculosum laminae terminalis (OVLT) and median eminence (ME). We also detected abundant kisspeptin fibres in the MBH and ME; kisspeptin cells were found in the arcuate nucleus (ARC), but not in rostral areas of the hypothalamus. Quantitative analysis of GnRH and kisspeptin fibres in the ME revealed a higher innervation density of kisspeptin than of GnRH fibres. The physiological significance of the anatomical findings reported here for the ovulatory mechanism in llamas is still to be determined.

Chapter 17 - The human hypothalamic kisspeptin system: Functional neuroanatomy and clinical perspectives

In mammals, kisspeptin neurons are the key components of the hypothalamic neuronal networks that regulate the onset of puberty, account for the pulsatile secretion of gonadotropin-releasing hormone (GnRH) and mediate negative and positive estrogen feedback signals to GnRH neurons. Being directly connected anatomically and functionally to the hypophysiotropic GnRH system, the major kisspeptin cell groups of the preoptic area/rostral hypothalamus and the arcuate (or infundibular) nucleus, respectively, are ideally positioned to serve as key nodes which integrate various types of environmental, endocrine, and metabolic signals that can influence fertility. This chapter provides an overview of the current state of knowledge on the anatomy, functions, and plasticity of brain kisspeptin systems based on the wide literature available from different laboratory and domestic species. Then, the species-specific features of human hypothalamic kisspeptin neurons are described, covering their topography, morphology, unique neuropeptide content, plasticity, and connectivity to hypophysiotropic GnRH neurons. Some newly emerging roles of central kisspeptin signaling in behavior and finally, clinical perspectives, are discussed.

Kisspeptin Neurons in the Infundibular Nucleus of Ovariectomized Cats and Dogs Exhibit Unique Anatomical and Neurochemical Characteristics.

Neurons co-synthesizing kisspeptin (KP), neurokinin B (NKB), and dynorphin (“KNDy neurons”) in the hypothalamic arcuate/infundibular nucleus (INF) form a crucial component of the gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) “pulse generator.” The goal of our study was to characterize KP neuron distribution, neuropeptide phenotype and connectivity to GnRH cells in ovariectomized (OVX) dogs and cats with immunohistochemistry on formalin-fixed hypothalamic tissue sections. In both species, KP and NKB neurons occurred in the INF and the two cell populations overlapped substantially. Dynorphin was detected in large subsets of canine KP (56%) and NKB (37%) cells and feline KP (64%) and NKB (57%) cells; triple-labeled (“KNDy”) somata formed ∼25% of all immunolabeled neurons. Substance P (SP) was present in 20% of KP and 29% of NKB neurons in OVX cats but not dogs, although 26% of KP and 24% of NKB neurons in a gonadally intact male dog also contained SP signal. Only in cats, cocaine- and amphetamine regulated transcript was also colocalized with KP (23%) and NKB (7%). In contrast with reports from mice, KP neurons did not express galanin in either carnivore. KP neurons innervated virtually all GnRH neurons in both species. Results of this anatomical study on OVX animals reveal species-specific features of canine and feline mediobasal hypothalamic KP neurons. Anatomical and neurochemical similarities to and differences from the homologous KP cells of more extensively studied rodent, domestic and primate species will enhance our understanding of obligate and facultative players in the molecular mechanisms underlying pulsatile GnRH/LH secretion.

Perinatal Exposure to Methoxychlor Affects Reproductive Function and Sexual Behavior in Mice.

Numerous chemicals derived from human activity are now disseminated in the environment where their exert estrogenic endocrine disrupting effects, and therefore represent major health concerns. The present study explored whether Methoxychlor (MXC), an insecticide with xenoestrogens activities, given during the perinatal period (from gestational day 11 to postnatal day 8) and at an environmentally dose [20 μg/kg (body weight)/day], would affect reproductive physiology and sexual behavior of the offspring in mice. While MXC exposure did not induce any differences in the weight gain of animals from birth to 4 months of age, a clear difference (although in opposite direction according to the sexes) was observed on the anogenital distance between intact and exposed animals. A similar effect was also observed on preputial separation and vaginal opening, which reflects, respectively, in males and females, puberty occurrence. The advanced puberty observed in females was associated with an enhanced expression of kisspeptin cells in the anteroventral periventricular region of the medial preoptic area. Exposure to MXC did not induce in adult females changes in the estrous cycle or in the weight of the female reproductive tract. By contrast, males showed reduced weight of the epididymis and seminiferous vesicles associated with reduced testosterone levels and seminiferous tubule diameter. We also showed that both males and females showed deficits in mate preference tests. As a whole, our results show that MXC impacts reproductive outcomes.