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.

RNA-sequencing of AVPV and ARH reveals vastly different temporal and transcriptomic responses to estradiol in the female rat hypothalamus.

In females, estrogens have two main modes of action relating to gonadotropin secretion: positive feedback and negative feedback. Estrogen positive and negative feedback are controlled by different regions of the hypothalamus: the preoptic area/anterior portion (mainly the anteroventral periventricular nucleus, AVPV) of the hypothalamus is associated with estrogen positive feedback while the mediobasal hypothalamus (mainly the arcuate nucleus of the hypothalamus, ARH), is associated with estrogen negative feedback. In this study, we examined the temporal pattern of gene transcription in these two regions following estrogen treatment. Adult, ovariectomized, Long Evans rats received doses of estradiol benzoate (EB) or oil every 4 days for 3 cycles. On the last EB priming cycle, hypothalamic tissues were dissected into the AVPV+ and ARH+ at 0 hrs (baseline/oil control), 6 hrs, or 24 hrs after EB treatment. RNA was extracted and sequenced using bulk RNA sequencing. Differential gene analysis, gene ontology, and weighted correlation network analysis (WGCNA) was performed. Overall, we found that the AVPV+ and ARH+ respond differently to estradiol stimulation. In both regions, estradiol treatment resulted in more gene up-regulation than down-regulation. S100g was very strongly up-regulated by estradiol in both regions at 6 and 24 hrs after EB treatment. In the AVPV+ the highest number of differentially expressed genes occurred 24 hrs after EB. In the ARH+, the highest number of genes differentially expressed by EB occurred between 6 and 24 hrs after EB, while in the AVPV+, the fewest genes changed their expression between these time points, demonstrating a temporal difference in the way that EB regulates transcription these two areas. Several genes strongly implicated in gonadotropin release were differentially affected by estradiol including Esr1, encoding estrogen receptor-α and Kiss1, encoding kisspeptin. As an internal validation, Kiss1 was up-regulated in the AVPV+ and down-regulated in the ARH+. Gene network analysis revealed the vastly different clustering of genes modulated by estradiol in the AVPV+ compared with the ARH+. These results indicate that gene expression in these two hypothalamic regions have specific responses to estradiol in timing and direction.

Adolescent alcohol exposure increases orexin-A/hypocretin-1 in the anterior hypothalamus.

Adolescence is a time of marked changes in sleep, neuromaturation, and alcohol use. While there is substantial evidence that alcohol disrupts sleep and that disrupted sleep may play a role in the development of alcohol use disorders (AUD), there is very little known about the brain mechanisms underlying this phenomenon. The orexin (also known as hypocretin) system is fundamental for a number of homeostatic mechanisms, including the initiation and maintenance of wakefulness that may be impacted by adolescent alcohol exposure. The current study investigated the impact of adolescent ethanol exposure on adult orexin-A/hypocretin-1 immunoreactive (orexin-A + IR) cells in hypothalamic nuclei in two models of adolescent intermittent ethanol (AIE) exposure. Both models assess adult hypothalamic orexin following either an AIE vapor exposure paradigm, or an AIE intragastric gavage paradigm during adolescence. Both AIE exposure models found that binge levels of ethanol intoxication during adolescence significantly increased adult orexin-A + IR expression in the anterior hypothalamic nucleus (AHN). Further, both AIE models found no change in orexin-A + IR in the posterior hypothalamic area (PH), perifornical nucleus (PeF), dorsomedial hypothalamic nucleus dorsal part (DMD) or lateral hypothalamic area (LH). However, AIE vapor exposure reduced orexin-A + IR in the paraventricular nucleus (PVN), but AIE gavage exposure did not. These findings suggest that the AHN orexinergic system is increased in adults following binge-like patterns of intoxication during adolescence. Altered adult AHN orexin could contribute to long-lasting changes in sleep.

Chronic exposure to low dose of bisphenol A causes follicular atresia by inhibiting kisspeptin neurons in anteroventral periventricular nucleus in female mice.

Bisphenol-A (BPA) is an estrogenic chemical extensively used in industrial and household applications. The present study was conducted to investigate the effect of chronic exposure to BPA on the adult female neuroendocrine system. Herein, we found that expose of adult female mice to BPA (50 µg/kg) by oral gavage for 60 days (BPA mice) prolonged diestrus and decreased serum 17ß-estradiol (E2) concentration by reducing the number of antral follicles and corpora luteum. In comparison with controls, the levels of serum luteinizing hormone (LH), follicle stimulating hormone (FSH), hypothalamic gonadotrophin releasing hormone (GnRH) and the expression of kisspeptin in anteroventral periventricular nucleus (AVPV) decreased in BPA mice, which could be reversed by injecting kisspeptin-10 (i.c.v.). Treatment with BPA or estrogen receptor α (ERα) antagonist MPP, but not ERß antagonist PHTPP inhibited E2-induced AVPV-kisspeptin expression in ovariectomized mice. Use of ERα agonist PPT rather than ERß agonist DPN enhanced AVPV-kisspepetin expression, which decreased after treatment with BPA. The amplitude of the proestrus LH surge decreased in mice exposed to BPA, but was recovered by administering kisspeptin-10. The present study provides in vivo evidence that chronic exposure to a low dose of BPA suppressed ERα-induced activation of AVPV-kisspeptin neurons, leading to prolonged diestrus and reduced ovulation in adult female mice.

GABAergic input through GABAB receptors is necessary during a perinatal window to shape gene expression of factors critical to reproduction such as Kiss1.

Lack of GABAB receptors in GABAB1 knockout mice decreases neonatal ARC kisspeptin 1 (Kiss1) expression in the arcuate nucleus of the hypothalamus (ARC) in females, which show impaired reproduction as adults. Our aim was to selectively impair GABAB signaling during a short postnatal period to evaluate its impact on the reproductive system. Neonatal male and female mice were injected with the GABAB antagonist CGP 55845 (CGP, 1 mg/kg body wt sc) or saline from postnatal day 2 (PND2) to PND6, three times per day (8 AM, 1 PM, and 6 PM). One group was killed on PND6 for collection of blood samples (hormones by radioimmunoassay), brains for gene expression in the anteroventral periventricular nucleus-periventricular nucleus continuum (AVPV/PeN), and ARC micropunches [quantitative PCR (qPCR)] and gonads for qPCR, hormone contents, and histology. A second group of mice was injected with CGP (1 mg/kg body wt sc) or saline from PND2 to PND6, three times per day (8 AM, 1 PM, and 6 PM), and left to grow to adulthood. We measured body weight during development and parameters of sexual differentiation, puberty onset, and estrous cycles. Adult mice were killed, and trunk blood (hormones), brains for qPCR, and gonads for qPCR and hormone contents were obtained. Our most important findings on PND6 include the CGP-induced decrease in ARC Kiss1 and increase in neurokinin B (Tac2) in both sexes; the decrease in AVPV/PeN tyrosine hydroxylase (Th) only in females; the increase in gonad estradiol content in both sexes; and the increase in primordial follicles and decrease in primary and secondary follicles. Neonatally CGP-treated adults showed decreased ARC Kiss1 and ARC gonadotropin-releasing hormone (Gnrh1) and increased ARC glutamic acid decarboxylase 67 (Gad1) only in males; increased ARC GABAB receptor subunit 1 (Gabbr1) in both sexes; and decreased AVPV/PeN Th only in females. We demonstrate that ARC Kiss1 expression is chronically downregulated in males and that the normal sex difference in AVPV/PeN Th expression is abolished. In conclusion, neonatal GABAergic input through GABAB receptors shapes gene expression of factors critical to reproduction.

Dynorphin and GABAA Receptor Signaling Contribute to Progesterone's Inhibition of the LH Surge in Female Mice.

Progesterone can block estrogen-induced luteinising hormone (LH) surge secretion and can be used clinically to prevent premature LH surges. The blocking effect of progesterone on the LH surge is mediated through its receptor in the anteroventral periventricular nucleus (AVPV) of the hypothalamus. However, the underlying mechanisms are unclear. The preovulatory LH surge induced by estrogen is preceded by a significant reduction in hypothalamic dynorphin and gamma-aminobutyric acid (GABA) release. To test the detailed roles of dynorphin and GABA in an LH surge blockade by progesterone, ovariectomized and 17ß-estradiol capsule-implanted (OVX/E2) mice received simultaneous injections of estradiol benzoate (EB) and progesterone (P) or vehicle for 2 consecutive days. The LH level was monitored from 2:30 pm to 8:30 pm at 30-minute intervals. Progesterone coadministration resulted in the LH surge blockade. A continuous microinfusion of the dynorphin receptor antagonist nor-BNI or GABAA receptor antagonist bicuculline into the AVPV from 3:00 pm to 7:00 pm reversed the progesterone-mediated blockade of the LH surge in 7 of 9 and 6 of 10 mice, respectively. In addition, these LH surges started much earlier than the surge induced by estrogen alone. However, 5 of 7 progesterone-treated mice did not show LH surge secretion after microinfusion with the GABAB receptor antagonist CGP-35348. Additionally, peripheral administration of kisspeptin-54 promotes LH surge-like release in progesterone treated mice. These results demonstrated that the progesterone-mediated suppression of the LH surge is mediated by an increase in dynorphin and GABAA receptor signaling acting though kisspeptin neurons in the AVPV of the hypothalamus in female mice.

GnRH(1-5), a metabolite of gonadotropin-releasing hormone, enhances luteinizing hormone release via activation of kisspeptin neurons in female rats.

Accumulating evidence suggests that kisspeptin neurons in the arcuate nucleus (ARC), which coexpress neurokinin B and dynorphin, are involved in gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) pulse generation, while the anteroventral periventricular nucleus (AVPV) kisspeptin neurons are responsible for GnRH/LH surge generation. The present study aims to examine whether GnRH(1-5), a GnRH metabolite, regulates LH release via kisspeptin neurons. GnRH(1-5) was intracerebroventricularly injected to ovariectomized and estrogen-treated Wistar-Imamichi female rats. Immediately after the central GnRH(1-5) administration at 2 nmol, plasma LH concentration increased, resulting in significantly higher levels of the area under the curve and baseline of plasma LH concentrations compared to vehicle-injected controls. On the other hand, in Kiss1 knockout rats, GnRH(1-5) administration failed to affect LH secretion, suggesting that the facilitatory effect of GnRH(1-5) on LH release is mediated by kisspeptin neurons. Double in situ hybridization (ISH) for Kiss1 and Gpr101, a GnRH(1-5) receptor gene, revealed that few Kiss1-expressing cells coexpress Gpr101 in both ARC and AVPV. On the other hand, double ISH for Gpr101 and Slc17a6, a glutamatergic marker gene, revealed that 29.2% of ARC Gpr101-expressing cells coexpress Slc17a6. Further, most of the AVPV and ARC Kiss1-expressing cells coexpress Grin1, a gene encoding a subunit of NMDA receptor. Taken together, these results suggest that the GnRH(1-5)-GPR101 signaling facilitates LH release via indirect activation of kisspeptin neurons and that glutamatergic neurons may mediate the signaling. This provides a new aspect of kisspeptin- and GnRH-neuronal communication with the presence of stimulation from GnRH to kisspeptin neurons in female rats.

Retinoblastoma binding protein 7 is involved in Kiss1 mRNA upregulation in rodents.

Kisspeptin, encoded by Kiss1, is essential for reproduction in mammals. Kiss1 expression is regulated by estrogen via histone acetylation in the Kiss1 promotor region. Thus, elucidation of histone modification factor(s) involved in the regulation of Kiss1 expression is required to gain further understanding of the mechanisms of its control. The RNA-seq analysis of isolated kisspeptin neurons, obtained from the arcuate nucleus (ARC) of female rats, revealed that Rbbp7, encoding retinoblastoma binding protein 7 (RBBP7), a member of histone modification and chromatin remodeling complexes, is highly expressed in the ARC kisspeptin neurons. Thus, the present study aimed to investigate whether RBBP7 is involved in Kiss1 expression. Histological analysis using in situ hybridization (ISH) revealed that Rbbp7 expression was located in several hypothalamic nuclei, including the ARC and the anteroventral periventricular nucleus (AVPV), where kisspeptin neurons are located. Double ISH for Rbbp7 and Kiss1 showed that a majority of kisspeptin neurons (more than 85%) expressed Rbbp7 mRNA in both the ARC and the AVPV of female rats. Further, Rbbp7 mRNA knockdown significantly decreased in vitro expression of Kiss1 in a mouse immortalized kisspeptin neuronal cell line (mHypoA-55). Estrogen treatment significantly decreased and increased Kiss1 mRNA levels in the ARC and AVPV of ovariectomized female rats, respectively, but failed to affect Rbbp7 mRNA levels in both the nuclei. Taken together, these findings suggest that RBBP7 is involved in the upregulation of Kiss1 expression in kisspeptin neurons of rodents in an estrogen-independent manner.

Comparative study of environmental pollutants bisphenol A and bisphenol S on sexual differentiation of anteroventral periventricular nucleus and spermatogenesis.

BACKGROUND: Bisphenol A is well known endocrine-disrupting chemical while Bisphenol S was considered a safe alternative. The present study aims to examine the comparative effects of xenobiotic bisphenol-A (BPA) and its substitute bisphenol-S (BPS) on spermatogenesis and development of sexually dimorphic nucleus population of dopaminergic neurons in the anteroventral periventricular nucleus (AVPV) of the hypothalamus in male pups. METHODS: Sprague Dawley rat's pups were administered subcutaneously at the neonatal stage from postnatal day PND1 to PND 27. Thirty animals were divided into six experimental groups (6 animals/group). The first group served as control and was provided with normal olive oil. The four groups were treated with 2 µg/kg and 200 µg/kg of BPA and BPS, respectively. The sixth group was given with 50 µg/kg of estradiol dissolved in olive oil as a standard to find the development of dopaminergic tyrosine hydroxylase neurons in AVPV regions. Histological analysis for testicular tissues and immunohistochemistry for brain tissues was performed. RESULTS: The results revealed adverse histopathological changes in testis after administration of different doses of BPA and BPS. These degenerative changes were marked by highly significant (p < 0.001) decrease in tubular and luminal diameters of seminiferous tubule and epithelial height among bisphenols treated groups as compared to control. Furthermore, significantly increased (p < 0.001) TH-ir cell bodies in the AVPV region of the brain with 200 µg/kg dose of BPA and BPS was evident. CONCLUSION: It is concluded that exposure of BPA and BPS during a critical developmental period can structural impairments in testes and affects sexual differentiation of a dimorphic dopaminergic population of AVPV region of hypothalamus in the male brain.

Alterations of estradiol-induced histone H3 acetylation in the preoptic area and anteroventral periventricular nucleus of middle-aged female rats.

In this study we investigated the characteristics of histone H3 acetylation in the anterior hypothalamus under E2 positive feedback to gain a better understanding of the mechanism underlying reduced GnRH neuron activation and altered gene expression in female reproductive aging. Young and middle-aged female rats were ovariectomized (OVX) and treated with estradiol (E2) or oil. C-Fos expression, the number of GnRH neurons co-localized with c-Fos in the preoptic area (POA), and the number of acetylated histone H3 cells in the POA and anteroventral periventricular nucleus (AVPV) were quantified at the time of the expected GnRH neuron activation. We used real-time PCR to evaluate the expression of Esr1 target genes including Kiss1 and VGluT2 and genes known as Esr1 coregulators in the anterior hypothalamus. Our results show that in the young females, E2 markedly increased histone H3 acetylation in the POA and AVPV, coincident with increased c-Fos and GnRH neuron activation in the POA. In middle-aged females, E2-induced histone H3 acetylation was reduced in the POA but was not significantly altered in the AVPV. This occurred in association with a reduction of c-Fos expression and the number of GnRH cells expressing c-Fos in the POA as well as a down-regulation of Kiss1 and VGluT2 mRNA expression in the anterior hypothalamus of the animals. E2 caused significant decreases in Ncoa2 and Crebbp mRNA expression in the anterior hypothalamus of young, but not middle-aged females. Taken together, these data suggest that alterations of histone H3 acetylation in the POA and AVPV and the inability of Ncoa2 and Crebbp to respond to E2 in the middle-aged anterior hypothalamus partially contribute to the decline of GnRH neuron activation and E2 target gene expression changes that occur in female along with reproductive aging.

Reduction of Kiss1 expression in the anteroventral periventricular nucleus is associated with atrazine-induced attenuation of the luteinizing hormone surge in female rats.

Atrazine, a commonly used herbicide, suppresses the luteinizing hormone (LH) surge in female rats, although the underlying mechanism remains unclear. Kisspeptin, encoded by the Kiss1 gene, is a hypothalamic peptide that controls gonadotropin-releasing hormone (GnRH) release from the GnRH neurons. Kisspeptin neurons in the anteroventral periventricular nucleus (AVPV) are involved in regulating pre-ovulatory GnRH and LH surge. To clarify the effect of atrazine on the LH surge in female rats, we investigated its effects on hypothalamic GnRH and kisspeptin. Ovariectomized female rats in a high-dose estradiol supplementation model were orally administered vehicle or 100 mg/kg of atrazine once daily for 5 days. This attenuated the LH surge but did not affect baseline LH levels, with no difference in hypothalamic GnRH levels between the vehicle-treated and atrazine-treated animals. After the fifth treatment, subcutaneous administration of kisspeptin (at 0, 0.1, 1, and 10 nmol/kg) induced a dose-dependent LH release almost equivalent in the vehicle- and atrazine-treated animals, suggesting that GnRH neurons maintain normal responsiveness to kisspeptin. However, Kiss1 mRNA expression levels in the AVPV were significantly reduced in the atrazine-treated animals. Given the normal response of GnRH neurons to exogenously administered kisspeptin, the suppressive effect of atrazine may be explained by suppression of Kiss1 expression in the AVPV leading to the attenuation of kisspeptin release from kisspeptin neurons in the AVPV. Further studies are warranted to elucidate more precisely the mechanism of atrazine's involvement in the suppression of Kiss1 mRNA expression in the AVPV.

Crocetin attenuates DHT-induced polycystic ovary syndrome in mice via revising kisspeptin neurons.

Polycystic ovarian syndrome (PCOS), the most common endocrine disorder of women in reproductive age, is typical with hyperandrogenism and disturbance of the hypothalamus-pituitary-ovary (HPO) axis, i.e. abnormal expression of hypothalamic gonadotropin-releasing hormone (GnRH) followed by the elevated ratio of serum luteinizing hormone (LH) level to follicle-stimulating hormone (FSH) level. This derangement might have a close relationship with hypothalamic kisspeptin expression that is thought to be a key regulator of GnRH. Crocetin, one of the main components of Saffron clinically used as traditional medicine in gynecology diseases, was evaluated for its therapeutic effects on PCOS induced by prenatally exposure to dihydrotestosterone (DHT) in mice. Herein, we found that DHT-treated mice showed a similar phenotype to human PCOS such as heavier ovary, prolonged diestrus, multiple enlarged follicles with fewer corpus luteum, and higher LH and testosterone levels. Kisspeptin expression was lower in anteroventral periventricular nucleus (AVPV) but higher in arcuate nucleus (ARC). Treatment of crocetin prevented the prolongation of diestrus and reduction in corpora luteum, recover the levels of GnRH, FSH, LH, progesterone (P4), estradiol (E2) and testosterone (T), and increase the kisspeptin level in AVPV but reduce that in ARC. The present study provides in vivo evidence that crocetin improved the PCOS in mice via increasing AVPV-kisspeptin and reducing ARC-kisspeptin expression.

Impact of Perfluorooctane Sulfonate on Reproductive Ability of Female Mice through Suppression of Estrogen Receptor α-Activated Kisspeptin Neurons.

Perfluorooctane sulfonate (PFOS) is used extensively in industrial and household applications. High exposure to PFOS has been associated with increased odds of irregular and long menstrual cycles in women. However, the underlying mechanisms remain to be elucidated. Herein, we show that adult female mice appeared prolongation of diestrus and reduction of corpora luteum within a week of oral administration of PFOS (10 mg/kg), which are associated with decreases in the levels of serum progesterone, LH and hypothalamic GnRH. The number of AVPV-kisspeptin neurons and the AVPV-kisspeptin expression were increased in proestrus mice or OVX-mice treated with high-dose estradiol benzoate (0.05 mg/kg), which were suppressed by the administration of PFOS. The administration of PFOS or GPR54 antagonist P234 prevented the generation of LH-surge in OVX-mice treated with high-dose E2. In hypothalamic slices incubated in 100 nM E2 for 4 h, the AVPV-kisspeptin expression was significantly enhanced, which was inhibited by PFOS in a dose-dependent manner or estrogen receptor α (ERα) antagonist MPP, but not ERß antagonist PHTPP. The incubation of ERα agonist PPT rather than ERß agonist DPN could increase the level of AVPV-kisspeptin expression, which was sensitive to the treatment with PFOS. The administration of GPR54 agonist kisspeptin-10 in PFOS-mice could correct the prolongation of diestrus and reduction of corpora luteum, and recover the LH-surge and the levels of LH and GnRH. The results indicate that exposure to PFOS suppressed ERα-induced activation of AVPV-kisspeptin neurons leads to diestrus prolongation and ovulation reduction.

Inhibiting Production of New Brain Cells during Puberty or Adulthood Blunts the Hormonally Induced Surge of Luteinizing Hormone in Female Rats.

New cells are added during both puberty and adulthood to hypothalamic regions that govern reproduction, homeostasis, and social behaviors, yet the functions of these late-born cells remain elusive. Here, we pharmacologically inhibited cell proliferation in ventricular zones during puberty or in adulthood and determined subsequent effects on the hormone-induced surge of luteinizing hormone (LH) in female rats. Initial neuroanatomical analyses focused on verifying incorporation, activation, and pharmacological inhibition of pubertally or adult born cells in the anteroventral periventricular nucleus (AVPV) of the hypothalamus because of the essential role of the AVPV in triggering the preovulatory LH surge in females. We first showed that approximately half of the pubertally born AVPV cells are activated by estradiol plus progesterone (P) treatment, as demonstrated by Fos expression, and that approximately 10% of pubertally born AVPV cells express estrogen receptor alpha (ERα). Next, we found that mitotic inhibition through intracerebroventricular (ICV) administration of cytosine ß-D-arabinofuranoside (AraC), whether during puberty or in adulthood, decreased the number of new cells added to the AVPV and the suprachiasmatic nucleus (SCN), and also blunted and delayed the hormone-induced LH surge. These studies do not prove, but are highly suggestive, that ongoing postnatal addition of new cells in periventricular brain regions, including the AVPV and SCN, may be important to the integrity of female reproduction.

Sex Differences in Steroid Receptor Coexpression and Circadian-Timed Activation of Kisspeptin and RFRP-3 Neurons May Contribute to the Sexually Dimorphic Basis of the LH Surge.

In rodents, the ovulation-inducing luteinizing hormone (LH) surge is sexually dimorphic, occurring only in females, but the reasons for this sex difference are unclear. Two neuropeptides, kisspeptin and RFamide-related peptide 3 (RFRP-3), are hypothesized to regulate the gonadotropin-releasing hormone (GnRH)/LH surge. In females, both of these systems show circadian changes coincident with the LH surge, but whether males show similar temporal changes under comparable hormonal conditions is unknown. Here, we evaluated circadian time (CT)-dependent changes in gene expression and neuronal activation of Kiss1 and Rfrp neurons of female and male mice given identical LH surge-inducing estrogen regimens. As expected, females, but not males, displayed a late afternoon LH surge and GnRH neuronal activation. Kiss1 expression in the anteroventral periventricular nucleus (AVPV) was temporally increased in females in the late afternoon, whereas males demonstrated no temporal changes in AVPV Kiss1 expression. Likewise, neuronal activation of AVPV Kiss1 neurons was dramatically elevated in the late afternoon in females but was low at all circadian times in males. Estrogen receptor α levels in AVPV Kiss1 neurons were sexually dimorphic, being higher in females than males. AVPV progesterone receptor levels were also higher in females than males. Hypothalamic Rfrp messenger RNA levels showed no CT-dependent changes in either sex. However, Rfrp neuronal activation was temporally diminished in the afternoon/evening in females but not males. Collectively, the identified sex differences in absolute and CT-dependent AVPV Kiss1 levels, AVPV sex steroid receptor levels, and circadian-timed changes in neuronal activation of both Kiss1 and Rfrp neurons suggest that multiple sexually dimorphic processes in the brain may underlie proper LH surge generation.

Role of amygdala kisspeptin in pubertal timing in female rats.

To investigate the mechanism by which maternal obesity disrupts reproductive function in offspring, we examined Kiss1 expression in the hypothalamic arcuate (ARC) and anteroventral periventricular (AVPV) nuclei, and posterodorsal medial amygdala (MePD) of pre-pubertal and young adult offspring. Sprague-Dawley rats were fed either a standard or energy-dense diet for six weeks prior to mating and throughout pregnancy and lactation. Male and female offspring were weaned onto normal diet on postnatal day (pnd) 21. Brains were collected on pnd 30 or 100 for qRT-PCR to determine Kiss1 mRNA levels. Maternal obesity increased Kiss1 mRNA expression in the MePD of pre-pubertal male and female offspring, whereas Kiss1 expression was not affected in the ARC or AVPV at this age. Maternal obesity reduced Kiss1 expression in all three brain regions of 3 month old female offspring, but only in MePD of males. The role of MePD kisspeptin on puberty, estrous cyclicity and preovulatory LH surges was assessed directly in a separate group of post-weanling and young adult female rats exposed to a normal diet throughout their life course. Bilateral intra-MePD cannulae connected to osmotic mini-pumps for delivery of kisspeptin receptor antagonist (Peptide 234 for 14 days) were chronically implanted on pnd 21 or 100. Antagonism of MePD kisspeptin delayed puberty onset, disrupted estrous cyclicity and reduced the incidence of LH surges. These data show that the MePD plays a key role in pubertal timing and ovulation and that maternal obesity may act via amygdala kisspeptin signaling to influence reproductive function in the offspring.

Hypothalamic effects of progesterone on regulation of the pulsatile and surge release of luteinising hormone in female rats.

Progesterone can block the oestradiol-induced GnRH/LH surge and inhibit LH pulse frequency. Recent studies reported that progesterone prevented premature LH surges during ovarian hyperstimulation in women. As the most potent stimulator of GnRH/LH release, kisspeptin is believed to mediate the positive and negative feedback effects of oestradiol in the hypothalamic anteroventral periventricular (AVPV) and arcuate (ARC) nuclei, while the region-specific role of progesterone receptors in these nuclei remains unknown. This study examined the hypothesis that progesterone inhibits LH surge and pulsatile secretion via its receptor in the ARC and/or AVPV nuclei. Adult female rats received a single injection of pregnant mare serum gonadotropin followed by progesterone or vehicle. Progesterone administration resulted in a significant prolongation of the oestrous cycle and blockade of LH surge. However, microinjection of the progesterone receptor antagonist, RU486, into the AVPV reversed the prolonged cycle length and rescued the progesterone blockade LH surge, while RU486 into the ARC shortened LH pulse interval in the progesterone treated rats. These results demonstrated that progesterone's inhibitory effect on the GnRH/LH surge and pulsatile secretion is mediated by its receptor in the kisspeptin enriched hypothalamic AVPV and ARC respectively, which are essential for progesterone regulation of oestrous cyclicity in rats.

The role of estrogen receptor subtypes for induction of delayed effects on the estrous cycle and female reproductive organs in rats.

It has been reported that neonatal exposure to estrogens at relatively low doses can induce early onset anovulation as a delayed effect in female rats. Dysfunction of kisspeptin neurons in the anteroventral periventricular nucleus (AVPV) was proposed to be a trigger for this effect. To determine the roles of estrogen receptor (ER) subtypes in the induction of delayed effects, we conducted a series of experiments using Donryu rats to examine whether neonatal injection of an ERα agonist (PPT), an ERß agonist (DPN) or an ERα antagonist (ICI) could induce delayed effects. Also, involvement of the kisspeptin neurons in the AVPV for induction of delayed effect by PPT and DPN was investigated. We observed that neonatal exposure to PPT, DPN and ICI induced the early onset of abnormal estrous cyclicity after sexual maturation, suggesting that the compounds capable of inducing delayed effects are not limited to ERα agonists. On the other hand, the data suggested the possibility that DPN and ICI functioned partially as ERα agonists in the neonatal brain. Regardless of the agents used, there is a possibility that dysfunction of kisspeptin neurons in the AVPV might contribute to induction of early onset anovulation.

Loss of Fertility in the Absence of Progesterone Receptor Expression in Kisspeptin Neurons of Female Mice.

Ovarian steroids, estradiol and progesterone, play central roles in regulating female reproduction by acting as both positive and negative regulators of gonadotropin-releasing hormone (GnRH) secretion in the hypothalamus. Recent studies have identified kisspeptin neurons of the hypothalamus as the target of estrogenic regulation of GnRH secretion. In this study, we aimed to determine the significance of progesterone receptor (PGR) expression in the kisspeptin neurons. To this end, the Pgr gene was selectively ablated in mouse kisspeptin neurons and the reproductive consequence assessed. The hypothalamus of the Pgr deficient female mouse expressed kisspeptin, the pituitary released LH in response to GnRH stimulation, and the ovary ovulated when stimulated with gonadotropins. However, the mutant mouse gradually lost cyclicity, was unable to generate a LH surge in response to rising estradiol, and eventually became infertile. Taken together, these results indicate that the loss of PGR impairs kisspeptin secretory machinery and therefore that PGR plays a critical role in regulating kisspeptin secretion.

Excitability and Burst Generation of AVPV Kisspeptin Neurons Are Regulated by the Estrous Cycle Via Multiple Conductances Modulated by Estradiol Action.

The preovulatory secretory surge of gonadotropin-releasing hormone (GnRH) is crucial for fertility and is regulated by a switch of estradiol feedback action from negative to positive. GnRH neurons likely receive estradiol feedback signals via ERα-expressing afferents. Kisspeptin neurons in anteroventral periventricular nucleus (AVPV) are thought to be critical for estradiol-positive feedback induction of the GnRH surge. We examined the electrophysiological properties of GFP-identified AVPV kisspeptin neurons in brain slices from mice on the afternoon of diestrus (negative feedback) and proestrus (positive feedback, time of surge). Extracellular recordings revealed increased firing frequency and action potential bursts on proestrus versus diestrus. Whole-cell recordings were used to study the intrinsic mechanisms of bursting. Upon depolarization, AVPV kisspeptin neurons exhibited tonic firing or depolarization-induced bursts (DIB). Both tonic and DIB cells exhibited bursts induced by rebound from hyperpolarization. DIB occurred similarly on both cycle stages, but rebound bursts were observed more often on proestrus. DIB and rebound bursts were both sensitive to Ni(2+), suggesting that T-type Ca(2+) currents (I Ts) are involved. I T current density was greater on proestrus versus diestrus. In addition to I T, persistent sodium current (I NaP) facilitated rebound bursting. On diestrus, 4-aminopyridine-sensitive potassium currents contributed to reduced rebound bursts in both tonic and DIB cells. Manipulation of specific sex steroids suggests that estradiol induces the changes that enhance AVPV kisspeptin neuron excitability on proestrus. These observations indicate cycle-driven changes in circulating estradiol increased overall action potential generation and burst firing in AVPV kisspeptin neurons on proestrus versus diestrus by regulating multiple intrinsic currents.