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.

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.

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.

Characterization and gonadal hormone regulation of a sexually dimorphic corticotropin-releasing factor receptor 1 cell group.

Corticotropin-releasing factor binds with high affinity to CRF receptor 1 (CRFR1) and is implicated in stress-related mood disorders such as anxiety and depression. Using a validated CRFR1-green fluorescent protein (GFP) reporter mouse, our laboratory recently discovered a nucleus of CRFR1 expressing cells that is prominent in the female rostral anteroventral periventricular nucleus (AVPV/PeN), but largely absent in males. This sex difference is present in the early postnatal period and remains dimorphic into adulthood. The present investigation sought to characterize the chemical composition and gonadal hormone regulation of these sexually dimorphic CRFR1 cells using immunohistochemical procedures. We report that CRFR1-GFP-ir cells within the female AVPV/PeN are largely distinct from other dimorphic cell populations (kisspeptin, tyrosine hydroxylase). However, CRFR1-GFP-ir cells within the AVPV/PeN highly co-express estrogen receptor alpha as well as glucocorticoid receptor. A single injection of testosterone propionate or estradiol benzoate on the day of birth completely eliminates the AVPV/PeN sex difference, whereas adult gonadectomy has no effect on CRFR1-GFP cell number. These results indicate that the AVPV/PeN CRFR1 is regulated by perinatal but not adult gonadal hormones. Finally, female AVPV/PeN CRFR1-GFP-ir cells are activated following an acute 30-min restraint stress, as assessed by co-localization of CRFR1-GFP cells with phosphorylated (p) CREB. CRFR1-GFP/pCREB cells were largely absent in the male AVPV/PeN. Together, these data indicate a stress and gonadal hormone responsive nucleus that is unique to females and may contribute to sex-specific stress responses.

Integration of Circadian and Metabolic Control of Reproductive Function.

Optimal fertility in humans and animals relies on the availability of sufficient metabolic fuels, information about which is communicated to the brain via levels of the hormones leptin and insulin. The circadian clock system is also critical; this input is especially evident in the precise timing of the female-specific surge of GnRH and LH secretion that triggers ovulation the next day. Chronodisruption and metabolic imbalance can both impair reproductive activity, and these two disruptions exacerbate each other, such that they often occur simultaneously. Kisspeptin neurons located in the anteroventral periventricular nucleus of the hypothalamus are able to integrate both circadian and metabolic afferent inputs and use this information to modulate the timing and magnitude of the preovulatory GnRH/LH surge. In an environment in which exposure to high caloric diets and chronodisruptors such as artificial night lighting, shift work, and transmeridian travel have become the norm, the implications of these factors for couples struggling to conceive deserve closer attention and more public education.

The bovine anterior hypothalamus: Characterization of the vasopressin-oxytocin containing nucleus and changes in relation to sexual differentiation.

In an effort to systematically describe the neurochemical anatomy of the bovine anterior hypothalamus, we used a series of immunocytochemical markers such as acetylcholine esterase (AChE), arginine-vasopressin (AVP), calbindin (Calb), galanin (Gal), neuropeptide-Y (NPY), oxytocin (OXT), somatostatin (SST), and vasoactive intestinal peptide (VIP). We also investigated the potential sex difference present in the suprachiasmatic nucleus (SCN) and the vasopressin-oxytocin containing nucleus (VON) of six male and six female Bos taurus. Our study revealed that the cytochemical structure of the cattle anterior hypothalamus follows the blueprint of other mammals. The VON, which was never described before in cattle, showed a sex difference with a 33.7% smaller volume and 23.2% fewer magnocellular neurons (approximately 20-30 µm) in the male. The SCN also did show a sex difference in VIP neurons and volume with a 36.1% larger female nucleus with 28.1% more cells. Additionally, we included five heifers with freemartin syndrome as a new animal model relevant to sexual differentiation in the brain. This is, to the best of our knowledge, the first freemartin study in relation to the brain. Surprisingly, the SCN of freemartin heifers was 32.5% larger than its control male and female counterparts with 29% more VIP cells. Conversely, the freemartin VON had an intermediary size between male and female. To analyze our data, a classical statistical analysis and a novel multivariate and multi-aspect approach were applied. These findings shed new light on sexual dimorphism in the bovine brain and present this species with freemartins as a valuable animal model in neuroscience.

Synaptic Innervation of the GnRH Neuron Distal Dendron in Female Mice.

GnRH neuron cell bodies are scattered throughout the basal forebrain but funnel their projections to the median eminence to release GnRH into the pituitary portal system to control fertility. Prior studies have shown that GnRH neurons located in the anterior hypothalamus send projections to the median eminence that have characteristics of both dendrites and axons. These unusual structures have been termed "dendrons." To address whether the dendron is unique to anterior hypothalamic GnRH neurons or is also a characteristic of more rostral GnRH neurons, we used viral vector‒mediated GnRH neuron‒specific tract-tracing coupled with CLARITY optical clearing. Individual rostral preoptic area GnRH neurons in female mice were identified to elaborate processes up to 4 mm in length that exhibited spines and projected all the way to the median eminence before branching into multiple short axons. The synaptic innervation patterns of distal GnRH neuron dendrons and their short axons in the vicinity of the median eminence were examined using electron microscopy. This revealed the presence of a high density of synaptic inputs to distal dendrons at the border of the median eminence. In contrast, no synapses were detected on any GnRH neuron axons. These studies demonstrate that GnRH neurons in the rostral preoptic area project dendrons to the edge of the median eminence, whereupon they branch into multiple short axons responsible for GnRH secretion. The dense synaptic innervation of these distal dendrons likely represents an efficient mechanism for controlling GnRH secretion required for fertility.

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.

STAT5 signaling in kisspeptin cells regulates the timing of puberty.

Previous studies have shown that kisspeptin neurons are important mediators of prolactin's effects on reproduction. However, the cellular mechanisms recruited by prolactin to affect kisspeptin neurons remain unknown. Using whole-cell patch-clamp recordings of brain slices from kisspeptin reporter mice, we observed that 20% of kisspeptin neurons in the anteroventral periventricular nucleus was indirectly depolarized by prolactin via an unknown population of prolactin responsive neurons. This effect required the phosphatidylinositol 3-kinase signaling pathway. No effects on the activity of arcuate kisspeptin neurons were observed, despite a high percentage (70%) of arcuate neurons expressing prolactin-induced STAT5 phosphorylation. To determine whether STAT5 expression in kisspeptin cells regulates reproduction, mice carrying Stat5a/b inactivation specifically in kisspeptin cells were generated. These mutants exhibited an early onset of estrous cyclicity, indicating that STAT5 transcription factors exert an inhibitory effect on the timing of puberty.

Amygdala Kisspeptin Neurons: Putative Mediators of Olfactory Control of the Gonadotropic Axis.

Kisspeptins and their receptors are potent regulators of the gonadotropic axis. Kisspeptin neurons are found mainly in the hypothalamic arcuate nucleus and the anteroventral periventricular nucleus. However, there is also a third population of kisspeptin neurons, located in the amygdala. We used fluorescence immunohistochemistry to quantify and localize the amygdala kisspeptin neurons and to reveal close apposition and putative innervations by vasopressinergic and tyrosine hydroxylase-positive dopaminergic neurons. Using microinjections of retro- and anterograde tracers, and viral transfection systems in rats and transgenic mice, we showed reciprocal connectivity between the accessory olfactory bulb and the amygdala kisspeptin neurons. In vitro recordings indicate an inhibitory action of kisspeptin on mitral cells in the accessory olfactory bulb. Using viral specific-cell gene expression in transgenic mice in combination with double immunofluorescence histochemistry, we found that the amygdala kisspeptin neurons also project to gonadotropin-releasing hormone (GnRH) neurons in the preoptic area. Our neuroanatomical and electrophysiological data suggest that amygdala kisspeptin neurons integrate social behaviour and odour information into GnRH neurons in the preoptic area to coordinate the gonadotropic axis and the appropriate output behaviour to odour cues.

Insights from extracellular matrix studies in the hypothalamus: structural variations of perineuronal nets and discovering a new perifornical area of the anterior hypothalamus.

The hypothalamus controls metabolism, stress responses, and instinctive behaviors for individual survival and species preservation. Recent studies suggest that hypothalamic neurons retain plasticity throughout adulthood, which enables these neurons to respond to various kinds of changes in environment, nutrients, and fluctuating hormones. One of the mechanisms underlying the regulation of neural plasticity is the formation of a stable extracellular matrix (ECM) structure called perineuronal nets (PNNs). PNNs are large aggregates of heterogeneous ECM molecules such as chondroitin sulfate proteoglycans (CSPGs), hyaluronan, their link proteins, and tenascin-R. PNNs surround the cell body and proximal dendrites of a subset of neurons and limit adult neural plasticity. This review describes the CSPG-based ECM, including the PNNs, with a special focus on the hypothalamus of mice. We first provide an overview of PNNs in terms of their structure, molecular components, and functions, most of which have been demonstrated by extrahypothalamic studies. Second, we show the presence or absence of PNNs within individual hypothalamic regions and then describe non-PNN-formed ECM containing CSPGs that can be observed in particular hypothalamic regions. Finally, we will introduce a newly identified mouse hypothalamic area that we named the perifornical area of the anterior hypothalamus (PeFAH), which contains a cluster of PNN-positive neurons. PeFAH neurons express enkephalin and have bidirectional connections with the lateral septum. The anterior hypothalamus and lateral septum are thought to regulate defensive behaviors; therefore, the PeFAH neurons and PNNs around them could be involved in the regulation of defensive behaviors.

Visualisation of Kiss1 Neurone Distribution Using a Kiss1-CRE Transgenic Mouse.

Kisspeptin neuropeptides are encoded by the Kiss1 gene and play a critical role in the regulation of the mammalian reproductive axis. Kiss1 neurones are found in two locations in the rodent hypothalamus: one in the arcuate nucleus (ARC) and another in the RP3V region, which includes the anteroventral periventricular nucleus (AVPV). Detailed mapping of the fibre distribution of Kiss1 neurones will help with our understanding of the action of these neurones in other regions of the brain. We have generated a transgenic mouse in which the Kiss1 coding region is disrupted by a CRE-GFP transgene so that expression of the CRE recombinase protein is driven from the Kiss1 promoter. As expected, mutant mice of both sexes are sterile with hypogonadotrophic hypogonadism and do not show the normal rise in luteinising hormone after gonadectomy. Mutant female mice do not develop mature Graafian follicles or form corpora lutea consistent with ovulatory failure. Mutant male mice have low blood testosterone levels and impaired spermatogenesis beyond the meiosis stage. Breeding Kiss-CRE heterozygous mice with CRE-activated tdTomato reporter mice allows fluorescence visualisation of Kiss1 neurones in brain slices. Approximately 80-90% of tdTomato positive neurones in the ARC were co-labelled with kisspeptin and expression of tdTomato in the AVPV region was sexually dimorphic, with higher expression in females than males. A small number of tdTomato-labelled neurones was also found in other locations, including the lateral septum, the anterodorsal preoptic nucleus, the amygdala, the dorsomedial and ventromedial hypothalamic nuclei, the periaquaductal grey, and the mammillary nucleus. Three dimensional visualisation of Kiss1 neurones and fibres by CLARITY processing of whole brains showed an increase in ARC expression during puberty and higher numbers of Kiss1 neurones in the caudal region of the ARC compared to the rostral region. ARC Kiss1 neurones sent fibre projections to several hypothalamic regions, including rostrally to the periventricular and pre-optic areas and to the lateral hypothalamus.

Evaluation of Immortalized AVPV- and Arcuate-Specific Neuronal Kisspeptin Cell Lines to Elucidate Potential Mechanisms of Estrogen Responsiveness and Temporal Gene Expression in Females.

In females, ovarian estradiol modulates kisspeptin (Kiss-1) synthesis to act as an obligatory regulator of downstream gonadotropin release in vivo, via stimulation of GnRH neurons. Changes in the ovarian condition are relayed to the neuroendocrine hypothalamus via two sexually dimorphic Kiss-1 populations, located in the anteroventral periventricular (AVPV) and arcuate nuclei, conveying estradiol-positive and -negative feedback, respectively. To elucidate how differential responsiveness to estradiol is mediated in these populations, we generated two kisspeptin-secreting cell lines from an adult kiss1-green fluorescent protein (GFP) female mouse. These lines recapitulate in vivo responsiveness to estradiol, with KTaV-3 (AVPV) cells demonstrating significantly increased kiss1 expression under high physiological estradiol exposure, whereas KTaR-1 (arcuate) cells exhibit kiss1 suppression after lower estradiol exposure. Baseline expression of estrogen receptor-α (esr1) differs significantly between KTaV-3 and KTaR-1 cells, with KTaR-1 cells demonstrating higher basal expression of esr1. Estradiol stimulation of kiss1 expression in KTaV-3 cells is modulated in a dose-dependent manner up to 25.0 pM, with less responsiveness observed at higher doses (>50.0 pM). In contrast, KTaR-1 kiss1 attenuates at lower estradiol doses (2.0-5.0 pM), returning to baseline levels at 25.0 pM and greater. Furthermore, the expression of the core clock genes bmal1 and per2 show normal rhythms in KTaV-3 cells, regardless of estradiol treatment. Conversely, KTaR-1 antiphasic transcription of bmal1 and per2 is phase delayed by low estradiol treatment. Strikingly, estradiol induces circadian rhythms of kiss1 expression only in KTaV-3 cells. Further exploration into estradiol responsiveness will reveal mechanisms responsible for the differential expression pattern demonstrated in vivo between these cell types.

Neurons and Glial Cells Are Added to the Female Rat Anteroventral Periventricular Nucleus During Puberty.

The anteroventral periventricular nucleus (AVPV) orchestrates the neuroendocrine-positive feedback response that triggers ovulation in female rodents. The AVPV is larger and more cell-dense in females than in males, and during puberty, only females develop the capacity to show a positive feedback response. We previously reported a potential new mechanism to explain this female-specific gain of function during puberty, namely a female-biased sex difference in the pubertal addition of new cells to the rat AVPV. Here we first asked whether this sex difference is due to greater cell proliferation and/or survival in females. Female and male rats received the cell birthdate marker 5-bromo-2'-deoxyuridine (BrdU; 200 mg/kg, ip) on postnatal day (P) 30; brains were collected at short and long intervals after BrdU administration to assess cell proliferation and survival, respectively. Overall, females had more BrdU-immunoreactive cells in the AVPV than did males, with no sex differences in the rate of cell attrition over time. Thus, the sex difference in pubertal addition of AVPV cells appears to be due to greater cell proliferation in females. Next, to determine the phenotype of pubertally born AVPV cells, daily BrdU injections were given to female rats on P28-56, and tissue was collected on P77 to assess colocalization of BrdU and markers for mature neurons or glia. Of the pubertally born AVPV cells, approximately 15% differentiated into neurons, approximately 19% into astrocytes, and approximately 23% into microglia. Thus, both neuro- and gliogenesis occur in the pubertal female rat AVPV and potentially contribute to maturation of female reproductive function.

Pre- and postnatal bisphenol A treatment does not alter the number of tyrosine hydroxylase-positive cells in the anteroventral periventricular nucleus (AVPV) of weanling male and female rats.

Exposure to Bisphenol A (BPA) may interfere with brain sexual differentiation. Altered numbers of tyrosine hydroxylase (TH) cells in the rodent anteroventral periventricular nucleus (AVPV) after developmental BPA treatment have been reported; however, definitive conclusions are lacking. The current study incorporated many of the guidelines suggested for endocrine disrupter research. Specifically, ethinyl estradiol (EE2) served as a reference estrogen, exogenous environmental estrogen exposure was controlled, BPA was administered orally, and subjects consumed a low phytoestrogen diet. Here, on gestational days 6-21, Sprague-Dawley rats (10-15/group) were gavaged with 2.5 or 25.0 µg BPA/kg/day or 5.0 or 10.0 µg EE2/kg/day or the vehicle (5 ml of 0.3% aqueous carboxymethylcellulose/kg/day). A naïve control group was weighed and restrained, but not gavaged. Beginning on postnatal day (PND) 1 and continuing until PND 21, the 4 pups/sex/litter were orally treated with the same dose their dam had received. On PND 21, 1/sex/litter was perfused and the brain removed. TH immunoreactivity (TH-ir) was counted in 8 images/pup by a technician blind to treatment status. ANOVA results indicated significantly higher TH-ir cells/mm(2) in females (main effect of sex: p<0.01); however, there was no significant effect of treatment or a significant interaction of treatment with sex. In a separate untreated group of PND 21 Sprague-Dawley pups, AVPV volume was quantified and no significant sexual dimorphism was apparent. Similar to our reported results of behavioral assessments, the BPA treatment paradigm used here (2.5 or 25.0 µg BPA/kg/day administered orally) does not appear to cause significant alterations in AVPV TH-ir.

Absence of Female-Typical Pheromone-Induced Hypothalamic Neural Responses and Kisspeptin Neuronal Activity in α-Fetoprotein Knockout Female Mice.

Pheromones induce sexually dimorphic neuroendocrine responses, such as LH secretion. However, the neuronal network by which pheromones are converted into signals that will initiate and modulate endocrine changes remains unclear. We asked whether 2 sexually dimorphic populations in the anteroventral periventricular and periventricular nuclei that express kisspeptin and tyrosine hydroxylase (TH) are potential candidates that will transduce the olfactory signal to the neuroendocrine system. Furthermore, we assessed whether this transduction is sensitive to perinatal actions of estradiol by using female mice deficient in α-fetoprotein (AfpKO), which lack the protective actions of Afp against maternal estradiol. Wild-type (WT) and AfpKO male and female mice were exposed to same- versus opposite-sex odors and the expression of Fos (the protein product of the immediate early gene c-Fos) was analyzed along the olfactory projection pathways as well as whether kisspeptin, TH, and GnRH neurons are responsive to opposite-sex odors. Male odors induced a female-typical Fos expression in target forebrain sites of olfactory inputs involved in reproduction in WT, but not in AfpKO females, whereas female odors induced a male-typical Fos expression in males of both genotypes. In WT females, opposite-sex odors induced Fos in kisspeptin and TH neurons, whereas in AfpKO females and WT males, only a lower, but still significant, Fos expression was observed in TH but not in kisspeptin neurons. Finally, opposite-sex odors did not induce any significant Fos expression in GnRH neurons of both sexes or genotypes. Our results strongly suggest a role for fetal estrogen in the sexual differentiation of neural responses to sex-related olfactory cues.

Relative Importance of the Arcuate and Anteroventral Periventricular Kisspeptin Neurons in Control of Puberty and Reproductive Function in Female Rats.

Kisspeptin plays a critical role in pubertal timing and reproductive function. In rodents, kisspeptin perikarya within the hypothalamic arcuate (ARC) and anteroventral periventricular (AVPV) nuclei are thought to be involved in LH pulse and surge generation, respectively. Using bilateral microinjections of recombinant adeno-associated virus encoding kisspeptin antisense into the ARC or AVPV of female rats at postnatal day 10, we investigated the relative importance of these two kisspeptin populations in the control of pubertal timing, estrous cyclicity, and LH surge and pulse generation. A 37% knockdown of kisspeptin in the AVPV resulted in a significant delay in vaginal opening and first vaginal estrous, abnormal estrous cyclicity, and reduction in the occurrence of spontaneous LH surges, although these retained normal amplitude. This AVPV knockdown had no effect on LH pulse frequency, measured after ovariectomy. A 32% reduction of kisspeptin in the ARC had no effect on the onset of puberty but resulted in abnormal estrous cyclicity and decreased LH pulse frequency. Additionally, the knockdown of kisspeptin in the ARC decreased the amplitude but not the incidence of LH surges. These results might suggest that the role of AVPV kisspeptin in the control of pubertal timing is particularly sensitive to perturbation. In accordance with our previous studies, ARC kisspeptin signaling was critical for normal pulsatile LH secretion in female rats. Despite the widely reported role of AVPV kisspeptin neurons in LH surge generation, this study suggests that both AVPV and ARC populations are essential for normal LH surges and estrous cyclicity.

Conditional Viral Tract Tracing Delineates the Projections of the Distinct Kisspeptin Neuron Populations to Gonadotropin-Releasing Hormone (GnRH) Neurons in the Mouse.

Kisspeptin neurons play an essential role in the regulation of fertility through direct regulation of the GnRH neurons. However, the relative contributions of the two functionally distinct kisspeptin neuron subpopulations to this critical regulation are not fully understood. Here we analyzed the specific projection patterns of kisspeptin neurons originating from either the rostral periventricular nucleus of the third ventricle (RP3V) or the arcuate nucleus (ARN) using a cell-specific, viral-mediated tract-tracing approach. We stereotaxically injected a Cre-dependent recombinant adenovirus encoding farnesylated enhanced green fluorescent protein into the ARN or RP3V of adult male and female mice expressing Cre recombinase in kisspeptin neurons. Fibers from ARN kisspeptin neurons projected widely; however, we did not find any evidence for direct contact with GnRH neuron somata or proximal dendrites in either sex. In contrast, we identified RP3V kisspeptin fibers in close contact with GnRH neuron somata and dendrites in both sexes. Fibers originating from both the RP3V and ARN were observed in close contact with distal GnRH neuron processes in the ARN and in the lateral and internal aspects of the median eminence. Furthermore, GnRH nerve terminals were found in close contact with the proximal dendrites of ARN kisspeptin neurons in the ARN, and ARN kisspeptin fibers were found contacting RP3V kisspeptin neurons in both sexes. Together these data delineate selective zones of kisspeptin neuron inputs to GnRH neurons and demonstrate complex interconnections between the distinct kisspeptin populations and GnRH neurons.

Classical and membrane-initiated estrogen signaling in an in vitro model of anterior hypothalamic kisspeptin neurons.

The neuropeptide kisspeptin is essential for sexual maturation and reproductive function. In particular, kisspeptin-expressing neurons in the anterior rostral periventricular area of the third ventricle are generally recognized as mediators of estrogen positive feedback for the surge release of LH, which stimulates ovulation. Estradiol induces kisspeptin expression in the neurons of the rostral periventricular area of the third ventricle but suppresses kisspeptin expression in neurons of the arcuate nucleus that regulate estrogen-negative feedback. To focus on the intracellular signaling and response to estradiol underlying positive feedback, we used mHypoA51 cells, an immortalized line of kisspeptin neurons derived from adult female mouse hypothalamus. mHypoA51 neurons express estrogen receptor (ER)-α, classical progesterone receptor (PR), and kisspeptin, all key elements of estrogen-positive feedback. As with kisspeptin neurons in vivo, 17ß-estradiol (E2) induced kisspeptin and PR in mHypoA51s. The ERα agonist, 1,3,5-Tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole, produced similar increases in expression, indicating that these events were mediated by ERα. However, E2-induced PR up-regulation required an intracellular ER, whereas kisspeptin expression was stimulated through a membrane ER activated by E2 coupled to BSA. These data suggest that anterior hypothalamic kisspeptin neurons integrate both membrane-initiated and classical nuclear estrogen signaling to up-regulate kisspeptin and PR, which are essential for the LH surge.