Anteroventral Periventricular Research Articles

Maternal oral exposure to low-dose BPA accelerates the onset of puberty by promoting prepubertal Kiss1 expression in the AVPV nucleus of female offspring

As the incidence of precocious puberty has risen in recent years and the age at puberty onset is younger, children may be at increased risk for health consequences associated with the early onset of puberty. Bisphenol A (BPA) is recognized as an endocrine disruptor chemical that is reported to induce precocious puberty. The effect of BPA exposure modes, times, and doses (especially low dose) were controversial. In the present study, we evaluated the potential effects of maternal exposure to low-dose BPA on the hypothalamus, particularly on the arcuate (ARC) nucleus and anteroventral periventricular (AVPV) nucleus during peri-puberty in offspring of BPA-treated rats. Pregnant rats were exposed to corn oil vehicle, 0.05 mg·kg−1·day−1 BPA, or 5 mg·kg−1·day−1 from gestation day 1 (GD1) to postnatal day 21 (PND21) by daily gavage. Body weight (BW), vaginal opening (VO), ovarian follicular luteinization, and relevant hormone concentrations were measured; hypothalamic Kiss1 and GnRH1 levels by western immunoblot analysis were also assessed as indices of puberty onset. During or after exposure, low-dose BPA restricted BW after birth (at PND1 and PND5), and subsequently accelerated puberty onset by promoting the expression of prepubertal Kiss1 and GnRH1 in the AVPV nucleus on PND30, leading to advanced VO, an elevation in LH and FSH concentrations (on PND30). We also noted increased BW on PND30 and PND35. Maternal oral exposure to low-dose BPA altered the BW curve during the neonatal and peripubertal periods, and subsequently accelerated puberty onset by promoting prepubertal Kiss1 expression in the AVPV nucleus.

Kisspeptin-neuron control of LH pulsatility and ovulation.

Feedback from oestradiol (E2) plays a critical role in the regulation of major events in the physiological menstrual cycle including the release of gonadotrophins to stimulate follicular growth, and the mid-cycle luteinising hormone (LH) surge that leads to ovulation. E2 predominantly exerts its action via oestrogen receptor-alpha (ERα), however, as gonadotrophin releasing hormone (GnRH) neurons lack ERα, E2-feedback is posited to be indirectly mediated via upstream neurons. Kisspeptin (KP) is a neuropeptide expressed in hypothalamic KP-neurons that control GnRH secretion and plays a key role in the central mechanism regulating the hypothalamic-pituitary-gonadal (HPG) axis. In the rodent arcuate (ARC) nucleus, KP is co-expressed with Neurokinin B and Dynorphin; and thus, these neurons are termed 'Kisspeptin-Neurokinin B-Dynorphin' (KNDy) neurons. ARC KP-neurons function as the 'GnRH pulse generator' to regulate GnRH pulsatility, as well as mediating negative feedback from E2. A second KP neuronal population is present in the rostral periventricular area of the third ventricle (RP3V), which includes anteroventral periventricular (AVPV) nucleus and preoptic area neurons. These RP3V KP-neurons mediate positive feedback to induce the mid-cycle luteinising hormone (LH) surge and subsequent ovulation. Here, we describe the role of KP-neurons in these two regions in mediating this differential feedback from oestrogens. We conclude by considering reproductive diseases for which exploitation of these mechanisms could yield future therapies.

IGF1 gene therapy in middle-aged female rats delays reproductive senescence through its effects on hypothalamic GnRH and kisspeptin neurons.

The process of aging is the result of progressive loss of homeostasis and functional body impairment, including the central nervous system, where the hypothalamus plays a key role in regulating aging mechanisms. The consequences of aging include a chronic proinflammatory environment in the hypothalamus that leads to decreased secretion of gonadotropin-releasing hormone (GnRH) and impairs kisspeptin neuron functionality. In this work, we investigated the effect of insulin-like growth factor 1 (IGF1) gene therapy on hypothalamic kisspeptin/GnRH neurons and on microglial cells, that mediate the inflammatory process related with the aging process. The results show that IGF1 rats have higher kisspeptin expression in the anteroventral periventricular (AVPV) nucleus and higher immunoreactivity of GnRH in the arcuate nucleus and median eminence. In addition, IGF1-treated animals exhibit increased numbers of Iba1+ microglial cells and MHCII+/Iba1+ in the AVPV and arcuate nuclei. In conclusion, IGF1 gene therapy maintains kisspeptin production in the AVPV nucleus, induces GnRH release in the median eminence, and alters the number and reactivity of microglial cells in middle-aged female rats. We suggest that IGF1 gene therapy may have a protective effect against reproductive decline.

PMON12 Effects of Endocrine Disruptors on Autophagy Markers Gene Expression in Immortalized GnRH Neurons and Hypothalami of Adult Male Mice

Abstract Benzophenones (BP) and Bisphenol A (BPA) are endocrine disrupting chemicals (EDC). Previously, we showed that the in-vitro exposure to EDC inhibited Kiss-induced gene expression in GT1-7 and GN11 cells (donated by Pamela Mellon, UCSD), and altered expression of inflammatory markers in GT1-7 cells and hypothalami of adult male mice.In the present study, we evaluated the effects of the in-vivo exposure to BP2 and BP3 (250 µg/kg/day, orally, for 5 days) on the expression of autophagy markers in hypothalamic nuclei in C57Bl/6 adult male mice, and the in-vitro exposure to BPA, BP2 or BP3 in immortalized GnRH neurons. Animals were sacrificed, brains rapidly dissected and conserved -80°C until processed. Micropunches containing Anteroventral Periventricular (AVPV) or Arcuate (ARC) nucleus were obtained. Effects of the in-vitro exposure for 12 or 24 hours to BPA, BP2 or BP3 (1×10-9M, Sigma), or DMSO as control, were evaluated in GT1-7 and GN11 cell lines, mature and immature GnRH neurons. ARN was obtained using Tri-Reagent (Molecular Research Center, Inc), reverse-transcribed and gene expression analyzed by qPCR, using Ppib as control gene. Results were presented as Media±SE and analyzed by ANOVA (Statistica, StatSoft Inc, USA).In-vivo, five-day exposure to BP2 or BP3 did not alter Lamp2 or p62 gene expression in the AVPV or ARC in adult male mice (ANOVA: ns). In GT1-7 cells, Ulk1 was not significantly altered by any of the stimuli (ANOVA: ns; n=5), whereas in GN11 cells, BP3 significantly increased Ulk1 gene expression after 12 and 24 h exposure [DMSO-24h: 0.93±0.21, DMSO-12h: 0.73± 0.19, BPA-24h: 1.78±0.31, BPA-12h: 1.47±0.33, BP2-24h: 1.51±0.49, BP2-12h: 1.06±0.23, BP3-24h: 2.41±0.89, BP3-12h: 2.45±0.82, Repeated Measures ANOVA: Main effect "TREATMENT" p<0.05, BP3 different from DMSO, p<0.05, n=4]. Our results reinforce the notion that the exposure to EDC have different effects depending on the experimental model and time of exposure, with developing cells being more susceptible to disruption that mature cells. We need more experiments to fully understand if exposure to EDC alters other markers of the autophagy process in mouse hypothalamus and GnRH neurons.Funding: CONICET, ANPCYT, International Society for Neurochemistry, Fund. Williams, Fund. R. Barón. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Bisphenol A exposure advances puberty onset by changing Kiss1 expression firstly in arcuate nucleus at juvenile period in female rats

Bisphenol A (BPA) is ubiquitous in the environment and its adverse effects on precocious puberty have been reported. But its mechanism is not clear. In the present study, the potential effects of BPA on endocrine functions of hypothalamus, especially in the arcuate (ARC) nucleus and anteroventral periventricular (AVPe) nucleus, were studied from postnatal day 15 (PND15) to PND35 in female Sprague-Dawley (SD) rats. Neonatal rats were exposed to 0.5 mg·kg−1·day−1 BPA or corn oil vehicle from PND1 to PND14 via intramuscular injection. From PND20 to PND 25, BPA caused enrichment of H3K4me2 and H3K4me3 at Kiss1 promoter, concurrent with elevated gene expressions of Kiss1 and GnRH1 in ARC and strikingly increased serum E2 levels in BPA group on PND25. Until PND30, BPA induced obviously overexpression of Kiss1 and GnRH1 in AVPe nucleus. Subsequently, the vagina opening and first ovulation had occurred earlier in rats with BPA exposure in respect to vehicle by PND35. In this study, it is suggested that the effects of BPA on precocious puberty may be due to its action to activate Kiss1 gene in ARC during the juvenile period (from PND20 to PND25) firstly, subsequently to evoke the AVPe neurons, resulting in precocious puberty in the end.

Distribution of kisspeptin system and its relation with gonadotropin-releasing hormone in the hypothalamus of the South American plains vizcacha, Lagostomus maximus

Kisspeptin (KISS), a key hormone involved in the regulation of the hypothalamic-pituitary-ovarian (HPO) axis, has been localized in the anteroventral periventricular (AVPV) nucleus and the neighboring rostral periventricular nucleus (PeVN), and in the arcuate (ARC) nucleus of the mammalian hypothalamus. In the ARC, the KISS neurons that co-express neurokinin B (NKB) and dynorphin A (Dyn) are named KNDy cells. The South American plains vizcacha is a rodent with peculiar reproductive traits. Around mid-pregnancy, vizcacha shows the reactivation of its HPO axis with the pulsatile release of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH), an essential event for the success of gestation. Considering the role of KISS system in GnRH modulation, the aim of this work was to study their neuroanatomical distribution in adult vizcachas. AVPV showed sexual dimorphism with a significant smaller area in males (t-Test, p < 0.05), and KISS immunoreactivity was detected in somas and varicosities homogenously distributed in the AVPV with a concordant sex-related expression pattern. NKB and Dyn expression was also observed in cytoplasm of neurons scattered in the AVPV. Three subpopulations of neurons were detected in the AVPV: neurons expressing Dyn and NKB (DyNK cells), neurons expressing KISS and NKB (KiNK cells), and single NKB expressing neurons. Strikingly, KISS and Dyn were always expressed in different cells. In addition, in the ARC nucleus, KNDy cells were detected. On the other hand, KISS and GnRH expression was detected in different subpopulations of neurons, GnRH cells showed KISS receptor (KISSR or GPR-54) expression, and KISS immunoreactive afferent contacts were detected making close appositions onto somas and dendrites of GnRH cells. These results show similarities and differences between the KISS system in the hypothalamus of the vizcacha and other mammals, and constitute crucial observations about KISS and GnRH relation. Considering the peculiarity of HPO axis regulation in this species, the present work provides a neuroanatomical framework for the further elucidation of molecular mechanisms underlying GnRH expression and secretion.

Optogenetic Activation of Arcuate Kisspeptin Neurons Generates a Luteinizing Hormone Surge-Like Secretion in an Estradiol-Dependent Manner.

Traditionally, the anteroventral periventricular (AVPV) nucleus has been the brain area associated with luteinizing hormone (LH) surge secretion in rodents. However, the role of the other population of hypothalamic kisspeptin neurons, in the arcuate nucleus (ARC), has been less well characterized with respect to surge generation. Previous experiments have demonstrated ARC kisspeptin knockdown reduced the amplitude of LH surges, indicating that they have a role in surge amplification. The present study used an optogenetic approach to selectively stimulate ARC kisspeptin neurons and examine the effect on LH surges in mice with different hormonal administrations. LH level was monitored from 13:00 to 21:00 h, at 30-minute intervals. Intact Kiss-Cre female mice showed increased LH secretion during the stimulation period in addition to displaying a spontaneous LH surge around the time of lights off. In ovariectomized Kiss-Cre mice, optogenetic stimulation was followed by a surge-like secretion of LH immediately after the stimulation period. Ovariectomized Kiss-Cre mice with a low dose of 17β-estradiol (OVX+E) replacement displayed a surge-like increase in LH release during period of optic stimulation. No LH response to the optic stimulation was observed in OVX+E mice on the day of estradiol benzoate (EB) treatment (day 1). However, after administration of progesterone (day 2), all OVX+E+EB+P mice exhibited an LH surge during optic stimulation. A spontaneous LH surge also occurred in these mice at the expected time. Taken together, these results help to affirm the fact that ARC kisspeptin may have a novel amplificatory role in LH surge production, which is dependent on the gonadal steroid milieu.

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.

Differential Expression of Estrogen Receptors in the Hypothalamus Underlies the Bimodal Effects of Estradiol on Luteinizing Hormone Release

Low 17β-estradiol (E2) levels suppress luteinizing hormone (LH) release, while high E2 stimulates an LH surge in the positive-feedback required for ovulation. Kisspeptin (Kp) neurons in the anteroventral periventricular (AVPV) and arcuate (ARC) nuclei of hypothalamus have been implicated in E2 positive- and negative-feedback effects, respectively. However, how E2 differentially regulates these two neuronal populations remains unknown. We investigated whether neurons in the AVPV and ARC are differently responsive to changes in E2 levels and the regulatory role of estrogen receptors (ERs). Ovariectomized (OV) rats received oil or E2 at doses of 4 (OVE-4), 40 (OVE-40), or 80 (OVE-80) µg/kg. Rats on diestrus (DI) and proestrus (PRO) were used as physiological controls. OV rats were also treated with 0.02, 0.2 or 2.0 mg/kg of propyl pyrazole triol (PPT), or 6 mg/kg of diarylpropionitrile (DPN). Serial blood samples were collected for hormonal measurements. Brains were processed for immunohistochemistry and qPCR analyses in the AVPV and ARC. The E2 doses gradually increased plasma E2, with PRO levels being attained in OVE-80 rats. OVE-80 rats displayed a PRO-like LH surge, while LH levels were constantly suppressed in OVE-4 rats. Progesterone receptor (PR) was used as an index of E2 responsiveness. PR expression was increased in the AVPV of PRO and OVE-80 rats, associated with c-Fos expression and occurrence of LH surge. In the ARC, both low and high E2 induced PR expression and reduced the number of Kp-immunoreactive (ir) neurons, consistent with the negative feedback effects on LH. E2 at 4 or 80 µg/kg equally induced PR expression in 90% of ARC Kp-ir neurons. Despite the higher sensitivity to E2 in the ARC, the percentage of neurons expressing ERα was lower in the ARC compared with AVPV. However, Esr1 expression was 2-fold higher in the ARC than in AVPV for low E2 levels, whereas both Esr1 and Esr2 were more expressed in the AVPV under high E2 status. Notably, Esr1/Esr2 ratio was twice as high in the ARC as in the AVPV regardless of E2 levels, suggesting a stronger ERβ inhibition over ERα in the AVPV. Accordingly, ERα selective activation with PPT increased PR in the ARC at the doses of 0.2 and 2.0 mg/kg, reducing plasma LH, while only the highest dose was able to stimulate PR expression in the AVPV. ERβ activation with DPN, in turn, had no effect in OV rats but amplified the induction of AVPV PR and the size of the LH surge in OVE-80 rats. Thus, we provide evidence that ARC and AVPV neurons are responsive to low and high E2 levels, respectively. ARC is 10 times more sensitive to ERα activation than AVPV, whereas ERβ positively modulates AVPV responsiveness to high E2. These differential responses to E2 seem to be related to differences in the relative Esr1 and Esr2 expression in the ARC and AVPV. Our findings suggest that hypothalamic differences in the relative expression of ERs play a key role in the bimodal regulation of LH release by E2.

Dissecting the Involvement of Arcuate Nucleus Kisspeptin Neurons in Puberty Onset and LH Secretion

Puberty is a crucial period of transition to adulthood, marked by an increased activation of gonadotropin-releasing hormone (GnRH) neurons that drives increased pulsatile secretion of pituitary luteinizing hormone (LH). The mechanisms governing GnRH neuron activation at puberty remain unclear but are likely due to enhanced signaling from upstream neuron populations, including kisspeptin neurons. Kisspeptin (encoded by Kiss1) directly stimulates GnRH neurons to drive GnRH release and downstream LH secretion. Humans and animals with Kiss1 mutations fail to reach puberty, demonstrating kisspeptin’s importance in puberty onset. Nonetheless, the specific brain area(s) from where kisspeptin signaling arises to trigger puberty remain undetermined. Kisspeptin is primarily expressed in two hypothalamic areas, the arcuate nucleus (ARC) and anteroventral periventricular (AVPV) region. ARC Kiss1 neurons are known to drive pulsatile GnRH/LH secretion in both sexes whereas AVPV Kiss1 neurons are sexually dimorphic and mediate the preovulatory GnRH/LH surge in females. We previously showed that Kiss1 gene expression increases in both the ARC and AVPV across the peri-pubertal period, yet it still remains to be determined whether just one or both of these populations is essential for proper pubertal timing. Indeed, the relative involvement of either ARC or AVPV Kiss1 neurons in the pubertal process still remains unknown. Here, we hypothesized that ARC Kiss1 neurons are required for normal puberty timing in both sexes and, conversely, AVPV Kiss1 neurons are not sufficient on their own to trigger normal puberty. To test this hypothesis, we used transgenic mice expressing diphtheria toxin receptor (DTR) exclusively in Kiss1 cells (Kiss Cre/iDTR flox) and took advantage of the differential ontogeny of ARC and AVPV Kiss1 neurons to selectively ablate ARC kisspeptin neurons before puberty, while leaving AVPV neurons intact. We found that targeted deletion of just ARC Kiss1 neurons during the juvenile period (which does not alter AVPV Kiss1 cell number) significantly delays puberty onset in both sexes, as measured by vaginal opening, first estrous, and preputial separation. In addition, these mice also exhibit decreased basal and pulsatile LH secretion in adulthood, further supporting a role for ARC kisspeptin neurons in GnRH pulse generation. By contrast, females with ablated ARC Kiss1 cells still exhibit full estradiol-induced LH surges, ruling out a necessary role of ARC kisspeptin neurons in that process and further supporting AVPV kisspeptin as the primary regulator of the surge. Collectively, our findings demonstrate that ARC Kiss1 neurons are required for both properly timed activation of the reproductive axis during puberty and proper pulsatile LH secretion in adulthood, while AVPV Kiss1 neurons are not sufficient to drive normal puberty onset but are sufficient for the preovulatory LH surge.

Mice With Targeted Deletion of ARC Kisspeptin Exhibit Immature Gametogenesis and Impaired Fertility

Hypothalamic kisspeptin is primarily synthesized in two discrete nuclei - the anteroventral periventricular (AVPV) and the arcuate (ARC) nuclei. We have previously developed a selective, conditional ARC kisspeptin knock-out (KO) mouse line, namely the Pdyn-Cre/Kissfl/fl KO mice, that exhibited normal puberty onset in both sexes, but impaired estrous cyclicity and LH pulsatility in Pdyn-Cre/Kissfl/fl KO females. To examine the end-organ effect of the lack of ARC kisspeptin, we examined gametogenesis, gonad morphology, and fertility. Hematoxylin and eosin (H&E) staining of serial-sectioned whole ovaries demonstrated that Pdyn-Cre/Kissfl/fl KO female mice lacked corpora lutea - their ovarian folliculogenesis did not progress beyond antral follicle development, suggesting an ovulatory defect in Pdyn-Cre/Kissfl/fl KO females. 75% of the Pdyn-Cre/Kissfl/fl KO male mice had testes exhibiting a striking decrease in mature sperm in the seminiferous tubules. The remaining 25% showed evidence of mature sperm. Further evidence of a hypogonadal phenotype of the Pdyn-Cre/Kissfl/fl KO mice included the significantly low weight and small size of the ovaries, uteri, and testes when compared to control littermates. In a controlled, continuous mating paradigm with proven WT males, 2-4-month-old Pdyn-Cre/Kissfl/fl KO female mice failed to become pregnant or produce any pups, whereas age-matched WT females exhibited normal pregnancies to term. Thus, Pdyn-Cre/Kissfl/fl KO females have complete infertility. Ongoing studies of male fertility data suggest that Pdyn-Cre/Kissfl/fl KO males are subfertile, in accordance with their variable spermatogenesis phenotype - some KO males sired pups when paired with proven, WT females, whereas other KO males are infertile. Future experiments include assessing the capability of Pdyn-Cre/Kissfl/fl KO mice to respond to chronic, exogenous kisspeptin and GnRH administration to rescue abnormal LH pulsatility and estrous cyclicity in females, as well as the impaired fertility in both sexes.

SUN-261 Deletion of Hepatic Kisspeptin Results in Abnormal Glucose Metabolism in Female Mice

Kisspeptin is a hypothalamic protein critical for neuroendocrine control of pubertal development and fertility and is modulated by nutritional signals. Kisspeptin has been localized to specific neurons located in the arcuate and anteroventral periventricular (AVPV) nuclei of the hypothalamus and is secreted to control GnRH mediated pubertal maturation and reproduction. Kisspeptin has also been localized to peripheral tissues including the liver, fat, gonads, intestine and placenta, although its role in these tissues is unclear. The objective of current study is to define the role of hepatic kisspeptin as a metabolic sensor. A floxed Kiss1 mouse has been developed, and ablation of liver-specific Kiss1 was achieved in two to three month old Kiss1f/f male and female mice given a single tail vein injection of thyroid hormone-binding globulin (TBG) promoter-driven Cre recombinase adeno-associated virus (AAV-CRE). A control group of Kiss1f/f male and female mice received an injection of AAV-GFP, expressing green fluorescent protein. Two weeks after injection, a glucose tolerance test (GTT) was performed followed by an insulin tolerance test. To determine whether changes had occurred in the reproductive axis, estrous cyclicity was assessed by daily vaginal smears and estrous cycle phases determined by vaginal cytology. Mice were euthanized four weeks post-injection and tissues were collected for RNA extraction and gene expression analysis via qRT-PCR. As expected, qRT-PCR data showed absence of Kiss1 expression in the liver of AAV-CRE mice compared to AAV-GFP mice with no changes in kisspeptin gene expression were noted in the ovary, testes, spleen, pancreas, arcuate or AVPV. Estrous cyclicity was also not affected by viral ablation of hepatic Kiss1. Elevated fasting glucose and glucose intolerance in the GTT were found in AAV-CRE compared to AAV-GFP females (P < 0.05). No differences in AAV-CRE and AAV-GFP male mice were found, indicating the importance of Kiss1 in glucose homeostasis in females. The insulin tolerance test was not statistically different between groups or treatments. Further research is required to elucidate the mechanism by which hepatic kisspeptin alters glucose metabolism in mice in a sexually-dimorphic fashion.

SUN-LB57 Assessing the Requirement of Arcuate Nucleus Kisspeptin Neurons in Puberty Onset and Adult LH Secretion

Puberty is a critical developmental period marking the transition to adulthood and attainment of reproductive capability. A hallmark of puberty is increased pulsatile secretion of pituitary luteinizing hormone (LH) which is itself driven by increased gonadotropin-releasing hormone (GnRH) from the forebrain. The mechanisms governing GnRH neuron activation at puberty still remain unclear, but likely include enhanced stimulation from upstream reproductive neural circuits, including kisspeptin neurons. Kisspeptin is a potent stimulator of GnRH and is required for proper puberty onset. However, the specific brain site(s) from where kisspeptin signaling arises to trigger puberty remain unclarified. Kisspeptin is expressed in two primary nuclei in the hypothalamus, the arcuate nucleus (ARC) and anteroventral periventricular (AVPV) region. Studies suggest that, in adulthood, ARC Kiss1 neurons are involved in driving pulsatile secretion of GnRH (and hence, LH) in both sexes whereas AVPV Kiss1 neurons participate in the preovulatory GnRH/LH surge in females. However, the specific role of either kisspeptin neuron population in puberty onset still remains unknown. We previously showed that both kisspeptin populations show increased Kiss1 gene expression across the pubertal period, yet whether just one or both (or neither) population is needed for puberty to occur has not been determined. Here, we sought to tease out the role—if any—of ARC and AVPV Kiss1 neurons in the pubertal onset process. Since ARC Kiss1 neurons are abundant in both sexes and drive pulsatile GnRH secretion in adulthood, we hypothesized that ARC Kiss1 neurons are necessary for normal puberty onset and, conversely, that AVPV Kiss1 neurons are not sufficient on their own to induce normal puberty. To test this hypothesis, we used a Cre-specific diphtheria toxin approach to ablate just ARC Kiss1 neurons in juvenile mice (~ 2 weeks old) while leaving AVPV Kiss1 neurons intact. Preliminary data thus far indicates that site specific ablation of just ARC Kiss1 neurons during the juvenile period significantly delays puberty onset in both sexes, as measured by vaginal opening, first estrous, and preputial separation. In addition, selective ARC Kiss1 neuron ablation in juvenile life diminishes pulsatile LH secretion levels measured in adulthood, but does not alter LH surge generation in adult females. These preliminary findings empirically demonstrate that, in mice, ARC Kiss1 neurons are required for proper activation of the reproductive axis during puberty but not the LH surge in adulthood, and AVPV Kiss1 neurons are not sufficient to trigger normal pubertal onset.

Effects of the Fertility Drugs Clomiphene Citrate and Letrozole on Kiss-1 Expression in Hypothalamic Kiss-1-Expressing Cell Models.

Clomiphene citrate (CC) and letrozole stimulate the hypothalamic-pituitary-ovarian axis and are used widely as oral fertility drugs to induce folliculogenesis. We examined whether these drugs increase Kiss-1 expression in hypothalamic cell models. We utilized two hypothalamic cell models, mHypoA-50 and mHypoA-55, which originated from Kiss-1 neurons in the anteroventral periventricular (AVPV) nucleus and arcuate (ARC) nucleus of the mouse hypothalamus, respectively. The cells were stimulated with CC or letrozole, after which Kiss-1 mRNA expression was determined. CC stimulated Kiss-1 gene expression in mHypoA-50 and mHypoA-55 cells. The basal expression of Kiss-1 was significantly increased in the presence of estradiol (E2) in mHypoA-50 cells, and the CC-induced increase in Kiss-1 expression was not observed in the presence of E2 in these cells. In contrast, E2 did not modify the basal expression of Kiss-1 in mHypoA-55 cells, and CC-induced Kiss-1 expression was still observed in the presence of E2. The significant increase in Kiss-1 gene expression in mHypoA-50 and mHypoA-55 cells was blunted in the presence of estrogen receptor antagonists. Aromatase was expressed in mHypoA-50 and mHypoA-55 cells. Letrozole, an aromatase inhibitor, increased Kiss-1 expression in mHypoA-55 ARC cells but not in mHypoA-50 AVPV cells. Although the basal expression of Kiss-1 was increased by E2, letrozole did not modulate Kiss-1 expression in mHypoA-50 cells. Letrozole-induced Kiss-1 gene expression in mHypoA-55 cells was not modulated in the presence of E2. The fertility drugs CC and letrozole modulated Kiss-1 expression in hypothalamic cell models.

Periodic Remodeling in a Neural Circuit Governs Timing of Female Sexual Behavior

Behaviors are inextricably linked to internal state. We have identified a neural mechanism that links female sexual behavior with the estrus, the ovulatory phase of the estrous cycle. We find that progesterone-receptor (PR)-expressing neurons in the ventromedial hypothalamus (VMH) are active and required during this behavior. Activating these neurons, however, does not elicit sexual behavior in non-estrus females. We show that projections of PR+ VMH neurons to the anteroventral periventricular (AVPV) nucleus change across the 5-day mouse estrous cycle, with ∼3-fold more termini and functional connections during estrus. This cyclic increase in connectivity is found in adult females, but not males, and regulated by estrogen signaling in PR+ VMH neurons. We further show that these connections are essential for sexual behavior in receptive females. Thus, estrogen-regulated structural plasticity of behaviorally salient connections in the adult female brain links sexual behavior to the estrus phase of the estrous cycle.

Role of RFRP-3 in the Regulation of Kiss-1 Gene Expression in the AVPV Hypothalamic Cell Model mHypoA-50.

Kisspeptin, encoded by the Kiss-1 gene, plays a crucial role in reproductive function by governing the hypothalamic-pituitary-gonadal axis. The recently established Kiss-1-expressing cell model mHypoA-50 displays characteristics of neuronal cells of the anteroventral periventricular (AVPV) region of the mouse hypothalamus. Because Kiss-1 gene expression in these cells is upregulated by estradiol (E2), mHypoA-50 cells are regarded as a valuable model for the study of Kiss-1-expressing neurons in the AVPV region. These cells also express RFamide-related peptide-3 (RFRP-3), a mammalian homolog of gonadotropin inhibitory hormone. The RFRP-3 expression in mHypoA-50 cells was increased by melatonin stimulation. In addition, E2 stimulation increased RFRP-3 expression in these cells. Treatment of the mHypoA-50 cells with exogenous RFRP-3 resulted in the increase of Kiss-1 messenger RNA expression within the cells; however, RFRP-3 did not modify gonadotropin-releasing hormone or kisspeptin-induced Kiss-1 gene expression in these cells. In addition, we found that RFRP-3 stimulation increased the expression of corticotropin-releasing hormone, which may be involved in E2-induced positive feedback in mHypoA-50 cells. Our observations suggest that RFRP-3 might be involved in positive feedback regulation by directly or indirectly increasing Kiss-1 gene expression.

SUN-200 Computational Model for Energy Sensing of PMv Neuronal Circuitry

The energy sensing in reproduction is mainly modulated by the ventral premammillary nucleus (PMv) neurons in hypothalamus. They are sensitive to the adipocyte-derived hormone leptin; some of them are excitatory to leptin, and the others are inhibitory to leptin. Recently we have characterized PMv neurons expressing dopamine transporters (DAT) suppress female reproductive function, and this is inhibited by leptin. We hypothesize that 1) PMv DAT neurons depolarize AgRP neurons. Some PMv non-DAT neurons depolarize Kiss1 neurons, and this pathway is independent of GABAergic projection from AgRP to Kiss1 neurons. The other PMv non-DAT neurons innervate directly to GnRH neurons. The size of PMv DAT sub-population projecting to AgRP neurons is main determinant of suppressing GnRH bursting spikes. 2) The direct innervation from PMv non-DAT to GnRH neurons sustains GnRH pulsatility even with low level of kisspeptin secretion. To construct a minimal neuronal circuit for energy sensing, we developed a computational model of conductance-based electrophysiology; input signal to PMv by dopamine transporter and leptin, as well as estradiol to Kiss1 neurons, and the output response of bursting spike frequency by GnRH neurons. For excitatory PMv neurons, leptin-sensitive TRPC channels are incorporated. Leptin-dependent KATP as well as hypothesized dopamine-sensitive channels are applied for DAT neurons inhibited by leptin. Both subpopulation neurons release glutamate neurotransmitters. For Kiss1 neurons, we employed T-type calcium channels and hyperpolarization-activated rebounding channels with kisspeptin neuropeptide release. We have incorporated Kiss1 sub-populations in anteroventral periventricular (AVPV) with estradiol-positive feedback and those in the arcuate with estradiol-negative feedback. For AgRP neurons, leptin-dependent KATP channels are considered with GABAA release for projection to Kiss1 neurons. The computational neuronal circuitry model based on the regulation of PMv DAT neurons by viral DREADDs demonstrates that Kiss1 and AgRP neurons mediate for the suppression of GnRH neurons and regulation of female fertility. The direct relay from leptin-activated PMv to GnRH with restriction of kisspeptin secreted from Kiss1 shows a moderate contribution of this pathway to sexual maturation and fertility independent of steroidogenic feedback.

SUN-473 The Contribution of Bmal1 Expression in the Suprachiasmatic Nucleus to Female Fertility

The preovulatory gonadotropin-releasing hormone (GnRH) and resulting luteinizing hormone (LH) surge is gated to the onset of the active period in mice by a circadian and steroid signal. The temporal signal that initiates this process is believed to arrive from the suprachiasmatic nucleus (SCN), the hypothalamic circadian pacemaker. The SCN may contribute to reproduction through two discrete projections: vasoactive inhibitory peptide (VIP) neurons in the SCN project to gonadotropin-releasing hormone (GnRH) neurons in the preoptic area, and arginine vasopressin (AVP) neurons in the SCN project to kisspeptin (Kiss1) neurons in the anteroventral periventricular (AVPV). Genomic deletion of the critical circadian clock component, Bmal1, results in infertility in females. We sought to determine whether Bmal1 was critical in the SCN VIP and AVP neurons by using cre/lox technology to create three lines of mutant mice: AVP-Bmal1-/-, VIP-Bmal1-/-, and AVP-VIP-Bmal1-/-. We found no significant difference in LH levels following a kisspeptin challenge (2 mg/kg) between AVP-Bmal1-/- and Bmal1flox/flox (617.4 ± 216.9 vs 629.6 ± 110.6 pg/mL) or GnRH challenge (10 μg/kg) (1029.0 ± 246.2 vs 712.7 ± 142.6 pg/mL). Preliminary findings of an induced LH surge found AVP-Bmal1-/- and VIP-Bmal1-/- mice are able to mount a surge at the time of lights off (Bmal1flox/flox: 3 of 5; AVP-Bmal1-/-: 1 of 2, VIP-Bmal1:-/- 4 of 4; AVP-VIP-Bmal1-/- 0 of 2). Additionally, we were able to detect an endogenous surge at the time of lights off in both AVP-Bmal1-/-, VIP-Bmal1-/- mice, but not AVP-VIP-Bmal1-/-. However, AVP-Bmal1-/-, VIP-Bmal1-/-, and AVP-VIP-Bmal1-/- females carried litters to term and show no differences in the time to first litter compared to Bmal1flox/flox control (AVP-Bmal1-/-: 48 ± 4.8 vs 44.3 ± 0.3 days; VIP-Bmal1-/-: 29.6 ± 7.7 vs 31.0 ± 4.2 days; AVP-VIP-Bmal1-/-;27.7 ± 7 vs 23 ± 0 days); number of litters in 100 days (AVP-Bmal1-/-: 3.7 ±0.6 vs 4 ± 0.6; VIP-Bmal1-/-: 3.0 ± 1.0 vs 3.0 ± 0.0; AVP-VIP-Bmal1-/- 3.5 ± 0.7 vs 3.5 ± 0.7 days); or pups per litter (AVP-Bmal1-/-: 7.2 ± 0.7 vs 7.3 ± 0.7; VIP-Bmal1-/-: 6.2 ± 1.6 vs 7.6 ± 2.9; AVP-VIP-Bmal1-/- 8.1 ± 0.9 vs 9 ± 1.4 pups). Overall, these findings suggest that Bmal1 expression in the SCN AVP or VIP neurons is not required for fertility in a 12:12 light:dark cycle.

Genetic dissection of the different roles of hypothalamic kisspeptin neurons in regulating female reproduction.

The brain regulates fertility through gonadotropin-releasing hormone (GnRH) neurons. Estradiol induces negative feedback on pulsatile GnRH/luteinizing hormone (LH) release and positive feedback generating preovulatory GnRH/LH surges. Negative and positive feedbacks are postulated to be mediated by kisspeptin neurons in arcuate and anteroventral periventricular (AVPV) nuclei, respectively. Kisspeptin-specific ERα knockout mice exhibit disrupted LH pulses and surges. This knockout approach is neither location-specific nor temporally controlled. We utilized CRISPR-Cas9 to disrupt ERα in adulthood. Mice with ERα disruption in AVPV kisspeptin neurons have typical reproductive cycles but blunted LH surges, associated with decreased excitability of these neurons. Mice with ERα knocked down in arcuate kisspeptin neurons showed disrupted cyclicity, associated with increased glutamatergic transmission to these neurons. These observations suggest that activational effects of estradiol regulate surge generation and maintain cyclicity through AVPV and arcuate kisspeptin neurons, respectively, independent from its role in the development of hypothalamic kisspeptin neurons or puberty onset.

Neonatal and juvenile exposure to perfluorooctanoate (PFOA) and perfluorooctane sulfonate (PFOS): Advance puberty onset and kisspeptin system disturbance in female rats

Perfluorooctanoate (PFOA) and perfluorooctane sulfonate (PFOS) are widespread and persistent chemicals in the environment, and limited data about their effects on puberty development are available. In order to explore the effects of neonatal and juvenile PFOA/PFOS exposure on puberty maturation, female rats were injected with PFOA or PFOS at 0.1, 1 and 10 mg/kg/day during postnatal day (PND) 1–5 or 26–30. The day of vaginal opening (VO) and first estrus were significantly advanced in 10 mg/kg PFOA, 1 and 10 mg/kg PFOS groups after neonatal and juvenile exposure. Besides, neonatal PFOA/PFOS exposure increased body weight and anogenital distance (AGD) in a non-dose-dependent manner. Estradiol and luteinizing hormone levels were also increased with more frequent occurrences of irregular estrous cycles in 0.1 and 1 mg/kg PFOA/PFOS exposure groups. Although no altered ovarian morphology was observed, follicles numbers were reduced in neonatal groups. Kiss1, Kiss1r and ERα mRNA expressions were downregulated after two periods’ exposure in the hypothalamic anteroventral periventricular (AVPV) and arcuate (ARC) nuclei. PFOA/PFOS exposure also suppressed kisspeptin fiber intensities, especially at the high dose. In conclusion, neonatal and juvenile are critical exposure periods, during which puberty maturation may be vulnerable to environmental exposure of PFOA/PFOS, and kisspeptin system plays a key role during these processes.