GnRH Pulse Research Articles

9228 Insulin-Like Growth Factor-1 Modulates Arcuate Kisspeptin Neuron Activity

Abstract Disclosure: J.N. Silva: None. R. Frazao: None. The kisspeptin-producing cells located at the arcuate nucleus of the hypothalamus (ARHKisspeptin) are considered the GnRH pulse generator necessary for fertility. In addition, ARHKisspeptin cells act as a metabolic sensor to control the hypothalamic-pituitary-gonadal (HPG) axis. The insulin-like growth factor-1 (IGF-1) is considered a component contributing to puberty onset and fertility. Peripherical or intracerebroventricular IGF-1 administration is sufficient to increase hypothalamic Kiss1 expression. However, whether IGF-1 acts directly on ARHKisspeptin cells to modulate the HPG axis is not known. To determine whether IGF-1 modulates ARHKisspeptin neuron activity, we used mice expressing the green fluorescent protein “humanized renilla” under the transcriptional control of the Kiss1 gene (8-10 weeks old). Fura-2 fluorescent probe was used to evaluate the state coupled or uncoupled to calcium in the face of IGF- 1 administration (0.5 μg/ml). Furthermore, whole-cell patch-clamp was employed to determine IGF-1 effects on ARHKisspeptin neuron's resting membrane potential (RMP). FURA-2 was incorporated by 64 ARHKisspeptin neurons out of 424 cells in the ARH. IGF-1 administration to the bath led to a significant increase of the intracellular calcium levels ([Ca2+]i) in 53% of ARHKisspeptin cells evaluated in male mice (34 out of 64 cells; IGF-1, 203.0 ± 26.1 nM; baseline period, 68.8 ± 9.6 nM; P< 0.0001). Non-responsive cells (30 out of 64 ARHKisspeptin neurons) exhibited average [Ca2+]i of 53.2 ± 6.3 nM after IGF-1 application vs 46.2 ± 5.6 nM in the baseline period (P= 0.3). Of note, IGF-1 also increased [Ca2+]i in 25% of non-kisspeptin cells in the ARH (93 out of 364 cells, IGF-1, 193.8 ± 12.4 nM; vs baseline period, 73.0 ± 5.6 nM, P< 0.0001). By evaluating the IGF-1 effects on ARHKisspeptin neuron’s RMP, we observed that IGF-1 tends to depolarize the RMP of 18% of cells recorded from males (4 out of 22 cells, IGF-1, -58.0 ± 3.0 mV; baseline, -64.1 ± 4.4 mV; P= 0.05), and depolarized the RMP of 44% of cells recorded from female mice (7 out of 16 cells, IGF-1, -61.1 ± 3.3 mV; baseline, -66.3 ± 2.7 mV; P= 0.002). Importantly, IGF-1-induced depolarization of ARHKisspeptin cells RMP was blocked when the recordings were performed in the presence of tetrodotoxin and ionotropic amino acid antagonists. Therefore, IGF-1-induced effects on ARHKisspeptin cells RMP depend on action potentials mediated by synaptic transmission. Our results suggest that IGF-1 indirectly modulates the activity of a large number of ARHKisspeptin neurons. Thus, situations of metabolic stress that induce changes in growth hormone secretion and, consequently, IGF-1 circulating levels, (e.g., fasting), can modulate the HPG axis through the indirect modulation of the activity of ARHKisspeptin cells. Presentation: 6/2/2024

8388 Insulin-Like Growth Factor-1 Modulates Arcuate Kisspeptin Neuron Activity

Abstract Disclosure: J.N. Silva: None. R. Frazao: None. The kisspeptin-producing cells located at the arcuate nucleus of the hypothalamus (ARHKisspeptin) are considered the GnRH pulse generator necessary for fertility. In addition, ARHKisspeptin cells act as a metabolic sensor to control the hypothalamic-pituitary-gonadal (HPG) axis. The insulin-like growth factor-1 (IGF-1) is considered a component contributing to puberty onset and fertility. Peripherical or intracerebroventricular IGF-1 administration is sufficient to increase hypothalamic Kiss1 expression. However, whether IGF-1 acts directly on ARHKisspeptin cells to modulate the HPG axis is not known. To determine whether IGF-1 modulates ARHKisspeptin neuron activity, we used mice expressing the green fluorescent protein “humanized renilla” under the transcriptional control of the Kiss1 gene (8-10 weeks old). Fura-2 fluorescent probe was used to evaluate the state coupled or uncoupled to calcium in the face of IGF- 1 administration (0.5 μg/ml). Furthermore, whole-cell patch-clamp was employed to determine IGF-1 effects on ARHKisspeptin neuron's resting membrane potential (RMP). FURA-2 was incorporated by 64 ARHKisspeptin neurons out of 424 cells in the ARH. IGF-1 administration to the bath led to a significant increase of the intracellular calcium levels ([Ca2+]i) in 53% of ARHKisspeptin cells evaluated in male mice (34 out of 64 cells; IGF-1, 203.0 ± 26.1 nM; baseline period, 68.8 ± 9.6 nM; P< 0.0001). Non-responsive cells (30 out of 64 ARHKisspeptin neurons) exhibited average [Ca2+]i of 53.2 ± 6.3 nM after IGF-1 application vs 46.2 ± 5.6 nM in the baseline period (P= 0.3). Of note, IGF-1 also increased [Ca2+]i in 25% of non-kisspeptin cells in the ARH (93 out of 364 cells, IGF-1, 193.8 ± 12.4 nM; vs baseline period, 73.0 ± 5.6 nM, P< 0.0001). By evaluating the IGF-1 effects on ARHKisspeptin neuron’s RMP, we observed that IGF-1 tends to depolarize the RMP of 18% of cells recorded from males (4 out of 22 cells, IGF-1, -58.0 ± 3.0 mV; baseline, -64.1 ± 4.4 mV; P= 0.05), and depolarized the RMP of 44% of cells recorded from female mice (7 out of 16 cells, IGF-1, -61.1 ± 3.3 mV; baseline, -66.3 ± 2.7 mV; P= 0.002). Importantly, IGF-1-induced depolarization of ARHKisspeptin cells RMP was blocked when the recordings were performed in the presence of tetrodotoxin and ionotropic amino acid antagonists. Therefore, IGF-1-induced effects on ARHKisspeptin cells RMP depend on action potentials mediated by synaptic transmission. Our results suggest that IGF-1 indirectly modulates the activity of a large number of ARHKisspeptin neurons. Thus, situations of metabolic stress that induce changes in growth hormone secretion and, consequently, IGF-1 circulating levels, (e.g., fasting), can modulate the HPG axis through the indirect modulation of the activity of ARHKisspeptin cells. Presentation: 6/2/2024

Exploring the connection: endocrine disruptors and polycystic ovarian syndrome

Polycystic ovarian syndrome (PCOS) is heterogeneous endocrine disorder in females manifesting reproductive dysfunction and metabolic abnormalities. Endocrinopathy in the form of hyperandrogenism leading to alteration in clinical phenotype and fertility seen. Atherogenic dyslipidaemia and insulin resistance as a result of metabolic disturbance also encountered. Recent years, endocrine disrupting chemicals (EDCs) are widely studied and linked for their alleged role in the development of PCOS. EDCs like bisphenol A (BPA), Phthalate, methoxychlor and chlorpyrifos which are present in many industrial as well as daily use products poses risk of development of various diseases. This review discusses the role of EDCS specially BPA in the pathogenesis of PCOS with study of interest identified and extracted from databases like Pub Med and Google scholars using MeSH keywords. BPA has estrogenic property and binds to oestrogen receptors α and β. Stimulation of ovarian theca cells and dysregulation of steroid biosynthesis leads to androgen overproduction. It stimulates GnRH Pulse generator, decreasing the level of LH hence fertility is affected. BPA also interact with adipose tissue receptors and causes differentiation, lipid deposition and inhibition of adiponectin. Its serum and urinary levels are found to be elevated in PCOS patient. In animal studies, it is found that BPA exposure causes impaired folliculogenesis, insulin resistance and DNA methylation. EDC exposure, especially BPA which is an integral constituent of many industrial and daily use items may cause PCOS possibly by altering androgen synthesis, adipocyte stimulation and epigenetic modification.

Chemogenetic activation of PVN CRH neurons disrupts the estrous cycle and LH dynamics in female mice.

The impact of stress on reproductive function is significant. Hypothalamic paraventricular nucleus (PVN) corticotrophin-releasing hormone (CRH) plays a major role in regulating the stress response. Understanding how the hypothalamic-pituitary-adrenal (HPA) axis and the hypothalamic-pituitary-gonadal (HPG) axis interact is crucial for comprehending how stress can lead to reproductive dysfunction. However, whether stress influences reproductive function via modulating PVN CRH or HPA sequelae is not fully elucidated. In this study, we investigated the impact of chemogenetic activation of PVN CRH neurons on reproductive function. We chronically and selectively stimulated PVN CRH neurons in female CRH-Cre mice using excitatory designer receptor exclusively activated by designer drugs (DREADDs) viral constructs, which were bilaterally injected into the PVN. The agonist compound-21 (C21) was delivered through the drinking water. We determined the effects of DREADDs activation of PVN CRH neurons on the estrous cycles, LH pulse frequency in diestrus and metestrus and LH surge in proestrus mice. The effect of long-term C21 administration on basal corticosterone secretion and the response to acute restraint stress during metestrus was also examined. Additionally, computer simulations of a mathematical model were used to determine the effects of DREADDs activation of PVN CRH neurons, simulating chronic stress, on the physiological parameters examined experimentally. As a result, and consistent with our mathematical model predictions, the length of the estrous cycle was extended, with an increase in the time spent in estrus and metestrus, and a decrease in proestrus and diestrus. Additionally, the frequency of LH pulses during metestrus was decreased, but unaffected during diestrus. The occurrence of the preovulatory LH surge during proestrus was disrupted. The basal level of corticosterone during metestrus was not affected, but the response to acute restraint stress was diminished after long-term C21 application. These data suggest that PVN CRH neurons play a functional role in disrupting ovarian cyclicity and the preovulatory LH surge, and that the activity of the GnRH pulse generator remains relatively robust during diestrus but not during metestrus under chronic stress exposure in accordance with our mathematical model predictions.

The GnRH pulse generator - shining a light into the black box

The GnRH pulse generator - shining a light into the black box

FRI286 Stimulation Of Egr1 Expression Is Mediated By A Gαq/11-signaling Pathway In A Pattern Distinct From That Of MEK/ERK At High And Low GnRH Pulse Frequencies

Abstract Disclosure: G.A. Stamatiades: None. H.K. Kim: None. R.S. Carroll: None. U.B. Kaiser: None. Introduction: The pulsatile release of the hypothalamic decapeptide, GnRH, activates signal transduction cascades in pituitary gonadotropes to control the synthesis and secretion of LH and FSH, hormones critical for normal reproductive function and fertility. LH and FSH release are differentially regulated by varying GnRH pulse patterns, with LH secretion being preferentially stimulated at high (e.g., every 30 min) GnRH pulse frequencies, while FSH secretion is preferentially induced at low (e.g., every 2 hour) GnRH pulses. LH is a heterodimer comprised of a common α subunit and a distinct LHβ subunit, with Lhb gene (encoding LHβ) transcription being preferentially stimulated at the higher GnRH pulse frequencies. It is well established that Egr1 is a key inducer of Lhb expression upon pulsatile GnRH stimulation. In addition, it has been shown that MEKI/II inhibition abrogates the induction of Lhb mRNA levels at both high and low GnRH pulse frequencies. Hypothesis:Egr1 expression is mediated by a Gαq/11-signaling pathway that includes MEK/ERK at both high and low GnRH pulse frequencies. Methods: To determine whether a Gαq/11-mediated pathway regulates Egr1 expression upon GnRH stimulation, LβT2 cells transduced with lentiviruses encoding scrambled or Gnaq/11 shRNA were perifused and treated with pulsatile GnRH at either high (q30min) or low (q2h) pulse frequencies, or with medium only, for 20 hours. Following the same perifusion paradigm, ERK1/2 phosphorylation was also assessed. Results: LβT2 cells transduced with a scrambled control shRNA showed a GnRH pulse frequency-dependent pattern of induction of Egr1 expression, with significantly greater induction at high than at low GnRH pulse frequency, compared to cells perifused with media only. In addition, Gαq/11 depletion reduced Egr1 mRNA levels at both high and low GnRH pulse frequencies, but the frequency dependence was maintained. A GnRH pulse frequency-dependent pattern of induction of ERK1/2 phosphorylation was observed, with significantly greater induction at low than at high GnRH pulse frequency in control cells transduced with scrambled control shRNA, consistent with prior studies from our group. Following Gnaq/11 KD, the induction of ERK1/2 phosphorylation was reduced at both GnRH pulse frequencies, with loss of the pulse frequency dependence. Conclusion: Taken together, these findings suggest that a Gαq/11-signaling pathway involving Egr1 induces Lhb expression at both pulse frequencies. However, the link between MEK/ERK and Egr1 needs to be further investigated, given the differences in the GnRH pulse frequency dependent patterns of induction. Presentation: Friday, June 16, 2023

SAT608 Gonadotrope-specific Deletion Of Icer Results In Increased Lh Pulsatility But Reduced Fsh Levels In Mice

Abstract Disclosure: N.P. Boris: None. S.A. Pereira: None. G.A. Stamatiades: None. K.N. Hausken: None. H. Kim: None. R.S. Carroll: None. U.B. Kaiser: None. Background: The hypothalamic-pituitary-gonadal (HPG) axis is regulated by pulsatile secretion of hypothalamic GnRH, which acts on anterior pituitary gonadotropes to control synthesis and secretion of LH and FSH. High and low GnRH pulse frequencies preferentially stimulate Lhb and Fshb expression, respectively, through mechanisms that are not completely understood. The mouse Fshb gene promoter contains a half-AP-1/half-cAMP response element (CRE) motif; we have shown previously that this element is bound by CRE-binding protein (Creb) in vitro to increase expression in response to low GnRH pulse frequencies, but is antagonized by inducible cAMP early repressor (Icer) at higher GnRH pulse frequencies, resulting in a lower level of Fshb transcription. In this study, we aimed to identify the role of Icer in gonadotropin regulation in vivo in mice. We hypothesized that gonadotrope-specific deletion of Icer may influence pubertal timing and fertility due to an increase in Fshb expression. Methods and Results: Gonadotrope-specific deletion of Icer (GnrhrGRIC/+/Icerfl/fl; Icer KO) and control (Icerfl/fl) mice were generated, and pubertal and reproductive phenotypes were evaluated. There was no difference in body weight or in pubertal timing, evaluated by age of preputial separation in males and by vaginal opening and first estrus in females. In post-pubertal male mice, neither basal LH and FSH levels nor the LH or FSH response to acute GnRH stimulation were significantly different in Icer KO mice, compared to littermate Icerfl/fl controls. Male Icer KO mice 14 days post-gonadectomy (GDX) had significantly more LH pulses within a three-hour period than controls (Icer KO 5.83 ± 0.4 pulses vs Icerfl/fl 4.66 ± 0.3 pulses; p=0.049), but no differences in basal LH or LH pulse amplitude. Interestingly, serum FSH levels were significantly lower in Icer KO mice compared to Icerfl/fl controls 2 days post-GDX (Icer KO 20.0 ± 1.0 vs Icerfl/fl 26.0 ± 0.8; p=0.003) and 7 days post-GDX (Icer KO 18.0 ± 0.4 vs Icerfl/fl 22.5 ± 0.8, p=0.002). There were no significant differences in pituitary Fshb, Lhb, or Gnrhr mRNA levels between Icer KO and Icerfl/fl control mice 14 days post-GDX. Conclusions: Deletion of Icer from pituitary gonadotropes in mice did not affect body weight or timing of pubertal onset. However, in GDX male mice, LH pulse frequency was increased, whereas the post-GDX increase in serum FSH levels was blunted, in Icer KO mice compared to controls. These findings support a role for Icer in both LH and FSH regulation in vivo and highlight the importance of extending in vitro observations to in vivo models. Presentation: Saturday, June 17, 2023

FRI284 The GnRH Pulse Generator Activity In Mouse Models Of Polycystic Ovary Syndrome

Abstract Disclosure: Z. Zhou: None. M. Pardo-Navarro: None. S.Y. Han: None. A.E. Herbison: None. Up to one in five women worldwide suffer from polycystic ovary syndrome (PCOS), a condition typified by problems with fertility, high androgen levels, and altered metabolism. Primarily due to the unknown aetiology of this disorder, limited strategies are available for PCOS treatment. The hypothalamic GnRH pulse generator is responsible for driving the pulsatile release of LH to control ovarian function. As PCOS patients show profound increase in LH pulse frequency, it is thought that over-activity of the GnRH pulse generator may have a major role in generating the sub-fertility of women with PCOS. Recent studies involving the real-time imaging of neural activity in genetic mouse models have revealed that a population of kisspeptin neurons located in the arcuate nucleus of the hypothalamus is the long-sought after “GnRH pulse generator”. These arcuate kisspeptin neurons exhibit synchronization events (SEs) that correlate perfectly with pulsatile LH secretion. With this technique, we aimed to characterize the activity pattern of the arcuate kisspeptin neurons in two most-widely used mouse models of PCOS: prenatal androgen (PNA) and peripubertal androgen models. In the first model, PNA female mice exhibited near-acyclic estrous cycles as previously reported, but surprisingly exhibited only a slightly (∼30%) increased frequency of arcuate kisspeptin neuron SEs over a 24-hour recording period compared to the controls. The profiles and dynamics of individual SEs were not different between PNA and control animals. Concurrent fiber photometry recording and serial blood sampling showed that each SE is tightly followed by an LH pulse in all animals. Three weeks following ovariectomy, the SE frequency and profile were not different between PNA and control mice. In the second peripubertal model, female mice implanted with dihydrotestosterone capsules at 3-weeks of age exhibited significant weight gain but decreased arcuate kisspeptin neuron activity over a 24-hour recording period also without changes in SE dynamics compared to the controls. In conclusion, it appears that neither of the two most-commonly used PCOS mouse models mirror the human condition in which many PCOS women exhibit robust increases in pulsatile LH secretion. Presentation: Friday, June 16, 2023

FRI419 Progesterone Regulation Of The GnRH Pulse Generator

Abstract Disclosure: E.G. Wall: None. A group of kisspeptin neurons located in the arcuate nucleus (ARN) of the hypothalamus represent the GnRH pulse generator and their activity is thought to be controlled primarily by the feedback actions of estradiol and progesterone (P4). In a healthy menstruating woman, following the mid-cycle ovulation, P4 levels dramatically increase and exert a negative feedback action onto the hypothalamus to slow pulsatile LH secretion. However, the exact mechanism by which P4 negatively feeds back to the hypothalamus remains unknown. The ARN kisspeptin neurons display synchronized episodes (SEs) of activity approximately every hour for most of the cycle but these dramatically reduce in number following the LH surge. As these ARN kisspeptin neurons express the progesterone receptor (PR) it is hypothesized that they may be the site at which P4 negative feedback occurs. Given the lack of detailed information in the mouse, we first assessed the profile of circulating P4 levels in mice at 8-hr intervals throughout the estrous cycle. Terminal blood samples were taken from individual mice along the 4 stages of the estrous cycle at 3-time points 8 hrs apart and the gonadal hormones were analyzed using LC-MS. The concentration of P4 was found to peak around the time of the LH surge at 6 pm on proestrus (21.94 ± 3.78 ng/ml) and was at its lowest at diestrus 10 am (0.42 ± 0.11 ng/ml). We next assessed the impact of circulating P4 on the activity of the ARN kisspeptin pulse generator using GCaMP fibre photometry in freely behaving mice. A prior study found that IP administration of 8 mg/kg P4 resulted in circulating P4 concentrations reaching 34 ng/ml (Wong et al., J Pharm Pharmacol 64, 2012). Hence, in this study, diestrus mice equipped for monitoring the activity of the kisspeptin pulse generator were injected with either 8 or 4 mg/kg P4 at 10 am and SEs monitored for 24 hrs. Both doses resulted in a significantly decreased frequency of SEs compared to the vehicle (p<0.05), which persisted for 6 hrs. To next examine whether P4 may be acting directly on the ARN kisspeptin neurons to slow pulse generation, P4 (25 ng/ml) was infused directly into the ARN while the activity of the pulse generator was monitored. Again, this resulted in a decreased frequency of SEs compared to the vehicle with the effects lasting approximately 5 hrs (p<0.05).These results indicate that P4 exerts powerful inhibitory control over the activity of the kisspeptin pulse generator and that these effects may be direct. Elucidating the mechanisms of P4 negative feedback may provide a platform for the development of new therapeutic strategies in the infertility clinic. Presentation: Friday, June 16, 2023

OR09-02 Chemogenetic Manipulation Of Brainstem Noradrenergic Neurons Modulates The Activity Of The Kisspeptin GnRH Pulse Generator In Mice

Abstract Disclosure: S. Vas: None. M. Ruiz-Cruz: None. S. Han: None. S. Hwa-Yeo: None. A.E. Herbison: None. The kisspeptin neurons in the arcuate nucleus (ARNKISS) represent the GnRH pulse generator that drives pulsatile luteinizing hormone secretion. These neurons serve as a hub to integrate multiple cues, such as the negative feedback control from steroid hormones, as well as metabolic, and stress-related inputs. Brainstem noradrenergic (NA) neurons have long been implicated in the control of GnRH pulse generator frequency and fertility but the precise mechanisms through which they operate, and their roles remain unclear. Here we used intersectional Cre-lox and FLP-FRT recombination approaches to couple ARNKISS neuron GCaMP6 fiber photometry with the brainstem NA DREADD (Designer Receptor Exclusively Activated by Designer Drug) modulation to investigate the role that NA may play in controlling ARNKISS pulse generator activity. Kiss1-Cre,GCaMP6s mice were crossed with a mouse line in which FLP recombinase is expressed selectively in dopamine β-hydroxylase (NA) neurons. Adult female mice were injected bilaterally with a FLP-dependent retrograde viral vector (AAVrg-hSyn-fDIO-hM3D(Gq)-mCherry-WPREpA) into the mid-caudal ARN to express excitatory (hM3Dq) DREADD receptors selectively in brainstem NA neurons that project to the ARN. An optical fiber was then implanted to detect the calcium-dependent population activity of KISSARN neurons. Histology revealed m-Cherry/DREADD expression in small sub-populations of tyrosine-hydroxylase neurons located in the brainstem A1, A2 and A6, confirming direct innervation from these regions to the ARN. Selective activation of these NA neurons with 1.5 and 3 mg/kg CNO (s.c.) in diestrous-stage mice, dose-dependently inhibited the frequency of the pulse generator synchronization episodes by 48-76% over 4 hours. These observations demonstrate that, when activated, brainstem NA neurons exert an inhibitory influence on the kisspeptin GnRH pulse generator in intact female mice. The new data may help to dissect how key components of the stress-responsive circuitry impact the central regulation of fertility. Presentation: Friday, June 16, 2023

SAT607 Targeted Modulation Of Kisspeptin Neurons Improves Hyperandrogenism And Hyperactive LH Secretion In A Pre-clinical PCOS-like Mouse Model: Implications For Novel Therapeutics In PCOS Women

Abstract Disclosure: E. Coutinho: Employee; Self; Crinetics. L.A. Esparza: None. A.S. Kauffman: Consulting Fee; Self; Crinetics. Polycystic ovary syndrome (PCOS), a common reproductive disorder in women, is characterized by hyperandrogenemia, chronic anovulation, and/or cystic ovarian follicles. Another common hallmark of PCOS is LH hyper-pulsatility, believed to be driven by hyperactive GnRH neurons, but the reason for the increased GnRH secretion still remains unclear. We previously developed a PCOS-like mouse model using chronic letrozole (LET; aromatase inhibitor) and reported that LET females demonstrated multiple PCOS-like phenotypes, including polycystic ovaries, anovulation, hyperandrogenemia, enlarged ovaries, and hyperactive LH pulse secretion. Given the proposed role of hypothalamic arcuate kisspeptin neurons as the GnRH pulse generator, we determined that LET females have markedly elevated Kiss1 and Tac2 expression and increased Kiss1 neuronal activation in the arcuate nucleus, correlating with their hyper-pulsatile LH secretion. Thus, in the LET PCOS-like condition, and perhaps in PCOS women, increased hypothalamic kisspeptin and NKB neuron activity may be fundamental contributors to the hyperactive LH secretion and downstream hyperandrogenemia. We next tested this possibility using chemogenetics in transgenic PCOS-like mice to acutely and chronically suppress kisspeptin neuron activity. Acute inhibition of kisspeptin neurons successfully lowered abnormally high, rapid in vivo LH pulse secretion in PCOS-like females. Likewise, chronic in vivo suppression of endogenous kisspeptin neuron activity for several weeks dramatically lowered both circulating LH and T levels, and reduced ovarian weights, in females previously displaying a PCOS-like phenotype, with a trend towards improvement of overweight phenotype. These data support the hypothesis that overactive kisspeptin neurons may be one possible driver of neuroendocrine PCOS-like phenotypes. Importantly, these findings suggest that modulation of kisspeptin neurons may be a viable, novel therapeutic target to treat hyperandrogenism and hyperactive LH pulsatility in PCOS women. Indeed, although identical studies have not yet been done in humans, our findings complement recent clinical investigations using NKB receptor antagonists or dynorphin analogs to lower LH pulse secretion in PCOS women and other PCOS-like animal models. Presentation: Saturday, June 17, 2023

FRI273 Generation And In Vitro Characterization Of A Novel Immortalized Ovine Neuronal KNDy Cell Line

Abstract Disclosure: R. Toyoizumi: None. C.Y. Markgraf: None. A. Starman: None. E. Rodriguez: None. B.L. Lee: None. R. Bahn-Bales: None. O. Stricklin: None. C. Estill: None. C.E. Roselli: None. P.E. Chappell: None. The multiphenotypic hypothalamic KNDy neurons play a critical role in the neuroendocrine regulation of reproduction, and via their stimulation of GnRH release, are requisite for fertility and pubertal progression. In vivo evidence has accumulated identifying this more caudal population of Kisspeptin neurons in mediating predominantly negative feedback effects of gonadal steroids, and their ability to synthesize and secrete kisspeptin (KP), neurokinin B (NKB/Tac3), and dynorphin (Dyn) implicates them as crucial regulators of GnRH pulse generation. We have previously generated murine immortalized arcuate KNDy neuronal cell lines to explore molecular mechanisms underlying the steroid hormone regulation of these cells, and in the current study, we characterize a novel immortalized ovine neuronal cell line model of KNDy (oKNDy) cells derived from fetal female sheep brain (∼133 days of fetal development). Clonal lines were derived from isolated mediobasal hypothalamic explants using lentiviral infection of neurons with plasmids encoding SV40 large T-antigen. These neurons express ovine kiss1, tac3, and pdyn, as well as steroid receptors esr1, esr2, and pgr. Similar to KNDy neurons in vivo, receptors for Tac3 (tacr3) and Dyn (oprk1) were also found to be expressed in our oKNDy cells. Further, low physiological concentrations (5-50pM) of 17β-estradiol (E2) were found to significantly repress kiss1 and tac3 expression, while having no effect on tacr3. Concentrations of E2 and progesterone (P4) approximating proestrous levels maximally repressed kiss1 and tac3 after 24h, with elevations in pdyn noted under the same conditions. While cells exposed to higher E2 levels exhibited greater kiss1/tac3 repression, P4 exposure alone revealed stimulation of expression with increasing P4. Since these neurons were derived from the short-day seasonally breeding sheep, they can also be used to explore how circannual timing cues derived from the pituitary pars tuberalis may exert gating signals on the reproductive axis as previously modeled in vivo. Consistent with this, we have found high levels of thyroid hormone receptor α (thra) expression in our oKNDy neurons, suggesting they can respond to locally-synthesized T3. We are continuing to characterize these oKNDy neuronal cells in culture in order to provide insight into GnRH pulse generation and construct molecular models of neuronal responsiveness to steroids and other regulatory inputs. Presentation: Friday, June 16, 2023

Lesions of KNDy and Kiss1R Neurons in the Arcuate Nucleus Produce Different Effects on LH Pulse Patterns in Female Sheep.

The current model for the synchronization of GnRH neural activity driving GnRH and LH pulses proposes that a set of arcuate (ARC) neurons that contain kisspeptin, neurokinin B, and dynorphin (KNDy neurons) is the GnRH pulse generator. This study tested the functional role of ovine KNDy neurons in pulse generation and explored the roles of nearby Kiss1 receptor (Kiss1R)-containing cells using lesions produced with saporin (SAP) conjugates. Injection of NK3-SAP ablated over 90% of the KNDy cells, while Kiss-SAP (saporin conjugated to kisspeptin-54) lesioned about two-thirds of the Kiss1R population without affecting KNDy or GnRH cell number. Both lesions produced a dramatic decrease in LH pulse amplitude but had different effects on LH pulse patterns. NK3-SAP increased interpulse interval, but Kiss-SAP did not. In contrast, Kiss-SAP disrupted the regular hourly occurrence of LH pulses, but NK3-SAP did not. Because Kiss1R is not expressed in KNDy cells, HiPlex RNAScope was used to assess the colocalization of 8 neurotransmitters and 3 receptors in ARC Kiss1R-containing cells. Kiss1R cells primarily contained transcript markers for GABA (68%), glutamate (28%), ESR1 (estrogen receptor-α) mRNA, and OPRK1 (kappa opioid receptor) mRNA. These data support the conclusion that KNDy neurons are essential for GnRH pulses in ewes, whereas ARC Kiss1R cells are not but do maintain the amplitude and regularity of GnRH pulses. We thus propose that in sheep, ARC Kiss1R neurons form part of a positive feedback circuit that reinforces the activity of the KNDy neural network, with GABA or glutamate likely being involved.

Hypothalamic PVN CRH Neurons Signal Through PVN GABA Neurons to Suppress GnRH Pulse Generator Frequency in Female Mice.

Corticotropin-releasing hormone (CRH) neurons in the paraventricular nucleus of the hypothalamus (PVN) are central to the stress response. Chemogenetic activation of PVN CRH neurons decreases LH pulse frequency but the mechanism is unknown. In the present study, optogenetic stimulation of PVN CRH neurons suppressed LH pulse frequency in estradiol-replaced ovariectomized CRH-cre mice, and this effect was augmented or attenuated by intra-PVN GABAA or GABAB receptor antagonism, respectively. PVN CRH neurons signal to local GABA neurons, which may provide a possible indirect mechanism by which PVN CRH neurons suppress LH pulse frequency. Optogenetic stimulation of potential PVN GABAergic projection terminals in the hypothalamic arcuate nucleus in ovariectomized estradiol-replaced Vgat-cre-tdTomato mice via an optic fiber implanted in the arcuate nucleus suppressed LH pulse frequency. To further determine whether PVN CRH neurons signal through PVN GABA neurons to suppress LH pulsatility, we combined recombinase mice with intersectional vectors to selectively target these neurons. CRH-cre::Vgat-FlpO mice expressing the stimulatory opsin ChRmine in non-GABAergic CRH neurons alone or in combination with the inhibitory opsin NpHR3.3 in non-CRH-expressing GABA neurons in the PVN were used. Optogenetic stimulation of non-GABAergic CRH neurons suppressed pulsatile LH secretion; however, LH pulse frequency was not affected when CRH neurons were stimulated and PVN GABA neurons were simultaneously inhibited. Together, these studies demonstrate that suppression of LH pulse frequency in response to PVN CRH neuronal activation is mediated by GABAergic signalling intrinsic to the PVN and may incorporate PVN GABAergic projection to the hypothalamic GnRH pulse generator.

Perinatal exposure to the fungicide ketoconazole alters hypothalamic control of puberty in female rats.

Estrogenic endocrine disrupting chemicals (EDCs) such as diethylstilbestrol (DES) are known to alter the timing of puberty onset and reproductive function in females. Accumulating evidence suggests that steroid synthesis inhibitors such as ketoconazole (KTZ) or phthalates may also affect female reproductive health, however their mode of action is poorly understood. Because hypothalamic activity is very sensitive to sex steroids, we aimed at determining whether and how EDCs with different mode of action can alter the hypothalamic transcriptome and GnRH release in female rats. Female rats were exposed to KTZ or DES during perinatal (DES 3-6-12μg/kg.d; KTZ 3-6-12mg/kg.d), pubertal or adult periods (DES 3-12-48μg/kg.d; KTZ 3-12-48mg/kg.d). Ex vivo study of GnRH pulsatility revealed that perinatal exposure to the highest doses of KTZ and DES delayed maturation of GnRH secretion before puberty, whereas pubertal or adult exposure had no effect on GnRH pulsatility. Hypothalamic transcriptome, studied by RNAsequencing in the preoptic area and in the mediobasal hypothalamus, was found to be very sensitive to perinatal exposure to all doses of KTZ before puberty with effects persisting until adulthood. Bioinformatic analysis with Ingenuity Pathway Analysis predicted "Creb signaling in Neurons" and "IGF-1 signaling" among the most downregulated pathways by all doses of KTZ and DES before puberty, and "PPARg" as a common upstream regulator driving gene expression changes. Deeper screening ofRNAseq datasets indicated that a high number of genes regulating the activity of the extrinsic GnRH pulse generator were consistently affected by all the doses of DES and KTZ before puberty. Several, including MKRN3, DNMT3 or Cbx7, showed similar alterations in expression at adulthood. nRH secretion and the hypothalamic transcriptome are highly sensitive to perinatal exposure to both DES and KTZ. The identified pathways should be exploredfurther to identify biomarkers for future testing strategies for EDC identification and when enhancing the current standard information requirements in regulation.

KNDy neurons as the GnRH pulse generator: Recent studies in ruminants

This review considers three aspects of recent work on the role of KNDy neurons in GnRH pulse generation in ruminants. First, work on basic mechanisms of pulse generation includes several tests of this hypothesis, all of which support it, and evidence that Kiss1r-containing neurons form a positive feedback circuit with the KNDy neural network that strengthen the activity of this network. The second section on pathways mediating external inputs focuses on the influence of nutrition and photoperiod, and describes the evidence supporting roles for proopiomelanocortin (POMC) and agouti-related peptide (AgRP) afferents to KNDy cells in each of these. Finally, we review studies exploring the potential applications of manipulating signaling by kisspeptin, and the other KNDy peptides, to control reproductive function in domestic animals and conclude that, although these approaches show some promise, they do not have major advantages over current practices at this time.

Circulating levels and the bioactivity of miR-30b increase during pubertal progression in boys.

Puberty marks the transition from childhood to adulthood and is initiated by activation of a pulsatile GnRH secretion from the hypothalamus. MKRN3 functions as a pre-pubertal break on the GnRH pulse generator and hypothalamic expression and circulating levels of MKRN3 decrease peri-pubertally. In rodents, microRNA miR-30b seems to directly target hypothalamic MKRN3 expression - and in boys, circulating levels of miR-30b-5p increase when puberty is pharmacologically induced. Similarly, miR-200b-3p and miR-155-5p have been suggested to inhibit expression of other proteins potentially involved in the regulation of GnRH secretion. Here we measure circulating levels of these three miRNAs as boys progress through puberty. Forty-six boys from the longitudinal part of the Copenhagen Puberty Study were included. All boys underwent successive clinical examinations including estimation of testis size by palpation. miR-30b-5p, miR-200b-3p, and miR-155-5p were measured in serum by RT-qPCR using a kit sensitive to the phosphorylation status of the miRNAs. Thirty-nine boys had miRNA levels measured in three consecutive samples (pre-, peri-, and post-pubertally) and seven boys had miR-30b-5p levels measured in ten consecutive samples during the pubertal transition. When circulating levels of miR-30b-5p in pre- and peri-pubertal samples were compared with post-pubertal levels, we observed a significant increase of 2.3 and 2.2-fold (p-value<6.0×10-4), respectively, and a larger fraction of miR-30b-5p appeared to be phosphorylated post-pubertally indicating an increase in its bioactivity. We also observed a negative correlation between circulating levels of miR-30b-5p and MKRN3. The inter-individual variation in circulating miR-30b levels was substantial and we could not define a clinical threshold for miR-30b-5p suggestive of imminent puberty. Also, miR-155-5p showed significantly increasing levels from the peri- to the post-pubertal stage (p=3.0×10-3), whereas miR-200b-3p did not consistently increase. Both circulating levels of miR-30b-5p and its bioactivity increase during the pubertal transition in boys supporting its role in the activation of the HPG axis at the onset of physiologically normal puberty.

GnRH Pulse Generation in Rodents: Time to Terminate the Role of Dynorphin?

GnRH Pulse Generation in Rodents: Time to Terminate the Role of Dynorphin?

Uncovering how the GnRH pulse generator is controlled in female mice.

Uncovering how the GnRH pulse generator is controlled in female mice.

Tachykinins, new players in the control of reproduction and food intake: A comparative review in mammals and teleosts.

In vertebrates, the tachykinin system includes tachykinin genes, which encode one or two peptides each, and tachykinin receptors. The complexity of this system is reinforced by the massive conservation of gene duplicates after the whole-genome duplication events that occurred in vertebrates and furthermore in teleosts. Added to this, the expression of the tachykinin system is more widespread than first thought, being found beyond the brain and gut. The discovery of the co-expression of neurokinin B, encoded by the tachykinin 3 gene, and kisspeptin/dynorphin in neurons involved in the generation of GnRH pulse, in mammals, put a spotlight on the tachykinin system in vertebrate reproductive physiology. As food intake and reproduction are linked processes, and considering that hypothalamic hormones classically involved in the control of reproduction are reported to regulate also appetite and energy homeostasis, it is of interest to look at the potential involvement of tachykinins in these two major physiological functions. The purpose of this review is thus to provide first a general overview of the tachykinin system in mammals and teleosts, before giving a state of the art on the different levels of action of tachykinins in the control of reproduction and food intake. This work has been conducted with a comparative point of view, highlighting the major similarities and differences of tachykinin systems and actions between mammals and teleosts.