Pulsatile LH Secretion Research Articles

289 Postnatal Obesity Amplifies the Effect of Prenatal Testosterone Excess in Reducing the Progesterone Negative Feedback Sensitivity in First-Generation Ewes

Abstract Polycystic ovary syndrome (PCOS) is the leading cause of anovulatory infertility in women of reproductive age. Women with PCOS exhibit reduced sensitivity of the GnRH neuronal system to the inhibitory effects of both progesterone and estradiol. Additionally, obesity amplifies the severity of the PCOS phenotype. In sheep, testosterone (T) excess during gestational days (GD) 30-90 and 60-90 reduces neuroendocrine sensitivity to the progesterone negative feedback and recapitulates the PCOS phenotype. This study investigated the contribution of postnatal obesity in amplifying or mitigating the effects of mid-gestational T excess in disrupting the responsiveness to progesterone negative feedback in sheep. Suffolk ewes received T propionate (T; 100 mg i.m.) or corn oil (CO; vehicle) twice weekly from GD 60-90 (term = 147 d). At 5 mo of age, F1 T lambs were assigned randomly to either a maintenance (100% of NRC requirements) or overfed diet (130% of NRC requirements) and control lambs were fed the maintenance diet. At 22 mo of age, F1 ewes (n = 8/group) were synchronized with two injections of PGF2α 11 d apart. After the second PGF2α administration, blood samples were collected daily for 14 d and progesterone concentrations were determined via radioimmunoassay. Ewes that were confirmed to be cycling and in the mid-luteal phase based on progesterone concentrations (progesterone ≥ 1 ng/ml on day 10 and ≥ 0.5 ng/ml on days 8-9 and 11-12) were used for LH pulse characterization. On day 10 after PGF2α synchronization (mid-luteal phase), blood samples were collected every 15 min for 6 h to characterize LH pulsatile secretion via radioimmunoassay. Data were analyzed as one-way ANOVA with Tukey's HSD post-hoc analysis. Frequency of LH pulses was greater (P < 0.05) in T maintenance (3.75 ± 0.41 pulses/6h) and T overfed ewes (3.50 ± 0.34 pulses/6h) compared with controls (2.00 ± 0.37 pulses/6 h). Luteinizing hormone pulse amplitude was greater (P < 0.05) in T overfed (2.03 ± 0.15 ng/mL) compared with T maintenance (1.44 ± 0.17 ng/mL), but not to controls (1.74 ± 0.21 ng/mL). Also, average LH concentration was greater (P < 0.05) in T overfed (2.03 ± 0.24 ng/mL), but not T maintenance (1.31 ± 0.35 ng/mL), compared with controls (0.93 ± 0.30 ng/mLl). In conclusion, these findings demonstrate the activational role of postnatal obesity in amplifying prenatal T-induced alterations in tonic LH secretion during progesterone negative feedback. The activational effect of postnatal obesity in increasing LH secretion and consequently thecal androgen production is likely a contributor to the severity of hyperandrogenic status in women with PCOS. Research support: NIH-NICHD (R01HD099096).

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

FRI408 Metabolic Hormone- And Energy-related-factors Are Associated With The Pulsatile LH Secretion Response To Short-term Energy Restriction In Healthy Women

Abstract Disclosure: S. Shekhar: None. J. Tessa Tonleu: None. E.D. Brown: None. C. Okigbo: None. L. Helen: None. A.E. Kim: None. B. Purse: None. D. Zaccaro: None. S.J. Soldin: None. K. Hirsch: None. A. Smith-Ryan: None. J.E. Hall: None. Introduction: Functional hypothalamic amenorrhea is characterized by reproductive dysfunction in a deficient energy state mediated through suppressed secretion of pulsatile GnRH. We investigated the relationship between metabolic, appetite, energy, body composition and attitude variables with LH pulse frequency responses. Methods: Two 5-day dietary interventions were administered to nineteen regularly cycling, healthy women (mean age (SD): 23.36 (2.08) y) with unchanged exercise in the early follicular phases of two menstrual cycles. The neutral energy availability diet (NEA, 45 kCal/kg*LBM/d, ±0%) was followed by a deficient energy availability (DEA, -55%) intervention. On enrollment, we administered the following questionnaires: almost perfect, perceived stress, dysfunctional attitude, eating attitudes, exercise (IPAQ), appetite, and palatability. Resting energy expenditure (REE), maximum oxygen uptake (VO2 max) and body composition were assessed at baseline. After each intervention, blood was sampled every 10 min for LH and metabolic hormones (thyroid hormones, GH, cortisol, glucose, insulin, leptin, and orexin) was measured at varying intervals (0800 — 1600hrs). Outcomes included 1) DEA LH pulse frequency expressed as an absolute number (LHPF), 2) change from NEA (αLHPF) and 3) % change from NEA (%αLHPF). Principal components (PC) analysis tested the association between predictor variables separated into relevant domains. As expected from the degree of energy restriction, LHPF decreased in response to DEA in ∼53% of women (‘responders’, n=10) for whom PC analysis was separately performed. Results: For the entire group, the PC for energy expenditure domain highlighted VO2 max and REE and was significantly associated with αLHPF (p=0.04) and %αLHPF (p=0.05). There was no relationship between metabolic or thyroid hormone, or body composition domains with DEA LHPF, αLHPF and %αLHPF. In responders, the fasting metabolic domain primarily consisted of insulin and leptin and was associated with αLHPF (p=0.008) and %αLHPF (p=0.08). The thyroid domain, which was a primary composite of T3, reverse T3 and T4 had a trend of association with DEA LHPF (p=0.06) and %αLHPF (p=0.05). The metabolic domain primarily consisted of samples from the across-the-day growth hormone and cortisol and was associated with αLHPF (p=0.05) and %αLHPF (p=0.049). There was no association between attitude, anthropometry, or energy domains and outcome variables in this subgroup. Conclusion: Our data suggest that in normal women higher resting energy expenditure and a greater degree of fitness predict a greater inhibitory effect of energy restriction on the reproductive axis. In those who demonstrate this inhibition, the degree of response to ER can be predicted by the fasting metabolic (insulin and leptin) and thyroid domains. Presentation: Friday, June 16, 2023

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.

Long-term Glucose Infusions had no Influence on Gonadotropins Hormone Secretion in Ovariectomized Goats

‌Background: It is reported that high nutritional supplementation treatment for seven days stimulated pulsatile LH secretion attended by increased in the plasma glucose and insulin concentrations in goat. The present study tested the hypothesis that whether providing peripheral glucose to goats associated with digestion of high nutrition supplementation increase gonadotropin secretion in ovariectomized goats. Materials and Methods: There was two experimental groups. The treatment animals from both experiences received glucose infusion. In the first experiment (constant glucose infusion; 50% glucose + saline at a rate of 7.5 ml/h for 7 days) and in the second experiment (progressive glucose infusion; 20% glucose + saline by increasing 5% glucose in 24hr intervals for 7 days at a rate of 15 ml/h for 7 days). The control animals from both experiences received saline infusion in the same manner. Findings: In the first experiment, there was no significant diversity in the concentrations of glucose and insulin among the treatment period as compared with mean pre-infusion period or compared to control groups except for Day 2 (p<0.05). In the second experiment, concentrations of both the glucose and insulin started increasing significantly from Day 2 and remained with elevated levels until day 7 as compared with mean pre-infusion period or compared to control groups (p<0.05). The mean Follicle stimulating hormone concentration in treatment group was not affected by glucose infusion as compared with pre-infusion periods or control group. The mean LH (concentration and pulse frequency) was not affected by glucose or saline infusion in both experiments. Conclusion: The present studies suggest that an increase in the glucose availability does not control the gonadotropin secretion as a stimulatory mediator in ovariectomized goats.

GABA Signaling in the Posterodorsal Medial Amygdala Mediates Stress-induced Suppression of LH Pulsatility in Female Mice.

Psychological stress is linked to infertility by suppressing the hypothalamic GnRH pulse generator. The posterodorsal subnucleus of the medial amygdala (MePD) is an upstream regulator of GnRH pulse generator activity and displays increased neuronal activation during psychological stress. The MePD is primarily a GABAergic nucleus with a strong GABAergic projection to hypothalamic reproductive centers; however, their functional significance has not been determined. We hypothesize that MePD GABAergic signalling mediates psychological stress-induced suppression of pulsatile LH secretion. We selectively inhibited MePD GABA neurons during psychological stress in ovariectomized (OVX) Vgat-cre-tdTomato mice to determine the effect on stress-induced suppression of pulsatile LH secretion. MePD GABA neurons were virally infected with inhibitory hM4DGi-designer receptor exclusively activated by designer drugs (DREADDs) to selectively inhibit MePD GABA neurons. Furthermore, we optogenetically stimulated potential MePD GABAergic projection terminals in the hypothalamic arcuate nucleus (ARC) and determined the effect on pulsatile LH secretion. MePD GABA neurons in OVX female Vgat-cre-tdTomato mice were virally infected to express channelrhodopsin-2 and MePD GABAergic terminals in the ARC were selectively stimulated by blue light via an optic fiber implanted in the ARC. DREADD-mediated inhibition of MePD GABA neurons blocked predator odor and restraint stress-induced suppression of LH pulse frequency. Furthermore, sustained optogenetic stimulation at 10 and 20 Hz of MePD GABAergic terminals in the ARC suppressed pulsatile LH secretion. These results show for the first time that GABAergic signalling in the MePD mediates psychological stress-induced suppression of pulsatile LH secretion and suggest a functionally significant MePD GABAergic projection to the hypothalamic GnRH pulse generator.

Reversible hypogonadotropic hypogonadism in men with the fertile eunuch/Pasqualini syndrome: A single-center natural history study.

Congenital hypogonadotropic hypogonadism (HH) is a heterogeneous genetic disorder characterized by disrupted puberty and infertility. In most cases, HH is abiding yet 10-15% undergo reversal. Men with HH and absent and partial puberty (i.e., testicular volume <4mL and >4mL respectively) have been well-studied, but the rare fertile eunuch (FE) variant remains poorly characterized. This natural history study of 240 men with HH delineates the clinical presentation, neuroendocrine profile, rate of reversal and genetics of the FE variant. We compared three HH groups: FE (n=38), absent puberty (n=139), and partial puberty (n=63). The FE group had no history of micropenis and 2/38 (5%) had cryptorchidism (p<0.0001 vs. other groups). The FE group exhibited higher rates of detectable gonadotropins, higher mean LH/FSH levels, and higher serum inhibin B levels (all p<0.0001). Neuroendocrine profiling showed pulsatile LH secretion in 30/38 (79%) of FE men (p<0.0001) and 16/36 (44%) FE men underwent spontaneous reversal of HH (p<0.001). The FE group was enriched for protein-truncating variants (PTVs) in GNRHR and FGFR1 and 4/30 (13%) exhibited oligogenic PTVs. Findings suggest men with the FE variant exhibit the mildest neuroendocrine defects of HH men and the FE sub-type represents the first identified phenotypic predictor for reversible HH.

RF10 | PMON215 Greater changes in metabolic hormones are associated with a smaller decrease in pulsatile LH secretion in response to short term moderate energy restriction in healthy, young women

Abstract Background Energy restriction may result in hypothalamic amenorrhea by inhibiting GnRH secretion and downstream pulsatile LH secretion. Energy restriction also leads to adaptive changes in metabolic hormones. We sought to determine whether metabolic changes in response to energy restriction predict or mitigate inhibition of GnRH secretion. Methods Nineteen healthy women, (mean age ± SD; 23.36 ± 2.08 yrs) with regular ovulatory cycles and no evidence of energy restriction or excessive exercise, underwent two 5-day dietary interventions with identical exercise in the early follicular phase of two menstrual cycles. A neutral energy availability (NEA; 45 kCal/kg*LBM/d) was followed by a deficient energy availability (DEA; 20 kCal/kg*LBM/d) diet. On day five of each intervention, body composition was analyzed (BodPod®), and blood was sampled between 0800 —1600 hours for LH, TSH and GH, every 10 min, cortisol every 30 min, T3, reverse T3 (rT3) and T4 every 60 min, free T3 (FT3), free T4 (FT4) and TBG at 0800 h and 1600 h. We correlated means of pulsatile LH pulse frequency (LHPF) with integrated metabolic hormone changes across the day in the subset of women with reduced or unchanged LHPF after DEA (n=10) using linear regression. Results In ∼50% of healthy young women, LHPF decreased or was unchanged in response to short-term moderate energy deprivation. BMI and bodyweight declined after DEA in both groups while % fat mass was unchanged. TSH, T3, leptin, insulin declined, and T4 increased from NEA to DEA while glucose, cortisol and GH were unchanged. In subjects with reduced/unchanged LHPF, % delta T3 correlated inversely with delta LHPF (r=-0.727, p=0.017). Similarly, non-fasting delta TSH correlated inversely with % delta LHPF (r=-0.643, p=0.045), and % delta AUC TSH (r=-0.642, p=0.045) correlated inversely with DEA LHPF. Percent delta insulin (r= -0.722, p=0.018) and % delta insulin AUC (r= – 0.688, p=0.028) correlated inversely with delta LHPF. Percent delta insulin-glucose ratio (r=-0.772, p=0.009) and % change in AUC insulin-glucose ratio (r=-0.759, p=0.011) also correlated inversely with delta LHPF. This inverse relationship was preserved when insulin was normalized for caloric intake (n-insulin) was correlated with % delta LHPF (r=-0.722, p=0.018) and % delta AUC n-insulin correlated with delta LHPF (r= -0.631 and p= 0.050). Delta AUC leptin (r=-0.684, p=0.029) and % delta AUC leptin (r=-0.670, p=0.034) also correlated inversely with delta LHPF. Mean cortisol at NEA correlated inversely with LHPF during DEA (r=-0.816, p=0.007), implying that higher baseline cortisol levels may predict a greater relative fall in LHPF. Cortisol AUC in DEA also correlated inversely with LHPF in DEA (r=-0.722, p=0.028). Conclusion Our data suggest that greater changes in metabolic hormones may protect against the inhibitory effect of moderate energy deprivation on GnRH secretion in healthy young women with normal reproductive function. Presentation: Saturday, June 11, 2022 1:30 p.m. - 1:35 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Reproductive Phenotypes and Genotypes in Men With IHH.

Isolated hypogonadotropic hypogonadism (IHH) is phenotypically and genetically heterogeneous. This work aimed to determine the correlation between genotypic severity with pubertal and neuroendocrine phenotypes in IHH men. A retrospective study was conducted (1980-2020) examining olfaction (Kallmann syndrome [KS] vs normosmic IHH [nHH]), baseline testicular volume (absent vs partial puberty), neuroendocrine profiling (pulsatile vs apulsatile luteinizing hormone [LH] secretion), and genetic variants in 62 IHH-associated genes through exome sequencing (ES). In total, 242 men (KS: n = 131 [54%], nHH: n = 111 [46%]) were included. Men with absent puberty had significantly lower gonadotropin levels (P < .001) and were more likely to have undetectable LH (P < .001). Logistic regression showed partial puberty as a statistically significant predictor of pulsatile LH secretion (R2 = 0.71, P < .001, OR: 10.8; 95% CI, 3.6-38.6). Serum LH of 2.10 IU/L had a 95% true positive rate for predicting LH pulsatility. Genetic analyses in 204 of 242 IHH men with ES data available revealed 36 of 204 (18%) men carried protein-truncating variants (PTVs) in 12 IHH genes. Men with absent puberty and apulsatile LH were enriched for oligogenic PTVs (P < .001), with variants in ANOS1 being the predominant PTV in this genotype-phenotype association. Men with absent puberty were enriched for ANOS1 PTVs compared to partial puberty counterparts (P = .002). PTVs in other IHH genes imparted more variable reproductive phenotypic severity. Partial puberty and LH greater than or equal to 2.10 IU/L are proxies for pulsatile LH secretion. ANOS1 PTVs confer severe reproductive phenotypes. Variable phenotypic severity in the face of severe genetic variants in other IHH genes point to significant neuroendocrine plasticity of the HPG axis in IHH men.

Role of Posterodorsal Medial Amygdala Urocortin-3 in Pubertal Timing in Female Mice.

Post-traumatic stress disorder impedes pubertal development and disrupts pulsatile LH secretion in humans and rodents. The posterodorsal sub-nucleus of the medial amygdala (MePD) is an upstream modulator of the hypothalamic gonadotropin-releasing hormone (GnRH) pulse generator, pubertal timing, as well as emotional processing and anxiety. Psychosocial stress exposure alters neuronal activity within the MePD increasing the expression of Urocortin3 (Ucn3) and its receptor corticotropin-releasing factor type-2 receptor (CRFR2) while enhancing the inhibitory output from the MePD to key hypothalamic reproductive centres. We test the hypothesis that psychosocial stress, processed by the MePD, is relayed to the hypothalamic GnRH pulse generator to delay puberty in female mice. We exposed C57Bl6/J female mice to the predator odor, 2,4,5-Trimethylthiazole (TMT), during pubertal transition and examined the effect on pubertal timing, pre-pubertal LH pulses and anxiety-like behaviour. Subsequently, we virally infected Ucn3-cre-tdTomato female mice with stimulatory DREADDs targeting MePD Ucn3 neurons and determined the effect on pubertal timing and pre-pubertal LH pulse frequency. Exposure to TMT during pubertal development delayed puberty, suppressed pre-pubertal LH pulsatility and enhanced anxiety-like behaviour, while activation of MePD Ucn3 neurons reduced LH pulse frequency and delayed puberty. Early psychosocial stress exposure decreases GnRH pulse generator frequency delaying puberty while inducing anxiety-behaviour in female mice, an effect potentially involving Ucn3 neurons in the MePD.

Acute progesterone feedback on gonadotropin secretion is not demonstrably altered in estradiol-pretreated women with polycystic ovary syndrome.

Women with polycystic ovary syndrome (PCOS) demonstrate gonadotropin‐releasing hormone (GnRH) pulse generator resistance to suppression with 7 days of progesterone and estradiol administration. It remains unknown whether such women demonstrate impairments in acute progesterone negative feedback on LH pulse frequency or progesterone positive feedback on gonadotropin release. This was a randomized, double‐blind, placebo‐controlled crossover study designed to test the hypothesis that acute, progesterone‐related suppression of LH pulse frequency and progesterone‐related augmentation of gonadotropin release are impaired in PCOS. Twelve normally cycling women and 12 women with PCOS completed study. Volunteers were pretreated with transdermal estradiol (0.2 mg/day) for 3 days and then underwent a frequent blood sampling study (20:00–20:00 h), during which they received micronized progesterone (100 mg) or placebo at 06:00 h. In a second study admission, volunteers received the intervention they did not receive during the first admission, but the protocol was otherwise identical. The primary outcome measures were LH secretory characteristics and circulating gonadotropin concentrations. Exogenous progesterone did not reduce LH pulse frequency in either group. Mean LH, pulsatile LH secretion, LH pulse mass, and mean FSH increased more with progesterone compared to placebo in both groups. Although trends toward less pronounced changes in LH pulse mass and pulsatile LH secretion were observed in the PCOS group, these differences were not statistically significant. In summary, exogenous progesterone did not suppress LH pulse frequency within 12 hours in estradiol‐pretreated women, and the positive feedback effect of progesterone on gonadotropin release was not demonstrably impaired in PCOS.New & NoteworthyThis study indicated that exogenous progesterone does not reduce LH pulse frequency within 12 h in women with PCOS, but progesterone acutely increased gonadotropin in these women. This study suggested that progesterone‐related augmentation of gonadotropin release may be impaired in PCOS compared to normally cycling women, but this finding was not statistically significant.

Vasoactive intestinal peptide exerts an excitatory effect on hypothalamic kisspeptin neurons during estrogen negative feedback

Hypothalamic kisspeptin neurons are the primary modulators of gonadotropin-releasing hormone (GnRH) neurons. It has been shown that circadian rhythms driven by the suprachiasmatic nucleus (SCN) contribute to GnRH secretion. Kisspeptin neurons are potential targets of SCN neurons due to reciprocal connections with the anteroventral periventricular and rostral periventricular nuclei (AVPV/PeN) and the arcuate nucleus of the hypothalamus (ARH). Vasoactive intestinal peptide (VIP), a notable SCN neurotransmitter, modulates GnRH secretion depending on serum estradiol levels, aging or time of the day. Considering that kisspeptin neurons may act as interneurons and mediate VIP's effects on the reproductive axis, we investigated the effects of VIP on hypothalamic kisspeptin neurons in female mice during estrogen negative feedback. Our findings indicate that VIP induces a TTX-independent depolarization of approximately 30% of AVPV/PeN kisspeptin neurons in gonad-intact (diestrus) and ovariectomized (OVX) mice. In the ARH, the percentage of kisspeptin neurons that were depolarized by VIP was even higher (approximately 90%). An intracerebroventricular infusion of VIP leds to an increased percentage of kisspeptin neurons expressing the phosphoSer133 cAMP-response-element-binding protein (pCREB) in the AVPV/PeN. On the other hand, pCREB expression in ARH kisspeptin neurons was similar between saline- and VIP-injected mice. Thus, VIP can recruit different signaling pathways to modulate AVPV/PeN or ARH kisspeptin neurons, resulting in distinct cellular responses. The expression of VIP receptors (VPACR) was upregulated in the AVPV/PeN, but not in the ARH, of OVX mice compared to mice on diestrus and estradiol-primed OVX mice. Our findings indicate that VIP directly influences distinct cellular aspects of the AVPV/PeN and ARH kisspeptin neurons during estrogen negative feedback, possibly to influence pulsatile LH secretion.

252 Impact of Pre- and Postnatal Nutritional Extremes on Tonic Secretion of Gonadotropins and Feedback Responsiveness to Estradiol in Sexually Mature Heifers

Abstract Objectives were to test the hypotheses that nutritional extremes during prenatal development interact with postnatal nutrition during the juvenile period of heifers to impact 1) tonic secretion of gonadotropins, and 2) estradiol-17β (E2) negative and positive feedback responsiveness in adulthood. Heifers were selected from a larger population programmed nutritionally using a 3 x 2 factorial arrangement of pre- and postnatal diets. Beginning at 90 days of pregnancy, Bos indicus-influenced cows (n = 95) were fed to achieve body condition scores (BCS; 1–9 scale) of 3–3.5 (L; thin), 5.5–6 (M; moderate), or 7.5–8 (H; obese) by onset of the third trimester and maintained thereafter. Heifer offspring were weaned at 3–3.5 months of age and assigned to either a low- (L; 0.5 kg/day) or high-gain (H; 1.0 kg/day) diet until 8 mo. of age, then fed a common diet until puberty. Heifers (n = 18; 6/grp) representing HH, MH and LL combinations were ovariectomized postpubertally (17.1 mo. of age) and received E2 replacement. In Exp. 1, blood samples were collected at 10-min intervals to evaluate pulsatile secretion of LH and FSH for 5.5 hours. In Exp. 2, heifers received E2 (2.4 ug/kg I.M.), with blood sampling at 30-min to 1-h intervals for 30 h. Heifers in the MH (1.25 ± 0.11 ng/mL) group tended (P &amp;lt; 0.09) to have greater LH pulse amplitude compared to HH (0.91 ± 0.14 ng/mL) and LL (0.96 ± 0.09 ng/mL); otherwise, frequency, amplitude, and mean concentrations of LH and FSH did not differ among groups. Exogenous E2 (Exp. 2) suppressed (P &amp;lt; 0.0001) mean plasma concentrations of LH and FSH equally among groups, then stimulated equivalent surges of LH beginning at 14 ± 0.02 h, with only two FSH surges detected. Neither pre- nor postnatal nutrition affected E2 negative or positive feedback in this study.

Urocortin3 in the Posterodorsal Medial Amygdala Mediates Stress-induced Suppression of LH Pulsatility in Female Mice.

Psychosocial stress disrupts reproduction and interferes with pulsatile LH secretion. The posterodorsal medial amygdala (MePD) is an upstream modulator of the reproductive axis and stress. Corticotropin-releasing factor type 2 receptors (CRFR2s) are activated in the presence of psychosocial stress together with increased expression of the CRFR2 ligand Urocortin3 (Ucn3) in the MePD of rodents. We investigate whether Ucn3 signalling in the MePD is involved in mediating the suppressive effect of psychosocial stress on LH pulsatility. First, we administered Ucn3 into the MePD and monitored the effect on LH pulses in ovariectomized mice. Next, we delivered Astressin2B, a selective CRFR2 antagonist, intra-MePD in the presence of predator odor, 2,4,5-trimethylthiazole (TMT) and examined the effect on LH pulses. Subsequently, we virally infected Ucn3-cre-tdTomato mice with inhibitory designer receptor exclusively activated by designer drugs (DREADDs) targeting MePD Ucn3 neurons while exposing mice to TMT or restraint stress and examined the effect on LH pulsatility as well as corticosterone release. Administration of Ucn3 into the MePD dose-dependently inhibited LH pulses and administration of Astressin2B blocked the suppressive effect of TMT on LH pulsatility. Additionally, DREADDs inhibition of MePD Ucn3 neurons blocked TMT and restraint stress-induced inhibition of LH pulses and corticosterone release. These results demonstrate for the first time that Ucn3 neurons in the MePD mediate psychosocial stress-induced suppression of the GnRH pulse generator and corticosterone secretion. Ucn3 signalling in the MePD plays a role in modulating the hypothalamic-pituitary-gonadal and hypothalamic-pituitary-adrenal axes, and this brain locus may represent a nodal center in the interaction between the reproductive and stress axes.

Progesterone Positive Feedback on Pulsatile LH Secretion and FSH Release May Be Blunted in Estradiol-Pretreated Women With PCOS

In women pretreated with estradiol (E2), exogenous progesterone (P4) acutely augments LH and FSH release (P4 positive feedback). Women with PCOS exhibit impaired P4 negative feedback on LH pulse frequency, but it remains unclear whether such women exhibit impaired P4 positive feedback on LH/FSH release. We sought to explore the latter notion as an a priori secondary hypothesis in a study primarily designed to assess whether P4 acutely suppresses LH pulse frequency. We studied 12 women with PCOS and 12 normally-cycling, non-hyperandrogenic controls. After 3 days of transdermal E2 pretreatment (0.2 mg/day), subjects were admitted to the Clinical Research Unit (CRU) for a 24-hour frequent blood sampling protocol starting at 2000 h. (CRU admissions occurred no earlier than cycle day 7 in PCOS and between days 7 and 11 inclusive in controls.) At 0600 h, subjects received either 100 mg oral micronized P4 or placebo (PBO). In a subsequent menstrual cycle, subjects underwent an identical CRU protocol except that P4 was exchanged for PBO or vice versa. LH secretion was analyzed using Autodecon, a deconvolution program that provides estimates of LH pulse frequency, pulsatile LH secretion (amount of LH secreted as pulses), and basal (non-pulsatile) LH secretion. Results were analyzed using 2-period crossover design analysis of covariance. In both groups, neither LH pulse frequency nor basal LH secretion changed significantly with P4 (compared to changes with PBO). Mean LH increased with P4 in both groups—3.1-fold (95% CI, 2.4–4.0) in controls and 2.7-fold (95% CI, 2.1–3.5) in PCOS; in both groups, P4-related changes were significantly greater than PBO-related changes (Bonferroni-corrected p=0.012 and 0.010, respectively). In controls, pulsatile LH secretion increased 3.5-fold (95% CI, 2.3–5.2) with P4—significantly more than with PBO (p=0.029); while in PCOS, a 2.6-fold (95% CI, 1.8–3.9) increase with P4 was not significantly different from changes with PBO (p=0.911). In controls, mean FSH increased 2.0-fold (95% CI, 1.7–2.3) with P4—significantly more than with PBO (p=0.004); but in PCOS, a 1.5-fold (95% CI, 1.3–1.8) increase was not significantly different from changes with PBO (p=0.072). Despite the above, between-group (PCOS vs. controls) differences in P4-induced changes in pulsatile LH secretion and mean FSH were not formally (statistically) demonstrable. Between-group differences representing potential confounders included age (median 25.5 vs. 19.0 y; p=0.029), body mass index (29.9 vs. 21.8 kg/m2; p=0.006), and cycle day of CRU admissions (day 45.0 vs. 10.4 for P4 admissions; 30.0 vs. 10.0 for PBO admissions). In summary, these data suggest that P4-induced increases in pulsatile LH secretion and mean FSH may be blunted in PCOS compared to controls, which could contribute to ovulatory dysfunction in PCOS. However, our results do not confirm this possibility, and further study is needed.

SUN-239 Effects of High-Fat Diet-Induced Obesity on Pulsatile LH Secretion and Hypothalamic KISS1 Expression in Female Rats

Female obesity is associated with menstrual dysfunction leading to anovulation and infertility. It has recently been reported obesity-induced infertility is involved in the dysfunction of a kisspeptin neuron, a key player in reproduction via direct stimulation of gonadotropin releasing hormone (GnRH) and subsequent gonadotropin release in mammalian species. Previous studies reported that obesity due to high-fat diet (HFD) for 8 months induced a disruption in estrous cyclicity, caused by a decrease in Kiss1 (coding kisspeptin) expression in the hypothalamic arcuate nucleus (ARC) in female rodents. Here we showed the effects of shorter-term (4 months) HFD on pulsatile LH secretion and hypothalamic Kiss1 expression to show pathogenic mechanism underlying obesity-induced infertility. Female Wistar-Imamichi strain rats (7 weeks old) fed on either a standard diet (10% calories from fat) or a high-fat diet (45% calories from fat) for 4 months. Estrous cyclicity and body weight were monitored regularly. All animals were implanted with a jugular catheter and collected blood samples to analyze pulsatile LH secretion, after a week of the ovariectomy with low-dose replacement estradiol to negate influence of changes in ovarian steroid levels and mimic diestrous levels of plasma estrogen. On the next day of the blood sampling, rats were perfused with 0.05 M PBS followed by 4% paraformaldehyde and their brains were collected for in situ hybridization of Kiss1 and Gnrh1. The HFD-fed rats showed progressive increases in body weight, along with hyperphagia and adipose tissue accumulation, compared with control animals. Fifty-eight percent of the HFD-fed rats exhibited irregular estrous cycles, whereas remaining HFD-fed rats showed regular cycles. Two out of 7 rats showing HFD-induced irregular estrous cycles exhibited profound suppression of the LH pulse frequency and the number of Kiss1-expressing cells in the ARC, whereas remaining HFD-fed rats showed normal LH pulses and ARC Kiss1 expressions. The number of Kiss1-expressing cells in the ARC had close positive correlation with LH pulse frequency (R2=0.6872, P<0.001) in both groups. Additionally, the number of Kiss1- or Gnrh1-expressing cells in the anteroventral periventricular nucleus or the preoptic area, were comparable between groups. Taken together, our finding reveals the possibility that irregular menstruation was also induced by changes in the kisspeptin-GnRH independent pathway during the incipient stage of obese infertility.

SUN-LB50 Increased In Vivo Pulsatile LH Secretion and Hypothalamic Kisspeptin, NKB, and Dynorphin RNA Levels in a PCOS-Like Mouse Model

Polycystic ovary syndrome (PCOS) is a reproductive disorder in women characterized by hyperandrogenemia, anovulation, cystic ovaries, and LH hyper-pulsatility, but the mechanisms causing the pathophysiology remain incompletely understood. We recently reported a novel mouse model that recapitulates the majority of PCOS phenotypes in adulthood. Females given constant, long-term letrozole to reduce aromatase activity demonstrate PCOS-like phenotypes, including polycystic ovaries, anovulation, elevated circulating testosterone, and increased LH. In vivo LH pulsatile secretion, which is greatly elevated in PCOS women, was not previously studied, nor were possible changes in reproductive neurons known to control GnRH/LH secretion. Here, we used recent technical advances in the field to examine in vivo LH pulse dynamics of freely-moving LET female mice versus control and ovariectomized (OVX) mice. We also studied whether hypothalamic gene expression of several important reproductive regulators, kisspeptin, neurokinin B (NKB), and dynorphin, is altered in LET females. Compared to controls, LET females exhibited very rapid, elevated in vivo LH pulsatility, with increased pulse frequency, amplitude, and basal levels, similar to PCOS women. LET mice also had markedly elevated Kiss1, Tac2, and Pdyn expression along with increased Kiss1 neuron activation in the hypothalamic arcuate nucleus. Although elevated, most hyperactive LH pulse parameters and increased arcuate mRNA measures of LET mice were significantly lower than in OVX littermates. Our findings demonstrate that LET mice, like PCOS women, have markedly elevated LH pulsatility which likely drives increased ovarian androgen secretion. Increased arcuate kisspeptin and NKB levels may be fundamental contributors to the enhanced stimulation of LH pulse secretion in this PCOS-like condition, and perhaps, in some PCOS women.

The Origin of GnRH Pulse Generation: An Integrative Mathematical-Experimental Approach.

Fertility critically depends on the gonadotropin-releasing hormone (GnRH) pulse generator, a neural construct comprised of hypothalamic neurons coexpressing kisspeptin, neurokoinin-B and dynorphin. Here, using mathematical modeling and in vivo optogenetics we reveal for the first time how this neural construct initiates and sustains the appropriate ultradian frequency essential for reproduction. Prompted by mathematical modeling, we show experimentally using female estrous mice that robust pulsatile release of luteinizing hormone, a proxy for GnRH, emerges abruptly as we increase the basal activity of the neuronal network using continuous low-frequency optogenetic stimulation. Further increase in basal activity markedly increases pulse frequency and eventually leads to pulse termination. Additional model predictions that pulsatile dynamics emerge from nonlinear positive and negative feedback interactions mediated through neurokinin-B and dynorphin signaling respectively are confirmed neuropharmacologically. Our results shed light on the long-elusive GnRH pulse generator offering new horizons for reproductive health and wellbeing.SIGNIFICANCE STATEMENT The gonadotropin-releasing hormone (GnRH) pulse generator controls the pulsatile secretion of the gonadotropic hormones LH and FSH and is critical for fertility. The hypothalamic arcuate kisspeptin neurons are thought to represent the GnRH pulse generator, since their oscillatory activity is coincident with LH pulses in the blood; a proxy for GnRH pulses. However, the mechanisms underlying GnRH pulse generation remain elusive. We developed a mathematical model of the kisspeptin neuronal network and confirmed its predictions experimentally, showing how LH secretion is frequency-modulated as we increase the basal activity of the arcuate kisspeptin neurons in vivo using continuous optogenetic stimulation. Our model provides a quantitative framework for understanding the reproductive neuroendocrine system and opens new horizons for fertility regulation.

Acute suppression of LH secretion by prolactin in female mice is mediated by kisspeptin neurons in the arcuate nucleus

Elevated prolactin levels suppress pulsatile release of GnRH from the hypothalamus, with a consequent reduction in pulsatile LH secretion from the pituitary. In this paper, the authors showed in mice that kisspeptin neurons located in the arcuate nucleus (ARC) respond to prolactin and conditional knockout of the prolactin receptor specifically in ARC kisspeptin neurons prevented prolactin-induced suppression of LH secretion.