Luteinizing Hormone Pulse Research Articles

8602 Classical progesterone signaling in kisspeptin neurons is not required for negative feedback regulation of the GnRH pulse generator in mice

Abstract Disclosure: K.M. Dillon: None. D.B. Lohr: None. A.G. Novak: None. A.V. Petriv: None. N.T. Neifert: None. A.M. Moore: None. Mammalian reproductive function depends on the ability of progesterone to suppress pulsatile gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion in a homeostatic negative feedback loop. Impairments in progesterone negative feedback are associated with the development of polycystic ovary syndrome (PCOS), the leading cause of infertility for people with ovaries. Although previous research indicates cells upstream from GnRH neurons expressing the classical progesterone receptor (PR) are required for negative feedback, the identity of these cells and the mechanism by which they reduce GnRH/LH pulsatile secretion remain unclear. In this study, we aimed to address the hypothesis that PR expressed by a neuronal population in the arcuate nucleus expressing Kisspeptin, Neurokinin B, and Dynorphin, recently defined as the GnRH pulse generator, is required for negative feedback regulation of LH pulses. To achieve this, we utilized Cre-lox technology in mice to conditionally delete PR from kisspeptin cells (KPRKO mice) and assessed fecundity, LH pulsatility, and the mRNA expression of KNDy neuropeptides. Female KPRKO mice paired with B6 stud males for 3 months produced significantly fewer litters (p<0.05, n=5) and pups per litter (p<0.05) compared to WT controls (n=5) (students t-tests), recapitulating a previously reported subfertile phenotype in this model. In a separate cohort of animals, serial blood samples were collected every 6 minutes for 2 hours from the tail-tip of habituated WT and KPRKO mice in diestrus (n=6/group) and estrus (n=6/group), and an ultra-sensitive ELISA was used to detect LH pulses. However, compared to WT controls, KPRKO mice did not exhibit any significant changes in total and baseline LH release, pulse frequency or pulse amplitude in either cycle stages (p>0.05, students t-tests). At the completion of serial blood sampling, brains were collected from WT and KPRKO mice and RNAscope in situ hybridization was used to quantify mRNA for kisspeptin (KISS1) and dynorphin (PDYN), which are excitatory and inhibitory to GnRH/LH secretion, respectively. No differences in the number of KISS1- and PDYN-expressing cells or the number of KISS1 and PDYN mRNA transcripts were observed between WT and KPRKO mice (n=4/group, p>0.05, students t-tests). These data suggest that progesterone signaling in kisspeptin cells through PR is not essential for negative feedback regulation of LH pulses and is unlikely to contribute to the infertile phenotype of KPRKO mice. These results suggest that negative feedback may be maintained via non-classical mechanisms or an unidentified PR-positive neuronal population. Presentation: 6/1/2024

7940 Is Urocortin 2 A Mediator Of Stress-induced Suppression Of The Luteinizing Hormone Surge?

Abstract Disclosure: H.F. Bell: None. R.A. Carrasco: None. M.J. Kreisman: None. R.B. McCosh: None. K.M. Breen Church: None. Stress is thought to be a contributing factor to the reduction of reproductive function and infertility observed in many different species. Although this is a well-documented phenomenon, the neural mechanisms mediating the suppression of reproductive neuroendocrine activity in response to stress are not yet fully understood. The paraventricular nucleus (PVN) of the hypothalamus is an integral part of the neural response to stress. The PVN initiates activation of Corticotropin Releasing Hormone (CRH) neurons and, in addition to CRH, the PVN contains a population of neurons expressing Urocortin 2 (UCN2), a CRH-like family member, with high affinity for CRH receptor 2. In females, stress has been shown to inhibit both the pulse and surge modes of Luteinizing Hormone (LH) secretion. We have preliminary evidence that kisspeptin neurons, controlling either type of LH secretion, contain CRH receptor 2. In this study we tested the sufficiency of UCN2 to disrupt the estradiol-induced LH surge. Female mice were ovariectomized and given a high estradiol treatment to induce a circadian-timed LH surge. Two days later, UCN2 or Vehicle was administered into the lateral ventricle via ICV injection on the morning of the expected surge (10 AM). Blood and brains were collected in the evening (6 PM), at the time of the expected surge. Whole blood from the tail tip was measured by LH ELISA and a surge was defined as > 0.5 ng/mL. A majority of saline-treated animals expressed an LH surge (8 of 9 females). In contrast, 0% of UCN2-treated animals surged (n=7), suggesting that UCN2 impaired the positive feedback response to estradiol. Additionally, immunohistochemistry was performed to observe colocalization of c-Fos within Kisspeptin neurons in the rostral periventricular region of the third ventricle (RP3V) which are responsible for surge generation. Lower Kisspeptin/C-Fos co-expression in animals treated with UCN2 demonstrated the efficacy of UCN2 to impair estradiol-induced activation of RP3V Kisspeptin neurons which are critical for LH surge generation. These results support the hypothesis that UCN2 signaling plays a mediatory role in the stress-induced reduction of ovulatory function in the female mouse. Presentation: 6/1/2024

Central δ/κ opioid receptor signaling pathways mediate chronic and/or acute suckling-induced LH suppression in rats during late lactation.

In mammals, secretion of tonic (pulsatile) gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) is often suppressed during lactation. Suppression of GnRH/LH pulses in lactating dams is assumed to be caused by suckling stimuli and a chronic negative energy balance due to milk production. The present study aimed to investigate whether the central enkephalin-δ opioid receptor (DOR) signaling mediated the suppression of LH secretion by acute suckling stimuli and/or chronic negative energy balance due to milk production in rats during late lactation when dams were under a heavy energy demand. On postpartum day 16, the number of Penk (enkephalin mRNA)-expressing cells in the arcuate nucleus was significantly higher in lactating rats than in non-lactating control rats. Pulsatile LH secretion was suppressed in rats with chronic suckling or acute 1-h suckling stimuli 6 h after pup removal on day 16 of lactation. Central DOR antagonism significantly increased the mean LH concentrations and the baseline of LH pulses in rats with chronic suckling but not with acute suckling stimuli on day 16 of lactation. Besides, central κ opioid receptor (KOR) antagonism increased the amplitude of LH pulses in rats with the acute suckling stimuli on day 16 of lactation. These results suggest that central DOR signaling mediates the suppression of LH secretion caused by a negative energy balance in rats receiving chronic suckling during late lactation. On the other hand, central KOR signaling likely mediates acute suckling stimuli-induced suppression of LH secretion in rats during late lactation.

Chemogenetic Activation of RFRP Neurons Reduces LH Pulse Frequency in Female but not Male Mice.

The neuropeptide RFRP-3 (RFamide-related peptide-3) is thought to play a role in the negative regulation of fertility. However, the exogenous administration of RFRP-3 yields varying results depending on the dose and route of administration, sex of the subject, and many other variables. Manipulation of in vivo neuronal activity using DREADDs (designer receptor exclusively activated by designer drugs) technology enables investigation of cell type-specific neuronal activation in a manner that better reflects endogenous neuronal activity. To test the effects of RFRP neuronal activation on pulsatile luteinizing hormone (LH) secretion. We generated mice expressing the stimulatory hM3Dq designer receptor exclusively in RFRP cells using 2 different Cre-loxP-mediated approaches: (1) we bred mice to express hM3Dq in all Rfrp-Cre-expressing cells, including some that transiently expressed Rfrp-Cre neonatally (RFRP × hM3Dq mice), and (2) we stereotaxically injected Cre-dependent hM3Dq into the dorsomedial nucleus of RFRP-Cre mice to drive hM3Dq expression exclusively in a subpopulation of adult Rfrp-Cre neurons (RFRP-AAV-hM3Dq mice). We then investigated the effects of acute hM3Dq activation on LH pulse frequency in RFRP × hM3Dq mice, RFRP-AAV-hM3Dq mice, and their respective controls. In both female RFRP × hM3Dq and RFRP-AAV-hM3Dq mice, chemogenetic activation of Cre-driven hM3Dq led to a significant 35% to 50% reduction in LH pulse frequency compared with controls, while no differences in pulse amplitude or mean LH concentration were observed. In marked contrast, RFRP activation did not cause any changes to LH pulse dynamics in male mice. These data show for the first time that activation of neurons that have expressed Rfrp, or of a subset of adult RFRP neurons, can independently suppress LH pulsatility in female, but not male mice.

The deletion of nuclear progesterone receptors from kisspeptin cells does not impair negative feedback in female mice.

Reproductive function in mammals depends on the ability of progesterone to suppress pulsatile gonadotrophin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion in a homeostatic negative feedback loop. Previous research identified that cells upstream from GnRH neurons expressing the nuclear progesterone receptor (PGR) are required for progesterone-negative feedback. However, the identity of these cells and the mechanism by which they reduce GnRH/LH pulsatile secretion is unknown. We aimed to address the hypothesis that PGR expressed by a neural population in the arcuate nucleus recently identified as the GnRH pulse generator, cells expressing Kisspeptin, Neurokinin B, and Dynorphin (KNDy cells), mediate progesterone negative feedback. To achieve this, we utilized female mice with the PGR gene conditionally deleted from kisspeptin cells (KPRKO mice) and observed a substantial decrease in the percentage of KNDy neurons co-expressing PGR mRNA (11% in KPRKO mice versus 86% in wildtype mice). However, KPRKO mice did not display changes in the frequency or amplitude of LH pulses in diestrus or estrus, nor in the ability of exogenous progesterone to blunt a post-castration rise in LH. Further, mRNA expression of arcuate kisspeptin and dynorphin, which are excitatory and inhibitory to GnRH secretion, respectively, remained unaltered in KPRKO mice compared to wildtype controls. Together, these findings show that the near-complete loss of PGR signaling from KNDy cells does not impact negative feedback regulation of GnRH pulse generation in mice, suggesting that feedback through this receptor can occur via a small number of KNDy cells or a yet unidentified cell population.

Peripheral administration of a κ-opioid receptor agonist nalfurafine inactivates gonadotropin-releasing hormone pulse generator activity in goats

Neurons co-expressing kisspeptin, neurokinin B, and dynorphin A (KNDy neurons), located in the arcuate nucleus (ARC) of the hypothalamus, are indicated to be the gonadotropin-releasing hormone (GnRH) pulse generator. Dynorphin A is reported to suppress GnRH pulse generator activity. Nalfurafine is a selective agonist of the κ-opioid receptor (KOR), a receptor for dynorphin A, clinically used as an anti-pruritic drug. This study aimed to evaluate the effects of nalfurafine on GnRH pulse generator activity and luteinizing hormone (LH) pulses using female goats. Nalfurafine (0, 2, 4, 8, or 16 μg/head) was intravenously injected into ovariectomized Shiba goats. The multiple unit activity (MUA) in the ARC area was recorded, and plasma LH concentrations were measured 2 and 48 h before and after injection, respectively. The MUA volley interval during 0–2 h after injection was significantly increased in the nalfurafine 8 and 16 μg groups compared with the vehicle group. In 0–2 h after injection, the number of LH pulses was significantly decreased in the nalfurafine 8 and 16 μg groups, and the mean and baseline LH were significantly decreased in all nalfurafine-treated groups (2, 4, 8, and 16 μg) compared with the vehicle group. These results suggest that nalfurafine inhibits the activity of the GnRH pulse generator in the ARC, thus suppressing pulsatile LH secretion. Therefore, nalfurafine could be used as a reproductive inhibitor in mammals.

Norepinephrine Neurons in the Nucleus of the Solitary Tract Suppress Luteinizing Hormone Secretion in Female Mice.

Stress impairs fertility, at least in part, via inhibition of gonadotropin secretion. Luteinizing hormone (LH) is an important gonadotropin that is released in a pulsatile pattern in males and in females throughout the majority of the ovarian cycle. Several models of stress, including acute metabolic stress, suppress LH pulses via inhibition of neurons in the arcuate nucleus of the hypothalamus that coexpress kisspeptin, neurokinin B, and dynorphin (termed KNDy cells) which form the pulse generator. The mechanism for inhibition of KNDy neurons during stress, however, remains a significant outstanding question. Here, we investigated a population of catecholamine neurons in the nucleus of the solitary tract (NTS), marked by expression of the enzyme dopamine beta-hydroxylase (DBH), in female mice. First, we found that a subpopulation of DBH neurons in the NTS is activated (express c-Fos) during metabolic stress. Then, using chemogenetics, we determined that activation of these cells is sufficient to suppress LH pulses, augment corticosterone secretion, and induce sickness-like behavior. In subsequent studies, we identified evidence for suppression of KNDy cells (rather than downstream signaling pathways) and determined that the suppression of LH pulses was not dependent on the acute rise in glucocorticoids. Together these data support the hypothesis that DBH cells in the NTS are important for regulation of neuroendocrine and behavioral responses to stress.

Chronic inflammation decreases arcuate kisspeptin expression in male sheep

Lipopolysaccharide (LPS) from Gram-negative bacteria induces an immune response and impairs reproduction through suppression of gonadotropin releasing hormone (GnRH), subsequently luteinizing hormone (LH) secretion. While there is evidence that acute inflammation inhibits kisspeptin, little is known about the impact of chronic inflammation on this key reproductive neuropeptide in livestock species. Thus, we sought to examine a central mechanism whereby LPS suppresses LH secretion in sheep. Twenty wethers were randomly assigned to one of five treatment groups: control (CON; n=4), single acute IV LPS dose (SAD; n=4), daily acute IV LPS dose (DAD; n=4), daily increasing IV LPS dose (DID; n=4), and chronic subcutaneous LPS dose (CSD; n=4). On Days 1 and 7, blood samples were collected every 12 minutes for 360 minutes using jugular venipuncture. Following blood collection on Day 7, all animals were euthanized, brain tissue was perfused with 4% paraformaldehyde, and hypothalamic blocks were removed and processed for immunohistochemistry. On Day 1, LH pulse frequency was significantly lower (p=0.02) in SAD (0.25 ± 0.1 pulses/hour), DAD (0.25 ± 0.1 pulses/hour), DID (0.35 ± 0.1 pulses/hour), and CSD (0.40 ± 0.1 pulses/hour) compared to CON (0.70 ±0.1 pulses/hour). On Day 7, only DID animals (0.35 ± 0.1 pulses/hour) had significantly lower (p=0.049) LH pulse frequency compared to controls (0.85 ± 0.1 pulse/hour). Furthermore, only DID animals (33.3 ± 10.9 cells/section/animal) had significantly fewer (p=0.001) kisspeptin-immunopositive cells compared to controls (82.6 ± 13.6 cells/section/animal). Taken together, we suggest that daily increasing doses of LPS is a powerful inhibitor of kisspeptin neurons in young male sheep and a physiologically relevant model to examine the impact of chronic inflammation on the reproductive axis in livestock.

Obesity Alters POMC and Kisspeptin Neuron Cross Talk Leading to Reduced Luteinizing Hormone in Male Mice.

Obesity is associated with hypogonadism in males, characterized by low testosterone and sperm number. Previous studies determined that these stem from dysregulation of hypothalamic circuitry that regulates reproduction, by unknown mechanisms. Herein, we used mice fed chronic high-fat diet, which mimics human obesity, to determine mechanisms of impairment at the level of the hypothalamus, in particular gonadotropin-releasing hormone (GnRH) neurons that regulate luteinizing hormone (LH), which then regulates testosterone. Consistent with obese humans, we demonstrated lower LH, and lower pulse frequency of LH secretion, but unchanged pituitary responsiveness to GnRH. LH pulse frequency is regulated by pulsatile GnRH secretion, which is controlled by kisspeptin. Peripheral and central kisspeptin injections, and DREADD-mediated activation of kisspeptin neurons, demonstrated that kisspeptin neurons were suppressed in obese mice. Thus, we investigated regulators of kisspeptin secretion. We determined that the LH response to NMDA was lower in obese mice, corresponding to fewer glutamate receptors in kisspeptin neurons, which may be critical for kisspeptin synchronization. Given that kisspeptin neurons also interact with anorexigenic POMC neurons, which are affected by obesity, we examined their cross talk, and determined that the LH response to either DREADD-mediated activation of POMC neurons or central injection of αMSH, a product of POMC, is abolished in obese mice. This was accompanied by diminished levels of αMSH receptor, MC4R, in kisspeptin neurons. Together, our studies determined that obesity leads to the downregulation of receptors that regulate kisspeptin neurons, which is associated with lower LH pulse frequency, leading to lower LH and hypogonadism.

Raphe glucose-sensing serotonergic neurons stimulate KNDy neurons to enhance LH pulses via 5HT2CR: rat and goat studies

Dysfunction of central serotonergic neurons is known to cause depressive disorders in humans, who often show reproductive and/or glucose metabolism disorders. This study examined whether dorsal raphe (DR) serotonergic neurons sense high glucose availability to upregulate reproductive function via activating hypothalamic arcuate (ARC) kisspeptin neurons (= KNDy neurons), a dominant stimulator of gonadotropin-releasing hormone (GnRH)/gonadotropin pulses, using female rats and goats. RNA-seq and histological analysis revealed that stimulatory serotonin-2C receptor (5HT2CR) was mainly expressed in the KNDy neurons in female rats. The serotonergic reuptake inhibitor administration into the mediobasal hypothalamus (MBH), including the ARC, significantly blocked glucoprivic suppression of luteinizing hormone (LH) pulses and hyperglycemia induced by intravenous 2-deoxy-D-glucose (2DG) administration in female rats. A local infusion of glucose into the DR significantly increased in vivo serotonin release in the MBH and partly restored LH pulses and hyperglycemia in the 2DG-treated female rats. Furthermore, central administration of serotonin or a 5HT2CR agonist immediately evoked GnRH pulse generator activity, and central 5HT2CR antagonism blocked the serotonin-induced facilitation of GnRH pulse generator activity in ovariectomized goats. These results suggest that DR serotonergic neurons sense high glucose availability to reduce gluconeogenesis and upregulate reproductive function by activating GnRH/LH pulse generator activity in mammals.

NK3R signalling in the posterodorsal medial amygdala is involved in stress-induced suppression of pulsatile LH secretion in female mice.

Psychosocial stress negatively impacts reproductive function by inhibiting pulsatile luteinizing hormone (LH) secretion. The posterodorsal medial amygdala (MePD) is responsible in part for processing stress and modulating the reproductive axis. Activation of the neurokinin 3 receptor (NK3R) suppresses the gonadotropin-releasing hormone (GnRH) pulse generator, under hypoestrogenic conditions, and NK3R activity in the amygdala has been documented to play a role in stress and anxiety. We investigate whether NK3R activation in the MePD is involved in mediating the inhibitory effect of psychosocial stress on LH pulsatility in ovariectomised female mice. First, we administered senktide, an NK3R agonist, into the MePD and monitored the effect on pulsatile LH secretion. We then delivered SB222200, a selective NK3R antagonist, intra-MePD in the presence of predator odour, 2,4,5-trimethylthiazole (TMT) and examined the effect on LH pulses. Senktide administration into the MePD dose-dependently suppresses pulsatile LH secretion. Moreover, NK3R signalling in the MePD mediates TMT-induced suppression of the GnRH pulse generator, which we verified using a mathematical model. The model verifies our experimental findings: (i) predator odour exposure inhibits LH pulses, (ii) activation of NK3R in the MePD inhibits LH pulses and (iii) NK3R antagonism in the MePD blocks stressor-induced inhibition of LH pulse frequency in the absence of ovarian steroids. These results demonstrate for the first time that NK3R neurons in the MePD mediate psychosocial stress-induced suppression of the GnRH pulse generator.

HormoneBayes: A novel Bayesian framework for the analysis of pulsatile hormone dynamics.

The hypothalamus is the central regulator of reproductive hormone secretion. Pulsatile secretion of gonadotropin releasing hormone (GnRH) is fundamental to physiological stimulation of the pituitary gland to release luteinizing hormone (LH) and follicle stimulating hormone (FSH). Furthermore, GnRH pulsatility is altered in common reproductive disorders such as polycystic ovary syndrome (PCOS) and hypothalamic amenorrhea (HA). LH is measured routinely in clinical practice using an automated chemiluminescent immunoassay method and is the gold standard surrogate marker of GnRH. LH can be measured at frequent intervals (e.g., 10 minutely) to assess GnRH/LH pulsatility. However, this is rarely done in clinical practice because it is resource intensive, and there is no open-access, graphical interface software for computational analysis of the LH data available to clinicians. Here we present hormoneBayes, a novel open-access Bayesian framework that can be easily applied to reliably analyze serial LH measurements to assess LH pulsatility. The framework utilizes parsimonious models to simulate hypothalamic signals that drive LH dynamics, together with state-of-the-art (sequential) Monte-Carlo methods to infer key parameters and latent hypothalamic dynamics. We show that this method provides estimates for key pulse parameters including inter-pulse interval, secretion and clearance rates and identifies LH pulses in line with the widely used deconvolution method. We show that these parameters can distinguish LH pulsatility in different clinical contexts including in reproductive health and disease in men and women (e.g., healthy men, healthy women before and after menopause, women with HA or PCOS). A further advantage of hormoneBayes is that our mathematical approach provides a quantified estimation of uncertainty. Our framework will complement methods enabling real-time in-vivo hormone monitoring and therefore has the potential to assist translation of personalized, data-driven, clinical care of patients presenting with conditions of reproductive hormone dysfunction.

Neonatal Aromatase Inhibition Blocked Defeminization of AVPV Kiss1 Neurons and LH Surge-Generating System in Male Rats.

The neuroendocrine system that controls the preovulatory surge of gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH), which triggers ovulation in female mammals, is sexually differentiated in rodents. A transient increase in circulating testosterone levels in male rats within a few hours of birth is primarily responsible for the defeminization of anteroventral periventricular nucleus (AVPV) kisspeptin neurons, which are critical regulators of the GnRH/LH surge. The present study aimed to determine whether neonatal estradiol-17β (E2) converted from testosterone by aromatase primarily causes the defeminization of AVPV kisspeptin neurons and the surge of GnRH/LH in male rodents. The results of the present study showed that the neonatal administration of letrozole (LET), a nonsteroidal aromatase inhibitor, within 2 hours of birth rescued AVPV Kiss1 expression and the LH surge in adult male rats, while the neonatal administration of testosterone propionate (TP) irreversibly attenuated AVPV Kiss1 expression and the LH surge in adult female rats. Furthermore, the neonatal LET-treated Kiss1-Cre-activated tdTomato reporter males exhibited a comparable number of AVPV Kiss1-Cre-activated tdTomato-expressing cells to that of vehicle-treated female rats, while neonatal TP-treated females showed fewer AVPV Kiss1-Cre-activated tdTomato-expressing cells than vehicle-treated females. Moreover, neonatal TP administration significantly decreased the number of arcuate Kiss1-expressing and Kiss1-Cre-activated tdTomato-positive cells and suppressed LH pulses in adult gonadectomized female rats; however, neonatal LET administration failed to affect them. These results suggest that E2 converted from neonatal testosterone is primarily responsible for the defeminization of AVPV kisspeptin neurons and the subsequent GnRH/LH surge generation in male rats.

Modulatory Effects of Ethinyl Estradiol Plus Drospirenone Contraceptive Pill on Spontaneous and GnRH-Induced LH Secretion

Background: Combined oral contraceptives (COCs) work mostly by preventing the pre-ovulatory gonadotropin surge, but the action of COCs on spontaneous episodic and GnRH (gonadotropin-releasing hormone)-induced LH (luteinizing hormone) release has been poorly evaluated. Oral contraceptives are known to act on the spontaneous hypothalamic–pituitary functions reducing both GnRH and gonadotropin release and blocking ovulation. Aim: To evaluate spontaneous and GnRH-induced LH release during both phases of the menstrual cycle or under the use of the contraceptive pill. Methods: A group of 12 women, subdivided into two groups, volunteered for the study. Group A (n = 6, controls) received no treatments, while Group B (n = 6) received a 21 + 7 combination of ethinyl-estradiol (EE) 30 µg + drospirenone (DRSP) 3 mg. Both groups were evaluated twice: Group A during follicular and luteal phases, Group B during pill assumption and during the suspension interval, performing a pulsatility test, GnRH stimulation test, and hormonal parameters evaluation. Spontaneous and GnRH-induced secretory pulses were evaluated, as well as the instantaneous secretory rate (ISR). Results: COC treatment lowered LH and FSH (follicle stimulating hormone) levels significantly if compared to the follicular phase of spontaneous cycles. During the suspension interval, hormone levels rapidly rose and became comparable to those of the follicular phase of the control group. The LH pulse frequency under COC administration during the suspension interval was similar to that observed during the follicular phase (2.6 ± 0.3 pulses/180 min and 2.3 ± 0.2 pulses/180 min, respectively). The GnRH-induced LH peaks were greater in amplitude and duration than those observed after ISR computation in both groups. The GnRH-induced LH release during the luteal phase of the control subjects was higher than in the follicular phase (51.2 ± 12.3 mIU/mL and 14.9 ± 1.8 mIU/mL, respectively). Conversely, subjects under COC showed a GnRH-induced LH response similar during COC and during the suspension interval. Conclusions: Our data support that the EE + DRSP preparation acts on both spontaneous pulsatile release and GnRH-induced LH release during the withdrawal period of the treatment, and that after 5–7 days from the treatment suspension, steroidal secretion from the ovary is resumed, such as that of androgens. This suggests that in hyperandrogenic patients, a suspension interval as short as 4 days might be clinically better.

Are menstrual disturbances associated with an energy availability threshold? A critical review of the evidence.

Exercising women have a high prevalence of menstrual disturbances. In 2003, it was suggested that disruption in luteinizing hormone (LH) pulsatility occurs below a threshold of energy availability (EA) of 30kcal/kg lean body mass (LBM)/day. This paper is a critical review of the evidence regarding the theory that disruptions to the reproductive axis and menstrual disturbances occur below the proposed threshold. Short-term laboratory studies demonstrated that 4-5 days of an EA below 30kcal/kg LBM/day, induced with or without exercise, decreased serum triiodothyronine and LH pulse frequency, and increased LH pulse amplitude in sedentary, regularly menstruating women. Fewer studies have investigated downstream ovarian effects after long-term exposure to low EA. The Sargent Camp Study was the first randomized trial that induced luteal phase defects, delayed menses, and anovulation by causing weight loss (-4± 0.3kg) with an abrupt increase in exercise volume for two menstrual cycles. The BioEnergetics Study was a randomized controlled trial that induced varying levels of energy deficits by manipulating energy intake and expenditure for three menstrual cycles. LH pulse frequency and triiodothyronine decreased, and 57% of women developed luteal phase defects, anovulation, and/or oligomenorrhea. An EA below 30kcal/kg fat free mass/day increased the chance of experiencing a menstrual disturbance by 50%. However, menstrual disturbances were observed above and below that EA threshold, and changes in LH pulse frequency predicted only luteal phase defects, not oligomenorrhea or anovulation. The proposed EA threshold is not a cut-off below which menstrual disturbances occur, but represents an increased risk of experiencing menstrual disturbances.

A high-fat eucaloric diet induces reprometabolic syndrome of obesity in normal weight women.

We examined the effects of 1 month of a eucaloric, high-fat (48% of calories) diet (HFD) on gonadotropin secretion in normal-weight women to interrogate the role of free fatty acids and insulin in mediating the relative hypogonadotropic hypogonadism of obesity. Eighteen eumenorrheic women (body mass index [BMI] 18-25 kg/m2) were studied in the early follicular phase of the menstrual cycle before and after exposure to an HFD with frequent blood sampling for luteinizing hormone (LH) and follicle-stimulating hormone (FSH), followed by an assessment of pituitary sensitivity to gonadotropin-releasing hormone (GnRH). Mass spectrometry-based plasma metabolomic analysis was also performed. Paired testing and time-series analysis were performed as appropriate. Mean endogenous LH (unstimulated) was significantly decreased after the HFD (4.3 ± 1.0 vs. 3.8 ± 1.0, P < 0.01); mean unstimulated FSH was not changed. Both LH (10.1 ± 1.0 vs. 7.2 ± 1.0, P < 0.01) and FSH (9.5 ± 1.0 vs. 8.8 ± 1.0, P < 0.01) responses to 75 ng/kg of GnRH were reduced after the HFD. Mean LH pulse amplitude and LH interpulse interval were unaffected by the dietary exposure. Eucaloric HFD exposure did not cause weight change. Plasma metabolomics confirmed adherence with elevation of fasting free fatty acids (especially long-chain mono-, poly-, and highly unsaturated fatty acids) by the last day of the HFD. One-month exposure to an HFD successfully induced key reproductive and metabolic features of reprometabolic syndrome in normal-weight women. These data suggest that dietary factors may underlie the gonadotrope compromise seen in obesity-related subfertility and therapeutic dietary interventions, independent of weight loss, may be possible.

Conditional Oprk1-dependent Kiss1 deletion in kisspeptin neurons caused estrogen-dependent LH pulse disruption and LH surge attenuation in female rats

The gonadotropin-releasing hormone (GnRH) pulse and surge are considered to be generated by arcuate kisspeptin/neurokinin B/dynorphin A (KNDy) neurons and anteroventral periventricular nucleus (AVPV) kisspeptin neurons, respectively, in female rodents. The majority of KNDy and AVPV kisspeptin neurons express κ-opioid receptors (KORs, encoded by Oprk1) in female rodents. Thus, this study aimed to investigate the effect of a conditional Oprk1-dependent Kiss1 deletion in kisspeptin neurons on the luteinizing hormone (LH) pulse/surge and fertility using Kiss1-floxed/Oprk1-Cre rats, in which Kiss1 was deleted in cells expressing or once expressed the Oprk1/Cre. The Kiss1-floxed/Oprk1-Cre female rats, with Kiss1 deleted in a majority of KNDy neurons, showed normal puberty while having a one-day longer estrous cycle and fewer pups than Kiss1-floxed controls. Notably, ovariectomized (OVX) Kiss1-floxed/Oprk1-Cre rats showed profound disruption of LH pulses in the presence of a diestrous level of estrogen but showed apparent LH pulses without estrogen treatment. Furthermore, Kiss1-floxed/Oprk1-Cre rats, with Kiss1 deleted in approximately half of AVPV kisspeptin neurons, showed a lower peak of the estrogen-induced LH surge than controls. These results suggest that arcuate and AVPV kisspeptin neurons expressing or having expressed Oprk1 have a role in maintaining normal GnRH pulse and surge generation, the normal length of the estrous cycle, and the normal offspring number in female rats.

PSIV-17 Postnatal Obesity Amplifies Gnrh-Induced Lh Release in Prenatal Testosterone-Treated Female Sheep

Abstract Polycystic ovary syndrome (PCOS) is the most common hormonal disorder in females of reproductive age. Hypersecretion of luteinizing hormone (LH), partly due to increased pituitary sensitivity to gonadotrophin-releasing hormone (GnRH), is a significant cause of infertility in women with PCOS. Moreover, obesity amplifies the severity of reproductive dysfunction in PCOS. In sheep, testosterone (T) excess during gestational days (GD) 30 to 90 results in GnRH-mediated LH hypersecretion and recapitulates the PCOS phenotype. This study investigated 1) if mid-gestation (GD 60 to 90) is the susceptibility window for programming pituitary hypersensitivity to GnRH, and 2) the contribution of postnatal obesity in modulating LH responsiveness to GnRH. Suffolk ewes received T propionate (T; 100 mg i.m.) or corn oil (C; vehicle) twice weekly from GD 60 to 90 (term = 147 d). At 5 mo of age, 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. During the anestrous period (late summer), ewes (n = 8/group; ~17 mo of age) received a GnRH bolus injection (2 ng/kg) intravenously every 1.5 h and blood samples were collected at 15-min intervals for a 6-h period. Despite animals being in seasonal anestrous, few endogenous LH pulses were evident during the 6 h-sampling period. To avoid confounding, only clear responses to GnRH challenge that occurred within 15 to 45 min after each GnRH injection were considered for further analyses. Mean number of exogenous GnRH-induced LH responses that met these criteria did not differ between groups and averaged 1.81 ± 0.27 pulses/animal. The parameters of these GnRH-induced responses were analyzed as one-way ANOVA with Tukey’s HSD post-hoc analysis. Mean LH pulse peak was greater (P &amp;lt; 0.05) in T overfed ewes (4.59 ± 0.49 ng/mL) compared with T maintenance (2.19 ± 0.61 ng/mL) and Control ewes (2.58 ± 0.53 ng/mL). Additionally, amplitude of LH pulses was greater (P &amp;lt; 0.05) in T overfed ewes (2.66 ± 0.39 ng/mL) compared with T maintenance (1.03 ± 0.48 ng/mL) and tended (P = 0.089) to be greater in T overfed compared with Control ewes (1.41 ± 0.42 ng/mL). LH pulse peak and amplitude in T maintenance did not differ from Control ewes. In conclusion, failure of GD 60 to 90 T treatment to increase GnRH responsiveness as opposed to our earlier finding of increased responsiveness in GD 30 to 90 T-treaded sheep indicates that the susceptibility window for programming pituitary hypersensitivity to GnRH lies between GD 30 to 60. Postnatal obesity may act as an independent activational signal in amplifying the pituitary sensitivity to GnRH in female sheep. This premise is supported by observations that obesity exacerbates pituitary-mediated LH hypersecretion in women with PCOS. Research support: NIH-NICHD (R01HD099096).

25 Role of Kndy and Arcuate Kiss1r-Containing Neurons in the Preovulatory Luteinizing Hormone Surge and Puberty Onset of Female sheep

Abstract Neurons within the arcuate nucleus (ARC) of the hypothalamus containing kisspeptin, neurokinin B (NKB), and dynorphin (KNDy neurons) have an important role in regulating the pulsatile secretion of gonadotropin releasing hormone (GnRH) and luteinizing hormone (LH). In sheep, kisspeptin neurons also contribute to the LH surge, as kisspeptin receptor (Kiss1r) antagonist administration reduces surge amplitude by 50% and KNDy neurons are likely involved, based on increased Fos expression at the time of the surge. However, the extent to which kisspeptin acts within the ARC regulate the GnRH/LH surge remains unclear. Thus, herein we tested the hypothesis that deletion of KNDy or ARC Kiss1r-containing neurons would impair the LH surge. Adult female sheep received bilateral injections targeting the ARC of NKB-saporin (NKB-SAP, n = 8), kisspeptin-saporin (Kiss-SAP, n = 10), or blank-saporin (Blank-SAP, n = 7) as a control. In other work, NKB-SAP lesioned over 90% of ovine KNDy neurons, while Kiss-SAP lesioned 67% of Kiss1r-containing cells without affecting KNDy or GnRH cell number. Ewes were also ovariectomized and a subcutaneous silastic estradiol (E2) implant was inserted at the time of neurosurgery. Two artificial luteal phases were simulated with progesterone-containing CIDRs, immediately followed by E2 treatment via implants to induce an LH surge. Blood samples were collected every two to four hours over two days and analyzed for LH via radioimmunoassay. LH surge amplitude in six of eight NKB-SAP ewes (49.5 ± 11.7 ng/mL) was significantly reduced compared with Blank-SAP control ewes (156.7 ± 20.2 ng/mL, p = 0.0001), a reduction similar to that produced by treatment with a Kiss1r antagonist. Nine of ten Kiss-SAP treated ewes displayed little to no increase of LH at the time of the expected surge (16.6 ± 5.3 ng/mL, p &amp;lt; 0.0001). Lesion effectiveness is currently being assessed by RNAscope, however all Kiss-SAP animals examined to date have significantly reduced ARC Kiss1r cell numbers except a single ewe which exhibited a normal LH surge. Based on these data, we propose that in ewes, KNDy neurons contribute to, but are not required for, the LH surge. In contrast, ARC Kiss1r-containing cells are essential for a functional LH surge. Given these results, we are currently assessing the role of ARC Kiss1r neurons in ovine puberty using a similar approach. Our data to date shows that time to puberty onset is similar for Kiss-SAP, Blank-SAP, and non-surgical control animals as measured by an increase in progesterone (p = 0.35). Blood samples to detect LH pulses and the LH surge are currently being analyzed, as are ARC Kiss1r cell numbers.

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 &amp;lt; 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 &amp;lt; 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 &amp;lt; 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).