Pulsatile Luteinizing Hormone Secretion Research Articles

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.

CRISPR-Cas9 knockdown of ESR1 in preoptic GABA-kisspeptin neurons suppresses the preovulatory surge and estrous cycles in female mice.

Evidence suggests that estradiol-sensing preoptic area GABA neurons are involved in the preovulatory surge mechanism necessary for ovulation. In vivo CRISPR-Cas9 editing was used to achieve a 60-70% knockdown in estrogen receptor alpha (ESR1) expression by GABA neurons located within the regions of the rostral periventricular area of the third ventricle (RP3V) and medial preoptic nuclei (MPN) in adult female mice. Mice exhibited variable reproductive phenotypes with the only significant finding being mice with bilateral ESR1 deletion in RP3V GABA neurons having reduced cFos expression in gonadotropin-releasing hormone (GnRH) neurons at the time of the surge. One sub-population of RP3V GABA neurons expresses kisspeptin. Re-grouping ESR1-edited mice on the basis of their RP3V kisspeptin expression revealed a highly consistent phenotype; mice with a near-complete loss of kisspeptin immunoreactivity displayed constant estrus and failed to exhibit surge activation but retained pulsatile luteinizing hormone (LH) secretion. These observations demonstrate that ESR1-expressing GABA-kisspeptin neurons in the RP3V are essential for the murine preovulatory LH surge mechanism.

New methods to investigate the GnRH pulse generator.

The exact neural construct underlying the dynamic secretion of gonadotrophin-releasing hormone (GnRH) has only recently been identified despite the detection of multiunit electrical activity volleys associated with pulsatile luteinising hormone (LH) secretion four decades ago. Since the discovery of kisspeptin/neurokinin B/dynorphin neurons in the mammalian hypothalamus, there has been much research into the role of this neuronal network in controlling the oscillatory secretion of gonadotrophin hormones. In this review, we provide an update of the progressive application of cutting-edge techniques combined with mathematical modelling by the neuroendocrine community, which are transforming the functional investigation of the GnRH pulse generator. Understanding the nature and function of the GnRH pulse generator can greatly inform a wide range of clinical studies investigating infertility treatments.

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 < 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.

SAT601 The Role Of The Melanocortin System In Ovine Puberty

Abstract Disclosure: E.G. Aerts: None. M.J. Griesgraber: None. A. Thomson: None. M. Brown: None. A. Seman: None. S.L. Hardy: None. R.L. Goodman: None. C. Nestor: None. S.M. Hileman: None. The neural mechanisms that regulate the timing of puberty onset are not completely known. In several species, including sheep, puberty onset relies on an increase in gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion as a result of decreased sensitivity to estradiol (E2) inhibition. Additionally, it is unclear how E2 acts in the hypothalamus to inhibit GnRH/LH secretion, as GnRH neurons do not contain estrogen receptor alpha (ERα). Neurons within the arcuate nucleus (ARC) of the hypothalamus that contain kisspeptin, neurokinin B, and dynorphin (KNDy neurons) are critical for the generation of GnRH pulses and contain ERα. Data from our laboratory suggests that KNDy neurons, while critical, may be responding to other inputs that time puberty onset. Proopiomelanocortin (POMC) and Agouti-related peptide (AgRP) neurons in the ARC have been shown to contain ERα and provide input to KNDy neurons, which contain the melanocortin 3 and 4 receptor (MC3R, MC4R). The goal of this work was to assess POMC- and AgRP-positive cell numbers, input onto KNDy neurons, and neuronal activity changes in a puberty-specific manner. Female sheep were ovariectomized and given subcutaneous E2 implants at 5 months (prepubertal, n=4), 8 months (peripubertal, n=5), and 10 months (postpubertal, n=5) of age. Two weeks after surgery, blood samples were collected via jugular venipuncture every 12 minutes for 4 hours and assessed for LH. The sheep were then sacrificed and hypothalamic tissue was perfused, collected, and prepared for staining. The tissue was assessed via immunohistochemistry for POMC, AgRP, kisspeptin, NKB, and c-Fos, a known marker of neuronal activity. Pulsatile LH secretion was, as expected, reduced at 5 months and highest at 10 months. ARC POMC cell numbers did not significantly change over development (p>0.10). The number of ARC POMC close contacts onto ARC kisspeptin neurons increased significantly with development (p<0.05), as well as the percentage of ARC POMC neurons expressing c-Fos (p<0.05). ARC AgRP cell numbers and the percentage of AgRP cells colocalized with c-Fos decreased significantly over time (p<0.05). The number of AgRP close contacts onto ARC NKB cells is currently being analyzed. The percentage of NKB cells colocalizing c-Fos throughout pubertal development also increased significantly (p<0.05). This data supports the hypothesis that the ARC melanocortin system impacts KNDy neurons to regulate GnRH/LH secretion throughout pubertal development. Presentation: Saturday, June 17, 2023

SAT375 Impact of Flutamide on Wake vs. Sleep LH Pulse Frequency in PCOS: An Interim Analysis

Abstract Disclosure: C.K. Matingou: None. S. Kim: None. M. Gilrain: None. C.M. Burt Solorzano: None. C.R. McCartney: None. Polycystic ovary syndrome (PCOS) is characterized by hyperandrogenism and ovulatory dysfunction. PCOS is also associated with luteinizing hormone (LH) excess, in part related to persistently high gonadotropin-releasing hormone (GnRH) pulse frequency. Furthermore, late pubertal girls with obesity and hyperandrogenemia did not exhibit sleep-associated decreases in LH (GnRH) pulse frequency when studied during the follicular phase, in contrast to non-hyperandrogenemic controls with or without obesity (JCEM 2014;99:2887-96). We therefore aimed to test two hypotheses: (1) sleep-associated reductions in LH pulse frequency are less pronounced in adult PCOS compared to late-follicular controls; and (2) sleep-associated reductions in LH pulse frequency are normalized in PCOS with 4 weeks of flutamide (androgen-receptor antagonist) pretreatment. To date, we have studied 5 women with PCOS (median age 30 y, BMI 42.6 kg/m2) and 5 ovulatory controls (median age 22 y, BMI 23.2 kg/m2). Subjects underwent two separate 16-hour frequent-blood-sampling studies to characterize pulsatile LH secretion, once after flutamide pretreatment (250 mg BID x 4 weeks) and once after placebo (PBO) pretreatment (BID for 4 weeks). In each study, 1500-2300 h and 2300-0700 h were designated the wake and sleep periods, respectively. The order of flutamide and PBO administration was randomized, and treatment allocation was switched after the first admission per a cross-over design. Controls were studied on cycle days 7-10, while those with PCOS were studied no earlier than cycle day 7. Serum progesterone concentrations were < 1.5 ng/dl for all admissions. With PBO pretreatment, participants with PCOS and controls exhibited the following: wake LH pulse frequency 1.19 (1.08-1.33) (median [interquartile range]) and 1.22 (0.97-1.27) pulses/h, respectively; sleep LH pulse frequency 0.93 (0.78-1.28) and 0.83 (0.78-0.99) pulses/h, respectively; and wake-to-sleep change in LH pulse frequency of -19.4 (-21.9 to +2.9) and -15.5 (-31.8 to -12.0) percent, respectively. For participants with PCOS, pretreatment with flutamide was associated with the following changes compared to PBO: wake LH pulse frequency +0.01 (-0.06 to +0.03) pulses/h; sleep LH pulse frequency +0.06 (+0.01 to +0.07) pulses/h; and wake-to-sleep LH pulse frequency change +5 (+7 to -12) percent. None of these differences were statistically significant by conservative nonparametric testing. We conclude that this interim analysis does not offer any indication that wake- and sleep-associated LH pulse frequencies are different between PCOS and late-follicular controls, nor does it suggest clear differences in wake-to-sleep changes in late-follicular LH pulse frequency between groups. Furthermore, this analysis does not seem to offer any indication that flutamide alone alters wake- and/or sleep-associated LH pulse frequencies in PCOS. Presentation Date: Saturday, June 17, 2023

FRI279 Submaximal Loss Of KNDy Neurons Accelerates And Amplifies Pulsatile LH Secretion In Female Rats

Abstract Disclosure: A.C. Campideli-Santana: None. K.S. da Costa Silva: None. R. Araújo-Lopes: None. L.M. Antunes: None. R.E. Szawka: None. Kisspeptin is a powerful stimulator neuropeptide in the hypothalamus-pituitary-gonadal axis and is essential for fertility. Neurons in the arcuate nucleus (ARC) of the hypothalamus that coexpress kisspeptin, neurokinin B, and dynorphin (KNDy) play a crucial role in the control of luteinizing hormone (LH) secretion, but the neuroendocrine mechanisms involved in this regulation are not fully understood. Our previous study demonstrated that the moderate loss of KNDy neurons amplifies the LH surge induced by estradiol in ovariectomized rats (1). The current study aimed to perform a longitudinal evaluation of female rats in which KNDy neurons were ablated to determine the integrated role of this neuronal population in the control of pulsatile LH secretion. Adult female rats underwent a neurochemical ablation of KNDy neurons via intra-ARC stereotaxic injections of neurokinin-3 receptor agonist conjugated with saporin (NK3-SAP), while control animals received blank-saporin (Control). Estrous cyclicity was monitored over 21 days before and after the stereotaxic surgery. Three weeks after surgery, blood samples (10 µL) were withdrawn from the tail tip at 6-minute intervals for 180 minutes on the day of diestrus. Rats were then ovariectomized and the ovaries were processed for histological analysis. Whole blood LH levels were measured by ELISA and brains were immunohistochemically labeled for neurokinin B in the ARC. NK3-SAP rats bearing a submaximal loss of KNDy neurons (between 55-95%; n = 8) displayed irregular estrous cycles. The ovarian weight and the number of corpora lutea and atretic follicles were unaltered, whereas the number of healthy early antral follicles decreased in NK3-SAP rats. The frequency of LH pulses was increased and both pulse amplitude and mean LH levels were higher in NK3-SAP compared with Control rats. Thus, the submaximal loss of KNDy neurons affects the regularity of estrous cyclicity but does not preclude ovulation, while seems to stimulate the maturation of early antral follicles. Notably, KNDy neuron loss accelerates and amplifies LH pulsatile secretion during the rat estrous cycle. These results suggest a new role for KNDy neurons in restraining the frequency and amplitude of LH pulses in ovary-intact animals, with possible implications in the rate of recruitment of early antral follicles. Reference: Campideli-Santana et al., J Neuroendocrinol. 2022 Nov;34(11):e13204. Support: FAPEMIG, CNPq, CAPES, and Pro-reitoria de Pesquisa-UFMG. 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

Hypothalamic neuroglial plasticity is regulated by anti-Müllerian hormone and disrupted in polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is the most common reproductive-endocrine disorder affecting between 5 and 18% of women worldwide. An elevated frequency of pulsatile luteinizing hormone (LH) secretion and higher serum levels of anti-Müllerian hormone (AMH) are frequently observed in women with PCOS. The origin of these abnormalities is, however, not well understood. We studied brain structure and function in women with and without PCOS using proton magnetic resonance spectroscopy (MRS) and diffusion tensor imaging combined with fiber tractography. Then, using a mouse model of PCOS, we investigated by electron microscopy whether AMH played a role on the regulation of hypothalamic structural plasticity. Increased AMH serum levels are associated with increased hypothalamic activity/axonal-glial signalling in PCOS patients. Furthermore, we demonstrate that AMH promotes profound micro-structural changes in the murine hypothalamic median eminence (ME), creating a permissive environment for GnRH secretion. These include the retraction of the processes of specialized AMH-sensitive ependymo-glial cells called tanycytes, allowing more GnRH neuron terminals to approach ME blood capillaries both during the run-up to ovulation and in a mouse model of PCOS. We uncovered a central function for AMH in the regulation of fertility by remodeling GnRH terminals and their tanycytic sheaths, and provided insights into the pivotal role of the brain in the establishment and maintenance of neuroendocrine dysfunction in PCOS. INSERM (U1172), European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement n° 725149), CHU de Lille, France (Bonus H).

Mechanisms of Lactation-induced Infertility in Female Mice.

Lactation in mammals is associated with a period of infertility, which serves to direct maternal metabolic resources toward caring for the newborn offspring rather than supporting another pregnancy. This lactational infertility is characterized by reduced pulsatile luteinizing hormone (LH) secretion and lack of ovulation. The mechanisms mediating suppression of LH secretion during lactation are unclear. There are potential roles for both hormonal cues such as prolactin and progesterone, and pup-derived cues such as suckling, on the inhibition of reproduction. To enable future studies using transgenic animals to investigate these mechanisms, in the present study our aim was to characterize lactational infertility in mice, and to investigate the effect of removing pup-derived cues on LH secretion, time to ovulation, and kisspeptin immunoreactivity. We first confirmed that C57BL/6J mice experience prolonged anestrus during lactation, which is dependent on establishment of lactation, as removal of pups the day of parturition led to immediate resumption of pulsatile LH secretion and normal estrous cycles. Once lactation is established, however, the lactational anestrus persisted for several days even after premature removal of pups. Pharmacological suppression of prolactin following premature weaning significantly reduced this period of lactational infertility. Progesterone does not appear to play a significant role in the suppression of fertility during lactation in mice, as levels measured during lactation were not different from nonpregnant mice. These data suggest that prolactin plays a key role in mediating anestrus during early lactation in mice, even in the absence of the suckling stimulus.

Long-term effects of prenatal undernutrition on female rat hypothalamic KNDy neurons.

The nutritional environment during development periods induces metabolic programming, leading to metabolic disorders and detrimental influences on human reproductive health. This study aimed to determine the long-term adverse effect of intrauterine malnutrition on the reproductive center kisspeptin-neurokinin B-dynorphin A (KNDy) neurons in the hypothalamic arcuate nucleus (ARC) of female offspring. Twelve pregnant rats were divided into ad-lib-fed (control, n = 6) and 50% undernutrition (UN, n = 6) groups. The UN group was restricted to 50% daily food intake of the control dams from gestation day 9 until term delivery. Differences between the two groups in terms of various maternal parameters, including body weight (BW), pregnancy duration, and litter size, as well as birth weight, puberty onset, estrous cyclicity, pulsatile luteinizing hormone (LH) secretion, and hypothalamic gene expression of offspring, were determined. Female offspring of UN dams exhibited low BW from birth to 3 weeks, whereas UN offspring showed signs of precocious puberty; hypothalamic Tac3 (a neurokinin B gene) expression was increased in prepubertal UN offspring, and the BW at the virginal opening was lower in UN offspring than that in the control group. Interestingly, the UN offspring showed significant decreases in the number of KNDy gene-expressing cells after 29 weeks of age, but the number of ARC kisspeptin-immunoreactive cells, pulsatile LH secretions, and estrous cyclicity were comparable between the groups. In conclusion, intrauterine undernutrition induced various changes in KNDy gene expression depending on the life stage. Thus, intrauterine undernutrition affected hypothalamic developmental programming in female rats.

Short-term depletion of plasma estrogen affects hypothalamic kisspeptin-neurokinin B-dynorphin A neurons, gonadotrophs, and pulsatile luteinizing hormone secretion in female rats

Kisspeptin-neurokinin B-dynorphin A (KNDy) neurons in the arcuate nucleus (ARC) regulate pulsatile luteinizing hormone (LH) secretion. These neurons express estrogen receptors and are negatively regulated by estrogen. This study aimed to determine whether estrogen supplementation after short-term ovariectomy-induced estrogen depletion has different effects on KNDy neurons depending on the timing of the supplementation. To decrease endogenous estradiol (E2) for a short time, adult female rats received a tube filled with E2 one week after ovariectomy and utilized it one week later (O1w + E). From the results of immunohistochemistry, the response to E2 was attenuated in KNDy neurons of O1w + E rats. Enlarged LH-secreting cells in the anterior pituitary were found in O1w + E rats; however, such enlarged LH cells were not found in ones without previous short-term E2 depletion. From the analysis of LH pulses, plasma LH levels were increased in O1w + E rats relative to ones without previous short-term E2 depletion. These results suggested that once endogenous sex steroids were depleted, the response to E2 in hypothalamic KNDy neurons did not fully recover in one week. Thus, short-term sex steroid depletion due to gonadectomy could alter the response to the sex steroids in KNDy neurons even though the period without sex steroids is only one week, and the alteration is likely to affect plasma hormone levels.

Posterodorsal Medial Amygdala Urocortin-3, GABA, and Glutamate Mediate Suppression of LH Pulsatility in Female Mice.

The posterodorsal subnucleus of the medial amygdala (MePD) is an upstream modulator of the hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary-adrenal (HPA) axes. Inhibition of MePD urocortin-3 (Ucn3) neurons prevents psychological stress-induced suppression of luteinizing hormone (LH) pulsatility while blocking the stress-induced elevations in corticosterone (CORT) secretion in female mice. We explore the neurotransmission and neural circuitry suppressing the gonadotropin-releasing hormone (GnRH) pulse generator by MePD Ucn3 neurons and we further investigate whether MePD Ucn3 efferent projections to the hypothalamic paraventricular nucleus (PVN) control CORT secretion and LH pulsatility. Ucn3-cre-tdTomato female ovariectomized (OVX) mice were unilaterally injected with adeno-associated virus (AAV)-channelrhodopsin 2 (ChR2) and implanted with optofluid cannulae targeting the MePD. We optically activated Ucn3 neurons in the MePD with blue light at 10Hz and monitored the effect on LH pulses. Next, we combined optogenetic stimulation of MePD Ucn3 neurons with pharmacological antagonism of GABAA or GABAB receptors with bicuculline or CGP-35348, respectively, as well as a combination of NMDA and AMPA receptor antagonists, AP5 and CNQX, respectively, and observed the effect on pulsatile LH secretion. A separate group of Ucn3-cre-tdTomato OVX mice with 17β-estradiol replacement were unilaterally injected with AAV-ChR2 in the MePD and implanted with fiber-optic cannulae targeting the PVN. We optically stimulated the MePD Ucn3 efferent projections in the PVN with blue light at 20Hz and monitored the effect on CORT secretion and LH pulses. We reveal for the first time that activation of Ucn3 neurons in the MePD inhibits GnRH pulse generator frequency via GABA and glutamate signaling within the MePD, while MePD Ucn3 projections to the PVN modulate the HPG and HPA axes.

OR17-2 GABAergic Signalling in the Posterodorsal Medial Amygdala Mediates Psychological Stress-induced Suppression of the GnRH Pulse Generator in Female Mice

Abstract Psychological stress is linked to infertility and reproductive dysfunction by suppressing the hypothalamic gonadotropin-releasing hormone (GnRH) pulse generator and pulsatile luteinising hormone (LH) secretion. The posterodorsal subnucleus of the medial amygdala (MePD) is a key upstream regulator of GnRH pulse generator activity and displays increased neuronal activation in response to psychological stressors. The MePD is primarily a GABAergic nucleus with a strong GABAergic projection to hypothalamic reproductive centres, however, their functional significance has not been determined. We hypothesis that MePD GABAergic signalling is a crucial mediator of psychological stress-induced suppression of pulsatile LH secretion. We used a chemogenetic approach to selectively silence MePD GABA neurones during psychological stress to determine the effect on pulsatile LH secretion. Female ovariectomised Vgat-cre mice (n=7) were virally infected to selectively express inhibitory hM4DGi-DREADDs in MePD GABA neurones. Mice were exposed to either predator odour (PO) or restraint stress and given an intraperitoneal injection of either saline or the DREADD chemical activator clozapine-N-oxide (CNO, 5mg/kg). Statistical significance was determined using a one-way ANOVA and post-hoc Tukey test. Data are presented as mean ± SEM and p<0.05 was considered significant. PO exposure significantly increased LH inter-pulse interval (IPI) in the saline group (prestress: 19.05 ± 1.16 min vs. PO: 29.28 ± 3.16 min, p<0.05, n=7) and this effect was blocked by DREADD-mediated inhibition of MePD GABA neurones (prestress: 20.59 ± 1.12 min vs. PO: 21.31 ± 1.76 min, p>0.05, n=7). Restraint stress dramatically increased LH IPI in the saline group (prestress: 17.78 ± 1.02 min vs. restraint: 45.71 ± vs 5.50 min, p<0.05, n=7) and DREADD-mediated inhibition of MePD GABA neurones blocked this effect (prestress: 18.57 ± 1.97 min vs. restraint: 27.26 ± 4.02 min, p>0.05, n=7). Direct projections from the MePD to the GnRH pulse generator in the hypothalamic arcuate nucleus (ARC) have been identified, however, their phenotype is unknown. In this study, we optogenetically stimulated potential MePD GABAergic projection neurone terminals in the ARC and determined the effect on LH IPI. MePD GABA neurones in female Vgat-cre ovariectomised mice (n=6) were virally infected to express channelrhodopsin 2 protein and MePD GABAergic terminals in the ARC were selectively stimulated by blue light via a chronically implanted fibre-optic cannula in the ARC. Sustained optogenetic stimulation at 10 and 20Hz of MePD GABAergic terminals in the ARC dose-dependently suppressed pulsatile LH secretion (LH IPI; control: 23.34 ± 2.11 min vs. 10Hz: 38.08 ± 3.61 min; 20Hz: 56.67 ± 3.33 min, p<0.05, n=6). These findings confirm a functionally significant MePD GABAergic projection to the ARC and highlight the importance of GABA signalling in the MePD in mediating stress-induced suppression of reproductive function. Collectively, these findings provide novel insight into the amygdala neural circuitry underpinning stress-induced reproductive function. Presentation: Sunday, June 12, 2022 11:15 a.m. - 11:30 a.m.

RF01 | PMON45 Urocortin 3 Interaction with GABA and Glutamate in the Posterodorsal Subnucleus of The Medial Amygdala Mediates Suppression of GnRH Pulse Generator Frequency in Female Mice

Abstract Stress exerts a profound impact on reproductive function and disrupts pulsatile luteinizing hormone (LH) secretion. The posterodorsal subnucleus of the medial amygdala (MePD) is an upstream modulator of the hypothalamus pituitary gonadal axis. Stress alters neuronal activity within the MePD, increasing the expression of Urocortin3 (Ucn3) while enhancing the inhibitory output from the MePD to key hypothalamic reproductive centres. Designer receptor exclusively activated by designer drugs (DREADDs) inhibition of MePD Ucn3 neurons prevents psychological stress-induced suppression of LH pulses in female mice. The neurochemical pathways in the MePD responsible for regulating the GnRH pulse generator in the presence of stress remain unknown. We investigate the neurotransmission involved in the neural circuitry responsible for the suppression of GnRH pulse generator activity by MePD Ucn3 neuronal activation. Ucn3-Cre-tdTomato female ovariectomised (OVX) mice were unilaterally injected with AAV-ChR2 and implanted with optofluid cannulae targeting the MePD. Firstly, we optically stimulated MePD Ucn3 neurons with blue light at 10 Hz, 10 mW and monitored the effect on LH pulses. Next, we combined optogenetic activation of MePD Ucn3 neurons with pharmacological antagonism of GABAA or GABAB receptors with bicuculline or CGP, respectively, and observed the effect on LH pulsatility. Finally, during optical activation of MePD Ucn3 neurons we administered a combination of NMDA and AMPA receptor antagonists, AP5 and CNQX respectively, and monitored the effect on pulsatile LH secretion. Optogenetic stimulation of MePD Ucn3 neurons suppressed pulsatile LH secretion compared to controls and administration of aCSF had no effect on LH pulse interval (MePD Ucn3 activation: 36.04 ± 2.49 vs. pre-treatment control period: 17.50 ± 0.67 vs control virus: 21.67 ± 1.67 min; mean ± SEM; p<0.0001; n=12). Intra-MePD infusion of bicuculline (BIC) or CGP during optogenetic stimulation of MePD Ucn3 neurons completely blocked the suppressive effect of MePD Ucn3 neuronal activation on LH pulsatility (MePD Ucn3 activation and BIC: 20.50 ± 2.03 vs. pre-treatment control period: 17.58 ± 0.75 min; MePD Ucn3 activation and CGP: 16.30 ± 0.63 vs. pre-treatment control period: 17.50 ± 0.77 min; n=7-10). Intra-MePD infusion of AP5 + CNQX during optogenetic stimulation of MePD Ucn3 neurons similarly completely blocked the suppressive effect of MePD Ucn3 neuronal activation on LH pulsatility (MePD Ucn3 activation and AP5 + CNQX: 19.58 ± 1.42 min; n=6). Our findings show for the first time that optogenetic stimulation of MePD Ucn3 neurons inhibits GnRH pulse generator activity via GABA and glutamate signalling within the MePD. Presentation: Saturday, June 11, 2022 1:12 p.m. - 1:17 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Prenatal androgen treatment impairs the suprachiasmatic nucleus arginine-vasopressin to kisspeptin neuron circuit in female mice.

Polycystic ovary syndrome (PCOS) is associated with elevated androgen and luteinizing hormone (LH) secretion and with oligo/anovulation. Evidence indicates that elevated androgens impair sex steroid hormone feedback regulation of pulsatile LH secretion. Hyperandrogenemia in PCOS may also disrupt the preovulatory LH surge. The mechanisms through which this might occur, however, are not fully understood. Kisspeptin (KISS1) neurons of the rostral periventricular area of the third ventricle (RP3V) convey hormonal cues to gonadotropin-releasing hormone (GnRH) neurons. In rodents, the preovulatory surge is triggered by these hormonal cues and coincident timing signals from the central circadian clock in the suprachiasmatic nucleus (SCN). Timing signals are relayed to GnRH neurons, in part, via projections from SCN arginine-vasopressin (AVP) neurons to RP3VKISS1 neurons. Because rodent SCN cells express androgen receptors (AR), we hypothesized that these circuits are impaired by elevated androgens in a mouse model of PCOS. In prenatally androgen-treated (PNA) female mice, SCN Ar expression was significantly increased compared to that found in prenatally vehicle-treated mice. A similar trend was seen in the number of Avp-positive SCN cells expressing Ar. In the RP3V, the number of kisspeptin neurons was preserved. Anterograde tract-tracing, however, revealed reduced SCNAVP neuron projections to the RP3V and a significantly lower proportion of RP3VKISS1 neurons with close appositions from SCNAVP fibers. Functional assessments showed, on the other hand, that RP3VKISS1 neuron responses to AVP were maintained in PNA mice. These findings indicate that PNA changes some of the neural circuits that regulate the preovulatory surge. These impairments might contribute to ovulatory dysfunction in PNA mice modeling PCOS.

A Modified Ultra-Sensitive ELISA for Measurement of LH in Mice.

A major obstacle to monitoring pulsatile luteinizing hormone (LH) secretion in mice has been an assay with sufficient sensitivity in small blood volumes. In 2013, Steyn and colleagues published a highly sensitive enzyme-linked immunosorbent assay (ELISA) that overcame this barrier by coupling a duo of LH antibodies effective in accurately measuring LH in 4-µL whole-blood aliquots. To address the unavailability of the original detection antibody, AFP240580Rb, we validated a replacement detection antibody, biotinylated-5303 SPRN-5, to be used within the established ELISA. This modified LH ELISA demonstrated a minimum detection limit of 0.0028 ng/mL and a limit of quantification of 0.0333 ng/mL or 0.0666 ng/mL in diluted whole-blood samples of volume 6.4 µL (1:10) or 3.2 µL (1:20), respectively. Detection antibody 5303 SPRN-5 demonstrated parallelism, high precision, and accuracy across the standard curve. LH concentrations in comparison assays, using either 5303 SPRN-5 or AFP240580Rb, were highly correlated (R2 = 0.9829) and demonstrated LH pulse profiles from gonadectomized mice that were nearly superimposable. Pulsatile LH secretion was demonstrated in gonad-intact males and diestrous females and basal LH levels measured with 5303 SPRN-5 were approximately 5-fold higher than the limit of quantification. In addition, we document utility of this new LH ELISA to accurately measure LH in whole blood or serum across multiple sampling sites, as well as in pituitary extracts, LβT2 cells, or media. In summary, the modified LH ELISA described here is highly effective in measuring LH across a range of sample types and small volumes in mice.

Chronic androgen excess in female mice does not impact luteinizing hormone pulse frequency or putative GABAergic inputs to GnRH neurons.

Polycystic ovary syndrome (PCOS) is associated with androgen excess and, frequently, hyperactive pulsatile luteinizing hormone (LH) secretion. Although the origins of PCOS are unclear, evidence from pre‐clinical models implicates androgen signalling in the brain in the development of PCOS pathophysiology. Chronic exposure of female mice to dihydrotestosterone (DHT) from 3 weeks of age drives both reproductive and metabolic impairments that are ameliorated by selective androgen receptor (AR) loss from the brain. This suggests centrally driven mechanisms in hyperandrogen‐mediated PCOS‐like pathophysiology that remain to be defined. Acute prenatal DHT exposure can also model the hyperandrogenism of PCOS, and this is accompanied by increased LH pulse frequency and increased GABAergic innervation of gonadotrophin‐releasing hormone (GnRH) neurons. We aimed to determine the impact of chronic exposure of female mice to DHT, which models the hyperandrogenism of PCOS, on pulsatile LH secretion and putative GABAergic input to GnRH neurons. To do this, GnRH‐green fluorescent protein (GFP) female mice received either DHT or blank capsules for 90 days from postnatal day 21 (n = 6 or 7 per group). Serial tail‐tip blood sampling was used to measure LH dynamics and perfusion‐fixed brains were collected and immunolabelled for vesicular GABA transporter (VGAT) to assess putative GABAergic terminals associated with GFP‐labelled GnRH neurons. As expected, chronic DHT resulted in acyclicity and significantly increased body weight. However, no differences in LH pulse frequency or the density of VGAT appositions to GnRH neurons were identified between ovary‐intact DHT‐treated females and controls. Chronic DHT exposure significantly increased the number of AR expressing cells in the hypothalamus, whereas oestrogen receptor α‐expressing neuron number was unchanged. Therefore, although chronic DHT exposure from 3 weeks of age increases AR expressing neurons in the brain, the GnRH neuronal network changes and hyperactive LH secretion associated with prenatal androgen excess are not evident. These findings suggest that unique central mechanisms are involved in the reproductive impairments driven by exposure to androgen excess at different developmental stages.

Kisspeptin Stimulates the Pulsatile Secretion of Luteinizing Hormone (LH) during Postpartum Anestrus in Ewes Undergoing Continuous and Restricted Suckling.

Simple SummaryOne of the major impediments to improving the efficiency of sheep production systems is the difficulty of breeding the females before their young are weaned. A major physiological barrier is suckling, because it prevents the initiation of a new reproductive cycle by inhibiting the pulsatile secretion of gonadotropin-releasing hormone (GnRH) and thus the secretion of LH pulses. It has now become clear that, at brain level, the secretion of GnRH is controlled primarily by the neuropeptide kisspeptin (Kp), a central player in the ‘KNDy’ system that generates GnRH pulses. Here, we report that intravenous administration of Kp stimulates pulsatile LH secretion in ewes during postpartum anestrus. Moreover, the response was greater when suckling was restricted to 30 min twice a day. We conclude that kisspeptin application increases pulsatile LH secretion in suckling ewes, suggesting that suckling inhibits ovulation by reducing the activity of kisspeptin neurons.This study tested whether the intravenous application of kisspeptin can stimulate the pulsatile secretion of LH in suckling ewes during postpartum anestrus. Ten days after lambing, Pelibuey ewes were allocated among two groups: (1) continuous suckling (n = 8), where the lambs remained with their mothers; and (2) restricted suckling (n = 8), where the mothers suckled their lambs twice daily for 30 min. On Day 19 postpartum, the ewes were individually penned with ad libitum access to water and feed and given an indwelling catheter in each jugular vein. On Day 20, 4 mL of blood was sampled every 15 min from 08:00 to 20:00 h to determine LH pulse frequency. At 14:00 h, four ewes in each group received 120 μg of kisspeptin diluted in 3 mL of saline as a continuous infusion for 6 h; the remaining four ewes in each group received only saline. The interaction between kisspeptin and suckling type did not affect LH pulse frequency (p > 0.05). Before kisspeptin administration, pulse frequency was similar in all groups (1.50 ± 0.40 pulses per 6 h; p > 0.05). With the application of kisspeptin, pulse frequency increased to 3.50 ± 0.43 pulses per 6 h (p ≤ 0.014), so the concentration of LH (1.11 ± 0.14 ng mL−1) was greater in kisspeptin-treated ewes than in saline-treated ewes (0.724 ± 0.07 ng mL−1; p ≤ 0.040). The frequency of LH pulses was greater with restricted suckling than with continuous suckling (2.44 ± 0.29 versus 1.69 ± 0.29 pulses per 6 h; p ≤ 0.040). We conclude that intravenous application of kisspeptin increases the pulsatile secretion of LH in suckling ewes and that suckling might reduce kisspeptin neuronal activity, perhaps explaining the suppression of ovulation. Moreover, the effects of kisspeptin and suckling on pulsatile LH secretion appear to be independent, perhaps operating through different neural pathways.

Reformulation of PULSAR for Analysis of Pulsatile LH Secretion and a Revised Model of Estrogen-Negative Feedback in Mice.

The recent use of the tail-tip bleeding approach in mice has enabled researchers to generate detailed pulse and surge profiles of luteinizing hormone (LH) secretion in mice. However, the analysis of pulsatile LH secretion is piecemeal across the field with each laboratory using their own methodology. We have reformulated the once-popular PULSAR algorithm of Merriam and Wachter to operate on contemporary computer systems and provide downloadable and online pulse analysis platforms. As it is now possible to record the activity of the gonadotropin-releasing hormone pulse generator in freely behaving mice, we have been able to unambiguously define LH pulses in intact and gonadectomized male and female mice. These data sets were used to determine the appropriate PULSAR parameter sets for analyzing pulsatile LH secretion in the mouse. This was then used to establish an accurate model of estrogen negative feedback in the mouse. Intact and ovariectomized mice given Silastic capsules containing 1, 2, and 4 μg 17-β-estradiol/20 g body weight were tail-tip bled at 6-min intervals, and the resultant LH profiles were analyzed with PULSAR. Only the 4 μg 17-β-estradiol capsule treatment was found to return LH pulse amplitude and frequency to that of intact diestrous mice. Ultrasensitive mass spectrometry analysis showed that the 4 μg 17-β-estradiol capsule generated circulating estradiol levels equivalent to that of diestrous mice. It is hoped that the reformulation of PULSAR and generation of a realistic model of estrogen-negative feedback will provide a platform for the more uniform assessment of pulsatile hormone secretion in mice.