GnRH Pulse Frequency Research Articles

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

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

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

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

ODP405 Effects of Gonadotrope-Specific Knockout of Creb on HPG Axis Function in Mice

Abstract Background and Objective Reproduction is regulated by FSH and LH, which are synthesized and secreted by pituitary gonadotropes in response to pulsatile hypothalamic GnRH in a frequency dependent manner. Low and high GnRH pulse frequencies preferentially increase Fshb and Lhb expression, respectively, mediated by preferential coupling to Gαs-PKA-Creb and Gαq-PKC-MAPK-AP1 signaling cascades in vitro. The mouse Fshb proximal promoter contains a half-AP1/half-CRE recognition motif that binds Creb in vitro, and mutation of this half-CRE results in loss of preferential GnRH stimulation of Fshb at low pulse frequencies in immortalized gonadotrope LβT2 cells. Therefore, the purpose of this study was to identify the direct role of Creb on gonadotropin regulation in vivo. We hypothesized that gonadotrope-specific deletion of Creb would influence pubertal timing and/or fertility due to reduced Fshb expression. Methods and Results Gonadotrope-specific Creb knockout (Creb1 fx/fx / Gnrhr GRIC/+; Creb KO) and control (Creb1 fx/fx) mice were generated by Cre-Lox recombination and markers for pubertal onset were evaluated. We observed no differences in body weights of Creb KO and littermate control mice in either females or males from postnatal day (PND) 21 to 3 months of age (n=9/genotype/sex). The age of vaginal opening, a marker of puberty onset in female mice, was not significantly different between female Creb KO (PND 28.4±0.5; n=21) and littermate control (PND 29.5±0.6; n=17) mice. Similarly, the age of first estrus, determined by vaginal lavage and cytology, was not significantly different between Creb KO mice (PND 37.6±1.9; n=9) and littermate controls (PND 36.6±2.2; n=8). Estrous cyclicity was normal over a three-week period in Creb KO and control mice, which spent similar amounts of time in diestrus/metestrus, proestrus, or estrus. Male Creb KO mice tended to show preputial separation at a younger age (PND 28.8±0.3; n=19) compared to littermate control (PND 29.6±0.3; n=22) mice, but the difference did not reach statistical significance (p=0. 06). Conclusions Deletion of Creb from pituitary gonadotropes did not affect the timing of pubertal onset in female mice, but we did observe a trend for male Creb KO mice to have advanced puberty, as measured by preputial separation. Normal pubertal timing does not preclude potential effects of Creb-mediated gonadotropin regulation on folliculogenesis, spermatogenesis, or fertility (including litter size), for which studies are ongoing. Presentation: No date and time listed

LBMON274 Total Osteocalcin Levels Are Independently Associated With Worse Testicular Function And A Higher Degree Of HPG Axis Activation In Klinefelter Syndrome

Abstract Context. Osteocalcin (OCN) is an osteoblast-produced polypeptide, emerging as the core element of the bone-testicular axis toghether with its undercarboxylated form (uOCN), proposed to increase testosterone (Te) levels in healthy men, by binding the Gpcr6a receptor on Leydig cells and modulating the GnRH pulse frequency and amplitude. However little is known with regards to its role in pubertal development and male hypogonadism. Objective and Design We investigated OCN concentrations in 47,XXY men affected by Klinefelter syndrome (KS), a model of adult hypergonadotropic hypogonadism, in a retrospective longitudinal study between 2007 and 2021 at an academic referral center. Patients and Methods 254 KS subjects, divided into the following groups: 1) pre-pubertal (n = 48, from 1 year of age until Tanner stage II), 2) pubertal (n = 46, Tanner stages II through V, <18 years), and 3) adults (n = 160, Tanner stage V, ≥18 years). All (pre-)pubertal patients were Te-naïve. Adult patients were categorized as: 1) eugonadal (total Te > 10.4 nmol/L; n = 47), 2) hypogonadal (Te < 10.4 nmol/L; n = 39), and 3) receiving testosterone replacement therapy (TRT) (n = 74). Data are presented as means ± SD, were tested with Brown-Forsythe and Welch ANOVA tests for unequal variances, corrected for multiple comparisons (Dunnett T3), and with partial correlations, after bootstrapping on 2000 samples. Main outcomes. Total serum OCN, hypothalamic-pituitary-gonadal (HPG) axis hormones (LH, FSH, total Te, 11β-estradiol, SHBG), and derived indexes. Results OCN levels varied throughout the life span, with a mean of 85.9±30.4 ng/mL in pre-pubertal infants, peaking at 130. 0±77.2 ng/mL in pubertal children (p = 0.243 vs pre-pubertal) and then declining to 22.9±9. 0 ng/mL in adults (p < 0. 001 vs pre-pubertal and pubertal). In (pre-)pubertal boys no correlation with HPG axis hormones was found. When comparing adult KS, OCN values were highest in eugonadal (26.5±10.4 ng/mL), slightly lower in hypogonadal (24.5±8.1 ng/mL, p = 0.268 vs eugonadal) and significantly lower in TRT subjects (20.4±8. 0 ng/mL, p = 0. 008 vs. eugonadal and = 0. 013 vs. hypogonadal). In adults, OCN correlated with both LH (r = 0.23, p = 0. 017) and FSH levels (r = 0.28, p = 0. 004). These significancies were maintained after the exclusion of subjects on TRT. Surprisingly, adjusting for age and BMI revealed significant inverse correlations with total Te (r = -0.44, p = 0. 004), calculated free Te (r = -0.37, p = 0. 016), the Te/LH (r = -0.40, p = 0. 010) and the calculated free Te/LH ratios (r = -0.33, p = 0. 031). These results were confirmed on a smaller sample with available uOCN levels. Conclusions In an experimental model of hypergonadotropic hypogonadism, OCN showed no association with gonadal function during normal pre-puberty and pubertal development. In adults, OCN levels were unexpectedly associated with worse testicular function and a higher degree of HPG stimulation. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Central Regulation of PCOS: Abnormal Neuronal-Reproductive-Metabolic Circuits in PCOS Pathophysiology.

Polycystic ovary syndrome (PCOS) is a common reproductive endocrine disease. PCOS patients are characterized by hyperandrogenemia, anovulation, and metabolic dysfunction. Hypothalamus–pituitary–ovary axis imbalance is considered as an important pathophysiology underlying PCOS, indicating that central modulation, especially the abnormal activation of hypothalamic GnRH neurons plays a vital role in PCOS development. Increased GnRH pulse frequency can promote LH secretion, leading to ovarian dysfunction and abnormal sex steroids synthesis. By contrast, peripheral sex steroids can modulate the action of GnRH neurons through a feedback effect, which is impaired in PCOS, thus forming a vicious cycle. Additionally, hypothalamic GnRH neurons not only serve as the final output pathway of central control of reproductive axis, but also as the central connection point where reproductive function and metabolic state inter-regulate with each other. Metabolic factors, such as insulin resistance and obesity in PCOS patients can regulate GnRH neurons activity, and ultimately regulate reproductive function. Besides, gut hormones act on both brain and peripheral organs to modify metabolic state. Gut microbiota disturbance is also related to many metabolic diseases and has been reported to play an essential part in PCOS development. This review concludes with the mechanism of central modulation and the interaction between neuroendocrine factors and reproductive or metabolic disorders in PCOS development. Furthermore, the role of the gut microenvironment as an important part involved in the abnormal neuronal–reproductive–metabolic circuits that contribute to PCOS is discussed, thus offering possible central and peripheral therapeutic targets for PCOS patients.

Beyond GnRH, LH and FSH: The role of kisspeptin on hypothalalmic-pituitary gonadal (HPG) axis pathology and diagnostic consideration.

Kisspeptin as a neuropeptide was established initially as regulator for gonadotropin-releasing hormone for pulse frequency and intensity. In the current review, initial search on PubMed was done with key word "Kisspeptin" with further filters to include reviews and trials from the last 10 years. Of the 313 articles shortlisted, 160(51%) dealt with kisspeptin pathology and diagnostic evaluation in various physiological conditions like puberty, while 57(18.2%) dealt with pathological conditions like hypogonadism, 53(17%) infertility, and 43(13.7%) with polycystic ovarian syndrome. This review explored existing data regarding understanding of the negative and positive influences on the kisspeptin hormone-release kinetics. It highlighted the recently identified ligands and pathways which could affect the gonadal steroids, including various metabolic alterations and environmental triggers. Also, the review highlighted the kisspepetin/G-protein coupled receptor-54 interaction which were influenced by neighbouring endocannabinoid system, Gamma aminobutyric acid (GABA)-ergic neuronal outputs and other chemical agents. It was also highlighted that the release of kisspepetin was identified as a group of neurons termed kisspeptin, neurokinin B, and dynorphin, or KNDy, in the arcuate nuclei. The data indicated the use of kisspepetin as a diagnostic marker for precocious puberty, puberty confirmation, hypogonadism, infertility and polycystic ovarian syndrome.

Plasticity of Anterior Pituitary Gonadotrope Cells Facilitates the Pre-Ovulatory LH Surge.

Gonadotropes cells located in the anterior pituitary gland are critical for reproductive fitness. A rapid surge in the serum concentration of luteinizing hormone (LH) secreted by anterior pituitary gonadotropes is essential for stimulating ovulation and is thus required for a successful pregnancy. To meet the requirements to mount the LH surge, gonadotrope cells display plasticity at the cellular, molecular and morphological level. First, gonadotrope cells heighten their sensitivity to an increasing frequency of hypothalamic GnRH pulses by dynamically elevating the expression of the GnRH receptor (GnRHR). Following ligand binding, GnRH initiates highly organized intracellular signaling cascades that ultimately promote the synthesis of LH and the trafficking of LH vesicles to the cell periphery. Lastly, gonadotrope cells display morphological plasticity, where there is directed mobilization of cytoskeletal processes towards vascular elements to facilitate rapid LH secretion into peripheral circulation. This mini review discusses the functional and organizational plasticity in gonadotrope cells including changes in sensitivity to GnRH, composition of the GnRHR signaling platform within the plasma membrane, and changes in cellular morphology. Ultimately, multimodal plasticity changes elicited by gonadotropes are critical for the generation of the LH surge, which is required for ovulation.

Understanding the trade-off between the environment and fertility in cows and ewes.

The environment contributes to production diseases that in turn badly affect cow performance, fertility and culling. Oestrus intensity is lower in lame cows, and in all cows 26% potential oestrus events are not expressed (to avoid getting pregnant). To understand these trade-offs, we need to know how animals react to their environment and how the environment influences hypothalamus-pituitary-adrenal axis (HPA) interactions with the hypothalamus-pituitary-ovarian axis (HPO). Neurotransmitters control secretion of GnRH into hypophyseal portal blood. GnRH/LH pulse amplitude and frequency drive oestradiol production, culminating in oestrus behaviour and a precisely-timed GnRH/LH surge, all of which are disrupted by poor environments. Responses to peripheral neuronal agents give clues about mechanisms, but do these drugs alter perception of stimuli, or suppress consequent responses? In vitro studies confirm some neuronal interactions between the HPA and HPO; and immuno-histochemistry clarifies the location and sequence of inter-neurone activity within the brain. In both species, exogenous corticoids, ACTH and/or CRH act at the pituitary (reduce LH release by GnRH), and hypothalamus (lower GnRH pulse frequency and delay surge release). This requires inter-neurones as GnRH cells do not have receptors for HPA compounds. There are two (simultaneous, therefore fail-safe?) pathways for CRH suppression of GnRH release via CRH-Receptors: one being the regulation of kisspeptin/dynorphin and other cell types in the hypothalamus, and the other being the direct contact between CRH and GnRH cell terminals in the median eminence. When we domesticate animals, we must provide the best possible environment otherwise animals trade-off with lower production, less intense oestrus behaviour, and impaired fertility. Avoiding life-time peri-parturient problems by managing persistent lactations in cows may be a worthy trade-off on both welfare and economic terms – better than the camouflage use of drugs/hormones/feed additives/intricate technologies? In the long term, getting animals and environment in a more harmonious balance is the ultimate strategy.

Assessment of relationship between hormones and insulin resistance in PCOS

Introduction: Polycystic ovarian syndrome (PCOS) is the most common heterogenous disorder of reproductive age. Increased frequency of GnRH pulses from hypothalamus elevates LH levels this in turn causes increased androgen production. The chronic hyperandrogenic state have multiple long and short term complications which includes DM and CVD. Insulin resistance can be characterized as impaired action of insulin on glucose metabolism which increases risk of developing T2DM. Hyperandrogenism with hyperinsulinemia also leads to dyslipidemia. Aim: To estimate LH/FSH ratio, testosterone in PCOS patients and to correlate its significance with the insulin resistance. Materials and Methods: This case-control study was conducted on clinically, diagnosed 50 PCOS patients, aged 15 to 35 years were included as cases. Age-matched 50 apparently heal thy women were included as controls. Serum leutinising hormone(LH), Follicle stimulating hormone(FSH), testosterone, serum insulin were analysed by chemiluminiscence immunoassay(CLIA) on Maglumi 1000. Mindray BS 300, fully automated analyser was used for estimation of Total cholesterol(TC), High density lipoprotein(HDL), Triglyceride(TG), Fasting Blood Glucose (FBG), Low density lipoprotein (LDL) was calculated using Friedewald’s formula. Insulin resistance was assessed by HOMA IR. Descriptive statistics analysis was done using unpaired student’s t-test. Chi-square/ Fisher Exact test has been used to find the significance of study parameters on categorical scale. Pearson’s correlation coefficient was applied to analyse the correlation. p value Results: In our study the LH /FSH ratio, HOMA IR and Testosterone was increased in cases compared to controls with p 0.01 and for testosterone p 0.001. In contrast HDL decreased in cases compared to controls (p and Total cholesterol were not significantly increased in cases however we can find that LDL was still increased in cases. Average BMI was within the normal range (p=0.06). On correl

Hypothalamic Reproductive Endocrine Pulse Generator Activity Independent of Neurokinin B and Dynorphin Signaling.

Kisspeptin-neurokinin B (NKB)-dynorphin neurons are critical regulators of the hypothalamic-pituitary-gonadal axis. NKB and dynorphin are hypothesized to influence the frequency of GnRH pulses, whereas kisspeptin is hypothesized to be a generator of the GnRH pulse. How these neuropeptides interact remains unclear. To probe the role of NKB in GnRH pulse generation and to determine the interactions between NKB, kisspeptin, and dynorphin in humans and mice with a complete absence of NKB. Case/control. Academic medical center. Members of a consanguineous family bearing biallelic loss-of-function mutations in the gene encoding NKB and NKB-deficient mice. Frequent blood sampling to characterize neuroendocrine profile and administration of kisspeptin, GnRH, and naloxone, a nonspecific opioid receptor antagonist used to block dynorphin. LH pulse characteristics. Humans lacking NKB demonstrate slow LH pulse frequency, which can be increased by opioid antagonism. Mice lacking NKB also demonstrate impaired LH secretion, which can be augmented with an identical pharmacologic manipulation. Both mice and humans with NKB deficiency respond to exogenous kisspeptin. The preservation of LH pulses in the absence of NKB and dynorphin signaling suggests that both peptides are dispensable for GnRH pulse generation and kisspeptin responsiveness. However, NKB and dynorphin appear to have opposing roles in the modulation of GnRH pulse frequency.

SAT-412 Effects of the Ovary and Dihydrotestosterone on Firing Rate of GnRH Neurons in Prenatally Androgenized Mice

GnRH pulse frequency drives downstream reproductive function by signaling for differential release of gonadotropins, which subsequently control ovarian function. In patients with polycystic-ovary syndrome (PCOS), neuroendocrine defects, including persistent high-frequency LH (and presumably GnRH) pulses, are coupled with dysregulated ovarian function. Prenatally androgenized (PNA) animal models exhibit features similar to PCOS, including increased androgens and high-frequency LH pulses. Adult PNA mice exhibit these phenotypes and also have increased GnRH neuron firing rate and increased excitatory GABAergic transmission to GnRH neurons. In contrast to adults, GnRH neuron firing rate at 3 weeks of age is reduced in PNA females. The causes of this age-dependent difference in firing rate are not known, however puberty occurs between these ages and androgens can increase GnRH neuron firing rate in adulthood. Consistent with this, ovariectomy in adult PNA mice decreased firing rate to that in control diestrous mice. Here we tested the hypothesis that a lack of ovarian androgens mediated this decrease. We also investigated if ovariectomy changed firing rate in 3-week old PNA mice. PNA mice were born from dams injected daily on days 16-18 of gestation with 225µg dihydrotestosterone (DHT). Ovariectomy (OVX) or sham surgery was performed and at this time some mice received constant-release DHT implants that produce sub-male levels of androgen (400 µg/implant) 5-7 days before recordings (surgery at 14-16 days of age for prepubertal mice). Hour-long extracellular recordings were made to ascertain spontaneous firing rate of GFP-identified GnRH neurons in acutely-prepared brain slices. In adult OVX PNA mice, DHT increased firing rate of GnRH neurons relative to OVX PNA, but firing rate was still less than in sham-operated PNA animals (two-way ANOVA/Fisher’s LSD, n= 7-11 cells/group). In adult OVX control mice, preliminary data indicate that DHT replacement alone does not alter firing rate, in contrast to previous results with DHT plus estradiol. At three weeks of age, there was no difference in firing rate between PNA sham and OVX mice (unpaired two-tailed Student’s t-test, n=7-8 cells/group). These studies suggest that increased GnRH firing rate in adult PNA females is driven at least in part by ovarian androgens, whereas changes in firing rate in 3-week-old PNA females are driven by a different mechanism, such as prenatal programming, rather than ovarian factors.

GnRH Transactivates Human AMH Receptor Gene via Egr1 and FOXO1 in Gonadotrope Cells

Background/Objectives: Anti-Müllerian hormone (AMH) signaling is critical for sexual differentiation and gonadal function. AMH receptor type 2 (AMHR2) is expressed in extragonadal sites such as brain, and pituitary and emerging evidence indicates that AMH biological action is much broader than initially thought. We recently reported that AMH signaling enhances follicle-stimulating hormone synthesis in pituitary gonadotrope cells. However, mechanisms regulating AMHR2 expression in these extragonadal sites remain to be explored. Method/Results: Here, we demonstrated in perifused murine LβT2 gonadotrope cells that Amhr2 expression is differentially regulated by GnRH pulse frequency with an induction under high GnRH pulsatility. Furthermore, we showed that GnRH transactivates the human AMHR2 promoter in LβT2 cells. Successive deletions of the promoter revealed the importance of a short proximal region (–53/–37 bp) containing an Egr1 binding site. Using site-directed mutagenesis of Egr1 motif and siRNA mediated-knockdown of Egr1, we demonstrated that Egr1 mediates basal and GnRH-dependent activity of the promoter, identifying Egr1 as a new transcription factor controlling hAMHR2 expression. We also showed that SF1 and β-catenin are required for basal promoter activity and demonstrated that both factors contribute to the GnRH stimulatory effect, independently of their respective binding sites. Furthermore, using a constitutively active mutant of FOXO1, we identified FOXO1 as a negative regulator of basal and GnRH-dependent AMHR2 expression in gonadotrope cells. Conclusions: This study identifies GnRH as a regulator of human AMHR2 expression, further highlighting the importance of AMH signaling in the regulation of gonadotrope function.

Modeling the Male Reproductive Endocrine Axis: Potential Role for a Delay Mechanism in the Inhibitory Action of Gonadal Steroids on GnRH Pulse Frequency.

We developed a compartmental model so we could test mechanistic concepts in the control of the male reproductive endocrine axis. Using SAAM II computer software and a bank of experimental data from male sheep, we began by modeling GnRH-LH feed-forward and LH-T feedback. A key assumption was that the primary control signal comes from a hypothetical neural network (the PULSAR) that emits a digital (pulsatile) signal of variable frequency that drives GnRH secretion in square wave-like pulses. This model produced endocrine profiles that matched experimental observations for the testis-intact animal and for changes in GnRH pulse frequency after castration and T replacement. In the second stage of the model development, we introduced a delay in the negative feedback caused by the aromatization of T to estradiol at the brain level, a concept supported by empirical observations. The simulations showed how changes in the process of aromatization could affect the response of the pulsatile signal to inhibition by steroid feedback. The sensitivity of the PULSAR to estradiol was a critical factor, but the most striking observation was the effect of time delays. With longer delays, there was a reduction in the rate of aromatization and therefore a decrease in local estradiol concentrations, and the outcome was multiple-pulse events in the secretion of GnRH/LH, reflecting experimental observations. In conclusion, our model successfully emulates the GnRH-LH-T-GnRH loop, accommodates a pivotal role for central aromatization in negative feedback, and suggests that time delays in negative feedback are an important aspect of the control of GnRH pulse frequency.

GnRH Pulse Frequency Control of Fshb Gene Expression Is Mediated via ERK1/2 Regulation of ICER.

The pulsatile release of GnRH regulates the synthesis and secretion of pituitary FSH and LH. Two transcription factors, cAMP-response element-binding protein (CREB) and inducible cAMP early repressor (ICER), have been implicated in the regulation of rat Fshb gene expression. We previously showed that the protein kinase A pathway mediates GnRH-stimulated CREB activation. We hypothesized that CREB and ICER are activated by distinct signaling pathways in response to pulsatile GnRH to modulate Fshb gene expression, which is preferentially stimulated at low vs high pulse frequencies. In the LβT2 gonadotrope-derived cell line, GnRH stimulation increased ICER mRNA and protein. Blockade of ERK activation with mitogen-activated protein kinase kinase I/II (MEKI/II) inhibitors significantly attenuated GnRH induction of ICER mRNA and protein, whereas protein kinase C, calcium/calmodulin-dependent protein kinase II, and protein kinase A inhibitors had minimal effects. GnRH also stimulated ICER in primary mouse pituitary cultures, attenuated similarly by a MEKI/II inhibitor. In a perifusion paradigm, MEKI/II inhibition in LβT2 cells stimulated with pulsatile GnRH abrogated ICER induction at high GnRH pulse frequencies, with minimal effect at low frequencies. MEKI/II inhibition reduced GnRH stimulation of Fshb at high and low pulse frequencies, suggesting that the ERK pathway has additional effects on GnRH regulation of Fshb, beyond those mediated by ICER. Indeed, induction of the activating protein 1 proteins, cFos and cJun, positive modulators of Fshb transcription, by pulsatile GnRH was also abrogated by inhibition of the MEK/ERK signaling pathway. Collectively, these studies indicate that the signaling pathways mediating GnRH activation of CREB and ICER are distinct, contributing to the decoding of the pulsatile GnRH to regulate FSHβ expression.

The role of kisspeptin signalling in the hypothalamic-pituitary-gonadal axis--current perspective.

The discovery of kisspeptins in the recent past remoulded current understanding of the neuroendocrine axis relating to the regulation of human puberty and reproduction. Kisspeptins have been recognised to act upstream of GnRH and have been shown to play a vital role in the control of the hypothalamic-pituitary-gonadal axis via regulation of gonadotrophin secretion, onset of puberty, and control of fertility. KNDy (kisspeptin/neurokinin-B/dynorphin) neurons have been suggested to modulate GnRH pulsatile secretion, which is required to support reproductive function in both sexes. They have also been involved in mediating both positive and negative sex steroid feedback signals to GnRH neurons and serve as a vital connection between reproduction and metabolic status of the body. When kisspeptin is administered to healthy humans, and in patients with reproductive disorders, it strongly and directly stimulates GnRH and subsequent LH secretion and enhances LH pulse frequency. These observations suggest that kisspeptins are a potential novel therapeutic approach for treating disorders with either pathologically reduced or augmented gonadotrophins pulsatile secretion and is currently a focus of translational research. Kisspeptins have also been identified in several peripheral reproductive organs, indicating their role in modulation of ovarian function, embryo implantation, and placentation, but a great deal of work remains to be done to explore further in this regard, and the evidence is only available from studies done on animal models. In this review we will mainly focus on current available evidence related to the role of kisspeptins in controlling GnRH pulse frequency, specifically their role in puberty, fertility, and reproduction. We will also be appraising other factors that regulate the kiSS1/Kisspeptin/GPR-54 system.

Feedback and GnRH pulse frequency decoding: a mathematical model for GnRH signalling in gonadotrophs

Feedback and GnRH pulse frequency decoding: a mathematical model for GnRH signalling in gonadotrophs

Kisspeptin, Neurokinin B, and Dynorphin Act in the Arcuate Nucleus to Control Activity of the GnRH Pulse Generator in Ewes

Recent work has led to the hypothesis that kisspeptin/neurokinin B/dynorphin (KNDy) neurons in the arcuate nucleus play a key role in GnRH pulse generation, with kisspeptin driving GnRH release and neurokinin B (NKB) and dynorphin acting as start and stop signals, respectively. In this study, we tested this hypothesis by determining the actions, if any, of four neurotransmitters found in KNDy neurons (kisspeptin, NKB, dynorphin, and glutamate) on episodic LH secretion using local administration of agonists and antagonists to receptors for these transmitters in ovariectomized ewes. We also obtained evidence that GnRH-containing afferents contact KNDy neurons, so we tested the role of two components of these afferents: GnRH and orphanin-FQ. Microimplants of a Kiss1r antagonist briefly inhibited LH pulses and microinjections of 2 nmol of this antagonist produced a modest transitory decrease in LH pulse frequency. An antagonist to the NKB receptor also decreased LH pulse frequency, whereas NKB and an antagonist to the receptor for dynorphin both increased pulse frequency. In contrast, antagonists to GnRH receptors, orphanin-FQ receptors, and the N-methyl-D-aspartate glutamate receptor had no effect on episodic LH secretion. We thus conclude that the KNDy neuropeptides act in the arcuate nucleus to control episodic GnRH secretion in the ewe, but afferent input from GnRH neurons to this area does not. These data support the proposed roles for NKB and dynorphin within the KNDy neural network and raise the possibility that kisspeptin contributes to the control of GnRH pulse frequency in addition to its established role as an output signal from KNDy neurons that drives GnRH pulses.

How Much Kissing Is Required for Fertility?

The mammalian reproductive axis is regulated by neuroendocrine signaling pathways within the hypothalamus that control pulsatile secretion of GnRH into the hypophyseal portal system. GnRH stimulates gonadotropic hormone secretion from the anterior pituitary to activate pubertal maturation of the gonads and regulate fertility. Thus, substancesandphysiological inputs thataffectGnRHrelease provide important control mechanisms for regulation of the reproductive axis. Within the past decade, kisspeptins have beenidentifiedasakeyregulatorofGnRHrelease(forreview see Ref. 1). Kisspeptins are a family of overlapping amidated neuropeptides encoded by the Kiss1 gene that act as potent agonists of GnRH release by binding to their membrane receptor (kisspeptin receptor, KISS1R, also known as G-protein coupled receptor 54, GPR54) expressed by GnRH neurons (2). Kiss1 neurons reside within 2 regions of the hypothalamus; the arcuate (ARC) and the anteroventral periventricular (AVPV) nuclei. The Kiss1 neurons in the ARC are thought to regulate GnRH pulse frequency, whereas the AVPV neurons, which are sexually dimorphic with greater numbers in females, are required for inducing the preovulatory LH surge. Thecriticalrolethatkisspeptinsplayinmaintainingmammalian fertility was highlighted by the effect of loss of expression of these neuropeptides in transgenic mice (for review see Ref. 3). Disruption of the Kiss1 gene in mice results in failure of sexual maturation at puberty and infertility. The mutantmicehavehypogonadotropichypogonadism,similar to that found in patients with inactivating mutations of the kisspeptin receptor (4, 5). Mutant female mice do not show normal estrous cycling but have times of persistent estrus, probably reflecting their failure to ovulate. Consistent with this phenotype, the mutant female mice were incapable of eliciting an estradiol-induced LH surge required for ovulation (6). In contrast, however, female mice in which Kiss1 neurons were chronically ablated during development by cell-restricted expression of a diphtheria toxin entered puberty at the normal time and were fertile (7). This was a completely unexpected finding given that loss of Kiss1 neurons produced a less severe phenotype than loss of the kisspeptin protein and led to the controversial suggestion that kisspeptin signaling is not essential for female fertility. To account for these differences, it was suggested that compensatory changes occur during brain development that overcome the loss of the Kiss1 neurons. In support of this, it was shown that temporal ablation of Kiss1 neurons in 20-weekold adult females caused infertility, possibly because neuronalcompensationcouldnotoccur.Female infertilitywasalso found when Kiss1 neurons were ablated at postnatal day 20, suggesting that compensation occurs before this time point. The paper published in this issue of Endocrinology by Popa and colleagues (8) is an important contribution to this field because it provides data regarding the minimum amount of kisspeptin protein that is required to maintain fertility in mice. The authors report the reproductive phenotype of a transgenic mouse line that carries a hypomorphic mutation in the Kiss1 gene. These mice contain a CRE-GFP transgene inserted between the transcriptional start site and the coding region of the Kiss1 gene (9), which reduces expression of Kiss1 transcripts and greatly diminishes kisspeptin protein levels. Male mice with only a 5% level of Kiss1 transcripts are fertile and give rise to normal-sized litters. Female mice are also fertile but show a more severe effect than in males with reduced fertility rates and smaller litter sizescomparedwithwild-typemice.This sexuallydimorphic difference in the severity of the reproductive defect may indicate that male mice can cope with lower kisspeptin levels than females. It is likely that the mutant males will have a reduced sperm count because they have smaller testes and lowerFSHlevels thannormalbut that the spermnumbersdo

Editorial: Molecular Endocrinology Articles in the Spotlight for April 2013

We are pleased to feature the following articles in this month’s issue of Molecular Endocrinology. Interestingly, the 3 articles provide novel insights into various hypothalamic pathways that regulate important aspects of reproduction and metabolism. In “GnRH Pulse Frequency-Dependent Stimulation of FSH Transcription Is Mediated via Activation of PKA and CREB,” Thompson et al provide a mechanism of GnRH pulse frequency-dependent activation of FSH involving differential activation of protein kinase A (PKA) and subsequent differential levels of cAMP-responsive element-binding protein (CREB) phosphorylation. LH gene expression is unaffected by this pathway. Furthermore, GnRH preferentially stimulates CREB phosphorylation at slow pulse frequencies, whereas PKA (the main regulator of CREB phosphorylation in the murine LT2 gonadotrope cell model) activity increases to a greater extent at slow GnRH pulse frequencies, correlating with the increase in CREB phosphorylation. Because pulsatile GnRHregulatestheexpressionandsecretionofbothFSH and LH, this work identifies the intracellular mechanism downstream of GnRH receptor activation responsible for triggering the diverse reproductive consequences of the individual gonadotropins. “Nuclear Receptor LRH-1 Induces the Reproductive Neuropeptide Kisspeptin in the Hypothalamus” by Atkin et al (freely available at http://mend.endojournals.org/) provides novel insights into the molecular mechanism controlling kisspeptin’s differential secretion. The authors show that mice lacking LRH-1 in kisspeptin neurons have reduced plasma FSH levels and corresponding defects in follicle maturation and ovulation in the ovary. These knockout mice have a prolonged estrous cycle and deliver fewer pups per litter. On a molecular level, these findings help explain why kisspeptin is differentially regulatedindistinctregionsofthehypothalamus,thearcuate and anteroventral periventricular nuclei, and have revealed an important role for LRH-1 in regulating the female reproductive axis. Groba et al’s “Hypothyroidism Compromises Hypothalamic Leptin Signaling in Mice” reveals that leptin signaling in hypothalamic arcuate neurons is compromised under hypothyroid conditions. Activation of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathways by leptin is reduced in hypothyroid obese (ie, ob/ob) mice as evidenced by reduced phopho-STAT3 immunoreactivity and decreased SOCS3 expression. A diminished reduction in body weight and food intake in hypothyroid ob/ob mice was observed after leptin treatment. Importantly, hypothyroidism is associated with elevated transcript levels of shorter leptin receptor isoforms. Consequently, up-regulation of the shorter isoforms of the leptin receptor may represent a major mechanism by which hypothyroidism influences the leptin signaling pathway in hypothyroid mice.

GnRH Pulse Frequency-Dependent Stimulation of FSHβ Transcription Is Mediated via Activation of PKA and CREB

Expression of pituitary FSH and LH, under the control of pulsatile GnRH, is essential for fertility. cAMP response element-binding protein (CREB) has been implicated in the regulation of FSHβ gene expression, but the molecular mechanisms by which pulsatile GnRH regulates CREB activation remain poorly understood. We hypothesized that CREB is activated by a distinct signaling pathway in response to pulsatile GnRH in a frequency-dependent manner to dictate the FSHβ transcriptional response. GnRH stimulation of CREB phosphorylation (pCREB) in the gonadotrope-derived LβT2 cell line was attenuated by a protein kinase A (PKA) inhibitor, H89. A dominant negative PKA (DNPKA) reduced GnRH-stimulated pCREB and markedly decreased GnRH stimulation of FSHβ mRNA and FSHβLUC activity, but had little effect on LHβLUC activity, indicating relative specificity of this pathway. In perifusion studies, FSHβ mRNA levels and FSHβLUC activities were increased by pulsatile GnRH, with significantly greater increases at low compared with high pulse frequencies. DNPKA markedly reduced these GnRH-stimulated FSHβ responses at both low and high pulse frequencies. Correlating with FSHβ activation, both PKA activity and levels of pCREB were increased to a greater extent by low compared with high GnRH pulse frequencies, and the induction of pCREB was also attenuated by overexpression of DNPKA at both low and high pulse frequencies. Taken together, these data indicate that a PKA-mediated signaling pathway mediates GnRH activation of CREB at low-pulse frequencies, playing a significant role in the decoding of the hypothalamic GnRH signal to result in frequency-dependent FSHβ activation.