GnRH Action Research Articles

Genetic architecture of congenital hypogonadotropic hypogonadism: insights from analysis of a Portuguese cohort.

What is the contribution of genetic defects in Portuguese patients with congenital hypogonadotropic hypogonadism (CHH)? Approximately one-third of patients with CHH were found to have a genetic cause for their disorder, with causal pathogenic and likely pathogenic germline variants distributed among 10 different genes; cases of oligogenic inheritance were also included. CHH is a rare and genetically heterogeneous disorder characterized by deficient production, secretion, or action of GnRH, LH, and FSH, resulting in delayed or absent puberty, and infertility. Genetic screening was performed on a cohort of 81 Portuguese patients with CHH (36 with Kallmann syndrome and 45 with normosmic hypogonadotropic hypogonadism) and 263 unaffected controls. The genetic analysis was performed by whole-exome sequencing followed by the analysis of a virtual panel of 169 CHH-associated genes. The main outcome measures were non-synonymous rare sequence variants (population allele frequency <0.01) classified as pathogenic, likely pathogenic, and variants of uncertain significance (VUS). A genetic cause was identified in 29.6% of patients. Causal pathogenic and likely pathogenic variants were distributed among 10 of the analysed genes. The most frequently implicated genes were GNRHR, FGFR1, ANOS1, and CHD7. Oligogenicity for pathogenic and likely pathogenic variants was observed in 6.2% of patients. VUS and oligogenicity for VUS variants were observed in 85.2% and 54.3% of patients, respectively, but were not significantly different from that observed in controls. N/A. The identification of a large number of VUS presents challenges in interpretation and these may require reclassification as more evidence becomes available. Non-coding and copy number variants were not studied. Functional studies of the variants were not undertaken. This study highlights the genetic heterogeneity of CHH and identified several novel variants that expand the mutational spectrum of the disorder. A significant proportion of patients remained without a genetic diagnosis, suggesting the involvement of additional genetic, epigenetic, or environmental factors. The high frequency of VUS underscores the importance of cautious variant interpretation. These findings contribute to the understanding of the genetic architecture of CHH and emphasize the need for further studies to elucidate the underlying mechanisms and identify additional causes of CHH. This research was funded by the Portuguese Foundation for Science and Technology (grant numbers PTDC/SAU-GMG/098419/2008, UIDB/00709/2020, CEECINST/00016/2021/CP2828/CT0002, and 2020.04924.BD) and by Sidra Medicine-a member of the Qatar Foundation (grant number SDR400038). The authors declare no competing interests.

Molecular characterization of gonadotropin-releasing hormone receptor and its expression profiling in response to HCG stimulation in striped murrel, Channa striata (Bloch, 1793)

The gonadotropin releasing hormone receptors (GnRHR) play an important role in the activation of GnRH and elicit the release of gonadotropins that plays an important role in maturation and spawning. In the present work, molecular characterization of the GnRHR gene of snakehead murrel, Channa striata was carried out. The full-length cDNA of GnRH receptors of the 1581 bp sequence, which encodes 440 amino acids was identified by RACE-PCR. Signal peptide analysis confirmed that GnRHR is a non-secretory protein, and hydropathy profiles indicated its hydrophobic nature. The deduced amino acid sequences of the GnRHR showed the expected conserved domains, which include seven membrane-spanning domains, two potential N-linked glycosylation sites (631 Asp, 561 Asp), and 32 sites phosphorylation sites of serine and threonine residues. A homology search of GnRHR cDNA sequence revealed 88 % identity with Channa argus, followed by 85 % and 83 % sequence identities with Lates calcarifer, and Anabas testudineus at the protein level. Phylogenetic analysis depicted a close association with Channa argus and other fish species. Our findings showed differential GnRHR expression during different reproductive stages, with higher expression after HCG administration. Expression profiling revealed that GnRHR is highly expressed in the brain and testis at 16 h post-HCG injection, while it was neutrally regulated in female ovarian tissues. This study provides full-length coding sequence information of the GnRHR gene and will help to understand the reproductive biology of murrel using expression studies and devise strategies for induced breeding.

FRI277 Temporal Coordination Of Female Reproductive Axis Activity By Vasoactive Intestinal Polypeptide (VIP) Expressing Cells In The Suprachiasmatic Nucleus

Abstract Disclosure: C. Phumsatitpong: None. S.R. Bever: None. S.L. Hu: None. M.C. Ambrose: None. L.J. Kriegsfeld: None. Circadian rhythms orchestrated by a master clock in the suprachiasmatic nucleus (SCN) are essential for ovulation in spontaneously-ovulating rodents. SCN signaling integrates with estradiol positive feedback to generate the preovulatory luteinizing hormone (LH) surge. Additionally, subordinate clocks in reproductively-relevant SCN targets temporally gate responsiveness to upstream signaling to coordinate ovulation with the active time of day. We have previously shown that the SCN communicates directly to kisspeptin (Kp) and RFamide-related peptide-3 (RFRP-3) neurons to temporally balance key stimulatory and inhibitory input to GnRH neurons required for LH surge generation. Because VIP-expressing neurons in the SCN have been implicated in the LH surge, we employed a cell-specific chemogenetic approach to determine the functional role of this cell phenotype in female reproductive axis regulation required for surge generation. We hypothesized that temporal gating by subordinate cellular clocks will prevent morning stimulation of VIP-expressing cells from increasing Kp and GnRH, and decreasing RFRP-3, cellular activity. In the afternoon, we expected inhibition of VIP-expressing cells to decrease Kp and GnRH, and increase RFRP-3, cellular activity. Specifically, female VIP-Cre mice were stereotaxically injected with either AAV-hM3Dq or hM4Di to express activating (3Dq) or inhibitory (4Di) DREADDs in VIP-expressing SCN cells. Following recovery, mice were injected clozapine n-oxide (CNO) either in the morning, prior to the LH surge (3Dq experiment), or the afternoon, around the time of the LH surge (4Di experiment). Brains were collected to examine neuronal activation in Kp, GnRH, and RFRP-3 neurons using FOS immunofluorescence as a marker. Morning stimulation of SCN VIP neurons decreased RFRP-3 neuronal activation, whereas Kp and GnRH neurons were unaffected at this time, suggesting a temporal gating mechanism at GnRH and/or Kp cells prevents premature ovulation. In contrast, afternoon inhibition of SCN VIP-expressing neurons led to an increase in neuronal activation in RFRP-3 neurons concomitant with a decrease in GnRH and Kp neurons, suggesting that VIP stimulation is required to remove RFRP-3 inhibition on the GnRH and Kp systems. We confirmed this possibility by localizing afternoon injections of CNO to RFRP-3 cells, which led to comparable decreases in GnRH and Kp cellular activity. These data indicate the importance of SCN VIP neurons in the temporally coordinated disinhibition of RFRP-3 neuronal activity required for stimulation of GnRH and Kp neurons around the time of LH surge. Supported by NIH Grant HD-050470. Presentation: Friday, June 16, 2023

Use of oral GnRH antagonists combined therapy in the management of symptomatic uterine fibroids.

Uterine fibroids have an impact on women's lives due to their high prevalence, physical symptoms, their consequences on patients' emotional and psychological well-being and loss of work productivity. The choice of therapeutical approaches varies depending on several factors, and therefore should be applied individually. Currently, there is an unmet need for good, reliable, uterine-sparing options. The oral GnRH antagonists (Elagolix, Relugolix, Linzagolix) represent a new alternative for the medical management of hormone-dependent gynaecological diseases such as uterine fibroids or endometriosis. They rapidly bind to the GnRH receptor, block endogenous GnRH activity and directly suppress LH and FSH production, avoiding unwanted flare-up effects. Some GnRH antagonists are marketed in combination with hormone replacement therapy add-back to counteract hypo-oestrogenic side effects. According to the registration trials, once-daily GhRH antagonist combination therapy results in a significant reduction in menstrual bleeding, as compared with placebo, and preserves bone mineral density, for up to 104 weeks. Further studies in the long term are needed to evaluate the whole impact of medical treatment of uterine fibroids on the management of this common women's disease.

GnRH Neuron Excitability and Action Potential Properties Change with Development But Are Not Affected by Prenatal Androgen Exposure.

Gonadotropin-releasing hormone (GnRH) neurons produce the final output from the brain to control pituitary gonadotropin secretion and thus regulate reproduction. Disruptions to gonadotropin secretion contribute to infertility, including polycystic ovary syndrome (PCOS) and idiopathic hypogonadotropic hypogonadism. PCOS is the leading cause of infertility in women and symptoms resembling PCOS are observed in girls at or near the time of pubertal onset, suggesting that alterations to the system likely occurred by that developmental period. Prenatally androgenized (PNA) female mice recapitulate many of the neuroendocrine phenotypes observed in PCOS, including altered time of puberty, disrupted reproductive cycles, increased circulating levels of testosterone, and altered gonadotropin secretion patterns. We tested the hypotheses that the intrinsic properties of GnRH neurons change with puberty and with PNA treatment. Whole-cell current-clamp recordings were made from GnRH neurons in brain slices from control and PNA females before puberty at three weeks of age and in adulthood to measure GnRH neuron excitability and action potential (AP) properties. GnRH neurons from adult females were more excitable and required less current to initiate action potential firing compared with three-week-old females. Further, the afterhyperpolarization (AHP) potential of the first spike was larger and its peak was delayed in adulthood. These results indicate development, not PNA, is a primary driver of changes to GnRH neuron intrinsic properties and suggest there may be developmentally-induced changes to voltage-gated ion channels in GnRH neurons that alter how these cells respond to synaptic input.

Mutual Effects of Orexin and Bone Morphogenetic Proteins on Gonadotropin Expression by Mouse Gonadotrope Cells.

Orexin plays a key role in the regulation of sleep and wakefulness and in feeding behavior in the central nervous system, but its receptors are expressed in various peripheral tissues including endocrine tissues. In the present study, we elucidated the effects of orexin on pituitary gonadotropin regulation by focusing on the functional involvement of bone morphogenetic proteins (BMPs) and clock genes using mouse gonadotrope LβT2 cells that express orexin type 1 (OX1R) and type 2 (OX2R) receptors. Treatments with orexin A enhanced LHβ and FSHβ mRNA expression in a dose-dependent manner in the absence of GnRH, whereas orexin A in turn suppressed GnRH-induced gonadotropin expression in LβT2 cells. Orexin A downregulated GnRH receptor expression, while GnRH enhanced OX1R and OX2R mRNA expression. Treatments with orexin A as well as GnRH increased the mRNA levels of Bmal1 and Clock, which are oscillational regulators for gonadotropin expression. Of note, treatments with BMP-6 and -15 enhanced OX1R and OX2R mRNA expression with upregulation of clock gene expression. On the other hand, orexin A enhanced BMP receptor signaling of Smad1/5/9 phosphorylation through upregulation of ALK-2/BMPRII among the BMP receptors expressed in LβT2 cells. Collectively, the results indicate that orexin regulates gonadotropin expression via clock gene expression by mutually interacting with GnRH action and the pituitary BMP system in gonadotrope cells.

Leydig Cells in Immunocastrated Polish Landrace Pig Testis: Differentiation Status and Steroid Enzyme Expression Status.

Porker immunocastration against gonadoliberin (GnRH) secretion has been utilized since 2009; however, consumers are still skeptical of it. This is due to not having full information available on the problem of a boar taint, as well as a lack of research on morphological and molecular changes that may occur in the animal reproductive system and other body systems. The present study aimed to explore the functional status of steroidogenic Leydig cells of the testicular interstitial tissue in immunocastrated Polish Landrace pigs. Analyses were performed using Western blot, immunohistochemistry for relaxin (RLN), insulin-like 3 protein (INSL3), pelleted growth factor receptor α (PDGFRα), cytochrome P450scc, 3β- and 17β-hydroxysteroid dehydrogenases (3β-HSD, 17β-HSD), cytochrome P450arom, and 5α-reductase (5α-RED). Immunoassay ELISA was used to measure the androstenone, testosterone, and estradiol levels in the testis and serum of immunocastrates. We revealed disturbances in the distribution and expression of (i) RLN, indicating an inflammatory reaction in the interstitial tissue; (ii) INSL3 and PDGFRα, indicating alterations in the differentiation and function of fetal, perinatal, or adult Leydig cell populations; (iii) P450scc, 3β-HSD, 17β-HSD, P450arom, and 5α-RED, indicating disturbances in the sex steroid hormone production and disturbed functional status of Leydig cells; as well as (iv) decreased levels of androstenone, testosterone, and estradiol in testicular tissue and serum, indicating the dedicated action of Improvac to reduce boar taint at both the hypothalamic–hypophysis–gonadal axis and local level (Leydig cells). In summary, our study provides a significant portion of knowledge on the function of Leydig cells after immunocastration, which is also important for the diagnosis and therapy of testis dysfunction due to GnRH action failure and/or Leydig cell differentiational–functional alterations.

Adult Onset Isolated Hypogonadotropic Hypogonadism- a Cause of Secondary Amenorrhea

A 23-year-old African American female was referred for secondary amenorrhea evaluation. She attained menarche at 12 years and had regular menses. At 18 years, she used OCPs for few months, and used plan B, after which her menses stopped. She had hot flashes and sweating. She was placed on progesterone, but never had withdrawal bleeding. Review of systems was positive for intentional weight loss after her menses stopped, hair loss and nipple discharge. She denied any loss of sensation of smell. She has a PMH of asthma, anxiety and OSA. Family history was not significant for any fertility issues. She smoked cannabis after menses stopped. On physical examination, vitals were stable, BMI of 35 kg/meter2, well-developed secondary sexual characteristics, no thyromegaly, acne or hirsutism. Upon work up, CBC, CMP were normal, Urine pregnancy test was negative, gonadotropins were undetectable (FSH- <0.7mIU/ml, LH- <0.2mIU/ml), anti-mullerian hormone was 3.82ng/ml (WNL), Estradiol was also absent (<15pg/ml), with a low Total testosterone (11ng/dl), TSH was 1.17uIU/ml, Free T4 was 1.1ng/dl, ACTH was 9.58pg/ml, Cortisol was 14.8mcg/dl, and Prolactin was 1.5ng/ml. MRI brain was normal with normal pituitary gland, no focal lesion visualized. Pelvic ultrasound showed ovaries 5.9mL and 4.6mL with multiple follicles present bilaterally. Diagnosis of adult-onset isolated hypogonadotropic hypogonadism (IHH) was made. Patient was started on estradiol patches and progesterone. IHH is a genetic disorder of defective production or action of GnRH. IHH when associated with anosmia is called Kallmann syndrome. It was first described by German American geneticist Joseph Kallmann in 1944. GnRH is a decapeptide, produced in arcuate nucleus and pre-optic nucleus of hypothalamus. GnRH stimulates anterior pituitary to secrete FSH and LH. IHH is caused due to impaired migration of GnRH neurons to brain during embryogenesis. It is inherited as autosomal or X-linked dominant or recessive. Gene mutations associated are ANOS-1, FGFR, PROK-2. It is rare in females. IHH has a broad spectrum of clinical presentation from complete absence of sexual development to partial completion of puberty. It presents with microphallus, cryptorchidism, cleft lip/palate, syndactyly, renal aplasia. In childhood presents with anosmia, hearing deficits, dental agenesis, mirror movements, short stature. During puberty, absent pubertal growth spurt, amenorrhea, lack of virilization, no secondary sexual characteristics, infertility. In partial forms, known as Adult onset or acquired form of IHH, patients have slight testicular growth, thelarche, menarche. Goals of treatment are- pubertal induction, maintenance of sexual maturation and restoration of fertility. In females, pre-puberty only estrogen is given, after puberty both estrogen and progesterone are used, for fertility, pulsatile gonadotropins or GnRH analogues are used.

Mutation of the GnRHR Proximal Promoter AP-1 Element in Mice Results in Suboptimal GnRH Induction of LH and an Abnormal Reproductive Phenotype

Reproduction is regulated by the gonadotropins, LH and FSH, which are synthesized and secreted by pituitary gonadotrophs in response to hypothalamic GnRH in a pulse frequency dependent manner. The gonadotroph decodes GnRH pulsatility via the GnRH receptor (GnRHR), which increases in expression and cell surface density before estrus and is responsible for downstream signaling cascades that differentially favor gonadotropin expression. The gonadotroph Gnrhr promoter contains a tripartite enhancer, including an AP-1 element that is necessary for full GnRH induction of Gnrhr expression in vitro. We previously generated an AP-1 knock-in (KI) mouse model with a single point mutation (C-269T) in the Gnrhr promoter AP-1 binding motif that resulted in an abnormal reproductive phenotype in female mice. Compared to wildtype (WT) littermates, female KI mice had a significant delay in first estrus, disrupted estrous cyclicity, fewer corpora lutea, and smaller litters. Males had no apparent reproductive phenotype. Basal serum gonadotropin levels were similar between WT and KI mice, but gonadectomy induced a significantly lower rise in serum LH levels of KI mice relative to WT mice, concomitant with significantly lower pituitary Gnrhr, Lhb, and Fshb mRNA levels in both sexes. We have now extended the characterization of these mice by measuring LH pulsatility and assessing GnRH induction of LH in vivo and in vitro. The frequency and amplitude of LH pulses over three hours were similar in ovariectomized WT and KI mice; however, KI mice had significantly reduced LH secretion, as measured by area under the curve. Similarly, GnRH treatment induced a diminished LH response in intact KI compared to WT males. In vitro cultures of hemi-pituitaries from gonadectomized WT and KI males were exposed to 0.01 nM GnRH and LH secretion into culture media was measured by ELISA at 0, 0.5, 1, 2, and 4 hours. There was no difference in basal LH secretion between WT and KI pituitaries but GnRH induction of LH was significantly lower in cultures from AP-1 mutant mice, indicating a direct impairment of GnRH action at the level of the pituitary. Taken together, these data indicate that the gonadotroph Gnrhr AP-1 promoter motif is critical for normal reproductive function. Prevention of AP-1 binding to the Gnrhr proximal promoter element decreases GnRH-induced Gnrhr, Lhb, and Fshb levels, impairs GnRH-stimulated LH secretion, and disrupts pubertal development and reproductive cyclicity in female mice.

Gonadotropin-Releasing Hormone Receptors in Prostate Cancer: Molecular Aspects and Biological Functions.

Pituitary Gonadotropin-Releasing Hormone receptors (GnRH-R) mediate the activity of the hypothalamic decapeptide GnRH, thus playing a key role in the regulation of the reproductive axis. Early-stage prostate cancer (PCa) is dependent on serum androgen levels, and androgen-deprivation therapy (ADT), based on GnRH agonists and antagonists, represents the standard therapeutic approach for PCa patients. Unfortunately, the tumor often progresses towards the more aggressive castration-resistant prostate cancer (CRPC) stage. GnRH receptors are also expressed in CRPC tissues, where their binding to both GnRH agonists and antagonists is associated with significant antiproliferative/proapoptotic, antimetastatic and antiangiogenic effects, mediated by the Gαi/cAMP signaling cascade. GnRH agonists and antagonists are now considered as an effective therapeutic strategy for CRPC patients with many clinical trials demonstrating that the combined use of these drugs with standard therapies (i.e., docetaxel, enzalutamide, abiraterone) significantly improves disease-free survival. In this context, GnRH-based bioconjugates (cytotoxic drugs covalently linked to a GnRH-based decapeptide) have been recently developed. The rationale of this treatment is that the GnRH peptide selectively binds to its receptors, delivering the cytotoxic drug to CRPC cells while sparing nontumor cells. Some of these compounds have already entered clinical trials.

The Role of GnRH Receptor Autoantibodies in Polycystic Ovary Syndrome.

ObjectiveIs polycystic ovary syndrome (PCOS) associated with activating autoantibodies (AAb) to the second extracellular loop (ECL2) of gonadotropin-releasing hormone receptor (GnRHR)?Design and MethodsWe retrospectively screened sera from 40 patients with PCOS and 14 normal controls (NCs) with regular menses using enzyme-linked immunosorbent assay (ELISA) for the presence of GnRHR-ECL2-AAb. We obtained similar data from 40 non-PCOS ovulatory but infertile patients as a control group (OIC) of interest. We analyzed GnRHR-ECL2-AAb activity in purified immunoglobulin (Ig)G using a cell-based GnRHR bioassay.ResultsThe mean ELISA value in the PCOS group was markedly higher than the NC (P = .000036) and the OIC (P = .0028) groups. IgG from a sample of 5 PCOS subjects, in contrast to a sample of 5 OIC subjects, demonstrated a dose-dependent increase in GnRHR-stimulating activity qualitatively similar to the acute action of the natural ligand GnRH and the synthetic agonist leuprolide. The GnRHR antagonist cetrorelix significantly suppressed (P < .01) the elevated GnRHR activity induced by IgG from 7 PCOS patients while the IgG activity level from 7 OIC subjects was unchanged. Five other OIC subjects had relatively high ELISA values at or above the 95% confidence limits. On further study, 3 had normal or low activity while 2 had elevated IgG-induced GnRHR activity. One suppressed with cetrorelix while the other did not. The copresence of PCOS IgG increased the responsiveness to GnRH and shifted the dosage response curve to the left (P < .01).ConclusionsGnRHR-ECL2-AAb are significantly elevated in patients with PCOS compared with NCs. Their presence raises important etiological, diagnostic, and therapeutic implications.

SAT-298 Integrative Single-Cell Transcriptomic and Epigenomic Landscape of Mouse Anterior Pituitary Cell Types

The pituitary gland is a critical regulator of the neuroendocrine system. To further our understanding of the classification, cellular heterogeneity, and regulatory landscape of pituitary cell types, we performed and computationally integrated single cell (SC)/single nucleus (SN) resolution experiments capturing RNA expression, chromatin accessibility, and DNA methylation state from mouse dissociated whole pituitaries. Both SC and SN transcriptome analysis and promoter accessibility identified the five classical hormone-producing cell types (somatotropes, gonadotropes (GT), lactotropes, thyrotropes, and corticotropes). GT cells distinctively expressed transcripts for Cga, Fshb, Lhb, Nr5a1, and Gnrhr in SC RNA-seq and SN RNA-seq. This was matched in SN ATAC-seq with GTs specifically showing open chromatin at the promoter regions for the same genes. Similarly, the other classically defined anterior pituitary cells displayed transcript expression and chromatin accessibility patterns characteristic of their own cell type. This integrated analysis identified additional cell-types, such as a stem cell cluster expressing transcripts for Sox2, Sox9, Mia, and Rbpms, and a broadly accessible chromatin state. In addition, we performed bulk ATAC-seq in the LβT2b gonadotrope-like cell line. While the FSHB promoter region was closed in the cell line, we identified a region upstream of Fshb that became accessible by the synergistic actions of GnRH and activin A, and that corresponded to a conserved region identified by a polycystic ovary syndrome (PCOS) single nucleotide polymorphism (SNP). Although this locus appears closed in deep sequencing bulk ATAC-seq of dissociated mouse pituitary cells, SN ATAC-seq of the same preparation showed that this site was specifically open in mouse GT, but closed in 14 other pituitary cell type clusters. This discrepancy highlighted the detection limit of a bulk ATAC-seq experiment in a subpopulation, as GT represented ~5% of this dissociated anterior pituitary sample. These results identified this locus as a candidate for explaining the dual dependence of Fshb expression on GnRH and activin/TGFβ signaling, and potential new evidence for upstream regulation of Fshb. The pituitary epigenetic landscape provides a resource for improved cell type identification and for the investigation of the regulatory mechanisms driving cell-to-cell heterogeneity.Additional authors not listed due to abstract submission restrictions: N. Seenarine, M. Amper, N. Jain (ISMMS).

The interference of DEHP in precocious puberty of females mediated by the hypothalamic IGF-1/PI3K/Akt/mTOR signaling pathway

DEHP is reported to cause precocious puberty of females in both humans and rodents, but the underlying mechanisms were largely unknown. This study was designed to clarify the effects and the mechanisms of DEHP on the pathogenesis of sexual precocity. Prepubertal female rats were treated with DEHP for 4 weeks. Key organs were analyzed in control conditions and after exposure to 0.2, 1, and 5 mg/kg/day DEHP in pubertal female rats. To determine the role of the IGF-1/PI3K/Akt/mTOR signaling pathway in DEHP-induced female precocious puberty, 36 rats were treated with 5 mg/kg/day DEHP to establish a model of female precocious puberty. And we investigated the expression of genes and proteins related to IGF-1 pathway in rat hypothalamus after treatment with inhibitors. In the present study, we observed that DEHP treatment resulted in earlier vaginal opening time, higher number of Nissl bodies in the hypothalamus neurons, lower apoptosis of hypothalamic cells, higher IGF-1 and GnRH levels in the serum and hypothalamus. DEHP could also upregulated the expression of IGF-1/PI3K/Akt/mTOR pathway and GnRH in the hypothalamus of adolescent female rats, and inhibition of IGF-1R and mTOR in hypothalamus could block the activation of Kiss-1, GPR54, and GnRH by DEHP. In summary, our study suggested that DEHP might activate the hypothalamic GnRH neurons prematurely through the IGF-1 signaling pathway and promote GnRH release, leading to the initiation of female sexual development. Our results provide a new molecular mechanism underlying reproductive and developmental toxicity in pubertal female rats induced by DEHP.

Gonadoliberin – Synthesis, Secretion, Molecular Mechanisms and Targets of Action

Decapeptide gonadoliberin (GnRH) is the most important regulator of the hypothalamic-pituitary-gonadal (HPG) axis that controls the synthesis and secretion of the luteinizing and follicle-stimulating hormones by gonadotrophs in the adenohypophysis. GnRH is produced by the specialized hypothalamic neurons using the site-specific proteolysis of the precursor protein and is secreted into the portal pituitary system, where it binds to the specific receptors. These receptors belong to the family of G protein-coupled receptors, and they are located on the surface of gonadotrophs and mediate the regulatory effects of GnRH on the gonadotropins production. The result of GnRH binding to them is the activation of phospholipase C and the calcium-dependent pathways, the stimulation of different forms of mitogen-activated protein kinases, as well as the activation of the enzyme adenylyl cyclase and the triggering of cAMP-dependent signaling pathways in the gonadotrophs. The gonadotropins, kisspeptin, sex steroid hormones, insulin, melatonin and a number of transcription factors have an important role in the regulation of GnRH1 gene expression, which encodes the GnRH precursor, as well as the synthesis and secretion of GnRH. The functional activity of GnRH-producing neurons depends on their migration to the hypothalamic region at the early stages of ontogenesis, which is controlled by anosmin, ephrins, and lactosamine-rich surface glycoconjugate. Dysregulation of the migration of GnRH-producing neurons and the impaired production and secretion of GnRH, lead to hypogonadotropic hypogonadism and other dysfunctions of the reproductive system. This review is devoted to the current state of the problem of regulating the synthesis and secretion of GnRH, the mechanisms of migration of hypothalamic GnRH-producing neurons at the early stages of brain development, the functional activity of the GnRH-producing neurons in the adult hypothalamus and the molecular mechanisms of GnRH action on the pituitary gonadotrophs. New experimental data are analyzed, which significantly change the current understanding of the functioning of GnRH-producing neurons and the secretion of GnRH, which is very important for the development of effective approaches for correcting the functions of the HPG axis.

Negative feedback regulation of the secretion and actions of GnRH in male ruminants

The roles of testicular hormones in the negative feedback regulation of the secretion and actions of GnRH in male domestic ruminants are reviewed, concentrating mainly on research conducted with rams. Testicular steroids have major feedback actions directly at the hypothalamus to inhibit the secretion of GnRH, although it is apparent that, under certain circumstances, the steroids also have actions directly at the pituitary gland. Further research is necessary to delineate these actions and to determine the contribution of testosterone and its primary metabolites, dihydrotestosterone and oestradiol, to negative feedback on the hypothalamo-pituitary unit. Since GnRH neurones do not possess receptors for steroids, testicular steroids must evoke other neuronal pathways to influence GnRH producing neurones. While the opioids may be important in this regard, it is necessary to determine which other neuronal pathways may also be involved to understand fully the mechanism of action of testicular steroids. It appears that the feedback regulation of the secretion of LH can be accounted for by testicular steroids, whereas the secretion of FSH is influenced by inhibin and steroids, and possibly the recently isolated proteins follistatin and activin. The actions of inhibin to suppress the secretion of FSH occur at the pituitary gland and not on the synthesis or secretion of GnRH. There is a complex interaction between testosterone and inhibin in the control of FSH secretion that results in synergistic effects during the non-breeding season but not during the breeding season. Activin has been shown to have FSH-stimulating properties and follistatin has been shown to have FSH-inhibiting properties, but it is unknown if these proteins play a physiological role in the feedback regulation of FSH in male domestic ruminants.

Induction of Ovulation in Mangalarga Marchador Mares by hCG or GnRH

Background: Induction of ovulation is a key procedure for horse assisted reproduction technologies, such as for artificial insemination (AI) and embryo transfer. The application of hCG remains as the primary ovulation-inducing agent for horse assisted reproduction, but alternatives are in demand to avoid its adverse effects, such as loss of ovulation-inducing ef­ficiency over multiple applications by hCC-antibody production. Despite reports on alternative ovulation-inducing agents, pair-wise comparisons of such agents under similar conditions have been limited. Under such scenario, the work was aimed to determine the efficiency of both hCG and Buserelin at inducing ovulation in Mangalarga Marchador mares raised in the Northeast of Brazil under an AI program.Materials, Methods &amp; Results: Mares were initially selected based on their reproductive performance, the absence of clinical-reproductive alterations and adequate body condition score. Mares in diestrus were randomly distributed in three experimental conditions, received 5 mg of Dinoprost and were monitored daily for estrus detection. After estrus detection, ovaries were monitored by ultrasonography, in 12-h intervals, until the follicle reached 35 mm. At this time-point, ovula­tion was induced with 0.042 mg of Buserelin endovenously, with 3,000 IU hCG by an intramuscular shot, and control mares received 2 mL of saline solution, also by an intramuscular shot. Both hCG and Buserelin displayed similar efficien­cies (P &gt; 0.05) for induction of ovulation and that both agents were effective (P &lt; 0.05) for such purpose, since greater percentages (P &lt; 0.05) of induction on mares treated from those of the control. Moreover, the total number of ovulations in mares treated at the end of the experiment was not different (P &gt; 0.05) from those found in the Control. All ovulations occurred within a 72-h period after treatment. It can be observed that in mares treated with hCG or Buserelin, ovulations occurred both in more mares (P &lt; 0.05) and at earlier time-points than mares from the control. It is also possible to note that pregnancy was not different (P &gt; 0.05) between hCG and Buserelin groups, and that pregnancy of mares treated with ovulation-inducing factors was similar to the control.Discussion: The majority of ovulations in mares occurred within initial 48-h after treatment for both hCG and GnRH, suggesting a similar potential for horse assisted reproduction. Both hCG and Buserelin are two commonly used agents for induction of ovulation in mares. As described here, the majority of ovulations occurred within initial 48-h after treatment, a fact which can be attributed to hCG and GnRH activity, since it can happen in intervals from 36 to 48-h after treatment. Pregnancy rates did not differ among groups. These results are under the working hypothesis that hCG and Buserelin would display similar efficiencies on pregnancy rates. Despite the understanding of hCG activity on induction of ovulation due to its high specificity toward LH receptors and results from a direct effect on diminishing estradiol concentration, increasing LH, and further inducing ovulation within 48-h after treatment. In contrast, Buserelin has a similar functional property but acts upon LH synthesis and its release. In conclusion, ovulation in mares can be induced with both hCG and Buserelin, and both ovulation-inducing agents do not affect pregnancy rates.Keywords: gonadotropin, releasing factor, ovary, follicle, corpus luteum.

Induction of Ovulation in Mangalarga Marchador Mares by hCG or GnRH

Background: Induction of ovulation is a key procedure for horse assisted reproduction technologies, such as for artificial insemination (AI) and embryo transfer. The application of hCG remains as the primary ovulation-inducing agent for horse assisted reproduction, but alternatives are in demand to avoid its adverse effects, such as loss of ovulation-inducing efficiency over multiple applications by hCC-antibody production. Despite reports on alternative ovulation-inducing agents, pair-wise comparisons of such agents under similar conditions have been limited. Under such scenario, the work was aimed to determine the efficiency of both hCG and Buserelin at inducing ovulation in Mangalarga Marchador mares raised in the Northeast of Brazil under an AI program.Materials, Methods &amp; Results: Mares were initially selected based on their reproductive performance, the absence of clinical-reproductive alterations and adequate body condition score. Mares in diestrus were randomly distributed in three experimental conditions, received 5 mg of Dinoprost and were monitored daily for estrus detection. After estrus detection, ovaries were monitored by ultrasonography, in 12-h intervals, until the follicle reached 35 mm. At this time-point, ovulation was induced with 0.042 mg of Buserelin endovenously, with 3,000 IU hCG by an intramuscular shot, and control mares received 2 mL of saline solution, also by an intramuscular shot. Both hCG and Buserelin displayed similar efficiencies (P &gt; 0.05) for induction of ovulation and that both agents were effective (P &lt; 0.05) for such purpose, since greater percentages (P &lt; 0.05) of induction on mares treated from those of the control. Moreover, the total number of ovulations in mares treated at the end of the experiment was not different (P &gt; 0.05) from those found in the Control. All ovulations occurred within a 72-h period after treatment. It can be observed that in mares treated with hCG or Buserelin, ovulations occurred both in more mares (P &lt; 0.05) and at earlier time-points than mares from the control. It is also possible to note that pregnancy was not different (P &gt; 0.05) between hCG and Buserelin groups, and that pregnancy of mares treated with ovulation-inducing factors was similar to the control.Discussion: The majority of ovulations in mares occurred within initial 48-h after treatment for both hCG and GnRH, suggesting a similar potential for horse assisted reproduction. Both hCG and Buserelin are two commonly used agents for induction of ovulation in mares. As described here, the majority of ovulations occurred within initial 48-h after treatment, a fact which can be attributed to hCG and GnRH activity, since it can happen in intervals from 36 to 48-h after treatment. Pregnancy rates did not differ among groups. These results are under the working hypothesis that hCG and Buserelin would display similar efficiencies on pregnancy rates. Despite the understanding of hCG activity on induction of ovulation due to its high specificity toward LH receptors and results from a direct effect on diminishing estradiol concentration, increasing LH, and further inducing ovulation within 48-h after treatment. In contrast, Buserelin has a similar functional property but acts upon LH synthesis and its release. In conclusion, ovulation in mares can be induced with both hCG and Buserelin, and both ovulation-inducing agents do not affect pregnancy rates.

Apoptotic mechanism behind the testicular atrophy in photorefractory and scotosensitive quail: Involvement of GnIH induced p-53 dependent Bax-Caspase-3 mediated pathway

In most of the avian species, daylength or photoperiod is the main environmental factor regulating reproduction. During their annual gonadal cycle, birds once sensitive to short or long day effect develop refractoriness to the same daylength and gonad develop or regress accordingly. The present study investigated the effects of photoperiodic alterations on apoptosis mediated testicular responses of photosensitive/photorefractory and scotosensitive/scotorefractory quail, Coturnix coturnix japonica. Testicular apoptosis in the quail of different photoperiodic conditions was assessed by monitoring the alterations in the local testicular expression of GnRH-I, GnIH, pro-apoptotic proteins (p53 and Bax), inactive caspase (pro-Caspase-3), executioner active-Caspase-3 and inactive/uncleaved PARP-1 (DNA repair enzyme) and TUNEL analysis. Alterations in these parameters indicate that testicular quiescence/regression in scotosensitive and photorefractory quail is mediated by apoptosis of testicular cells and hence apoptosis appears to be the key mechanism of testicular regression in Japanese quail.Present findings demonstrated the underlying molecular mechanism of how avian testes respond differentially to same photoperiodic conditions and exhibit scoto-/photo-sensitivity and refractoriness. It is concluded that photoperiod induced testicular stimulation in photosensitive/scotorefractory quail may be due to apoptotic inhibition and testicular regression in scotosensitive/photorefractory quail is guided by apoptosis, an effect invariably regulated by local action of GnRH and GnIH.

The mystery of puberty initiation: genetics and epigenetics of idiopathic central precocious puberty (ICPP).

Puberty is a major developmental stage. Damaging mutations, considered as "mistakes of nature", have contributed to the unraveling of the networks implicated in the normal initiation of puberty. Genes involved in the abnormal hypothalamic-pituitary-gonadal (HPG) axis development, in the normosmic idiopathic hypogonadotropic hypogonadism (nIHH), in the X-linked or autosomal forms of Kallmann syndrome and in precocious puberty have been identified (GNRH1, GNRHR, KISS1, GPR54, FGFR1, FGF8, PROK2, PROKR2, TAC3, TACR3, KAL1, PROK2, PROKR2, CHD7, LEP, LEPR, PC1, DAX1, SF-1, HESX-1, LHX3, PROP-1). Most of them were found to play critical roles in HPG axis development and regulation, the embryonic GnRH neuronal migration and secretion, the regulation and action of the hypothalamic GnRH. However, the specific neural and molecular mechanisms triggering GnRH secretion remain one of the scientific enigmas. Although GnRH neurons are probably capable of autonomously generating oscillations, many gonadal steroid-dependent and -independent mechanisms have also been proposed. It is now well proven that the secretion of GnRH is regulated by kisspeptin as well as by permissive or opposing signals mediated by neurokinin B and dynorphin. These three supra-GnRH regulators compose the kisspeptin-neurokinin B-dynorphin neuronal (KNDy) system, a key player in pubertal onset and progression. Moreover, an ongoing increasing number of inhibitory, stimulatory and permissive networks acting upstream on GnRH neurons, such as GABA, NPY, LIN28B, MKRN3 and others integrate diverse hormonal and peripheral signals and have been proposed as the "gate-keepers" of puberty, while epigenetic modifications play also an important role in puberty initiation.

Expanding the genetic spectrum of ANOS1 mutations in patients with congenital hypogonadotropic hypogonadism.

What is the prevalence and functional consequence of ANOS1 (KAL1) mutations in a group of men with congenital hypogonadotropic hypogonadism (CHH)? Three of forty-two (7.1%) patients presented ANOS1 mutations, including a novel splice site mutation leading to exon skipping and a novel contiguous gene deletion associated with ichthyosis. CHH is characterized by lack of pubertal development and infertility, due to deficient production, secretion or action of GnRH, and can be associated with anosmia/hyposmia (Kallmann syndrome, KS) or with a normal sense of smell (normosmic CHH). Mutations in the anosmin-1 (ANOS1) gene are responsible for the X-linked recessive form of KS. This cross-sectional study included 42 unrelated men with CHH (20 with KS and 22 with normosmic CHH). Patients were screened for mutations in the ANOS1 gene by DNA sequencing. Identified mutations were further investigated by RT-PCR analysis and multiplex ligation-dependent probe amplification (MLPA) analysis. Hemizygous mutations were identified in three (7.1%) KS cases: a novel splice acceptor site mutation (c.542-1G>C), leading to skipping of exon 5 in the ANOS1 transcript in a patient with self-reported normosmia (but hyposmic upon testing); a recurrent nonsense mutation (c.571C>T, p.Arg191*); and a novel 4.8 Mb deletion involving ANOS1 and eight other genes (VCX3B, VCX2, PNPLA4, VCX, STS, HDHD1, VCX3A and NLGN4X) in KS associated with ichthyosis. Objective olfactory testing was not performed in all cases of self-reported normosmia and this may have underestimated the olfactory deficits. This study further expands the spectrum of known genetic defects associated with CHH and suggests that patients with self-reported normal olfactory function should not be excluded from ANOS1 genetic testing. This study was funded by the Portuguese Foundation for Science and Technology. The authors have no conflicts of interest. N/A.