Chronic estradiol exposure suppresses luteinizing hormone surge without affecting kisspeptin neurons and estrogen receptor alpha in anteroventral periventricular nucleus†.

Mammalian ovulation is induced by a luteinizing hormone surge, which is triggered by elevated plasma estrogen levels; however, chronic exposure to high levels of estradiol is known to inhibit luteinizing hormone secretion. In the present study, we hypothesized that the inhibition of the luteinizing hormone surge by chronic estradiol exposure is due to the downregulation of the estrogen receptor alpha in kisspeptin neurons at hypothalamic anteroventral periventricular nucleus, which is known as the gonadotropin-releasing hormone/luteinizing hormone surge generator. Animals exposed to estradiol for 2 days showed an luteinizing hormone surge, whereas those exposed for 14 days showed a significant suppression of luteinizing hormone. Chronic estradiol exposure did not affect the number of kisspeptin neurons and the percentage of kisspeptin neurons with estrogen receptor alpha or c-Fos in anteroventral periventricular nucleus, but it did affect the number of kisspeptin neurons in arcuate nucleus. Furthermore, chronic estradiol exposure did not affect gonadotropin-releasing hormone neurons. In the pituitary, 14-day estradiol exposure significantly reduced the expression of Lhb mRNA and LHß-immunoreactive areas. Gonadotropin-releasing hormone-induced luteinizing hormone release was also reduced significantly by 14-day estradiol exposure. We revealed that the suppression of an luteinizing hormone surge by chronic estradiol exposure was induced in association with the significant reduction in kisspeptin neurons in arcuate nucleus, luteinizing hormone expression in the pituitary, and pituitary responsiveness to gonadotropin-releasing hormone, and this was not caused by changes in the estrogen receptor alpha-expressing kisspeptin neurons in anteroventral periventricular nucleus and gonadotropin-releasing hormone neurons, which are responsible for estradiol positive feedback.

Gonadotropin Activity during Early Folliculogenesis and Implications for Polycystic Ovarian Syndrome and Premature Ovarian Insufficiency: A Narrative Review.

Female fertility depends on the ovarian reserve of follicles, which is determined at birth. Primordial follicle development and oocyte maturation are regulated by multiple factors and pathways and classified into gonadotropin-independent and gonadotropin-dependent phases, according to the response to gonadotropins. Folliculogenesis has always been considered to be gonadotropin-dependent only from the antral stage, but evidence from the literature highlights the role of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) during early folliculogenesis with a potential role in the progression of the pool of primordial follicles. Hormonal and molecular pathway alterations during the very earliest stages of folliculogenesis may be the root cause of anovulation in polycystic ovary syndrome (PCOS) and in PCOS-like phenotypes related to antiepileptic treatment. Excessive induction of primordial follicle activation can also lead to premature ovarian insufficiency (POI), a condition characterized by menopause in women before 40 years of age. Future treatments aiming to suppress initial recruitment or prevent the growth of resting follicles could help in prolonging female fertility, especially in women with PCOS or POI. This review will briefly introduce the impact of gonadotropins on early folliculogenesis. We will discuss the influence of LH on ovarian reserve and its potential role in PCOS and POI infertility.

Effect of basal luteinizing hormone (bLH) level on in vitro fertilization/intra-cytoplasmic injections (IVF/ICSI) outcomes in polycystic ovarian syndrome (PCOS) patients.

OBJECTIVE: To evaluate the effect of basal luteinizing hormone (bLH) levels on In Vitro Fertilization/Intra-Cytoplasmic Injections (IVF/ICSI) outcomes in polycystic ovary syndrome (PCOS). METHODS: A total of 256 PCOS patients who underwent IVF/ICSI treatment in our center from January 2018 to January 2022 were analyzed retrospectively. The patients were based on the third quartile (12.455) of the basal LH value was taken as the cut-off value and was divided into high and low LH group: high LH group (LH ≥ 12.455 IU / L) and low LH group (LH < 12.455 IU / L) and the OC group was pretreated with oral contraceptives. The outcomes in ovulation induction and embryo transfer cycles of the three groups were then compared. In addition, factors influencing the number of good quality embryos and the early onset LH peak were analyzed. RESULTS: Ages, infertility duration, body mass index (BMI), and basal follicle-stimulating hormone (FSH), and progesterone (P), testosterone (T) levels were not significantly different among the three groups (p > 0.05). However,there were significant differences in basal LH and basal E2 between low LH group and high LH group, and there were significant differences in basal LH between high LH group and OC group (p < 0.05). LH on the antagonist day was significantly different between low LH group and high LH group and between high LH group and OC group (p < 0.05). LH on the hCG (human Chorionic Gonadotropin) day there were significant differences between low LH group and OC group, high LH group and OC group (p < 0.05). The Mode of triggering between the three groups had significant differences between the two groups (p < 0.05). In addition, the number of days from gonadotropin (Gn) initiation to antagonist addition were significantly different among the three groups (p < 0.05). In addition, total Gn doses,the number of oocytes retrieved, the number of Gn days, 2pronucleus (2PN) numbers, number of good quality embryos, and number of high risk OHSS (Ovarian Hyper-stimulation Syndrome), cases with OHSS occurrences were not significantly different among the three groups (p > 0.05). Moreover, the cycle and clinical pregnancy outcomes and the cumulative clinical pregnancy rate and the cumulative live birth rate were not significantly different among the three groups (p > 0.05). LH levels on the day of antagonist addition affected the number of good-quality embryos (B < 0, p < 0.05). However, LH levels on the day antagonist was added were not significantly correlated with basal LH levels (Pearson correlation coefficient = 0.259), the ROC curve was constructed for the logistic prediction model of the early onset LH peak, and the AUC value was 0.747, indicating that the logistic combined model we constructed had a good ability to predict the early onset LH peak. CONCLUSION: Basal LH levels do not affect the pregnancy outcomes in PCOS patients after antagonist protocols. Besides, LH levels on the day of antagonist addition affect the number of good quality embryos for PCOS patients undergoing IVF /ICSI.

Using Quantitative Hormonal Fertility Monitors to Evaluate the Luteal Phase: Proof of Concept Case Study.

Several new quantitative fertility monitors are now available for at-home use that measure estrogen, luteinizing hormone (LH), and progesterone (PDG) in urine. This case report compares the Mira and Inito quantitative fertility monitors with the well-established qualitative ClearBlue fertility monitor. Three clinical scenarios were evaluated: a normal cycle, a prolonged luteinization cycle, and an anovulatory cycle. The identification of the luteal phase (or lack thereof in the case of anovulation) and the transition through the three processes of luteinization, progestation, and luteolysis were clearly demarcated with the help of quantitative LH and PDG. Quantitative fertility monitors have the potential to identify details of the luteal phase to help women with regular cycles and abnormal luteal phases to help target interventions for optimizing fertility.

Kobayashi Award 2019: The neuroendocrine regulation of the mammalian reproduction.

Mammalian reproductive function is a complex system of many players orchestrated by the hypothalamus-pituitary-gonadal (HPG) axis. The hypothalamic gonadotropin-releasing hormone (GnRH) and the consequent pituitary gonadotropin release show two modes of secretory patterns, namely the surge and pulse modes. The surge mode is triggered by the positive feedback action of estrogen secreted from the mature ovarian follicle to induce ovulation in females of most mammalian species. The pulse mode of GnRH release is required for stimulating tonic gonadotropin secretion to drive folliculogenesis, spermatogenesis and steroidogenesis and is negatively fine-tuned by the sex steroids. Accumulating evidence suggests that hypothalamic kisspeptin neurons are the master regulator for animal reproduction to govern the HPG axis. Specifically, kisspeptin neurons located in the anterior hypothalamus, such as the anteroventral periventricular nucleus (AVPV) in rodents and preoptic nucleus (POA) in ruminants, primates and others, and the neurons located in the arcuate nucleus (ARC) in posterior hypothalamus in most mammals are considered to play a key role in generating the surge and pulse modes of GnRH release, respectively. The present article focuses on the role of AVPV (or POA) kisspeptin neurons as a center for GnRH surge generation and of the ARC kisspeptin neurons as a center for GnRH pulse generation to mediate estrogen positive and negative feedback mechanisms, respectively, and discusses how the estrogen epigenetically regulates kisspeptin gene expression in these two populations of neurons. This article also provides the mechanism how malnutrition and lactation suppress GnRH/gonadotropin pulses through an inhibition of the ARC kisspeptin neurons. Further, the article discusses the programming effect of estrogen on kisspeptin neurons in the developmental brain to uncover the mechanism underlying the sex difference in GnRH/gonadotropin release as well as an irreversible infertility induced by supra-physiological estrogen exposure in rodent models.

Presence of GnRH3 in sturgeon and the roles of GnRH1 and GnRH2 on the regulation of LH/FSH in mature female sterlet Acipenser ruthenus in vitro.

Three forms of gonadotropin-releasing hormones (GnRHs), ArGnRH1, ArGnRH2, and ArGnRH3, were identified in sterlet. Compared with their orthologue, ArGnRH1 and ArGnRH2 have conserved core decapeptide but show low identity in the signal peptide and the rest of the sequences. The existence of the GnRH3 paralogue of sturgeon was predicted for the first time with TBLASTN by using the amino acid sequences of catshark and whale shark GnRH3 precursor as queries against the whole genome and transcript data of sterlet. The predicted ArGnRH3 cDNA sequence was composed of three exons containing all the elements of the GnRH family. The successful molecular cloning of GnRH3 from sterlets verified its expression in the brain of sturgeons. The analysis of the ArGnRH3 amino acid sequence revealed a completely conserved decapeptide sequence that shows 100% identity with the sequence of teleosts and differs in one amino acid with that of the cartilaginous fish (catshark and whale shark) at the 5th position. The structure of the phylogenetic tree showed that a total of 52 vertebrate GnRH sequences were clustered into three main clades corresponding to GnRH1, GnRH2, and GnRH3. The ArGnRH3 sequence is the oldest GnRH3 identified in teleosts. The tissue distribution analysis showed that ArGnRH1 was expressed in all the 13 examined tissues of females and in most of the tested tissues of male fish, with the highest expression in the pituitary and hypothalamus. ArGnRH2 is only expressed in the pituitary, hypothalamus, and gonads of both female and male sterlets. ArGnRH3 mRNA could be detected in the pituitary, hypothalamus, and gonad in both female and male fish. It is also present in the spleen, head kidney, and gill in female fish and in kidney and heart in male fish. However, the ArGnRH3 only showed weak expression in all the positive tissues. ArGnRH1 and ArGnRH2 active decapeptides were synthesized to investigate their roles on the regulation of LH/FSH using a mixed brain cell line from a sexually mature female sterlet. The results showed that ArGnRH1 and ArGnRH2 exerted different effects on the gene expression and release of gonadotropins. ArGnRH1 promoted the expression of fshß significantly around 48 h, and the expression was suppressed when the treatment time was extended to 72 h. ArGnRH1 had no significant effects on the level of either mRNA or secreted lh in any of the tested treatment length or concentrations. Moreover, ArGnRH1 did not stimulate the activity of gonadotropins in the maturation stage of female sturgeons. ArGnRH2 promoted the expression of fshß at 24 h and 48 h and increased mRNA level of lhß at 6 h and 48 h, accompanied by the significant secretion of LH at 72 h, although the high mRNA level of fsh did not correlate with the secretion of FSH in ArGnRH2-treated groups. In conclusion, ArGnRH2 plays an important role in the maturation stage of female sterlets. Therefore, ArGnRH2 has the potential to induce ovulation and spermiation in sturgeons.

Prediction, assessment, and management of suboptimal GnRH agonist trigger: a systematic review.

PURPOSE: This systematic review aimed to identify baseline patient demographic and controlled ovarian stimulation characteristics associated with a suboptimal response to GnRHa triggering, and available options for prevention and management of suboptimal response. METHODS: PubMed, Google Scholar, Medline, and the Cochrane Library were searched for keywords related to GnRHa triggering, and peer-reviewed articles from January 2000 to September 2021 included. RESULTS: Thirty-seven studies were included in the review. A suboptimal response to GnRHa triggering was more likely following long-term or recent oral contraceptive use and with a low or high body mass index. Low basal serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol serum levels were correlated with suboptimal oocyte yield, as was a low serum LH level on the day of triggering. A prolonged stimulation period and increased gonadotropin requirements were correlated with suboptimal response to triggering. Post-trigger LH < 15 IU/L best correlated with an increased risk for empty follicle syndrome and a lower oocyte retrieval rate. Retriggering with hCG may be considered in patients with suboptimal response according to post-trigger LH, as in cases of failed aspiration. CONCLUSION: Pre-treatment assessment of patient characteristics, with pre- and post-triggering assessment of clinical and endocrine cycle characteristics, may identify cases at risk for suboptimal response to GnRHa triggering and optimize its utilization.

Localization of kisspeptin, NKB, and NK3R in the hypothalamus of gilts treated with the progestin altrenogest.

Mechanisms in the brain controlling secretion of gonadotropin hormones in pigs, particularly luteinizing hormone (LH), are poorly understood. Kisspeptin is a potent LH stimulant that is essential for fertility in many species, including pigs. Neurokinin B (NKB) acting through neurokinin 3 receptor (NK3R) is involved in kisspeptin-stimulated LH release, but organization of NKB and NK3R within the porcine hypothalamus is unknown. Hypothalamic tissue from ovariectomized (OVX) gilts was used to determine the distribution of immunoreactive kisspeptin, NKB, and NK3R cells in the arcuate nucleus (ARC). Almost all kisspeptin neurons coexpressed NKB in the porcine ARC. Immunostaining for NK3R was distributed throughout the preoptic area (POA) and in several hypothalamic areas including the periventricular and retrochiasmatic areas but was not detected within the ARC. There was no colocalization of NK3R with gonadotropin-releasing hormone (GnRH), but NK3R-positive fibers in the POA were in close apposition to GnRH neurons. Treating OVX gilts with the progestin altrenogest decreased LH pulse frequency and reduced mean circulating concentrations of LH compared with OVX control gilts (P < 0.01), but the number of kisspeptin and NKB cells in the ARC did not differ between treatments. The neuroanatomical arrangement of kisspeptin, NKB, and NK3R within the porcine hypothalamus confirms they are positioned to stimulate GnRH and LH secretion in gilts, though differences with other species exist. Altrenogest suppression of LH secretion in the OVX gilt does not appear to involve decreased peptide expression of kisspeptin or NKB.

A mechanistic evaluation of the potential for octamethylcyclotetrasiloxane to produce effects via endocrine modes of action.

This review is a hypothesis driven, mechanistic evaluation of the potential for octamethylcyclotetrasiloxane (D4) to produce any effects via endocrine modes of action. D4 is a volatile, lipophilic liquid used in the production of high molecular weight dimethylsiloxane polymers. These are used in a variety of industrial, medical, cleaning, and personal care products, and they may contain low levels of residual D4. Low concentrations of D4 are found in the environment and there is potential for low level human exposure. All of the measured environmental and workplace levels of D4 fall below no observed effect levels (NOEL). Most of the effects of high dose D4 involve the female reproductive system. In the mature intact female rat following chronic high dose exposure, D4 may cause inhibition of mating and ovulation, decreased live litter sizes, small increases in the estrogen to progesterone ratio primarily through decreases in progesterone, and increases in uterine hyperplasia. When endogenous estrogens are very low, high dose D4 causes increases in some uterine parameters. To assess whether these high dose effects can be attributed to an endocrine mode of action, endpoints are ranked for relevance and strength, consistent with published concepts. When sufficient information is available the level of activity of D4 for producing the observed effect is compared with that of potent endocrines. The conclusions reached are that all of the effects of D4 fall well short of any established criteria for D4 to be capable of producing any adverse effect via an endocrine mode of action.

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.

Effect of neuronal nitric oxide synthase serine-1412 phosphorylation on hypothalamic-pituitary-ovarian function and leptin response.

Hypothalamic neuronal nitric oxide synthase (nNOS) potentiates adult female fertility in rodents by stimulating gonadotropin releasing hormone (GnRH) secretion, which in turn promotes luteinizing hormone (LH) release and ovulation. The mechanism of hypothalamic nNOS activation is not clear but could be via nNOS serine1412 (S1412) phosphorylation, which increases nNOS activity and physiologic NO effects in other organ systems. In female rodents, hypothalamic nNOS S1412 phosphorylation reportedly increases during proestrus or upon acute leptin exposure during diestrus. To determine if nNOS S1412 regulates female reproduction in mice, we compared the reproductive anatomy, estrous cycle duration and phase proportion, and fecundity of wild-type and nNOS serine1412➔alanine (nNOSS1412A) knock-in female mice. We also measured hypothalamic GnRH and serum LH, follicle stimulating hormone (FSH), estradiol, and progesterone in diestrus mice after intraperitoneal leptin injection. Organ weights and histology were not different by genotype. Ovarian primordial follicles, antral follicles, and corpora lutea were similar for wild-type and nNOSS1412A mice. Likewise, estrous cycle duration and phase length were not different, and fecundity was unremarkable. There were no differences among genotypes for LH, FSH, estradiol, or progesterone. In contrast to prior studies, our work suggests that nNOS S1412 phosphorylation is dispensable for normal hypothalamic-pituitary-ovarian function and regular estrous cycling. These findings have important implications for current models of fertility regulation by nNOS phosphorylation.

Role of anti-Müllerian hormone and testosterone in follicular growth: a cross-sectional study.

BACKGROUND: Anti-Müllerian hormone (AMH) is now considered the best serum biomarker of ovarian reserve, while basal sex hormones are classic markers used for assessing ovarian reserve. The interaction between AMH and sex hormones are complicated and not sufficiently addressed. In this study, we took diminished ovarian reserve (DOR) and polycystic ovarian syndrome (PCOS) as two extremes of ovarian reserve (deficient and excessive respectively) to investigate the role of AMH and sex hormones in follicular growth. METHODS: A retrospective cross-sectional survey was performed. The patients assessed AMH and basal sex hormones in the Second Hospital of Zhejiang University from April 2016 to March 2019 were involved in this study. Serum AMH and sex hormone concentrations were tested with electrochemiluminescence method. Stepwise linear regression and binary logistic regression was used to determine the predictors of AMH level and to explore the involved factors determining DOR and PCOS. RESULTS: In the present study, we found that age and follicle-stimulating hormone (FSH) were main negative correlation factors, and luteinizing hormone (LH) and testosterone (T) were main positive factors of AMH. In DOR group, age, FSH and estradiol (E2) increased and T decreased, while in PCOS group, LH and T increased. Binary logistic regression found that age, weight, FSH, E2, and T were the significant factors which independently predicted the likelihood of DOR, and that age, body mass index (BMI), AMH, LH, and T predicted the likelihood of PCOS. CONCLUSIONS: Our study demonstrated that age, FSH, and T were factors that most closely correlated with AMH level, and T was involved in both DOR and PCOS. Since DOR and PCOS are manifested with insufficient AMH and excessive AMH respectively, it is suggested that total testosterone correlated with AMH closely and plays an important role in follicular growth. More attention should be given to testosterone level during controlled ovarian hyperstimulation (COH) process.

AMP-activated protein kinase activation reduces the transcriptional activity of the murine luteinizing hormone ß-subunit gene.

Malnutrition is one of the factors that induces reproductive disorders. However, the underlying biological processes are unclear. AMP-activated protein kinase (AMPK) is an enzyme that plays crucial role as a cellular energy sensor. In the present study, we examined the effects of AMPK activation on the transcription of the murine gonadotropin subunit genes Cga, Lhb, and Fshb, and the gonadotropin-releasing hormone receptor Gnrh-r. Real-time PCR and transcription assay using LßT2 cells demonstrated that 5-amino-imidazole carboxamide riboside (AICAR), a cell-permeable AMP analog, repressed the expression of Lhb. Next, we examined deletion mutants of the upstream region of Lhb and found that the upstream regulatory region of Lhb (-2527 to -2198 b) was responsible for the repression by AICAR. Furthermore, putative transcription factors (SP1, STAT5a, and TEF) that might mediate transcriptional control of the Lhb repression induced by AICAR were identified. In addition, it was confirmed that both AICAR and a competitive inhibitor of glucose metabolism, 2-deoxy-D-glucose, induced AMPK phosphorylation in LßT2 cells. Therefore, the upstream region of Lhb is one of the target sites for glucoprivation inducing AMPK activation. In addition, AMPK plays a role in repressing Lhb expression through the distal -2527 to -2198 b region.

Short-term but not long-term high-fat diet induces an increase in gene expression of gonadotropic hormones and GPR120 in the male mouse pituitary gland.

High-fat diet (HFD) is associated with the regulation of reproductive functions. This study aimed to investigate the effects of short-term HFD on the mRNA expression levels of follicle-stimulating hormone ß subunit (FSHß), luteinizing hormone ß subunit (LHß), gonadotropin-releasing hormone receptor, and long-chain fatty acid receptor, GPR120, in the matured male mouse pituitary gland. Adult male mice were fed either control chow or HFD for 1, 2, 5, 10, 30 and 150 days. Fshb and Gpr120 mRNA expression levels in the pituitary glands were significantly increased during 2 to 30 days of HFD feeding. Gnrh-r mRNA in the 30 days HFD fed group and body weight in the 30 and 150 days HFD fed groups were higher than control. However, there were no significant differences in plasma non-esterified fatty acids or glucose levels during the 150 days of HFD feeding. These results suggest that male mice feeding a short-term HFD induces FSHß synthesis and GPR120 expression in their pituitary gonadotropes.

Using Aptamers as a Novel Method for Determining GnRH/LH Pulsatility.

Aptamers are a novel technology enabling the continuous measurement of analytes in blood and other body compartments, without the need for repeated sampling and the associated reagent costs of traditional antibody-based methodologies. Aptamers are short single-stranded synthetic RNA or DNA that recognise and bind to specific targets. The conformational changes that can occur upon aptamer-ligand binding are transformed into chemical, fluorescent, colour changes and other readouts. Aptamers have been developed to detect and measure a variety of targets in vitro and in vivo. Gonadotropin-releasing hormone (GnRH) is a pulsatile hypothalamic hormone that is essential for normal fertility but difficult to measure in the peripheral circulation. However, pulsatile GnRH release results in pulsatile luteinizing hormone (LH) release from the pituitary gland. As such, LH pulsatility is the clinical gold standard method to determine GnRH pulsatility in humans. Aptamers have recently been shown to successfully bind to and measure GnRH and LH, and this review will focus on this specific area. However, due to the adaptability of aptamers, and their suitability for incorporation into portable devices, aptamer-based technology is likely to be used more widely in the future.

Identification of Key Receptor Residues Discriminating Human Chorionic Gonadotropin (hCG)- and Luteinizing Hormone (LH)-Specific Signaling.

(1) The human luteinizing hormone (LH)/chorionic gonadotropin (hCG) receptor (LHCGR) discriminates its two hormone ligands and differs from the murine receptor (Lhr) in amino acid residues potentially involved in qualitative discerning of LH and hCG. The latter gonadotropin is absent in rodents. The aim of the study is to identify LHCGR residues involved in hCG/LH discrimination. (2) Eight LHCGR cDNAs were developed, carrying "murinizing" mutations on aminoacidic residues assumed to interact specifically with LH, hCG, or both. HEK293 cells expressing a mutant or the wild type receptor were treated with LH or hCG and the kinetics of cyclic adenosine monophosphate (cAMP) and phosphorylated extracellular signal-regulated kinases 1/2 (pERK1/2) activation was analyzed by bioluminescence resonance energy transfer (BRET). (3) Mutations falling within the receptor leucine reach repeat 9 and 10 (LRR9 and LRR10; K225S +T226I and R247T), of the large extracellular binding domain, are linked to loss of hormone-specific induced cAMP increase, as well as hCG-specific pERK1/2 activation, leading to a Lhr-like modulation of the LHCGR-mediated intracellular signaling pattern. These results support the hypothesis that LHCGR LRR domain is the interaction site of the hormone ß-L2 loop, which differs between LH and hCG, and might be fundamental for inducing gonadotropin-specific signals. (4) Taken together, these data identify LHCGR key residues likely evolved in the human to discriminate LH/hCG specific binding.

Molecular function of gonadotrophins and their receptors in the ovarian development of turbot (Scophthalmus maximus).

Gonadotropins (GtHs) and their receptors (follicle-stimulating hormone receptor, FSHR; luteinizing hormone receptor, LHR) are involved in the regulation of gametogenesis and play important roles during the reproductive cycles in vertebrate species, including fish. This minireview focuses on the molecular characterization and quantification of GtHs (common glycoprotein α subunit CGα, FSHß, and LHß) and their receptors (FSHR and LHR) throughout the reproductive cycle of female turbot Scophthalmus maximus. Information about GtHs, FSHR, LHR as well as other ligand-receptors interaction from different teleosts are also included in this review for the implications they may have on the functions of GtHs, FSHR and LHR in the reproductive development of turbot. These findings may enhance our understanding of the physiological roles of the GtHs, FSHR and LHR in controlling of flatfish ovarian development during the reproductive cycle and contributing to the improvement of management strategies for turbots in captivity.

Short communication: Sensitivity of estrus alerts and relationship with timing of the luteinizing hormone surge.

Both the sensitivity of an estrus detection system and the consistency of alarms relative to ovulation determine its value for a farmer. The objective of this study was to compare an activity-based system and a milk progesterone-based system for their ability to detect estrus reliably, and to investigate how their alerts are linked to the time of the LH surge preceding ovulation. The study was conducted on an experimental research farm in Flanders, Belgium. The activity alerts were generated by a commercial activity meter (ActoFIT, DeLaval, Tumba, Sweden), and milk progesterone was measured using a commercial ELISA kit. Sensitivity and positive predictive value of both systems were calculated based on 35 estrus periods over 43 d. Blood samples were taken for determination of the LH surge, and the intervals between timing of the alerts and the LH surge were investigated based on their range and standard deviation (SD). Activity alerts had a sensitivity of 80% and a positive predictive value of 65.9%. Alerts were detected from 39 h before until 8 h after the LH surge (range: 47 h, SD: 16 h). Alerts based on milk progesterone were obtained from a recently developed monitoring algorithm using a mathematical model and synergistic control. All estruses were correctly identified by this algorithm, and the LH surge followed, on average, 62 h later. Using the mathematical model, model-based indicators for the estimation of ovulation time can be calculated. Depending on which model-based indicator was used, ranges of 33 to 35 h and SD of about 11 h were obtained. Because detection of the LH surge was very labor intensive, only a limited number of potential estrus periods could be studied.

FSH/LH-Dependent Upregulation of Ahr in Murine Granulosa Cells Is Controlled by PKA Signaling and Involves Epigenetic Regulation.

The aryl hydrocarbon receptor (Ahr) is a ligand-activated transcription factor primarily known for its toxicological functions. Recent studies have established its importance in many physiological processes including female reproduction, although there is limited data about the precise mechanisms how Ahr itself is regulated during ovarian follicle maturation. This study describes the expression of Ahr in ovarian granulosa cells (GCs) of immature mice in a gonadotropin-dependent manner. We show that Ahr upregulation in vivo requires both follicle stimulating hormone (FSH) and luteinizing hormone (LH) activities. FSH alone increased Ahr mRNA, but had no effect on Ahr protein level, implicating a possible LH-dependent post-transcriptional regulation. Also, the increase in Ahr protein is specific to large antral follicles in induced follicle maturation. We show that Ahr expression in GCs of mid-phase follicular maturation is downregulated by protein kinase A (PKA) signaling and activation of Ahr promoter is regulated by chromatin remodeling.

GnRH Antagonists Produce Differential Modulation of the Signaling Pathways Mediated by GnRH Receptors.

Commercial gonadotropin-releasing hormone (GnRH) antagonists differ by 1-2 amino acids and are used to inhibit gonadotropin production during assisted reproduction technologies (ART). In this study, potencies of three GnRH antagonists, Cetrorelix, Ganirelix and Teverelix, in inhibiting GnRH-mediated intracellular signaling, were compared in vitro. GnRH receptor (GnRHR)-transfected HEK293 and neuroblastoma-derived SH-SY5Y cell lines, as well as mouse pituitary LßT2 cells endogenously expressing the murine GnRHR, were treated with GnRH in the presence or absence of the antagonist. We evaluated intracellular calcium (Ca2+) and cAMP increases, cAMP-responsive element binding-protein (CREB) and extracellular-regulated kinase 1 and 2 (ERK1/2) phosphorylation, ß-catenin activation and mouse luteinizing-hormone ß-encoding gene (Lhb) transcription by bioluminescence resonance energy transfer (BRET), Western blotting, immunostaining and real-time PCR as appropriate. The kinetics of GnRH-induced Ca2+ rapid increase revealed dose-response accumulation with potency (EC50) of 23 nM in transfected HEK293 cells, transfected SH-SY5Y and LßT2 cells. Cetrorelix inhibited the 3 × EC50 GnRH-activated calcium signaling at concentrations of 1 nM-1 µM, demonstrating higher potency than Ganirelix and Teverelix, whose inhibitory doses fell within the 100 nM-1 µM range in both transfected HEK293 and SH-SY5Y cells in vitro. In transfected SH-SY5Y, Cetrorelix was also significantly more potent than other antagonists in reducing GnRH-mediated cAMP accumulation. All antagonists inhibited pERK1/2 and pCREB activation at similar doses, in LßT2 and transfected HEK293 cells treated with 100 nM GnRH. Although immunostainings suggested that Teverelix could be less effective than Cetrorelix and Ganirelix in inhibiting 1 µM GnRH-induced ß-catenin activation, Lhb gene expression increase occurring upon LßT2 cell treatment by 1 µM GnRH was similarly inhibited by all antagonists. To conclude, this study has demonstrated Cetrorelix-, Ganirelix- and Teverelix-specific biased effects at the intracellular level, not affecting the efficacy of antagonists in inhibiting Lhb gene transcription.