GnRH Receptor Research Articles

Gonadotropin releasing hormone - gonadotropes interactions revealed by pituitary single-cell transcriptomics in zebrafish.

Gonadotropin-releasing hormone (GnRH) governs reproduction by regulating pituitary gonadotropins. Unlike most vertebrates, gnrh-/- zebrafish are fertile. To elucidate the role of the hypophysiotropic-Gnrh3 and other mechanisms regulating pituitary gonadotropes, we profiled the gene expression of all individual pituitary cells of wild-type and gnrh3-/- adult female zebrafish. The single-cell RNA-Seq showed that Lh and Fsh gonadotropes express the two gonadotropin beta subunits with a ratio of 140:1 (lhb:fshb) and 4:1 (fshb:lhb), respectively. Lh gonadotropes predominantly express genes encoding receptors for Gnrh (gnrhr2), thyroid hormone, estrogen, and steroidogenic factor 1 (SF1). No Gnrh receptor transcript was enriched in Fsh gonadotropes. Instead, cholecystokinin receptor-b and galanin receptor-1b transcripts were enriched in these cells. The loss of Gnrh3 gene in gnrh3-/- zebrafish resulted in downregulation of fshb in Lh gonadotropes and upregulation of pituitary hormones like thyroid-stimulating hormone, growth hormone, prolactin and proopiomelanocortin-a. Likewise, targeted chemogenetic ablation of Gnrh3 neurons led to a decrease in the number of fshb+, lhb+ and fshb+/lhb+ cells. Our studies suggest that Gnrh3 directly acts on Lh gonadotropes through Gnrhr2, but the outcome of this interaction is still unknown. Gnrh3 also regulates fshb expression in both gonadotropes, most likely via a non-Gnrh receptor route. Altogether, while Lh secretion and synthesis are likely regulated in a Gnrh-independent manner, Gnrh3 seems to play a role in the cellular organization of the pituitary. Moreover, the co-expression of lhb and fshb in both gonadotropes provides a possible explanation as to why gnrh3-/- zebrafish are fertile.

A systematic review and meta-analysis of cardiovascular disease risk with degarelix and GnRH agonists in prostate cancer.

Degarelix is a third-generation GnRH receptor antagonist approved for the treatment of prostate cancer, however, the decision to use a GnRH agonist or an antagonist depends on several factors. We aimed to perform a meta-analysis comparing the cardiovascular disease risk between degarelix and gonadotropin-releasing hormone agonists in patients with all stages of prostate cancer. Databases were searched for randomized control trials (RCTs) and observational studies that compared the risk of cardiovascular disease between degarelix and GnRH agonists in patients with prostate cancer. We computed for binary endpoints risk ratio (RR) or hazard ratio (HR) with 95% confidence intervals (CI) which were analyzed using a random-effects model. A total of 15 studies were included with 123,969 patients and follow-up ranging from 3 to 13months. Degarelix was associated with a significantly lower incidence of major adverse cardiovascular events (RR 0.59; 95% CI 0.41-0.84; p = 0.003; I2 = 84%). Incidence of stroke (RR 0.89; 95% CI 0.56-1.42; p = 0.62; I2 = 0%), all-cause mortality (RR 0.64; 95% CI 0.37-1.13; p = 0.12; I2 = 41%), hypertension (RR 0.71; 95% CI 0.48, 1.04; p = 0.08; I2 = 0%), myocardial infarction (HR 1.04; 95% CI 0·59-1·84; p = 0·86; I2 = 66%), heart failure (HR 0.79; 95% CI 0.38-1.62; p = 0.52; I2 = 79%) and arrhythmia (RR 0.63; 95% CI 0.28-1.41; p = 0.86; I2 = 37%), did not reach a statistically significant difference between groups. In patients with prostate cancer, degarelix is associated with a significantly lower incidence of major adverse cardiovascular events.

Endometriosis in Focus: Modern Therapeutic Approaches for Enhanced Patient Outcomes - Review

Endometriosis (EM) is a chronic condition affecting millions of women globally, characterized by the growth of endometrial-like tissue outside the uterus. This review highlights the latest advancements in understanding and treating endometriosis, focusing on new therapeutic strategies aimed at improving patient care. The article briefly discusses the pathogenesis of endometriosis and reviews traditional treatment methods, including hormonal therapies (progestins, GnRH agonists, and oral contraceptives) and surgical interventions. It then explores the limitations of these conventional therapies and delves into newer strategies targeting specific molecular pathways, offering more effective symptom relief with fewer side effects. Emerging treatments that modulate the immune response and reduce inflammation are also examined, along with innovative approaches to pain management, both pharmacological and non-pharmacological. The review further considers alternative and supportive therapies, such as dietary changes, gut microbiota interventions, and complementary treatments like acupuncture, which may improve the overall management of endometriosis. This review emphasizes the shift toward more personalized, holistic care in treating endometriosis, with a focus on emerging therapies like Aromatase Inhibitors, GnRH receptor antagonists, and hormone receptor modulators. These approaches represent a significant advancement in improving the quality of life for women with this condition.

Deciphering Aflatoxin B1 affected critical molecular pathways governing cancer: A bioinformatics study using CTD and PANTHER databases.

Aflatoxin B1 (AFB1) is a fungal toxin consistently found as a contaminant in food products such as cereals, nuts, spices, and oilseeds. AFB1 exposure can lead to hepatotoxicity, cancer, immune suppression, reproductive deficiency, nutritional dysfunction, and growth impairment. AFB1 has also been listed as one of the most potent human carcinogens by the International Agency for Research on Cancer. Although the correlation between AFB1 exposure and cancer initiation and progression is already reported in the literature, very little information is available about what molecular pathways are affected during cancer development. Considering this, we first selected AFB1-responsive genes involved in five deadliest cancer types including lung, colorectal, liver, stomach, and breast cancers from the Comparative Toxicogenomics Database (CTD). Then, using the PANTHER database, a statistical overrepresentation test was performed to identify the significantly affected pathways in each cancer type. The gonadotropin-releasing hormone receptor (GnRHR) pathway, the CCKR signaling pathway, and angiogenesis were found to be the most affected pathways in lung, breast, liver, and stomach cancers. In addition, AFB1 toxicity majorly impacted apoptosis and Wnt signaling pathways in liver and stomach cancers, respectively. Moreover, the most affected pathways in colorectal cancer were the Wnt, CCKR, and GnRHR pathways. Furthermore, gene analysis was also performed for the most affected pathways associated with each cancer and identified thirteen key genes (e.g., FOS, AKT1) that may serve as biological markers for a particular type of AFB1-induced cancer as well as for in vitro AFB1 toxicological studies using specific cancer cell lines.

7211 Clinical and Molecular Characterization Of An Aldosterone-Producing Adenoma Harboring Double Somatic Mutations In CTNNB1 and GNA11

Abstract Disclosure: A. Blinder: None. K. Nanba: Grant Recipient; Self; AstraZeneca. A. Udager: None. Y. Hirokawa: None. T. Miura: None. H. Okuno: None. K. Moriyoshi: None. Y. Yamazaki: None. H. Sasano: None. A. Yasoda: None. N. Satoh-Asahara: None. W.E. Rainey: None. T. Tagami: None. Background: Somatic activating mutations in CTNNB1, encoding β-catenin, have been detected in a small subset of aldosterone-producing adenomas (APAs). As a possible pathogenesis, an association of CTNNB1-mutated APA with pregnancy and menopause has been proposed. A recent study reported double somatic mutations in CTNNB1 and GNA11/Q, encoding guanine nucleotide-binding protein subunits alpha-11 and alpha-Q, in APA. However, due to its rare incidence, clinical and molecular features of APA associated with these mutations have not been well characterized. Herein, we report a case of APA harboring somatic CTNNB1 and GNA11 mutations. Clinical Case: A 46-year-old Japanese woman presented with hypertension and hypokalemia. She had two pregnancies in the past but had no history of pregnancy-induced hypertension. She had regular menstrual cycles at presentation. Laboratory testing showed elevated plasma aldosterone concentration with suppressed plasma renin activity. She was diagnosed as having primary aldosteronism based on the results of a captopril challenge test. Adrenal computed tomography revealed a 2 cm right adrenal mass. Adrenal venous sampling indicated excess aldosterone production from the right adrenal gland. She underwent right laparoscopic adrenalectomy. Histologic diagnosis of the resected tumor was adrenocortical adenoma based on the criteria of Weiss. Notably, Ki-67 labeling index was high (6% at hotspots). After surgery, her blood pressure and serum potassium both normalized. According to the primary aldosteronism surgical outcome (PASO) study criteria, PA remained clinically and biochemically cured at follow-up 2.5 years after surgery. Results: Immunohistochemistry (IHC) showed high and diffuse expression of aldosterone synthase (CYP11B2) within the tumor, whereas immunoreactivity of 17α-hydroxylase (CYP17A1) and 11β-hydroxylase (CYP11B1), both required for cortisol biosynthesis, was markedly low. VSNL1, a marker for the zona glomerulosa (ZG) where physiologic aldosterone biosynthesis occurs, was highly expressed within the tumor. CYP11B2 IHC-guided tumor capture followed by targeted next-generation sequencing identified double somatic CTNNB1 (p.D32Y) and GNA11 (p.Q209H) mutations. Quantitative real-time RT-PCR revealed high tumor expression of LHCGR (LH receptor) and GNRHR (GNRH receptor) mRNA levels compared with those in adjacent normal adrenal (148-fold and 56-fold, respectively). Immunofluorescence staining of the tumor revealed the presence of activated β-catenin, consistent with features of the normal ZG. Conclusions: Our study indicates that an APA with double somatic mutations in CTNNB1 and GNA11 has characteristics of the ZG and could occur in adults with no clear association with pregnancy or menopause. Presentation: 6/3/2024

N-acetylcysteine and zinc sulphate abate di-2-ethylhexyl phthalate-mediated reproductive dysfunction in rats: Focus on oxidative and sex hormone receptors mechanisms

Objective: To investigate the potential of N-acetylcysteine (NAC) and zinc sulphate (ZnSO4) in mitigating reproductive dysfunction caused by di-2-ethylhexyl phthalate (DEHP) in rats and to understand the underlying mechanisms, specifically oxidative stress and sex hormone receptor activity. Methods: Thirty-five male Wistar rats were randomly divided into five equal groups (n=7 per group). Group 1 was administered 0.5 mL of distilled water and served as the control group. Group 2 was given only DEHP (750 mg/kg/day), while group 3, 4 and 5 were given DEHP (750 mg/kg/day) plus NAC (100 mg/kg/day), DEHP (750 mg/kg/day) plus ZnSO4 (0.5 mg/kg/day), and DEHP (750 mg/kg/day) plus NAC (100 mg/kg/day) as well as ZnSO4 (0.5 mg/kg/day), respectively. All treatments lasted for 21 days. Samples were obtained after the rats were sacrificed, and hormones levels in the serum and markers of oxidative stress in the testicles were analyzed using the enzyme-linked immunosorbent assay. The amount of androgen receptors in the testicles was determined by immunohistochemistry, and the susceptibility of testosterone and DEHP to bind to androgen receptor and 5α-reductase was determined by molecular docking studies. Results: DEHP decreased reproductive hormones, testicular antioxidant enzymes, increased malondialdehyde levels, and negatively impacted histology of the pituitary and testes. NAC or ZnSO4 treatment showed a marked improvement in testicular antioxidant status and hormone levels, as well as a positive effect on the histology of the pituitary and testes. The combination of both treatments appeared to be more effective. The affinity of DEHP to bind to androgen receptors may lead to disruption of androgen receptor signaling, which can further result in dysfunction of hormones related to androgen. However, NAC is more likely to form stronger binding interactions with follicle stimulating hormone and luteinizing hormone receptors, as well as gonadotropin-releasing hormone receptors, when compared to DEHP. Conclusions: The possibility that NAC and ZnSO4 could downregulate DEHP-induced sex hormone changes is suggested by their potential to reduce toxicity.

Genome sequencing projects reveal new insights into the mammalian Gonadotropin-releasing Hormone II system.

The type II gonadotropin-releasing hormone (GnRH-II) was first discovered in chicken (Gallus gallus) brain and then shown to be present in many vertebrates. Indeed, its structure is conserved unchanged throughout vertebrate evolution from teleost fish through to mammals suggesting a crucial function. Yet the functional significance has been largely unexplored. Studies in comparative endocrinology show that the GnRH-II system is differentially functional in mammalian species. Intact GnRH-II neuropeptide and receptor genes (GnRH2 and GnRH receptor 2 GnRHR2) occur in marmoset monkeys (Callithrix jacchus), musk shrews (Suncus murinus) and pigs (Sus scrofa). However, one or other or both of these genes are inactivated in other species, where mutations or remnants affecting GnRH2 neuropeptide and/or type II GnRHR exons are retained in conserved genomic loci. New data from DNA sequencing projects facilitate extensive analysis of species-specific variation in these genes. Here, we describe GnRH2 and GnRHR2 genes spanning a collection of 21 taxonomic orders, encompassing around 140 species from Primates, Scandentia, Eulipotyphla, Rodentia, Lagomorpha, Artiodactyla, Carnivora, Perissodactyls, Pholidota, Chiroptera, Afrotheria, Xenarthra and Marsupialia. Intact coding exons for both GnRH2 and GnRHR2 occur in monkeys, tree shrews, shrews, moles, hedgehogs, several rodents (degu, kangaroo-rat, pocket mouse), pig, pecarry and warthog, camels and alpaca, bears, Weddell seal, hyena, elephant, aardvark and marsupials. Inactivating mutations affecting GnRH2 and GnRHR2, some located at conserved sites within exons, occur in species of primates, most rodents, lagomorphs, bovidae, cetaceans, felidae, canidae and other carnivora, pangolins, most bats, armadillo, brushtail and echidna. A functional GnRH-II system appears retained within several taxonomic families of mammals, but intact retention does not extend to whole taxonomic orders. Defining how endogenous GnRH-II neuropeptide operates in different mammals may afford functional insight into its actions in the brain, especially as, unlike the type I GnRH system, it is expressed in the mid brain and not the hypothalamus.

Novel non-peptide uracil-derived human gonadotropin-releasing hormone receptor antagonists

Gonadotropin-releasing hormone (GnRH) is the main regulator of the reproductive system, acting on gonadotropic cells by binding to the GnRH1 receptor (GnRH1R). Traditionally, therapies targeting this receptor have relied on peptide modulators, which required subcutaneous or intramuscular injections. Due to the limitations of the parenteral administrations, there is a growing interest in developing oral small molecule modulators of GnRH1R as more convenient therapeutic alternatives.In this study, we examined the potential of chemically modifying elagolix, the first approved non-peptide, orally active GnRH1R antagonist, to increase its atropisomeric properties by introducing new moieties. We designed and synthesized the thio-uracil (1) and cytosine (2) derivatives of elagolix, both demonstrating GnRH1R antagonistic activities, with EC50 values of 39 and 110 nM, respectively. The atropisomers of 1 and 2 were efficiently separated using silica gel chromatography, and extensive NMR investigation, supported by Density Functional Theory (DFT) calculations, allowed us to define their conformations and rotational barriers. Docking and Molecular Dynamics (MD) studies revealed that 1 and 2 bind to GnRH1R with ΔG values comparable to elagolix, but through distinct binding modes. These results highlight the potential of non-peptide modulators to effectively modulate GnRH1R activity and pave the way for developing novel modulators.

GnRH protective effects against long-term potentiation impairment induced by AANAT-siRNA

There is an interplay between the gonadotropin-releasing hormone (GnRH) and melatoninergic systems. The key enzyme of melatonin synthesis (arylalkylamine N-acetyltransferase, AANAT), and GnRH receptors are expressed in the hippocampus. While it has been shown that hippocampal AANAT enzyme activity is necessary for proper hippocampal cognitive function, their role in long-term potentiation (LTP) induction is not fully understood. In current study, the impact of GnRH on LTP induction was investigated, while hippocampal melatonin synthesis had been inhibited. The melatonin synthesis was inhibited by AANAT-siRNA administration, and LTP was induced using in vivo field potential electrophysiological recording.Animals were divided into 5 groups: Intact, vehicle, siRNA, GnRH and siRNA+GnRH. All animals, except intact group, experienced the stereotaxic surgery and intra-hippocampal cannulation to receive vehicle agent, AANAT siRNA (0.5 μg/hip), GnRH (1 ng/rat), and AANAT siRNA+GnRH. The recognition memory was assessed by Novel object recognition test. The field potential electrophysiology experiment was conducted by stimulating the Schaffer collateral pathway, and LTP induction was carried out through high-frequency stimulation (HFS). After recording, animals' brain was isolated and quickly frozen for further hippocampal melatonin levels measurement by LC-MS and AANAT mRNA levels by qRT-PCR.GnRH injection in the hippocampus increased local AANAT-mRNA expression and melatonin levels. GnRH-treated animals displayed higher LTP amplitude compared to intact, vehicle and siRNA groups. While the reduction in hippocampal melatonin levels by AANAT-siRNA inhibited LTP and impaired recognition memory, the GnRH prevented these adverse effects. The data suggests that GnRH have protective effects against AANAT-siRNA-induced LTP decline. The protective mechanism at least partially, may be related to the increased expression of local AANAT-mRNA.

A GnRH neuronal population in the olfactory bulb translates socially relevant odors into reproductive behavior in male mice.

Hypothalamic gonadotropin-releasing hormone (GnRH) neurons regulate fertility and integrate hormonal status with environmental cues to ensure reproductive success. Here we show that GnRH neurons in the olfactory bulb (GnRHOB) of adult mice can mediate social recognition. Specifically, we show that GnRHOB neurons extend neurites into the vomeronasal organ and olfactory epithelium and project to the median eminence. GnRHOB neurons in males express vomeronasal and olfactory receptors, are activated by female odors and mediate gonadotropin release in response to female urine. Male preference for female odors required the presence and activation of GnRHOB neurons, was impaired after genetic inhibition or ablation of these cells and relied on GnRH signaling in the posterodorsal medial amygdala. GnRH receptor expression in amygdala kisspeptin neurons appear to be required for GnRHOB neurons' actions on male mounting behavior. Taken together, these results establish GnRHOB neurons as regulating fertility, sex recognition and mating in male mice.

Cytotoxic Activity of Novel GnRH Analogs Conjugated with Mitoxantrone in Ovarian Cancer Cells.

The gonadotropin-releasing hormone (GnRH) receptor (GnRH-R) is highly expressed in ovarian cancer cells (OCC), and it is an important molecular target for cancer therapeutics. To develop a new class of drugs targeting OCC, we designed and synthesized Con-3 and Con-7 which are novel high-affinity GnRH-R agonists, covalently coupled through a disulfide bond to the DNA synthesis inhibitor mitoxantrone. We hypothesized that Con-3 and Con-7 binding to the GnRH-R of OCC would expose the conjugated mitoxantrone to the cellular thioredoxin, which reduces the disulfide bond of Con-3 and Con-7. The subsequent release of mitoxantrone leads to its intracellular accumulation, thus exerting its cytotoxic effects. To test this hypothesis, we determined the cytotoxic effects of Con-3 and Con-7 using the SKOV-3 human OCC. Treatment with Con-3 and Con-7, but not with their unconjugated GnRH counterparts, resulted in the accumulation of mitoxantrone within the SKOV-3 cells, increased their apoptosis, and reduced their proliferation, in a dose- and time-dependent manner, with half-maximal inhibitory concentrations of 0.6-0.9 µM. It is concluded that Con-3 and Con-7 act as cytotoxic "prodrugs" in which mitoxantrone is delivered in a GnRH-R-specific manner and constitute a new class of lead compounds for use as anticancer drugs targeting ovarian tumors.

Identification of Environmental Compounds That May Trigger Early Female Puberty by Activating Human GnRHR and KISS1R.

There has been an alarming trend toward earlier puberty in girls, suggesting the influence of an environmental factor(s). As the reactivation of the reproductive axis during puberty is thought to be mediated by the hypothalamic neuropeptides kisspeptin and gonadotropin-releasing hormone (GnRH), we asked whether an environmental compound might activate the kisspeptin (KISS1R) or GnRH receptor (GnRHR). We used GnRHR or KISS1R-expressing HEK293 cells to screen the Tox21 10K compound library, a compendium of pharmaceuticals and environmental compounds, for GnRHR and KISS1R activation. Agonists were identified using Ca2+ flux and phosphorylated extracellularly regulated kinase (p-ERK) detection assays. Follow-up studies included measurement of genes known to be upregulated upon receptor activation using relevant murine or human cell lines and molecular docking simulation. Musk ambrette was identified as a KISS1R agonist, and treatment with musk ambrette led to increased expression of Gnrh1 in murine and human hypothalamic cells and expansion of GnRH neuronal area in developing zebrafish larvae. Molecular docking demonstrated that musk ambrette interacts with the His309, Gln122, and Gln123 residues of the KISS1R. A group of cholinergic agonists with structures similar to methacholine was identified as GnRHR agonists. When applied to murine gonadotrope cells, these agonists upregulated Fos, Jun, and/or Egr1. Molecular docking revealed a potential interaction between GnRHR and 5 agonists, with Asn305 constituting the most conservative GnRHR binding site. In summary, using a Tox21 10K compound library screen combined with cellular, molecular, and structural biology techniques, we have identified novel environmental agents that may activate the human KISS1R or GnRHR.

Discovery and computational exploration of SNPs in GNRHR gene and their influence on protein structure and function in Indian goat breeds

This study focuses on identifying functional single nucleotide polymorphisms (SNPs) within the gonadotropin-releasing hormone receptor (GNRHR) gene and conducting subsequent in-silico analysis of their effects on protein structure and function in two distinct South Indian goat breeds, namely Attapady Black (n = 120) and Malabari goats (n = 180), known for their divergent prolificacy traits. Utilizing a DNA pool sequencing assay, ten SNPs were uncovered in the study population: c.-1129T>G, c.-1069A>G, c.-978A>C, c.-605A>G, c.-33A>G, c.-29T>G, c.48G>A, c.75G>A, c.209T>G, and c.*212A>G. Notably, two polymorphisms, c.-1129T>G and c.-33A>G, were novel. Additionally, two polymorphisms, c.-33A>G and c.-978A>C, were exclusive to Malabari goats. Analysis of upstream variants revealed modifications to transcription factor and micro-RNA (miRNA) binding sites, suggesting potential alterations in GNRHR expression. Of particular significance was the non-synonymous exonic variant at c.209T>G locus, resulting in methionine to arginine substitution at the 70th position within the first intracellular loop of the receptor protein. This amino acid change may have implications for the functional dynamics of the receptor as GnRHR intracellular loops are involved in G protein coupling thereby facilitation of downstream signalling pathways. The identified SNPs and their in-silico impact analysis contribute to our understanding of the molecular mechanisms underlying reproductive traits in these goat populations, with implications for future breeding strategies and genomic selection programs.

The impact of neoadjuvant relugolix on multi-dimensional patient-reported fatigue.

Androgen deprivation therapy has been shown to improve cancer control when combined with radiotherapy. Relugolix is an oral GnRH receptor antagonist that achieves rapid profound testosterone suppression, which may increase the perception and/or impact of fatigue. This study sought to evaluate neoadjuvant relugolix-induced fatigue in prostate cancer patients prior to the start of stereotactic body radiation therapy (SBRT). Relugolix was initiated at least two months before SBRT. The 13-item Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-F) questionnaire was collected at baseline and one hour prior to SBRT initiation. A five-point scale was used to score individual items. Overall scores range from 0-52 and individual item scores were converted to 0-100, with higher scores reflecting less fatigue. Five "experience" items explored self-perceptions of fatigue, and eight "impact" items sought to evaluate the effect of fatigue on daily activities. Items were evaluated for statistical significance (paired t-test, p < 0.05) and clinical significance (minimally important difference (MID); 0.5 standard deviation from baseline). Between March 2021 to December 2023, 89 men were treated at Georgetown with neoadjuvant relugolix and SBRT. Mean age was 71 years (range: 49-87). Median initiation of relugolix was 4.5 months prior to SBRT (range: 2-14.2 months). 93% patients achieved castration (testosterone levels ≤ 50 ng/dL) and 85% patients achieved profound castration (testosterone levels ≤ 20 ng/dL). 87 patients completed the FACIT-F questionnaire, with an average overall score of 45.6 at baseline and 41.0 at SBRT initiation. This difference was statistically and clinically significant (p < 0.01, MID = 3.55). Patients experienced an increase in fatigue for 12 of 13 items, with statistically significant changes for 11 items. Three of five experience items showed a clinically significant increase in fatigue. Only two of eight impact items were clinically significant. Our study shows that relugolix significantly increases fatigue, affecting multiple areas of life. While the fatigue does not appear to generally impact a patient's ability to carry out normal activities, patients demonstrate frustration with being too tired for these activities. It is essential for clinicians to counsel prostate cancer patients on the impact of neoadjuvant relugolix on quality-of-life issues like fatigue.

Fetal endocrine axes mRNA expression levels are related to sex and intrauterine position

BackgroundThe hypothalamic–pituitary–adrenal (HPA) and -gonadal (HPG) axes are two major pathways that connect the neural and endocrine systems in vertebrates. Factors such as prenatal stress and maternal exposure to exogenous steroids have been shown to affect these pathways during fetal development. Another less studied factor is the transfer of hormones across fetuses in multifetal pregnancies. This form of transfer has been shown to influence the morphology, anatomy, physiology, and behavior of the offspring in litter-bearing mammals, an influence termed the intrauterine position (IUP) effect. In this study, we sought to delineate how the IUP effects HPA and HPG brain receptors, peptides, and enzymes (hereafter components) in utero and how these influences may differ between males and females.MethodsWe utilized the unconventional model of culled free-ranging nutria (Myocastor coypus), with its large natural variation. We collected brain tissues from nutria fetuses and quantified the expression of key HPA and HPG components in three brain regions: prefrontal cortex, hypothalamus, and striatum.ResultsWe found an interaction between sex and IUP in the mineralocorticoid receptor (MR), gonadotropin-releasing hormone receptor (GNRHR), androgen receptor (AR), and estrogen receptor alpha (ESR1). IUP was significant in both gonadotropin-releasing hormone (GnRH) and its receptor GNRHR, but in different ways. In the hypothalamus, fetuses adjacent to same-sex neighbors had higher expression of GnRH than fetuses neighboring the opposite sex. Conversely, in the cortex, GNRHR exhibited the inverse pattern, and fetuses that were neighboring the opposite sex had higher expression levels than those neighboring the same sex. Regardless of IUP, in most components that showed significant sex differences, female fetuses had higher mRNA expression levels than male fetuses. We also found that HPA and HPG components were highly related in the early stages of gestation, and that there was an interaction between sex and developmental stage. In the early stages of pregnancy, female component expression levels were more correlated than males’, but in the last trimester of pregnancy, male components were more related to each other than female’s.ConclusionsThis study suggests that there are sexually different mechanisms to regulate the HPA and HPG axes during fetal development. Higher mRNA expression levels of endocrine axes components may be a mechanism to help females cope with prolonged androgen exposure over a long gestational period. Additionally, these findings suggest different coordination requirements of male and female endocrine axes during stages of fetal development.

Identification and Functional Analysis of Adipokinetic Hormone Receptor in Ostrinia furnacalis Guenée Larvae Parasitized by Macrocentrus cingulum.

As a typical G protein-coupled receptor, the adipokinetic hormone receptor (AKHR) has seven transmembrane domains (TMDs), and its structure and function are similar to the gonadotropin-releasing hormone receptor (GnRHR) in vertebrates. However, there is a dearth of information on other components of the AKHR signaling pathway and how it functions in the interaction between insect hosts and parasitoids. In this study, we cloned and analyzed the multifunctional Ostrinia furnacalis AKHR (OfAKHR) cDNA (GenBank accession number MF797868). OfAKHR has a 2206 bp full-length cDNA, which includes an open reading frame containing 1194 bp. OfAKHR contains the typical seven TMDs, and a "DRY" motif. OfAKHR has the highest relative expression in the fat body and the fifth instar larvae. The results revealed that ApoLpⅢ, PPO2, GS, TPS, Cecropin, and Moricin decreased the transcription levels from 48 to 72 h after the knockdown of OfAKHR expression by dsOfAKHR injection in the fourth instar O. furnacalis larvae. The parasitization of Macrocentrus cingulum selectively upregulated the expression levels of nutrition metabolism and immune-related genes in parasitized O. furnacalis larvae, stimulated lysozyme activity, and obviously raised the concentrations of triglyceride and trehalose in the hemolymph of O. furnacalis larvae. However, they inhibited the activities of PO and trehalase. This study is conducive to a deeper cognition of the roles of OfAKHR in nutrition and immune homeostasis, coevolution, and coexistence between parasitic wasps and hosts. It also sheds light on the potential as the target of pest control reagents.

The Effects of Human Chorionic Gonadotropin and Gonadotropin-Releasing Hormone Receptor Antagonist on Testicular Steroidogenesis in Normal and Obese Rats.

We studied the effect of a high-fat, high-carbohydrate diet (HFHCD) on basal testosterone levels in the blood and testosterone, its precursors, and expression of steroidogenic genes in the testes of rats treated with human chorionic gonadotropin (hCG, 10 IU/rat, subcutaneously, once), gonadotropin-releasing hormone receptor antagonist cetrorelix (75 μg/kg, subcutaneously, 3 days), and their combination. In HFHCD rats, no obvious signs of androgen deficiency were observed and the response of the testes to hCG stimulation was preserved. Unlike control rats (normal diet), the expression of the luteinizing hormone receptor gene in these rats did not change in response to hCG stimulation and cetrorelix administration; they also showed a paradoxical, more pronounced response to hCG administration under conditions of suppression of the gonadotropin secretion by cetrorelix. This suggests that the etiology and pathogenesis of obesity may have different effects on the hormonal status of the male reproductive system.

Divergent folding-mediated epistasis among unstable membrane protein variants.

Many membrane proteins are prone to misfolding, which compromises their functional expression at the plasma membrane. This is particularly true for the mammalian gonadotropin-releasing hormone receptor GPCRs (GnRHR). We recently demonstrated that evolutionary GnRHR modifications appear to have coincided with adaptive changes in cotranslational folding efficiency. Though protein stability is known to shape evolution, it is unclear how cotranslational folding constraints modulate the synergistic, epistatic interactions between mutations. We therefore compared the pairwise interactions formed by mutations that disrupt the membrane topology (V276T) or tertiary structure (W107A) of GnRHR. Using deep mutational scanning, we evaluated how the plasma membrane expression of these variants is modified by hundreds of secondary mutations. An analysis of 251 mutants in three genetic backgrounds reveals that V276T and W107A form distinct epistatic interactions that depend on both the severity and the mechanism of destabilization. V276T forms predominantly negative epistatic interactions with destabilizing mutations in soluble loops. In contrast, W107A forms positive interactions with mutations in both loops and transmembrane domains that reflect the diminishing impacts of the destabilizing mutations in variants that are already unstable. These findings reveal how epistasis is remodeled by conformational defects in membrane proteins and in unstable proteins more generally.

Divergent folding-mediated epistasis among unstable membrane protein variants

Many membrane proteins are prone to misfolding, which compromises their functional expression at the plasma membrane. This is particularly true for the mammalian gonadotropin-releasing hormone receptor GPCRs (GnRHR). We recently demonstrated that evolutionary GnRHR modifications appear to have coincided with adaptive changes in cotranslational folding efficiency. Though protein stability is known to shape evolution, it is unclear how cotranslational folding constraints modulate the synergistic, epistatic interactions between mutations. We therefore compared the pairwise interactions formed by mutations that disrupt the membrane topology (V276T) or tertiary structure (W107A) of GnRHR. Using deep mutational scanning, we evaluated how the plasma membrane expression of these variants is modified by hundreds of secondary mutations. An analysis of 251 mutants in three genetic backgrounds reveals that V276T and W107A form distinct epistatic interactions that depend on both the severity and the mechanism of destabilization. V276T forms predominantly negative epistatic interactions with destabilizing mutations in soluble loops. In contrast, W107A forms positive interactions with mutations in both loops and transmembrane domains that reflect the diminishing impacts of the destabilizing mutations in variants that are already unstable. These findings reveal how epistasis is remodeled by conformational defects in membrane proteins and in unstable proteins more generally.

Characterization of corazonin signaling in a molluscan model species, Lymnaea stagnalis

In recent years, new concepts have emerged regarding the nomenclature, functions, and relationships of different peptide families of the gonadotropin-releasing hormone (GnRH) superfamily. One of the main driving forces for this originated from the emerging evidence that neuropeptides previously called molluscan GnRH are multifunctional and should be classified as corazonin (CRZ). However, research articles still appear that use incorrect nomenclature and attribute the same function to molluscan CRZs as vertebrate GnRHs. The aim of the present study was to further support the recent interpretation of the origin and function of the GnRH superfamily. Towards this goal, we report the characterization of CRZ signaling system in the molluscan model species, the great pond snail (Lymnaea stagnalis). We detected a CRZ-receptor-like sequence (Lym-CRZR) by homology-searching in the Lymnaea transcriptomes and the deduced amino acid sequence showed high sequence similarity to GnRH receptors and CRZ receptors. Molecular phylogenetic tree analysis demonstrated that Lym-CRZR is included in the cluster of molluscan CRZRs. Lym-CRZR transiently transfected into HEK293 cells was found to be localized at the plasma membrane, confirming that it functions as a membrane receptor, like other G protein-coupled receptors. The signaling assays revealed that the previously identified Lym-CRZ neuropeptide stimulated intracellular Ca2+ mobilization in a dose-dependent manner, but not cyclic AMP production, in HEK293 cells transfected with Lym-CRZR. Finally, we demonstrated a wide tissue distribution of Lym-CRZR. These results suggest that Lym-CRZ is a multifunctional peptide and provide further insights into the evolution of the GnRH neuropeptide superfamily. The present study also supports the notion that previously termed molluscan “GnRH” should be classified as “CRZ”.