LHbeta Research Articles

Effects of Metformin in High Concentrations on the Fertility of Female Sailfin Molly Poecilia latipinna

Metformin is a medicine that is used to treat type 2 diabetes and polycystic ovary syndrome (PCOS). This study investigated the effects of exposing the sailfin molly fish to sub-lethal concentrations of 0.008 mol.L-1 of metformin. The results showed that metformin caused disturbances in the endocrine glands, especially the pituitary hormones (GTHs). Metformin increased the expression of the Fshb and inhibited the Lhb genes in the pituitary gland, which led to higher levels of FSH and lower levels of LH hormones in the blood of fish. These hormonal changes led to irregularities in the ovarian development of female fish, such as an increase in the first stage of primary eggs and a delay in the final stage of embryo production.

Dual effects of gonadotropin-inhibitory hormone on testicular development in prepubertal Minxinan Black rabbits (Oryctolagus cuniculus).

Gonadotropin-inhibitory hormone (GnIH) is a neurohormone that not only suppresses reproduction at the brain level but also regulates steroidogenesis and gametogenesis at the gonad level. However, its function in gonadal physiology has received little attention in rabbits. The main objective of this study was to evaluate the effects of GnIH on testicular development and function in prepubertal Minxinan Black rabbits (Oryctolagus cuniculus). In the present study, we investigated the serum reproductive hormone concentration, testicular parameters, morphology of seminiferous tubules, apoptosis of testicular cells, and expression of reproductive-related genes in male prepubertal Minxinan Black rabbits intraperitoneally administered with 0, 0.5, 5, or 50 μg quail GnIH-related peptides (qGnIH) for 10 days. Compared with the vehicle, administration with 5 μg of qGnIH downregulated the serum testosterone concentration and mRNA levels of spermatogenic genes (PCNA, FSHR, INHβA, HSF1, and AR) and upregulated the apoptosis rate of testicular cells; administration with 50 μg of qGnIH decreased the serum testosterone concentration and hypothalamic GnIH gene mRNA level and increased the serum LH concentration, pituitary LHβ gene mRNA level, testicular weight, gonadosomatic index (GSI), and spermatogenic cell layer thickness. It is concluded that GnIH could exert dual actions on testicular development depending on the male prepubertal rabbits receiving different intraperitoneal doses.

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

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

OR2801 Novel Cis-Regulatory Mechanisms Of Lhb Transcription By Non-Coding RNAs And Non-Canonical DNA Structures

Abstract Disclosure: T. Refael: None. H. Gruber: None. G. Golan: None. L. Pnueli: None. P. Melamed: None. Expression of the Lhb gene was shown previously to be directed by synergistic actions of lineage-specific transcription factors at conserved regions of the proximal promoter. However extensive genomic analysis during the last few decades has expanded the repertoire of factors we now know to be involved in transcriptional regulation, revealing roles for distal transcriptional enhancers and long non-coding RNAs (lncRNAs), whose modes of action are only beginning to be elucidated1. Recently it has also become evident that regulatory regions of the genome are enriched with non-canonical DNA structures. These include G-quadruplexes (G4s) which form on guanine-rich stretches of single-stranded DNA and iMotifs (iMs) on the complementary strand, and are reported to bind various proteins which presumably regulate and mediate their actions. We identified in available mice pituitary scATAC-seq data a region of gonadotrope-specific open chromatin upstream of the Lhb gene, and chromatin conformation capture (3C) in murine gonadotrope cell lines indicated the distal sites that are in physical proximity to the Lhb promoter. Further, we show by chromatin immunoprecipitation (ChIP) a peak of H3K4 monomethylation (H3K4me1) ∼3 kbp upstream of the Lhb TSS, and transcription of two bi-directional non-coding enhancer RNAs (eRNAs), both of which are characteristic of transcriptional enhancers. One of the eRNAs was found to direct open chromatin at the Lhb promoter and is required for Lhb transcription, while guide RNA-mediated recruitment of dCas9- KRAB or dCas9-VP64 to this locus altered Lhb mRNA levels accordingly, confirming its functional enhancer activity. However this enhancer activity is not sufficient for optimal Lhb transcription, and we found that splicing in situ of a distinct downstream lncRNA is also required. The DNA at the central non-coding region of the enhancer, and at the promoter of this lncRNA, forms G4 and iM structures, seen by circular dichroism and ChIP. These DNA structures are affected differentially by local conditions including changes in pH or hormonal treatments, and appear to have diverse effects. HMGB2, which is known to bind structured DNA, was found to bind the enhancer iM in cells and in vitro. Further, HMGB2 knockdown reduced the iM signal and Lhb mRNA, eRNA and lncRNA expression levels quite dramatically. We thus present novel elements involved in the regulation of Lhb transcription through various classes of non-coding RNAs and DNA structures, while emphasizing the complexity of transcriptional regulation via context-dependent cis-acting elements.

O-187 The combination of machine learning and pharmacogenetics for the prediction of sub-optimal ovarian response

Abstract Study question Can an artificial intelligence (AI)-based model predict the risk of suboptimal ovarian response using patient genetic data? Summary answer AI-based predictive models can identify relevant predicting factors for suboptimal ovarian response, including several genetic variants. What is known already A substantial proportion of women classified as “normo-responders” may not reach the optimal range of oocytes retrieved after COS. Polyzos and Sunkara (2015) defined a new category of women (15-36%) with a worse prognosis and a “suboptimal” level of ovarian response in which the number of oocytes retrieved ranges between 4 and 9 oocytes. The explanation for this behavior has been suggested to be genetic and indeed variants in FSH receptor and LH subunit-B have been identified that predispose to lower ovarian sensitivity to stimulation. Therefore, these women might require higher doses of gonadotropins or longer stimulations. Study design, size, duration This observational study included a retrospective analysis of 1370 ovarian stimulations from egg donors performed between March 2018 and April 2022. The oocyte donor candidates were selected according to our clinic donation program requirements and ASRM and ESHRE guidelines for oocyte donation. They were stimulated following a progesterone-primed ovarian stimulation protocol. All donors started stimulation with 150-300 IU/day of FSH according to AFC and BMI. A GnRH agonist was used for final oocyte maturation. Participants/materials, setting, methods Oocyte donors were healthy women between 18 and 35 years old (n = 504). In order to establish the predictive machine learning models, patient characteristics and controlled ovarian stimulation data were recorded in a data frame. In addition, donors were genotyped for 31 variants corresponding to 16 genes associated with ovarian response. The association between the different variables and risk of suboptimal ovarian response was analysed using SPSS (v23.0) and R (v. 4.2.0) statistical software. Main results and the role of chance The oocyte donors had a mean age of 25.4±4.0 and a high ovarian reserve (AFC: 18.4±7). The mean number of oocytes retrieved after ovarian stimulation was 16.3±7.5, of which 13.1±6.4 were mature. Despite being young women with good ovarian reserve, 16.4% of the stimulations were suboptimal (4-9 oocytes retrieved). Classical statistical method (binary logistic regression) and 5 different supervised classification machine learning algorithms (multi-layer perceptron, support vector-machines, k-nearst neighbors, random forest and eXtreme Gradient Boosting (XGBoost)) were used to establish a prediction model for suboptimal ovarian response. The model with the highest AUC value was the XGBoost (0.876). The accuracy, sensitivity and specificity were 0.798, 0.809 and 0.787 respectively. The variables that had the greatest predictive power in the best machine learning algorithm (XGBoost) were: age, BMI, AFC and total gonadotrophin dose. In addition, four genetic variants were identified that modified the risk of a suboptimal ovarian response: -ESR2 (oestrogen receptor; c.*39G>A). -LHB (LH beta subunit; c.82T>C/p.Trp28Arg). -SOD2 (superoxide dismutase; c.47T>C/p.Val16Ala). A mitochondrial enzyme that catalyses the detoxification and protection against redox damage that can occur in COS. -and TP53 (tumour suppressor protein; c.215C>T/p.Pro72Leu) that exerts a protective effect on DNA damage in folliculogenesis. Limitations, reasons for caution Our investigation was performed with retrospectively collected data, and hence it will be of importance to collect data prospectively to confirm that the new identified variants are associated with suboptimal ovarian response also in the IVF population. Wider implications of the findings The combination of AI and pharmacogenetics has led to the identification of genetic variants that might predispose to suboptimal ovarian response. Women who carry these variants (erroneously considered as normo-responders) could be candidates for personalization of their ovarian stimulation treatment with higher doses of gonadotrophins and/or longer stimulations. Trial registration number Not applicable

Salmonid Pituitary Cells as a Test System for Identifying Endocrine-Disrupting Compounds.

The pituitary gland is a central regulator of reproduction, producing two gonadotropins, follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh), which regulate gonadal development, sex steroid synthesis, and gamete maturation. The present study sought to optimize an in vitro test system using pituitary cells isolated from previtellogenic female coho salmon and rainbow trout, focusing on fshb and lhb subunit gene expression. Initially, we optimized culture conditions for duration and benefits of culturing with and without addition of endogenous sex steroids (17β-estradiol [E2] or 11-ketotestosterone) or gonadotropin-releasing hormone (GnRH). The results suggest that culturing with and without E2 was valuable because it could mimic the (+) feedback effects on Lh that are observed from in vivo studies. After optimizing assay conditions, a suite of 12 contaminants and other hormones was evaluated for their effects on fshb and lhb gene expression. Each chemical was tested at four to five different concentrations up to solubility limitations in cell culture media. The results indicate that more chemicals alter lhb synthesis than fshb. The more potent chemicals were estrogens (E2 and 17α-ethynylestradiol) and the aromatizable androgen testosterone, which induced lhb. The estrogen antagonists 4-OH-tamoxifen and prochloraz decreased the E2-stimulated expression of lhb. Among several selective serotonin reuptake inhibitors tested, the sertraline metabolite norsertraline was notable for both increasing fshb synthesis and decreasing the E2 stimulation of lhb. These results indicate that diverse types of chemicals can alter gonadotropin production in fish. Furthermore, we have shown that pituitary cell culture is useful for screening chemicals with potential endocrine-disrupting activity and can support the development of quantitative adverse outcome pathways in fish. Environ Toxicol Chem 2023;42:1730-1742. © 2023 SETAC.

Influence of magnetic iron oxide nanoparticles in reproductive efficiency of adult male rats

This study aimed to investigate the dose-dependent and time-dependent effect of magnetic iron oxide nanoparticles (MIONPs) on reproduction in male rats. Following their synthesis, the physicochemical properties of MIONPs were determined. Sixty-four adult male rats, aged 90 days were randomly assigned to control (C), orally administered with distilled water, and three treated groups, orally administered with 1, 5, 10 mg/kg/day of MIONPs solution (TL, TM, and TH groups, respectively), for 28 days. Each group was allocated to two subgroups, sacrificed after 14 and 28 days of treatment. After each period, the males were weighed and sacrificed. Decreased body weight and genital organ weights were shown in TM and TH groups, at both experimental periods, compared with control in a dose-dependent manner. The serum concentration of GnRH, FSH, LH, and testosterone increased in the TL group and decreased in TM and TH groups compared with control in a dose-dependent and time-dependent manner. At both periods, the lowest expression levels of pituitary FSHβ and LHβ genes and testicular inh-α and LHR genes were recorded in TM and TH groups, and the highest levels were expressed in the TL group. The testis sections of TL males, showed normal architecture, but those from TM and TH groups showed degenerative and necrotic changes, reduced germinal epithelium, vacuolation, and decreased number of spermatocytes apparent. It is concluded that a low dose of MIONPS has a beneficial effect, whereas moderate or high doses have pathological effects on male reproduction.

ODP297 Analysis of the responsible region of TRH in the hypothalamus-pituitary-thyroid axis using the Paraventricular nucleus-specific TRH deficient mice.

Abstract Objectives Thyrotropin-releasing hormone (TRH) was the first hypothalamic hormone isolated that stimulates pituitary thyroid-stimulating hormone (TSH) secretion. TRH was later found to be distributed throughout the brain, gastrointestinal tract and pancreaticβ-cells. We previously reported the TRH null mice (conventional TRHKO) which exhibit characteristic tertiary hypothyroidism, however the responsible regionfor the hypothalamic-pituitary-thyroid (HPT) axis remained obscure because of the broad expression of TRH in hypothalamus. Previous studies suggested that TRH in the hypothalamic paraventricular nucleus (PVN) was secreted through the median eminence (ME), however it was not directly demonstrated . To determine the region functionally responsible for the HPT axis, we established and analyzed PVN-specific TRH knock-out (PVN-TRHKO) mice. Methods For the targeting vector, a Neo-loxP cassette was inserted into the 3′-UTR of the Trh gene in ES cells. Another loxP site was placed at intron 1. After we obtained the Trh +/lox [neo+] founder mice, we crossed them with the CAG-FLPe transgenic mice to remove the Neo cassette. The offspring were backcrossed to wild-type mice, producing conditional knock-out mice without the Neo cassette, which were labeled as Trh +/lox . We crossed Trh +/lox with transgenic mice expressing Single-minded homolog (Sim)1-Cre recombinase and created PVN-TRHKO mice (Sim1-Cre; Trh lox/lox mice). We confirmed that most Sim1 was expressed in the PVN using Sim1-Cre/tdTomato mice. We performed the following experiments using PVN-TRHKO andTrh lox/lox mice as control (Ct). 1) immunostaining of TRH using antibody forproTRH in cerebrum, 2)qPCR analysis detecting the expression of preproTrh mRNA in hypothalamic PVN, 3) measurement of the level of serum free-T4 and TSH, 4)qPCR analysis detecting theexpression levels of Tshβ, Prl, Cga, Gh, Pomc and Lhβ gene mRNAin the anterior pituitary and 5)immunohistochemical analysis with antibodies for TSHβ and Prolactin in anterior pituitary. Results In the PVN-TRHKO mice, 1) ProTRH-immunopositive cells were lost in the area of PVN and ME, whereas proTRH detected in the medial preoptic area(MPA) and medial preoptic nucleus (MPO) in the hypothalamus.2)The expression level of proTRHmRNA was decreased to7.7% compared with Ct.3) Free-T4 levels decreased to 50% and the TSH levels increased to 170% of those in Ct. 4) TshβmRNA levels were decreased by 65% and thePrlmRNA levels were reduced to 55% of those in Ct. 5) The number of TSH-positive andProlactin-positive cells in the pituitary significantly decreased to 50% and 60% of those in Ct. Conclusions PVN-TRHKO mice exhibited tertiary hypothyroidism similar to conventional TRHKO. The pathway of TRH neuron from PVN to ME was lost in the PVN-TRHKO mice. These findings are conclusive evidence that the TRH neuron in the PVN is the center of the HPT axis. Presentation: No date and time listed

PMON56 LH synthesis is regulated by RNA-binding protein ZFP36 bound with AU-rich elements in gonadotropes

Abstract Gonadotropin secretion, which is stimulated by gonadotropin-releasing hormone (GnRH), is a critical feature of reproductive control and maintenance of fertility. GnRH stimulation increases LH secretion 50-100-fold over baseline per pulse, whereas Lhb gene expression increases a modest 1.4-fold. This observation shows that significant post-transcriptional regulation of gonadotropin synthesis and secretion must occur. We have previously shown that GnRH-induced Dusp1 expression, a factor central for feedback control of GnRH signaling in gonadotropes, depends on interaction of the 3'UTR of its mRNA with the RNA binding protein (RBP) ELAVL1, which binds AU-rich elements (ARE) in target mRNAs (1). Recently, using RIP-Chip analysis, we have shown that GnRH receptor (Gnrhr) expression depends on interaction of its mRNA with ELAVL1. Gnrhr mRNA is among the most highly represented ELAVL1-associated mRNAs (2). Further, many mRNAs encoding proteins essential to gonadotropin gene expression, including the immediate-early transcription factor genes Egr1, 2, and 4, Fos, Fosb, Jun, and Junb, as well as Dusp1 and 2, show increased binding to ELAVL1 after GnRH treatment, although gonadotropin Lhb mRNA is not, due to the absence of ARE site to be bound. Typically, association with ELAVL1 stabilize expression of target mRNAs, promoting their translation. Also, of interest is the role of the RBP ZFP36, which competes with ELAVL1 for binding to ARE's but has the opposing effect of directing mRNA to the degradation pathway. Significantly, 5 minutes pulsatile GnRH stimulated ZFP36 protein production in 30-60 minutes in LbetaT2 cells. And, knockdown of ELAVL1 results in the deterioration of Gnrhr expression and of the immediate-early gene Egr1 a transcription factor central to Lhb expression. We have examined the direct role of ZFP36 protein in GnRH-induced gene expression. Interestingly, Gnrhr and Lhb mRNA expression levels were decreased by ZFP36 overexpression in LbetaT2 cells compared with control cells. Also, GnRH-stimulated EGR1 production by was decreased by ZFP36 overexpression. These observations are consistent with previous results with ELAVL1 knockdown showing decreased mRNA stability. We cloned Egr1, Gnrhr and Zfp36 3'UTRs which contain AREs bound by ELAVL1 into luciferase reporter genes in pmirGLO vector to evaluate whether the 3'UTR is directly affected by ZFP36. These Egr1, Gnrhr and Zfp36 3'UTR contained mirGLOs were transfected in both ZFP36 overexpression and control cells and luciferase activity was monitored. The results showed that luciferase expression from reporters bearing the target 3'UTR were significantly reduced in ZFP36 overexpressed cells. Collectively, our studies show that ELAVL1, ZFP36 and their target mRNAs are regulated by GnRH and this indirectly regulates Lhb gene expression in gonadotropes. ) Do MT, T Kim et al. Mol Cell Endocrinology 382 (1): 1346-1357, 2014 ) Terasaka T et al. Endocrinology 160(8): 1999-2014, 2019 Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Ciprofloxacin and enrofloxacin can cause reproductive toxicity via endocrine signaling pathways.

Ciprofloxacin (CIP) and enrofloxacin (ENR) are veterinary antibiotics commonly utilized to treat and prevent animal diseases. Environmental and dietary antibiotic residues can directly and indirectly affect the reproductive development of animals and humans. This article investigated the reproductive toxicity of CIP in male zebrafish, showing that it could decrease the spermatogonial weight and damage the spermatogonial tissue. The sex hormone assays showed that CIP decreased fshb and lhb gene expression and plasma testosterone (T). In addition, transcriptome analysis indicated that the effect of CIP on zebrafish might be related to the endocrine signaling pathways. ENR, which was selected for further study, inhibited mouse Leydig (TM3) and Sertoli (TM4) cell proliferation and caused cell cycle arrest. The sperm concentration, serum luteotropic hormone (LH) and follicle-stimulating hormone (FSH), and T levels decreased in adolescent mice after ENR treatment for 30d in vivo. Hematoxylin and eosin (H&E) staining showed that ENR exposure potentially induced testicular injury, while the real-time quantitative PCR (qPCR) results indicated that ENR inhibited the mRNA expression of key genes in the Leydig cells (cyp11a1, 3β-HSD, and 17β-HSD), Sertoli cells (Inhbβ and Gdnf) and spermatogenic cells (Plzf, Stra8 and Dmc1). In conclusion, these findings indicated that ENR exposure might influence the development of the testes of pubescent mice.

Gonadotropin profile in experimentally induced hypothyroid and hyperthyroid cyclic female rats

The current study examined the association of thyroid disorders with reproductive dysfunction by determining its effect on gonadotropin secretion in cyclic female rats. Sixty cyclic females were assigned to three groups (20 each) and supplemented, for 30 days plus two consequent estrous cycles, with drinking water (control), methimazole in drinking water (0.02% w/v) (hypothyroid group), and thyroxine in drinking water (0.002% w/v) and gastric gavage of 200 μg/kg body weight (hyperthyroid group). At late proestrus, ten females from each group (for each cycle) were anesthetized and dissected. Blood samples were obtained to assess thyroid-stimulating hormone, free and total triiodothyronine, free and total thyroxin, follicle-stimulating hormone, luteinizing hormone, and prolactin concentrations. Ovarian and pituitary tissue samples were obtained for molecular analysis of ovarian thyroid receptor genes and pituitary TSH, FSHβ, and LHβ genes. In comparison with control, the Hypo group revealed increased serum concentrations of TSH and PRL and the expression levels of pituitary TSH and ovarian TRsTRs genes and significant decrease of FT3, TT3, FT4, TT4, FSH, and LHLH concentrations and the expression levels of pituitary FSHβ and LHβ genes. In contrast, the Hyper group showed increased serum FT3, TT3, FT4, TT4, and LHLH concentrations and the expression levels of pituitary LHβ and ovarian TRsTRs genes and decreased serum TSH FSH and PRL concentrations and pituitary FSHβ and TSH gene expression levels. It is concluded that thyroid dysfunction is associated with altered serum gonadotropin secretion and reproductive failure.

Investigation of biomarkers in Endometriosis-associated infertility: Systematic Review

The relationship between endometriosis and infertility is still unknown, but it is possible that genetic polymorphisms influence these two variables. This study aims to identify, in the literature, which polymorphisms are related to infertility in women with endometriosis. A search was performed in databases using the descriptors: polymorphisms genetics and infertility and endometriosis. 386 articles were identified, and after applying the inclusion and exclusion criteria, 33 case-control studies were included. Genes and their respective polymorphisms, which exhibited statistically significant values, were classified into three categories: related to metabolic/cellular processes, steroidogenesis and sex hormone receptors, inflammation and immune response. In summary, the results of these studies suggest that the polymorphisms rs882605 of MUC4 gene, rs16826658 of WNT4 gene, rs10953316 of MUC17 gene, rs10928050 of KAZN gene, rs1799889 of PAI-1 gene, (TA)n repeats of ESR1 gene, (CA)n repeats of ESR2 gene, rs605059 of HSD17B1 gene, rs743572 of CYP17A1 gene, insLQ of LHR gene, p.Ile49Ser of AMH gene, rs12700667 of NPVF/NFE2L3 gene, G1502A of LHβ gene, G + 1730A of ERβ gene, rs7528684 of FCRL3 gene, rs3761549 of FOXP3 gene and rs28362491 of NFKβ1 gene are implicated in the etiology of infertility in women with endometriosis.

Male hypogonadism caused by a homozygous missense mutation of the LHB gene.

Male hypogonadism caused by a homozygous missense mutation of the LHB gene.

Single molecule characterization of the binding kinetics of a transcription factor and its modulation by DNA sequence and methylation.

The interaction of transcription factors with their response elements in DNA is emerging as a highly complex process, whose characterization requires measuring the full distribution of binding and dissociation times in a well-controlled assay. Here, we present a single-molecule assay that exploits the thermal fluctuations of a DNA hairpin to detect the association and dissociation of individual, unlabeled transcription factors. We demonstrate this new approach by following the binding of Egr1 to its consensus motif and the three binding sites found in the promoter of the Lhb gene, and find that both association and dissociation are modulated by the 9 bp core motif and the sequences around it. In addition, CpG methylation modulates the dissociation kinetics in a sequence and position-dependent manner, which can both stabilize or destabilize the complex. Together, our findings show how variations in sequence and methylation patterns synergistically extend the spectrum of a protein's binding properties, and demonstrate how the proposed approach can provide new insights on the function of transcription factors.

GnRHa implants and temperature modulate Lambari‐do‐rabo‐amarelo, Astyanax altiparanae (Characiformes: Characidae) induced reproduction out of the reproductive season

We evaluated the effect of gonadotropin in controlled-release implant together with temperature manipulation in the reproduction of Astyanax altiparanae (lambari) during the non-breeding season. Three groups of adult females were maintained at 20°C and three at 28°C. For each temperature, one group was treated with 4 μg GnRHa g−1, other with 8 μg GnRHa g−1 and other was sham implanted as a control during 4 weeks. Afterwards, 4 females/experimental group were induced to spawn with human chorionic gonadotropin, while 4 females/group were injected with saline. GnRHa implants increased fshβ and lhβ gene expression in females maintained at 28°C. The vtgA gene expression increased in the high GnRHa dose at 20°C, while this higher GnRHa dose associated with the higher temperature increased Gonadosomatic Index. The hCG injection increased the plasma Mature Induction Steroid levels and trigger ovulation, except in control at 28°C. The control females at 20°C spawned but with low reproductive performance, confirming that this is not a limiting temperature for reproduction. The administration of GnRHa as implant increased gonadotropin gene expression at 28°C, temperature at which GSI also increased. Qualitatively, the implants improved reproduction at 20°C, providing offspring out of the reproductive season, improving lambari production throughout the year.

Exogenous orexin-A downregulates luteinizing hormone secretory activity in prepubertal female rats.

Orexin-A is a neuropeptide synthesized in the lateral hypothalamus. Orexin-A immunoreactive fibres overlap distribution with GnRH neurons. In adult rats, orexin A is known to affect LH secretion via GnRH release modulation. Because data concerning the impact of orexin-A on the hypothalamo-pituitary axis activity are limited, we focused on the involvement of orexin-A and receptors of NPY in the modulation of LH release and LH subunit b (Lhb) mRNA expression in prepubertal female rats. Forty immature female Wistar rats were divided into 4 groups and received 2 intracerebroventricular (icv) microinjections of: 1 - artificial cerebrospinal fluid (CSF) (controls); 2 - CSF followed by orexin A; 3 - selective NPY receptor antagonist (BIBP) followed by CSF; 4 - BIBP followed by orexin A. One hour after the last microinjection, all rats were decapitated. Trunk blood was collected, and serum was stored at -20°C for the LH RIA examination. The adenohypophysis was immediately excised, flash-frozen, and kept at -80°C for RNA extraction. Real-time PCR amplification was carried out, and relative Lhb gene expression was calculated. In comparison to the CSF-treated controls with a mean LH serum concentration of 0.40 ± 0.02 ng/mL, the mean LH serum level was diminished both after orexin-A (0.27 ± 0.01 ng/mL) and after BIBP (0.30 ± 0.02 ng/mL) icv microinjections. In the presence of BIBP, orexin-A more effectively inhibited LH release (0.20 ± 0.01 ng/mL) when compared to the BIBP-treated group. Orexin-A and BIBP exerted a consistent inhibitory effect on Lhb mRNA expression levels in the anterior pituitary gland. In comparison to the CSF-treated controls, orexin-A, and BIBP-treated females responded with, respectively, 35% and 40% reduction of Lhb mRNA expression. Orexin-A and BIBP co-administration evoked a further reduction of Lhb gene transcriptional activity. Orexin-A exerts a down-regulatory effect on LH synthesis and release in immature female rats. Considering that Y1R-oriented down-regulation of endogenous NPY activity did not reverse the suppressive effect of exogenous orexin-A, it might be suggested that NPY and orexin A systems can operate independently to affect gonadotropin activity in the anterior pituitary of the immature female rats.

Multifactorial control of gonadotropin release for induction of oocyte maturation: Influence of gonadotropin-releasing hormone, gonadotropin release-inhibiting factor and dopamine receptors in the catfish, Heteropneustes fossilis

Several external and internal factors contribute to the reproductive success of teleosts, which makes the reproductive process complex and unique. In the Indian freshwater catfish, Heteropneustes fossilis, monsoon plays a crucial role as it fine tunes the neuroendocrine axis, culminating in oocyte maturation. Therefore, induction of oocyte maturation requires the coordinated interaction among hypothalamic, hypophyseal, and peripheral hormones. In the present investigation, dual neuroendocrine control of oocyte maturation has been demonstrated in the catfish, H. fossilis. The maturational response in gravid catfish is inhibited in the presence of dopamine but GnRH evokes the oocyte maturation and ovulation. GnRH upregulates the expression of lhb gene as well as increases plasma levels of LH significantly within 30 minutes of its administration. Destruction of the preoptic region in gravid catfish by electrolytic or chemical lesions also causes oocyte maturation and ovulation. But this response is inhibited if dopamine is injected into the nucleus preopticus periventricularis-lesioned fishes. These observations support the role of dopamine as an inhibitory factor, therefore specific receptors of dopamine have been characterized in catfish and their expression in the brain has been quantified. Dopamine receptors are upregulated in dopamine-treated fishes and downregulated if a dopamine antagonist (pimozide) is injected. The present study suggests the presence of inhibitory mechanism for LH secretion in gravid catfish. Abolition of this inhibition is necessary to release LH surge, which in turn stimulates resumption of meiosis and ovulation. Thus peptidergic as well as aminergic systems regulate oocyte maturation in H. fossilis. Neuroendocrine regulation of oocyte maturation and ovulation has major implications for inducing spawning in aquaculture.

Effect of dietary pomegranate peel powder on productive traits, blood chemistry, economic efficiency and the expression of FSHR and LH-β genes in two strains of laying Japanese quail.

The current experiment was designed to study the response of two strains of laying Japanese quail to diet containing different levels (0%, 3%, 6%, and 9%) of pomegranate peel (PP) powder for a period of 6weeks, on egg production, blood chemistry, carcass traits, expression of FSHR and LH-β genes, and economic efficiency. A total of 576 6-week-old Japanese quails were made up of two strains: white quails (n=288) and brown quails (n=288). Four treatment groups were randomly distributed for each strain. Each group was subdivided into 6 replicates of 10 birds each. Results revealed that the white strain showed significant (P<0.05) higher values in final body weight (BW), egg quality parameters, weights of dressed carcass, and total return (TR) compared with the brown strain. Brown strain had higher hen day egg production % (HDEP%). A significant (P<0.05) interaction effects between genetic type and the dietary PP powder levels was recorded in growth traits, some egg production traits, and mRNA expression of FSHR and LH-β genes. Results concluded that all the dietary levels of PP powder up to 9% improved growth traits, egg production traits, fertility% (P=0.001), and hatchability% (P=0.007). Moreover, they have a safely biochemical effect on the level of urea (P=0.002) and the concentration of aspartate amino transferase (AST) (P<0.001). It also enhanced mRNA expression of FSHR and LH-β genes, but showed non-significant (P>0.05) influence on carcass traits. Economically, the net return (NR) was increased in quails fed on 6% and 9% PP powder diets.

Characterization of the Luteinizing Hormone Beta (LH-β) Subunit Gene in the Goat

Background: Luteinizing hormone (LH) plays a critical role in ovulation and maintenance of pregnancy in female and gamete production in male during fertile phase of life. Physiological disturbance of this hormone leads to conditions like delayed ovulation, anovulation, cystic ovarian disease and lack of sexual desire in male. Since, there had been no report of molecular characterization of β-subunit of luteinizing hormone (LH) of Indian goat, the present study aimed to clone and characterize genomic DNA encoding LHβ subunit.Methods: Genomic DNA was extracted from goat blood and amplified using specific LHβ gene primers. After cloning and transformation, plasmids were isolated from randomly selected white colonies. Presence of insert was confirmed by restriction enzyme digestion of plasmids. After confirmation by PCR, plasmids were sent for DNA sequencing. Result: Analysis of sequence revealed an insert of 1006 bp size as expected. Comparison of nucleotide sequence revealed the cloned gene to be is LHβ encoding 141 amino acids. It showed 97.3 and 91.7% similarity with sheep and cattle respectively. Inferred amino acid sequence showed absolute similarity (100%) with sheep and buffalo. The common and essential features such as twelve cysteine molecules, a single potential N-glycosylation site, the CAGY region and another tetrapeptide CGPC are all found in the goat sequence too.

Characterization of Gonadotroph Pituitary Adenomas Based on the Recent 2017 WHO Pituitary Tumor Classification

Abstract Introduction: More than 20% of pituitary adenomas are nonfunctional, the majority of which are of gonadotroph origin. Whereas previously, immunohistochemistry of pituitary hormones was used to classify adenoma subtypes, in 2017 the World Health Organization (WHO) reclassified pituitary adenomas using transcription factor expression in addition to immunohistochemistry. With this change, clinically nonfunctional gonadotroph adenomas can be distinguished among: (1) those staining for the transcription factor SF-1 and gonadotropins FSH and/or LH (FSH/LH+), (2) those that stain for SF-1 but not for FSH or LH (FSH/LH- SF1+), and (3) true null cell adenomas. It is unclear whether these three subgroups behave similarly clinically, or if they have distinct manifestations or outcomes. Our aim was to characterize these subgroups in regard to tumor size, recurrence and pituitary insufficiency. Methods: In a retrospective chart review, 71 patients from 2017-2020 who presented to the hospital for transsphenoidal resection of clinically nonfunctioning pituitary adenomas were reviewed. All patients with pituitary adenomas that stained positive for SF-1 and negative for T-PIT and PIT-1, and tumors that were negative for all three transcription factors were evaluated. Those lacking clinical data were excluded. Clinical characteristics examined include: demographics, tumor size, invasion of cavernous sinus, and hormone deficiencies. Results: Of the 71 pituitary tumors, 45% (n=32) stained positive for the beta subunit FSH and/or LH (FSH/LH+) and SF-1, 44% (n=31) stained for SF-1 with negative pituitary hormone stains (FSH/LH- SF1+), and 11% (n=8) were negative for all transcription factors and hormones (true null). All tumors were macroadenomas (&amp;gt;1 cm). While there were &amp;gt;50% males in the FSH/LH+ and FSH/LH- SF1+ groups, in the true null group only 25% of patients were male. Most patients were &amp;gt;50 years old in all 3 groups (81% FSH/LH+, 75% FSH/LH- SF1+, 88% true null). The prevalence of cavernous sinus involvement was 36% in both groups that stained for SF-1, but was 62% in the true null group. Both SF-1+ groups had similar tumor sizes and prevalence of panhypopituitarism (15-21%), but there were more episodes of recurrence since last known follow up in the FSH/LH- SF1+ group (20%), compared to FSH/LH+ tumors (7%). The true null group had ≥50% rates for both panhypopituitarism and recurrence. Conclusions: In this study, we highlighted the category of FSH/LH- SF1+ gonadotroph adenomas and compared these to FSH/LH+ and true null cell tumors. Based on clinical features, FSH/LH- SF1+ gonadotroph adenomas are similar to FSH/LH+ staining pituitary adenomas in regard to age, sex, size, and degree of cavernous sinus invasion, although there were more recurrences in the FSH/LH- SF1+ group. Though less common, our cohort suggests more aggressive tendencies in the true null group compared to SF-1 staining tumors.