Follicle-stimulating hormone promotes growth of human prostate cancer cell line-derived tumor xenografts.

Chemical castration in prostate cancer can be achieved with gonadotropin-releasing hormone (GnRH) agonists or antagonists. Their effects differ by the initial flare of gonadotropin and testosterone secretion with agonists and the immediate pituitary-testicular suppression by antagonists. While both suppress luteinizing hormone (LH) and follicle-stimulating hormone (FSH) initially, a rebound in FSH levels occurs during agonist treatment. This rebound is potentially harmful, taken the expression of FSH receptors (R) in prostate cancer tissue. We herein assessed the role of FSH in promoting the growth of androgen-independent (PC-3, DU145) and androgen-dependent (VCaP) human prostate cancer cell line xenografts in nude mice. Gonadotropins were suppressed with the GnRH antagonist degarelix, and effects of add-back human recombinant FSH were assessed on tumor growth. All tumors expressed GnRHR and FSHR, and degarelix treatment suppressed their growth. FSH supplementation reversed the degarelix-evoked suppression of PC-3 tumors, both in preventive (degarelix and FSH treatment started upon cell inoculation) and therapeutic (treatments initiated 3 weeks after cell inoculation) setting. A less marked, though significant FSH effect occurred in DU145, but not in VCaP xenografts. FSHR expression in the xenografts supports direct FSH stimulation of tumor growth. Testosterone supplementation, to maintain the VCaP xenografts, apparently masked the FSH effect on their growth. Treatment with the LH analogue hCG did not affect PC-3 tumor growth despite their expression of luteinizing hormone/choriongonadotropin receptor. In conclusion, FSH, but not LH, may directly stimulate the growth of androgen-independent prostate cancer, suggesting that persistent FSH suppression upon GnRH antagonist treatment offers a therapeutic advantage over agonist.

TRIB3 regulates FSHR expression in human granulosa cells under high levels of free fatty acids.

BACKGROUND: Granulosa cells (GCs) in cumulus oophorus highly express follicle stimulating hormone receptor (FSHR), which is the most important mediator of both estradiol synthesis and oocyte maturation. Obese women have elevated free fatty acids (FFAs) levels in their follicular fluids and decreased FSHR expression in GCs, which is related to an altered protein kinase B/glycogen synthase kinase 3ß (Akt/GSK3ß) signaling pathway. Such FFA increases accompany 3-fold rises in pseudokinase 3 (TRIB3) expression and reduce the Akt phosphorylation status in both the human liver and in insulinoma cell lines. Therefore, in a high FFA environment, we determined if TRIB3 mediates regulation of FSHR via the Akt/GSK3ß signaling pathway in human GCs. METHODS: GCs from women undergoing in vitro fertilization were collected and designated as high and low FFAs cohorts based on their follicular fluid FFA content. GCs with low FFA levels and a human granulosa-like tumor (KGN) cell line were exposed to palmitic acid (PA), which is a dominate FFA follicular fluid constituent. The effects were assessed of this substitution on the Akt/GSK3ß signaling pathway activity as well as the expressions of TRIB3 and FSHR at both the gene and protein levels by qPCR, Western blot and immunofluorescence staining analyses. Meanwhile, the individual effects of TRIB3 knockdown in KGN cells and p-AKT inhibitors were compared to determine the mechanisms of FFA-induced FSHR downregulation. RESULTS: The average FSH dose consuming per oocyte (FSH dose/oocyte) was elevated and Top embryo quality ratio was decreased in women with high levels of FFAs in their follicular fluid. In these women, the GC TRIB3 and ATF4 protein expression levels were upregulated which was accompanied by FSHR downregulation. Such upregulation was confirmed based on corresponding increases in their gene expression levels. On the other hand, the levels of p-Akt decreased while p-GSK3ß increased in the GCs. Moreover, TRIB3 knockdown reversed declines in FSHR expression and estradiol (E2) production in KGN cells treated with PA, which also resulted in increased p-Akt levels and declines in the p-GSK3ß level. In contrast, treatment of TRIB3-knockdown cells with an inhibitor of p-Akt (Ser473) resulted in rises in the levels of both p-GSK3ß as well as FSHR expression whereas E2 synthesis fell. CONCLUSIONS: During exposure to a high FFA content, TRIB3 can reduce FSHR expression through stimulation of the Akt/GSK3ß pathway in human GCs. This response may contribute to inducing oocyte maturation.

Transcript abundance of anti-Mullérian hormone and follicle-stimulating hormone receptor predicted superstimulatory response in embryo donor Holstein cows.

The goal was to investigate the relationship among mRNA expressions of anti-Mullérian hormone (AMH), follicle-stimulating hormone receptor (FSHR) and responses to superovulation (SO) in embryo donor dairy cows. Holstein cows (n = 19) were submitted to a standard SO protocol, with twice daily FSH treatments, and artificially inseminated. Prior to SO (Day 0), relative mRNA expressions of AMH and FSHR in blood were determined for all cows. Day 7 embryos were collected and were graded to determine superovulatory response for each donor. Results showed that relative mRNA expressions of AMH and FSHR were positively correlated (R2  = 0.94). Relative mRNA expressions of both AMH and FSHR were positively correlated with total embryos (R2  = 0.68 and 0.69, respectively), total transferable embryos (R2  = 0.92 and 0.97, respectively) and total grade 1 embryos (R2  = 0.54 and 0.59, respectively). Further, transcript abundances of AMH and FSHR positively associated with milk production of donor cows, and meanwhile, they were negatively associated with days in milk (DIM) at submission of cows to SO (p < .05) protocol. The relative mRNA expression of AMH was higher (p < .05) in donor cows <5 years of age. However, age of donor at superovulation did not influence mRNA expression of FSHR. Collectively, we infer that the mRNA expressions of AMH and FSHR prior to superovulation can predict donor cows' positive response to superovulation.

Mechanistic insight into how gonadotropin hormone receptor complexes direct signaling†.

Gonadotropin hormones and their receptors play a central role in the control of male and female reproduction. In recent years, there has been growing evidence surrounding the complexity of gonadotropin hormone/receptor signaling, with it increasingly apparent that the Gαs/cAMP/PKA pathway is not the sole signaling pathway that confers their biological actions. Here we review recent literature on the different receptor-receptor, receptor-scaffold, and receptor-signaling molecule complexes formed and how these modulate and direct gonadotropin hormone-dependent intracellular signal activation. We will touch upon the more controversial issue of extragonadal expression of FSHR and the differential signal pathways activated in these tissues, and lastly, highlight the open questions surrounding the role these gonadotropin hormone receptor complexes and how this will shape future research directions.

Engineered DNA Vaccination against Follicle-Stimulating Hormone Receptor Delays Ovarian Cancer Progression in Animal Models.

Ovarian cancer presents in 80% of patients as a metastatic disease, which confers it with dismal prognosis despite surgery and chemotherapy. However, it is an immunogenic disease, and the presence of intratumoral T cells is a major prognostic factor for survival. We used a synthetic consensus (SynCon) approach to generate a novel DNA vaccine that breaks immune tolerance to follicle-stimulating hormone receptor (FSHR), present in 50% of ovarian cancers but confined to the ovary in healthy tissues. SynCon FSHR DNA vaccine generated robust CD8+ and CD4+ cellular immune responses and FSHR-redirected antibodies. The SynCon FSHR DNA vaccine delayed the progression of a highly aggressive ovarian cancer model with peritoneal carcinomatosis in immunocompetent mice, and it increased the infiltration of anti-tumor CD8+ T cells in the tumor microenvironment. Anti-tumor activity of this FSHR vaccine was confirmed in a syngeneic murine FSHR-expressing prostate cancer model. Furthermore, adoptive transfer of vaccine-primed CD8+ T cells after ex vivo expansion delayed ovarian cancer progression. In conclusion, the SynCon FSHR vaccine was able to break immune tolerance and elicit an effective anti-tumor response associated with an increase in tumor-infiltrating T cells. FSHR DNA vaccination could help current ovarian cancer therapy after first-line treatment of FSHR+ tumors to prevent tumor recurrence.

Characterization of gonadotropin receptors Fshr and Lhr in Japanese medaka, Oryzias latipes.

Reproduction in vertebrates is controlled by the brain-pituitary-gonad axis, where the two gonadotropins follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) play vital parts by activating their cognate receptors in the gonads. The main purpose of this work was to study intra- and interspecies ligand promiscuity of teleost gonadotropin receptors, since teleost receptor specificity is unclear, in contrast to mammalian receptors. Receptor activation was investigated by transfecting COS-7 cells with either Fsh receptor (mdFshr, tiFshr) or Lh receptor (mdLhr, tiLhr), and tested for activation by recombinant homologous and heterologous ligands (mdFshßα, mdLhßα, tiFshßα, tiLhßα) from two representative fish orders, Japanese medaka (Oryzias latipes, Beloniformes) and Nile tilapia (Oreochromis niloticus, Cichliformes). Results showed that each gonadotropin preferentially activates its own cognate receptor. Cross-reactivity was detected to some extent as mdFshßα was able to activate the mdLhr, and mdLhßα the mdFshr. Medaka pituitary extract (MPE) stimulated CRE-LUC activity in COS-7 cells expressing mdlhr, but could not stimulate cells expressing mdfshr. Recombinant tiLhßα, tiFshßα and tilapia pituitary extract (TPE) could activate the mdLhr, suggesting cross-species reactivity for mdLhr. Cross-species reactivity was also detected for mdFshr due to activation by tiFshßα, tiLhßα, and TPE, as well as for tiFshr and tiLhr due to stimulation by mdFshßα, mdLhßα, and MPE. Tissue distribution analysis of gene expression revealed that medaka receptors, fshr and lhr, are highly expressed in both ovary and testis. High expression levels were found for lhr also in brain, while fshr was expressed at low levels. Both fshr and lhr mRNA levels increased significantly during testis development. Amino acid sequence alignment and three-dimensional modelling of ligands and receptors highlighted conserved beta sheet domains of both Fsh and Lh between Japanese medaka and Nile tilapia. It also showed a higher structural homology and similarity of transmembrane regions of Lhr between both species, in contrast to Fshr, possibly related to the substitution of the conserved cysteine residue in the transmembrane domain 6 in medaka Fshr with glycine. Taken together, this is the first characterization of medaka Fshr and Lhr using homologous ligands, enabling to better understand teleost hormone-receptor interactions and specificities. The data suggest partial ligand promiscuity and cross-species reactivity between gonadotropins and their receptors in medaka and tilapia.

The regulatory effect of genistein on P450 aromatase and follicle-stimulating hormone receptor in mouse experimental model of menopausal metabolic syndrome.

In order to investigate the mechanism of genistein (Gen) in the treatment of climacteric syndrome, an in vivo study was performed to investigate the beneficial effects of genistein on the expression of P450 aromatase (P450 arom) and follicle-stimulating hormone receptor (FSHR) in the mouse ovary and uterus. Fifty female ICR mice (45 ± 5g, n = 50), aged 12 months, were divided into the following five groups with 10 animals in each: blank control group (CG), low-dose genistein group (L-Gen), middle-dose genistein group (M-Gen) and high-dose genistein group (H-Gen) (received 15, 30 and 60 mg/kg of genistein, respectively), and oestrogen group (EG; received 0.5 mg/kg diethylstilbestrol). The expression levels of the FSHR protein were determined by an immunohistochemical staining method. The expression of P450 arom, Cytochrome P450 19 (CYP19) and FSHR was quantified by real-time PCR. Immunohistochemical results showed that the expression levels of the FSHR protein in the M-Gen (average stained area: 20.79) and the H-Gen (average stained area: 21.21) groups were significantly stronger than in the CG (average area was 17.24) group (p < .05). The expression levels of CYP19 mRNA and P450 arom were positively correlated with the dose of genistein. Specifically, the relative expression levels in the H-Gen and EG groups were more than 1.5 times higher than in the CG group (p < .05). Genistein played a significant role in regulating aromatase and FSHR gene expression to improve perimenopausal ovarian and uterine function.

D224V and S128Y mutation in FSHRED influence thumb movement differentially: An intricate insight gained by short-term molecular dynamics simulation.

Follicle-stimulating hormone-follicle-stimulating hormone receptor (FSH-FSHR) interaction is one of the most thoroughly studied signaling pathways primarily because of being implicated in sexual reproduction in mammals by way of maintaining gonadal function and sexual fertility. Despite material advances in understanding the role of point mutations, their mechanistic basis in FSH-FSHR signaling is still confined to mystically altered behavior of sTYS335 (sulfated tyrosine) yet lacking a substantial theory. To understand the structural basis of receptor modulation, we choose two behaviorally contradicting mutations, namely S128Y (activating) and D224Y (inactivating), found in FSH receptor responsible for ovarian hyperstimulation syndrome and ovarian dysgenesis, respectively. Using short-term molecular dynamics simulations, the atomic scale investigations reveal that the binding pattern of sTYS with FSH and movement of the thumb region of FSHR show distinct contrasting patterns in the two mutants, which supposedly could be a critical factor for differential FSHR behavior in activating and inactivating mutations.

Effects of FSHR polymorphisms on premature ovarian insufficiency in human beings: a meta-analysis.

BACKGROUND: Whether follicle-stimulating hormone receptor (FSHR) polymorphisms are implicated in premature ovarian insufficiency (POI) remains controversial. Thus, we performed this study to explore correlation between FSHR polymorphisms and POI in human beings. METHODS: Literature retrieve was conducted in PubMed, Medline, Embase and CNKI. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. RESULTS: Sixteen studies were enrolled for analyses. No significant relationship with POI was found for rs6165 and rs6166 polymorphisms in overall analyses. Further subgroup analyses revealed that rs6166 polymorphism was significantly associated with the risk of POI in Asians with both FEM and REM. Nevertheless, we failed to detect any significant associations with POI for other ethnicities. CONCLUSIONS: Our findings indicated that FSHR rs6166 polymorphism may serve as a potential genetic biomarker of POI in Asians, but not in other ethnicities.

Correlation of the Aryl Hydrocarbon Receptor with FSHR in Ovarian Cancer Patients.

Expression of the aryl hydrocarbon receptor (AhR) has been described in various tumor entities from different organs. However, its role in ovarian cancer has not been thoroughly investigated. We aimed to elucidate the prognostic impact of AhR, its correlation with the follicle-stimulating hormone receptor (FSHR), and their functional role in ovarian cancer. By immunohistochemistry, AhR staining was analyzed in a subset of 156 samples of ovarian cancer patients. AhR staining was assessed in the nucleus and the cytoplasm using the semi-quantitative immunoreactive score (IRS), and the scores were grouped into high- and low-level expression. AhR expression was detected in all histological subtypes, with clear cell ovarian cancer displaying the highest staining intensity. Low cytoplasmic expression of AhR was associated with longer overall survival (median 183.46 vs. 85.07 months; p = 0.021). We found a positive correlation between AhR and FSHR (p = 0.005). Ovarian cancer patients with high cytoplasmic AhR and concurrent FSHR expression had the worst outcome (median 69.72 vs. 43.32 months; p = 0.043). Consequently, low cytoplasmic AhR expression seems to be associated with improved survival in ovarian cancer patients. Our data suggest that AhR and FSHR levels correlate with each other, and their concurrent expression was observed in ovarian cancer patients with the worst outcome. Further investigation of the interaction of both receptors and their functional role might better predict the impact of endocrine therapy in ovarian cancer.

Expression of FSHR in chondrocytes and the effect of FSH on chondrocytes.

OBJECTIVE: Chondrocytes express many kinds of hormone receptors. The function of Follicle stimulating hormone (FSH) in the ovary is mediated by FSH receptor (FSHR). FSH receptor (FSHR) is found in many non-ovarian tissues, however it has been unclear if chondrocytes express FSHR. The purpose of this study is to determine it. METHODS: Mouse primary chondrocytes and human articular cartilage tissues were examined. The expression and sequence of FSHR mRNA by reverse transcription polymerase chain reaction (RT-PCR) and sequenced, respectively, and its protein expression was tested using western blotting and location was observed under immunofluorescence microscopy. Ovarian tissue was as a positive control. After FSH stimulated mouse chondrocytes, intracellular cAMP levels were assessed by ELISA, and gene expression relative to Mouse WNT Signaling Pathway was tested by RT2 Profiler PCR Arrays. RESULTS: FSHR was detected at the transcriptional level and confirmed to have the same sequence as that of ovary-derived mRNA of FSHR. FSHR proteins presented at the same line as the positive proteins of ovary, in mouse chondrocytes and human cartilage tissue, respectively. FSHR proteins were located at the cell membrane. Intracellular cAMP contents were significantly elevated up to 7-fold in mouse chondrocytes by forskolin (100 mM) (P < 0.001); however, different doses of FSH did not change the cAMP contents in mouse primary chondrocytes. RT2 Profiler PCR Arrays demonstrated that FSH could cause changes in gene expression among the 84 preordained genes, such as Fosl1, Rhou, and Dkk1, in mouse chondrocytes relative to the control. CONCLUSION: Mouse chondrocytes and human articular cartilage express functional FSHR. Moreover, FSH can act on chondrocytes and cause genetic changes.

Pharmacoperones for Misfolded Gonadotropin Receptors.

The gonadotropin receptors (luteinising hormone receptor; LHR and follicle-stimulating hormone receptor; FSHR) are G protein-coupled receptors (GPCRs) that play an important role in the endocrine control of reproduction. Thus genetic mutations that cause impaired function of these receptors have been implicated in a number of reproductive disorders. Disease-causing genetic mutations in GPCRs frequently result in intracellular retention and degradation of the nascent protein through misfolding and subsequent recognition by cellular quality control machinery. The discovery and development of novel compounds termed pharmacological chaperones (pharmacoperones) that can stabilise misfolded receptors and restore trafficking and plasma membrane expression are therefore of great interest clinically, and promising in vitro data describing the pharmacoperone rescue of a number of intracellularly retained mutant GPCRs has provided a platform for taking these compounds into in vivo trials. Thienopyrimidine small molecule allosteric gonadotropin receptor agonists (Org 42599 and Org 41841) have been demonstrated to have pharmacoperone activity. These compounds can rescue cell surface expression and in many cases, hormone responsiveness, of a range of retained mutant gonadotropin receptors. Should gonadotropin receptor selectivity of these compounds be improved, they could offer therapeutic benefit to subsets of patients suffering from reproductive disorders attributed to defective gonadotropin receptor trafficking.

Cloning, characterization, and molecular expression of gonadotropin receptors in European hake (Merluccius merluccius), a multiple-spawning species.

Teleosts have many spawning strategies and the hormonal control of gametogenesis is not well defined among the species or even, between sexes. To increase the knowledge of gonadotropin hormones, we studied the trend by gene expression of gonadotropin receptors in the follicles and testis at different maturity stages in the European hake (Merluccius merluccius), a multiple-spawning species. With this aim, fshr and lhr were sequenced, characterized, and their gene expression was quantified in oocytes and in testes at different maturity stages. The deduced amino acid sequences were used to phylogenetic studies and evidenced that both receptors are phylogenetically closed to other gadoid species. The gene expression of both receptors was poorly expressed in primary follicles, increased in vitellogenic follicles and to later decrease in hydrated oocytes. In testis, highest levels of lhr were detected during spermiation, while levels of fshr were constant. For the first time, a histological analysis was performed in European hake testes showing an unrestricted lobular testis. To better elucidate the mechanisms involved in the oogenesis of the European hake, the expression of estrogen receptor and cyp19a was also investigated displaying high levels in all classes of follicles. All these data allow to increase the knowledge on reproductive physiology of an important socioeconomical species and it seeks to shed more light on the role of the receptors here studied during gametogenesis of multiple-spawning fish.

Expression of the gonadotropin receptors during follicular development.

Background: Gonadotropins induce follicular development that leads to ovulation and luteinization. In women, the level of gonadotropins, along with the expression of their receptors, changes dynamically throughout the menstrual cycle. This study aimed to clarify the mechanisms underlying these phenomena. Methods: The literature was reviewed, including that published by the authors. Main findings Results: Follicle-stimulating hormone receptor expression in the granulosa cells was induced by androgens that were derived from growth differentiation factor-9-stimulated theca cells. In the theca cells, luteinizing hormone receptor (LHR) expression was noted from their appearance. In the granulosa cells, follicle-stimulating hormone (FSH) stimulation was essential for LHR expression. However, FSH alone was not sufficient to respond to the luteinizing hormone (LH) surge for oocyte maturation, ovulation, and subsequent luteinization. To achieve these stages, various local factors that were derived from the granulosa and theca cells in response to FSH and LH stimulation had to work synergistically in an autocrine/paracrine manner to strongly induce LHR expression. Following the LH surge, the LHR expression decreased markedly; miRNAs were involved in this transient LHR downregulation. Following ovulation, LHR expression drastically increased again toward luteinization. Conclusion: The expression of gonadotropin receptors is controlled by sophisticated and complicated systems; a breakdown of this system could lead to ovulation disorders.

Seasonal expressions of follicle-stimulating hormone receptor and luteinizing hormone receptor in the scented gland of the male muskrat (Ondatra zibethicus).

Accumulating evidence has shown that follicle-stimulating hormone (FSH) and luteinizing hormone (LH) may influence the functions of nongonadal tissues in addition to their classic target gonads. Our previous studies revealed that the scented glands of male muskrats expressed prolactin receptor, steroidogenic enzymes, and inhibin/activin subunits. To further seek the evidence of the activities of pituitary gonadotropins in scented glands, we investigated the seasonal expression patterns of FSH receptor (FSHR) and LH/choriogonadotropin receptor (LHCGR). The weight and size of scented glands during the breeding season were significantly higher than those during the nonbreeding season. Immunohistochemical studies showed that FSHR was present in the serous cells of scented glands, whereas LHCGR was present in the interstitial cells. The protein and mRNA expression levels of FSHR and LHCGR were significantly higher in the scented glands during the breeding season than those during the nonbreeding season. Importantly, the levels of circulating FSH and LH were remarkably higher during the breeding season. Taken together, these results suggested that gonadotropins may affect the function of muskrat scented gland via the locally expressed receptors in a season-dependent manner.

Effects of different dietary DHA:EPA ratios on gonadal steroidogenesis in the marine teleost, tongue sole (Cynoglossus semilaevis).

The present study was conducted to investigate the effects of dietary DHA and EPA on gonadal steroidogenesis in mature females and males, with a feeding trial on tongue sole, a typical marine teleost with sexual dimorphism. Three experimental diets differing basically in DHA:EPA ratio, that is, 0·68 (diet D:E-0·68), 1·09 (D:E-1·09) and 2·05 (D:E-2·05), were randomly assigned to nine tanks of 3-year-old tongue sole (ten females and fifteen males in each tank). The feeding trail lasted for 90 d before and during the spawning season. Fish were reared in a flowing seawater system and fed to apparent satiation twice daily. Compared with diet D:E-0·68, diet D:E-1·09 significantly enhanced the oestradiol production in females, whereas diet D:E-2·05 significantly enhanced the testosterone production in males. In ovaries, diet D:E-1·09 induced highest mRNA expression of follicle-stimulating hormone receptor (FSHR), steroidogenic acute regulatory protein, 17α-hydroxylase (P450c17) and 3ß-hydroxysteroid dehydrogenase (3ß-HSD). In testes, diet 2·05 resulted in highest mRNA expression of FSHR, cholesterol side-chain cleavage enzyme, P450c17 and 3ß-HSD. Fatty acid profiles in fish tissues reflected closely those of diets. Female fish had more gonadal EPA content but less DHA content than male fish, whereas there was a reverse observation in liver. In conclusion, the dietary DHA:EPA ratio, possibly combined with the dietary EPA:arachidonic acid ratio, differentially regulated sex steroid hormone synthesis in mature female and male tongue soles. Females seemed to require more EPA but less DHA for the gonadal steroidogenesis than males. The results are beneficial to sex-specific nutritive strategies in domestic teleost.

Disruption of the Reproductive Axis in Freshwater Fish, Catla catla, After Bisphenol-A Exposure.

Environmental estrogens such as bisphenol-A (BPA) cause reproductive disorders in many vertebrate species, especially fish. BPA is used extensively in the manufacture of plastic and plastic products, epoxy resins, and dental sealants. The presence of BPA in sewage and surface water raises a potential threat to aquatic populations. In the present study, we investigated the effects of BPA on ovarian histology and transcription of key genes involved in reproduction. Adult female Catla catla were exposed to graded concentrations of BPA (10, 100, 1000 µg/l) for 14 days. Ovary histology and expression of steroidogenic acute regulatory protein (star), ovary aromatase (cyp19a), follicle-stimulating hormone receptor (fshr), and luteinizing hormone receptor (lhr) were evaluated in ovary of female fish after 14 days. Fish ovaries from the control and 10 µg/l BPA exposed groups included primary oocytes (POCs), while fish exposed to higher concentrations of BPA (100 and 1000 µg/l) contained tertiary and mature oocytes with increased numbers of atretic follicles. Significant increases in mRNA transcripts of star were observed in fish exposed to 100 and 1000 µg/l BPA. A 15-fold increase in the expression of ovary aromatase (cyp19a) was detected in fish exposed to 100 µg/l BPA. fshr increased in a dose-dependent manner. Increases in the expression of lhr, although not statistically significant, were observed in fish exposed to 100 and 1000 µg/l BPA when compared to control. The results of the present study indicate that BPA causes alterations in the expression patterns of genes involved in the reproductive pathway, which may lead to negative effects on the reproductive system in female fish.

The role of gene polymorphisms and AMH level in prediction of poor ovarian response in Egyptian women undergoing IVF procedure.

OBJECTIVE: The aim of this study is to assess the role of AMH in prediction of poor ovarian response as well as the relation between ESR 2 (+ 1730G>A) (rs4986938) and FSHR p.Thr307Ala (c.919A>G, rs6165) SNPs and the poor ovarian response in Egyptian women undergoing IVF procedure. Discovering the genetic variants associated with ovarian response is an important step towards individualized pharmacogenetic protocols of ovarian stimulation. METHODS: We performed a prospective study on 216 young women with unexplained infertility. Ovarian stimulation was performed according to the GnRH antagonist protocol with a fixed daily morning dose of human menopausal gonadotrophin (HMG). The estrogen receptor (ESR2) (+ 1730G>A) (rs4986938) and FSH receptor p.Thr307Ala (c.919A>G, rs6165) single nucleotide polymorphisms (SNPs) were detected by real-time polymerase chain reaction. Serum FSH, Estradiol (E2) and anti-Müllerian hormone (AMH) levels were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: This study revealed that the low AMH level was highly significantly related to the poor ovarian response (p < 0.001). Furthermore, the frequency of the ESR2 (AA) genotype and the FSHR (Ala307Ala) genotypes were highly significantly associated with the poor ovarian response (p < 0.001). CONCLUSION: The AMH level in combination with the ESR2 and the FSHR gene polymorphisms predict the poor ovarian response to COH in Egyptian women. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02640976.

Protein and mRNA expression of follicle-stimulating hormone receptor and luteinizing hormone receptor during the oestrus in the yak (Bos grunniens).

Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) have a central role in follicle growth, maturation and oestrus, but no clear pathway in the seasonal oestrus of yak (Bos grunniens) has been found. To better understand the role of FSH and LH in seasonal oestrus in the yak, six yaks were slaughtered while in oestrus, and the pineal gland, hypothalamus, pituitary gland, and gonads were collected. Using real-time PCR and immunohistochemical assays, we determined the mRNA and protein expression of the FSH and LH receptors (FSHR and LHR) in these organs. The analysis showed that the FSHR mRNA expression level was higher in the pituitary gland tissue compared with LHR (p < .01) during oestrus. By contrast, there was low expression of FSHR and LHR mRNA in the pineal gland and hypothalamus. FSHR mRNA expression was higher than that of LHR (p < .05) in the ovary, whereas LHR mRNA expression was higher than that of FSHR (p < .01) in the uterus. FSHR and LHR proteins were located in the pinealocyte, synaptic ribbon and synaptic spherules of the pineal gland and that FSH and LH interact via nerve fibres. In the hypothalamus, FSHR and LHR proteins were located in the magnocellular neurons and parvocellular neurons. FSHR and LHR proteins were localized in acidophilic cells and basophilic cells in the pituitary gland, and in surface epithelium, stromal cell and gland epithelium in the uterus. In the ovary, FSHR and LHR protein were present in the ovarian follicle. Thus, we concluded that FSHR and LHR are located in the pineal gland, hypothalamus, pituitary and gonad during oestrus in the yak. However, FSHR was mainly expressed in the pituitary gland and ovaries, whereas LHR was mainly expressed in the pituitary gland and uterus.

A novel follicle-stimulating hormone receptor mutation causing primary ovarian failure: a fertility application of whole exome sequencing.

STUDY QUESTION: Can whole exome sequencing (WES) and in vitro validation studies be used to find the causative genetic etiology in a patient with primary ovarian failure and infertility? SUMMARY ANSWER: A novel follicle-stimulating hormone receptor (FSHR) mutation was found by WES and shown, via in vitro flow cytometry studies, to affect membrane trafficking. WHAT IS KNOWN ALREADY: WES may diagnose up to 25-35% of patients with suspected disorders of sex development (DSD). FSHR mutations are an extremely rare cause of 46, XX gonadal dysgenesis with primary amenorrhea due to hypergonadotropic ovarian failure. STUDY DESIGN, SIZE, DURATION: A WES study was followed by flow cytometry studies of mutant protein function. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study subjects were two Turkish sisters with hypergonadotropic primary amenorrhea, their parents and two unaffected sisters. The affected siblings and both parents were sequenced (trio-WES). Transient transfection of HEK 293T cells was performed with a vector containing wild-type FSHR as well as the novel FSHR variant that was discovered by WES. Cellular localization of FSHR protein as well as FSH-stimulated cyclic AMP (cAMP) production was evaluated using flow cytometry. MAIN RESULTS AND THE ROLE OF CHANCE: Both affected sisters were homozygous for a previously unreported missense mutation (c.1222G>T, p.Asp408Tyr) in the second transmembrane domain of FSHR. Modeling predicted disrupted secondary structure. Flow cytometry demonstrated an average of 48% reduction in cell-surface signal detection (P < 0.01). The mean fluorescent signal for cAMP (second messenger of FSHR), stimulated by FSH, was reduced by 50% in the mutant-transfected cells (P < 0.01). LIMITATIONS, REASONS FOR CAUTION: This is an in vitro validation. All novel purported genetic variants can be clinically reported only as 'variants of uncertain significance' until more patients with a similar phenotype are discovered with the same variant. WIDER IMPLICATIONS OF THE FINDINGS: We report the first WES-discovered FSHR mutation, validated by quantitative flow cytometry. WES is a valuable tool for diagnosis of rare genetic diseases, and flow cytometry allows for quantitative characterization of purported variants. WES-assisted diagnosis allows for treatments aimed at the underlying molecular etiology of disease. Future studies should focus on pharmacological and assisted reproductive treatments aimed at the disrupted FSHR, so that patients with FSH resistance can be treated by personalized medicine. STUDY FUNDING/COMPETING INTERESTS: E.V. is partially funded by the DSD Translational Research Network (NICHD 1R01HD068138). M.S.B. is funded by the Neuroendocrinology, Sex Differences and Reproduction training grant (NICHD 5T32HD007228). The authors have no competing interests to disclose.