Allosteric modulation of gonadotropin receptors.

Gonadotropins regulate reproductive functions by binding to G protein-coupled receptors (FSHR and LHCGR) expressed in the gonads. They activate multiple, cell-specific signalling pathways, consisting of ligand-dependent intracellular events. Signalling cascades may be modulated by synthetic compounds which bind allosteric sites of FSHR and LHCGR or by membrane receptor interactions. Despite the hormone binding to the orthosteric site, allosteric ligands, and receptor heteromerizations may reshape intracellular signalling pattern. These molecules act as positive, negative, or neutral allosteric modulators, as well as non-competitive or inverse agonist ligands, providing a set of new compounds of a different nature and with unique pharmacological characteristics. Gonadotropin receptor allosteric modulation is gathering increasing interest from the scientific community and may be potentially exploited for clinical purposes. This review summarizes the current knowledge on gonadotropin receptor allosteric modulation and their potential, clinical use.

Sheep with ovarian androgen excess have fibrosis and follicular arrest with increased mRNA abundance for steroidogenic enzymes and gonadotropin receptors.

An androgen excess ovarian micro-environment may limit follicle progression in sheep. Two populations of ewes with divergent follicular fluid androstenedione (A4) were identified in a flock in Jordan: High A4; (A4) ≥ 30 ng/mL, (N = 12) or Control A4 (Control); A4 ≤ 15 ng/mL; (N = 12). We hypothesized High A4 ewes would have increased steroidogenic enzyme mRNA abundance, inflammation, and follicular arrest. Messenger RNA abundance for steroidogenic enzymes StAR, CYP17A1, CYP11A1, and HSD3B1 were increased in theca cells while CYP17A1, CYP19A1, and HSD3B1 were increased in granulosa cells in High A4 ewes compared to Control. Gonadotropin receptor mRNA expression for LHCGR was increased in theca and FSHR in granulosa in High A4 ewes. Messenger RNA expression of FOS when reduced, increases expression of CYP17A1 which was observed in High A4 granulosa cells compared to Control. Furthermore, High A4 ewes had greater numbers of primordial follicles (P < 0.001) and fewer developing follicles compared to Control before, and after 7 d of culture, indicating follicular arrest was not alleviated by cortex culture. Increased fibrosis in the ovarian cortex was detected in High A4 ewes relative to Control (P < 0.001) suggesting increased inflammation and altered extracellular matrix deposition. Thus, this High A4 ewes population has similar characteristics to High A4 cows and women with polycystic ovary syndrome suggesting that naturally occurring androgen excess occurs in multiple species and may be a causative factor in follicular arrest and subsequent female sub- or infertility.

Excess androgen (androstenedione; A4) in ewes can result in ovarian follicular arrest and fibrosis contributing to anovulation in sheep. We have identified a naturally occurring ovarian A4 excess in a sheep population with similar characteristics to High A4 cows, both of which are similar to that in women with polycystic ovary syndrome indicating that several mammalian species experience naturally occurring androgen excess resulting in infertility or follicle arrest. Somatic cells, theca and granulosa, surrounding the egg in High A4 ewes had increased expression of steroidogenic enzymes, similar to that seen in High A4 cows, permitting more ovarian cells to manufacture androgens, which may be the cause of androgen excess. Thus, naturally occurring androgen-excess in domestic livestock females can be utilized as models to research the causes of androgen excess and determine the mechanisms that result in follicular arrest and sub- or infertility.

Regulation of antral follicular growth by an interplay between gonadotropins and their receptors.

Knowledge of the growth and maturation of human antral follicles is based mainly on concepts and deductions from clinical observations and animal models. To date, new experimental approaches and in vitro data contributed to a deep comprehension of gonadotropin receptors' functioning and may provide new insights into the mechanisms regulating still unclear physiological events. Among these, the production of androgen in the absence of proper LH levels, the programming of follicular atresia and dominance are some of the most intriguing. Starting from evolutionary issues at the basis of the gonadotropin receptor signal specificity, we draw a new hypothesis explaining the molecular mechanisms of the antral follicular growth, based on the modulation of endocrine signals by receptor-receptor interactions. The "heteromer hypothesis" explains how opposite death and life signals are delivered by gonadotropin receptors and other membrane partners, mediating steroidogenesis, apoptotic events, and the maturation of the dominant follicle.

Roles of Gonadotropin Receptors in Sexual Development of Medaka.

The gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), are secreted from the pituitary and bind to the FSH receptor (FSHR) and LH receptor (LHR) to regulate gonadal development in vertebrates. Previously, using fshr-knockout (KO) medaka (Oryzias latipes), we demonstrated that FSH regulates ovarian development by elevating estrogen levels. However, the lhr-KO phenotype in medaka is poorly characterized. Here, we generated lhr-KO medaka using the transcription activator-like effector nuclease (TALEN) technique. We analyzed its phenotype and that of fshr-KO, lhr;fshr double-heterozygotes (double-hetero), and double-KO fish. All genetically male medaka displayed normal testes and were fertile, whereas fshr-KO and double-KO genetically female fish displayed small ovaries containing many early pre-vitellogenic oocytes and were infertile. Although lhr-KO genetically female fish had normal ovaries with full-grown oocytes, ovulation did not occur. Levels of 17α,20ß-dihydroxy-4-pregnen-3-one, which is required for meiotic maturation of oocytes and sperm maturation in teleost fish, were significantly decreased in all KO female medaka ovaries except for double-heteros. Further, 17ß-estradiol levels in fshr-KO and double-KO ovaries were significantly lower than those in double-heteros. These findings indicate that LH is necessary for oocyte maturation and FSH is necessary for follicle development, but that neither are essential for spermatogenesis in medaka.

Early preantral follicles of the domestic cat express gonadotropin and sex steroid signaling potential.

Key biomolecular processes, which regulate primordial ovarian follicle dormancy and early folliculogenesis in mammalian ovaries, are not fully understood. The domestic cat is a useful model to study ovarian folliculogenesis and is the most relevant for developing in vitro growth methods to be implemented in wild felid conservation breeding programs. Previously, RNA-sequencing of primordial (PrF), primary (PF), and secondary follicle (SF) samples from domestic cat implicated ovarian steroidogenesis and steroid reception during follicle development. Here, we aimed to identify which sex steroid biosynthesis and metabolism enzymes, gonadotropin receptors, and sex steroid receptors are present and may be potential regulators. Differential gene expression, functional annotation, and enrichment analyses were employed and protein localization was studied too. Gene transcripts for PGR, PGRMC1, AR (steroid receptors), CYP11A1, CYP17A1, HSD17B1 and HSD17B17 (steroidogenic enzymes), and STS (steroid metabolizing enzyme) were significantly differentially expressed (Q values of ≤0.05). Differential gene expression increased in all transcripts during follicle transitions apart from AR which decreased by the secondary stage. Immunohistochemistry localized FSHR and LHCGR to oocytes at each stage. PGRMC1 immunostaining was strongest in granulosa cells, whereas AR was strongest in oocytes throughout each stage. Protein signals for steroidogenic enzymes were only detectable in SFs. Products of these significantly differentially expressed genes may regulate domestic cat preantral folliculogenesis. In vitro growth could be optimized as all early follicles express gonadotropin and steroid receptors meaning hormone interaction and response may be possible. Protein expression analyses of early SFs supported its potential for producing sex steroids.

Misfolded G Protein-Coupled Receptors and Endocrine Disease. Molecular Mechanisms and Therapeutic Prospects.

Misfolding of G protein-coupled receptors (GPCRs) caused by mutations frequently leads to disease due to intracellular trapping of the conformationally abnormal receptor. Several endocrine diseases due to inactivating mutations in GPCRs have been described, including X-linked nephrogenic diabetes insipidus, thyroid disorders, familial hypocalciuric hypercalcemia, obesity, familial glucocorticoid deficiency [melanocortin-2 receptor, MC2R (also known as adrenocorticotropin receptor, ACTHR), and reproductive disorders. In these mutant receptors, misfolding leads to endoplasmic reticulum retention, increased intracellular degradation, and deficient trafficking of the abnormal receptor to the cell surface plasma membrane, causing inability of the receptor to interact with agonists and trigger intracellular signaling. In this review, we discuss the mechanisms whereby mutations in GPCRs involved in endocrine function in humans lead to misfolding, decreased plasma membrane expression of the receptor protein, and loss-of-function diseases, and also describe several experimental approaches employed to rescue trafficking and function of the misfolded receptors. Special attention is given to misfolded GPCRs that regulate reproductive function, given the key role played by these particular membrane receptors in sexual development and fertility, and recent reports on promising therapeutic interventions targeting trafficking of these defective proteins to rescue completely or partially their normal function.

Ovarian gene expression in collared peccary (Pecari tajacu Linnaeus, 1758) subjected to gonadotropin stimulation protocols.

Ovarian response of collared peccaries (Pecari tajacu), after hormonal stimulation with gonadotropin association (eCG/hCG), was accessed by both gene expression and follicular development. Thus, collared peccaries (n = 8) were treated with the dose used for sows (swine dose, SWD) or with dose adjusted for peccary's weight (allometric dose, ALD). The gene expression of receptors was evaluated for both gonadotropins (FSHR and LHCGR) and growth factors (proteins codified by TGFßR-1, BMPR1-A and BMPR2 genes) in antral follicles, cortex and corpora haemorrhagica (CH). Five days after gonadotropin injection, all females presented CH. The ovulation rate was similar (p > .05) between SWD (4.00 ± 1.17) and ALD (2.50 ± 0.43) group. The total number of follicles per animal and amounts of small (<3 mm), medium (3-5 mm) and large (>5 mm) follicles was similar among groups. However, SWD produced large follicles heavier than ALD group, as accessed by weight of follicular wall biopsies. Ovarian follicles expressed both gonadotropin and growth factor receptors at levels which are independent from gonadotropin dose. In conclusion, the two gonadotropin doses (SWD and ALD) can be used for ovarian stimulation of collared peccary. Additionally, FSH and growth factors (TGFßR-1, BMPR1-A and BMPR2) receptors are more expressed in the early follicle development, while LH receptor seems to be more important in the final of follicular growth.

ß-arrestin 2 Is a Prognostic Factor for Survival of Ovarian Cancer Patients Upregulating Cell Proliferation.

Establishing reliable prognostic factors as well as specific targets for new therapeutic approaches is an urgent requirement in advanced ovarian cancer. For several tumor entities, the ubiquitously spread scaffold protein ß-arrestin 2, a multifunctional scaffold protein regulating signal transduction and internalization of activated G protein-coupled receptors (GPCRs), has been considered with rising interest for carcinogenesis. Therefore, we aimed to elucidate the prognostic impact of ß-arrestin 2 and its functional role in ovarian cancer. ß-arrestin 2 expression was analyzed in a subset of 156 samples of ovarian cancer patients by immunohistochemistry. Cytoplasmic expression levels were correlated with clinical as well as pathological characteristics and with prognosis. The biologic impact of ß-arrestin 2 on cell proliferation and survival was evaluated, in vitro. Following transient transfection by increasing concentrations of plasmid encoding ß-arrestin 2, different cell lines were evaluated in cell viability and death. ß-arrestin 2 was detected in all histological ovarian cancer subtypes with highest intensity in clear cell histology. High ß-arrestin 2 expression levels correlated with high-grade serous histology and the expression of the gonadotropin receptors FSHR and LHCGR, as well as the membrane estrogen receptor GPER and hCGß. Higher cytoplasmic ß-arrestin 2 expression was associated with a significantly impaired prognosis (median 29.88 vs. 50.64 months; P = 0.025). Clinical data were confirmed in transfected HEK293 cells, human immortalized granulosa cell line (hGL5) and the ovarian cancer cell line A2780 in vitro, where the induction of ß-arrestin 2 cDNA expression enhanced cell viability, while the depletion of the molecule by siRNA resulted in cell death. Reflecting the role of ß-arrestin 2 in modulating GPCR-induced proliferative and anti-apoptotic signals, we propose ß-arrestin 2 as an important prognostic factor and also as a promising target for new therapeutic approaches in advanced ovarian cancer.

Molecular characterization of two Russian sturgeon gonadotropin receptors: Cloning, expression analysis, and functional activity.

Sturgeons are being used in aquaculture because wild populations are now endangered due to overfishing for caviar. A challenge in working with sturgeon as an aquacultured species is its long and slow reproductive development. Reproduction is a hormonally regulated process that involves hierarchical signaling between the brain, pituitary gland, and gonads. In an effort to better understand the hormonal regulation of sturgeon reproduction, we have cloned the Russian sturgeon (st), Acipenser gueldenstaedtii, luteinizing hormone receptor (stLHR) and follicle stimulating hormone receptor (stFSHR) and measured their expression from previtellogenic to mature ovarian follicles. Sturgeon LHR and FSHR expression was elevated in early-vitellogenic and mature follicles compared with pre-vitellogenic and mid-vitellogenic follicles, and only LHR expression increased during late-vitellogenesis. Recombinant sturgeon FSH and LH both activated sturgeon LHR and FSHR in a cAMP reporter assay. Further molecular characterization of these receptors was accomplished by in silico modeling and cAMP reporter assays using heterologous recombinant gonadotropins from human and piscine species. There was no apparent trend in heterologous LH and/or FSH activation of the sturgeon LHR or FSHR. These data suggest that permissive activation of LHR and FSHR are a consequence of some yet undetermined biological characteristic(s) of different piscine species.

The effects of 11-ketotestosterone implants on transcript levels of gonadotropin receptors, and foxl2 and dmrt1 genes in the Previtellogenic ovary of cultured beluga (Huso huso).

The in vivo effect of 11-ketotestosterone (11KT) on transcript levels of the gonadotropin receptors (fshr and lhr) and sex differentiation-related genes (dmrt1 and foxl2) was examined in the ovaries of immature female beluga. For this purpose, six fish were treated with implants containing 2.5 mg 11KT and a placebo group of six females of the same age and gametogenic stage were given a blank implant. The implants were intraperitoneally inserted into 4-year-old females at the previtellogenic stage (mean body weight 5580 ± 165 g) and maintained under culture conditions for 8 weeks. Ovary samples for gene expression analysis of lhr, fshr, dmrt1 and foxl2 were collected by biopsy at 3 and 8 weeks post implantation. Diameters of oocytes increased in response to 11KT treatment, both at 3 and at 8 weeks post implantation, but no obvious changes were evident in cytology. Three weeks of 11KT treatment did not affect target gene expression, but a tendency for a time-dependent decrease of lhr and dmrt1 mRNA levels was observed in both treatment and placebo groups. By 8 weeks of treatment, however, 11KT implants provoked the upregulation of fshr and foxl2 transcript levels. Furthermore, lhr and dmrt1 transcript abundances recovered by 8 weeks of exposure in both blank- and 11KT-implanted beluga. These results suggest that 11KT, either directly or indirectly, may affect gametogenesis and regulate some key components of the reproductive axis in female beluga.

Ontogenetic and tissue-specific expression of gonadotropin-inhibitory hormone (GnIH) and its receptors in Catla catla.

Gonadotropin-inhibitory hormone (GnIH) is an RFamide peptide, and its role in reproduction is well studied from fish to mammals, but very few reports are available about the function of GnIH during larval development. In this study, we examined the GnIH and GnIH receptors (GnIHRs) expression from embryogenesis to adult stage and tissue-specific expression in adult Catla catla using quantitative real-time (qRT) PCR. The qRT PCR analysis of GnIH mRNA during ontogenetic development showed the increasing trend from early developmental stages to the adult stage with the highest expression in 24 months fish. However, the expression of two GnIH receptors, GnIHR1 and GnIHR2 also increased from larval stages to the adults with a peak at 17 days post-hatching, while GnIHR3 showed the higher mRNA expression during embryogenesis and then decreasing gradually. Tissue distribution analysis of GnIH showed the highest mRNA expression of GnIH in the brain, followed by gonads of both the sexes. GnIHR1 and GnIHR2 were also highly expressed in the brain and gonads of both the sexes, while GnIHR3 showed the highest expression in gonads of both the sexes without any expression in the brain. These results suggest that the brain is the primary site of action for GnIH, GnIHR1 and GnIHR2, while gonads for GnIHR3.

Role of vaspin in porcine ovary: effect on signaling pathways and steroid synthesis via GRP78 receptor and protein kinase A†.

Vaspin, visceral-adipose-tissue-derived serine protease inhibitor, is involved in the development of obesity, insulin resistance, inflammation, and energy metabolism. Our previous study showed vaspin expression and its regulation in the ovary; however, the role of this adipokine in ovarian cells has never been studied. Here, we studied the in vitro effect of vaspin on various kinase-signaling pathways: mitogen-activated kinase (MAP3/1), serine/threonine kinase (AKT), signal transducer and activator of transcription 3 (STAT3) protein kinase AMP (PRKAA1), protein kinase A (PKA), and on expression of nuclear factor kappa B (NFKB2) as well as on steroid synthesis by porcine ovarian cells. By using western blot, we found that vaspin (1 ng/ml), in a time-dependent manner, increased phosphorylation of MAP3/1, AKT, STAT3, PRKAA1, and PKA, while it decreased the expression of NFKB2. We observed that vaspin, in a dose-dependent manner, increased the basal steroid hormone secretion (progesterone and estradiol), mRNA and protein expression of steroid enzymes using real-time PCR and western blot, respectively, and the mRNA of gonadotropins (FSHR, LHCGR) and steroids (PGR, ESR2) receptors. The stimulatory effect of vaspin on basal steroidogenesis was reversed when ovarian cells were cultured in the presence of a PKA pharmacological inhibitor (KT5720) and when GRP78 receptor was knocked down (siRNA). However, in the presence of insulin-like growth factor type 1 and gonadotropins, vaspin reduced steroidogenesis. Thus, vaspin, by activation of various signaling pathways and stimulation of basal steroid production via GRP78 receptor and PKA, could be a new regulator of porcine ovarian function.

Traditional Medicine (Mahuang-Tang) Improves Ovarian Dysfunction and the Regulation of Steroidogenic Genes in Letrozole-Induced PCOS Rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Mahuang-Tang (MHT) has traditionally been used in Asia to treat a variety of diseases, such as fever without sweating, joint pain, lower back pain, asthma, and gynecological conditions. Polycystic ovary syndrome (PCOS) is a kind of gynecological disease that causes amenorrhea, infertility, and menopausal and urogenital disorders that could benefit from MHT treatment. AIM OF THE STUDY: In this study, we examined the effects of MHT on ovarian hormones and steroidogenic enzymes in female PCOS rats. METHODS AND RESULTS: The PCOS rat model was induced by Letrozole, and an in vivo evaluation of whether the dietary consumption of MHT improved the PCOS-like symptoms was conducted. The luteinizing hormone (LH) level and luteinizing hormone/follicular-stimulating hormone (LH/FSH) ratio increased in PCOS rats but decreased following MHT treatment. In the PCOS rats, the reduced estrogen level was restored to that of normal controls with MHT treatment in serum. The transcription level(s) of gonadotropin receptors (Fshr and Lhr), steroid receptors (Pgr, and Esr1) and steroidogenic enzymes (Cyp19a1, Hsd3b1, Hsd17a1, and Cyp11a1) changed under the PCOS condition, and were regulated by MHT treatment in the ovaries of PCOS rats. The reproductive tissues of Letrozole-induced PCOS rats were restored into estrogenic condition from androgen environments. CONCLUSION: These results suggest that MHT ameliorates the symptoms of PCOS by improving the dysregulation of ovarian steroids and steroidogenic enzymes in PCOS rats.

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.

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.

Association between duration of obesity and severity of ovarian dysfunction in rat-cafeteria diet approach.

Consumption of unhealthy, energy-dense palatable food during early age leads to obesity in children and the onset of obesity during childhood has a profound effect on the reproductive health of women. In this study, the mechanism underlying diet-induced obesity on ovarian dysfunction was studied by exposing rats to cafeteria diet (CAFD) for two different durations. For that purpose, 21-day-old female Sprague Dawley rats were fed ad libitum with a standard diet (control group) and a cafeteria diet (CAFD group) for a period of 20 weeks (20 W) and 32 weeks (32 W). We observed obesity, hyperglycemia, hyperlipidemia, hyperleptinemia and hypoadiponectinemia in CAFD fed groups. Hyperinsulinemia, hypergonadotrophism, hypertestosteronemia and hyperprogesteronemia were observed in the 20 W-CAFD group. Conversely, in the 32 W-CAFD group hypersecretion declined to hyposecretion. The levels of estradiol remained low during both time periods. The duration of estrous cycle was extended in the CAFD fed rats. The ovary weight was higher in the 20 W-CAFD fed rats but it was drastically reduced over a longer duration cafeteria diet feeding. In the 20 W-CAFD fed rats, the protein levels of LHR, StAR, CYP11A1, 3ß-HSD and 17ß-HSD were increased but FSHR and CYP19A1 levels were decreased in the ovary. On the other hand, gonadotropin receptor and the protein levels of steroidogenic enzymes were decreased in the ovary of 32 W-CAFD fed rats. We conclude that the duration of energy-dense diet consumption has differential regulatory mechanism in altering the ovarian steroid production. In 20 W-CAFD fed rats, hypergonadotropic condition was observed whereas, 32 W-CAFD consumption induced hypogonadotropic hypogonadism.

Effects of high and low temperature on expression of GnIH, GnIH receptor, GH and PRL genes in the male grass puffer during breeding season.

Gonadotropin-inhibitory hormone (GnIH) is a multifunctional hypophysiotropic neurohormone and has a stimulatory role in the control of reproduction in the grass puffer. To clarify the neuroendocrine mechanisms underlying the effect of changes in water temperature on reproduction in fish, we previously revealed that, in parallel to gonadal regression, both low and high temperature significantly decreased the expressions of the genes encoding kisspeptin (kiss2), kisspeptin receptor (kiss2r), gonadotropin-releasing hormone 1 (gnrh1) in the brain and gonadotropin (GTH) subunits (fshb and lhb) in the pituitary of sexually mature male grass puffer. In this study, we examined the changes in expression of gnih and GnIH receptor gene (gnihr) in the brain and pituitary along with the genes for growth hormone (gh) and prolactin (prl) in the pituitary of male grass puffer exposed to low temperature (14 °C), normal temperature (21 °C, as initial control) and high temperature (28 °C) conditions for 7 days. The levels of gnih and gnihr mRNAs were significantly decreased in both low and high temperature conditions compared to normal temperature in the brain and pituitary. Similarly, the gh mRNA levels were significantly decreased in both low and high temperature conditions. The prl mRNAs showed no significant changes at high temperature, whereas drastically decreased at low temperature possibly by dysfunctional cold stress. Taken together, the present results suggest that, in addition to the inhibitory effect of temperature changes on the Kiss2/GnRH1/GTH system, the suppression of GnIH/GH system may also be involved in the termination of reproduction by high temperature at the end of breeding season.

Expressional regulation of gonadotropin receptor genes and androgen receptor genes in the eel testis.

Receptors for follicle-stimulating hormone (Fshr), luteinizing hormone (Lhcgr1 and Lhcgr2) and androgens (Ara and Arb) transduce the hormonal signals that coordinate spermatogenesis, but the factors that regulate the abundance of these transducers in fish testes remain little-understood. To mend this paucity of information, we first determined changes in transcript abundance for these receptors (fshr, lhcgr1, ara and arb) during spermatogenesis induced by human chorionic gonadotropin (hCG) injection in the eel, Anguilla australis. We related our findings to testicular production of the fish androgen, 11-ketotestosterone (11-KT), and to the levels of the transcripts encoding steroidogenic acute regulatory protein (star) and 11ß-hydroxylase (cyp11b), and subsequently evaluated the effects of hCG or 11-KT on mRNA levels of these target genes in vitro. Testicular 11-KT production was greatly increased by hCG treatment, both in vivo and in vitro, and associated with up-regulation of star and cyp11b transcripts. In situ hybridization indicated that testicular fshr mRNA levels were higher in the early stages of hCG-induced spermatogenesis, while lhcgr1 transcripts were most abundant later, once spermatids were observed. In vitro experiments further showed that hCG and its steroidal mediator 11-KT significantly increased fshr transcript abundance. These data provide new angles on the interactions between gonadotropin and androgen signaling during early spermatogenesis. Increases in levels of 11-KT following hCG injection elevated testicular fshr mRNA levels augmenting Fsh sensitivity in the testis. This evidence is suggestive of a positive feedback loop between gonadotropins and 11-KT that may be key to regulating early spermatogenesis in fish.

Interaction of starfish gonadotropin with its receptor: Effect of chimeric relaxin-like gonad-stimulating peptides.

A relaxin-like gonad-stimulating peptide (RGP) of starfish Patiria (Asterina) pectinifera is the first identified invertebrate gonadotropin for final gamete maturation. Recently, we found three orthologs of RGP in the class Asteroida; PpeRGP in P. pectinifera, AamRGP in Asterias amurensis, and AjaRGP in Aphelasterias japonica. In this study, nine kinds of RGP derivatives with exchanged each A- and B-chain were synthesized chemically to analyze the interaction of RGP with its receptor. Among these RGP derivatives, PpeRGP and its chimeric RGPs with B-chains from AamRGP or AjaRGP could induce oocyte maturation and ovulation in P. pectinifera ovaries. In contrast, other RGP derivatives were failed to induce spawning in P. pectinifera ovaries. Circular dichroism spectra of PpeRGP were similar to those of chimeric RGPs with the B-chains from AamRGP or AjaRGP. Furthermore, the predicted three-dimensional structure models of the B-chains from RGP derivatives have almost the same conformation. These findings suggest that the B-chain of PpeRGP is involved in binding to its receptor. Thus, it is likely that the A-chain of AamRGP or AjaRGP disturbs the binding of the PpeRGP B-chain to its receptor.

Expression of gonadotropin subunit and gonadotropin receptor genes in wild female New Zealand shortfinned eel (Anguilla australis) during yellow and silver stages.

Despite tremendous importance of follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) as primary controllers of reproductive development, information on the expression profiles of the genes encoding gonadotropin subunits and gonadotropin receptors (Fshr and Lhr) in wild eels are essentially non-existent. This study investigated pituitary fshb and lhb mRNA levels and ovarian fshr and lhr mRNA levels of wild shortfinned eels, Anguilla australis at different stages of oogenesis. Protein expression of Fsh in the pituitary was also quantified and visualized using slot blot and immunohistochemistry. Pituitary fshb and lhb mRNA levels showed a differential expression pattern, fshb mRNA levels increasing significantly from the perinucleolus (PN) to the oil droplet stage (OD) before slightly decreasing (not significantly) in the early vitellogenic stage (EV). A similar trend was observed in relative Fsh protein levels analyzed by slot blot and immunohistochemistry, but this trend was not reflected in the plasma levels of sex steroids. In contrast, pituitary lhb mRNA levels increased significantly from the PN to EV stage. A higher expression of Fsh at both mRNA and protein levels in the pituitary of eels at the OD stage compared to other investigated stages suggests that synthesis of Fsh production in the pituitary may reach a peak at the OD stage. In the ovary, transcript abundances of fshr and lhr gradually increased during previtellogenic follicle growth, but markedly and significantly increased thereafter. Taken together, our data suggest i) that Fsh release may be very limited, or absent, prior to onset of puberty in shortfinned eels and ii) that Lh is not functionally important in this fish during the EV stage.