Follicle-stimulating Hormone Beta Research Articles

Effects of melanocortin-4 receptor agonists and antagonists on expression of genes related to reproduction in spotted scat, Scatophagus argus.

Melanocortin-4 receptor (Mc4r) function related to reproduction in fish has not been extensively investigated. Here, we report on gene expression changes by real-time PCR following treatment with Mc4r agonists and antagonists in the spotted scat (Scatophagus argus). Using in vitro incubated hypothalamus, the Mc4r nonselective agonist NDP-MSH ([Nle4, D-Phe7]-α-melanocyte stimulating hormone; 10-6 M) and selective agonist THIQ (N-[(3R)-1, 2, 3, 4-Tetrahydroisoquinolinium-3-ylcarbonyl]- (1R)-1-(4-chlorobenzyl)-2-[4-cyclohexyl-4-(1H-1,2,4-triazol-1-ylmethyl) piperidin-1-yl]-2-oxoethylamine; 10-7 M) significantly increased the expression of gnrh (Gonadotropin releasing hormone), while the Mc4r nonselective antagonist SHU9119 (Ac-Nle-[Asp-His-DPhe/DNal(2')-Arg-Trp-Lys]-NH2; 10-6 M) and selective antagonist Ipsen 5i (compound 5i synthesized in Ipsen Research Laboratories; 10-6 M) significantly inhibited gnrh expression after 3h of incubation. In incubated pituitary tissue, NDP-MSH and THIQ significantly increased the expression of fshb (Follicle-stimulating hormone beta subunit) and lhb (Luteinizing hormone beta subunit), while SHU9119 and Ipsen 5i significantly decreased fshb and lhb expression after 3h of incubation. During the in vivo experiment, THIQ (1mg/kgbw) significantly increased gnrh expression in hypothalamic tissue, as well as the fshb and lhb expression in pituitary tissue 12h after abdominal injection. Furthermore, Ipsen 5i (1mg/kgbw) significantly inhibited gnrh expression in hypothalamic tissue, as well as fshb and lhb gene expression in pituitary tissue 12h after abdominal injection. In summary, Mc4r singling appears to stimulate gnrh expression in the hypothalamus, thereby modulating the synthesis of Fsh and Lh in the pituitary. In addition, Mc4r also appears to directly regulate fshb and lhb levels in the pituitary in spotted scat. Our study suggests that Mc4r, through the hypothalamus and pituitary, participates in reproductive regulation in fish.

Molecular characterization and functional analysis of pituitary GnRH receptor in a commercial scombroid fish, chub mackerel (Scomber japonicus)

The gonadotropin-releasing hormone (GnRH) is essential during pubertal onset, for its regulation of the synthesis and release of pituitary gonadotropins. Its action is mediated by GnRH receptors (GnRHRs) in the pituitary gonadotrophs. Our previous study demonstrated that the chub mackerel brain expresses three GnRH forms (gnrh1, gnrh2, and gnrh3), and that only GnRH1 neurons innervate anterior pituitary regions. Furthermore, chub mackerel gnrh1 mRNA exhibited a significant increase at pubertal onset. The present study aimed to isolate the functional GnRHR form involved in chub mackerel puberty. The open reading frame of our cloned receptor encodes 428 amino acids and contains seven transmembrane domains. Phylogenetic analysis also indicated clustering with other teleost-type IIB GnRHRs, mainly those involved in reproduction. Reporter gene assay results showed that all four synthetic peptides (GnRH1, GnRH2, GnRH3, and GnRH analogue) bind to the cloned receptor. Three deduced GnRH ligands stimulated luteinizing hormone (LH) release from cultured pituitary cells in vitro. Receptor gene expression was mainly detected in the pituitary and showed an increasing trend in the developing gonadal stages of both sexes during the pubertal process; this process was synchronous with previous studies of follicle-stimulating hormone beta (fshβ) and lhβ gene expression in chub mackerel. These results suggest that the cloned receptor is likely involved in the regulation of pubertal onset in this species. Therefore, we have designated the receptor cmGnRHR1.

Genetic Evidence for Multifactorial Control of the Reproductive Axis in Zebrafish.

It is generally believed that kisspeptin and gonadotropin-releasing hormone (GnRH) are required for reproduction in vertebrates. In this study, we generated gnrh3-null zebrafish and found that gnrh3 mutation did not impair gonad development and reproductive capacity. Moreover, zebrafish triple knockout mutant lacking gnrh3 and the 2 kiss1s genes undergo normal puberty and gonad maturation. The expression of follicle-stimulating hormone beta (fshβ) and luteinizing hormone beta (lhβ) was not significantly altered whereas the expression of neuropeptide Y (npy), tachykinin 3 (tac3), and secretogranin-II (sgII) was significantly increased in the triple knockout mutant, suggesting that compensation mechanisms exist to stimulate the reproductive axis in the absence of kiss and gnrh. Our results challenge the prevailing view that GnRH is indispensable for reproduction across species. These data provide genetic evidence that different mechanisms have evolved for the neuroendocrine control of reproduction between mammals and fish: pulsatile release of GnRH to the portal system is the final gateway to stimulate the reproductive axis in mammals, whereas multiple factors act in parallel with GnRH to stimulate the reproductive axis in certain fish species.

Impact of severity of illness on the function of the hypothalamo-pituitary-gonadal axis in postmenopausal women with acute severe illness: Implications for predicting disease outcome

Background:While elevated levels of estradiol were predictive of mortality in critically ill surgical and trauma patients, their ability to predict outcome in nonsurgical patients has not been studied. We aimed to study the determinants of gonadotropin levels in acutely ill postmenopausal women with nonsurgical disease and the impact of changes in the gonadal axis on the outcome of these patients.Methods:Thirty-five postmenopausal women admitted to medical intensive care with acute severe illness and having a Simplified Acute Physiology Score (SAPS II score) ≥30 (in-hospital mortality rate ≥ 10%) were recruited. On the 5th day of hospitalization, fasting samples were collected at 8.00 am and tested for luteinizing hormone (LH), follicle-stimulating hormone (FSH), estradiol, free triiodothyronine, free thyroxine, thyrotropin, cortisol, prolactin, dehydroepiandrosterone, androstenedione, and sex hormone-binding globulin. Multiple linear regression analysis was performed to identify independent determinants if any of LH and FSH. Receiver operating characteristic (ROC) curves were drawn for different cutoffs of LH, FSH, and estradiol to diagnose mortality and prolonged hospitalization.Results:There was an independent negative association between the FSH and the SAPS II score (beta = −0.435; P = 0.014), but not with any of the other tested parameters (estradiol, prolactin, or cortisol). Among components of the SAPS II score, the total leukocyte count (TLC) was negatively associated with serum FSH (beta coefficient = −0.635, P = 0.013). None of these parameters were determinants of LH. On ROC analysis, neither estradiol nor gonadotropins were diagnostic for in-hospital mortality. However, among survivors, low estradiol was diagnostic for prolonged hospital stay (area under the curve = 0.785; P = 0.015).Conclusion:FSH, but not LH, is negatively associated with the severity of illness, particularly to its inflammatory component (TLC). Low estradiol in survivors was a predictor of prolonged hospital stay.

Delayed onset of puberty in male offspring from bisphenol A-treated dams is followed by the modulation of gene expression in the hypothalamic-pituitary-testis axis in adulthood.

Bisphenol A (BPA) is a synthetic endocrine-disrupting chemical of high prevalence in the environment, which may affect the function of the hypothalamic-pituitary-testis (HPT) axis in adult rats. The aim of the present study was to evaluate whether exposure to BPA during hypothalamic sexual differentiation at doses below the reproductive no observable adverse effect level of the World Health Organization causes changes in the regulation of the HPT axis. For this, 0.5 or 5mgkg-1 BPA was injected subcutaneously to the mothers from gestational day 18 to postnatal day (PND) 5. In adulthood (PND90), the mRNA expression of genes related to HPT axis was evaluated in hypothalamus, pituitary and testis. Hypothalamic expression of gonadotrophin-releasing hormone (Gnrh) and estrogen receptor 2 (Esr2) mRNA was increased in both BPA-treated groups compared to control group. In the pituitary, follicle stimulating hormone beta subunit (Fshb) and androgen receptor (Ar) mRNA expression was increased compared to control group in rats treated with 0.5mgkg-1 of BPA, whereas estrogen receptor 1 (Esr1) mRNA expression was only increased in the group treated with 5mgkg-1of BPA, compared to control group. In the testis, there was increased expression of FSH receptor (Fshr) and inhibin beta B subunit (Inhbb) transcripts only in rats treated with 0.5mgkg-1 of BPA. Serum testosterone and LH concentrations were increased in the group treated with 5mgkg-1of BPA. The results of the present study demonstrate for the first time that perinatal exposure to low doses of BPA during the critical period of hypothalamic sexual differentiation modifies the activity of the HPT axis in the offspring, with consequences for later life in adult rats.

Changes in plasma steroid levels and gene expression of pituitary gonadotropins, testicular steroidogenesis-related proteins and insulin-like growth factors during spermatogenesis of the yellowtail Seriola quinqueradiata

We have examined the annual cycle of spermatogenesis in captive yellowtail Seriola quinqueradiata, including the associated changes in both plasma 11-ketotestosterone (11-KT) level and gene expression of key endocrine factors, such as the pituitary gonadotropin subunits, testicular steroidogenesis-related proteins and insulin-like growth factors (IGFs), at different testicular developmental stages. Spermatogenesis was initiated in January, and all males had progressed to the spermiation stage in April. Plasma 11-KT levels and gene expression profiles were analyzed in relation to changes in histological findings. Plasma 11-KT levels increased during early to late spermatogenesis and were positively correlated with early mitotic divisions of spermatogonia leading to meiosis. The genes encoding the follicle stimulating hormone (FSH) beta subunit (fshb) and the luteinizing hormone (LH) beta subunit (lhb) both showed parallel expression patterns during spermatogenesis. A previous study revealed that FSH receptor mRNA in yellowtail testes is strongly expressed from early to late spermatogenesis, whereas levels of LH receptor mRNA peak during spermiation; only cyp17a1 [encoding cytochrome P450 17α-hydroxylase/C17-C20 lyase (P450c17)] transcript levels increased during early spermatogenesis. The expression patterns of the genes encoding testicular IGF-1 and -2 (igf-1 and igf-2) differed from each other. Altogether, early spermatogenesis was associated with the regulation of 11-KT, which was probably induced by FSH through stimulation of specific steroidogenic enzymes, such as P450c17, and spermiation was considered to be triggered by LH. In addition, IGF-1 and IGF-2 are individually involved in spermatogenesis via autocrine/paracrine mechanisms.

Genetic variations altering FSH action affect circulating hormone levels as well as follicle growth in healthy peripubertal girls.

Do variants of the genes encoding follicle stimulating hormone (FSH) beta subunit (B) and FSH receptor (R) impact circulating reproductive hormone levels and ovarian follicle maturation in healthy peripubertal girls? FSHB and FSHR genetic variants exert, alone or their combination, distinct effects on reproductive hormone levels as well as ovarian follicle maturation in healthy peripubertal girls. FSHB and FSHR genetic variants impact reproductive hormone levels as well as associated pathologies in women. While FSHR c. 2039A>G is known to alter gonadotrophin levels in women, FSHR c.-29G>A has not yet been shown to exert effect and there are conflicting results concerning FSHB c.-211G>T. This population-based study included 633 girls recruited as part of two cohorts, the COPENHAGEN Puberty Study (2006-2014, a cross-sectional and ongoing longitudinal study) and the Copenhagen Mother-Child Cohort (1997-2002, including transabdominal ultrasound (TAUS) of the ovaries in a subset of 91 peripubertal girls). Clinical examinations, including pubertal breast stage (Tanner's classification B1-B5) were performed. Circulating levels of FSH, luteinizing hormone (LH), estradiol, anti-Mullerian hormone (AMH) and inhibin-B were assessed by immunoassays. In a subset of the girls (n = 91), ovarian volume and the number/size of antral follicles were assessed by TAUS. Genotypes were determined by competitive PCR. FSHR c.2039A>G minor alleles were positively associated with serum FSH (β = 0.08, P = 0.004), LH (β = 0.06, P = 0.012) and estradiol (β = 0.06, P = 0.017) (adjusted for Tanner stages). In a combined model, FSHR c.-29G>A and FSHR c.2039A>G alleles were positively associated with FSH levels in early-pubertal girls (B2 + B3, n = 327, r = 0.1, P = 0.02) and in young adolescents (B4 + B5, n = 149, r = 0.2, P = 0.01). Serum AMH and inhibin B levels were not significantly influenced by the single nucleotide polymorphisms (SNPs). Single SNPs were not associated with follicles counts, however, a cumulative minor allele count (FSHB c.-211 G>T and FSHR c.-29G>A) was negatively associated with the number of large follicles (≥5 mm) (n = 91, P = 0.04) (adjusted for Tanner stages). Since we studied girls and young adolescents during pubertal transition, our study may not be fully comparable with previous studies on FSHB and FSHR variants in adult women. The group of young adolescents (Tanner B4 + B5) reflects the endocrine situation in adult women best, however, the group is not large enough to contribute substantially to the conflicting results concerning the influence of FSHB c.-211G>T in adult women. Furthermore, we have no information about the exact day of the menstrual cycle in the subgroup of girls with menarche. The sex-specific interaction of FSHB and FSHR genetic variants and physiological as well as pathological conditions is being increasingly elucidated. The variant triplet set might serve as diagnostic and pharmacogenetic marker. For the first time, we show an additional effect of FSHR c.-29G>A on serum FSH levels in healthy girls. Moreover, morphological data suggest impaired FSH-induced maturation of ovarian follicles in minor allele carriers of FSHB c.-211G>T and FSHR c.-29G>A. This may explain previous findings of delayed pubertal onset in these girls. Funding was provided by the Danish Agency for Science, Technology and Innovation (09-067180), Danish Ministry of the Environment, CeHoS (MST-621-00065), Capital Region of Denmark (December 2011), Ministry of Higher Education and Science (DFF-1331-00113) and EDMaRC (Danish Ministry of Health). A.S.B. was funded from December 2015 by ReproUnion (EU Interreg Öresund-Kattegat-Skagerrak). The authors declare no conflict of interest.

Molecular cloning and characterization of the gonadotropin subunits GPα, FSHβ, and LHβ genes in the stinging catfish Heteropneustes fossilis: phylogeny, seasonal expression and pituitary localization.

Gonadotropins are heterodimeric glycoproteins secreted by the pituitary, and consist of a common glycoprotein hormone alpha (GPα) and the function-specific follicle-stimulating hormone beta subunit (FSHβ) or luteinizing hormone beta subunit (LHβ). In the present study, the subunit protein genes were cloned and characterized from the pituitary of the catfish Heteropneustes fossilis. Full-length cDNAs of GPα, FSHβ, and LHβ are 511 base pairs (bp), 659 bp and 660 bp long, and encode 92, 108, and 112 aminoacids long mature proteins, respectively. GPα has 10 cysteines with 2 N-linked glycosylation sites while LHβ contains 12 cysteines with a single N-linked glycosylation site. In contrast, FSHβ has 13 cysteines, 1 additional over the conserved 12 cysteines of other vertebrates, and a single glycosylation site between Cys 3 and Cys 4. Phylogenetic analyses of the deduced proteins confirm their homology and relationships with the respective gonadotropin subunit proteins of gnathostome vertebrates. Tissue expression analysis by semi-quantitative RT-PCR shows that GPα mRNA is expressed only in the pituitary while both FSHβ and LHβ mRNA are expressed in extra-pituitary sites. The subunit mRNAs show both seasonal and sex dimorphic variations especially in the expression of FSHβ and LHβ transcripts. In the sexually quiescent phase, the transcript expression is low while in the recrudescent phase, the expressions are differential, high, and varied with regard to sex and reproductive phase. In situ hybridization of the mRNAs gave positive signals in gonadotropes in the pars distalis of the pituitary, which exhibited seasonal variation in staining intensity and numbers.

Effect of diet supplementation with a commercial probiotic containingPediococcus acidilactici(Lindner, 1887) on the expression of five quality markers in zebrafish (Danio rerio(Hamilton, 1822)) testis

The effects of a commercial probiotic diet supplement (Bactocell®), containing lactic acid bacteria Pediococcus acidilactici were evaluated by identifying five zebrafish (Danio rerio) sperm quality markers. P. acidilactici (106 colony-forming units per gram) was supplied to young zebrafish males (age 8 months) as a feed probiotic additive during a 10 days test period. Five transcripts, previously described as male zebrafish quality markers, were studied: brain-derived neurotrophic factor (bdnf), BCL2-interacting killer (bik), double-sex and mab-3 related transcription factor 1 (dmrt1), follicle-stimulating hormone beta subunit (fshb) and leptin a (lepa). After exposition, males fed on with the supplemented diet with a commercial probiotic containing P. acidilactici presented an over-expression on three fertility markers comparing to the control group: lepa, dmrt1 and bdnf. These results suggest that P. acidilactici has a potential use as probiotic supplement in zebrafish diet for the improvement of molecular parameters in testicular cells, indicating that probiotic supplementation could affect male reproductive performance.

Development of approaches to induce puberty in cultured female sablefish (Anoplopoma fimbria)

Efforts to establish sustainable and efficient aquaculture production of sablefish (Anoplopoma fimbria) have been constrained by delayed puberty in cultured females. This study integrates a series of experiments aimed at gaining an understanding of the reproductive physiology of puberty in female sablefish. We detected transcripts for the dopamine D2 receptor (drd2) in brain, pituitary and ovary of sablefish, and prepubertal females exhibited significantly elevated brain and pituitary drd2 expression relative to wild maturing females. Treatments with sustained-release cholesterol pellets containing testosterone (T) and the dopamine D2 receptor antagonist, metoclopramide (Met), stimulated expression of pituitary luteinizing hormone beta subunit (lhb) and follicle-stimulating hormone beta subunit (fshb), respectively, in prepubertal females, whereas a combination of T and gonadotropin-releasing hormone agonist (GnRHa) had a strong synergistic effect on lhb expression (2000-fold higher than control). Although T induced a significant increase in the maximum ovarian follicle volume, none of the treatments tested stimulated onset of vitellogenesis. Using liquid chromatography/tandem mass spectrometry, we demonstrated that Met stimulated production of T by previtellogenic ovarian follicles in vitro, whereas gonadotropin preparations enhanced 17α-hydroxyprogesterone, androstenedione (A4), T and 17β-estradiol (E2) production. Treatment with T increased production of A4, 11β-hydroxyandrostenedione, 11β-hydroxytestosterone, E2, 11-ketotestosterone, and 5α-dihydrotestosterone (DHT). Interestingly, in the presence of high doses of T the previtellogenic ovary preferentially produced A4 and DHT over any other metabolite. Our data suggest the existence of dopamine inhibition of the reproductive axis in female sablefish. Treatments with Met and T elevated gonadotropin mRNAs in prepubertal females but failed to stimulate the transition into vitellogenic growth, suggesting a possible failure in pituitary gonadotropin protein synthesis/release. Previtellogenic ovarian follicles of sablefish are equipped to synthesize steroids, including those required for vitellogenic growth, and DHT, a steroid hormone whose role in reproduction of fishes remains unknown.

Adult exposure to bisphenol A (BPA) in Wistar rats reduces sperm quality with disruption of the hypothalamic-pituitary-testicular axis.

Reproductive physiology involves complex biological processes that can be disrupted by exposure to environmental contaminants. The effects of bisphenol A (BPA) on spermatogenesis and sperm quality is still unclear. The objective of this study was to investigate the reproductive toxicity of BPA at dosages considered to be safe (5 or 25mg BPA/kg/day). We assessed multiple sperm parameters, the relative expression of genes involved in the central regulation of the hypothalamic-pituitary-testicular axis, and the serum concentrations of testosterone, estradiol, LH and FSH. BPA exposure reduced sperm production, reserves and transit time. Significant damage to the acrosomes and the plasma membrane with reduced mitochondrial activity and increased levels of defective spermatozoa may have compromised sperm function and caused faster movement through the epididymis. BPA exposure reduced the serum concentrations of testosterone, LH and FSH and increased the concentration of estradiol. The relative gene expression revealed an increase in gonadotropin releasing hormone receptor (Gnrhr), luteinizing hormone beta (Lhb), follicle stimulating hormone beta (Fshb), estrogen receptor beta (Esr2) and androgen receptor (Ar) transcripts in the pituitary and a reduction in estrogen receptor alpha (Esr1) transcripts in the hypothalamus. In this study, we demonstrated for the first time that adult male exposure to BPA caused a reduction in sperm production and specific functional parameters. The corresponding pattern of gene expression is indicative of an attempt by the pituitary to reestablish normal levels of LH, FSH and testosterone serum concentrations. In conclusion, these data suggest that at dosages previously considered nontoxic to reproductive function, BPA compromises the spermatozoa and disrupts the hypothalamic-pituitary-gonadal axis, causing a state of hypogonadotropic hypogonadism.

DS1, a delta subunit-containing GABA(A) receptor agonist, increases gonadotropin subunit gene expression in mouse pituitary gonadotrophs.

4-Chloro-N-[6,8-dibromo-2-(2-thienyl)imidazo[1,2-alpyridine-3-yl] (DS1) is a GABA(A) receptor agonist that selectively binds to delta subunit-containing GABA(A) alpha4beta3delta receptors. In the present study, we examined the effect of DS1 on pituitary gonadotropin subunit gene expression using the mouse pituitary gonadotroph cell line LbetaT2. DS1 increased the promoter activity of the gonadotropin subunits luteinizing hormone beta (LHbeta), follicle-stimulating hormone beta (FSHbeta), and alpha. Gonadotropin-releasing hormone (GnRH) receptor promoters were also activated by DS1. The effects of DS1 on gonadotropin subunit promoters were obvious, but they were less than those induced by stimulation with GnRH. GnRH-stimulated gonadotropin subunit promoters were enhanced in the presence of DS1. A prototypic specific agonist for GABAA receptors, muscimol, failed to increase LHbeta and FSHbeta subunit promoter activity and had no effect on GnRH-increased LHbeta and FSHbeta promoter activity. In addition, SKF97541, a specific agonist for GABAB receptors, did not modulate basal or GnRH-induced LHbeta and FSHbeta promoter activity. A natural GABA compound failed to increase gonadotropin promoter activity and potentiated the effect of GnRH on the FSHbeta promoter. DS1 increased the activity of serum response element (SRE) and cAMP response element (CRE) promoters, which reflect the activity of the extracellular signal-regulated kinase and cAMP/protein kinase A (PKA) pathways, and GnRH-increased SRE and CRE promoter activity was enhanced in the presence of DS1. A specific inhibitor of the ERK signaling pathway, U0126, prevented DS1-induced LHbeta and FSHbeta promoter activity almost completely; however, H89, a PKA inhibitor, did not modulate the effect of DS1. Our current observations demonstrate that the GABAA alpha4beta3delta receptor agonist DS1 can stimulate gonadotropin subunit gene expression in association with the ERK signaling pathway.

Extragonadal FSH receptor: is it real?

Follicle-stimulating hormone (FSH) is a pituitary gonadotroph-derived heterodimeric glycoprotein that binds G protein-coupled FSH receptors (FSHRs) on ovarian granulosa cells [1–3]. This FSH/FSHR signaling pathway is essential for female fertility because null mice lacking either the FSH ligand [4] or FSHR [5, 6] are sterile as a result of preantral stage block in ovarian folliculogenesis. These FSH ligand/receptor null mice phenocopy women harboring inactivating mutations (homozygous recessive) in corresponding alleles encoding the hormone-specific FSH beta subunit and FSHR [7, 8]. A number of in vitro and in vivo studies have documented the cascade of signaling events elicited by FSH binding to FSHRs on granulosa cells [2, 3, 9]. Although the predominant signaling pathway activated by FSH is cAMP-mediated phosphorylation of PKA and CREB, activation of other signaling pathways, including the mitogen-activated protein kinase pathway, has also been identified [8, 10]. Interestingly, several FSHR isoforms are expressed in ovary (presumably on granulosa cells of preantral follicles) prior to the well-known FSH actions during early folliculogenesis [11–14]. However, the functional significance of these FSHR isoforms is unknown. Although the classical prevailing view is that pituitary-derived FSH acts on ovarian granulosa cells in the female, emerging evidence suggests FSH may exert physiological effects on extragonadal tissues and organs as well. A series of studies document that FSH acts on osteoclasts by activating FSHRs via a nonclassical pathway and regulates bone mass in mice [15–19]. Consistent with this, women carrying a polymorphism in FSHR resulting in hyperactivation of FSHRs are more prone to bone loss [20]. However, FSH actions on bone osteoclasts remain controversial [21, 22]. Moreover, FSH actions on monocytes [23, 24] and human umbilical vein endothelial cells [25] and expression of FSHRs on endothelial cells of the tumor vasculature [26] have been reported. In a recent issue of Biology of Reproduction (BOR), Stilley and colleagues present their remarkable findings on FSHR expression in the human female reproductive tract and developing placenta and feto-placental defects in an Fshr mutant mouse model [27].

Molecular cloning, characterization and functional analysis of QRFP in orange-spotted grouper (Epinephelus coioides).

The peptide QRFP plays an important role in the regulation of vertebrate feeding behavior. In this study, we cloned the full length cDNA of a QRFP precursor in a teleost fish, the orange-spotted grouper (Epinephelus coioides). Sequence analysis has shown that the functional regions of QRFP in other vertebrates (QRFP-25 and QRFP-7) are conserved in orange-spotted grouper. RT-PCR demonstrated that the pre-processed mRNA of QRFP is widely expressed in orange-spotted grouper. Three days of food deprivation did not change the hypothalamic pre-processed QRFP expression. However, QRFP expression significantly increased when the fish were reefed after three days of fasting. Intraperitoneal injection of QRFP-25 peptide to orange-spotted grouper suppressed expression of orexin, but elevated expression of pro-opiomelanocortin (POMC) in the hypothalamus. We also investigated the effects of QRFP-25 on the expression of reproductive genes. The peptide suppressed the expression of seabream-type gonadotropin-releasing hormones (sbGnRH), luteinizing hormone beta subunit (LHβ) and follicle-stimulating hormone beta subunit (FSHβ) in vivo, as well as inhibited the expression of LHβ and FSHβ in pituitary cells in primary culture. Our results indicate that QRFP may play an inhibitory role in the regulation of feeding behavior and reproduction in orange-spotted grouper.

Seasonal expression of KiSS-1 and the pituitary gonadotropins LHβ and FSHβ in adult male Libyan jird (Meriones libycus)

The molecular mechanisms operating on a seasonal time-scale and regulating functions such as reproduction are poorly understood in animals living in desert environments. Kisspeptin, the product of the KiSS-1 gene, plays a critical role in control of the hypothalamic-pituitary-gonad axis that orchestrates the reproductive system in vertebrates. We report a sequence analysis of KiSS-1 and the pituitary luteinising hormone-beta (LHβ) and follicle-stimulating hormone-beta (FSHβ) in the Libyan jird (Meriones libycus), a seasonal breeding rodent that is sexually active during spring and quiescent in fall. We also assessed gene expression by quantitative real-time reverse transcription-polymerase chain reaction during the non-breeding and breeding seasons. The KiSS-1 cDNA sequence analysis showed high homology between M. libycus and all other rodents (94%) and humans (92%). KiSS-1 expression was higher during the breeding season than that during the non-breeding season. In contrast, LHβ and FSHβ expression levels were higher during the non-breeding season in autumn and varied in an opposite manner with testicular, seminal vesicle weights and plasma testosterone levels. Our results extend the role for KiSS-1 in activating the HPG axis in this desert rodent in its natural biotope by relaying environmental cues as in other seasonal non-desert rodent models.

Insulin‐like growth factor I regulation of FOXO1 in gonadotropes (1012.7)

The transcription factor FOXO1 is important for several aspects of reproductive function, including endometrial decidualization, ovarian follicular maturation, and spermatogenesis. We have previously reported that in pituitary gonadotropes FOXO1 functions as a repressor of both luteinizing hormone beta (Lhb) and follicle stimulating hormone beta (Fshb) transcription, which are critical for reproduction. Insulin‐like growth factor I (IGFI) is a negative regulator of FOXO1 transcriptional activity. Given that FOXO1 can repress Lhb and Fshb transcription and that IGFI stimulation of gonadotrope cells enhances Lhb and Fshb synthesis, we investigated how IGFI regulates FOXO1 activity in gonadotropes in the current study. In LβT2 gonadotrope cells, we determined by western analyses that IGFI induced Ser256 FOXO1 phosphorylation, causing FOXO1 inhibition and cytoplasmic localization as identified by immunofluorescence microscopy. In addition, inhibition of components of the IGFI signaling pathway, such as PI3K or AKT, blocked IGFI‐induced phosphorylation of FOXO1. As the in vivo gonadotrope hormone milieu includes GnRH along with IGFI, we next investigated the effect of GnRH on FOXO1 phosphorylation and subcellular localization. Stimulation of LβT2 cells with GnRH alone induced moderate cytoplasmic localization of FOXO1, although GnRH did not induce phosphorylation of FOXO1 Ser256. Moreover, the GnRH‐induced shuttling of FOXO1 to the cytoplasm was not blocked by inhibition of PI3K activity. We are currently investigating the effects of IGFI and GnRH co‐treatment.Grant Funding Source: Supported by NIH Grants T32 HD007203 and F32 HD074414 to DVS; R01 HD067448 and U54 HD012303 to VGT.

Effects of diethylstilbestrol on luteinizing hormone-producing cells in the mouse anterior pituitary

Gonadotrophs in the anterior pituitary secrete luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Neonatal diethylstilbestrol (neoDES) treatment affects reproductive function of male and female mice, but the effect of this treatment on the development as well as direct effects on pituitary gonadotrophs have not been ascertained. We investigated LH-secreting gonadotropes and the expression of genes involved in the synthesis and secretion of gonadotropins in the anterior pituitary of neoDES mice using immunohistochemistry and real-time reverse transcription-polymerase chain reaction (RT-PCR). The percentage of LH-secreting gonadotropes in 90-day-old female mice treated neonatally with an oil vehicle (neoOil) was significantly lower than in 30-day-old neoOil females but not in males, indicating a significant reduction after reproductive maturation in females. The percentage of LH-secreting gonadotropes in the medial area of 90-day-old neoDES females was significantly lower than that of 90-day-old neoOil females, ovariectomized neoOil females, and neoOil and neoDES males. The expression of the LH beta (Lhb) subunit in the anterior pituitary of 90-day-old neoDES females was similar to that in neoOil females, but it was significantly lower than that observed in 90-day-old males. Ovariectomy increased the expression of the alpha subunit of glycoprotein hormones, FSH beta (Fshb) subunit and Lhb subunit both in neoOil and neoDES females, suggesting that the anterior pituitary of neoDES female mice is regulated by ovarian hormones via negative feedback. In organ-cultured, anterior pituitaries exposed to DES exhibited no change in the number of LH-secreting gonadotropes but did reduced gene expression. These results suggest that LH-secreting gonadotropes in the female mice are not only directly affected by neoDES but also are influenced by the masculinization of the hypothalamus. That is, neonatal DES exposure can masculinize or defeminize the hypothalamus of female mice. However, the regulation of the pituitary gonadotropins by the hypothalamus could be different from that in intact male mice.

Plasma Nesfatin-1 Is Not Affected by Long-Term Food Restriction and Does Not Predict Rematuration among Iteroparous Female Rainbow Trout (Oncorhynchus mykiss)

The metabolic peptide hormone nesfatin-1 has been linked to the reproductive axis in fishes. The purpose of this study was to determine how energy availability after spawning affects plasma levels of nesfatin-1, the metabolic peptide hormone ghrelin, and sex steroid hormones in rematuring female rainbow trout (Oncorhynchus mykiss). To limit reproductive maturation, a group of female trout was food-restricted after spawning and compared with a control group that was fed a standard broodstock ration. The experiment was conducted twice, once using two-year-old trout (second-time spawners) and once using three-year-old trout (third-time spawners). During monthly sampling, blood was collected from all fish, and a subset of fish from each treatment was sacrificed for pituitaries. Pituitary follicle-stimulating hormone-beta (fsh-β) mRNA expression was analyzed with q-RT-PCR; plasma hormone levels were quantified by radioimmunoassay (17β-estradiol and ghrelin) and enzyme-linked immunosorbent assay (11-keto-testosterone and nesfatin-1). Although plasma nesfatin-1 levels increased significantly in the months immediately after spawning within both feeding treatments, plasma nesfatin-1 did not differ significantly between the two treatments at any point. Similarly, plasma ghrelin levels did not differ significantly between the two treatments at any point. Food restriction arrested ovarian development by 15–20 weeks after spawning, shown by significantly lower plasma E2 levels among restricted-ration fish. Pituitary fsh-β mRNA levels were higher among control-ration fish than restricted-ration fish starting at 20 weeks, but did not differ significantly between treatment groups until 30 weeks after spawning. Within both treatment groups, plasma 11-KT was elevated immediately after spawning and rapidly decreased to and persisted at low levels; starting between 20 and 25 weeks after spawning, plasma 11-KT was higher among control-ration fish than restricted-ration fish. The results from these experiments do not provide support for plasma nesfatin-1 as a signal for the initiation of reproductive development in rematuring female rainbow trout.

Steroid exposure during larval development of Xenopus laevis affects mRNA expression of the reproductive pituitary–gonadal axis in a sex- and stage-dependent manner

Steroids are known to influence the reproductive pituitary–gonadal axis in adult amphibians. Here, we studied the effects of hormones on pituitary and gonadal mRNA expression during the development of Xenopus laevis. Tadpoles at NF 58 (prometamorphosis) and at NF 66 (freshly metamorphosed) were exposed for three days to 17β-estradiol (E2), tamoxifen (TAM), testosterone (T), dihydrotestosterone (DHT) at 10−7M, and flutamide (FLU) at 10−6M. In both genders at NF 58 and 66, T and DHT decreased luteinizing hormone beta (lhβ), but increased follicle stimulating hormone beta (fshβ), while FLU induced lhβ specifically in males. In the testis steroidogenic genes (p450 side chain cleavage enzyme, p450scc; steroid acute regulatory protein, star) at NF 58 showed a similar pattern as for lhβ, while the response at NF 66 was only partially present. In females, TAM induced lhβ at NF 58, while E2 decreased lhβ and increased fshβ at NF 66. In the ovaries, no alterations were observed for the steroidogenic genes. Summarizing, gonadotropic and steroidogenic mRNA expression may indicate control of androgen level during testis differentiation in male tadpoles at NF 58. In females the non-responsiveness of steroidogenic genes could be a sign of gonadal quiescence during pre-pubertal stages.

Polymorphism of follicle stimulating hormone beta (FSHβ) subunit gene and its association with litter traits in giant panda

The different SSCP patterns of the follicle stimulating hormone beta (FSHβ) gene amplified by three pairs of primers were sequenced. Comparisons among the three nucleotide sequences of three genotypes indicated that three base substitutions (A213T, A91G, and A89C) were detected in FSHβ gene, which A213T substitution led to one amino acids mutation (Lys > Met), and the other two substitutions were synonymous mutations. The AA, AB and BB genotypes patterns obtained by FSHβ primer1 had evident relation with the litter traits, but the SSCP genotypes patterns obtained by FSHβ primer2 and primer3 had no evident relation with the litter traits in giant panda. The giant panda with AA and AB genotype had the largest litter size and multiparity rate compared with the BB genotypes (P < 0.05). We speculated that the giant pandas with the A allele have better litter traits than those with the B allele.