Transcription factor GATA2 may potentiate follicle-stimulating hormone production in mice via induction of the BMP antagonist gremlin in gonadotrope cells.

Mammalian reproduction depends on the gonadotropins, follicle-stimulating hormone (FSH), and luteinizing hormone, which are secreted by pituitary gonadotrope cells. The zinc-finger transcription factor GATA2 was previously implicated in FSH production in male mice; however, its mechanisms of action and role in females were not determined. To directly address GATA2 function in gonadotropes, we generated and analyzed gonadotrope-specific Gata2 KO mice using the Cre-lox system. We found that while conditional KO (cKO) males exhibited ∼50% reductions in serum FSH levels and pituitary FSHß subunit (Fshb) expression relative to controls, FSH production was apparently normal in cKO females. In addition, RNA-seq analysis of purified gonadotropes from control and cKO males revealed a profound decrease in expression of gremlin (Grem1), a bone morphogenetic protein (BMP) antagonist. We show Grem1 was expressed in gonadotropes, but not other cell lineages, in the adult male mouse pituitary. Furthermore, Gata2, Grem1, and Fshb mRNA levels were significantly higher in the pituitaries of WT males relative to females but decreased in males treated with estradiol and increased following ovariectomy in control but not cKO females. Finally, we found that recombinant gremlin stimulated Fshb expression in pituitary cultures from WT mice. Collectively, the data suggest that GATA2 promotes Grem1 expression in gonadotropes and that the gremlin protein potentiates FSH production. The mechanisms of gremlin action have not yet been established but may involve attenuation of BMP binding to activin type II receptors in gonadotropes, facilitating induction of Fshb transcription by activins or related ligands.

Advanced puberty triggered by bi-weekly changes in reproductive factors during the photolabile period in a male teleost fish, Dicentrarchus labrax L.

This study evaluated the impact of continuous light (LL) within the photolabile period on advanced puberty in juvenile male European sea bass. The exposure to an LL regime for 1 month, from August 15 to September 15 (LLa/s), was compared to a constant simulated natural photoperiod (NP) and constant continuous light conditions year-round (LLy). Somatic growth, hormone plasma levels, rates of testicular maturation and spermiation, as well as the mRNA levels of some reproductive genes were analyzed. Our results demonstrated that both LLa/s and LLy treatments, which include LL exposure during the photolabile period, were highly effective in inhibiting the gametogenesis process that affects testicular development, and clearly reduced the early sexual maturation of males. Exposure to an LL photoperiod affected body weight and length of juvenile fish during early gametogenesis and throughout the first year of life. Interestingly, LL induced bi-weekly changes in some reproductive factors affecting Gnrh1 and Gnrh2 content in the brain, and also reduced pituitary fshß expression and plasmatic levels of 11-KT, E2, Fsh throughout early gametogenesis. We suggest that low levels of E2 in early September in the LL groups, which would be concomitant with the reduced number of spermatogonial mitoses in these groups, might indicate a putative role for estrogens in spermatogonial proliferation during the early gonadal development of this species. Furthermore, a significant decrease in amh expression was observed, coinciding with low plasma levels of 11-KT under LL regimes, which is consistent with the idea that this growth factor may be crucial for the progress of spermatogenesis in male sea bass.

Sensitivity of pituitary gonadotropes to hyperglycemia leads to epigenetic aberrations and reduced follicle-stimulating hormone levels.

The connection between metabolism and reproductive function is well recognized, and we hypothesized that the pituitary gonadotropes, which produce luteinizing hormone and follicle-stimulating hormone (FSH), mediate some of the effects directly via insulin-independent glucose transporters, which allow continued glucose metabolism during hyperglycemia. We found that glucose transporter 1 is the predominant glucose transporter in primary gonadotropes and a gonadotrope precursor-derived cell line, and both are responsive to culture in high glucose; moreover, metabolite levels were altered in the cell line. Several of the affected metabolites are cofactors for chromatin-modifying enzymes, and in the gonadotrope precursor-derived cell line, we recorded global changes in histone acetylation and methylation, decreased DNA methylation, and increased hydroxymethylation, some of which did not revert to basal levels after cells were returned to normal glucose. Despite this weakening of epigenetic-mediated repression seen in the model cell line, FSH ß-subunit ( Fshb) mRNA levels in primary gonadotropes were significantly reduced, apparently due in part to increased autocrine/paracrine effects of inhibin. However, unlike thioredoxin interacting protein and inhibin subunit α, Fshb mRNA levels did not recover after the return of cells to normal glucose. The effect on Fshb expression was also seen in 2 hyperglycemic mouse models, and levels of circulating FSH, required for follicle growth and development, were reduced. Thus, hyperglycemia seems to target the pituitary gonadotropes directly, and the likely extensive epigenetic changes are sensed acutely by Fshb. This scenario would explain clinical findings in which, even after restoration of optimal blood glucose levels, fertility often remains adversely affected. However, the relative accessibility of the pituitary provides a possible target for treatment, particularly crucial in the young in which hyperglycemia is increasingly common and fertility most relevant.-Feldman, A., Saleh, A., Pnueli, L., Qiao, S., Shlomi, T., Boehm, U., Melamed, P. Sensitivity of pituitary gonadotropes to hyperglycemia leads to epigenetic aberrations and reduced follicle-stimulating hormone levels.

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

STUDY QUESTION: 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? SUMMARY ANSWER: 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. WHAT IS KNOWN ALREADY: 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. STUDY DESIGN, SIZE, DURATION: 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). PARTICIPANTS/MATERIALS, SETTING, METHODS: 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. MAIN RESULTS AND THE ROLE OF CHANCE: FSHR c.2039A>G minor alleles were positively associated with serum FSH (ß = 0.08, P = 0.004), LH (ß = 0.06, P = 0.012) and estradiol (ß = 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). LIMITATIONS, REASONS FOR CAUTION: 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. WIDER IMPLICATIONS OF THE FINDINGS: 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. STUDY FUNDING/COMPETING INTERESTS: 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 characterization of three gonadotropin subunits and their expression patterns during ovarian maturation in Cynoglossus semilaevis.

The endocrine regulation of reproduction in a multiple spawning flatfish with an ovary of asynchronous development remains largely unknown. The objectives of this study were to monitor changes in mRNA expression patterns of three gonadotropin hormone (GTH) subunits (FSHß, LHß and CGα) and plasma GTH levels during ovarian maturation of half-smooth tongue sole Cynoglossus semilaevis. Cloning and sequence analysis revealed that the cDNAs of FSHß, LHß and CGα were 541, 670 and 685 bp in length, and encode for peptides of 130, 158 and 127 amino acids, respectively. The number of cysteine residues and potential N-linked glycosylation sites of the flatfish GTHs were conserved among teleosts. However, the primary structure of GTHs in Pleuronectiformes appeared to be highly divergent. The FSHß transcriptional level in the pituitary remained high during the vitellogenic stage while plasma levels of FSH peaked and oocyte development was stimulated. The LHß expression in the pituitary and ovary reached the maximum level during oocyte maturation stages when the plasma levels of LH peaked. The brain GTHs were expressed at the different ovarian stages. These results suggested that FSH and LH may simultaneously regulate ovarian development and maturation through the brain-pituitary-ovary axis endocrine system in tongue sole.

Mechanisms in endocrinology: Genetics of FSH action: a 2014-and-beyond view.

OBJECTIVE: To assess the pharmacogenetic potential of FSH for infertility treatment. DESIGN: Review of the literature and genomic databases. METHODS: Single-nucleotide polymorphism (SNP) assessed: rs6166 (c.2039A>G, p.N680S), rs6165 (c.919A>G, p.T307A), rs1394205 (c.-29G>A) in FSHR, and rs10835638 (c.-211G>T) in FSHB. Literature search via PubMed. Blast analysis of genomic information available in the NCBI nucleotide database. Comparison of allele frequency and haplotype distribution using the http://spsmart.cesga.estool. RESULTS: All these SNPs appear first in Homo, result in reduced FSH action, and are present with variable frequencies and combinations worldwide. Stringent clinical studies demonstrate that the FSHR genotype influences serum FSH levels and gonadal response in both sexes. Serum FSH levels depend on the -211G>T SNP, influencing transcriptional activity of the FSHB promoter. Genotypes reducing FSH action are overrepresented in infertile subjects. CONCLUSIONS: Although the clinical relevance of the FSHR polymorphisms alone is limited, the combination of FSHR and FSHB genotypes has a much stronger impact than either one alone in both sexes. About 20% of people are carriers of the alleles associated with lower serum FSH levels/reduced FSHR expression or activity, possibly less favorable for reproduction. Prospective studies need to investigate whether stratification of infertile patients according to their FSHR-FSHB genotypes improves clinical efficacy of FSH treatment compared with the current, naïve approach. A relative enrichment of less favorable FSHR-FSHB genotypes may be related to changes in human reproductive strategies and be a marker of some health-related advantage at the cost of reduced fertility.

FSHB-211 and FSHR 2039 are associated with serum levels of follicle-stimulating hormone and antimüllerian hormone in healthy girls: a longitudinal cohort study.

OBJECTIVE: To investigate whether genetic polymorphisms in the FSH pathway (FSHB-211 G→T and FSHR 2039 A→G) affect serum levels of FSH, antimüllerian hormone (AMH), and age at pubertal onset. FSH secretion and FSH signal transduction are enhanced in carriers of FSHB GG and FSHR AA, respectively. Furthermore, the combined genotype FSHB GG+FSHR AA is the most favorable for male gonadal function, but the effect of this genotype has never been evaluated in peripubertal females. AMH is a marker of ovarian function and is negatively correlated with FSH in prepubertal girls. DESIGN: Secondary analyses of a prospective cohort study. SETTING: General community. PATIENT(S): We examined 78 healthy girls twice yearly for 6 years; the median age at baseline was 9.3 years. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Hormone levels were measured by immunoassays, and DNA was isolated from blood and genotyped by restriction fragment length polymorphism of polymerase chain reaction-amplified regions. RESULT(S): Carriers of FSHB GG+FSHR AA had higher FSH before pubertal onset (median 2.2 vs. 1.5 IU/L) and lower AMH (13.8 vs. 19.4 pmol/L) compared with carriers of other genotypes. In crude analysis, girls with FSHB GG+FSHR AA entered puberty earlier, 9.7 vs. 10.6 years. However, the difference was no longer statistically significant after including interval-, right-, and left-censored data in a probit analysis. CONCLUSION(S): The combined effect of FSHB GG+FSHR AA may potentiate the FSH pathway, which increases serum levels of FSH and reduces AMH. Common variations in genes regulating follicle growth may affect AMH levels independently of the number of resting primordial follicles.

Free fatty acids induce Lhb mRNA but suppress Fshb mRNA in pituitary LßT2 gonadotropes and diet-induced obesity reduces FSH levels in male mice and disrupts the proestrous LH/FSH surge in female mice.

Female obesity is associated with insulin resistance, hyperandrogenemia, and reproductive dysfunction. We hypothesized that elevated free fatty acids (FFAs) might directly modulate pituitary gonadotropin production. FFAs caused a time- and dose-dependent increase in phosphorylation of the MAPKs p38MAPK, c-Jun N-terminal kinase (JNK)-1/2, and ERK1/2 in LßT2 gonadotrope cells. Furthermore, FFAs up-regulated Lhb mRNA expression acutely, an effect that was blocked by JNK inhibition, but suppressed Fshb mRNA expression, an effect that was independent of MAPK signaling. FFAs enhanced the activation of the MAPKs in the presence of GnRH, although the cotreatment did not alter Lhb induction but did eliminate the GnRH induction of Fshb. FFAs also suppressed activin-induced Fshb expression. Knockdown experiments showed that the FFA effect on the inflammatory kinases p38MAPK and JNK and on Lhb, but not Fshb, mRNA expression is mediated via toll-like receptor-2 and toll-like receptor-4 and was mimicked by lipopolysaccharide stimulation. In vivo, male C57BL/6 mice on a high-fat diet showed reduced FSH levels consistent with the suppression of Fshb seen in vitro. Histological analysis of the testes showed an increased number of abnormal seminiferous tubules. Female mice on a high-fat diet lacked the expected proestrus LH and FSH surge and exhibited an increase in the number of days at estrus and a reduced number of days at proestrus, and ovaries had significantly fewer corpora lutea. Taken together, our findings suggest that lipid excess can lead to reproductive defects in both male and female mice.

The combination of genetic variants of the FSHB and FSHR genes affects serum FSH in women of reproductive age.

STUDY QUESTION: What is the effect of FSHB-211G>T together with the FSHR 2039 A>G on serum FSH in women? SUMMARY ANSWER: Serum FSH levels are affected by the combination of genetic polymorphisms in FSHR and FSHB. WHAT IS KNOWN ALREADY: The relationship between SNPs of the FSHR gene and serum FSH has not been completely clarified. Genetic variants of the FSHB gene have been associated with variation in gene transcription and serum FSH levels in men. No data have been published on the effect of the FSHB-211G>T in women, alone or in combination with the FSHR 2039 A>G. STUDY DESIGN, SIZE, DURATION: This study was a prospective study including 193 healthy women of reproductive age. PARTICIPANTS/MATERIALS, SETTING, METHODS: Infertile and otherwise healthy eumenorrheic women (n = 193) with normal BMI and serum FSH levels were recruited for the study. In all women early follicular phase FSH and AMH were measured by commercial assays, and antral follicle count was measured by transvaginal ultrasound. Genomic DNA was purified from total peripheral blood and genotyping for the two SNPs was performed. MAIN RESULTS AND THE ROLE OF CHANCE: No significant gradients of increasing or decreasing Day 3 FSH across the FSHR 2039 (AA/AG/GG) and FSHB-211 (GG/GT/TT) genotypes, respectively, were observed. When women were stratified according to the FSHR 2039, and FSHB-211 genotypes a statistically significant reduction of d3 FSH was shown in the group of women with the FSHB-211 GT + TT/FSHR2039 AA genotype compared with the FSHB-211 GG/FSHR2039 GG genotype, hence confirming a possible additive effect of the different SNPs in FSHR and FSHB on regulating serum FSH. LIMITATIONS, REASONS FOR CAUTION: This finding requires an independent confirmation. However, it confirms the relationship between serum FSH and FSHB together with FSHR gene polymorphisms already reported in males. WIDER IMPLICATIONS OF THE FINDINGS: The knowledge of the FSHB/FSHR genotype combination is fundamental for the proper interpretation of serum FSH levels in women of reproductive age. STUDY FUNDING/COMPETING INTERESTS: Merck Serono supported the study in the form of a research grant for the laboratory session. None of the authors have any competing interest to declare.

Lack of specificity of plasma concentrations of inhibin B and follicle-stimulating hormone for identification of azoospermic survivors of childhood cancer: a report from the St Jude lifetime cohort study.

PURPOSE: Many male survivors of childhood cancer are at risk for azoospermia. Although both the levels of follicle-stimulating hormone (FSH) and inhibin B are correlated with sperm concentration, their ability to predict azoospermia in survivors of childhood cancer remains uncertain. PATIENTS AND METHODS: Semen analysis was performed and serum levels of FSH and inhibin B were measured in 275 adult male survivors of childhood cancer who had received gonadotoxic therapy. Receiver operating characteristic (ROC) analysis was performed to determine the optimal inhibin B and FSH values for identifying patients with azoospermia. The patient sample was divided into a learning set and a validation set. Sensitivity, specificity, and positive and negative predictive value were calculated. RESULTS: Inhibin B was dichotomized as ≤ 31 ng/L or more than 31 ng/L and FSH was dichotomized as ≤ 11.5 mIU/mL or more than 11.5 mIU/mL based on results of the ROC analysis. Using these values, the specificity of the serum level of inhibin B for identifying azoospermic survivors was 45.0%, and the positive predictive value was 52.1%. The specificity for FSH was 74.1%, and the positive predictive value was 65.1%. CONCLUSION: Neither serum inhibin B nor FSH is a suitable surrogate for determination of sperm concentration in a semen sample. Young men and their physicians should be aware of the limitations of these measures for assessment of fertility potential.

Development of a homologous enzyme-linked immunosorbent assay for European sea bass FSH. Reproductive cycle plasma levels in both sexes and in yearling precocious and non-precocious males.

Since the late 1980s, gonadotropins have been isolated and characterized in several fish species, but specific immunoassays for the follicle-stimulating hormone (FSH) have only been developed for a few. The present study reports the development and use of a specific and homologous competitive ELISA for measuring FSH in European sea bass (Dicentrarchus labrax) using a recombinant FSH and its specific antiserum. Recombinant European sea bass FSHß and FSH heterodimer were produced in the methylotrophic yeast Pichia pastoris and a baculovirus expression system, respectively. Specific polyclonal antibodies, generated by rabbit immunization against recombinant FSHß, were used at a final dilution of 1:8000. Recombinant FSH heterodimer was used to generate a standard curve and for coating of microplates (166 µg/ml). The sensitivity of the assay was 0.5 ng/ml [B(0)-2SD], and the intra- and inter-assay coefficients of variation were 2.12% (n=10) and 5.44% (n=16) (B(i)/B(0) ∼45%), respectively. A high degree of parallelism was observed between the standard curve and serially diluted plasma and pituitary samples of European sea bass. The ELISA developed was used to study the plasma FSH profiles of mature males and females during the reproductive cycle, and those of immature juvenile males under different light regimes. The analysis showed that FSH increased significantly during the intermediate stages of spermatogenesis and during vitellogenesis. Analyses in immature juvenile males showed that the continuous light photoperiod significantly reduced plasma FSH levels, and consequently, testicular growth and precocious puberty. In conclusion, the immunoassay developed has proven to be sensitive, specific and accurate for measuring European sea bass FSH, and it represents a valuable tool for future studies on the reproductive endocrinology of this species.

Influence of temperature regime on endocrine parameters and vitellogenesis during experimental maturation of European eel (Anguilla anguilla) females.

We examined the effect of temperature in European silver eels during their maturation induced by injections of carp pituitary extract on endocrine parameters: pituitary fshß and lhß expression, plasma 17ß-estradiol (E2) and vitellogenin, estrogen receptor 1 (esr1), and vitellogenin 2 (vtg2) expression in liver. A variable thermal regime (T10) that increased from 10° to 14° and 17°C was compared with a constant 20°C regime (T20) during 12 weeks. T10 caused a faster development until week 8, higher fshß, lhß, esr1 expression, and higher E2 levels. The results strongly suggest that T10 is inducing a higher endogenous FSH level which increases the E2 circulating level during vitellogenesis. A variable thermal regime induced an fshß expression and E2 profile in vitellogenic hormonally matured eel females that were more similar to the profile observed in other naturally maturing fish.

Plasticity of the reproductive axis caused by social status change in an african cichlid fish: I. Pituitary gonadotropins.

Social position in a dominance hierarchy is often tightly coupled with fertility. Consequently, an animal that can recognize and rapidly take advantage of an opportunity to rise in rank will have a reproductive advantage. Reproduction in all vertebrates is controlled by the brain-pituitary-gonad axis, and in males of the African cichlid fish Astatotilapia burtoni, GnRH1 neurons at the apex of this axis are under social control. However, little is known about how quickly social information is transformed into functional reproductive change, or about how socially controlled changes in GnRH1 neurons influence downstream actions of the brain-pituitary-gonad axis. We created an opportunity for reproductively suppressed males to ascend in status and then measured how quickly the perception of this opportunity caused changes in mRNA and protein levels of the pituitary gonadotropins. mRNA levels of the ß-subunits of LH and FSH rose rapidly in the pituitary 30 min after suppressed males perceived an opportunity to ascend. In contrast, mRNA levels of GnRH receptor-1 remained unchanged during social transition but were higher in stable dominant compared with subordinate males. In the circulation, levels of both LH and FSH were also quickly elevated. There was a positive correlation between mRNA in the pituitary and circulating protein levels for LH and FSH, and both gonadotropins were positively correlated with plasma 11-ketotestosterone. Our results show that the pituitary is stimulated extremely rapidly after perception of social opportunity, probably to allow suppressed males to quickly achieve reproductive success in a dynamic social environment.

Increased Prevalance of the -211 T allele of follicle stimulating hormone (FSH) beta subunit promoter polymorphism and lower serum FSH in infertile men.

CONTEXT: The human FSHB promoter polymorphism (rs10835638; -211 G/T) has been associated with serum FSH in a cohort of young Estonian men. The minor allele carriers had reduced serum FSH (15.7% in GT heterozygotes; 40% in TT homozygotes) compared with GG homozygotes. OBJECTIVE: Because FSH is essential for normal spermatogenesis and fertility, we speculated that abnormalities in FSH action could contribute to male infertility. We sought to study whether genetically inherited constitutively reduced FSH levels may affect male reproduction and replicate the association between rs10835638 and serum FSH among infertile male patients. DESIGN: Genotyping of rs10835638 in a cohort of infertile men (n = 1029; Andrology Center of the Tartu University Clinics, Estonia), including idiopathic infertility cases (IIFC; n = 750). PATIENTS: Patients included male partners (sperm concentration <20 x 10(6)/ml) of infertile couples failing to conceive a child for 12 months or longer. RESULTS: A significant excess of TT homozygotes (1.1 vs. 2.4%) as well as GT heterozygotes (22.4 vs. 25.1%) was detected among infertile men compared with the young male cohort (chi(2) test, P < 0.05). The T allele of rs10835638 was associated with reduced serum FSH (analysis of covariance; full cohort: P = 1.20 x 10(-6), F = 13.8; IIFC: P = 7.70 x 10(-7), F = 14.3) as well as with low FSH to LH ratio (full cohort: P = 1.52 x 10(-11), F = 25.6; IIFC: P = 3.25 x 10(-9), F = 20.4). The median serum FSH levels differed between the GG and TT carriers by 48.5%. All IIFC with TT genotype exhibited low (<1.8) FSH to LH ratio. CONCLUSIONS: In perspective, this genetic marker may have clinical significance in molecular diagnostics of male reproductive success and a potential to identify positive responders to FSH treatment.

The intrapituitary endocrine events during maturation and timing of puberty in the female sheep.

The aim of this study was to determine the maturational activity of gonadotroph cells, the site of synthesis, storage and release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in Polish Merino female sheep born after the summer solstice. The actual time of puberty of these lambs was delayed until the following breeding season, when they were 14 months old. Changes were examined in 12 peripubertal (30-, 52-week-old) and pubertal (Days 15 and 17 of the second ovarian cycle) females. Histomorphological and functional changes in the gonadotroph population were assayed with hybridohistochemistry, immunohistochemistry, computer-assisted image analysis and radioimmunoassay. The percentage of the adenohypophyseal area (PAA) occupied by gonadotrophs containing LHbeta-mRNA was higher and the LH plasma concentration and pulse frequency were lower in the 52-week-old sheep in comparison with the 30-week-old sheep (P<0.05). The PAA occupied by immunoreactive (ir)-LHbeta-cells remained stable at the 30th and 52nd weeks of age and then increased at the pubertal follicular phase. The PAA occupied by ir-FSHbeta-cells was higher in the 52-week-old sheep compared with the 30-week-old sheep and then lower at the pubertal follicular phase (P<0.05). The PAA occupied by gonadotrophs containing LHbeta-mRNA or FSHbeta-mRNA was lower at the pubertal follicular phase in comparison with the 52nd week of age (P<0.05). In pubertal sheep, the PAA occupied by gonadotrophs containing LHbeta-mRNA or FSHbeta-mRNA was higher and the PAA occupied by ir-LHbeta or ir-FSHbeta-cells was lower at the preovulatory phase in comparison with the follicular phase of the cycle (P<0.05). In conclusion, the photoperiodic suspension of gonadotroph population's maturational functions has been observed at the level of LH storage and release but not at the level of LH synthesis during the expected time of puberty in female sheep of an aseasonal breed such as Merino. The findings show the heterogeneity in the patterns of LH and FSH post-transcriptional processing during the period of peripubertal/pubertal transition, explained by the different intrapituitary regulation at the level of post-transcriptional synthesis and storage rather, than at the level of release. Altogether, intrapituitary mechanisms of ovine maturation could have the histomorphological feature. Our observations prompt the hypothesis that the female lamb may be able to transduce changes in day length into the appropriate endocrine cues for sexual maturation after attainment by the pituitary gonadotroph population the full peripubertal efficiency, manifested by the sufficient storage of LH.

Urinary beta-luteinizing hormone and beta-follicle stimulating hormone immunoenzymometric assays for population research.

OBJECTIVE: We developed assays for measurement of urinary betaLH and betaFSH under collection and storage conditions typical of non-clinical research settings. DESIGN AND METHODS: IEMAs for free betaLH and total betaFSH were validated by standard methods. Stability of urinary betaLH and betaFSH was tested across freeze-thaws and stored long term at 4 degrees C or -20 degrees C, or short term at room temperature, and with heating to dissociate the subunits. RESULTS: The IEMAs exhibited acceptable parallelism, specificity, recovery (averaging 100% for betaLH, 97% for betaFSH), imprecision (maximum within-run and between run CVs, respectively, 4.8% and 25.7% for betaLH, 5.6% and 17.0% for betaFSH), and minimum detectable dose (2.5 pmol/L for betaLH, 6.8 pmol/L for betaFSH). Urine and serum measures were highly correlated (r=0.95 for LH, 0.86 for FSH). There was no consistent decline with any storage type. Dissociation of subunits by heating was needed for betaLH, but not betaFSH. CONCLUSION: These IEMAs measure free betaLH and total betaFSH, overcoming inter-individual variability in, and collection and storage effects on, subunit dissociation, without the need for urine preservatives.

Clinical and hormonal features of selective follicle-stimulating hormone (FSH) deficiency due to FSH beta-subunit gene mutations in both sexes.

OBJECTIVE: To report the clinical, hormonal, and molecular features of a female adolescent with selective FSH deficiency. In addition, a complete review of previous cases is provided, focusing on hormonal aspects. DESIGN: Clinical study. SETTING: University hospital. PATIENT(S): A 16-year-old girl with primary amenorrhea and poor breast development due to isolated FSH deficiency. INTERVENTION(S): Blood drawing before and after GnRH stimulation and pelvic ultrasound examination. MAIN OUTCOME MEASURE(S): Gonadotropin and E(2) measurements and sequencing of the FSH beta-subunit gene. RESULT(S): The patient was referred for primary amenorrhea and partial breast development (Tanner III). Her basal and GnRH-stimulated LH levels were elevated (31 IU/L and 98 IU/L, respectively), whereas her FSH levels were undetectable (<1 IU/L) in both conditions. Estradiol levels were low (<13 pg/mL). Automatic sequencing showed a nucleotide substitution of C for A in exon 3, resulting in a homozygous nonsense mutation in amino acid position 76 (Tyr76X) of the FSH beta-subunit. CONCLUSION(S): The Tyr76X mutation of the FSH beta-subunit was associated with a partial phenotype of FSH deficiency. To date, only four loss-of-function mutations of the FSH beta-subunit have been described in eight patients with undetectable serum FSH and high serum LH levels. Therefore, this unusual hormonal profile strongly suggests a defect in the FSH beta-subunit in both sexes.

Gonadotropin-releasing hormone receptor gene expression during pubertal development of female rats.

Appropriate expression of the GnRH receptor (GnRH-R) in gonadotrophs is critical for GnRH signaling and hence for gonadotropin secretion and sexual development. In the present work, we have studied the ontogeny of the steady-state GnRH-R mRNA levels in pituitaries of female rats from Day 5 to Day 55, when sexual maturity is attained. Developmental changes of gonadotropin subunit (alpha, FSHbeta, and LHbeta) mRNA levels were also assessed. In addition, the role of the endogenous GnRH on the maturational changes of GnRH-R and gonadotropin subunit gene expression was investigated. Messenger RNA levels were determined by Northern blot analysis of total RNA from anterior pituitaries. Amounts of the most abundant (5.0 kilobase [kb]) GnRH-R mRNA increased slowly from Day 5 through the infantile period, to peak at Day 20 ( approximately 4-fold increase vs. Day 5). Thereafter the levels of the GnRH-R mRNA decline abruptly by Day 25 (75% decrease vs. Day 20) and then fell slightly until Day 35. Parallel changes were observed on the 4.5-kb transcript of the GnRH-R gene. Alpha subunit mRNA was easily detected at Day 5 and its levels increased quickly through the beginning of the infantile period to peak at Day 10 (3.2-fold increase vs. Day 5); then it decreased by 85% at Day 35. FSHbeta and LHbeta mRNA levels rose slowly until Days 15-20, a short time before GnRH-R. Thereafter, the levels of both mRNAs fell until Day 35 (90% decrease vs. Day 15 for FSHbeta and 50% decrease vs. Day 20 for LHbeta). To ascertain whether developmental activation of the GnRH-R and gonadotropin subunit gene expression is GnRH dependent, we have studied the effect of blocking the endogenous GnRH action by treating developing female rats with the specific GnRH antagonist cetrorelix (1.5 mg/kg body weight/wk, s.c.) through the infantile (Days 5-20) and the juvenile period (Days 20-35). Cetrorelix completely blocked the rise of levels of the two most abundant species, 5.0 kb and 4.5 kb, of GnRH-R mRNA during the infantile phase and dropped them to almost undetectable levels during the juvenile prepubertal period. Cetrorelix also abolished the developmental rise of gonadotropin beta subunit mRNAs during the two periods of the study. In contrast, alpha subunit gene expression tended to decrease, but not significantly, with cetrorelix treatment during the two periods. These data demonstrate that sexual maturation of female rats is advanced by an early and strong induction of GnRH-R and gonadotropin subunit gene expression during the infantile period, followed by weaker persistent activation during puberty. Developmental GnRH-R and gonadotropin beta subunit gene expression is almost entirely GnRH dependent, not only in the juvenile prepubertal stage but also during the infantile period.

Pulsatile luteinizing hormone and follicle-stimulating hormone secretion and gonadotropin subunit mRNA levels in the ovariectomized GPR-4 transgenic rat.

Genetic targeting of the cAMP-specific phosphodiesterase 4D1 (PDE4D1) to gonadotropin-releasing hormone (GnRH) neurons in the GPR-4 transgenic rat resulted in decreased luteinizing hormone (LH) pulse frequency in castrated female and male rats. A similar decrease in the intrinsic GnRH pulse frequency was observed in GT1 GnRH cells expressing the PDE4D1 phosphodiesterase. We have extended these findings in ovariectomized (OVX) GPR-4 rats by asking what effect transgene expression had on pulsatile LH and follicle-stimulating hormone (FSH) secretion, plasma and pituitary levels of LH and FSH, and levels of the alpha-glycoprotein hormone subunit (alpha-GSU), LH-beta and FSH-beta subunit mRNAs. In OVX GPR-4 rats the LH pulse frequency but not pulse amplitude was decreased by 50% compared to wild-type littermate controls. Assaying the same samples for FSH, the FSH pulse frequency and amplitude were unchanged. The plasma and anterior pituitary levels of LH in the GPR-4 rats were significantly decreased by approximately 45%, while the plasma but not anterior pituitary level of FSH was significantly decreased by 25%. As measured by real-time RT-PCR, the mRNA levels for the alpha-GSU in the GPR-4 rats were significantly decreased by 41%, the LH-beta subunit by 38% and the FSH-beta subunit by 28%. We conclude that in the castrated female GPR-4 rats the decreased GnRH pulse frequency results in decreased levels of LH and FSH and in the alpha- and beta-subunit mRNA levels.

Normal reproductive function in InhBP/p120-deficient mice.

The inhibins are gonadal transforming growth factor beta superfamily protein hormones that suppress pituitary follicle-stimulating hormone (FSH) synthesis. Recently, betaglycan and inhibin binding protein (InhBP/p120, also known as the product of immunoglobulin superfamily gene 1 [IGSF1]) were identified as candidate inhibin coreceptors, shedding light on the molecular basis of how inhibins may affect target cells. Activins, which are structurally related to the inhibins, act within the pituitary to stimulate FSH production. Betaglycan increases the affinity of inhibins for the activin type IIA (ACVR2) receptor, thereby blocking activin binding and signaling through this receptor. InhBP/p120 may not directly bind inhibins but may interact with the activin type IB receptor, ALK4, and participate in inhibin B's antagonism of activin signaling. To better understand the in vivo functions of InhBP/p120, we characterized the InhBP/p120 mRNAs and gene in mice and generated InhBP/p120 mutant mice by gene targeting in embryonic stem cells. InhBP/p120 mutant male and female mice were viable and fertile. Moreover, they showed no alterations in FSH synthesis or secretion or in ovarian or testicular function. These data contribute to a growing body of evidence indicating that InhBP/p120 does not play an essential role in inhibin biology.