A cryptozoospermic infertile male with Y chromosome AZFc microdeletion and low FSH levels due to a simultaneous polymorphism in the FSHB gene: a case report.

Genetic causes account for 10-15% of male factor infertility, making the genetic investigation an essential and useful tool, mainly in azoospermic and severely oligozoospermic men. In these patients, the most frequent findings are chromosomal abnormalities and Y chromosome long arm microdeletions, which cause a primary severe spermatogenic impairment with classically increased levels of FSH. On the other hand, polymorphisms in the FSH receptor (FSHR) and FSH beta chain (FSHB) genes have been associated with different FSH plasma levels, due to variations in the receptor sensitivity (FSHR) or in the production of FSH from the pituitary gland (FSHB). Here, we describe an unusual patient with a combined genetic alteration (classic AZFc deletion of the Y chromosome and TT homozygosity for the -211G>T polymorphism in the FSHB gene (rs10835638)), presenting with cryptozoospermia, severe hypospermatogenesis, and normal LH and testosterone plasma concentrations, but low FSH levels. The patient partially benefitted from treatment with FSH (150 IU three times/week for 6 months) which allowed him to cryopreserve enough motile spermatozoa to be used for intracytoplasmic sperm injection. According to our knowledge, this is the first report of an infertile man with AZFc microdeletion with low FSH plasma concentrations related to homozygosity for the -211G>T polymorphism in the FSHB gene.

Laying performance, genetic parameters, and the expression of FSHß, LHß, FSHR, and LHR genes in Japanese quails selected for early egg production.

Investigating the impact of early egg production selection (the first 90 d of laying) on egg production features, cumulative selection response (CSR), and the mRNA expression of gonadotropins (FSHß and LHß), and their receptors (FSHR and LHR), in Japanese quails was the goal. The selection experiment involved 1293 females in all, 257 from the base group and 1036 from the 4 selected generations. Age and body weight at sexual maturity (ASM, BWSM), weight of the first egg (WFE), days to the first 10 eggs (DF10E), egg mass for the first 10 eggs (EMF10E), egg weight (EW), egg number at the first 90 d of laying (EN90D), and egg mass at the first 90 d of laying (EM90D) were all recorded. Most egg production traits had heritability estimates that were low to moderate and ranged from 0.17 to 0.33., where the highest estimates were reported for EN90D (0.33) and BWSM (0.32). With the exception of EN90D, low to moderate positive genetic correlations were observed between ASM and other egg production traits (0.17-0.44). The fourth generation showed significantly (P < 0.05) lower ASM and DF10E but higher BWSM, WFE, EN90D, EM10E, and EM90D when compared with the base generation. CSR were significant (P < 0.05) for ASM (-6.67 d), BWSM (27.13 g), WFE (0.93 g), DF10E (-1.25 d), EN90D (7.24 egg), EM10E (10.57 g), and EM90D (140.0 g). FSHß, LHß, FSHR, and LHR gene mRNA expression was considerably (P < 0.05) greater in the fourth generation compared to the base generation. In conclusion, selection programs depending on the efficiency of egg production (EN90D) could improve the genetic gain of egg production traits and upregulate the mRNA expression of FSHß, LHß, FSHR, and LHR genes in selected quails (fourth generation). These findings might help to enhance breeding plans and create commercial lines of high egg production Japanese quails.

FSHß links photoperiodic signaling to seasonal reproduction in Japanese quail.

Annual cycles in daylength provide an initial predictive environmental cue that plants and animals use to time seasonal biology. Seasonal changes in photoperiodic information acts to entrain endogenous programs in physiology to optimize an animal's fitness. Attempts to identify the neural and molecular substrates of photoperiodic time measurement in birds have, to date, focused on blunt changes in light exposure during a restricted period of photoinducibility. The objectives of these studies were first to characterize a molecular seasonal clock in Japanese quail and second, to identify the key transcripts involved in endogenously generated interval timing that underlies photosensitivity in birds. We hypothesized that the mediobasal hypothalamus (MBH) provides the neuroendocrine control of photoperiod-induced changes in reproductive physiology, and that the pars distalis of the pituitary gland contains an endogenous internal timer for the short photoperiod-dependent development of reproductive photosensitivity. Here, we report distinct seasonal waveforms of transcript expression in the MBH, and pituitary gland and discovered the patterns were not synchronized across tissues. Follicle-stimulating hormone-ß (FSHß) expression increased during the simulated spring equinox, prior to photoinduced increases in prolactin, thyrotropin-stimulating hormone-ß, and testicular growth. Diurnal analyses of transcript expression showed sustained elevated levels of FSHß under conditions of the spring equinox, compared to autumnal equinox, short (<12L) and long (>12L) photoperiods. FSHß expression increased in quail held in non-stimulatory short photoperiod, indicative of the initiation of an endogenously programmed interval timer. These data identify that FSHß establishes a state of photosensitivity for the external coincidence timing of seasonal physiology. The independent regulation of FSHß expression provides an alternative pathway through which other supplementary environmental cues, such as temperature, can fine tune seasonal reproductive maturation and involution.

Implication of FSHB rs10835638 variant in endometriosis in Brazilian women.

OBJECTIVE: The follicle-stimulating hormone subunit beta gene rs10835638 variant (c.-211G>T) may have detrimental effects on fertility and protective effects against endometriosis. A case-control analysis was performed, aiming to investigate the possible relationship between this variant and the development and/or progression of endometriosis. METHODS: This study included 326 women with endometriosis and 482 controls without endometriosis, both confirmed by inspection of the pelvic cavity during surgery. Genotyping was performed using a TaqMan real-time polymerase chain reaction assay. Genotype and allele frequencies and genetic models were compared between the groups. RESULTS: The genotype and allele frequencies of the rs10835638 variant did not differ between women with and those without endometriosis. Subdividing the endometriosis group into fertile and infertile groups did not result in a significant difference in these frequencies. However, the subgroup with minimal/mild endometriosis had a higher frequency of the GT genotype than the Control Group, regardless of fertility. The T allele was significantly more common in women with minimal/mild endometriosis than in the Control Group in the recessive model. CONCLUSION: The T allele is associated with the development of minimal/mild endometriosis in Brazilian women.

Chronological Changes in Gonadotropin-Releasing Hormone 1, Gonadotropins, and Sex Steroid Hormones along the Brain-Pituitary-Gonadal Axis during Gonadal Sex Differentiation and Development in the Longtooth Grouper, Epinephelus bruneus.

(1) Fshß and Lhß showed stronger signals and higher transcript levels from 590 to 1050 dph than at earlier stages, implying their active involvement during primary oocyte development. (2) Fshß and Lhß at lower levels were detected during the phases of ovarian differentiation and oogonial proliferation. (3) E2 concentrations increased significantly at 174, 333, and 1435 dph, while T concentrations exhibited significant increases at 174 and 333 dph. These findings suggest potential correlations between serum E2 concentrations and the phases of oogonial proliferation and pre-vitellogenesis.

Regulation of steroid production and key genes in catfish Heteropneustes fossilis using recombinant gonadotropins.

The two gonadotropins, FSH and LH, stimulate growth and development of the gonads through gonadal biosynthesis of steroid hormones and growth factors. To date, cDNA sequences encoding gonadotropin subunits have been isolated and characterized from a large number of fish species. Recently, we successfully cloned and characterized gonadotropins (LHß, FSHß, and GPα) from the pituitary glands of the catfish, Heteropneustes fossilis. In the present study, we describe herein the production of recombinant stinging catfish, H. fossilis (hf) FSH (rhfFSH) and LH (rhfLH) using the methylotrophic yeast P. pastoris expression system. We further explored the hypothesis that the recombinant gonadotropins can modulate the hypothalamus-pituitary-ovarian (HPO) axis genes (avt, it, gnrh2, kiss2, and cyp19a1a) and regulate their transcriptional profile and steroid levels in relation to their annual developmental stage during preparatory and pre-spawning phases under in-vitro conditions. We found that the different concentrations of recombinant rhfFSH and rhfLH significantly stimulated E2 levels in the preparatory and prespawning season, and also upregulated gonadal aromatase gene expression in a dose dependent manner. Our results demonstrate that the yeast expression system produced biologically active recombinant catfish gonadotropins, enabling the study of their function in the catfish.

EZH2 Gene Knockdown Inhibits Sheep Pituitary Cell Proliferation via Downregulating the AKT/ERK Signaling Pathway.

Pituitary gonadotropins perform essential functions in mammalian reproduction by stimulating gametogenesis and steroidogenesis in the ovaries and testicles. EZH2 is a histone methyltransferase that inhibits proliferation and aggravates apoptosis in stem cells subjected to pathological stimuli. However, the expression and molecular mechanisms of EZH2 in pituitary cells in vitro have not been extensively studied. In this study, the relative abundances of EZH2 mRNA (p < 0.01) and protein (p < 0.05) expression were larger in the pituitary cells of Hu sheep with relatively greater fecundity (GF) compared to those with lesser fecundity (LF). Loss-of-function examinations demonstrated that EZH2 gene knockdown led to an earlier induction of apoptosis in sheep pituitary cells (PCs). The relative abundance of CASP3, CASP9, and BAX was increased (p < 0.01), while BCL2's abundance was less decreased (p < 0.01) in PCs where there was EZH2 gene knockdown. Additionally, cell proliferation (p < 0.01) and viability (p < 0.01) were decreased in EZH2-knockdown sheep PCs, and the cell cycle was blocked compared to a negative control (NC). Notably, EZH2 gene knockdown led to reduced abundances of gonadotropin subunit gene transcripts (FSHß, p < 0.05) and reduced FSH release (p < 0.01) from PCs. EZH2 gene knockdown led to reduced phosphorylation of AKT, ERK, and mTOR (p < 0.01). The results suggest that EZH2 regulates pituitary cell proliferation, apoptosis, and FSH secretion through modulation of the AKT/ERK signaling pathway, providing a foundation for further study of pituitary cell functions.

RNA-seq analysis identifies age-dependent changes in expression of mRNAs - encoding N-glycosylation pathway enzymes in mouse gonadotropes.

Follicle-stimulating hormone (FSH) is a glycoprotein that is assembled as a heterodimer of α/ß subunits in gonadotropes. Each subunit contains two N-glycan chains. Our previous in vivo genetic studies identified that at least one N-glycan chain must be present on the FSHß subunit for efficient FSH dimer assembly and secretion. Moreover, macroheterogeneity observed uniquely on human FSHß results in ratiometric changes in age-specific FSH glycoforms, particularly during menopausal transition. Despite the recognition of many prominent roles of sugars on FSH including dimer assembly and secretion, serum half-life, receptor binding and signal transduction, the N-glycosylation machinery in gonadotropes has never been defined. Here, we used a mouse model in which gonadotropes are GFP-labeled in vivo and achieved rapid purification of GFP+ gonadotropes from pituitaries of female mice at reproductively young, middle, and old ages. We identified by RNA-seq analysis 52 mRNAs encoding N-glycosylation pathway enzymes expressed in 3- and 8-10-month-old mouse gonadotropes. We hierarchically mapped and localized the enzymes to distinct subcellular organelles within the N-glycosylation biosynthetic pathway. Of the 52 mRNAs, we found 27 mRNAs are differentially expressed between the 3- and 8-10-month old mice. We subsequently selected 8 mRNAs which showed varying changes in expression for confirmation of abundance in vivo via qPCR analysis, using more expanded aging time points with distinct 8-month and 14-month age groups. Real time qPCR analysis indicated dynamic changes in expression of N-glycosylation pathway enzyme-encoding mRNAs across the life span. Notably, computational analysis predicted the promoters of genes encoding these 8 mRNAs contain multiple high probability binding sites for estrogen receptor-1 and progesterone receptor. Collectively, our studies define the N-glycome and identify age-specific dynamic changes in mRNAs encoding N-glycosylation pathway enzymes in mouse gonadotropes. Our studies suggest the age-related decline in ovarian steroids may regulate expression of N-glycosylation enzymes in mouse gonadotropes and explain the age-related N-glycosylation shift previously observed on human FSHß subunit in pituitaries of women.

Gonadotropins and Sex Steroid Hormones in Captive-Reared Small Yellow Croaker (Larimichthys polyactis) and Their Role in Female Reproductive Dysfunction.

The seed production of small yellow croaker (SYC) is constrained by reproductive dysfunction in captive-reared females. Reproductive dysfunction is closely linked to endocrine reproductive mechanisms. To better understand the reproductive dysfunction in captive broodstock, functional characterization of gonadotropins (GtHs: follicle stimulating hormone ß subunit, fshß; luteinizing hormone ß subunit, lhß; and glycoprotein α subunit, gpα) and sex steroids (17ß-estradiol, E2; testosterone, T; progesterone; P) was performed using qRT-PCR, ELISA, in vivo, and in-vitro assay. The pituitary GtHs and gonadal steroids levels were significantly higher in ripen fish of both sexes. However, changes in lhß and E2 levels in females were not significant in the developing and ripen stages. Furthermore, GtHs and steroids levels were lower in females compared to males throughout the reproductive cycle. In vivo administration of gonadotropin releasing hormone analogue (GnRHa) significantly increased the expression of GtHs in both dose- and time-related manners. The lower and higher doses of GnRHa led to successful spawning in male and female SYC, respectively. Sex steroids in vitro significantly inhibited the expression of lhß in female SYC. Overall, GtHs were shown to play a vital role in final gonadal maturation, while steroids promoted negative feedback in the regulation of pituitary GtHs. Lower levels of GtHs and steroids might be key components in the reproductive dysfunction of captive-reared female SYC.

Activating Transcription Factor 3 Stimulates Follicle-Stimulating Hormone-ß Expression In Vitro But Is Dispensable for Follicle-Stimulating Hormone Production in Murine Gonadotropes In Vivo.

Follicle-stimulating hormone (FSH), a dimeric glycoprotein produced by pituitary gonadotrope cells, regulates spermatogenesis in males and ovarian follicle growth in females. Hypothalamic gonadotropin-releasing hormone (GnRH) stimulates FSHß subunit gene (Fshb) transcription, though the underlying mechanisms are poorly understood. To address this gap in knowledge, we examined changes in pituitary gene expression in GnRH-deficient mice (hpg) treated with a regimen of exogenous GnRH that increases pituitary Fshb but not luteinizing hormone ß (Lhb) messenger RNA levels. Activating transcription factor 3 (Atf3) was among the most upregulated genes. Activating transcription factor 3 (ATF3) can heterodimerize with members of the activator protein 1 family to regulate gene transcription. Co-expression of ATF3 with JunB stimulated murine Fshb, but not Lhb, promoter-reporter activity in homologous LßT2b cells. ATF3 also synergized with a constitutively active activin type I receptor to increase endogenous Fshb expression in these cells. Nevertheless, FSH production was intact in gonadotrope-specific Atf3 knockout [conditional knockout (cKO)] mice. Ovarian follicle development, ovulation, and litter sizes were equivalent between cKOs and controls. Testis weights and sperm counts did not differ between genotypes. Following gonadectomy, increases in LH secretion were enhanced in cKO animals. Though FSH levels did not differ between genotypes, post-gonadectomy increases in pituitary Fshb and gonadotropin α subunit expression were more pronounced in cKO than control mice. These data indicate that ATF3 can selectively stimulate Fshb expression in vitro but is not required for FSH production in vivo.

Genetic polymorphisms in ESR and FSHß genes and their association with litter traits in Large White pigs.

The estrogen receptor (ESR) gene and follicle-stimulating hormone ß (FSHß) gene are responsible for litter traits. The present study aimed to verify the polymorphisms of ESR and FSHß and assess their effects on the litter traits in 201 Large White pigs. Four SNPs (g.C669T, g.A1296G, g.C1665T and g.A1755G) were found in ESR. The TT genotype at g.C1665T locus and AA genotype at g.A1755G locus could significantly increase the total litter size of the first litter of American Large White pigs (p < 0.05). Eight SNPs were found in exon 3 of FSHß. The AA genotype at g.A511G locus, AA and AG genotypes at g.A617G locus, CC and CT genotypes at g.C630T locus, CT and TT genotypes at g.C652T locus, CT and TT genotypes at g.C735T locus, AA and AG genotypes at g.A746G, AA and AG genotypes at g.A921G and CT genotype at g.C678T could significantly increase the litter size of different strains of Large White pigs (p < 0.05). Our study revealed that the genetic variations of ESR and FSHß were closely related to the litter trait of Large White pigs. Therefore, ESR and FSHß genes could be used as molecular markers for the genetic selection of Large White pigs.

Inhibition of activin signalling reduces the growth of LßT2 gonadotroph pituitary tumours in mouse.

Pituitary tumours are benign neoplasms that derive from hormone-producing cells of the pituitary gland. While medical treatments have emerged for most subtypes, gonadotroph tumours that express follicle-stimulating hormone (FSH) and/or luteinizing hormone still lack therapeutic options apart from surgery and radiotherapy. Activin ligands are physiological regulators of production and secretion of FSH by gonadotroph cells, but their role in gonadotroph tumourigenesis remains little explored. Using the LßT2 mouse gonadotroph cell line which produces FSH under activin stimulation, we first tested whether subcutaneous xenografts of LßT2 cells resulted in tumour formation in Rag2KO mice. Histological analysis confirmed the presence of LßT2 tumours with endothelial cells and macrophages in their microenvironment. FSH expression was found in a subset of clusters of LßT2 cells in the tumours. We subsequently addressed the consequences of targeting activin signalling via injection of a soluble activin decoy receptor (sActRIIB-Fc). sActRIIB-Fc treatment resulted in significantly decreased LßT2 tumour volume. Reduced Smad2 phosphorylation as well as inhibition of tumour-induced FSH production confirmed the efficient targeting of activin-downstream signalling in treated tumours. More interestingly, treated tumours showed significantly fewer endothelial cells associated with reduced Vegfa expression. In vitro treatment of LßT2 cells with sActRIIB-Fc had no effect on cell proliferation or apoptosis, but Vegfa expression was inhibited, pointing to a likely paracrine effect of LßT2 cells on endothelial cells through activin-mediated Vegfa regulation. Further in vitro and in vivo studies are now needed to pinpoint the exact roles of activin signalling in these processes prior to translating these observations to the clinic.

Rerouting of follicle-stimulating hormone secretion and gonadal function.

Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are synthesized in the same pituitary cell, i.e., gonadotrope. They both consist of a common α-subunit that is noncovalently assembled with a hormone-specific ß-subunit in gonadotropes. The heterodimers exit gonadotropes through distinct modes of trafficking and secretion. The FSH is constitutively secreted, whereas LH is secreted in pulses through the regulated pathway that involves dense core granules. Based on several in vitro mutagenesis studies, the carboxy terminus heptapeptide of human LH-ß subunit is identified as a gonadotrope sorting determinant. When heptapeptide is genetically fused to human FSH-ß subunit and the mutant transgene expressed on a Fshb null genetic background, the rerouted FSH mutant dimer enters the LH secretory pathway, stored in dense core granules, coreleased with LH on gonadotropin releasing hormone stimulation and rescues Fshb null mice as efficiently as the constitutively secreted wild-type FSH. The rerouted FSH markedly suppresses follicle atresia and significantly enhances ovulations per cycle and prolongs the female reproductive life span. Gonadotropin rerouting is emerging as a novel paradigm to treat ovarian dysfunction in women, and may explain the origins of ovarian cyclicity as well as provide clues to understand gene and protein networks that maintain optimal ovarian function throughout the female reproductive life span.

Two Synthetic Peptides Corresponding to the Human Follicle-Stimulating Hormone ß-Subunit Promoted Reproductive Functions in Mice.

A follicle stimulating hormone (FSH) is widely used in the assisted reproduction and a synthetic peptide corresponding to a receptor binding region of the human (h) FSH-ß-(34−37) (TRDL) modulated reproduction. Furthermore, a 13-amino acid sequence corresponding to hFSH-ß-(37−49) (LVYKDPARPKIQK) was recently identified as the receptor binding site. We hypothesized that the synthetic peptides corresponding to hFSH-ß-(37−49) and hFSH-ß-(34−49), created by merging hFSH-ß-(34−37) and hFSH-ß-(37−49), modulate the reproductive functions, with the longer peptide being more biologically active. In male or female prepubertal mice, a single injection of 200 µg/g BW ip of hFSH-ß-(37−49) or hFSH-ß-(34−49) hastened (p < 0.05) puberty, whereas the same treatments given daily for 4 d promoted (p < 0.05) the gonadal steroidogenesis and gamete formation. In addition of either peptide to the in vitro cell cultures, promoted (p < 0.05) the proliferation of primary murine granulosa cells and the estradiol production by upregulating the expression of Ccnd2 and Cyp19a1, respectively. In adult female mice, 200 µg/g BW ip of either peptide during diestrus antagonized the FSH-stimulated estradiol increase and uterine weight gain during proestrus. Furthermore, hFSH-ß-(34−49) was a more potent (p < 0.05) reproductive modulator than hFSH-ß-(37−49), both in vivo and in vitro. We concluded that hFSH-ß-(37−49) and especially hFSH-ß-(34−49), have the potential for reproductive modulation.

Mutual Effects of Orexin and Bone Morphogenetic Proteins on Gonadotropin Expression by Mouse Gonadotrope Cells.

Orexin plays a key role in the regulation of sleep and wakefulness and in feeding behavior in the central nervous system, but its receptors are expressed in various peripheral tissues including endocrine tissues. In the present study, we elucidated the effects of orexin on pituitary gonadotropin regulation by focusing on the functional involvement of bone morphogenetic proteins (BMPs) and clock genes using mouse gonadotrope LßT2 cells that express orexin type 1 (OX1R) and type 2 (OX2R) receptors. Treatments with orexin A enhanced LHß and FSHß mRNA expression in a dose-dependent manner in the absence of GnRH, whereas orexin A in turn suppressed GnRH-induced gonadotropin expression in LßT2 cells. Orexin A downregulated GnRH receptor expression, while GnRH enhanced OX1R and OX2R mRNA expression. Treatments with orexin A as well as GnRH increased the mRNA levels of Bmal1 and Clock, which are oscillational regulators for gonadotropin expression. Of note, treatments with BMP-6 and -15 enhanced OX1R and OX2R mRNA expression with upregulation of clock gene expression. On the other hand, orexin A enhanced BMP receptor signaling of Smad1/5/9 phosphorylation through upregulation of ALK-2/BMPRII among the BMP receptors expressed in LßT2 cells. Collectively, the results indicate that orexin regulates gonadotropin expression via clock gene expression by mutually interacting with GnRH action and the pituitary BMP system in gonadotrope cells.

Effects of active immunization against a 13-amino acid receptor-binding epitope of FSHß on fertility regulation in female mice.

Follicle-stimulating hormone (FSH) is crucial for ovarian folliculogenesis and thus essential for female fertility. Here, we developed a novel FSH vaccine based on the tandem of a 13-amino acid receptor-binding epitope of FSHß (FSHß13AA-T) and used a mouse model to test its efficacy in female fertility regulation. Compared to placebo-immunized controls, FSHß13AA-T vaccination: induced a marked (P < 0.05) antibody generation; reduced (P < 0.05) serum concentrations of FSH, inhibin B and 17ß-estradiol; disrupted (P < 0.05) normal estrous cyclicity; delayed (P = 0.08) establishment of pregnancy; blocked (P < 0.05) folliculogenesis; and reduced (P < 0.05) litter size. Mechanistically, FSH vaccination reduced (P < 0.05) ovarian estrogen production by decreasing Lhcgr, Cyp19a1 and HSD3ß1 expression, and suppressed ovarian follicular development by decreasing ovarian Fshr, Inhα, Foxo3a, Bmp15 and Cdh1 expression. Overall, vaccination of female mice with FSHß13AA-T substantially disrupted FSH-dependent ovarian steroidogenesis and folliculogenesis, and caused subfertility. Therefore, vaccines based on FSHß13AA-T have potential as anti-fertility/contraceptive agents in females.

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.

Presence of GnRH3 in sturgeon and the roles of GnRH1 and GnRH2 on the regulation of LH/FSH in mature female sterlet Acipenser ruthenus in vitro.

Three forms of gonadotropin-releasing hormones (GnRHs), ArGnRH1, ArGnRH2, and ArGnRH3, were identified in sterlet. Compared with their orthologue, ArGnRH1 and ArGnRH2 have conserved core decapeptide but show low identity in the signal peptide and the rest of the sequences. The existence of the GnRH3 paralogue of sturgeon was predicted for the first time with TBLASTN by using the amino acid sequences of catshark and whale shark GnRH3 precursor as queries against the whole genome and transcript data of sterlet. The predicted ArGnRH3 cDNA sequence was composed of three exons containing all the elements of the GnRH family. The successful molecular cloning of GnRH3 from sterlets verified its expression in the brain of sturgeons. The analysis of the ArGnRH3 amino acid sequence revealed a completely conserved decapeptide sequence that shows 100% identity with the sequence of teleosts and differs in one amino acid with that of the cartilaginous fish (catshark and whale shark) at the 5th position. The structure of the phylogenetic tree showed that a total of 52 vertebrate GnRH sequences were clustered into three main clades corresponding to GnRH1, GnRH2, and GnRH3. The ArGnRH3 sequence is the oldest GnRH3 identified in teleosts. The tissue distribution analysis showed that ArGnRH1 was expressed in all the 13 examined tissues of females and in most of the tested tissues of male fish, with the highest expression in the pituitary and hypothalamus. ArGnRH2 is only expressed in the pituitary, hypothalamus, and gonads of both female and male sterlets. ArGnRH3 mRNA could be detected in the pituitary, hypothalamus, and gonad in both female and male fish. It is also present in the spleen, head kidney, and gill in female fish and in kidney and heart in male fish. However, the ArGnRH3 only showed weak expression in all the positive tissues. ArGnRH1 and ArGnRH2 active decapeptides were synthesized to investigate their roles on the regulation of LH/FSH using a mixed brain cell line from a sexually mature female sterlet. The results showed that ArGnRH1 and ArGnRH2 exerted different effects on the gene expression and release of gonadotropins. ArGnRH1 promoted the expression of fshß significantly around 48 h, and the expression was suppressed when the treatment time was extended to 72 h. ArGnRH1 had no significant effects on the level of either mRNA or secreted lh in any of the tested treatment length or concentrations. Moreover, ArGnRH1 did not stimulate the activity of gonadotropins in the maturation stage of female sturgeons. ArGnRH2 promoted the expression of fshß at 24 h and 48 h and increased mRNA level of lhß at 6 h and 48 h, accompanied by the significant secretion of LH at 72 h, although the high mRNA level of fsh did not correlate with the secretion of FSH in ArGnRH2-treated groups. In conclusion, ArGnRH2 plays an important role in the maturation stage of female sterlets. Therefore, ArGnRH2 has the potential to induce ovulation and spermiation in sturgeons.

Cloning of gonadotropin Gph-alpha, FSH-beta and LH-beta subunits and seasonal profiles of steroid hormones in wild-caught Nile perch, Lates niloticus.

The Nile perch (np; Lates niloticus) is a freshwater teleost species with a potential for aquaculture in freshwater surroundings. However, wild-caught breeders have persistently failed to spawn spontaneously in captivity. Cloning of the gonadotropin subunits and analysing seasonal variation in reproductive hormone levels for a 1-year period were done to gain knowledge on the physiological basis underlying the reproductive biology of np. The ß-follicle-stimulating hormone (FSH-ß) and ß-luteinizing hormone (LH-ß) subunits and their common α-glycoprotein (Gph-α) subunit were cloned using 3' and 5' RACE-PCR. The nucleotide sequences of the npgph-α, npfsh-ß, and nplh-ß subunits were 664, 580 and 675 nucleotides in length, encoding peptides of 124, 120 and 148 amino acids, respectively. The deduced amino acid sequence of each mature subunit showed high similarity with its counterparts in other teleost. Sequence analysis showed that npFSH-ß is more similar to higher vertebrate FSH-ßs than to higher vertebrate LH-ßs. Heterologous immunoassay was calibrated to analyse pituitary LH levels. While the LH immunoassay showed parallelism of npLH with that of tilapia (ta), no parallelism for FSH was found. Levels of pituitary LH were higher in females at gonadal stages of vitellogenic oocytes, mature secondary oocytes and mature tertiary oocytes with migrating nucleus than in pre-vitellogenic oocytes and early and late perinucleolus oocytes. Using competitive steroid ELISA, variations in the levels of the steroid hormones 11-ketotestosterone (11-KT) in males and E2 in females were characterized in relation to month and reproductive index of Nile perch. Our findings show that in females, gonadosomatic index and plasma E2 were highly correlated (R2 = 0.699, n = 172) and peaked from September to November while in males, the gonadosomatic index and plasma 11-KT peaked from October to November. In female fish, both steroid hormones were detected in the plasma but greatly varied in concentrations. E2 in particular, increased with the developmental stage of the gonads. The levels of steroid hormones, E2 and 11-KT in females and males respectively increased with fish size (total lengths) and suggest that females mature at a body length of 40-59 cm than their counter part males that mature at a total length of 60-70 cm. Taken together, we describe seasonal endocrine differences in wild-caught adult Nile perch which could potentially be exploited to manipulate the reproductive axis in cultured breeders.

Follicle-stimulating hormone beta subunit and receptor variations in infertile men in Central Black Sea Region of Turkey.

Genetic variants affecting the interaction of FSH-FSHR may negatively affect the male reproductive potential. The aim of this case-control study was to evaluate FSHB c.-211G>T and FSHR c.2039A>G variants in a cohort of infertile men from Central Black Sea Region in Turkey. One hundred and nine infertile men and 50 proven fertile controls were enrolled in the study. Genotyping was assessed by RFLP. The genotype frequencies of FSHB -211G>T and FSHR 2039A>G showed significant variation between infertile and fertile groups (χ2 , p = 0.046, GG vs. GT+TT, and p = 0.008, AA vs. AG+GG). FSHB -211GG was found to be higher in patients with OAT compared to fertile controls (82.3% vs. 64.0%, χ2 , p = 0.028). The distribution of FSHR 2039A>G alleles was different between infertile and fertile men (χ2 , p = 0.005, total infertile vs. fertile groups, p = 0.019, OAT vs. NOA vs. fertile groups). Further analysis showed that the frequencies of FSHR 2039AA wild-type genotype were higher in the oligoasthenoteratozoospermic and non-obstructive azoospermic groups compared with the controls (χ2 , 39.3% vs. 17.0%, p = 0.012, and 37.5% vs. 17.0%, p = 0.025 respectively). Our results showed wild-types of FSHB -211G>T and FSHR 2039A>G variants may cause susceptibility to male infertility in the Central Black Sea Region of Turkey.