Single molecule characterization of the binding kinetics of a transcription factor and its modulation by DNA sequence and methylation.

The interaction of transcription factors with their response elements in DNA is emerging as a highly complex process, whose characterization requires measuring the full distribution of binding and dissociation times in a well-controlled assay. Here, we present a single-molecule assay that exploits the thermal fluctuations of a DNA hairpin to detect the association and dissociation of individual, unlabeled transcription factors. We demonstrate this new approach by following the binding of Egr1 to its consensus motif and the three binding sites found in the promoter of the Lhb gene, and find that both association and dissociation are modulated by the 9 bp core motif and the sequences around it. In addition, CpG methylation modulates the dissociation kinetics in a sequence and position-dependent manner, which can both stabilize or destabilize the complex. Together, our findings show how variations in sequence and methylation patterns synergistically extend the spectrum of a protein's binding properties, and demonstrate how the proposed approach can provide new insights on the function of transcription factors.

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.

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.

The base pair-scale diffusion of nucleosomes modulates binding of transcription factors.

The structure of promoter chromatin determines the ability of transcription factors (TFs) to bind to DNA and therefore has a profound effect on the expression levels of genes. However, the role of spontaneous nucleosome movements in this process is not fully understood. Here, we developed a single-molecule optical tweezers assay capable of simultaneously characterizing the base pair-scale diffusion of a nucleosome on DNA and the binding of a TF, using the luteinizing hormone ß subunit gene (Lhb) promoter and Egr-1 as a model system. Our results demonstrate that nucleosomes undergo confined diffusion, and that the incorporation of the histone variant H2A.Z serves to partially relieve this confinement, inducing a different type of nucleosome repositioning. The increase in diffusion leads to exposure of a TF's binding site and facilitates its association with the DNA, which, in turn, biases the subsequent movement of the nucleosome. Our findings suggest the use of mobile nucleosomes as a general transcriptional regulatory mechanism.

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.

Di-n-butyl phthalate diminishes testicular steroidogenesis by blocking the hypothalamic-pituitary-testicular axis: relationship with germ cell apoptosis in Japanese quail.

Although di-n-butyl phthalate (DBP) induces germ cell apoptosis, the underlying mechanism is not yet clear in quail. In this study, prepubertal quails were given a single dose of 500mg kg-1 DBP by gavage and were then killed 3, 6 and 24h after treatment. There was a significant reduction in intratesticular testosterone (ITT) concentrations and testicular steroidogenic enzyme mRNA expression and a significant increase in germ cell apoptosis in DBP-treated compared with control quails at all time points. Maximum apoptosis was detected 6h after treatment and the maximum reduction in testosterone concentrations was at 3h. To investigate whether DBP suppressed testicular steroidogenesis by affecting the hypothalamic-pituitary-testicular axis, we analysed pituitary LH subunit ß (Lhb) mRNA expression and serum LH concentrations. At all time points, pituitary Lhb expression and serum LH concentrations were significantly decreased following DBP treatment. The present observations suggest the possibility that DBP blocked LH secretion from the hypothalamus and/or pituitary, thereby decreasing LH stimulation of Leydig cells and reducing ITT concentrations. DBP-induced decreases in ITT concentrations may cause changes to the physical structure of Sertoli cells, which, in turn, may induce germ cell apoptosis.

Effect of anti-Müllerian hormone on the regulation of pituitary gonadotropin subunit expression: roles of kisspeptin and its receptors in gonadotroph LßT2 cells.

Anti-Müllerian hormone (AMH) is primarily produced by ovarian granulosa cells and contributes to follicle development. AMH is also produced in other tissues, including the brain and pituitary; however, its roles in these tissues are not well understood. In this study, we examined the effect of AMH on pituitary gonadotrophs. We detected AMH and AMH receptor type 2 expression in LßT2 cells. In these cells, the expression of FSHß- but not α- and LHß-subunits increased significantly as the concentration of AMH increased. LßT2 cells expressed Kiss-1 and Kiss-1R. AMH stimulation resulted in decreases in both Kiss-1 and Kiss-1R. The siRNA-mediated knockdown of Kiss-1 in LßT2 cells did not alter the basal expression levels of α-, LHß-, and FSHß-subunits. In LßT2 cells overexpressing Kiss-1R, exogenous kisspeptin stimulation significantly increased the expression of all three gonadotropin subunits. However, kisspeptin-induced increases in these subunits were almost completely eliminated in the presence of AMH. In contrast, GnRH-induced increases in the three gonadotropin subunits were not modulated by AMH. Our observations suggested that AMH acts on pituitary gonadotrophs and induces FSHß-subunit expression with concomitant decreases in Kiss-1 and Kiss-1R gene expression. Kisspeptin, but not GnRH-induced gonadotropin subunit expression, was inhibited by AMH, suggesting that it functions in association with the kisspeptin/Kiss-1R system in gonadotrophs.

The Genetic Backdrop of Hypogonadotropic Hypogonadism.

The pituitary is an organ of dual provenance: the anterior lobe is epithelial in origin, whereas the posterior lobe derives from the neural ectoderm. The pituitary gland is a pivotal element of the axis regulating reproductive function in mammals. It collects signals from the hypothalamus, and by secreting gonadotropins (FSH and LH) it stimulates the ovary into cyclic activity resulting in a menstrual cycle and in ovulation. Pituitary organogenesis is comprised of three main stages controlled by different signaling molecules: first, the initiation of pituitary organogenesis and subsequent formation of Rathke's pouch; second, the migration of Rathke's pouch cells and their proliferation; and third, lineage determination and cellular differentiation. Any disruption of this sequence, e.g., gene mutation, can lead to numerous developmental disorders. Gene mutations contributing to disordered pituitary development can themselves be classified: mutations affecting transcriptional determinants of pituitary development, mutations related to gonadotropin deficiency, mutations concerning the beta subunit of FSH and LH, and mutations in the DAX-1 gene as a cause of adrenal hypoplasia and disturbed responsiveness of the pituitary to GnRH. All these mutations lead to disruption in the hypothalamic-pituitary-ovarian axis and contribute to the development of primary amenorrhea.

The effects of luteinising hormone gene polymorphism on the outcomes of in vitro fertilisation and embryo transfer.

Trp8Arg polymorphism of the LH beta gene has decreased bioactivity in vivo and previous studies showed conflicting data on the effect of LH beta gene polymorphism on the IVF outcome. In this study, 591 IVF patients were recruited. Patients with the variant allele(s) were the carrier group. In GnRH antagonist cycles, the clinical pregnancy rate was significantly lower in the carrier group (18.9%) than in the noncarrier group (37.1%). In long GnRH agonist cycles, the clinical pregnancy rate was comparable between both groups. To clarify the effect of COH protocols, IVF outcomes in the GnRH antagonist and long GnRH agonist protocol groups in carriers were analysed. Among carriers, the clinical pregnancy rate was significantly lower in the GnRH antagonist protocol group (18.9%) than in the long GnRH agonist protocol group (45.2%). Single nucleotide polymorphism analysis may contribute to the individualisation of COH protocols for each patient in the future.Impact StatementWhat is already known on this subject? Trp8Arg polymorphism of the LH beta gene is known to have decreased bioactivity in vivo. Previous studies have demonstrated hypo-sensitivity in the patients with the variant LH beta protein, while other study showed similar carrier frequency between the poor and the normal response group.What the results of this study add? The variant LH beta gene was associated with a lower clinical pregnancy rate in GnRH antagonist cycles but not in long GnRH agonist cycles.What the implications are of these findings for clinical practice and/or further research? Single nucleotide polymorphism analysis may contribute to the individualisation of COH protocols for each patient in the future.

Short-term but not long-term high-fat diet induces an increase in gene expression of gonadotropic hormones and GPR120 in the male mouse pituitary gland.

High-fat diet (HFD) is associated with the regulation of reproductive functions. This study aimed to investigate the effects of short-term HFD on the mRNA expression levels of follicle-stimulating hormone ß subunit (FSHß), luteinizing hormone ß subunit (LHß), gonadotropin-releasing hormone receptor, and long-chain fatty acid receptor, GPR120, in the matured male mouse pituitary gland. Adult male mice were fed either control chow or HFD for 1, 2, 5, 10, 30 and 150 days. Fshb and Gpr120 mRNA expression levels in the pituitary glands were significantly increased during 2 to 30 days of HFD feeding. Gnrh-r mRNA in the 30 days HFD fed group and body weight in the 30 and 150 days HFD fed groups were higher than control. However, there were no significant differences in plasma non-esterified fatty acids or glucose levels during the 150 days of HFD feeding. These results suggest that male mice feeding a short-term HFD induces FSHß synthesis and GPR120 expression in their pituitary gonadotropes.

AMP-activated protein kinase activation reduces the transcriptional activity of the murine luteinizing hormone ß-subunit gene.

Malnutrition is one of the factors that induces reproductive disorders. However, the underlying biological processes are unclear. AMP-activated protein kinase (AMPK) is an enzyme that plays crucial role as a cellular energy sensor. In the present study, we examined the effects of AMPK activation on the transcription of the murine gonadotropin subunit genes Cga, Lhb, and Fshb, and the gonadotropin-releasing hormone receptor Gnrh-r. Real-time PCR and transcription assay using LßT2 cells demonstrated that 5-amino-imidazole carboxamide riboside (AICAR), a cell-permeable AMP analog, repressed the expression of Lhb. Next, we examined deletion mutants of the upstream region of Lhb and found that the upstream regulatory region of Lhb (-2527 to -2198 b) was responsible for the repression by AICAR. Furthermore, putative transcription factors (SP1, STAT5a, and TEF) that might mediate transcriptional control of the Lhb repression induced by AICAR were identified. In addition, it was confirmed that both AICAR and a competitive inhibitor of glucose metabolism, 2-deoxy-D-glucose, induced AMPK phosphorylation in LßT2 cells. Therefore, the upstream region of Lhb is one of the target sites for glucoprivation inducing AMPK activation. In addition, AMPK plays a role in repressing Lhb expression through the distal -2527 to -2198 b region.

Gpr120 mRNA expression in gonadotropes in the mouse pituitary gland is regulated by free fatty acids.

GPR120 is a long-chain fatty acid (LCFA) receptor that is specifically expressed in gonadotropes in the anterior pituitary gland in mice. The aim of this study was to investigate whether GPR120 is activated by free fatty acids in the pituitary of mice and mouse immortalized gonadotrope LßT2 cells. First, the effects of palmitate on GPR120, gonadotropic hormone b-subunits, and GnRH-receptor expression in gonadotropes were investigated in vitro. We observed palmitate-induced an increase in Gpr120 mRNA expression and a decrease in follicle-stimulating hormone b-subunit (Fshb) expression in LßT2 cells. Furthermore, palmitate exposure caused the phosphorylation of ERK1/2 in LßT2 cells, but no significant changes were observed in the expression levels of luteinizing hormone b-subunit (Lhb) and gonadotropin releasing hormone-receptor (Gnrh-r) mRNA and number of GPR120 immunoreactive cells. Next, diurnal variation in Gpr120 mRNA expression in the male mouse pituitary gland was investigated using ad libitum and night-time restricted feeding (active phase from 1900 to 0700 h) treatments. In ad libitum feeding group mice, Gpr120 mRNA expression at 1700 h was transiently higher than that measured at other times, and the peak blood non-esterified fatty acid (NEFA) levels were observed from 1300 to 1500 h. These results were not observed in night-time-restricted feeding group mice. These results suggest that GPR120 is activated by LCFAs to regulate follicle stimulating hormone (FSH) synthesis in the mouse gonadotropes.

Nr5a1b promotes and Nr5a2 inhibits transcription of lhb in the orange-spotted grouper, Epinephelus coioides†.

Nr5a1 (Sf-1) up-regulates lhb expression across vertebrates; however, its regulatory roles on fshb remain to be defined. Moreover, the involvement of Nr5a2 in the regulation of gonadotropin expression is not clear either. In the present study, the involvement of Nr5a1b (a homologue of Nr5a1) and Nr5a2 in the regulation of lhb and fshb expression in the orange-spotted grouper was examined. Dual fluorescent immunohistochemistry using homologous antisera showed that in the pituitary of orange-spotted groupers, Lh cells contain both immunoreactive Nr5a1b and Nr5a2 signals, whereas Fsh cells contain neither of them. In LßT2 cells, Nr5a1b up-regulated basal activities of lhb and fshb promoters possibly via Nr5a sites, and synergistically (on lhb promoter) or additively (on fshb promoter) with forskolin. Surprisingly, Nr5a2 inhibited basal activities of lhb promoter possibly via Nr5a sites and attenuated the stimulatory effects of both forskolin and Nr5a1b. In contrast, Nr5a2 had no effects on fshb promoter. Chromatin immunoprecipitation analysis showed that both Nr5a1b and Nr5a2 bound to lhb promoter, but not fshb promoter in the pituitary of the orange-spotted grouper. The abundance of Nr5a1b bound to lhb promoter was significantly higher at the vitellogenic stage than the pre-vitellogenic stage, whereas that of Nr5a2 exhibited an opposite trend. Taken together, data of the present study demonstrated antagonistic effects of Nr5a1b and Nr5a2 on lhb transcription in the orange-spotted grouper and revealed novel regulatory mechanisms of differential expression of lhb and fshb genes through Nr5a homologues in vertebrates.

Steroidogenic acute regulatory protein and luteinizing hormone are required for normal ovarian steroidogenesis and oocyte maturation in zebrafish†.

In recent studies, luteinizing hormone (LH) was reported to play important roles in oocyte maturation. However, the mechanism by which LH signaling, especially regarding the steroidogenesis process, affects oocyte maturation has not been clarified. In this study, zebrafish models with a functional deficiency in luteinizing hormone beta (Lhb) or steroidogenic acute regulatory protein (Star), an enzyme that promotes the transport of cholesterol into the inner mitochondrial membrane for maturation-induced hormone (MIH) production, were generated using transcription activator-like effector nucleases (TALENs). Similar phenotypes of the maturation-arrested oocytes in both female mutants have been observed. The levels of MIH in the oocytes of the female mutants were clearly decreased in both the lhb and star knockout zebrafish. The expression of star was dramatically down-regulated in the lhb mutant follicles and was clearly promoted by forskolin and hCG in vitro. Furthermore, treatment with the MIH precursors, pregnenolone or progesterone, as well as with MIH itself rescued the maturation-arrested oocyte phenotypes in both lhb and star mutants. The plasma levels of other steroids, including testosterone, estradiol, and cortisol, were not affected in the lhb mutants, while the levels of gonad hormones testosterone and estradiol were significantly increased in the star mutants. The cortisol levels were decreased in the star mutants. Collectively, our results confirm that LH plays important roles in the initiation of MIH synthesis from cholesterol and maintains oocyte maturation in zebrafish, as well as provide evidence that Star might act downstream of LH signaling in steroidogenesis.

Thermal manipulation and GnRHa therapy applied to the reproduction of lambari-do-rabo-amarelo, Astyanax altiparanae females (Characiformes: Characidae) during the non-breeding season.

Lambari-do-rabo-amarelo Astyanax altiparanae in the wild reproduce during spring and summer, but females undergo vitellogenesis throughout the year, including the non-spawning winter period when water temperatures are low. The present study investigated the physiological role of temperature modulation on the hypothalamus-pituitary-gonads axis of lambari during winter, as well as the effects of gonadotropin releasing hormone agonist (GnRHa) therapy. Captive females were exposed to two different temperatures (20 °C and 27 °C) and were injected weekly with GnRHa for 21 days during winter (Control, CTR; Low dose; LD and high dose of GnRHa, HD). At the end of the 21-days period gonadosomatic index (GSI), oocyte stage of development and theoretical fecundity were evaluated, together with plasma levels of 17ß-estradiol (E2). Gene expression of the two pituitary gonadotropins follicle-stimulating hormone (fshß) and luteinizing hormone (lhß), as well as hepatic vitellogenin-A (vtgA) expression were also analyzed. At the end of the experimental period, females from the six different experimental conditions were induced to spawn using human chorionic gonadotropin (hCG). Spawning performance parameters and plasma levels of the maturation inducing steroid (MIS) were analyzed. Gene expression of fshß did not change with temperature manipulation, but females exposed to 27 °C and supplemented with a HD of GnRHa exhibited an increased fshß gene expression, associated with higher E2 levels. The higher water temperature alone was able to increase E2 levels. At both water temperatures GnRHa injections induced a decrease in E2 levels. GnRHa injected females had a lower vtgA gene expression levels at 20 °C. Even with differences in the gene expression of gonadotropins among the various temperature/GnRHa treatments, GSI and oocyte diameter did not change, but GnRHa enhanced the number of vitellogenic oocytes at 20 °C. The reproductive performance of lambari induced to spawn with hCG was better after the combined treatment with GnRHa and summer temperature.

Photoperiodic regulation of plasma gonadotropin levels in previtellogenic greater amberjack (Seriola dumerili).

In Seriola species, exposure to a long photoperiod regime is known to induce ovarian development. This study examined photoperiodic effects on pituitary gene expression and plasma levels of follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) in previtellogenic greater amberjack (Seriola dumerili). The fish were exposed to short (8L:16D) or long (18L:6D) photoperiod. The water temperature was maintained at 22 °C. Compared with the short-photoperiod group, plasma Fsh levels were higher on days 10 and 30 in the long-photoperiod group, but plasma Lh levels did not significantly differ. On day 30, pituitary Fsh- and Lh-ß subunit gene expressions were also higher in the long-photoperiod group than the short-photoperiod group, whereas α-subunit gene expressions were higher on days 20 and 30. Throughout the experiment, average gonadosomatic index and plasma E2 levels did not significantly differ between the two groups. This study clearly demonstrated that a long photoperiod induced Fsh release in the previtellogenic fish followed by upregulation of pituitary Fsh and Lh subunit gene expressions. An increase in plasma Fsh levels may be a key factor that mediates the photoperiodic effect on the initiation of ovarian development.

Neurokinin B signaling in hermaphroditic species, a study of the orange-spotted grouper (Epinephelus coioides).

Neurokinin B (NKB) plays important roles in the mammalian reproductive axis by modulating the release of gonadotropin-releasing hormone (GnRH) and gonadotropins. In the present study, the tac3 cDNA was cloned from a hermaphroditic species, the orange-spotted grouper. Sequence analysis showed that the grouper Tac3 precursor encoded two tachykinin peptides, NKB and NKB-related peptide (NKBRP). Expression analysis in different tissues revealed that tac3 mRNA was highly expressed in the brain of the orange-spotted grouper. In situ hybridization further revealed that it was localized in some hypothalamic nuclei associated with reproductive regulation. During ovarian development, an increase of tac3 expression in the hypothalamus was observed at vitellogenesis stage. Intraperitoneal administration of NKB could increase the gnrh1 and lhß mRNA levels, and enhance the serum estrogen levels, but did not significantly influence lhß expression in cultured pituitary cells, indicating that NKB does not directly exert its actions on the pituitary gland. However, it was found that NKBRP had no effect on the expression of two gnrhs and two gths in vivo and in vitro. Effects of sex steroids on tac3 expression were further investigated. During the 17-methyltestosterone-induced sex change in the orange-spotted grouper, hypothalamic tac3 expression showed no significant change. Interestingly, ovariectomy greatly stimulated tac3 expression, while the 17ß-estradiol treatment reversed this effect. In general, our data highly indicated that NKB signaling could activate the reproductive axis in the orange-spotted grouper. Our study is the first description of the NKB signaling in the hermaphroditic species.

Reproductive stage- and sex-dependant effects of neurohypophyseal nonapeptides on gonadotropin subunit mRNA expression in the catfish Heteropneustes fossilis: An in vitro study.

In the present study, in vitro effects of synthetic vasotocin (VT), isotocin (4Ser, 8Ile- oxytocin; ITb) and the recently cloned IT gene paralog product (8Val-Isotocin, ITa) were studied on the expression of pituitary gonadotropin (GtH) subunit mRNA levels. In male pituitaries of early (preparatory phase) and late (prespawning phase) recrudescing catfish, Heteropneustes fossilis, VT (10 nM, 100 nM and 1000 nM) stimulated fshß expression dose-dependently. But in females, the dose-dependent effect was found only in the preparatory phase. In males, VT stimulated lhß expression only at higher doses. In females, VT produced a significant dose-dependent increase of the lhß expression only in the prespawning phase. VT stimulated the expression of gpα, dose-dependently in the preparatory phase in males and in the prespawning phase in females. The incubation of the pituitaries with ITb did not alter the fshß expression in either sex in both preparatory and prespawning phases. In males, ITb stimulated the expression of lhß and gpα only at the highest concentration (1000 nM) in both phases. In females, ITb stimulated both lhß and gpα expression only at 1000 nM in the preparatory phase and dose-dependently in the prespawning phase. The incubation of the pituitaries with ITa produced effects similar to ITb on the expression of fshß, lhß, and gpα. The results show that the basic peptide VT modulates both fshß and lhß expressions, which are influenced by the sex and reproductive stage. The neutral peptide ITA/ITb exerts an insignificant effect on the fshß expression regardless of sex or season. Both VT and ITa/ITb elicit a significant effect on the lhß expression in late recrudescent phase especially in females.

Homozygous nonsense mutation Trp28X in the LHB gene causes male hypogonadism.

PURPOSE: The purpose of this study was to investigate a novel mutation in the luteinizing hormone beta-subunit (LHB) gene in one male patient with hypogonadism due to selective luteinizing hormone (LH) deficiency. METHODS: Sanger sequencing of one 28-year-old man born to consanguineous parents was performed. Treatment with human chorionic gonadotropin (hCG) (2000 IU, twice a week) was initiated for 3 months, followed by 5000 IU weekly to date. RESULTS: We identified a novel c.84G>A[p.W28X] nonsense LHB mutation. The W28X mutation produces a truncated LHB peptide of seven amino acids, which prevents the synthesis of intact LH. After 40 days of treatment with hCG, the patient exhibited a few spermatozoa in the semen. Treated for 6 months, the patient exhibited normal seminal parameters. CONCLUSIONS: We identified a novel mutation in the LHB gene in a male patient with hypogonadism and provided evidence that LHB nonsense mutation can cause selective LH deficiency. We reconfirmed hCG treatment may restore male fertility due to LHB mutation.

[Correlation of the single nucleotide polymorphisms rs34349826 and rs6521 of the LHB gene with male infertility in Chinese men].

Objective: To study the relationship of the single nucleotide polymorphisms (SNP) rs34349826 (c.104 A>G) and rs6521 (c.114 C>G) of the luteinizing hormone beta-subunit (LHB) gene with male infertility in Chinese men. METHODS: This case-control study included 405 males with primary infertility (the infertility group) and 424 normal fertile men (the control group), the former again divided into subgroups of oligospermia, severe oligozoospermia and azoospermia according to the sperm concentration. Clinical data were collected from all the subjects and genomic DNA obtained from their peripheral blood for genotyping rs34349826 and rs6521 of the LHB gene by Sequence MassArray. We analyzed the correlation of male infertility with the SNPs of the two loci using the logistic regression model as well as its association with their haplotype combination with the SHEsis online software. RESULTS: There were statistically significant differences between the control and infertility groups in the semen volume (ï¼»3.51 ± 1.36ï¼½ vs ï¼»3.74 ± 1.71ï¼½ ml, P <0.05), sperm concentration (ï¼»79.21 ± 61.60ï¼½ vs ï¼»27.37 ± 30.80ï¼½ ×106/ml, P <0.01), percentage of progressively motile sperm (ï¼»39.40 ± 9.64ï¼½ % vs ï¼»11.90 ± 14.72ï¼½ %, P <0.01), and levels of serum luteinizing hormone (LH) (ï¼»3.29 ± 1.39ï¼½ vs ï¼»6.25 ± 4.83ï¼½ IU/L, P <0.01) and follicle-stimulating hormone (FSH) (ï¼»4.56 ± 2.31ï¼½ vs ï¼»15.64 ± 17.03ï¼½ IU/L, P <0.01). Logistic regression analysis revealed no correlation between male infertility and the genotypes of the rs34349826 and rs6521 loci of the LHB gene, and similar results were found in the subgroups of the infertile males. SHEsis analysis on the haplotypes of the rs34349826 and rs6521 loci showed the GG genotype combination to be a protective factor against male infertility. CONCLUSIONS: The rs34349826 and rs6521 loci of the LHB gene were not related to male infertility, which can be further confirmed by larger-sample studies. The GG genotype combination is a protective factor against male infertility.