LHbeta Research Articles

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

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

Disruption of Zebrafish Follicle-Stimulating Hormone Receptor (fshr) But Not Luteinizing Hormone Receptor (lhcgr) Gene by TALEN Leads to Failed Follicle Activation in Females Followed by Sexual Reversal to Males.

Gonadotropins are primary hormones that control vertebrate reproduction. In a recent study, we analyzed the impacts of FSH and LH on zebrafish reproduction by disrupting FSH and LH-β genes (fshb and lhb) using transcription activator-like effector nuclease (TALEN) technology. Using the same approach, we successfully deleted FSH and LH receptor genes (fshr and lhcgr) in the present study. In contrast to the deficiency of its cognate ligand FSH, the fshr-deficient females showed a complete failure of follicle activation with all ovarian follicles arrested at the primary growth-previtellogenic transition, which is the marker for puberty onset in females. Interestingly, after blockade at the primary growth stage for varying times, all females reversed to males, and all these males were fertile. In fshr-deficient males, spermatogenesis was normal in adults, but the initiation of spermatogenesis in juveniles was retarded. In contrast to fshr, the deletion of the lhcgr gene alone caused no obvious phenotypes in both males and females; however, double mutation of fshr and lhcgr resulted in infertile males. In summary, our results in the present study showed that Fshr was indispensable to folliculogenesis and the disruption of the fshr gene resulted in a complete failure of follicle activation followed by masculinization into males. In contrast, lhcgr does not seem to be essential to zebrafish reproduction in both males and females. Neither Fshr nor Lhcgr deficiency could phenocopy the deficiency of their cognate ligands FSH and LH, which is likely due to the fact that Fshr can be activated by both FSH and LH in the zebrafish.

The effects of progesterone on transcriptional expression profiles of genes associated with hypothalamic–pituitary–gonadal and hypothalamic–pituitary–adrenal axes during the early development of zebrafish (Danio rerio)

Progesterone (P4) has been reported in surface water, and it may have adverse effects on aquatic organisms. This study provided the transcriptional effects of P4 during the early development of zebrafish. Zebrafish embryos were exposed for 144h post fertilization (hpf) to 0, 6, 45 and 90ngL−1 P4, and transcriptional expression profiles of the hypothalamic–pituitary–gonadal (HPG) and hypothalamic–pituitary–adrenal (HPA) axes were assessed every day. For the receptor signaling pathways, P4 significantly induced the transcript of Pgr gene above 45ngL−1 at 72 and 144hpf, but inhibited its transcript above 6ngL−1 at 96 and 120hpf. A significant up-regulation of Vtg1 mRNA was observed at 6ngL−1 P4 or higher at 24, 96 and 144hpf. For the steroidogenic pathways, the transcriptional expression of Cyp11a1 and Hsd17b3 mRNAs was mediated by 6ngL−1 P4 or higher according to different exposure time points. In addition, P4 resulted in a significant induction of Cyp19a1a and Cyp11b mRNA expression while it caused a significant inhibition of Hsd11b2 mRNA expression above 6ngL−1. For the other target genes related to hypothalamic and pituitary hormones, P4 mainly modulated the transcripts of Gnrh2, Fshb and Lhb genes at 6ngL−1 or higher. The overall results from the present study indicate that P4 at environmentally relevant concentrations could cause the potential effects on zebrafish reproductive and adrenal endocrine systems by interfering with the HPG and HPA axes.

Caffeine stimulates in vitro pituitary LH secretion in lipopolysaccharide-treated ewes

The study was designed to determine the effects of caffeine on luteinizing hormone (LH) secretion and gene expression of caffeine-associated receptors in anterior pituitary (AP) explants obtained from saline- and lipopolysaccharide (LPS)-treated ewes. Animals had been treated with LPS or saline daily for seven days. Three hours after the last injection of LPS/saline, the AP were collected and divided into four explants. The explants were incubated with: 1/medium-199 (control explants), 2/gonadotropin-releasing hormone (GnRH; 100pmol/mL; a positive control), 3/caffeine (10mmol/L), or 4/GnRH+caffeine. Caffeine stimulated (p<0.05) LH release by explants from both saline (19.7 vs. control 12.6ng/mg) and LPS (28.3 vs. control 13.9ng/mg) treated animals. The effect of caffeine on LH secretion was stronger in the LPS-treated group than in saline-treated group, and the observed LH release was similar to that induced by GnRH alone (27.2ng/mg). Caffeine increased (p<0.05) LHβ gene expression only in explants from LPS-treated animals. In conclusion, the results of the present study demonstrated a stimulatory in vitro effect of caffeine on LH secretion by ovine pituitary explants. The potency of the caffeine-induced LH secretion was affected by in vivo treatment of the animals with endotoxin.

Estudo de associação genômica de prenhez pós-parto em vacas de corte

The aim of this study was to evaluate genetic diversity of nine molecular markers, six short tandem repeats - STRs (BM4325, BMS3004, ILSTS002, IDVGA51, HEL5, AFZ1) and three single nucleotide polymorphisms (SNPs; LepSau3A1 A-B, LepSau3A1 1-2, and FSHRAlu1), linked to genes involved in reproductive function and their possible effect on reproductive performance. For this purpose, 81 crossbred beef cows were used in this study. The animals were classified into two groups (fertile and sub-fertile cows) based on their pregnancy status after two breeding seasons. High genetic diversity level was observed highlighted by the polymorphic content information ranging 0.23 to 0.87 and expected heterozygosity from 27 to 89%, with an average of 62%. Alleles BM4325 103, BMS3004 129, ILSTS002 137, IDVGA51 177, LEPSau3A1 A, LEPSau3A1 1, HEL5 149, AFZ1 119 and FSHRAlu1 G presented high frequencies. Two STRs (IDVGA51 and ILSTS002), linked to Leptin and LHβ genes, respectively, were associated to reproductive performance. These data support previous findings suggesting the potential use of IDVGA51 and ILSTS002 STRs for reproductive performance selection.

In vivo oestrogenic modulation of Egr1 and Pitx1 gene expression in female rat pituitary gland.

EGR1 and PITX1 are transcription factors required for gonadotroph cell Lhb promoter activation. To determine changes in Egr1 and Pitx1 mRNA levels in central and peripheral pituitary stimulations, an in vivo model based on i.c.v. pulsatile (1 pulse/0.5 h over 2 h) GnRH agonist (1.5 nM buserelin) or antagonist (2 nM antide) microinjections was used. The microinjections were given to ovariectomised and 17β-oestradiol (E2) (3×20 μg), ERA (ESR1) agonist propyl pyrazole triol (PPT) (3×0.5 mg), ERB (ESR2) agonist diarylpropionitrile (DPN) (3×0.5 mg) s.c. pre-treated rats 30 min after last pulse anterior pituitaries were excised. Relative mRNA expression was determined by quantitative RT-PCR (qRT-PCR). Results revealed a gene-specific response for GnRH and/or oestrogenic stimulations in vivo. Buserelin pulses enhanced Egr1 expression by 66% in ovariectomised rats, whereas the oestradiol-supplemented+i.c.v. NaCl-microinjected group showed a 50% increase in Egr1 mRNA expression. The oestrogenic signal was transmitted via ERA (ESR1) and ERB (ESR2) activation as administration of PPT and DPN resulted in 97 and 62%, respectively, elevation in Egr1 mRNA expression. A synergistic action of GnRH agonist and 17β-oestradiol (E2) stimulation of the Egr1 gene transcription in vivo were found. GnRHR activity did not affect Pitx1 mRNA expression; regardless of NaCl, buserelin or antide i.c.v. pulses, s.c. oestrogenic supplementation (with E2, PPT or DPN) consistently decreased (by -46, -48 and -41% respectively) the Pitx1 mRNA in the anterior pituitary gland. Orchestrated Egr1 and Pitx1 activities depending on specific central and peripheral regulatory inputs could be responsible for physiologically variable Lhb gene promoter activation in vivo.

Cellular distribution of Egr1 transcription in the male rat pituitary gland.

The transcription factor gene Egr1 is necessary for female fertility; EGR1 protein is an established molecular regulator of adult female gonadotroph function where it mediates GNRH-stimulated transcription of the Lhb gene. Recent studies have also implicated pituitary EGR1 in the mediation of other physiological signals indicating an integrative function. However, the role of EGR1 in males is less well defined and this uncertainty is compounded by the absence of cellular expression data in the male pituitary gland. The aim of this study, therefore, was to define the distribution of Egr1 gene expression in the adult male rat pituitary. To further this aim, we have evaluated cellular populations in a transgenic rat model (Egr1-d2EGFP), in which we demonstrate regulated green fluorescent protein (GFP) expression in EGR1+ pituitary cells. Cellular filling by GFP enabled morphological and molecular differentiation of different populations of gonadotrophs; Egr1 transcription and LHB were highly co-localised in a major population of large cells but only minimally co-localised in small GFP+ cells; the latter cells were shown to be largely (80%) composed of minority populations of GH+ somatotrophs (9% of total GH+) and PRL+ lactotrophs (3% of total PRL+). Egr1 transcription was not found in TSH+, ACTH+ or SOX2+ precursor cells and was only minimally co-localised in S-100β+ folliculostellate cells. Our demonstration that the Egr1 gene is actively and selectively transcribed in a major sub-population of male LHB+ cells indicates a largely conserved role in gonadotroph function and has provided a basis for further defining this role.

Polymorphisms of LHβ and GnRHR genes and their association with the number of embryos recovered in goats

The objective of the present study was to explore a predictor of superovulation response on the basis of associations between the number of embryos recovered and gene polymorphism. Variation in the goat LHβ and GnRHR genes was investigated using polymerase chain reaction–single-strand conformational polymorphism and DNA sequencing. Two single nucleotide polymorphisms (SNPs) were identified in the 5′-UTR of LHβ gene (A59C, P1 locus) and in the Exon 2 of GnRHR gene (T177A, P6 locus). At the P1 locus in both breeds, the frequencies of one allele were 0.46 and 0.51, respectively. At the P6 locus, the minor allele frequency was 0.23. Associations of both SNPs with the number of embryos recovered and the corpus luteum number were evaluated in Boer and Shaanbei goat breeds. Association analysis showed that both SNPs had significant (P &lt; 0.05) effects on the number of embryos recovered and corpus luteum number. These results indicate that LHβ and GnRHR genes are potential markers for the number of embryos recovered.

The role of the 3′ region of mammalian gonadotropin β subunit gene in the luteinizing hormone to chorionic gonadotropin evolution

CGβ subunits comprise a unique carboxyl-terminal peptide (CTP) that has multiple O-linked glycans and extends serum half-life of the protein. It has evolved by incorporating a previously untranslated region of the LHβ gene into the reading frame. Although CTP-like sequences are encrypted in the LHβ genes of several mammals, the CGβ subunit developed only in primates and equids. To study this restriction in evolution, we examined whether the cryptic CTP decoded from the bovine LHβ gene (boCTP) possesses key characteristics of the human (h) CGβ-CTP. The boCTP does not impede several crucial aspects of hormone biosynthesis, but compared to the hCGβ-CTP, the stretch lacks O-glycans and determinants for circulatory survival. O-glycan deficiency and the associated incapacity to extend serum half-life is a major drawback of the boCTP. This may explain why LH did not evolve into CG in ruminants and consequently alternative mechanisms evolved to delay luteolysis early in gestation.

Involvement of pituitary gonadotropins, gonadal steroids and breeding season in sex change of protogynous dusky grouper, Epinephelus marginatus (Teleostei: Serranidae), induced by a non-steroidal aromatase inhibitor

Two experiments were performed using the aromatase inhibitor (AI) letrozole (100mg/kg) to promote sex change, from female-to-male, in protogynous dusky grouper. One experiment was performed during the breeding season (spring) and the other at the end of the breeding season (summer). During the spring, AI promoted sex change after 9weeks and the sperm produced was able to fertilize grouper oocytes. During the summer, the sex change was incomplete; intersex individuals were present and sperm was not released by any of the animals. Sex changed gonads had a lamellar architecture; cysts of spermatocytes and spermatozoa in the lumen of the germinal compartment. In the spring, after 4weeks, 11ketotestosterone (11KT) levels were higher in the AI than in control fish, and after 9weeks, coincident with semen release, testosterone levels increased in the AI group, while 11KT returned to the initial levels. Estradiol (E2) levels remained unchanged during the experimental period. Instead of decreasing throughout the period, as in control group, 17 α-OH progesterone levels did not change in the AI-treated fish, resulting in higher values after 9weeks when compared with control fish. fshβ and lhβ gene expression in the AI animals were lower compared with control fish after 9weeks. The use of AI was effective to obtain functional males during the breeding season. The increase in androgens, modulated by gonadotropins, triggered the sex change, enabling the development of male germ cells, whereas a decrease in E2 levels was not required to change sex in dusky grouper.

AMP-Activated Protein Kinase Is a Key Intermediary in GnRH-Stimulated LHβ Gene Transcription

GnRH regulation of pituitary gonadotropin gene transcription is critical for fertility, and metabolic dysregulation is associated with reproductive disorders and altered hypothalamic-pituitary responses. Here, we examined signaling pathways in gonadotropes through which GnRH modulates gonadotropin levels, and potential common signaling pathways with insulin. Using LβT2 cells, we show that GnRH rapidly (5 minutes) triggers activating phosphorylation of AMP-activated protein kinase (AMPK) up to 5-fold; this stimulation is enhanced by insulin through increased total AMPKα levels and activity. GnRH also stimulated c-Jun N-terminal kinase (JNK) and ERK activation, whereas insulin alone stimulated Akt. Inhibition of AMPK activity by compound C, or diminishing AMPK levels by small interfering RNA against AMPKα, prevented GnRH-stimulated transcription of the endogenous LHβ gene and transfected LHβ promoter. Egr-1 (early growth response-1), a transcription factor required for LHβ expression, is synthesized in response to GnRH, and compound C prevents this induction. However, overexpression of Egr-1 in the presence of compound C did not restore GnRH stimulation of LHβ, suggesting that AMPK stimulation of transcription also occurs through additional mechanisms or signaling pathways. One such pathway may be JNK activation, because GnRH stimulation of JNK activity and LHβ transcription occurs more slowly than stimulation of AMPK activity, and AMPK inhibition by compound C or small interfering RNA also prevented GnRH-stimulated JNK phosphorylation. Finally, in primary mouse pituitary cells, GnRH also stimulates AMPK, and AMPK inhibition suppresses GnRH-stimulated LHβ transcription. These studies indicate a novel role for AMPK in GnRH-stimulated transcription in pituitary gonadotropes and a potential common mechanism for GnRH and metabolic modulation of fertility.

Hypothyroidism in Adult Male Rats Alters Posttranscriptional Mechanisms of Luteinizing Hormone Biosynthesis

Studies in men are not consistent regarding the effects of thyroid hormone on the production of gonadotropins. In hypothyroidism consequent to diverse causes, an increase or no change in serum luteinizing hormone (LH) have been reported. The attempt to explain the mechanisms involved in this pathology using rats as an experimental model also seems to repeat this divergence, since hypothyroidism has been shown to induce hypogonadotropic hypogonadism, a hypergonadotropic state, or not to affect the basal levels of LH. Notably, the promoter region of the gene encoding the Lh beta subunit and GnRH (gonadotropin-releasing factor) does not contain a thyroid responsive element. Therefore, we investigated the hypothesis that, in male rats, posttranscriptional mechanisms of LH synthesis are altered in hypothyroidism. We also attempted to determine if hypothyroidism directly affects testicular function in male rats. Male Wistar rats, 60 days old, were thyroidectomized or sham-operated. After 20 days, they were decapitated, and the pituitaries were collected and analyzed for Lh mRNA, LH content, poly(A) tail length, and polysome profile. The testes were collected and analyzed for Lh receptor mRNA, LH receptor content, and histology using morphometric analyses. The testis, epididymis, seminal vesicle, and ventral prostate were weighed, and serum concentrations of LH, testosterone, thyrotropin (TSH), and triiodothyronine (T3) were measured. Hypothyroidism was associated, in the pituitary, with an increase in Lh mRNA expression, a reduction in Lh mRNA poly(A) tail length, a reduction in the number of LH transcripts associated with polysomes. Pituitary LH was decreased but serum LH was increased from 102 to 543 pg/mL. Despite this, serum testosterone concentrations were decreased from 1.8 to 0.25 ng/mL. A decreased germinative epithelium height of the testes and a reduced weight of androgen-responsive tissues were observed (ventral prostrate: 74 vs. 23 mg/100 g body weight [BW]; seminal vesicle undrained: 280 vs. 70 mg/100 g BW; and seminal vesicle drained: 190 vs. 60 mg/100 g BW). Hypothyroidism in adult male rats has dual effects on the pituitary testicular axis. It alters posttranscriptional mechanisms of LH synthesis and probably has a direct effect on testicular function. However, these data suggest the possibility that reduced LH bioactivity may account in part for impaired testicular function.

The effect of rivastigmine on the LPS-induced suppression of GnRH/LH secretion during the follicular phase of the estrous cycle in ewes

This study was designed to determine the effect of a potent subcutaneously injected acetylcholinesterase inhibitor, rivastigmine (6mg/animal), on the gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) release during inflammation induced by an intravenous lipopolysaccharide (LPS) (400ng/kg) injection in ewes during the follicular phase of the estrous cycle. The results are expressed as the mean values from −2 to −0.5h before and +1 to +3h after treatment. Rivastigmine decreased the acetylcholinesterase concentration in the blood plasma from 176.9±9.5 to 99.3±15.1μmol/min/ml. Endotoxin suppressed LH (5.4±0.6ng/ml) and GnRH (4.6±0.4pg/ml) release; however, the rivastigmine injection restored the LH concentration (7.8±0.8ng/ml) to the control value (7.8±0.7ng/ml) and stimulated GnRH release (7.6±0.8pg/ml) compared to the control (5.9±0.4pg/ml). Immune stress decreased expression of the GnRH gene and its receptor (GnRH-R) in the median eminence as well as LHβ and GnRH-R in the pituitary. In the case of the GnRH and LHβ genes, the suppressive effect of inflammation was negated by rivastigmine. LPS stimulated cortisol and prolactin release (71.1±14.7 and 217.1±8.0ng/ml) compared to the control group (9.0±5.4 and 21.3±3.5ng/ml). Rivastigmine also showed a moderating effect on cortisol and prolactin secretion (43.1±13.1 and 169.7±29.5ng/ml). The present study shows that LPS-induced decreases in GnRH and LH can be reduced by the AChE inhibitor. This action of the AChE inhibitor could result from the suppression of pro-inflammatory cytokine release and the attenuation of the stress response. However, a direct stimulatory effect of ACh on GnRH/LH secretion should also be considered.

LPS-induced inflammation potentiates the IL-1β-mediated reduction of LH secretion from the anterior pituitary explants.

Acting at the level of the brain, interleukin- (IL-)1β is considered to be one of the most potent downregulators of reproduction processes during immune/inflammatory challenge. IL-1β suppresses gonadotropin-releasing hormone (GnRH) secretion from the hypothalamus resulting in the inhibition of the luteinizing hormone (LH) release from the anterior pituitary (AP). However, the presence of IL-1β receptors in the AP suggests the possible direct action of this cytokine on LH secretion. The study was designed to determine the effect of IL-1β on the LH secretion from the AP explants collected from saline and LPS-treated ewes in the follicular phase. It was found that IL-1β suppressed (P ≤ 0.01) GnRH-stimulated LH release and LHβ gene expression in AP explants in both groups. However, IL-1β action was more potent in the explants collected from LPS-treated animals. Pituitaries from LPS-treated animals were characterized by increased (P ≤ 0.01) IL-1 type I receptor and decreased (P ≤ 0.01) GnRH receptor gene expression level compared to the saline-treated group. IL-1β also affected the GnRH-R gene expression in explants collected from LPS-treated animals. Our results show that direct action of IL-1β on the pituitary gonadotropes could be one of the reasons of the reproductive processes disorders accompanying an inflammatory state.

Developmental tracing of luteinizing hormone β‐subunit gene expression using green fluorescent protein transgenic medaka (Oryzias latipes) reveals a putative novel developmental function

Luteinizing hormone (LH) and follicle stimulating hormone (FSH), produced in gonadotrope cells in the adenohypophysis are key regulators of vertebrate reproduction. The differential regulation of these hormones, however, is poorly understood and little is known about gonadotrope embryonic development. We developed a stable transgenic line of medaka with the LH beta subunit gene (lhb) promotor driving green fluorescent protein (gfp) expression to characterize development of LH-producing gonadotropes in whole larvae and histological sections. Additionally, developmental and tissue-specific gene expression was examined. The lhb gene is maternally expressed during early embryogenesis. Transcript levels increase by stage 21 (36 hours post fertilization [hpf]) and then decrease during continued larval development. Examination of the expression of pituitary marker genes show that LH-producing cells are initially localized outside the primordial pituitary, and they were localized to the developing gut tube by 32 hpf. At hatching, lhb-GFP is clearly detected in the gut epithelium and in the anterior digestive tract. lhb-GFP expression later consolidate in the developing pituitary by 2 weeks postfertilization. During embryonic development, lhb is primarily expressed outside the central nervous system and pituitary. The novel expression of lhb in the embryonic gut suggests that LH has a hitherto unidentified developmental function.

Immunoreactivity of Gonadotrophs (FSH and LH Cells) and Gonadotropin Subunit Gene Expression in the Male Chub MackerelScomber japonicusPituitary during the Reproductive Cycle

The gonadotropins (GtHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), are heterodimers composed of a common α subunit (GPα) and a unique β subunit (FSHβ or LHβ); they are synthesized in and secreted from gonadotrophs (FSH and LH cells) in the pituitary. Little is known about the roles of FSH and LH during spermatogenesis in perciform fishes. In this study, we examined immunoreactive changes in FSH and LH cells, and changes in the gene expression of the three gonadotropin subunits in the pituitary of male chub mackerel Scomber japonicus during testicular development. FSHβ-immunoreactive (ir) and LHβ-ir cell area were measured immuno-histochemically based on the FSH and LH cell-occupying area in the proximal pars distalis. The FSHβ-ir cell area increased significantly during spermiation, while FSHβ mRNA levels, already high at the beginning of spermatogenesis, increased further, peaking during spermiation. In contrast, LHβ-ir cell area and LHβ mRNA levels, which were low at the beginning of spermatogenesis, increased significantly during late spermatogenesis, peaking during spermiation. For both FSH and LH, GtHβ-ir cell area and GtHβ mRNA levels decreased until gonadal resting. GPα mRNA levels showed similar changes to LHβ mRNA levels. These results suggest that in the chub mackerel, FSH may play an important role in the early and late phases of spermatogenesis, and that LH may play a role during late spermatogenesis and spermiation. Moreover, our results demonstrate that changes in GtHβ-ir cell area were accompanied by similar changes in the expression of the FSHβ and LHβ genes, both of which increased during testicular development.

FOXO1 Transcription Factor Inhibits Luteinizing Hormone β Gene Expression in Pituitary Gonadotrope Cells

Synthesis of luteinizing hormone (LH) is tightly controlled by a complex network of hormonal signaling pathways that can be modulated by metabolic cues, such as insulin. One group of candidate genes that may be regulated by insulin signaling in pituitary gonadotrope cells is the FOXO subfamily of forkhead transcription factors. In this study we investigated whether FOXO1 is expressed in gonadotropes and if it can modulate LH β-subunit (Lhb) gene expression. We demonstrated that FOXO1 is expressed in murine gonadotrope cells and that insulin signaling increased FOXO1 phosphorylation and cytoplasmic localization in a PI3K-dependent manner. We also showed that FOXO1 repressed basal transcription and gonadotropin-releasing hormone (GnRH) induction of both the murine and human LHB genes in LβT2 cells, suggesting that FOXO1 regulation of LHB transcription may be conserved between rodents and humans. Although we did not detect FOXO1 binding to the proximal Lhb promoter, the FOXO1 DNA binding domain was necessary for the suppression, suggesting that FOXO1 exerts its effect through protein-protein interactions with transcription factors/cofactors required for Lhb gene expression. FOXO1 repression mapped to the proximal Lhb promoter containing steroidogenic factor 1 (SF1), pituitary homeobox 1 (PTX1), and early growth response protein 1 (EGR1) binding elements. Additionally, FOXO1 blocked induction of the Lhb promoter with overexpressed SF1, PTX1, and EGR1, indicating that FOXO1 repression occurs via these transcription factors but not through regulation of their promoters. In summary, we demonstrate that FOXO1 phosphorylation and cellular localization is regulated by insulin signaling in gonadotropes and that FOXO1 inhibits Lhb transcription. Our study also suggests that FOXO1 may play an important role in controlling LH levels in response to metabolic cues.

FOXO Regulation of Gonadotropin Hormone Synthesis in Pituitary Gonadotropes.

Synthesis of luteinizing hormone (LH) is tightly controlled by a complex network of hormonal signaling pathways that may be regulated by metabolic cues, such as insulin. One group of candidate genes that may act as metabolic sensors in pituitary gonadotrope cells is the FOXO subfamily of forkhead transcription factors. We investigated whether FOXO1 is expressed in gonadotropes and if it can modulate LH beta (Lhb) gene expression. We demonstrated that FOXO1 is expressed in murine gonadotrope cells and that insulin signaling increased FOXO1 phosphorylation and cytoplasmic localization. We also showed that FOXO1 repressed basal transcription and GnRH induction of both the murine and human LHB genes in LβT2 cells, indicating that FOXO1 regulation of LHB transcription may be conserved between rodents and humans. Although we did not detect FOXO1 binding to the proximal Lhb promoter, the FOXO1 DNA-binding domain was necessary for the suppression, suggesting that FOXO1 exerts its effect through protein-protein interactions with transcription factors/cofactors required for Lhb gene expression. FOXO1 repression mapped to the proximal Lhb promoter containing SF1, PTX1 and EGR1 binding elements. FOXO1 also blocked induction of the Lhb promoter with overexpressed SF1, PTX1 and EGR1, indicating that FOXO1 repression occurs via these transcription factors but not through regulation of their promoters. In summary, we demonstrate that FOXO1 phosphorylation and cellular localization is regulated by insulin signaling in gonadotropes and that FOXO1 inhibits Lhb transcription. Our studies suggest that FOXO1 may modulate LH hormone levels in response to metabolic cues.

CREB Binding Protein (CBP) Activation Is Required for Luteinizing Hormone Beta Expression and Normal Fertility in Mice

Normal function of the hypothalamic-pituitary-gonadal axis is dependent on gonadotropin-releasing hormone (GNRH)-stimulated synthesis and secretion of luteinizing hormone (LH) from the pituitary gonadotroph. While the transcriptional coactivator CREB binding protein (CBP) is known to interact with Egr-1, the major mediator of GNRH action on the Lhb gene, the role of CBP in Lhb gene expression has yet to be characterized. We show that in the LβT2 gonadotroph cell line, overexpression of CBP augmented the response to GNRH and that knockdown of CBP eliminated GNRH responsiveness. While GNRH-mediated phosphorylation of CBP at Ser436 increased the interaction with Egr-1 on the Lhb promoter, loss of this phosphorylation site eliminated GNRH-mediated Lhb expression in LβT2 cells. In vivo, loss of CBP phosphorylation at Ser436 rendered female mice subfertile. S436A knock-in mice had disrupted estrous cyclicity and reduced responsiveness to GNRH. Our results show that GNRH-mediated phosphorylation of CBP at Ser436 is required for Egr-1 to activate Lhb expression and is a requirement for normal fertility in female mice. As CBP can be phosphorylated by other factors, such as insulin, our studies suggest that CBP may act as a key regulator of Lhb expression in the gonadotroph by integrating homeostatic information with GNRH signaling.

G Proteins and Autocrine Signaling Differentially Regulate Gonadotropin Subunit Expression in Pituitary Gonadotrope

Gonadotropin-releasing hormone (GnRH) acts at gonadotropes to direct the synthesis of the gonadotropins, follicle-stimulating hormone (FSH), and luteinizing hormone (LH). The frequency of GnRH pulses determines the pattern of gonadotropin synthesis. Several hypotheses for how the gonadotrope decodes GnRH frequency to regulate gonadotropin subunit genes differentially have been proposed. However, key regulators and underlying mechanisms remain uncertain. We investigated the role of individual G proteins by perturbations using siRNA or bacterial toxins. In LβT2 gonadotrope cells, FSHβ gene induction depended predominantly on Gα(q/11), whereas LHβ expression depended on Gα(s). Specifically reducing Gα(s) signaling also disinhibited FSHβ expression, suggesting the presence of a Gα(s)-dependent signal that suppressed FSH biosynthesis. The presence of secreted factors influencing FSHβ expression levels was tested by studying the effects of conditioned media from Gα(s) knockdown and cholera toxin-treated cells on FSHβ expression. These studies and related Transwell culture experiments implicate Gα(s)-dependent secreted factors in regulating both FSHβ and LHβ gene expression. siRNA studies identify inhibinα as a Gα(s)-dependent GnRH-induced autocrine regulatory factor that contributes to feedback suppression of FSHβ expression. These results uncover differential regulation of the gonadotropin genes by Gα(q/11) and by Gα(s) and implicate autocrine and gonadotrope-gonadotrope paracrine regulatory loops in the differential induction of gonadotropin genes.