FSHbeta Gene Research Articles

Active immunization against AMH reveals its inhibitory role in the development of pre-ovulatory follicles in Zhedong White geese

The aim of this study was to investigate the effects of active immunization against recombinant Anti-Müllerian hormone (AMH) protein on the ovarian follicular development, egg production, and molecular regulatory mechanisms in broody-prone Zhedong White geese. For this, a recombinant goose AMH protein was expressed using a prokaryotic expression system. Fifty incubating geese from the same genetic background were selected and equally divided into two groups. The immunization group was actively immunized against the recombinant goose AMH protein, whereas the control group was immunized against bovine serum albumin (BSA). Immunization against AMH accelerated ovarian follicular development and increased clutch sizes by one to two eggs in two consecutive laying-incubation cycles. Furthermore, immunization against AMH upregulated the mRNA transcription levels of the FSH-beta gene in the pituitary gland, and FSHR, 3beta-HSD, and Smad4 genes in the granulosa layer of pre-ovulatory follicles; however, immunization downregulated the expression of the OCLN gene in the granulosa layer of pre-ovulatory follicles, and Smad5 and Smad9 genes in the granulosa layer of SYFs. These results suggest that AMH might hinder ovarian follicular development by decreasing both pituitary FSH secretion as well as ovarian follicular sensitivity to FSH. The latter molecular mechanism could be fulfilled by regulating Smad5 or Smad9 signals in SYFs, as well as the FSHR and Smad4 signals that affect progesterone synthesis and yolk deposition in the pre-ovulatory follicles.

Single Nucleotide Polymorphism Analysis in Exon 3 of FSHbeta Gene in Holstein Friesian and Gir Crossbred Bull

Prediction of male fertility is of prime importance in production animals. Follicle-stimulating hormone (FSH) plays a pivotal role in the reproduction of mammals. Single nucleotide polymorphisms (SNPs) in FSHB gene have been reported to affect the semen quality and fertility in cattle. The present study aimed to characterize and analyze the presence of SNPs in the FSHB-3 of HFxG 5043 Indian crossbred bull. Analysis revealed that HFxG 5043 FSHB-3 has 99.2% homology at nucleotide level and 100% homology at amino acid level with cattle FSHB-3 sequences. Two SNPs were detected in the FSHB-3 sequence of HFxG 5043 (4314A>G and 4489A>C) compared to cattle M83753 sequence. Both SNPs detected in HFxG 5043 FSHB-3 sequence were synonymous i.e. they did not result in amino acid change in FSHB-3. Findings of this study may provide basis for future research on genetic mechanisms affecting the semen traits and marker assisted selection of such traits in Indian crossbred cattle.

Association of FSH beta Gene Polymorphism on Testicular Parameters and Semen Quality in HF and Jersey Crossbred Bulls

Association of FSH beta Gene Polymorphism on Testicular Parameters and Semen Quality in HF and Jersey Crossbred Bulls

Searching for answers to the riddle of ovarian aging

Searching for answers to the riddle of ovarian aging

Runt-Related Transcription Factors Impair Activin Induction of the Follicle-Stimulating Hormone β-Subunit Gene

Synthesis of the FSH beta-subunit (FSHbeta) is critical for normal reproduction in mammals, and its expression within the pituitary gonadotrope is tightly regulated by activin. Here we show that Runt-related (RUNX) proteins, transcriptional regulators known to interact with TGFbeta signaling pathways, suppress activin induction of FSHbeta gene expression. Runx2 is expressed within the murine pituitary gland and dramatically represses activin-induced FSHbeta promoter activity, without affecting basal expression in LbetaT2 cells, an immortalized mouse gonadotrope cell line. This repressive effect is specific, because RUNX2 induces LHbeta transcription (with or without activin) and does not interfere with GnRH induction of either gonadotropin beta-subunit gene. Analysis of the murine FSHbeta promoter by transfection and gel shift assays reveals that RUNX2 repression localizes to a Runx-binding element at -159/-153, which is adjacent to a previously recognized region critical for activin induction. Mutation of this -153 activin-response element or, indeed, any of the five activin-responsive regions prevents activin induction and, in fact, RUNX2 suppression, instead converting RUNX2 to an activator of the FSHbeta gene. Although the Runx-binding element is required for RUNX2-mediated repression of FSHbeta induction by either activin or Smad3, confirming a functional role of this novel site, protein interactions in addition to those between RUNX2 and Smads are necessary to account for full repression of activin induction. In summary, the present study provides evidence for Runx2-mediated repression of activin-induced FSHbeta gene expression and reveals the context dependence of Runx2 action in hormonal regulation of the gonadotropin genes.

FoxL2 Is Required for Activin Induction of the Mouse and Human Follicle-Stimulating Hormone β-Subunit Genes

Activin is a major physiological regulator of FSH. We identify FoxL2 as a critical component in activin induction of FSHbeta, both for the mouse gene, induction of which is Sma- and Mad-related protein (Smad) dependent, and for the human gene that is Smad independent. FoxL2 has been shown to regulate gonadotrope gene expression (GnRH receptor, alpha-glycoprotein subunit, porcine FSHbeta, and follistatin), but the mechanisms of action are not well understood. We identify novel sites required for activin action in both the mouse and human FSHbeta promoters, some of which bind FoxL2, and show that the FoxL2-binding element encompasses a larger region (12 bp) than the previously identified forkhead-binding consensus (7 bp). Remarkably, although required for activin induction, FoxL2 sites neither contribute to basal FSHbeta promoter activity nor confer activin response to a heterologous promoter; thus, they are neither classical activin-response elements nor is their role solely to recruit Smads to the promoter. FoxL2 overexpression can potentiate activin induction in gonadotropes and can confer activin responsiveness to FSHbeta in heterologous cells where this promoter is normally refractory to activin induction. Although Smad3 requires the presence of FoxL2 sites to induce mouse FSHbeta, even through its consensus Smad-binding element; the human promoter, which is induced by activin independently of Smad3, also requires FoxL2 sites for its induction by activin; thus the actions of FoxL2 are not exclusively through interactions with the Smad pathway. Thus, FoxL2 plays a key role in activin induction of the FSHbeta gene, by binding to sites conserved across multiple species.

FSHbeta gene mutation in a female with delayed puberty and hypogonadism: response to recombinant human FSH.

We report a woman with primary amenorrhoea and infertility associated with an isolated deficiency of pituitary FSH that does not respond to GnRH administration. Serum inhibin B was undetectable and antimullerian hormone (AMH) was within the normal range. Ultra sound examination revealed a small uterus and small ovaries with few small follicles. We identified an homozygous 1-bp (G) deletion at codon 79 in FSHbeta gene suggesting a complete loss of function. The patient underwent studies of ovarian responsiveness to recombinant human FSH according to the following protocol: 150UI/d for five days following by 75 UI/d for 10 days. Estradiol plasma level started to increase from day 5 associated to a sharp increase of inhibine B and a decrease of LH. During the same time, we observed an excessive development of multiple follicles resulting in an arrest of the treatment to avoid hyperstimulation. The present study confirm that follicles up to 5 mm in diameter had developed in the absence of FSH and that FSH is required for the growth of follicles beyond the two-layer granulose stage.

The GnRH receptor and the response of gonadotrope cells to GnRH pulse frequency code. A story of an atypical adaptation of cell function relying on a lack of receptor homologous desensitization.

Brain control of the reproductive system is mediated through hypothalamic gonadotropin-releasing hormone (GnRH) which activates specific receptors (GnRHR) present at the surface of the pituitary gonadotropes to trigger secretion of the two gonadotropins LH and FSH. A unique feature of this system is the high dependence on the secretion mode of GnRH, which is basically pulsatile but undergoes considerable fluctuations in pulse frequency pattern in response to endogenous or external factors. How the physiological fluctuations of GnRH secretion that orchestrate normal reproduction are decoded by the gonadotrope cell machinery to ultimately control gonadotropin release and/or subunit gene transcription has been the subject of intensive studies during the past decades. Surprisingly, the mammalian GnRHR is unique among G protein-coupled receptor family as it lacks the carboxy-terminal tail usually involved in classical endocytotic process. Accordingly, it does not desensitize properly and internalizes very poorly. Both this atypical intrinsic property and post-receptor events may thus contribute to decode the GnRH signal. This includes the participation of a network of signaling pathways that differently respond to GnRH together with a growing amount of genes differentially sensitive to pulse frequency. Among these are two pairs of genes, the transcription factors EGR-1 and NAB, and the regulatory factors activin and follistatin, that function as intracellular autoregulatory feedback loops controlling respectively LHbeta and FSHbeta gene expression and hence, LH and FSH synthesis. Pituitary gonadotropes thus represent a unique model of cells functionally adapted to respond to a considerably fluctuating neuroendocrine stimulation, from short individual pulses to sustained GnRH as observed at the proestrus of ovarian cycle. Altogether, the data emphasize the adaptative reciprocal complementarity of hypothalamic GnRH neurones and pituitary gonadotropes to function as an original unit.

Synergistic Induction of Follicle-Stimulating Hormone β-Subunit Gene Expression by Gonadal Steroid Hormone Receptors and Smad Proteins

LH and FSH play crucial roles in mammalian reproduction by mediating steroidogenesis and gametogenesis. Gonadal steroid hormones influence gonadotropin production via feedback to the hypothalamus and pituitary. We previously demonstrated that progesterone and testosterone can stimulate expression of the FSH beta-subunit gene in immortalized gonadotrope-derived LbetaT2 cells. Herein, we investigate how these gonadal steroids modulate activin signaling in the gonadotrope. Cotreatment of LbetaT2 cells or mouse primary pituitary cells with steroids and activin results in a synergistic induction of FSHbeta gene expression. This synergy decreases when DNA-binding mutations are introduced into the steroid receptors or when mutations that reduce steroid hormone responsiveness are introduced into the FSHbeta promoter, indicating that synergy requires direct DNA binding of the steroid receptors. Furthermore, classical activin signaling via Smad proteins is necessary for this synergy. In addition, these steroid receptors physically interact with Smads and are sufficient for the synergism to occur on the FSHbeta promoter. Disruption of Smad binding to the promoter with a Smad protein lacking the DNA-binding domain or an FSHbeta promoter containing mutated activin-response elements prevents the synergistic enhancement of FSHbeta transcription. Collectively, our data demonstrate that the molecular mechanism for gonadal steroid hormone action on the FSHbeta promoter involves cross-talk between the steroid and activin signaling pathways. They also reveal that this synergism requires binding of both the steroid receptors and Smad proteins to their cognate DNA-binding elements and likely involves a direct protein-protein interaction between the two types of transcription factors.

Polymorphisms of three major genes for reproductive traits in Tibet pigs

Tibet pig is a special Chinese indigenous pig breed, which could survive in high-altitude areas of Tibet. In the present study, the genotypes of ESR, FSHbeta, and PRLR gene, the three known major genes of reproductive traits, were identified in 202 Tibet pig individuals. The results showed that the predominant genotypes of ESR, FSHbeta, and PRLR gene were BB, AB and AA, respectively. The genotype frequencies of the three genes were in Hardy-Weinberg equilibrium. The frequencies of desired alleles of the three genes were higher than those of other alleles, which indicated that Tibet pig had a good potential for improving reproductive traits.

Expression of Porcine FSH.BETA. Subunit Promoter-driven Herpes Simplex Virus Thymidine Kinase Gene in Transgenic Rats

A transgenic rat was established using the construct of porcine FSHbeta subunit promoter, the -852/+10 bp region, fused to a Herpes simplex virus thymidine kinase (HSV-TK) gene. Integration of the transgene was confirmed by PCR of tail DNA. RT-PCR of total RNAs of the pituitary, gonad, cerebellum, liver, kidney, adrenal gland, prostate, and uterus revealed that FSHbeta was only expressed in the pituitary. Analysis of the expression of reporter gene, HSV-TK, using two specific primer sets revealed that different transcripts were present in the pituitary and testis. The transcript initiated at the transcription initiation site of the porcine FSHbeta gene was detected in the pituitary, and another within the TK gene was found in the testis, indicating ectopic testis-specific expression. Immunohistochemistry of the pituitary glands of the transgenic rats for FSH and HSV-TK demonstrated that the FSH-producing cells also produced HSV-TK. The results indicated that the -852/+10 bp region of the FSHbeta promoter contains an element(s) that determines the tissue-specific expression. We succeeded in producing FSHbeta promoter-driven HSV-TK transgenic rats and were the first time to do so using an animal other than the mouse. The transgenic rats show male infertility that involves abnormal spermatogenesis. We also observed a decrease in the weight of the testis and epididymis, and both motile and living spermatozoa were absent in the epididymis. Consequently, the FSHbeta-HSV-TK transgenic rat will provide a useful model for studies on FSH function and male infertility.

High Level Expression of Prop-1 Gene in Gonadotropic Cell Lines

Prop-1 acts as an upstream regulator for the Pit-1 gene to induce development of Pit-1 lineage pituitary cell lines, GH-, PRL-, and TSH-producing cells, in the early stage of pituitary organogenesis. Furthermore, Prop-1 is presumed to be involved in the function of FSH/LH-producing cells, gonadotropes, since the defective Prop-1 gene shows hypogonadism. Recently, we reported evidence that Prop-1 directly regulates expression of the porcine FSHbeta gene, thus providing a novel advance in understanding the function of Prop-1 in FSH/LH production and hypogonadism. This study was intended to demonstrate the expressions of Prop-1 gene in pituitary tumor-derived cell lines. RT-PCR analyses were conducted of Pit-1, glycoprotein alpha subunit (alphaGSU), GnRH receptor, and cyclophilin A (a ubiquitously expressing gene). We observed expression of the Pit-1 gene in alphaT1-1, TalphaT1, MtT/S, GH3, and TtT/GF cells, expression of the alphaGSU gene in alphaT1-1, alphaT3-1, LbetaT2, LbetaT4, TalphaT1, and GH3 cells, and expression of GnRH receptor gene in alphaT3-1, LbetaT2, LbetaT4, and GH3 cells, respectively. These expression profiles were in accord with their cell lineages, with only a few exceptions. To accurately measure the expression level of the Prop-1 gene, a quantitative analysis was performed using the real-time PCR method. This analysis demonstrated that the LbetaT2 and LbetaT4 gonadotrope cell lines, which express the FSHbeta gene, express the Prop-1 gene. Taken together with our previous observation that Prop-1 is present in the adult porcine pituitary gonadotropes, Prop-1 might also be involved in development of gonadotropes and hormone production.

PCR-SSCP Polymorphism of FSHβ Gene and Its Relationship with Prolificacy of Jining Grey Goats

The follicle-stimulating hormone beta-subunit (FSHbeta) gene was studied as a candidate gene for the prolificacy in Jining Grey goats. According to the sequence of ovine FSHb gene, nine pairs of primers were designed to detect single nucleotide polymorphisms of 5' regulatory region, exon 1 and exon 2 of FSHbeta gene in both high fecundity breed (Jining Grey goat) and low fecundity breeds (Liaoning Cashmere goat, Boer goat and Angora goat) by PCR-SSCP. The results indicated that the homology of nucleotide sequence of this fragment between goat and sheep was 98 percent. Only the products amplified by primer P9 displayed polymorphism. Three genotypes (AA, AB and AC) were detected in Jining Grey goats and Liaoning Cashmere goats. Three genotypes (AA, CC and AC) were detected in Boer goats. Six genotypes (AA, BB, CC, AB, AC and BC) were detected in Angora goats. Sequencing revealed a G-->A mutation at 94 bp of exon 2 of FSHbeta gene in the BB genotype in comparison to the AA genotype and a C-->T mutation at 174 bp of exon 2 of FSHbeta gene in the CC genotype in comparison to the AA genotype. The former mutation resulted in an amino acid change: alanine-->threonine, and the latter mutation did not cause any amino acid change. Genotype frequency of AA, AB and AC was 0.686, 0.137 and 0.177 in Jining Grey goats, respectively. The does with genotype AA had 0.78 (P<0.05) or 0.64 (P<0.05) kids more than those with genotype AB or AC in Jining Grey goats, respectively.

Human FSHβ subunit gene is highly conserved

FSH is a pituitary gonadotropin that along with LH plays a key role in the regulation of gonadal function. The gonadotropic hormones are composed of two subunits, the common alpha subunit and the hormone-specific beta subunit, which determines the binding to specific receptors and induction of biological response. Unlike the LHbeta gene, shown in earlier studies to harbour several amino acid-altering polymorphisms and mutations, information about the eventual sequence variation of the human FSHbeta subunit is not available. In this study, we made sequence analysis and comparison of polymorphisms found in FSHbeta in two Caucasian populations, the Finns and the Danes. It was found that FSHbeta subunit is highly conserved in these populations. Compared with the published sequences, only three silent polymorphisms were detected in the coding regions of the gene, and the promoter sequence was completely identical with the reported sequence. Two of the polymorphisms found were novel, one in the Finnish and one in the Danish population. The results of the sequence analysis show that the human FSHbeta gene is highly conserved and amino acid changing mutations are apparently extremely rare, at least in the samples collected randomly from control populations. This may be due to the crucial role of normal FSH function in the regulation of fertility.

Diversity of microsatellites linked to the FSHbeta gene, their usefulness for individual identification and association with reproductive performance

The genetic diversity of three microsatellites (ILSTS027, MBO22, BM4325) mapped on the bovine chromosome 15 and linked to the follicle-stimulating hormone beta gene (FSHß) was investigated in cows of a Brangus Ibagé herd and the efficiency of these markers for individual identification and parentage control was estimated. Possible associations between each molecular marker and cow reproductive performance were also analyzed. Six alleles were detected in BM4325 and ILSTS027 and 12 in MB022, the most frequent being BM4325*101, BM4325*103, ILSTS027*169 and MB022*229. Polymorphic information content ranged from 0.58 to 0.88 while expected heterozygosity ranged from 65% to 89%, with an average mean value of 77%. Although only three markers were studied, the combined values indicate a high power of exclusion of a false parentage (94%) and of individual identification (3.8 x 10-4). The association analyses based on statistics parameters [MB022 (n=104, CI=545.3±127.0, WFC=349.9±53.4), BM4325 (n=106, CI=542.2±124.9, WFC=350.5±54.4) and ILSTS027 (n =105, CI=543.4 ± 124.5, WFC=350.1±54.5)] indicated no positive association between each microsatellite and weight at first calving. Calving interval (CI) also seemed not to be influenced by the ILSTS027 or MB022 system. However, carriers of at least one BM4325*101 allele presented a CI about 54 days shorter than the other animals (P=0.04; n=106). This marker could be useful for marker-assisted selection, allowing the improvement of reproductive performance, at least in the Brangus Ibagé herd.

CONTROL OF SPERMATOGENESIS IN PRIMATE AND PROSPECT OF MALE CONTRACEPTION

The present review is a summary of mechanisms of spermatogenesis in primates with emphasis on anti-spermatogenesis of testosterone (T), gossypol, and "testicular heat stress" for development of male contraception, Both FSH and testosterone stimulate all phases of spermatogenesis. FSH is capable of amplifying the population of the differential spermatogonia (B1, B2, B3 and B4) and controls the spermatogonia production rate, and, in synergy with testosterone, regulating spermatogenesis in adult monkeys. Pituitary FSH beta gene expression is governed by a feedback of Beta inhibin, which is a major component of the testicular negative feedback signals. Beta inhibin secreted by Sertoli cells is in turn inhibited by testosterone from Leydig cells under the control of LH. Disturbance of the normal interaction of pituitary FSH with Sertoli cell Beta inhibin is responsible for azoospermia or oligozoospermia induced by exogenous T. Three possible regimens of T, gossypol and "heat stress" have been suggested for male contraception. They act on different sites and stages of spermatogenesis in testis or sperm activity in epididymis. Apoptosis induced by testosterone occurs mainly at staged VII-VIII of spermatogenesis while that by testicular "heat stress" mostly occurs at stages I-IV and X-XII. Low dose of gossypol mainly influences the sperm activity in the epididymis although it also acts on testicular spermatids.

Synergy between activin A and gonadotropin-releasing hormone in transcriptional activation of the rat follicle-stimulating hormone-beta gene.

Both activin and GnRH can independently stimulate expression of the FSHbeta subunit gene. In this study, we used the gonadotrope-derived LbetaT2 cell line to investigate the potential interaction between activin and GnRH in regulating the transcriptional activity of the rat FSHbeta gene promoter. Activin A and GnRH synergistically enhanced rat FSHbeta transcriptional activity. Overexpression of SMAD3 (mediator of decapentaplegic-related protein 3), but not of SMAD2, increased transcriptional activation of the rat (r) FSHbeta gene promoter, which was further enhanced by the combined overexpression of SMAD3 and 4 (3+4). The stimulatory effects of SMAD3 overexpression were localized to -472/-256 of the rFSHbeta gene promoter, and activin- and GnRH-responsive proteins were shown to bind to region -284/-252. Sequence analysis identified a consensus palindromic SMAD-binding site at -266/-259 of the rFSHbeta gene promoter. Mutation of two bases located in the center of this palindrome effectively abrogated SMAD4 binding, markedly reduced SMAD3 and 3+4 stimulation of the rFSHbeta gene promoter, and significantly decreased the synergistic enhancement of promoter activity by both activin A and GnRH, and SMAD3 and GnRH. Blockade of the MAPK-signaling pathway did not significantly affect the response to combined stimulation with activin and GnRH. In contrast, interference with SMAD3 signaling caused a significant reduction in activin and GnRH synergy. The results indicate that SMAD3 plays an important role in the synergistic effects of activin and GnRH and demonstrate that this synergy is mediated by a palindromic cis-element located at -266/-259 of the rFSHbeta gene promoter.

Regulation of gonadotropins by inhibin and activin.

Inhibin, activin, and follistatin were first identified as gonadal hormones that could exert selective effects on follicle-stimulating hormone (FSH) secretion without affecting luteinizing hormone (LH). Although the primary source of inhibin remains the gonad, both activin and follistatin are produced in extragonadal tissues and can exert effects on FSH through an autocrine-paracrine mechanism. These proteins can effect the regulation of the gonadotropins at many levels. First, activin can directly stimulate FSH biosynthesis and release from the gonadotrope cells of the pituitary gland. Second, activin up-regulates gonadotropin-releasing hormone receptor (GnRHR) gene expression, leading to alterations in the synthesis and release of both gonadotropins in response to GnRH. Third, activin can stimulate GnRH release from GnRH neurons in the hypothalamus and thereby affect FSH and LH secretion. Both inhibin and follistatin can negatively regulate these effects by preventing activin binding to the activin receptor at the cell membrane and blocking activation of downstream signal transduction pathways. This review concentrates on the mechanisms through which inhibin, activin, and follistatin regulate the gonadotropins. We discuss the expression of inhibin/activin subunits and receptors throughout the hypothalamus and pituitary and their role in the regulation of FSH and LH. The mechanisms of inhibin and activin signaling are also reported, with particular attention to developments in our understanding of inhibin receptor action and activin-induced transcriptional regulation of the FSHbeta gene promoter. Finally, we present recent findings that other members of the transforming growth factor beta superfamily may also play an important role in transcriptional regulation of the pituitary gonadotropins.

Activin Regulation of the Follicle-Stimulating Hormone β-Subunit Gene Involves Smads and the TALE Homeodomain Proteins Pbx1 and Prep1

FSH is critical for normal reproductive function in both males and females. Activin, a member of the TGFbeta family of growth factors, is an important regulator of FSH expression, but little is known about the molecular mechanisms through which it acts. We used transient transfections into the immortalized gonadotrope cell line LbetaT2 to identify three regions (at -973/-962, -167, and -134) of the ovine FSH beta-subunit gene that are required for full activin response. All three regions contain homology to consensus binding sites for Smad proteins, the intracellular mediators of TGFbeta family signaling. Mutation of the distal site reduces activin responsiveness, whereas mutation of either proximal site profoundly disrupts activin regulation of the FSHbeta gene. These sites specifically bind LbetaT2 nuclear proteins in EMSAs, and the -973/-962 site binds Smad4 protein. Interestingly, the protein complex binding to the -134 site contains Smad4 in association with the homeodomain proteins Pbx1 and Prep1. Using glutathione S-transferase interaction assays, we demonstrate that Pbx1 and Prep1 interact with Smads 2 and 3 as well. The two proximal activin response elements are well conserved across species, and Pbx1 and Prep1 proteins bind to the mouse gene in vivo. Furthermore, mutation of either proximal site abrogates activin responsiveness of a mouse FSHbeta reporter gene as well, confirming their functional conservation. Our studies provide a basis for understanding activin regulation of FSHbeta gene expression and identify Pbx1 and Prep1 as Smad partners and novel mediators of activin action.

Extracellular signal-regulated kinase, Jun N-terminal kinase, p38, and c-Src are involved in gonadotropin-releasing hormone-stimulated activity of the glycoprotein hormone follicle-stimulating hormone beta-subunit promoter.

The role of ERK, Jun N-terminal kinase (JNK), p38, and c-Src in GnRH-stimulated FSHbeta-subunit promoter activity was examined in the LbetaT-2 gonadotroph cell line. Incubation of the cells with a GnRH agonist resulted in activation of ERK, JNK, p38, and c-Src. The peak of ERK activation was observed at 5 min, whereas that of JNK, p38, and c-Src at 30 min, declining thereafter. ERK activation by GnRH is dependent on protein kinase C (PKC), as evident by activation, inhibition, and depletion of 12-O-tetradecanoylphorbol-13-acetate-sensitive PKC subspecies. Ca(2+) influx, but not Ca(2+) mobilization, is required for ERK activation. GnRH signaling to ERK is partially mediated by dynamin and a protein tyrosine kinase, apparently c-Src. ERK activation by GnRH in LbetaT-2 cells does not involve transactivation of epidermal growth factor receptor or mediation via Gbetagamma or beta-arrestin. Once activated by GnRH, ERK translocates to the nucleus. We examined the role of ERK, JNK, p38, and c-Src in GnRH-stimulated ovine FSHbeta promoter, linked to a luciferase reporter gene (-4741oFSHbeta-LUC). The PKC activator 12-O-tetradecanoylphorbol-13-acetate, but not the Ca(2+) ionophore ionomycin, stimulated FSHbeta-luciferase (LUC) activity. Furthermore, down-regulation of PKC, but not removal of Ca(2+), inhibited the GnRH response. Cotransfection of FSHbeta-LUC and the constitutively active forms of Raf-1 and MEK stimulated FSHbeta-LUC activity, whereas the dominant negatives of Ras, Raf-1, and MEK and the selective MEK inhibitor PD98059, abolished GnRH-induced FSHbeta-LUC activity. The dominant negatives of CDC42 and JNK reduced the GnRH response by 36 and 49%, respectively. Incubation of the cells with the p38 or the c-Src inhibitors SB203580 and PP1 also reduced the GnRH response. Surprisingly, two proximal activator protein-1 sites contribute very little to the GnRH response. Thus, PKC, ERK, JNK, p38, and c-Src, but not Ca(2+), are involved in GnRH induction of the ovine FSHbeta gene.