LbetaT2 gonadotroph cells secrete follicle stimulating hormone (FSH) in response to active A.

Secretion of luteinizing hormone in response to gonadotropin releasing hormone (GnRH) has been described in the recently developed LbetaT2 gonadotroph cell line. We evaluated the expression of follicle stimulating hormone (FSH)beta mRNA and secretion of FSH from LbetaT2 cells in response to GnRH and activin A. LbetaT2 cells were treated with activin A in doses from 0 to 50 ng/ml, with or without a daily 10 nM GnRH pulse, or with GnRH alone. FSH secretion was stimulated over 6-fold by concomitant GnRH and activin A in a dose-responsive fashion at 72 h of treatment. FSHbeta mRNA was detectable by ribonuclease protection assay only in cells treated with activin A with or without GnRH. The demonstration of FSHbeta gene expression in LbetaT2 cells further validates these cells as mature, differentiated gonadotrophs and as an important tool for the study of gonadotroph physiology.     

A novel role for bone morphogenetic proteins in the synthesis of follicle-stimulating hormone

FSH is produced in pituitary gonadotropes as an alpha/beta heterodimer, and synthesis of the beta-subunit is the rate-limiting step in overall FSH production. Synthesis of FSHbeta can be regulated by activin and inhibin, both of which are members of the transforming growth factor-beta superfamily. Bone morphogenetic proteins (BMPs) also belong to the transforming growth factor-beta family and are multifunctional growth factors involved in many aspects of tissue development and morphogenesis, including regulation of FSH action in the ovary. Here we report a novel function for BMP-7 and BMP-6 in regulating FSH synthesis in the pituitary. Using primary pituitary cell cultures derived from transgenic mice that carry the ovine FSHbeta promoter linked to a luciferase reporter gene (oFSHbetaLuc), BMP-7 or BMP-6 was found to stimulate oFSHbetaLuc expression by 6-fold. Transient expression of the oFSHbetaLuc in a transformed gonadotrope cell line, LbetaT2, was induced 4-fold by BMP-7 or BMP-6 treatment. More importantly, BMP-7 and BMP-6 increased endogenous FSH secretion by 10- and 14-fold, respectively, from LbetaT2 cells, demonstrating for the first time that a functional signaling BMP system is present in gonadotropes. Two bioneutralizing antibodies to BMP-7, which cross-react with BMP-6, but not with activin A, decreased basal oFSHbetaLuc expression and FSH secretion from transgenic mouse pituitary cultures by 83-88% and 47-48%, respectively, suggesting an autocrine or paracrine role for BMP-7 or BMP-6 in FSH synthesis. Neither bioneutralizing antibody to activin A or activin B decreased basal oFSHbetaLuc expression or mouse FSH secretion significantly. Dose-dependent inhibition of FSH synthesis by anti-BMP7 was also observed in rat and sheep pituitary cultures. These results combined with the fact that the messenger RNAs for BMP-7 and BMP-6 were detected in mouse pituitaries and LbetaT2 cells indicate that BMP-7 and/or BMP-6 can function as FSH stimulators and may be significant physiological factors maintaining basal FSH expression in vivo.     

Cell-specific transcriptional regulation of follicle-stimulating hormone-beta by activin and gonadotropin-releasing hormone in the LbetaT2 pituitary gonadotrope cell model

FSH is secreted by gonadotropes of the anterior pituitary and plays a crucial role in mammalian reproduction. However, little is known about FSH gene regulation due to the lack of a gonadotrope cell line that synthesizes FSH. The LbetaT2 mouse pituitary cell line, isolated by targeted tumorigenesis in transgenic mice, has the characteristics of a mature gonadotrope, including expression of GnRH receptor, steroidogenic factor 1, and both the alpha- and beta-subunits of LH, but was thought not to express FSH. Using RT-PCR, we show that these cells synthesize FSH beta- subunit messenger RNA, which is induced by activin and inhibited by follistatin. Furthermore, in transient transfections an ovine FSHbeta 5'-regulatory region (5.5 kb) confers LbetaT2 cell-specific expression to a reporter gene compared with other pituitary and nonpituitary cell lines. This FSHbeta regulatory region responds to activin specifically in LbetaT2 cells, an effect that is blocked by follistatin. The LHbeta, alpha-subunit, and GnRH receptor regulatory regions are induced by activin and blocked by follistatin. Furthermore, LbetaT2 cells express the components of the activin system, and addition of follistatin alone reduces FSHbeta gene expression, demonstrating that an endogenous activin autocrine loop regulates FSH in these cells. In addition, GnRH stimulates both the FSHbeta and LHbeta regulatory regions, specifically in LbetaT2 cells. Surprisingly, GnRH induction is reduced by follistatin, suggesting its dependence on endogenous activin. As the mouse GnRH receptor promoter is inhibited by follistatin, reduction of GnRH receptor levels might be one mechanism by which follistatin interferes with GnRH induction of gonadotropin genes. In summary, LbetaT2 cells exhibit the characteristics of fully differentiated gonadotropes, including the expression of LH, FSH, GnRH receptor, and components of the activin/follistatin system, as well as display the appropriate responses to activin and GNRH:     

The activin receptor-like kinase 6 Booroola mutation enhances suppressive effects of bone morphogenetic protein 2 (BMP2), BMP4, BMP6 and growth and differentiation factor-9 on FSH release from ovine primary pituitary cell cultures

Bone morphogenetic proteins (BMPs) have been shown to influence the regulation of FSH synthesis and secretion at the level of the pituitary. Primary pituitary cells were harvested and cultured from Booroola ewes homozygous for a mutation in activin receptor-like kinase 6 (ALK6) also known as BMP receptor IB (BMPRIB), and from wild-type (WT) ewes to determine if the mutation caused alterations in FSH secretion in vitro. The cells were collected 24 h following induction of luteolysis and cultured for 72 h prior to being challenged for 24 h with BMP2, BMP4, BMP6, growth and differentiation factor-9 (GDF9), transforming growth factor-beta 1, activin-A and GnRH. The levels of FSH and LH were measured by RIA and then compared with the untreated controls. Primary pituitary cell cultures from Booroola ewes secreted less FSH than WT cells in the presence of BMP2, BMP4 and BMP6. These BMPs did not affect the FSH stores within the cells, or the levels of LH released. GDF9 appeared to act in a BMP-like manner by suppressing FSH secretion. The ALK6 receptor however, was not found to co-localise with gonadotroph cells in either Booroola or WT pituitary tissues. These findings imply that the increased sensitivity of Booroola cells to BMP2, BMP4, BMP6 and GDF9 cannot be due to the direct action of the ALK6 mutant Booroola receptor in the cells that synthesise FSH.     

Pituitary follistatin and activin gene expression, and the testicular regulation of FSH in the adult Rhesus monkey (Macaca mulatta).

In rats, FSHbeta gene expression and FSH secretion are increased and decreased, respectively, by pituitary activin and follistatin. Because little information is available on the paracrine control of FSH secretion in the primate, follistatin and activin/inhibin beta(B) messenger RNA (mRNA) levels were measured in pituitaries of adult male rhesus monkeys 6 weeks after castration or sham surgery (n = 5/group). Follistatin mRNA was determined by quantitative RT-PCR assay using oligonucleotide primers designed to span exons 3-5 of the human follistatin gene. Activin/inhibin beta(B) mRNA levels were measured by ribonuclease protection. Orchidectomy resulted in a 100-fold increase in plasma FSH concentrations and a 60-fold rise in those of LH. In castrated monkeys, levels of mRNA encoding FSHbeta, LHbeta, alpha- subunit, and GnRH receptor (GnRH-R) were increased 21-, 2.1-, 1.7-, and 1.7-fold, respectively (P < 0.01). Levels of pituitary follistatin and activin/inhibin beta(B) mRNAs, however, were similar in castrated and intact animals. These data suggest that the paracrine control of FSH secretion in the male differs substantially in primates and rodents. Specifically, the relatively greater postcastration rise in FSHbeta gene expression and FSH secretion in the adult male monkey may result because in this species pituitary follistatin gene expression does not increase after orchidectomy, as it does in the rat.     

Regulation of FSHbeta and GnRH receptor gene expression in activin receptor II knockout male mice

To examine in vivo, the local effects of inhibins and activins within the anterior pituitary, independent of their endocrine effects exerted from the gonad, in mediating FSH homeostasis, we used castrated knockout mice lacking either inhibin alpha or activin receptor II (ACVR2) alone or in combination. Compared to castrated wild-type (WT) mice, FSHbeta mRNA levels in the pituitaries of Acvr2 null mice were significantly downregulated in the absence of gonadal feedback. FSHbeta mRNA levels were not significantly higher in the pituitaries of castrated inhibin alpha null mice compared to those in Acvr2 null mice and remained the same in the pituitaries of castrated double mutant mice lacking both inhibin and ACVR2. In contrast to FSHbeta mRNA expression changes, pituitary FSH content was significantly reduced in Acvr2 null mice whereas it was only slightly upregulated in inhibin alpha null mice. Combined absence of both ACVR2 signaling and inhibins caused a decrease in FSH content compared to that in the absence of inhibins alone. These changes in pituitary content were in parallel to those in serum FSH levels in these three groups of castrated mice, suggesting that the unopposed actions of locally produced inhibins are dominant over those effects mediated by ACVR2 signaling to regulate FSH biosynthesis and secretion. Thus, our in vivo results demonstrate that within the pituitary, locally produced activins and inhibins exert their actions at distinct phases of FSH homeostasis. In an independent set of experiments, we tested whether in vivo signaling via ACVR2 is necessary for hypothalamic GnRH biosynthesis and for GnRH receptor expression. Our results demonstrate that in contrast to previous in vitro studies, signaling through ACVR2 is neither required for hypothalamic synthesis of GnRH peptide nor for expression of GnRH receptors in the anterior pituitary. We conclude that within the hypothalamic-pituitary short loop, ACVR2 signaling is critical only for FSH homeostasis and not for GnRH biosynthesis or induction of pituitary GnRH receptor expression. Our studies confirm the importance of using in vivo genetic models for studying regulation of the hypothalamic-pituitary-gonadal axis.     

mRNA expression of type I and type II receptors for activin, transforming growth factor-beta, and bone morphogenetic protein in the murine erythroleukemic cell line, F5-5.fl

OBJECTIVE: Intracellular signaling of activin and transforming growth factor-beta (TGF-beta) is thought to be mediated by the same molecules (Smad2/3 and Smad4). Although differentiation of murine erythroleukemia F5-5.fl cells is induced by activin, it is not induced by TGF-beta, suggesting that at some point TGF-beta signaling is defective. The aim of this study was to investigate the unresponsiveness of F5-5.fl cells to TGF-beta. DESIGN: mRNA expression of ligands, receptors, and signal mediators for the TGF-beta family was examined in F5-5.fl cells using RT-PCR. RESULTS: Activin induced erythrodifferentiation of F5-5.fl cells in a dose-dependent manner. Neither TGF-beta1 nor bone morphogenetic protein (BMP)-4 affected the differentiation of F5-5.fl cells in the presence or absence of activin. Although mRNAs of TGF-betas (TGF-beta1, TGF-beta2 and TGF-beta3) were detected, those of inhibin/activin (alpha-, betaA- and betaB-subunits) and BMPs (BMP-2, BMP-4 and BMP-7) could not be detected in the cells, suggesting that neither activins nor BMPs are produced in F5-5.fl cells. The expression of both type I (ALK-4/ActRIB) and type II (ActRII) receptors for activin was detected in F5-5.fl cells. In contrast, while the expression of type I receptor for TGF-beta (ALK-5/TbetaRI) was detected, that of type II receptor (TbetaRII) was not. The mRNA of all Smads examined was detected in F5-5.fl cells. CONCLUSIONS: A defect in the type II receptor might cause unresponsiveness to TGF-beta in F5-5.fl cells. An erythrodifferentiation assay using F5-5.fl cells would be useful for measuring net activin activity because it would not be necessary to consider endogenous activins and BMPs.     

Gonadotrophin-releasing hormone modulation of gonadotrophins in the ewe: evidence for differential effects on gene expression and hormone secretion.

While the regulation of gonadotrophin secretion by gonadotrophin-releasing hormone (GnRH) has been well documented in both rats and sheep, its role in the synthesis of gonadotrophin subunits remains unclear. We have investigated the effects of the specific inhibition of GnRH by a GnRH agonist on the expression of gonadotrophin subunit genes and the subsequent storage and release of both intact hormones and free alpha subunit. Treatment with GnRH agonist for 6 weeks abolished pulsatile LH secretion, reduced plasma concentrations of FSH and prevented GnRH-induced release of LH and FSH. This was associated with a reduction of pituitary LH-beta mRNA and FSH-beta mRNA levels (to 5 and 30% of luteal control values respectively), but not alpha mRNA which was significantly increased (75% above controls). While there was a small decrease in the pituitary content of FSH (30% of controls), there was a drastic reduction in LH pituitary content (3% of controls). In contrast to the observed rise in alpha mRNA, there was a decrease in free alpha subunit in both the pituitary and plasma (to 30 and 80% of control levels). These results suggest that, while GnRH positively regulates the expression of both gonadotrophin beta-subunit genes, it can, under certain circumstances, negatively regulate alpha-subunit gene expression. Despite the complete absence of LH and FSH in response to GnRH, there remained a basal level of beta-subunit gene expression and only a modest reduction (50%) in the plasma levels of both FSH and LH, suggesting that there is a basal secretory pathway.(ABSTRACT TRUNCATED AT 250 WORDS)