Transcriptional activation of the ovine follicle-stimulating hormone beta-subunit gene by gonadotropin-releasing hormone: involvement of two activating protein-1-binding sites and protein kinase C.

FSH is an alpha/beta heterodimeric glycoprotein, the formation of which is regulated primarily by expression of its beta-subunit. Recent studies on transcriptional regulation of the ovine FSH beta-subunit gene (oFSHbeta) have defined two functional activating protein-1 (AP-1) enhancers in the proximal promoter (located at -120 and -83 bp) that are probably physiologically important for FSHbeta expression. As GnRH is a major regulator of FSHbeta expression and is also known to stimulate the synthesis of Jun and Fos family members (AP-1), we investigated the possibility that oFSHbeta transcription may be regulated by GnRH through AP-1. Here we report the use of an in vitro cell system involving transient transfection of GnRH receptors (GnRHR) into HeLa cells to define regulatory elements involved in GnRH-mediated induction of oFSHbeta. This system was used to show that expression of luciferase constructs containing either the -4741/+759 region of the oFSHbeta gene (-4741oFSHbeta-Luc) or the -846/+44 region of the human alpha gene (alpha-Luc; a positive control) was stimulated 3.1 +/- 0.3- and 7.7 +/- 1.9-fold, respectively, by 100 nM GnRH. Another luciferase expression plasmid containing the Rous sarcoma virus promoter (a negative control) showed no response to GnRH. Similar results with these constructs were obtained in COS-7 cells. Studies with progressive 5'-deletion constructs and site-specific mutations demonstrated that this stimulation was dependent on each AP-1 site in the proximal promoter of oFSHbeta. Gel shift assays demonstrated the ability of GnRHR in HeLa cells to increase AP-1 binding activity. Responses in the HeLa cell system were dependent on GnRH (ED50 = 0.5 nM) and GnRHR, which was identified by photoaffinity labeling. In addition, GnRHR-expressing HeLa cells exhibited a normal GnRH-dependent mobilization of intracellular calcium. Finally, as protein kinase C (PKC) is a known target of GnRH action in gonadotropes, the role of PKC in transcriptional regulation of oFSHbeta and alpha-subunit genes by GnRH in HeLa cells was investigated. Although 12-O-tetradecanoyl 13-acetate induction of alpha-Luc and -215oFSHbeta-Luc could be completely blocked in a dose-dependent manner by the specific PKC inhibitor bisindolylmaleimide I, only 57-65% of the GnRH-mediated stimulation of these promoters was blocked, demonstrating the involvement of PKC as well as other signaling systems in GnRH induction. These data define a molecular action of GnRH on oFSHbeta gene transcription that involves two proximal AP-1 enhancer elements and PKC activation. Furthermore, these studies establish the usefulness of HeLa and COS-7 cells to investigate specific aspects of GnRH action on gonadotropin subunit gene expression, as similar signaling pathways and transcription factors that are activated by GnRH in gonadotropes (such as PKC, mitogen-activated protein kinase, Ca2+, and AP-1) exist in these cells.

Two proximal activating protein-1-binding sites are sufficient to stimulate transcription of the ovine follicle-stimulating hormone-beta gene.

FSH is an important regulator of mammalian gametogenesis and the female reproductive cycle. Although little is known about the transcriptional regulation of the beta-subunit (the rate-limiting subunit of FSH synthesis), sequence analysis of the ovine FSHbeta promoter has revealed a number of potential activating protein-1 (AP-1; Jun/Fos)-binding sites. To determine whether the gene encoding the beta-subunit of ovine FSH (oFSHbeta) is responsive to AP-1 transcriptional complexes, chimeric constructs containing deleted portions of the oFSHbeta promoter fused to a luciferase reporter were transiently transfected along with c-Jun and c-Fos expression constructs into JAR cells. Analysis of these deletion constructs revealed that the proximal promoter of oFSHbeta is highly stimulated by c-Jun and c-Fos proteins (typically 20-fold with a reporter construct containing oFSHbeta sequences from -215 to +759 bp). This stimulation was lost when a similar construct containing sequences from -84 to +759 bp was tested. Transcriptional start site analysis using reverse transcription-PCR verified that the transcriptional initiation of the -215-bp deletion construct, with or without cotransfected c-Jun/c-Fos, was the same as that observed in vivo. Computer analysis of oFSHbeta sequences from -215 to +1 bp identified four putative AP-1-like elements, located at -155, -120, -83, and -10 bp. Gel retardation experiments using oligonucleotides corresponding to the four putative AP-1-like sites revealed that only -120 and -83 sites in oFSHbeta bound AP-1 proteins in vitro. Site-directed mutagenesis of the -120 and -83 sites showed that each element was required for stimulation by c-Jun and c-Fos proteins as well as 12-O-tetradecanoyl phorbol-13-acetate in transient transfection assays. Finally, immunocytochemical dual labeling was used to show that more than 75% of all FSHbeta-containing cells in ovine pituitary sections from cycling ewes contained nuclear c-Jun, JunB, JunD, and Fos proteins. These data, taken together, show that oFSHbeta transcription can be stimulated by c-Jun and c-Fos proteins via two functionally linked AP-1-like sites in the oFSHbeta proximal promoter and that these sites are likely to be important regulators of FSH production in vivo.

Inhibin and estradiol alter gonadotropes differentially in ovine pituitary cultures: changing gonadotrope numbers and calcium responses to gonadotropin-releasing hormone.

Both inhibin (IN) and estradiol (E) use separate and distinct mechanisms to decrease the production of FSH 60%-80% while increasing receptors for GnRH (GnRH-Rec) 400%-600% in ovine pituitary cultures. To investigate how these hormones act to create outcomes that appear similar, individual cells in ovine pituitary cultures were analyzed for changes in GnRH-stimulated calcium signaling, GnRH binding, and gonadotropin content under IN or E treatments. Calcium mobilization studies showed that 10 nM GnRH increased intracellular calcium ([Ca++]i) in 9.2% +/- 0.8% of cells in control ovine pituitary cultures. After treatment with 10 nM E for 48 h, there was a small increase in the number of cells responding to GnRH (12.9% +/- 1.4% responded) and a major 5-fold increase in [Ca++]i response to GnRH as compared with untreated cells. By contrast, IN did not alter cellular calcium responses to GnRH but markedly increased the number of cells responding to GnRH (a 3.7-fold increase to 33.8 +/- 2.6%). Cytochemical studies measuring GnRH:GnRH-Rec complexes, FSH, and LH showed that E had no effect on the number of pituitary cells containing FSH, LH, or GnRH-Rec. In contrast, similar studies using IN showed that the number of cells containing GnRH-Rec and LH significantly increased (> 50%), whereas the number of FSH cells decreased (36%), and a lower than normal percentage of the remaining FSH cells carried GnRH-Rec. Thus, the net effect of IN on gonadotropes was to dramatically decrease the ratio of FSH:LH cells that contained GnRH-Rec, from 1:1 (untreated) to 1:8 (IN-treated) and to decrease the percentage of LH/FSH colocalization. In summary, these data indicate that E primarily operates on a fixed, preset number of ovine gonadotropes to inhibit FSH production and increase responsiveness to GnRH. IN, however, seems to change the very nature of ovine gonadotropes by completely turning off FSH synthesis in some cells, lowering GnRH-Rec in other FSH cells, and, finally, inducing LH and GnRH-Rec in newly recruited gonadotropes.

Gonadotropin-releasing hormone-stimulated calcium mobilization is altered in pituitary cultures from anestrous ewes.

GnRH transiently increases intracellular calcium ([Ca2+]i) in pituitary gonadotrophs in culture. This effect is observed within 10 sec of GnRH treatment, lasts 1-5 min, and is thought to be an important intracellular signal leading to the secretion of LH. Recent studies with ovine pituitary cultures uncovered seasonal differences in this transient calcium response to GnRH. This finding led to the hypothesis that anestrus alters the pituitary response to GnRH and prompted this study on seasonal effects on LH secretion and [Ca2+]i mobilization. Inhibin was used to enhance pituitary sensitivity to GnRH. Results showed that pituitary cultures from 16 cycling ewes secreted an average of 9% +/- 2% of stored LH after treatment with 10 nM GnRH (stored LH = 470 +/- 66 ng/million cells); 9% +/- 1% of pituitary cells increased the concentration of [Ca2+]i from 75-150 nM up to 400-1400 nM. When cultures were pretreated with inhibin, GnRH released an average of 15% +/- 3% of stored LH (stored LH = same as above), and 38% +/- 4% of cells transiently increased [Ca2+]i above 400 nM. Results also showed that pituitary cultures from 11 acycling ewes secreted similar percentages of LH although the cells contained less LH to begin with (stored LH = 190 +/- 24 ng/million cells), but absolutely none of these cells (500 counted) increased [Ca2+]i after GnRH treatment, with or without inhibin present. Levels of GnRH receptor mRNA were unchanged by season. These data show that LH secretion and the rapid, transient increase in [Ca2+]i caused by GnRH are uncoupled in the unique situation of anestrus. It is possible, in fact, that these two parameters are not necessarily coupled during estrous conditions and that the transient change in [Ca2+]i is connected with LH synthesis or some other gonadotroph function. The data also show that inhibin can increase GnRH-responding cells up to 38%, a fact suggesting that inhibin may be a differentiation factor capable of transforming multipotent pituitary cells into gonadotrophs in sheep.

Isolation of nine different biologically and immunologically active molecular variants of bovine follicular inhibin.

A combination of immunoaffinity chromatography, SDS-PAGE, and electroelution was used to simultaneously isolate 0.36-4.65 mg of nine different molecular forms of inhibin (pro alpha C-29 kDa; fully processed 34 kDa; and large inhibin forms 49, 53, 58, 77, 88, 110, and > 160 kDa) from 0.675 L of bovine follicular fluid (bFF). Each inhibin form, except pro alpha C, cross-reacted with inhibin alpha C 1-26-and beta A 82-114-subunit-directed antibodies during immunoblot analysis. Pro alpha C cross-reacted only with alpha-subunit antibodies. The inhibin forms consisted of 22-, 29-, 49-, or 58-kDa alpha subunits and 17- or 58-kDa beta subunits. During cultures of ovine pituitary cells, a 5-ng/ml dose of each inhibin form (except pro alpha C) suppressed basal accumulation of FSH 30% to 50% but increased GnRH-induced LH release 40% to 248%. The various inhibin forms cross-reacted in parallel fashion with standard curves generated during homologous and heterologous RIAs but with markedly different relative immunopotencies. In the RIAs, pro alpha cross-reacted 3- to 18-fold more than the fully processed inhibin form. The fully processed and the seven different large forms of inhibin cross-reacted with different relative immunopotencies in a two-site dimer-specific ELISA. We concluded that 1) a combination of immunoaffinity extraction, SDS-PAGE, and electroelution simultaneously isolated relatively large amounts of highly enriched preparations of nine different molecular forms of immunologically and biologically active inhibin from bFF; 2) eight different dimeric forms of bovine inhibin may regulate both basal FSH and GnRH-induced LH secretion by the pituitary gland, and 3) eight or nine different molecular forms of inhibin cross-react with different relative immunopotencies in the two-site dimer-specific assay or RIAs.