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Circulating androgens reach high concentrations in females of some reptiles and amphibians. We are testing the hypothesis that androgens can act directly in female reptilian reproductive tissues, via the androgen receptor. In this study, we sought to determine if androgen receptors are present in the oviduct of the turtle, Trachemys scripta, using radioligand-binding assays and immunological assays. An androgen-binding site was detected in turtle oviductal cytosol and oviductal nuclear extract by radioligand binding assay, using (3)H-dihydrotestosterone (DHT) as the ligand. This site was saturable (B(max)=11 pmol/g tissue), had a high affinity (10(-10) M), and showed specificity typical of androgen receptors (DHTtestosterone, progesteroneestradiol, cortisol). Western blotting using an anti-androgen receptor antibody revealed a band of immunoreactivity in oviductal cytosol at approximately 115 kDa, and a more prominent band at 50 kDa, possibly indicating a truncated form of the androgen receptor. Immunohistochemistry revealed crossreactivity of the androgen receptor antibody against oviductal glandular cells but not against oviductal luminal epithelial or muscularis cells. The presence of androgen receptor in the turtle oviduct suggests that androgens have a role in female reproduction and that their action can be mediated directly by androgen receptor.

Direct production of gonadal steroids from sulfated adrenal androgens may be an important alternative or complementary pathway for ovarian steroidogenesis. The conversion of sulfated adrenal androgens, present in serum at micromolar concentrations in adult women, into unconjugated androgens or estrogens requires steroid sulfatase (STS) activity. STS activity has not been characterized in the rat ovary. Substantial STS activity was present in homogenates of rat ovaries, primary cultures of rat granulosa cells, and a granulosa cell line, as determined by conversion of radiolabeled estrone sulfate (E1S) to unconjugated estrone. The potent inhibitor estrone sulfamate eliminated the STS activity. Using E1S as a substrate with microsomes prepared from a granulosa cell line, the K(m) of STS activity was approximately 72 microM, a value in agreement with previously published data for rat STS. Therefore, ovarian cells possess STS and can remove the sulfate from adrenal androgens such as dehydroepiandrosterone sulfate (DHEA-S). Using DHEA-S as a steroidogenic substrate represents an alternative model for the production of ovarian steroids versus the "two cell, two gonadotropin" model of ovarian estrogen synthesis, whereby thecal cells produce androgens from substrate cholesterol and granulosa cells convert the androgens into estrogens. The relative contribution of STS activity to ovarian steroidogenesis remains unclear but may have important physiological and pathophysiological implications.

The expression and function of estrogen receptor ERα/β subtypes and ERβ variants in granulosa cells have been determined using several integrated approaches: , Western blotting, indirect immunofluorescence, RT-PCR, and transient transfection assays. Each of these approaches has provided specific details concerning the dynamics of ER expression, ER functional activity, and estradiol (E) regulation of target genes in granulosa cells. Specifically, the studies presented herein document that messenger RNAs (mRNAs) encoding ERβ and its splice variants, as well as mRNA encoding ERα, are expressed in granulosa cells of immature rats before and during culture in serum-free medium. The results also provide the first documentation that functional (DNA binding and transcriptionally active) ER is present in cultured granulosa cells and that its ability to bind consensus estrogen response element (ERE) oligonucleotide and to transactivate an ERE promoter-reporter construct is associated with the level (type?) of recep...

Expression of progesterone receptor (PR) mRNA in granulosa cells of ovarian preovulatory follicles is induced by LH (1, 2) and is essential for ovulation (3). Although 17beta-estradiol (E) can induce PR mRNA and activate PR promoter-reporter constructs in other cell types, the effects of E in granulosa cells appear to be indirect. We show herein that E alone does not induce the expression of PR mRNA in preovulatory granulosa cells. Rather, induction of PR mRNA depends on the differentiation of granulosa cells in response to E and a physiological amount of FSH followed by exposure to agonists (elevated levels of LH, FSH, and forskolin) that markedly increase cAMP. Induction of PR mRNA by forskolin is blocked by the A-kinase inhibitor H89 and cycloheximide but not by the E antagonist, ICI 164,384. These results indicate that phosphorylation and synthesis of some regulatory factor(s) other than or in addition to the estrogen receptor (ER) are essential for transactivation of the PR gene. When distal and proximal PR promoter-reporter constructs that are responsive to E in other cell types were transiently transfected into differentiated granulosa cells, forskolin, but not E, induced activity. Likewise, when a vector containing the consensus vitellogenin B1 gene estrogen response element (ERE) was transfected into differentiated granulosa cells, forskolin, but not E, induced activity. Using electrophoretic mobility shift assays, the consensus ERE was shown to bind ERbeta, the predominant subtype present in rat granulosa cells, and ERalpha, the predominant subtype present in luteal cells, whereas the putative ERE-like region (ERE3) of the proximal PR promoter did not bind either ER subtype. Although the identity of the specific factors binding to the ERE3 site remain to be determined, mutation of this region abolished forskolin-induced activity of ERE3-PR-CAT constructs. The GC-rich region of the distal PR promoter bound Sp1 and Sp3 but not C/EBPalpha/beta, indicating that factors binding to ERE3 interact synergistically with Sp1/Sp3 to confer increased responsiveness of the distal promoter to forskolin. Taken together, these results indicate that activation of the A-kinase pathway leads to the phosphorylation of some transcription factor(s) other than or in addition to ER that is (are) critical for the transactivation of the PR gene and that this mechanism is selectively activated in differentiated granulosa cells possessing a preovulatory phenotype.

Previous studies have shown that inhibitors of protein tyrosine kinases, tyrphostins, can markedly attenuate the steady-state levels of mRNAs of hormone-induced genes expressed in ovarian cells. To further elucidate the mechanism of tyrphostin action, rat granulosa cells were electroporated with chimeric expression vectors containing the promoters of two key steroidogenic genes, cholesterol side chain cleavage cytochrome P450 (CYP11A; P450scc) and aromatase cytochrome P450 (CYP19; P450arom), ligated to the CAT reporter gene. The electroporation method of transfection documents that the respective promoter-reporter constructs, -379sccCAT and -534aromCAT, can confer greater than 10-fold FSH/cAMP responsiveness to the reporter genes expressed in naive granulosa cells. Furthermore, the electroporation approach allows transfection of DNA into small numbers of cells and facilitates the assay of expression in cells isolated from follicles at advanced stages of differentiation. In naive granulosa cells, the functional activities of -379sccCAT, -534aromCAT, and -169 alpha CGCAT were abolished by the A-kinase specific inhibitor, H89, supporting the notion that activation of protein kinase A is obligatory for transcriptional activation of the promoter regions within these genes. Similar inhibitory effects were also observed for tyrphostin AG18, thus implicating a tyrosine kinase in the regulation of the steroidogenic genes. As a result of eCG/hCG treatments, a gradual loss of transfection efficiency accompanied by decreasing forskolin induction of CAT expression was observed in the differentiating granulosa-lutein cells. Although the reason(s) for the apparent loss in the ability of hormones to regulate chimeric gene expression remains to be determined, cell and promoter refractoriness to hormone treatment appears to reflect a fundamental change in the mechanism of promoter activation in the differentiated cells compared to the naive granulosa cells.

The cholesterol side-chain cleavage cytochrome P450 (CYP11A; P450scc) gene is expressed in rat ovarian follicles in response to gonadotropins (FSH/LH) and cAMP. To identify functional regions within the rat P450scc promoter, 894 basepairs (bp) of 5'-flanking sequence and 5'-deletions (at -379, -101, -73, and -38 bp) were linked to the human GH reporter gene and transfected into cultured rat granulosa cells. cAMP inducibility of the rat promoter was localized to a region (between -73/-38 bp) that contains one of two AGGT/CC/TA motifs, designated SCC1 (-51/-43 bp) and SCC2 (-79/-71 bp), within the rat promoter. One of the nuclear proteins in granulosa cells that binds to SCC1 was identified as the orphan receptor, steroidogenic factor-1 (SF-1). In contrast, multiple protein-DNA complexes formed with SCC2, only one of which was clearly identified as SF-1. Nuclear extract binding was sequence specific; SCC1 bound SF-1 more strongly than did SCC2. Thus, the two AGGT/CC/TA motifs of the rat promoter appear to differ structurally and functionally. Furthermore, because the expression of SF-1 mRNA precedes hormonal/cAMP induction of P450scc mRNA and is not regulated in vitro by cAMP, the functional role of SF-1 in transcriptional regulation of the P450scc gene, including its induction by cAMP, is not entirely clear and is probably dependent on other factors and/or the modification (phosphorylation?) of SF-1.

BackgroundRelapse rates may be as high as 50% in people with major depressive disorder (MDD) previously treated to remission.AimsDuloxetine, an inhibitor of serotonin and noradrenaline reuptake that is licensed in Europe, the USA and elsewhere for the treatment of depressive episodes, was evaluated with regard to its efficacy, safety and tolerability in the prevention of relapse of MDD.MethodAdult out-patients with MDD received duloxetine (60 mg daily) for 12 weeks (n=533). Patients who responded to the drug were then randomised to duloxetine (60 mgdaily) (n=136) or placebo (n=142) for 26 weeks. The primary measure of efficacy was time to relapse.ResultsPatients who received duloxetine (60 mg daily) experienced significantly longer times to relapse of MDD, and better efficacy global well-being, and quality-of-life outcomes compared with patients who received placebo. It should be noted that adverse events which occur in discontinuation may mimic some signs of depressive relapse, and were not specifically elicited in this study.ConclusionsDuloxetine (60 mg daily) is effective in the prevention of relapse of MDD during continuation treatment.

Trophoblast giant cell differentiation is accompanied by transcriptional activation of the cytochrome P-450 side-chain cleavage (P450scc) gene. The Rcho-1 trophoblast cell line has the capacity to differentiate along the trophoblast giant cell lineage and has been used to study trophoblast-specific P450scc gene expression. In this report, P450scc gene promoter activities in trophoblast-specific P450scc gene expression. In this report, P450scc gene promoter activities in trophoblast cells have been mapped and the involvement of known modulators of steroid hydroxylase gene expression, the cyclic AMP/protein kinase A pathway and steroidogenic factor-1 (SF-1), evaluated. Comparisons were made with Y-1 adrenal and R2C Leydig cells. The cumulative results from transient and stable transfection experiments implicate the region between -428 and -511 bp of 5'-flanking DNA in the developmental activation of the P450scc promoter during trophoblast giant cell differentiation. Differences in basal activities of the P450scc promoter constructs were also observed in Y-1 adrenal and R2C Leydig cells; however, the magnitude of the differences was modest. Activators of the protein kinase A pathway stimulated P450scc promoter activity in Y-1 cells, whereas similar treatment of Rcho-1 trophoblast cells did not stimulate but actually inhibited P450scc promoter activity. The inhibitory activity was localized between -639 and -894 bp of the P450scc promoter. SF-1 mRNA and protein were detected in adrenal and gonadal cells but not in rat placenta or Rcho-1 trophoblast cells by Northern and Western blotting, respectively. Thus, P450scc gene activation during trophoblast cell differentiation involves an 83-bp region of its 5'-flanking DNA between -428 and -511 but does not appear to involve cyclic AMP-activated pathways or SF-1. In conclusion, the mechanism of P450scc gene activation during trophoblast cell differentiation appears different from the regulation of P450scc gene activation in other steroidogenic tissues.

Growth of ovarian follicles, ovulation, and luteinization involve sequential changes in the response of granulosa cells and theca cells to the gonadotropins, FSH and LH. Some of the genes that are differentially regulated by FSH/LH/cAMP as follicles develop are RIIβ, the regulatory subunit of A-kinase type II, aromatase cytochrome P450 (P450arom), cholesterol side-chain cleavage cytochrome P450 (P450scc), LH receptor (LH-R), and prostaglandin endoperoxide synthase (PGS). Whereas RIIβ(1), P450arom (2) and LH-R (3) are induced in growing follicles by low concentrations of hormone, P450scc is maximally induced by high concentrations of cAMP and under conditions in which the granulosa cells are stimulated to luteinize (4). In contrast, one isoform of PGS is rapidly but transiently induced by elevated gonadotropins/cAMP preceding ovulation (Fig. 7.1). To analyze the different molecular mechanisms by which FSH/LH/cAMP may be regulating some of these genes, we have isolated genomic 5’ flanking DNA of the RIIβ (5), P450scc (4), and P450arom (6) genes, determined the nucleotide sequences and the transcriptional start sites of each putative promoter-enhancer region, and are analyzing the ability of these promoters to confer cAMP regulation to reporter genes in transfected granulosa cells (4–8).

Two principal physiological events occur within preovulatory follicles as a consequence of the LH/FSH surge: ovulation and luteinization. The ovulation process culminates in the extrusion of the oocyte-cumulus complex and is dependent on a specific sequence of biochemical events. These biochemical changes include the rapid but transient increase in a novel, distinct isoform of prostaglandin endoperoxide synthase (PGS-2) (1–8), tissue plasminogen activator (tPA) (9, 10), and progesterone receptor (PR) (11–13). In contrast, luteinization is a process by which follicular granulosa and theca cells become nonmitotic and establish a specific, stable luteal cell phenotype. The biochemical changes that occur in response to the LH surge and are associated with luteinization include the marked and sustained induction of cholesterol side-chain cleavage cytochrome P450 (P450scc), as well as the dramatic and rapid decreases in mRNA for the LH receptor (LH-R), the regulatory subunit β of type II cyclic AMP-dependent protein kinase (RIIβ), aromatase, and 17α-hydroxylase (P45017α) (Fig. 10.1) (14). Once luteinization has occurred, genes regulated by FSH/LH/cAMP in the follicle are no longer regulated by these agonists in the corpus luteum. This apparent transition from cAMP-dependent regulation to cAMP-independent regulation of these genes has prompted us to ask why surge (as opposed to basal) concentrations of LH are required to initiate these two diverse processes, what might be unique about the genes induced by the LH surge, and what intracellular signaling pathways might be involved.