Novel interactions between human T-cell leukemia virus type I Tax and activating transcription factor 3 at a cyclic AMP-responsive element.

Human proenkephalin gene transcription is transactivated by human T-cell leukemia virus type I (HTLV-I) Tax in human Jurkat T lymphocytes. This transactivation was further enhanced in Jurkat cells treated with concanavalin A, cyclic AMP, or 12-O-tetradecanoylphorbol-13-acetate. Deletion and cis-element transfer analyses of the human proenkephalin promoter identified a cyclic AMP-responsive AP-1 element (-92 to -86) as both necessary and sufficient to confer Tax-dependent transactivation. Different AP-1 or cyclic AMP-responsive element-binding protein (CREB)/activating transcription factor (ATF) proteins which bind this element were expressed in murine teratocarcinoma F9 cells to identify those capable of mediating Tax-dependent transactivation of human proenkephalin gene transcription. Although CREB, c-Fos, c-Jun, and JunD did not have significant effects, JunB inhibited the Tax-dependent transactivation. In contrast, ATF3 dramatically induced Tax-dependent transactivation, which was further enhanced by protein kinase A. Electrophoretic mobility shift assays with recombinant fusion proteins expressed and purified from bacteria indicate that the DNA-binding activity of ATF3 is also dramatically enhanced by Tax. Chimeric fusion proteins consisting of the DNA-binding domain of the yeast transcription factor Gal4 and the amino-terminal domain (residues 1 to 66) of ATF3 were able to mediate Tax-dependent transactivation of a Gal4-responsive promoter, which suggests a direct involvement of this region of ATF3. Recombinant fusion proteins of glutathione S-transferase with either the amino- or carboxy-terminal (residues 139 to 181) domain of ATF3 were able to specifically interact with Tax. Furthermore, specific antisera directed against Tax coimmunoprecipitated ATF3 only in the presence of Tax.

Cross-reactivity of a monoclonal antibody directed against an estrogen-induced protein in MCF-7 human breast cancer cells with murine Leydig cell tumor proteins.

An estrogen-responsive murine Leydig cell tumor (M5480A) was examined for the presence of cross-reactive proteins to a monoclonal antibody directed against a Mr 24,000 estrogen-regulated protein in human breast cancer cells. Human breast tumor biopsies were used as controls for the cytosol preparations, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blot conditions used in these experiments. The estrogen-regulated Mr 24,000 protein was detected in sodium dodecyl sulfate-polyacrylamide gels of cytosols from four human breast tumor biopsies examined. Larger amounts of the Mr 24,000 protein were present in the two estrogen-progesterone receptor-positive tumor biopsies in comparison to the two estrogen-progesterone receptor-negative samples. In addition, the two receptor-positive samples demonstrated an additional, less intense immunoreactive band at Mr 21,000. Under identical conditions, the same monoclonal antibody bound to two major protein bands from sodium dodecyl sulfate-polyacrylamide gels of Leydig cell tumor cytosols at Mr 56,000 and Mr 86,000. Antibodies prepared from BALB/c mouse ascites fluid of animals bearing the parent myeloma cell line (P3X63NS1) exhibited no immunoreactivity against the human breast or Leydig cell tumor proteins. In light of the high degree of specificity which this monoclonal antibody exhibits, our results suggest that similar antigenic determinants may exist in these proteins from two distinct tumors.

Effect of 4-hydroxyandrostenedione on murine Leydig tumor cell steroidogenesis.

The murine Leydig cell tumor (M5480A) possesses high levels of estrogen receptor and is known to produce estrogens. In these studies we examined the effects of the potent aromatase inhibitor 4-hydroxyandrostenedione (4-OHA) on Leydig tumor cell steroidogenesis both in vitro and in vivo. The addition of 4-OHA to Leydig tumor cells in primary culture resulted in a dose- and a time-dependent decrease in media progesterone levels. The observed decrease was most likely due to impaired synthesis of progesterone, inasmuch as no alteration in progesterone metabolism was seen when progesterone levels were diminishing. However, 4-OHA inhibited progesterone conversion to testosterone following 1 h of incubation, but this effect disappeared coincident with 4-OHA metabolism. Analysis of pregnenolone production revealed a biphasic dose-dependent effect of 4-OHA. At low doses (0.01-0.1 microM), 4-OHA was found to decrease pregnenolone concentrations, while at higher doses (1-10 microM) pregnenolone levels were elevated. Therefore, the actions of 4-OHA on Leydig cell steroidogenesis in vitro appear to be multifocal. Other experiments were performed to evaluate the effects of 4-OHA on tumor-bearing male mice in vivo. In these studies, the predominant effects of 4-OHA were to act as an aromatase inhibitor and to inhibit progesterone production. Thus, while 4-OHA is a potent aromatase inhibitor, we have found that this compound may alter steroidogenesis in Leydig tumor cells at several sites prior to aromatization.

Inhibition of Leydig tumor cell steroidogenesis by 10-propargylestr-4-ene-3,17-dione, an irreversible aromatase inhibitor.

The murine Leydig cell tumor (M5480A) was assayed for the presence of aromatase activity and for the effects of 10-propargylestr-4-ene-3,17-dione (PED), an aromatase inhibitor, on steroidogenesis. Microsomal preparations from these tumors contained low levels of aromatase activity which was PED sensitive. In addition, these Leydig tumor cells were maintained in primary culture and incubated under basal and gonadotropin-stimulated conditions with various doses of PED. Medium levels of progesterone, a major product of these cells, were found to decrease in a dose- and time-dependent manner upon addition of PED. To determine whether the observed effect was due to reduced synthesis or to increased metabolism of progesterone, tritiated progesterone was added to these cell cultures. Analysis of culture medium by high-performance liquid chromatography suggested that PED dramatically reduced the conversion of labeled progesterone to testosterone. Furthermore, examination of medium pregnenolone levels revealed diminished amounts of this steroid as well. Taken together, these results suggest that PED or its metabolites inhibit Leydig tumor cell steroidogenesis at several sites. Thus, in addition to its role as an aromatase inhibitor, this agent also has effects prior to pregnenolone production, as well as other effects in the pathway between progesterone and testosterone.

Characterization of protein tyrosine kinase activity in murine Leydig tumor cells.

The activity of protein tyrosine kinase (EC 2.7.1.37) was characterized from Leydig tumor cells (M5480A) using the synthetic peptide NH2-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Arg-Arg-Gly-COOH as a substrate. Relatively high tyrosine-specific protein kinase activity (about 135 pmol/mg protein per min) was detected in a particulate fraction (30 000 X g pellet) and was found to be linear as a function of time and protein concentration. The enzymic activity in the particulate fraction was stimulated 1.4-fold by 0.02% Nonidet P-40 as judged by 32PO4 incorporated into the peptide. Phosphorylation of endogenous proteins in M5480A particulate fractions with [gamma-32P]ATP resulted in several alkali-resistant radiolabeled bands in polyacrylamide gels in the presence of sodium dodecyl sulfate. Included in this group was a major radiolabeled doublet with an apparent molecular-weight in the range of 50 000-54 000. Phosphoamino acid analysis of hydrolysates of these eluted proteins indicated the presence of phosphotyrosine. Several alkali-resistant radio-labeled bands, including a major doublet with an apparent molecular-weight of 32 000, were also detected after culturing M5480A cells in the presence of 32PO4. These studies demonstrate the presence of high levels of protein tyrosine kinase activity in Leydig tumor cells and of endogenous protein substrates for this enzyme activity.

Demonstration of distinct forms of testicular adenylate cyclases associated with germinal and Leydig cell fractions.

Decapsulated testes from adult rats were digested with collagenase, and the fraction enriched in germinal and Leydig cells was applied to a 0-4% continuous metrizamide gradient and centrifuged. This leads to separation of a germinal cell fraction and two putative Leydig cell populations that bind human choriogonadotropin, but only one of which responds to the gonadotropin with marked increase in testosterone production. Adenylate cyclase activity was present in these three fractions, and Mn2+ was more effective than Mg2+ as a divalent cation. The adenylate cyclase activity associated with the germinal cell fraction was just marginally stimulated by fluoride and by the non-hydrolyzable GTP analog 5'-guanylimidodiphosphate, while that associated with the Leydig cell populations was stimulated to a greater degree depending upon the type of divalent cation. Only the Leydig cell populations exhibited marked human choriogonadotropin-sensitive stimulation of adenylate cyclase activity in the presence of 5'-guanylimidodiphosphate above that observed with the GTP analog alone. These results suggest the presence of distinct adenylate cyclases in adult rat testis and indicate that both populations of Leydig cells are capable of producing cyclic AMP in response to gonadotropins such as human choriogonadotropin.

Transiently elevated levels of c-fos and c-myc oncogene messenger ribonucleic acids in cultured murine Leydig tumor cells after addition of human chorionic gonadotropin.

The gonadotropic hormones LH and human CG (hCG) normally function to stimulate steroidogenesis in testicular and ovarian cells through receptor-mediated activation of adenylate cyclase. These hormones are also important in regulating the development and growth of responsive cells. Such regulation requires tightly controlled gene expression. Herein we demonstrate that hCG induces increases in mRNAs encoding the competence oncogenes c-fos and c-myc in a murine Leydig cell tumor line (MA-10). When stimulated by hCG (40 ng/ml), the mRNA levels of both genes increase rapidly, peaking at 30 min for c-fos and 1 h for c-myc. Both mRNAs fall to near control levels by 3-6 h. This response to hCG is dose-dependent with half-maximal stimulation of these genes occurring at a concentration of 3 ng/ml, approximating the level required for 50% occupancy of the LH/hCG receptors and the ED50 for steroidogenesis. (Bu)2 cAMP (2 mM) elicits responses similar to those produced by hCG. The observation of oncogene control by the gonadotropin hCG provides further insight regarding the pathways by which such hormones may regulate steroidogenesis, growth, and differentiation of endocrine and neoplastic cells.

Stat5-mediated regulation of the human type II 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase gene: activation by prolactin.

Altered PRL levels are associated with infertility in women. Molecular targets at which PRL elicits these effects have yet to be determined. These studies demonstrate transcriptional regulation by PRL of the gene encoding the final enzymatic step in progesterone biosynthesis: 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase (3beta-HSD). A 9/9 match with the consensus Stat5 response element was identified at -110 to -118 in the human Type II 3beta-HSD promoter. 3beta-HSD chloramphenicol acetyltransferase (CAT) reporter constructs containing either an intact or mutated Stat5 element were tested for PRL activation. Expression vectors for Stat5 and the PRL receptor were cotransfected with a -300 --> +45 3beta-HSD CAT reporter construct into HeLa cells, which resulted in a 21-fold increase in reporter activity in the presence of PRL. Promoter activity showed an increased response with a stepwise elevation of transfected Stat5 expression or by treatment with increasing concentrations of PRL (max, 250 ng/ml). This effect was dramatically reduced when the putative Stat5 response element was removed by 5'-deletion of the promoter or by the introduction of a 3-bp mutation into critical nucleotides in the element. Furthermore, 32P-labeled promoter fragments containing the Stat5 element were shifted in electrophoretic mobility shift assay experiments using nuclear extracts from cells treated with PRL, and this complex was supershifted with antibodies to Stat5. These results demonstrate that PRL has the ability to regulate expression of a key human enzyme gene (type II 3beta-HSD) in the progesterone biosynthetic pathway, which is essential for maintaining pregnancy.

Cellular localization of ovarian proopiomelanocortin messenger RNA during follicular and luteal development in the rat.

Opioid peptides are expressed in the reproductive system and have been reported to regulate reproductive function. The present study used in situ hybridization to selectively localize ovarian cells containing high levels of proopiomelanocortin (POMC) mRNA, an opioid precursor, during different stages of ovarian development. Prepubertal rats were primed with PMSG to stimulate follicular development, followed by hCG to induce ovulation. Treatment groups consisted of control (no treatment), PMSG (2 days post-PMSG), 1 day corpus luteum (CL; 1 day post-hCG), and 8 day CL (8 days post-hCG). POMC mRNA-containing cells were present in antral follicles, CL, and the interstitial compartment. With gonadotropin treatment, the percentage of follicles containing heavily labeled cells increased in the PMSG and 1 day CL groups. The number of POMC mRNA-containing cells per follicle also increased in the 1 day CL group. In the CL, no difference was observed in the percentage of CL exhibiting labeled cells between the 1 day CL and 8 day CL groups; however, more labeled luteal cells per CL were present in the 1 day CL group. A marked increase in POMC mRNA-containing cells was observed in the interstitial compartment of the 1 day CL group. These results indicate that the number of POMC mRNA-containing cells increases with follicular development and CL formation; however, the ovarian distribution suggests that the labeled cells could be nonendocrine cells, possibly white blood cells. The in situ hybridization findings are indicative of low total concentrations of ovarian POMC mRNA, suggesting mainly an autocrine or paracrine role for POMC or POMC-derived peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Association of proenkephalin transcripts with polyribosomes in the heart.

Previous results have shown that the relative abundance of proenkephalin mRNA in the rat heart is comparable to the levels found in the brain; however, the extractable enkephalin-containing peptide levels are much lower in the heart. This lack of correspondence between the levels of transcript and peptide could arise from either the inefficient translation of proenkephalin transcripts or the translation of proenkephalin transcripts into peptides that are rapidly secreted or degraded. To distinguish between these possibilities, the translational status of proenkephalin mRNA in the rat heart was established by Northern blot analysis of sucrose density gradient-sedimented polysomal fractions and compared to the striatum, which is known to efficiently translate proenkephalin transcripts. In both tissues, we detected 1.5-kilobase transcripts, but an additional larger transcript of approximately 3.6 kilobases was detected in the heart. Both transcripts were associated primarily with polyribosomes, suggesting active translation of proenkephalin mRNA in the rat heart. RIA of the culture media and extracts from primary cultures of neonatal rat cardiomyocytes indicated the presence of immunoreactive Met-enkephalin-Arg6-Phe7, which was stimulated by 8-(4-chlorophenylthio)cAMP. These results suggest that proenkephalin transcripts are translated in the heart and that detectable levels of immunoreactive Met-enkephalin-Arg6-Phe7 are present in the media and cell extracts of primary cultures of neonatal rat cardiomyocytes.

Gonadotropin regulation of the rat proopiomelanocortin promoter: characterization by transfection of primary ovarian granulosa cells.

To characterize the transcriptional effects of human (h)FSH and hCG on the POMC gene, primary rat granulosa cells were transiently transfected with a chloramphenicol acetyltransferase (CAT) reporter plasmid under the control of the POMC promoter and 5' region. POMC-CAT contains a fragment of the rat POMC gene, extending from nucleotide -704 to nucleotide +63, fused to the CAT gene. Treatment of POMC-CAT-transfected cells with either hFSH (20 ng/ml) or hCG (10 ng/ml) significantly increased CAT enzyme activity; however, neither hCG nor hFSH increased CAT enzyme activity in cells transfected with pSV2-CAT, a reporter plasmid under the control of the SV40 virus promoter and 5' region. The phosphodiesterase inhibitor isobutylmethylxanthine or the nonhydrolyzable cAMP analog cAMP-chlorothiophenyl significantly increased CAT activity in POMC-CAT-transfected granulosa cells. Human FSH stimulated transcription 10, 20, and 40 h after treatment, but FSH stimulation at the two earlier time points was 2.5- to 5.5-fold greater than that at 40 h. Gonadotropin-stimulated steroidogenesis was equivalent in POMC-CAT-transfected granulosa cells, untransfected, and mock-transfected cells. This indicates that transfection left the physiological hormone response intact. These data demonstrate the following. 1) 767 basepairs of the rat POMC gene are enough to confer gonadotropin stimulation on the CAT marker gene in granulosa cells. 2) Although the POMC promotor lacks a well conserved cAMP response element, either of two different pharmacological manipulations of granulosa cells that raise intracellular cAMP can also stimulate POMC-driven CAT expression. 3) Transfected primary cultures of granulosa cells provide a nontransformed, physiologically relevant model with which to study hormone-regulated gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of prodynorphin gene expression in the ovary: distal DNA regulatory elements confer gonadotropin regulation of promoter activity.

Examination of the regulation of prodynorphin (pro-DYN) promoter activity is limited by the absence of a good cell model. The discovery of pro-DYN mRNA and derived peptides in the reproductive tract led us to examine the cellular localization and hormonal regulation of ovarian pro-DYN expression and to evaluate normal granulosa cells as a model for studying pro-DYN gene regulation. Ovarian pro-DYN mRNA levels were significantly elevated in PMSG-primed immature rats 12-24 h after receiving an ovulatory dose of hCG. Levels peaked 2 days after hCG, remained elevated throughout the ensuing pseudopregnancy, and rose again at the end of pseudopregnancy. In situ hybridization localized pro-DYN expression predominantly to granulosa and luteal cells. Transfection of primary cultures of granulosa cells revealed that the activity of the rat pro-DYN promoter [-1858 to 133 base pairs (bp)] was increased 18- to 19-fold by hCG and human FSH treatments and 7-fold by cAMP analog treatment. Deletion analysis identified a 358 bp fragment as the primary hormone-responsive sequence (-1858 to -1500 bp; containing three potential cAMP-responsive elements); its deletion resulted in severely reduced FSH responsiveness, and its ligation to hormone-unresponsive basal promoter sequences completely restored FSH responsiveness. This is an unusually distal position for cAMP-responsive elements compared to other cAMP-regulated genes. These data demonstrate specific expression of pro-DYN in granulosa and luteal cells, which is under sensitive gonadotropin regulation, and identify a distal hormone-responsive sequence within the promoter.

Proenkephalin gene expression in the primate uterus: regulation by estradiol in the endometrium.

Proenkephalin mRNA has previously been shown to be expressed in the rodent uterus with varying levels during the estrous cycle. To examine for the potential regulation of proenkephalin gene expression by steroid hormones in a primate displaying a menstrual cycle and to define the functional tissue within the uterus expressing this transcript, we have used Northern blot analysis of extracted RNA from isolated uterine tissue subtypes from normal adult rhesus macaques obtained during the menstrual cycle and from ovariectomized females under different physiological steroid hormone treatments. A strong band of proenkephalin mRNA of 1.3 kilobases was detected almost exclusively in the proliferative endometrium from monkeys in the follicular phase of the cycle. No proenkephalin mRNA was detected in secretory endometrium obtained from monkeys in the luteal phase. When ovariectomized macaques were implanted with silastic capsules of 17 beta-estradiol, proenkephalin mRNA was detected in the endometrium but not the myometrium of the estradiol-treated animals. No proenkephalin mRNA was detected in ovariectomized control animals. Under these conditions, we were unable to detect proenkephalin mRNA in ovariectomized macaques implanted with separate silastic capsules of 17 beta-estradiol and progesterone or in decidual tissue from early or late pregnancy. These results suggest that in the primate uterus 1) proenkephalin mRNA is expressed primarily in the endometrium of the uterus, 2) expression of the proenkephalin gene is regulated by 17 beta-estradiol in the endometrium, and 3) this effect of estradiol is antagonized by progesterone.

Depletion of the cytoplasmic estrogen receptor in gonadotropin-desensitized testes.

Recent studies have shown that hCG stimulates 17 beta-estradiol production in the testis, and a possible role for 17 beta-estradiol in hCG-induced desensitization of testosterone synthesis has been suggested. These studies were initiated to examine the testicular content of cytoplasmic estrogen receptor in relation to hCG-induced desensitization of testosterone production 24 h and 5 days after a sc injection of hCG. Twenty-four hours after the injection of 30, 300, and 3000 IU hCG, pronounced (82-88%) depletion of the cytoplasmic estrogen receptor was observed, while a 3-IU dose elicited a 41% depletion. A concomitant desensitization to hCG stimulation in vitro was observed after the injection of 30, 300, and 3000 IU hCG, as reflected by a lack of stimulation of testosterone production above that in matched controls. Testicular 17 beta-estradiol levels rose significantly after the injection of 300 or 3000 IU hCG. Five days after the injection of hCG, full replenishment of the cytoplasmic estrogen receptor had occurred in the 3- and 30-IU dose groups, while partial replenishment (22-56% depletion) had occurred with the 300- and 3000-IU dose groups. Only the 3000-IU dose group remained desensitized to hCG stimulation in vitro at this time point. Results indicated that occupancy by endogenous 17 beta-estradiol was not a factor, thus suggesting a true receptor depletion phenomenon. These results demonstrate that hCG-induced desensitization of testicular steroidogenesis is accompanied by depletion of the cytoplasmic estrogen receptor. Further, replenishment of the estrogen receptor at 5 days was accompanied by a return of the testicular response to hCG.

Induction of an estrogen-dependent early steroidogenic lesion in murine Leydig tumor cells.

The effects of low doses (37 pM to 3.7 nM) of 17 beta-estradiol on Leydig tumor cell steroidogenesis were studied in primary culture. This gonadotropin-responsive Leydig tumor line (M5480A) produces progesterone as the major steroid and lower levels of testosterone. It was found that these tumor cells possess a relatively high level of estradiol receptors, but only low levels of estradiol. We, therefore, maintained dispersed Leydig tumor cells in culture under basal or hCG-stimulated conditions for varying periods of time with or without graded doses of estradiol. The media from these cultures were analyzed for pregnenolone, progesterone, and testosterone by specific RIAs. Although testosterone levels were similar to control values, both pregnenolone and progesterone levels were significantly decreased by low doses of estradiol in a dose- and time-dependent manner. For example, basal progesterone levels were diminished 36% by 0.37 nM estradiol, and this effect could be reversed by the antiestrogen LY117018 [6-hydroxy-2-(p-hydroxyphenol)benzo-b-thien-3-yl-p-2-(1-pyrr olidinyl)- ethoxyphenyl ketone]. To evaluate whether the decreased medium progesterone level was due to increased metabolism, [3H] progesterone was added to estrogen-treated and control cells, and ether-extracted media were analyzed for steroid metabolites by HPLC. No significant difference in progesterone metabolism, including its conversion to testosterone, was detected between control and treated cells. Thus, the estradiol-mediated decrease in progesterone concentrations most likely reflects decreased synthesis rather than increased metabolism. These results provide the first indication of an estrogen-mediated effect at an early site in Leydig tumor cell steroidogenesis.

Demonstration of renin activity in purified rat Leydig cells: evidence for the existence of an endogenous inactive (latent) form of enzyme.

Previous histochemical studies demonstrated the specific localization of immunoreactive renin-like substance in Leydig cells of rat testes. The studies reported herein demonstrate a specific renin enzyme activity in the two purified populations of Leydig cells (I and II) of mature rat testes. Leydig cells of both populations also exhibited an inactive (latent) renin which was activated by the sulfhydryl reagents dithiothreitol, beta-mercaptoethanol, glutathione, and cysteine but not by limited proteolysis by trypsin, which is a characteristic activating agent for prorenin or inactive renin of the zymogen type. The activation of latent renin by dithiothreitol produced approximately 5-and 10-fold increases in renin activity in Leydig cell populations I and II, respectively. Active and latent renin showed strong affinity to an antirat renin immunoglobulin-Sepharose column, indicating a close immunological relationship of latent renin to active renin. Both active and latent renin from Leydig cell populations (I and II) exhibited the pH optimum of 6.0. The gel filtration of Leydig cell extracts characteristically revealed that the apparent mol wts of active and latent renin were 39,000 and 48,000, respectively. Both active and latent renin in Leydig cells remained almost at similar levels through four continuous subcultures. The activity of latent renin slightly increased during the four consecutive subcultures, while active renin levels remained almost constant.

Ovarian 3 beta-hydroxysteroid dehydrogenase and sulfated glycoprotein-2 gene expression are differentially regulated by the induction of ovulation, pseudopregnancy, and luteolysis in the immature rat.

The present studies were conducted to elucidate the effects of gonadotropin-induced alterations in ovarian status on expression of 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD), an enzyme which plays a crucial role in steroidogenesis, and sulfated glycoprotein-2 (SGP-2), a heterodimeric protein which is highly expressed by cells undergoing programmed death (i.e. apoptosis). Prepubescent female rats were used to reduce the influence of endogenous gonadotropins and to avoid the presence of preexisting, degenerating corpora lutea in the ovaries. 3 beta-HSD, cholesterol side-chain cleavage cytochrome P450, and SGP-2 messenger RNA (mRNA) levels were measured by Northern analysis of total ovarian RNA. Rats which received PMSG (20 IU) followed 48 h later by human CG (hCG) (10 IU) to induce ovulation and pseudopregnancy exhibited a significant increase in ovarian 3 beta-HSD mRNA 1 day later (164%, P less than 0.01 vs. saline control). The most dramatic change in 3 beta-HSD expression was the rise seen 2 days after hCG (262%, P less than 0.01), after which levels remain constantly elevated throughout pseudopregnancy. In contrast, ovarian cholesterol side-chain cleavage cytochrome P450 mRNA was greatly elevated (i.e. 15-fold) 48 h after PMSG treatment alone (P less than 0.01). Thus, gonadotropic stimulation which induces ovulation and luteogenesis is required to observe a potent stimulatory effect on ovarian 3 beta-HSD expression. The slow time course of induction is indicative of a differentiation-dependent expression. These observations are consistent with luteal cell expression of the 3 beta-HSD gene and suggest that this expression is correlated with the high progestin secretion and 3 beta-HSD activity characteristic of luteal cells. Interestingly, the pattern of regulation of ovarian SGP-2 expression was markedly different than that observed for 3 beta-HSD. PMSG treatment alone (48 h), and in combination with hCG, dramatically reduced SGP-2 mRNA to 12-27% of controls (P less than 0.01). SGP-2 levels were not elevated until 7 days after hCG; levels then remained constant through day 14 of pseudopregnancy. Since luteal progesterone secretion begins to diminish 5-7 days after hCG, the increased expression of SGP-2 on day 7 may be related to the initiation of the regression/degeneration of luteal cells which occurs during luteolysis. Thus, this study demonstrates that alterations in SGP-2 expression by the ovary may precede or occur simultaneously with cellular events initiating luteolysis and suggests a role for this glycoprotein as an early marker for luteolysis and implicates it in yet another instance of programmed cell death.

Differential regulation of proenkephalin expression in astrocytes by cytokines.

Astrocytes have previously been shown to respond to cytokines such as interleukin-1 beta, tumor necrosis factor-alpha, and gamma-interferon from multiple sources including microglia and astrocytes. Recently, astrocytes have also been shown to express the opioid precursor gene proenkephalin and proenkephalin-derived peptides. The objectives of the current study were to determine if immune cytokines regulate proenkephalin gene expression in primary cultures of neonatal rat cerebral astrocytes. Northern analysis of RNA from primary cultures of neonatal rat cerebral astrocytes indicated that proenkephalin transcript levels were decreased by approximately 50% with gamma-interferon treatment and increased approximately 100% by treatment with both tumor necrosis factor-alpha and interleukin-1 beta relative to untreated controls. Tumor necrosis factor-alpha treatment was unable to reverse the inhibitory effect of gamma-interferon pretreatment on proenkephalin messenger RNA levels in the astrocytes. In contrast, expression of the constitutively expressed glutamine synthetase gene was not altered by either tumor necrosis factor-alpha or gamma-interferon treatment. These cytokines also regulate the secretion of proenkephalin-derived peptides from astrocytes. The levels of immunoreactive Met-enkephalin-Arg6-Phe7 were increased by approximately 50% with tumor necrosis factor-alpha and decreased by approximately 40% with gamma-interferon relative to untreated controls. Tumor necrosis factor-alpha was again unable to reverse the inhibitory effect of gamma-interferon pretreatment on the secretion of proenkephalin-derived peptides. These results provide additional support for the hypothesis that rapidly proliferating astrocytes may serve an important and pivotal role in mediating the bi-directional neuroimmune interactions during central nervous system disease, infection, or trauma.

DAX-1 blocks steroid production at multiple levels.

DAX-1 is an unusual member of the nuclear hormone receptor superfamily whose expression is mainly, but not uniquely, restricted to steroidogenic tissues. We have recently shown that DAX-1 can block the first and rate-limiting step in steroid biosynthesis by repressing StAR (steroidogenic acute regulatory protein) expression. Here we show that DAX-1 blocks steroid production at multiple levels in the Y-1 mouse adrenocortical tumor cell line. Expression of DAX-1 in Y-1 cells significantly impairs both basal and cAMP-stimulated steroid production, without affecting the functionality of the cAMP-responsive PKA pathway. Experiments using an hydroxylated cholesterol derivative show that biochemical steps in steroidogenesis subsequent to cholesterol delivery to mitochondria are also impaired in Y-1 cells expressing DAX-1. This is explained by the repression of P450scc and 3beta-HSD expression, in addition to StAR. DAX-1 expression in Y-1 cells results in the inhibition of the activity of the StAR, P450scc and 3beta-HSD promoters. An inappropriate steroidogenic block in the male fetus might have an important role in the pathogenesis of sex reversal syndromes caused by a duplication of the genomic region of the X chromosome containing the DAX-1 gene.

Induction and activation of Stat 5 in the ovaries of pseudopregnant rats.

PRL acts in the ovary to promote the maintenance and function of the corpus luteum. However, the cellular signals that induce these responses have not been clearly defined. In the present report we demonstrate that Stat 5, previously identified as a transcription factor activated by PRL in the mammary gland, is also activated by PRL in the ovaries of pseudopregnant rats. Intraperitoneal injection of PRL into pseudopregnant rats results in the tyrosine phosphorylation and nuclear translocation of Stat 5. This activated Stat 5 possesses DNA-binding activity for a sequence containing the PRL-inducible element. In addition, we report that luteinization of the PMSG-primed ovary by the administration of hCG is accompanied by an induction of Stat 5 protein.