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M H Melner

Publications and source records attributed to M H Melner.

At least 37 records · Page 2Linked to original sources
PubMed1994

Progesterone receptor, but not estradiol receptor, messenger ribonucleic acid is expressed in luteinizing granulosa cells and the corpus luteum in rhesus monkeys.

Estrogens (i.e. estradiol) and progestins (i.e. progesterone) may act as local regulators of ovarian function in various species. This study tested the hypothesis that if progesterone and estradiol act via receptor-mediated pathways in the primate ovary, then receptor messenger RNAs (mRNAs) should be detectable in ovarian cells. The reverse transcription-polymerase chain reaction (RT-PCR) was employed to detect progesterone and estradiol receptor (PR and ER, respectively) mRNAs in the rhesus monkey ovary. Total RNA was isolated from macaque uterine myometrium (positive control), spleen (negative control), whole ovary, germinal (surface) epithelium-enriched cortical and medullary compartments of the ovary, granulosa cells in preovulatory follicles before and after an ovulatory stimulus, and corpora lutea from early (days 3-5), mid (days 7 and 8)-, and late (days 14 and 15) luteal phase of the menstrual cycle. Using primers to the hormone-binding region encoded by the receptor mRNAs, RT-PCR products of the expected sizes were detected for PR and ER from 1 microgram myometrial RNA, whereas products were not obtained from spleen. PR mRNA product was detected in all ovaries, germinal epithelium-enriched cortical and medullary compartments, and corpora lutea from all three stages of the luteal phase (n = 3/stage). PR mRNA product was detected as a strong band in one of three preparations obtained from granulosa cells before an ovulatory stimulus. In contrast, PR mRNA was detected in granulosa cells from all animals after an ovulatory dose of hCG. ER mRNA was detected in whole ovary and in germinal epithelium-enriched cortical compartments, with a barely visible product occasionally observed in medullary compartments of the ovary. In contrast to PR mRNA, ER mRNA was not detected in any corpora lutea throughout the luteal phase or in granulosa cells obtained before or after an ovulatory stimulus. To confirm the specificity of the RT-PCR products, restriction enzymes cleaved the PR product from myometrium, germinal epithelium-enriched cortical compartment, and corpus luteum into the predicted size fragments. Similarly, the ER product from the myometrium and the germinal epithelium-enriched compartment was cleaved into the expected size fragments. Sequence analysis of the PR and ER RT-PCR products revealed 99% homology to the complementary DNA for the hormone-binding region of human PR and ER, respectively. Thus, PR mRNA detection supports the hypothesis of progesterone action via classical receptor-mediated pathways in the luteinizing follicle and corpus luteum of the primate ovary.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1992

Differential regulation of anterior pituitary prodynorphin and gonadotropin-subunit gene expression by steroid hormones.

Prodynorphin is expressed by neurons of the hypothalamus and gonadotrophs of the anterior pituitary gland (AP) and plays a role in the negative feedback regulation of the reproductive neuroendocrine axis. The present study examined whether gonadal steroid hormones are capable of modulating pituitary prodynorphin expression in immature, female rats. Steroids were administered via subcutaneous Silastic implants and rats were killed at 29 days of age. Northern blot analysis was used to measure AP prodynorphin, luteinizing hormone-beta (LH beta), follicle-stimulating hormone-beta (FSH beta), and common alpha-subunit mRNA levels (normalized to 18S ribosomal RNA). Treatment groups (n = 5-6) consisted of control (CNT; empty implants), estradiol (E2; 4 days), E2 + progesterone (E2 + P4; 8 days and 4 days, respectively), and dihydrotestosterone (DHT; 4 days). Pituitary prodynorphin mRNA was significantly suppressed in only the DHT-treated animals (26 +/- 10% of CNT, p < 0.01). LH beta mRNA was suppressed by all steroid treatments (p < 0.01), FSH beta was lower in only the E2 group, and alpha-subunit was reduced in both the E2 + P4 and DHT groups (p < 0.01). Serum LH was suppressed by all steroid treatments but FSH was reduced in only the E2 and E2 + P4 groups (p < 0.01). Treatment of prepubescent rats with continuous high levels of gonadal steroids is known to severely reduce endogenous hypothalamic gonadotropin releasing hormone (GnRH) release and this is supported by our observation of reduced gonadotropin-subunit gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

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.

3-Hydroxysteroid Dehydrogenases↗
PubMed1992

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.

Animals↗
PubMed1992

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.

Animals↗
PubMed1991

A nonradioactive assay for transfected chloramphenicol acetyltransferase activity using fluorescent substrates.

Studies of the transcriptional activity of gene promoters have been greatly assisted by the widespread use of the chloramphenicol acetyltransferase (CAT) gene as a reporter gene. Previous techniques for assaying CAT enzymatic activity have utilized radioactive substrates or cofactors with the resulting complications of handling radioactive materials. We report here the development of fluorescent substrates for the CAT enzyme which form the basis of a CAT enzyme assay of enzyme kinetic parameters (Km and Vmax) and sensitivity similar to those based on radioactive substrates. Fluorescent substrates were designed as analogs of chloramphenicol and were based on the structure-function requirements of the enzyme. Several fluorophores were used to derivatize chloramphenicol base; one of the most effective was the borondipyrromethene difluoride (BODIPY) fluorophore. One BODIPY-chloramphenicol analog was found to have a Km for the purified CAT enzyme of 2 microM (compared to 12 microM for 14C-labeled chloramphenicol) and a Vmax of 120 pmole/min (compared to 180 pmol/min for the radioactive substrate). To verify its usefulness, a BODIPY--chloramphenicol-based CAT assay was used to measure transient transfection of primary cultures of ovarian granulosa cells in serum-free medium. This experimental system requires a highly sensitive assay for detecting transfected CAT gene activity. Robust expression of CAT activity was easily detected in crude cellular extracts using FluoReporter FAST CAT, a kit containing the BODIPY-chloramphenicol analog. The expression was precisely quantified by methanol extraction of the substrate and products from TLC plates and subsequent measurement of fluorescence using excitation-emission spectroscopy.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Specific inhibition of protein kinase A in granulosa cells abolishes gonadotropin regulation of the proopiomelanocortin promoter.

Gonadotropins (follicle-stimulating hormone (FSH), luteinizing hormone, and human chorionic gonadotropin) and beta-adrenergic agonists have been shown to stimulate expression of the proopiomelanocortin (POMC) gene in ovarian granulosa cells. The current studies investigate the intracellular mechanisms by which gonadotropins regulate gene expression. Primary cultures of rat granulosa cells were transfected with the plasmid POMC-CAT-150, which expresses the chloramphenicol acetyltransferase (CAT) reporter gene under the regulation of the rat POMC 5'-flanking region. CAT activity was stimulated by treatment of the cells with either 20 ng/ml FSH or 1 microM isoproterenol. To assess the role of protein kinase A (ATP:protein phosphotransferase; EC 2.7.1.37) in the gonadotropin and adrenergic response, an expression vector, MtR-AB, encoding a mutant RI regulatory subunit was cotransfected with POMC-CAT-150. The mutant protein kinase A regulatory subunit encoded by MtR-AB lacks functional cAMP-binding sites but effectively binds and specifically inhibits the catalytic activity of protein kinase A. The results of this analysis demonstrated that gonadotropin and adrenergic agonist stimulation of the POMC-CAT reporter construct in primary cultures of rat granulosa cells were abolished by cotransfection with MtR-AB; whereas a control SV40-promoter construct was unaffected by either gonadotropin treatment or cotransfection with MtR-AB. Basal expression directed by the POMC promoter was also decreased by cotransfection with the MtR-AB, implying that basal expression from the POMC promoter may also depend on protein kinase A. Deletion analysis of the POMC sequence indicated regions (-40 to -33 and +4 to +63) important for basal and FSH-stimulated expression. These studies suggest that both gonadotropin and adrenergic stimulation of the POMC promoter are mediated by protein kinase A and that regions proximal to the promoter are essential for gonadotropin-regulated expression from the promoter.

Animals↗
PubMed1991

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.

Androstenedione↗
PubMed1991

Intracellular mechanisms of gonadotropin-stimulated gene expression in granulosa cells.

Previous studies have shown that the gonadotropins follicle-stimulating hormone and luteinizing hormone stimulate proopiomelanocortin (POMC) promoter activity and mRNA levels in ovarian granulosa cells. The objective of these studies was to determine the role of cAMP-dependent protein kinases (pKA) in gonadotropin-stimulated gene expression. Primary cultures of rat granulosa cells were transfected with a gene construct consisting of the POMC promoter (-150 to +63; designated pOMC-CAT) fused to the chloramphenicol acetyltransferase (CAT) reporter gene either alone or cotransfected with an expression plasmid (designated mutant RI), which overexpresses a mutant form of the murine RI subunit incapable of binding cAMP and serving as an irreversible inhibitor of the catalytic subunit of pKA. Follicle-stimulating hormone or isoproterenol caused a significant stimulation of pOMC-CAT activity in transfected cells. Cotransfection of pOMC-CAT with mutant RI caused a significant inhibition of basal pOMC-CAT activity and abolished the gonadotropin stimulation. As a control, transfection of the SV-40 viral enhancer-promoter fused to CAT (pSV2-CAT) was unresponsive to follicle-stimulating hormone stimulation and cotransfection with mutant RI had no significant effect on pSV2-CAT activity. These studies suggest that gonadotropin regulation of the POMC promoter is mediated by pKA and that promoter activity is stringently controlled by pKA.

Animals↗
PubMed1991

Characterization of macaque 3 beta-hydroxy-5-ene steroid dehydrogenase/delta 5-delta 4 isomerase: structure and expression in steroidogenic and peripheral tissues in primate.

The conversion of 3 beta-hydroxy-5-ene steroids by the enzyme complex 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) is an obligatory step in the biosynthesis of all classes of hormonal steroids in classical steroidogenic as well as in peripheral tissues. To develop a model more closely related to the human, we have isolated and characterized cDNA clones encoding macaque 3 beta-HSD by screening a rhesus monkey ovary lambda gt11 cDNA library using a human 3 beta-HSD cDNA probe. Nucleotide sequence of 1629 bp from overlapping cDNA clones predicts a protein of 372 amino acids with a calculated molecular mass of 41,874 (excluding the first Met). The deduced amino acid sequence of macaque 3 beta-HSD displays 79.4% and 93.9% similarity with that of bovine and human 3 beta-HSD, respectively. RNA blot analysis performed under high stringency conditions of macaque poly(A)+ RNA samples using full-length 32P-labeled macaque 3 beta-HSD cDNA revealed the presence of an approximately 1.7 kb mRNA species in classical steroidogenic tissues, namely the ovary, testis and adrenal glands as well as in several peripheral tissues including the liver, kidney and epididymis. Computer analysis of the deduced macaque 3 beta-HSD protein sequence predicts the presence of an NH2-terminal membrane-associated segment as well as four additional membrane-spanning segments, thus suggesting that 3 beta-HSD is an integral protein. The availability of macaque cDNA should permit detailed studies concerning the tissue-specific expression as well as the hormonal regulation of 3 beta-HSD mRNA in classical steroidogenic glands as well as in peripheral tissues which are an important site of steroidogenesis in primates.

Adrenal Glands↗
PubMed1990

Cellular localization of proenkephalin mRNA and enkephalin peptide products in cultured astrocytes.

To identify the possible cellular sites of opioid gene expression during ontogeny, proenkephalin mRNA and enkephalin peptide expression were examined, respectively, by in situ hybridization and immunocytochemistry in organotypic explants of rat cerebellum and in astrocyte-enriched cultures of murine cerebral hemispheres. High levels of proenkephalin mRNA and enkephalin immunoreactivity were detected in immature cells identified as astrocytes. Double-labeling studies combining in situ hybridization and immunocytochemical localization of the astrocytic marker, glial fibrillary acidic protein, provided direct evidence that proenkephalin mRNA is expressed by astrocytes in culture. Based on previous studies that Met-enkephalin can inhibit astrocyte growth in vitro, the present results suggest that proenkephalin gene expression by astrocytes is important during central nervous system maturation.

Animals↗
PubMed1990

The regulation of proenkephalin expression in a distinct population of glial cells.

The expression of opioid genes was examined in isolated populations of glial cells in primary culture. Northern blot analysis of purified type I astrocytes, oligodendrocytes and mixed oligodendrocyte-type-2-astrocyte lineage cells derived from cerebral cortex demonstrated robust expression of proenkephalin mRNA exclusively in type I astrocytes. The expression of proenkephalin mRNA was stimulated by the beta-adrenergic agonist isoproterenol, and 8-(4-chlorophenyl thio)adenosine 3'-5'-cyclic monophosphate (cpt-cAMP). Both of these compounds regulated a proenkephalin-chloramphenicol acetyltransferase fusion gene transiently transfected into type I astrocytes. HPLC and immunoassay of the cell culture media revealed significant levels of unprocessed proenkephalin secreted by the cell and this secretion was stimulated by isoproterenol and cpt-cAMP. The relatively high levels of proenkephalin expressed suggest that enhanced expression in astrocytes may be important during neural development, in trauma-induced gliosis and in neuroimmune interactions.

Animals↗
PubMed1990

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)

Animals↗
PubMed1990

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.

Animals↗
PubMed1989

Androgens amplify beta-adrenergic and FSH stimulation of granulosa cells.

The effects of androgens on granulosa cell stimulation by isoproterenol and follicle-stimulating hormone (FSH) were determined. Two functional parameters of granulosa cell stimulation were monitored: (a) activity of a transfected proopiomelanocortin (POMC) promoter and (b) production of progesterone. Treatment with the beta-adrenergic agonist, isoproterenol, stimulated steroidogenesis, and both isoproterenol and FSH appeared to enhance POMC promoter activity. The non-aromatizable androgen, 5 alpha-dihydrotestosterone (DHT), produced no effect on either parameter, but it potentiated the steroidogenic response to isoproterenol. Preliminary data also indicated a potentiation by DHT of the FSH-mediated increase in POMC promoter activity; results with a combination of DHT and isoproterenol were suggestive of potentiation. A possible role for androgen amplification of adrenergic stimulation in polycystic ovarian syndrome is discussed.

Androgens↗
PubMed1989

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.

Animals↗
PubMed1989

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)

1-Methyl-3-isobutylxanthine↗