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Biomedical subjects

M H Melner

Publications and source records attributed to M H Melner.

At least 55 records · Page 3Linked to original sources
PubMed1989

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.

Animals↗
PubMed1986

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.

Amino Acids↗
PubMed1986

The regulation of granulosa cell proopiomelanocortin messenger ribonucleic acid by androgens and gonadotropins.

We have previously demonstrated the presence of proopiomelanocortin (POMC) messenger RNA (mRNA) in rat granulosa cells. This study examines the unique, tissue-specific regulation of granulosa cell POMC mRNA levels by hormones with established regulatory effects on these cells. Northern blot analysis of immature rat ovarian RNA revealed the presence of a single mRNA of approximately 900 base pairs in length. The levels of ovarian POMC mRNA increased approximately 6-fold 48 h after priming with PMSG when expressed per microgram of total RNA. Granulosa cells were isolated from immature PMSG-primed rats and cultured under serum-free conditions in the presence and absence of various hormones to determine their effect on POMC mRNA levels. Treatment of the cells for 48 h with LH (100 ng/ml), androstenedione (10(-7) M), or LH plus androstenedione elevated POMC mRNA levels. LH alone elicited a 5-fold increase in POMC mRNA, and androstenedione elicited a 10-fold increase; the combination treatment led to a 47-fold increase. The effects of the nonaromatizable androgen dihydrotestosterone (DHT) were also evaluated. Treatment for 48 h with DHT (10(-7) M) elicited a 40-fold increase in POMC mRNA levels. In vivo experiments measuring ovarian POMC mRNA from immature female rats corroborated the in vitro results. Animals injected sc with DHT (500 micrograms) demonstrated 5-fold increases in ovarian POMC mRNA when expressed per microgram of total RNA. These studies provide both in vitro and in vivo evidence that granulosa cell POMC mRNA is under unique hormonal regulation by both androgens and gonadotropins.

Androgens↗
PubMed1986

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.

Animals↗
PubMed1985

Detection of chloramphenicol acetyl transferase activity in transfected cells: a rapid and sensitive HPLC-based method.

An assay based on high-performance liquid chromatography (HPLC) has been developed for determining the expression of the acetyl coenzyme A: chloramphenicol 3-O-acetyltransferase gene in transfected cells. The assay is based on the separation of extracted, nonradioactive chloramphenicol and its acetylated derivatives by reverse-phase HPLC, using UV absorbance for detection. In addition to eliminating the need for costly [14C]chloramphenicol, this assay is as sensitive and much faster than the commonly used method involving thin-layer chromatography.

Acetyltransferases↗
PubMed1985

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.

Animals↗
PubMed1985

Hormonal induction of specific protein synthesis in murine Leydig tumor cells.

The M5480A murine Leydig cell tumor was used to investigate the effects of three hormones, which produce distinct biochemical actions, on cytoplasmic protein synthesis. Human choriogonadotropin treatment of tumor-bearing mice induced the synthesis of six proteins with relative molecular weights (Mr) of 135K, 82K, 60K, 19K, 18.2K and 17.3K (K = kilodaltons). Diethylstilbestrol induced one protein peak in common with the gonadotropin Mr = 135K) and three additional proteins with Mr's of 120K, 50K and 36K. Epidermal growth factor induced one major protein with Mr = 33K, which is similar to that of a protein induced in murine epidermal cells by tumor promoters. These studies demonstrate the induction of specific gene products in a hormone-responsive tumor.

Animals↗
PubMed1985

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.

Androstenedione↗
PubMed1984

Evidence for the synthesis of multiple pro-opiomelanocortin-like precursors in murine Leydig tumor cells.

The opiate peptide beta-endorphin has recently been localized in extrapituitary tissues and cells, including the Leydig cells of the testis, by immunohistochemical techniques. An intriguing question is whether this localization reflects an accumulation of the peptide through a specific uptake mechanism or is the result of synthesis within the cell in a large precursor form similar to pro-opiomelanocortin synthesized in the pituitary. Evidence is presented herein that specific antibodies against beta-endorphin and adrenocorticotrophic hormone precipitate identical precursor molecules from total cellular mRNA translation products of M5480A Leydig tumor cells. In addition, mRNA from these cells cross-hybridizes under stringent conditions with a cDNA coding for the rat pituitary pro-opiomelanocortin sequence. These data demonstrate the synthesis of a pro-opiomelanocortin-like mRNA in this Leydig cell tumor and strongly implicate biosynthesis within these cells.

Animals↗
PubMed1984

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.

Animals↗
PubMed1984

Effects of the antiestrogen CI-628 on hCG-induced desensitization of testosterone in purified Leydig cells.

The effect of CI-628 on human chorionic gonadotropin-induced desensitization of testosterone production was investigated. Purified Leydig cells were isolated from mature male rats 24 hr postinjection with (1) 30 of 300 IU hCG s.c., (2) 1 mg CI-628 i.p., or (3) CI-628 pretreatment plus hCG. Steroidogenic desensitization to Bt2 cAMP and hCG stimulation in vitro was observed with both doses of hCG administered in vivo. CI-628 treatment alone led to an increased basal production but an unaltered maximum production of testosterone obtained in the presence of hCG or Bt2 cAMP in vitro. CI-628 in combination with hCG treatment in vivo led to testosterone production in vitro that was similar to that observed with hCG treatment alone. CI-628 does not inhibit the hCG-induced desensitization of testosterone production, and the results do not support a role of estrogen receptors in this process.

Animals↗
PubMed1983

Tyrosine protein kinase activity in purified rat Leydig cells.

Tyrosine protein kinase activity has been estimated in purified testicular cells with the synthetic peptide substrate NH2-GLU-ASP-ALA-GLU-TYR-ALA-ALA-ARG-ARG-ARG-GLY-COOH. High levels of enzyme specific activity (56-165 pmol/mg/min) were found in the two populations of Leydig cells isolated by Metrizamide gradient centrifugation. Some activity was also detected in germinal cells, red cells and seminiferous tubules from testis but at levels 6-20 times lower than those found in the Leydig cell fractions. Higher levels of tyrosine protein kinase specific activity were found in population I than in population II Leydig cells.

Amino Acid Sequence↗
PubMed1983

Effects of human choriogonadotropin on mitochondrial and microsomal cytochrome P-450 levels in mouse testes.

The mitochondrial and microsomal cytochrome P-450 contents of C57B1/6 mouse testis have been measured using difference spectroscopy on stable enzyme preparations containing the ferrous-carbon monoxide complex. Results were obtained on control animals (52 +/- 3 days of age) and on animals injected subcutaneously with human choriogonadotropin (0.017 micrograms/g body weight 24 h prior to sacrifice). The high ratio of testicular mitochondrial cytochrome oxidase to P-450, which has previously precluded measurements of basal P-450 levels, was overcome by using N,N,N',N'-tetramethyl-p-phenylene diamine to bypass site II, in combination with antimycin A to prevent reverse electron flow. The basal levels of mitochondrial and microsomal P-450 in mouse testis were 37.9 +/- 3.5 and 28.9 +/- 1.6 pmol/mg protein, respectively. Following administration of a desensitizing dose of gonadotropin, the respective values were lowered to 19.9 +/- 1.4 and 19.6 +/- 2.1 pmol/mg protein in 24 h. This is the first report of a gonadotropin-mediated decrease in mitochondrial P-450 and thus demonstrates that desensitization leads to alterations in both microsomal and mitochondrial P-450 in mouse testis.

Animals↗
PubMed1983

Divergent effects of phenothiazines on Leydig tumor cell steroidogenesis and adenylate cyclase activity.

The dose and temporal (1-24 h) effects of two phenothiazines, chlorpromazine and trifluoperazine, on steroidogenesis and adenylate cyclase activity of gonadotropin-responsive Leydig tumor cells (M5480A) in primary culture were examined. At low doses (e.g. 0.1-1 microM) these antipsychotic drugs were slightly inhibitory (trifluoperazine) or without effect (chlorpromazine), while at 25 microM each drug was weakly stimulatory to basal testosterone production. Trifluoperazine was, in general, inhibitory to HCG-stimulated testosterone production, but chlorpromazine exhibited paradoxical effects. At 5 and 10 microM this neuroleptic agent increased HCG-stimulated steroidogenesis, while at 25 microM testosterone production was inhibited. In a particulate fraction prepared from the tumor the activity of adenylate cyclase was stimulated 3.4-fold in the presence of 10 microM 5'-guanylimidodiphosphate and 5-fold in the presence of HCG plus the non-hydrolyzable GTP analogue. Between doses of 1-100 microM neither drug altered the basal activity of adenylate cyclase. Trifluoperazine at doses of 1-100 microM inhibited 5'-guanylimidodiphosphate-stimulated adenylate cyclase activity both with and without added gonadotropin. At doses of 1-10 microM chlorpromazine had no effect on adenylate cyclase activity, but it stimulated activity in the dose range of 20-100 microM. Interestingly, in the presence of 5'-guanylimidodiphosphate this drug did not alter the stimulated enzymic activity achieved with a maximal dose of HCG. Therefore, these phenothiazines exhibit quite divergent dose-dependent effects and their actions must occur at multiple loci. Also, it seems unlikely that the effects of these agents on steroidogenesis and adenylate cyclase activity can be reconciled solely in terms of calmodulin-mediated processes.

Adenylyl Cyclases↗
PubMed1982

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.

Adenylyl Cyclases↗