Biomedical subjects

E P Murono

Publications and source records attributed to E P Murono.

At least 19 recordsLinked to original sources

PubMed1994

A Sertoli cell-secreted paracrine factor(s) stimulates proliferation and inhibits steroidogenesis of rat Leydig cells.

Previous studies have shown that disruption or damage to the seminiferous tubules by radiation, antiandrogen, vitamin A deficiency or experimental cryptorchidism causes Leydig cell hypertrophy and hyperplasia, suggesting that Sertoli cells secrete a mitogenic factor(s) that stimulates Leydig cell proliferation. To study the possible paracrine regulation of Leydig cell proliferation by Sertoli cells, highly purified Leydig cells and Sertoli cells were co-cultured in a two-chambered co-culture system. Our results revealed that co-culture of immature rat Sertoli cells with Leydig cells stimulated Leydig cell DNA synthesis by 19-fold, increased cell number by about 3.9-fold and increased the labeling index from 0.5% to 15.8%. In addition to these changes, co-culture reduced Leydig cell testosterone formation and luteinizing hormone (LH) receptor levels, and dramatically altered the morphology of Leydig cells. The addition of concentrates from Sertoli cell conditioned medium (SCCM) mimicked these biological effects. The Leydig cell mitogenic activity in SCCM was trypsin sensitive and inactivated by boiling for 2 h, suggesting that it is a protein. However, it was resistant to acid and dithiothreitol. The molecular weight of this putative factor(s) is above 10 kDa. The responsiveness of Leydig cells to this mitogenic protein(s) decreased with age, whereas the secretion of this protein(s) by Sertoli cells in culture did not change with age. The addition of 10 ng/ml of follicle stimulating hormone (FSH) dramatically decreased the mitogenic activity in SCCM, indicating that the secretion of this mitogenic factor(s) is inhibited by FSH. This paracrine factor(s) may be as yet an unidentified testicular growth factor(s) because it differs in molecular weight, stability and other characteristics from all previously reported Sertoli cell-produced or expressed growth factors.

Animals↗

PubMed1994

Enhanced stimulation of 5 alpha-reductase activity in cultured Leydig cell precursors by human chorionic gonadotropin.

Previous studies have demonstrated that the increase in number of Leydig cells during prepubertal maturation results, in part, from the differentiation of mesenchymal precursors between the second and fourth week of postnatal life. After conversion to immature Leydig cells, they actively synthesize testosterone, but this androgen does not accumulate because high 5 alpha-reductase activity rapidly converts testosterone to 5 alpha-reduced metabolites. The present studies examined whether the conversion of precursor cells to immature Leydig cells in vitro by human chorionic gonadotropin (hCG), as characterized by progressive increases in testosterone formation and 5-ene-3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta-HSD) activity, is associated similarly with an enhanced stimulation of 5 alpha-reductase activity. We also evaluated whether this conversion occurs following blockade of dihydrotestosterone (DHT) formation by the inclusion of a 5 alpha-reductase inhibitor during the entire treatment period. Precursor cells were isolated from immature rats using a multi-step procedure normally used to isolate highly purified Leydig cells from adult or immature rats. These cells localize in a region of lower density on Percoll gradients than Leydig cells. Although the acute (3h) response to hCG with respect to testosterone formation, and basal 3 beta-HSD and 5 alpha-reductase activities on day 1 of culture were much higher in purified Leydig cells than precursor cells from immature rats, the response of each parameter to chronic (6-day) treatment with hCG was much greater in precursor cells. Furthermore, the conversion of precursor cells to immature Leydig cells occurred in the presence of a 5 alpha-reductase inhibitor during the entire treatment period, suggesting that this conversion occurs in the absence of DHT. These results demonstrate for the first time that in addition to increased testosterone biosynthesis and 3 beta-HSD activity, the conversion of precursor cells to immature Leydig cells, in vitro, in response to chronic hCG treatment, involves enhanced 5 alpha-reductase activity.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗

PubMed1993

Biphasic effect of basic fibroblast growth factor on 125I-human chorionic gonadotropin binding to cultured immature Leydig cells.

The present studies examined the effects of basic fibroblast growth factor (bFGF or FGF-2) on 125I-human chorionic gonadotropin (hCG) binding to cultured immature rat Leydig cells. We found that low concentrations of bFGF (0.1-1.0 ng/ml) inhibited 125I-hCG binding to cultured immature Leydig cells in a dose- and time-dependent manner; however, this inhibition was reversed partially at higher bFGF concentrations (10-200 ng/ml). The decline in 125I-hCG binding by bFGF was due to a reduction in the number of binding sites per cell and not to a change in receptor affinity for the ligand. The inclusion of 10 micrograms/ml heparin (a concentration that is reported to block bFGF binding to heparan sulfate proteoglycans) with increasing bFGF concentrations had no effect on the inhibition of 125I-hCG binding by low bFGF concentrations, but completely blocked the secondary increase in binding by higher bFGF concentrations. In addition, neither varying heparin concentrations (0.1-25 micrograms/ml) nor insulin or insulin-like growth factor-I had any effect on the inhibition of 125I-hCG binding by 1 ng/ml bFGF. These studies suggest that receptor-mediated actions of bFGF (inhibition of hCG binding by low bFGF concentrations) on cultured immature Leydig cells are unaffected by heparin; however, the secondary increase in 125I-hCG binding observed with higher bFGF concentrations (mediated by bFGF binding to heparan sulfate proteoglycans) is blocked by heparin.

Animals↗

PubMed1993

Basic fibroblast growth factor-induced increase in 125I-human chorionic gonadotropin binding to luteinizing hormone receptors in cultured immature Leydig cells is mediated by binding to heparan sulfate proteoglycans.

Previous studies have shown that basic fibroblast growth (bFGF) has a biphasic effect on 125I-hCG binding to LH receptors in cultured Leydig cells from immature rats. Low concentrations of bFGF (0.1-1.0 ng/ml) progressively decreased binding, while higher concentrations (10-100 ng/ml) progressively increased binding above nadir levels. In the present studies, treatment of cultured immature Leydig cells with heparinase I and/or heparinase III, which enzymatically remove heparan sulfate proteoglycans, had no effect on basal binding of 125I-hCG to LH receptors or the decrease in binding due to treatment with low bFGF concentrations; however, this treatment dramatically reduced the secondary increase in binding following the addition of higher bFGF concentrations. These results strongly support the idea that the secondary increase in 125I-hCG binding to LH receptors elicited by treatment with higher bFGF concentrations is mediated by bFGF binding to heparan sulfate proteoglycans associated with the plasma membrane and/or extracellular matrix.

Animals↗

PubMed1993

Evidence that both receptor- and heparan sulfate proteoglycan-bound basic fibroblast growth factor are internalized by cultured immature Leydig cells.

The present studies examined how 125I-labeled basic fibroblast growth factor (bFGF) bound to high affinity receptors and with lower affinity to heparan sulfate proteoglycans (HSPG) of cultured immature rat Leydig cells was processed. Following incubation for 2 h at 4 degrees C with 125I-bFGF, cells were washed to remove unbound radioactivity. Fresh medium was added, and cells were incubated at 4 degrees and/or 37 degrees C. At time zero and at specific intervals over the next 6 h, the incubation medium was saved and cells washed to quantitate 125I-bFGF released into the medium, associated with HSPG of the cell surface or extracellular matrix (radioactivity released by washing cells with 2 M NaCl, pH 7.4), associated with cell surface receptors (radioactivity released by washing cells with 2 M NaCl, pH 4.0) or internalized (radioactivity resistant to high salt and acid washes, and solubilized with 0.5 M NaOH). Radioactivity released into the initial medium and the pooled washes was further divided into a trichloroacetic acid (TCA)-precipitated form (radioactivity precipitated by 10% TCA) and a TCA-soluble form (radioactivity remaining in the TCA supernatant). 125I-bFGF associated with both HSPG and surface receptors declined progressively during the first 4 h of incubation before stabilizing when cells were transferred to 37 degrees C. These declines were associated with a corresponding increase in intracellular 125I-bFGF. These changes were blocked by maintaining cells at 4 degrees C. The majority of internalized 125I-bFGF appeared to originate from the HSPG-bound fraction as there was a greater decline in HSPG-associated radioactivity and most of the increase in internalized radioactivity could be blocked by the inclusion of 10 micrograms/ml heparin (which mainly blocks 125I-bFGF binding to HSPG but not to high affinity receptors) during the initial incubation with 125I-bFGF for 2 h at 4 degrees C. Furthermore, HSPG-mediated internalization appeared to have two components: the major fraction was blocked by the inclusion of 10 micrograms/ml heparin, while a heparin-resistant fraction, appeared to be closely linked both quantitatively and temporarily to receptor-mediated internalization. A minor fraction of internalized 125I-bFGF was metabolized in lysosomes, as the inclusion of 50 microM chloroquine during the 6 h incubation at 37 degrees C inhibited most of the increase in TCA-soluble radioactivity appearing in the incubation medium.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1993

Effects of acidic fibroblast growth factor on 5-ene-3 beta-hydroxysteroid dehydrogenase-isomerase and 5 alpha-reductase activities and [125I]human chorionic gonadotrophin binding in cultured immature Leydig cells.

The present studies examined the effects of acidic fibroblast growth factor (aFGF) on 5-ene-3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta-HSD) and 5 alpha-reductase activities and [125I]human chorionic gonadotrophin ([125I]hCG) binding in cultured immature rat Leydig cells. Increasing concentrations of aFGF (0.1-20 ng/ml) progressively decreased basal 3 beta-HSD activity from 0.474 +/- 0.0335 to 0.093 +/- 0.0004 nmol progesterone/30 min/10(5) cells. This inhibition by aFGF (10 ng/ml) was partially reversed by 1 micrograms/ml insulin or 100 ng/ml insulin-like growth factor-I. Increasing aFGF concentrations (0.1-10 ng/ml) also inhibited hCG-stimulated 5 alpha-reductase activity in a dose-dependent manner, but had only a modest effect on basal enzyme activity. Increasing aFGF (0.1-200 ng/ml) also progressively inhibited [125I]hCG binding in cultured immature Leydig cells. These studies demonstrate a similarity in the inhibitive effects of aFGF with bFGF effects on 3 beta-HSD and 5 alpha-reductase activities and [125I]hCG binding to LH receptors, although, generally, higher aFGF concentrations were required to elicit maximal inhibitive effects. However, a FGF differed from the actions of bFGF on 3 beta-HSD activity and LH receptor levels in that a secondary increase with higher growth factor concentrations was not observed.

3-Hydroxysteroid Dehydrogenases↗

PubMed1993

Evidence that biphasic effects of basic fibroblast growth factor on 5-ene-3 beta-hydroxysteroid dehydrogenase-isomerase activity in cultured immature Leydig cells are mediated by binding to heparan sulfate proteoglycans.

The present studies examined the effects of heparin, heparinase, insulin or insulin-like growth factor-I on basic fibroblast growth factor actions on 5-ene-3 beta-hydroxysteroid dehydrogenase-isomerase activity of cultured immature rat Leydig cells. Treatment with basic fibroblast growth factor alone (0.025-20 ng/ml) for 2 days had a biphasic effect on enzyme activity, with lower concentrations (0.025-1 ng/ml) progressively inhibiting activity to approximately 20% of control, while higher concentrations (2.5-20 ng/ml) partially reversed the inhibitive effects. The inclusion of 10 micrograms/ml heparin, a concentration reported to inhibit growth factor binding to heparan sulfate proteoglycans, blocked the increase in enzyme activity elicited by higher growth factor concentrations, but had no effect on the progressive decline in activity due to lower concentrations. Concomitant treatment with heparinase I and III, which specifically hydrolyze heparan sulfate proteoglycans, had a similar effect. In addition, both insulin and insulin-like growth factor-I partially reversed the inhibition of enzyme activity due to treatment with 1 ng/ml basic fibroblast growth factor. These studies suggest that some basic fibroblast growth factor actions on cultured immature Leydig cells are mediated by binding to heparan sulfate proteoglycans, and that both insulin and insulin-like growth factor-I can reverse the inhibitive effects on 5-ene-3 beta-hydroxysteroid dehydrogenase-isomerase activity.

3-Hydroxysteroid Dehydrogenases↗

PubMed1992

Evidence for basic fibroblast growth factor receptors in cultured immature Leydig cells.

Previous studies have shown that basic fibroblast growth factor (bFGF) can modulate basal and luteinizing hormone/human chorionic gonadotropin (LH/hCG)-stimulated Leydig cell functions. It has not been ascertained whether these actions are due to direct or indirect effects on Leydig cells. To resolve this question, a multi-step procedure was used to isolate highly-purified Leydig cells from immature rats. 125I-bFGF binding studies were performed on cultured cells. Scatchard analysis of the data indicated a single binding site with an apparent Kd of 82 pM and a binding capacity of approximately 2800 sites per cell. Both bFGF and acidic FGF similarly were effective in displacing 125I-bFGF, suggesting that the receptor binds both bFGF and aFGF. However, neither hCG, follicle-stimulating hormone (FSH), insulin, insulin-like growth factor-1 (IGF-1), prolactin, platelet-derived growth factor (PDGF) or epidermal growth factor (EGF) were effective competitors. When binding studies were conducted on cultured testicular interstitial cellular fractions that are normally discarded during Leydig cell purification, bFGF receptors were identified in these fractions. These results demonstrate that bFGF can have direct effects on Leydig cells through specific receptors; however, because other interstitial cell type(s) also have bFGF receptors, they stress the importance of using highly purified cells when evaluating bFGF actions on Leydig cells.

Animals↗

PubMed1991

Delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity in two distinct density Leydig cells from immature rats. Differences in responsiveness to human chorionic gonadotropin or 8-bromoadenosine 3',5'-monophosphate.

The present studies examined the responsiveness to human chorionic gonadotropin (hCG) or 8-bromoadenosine 3',5'-monophosphate (8-Br-cAMP) of delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity of cultured immature Band 2 (low density) or Band 3 (high density) Leydig cells isolated on Percoll gradients. Enzyme activity increased in relation to the dose of hCG or 8-Br-cAMP in both bands; however, activity in Band 2 cells increased about 200% above control, while activity in Band 3 cells increased only about 30-60% above control following 6 days of treatment. Maximal responses were observed 4-6 days following exposure to hCG or 8-Br-cAMP in both bands. Because elevated 5 alpha-reductase activity prevents testosterone accumulation in immature Leydig cells, Band 2 or Band 3 cells were cultured in the presence of 4-methyl-4-aza-3-oxo-pregnan-(20S)-carboxylate, a 5 alpha-reductase inhibitor, to assess the relationship between changes in delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity and testosterone formation. Although hCG or 8-Br-cAMP-stimulated delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity began to decline by day 8 or 10 of treatment, testosterone levels progressively increased for 10 days before declining in both bands. Thus, changes in enzyme activity did not strictly correlate with testosterone synthesizing capacity of cultured Leydig cells. Enzyme activity also was measured in cells cultured with the 5 alpha-reductase inhibitor to determine whether the responsiveness of delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity of Band 2 or Band 3 cells was due, in part, to low testosterone levels. delta 5-3 beta-Hydroxysteroid dehydrogenase-isomerase activity in Band 2 or Band 3 cells cultured without or with the inhibitor was similar, suggesting that testosterone does not inhibit the enzyme in immature Leydig cells. The greater responsiveness of delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity in Band 2 cells was not due to Leydig cell replication as neither total DNA or [3H]thymidine incorporation into DNA was altered by hCG or cAMP.

5-alpha Reductase Inhibitors↗

PubMed1990

Evidence that Leydig precursors localize in immature band two cells isolated on Percoll gradients.

The present studies examined responses to hCG and/or insulin of 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase activity (3 beta-HSD) in cultured Band 2 and Band 3 cells from 25- to 40-day-old rats isolated on Percoll gradients. In Band 2 cells, from 25-day-old rats enzyme activity increased about 3- and 2.5-fold, after 6 days of exposure to hCG or insulin, respectively. However, hCG did not stimulate enzyme activity in Band 2 cells from 30-, 35- and 40-day-old animals, and responses to insulin alone or insulin plus hCG declined with age. In Band 3 cells only insulin increased enzyme activity at each age. Neither hCG or insulin altered DNA levels in Band 2 or Band 3 cells, suggesting that increased activity in Band 2 cells from 25-day-old rats was not due to cellular replication. However, hCG increased the number of cells staining positive for 3 beta-HSD about 4-fold in Band 2 cells from 25-day-old rats. Insulin did not increase the number of positive staining cells in Band 2 and Band 3 cells from 25-day-old rats, suggesting that its major effect was to increase enzyme activity in existing cells. These results suggest that during a limited period of maturation precursor cells in Band 2, which are undetected by histochemical staining for 3 beta-HSD, can be converted to Leydig cells in culture by hCG.

Animals↗

PubMed1990

Platelet derived growth factor inhibits 5 alpha-reductase and delta 5-3 beta-hydroxysteroid dehydrogenase activities in cultured immature Leydig cells.

Platelet derived growth factor inhibited both hCG- and 8-Br-cAMP-stimulated 5 alpha-reductase activity in cultured immature Leydig cells in a dose-dependent manner, while not significantly inhibiting basal enzyme activity. Platelet derived growth factor also inhibited basal delta 5-3 beta-hydroxysteroid dehydrogenase activity and hCG-stimulated testosterone formation. Maximal inhibitions were achieved with 10 ng/ml of platelet derived growth factor. These studies suggest that platelet derived growth factor should be included among the variety of locally produced regulatory factors which modulate Leydig cell function.

3-Hydroxysteroid Dehydrogenases↗

PubMed1990

Basic fibroblast growth factor inhibits delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity in cultured immature Leydig cells.

Basic fibroblast growth factor inhibited basal delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity in cultured Leydig cells from immature rats in a concentration- and time-dependent manner. Maximal inhibition was achieved with 5-10 ng/ml basic fibroblast growth factor following approximately 48 h of exposure. The inhibition of basal delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity was not altered by human chorionic gonadotropin; however, cycloheximide (0.5-2.0 micrograms/ml) partially reversed the effects of basic fibroblast growth factor in a dose-dependent manner. These studies suggest that locally-produced basic fibroblast growth factor may modulate Leydig cell testosterone formation by regulating delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity.

3-Hydroxysteroid Dehydrogenases↗

PubMed1990

Fibroblast growth factor inhibits 5 alpha-reductase activity in cultured immature Leydig cells.

The present studies examined the effects of basic fibroblast growth factor (bFGF) on 5 alpha-reductase activity of cultured Leydig cells from immature rats. Basic FGF inhibited both hCG- and 8-bromo-cyclic AMP-stimulated 5 alpha-reductase activity in a dose-dependent manner; however, it had little or no effect on basal enzyme activity. Inhibition was achieved with as little as 0.1 ng/ml bFGF, and maximal inhibition was observed with 10 ng/ml bFGF. These studies suggest that locally produced bFGF may play a role in modulating the age-dependent decline in 5 alpha-reductase activity in Leydig cells.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗

PubMed1990

Regulation of 5 alpha-reductase activity in cultured immature leydig cells by human chorionic gonadotropin.

The present studies examined the hormonal regulation of 5 alpha-reductase activity in cultured immature rat Leydig cells. Within the testis 5 alpha-reductase was concentrated in the interstitial cell compartment, and among interstitial cells, the enzyme was localized primarily in Band 3 of Percoll density gradients, which contains the majority of Leydig cells. Among various factors reported previously to stimulate testicular 5 alpha-reductase activity when administered in vivo to immature rats (LH/hCG, FSH, luteinizing hormone releasing hormone or prolactin), only LH/hCG directly stimulated 5 alpha-reductase activity of cultured immature Band 3 cells. Neither growth hormone which was reported previously to stimulate hepatic 5 alpha-reductase activity, nor insulin, insulin-like growth factor-I, or epidermal growth factor, which have been reported to modulate Leydig cell function, had any effect on 5 alpha-reductase activity of Band 3 cells. These studies suggest that the major factor directly stimulating 5 alpha-reductase activity in Leydig cells during early maturation is LH. However, it is possible that other factors acting indirectly may modulate the maturational rise in 5 alpha-reductase activity.

3-Hydroxysteroid Dehydrogenases↗

PubMed1990

Differential regulation of steroidogenic enzymes metabolizing testosterone or dihydrotestosterone by human chorionic gonadotropin in cultured rat neonatal interstitial cells.

The present studies examined the effects of hCG on steroidogenic enzyme activities involved in the metabolism of testosterone or dihydrostestosterone in cultured rat neonatal interstitial cells. 5 alpha-reductase and 17 beta-hydroxysteroid dehydrogenase activities, which are involved in the conversion of testosterone to dihydrotestosterone and androstenedione, respectively, were low in cultured neonatal interstitial cells, were unresponsive to hCG and declined to undetectable levels during 14 days of culture. However, delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity, which is involved in the biosynthesis of testosterone, and 5 alpha-androstane-3 alpha-hydroxysteroid dehydrogenase and 5 alpha-androstane-3 beta-hydroxysteroid dehydrogenase activities, which are involved in the conversion of dihydrotestosterone to 5 alpha-androstan-3 alpha, 17 beta-diol and 5 alpha-androstan-3 beta, 17 beta-diol, respectively, were maintained or increased by hCG during the same culture period. These results demonstrate that 5 alpha-reductase and 17 beta-hydroxysteroid dehydrogenase activities do not play a significant role in regulating testosterone accumulation in fetal/neonatal Leydig cells, and they suggest that these cells are adapted to maintain high testosterone but low dihydrotestosterone levels. The present results demonstrate also that fetal/neonatal Leydig cells differ from immature or adult Leydig cells with respect to the sensitivity of 5 alpha-reductase activity to LH/hCG.

17-Hydroxysteroid Dehydrogenases↗

PubMed1989

Maturational changes in steroidogenic enzyme activities metabolizing testosterone and dihydrotestosterone in two populations of testicular interstitial cells.

The present study examined changes in steroidogenic enzyme activities which metabolize testosterone or dihydrotestosterone between days 21-73 of maturation in Band 2 and Band 3 cells isolated by centrifugation of rat testicular interstitial cells on metrizamide density gradients. 5 alpha-reductase and 17 beta-hydroxysteroid dehydrogenase activities increased progressively in Band 2 and Band 3 cells between days 21-35 of maturation, then both enzyme activities declined to reach low levels in adult Band 2 and Band 3 cells. The significantly higher activities of both enzymes in Band 3, which contains a greater concentration of Leydig cells at each age, suggest their localization in Leydig cells. 5 alpha-androstane-3 alpha- and 3 beta-hydroxysteroid dehydrogenase activities increased in both Band 2 and Band 3 cells between days 21-50 of maturation and remained elevated; however, dihydrotestosterone was metabolized primarily to 5 alpha-androstane-3 beta,17 beta-diol in Band 2 cells, while 5 alpha-androstane-3 beta,17 beta-diol was the major metabolite of dihydrotestosterone in Band 3 cells. These studies suggest that testosterone accumulation during sexual maturation can be influenced by changing patterns of 5 alpha-reductase and 17 beta-hydroxysteroid dehydrogenase activities which metabolize testosterone, and of 5 alpha-androstane-3 alpha- and 3 beta-hydroxysteroid dehydrogenase activities which metabolize dihydrotestosterone in both Band 2 and Band 3 cells.

17-Hydroxysteroid Dehydrogenases↗

PubMed1989

5 alpha-reductase activity regulates testosterone accumulation in two bands of immature cultured Leydig cells isolated on Percoll density gradients.

The 5 alpha-reductase inhibitor, 4-methyl-4-aza-3-oxo-5 alpha-pregnan-20(s)-carboxylate was utilized to examine maturational changes in testosterone synthesizing capacity of Leydig cells localizing in Band 2 and Band 3 of Percoll density gradients. Immature Band 2 Leydig cells (from 25- to 39-day-old rats) cultured without the 5 alpha-reductase inhibitor, produced increased testosterone in response to hCG or 8-br-cAMP; however, basal, hCG- or 8-br-cAMP-stimulated testosterone accumulation was 4- to 12-fold higher when cells were cultured in the presence of inhibitor. Band 2 cells from older rats produced much less testosterone in response to hCG or 8-br-cAMP (less than 3.5 pmol/10(5], even when cultured with the inhibitor. Although immature Band 3 cells cultured in the absence of 5 alpha-reductase inhibitor produced increased testosterone in response to hCG or 8-br-cAMP, testosterone levels were relatively low, because elevated 5 alpha-reductase prevented appreciable androgen accumulation; however, Band 3 cells from older rats (over 39 days old) accumulated progressively more testosterone, because of the age-dependent decline in 5 alpha-reductase activity. Immature Band 3 cells cultured with the 5 alpha-reductase inhibitor accumulated 20- to 44-fold more testosterone in response to hCG of 8-br-cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗

PubMed1987

Microsomal ethanol-oxidizing system in purified rat Leydig cells.

These studies provide evidence for the presence of a microsomal ethanol oxidizing system in rat Leydig cells. Activity of the microsomal ethanol oxidizing system in Leydig cells was 47.4 +/- 4.1 nmol acetaldehyde per 20 min per mg protein, while activity in crude interstitial cells was 26.0 +/- 5.4 nmol. This suggests that among cells comprising interstitial cells, activity is concentrated in Leydig cells. Activity was linear with respect to protein concentration and incubation time. The highest specific activity was observed in the microsomal fraction. The most effective cofactor was NADPH. The apparent Km for ethanol was 4 mM, suggesting that this system could effectively metabolize ethanol at concentrations found in the blood of males who drink. The apparent Km for NADPH was 11 microM. The activity in Leydig cells was unaffected by 4-methylpyrazole or potassium cyanide, which inhibit alcohol dehydrogenase and catalase activities, respectively. These data provide strong evidence for an enzyme system in Leydig cell microsomes which is capable of metabolizing ethanol.

Alcohol Dehydrogenase↗