Rat Testicular Microsomes Research Articles

Effect of Hypophysectomy and Hormone Replacement on Fatty Elongation in Isolated Microsomes of Rat Testes

Effect of hypophysectomy on fatty acid elongation was investigated in isolated rat testicular microsomes incubated with [14C]malonyl CoA. Hypophystectomy resulted in a 40% decrease in total incorporation of 14C into fatty acids of microsomes. Several 18- and 20-carbon fatty acids of testicular microsomes from hypophysectomized rats had less 14C than did those from nonhypophysectomized ones, whereas only docosa-7,10,13,16-tetraenoic acid (22:4w6) had more 14C. Testosterone injected subcutaneously into hypophysectomized rats at a dose of 0.5 mg per day for eight days posthypophysectomy had no apparent effect on either the total 14C incorporated or the distribution of the 14C in the various fatty acids. Daily subcutaneous injections of 50 or 100 micrograms follicle stimulating hormone (FSH) had some effect on both total incorporation and distribution of 14C. Addition of 0.5 mg testosterone to the 50 micrograms FSH gave the same results as the FSH alone. Smaller amounts of stearic acid (18:0) and linoleic acid (18:2) and increased amounts of docosa-4,7,10,13,16-pentaenoic acid (22:5w6) were present in the microsomes of hypophysectomized compared to nonhypophysectomized rats. Testosterone replacement did not affect these differences, but FSH administration was partially effective in altering the values toward those observed in nonhypophysectomized rats. Results obtained in these experiments indicate that there is inhibition of the testicular microsomal fatty acid elongation system in hypophysectomized rats and that, although testosterone is not at all effective in relieving the inhibition, FSH administration is at least partly effective.

Production of testosterone from progesterone by rat testicular microsomes without release of the intermediates 17 alpha-hydroxyprogesterone and androstenedione.

It has been shown that during the in vitro conversion of progesterone to androstenedione, 17 alpha-hydroxyprogesterone is not an obligatory intermediate which equilibrates with freely diffusible steroids in the incubation medium. Recently a cytochrome P-450 was purified that catalyzed, in addition to hydroxylase/lyase activities, reduction of androstenedione to testosterone. In order to determine whether progesterone could be transformed to testosterone without both intermediates (17 alpha-hydroxyprogesterone and androstenedione) being equilibrated with steroids in the medium, several double-label double-substrate experiments were performed. When rat microsomes were incubated with an equimolar mixture of [14C]progesterone and 17 alpha-hydroxy[3H]progesterone, androstenedione was isolated with a 11-fold higher 14C/3H ratio than 17 alpha-hydroxyprogesterone, indicating that androstenedione could not be produced from free, diffusible 17 alpha-hydroxyprogesterone. Incubation of an equimolar mixture of 17 alpha-hydroxy[3H]progesterone and [14C]androstenedione with testicular microsomes resulted in the incorporation of 3-4-fold more 17 alpha-hydroxyprogesterone into testosterone than of androstenedione, although the latter is the immediate precursor of testosterone. In an experiment in which equimolar concentrations of [3H]progesterone and [14C]androstenedione were incubated with testicular microsomes, the large pool of progesterone inhibited competitively lyase activity, but still the label of progesterone was incorporated into testosterone to the same extent as that of androstenedione. These results indicate that testosterone can be produced by immature rat testicular microsomes from added progesterone on an organized unit without the intermediates equilibrating with the incubation medium.

Metabolic pathways for androstanediol formation in immature rat testis microsomes

Metabolic routes from progesterone to androstanediol in washed rat testicular microsomes were investigated, with special emphasis on the importance of 4-ene-3-oxosteroids, as well as the effect of a minimal effective dose of human chorionic gonadotropin on these transformations. Incubation of equimolar concentrations of a mixture of [ 14C]progesterone and 17α-hydroxy[ 3H]progesterone resulted in a large preference of 17α-hydroxyprogesterone over progesterone as substrate for androstanediol formation. Incubation of [ 3H]progesterone together with [ 14C]androstenedione resulted in the inhibition of C-17,20-lyase and in a low 14C/ 3H ratio in androstanediol, indicating the preference of progesterone over androstenedione as substrate for androstanediol production. When a mixture of 17α-hydroxyl[ 3H]progesterone and [ 14C]androstenedione was incubated with the microsomes, a more than 8-fold preference of 17α-hydroxyprogesterone as substrate for androstanediol production was found. The minimal dose of human chorionic gonadotropin stimulated testosterone production but inhibited androstanediol formation and effected, in some instances, a change in the metabolic routes. It is concluded that androstanediol is produced preferentially through 17-hydroxylated C-21 steroids, and also, to a lesser extent, through C-19 steroids.

Purification and properties of testicular 3β-hydroxy-5-ene-steroid dehydrogenase and 5-ene-4-ene isomerase

Through the treatment of rat testicular microsomes with sodium cholate, 3β-hydroxy-5-ene-steroid dehydrogenase and 5-ene-4-ene isomerase (abbreviated as the 3β -hydroxysteroid dehydrogenase and isomerase, respectively) were solubilized, and then purified by DEAE and hydroxylapatite column chromatographies. The findings were as follows: 1. With this purification procedure, the 3β-hydroxysteroid dehydrogenase activity could not be separated from the isomerase. 2. For 3-oxo-4-ene-steroid formation from 3β-hydroxy-5-ene-steroids, NAD + was required as a cofactor. While the 3β-hydroxysteroid dehydrogenase required NAD +, the isomerase also required NAD + or its reduced form, in contrast to the microbial enzyme. 3. On treatment of the purified enzyme with 5'- p-fluorosulfonyl-benzoyladenosine (FSBA), both enzyme activities were markedly reduced. 4. The enzyme, affinity labeled with [adenine-8- 14C]FSBA, showed a mol. wt of 46.8 K. 5. During 4-androstenedione production from DHA, 5-androstenedione was detected as an intermediate.

NADPH-Cytochrome P-450 Reductase from Untreated Rat Testicular and Liver Microsomes: Isolation and Comparison

NADPH-cytochrome P-450 reductase (EC 1.6.2.4), the enzyme involved in progesterone 17-hydroxylation, was purified to apparent homogeneity by detergent solubilization of the microsomal fractions of liver and testis from untreated rats. The enzymes from these two tissues were then compared with regard to several parameters. The liver and testicular reductases have the same subunit mol wt (79,000), determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and have similar specific activity for cytochrome c reduction (48.1 and 58.2 mumol min-1 mg-1, respectively). The absolute flavin absorption spectra of liver and testicular reductase were very similar. The spectra of native, reduced, and oxidized reductases were characteristic of flavoprotein with two flavin groups. The Km values for NADPH during cytochrome reduction were shown to be the same, within experimental error (5.29 +/- 0.46 microM-1 for liver and 4.37 +/- 0.53 microM-1 for testis). Monospecific antiserum was prepared against both rat liver and testicular reductases. There were no antigenic differences demonstrated by immunological tests using either antibody preparation. Antiliver reductase antiserum was shown to inhibit testicular microsomal progesterone 17-hydroxylase activity by 70%. Testicular cytochrome c reductase activity was inhibited by 94%, and liver cytochrome c reductase activity was inhibited by 85%. Peptide maps of both forms of reductase isolated from immunoprecipitates showed very similar patterns after enzymatic proteolysis. These results indicate that microsomal NADPH-cytochrome P-450 reductase involved in steroid hydroxylations in untreated rat liver and testis could not be distinguished from each other by these methods and, therefore, are probably the same protein.

Dolichol kinase activity in the developing rat testis

Dolichyl phosphate concentrations, a primary factor in regulating the rate of N-glycosidically linked glycoprotein synthesis, are dependent upon a cytidine triphosphate (CTP)-dependent dolichol kinase. This study examines dolichol kinase in rat testicular microsomes and defines assay conditions. As with dolichol kinases from other tissues, addition of 2-mercaptoethanol increased activity 60%. Inclusion of NaF, an inhibitor of testicular dolichyl phosphate phosphatase activity, also resulted in a 38% increase in activity. Triton X-100 was necessary for phosphorylation of both endogenous and exogenous dolichol; however, concentrations of detergent in excess of 0.25-0.35% were inhibitory. A 2- to 5-fold stimulation of kinase activity was obtained by addition of 50-100 microM exogenous dolichol. The high level of nucleoside triphosphatase activity in testicular microsomes mandated the inclusion of high levels of uridine triphosphate (UTP) to protect the [gamma-32 P] CTP. Increasing UTP concentrations up to 50 mM resulted in increased product formation. A clear requirement for divalent cations was observed; 5 mM ethylenediaminetetraacetate (EDTA) abolished activity. The following order of cation effectiveness was observed: Mn greater than or equal to Ca greater than Cd greater than Zn much greater than Mg. Ten mM optima were established for Ca2+ and Mn2+; the presence of UTP, however, results in significantly reduced concentrations of free Ca2+. Ion combination studies demonstrated interactive inhibitory effects between Ca2+ and other stimulatory divalent cations. Addition of 2 microM brain calmodulin, in the presence of 10 mM Ca2+, resulted in a 75-100% stimulation of activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Contribution of cytochrome b5 to androgen synthesis in rat testicular microsomes.

Cytochrome b5 was purified from porcine testicular microsomes, and its amino acid composition was determined. Rabbit antibody against the purified cytochrome b5 was prepared in order to study the contribution of cytochrome b5 to testicular microsomal oxygenases related to androgen production. In the presence of NADPH alone as the electron donor, the antibody against cytochrome b5 inhibited the activities of steroid 17 alpha-hydroxylase and C-17-C-20 lyase of rat testicular microsomal fraction. Addition of NADH to the NADPH-supported oxygenase assay system enhanced both steroid oxygenase activities, and addition of the antibody against cytochrome b5 decreased the NADH-caused stimulation of steroid 17 alpha-hydroxylase and C-17-C-20 lyase activities. When dehydroepiandrosterone and NAD+ were added as substrates for 3 beta-hydroxy-delta 5-steroid dehydrogenase in order to synthesize NADH by enzymatic reaction, the NADPH-supported activities of steroid 17 alpha-hydroxylase and C-17-C-20 lyase were further stimulated as compared with the addition of NADH, and this stimulation was suppressed by the antibody against cytochrome b5. These results suggest that cytochrome b5, together with 3 beta-hydroxy-delta 5-steroid dehydrogenase, contributes to the activities of steroid 17 alpha-hydroxylase and C-17-C-20 lyase in the testicular microsomal fraction.

Effect of chronic ethanol feeding on testicular content of enzymes required for testosteronogenesis.

Ethanol is a known inducer of microsomal enzymes as well as a testicular toxin. In order to evaluate the effect of chronic ethanol ingestion upon the microsomal enzymes required for testosterone synthesis, we examined the activity of four testicular enzymes (3 beta-hydroxysteroid dehydrogenase/isomerase, 17 alpha-hydroxylase, 17,20-lyase, and 17 beta-hydroxysteroid dehydrogenase) in 14 pairs of adult chronic alcohol-fed rats and their age-matched isocaloric controls. Ethanol feeding enhanced the activity of 17,20-lyase when expressed as either activity/mg of protein (p less than 0.05) or activity/g of testis (P less than 0.025). Similarly, the activity of 17 alpha-hydroxylase was increased in testes of the alcohol-fed animals (p less than 0.025) compared to controls. In contrast, chronic ethanol feeding reduced total activity of 3 beta-hydroxysteroid dehydrogenase/isomerase in alcohol-fed animals (p less than 0.05) compared to controls. No effect of ethanol feeding was seen on activity of 17 beta-hydroxysteroid dehydrogenase. Based upon these studies we conclude that chronic ethanol ingestion (1) increases testicular 17 alpha-hydroxylase and 17,20-lyase and (2) reduces 3 beta-hydroxysteroid dehydrogenase/isomerase in rat testicular microsomes. Therefore, we would propose that the major effect of chronic ethanol ingestion upon the enzymes required for testosterogenesis is the reduction of 3 beta-hydroxysteroid dehydrogenase/isomerase activity, the rate limiting step in sex steroid production from pregnenolone.

Conversion of a C-20-deoxy-C21, steroid, 5-pregnen-3beta-ol, into testosterone by rat testicular microsomes.

5-[7ALPHA-3H] Pregnen-3beta-ol, a C-20-deoxy analog of pregnenolone, was synthesized and tested as a substrate for the enzyme system occurring in testes that cleaves the side chain of C21 steroids between C-17 and C-20. This C-20-deoxy C21 steroid was incubated with a microsomal preparation obtained from rat testis and was converted into testosterone in 5% yield. Another C-20-deoxy analog of pregnenolone, 5,20-pregnadien-3beta-ol, was not converted into testosterone by this enzyme system. The significance of this finding for the natural processes by which pregnenolone is converted by the same subcellular fraction into the male sex hormone is examined in the light of the hypothesis that intermediates involved in steroidogenesis are transient, reactive complexes of the appropriate reactants (steroids, oxygen, etc.) with specific enzymes.

A mathematical analysis of the substrate effect observed in 3beta-hydroxysteroid dehydrogenase reactions of rat testicular microsomes.

The substrate effect in enzyme reactions has been explained mostly in terms of an additional substrate binding site on the enzyme other than the catalytic site. A rate equation for the reaction is introduced according to the steady state mechanism as follows: v = (Ps3+Qs2+Rs)/(s3+Ls2+Ms+N), were the six parameters, L,M,N,P,Q, and R, can be determined by the least-squares method from the experimental points. The v vs. s curve has an asymptote parallel to the s abscissa, and can be classified into one of four types. The type A curve has an intersection with the asymptote and an apparent maximum velocity; the curve descends toward the asymptote. Type B has no intersection and no stationary point; the curve ascends toward the asymptote. Type C has two intersections and two stationary points, an apparent maximum velocity and a minimum velocity; the curve ascends toward the asymptote. Type D has no intersection and two stationary points; the curve ascends toward the asymptote. The equation was applied to the 3beta-hydroxysteroid dehydrogenase [EC 1.1.1.145] reaction of rat testicular microsomes. The conversion of 3beta-hydroxyandrost-5-ene-17-one was represented by type C, with an apparent maximum velocity of 0.338 nmole/min/mg protein at 0.912 muM of the substrate concentration, minimum velocity of 0.108 nmole/min at 16.6 muM, and saturating velocity of 0.169 nmole/min at infinite concentration of the substrate. The converson of 3beta-hydroxypregn-5-ene-20-one was of type B, having two inflexion points, 0.320 nmole/min at 2.735 muM and 0.814 nmole/min at 12.39 muM, and a saturating velocity of 3.80 nmoles/min at infinite concentration of the substrate.

Inhibition of masculine differentiation in male rat fetuses by two candidates for active-site-directed-irreversible (ASDI) inhibitors of 17α hydroxylase and C17–20 Lyase

ASDI inhibitors stoichiometrically bind to enzyme active-sties have a high degree of “lock and key” specificity, cannot be removed from the active site by dilution and should have few side effects. Two synthetic steroids selected as ASDI candidates, 17β-ureidoandrosta-I, 4-dien-3-one and 16β-bromo-5α-pregnan-3β, 17α-diol-11, 20-dione were found to inhibit the conversion of pregnenolone or progesterone to testosterone by adult rat testicular microsomes specifically at the level of both 17α hydroxylase and C17-20 lyase. When administered to pregnant rats at 30mg/kg daily from day 13 to day 21 each inhibitor significantly blocked penis formation in male offspring by reducing anogenital distance from 3.69 ± 0.24mm. (control) to 3.26 ± 0.22 (17β-ureide) and 3.32 ± 0.23mm. (16β-bromide). Testosterone production in vitro by experimental tests was only 25% of that of controls with either pregnenolone or progesterone as substrates. Abnormal accumulations of progesterone and 17-hydroxyprogesterone were present in incubations of experimental tests. Female fetuses and maternal and fetal adrenal size and steroid synthesis were unaffected. Each inhibitor obliterated C19 steroid excretion in urines and feces of adult female rats as analyzed by gas-liquid chromatography and mass spectrometry. Thus, these inhibitors appear to block sex hormone without affecting glucorcorticoid synthesis in adult and fetal rats.

TESTOSTERONE FORMATION BY SUBCELLULAR PARTICLES OF RAT TESTES.

Testicular tissue of normal adult rats was homogenized and fractionated by differential centrifugation. The microsomal fraction contained the most active enzymic systems for the cleavage of the side chain of 17α-hydroxyprogesterone-4-14C. Washed microsomes transformed progesterone-4-l4C, 17α-hydroxyprogesterone- 4-14C and Δ4-androstene- 3,17-dione-4-14C to testosterone-14C, indicating that the 17α-hydroxylase, the 17α-hydroxyprogesterone side chain cleavage enzyme and the 17β-hydroxysteroid dehydrogenase activities are all contained within the microsomes. NADPH was required for side chain cleavage of 17α-hydroxyprogesterone as well as for the convei-sion of androstenedione to testosterone by the rat testicular microsomes. Addition of the 105,000 × g supernatant fluid of the testicular homogenate to suspensions of testicular microsomes significantly increased the C,9 steroid formation from progesterone-4-14C and 17α-hydroxyprogesterone-4-14C. The stimulation was observed in the presence of excess NADPH or an NADPH-generating system. Long-term treatment of the microsomal suspension with sonic waves (10 kc) or addition of an anionic detergent to the suspension caused inactivation of the 17α-hydroxyprogesterone side chain cleavage enzyme. Neither washing the microsomes with hypertonic NaCl solution nor freezing and thawing the microsomal suspension released the enzyme activity from the microsomes. The results suggest that the enzyme is firmly combined with cytoplasmic particles. (Endocrinology76: 563, 1965)

Effect of inhibitors on testicular microsomal steroid 17 α-hydroxylase and 17 α-hydroxypregnene C 17–C 20 lyase

3-(6-Chloro-3-methyl-2-indenyl)pyridine (SU 8000) and 3-(1,2,3,4-tetra-hydro-4-oxo-7-chloro-2-naphthyl)pyridine (SU 10603) competitively inhibited 17α-hydroxylation of [4-14C]progesterone by rat-testicular microsomes. The compounds also strongly but non-competitively inhibited the cleavage of the side chain of [4-14C]17α-hydroxyprogesterone by the microsomes. The apparent inhibitor constants of SU 8000 were determined as 1.35 μM and 1.00 μM against the 17α-hydroxylase and the 17α-hydroxypregnene C17–C20 lyase respectively. Corresponding K1 values for SU 10603 were 0.86 μM. and 0.54 μM. 17β-Hydroxysteroid dehydrogenase and 20α-hydroxysteroid dehydrogenase of the rat testes were not influenced by the compounds.

The pathway of formation of testosterone from 3β-hydroxypregn-5-en-20-one by rat testicular microsomes

3β-Hydroxypregn-5-en-20-one (pregnenolone)-7α- 3H was transformed to testosterone by rat testicular microsomes in the presence of NAD and NADPH. Addition of 105,000 × g supernatant fluid of the testicular homogenates signifincantly enhanced the formation of testosterone. Progesterone, 17α- hydroxyprogesterone and androst-4-ene-3, 17-dione (androstenedione) were obtained as the intermediates, whereas 3β, 17α-dihydroxypregn-5-en-20-one (17α-hydroxypregnenolone) and 3β-hydroxyandrost-5-en-17-one were found only in negligible amounts. A mixture of progesterone-4- 14C and pregnenolone- 7α- 3H was incubated with the microsomes for 5 to 20 minutes. The results showed that progesterone was more quickly converted to testosterone than was pregnenolone by the microsomes and that androstenedione was an intermediate substance between progesterone and testosterone as well as between pregnenolone and testosterone. A similar dual tracer study using 17α-hydroxyprogesterone-4- 14C and 17α-hydroxypregrenolone-7α- 3H showed that the former steroid was more quickly transformed to testosterone by the microsomes. It is concluded that testosterone is formed from pregnenolone by the microsomes mainly by way of progesterone and androstenedione.