Purification and characterization of 17 beta-hydroxysteroid dehydrogenase from Cylindrocarpon radicicola

An NAD+-linked 17 beta-hydroxysteroid dehydrogenase was purified to homogeneity from a fungus, Cylindrocarpon radicicola ATCC 11011 by ion exchange, gel filtration, and hydrophobic chromatographies. The purified preparation of the dehydrogenase showed an apparent molecular weight of 58,600 by gel filtration and polyacrylamide gel electrophoresis. SDS-gel electrophoresis gave Mr = 26,000 for the identical subunits of the protein. The amino-terminal residue of the enzyme protein was determined to be glycine. The enzyme catalyzed the oxidation of 17 beta-hydroxysteroids to the ketosteroids with the reduction of NAD+, which was a specific hydrogen acceptor, and also catalyzed the reduction of 17-ketosteroids with the consumption of NADH. The optimum pH of the dehydrogenase reaction was 10 and that of the reductase reaction was 7.0. The enzyme had a high specific activity for the oxidation of testosterone (Vmax = 85 mumol/min/mg; Km for the steroid = 9.5 microM; Km for NAD+ = 198 microM at pH 10.0) and for the reduction of androstenedione (Vmax = 1.8 mumol/min/mg; Km for the steroid = 24 microM; Km for NADH = 6.8 microM at pH 7.0). In the purified enzyme preparation, no activity of 3 alpha-hydroxysteroid dehydrogenase, 3 beta-hydroxysteroid dehydrogenase, delta 5-3-ketosteroid-4,5-isomerase, or steroid ring A-delta-dehydrogenase was detected. Among several steroids tested, only 17 beta-hydroxysteroids such as testosterone, estradiol-17 beta, and 11 beta-hydroxytestosterone, were oxidized, indicating that the enzyme has a high specificity for the substrate steroid. The stereospecificity of hydrogen transfer by the enzyme in dehydrogenation was examined with [17 alpha-3H]testosterone.     

Purification and properties of a new testosterone 17beta-dehydrogenase (NADP+) from guinea-pig liver.

As a result of studies of guinea-pig live testosterone 17beta-dehydrogenase (NADP+) (EC 1.1.1.64), a new testosterone 17beta-dehydrogenase was discovered. The new enzyme was purified to a single homogeneous protein from the 105 000 g-supernatant fraction of guinea-pig liver by (NH4)2SO4 fractional precipitation and two gel-filtration stages, DEAE-cellulose column chromatography and hydroxyapatite column chromatography. It was characterized by many properties. The enzyme has almost the same properties as the classical testosterone 17beta-dehydrogenase (NADP+) (EC 1.1.1.64), with respect to cofactor requirement, pH optima for dehydrogenation, effect of phosphate ion on the NAD+-dependent reaction and molecular weight, but characteristic differences were observed in substrate-specificity between the two dehydrogenases. With various androstane derivatives, the configuration of the A/B-ring junction was closely connected with enzyme activity. 5alpha-Androstanes, such as 5alpha-androstane-3alpha,17beta-diol, 5alpha-androstane-3beta,17beta-diol and 17beta-hydroxy-5alpha-androstan-3-one, and 5beta-congeners, such as 5beta-androstane-3alpha,17beta-diol, 5beta-androstane-3beta,17beta-diol and 17beta-hydroxy-5beta-androstan-3-one, served as substrates for both the EC 1.1.1.64 enzyme and the new enzyme. The EC 1.1.1.64 enzyme oxidized testosterone more rapidly than did the new enzyme. These comparisons were based on the relative activities, apparent Km values and apparent Vmax values.     

Isolation of novel microbial 3 alpha-, 3 beta-, and 17 beta-hydroxysteroid dehydrogenases. Purification, characterization, and analytical applications of a 17 beta-hydroxysteroid dehydrogenase from an Alcaligenes sp

By selecting for growth on testosterone or estradiol-17 beta as the only source of organic carbon, we have isolated a number of soil microorganisms which contain highly active and novel, inducible, NAD-linked 3 alpha-, 3 beta-, and 17 beta-hydroxysteroid dehydrogenases. Such enzymes are suitable for the microanalysis of steroids and of steroid-transforming enzymes, as well as for performing stereoselective oxidations and reductions of steroids. Of particular interest among these organisms is a new species of Alcaligenes containing 17 beta-hydroxysteroid dehydrogenase, easily separable from 3 beta-hydroxysteroid dehydrogenase. Unlike any of the other isolated organisms, this Alcaligenes sp. contained no 3 alpha-hydroxysteroid dehydrogenase activity. A large-scale purification (763-fold) to homogeneity of the major induced 17 beta-hydroxysteroid dehydrogenase was achieved by ion-exchange, hydrophobic, and affinity chromatographies. The enzyme has high specific activity for the oxidation of testosterone (Vmax = 303 mumol/min/mg of protein; Km = 3.6 microM) and reacts almost equally well with estradiol-17 beta (Vmax = 356 mumol/min/mg; Km = 6.4 microM). It consists of apparently identical subunits (Mr = 32,000) and exists in polymeric form under nondenaturing conditions (Mr = 68,000 by gel filtration and 86,000 by polyacrylamide gel electrophoresis). The isoelectric point is pH 5.1. The enzyme is almost completely specific for 17 beta-hydroxysteroids which may be delta 5-olefins or ring A phenols or have cis or trans A/B ring fusions. Substituents at other positions are tolerated, although the presence of a 16 alpha- or 16 beta-hydroxyl group blocks the oxidation of the 17 beta-hydroxyl function. 3 beta-Hydroxysteroids (A/B ring fusion trans, but not cis, or delta 5-olefins) are very poor substrates. The application of this highly active, specific, and stable 17 beta-hydroxysteroid dehydrogenase to the microestimation of steroids by enzymatic cycling of nicotinamide nucleotides and for the stereospecific oxidation of steroids is demonstrated.     

Solubilisation and reconstitution of acylcoenzyme A:estradiol-17 beta acyltransferase

Acylcoenzyme A:estradiol-17 beta acyltransferase in microsomes of bovine placenta cotyledons was strongly membrane bound. The enzyme was solubilised from microsomes by sodium cholate and was reconstituted into phospholipid vesicles. The apparent Km for estradiol-17 beta was 11 microM which was close to the value of 8 microM previously found with the membrane-bound enzyme. Testosterone was also a substrate for the reconstituted enzyme (apparent Km 62 microM) and was a competitive inhibitor (Ki 74 microM) of the acylation of estradiol-17 beta. Although various long-chained fatty acyl CoAs acted as acyl donors, these proved to have widely differing apparent Km values with palmitoleoyl CoA having the highest affinity (Km 24 microM) and arachidonoyl CoA the lowest affinity (Km 330 microM).     

The solubilisation of some steroids by phosphatidylcholine and phosphatidylcholine-cholesterol vesicles.

The solubility of the three steroid hormones, progesterone, testosterone, and estradiol-17 beta in water and phosphatidylcholine vesicles was measured after shaking and ultrasonication. All three steroids have low water solubility, which increases considerably at sonication for testosterone and estradiol-17 beta. The phosphatidylcholine vesicles have a very small solubilising capacity for the steroids; about 20 mumol/mol. This increases at sonication for estradiol-17 beta and decreases for testosterone. The capacity for progesterone is almost unaltered. The incorporation of cholesterol in the vesicles decreased the solubilisation capacity for testosterone and estradiol-17 beta but increased that for progesterone of shaked preparations. For the sonicated systems the cholesterol decreased the solubilising capacity for estradiol-17 beta but increased that for testosterone. The solubilisation experiments indicate that the steroid hormones are solubilised in the hydrocarbon part of the phosphatidylcholine bilayer and also 13CNMR results support this conclusion.     

17 beta-hydroxysteroid and 20 alpha-hydroxysteroid dehydrogenase activities of human placental microsomes: kinetic evidence for two enzymes differing in substrate specificity

During storage at 4 degrees C, the 17 beta-hydroxysteroid dehydrogenase activity of human placental microsomes with estradiol-17 beta was more stable than that with testosterone. In order to evaluate the basis for this difference, kinetics with C18-, C19-, and C21- steroids as substrates and/or inhibitors was studied in conjunction with an analysis of the effects of detergents. Both 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) and 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) activities were detected. At pH 9.0, apparent Michaelis constants were 0.8, 1.3, and 2.3 microM for estradiol-17 beta, testosterone, and 20 alpha-dihydroprogesterone, respectively, 17 beta-HSD activity with testosterone was inhibited by estradiol-17 beta, 5 alpha-dihydrotestosterone, 5 beta-dihydrotestosterone, 20 alpha-dihydroprogesterone, and progesterone. In each case 90 to 100% inhibition was observed at 50 to 200 microM steroid. Activity with 20 alpha-dihydroprogesterone was similarly sensitive to inhibition by C19-steroids. By contrast, 25 to 45% of the activity with estradiol-17 beta was not inhibited by high concentrations of C19- or C21-steroids and differed from the 17 beta-HSD activity with testosterone and the major fraction of that with estradiol-17 beta by being insensitive to solubilization by detergent. These results are consistent with an association of two dehydrogenase activities with human placental microsomes. One recognizes C18-, C19-, and C21-steroids as substrates with comparable affinities. The second appears to be highly specific for estradiol-17 beta. The former activity may account for most if not all of the oxidation-reduction at C-17 of C19-steroids and at C-20 of C21-compounds at physiological concentrations by term placental tissue.     

Inactivation of human placental 17 beta,20 alpha-hydroxysteroid dehydrogenase by 16-methylene estrone, an affinity alkylator enzymatically generated from 16-methylene estradiol-17 beta

The substrate 16-methylene estra-1,3,5(10)-triene-3,17 beta-diol (16-methylene estradiol-17 beta) and its enzyme-generated alkylating product, 3-hydroxy-16-methylene estra-1,3,5(10)-triene-17-one (16-methylene estrone), were synthesized to study the 17 beta- and 20 alpha-hydroxysteroid dehydrogenase activities which coexist in homogeneous enzyme purified from human placental cytosol. 16-Methylene estradiol, an excellent substrate (Km = 8.0 microM; Vmax = 2.8 mumol/mg/min) when enzymatically oxidized to 16-methylene estrone in the presence of NAD+ (256 microM), inactivates simultaneously the 17 beta- and 20 alpha-activities in a time-dependent and irreversible manner following pseudo-first order kinetics (t1/2 = 1.0 h, 100 microM, pH 9.2). 16-Methylene estradiol does not inactivate the enzyme in the absence of NAD+. 16-Methylene estrone (Km = 2.7 microM; Vmax = 2.9 mumol/mg/min) is an affinity alkylator (biomolecular rate constant k'3 = 63.3 liters/mol-s, pH 9.2; KI = 261 microM; k3 = 8.0 X 10(-4) S-1, pH 7.0) which also simultaneously inhibits both activities in an irreversible time-dependent manner (at 25 microM; t1/2 = 7.2 min, pH 9.2; t1/2 = 2.7 h, pH 7.0). Substrates (estradiol-17 beta, estrone, and progesterone) protect against inhibition of enzyme activity by 16-methylene estrone and 16-methylene estradiol. Affinity radioalkylation studies using 16-methylene [6,7-3H]estrone demonstrate that 1 mol of alkylator binds per mol of inactivated enzyme dimer. Thus, 16-methylene estradiol functions as a unique substrate for the enzymatic generation of a powerful affinity alkylator of 17 beta,20 alpha-hydroxysteroid dehydrogenase and should be a useful pharmacological tool.     

Comparison of sex hormone dependent induction of delta-aminolevulinic acid dehydratase in chick liver and oviduct

1. Comparative study on induction of hepatic and oviduct delta-aminolevulinic acid dehydratase (EC 4.2.1.24, ALAD) was performed following estradiol-17 beta and/or testosterone administration in immature female chicken (Gallus domesticus). 2. The lowest amount of estradiol for maximal induction of hepatic and oviduct ALAD activity was 2 mg/day/bird. 3. When estradiol of 2 mg/day/bird was administered for 15 days successively, induction extent of oviduct ALAD molecule was markedly larger and became approximately 144-fold in comparison with that of liver. Testosterone (2 mg/day/bird) alone did not induce both hepatic and oviduct ALAD activity. 4. Synergistic and antagonistic effect of testosterone on estradiol-induced total ALAD activity in oviduct was dependent on testosterone amount administered, whereas testosterone antagonized the inductive effect of estradiol on liver ALAD activity, independently of its amount.     

17Beta-hydroxysteroid dehydrogenase activity in Streptomyces hydrogenans.

Streptomyces hydrogenans converts 17beta-hydroxyandrost-4-ene-3-one (testosterone) to androst-4-ene-3,17-dione (androstenedione) in good yields. Time-dependence of the conversion, steroid uptake and release have been studied in vivo. Steroid analysis was done by thin-layer chromatography and recrystallization to constant specific radioactivity. After sonification of the cells the postulated 17beta-hydroxysteroid dehydrogenase activity was recovered in the 105 000 g supernatant. The enzyme was enriched by gel filtration on Sephadex G-200. It required NAD+ as cofactor. Its activity could be studied photometrically, because there are no further testosterone-netabolites. If S. hydrogenans was cultured in the presence of testosterone, estradiol or 5alphaH-dihydrotestosterone, the activity of 17beta-hydroxysteroid dehydrogenase increased.     

The effects of adrenal and gonadal steroids and K+-canrenoate on the metabolism of aldosterone by rat liver microsomes

The synthesis of polar aldosterone metabolites by rat liver microsomes at physiological concentrations of aldosterone (21.5 nM), was markedly inhibited by progesterone, testosterone, corticosterone, K+-canrenoate and estradiol-17 beta. In contrast, corticosterone and estradiol-17 beta significantly increased the synthesis of reduced aldosterone metabolites by 8- and 15-fold respectively, the majority of which were 5 alpha-reduced products of aldosterone. In experiments at higher substrate (aldosterone) concentrations (20-200 microM) the synthesis of ring A-reduced aldosterone metabolites by liver microsomes followed Michaelis-Menten kinetics with a Km[app] for aldosterone of 160 microM and Vmax[app] of 12.2 nmoles/mg protein/5 min. In these experiments progesterone, testosterone and K+-canrenoate all competitively inhibited the synthesis of reduced metabolites with inhibition constants (Ki [app]) of 70, 85 and 55 microM respectively; however, corticosterone did not. In contrast, estradiol-17 beta increased the rate of synthesis of reduced products by 40%, lowering the Km[app] to 83 microM.     

The determination of five steroids in avian plasma by radioimmunoassay and competitive protein-binding.

A method has been developed for the simultaneous determination of testosterone, 5alpha-dihydrotestosterone and corticosterone, or of estrone, estradiol-17beta and corticosterone, after separation on a Celite:propylene glycol:ethylene glycol column (6:1.5:1.5 w/v/v). The lower quarter of the column was packed with a Celite: water mixture (3:1 w/v) as a stationary phase (glycol) 'trap'. This effectively prevented leaching of the glycols into the eluate as the concentration of ethyl acetate in the mobile phase was increased to elute the more polar steroids. In addition, a second system utilizing a Celite: ethylene glycol column (2:1 w/v) for the separation of estrone and estradiol-17beta is described. Testosterone, 5alpha-dihydrotestosterone, estrone and estradiol-17beta were measured by radioimmunoassay and corticosterone by a competitive protein-binding technique. Reliability criteria are presented showing that the assay systems used are accurate and reproducible. Plasma-steroid levels of eight avian species are also presented and compared with those found by other investigators.     

Formation and turnover of long-chain fatty acid esters of 5-androstene-3 beta, 17 beta -diol in estrogen receptor positive and negative human mammary cancer cell lines in culture

Microsomal preparations derived from bovine placenta cotyledons, previously investigated as a convenient source of fatty acyl coenzyme A: estradiol-17 beta-acyl transferase, have been shown to acylate other steroids bearing 3 beta- or 17 beta-hydroxyl groups. In the presence of 0.1 mM oleoyl CoA, the apparent Km values for dehydroepiandrosterone, testosterone, and 5-androstene-3 beta,17 beta-diol (delta 5-DIOL) were 45, 67, and 20 microM, respectively. Acylation of delta 5-DIOL occurred at either the 3 beta- or 17 beta-positions to give monoesters. Testosterone, estradiol-17 beta, and delta 5-DIOL acted as competitive inhibitors for the acylation of the 3 beta-hydroxyl group of dehydroepiandrosterone (Ki values 71, 75, and 41 microM, respectively). Such data indicate that a single enzyme of wide substrate specificity may be involved in these acylation reactions. When estrogen receptor (ER) positive and negative human mammary cancer cell lines were incubated with 10 nM [3H]delta 5-DIOL, intracellular accumulation of delta 5-DIOL long-chain fatty acid esters occurred; rates being higher (p less than 0.001) in ER negative cells (MDA-MB-231 and MDA-MB-330) compared to MCF-7 cells (ER positive), and higher (P less than 0.005) in MDA-MB-231 cells compared to ZR-75-1 cells (ER positive). After exposure to 10 nM [3H]delta 5-DIOL for 16 h, the total labeled steroid fatty acid fraction was composed predominantly of delta 5-DIOL-3 beta- and 17 beta-monoesters (approximately 85%), the remainder containing approximately equal amounts of delta 5-DIOL-diesters and dehydroepiandrosterone-3 beta-esters. Subsequent transfer to medium lacking delta 5-DIOL was accompanied by a breakdown of the labeled esters, which was more rapid in the ER positive cell lines. During this period, intracellular free delta 5-DIOL levels rapidly declined in MDA-MB-330 cells but were maintained in MCF-7 cells, presumably by binding to ER. This behavior parallels that of estradiol-17 beta previously observed in these cell lines and further emphasizes the potential importance of the adrenal-derived estrogen delta 5-DIOL in consideration of a hormone-based etiology of human breast cancer.     

Inhibition of hepatic 17 beta- and 3 alpha-hydroxysteroid dehydrogenases by antiinflammatory drugs and nonsteroidal estrogens

Antiinflammatory agents and estrogens have been tested as inhibitors of two isozymes of guinea pig liver testosterone 17 beta-dehydrogenase (NADP) 1.1.1.64) and rat liver 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50). Antiinflammatory steroids and estradiols were highly inhibitory to 3 alpha-hydroxysteroid dehydrogenase and one isozyme of testosterone 17 beta-dehydrogenase, respectively, but nonsteroidal antiinflammatory agents and nonsteroidal estrogens such as hexestrol, dienstrol, diethylstilbestrol and zearalenone showed potent inhibitions on all the enzymes. Although the inhibitory potency of indomethacin for one isozymes of testosterone 17 beta-dehydrogenase and 3 alpha-hydroxysteroid dehydrogenase decreased with changing pH from 9.7 to 7.0, that of the nonsteroidal estrogens for all the enzymes was little affected by pH. No additive effect in double inhibitor experiments with indomethacin and the nonsteroidal estrogens was observed, and the compounds were all competitive inhibitors with respect to steroidal substrate. The results suggest that there is a very similar region in substrate binding sites of the enzymes.     

Regulation of Chorismate mutase-prephenate dehydratase and prephenate dehydrogenase from alcaligenes eutrophus.

Highly purified enzymes from Alcaligenes eutrophus H 16 were used for kinetic studies. Chorismate mutase was feedback inhibited by phenylalanine. In the absence of the inhibitor, the double-reciprocal plot was linear, yielding a Km for chorismate of 0.2 mM. When phenylalanine was present, a pronounced deviation from the Michaelis-Menten hyperbola occurred. The Hill coefficient (n) was 1.7, and Hill plots of velocity versus inhibitor concentrations resulted in a value of n' = 2.3, indicating positive cooperativity. Chorismate mutase was also inhibited by prephenate, which caused downward double-reciprocal plots and a Hill coefficient of n = 0.7, evidence for negative cooperativity. The pH optimum of chorismate mutase ranged from 7.8 to 8.2; its temperature optimum was 47 C. Prephenate dehydratase was competitively inhibited by phenylalanine and activated by tyrosine. Tyrosine stimulated its activity up to 10-fold and decreased the Km for prephenate, which was 0.67 mM without effectors. Tryptophan inhibited the enzyme competitively. Its inhibition constant (Ki = 23 muM) was almost 10-fold higher than that determined for phenylalanine (Ki = 2.6 muM). The pH optimum of prephenate dehydratase was pH 5.7; the temperature optimum was 48 C. Prephenate dehydrogenase was feedback inhibited by tyrosine. Inhibition was competitive with prephenate (Ki = 0.06 mM) and noncompetitive with nicotinamide adenine dinucleotide. The enzyme was further subject to product inhibition by p-hydroxyphenylpyruvate (Ki = 0.13 mM). Its Km for prephenate was 0.045 mM, and that for nicotinamide adenine dinucleotide was 0.14 mM. The pH optimum ranged between 7.0 and 7.6; the temperature optimum was 38 C. It is shown how the sensitive regulation of the entire enzyme system leads to a well-balanced amino acid production.     

Specificity of inhibition by steroids of porcine oocyte maturation in vitro.

In order to examine the influence of several steroids on the process of oocyte maturation, denuded (adherent cumulus granulosa cells mechanically removed) and intact (cumulus granulosa cells left attached) porcine oocytes were cultured in the presence or absence of estradiol-17 beta, estradiol-17 alpha, testosterone, cortisol, progesterone, or the nonsteroidal estrogen diethyl stilbestrol (all at 10 microgram/ml) in defined medium that contained either BSA or dextran. Estradiol-17 beta was the only steroid to exert a significant inhibitory effect on the maturation of denuded oocytes, and did so only in BSA supplemented medium. The inhibition was reversible in that oocytes, cultured in steroid-free medium after initial culture in estradiol-17 beta medium, resumed meiotic maturation. Oocytes took up 3H-estradiol-17 beta in both media, although less radiolabel entered oocytes in BSA supplemented medium. The majority of label in the oocytes, when cultured with either medium, was not displaced by excess radioinert estradiol-17 beta or progesterone, nor were the oocytes saturated even when cultured in 10(-6) M estradiol-17 beta. Autoradiography of sectioned oocytes after culture in 3H-estradiol-17 beta has shown that there was no selective accumulation of silver grains over the germinal vesicle as was the case with granulosa cell nuclei. This observation suggests that estradiol-17 beta may not act at the level of the oocyte nucleus.     

In vitro metabolism of testosterone by cultured Sertoli cells and the effect of FSH.

Cultures of Sertoli cells isolated from testes of 18-and 36-day-old Long Evans rats were used to investigate their capacity to metabolize testosterone and the effect of FSH on such metabolism. Three different approaches were used: 1) investigation of the metabolism of radiolabeled testosterone under saturating substrate conditions; 2) study of the metabolism of radiolabeled testosterone utilizing trace amounts of high specific activity substrates; 3) the utilization of radioimmunoassay for measurement of estradiol-17 beta. The following steroids were isolated and identified by recrystallization to constant specific acitvity from the control and FSH-treated cultures; testosterone (unconverted substrate), androstenedione, dihydrotestosterone, 3 alpha-hydroxy-5 alpha-androstan-17-one and 5 alpha-androstane-3 alpha, 17 beta-diol. Radioimmunoassay data suggests that the Sertoli cells produce an estradiol-17 beta-like compound from unlabeled testosterone and that this production is stimulated by FSH. However, the radioactive metabolite from all our studies that behaved chromatographically like estradiol--17 beta failed to crystallize to constant specific activity, while in each experiment, authentic radiolabeled estradiol-17 beta added as recovery tracer did. The data demonstrate that : 1) cultures of Sertoli cells from immature rats have 5 alpha-reductase, 3 alpha- and 17 beta-hydroxysteroid oxidoreductase activities; 2) these enzymes may be affected by FSH; 3) based on radiolabeled metabolic techniques, Sertoli cells were unable to biotransform testosterone to estradiol-17 beta even in the presence of FSH.     

Variation of steroid concentrations during the reproductive cycle of the clam Ruditapes decussatus: a one year study in the gulf of Gabès area

Progesterone, testosterone and estradiol-17beta were measured by radio-immunoassay (RIA) in the gonads of the clam Ruditapes decussatus. The reproductive cycle was also investigated. Our study covered a period of one year, from September 2003 to August 2004. The chosen site "Kerkennah", located out of industrial effluents, belongs to the gulf of Gabès area (Tunisia). Steroids varied from 178 to 2459 pg g(-1) wet mass for progesterone, from 40 to 326 pg g(-1) wet mass for testosterone and from 10 to 235 pg g(-1) wet mass for estradiol-17beta in females. However in males, these steroids ranged from 304 to 2303 pg g(-1) wet mass for progesterone, from 81 to 381 pg g(-1) wet mass for testosterone and from 48 to 168 pg g(-1) wet mass for estradiol-17beta. The reproductive cycle of R. decussatus, investigated by histological examination of gonadic sections, showed that gametogenesis occurred from April to February in males and from April to November in females. Progesterone and testosterone increased at the end of gametogenesis in both sexes. The highest estradiol-17beta was recorded at the beginning of vitellogenesis in females. Fluctuations in the levels of sex steroids during the reproductive cycle suggest their possible role as endogenous modulators of gametogenesis in R. decussatus. Although this species is considered as gonochoristic, 0.83% of hermaphrodites were observed.     

Estradiol-17 beta interference with meiotic maturation in Rana pipiens ovarian follicles: evidence for inhibition of 3 beta-hydroxysteroid dehydrogenase.

Increasing doses of estradiol-17 beta added to in vitro incubations inhibited pregnenolone-induced germinal vesicle breakdown in Rana pipiens ovarian follicles. The inhibition was reversed with increasing concentrations or pregnenolone added to the medium. Because no evidence of estradiol-17 beta inhibition or interaction with progesterone-induced GVBD was observed, the effect of estradiol-17 beta on the conversion of 3H-pregnenolone to 3H-progesterone was investigated. Estradiol-17 beta in doses as low as 10(-7) M significantly inhibited the conversion of 3H-pregnenolone to 3H-progesterone in follicles incubated in vitro. It is suggested that estradiol-17 beta is a feedback inhibitor of 3 beta-hydroxysteroid dehydrogenase-isomerase, the enzyme complex that converts pregnenolone to progesterone, a necessary step in the initiation of GVBD.     

Validation of radioimmunoassay systems for the measurement of 11-keto- and 11 beta-hydroxytestosterone in teleost blood

Highly specific antisera for 11-keto- and 11 beta-hydroxytestosterone have been raised in sheep. Assay systems for the simultaneous measurement of 11-ketotestosterone, 11 beta-hydroxytestosterone, testosterone, progesterone and estradiol-17 beta were validated for Ictalurus nebulosus plasma and Carassius auratus serum. In males of both species 11-ketotestosterone and testosterone were the major steroids detected. In females, testosterone and estradiol-17 beta were the predominant steroids measured. Data from samples taken at different stages of the annual cycle suggest that seasonal fluctuations in gonadal steroid secretion occur in I. nebulosus and C. auratus.