The skin of the male African catfish, Clarias gariepinus: a source of steroid glucuronides

Steroid metabolism in the skin of mature male African catfish, Clarias gariepinus, reared in the laboratory, was studied in vitro by tissue incubations with [3H]pregnenolone, [3H]dehydroepiandrosterone, [3H]17 alpha-hydroxyprogesterone, [3H]androstenedione, [14C]11 beta-hydroxyandrostenedione, and [3H]testosterone as precursors. While pregnenolone was not converted to any other steroid, dehydroepiandrosterone was transformed mainly to 5-androstene-3 beta, 17 beta-diol. The products of 17 alpha-hydroxyprogesterone incubations were 5 beta-pregnane-3 alpha,17 alpha-diol-20-one, 5 beta-pregnane-3 alpha,17 alpha, 20 beta-triol, and 5 beta-pregnan-17 alpha-o1-3,20-dione. The major steroids of androstenedione incubations were etiocholanolone, testosterone, and androsterone. Testosterone was converted mainly to etiocholanolone and androstenedione, and only small quantities of 11 beta-hydroxytestosterone, 11-ketotestosterone, and 11-ketoandrostenedione were the metabolites found in 11 beta-hydroxyandrostenedione incubation. These results demonstrated the presence of the enzymes 5 alpha- and 5 beta-reductases and 3 alpha-, 11 beta-, 17 beta-, and 20 beta-hydroxysteroid dehydrogenases in the skin. From enzymehistochemical results it appeared that the steroid conversions take place in the epithelial cells. Moreover, the presence of UDP-glucose dehydrogenase, an enzyme involved in the synthesis of glucuronic acid, in these cells indicates the possibility of steroid glucuronide formation. Indeed significant amounts of water-soluble steroid conjugates, particularly 5 beta-dihydrotestosterone- and testosterone-glucuronide, were found in the incubations with androstenedione and testosterone, indicating the presence of the UDP-glucuronosyl transferase in the catfish skin. In the light of these results, a role of the skin of African catfish in the production of semiochemicals having pheromonal properties is discussed.     

Steroid metabolism in the testes of the breeding and nonbreeding three-spined stickleback, Gasterosteus aculeatus

Steroid metabolism in the testes of sticklebacks was studied in vitro by tissue incubations with [3H]pregnenolone or [3H]androstenedione as precursors. In males in full reproductive condition (nesting), [3H]pregnenolone was mainly converted via progesterone and 17 alpha-hydroxyprogesterone into androstenedione, 11 beta-hydroxyandrostenedione, and 11-ketoandrostenedione. The latter was the largest product formed. The main products from the [3H]androstenedione incubation, 11 beta-hydroxyandrostenedione and 11-ketoandrostenedione, confirm these findings. The rate of androgen synthesis, especially of 11-ketoandrostenedione, was much lower after the end of the breeding season.     

Steroid metabolism in the seminal vesicles of the African catfish, Clarias gariepinus (Burchell), during the spawning season, under natural conditions, and kept in ponds

Steroid metabolism in the seminal vesicles of Clarias gariepinus, collected in the Hula nature reserve and in a fish pond in North Israel during the spawning season, was studied in vitro by homogenate and tissue incubations with [3H]pregnenolone, [3H]dehydroepiandrosterone, or [3H]androstenedione as precursor. The seminal vesicles were able to synthesize androgens, 11-oxygenated androgens, and 5 beta-reduced C19- and C21-steroids; a pregnenolone ester was also formed. Furthermore a potent synthesis of steroid glucuronides, i.e., the glucuronides of testosterone, 5 beta-dihydrotestosterone, 5 beta-androstan-3 alpha, 17 beta-diol, and etiocholanolone, was observed. When reared in ponds, the African catfish fails to spawn. This might be caused by a change in steroid glucuronide (pheromone) production by the seminal vesicles, as it appeared that the synthesis of glucuronides differs in "wild" and "pond" animals. Etiocholanolone glucuronide is completely absent in pond animals.     

A quantitative study of steroid bioconversions in the testis of the African catfish, Clarias gariepinus (Burchell), under natural spawning and natural and cultivated non-spawning conditions

Quantitative aspects of bioconversions in the testes of the African catfish (Clarias gariepinus) were studied in vitro by incubation of tissue with [3H]pregnenolone or [3H]androstenedione. During the breeding period, spawning and non-spawning animals were collected from their natural habitat, the Hula nature reserve, in northern Israel. In the same period, non-spawning animals were collected from a fish pond in the same region. It was shown that spawning was accompanied by significant changes in steroid bioconversions, i.e. a reduction in androgen synthesis, especially of 11 beta-hydroxyandrostenedione and 11 beta-hydroxytestosterone and an increase in the production of C21-steroids, especially progesterone, 17 alpha-hydroxyprogesterone and a pregnenolone ester. These changes resulted from a decreased contribution of the cytochrome P-450 enzymes 17 alpha-hydroxylase, C17-20-lyase and 11 beta-hydroxylase. A rise in plasma gonadotrophin concentration was observed only in spawning catfish. In the absence of such an increase in plasma gonadotrophin, steroid synthesis in the testes of non-spawning feral and pond catfish was primarily directed towards the production of 11-oxygenated androgens and 5 beta-pregnane-3 alpha,17 alpha,20 alpha-triol. It is suggested that spawning is induced by gonadotrophin and the ensuing change in steroidogenesis. It is possible that husbandry conditions inhibit the necessary increase in gonadotrophin release.     

The biosynthesis of 11-ketotestosterone by the testis of the Siamese fighting fish Betta splendens Regan (Anabantoidei, Belontiidae)

Testicular tissues of the Siamese fighting fish were incubated with [14C]pregnenolone for 10, 20, 30, 50, 80, and 120 min, and with [14C]progesterone, [14C]11 beta-hydroxyandrostenedione, [14C]11 beta-hydroxytestosterone, and [14C]androstenetrione for 120 min. 11-Ketotestosterone was the main metabolite in all 120-min incubations. No 11-oxygenated C21 steroids were found as metabolites of either pregnenolone or progesterone. The biosynthesis of 11-ketotestosterone proceeded through both the delta 5- and the delta 4- pathways as judged from the shape of the yield-time curves of the metabolites of pregnenolone. 11-Ketotestosterone formation from 11-oxygenated precursors increased in the order 11 beta-hydroxytestosterone less than 11 beta-hydroxyandrostenedione less than androstenetrione.     

Studies on the reproductive cycle on the female cobra, Naja naja. II. In vitro ovarian steroid synthesis.

The synthesis of steroids in vitro by minced ovarian tissue from the cobra, Naja naja, using [³H]pregnenolone and [³H]dehydroepiandrosterone([³H]DHA) as precursors was studied. From [³H]pregnenolone the major products were progesterone, pregnanolone (3α-hydroxy-5β-pregnan-20-one), 17α-hydroxyprogesterone, androstenedione, and testosterone. DHA and 17α-hydroxypregnenolone were tentatively identified, but insufficient material was available for positive characterization. From incubations using [³H]DHA as precursor, the only products identified were testosterone, androstenedione, and estradiol-17β. Significant amounts of radioactivity were associated with an estriol fraction from both the pregnenolone and the DHA incubations but were not further characterized. Time-lapse studies revealed an extremely rapid conversion of [³H] pregnenolone to progesterone, with a maximum occurring after 15 min in tissue taken from a cobra in April at the height of the reproductive period. Addition of cofactors to the medium markedly stimulated the synthesis of progesterone and pregnanolone from [³H]pregnenolone, but appeared to inhibit the production of other ovarian steroids. Mammalian LH, when added to the incubation medium, was found to stimulate the biosynthesis of 17α-hydroxyprogesterone, androstenedione, and testosterone from [³H]pregnenolone. Addition of fresh, homogenized snake pituitary or mammalian FSH appeared to increase the yield of testosterone but none of the precursors in the pathway, and there was a suggestion that FSH alone increased the rate of aromatization.     

The influence of mammalian and avian gonadotropins on in vitro ovarian steroid synthesis in the turtle (Chrysemys picta).

The synthesis of steroids from 7α[³H]cholesterol and 7α[³H]pregnenolone by turtle ovarian tissues in vitro was studied. Pregnenolone, 17α-hydroxypregnenolone, progesterone, 17α-hydroxyprogesterone, androstenedione, testosterone, dehydroepiandrosterone, estrone, estradiol 17β, estriol, and 16-epiestriol were identified as products. All estrogens were detectable in incubates of preovulatory follicular tissue, but only small quantities of estrone were found in incubates of follicular tissue from postovulatory animals and luteal tissue. The effects of mammalian and avian gonadotropins on the metabolism of tritiated precursors were studied. Both mammalian and avian LH were stimulatory when conversion of cholesterol or pregnenolone to major steroid products was examined. In particular, enhancement of estrogen biosynthesis predominated in preovulatory follicular tissue, whereas increased progestin yield was the major effect in follicular and luteal tissue from postovulatory animals. The effects of FSH were minimal compared to the same dose of LH. Thus, a slight increase in estrogen yield was only noted when preovulatory follicular tissue was incubated with cholesterol and mammalian FSH, and neither mammalian nor avian FSH had an effect on pregnenolone conversion by follicles from postovulatory animals. Prolactin had no effect on luteal progesterone synthesis when used alone, but reduced the stimulatory effect of mammalian LH on progesterone synthesis. 11-Desoxycorticosterone was not found to be a product of the turtle ovary under normal conditions or after in vitro ACTH stimulation.     

Steroid biosynthesis by the testes of the dogfish Scyliorhinus caniculus

Dogfish testes were incubated with radioactive progesterone, pregnenolone, and testosterone, and both free and conjugated metabolites were examined. In the free fraction, which contained 42–70% of the incubated radioactivity, progesterone, androstenedione, and testosterone were identified as incubation products of both progesterone and pregnenolone. In addition, a small amount of 17α-hydroxyprogesterone was identified as a metabolite of progesterone in one fish. Testosterone and androstenedione were the only free steroids isolated from incubations of testosterone. Although steroid glucuronide formation was insignificant, very large amounts of solvolysable steroids were isolated from all incubations. With pregnenolone and progesterone, 10–30% of the incubated radioactivity was recovered in this solvolysable fraction, in which the major products were identified as testosterone and 17α,20β-dihydroxy-4-pregnen-3-one. With two fish incubated with [¹⁴C]testosterone, 5α-androstane-3β, 17β-diol was isolated in low yield from the solvolysable fraction in addition to testosterone, but in one incubation with [³H]testosterone, the sole component of this fraction was testosterone which accounted for 21% of the initial radioactivity.     

Biochemical, histochemical, and ultrastructural analyses of presumed steroid-producing tissues in the sexually mature sea lamprey, Petromyzon marinus L

In vitro incubations of testicular, ovarian, and presumed adrenocortical tissues (PAT) from the mature sea lamprey, Petromyzon marinus, with [1,2-³H]cholesterol failed to form cortisol, cortisone, corticosterone, 11-deoxycortisol, 11-deoxycorticosterone, 17α-hydroxypregnenolone, 17α-hydroxyprogesterone, pregnenolone, and progesterone. “Isopolarity” and “isomorphicity” were establidhed for testosterone and androstenedione from the PAT incubation, but subsequent attempts at derivative formation indicated that no testosterone or androstenedione was formed. The interstitial cells of the testes and the cells within the ovarian follicles of P. marinus have a fine structure similar to those in other species of lamprey. The appearance of these tissues and of PAT remains unaltered after 4 hr of incubation. Histochemical procedures did not provide evidence for 3β-hydroxysteroid dehydrogenase (3β-HSD) activity in ovary and PAT, and only a weak 3β-HSD activity was observed over the interstitial tissue of the testes. Spectrophotometric evidence for 3β-HSD activity was obtained from PAT and testicular tissue homogenates. No 3β-HSD activity was observed in mature ovarian tissue homogenates.     

Testicular steroid metabolism during development in the normal and hypogonadal mouse

The patterns of testicular steroidogenesis were investigated during postnatal development in the normal mouse and in the hypogonadal (hpg) mouse from 20 days. The hpg mouse lacks GnRH and may be used to examine the function of this peptide in normal gonadal development. Testicular tissue was incubated with [3H]pregnenolone and metabolites were separated by thin-layer chromatography and high-performance liquid chromatography. In the normal mouse from 1 to 10 days, metabolism occurred predominantly through the delta 4 pathway, and progesterone, 17 alpha-hydroxyprogesterone, androstenedione and testosterone were the main metabolites formed, together with significant amounts of an unidentified polar steroid. Between 15 and 25 days, androstenedione became the major metabolite formed from pregnenolone. There was also a marked increase in 5 alpha-reductase activity during this age range, and 5 alpha-dihydrotestosterone and 5 alpha-androstane-3 alpha, 17 beta-diol were significant metabolites. In normal animals older than 30 days, testosterone became the major metabolite, and between 30 days and adulthood the pattern of metabolism changed significantly due to increased formation of intermediates from the delta 5 pathway. In the hpg mouse between 20 and 30 days, the pattern of steroid metabolism was unlike that of any age of the normal animal. Progesterone was the major metabolite formed and dehydroisoandrosterone was the major C19 steroid formed, although significant levels of androstenedione and testosterone were also formed. After 30 days there was a marked decrease in steroid metabolism, with androstenedione (the major androgen) being formed mainly through the delta 4 pathway.(ABSTRACT TRUNCATED AT 250 WORDS)     

Steroid metabolism in the yolk sac placenta and endometrium of the tammar wallaby, Macropus eugenii

Yolk sac and endometrial tissue were obtained from tammar wallabies between 11 and 25 days after the removal of pouch young. Tissues were examined histologically and steroid-metabolizing enzymes were identified by incubation for 3 h at 37 degrees C in Medium 199 containing labelled steroid precursors. Yolk sac membrane (YSM) incubated with labelled pregnenolone produced a small amount of progesterone and pregnanediols; 80.5 +/- 8.4 (S.E.M.) % of the original substrate remained unmetabolized. Labelled androstenedione was metabolized to 5 alpha-androstane-3,17-dione and androsterone, and only 5.8 +/- 3.8% of the original substrate remained at the end of incubation. Incorporation of androstenedione or dehydroepiandrosterone (DHA) into phenolic compounds was low (0.5 +/- 0.1%). There was no evidence for the enzymes, arylsulphatase or sulphotransferase, in YSM. Endometrial tissue from the same animals metabolized pregnenolone, DHA and androstenedione, converted progesterone to androstenedione, and produced aqueous-soluble steroid conjugates. The results demonstrated that YSM contains enzymes associated predominantly with steroid catabolism and with incipient progesterone synthesis. The findings are discussed in relation to the histological appearance of the tissues and compared with placental steroid synthesis in eutherian mammals.     

In vitro steroidogenesis by the nonzoned adrenocortical tissue of the skink, Tiliqua rugosa.

Steroid formation by adrenocortical tissue from the shink, Tiliqua rugosa, has been studied using established in vitro techniques. Both in conventional incubations, with timed sampling, and in incubations with dialysis, aldosterone, and corticosterone were major products. From endogenous precursors, and from [¹⁴C]acetate, yields of the two products were of the same order, whereas from [³H]pregnenolone maximal yields of corticosterone were at least tenfold greater than aldosterone. Maximal rates of steroid formation from the radioactive precursors occurred within the first few minutes of incubation, but maximal rates of steroid formation from endogenous precursors occurred significantly later, between 1–2 hr.In incubations with dialysis [¹⁴C]aldosterone was significantly less dialysable than [³H]aldosterone under all conditions, whereas [¹⁴C] and [³H]corticosterone were homogeneous. In contrast, neither aldosterone nor corticosterone formed from endogenous precursors were bound under control conditions, although binding was increased following dexamethasone pretreatment, and decreased following stimulation with Tiliqua pituitary extract (but not Synacthen), with concomitant changes in yields and specific activities.Inter alia the results suggest that products formed from [¹⁴C]acetate and from [³H]pregnenolone may be maintained in separate pools within the tissue, and this accounts for their different metabolic fates. The bound pool, penetrated only by [³H]acetate, yields more aldosterone than the free, and may be termed a “biosynthetic pool.” In addition there exists a “secretory reserve pool.” This is suggested by the difference between rates of steroid secretion from endogenous and added precursors, and also from the changes in dialysibility seen in steroids formed from endogenous precursors under different conditions of stimulation.In both the compartmental arrangement of steroids, and the production of large yields of aldosterone the adrenocortical tissue of Tiliqua shows similarities to the zona glomerulosa, but not the inner zones of the rat adrenal cortex.     

Suggestion of abnormal testicular steroidogenesis in some oligospermic men

Androgen biosynthesis in the testis may be analyzed in some detail by means of techniques of in vitro incubation of small testicular biopsy specimens with suitable radiolabelled precursors. Sixty-six tissue specimens from 33 patients who underwent bilateral testicular biopsies because of infertility were incubated in vitro with [3H]pregnenolone in order to investigate the possibility of abnormalities in their steroid biosynthetic activity. As a normal control, testicular tissue obtained by testicular biopsy from a young normal volunteer was used. The distribution of metabolites in the incubates of testes from 8 infertile men differed greatly from the remaining 25 patients and the normal control. The major steroids formed from pregnenolone by the testes of those 8 men were 17-hydroxypregnenolone, dehydroepiandrosterone, 20alpha-dihydropregnenolone and and 20alpha-dihydro-17-hydroxypregnenolone. Very small amounts of delta4-3 oxo products (progesterone, 17-hydroxyprogesterone, androstenedione and testosterone) were formed suggesting a deficiency of 3beta-hydroxy-steroid-dehydrogenase activity in the testes of these 8 men, possibly related to the derangement of their spermatogenic function.     

Activity of steroid C-17,20 lyase in the ovine placenta: effect of exposure to foetal glucocorticoid.

Microsomal fractions isolated from post-partum ovine placentae catalysed the synthesis of [3H]oestrone and [3H]oestradiol from [3H]17alpha-hydroxyprogesterone and NADPH; oestrone and oestradiol were formed in a ratio of approximately 50:1. The expected intermediate, [3H]androstenedione, did not accumulate during these incubations but was shown by trapping experiments to be the intermediate involved. Mean (+/- s.d.) uields of [3H]oestrone (% conversion of substrate) during incubation for 1 h of placentae from five animals in late pregnancy before the onset of labour, from five animals which delivered spontaneously at term and from four animals in which labour was induced by administration dexamethasone to the foetus were: in tissue obtained before labour, 3-2+/-0-44; in tissue obtained after the spontaneous onset of labour, 20-6+/-10-2 (P less than 0-01) and in tissue obtained after dexamethasone-induced labour, 24-4+/-2-13 (P less than 0-0001). This increase in oestrone synthesis suggests activation of steroid C-17,20 lyase, since this is the step limiting the rate of synthesis of oestrone in vitro. The enzyme is probably activated by foetal glucocorticoid. The findings are discussed in relation to the site of synthesis of oestrogens which in the sheep increase in concentration in the peripheral circulation at term, and with reference to a possible mechanism by which foetal glucocorticoid may control the onset of labour in this species.     

Steroidogenesis in the yellow corpuscles (adrenocortical homolog) in a holostean fish, the bowfin, Amia calva L

Yellow corpuscles from the ventral surface of the anterior kidney in bowfins (Amia calva L.) converted [7-3H]pregnenolone to radioactive 11-deoxycortisol, cortisol, and corticosterone in vitro. Aldosterone was not detected. Cortisol was the predominant steroid at the end of a 3-hr incubation period (20 degrees C). These experiments are the first to demonstrate steroidogenesis in holostean yellow bodies and they are the first incubations with pure adrenocortical tissue, free of head kidney, in any bony fish. White corpuscles of Stannius located along the total length of the kidneys were incubated under identical conditions but adrenocortical steroids were not found.     

Testicular steroidogenesis in the testicular feminized (Tfm) mouse: loss of 17 alpha-hydroxylase activity.

Testicular feminized (Tfm) mice are totally insensitive to androgen and may be used to study the role of the androgen receptor in normal development and function. We have examined testicular and Leydig cell steroidogenesis in Tfm mice. Serum bioactive LH was high in Tfm mice but serum testosterone was low and this was associated with a severe reduction in testicular testosterone production in vitro. Examination of [3H]pregnenolone metabolism by testes of Tfm mice indicated that progesterone, rather than testosterone, was the major steroid produced. Leydig cells were isolated from normal and Tfm mice and from normal mice in which testicular descent was surgically prevented before puberty. As in whole testes, androgen production in response to human chorionic gonadotrophin was severely reduced in Leydig cells from testes of Tfm mice compared with normal or cryptorchid groups. In contrast, progesterone production by Leydig cells from testes of Tfm mice was markedly increased in comparison with other groups. Total steroid production (progesterone plus androstenedione plus testosterone), however, was only 24% of normal in Leydig cells from Tfm mice. The pattern of steroid production by Leydig cells from cryptorchid testes was similar to control, although total steroid production was reduced to about 50% (this was significantly higher than the Tfm group, P less than 0.05). The high progesterone/androgen ratio in testes from Tfm mice suggested that 17 alpha-hydroxylase was depleted in these animals. To confirm this, activity of the four major steroidogenic enzymes associated with the smooth endoplasmic reticulum was measured.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro biosynthesis of 17 alpha,20 alpha,20 beta-dihydroxy-4-pegnen-3-one by the ovaries, testes, and head kidneys of the Atlantic salmon Salmo salar

Ovaries, testes, and head kidneys of sexually mature Atlantic salmon, Salmo salar, biosynthesized 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-diOHP) from equimolar amounts of [3H]pregnenolone plus [4-14C]progesterone in vitro. The 3H:14C isotope ratios of steroid metabolites indicated that the biosynthetic pathways to 17 alpha,20 beta-diOHP in the testes differed from those observed in the ovaries and head kidneys. [4-14C]Progesterone appeared to be the principal precursor of 17 alpha,20 beta-diOHP in the testes, whereas both precursors were efficiently biotransformed to 17 alpha,20 beta-diOPH in the ovaries and head kidneys. 17 alpha-Hydroxy-4-pregnen-3-one (17 alpha-OHP) was the immediate precursor to 17 alpha,20 beta-diOHP in all tissues. However, appreciable amounts of 17 alpha,20 beta-diOHP accumulated in vitro in the testes only in the presence of exogenous [14C]progesterone. Incubation of the testes, ovaries, and head kidneys with [14C]pregnenolone resulted in high yields of 17 alpha,20 beta-diOHP in the ovaries and head kidneys but no detectable amounts of the steroid in the testes. The results confirm that progesterone is the favored precursor to 17 alpha,20 beta-diOHP in the testes. The results also suggest that the head kidneys may be an excellent cellular source of 17 alpha,20 beta-diOHP in both male and female. Atlantic salmon and may play an important role in the sexual maturation process in this fish. It is suggested that biosynthetic control mechanism affecting 17 alpha,20 beta-diOHP synthesis and/or spermiation and ovulation may differ in male and female Atlantic salmon.     

The nature of the defect in familial male pseudohermaphroditism in Arabs of Gaza

Studies in six Arab individuals from Gaza with familial male pseudohermaphroditism (MPH) due to 17-ketoreductase deficiency revealed several metabolic aberrations associated with the disorder. Plasma LH, FSH, testosterone, and androstenedione concentrations were low in the two prepubertal patients. After hCG administration plasma androstenedione increased markedly. The four postpubertal MPH patients had very high plasma gonadotropin and androstenedione concentrations, the latter increasing further after hCG administration. Plasma testosterone concentrations in all six patients were moderately low or normal for age and increased little after hCG administration. Spermatic venous testosterone concentrations, measured in three adults, were within the normal range in two and low in one, while androstenedione concentrations were markedly elevated (15- to 32-fold) in all three patients. Kinetic analyses of progesterone and androstenedione metabolism were performed in testicular tissue of these patients and compared to the results in two control subjects. While testicular tissue from the two prepubertal patients metabolized progesterone only to androstenedione, and that to a limited extent, the tissue from the four postpubertal patients metabolized progesterone to 16 alpha- and 16 beta-hydroxyprogesterone, 17 alpha-hydroxyprogesterone, androstenedione, and testosterone and metabolized androstenedione to testosterone. The Michaelis constants of these reactions were similar in the tissue from the MPH and the control subjects. The production of 16 alpha- plus 16 beta-hydroxyprogesterone was 5.4- to 10.3-fold greater, and 17-hydroxylase activity was 5.8- to 8.1-fold lower in the testes of the postpubertal MPH patients compared to values in the control subjects. The preference of androstenedione production through the delta 4- or delta 5-pathways was examined in the testes of two adult MPH patients using an equimolar concentration of [14C]progesterone and [3H]pregnenolone as substrates. While the flow of substrates in the control testes was equal or slightly greater through the delta 4-pathway, the delta 5-pathway predominated in the testes of the MPH patients. A large amount of dehydroepiandrosterone accumulated when NAD, the cofactor for 3 beta-hydroxysteroid dehydrogenase-isomerase, was omitted, supporting the contention that androstenedione was produced in the testes of the MPH patients mainly through the delta 5-pathway. Additional support for this suggestion was the finding that the 3H/14C ratio in androstenedione and testosterone produced from both substrates was 8 times higher in the testes from MPH patients than in those from the control subjects.(ABSTRACT TRUNCATED AT 250 WORDS)     

The possible roles of membrane organization in the activity of androgen biosynthetic enzymes associated with normal or tumorous mouse Leydig cell microsomes.

The enzymes involved in conversion of pregnenolone to testosterone in Leydig cell tumors showed a wide distribution among smooth endoplasmic reticulum (SER), rough endoplasmic reticulum (RER), and cytosol, while these enzymatic activities in normal testes were associated primarily with smooth endoplasmic reticulum. Progesterone, used as a substrate in the presence of an NADPH-generating system, was metabolized to androstenedione and finally to testosterone by microsomes from some strains of tumor which did not form testosterone from exogenous labeled androstenedione. Treatment of microsomal membranes from normal testes with 0.1 M Ca++ and Mg++ caused a marked decrease in 17 beta-dehydrogenase activity, measured as conversion of exogenous [3H]androstenedione to [3H]-testosterone, without serious effects on activities of 3 beta-ol-dehydrogenase or 17 alpha-hydroxylase. Studies of initial velocity kinetics showed that treatment with magnesium ion resulted in a marked reduction in affinity of androstenedione for 17 beta-dehydrogenase while the maximum velocity was the same as in untreated microsomes. Also, experiments using [14C]progesterone and [3H]androstenedione simultaneously as substrates demonstrated that treatment with Mg++ ion made it more difficult for exogenous [3H]androstenedione to reach the active site of 17 beta-ol-dehydrogenase than [14C]androstenedione formed in the microsomal membrane from [14C]progesterone. Microsomal proteins were more easily solubilized and 3 beta-ol-dehydrogenase was more severely influenced by Mg++ ion in tumor membranes than in normal microsomes.     

In VitroBiosynthesis of Androgens in the Australian Lungfish,Neoceratodus forsteri

The serum concentration of testosterone was estimated from a population of wild lungfish over 6–7 years of sampling. Male lungfish were found to have high circulating levels of testosterone (∼50 ng/ml) which varied seasonally and could be correlated with spermatogenesis as judged by testis histology. Incubation of testis tissue slices with [³H]progesterone, [³H]17-hydroxyprogesterone, or [³H]testosterone confirmed that testosterone is the major androgen inNeoceratodus.Not even trace amounts of 11-keto- or 11β-hydroxytestosterone or 5α-dihydrotestosterone could be identified by TLC separations. There was little or no conjugation of steroids by the testes, except during the spawning season, when glucuronides of androstenedione and testosterone were produced.