Hormonal regulation of activities of 4-ene-5 beta and 5 alpha-reductases and 17 beta-ol-dehydrogenase in immature golden hamster ovary

Diethylstilbestrol (DES) pellets were implanted in female golden hamsters on day 22 after birth. Hamsters with or without the DES pellet were hypophysectomized on day 23. Starting from day 26, the hypophysectomized hamsters were injected daily with 2.3-40 micrograms NIH-LH-S19, 6 or 18 micrograms NIAMD-oFSH-13, 50 micrograms NIAMD-Rat-FSH-B-1, or saline for 3 days. Ovarian homogenates from these hamsters on day 29 were incubated with [14C]-4-androstene-3,17-dione and enzyme activity (nmol/g/h) was estimated. The 5 alpha- and 5 beta-reductase activities decreased significantly following hypophysectomy. In the hypophysectomized hamster ovary, a distinct response to LH but not to FSH or DES in the 5 alpha-reductase activity was found. On the other hand, the 17 beta-ol-dehydrogenase activity was stimulated by FSH but not by LH or DES. The 5 beta-reductase activity was stimulated by DES, FSH or 2.3 micrograms LH but not by 7-40 micrograms LH. In the DES-treated, hypophysectomized hamster ovary, LH and FSH stimulated the 5 alpha-reductase and 17 beta-ol-dehydrogenase activities, respectively, but FSH or LH treatment had no significant effect on the 5 beta-reductase activity. These results show that the 5 alpha-reductase activity is regulated by LH, while the 17 beta-ol-dehydrogenase activity is stimulated by FSH in immature golden hamster ovary. The 5 beta-reductase activity seems to be regulated predominantly by FSH but the effect of FSH is largely mediated by estrogen.     

Effects of hyperprolactinemia on FSH- or LH-induced activities of 17 beta-oxidoreductase, 5 alpha-reductase, and 17-hydroxylase in hypophysectomized rat testes

Two pituitaries from 7-week-old female rats (Sprague-Dawley strain) were grafted under the capsule of the left kidney of a 49-day old male rat. The pituitary grafted and sham-operated rats were hypophysectomized at 56 days of age. The hypophysectomized rats were given daily injections of NIAMDD-oFSH-13 (20 micrograms/0.5 ml saline), NIAMDD-oLH-23 (9 micrograms/0.5 ml saline) or saline for 4 days starting from day 58. The treated rats and normal male rats were killed at 61 days of age. Testicular homogenates were incubated with [14C]4-androstene-3, 17-dione or [3H] progesterone, and enzyme activities per testes were estimated. Hypophysectomy caused significant decreases in activities of testicular 17 beta-oxidoreductase and 17-hydroxylase. The decreased activity of 17 beta-oxidoreductase was significantly stimulated by FSH or LH treatment, whereas the decreased 17-hydroxylase activity was stimulated only by LH treatment. Although pituitary grafts alone showed little or no effect on these enzyme activities in the hypophysectomized rats, the grafts significantly inhibited FSH-stimulated 17 beta-oxidoreductase activity and the LH-stimulated 17 beta-oxidoreductase and 17-hydroxylase activities but enhanced LH-induced 5 alpha-reductase activity. The present results confirm previous findings that an excess of prolactin directly inhibits LH-stimulated 17-hydroxylase activity but enhances LH-induced 5 alpha-reductase activity in the rat testis. The present results also demonstrate that the same grafts directly inhibit FSH-stimulated 17 beta-oxidoreductase activity but have no effect on FSH-induced 5 alpha-reductase activity.     

Hyperprolactinemia enhances LH-stimulated 4-ene-5 alpha-reductase activity but inhibits LH-induced 17-hydroxylase activity in testes of hypophysectomized immature rats

Two pituitaries from 7-week old female rats (Sprague-Dawley strain) were grafted under the capsule of the left kidney of 21-day old male rat. The pituitary grafted and sham-operated rats were hypophysectomized at 27 days of age. The hypophysectomized rats, in groups of 4, were given daily injections of 9 micrograms NIAMDD-oLH-23 (minimum effective dose) or saline for 3 days starting from day 29. Testicular homogenates were incubated with [3H]progesterone or [14C]4-androstene-3,17-dione, and enzyme activities per testes were estimated. Testicular HCG-binding sites were also measured. Hypophysectomy caused significant decreases in activities of testicular 5 alpha-reductase, 17-hydroxylase, and 17 beta-hydroxysteroid oxidoreductase. These decreased enzyme activities were significantly stimulated by LH treatment. Although pituitary grafts alone showed no effects on these enzyme activities in the testes of the hypophysectomized rats, the grafts significantly enhanced LH-stimulated 5 alpha-reductase activities but inhibited LH-stimulated 17-hydroxylase activity. Testicular LH/HCG receptors were significantly increased by the grafts, especially in the presence of LH, without affecting affinity for HCG. The present results demonstrate for the first time that hyperprolactinemia directly stimulates LH-stimulated 5 alpha-reductase activity in rat testes. The results also show that the same grafts directly inhibit LH-stimulated 17-hydroxylase activity, probably via postreceptor mechanisms.     

Effects of hyperprolactinemia on activities of 17-hydroxylase, 17 beta-ol-dehydrogenase and 5 alpha-reductase in neonatally grafted and host testes in mice

Male and female (WB X C57BL/6)F1 hybrid mice were used. Two pituitaries from 60-80-day-old female mice were grafted under the capsule of the left kidney of 60-80-day-old male mice. One week after grafting, 2 testes from neonatal mice were grafted under the capsule of the right kidney of the grafted mice and 70-90-day-old intact male mice. The grafted and host testes, in groups of 10-26, were removed 15, 30, 40, 60 and 120 days after transplantation of the neonatal testes. Testicular homogenates were incubated with [3H]progesterone or [14C]4-androstene-3,17-dione, and enzyme activities per g tissue were estimated. Significantly elevated prolactin levels, slightly lower LH levels and normal testosterone levels were found in the mice with pituitary grafts, compared with those in the mice without pituitary grafts. Activities of 17-hydroxylase and 17 beta-ol-dehydrogenase increased clearly with age in the grafted testes in the mice without pituitary grafts, though the increases were inhibited significantly by the pituitary grafts. However, the pituitary grafts had no significant effect on activities of 17-hydroxylase and 17 beta-ol-dehydrogenase in the host testes under similar gonadotrophic stimulation. 5 alpha-Reductase activities in the grafted and host testes were unaffected by the pituitary grafts. These results show that hyperprolactinemia may directly inhibit increases in activities of 17-hydroxylase and 17 beta-ol-dehydrogenase with testicular age in neonatally grafted testes in mice.     

Hormonal regulation of activities of 4-ene-5 beta- and 5 alpha-reductases and 17 beta-hydroxy-dehydrogenase in immature golden hamster testis

We have reported [1-3] in immature golden hamster testis that 5 beta-reductase is localized in the tubular nongerm cells, while 5 alpha-reductase is present in the interstitial tissue and that the 17 beta-hydroxy-dehydrogenase activity is found predominantly in the tubular nongerm cells. Hormonal regulation of these enzyme activities was examined in the present study. Male golden hamsters were hypophysectomized on day 22 after birth. The hypophysectomized hamsters in groups of 3-8 were injected daily with 10 micrograms NIH-LH-S19, 50 micrograms NIAMD-Rat-FSH-B-1, 8 or 16 micrograms NIAMD-oFSH-13, 8 micrograms NIAMD-oFSH-13 plus 5 or 10 micrograms NIH-LH-S19, 1 mg testosterone propionate or saline for 5 days starting from day 23. Testicular homogenates of the treated hamsters and intact hamsters on day 28 were incubated with [14C]4-androstene-3,17-dione and NADPH, and enzyme activity (nmol/testes/h) was estimated. The activities of 5 beta- and 5 alpha-reductases and 17 beta-hydroxy-dehydrogenase decreased significantly 6 days after hypophysectomy. In the hypophysectomized hamster testis, a distinct response to FSH but not to LH in the activities of 5 beta-reductase and 17 beta-hydroxy-dehydrogenase was found. The injection of LH in addition to FSH showed no significant additive effects on these enzyme activities. The 5 alpha-reductase activity was stimulated significantly by LH plus FSH but not by LH alone, FSH alone or androgen. These results show that 5 beta-reduction of 4-ene-3-ketosteroids takes place in the Sertoli cells under the influence of FSH while 5 alpha-reduction occurs in the interstitial cells under the influence of LH and FSH in immature hamster testis.     

Intratubular localization of 4-ene-5 beta-reductase and 17 beta-hydroxy-dehydrogenase in immature golden hamster testis

We have reported [1,2] in immature golden hamster testis that 5 beta-reductase is localized in the seminiferous tubules, while 5a-reductase is present in the interstitial tissue and that the 17 beta-ol-dehydrogenase activity is found predominantly in the seminiferous tubules. In the present study, we show the intratubular localization of these enzymes. The left testis of golden hamster was irradiated with 2000R or 8000R of X-rays at 22 days of age. The hamsters were killed at 28 days of age. Homogenates of the left irradiated and right intact testes were incubated with [14C]-4-androstone-3,17-dione and NADPH, and enzyme activity was estimated. Both testes were also examined histologically. The X-irradiation of the testis resulted in an almost complete disappearance of germ cells with a significant decrease in testis weight, but the interstitial tissue and tubular nongerm cells including Sertoli cells remained almost unchanged. However, the activities of 5 beta-reductase and 17 beta-ol-dehydrogenase expressed as nmol formed/testis/h did not decrease at all. These results show that 5 beta-reductase is localized in the tubular nongerm cells including the Sertoli cells and 17 beta-ol-dehydrogenase is present in the tubular nongerm cells and interstitial tissue in immature golden hamster testis.     

Delay in the age of balano-preputial skinfold cleavage and alterations in serum profiles of testosterone, 5 alpha-androstane-3 alpha,17 beta-diol, and gonadotropins in adult rats treated during puberty with luteinizing hormone releasing hormone

Previous work has shown that chronic treatment of intact, immature male rats with luteinizing hormone releasing hormone (LHRH) decreases sex accessory gland weights and results in retardation of the normal developmental increase in the ratio of serum testosterone (T)/5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-Diol) via an apparent enhancement of testicular 5 alpha-reductase or 3 alpha-hydroxysteroid oxidoreductase activities. In the present work, androgen dependent balano-preputial skinfold cleavage was significantly delayed by approximately one week in intact, immature male rats which were treated daily for two weeks with either 1.0 micrograms, 2.5 micrograms or 5.0 micrograms of LHRH during a discrete phase of pubertal development (28-41 days of age). In intact, adult (62 day old) animals which received LHRH treatments during pubertal development, serum T concentrations and sex accessory gland weights were reduced compared to control animal values. Serum 3 alpha-Diol content in the adult rats was either unaltered or increased significantly depending on the LHRH dosage employed during sexual development. Serum luteinizing hormone concentrations were not different between control and LHRH-pretreated adult rats whereas the highest dosage of LHRH employed (5.0 micrograms) during puberty resulted in a significant elevation of adult serum follicle stimulating hormone levels. It is suggested that chronic LHRH treatment of the male rat during puberty results in a perturbation in testicular androgen biosynthetic activities and an impairment of pituitary-testicular hormone feedback mechanisms which persist at least through early adulthood.     

Time-studies of the changes in the sex-dependent activities of enzymes of hepatic steroid metabolism in the rat following gonadectomy.

The activities of cytoplasmic 3 alpha- and 17 beta-hydroxysteroid dehydrogenase, microsomal 3 alpha- and 3 beta-hydroxysteroid dehydrogenase and microsomal 5 alpha-reductase of rat liver were determined at different time points after gonadectomy on day 75 of life. Following testectomy the activities in male rats assume female values. However this change is relatively slow, 10--14 days being necessary for significant trends in individual activities to develop, and 40--60 days before the final level of activity is reached. The changes in enzyme activities after ovariectomy are only slight. The change in microsomal 5 alpha-reductase activity following gonadectomy of male rats is biphasic, the activity increasing initially to the normal female level before falling to the intermediate "neonatally androgen-imprinted" level. The reaction of 17 beta-hydroxysteroid dehydrogenase activity to testectomy and ovariectomy indicates that in the course of several years, during which we have investigated the behaviour of this enzyme in Chbb/THOM rats, the regulation of its activity has changed from one of oestrogen dependency to one of androgen dependency.     

Effects of gold on testicular steroidogenic and gametogenic functions in immature male albino rats

The present study was undertaken to evaluate the effects of gold chloride, a metallic earth salt, on steroidogenic and gametogenic functions of testis in immature rats. Immature rats of Wistar strain, were injected (s.c.) with gold chloride at the dose of 0.3 mg and 0.5 mg/kg body weight/day for 26 days. All the treated animals along with the vehicle-treated controls were sacrificed 24 hours after last injections. Testicular steroidogenic activity was evaluated by measuring the activities of two steroidogenic key enzymes, Delta5-3beta-hydroxysteroid dehydrogenase (Delta5-3beta-HSD) and 17-beta hydroxysteroid dehydrogenase (17-beta HSD). Gametogenic capacity was determined by counting the number of germ cells at stage VII of seminiferous cycle. Plasma levels of testosterone (T) was measured by radioimmunoassay (RIA). Administration of gold chloride at a dose of 0.3 mg/ kg body weight for 26 days led to insignificant changes of testicular Delta5-3beta-HSD,17beta-HSD activities and gametogenesis along with plasma T. In contrast 0.5 mg gold chloride treatment for 26 days caused a significant increase in plasma T (p < 0.001) along with stimulation of testicular Delta5-3beta-HSD activity (p < 0.001) and 17beta-HSD activity (p < 0.001). Gametogenic activity exhibited a significant increase in the number of step 7 spermatids (7Sd) (p < 0.001) at stage VII of seminiferous cycle when compared to control. The results of our experiment suggest that gold chloride treatment might be associated with significant stimulatory effects on testicular activities. Furthermore, since hormonal changes, altered steroidogenic enzymes and gametogenic activities were evident to a specific dose of gold chloride treatment, our data may have some clinical implication on the stimulation of fertility.     

Study of the direct effect of LHRH agonist on testicular 17-hydroxylase and 5 alpha-reductase activities in non-hypophysectomized adult rats treated with an anti-luteinizing hormone serum

In order to study both direct and pituitary-mediated mechanisms of action of the LHRH analogue [D-Ser(TBU)6, des-Gly-NH2(10)]LHRH ethylamide upon testicular steroidogenesis in adult rat, we compared the effects of the agonist when administered alone or concomitantly with an anti-LH serum to non-hypophysectomized rats. Testicular steroid contents and in vitro progesterone and testosterone metabolism were determined. Anti-LH serum administration was able to prevent 5 alpha-reductase stimulation by the agonistic peptide, but not the inhibition of 17-hydroxylase activity. These data suggest that modulation of 17-hydroxylase involves both direct and pituitary-mediated processes, while 5 alpha-reductase stimulation is mainly if not only due to a pituitary-mediated mechanism.     

Prolactin involvement in regulation of testicular 5 alpha-reductase activity in the immature rat

Treatment of intact immature (25-day-old) rats with bromoergocryptine (BR), which suppressed prolactin (Prl) secretion, decreased testicular 5 alpha-reductase activity, whereas treatment with Prl increased the enzyme activity in BR-treated animals. Serum luteinizing hormone (LH) concentrations were not reduced by BR treatment or elevated by Prl, suggesting that the BR and Prl effects on enzyme activity were not due to alterations in LH secretion. Hypophysectomy (at 21 days of age) caused a dramatic decrease in testicular 5 alpha-reductase activity, and treatment with LH partially reversed this effect. Treatment of hypophysectomized animals with Prl alone had no effect on the enzyme activity but enhanced the effect of LH. Testosterone propionate, given to hypophysectomized animals in a regimen that increased testicular testosterone to concentrations at least as high as those in intact (sham-hypophysectomized) controls, had no effect on enzyme activity, whether given alone or in combination with LH. These results indicate that Prl is involved, along with LH, in maintaining the high 5 alpha-reductase activity of the prepubertal rat testis; the action of Prl, apparently requiring the presence of LH, may be to decrease the rate of degradation of the enzyme. The data also suggest that the action of LH on testicular 5 alpha-reductase activity is not mediated by its stimulation of testosterone production.     

Effect of injection of gonadotrophin-releasing hormone on testicular steroidogenesis in the hypogonadal (hpg) mouse

Hypogonadal (hpg) mice were injected once daily with 10 ng, 50 ng or 1 microgram GnRH for 5, 10 or 20 days or 12 times daily with 4.2 ng GnRH for 5 days. Basal and hCG-stimulated production in vitro of androstenedione, testosterone and 5 alpha-androstane-3 alpha,17 beta-diol (androstanediol) were measured by radioimmunoassay. All doses of GnRH increased testicular weight and in-vitro androgen production although seminal vesicle weights were unchanged and serum testosterone concentrations remained undetectable. After 5 days' treatment androstenedione and androstanediol were the dominant androgens produced, the latter indicating the presence of high levels of 5 alpha-reductase. By 20 days testosterone production was predominant after treatment with higher doses of GnRH. Total androgen production (androstenedione + testosterone + androstanediol) after 5 and 10 days was similar at all concentrations of GnRH used. After 20 days' treatment total androgen production was significantly greater with 50 ng GnRH/day than with 10 or 1000 ng/day. Multiple daily injections of 4.2 ng GnRH (total dose 50 ng/day) had no greater effect on androgen production in vitro compared to single daily injections of 50 ng. This suggests that under the conditions used in this study the testis does not require pulsatile release of the gonadotrophins. The pattern of [3H]pregnenolone metabolism was measured after 5 days injection of 50 ng GnRH/day. Compared to control hpg animals there was a significant increase in formation of C19 steroids, synthesis being solely through the 4-ene pathway. These results show that GnRH treatment of hpg mice will induce testicular steroidogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

The action of 5 alpha-dihydrotestosterone and antiandrogens on the activities of 5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase in the pituitary gland of gonadectomized rats

In gonadectomized rats of either sex s.c. administration of 5 alpha-dihydrotestosterone (DHT) reversed, in a dose dependent manner, effects brought about by gonadectomy: it decreased pituitary wet weight and serum levels of LH and FSH and suppressed microsomal enzyme activities involved in testosterone and progesterone metabolism in the pituitary gland, NADPH-linked 5 alpha-reductase and NADH-linked 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSDH). Concomitantly administered nonsteroidal antiandrogen, flutamide (5 mg/day), antagonized some of the suppressive effects induced by a 14-day treatment of gonadectomized rats with high dose (1 mg/day) of DHT. It completely blocked DHT action on pituitary 5 alpha-reductase activity in the female rat and, in the male, inhibition was found to be 30-35%. In male, but not female rats, it completely blocked DHT suppression of serum FSH level whereas it slightly, but significantly inhibited DHT suppression of serum LH in rats of either sex. However, flutamide did not prevent DHT suppression of pituitary wet weight or NADH-linked 3 alpha-HSDH activity. Concomitantly administered progestational antiandrogen, cyproterone acetate (5 mg/day), inhibited DHT-induced weight increase of seminal vesicles by 50-55% and completely blocked the weight decrease of pituitary gland but did not antagonize DHT suppression of serum gonadotropins or pituitary enzyme activities. The results obtained with flutamide suggest that DHT-induced suppression of pituitary NADPH-linked 5 alpha-reductase, but not NADH-linked 3 alpha-HSDH activity, might involve an androgen receptor mechanism.     

Characteristics of flutamide action on prostatic and testicular functions in the rat

The effect of daily treatment with the pure antiandrogen Flutamide has been studied either alone or in combination with the LHRH agonist [D-Trp6, des-Gly-NH2(10)]LHRH ethylamide (LHRH-A), on testicular and prostatic functions in adult male rats. Treatment for 10 days with Flutamide (5 mg/rat, twice daily) caused a marked stimulation of plasma testosterone (T) associated with a significant increase in plasma gonadotropin concentrations and inhibited plasma PRL levels. Testicular weight is not changed following antiandrogen administration but testicular LH/hCG receptor levels are markedly decreased with no change in FSH receptor levels. Moreover, Flutamide treatment alone produces an important inhibition of ventral prostate and seminal vesicle weights associated with a significant decrease in prostatic beta-adrenergic receptor levels but no change is observed in specific ornithine decarboxylase (ODC) activity. Daily LHRH-A treatment at the dose of 1 microgram/day for 10 days decreases plasma T to levels comparable to those found in orchiectomized men (0.30 +/- 0.5 ng/ml). This effect is associated with an almost complete loss of testicular LH/hCG receptors, a decrease in testicular weight, a significant increase in plasma gonadotropins and a marked inhibition of plasma PRL concentration. A relatively smaller inhibition of ventral prostate and seminal vesicle weights follows treatment with the LHRH agonist alone, this effect being accompanied by a significant reduction in beta-adrenergic receptor concentration but no change in prostatic ODC activity. Combination of the two drugs, however, caused a potent inhibitory effect on both ventral prostate and seminal vesicle weight to values similar to those found in castrated rats. The prostatic weight loss is accompanied by a marked fall in ODC activity and in the concentration of beta-adrenergic receptors. The present data clearly show that combined treatment with an LHRH agonist and a pure antiandrogen is highly effective in inhibiting, not only prostatic growth, but also two androgen-sensitive parameters of prostatic activity.     

Solubilization and partial characterization of rat epididymal delta 4-steroid 5 alpha-reductase (cholestenone 5 alpha-reductase).

Epididymal delta 4-steroid 5 alpha-reductase (cholestenone 5 alpha-reductase), the enzyme that catalyses the conversion of testosterone into the biologically active metabolite dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one), is a membrane-bound enzyme found in both nuclear and microsomal subcellular fractions. In order to characterize epididymal delta 4-steroid 5 alpha-reductase, it was first necessary to solubilize the enzymic activity. Of the various treatments tested, a combination of 0.5% (w/v) Lubrol WX, 0.1 M-sodium citrate and 0.1 M-KCl maintained enzymic activity at control values and solubilized 66% of total epididymal delta 4-steroid 5 alpha-reductase activity in an active and stable form. The sedimentation coefficient of solubilized delta 4-steroid 5 alpha-reductase, as determined in continuous sucrose density gradients, was greater for the microsomal than for the nuclear enzyme (11.6S compared with 10.1S). Although the apparent Km values of the enzyme for testosterone were similar in nuclear and microsomal subcellular fractions (range 1.75 x 10(-7) - 4.52 x 10(-7)M), the apparent Km of the enzyme for NADPH was about 30-fold greater for the microsomal enzyme than for the nuclear enzyme. The apparent Km of the enzyme for either substrate was not significantly altered after solubilization. The relative capacity of steroids to inhibit the enzymic activity, the pH optima and the effects of Ca2+ and Mg2+ were similar for membrane-bound and solubilized delta 4-steroid 5 alpha-reductase in both the nuclear and the microsomal fractions. The results reported demonstrate that epididymal delta 4-steroid 5 alpha-reductase can be solubilized in an active and stable form with no significant changes in the kinetic characteristics of the enzyme after solubilization; furthermore, kinetic and molecular-size differences observed for the nuclear and the microsomal forms of the enzyme suggest that there may exist at least two forms of epididymal delta 4-steroid 5 alpha-reductase.     

Properties of 4-ene-5 alpha-reductase and studies on its solubilization from porcine testicular microsomes

The activity of 4-ene-5 alpha-reductase was assayed in porcine testis homogenates and subcellular fractions, using testosterone as substrate. 'Marker' enzyme activities were utilized to indicate the purity of the subcellular fractions. 4-Ene-5 alpha-reductase activity was associated with the microsomal fraction; there was no activity in the purified nuclear fraction. Enzyme activity was higher in the testes of 6 week old pigs than those of 3 and 17 week old animals, and a range of activity was found. The enzyme was unstable when stored at -20 degrees C but the addition of albumin (0.1%, w/v) or glycerol (20%, v/v) to the buffer and storage at -70 degrees C or in liquid nitrogen ensured that maximal activity was retained for at least 35 days. In addition to 5 alpha-DHT, other 5 alpha-reduced metabolites and 4-androstenedione were formed in this reaction; NADPH was the preferred cofactor, but 40% of the 4-ene-5 alpha-reductase activity was retained when NADH was used. Solubilization of the microsomal enzyme was achieved using sodium citrate (0.1 M); 4-ene-5 alpha-reductase activity was enhanced to greater than 120% and 60% of this activity was in the soluble fraction. The optimum pH and temperature for both soluble and membrane-bound 4-ene-5 alpha-reductase were 6.9 and 32 degrees C, respectively. The mean apparent Km and Vmax were 0.6 mumol/l and 158 pmol/min/mg microsomal protein for the microsomal enzyme and 1.42 mumol/l and 212.0 pmol/min/mg soluble protein for the solubilized 4-ene-5 alpha-reductase. The estimated sedimentation coefficient was 11.6.     

Fetal lamb 3 beta, 20 alpha-hydroxysteroid oxidoreductase: dual activity at the same active site examined by affinity labeling with 16 alpha-(bromo[2'-14C]acetoxy)progesterone

3 beta,20 alpha-Hydroxysteroid oxidoreductase was purified to homogeneity from fetal lamb erythrocytes. The Mr 35,000 enzyme utilizes NADPH and reduces progesterone to 4-pregnen-20 alpha-ol-3-one [Km = 30.8 microM and Vmax = 0.7 nmol min-1 (nmol of enzyme)-1] and 5 alpha-dihydrotestosterone to 5 alpha-androstane-3 beta, 17 beta-diol [Km = 74 microM and Vmax = 1.3 nmol min-1 (nmol of enzyme)-1]. 5 alpha-Dihydrotestosterone competitively inhibits (Ki = 102 microM) 20 alpha-reductase activity, suggesting that both substrates may be reduced at the same active site. 16 alpha-(Bromoacetoxy)progesterone competitively inhibits 3 beta- and 20 alpha-reductase activities and also causes time-dependent and irreversible losses of both 3 beta-reductase and 20 alpha-reductase activities with the same pseudo-first order kinetic t1/2 value of 75 min. Progesterone and 5 alpha-dihydrotestosterone protect the enzyme against loss of the two reductase activities presumably by competing with the affinity alkylating steroid for the active site of 3 beta,20 alpha-hydroxysteroid oxidoreductase. 16 alpha-(Bromo[2'-14C]acetoxy) progesterone radiolabels the active site of 3 beta,20 alpha-hydroxysteroid oxidoreductase wherein 1 mol of steroid completely inactivates 1 mol of enzyme with complete loss of both reductase activities. Hydrolysis of the 14C-labeled enzyme with 6 N HCl at 110 degrees C and analysis of the amino acid hydrolysate identified predominantly N pi-(carboxy[2'-14C]methyl)histidine [His(pi-CM)].(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of gonadotropins on adrenalectomy induced changes in the testis of albino mouse

Effects of adrenalectomy and administration of gonadotropins on cell counts of different cell types of spermatogenesis and morphology of the Leydig cells were studied in 30 day old mice. Adrenalectomy (duration, 12 days; age at autopsy 42 days) caused a significant decrease in the diameters of seminiferous tubules and Leydig cell nucleus and, cell counts of intermediate spermatogonia, round and elongated spermatids. Administration of FSH (75 micrograms/0.1 ml saline) + LH (25 micrograms/0.1 ml saline) everyday for 12 days to adrenalectomized mice restored testicular activity as revealed by significant increases in mean diameter of the Leydig cell nuclei and cell counts of intermediate spermatogonia and elongated spermatids over those of adrenalectomized mice. The results indicate that (i) testis of adrenalectomized mouse responds to gonadotropin treatment and (ii) impairment in gonadotropin secretion is possibly a major factor in inducing testicular regression following adrenalectomy.     

Influence of experimental cryptorchidism on cholesterol side-chain cleavage enzyme and delta5-3beta-hydroxysteroid dehydrogenase activities in rat testes.

Testicular cholesterol side-chain cleavage enzyme (CSCCE) and delta5-3beta-hydroxysteroid dehydrogenase (delta5-3beta-HSD) activities were assessed 12 hours and 2, 4, 8, 16, and 32 days after surgical induction of bilateral cryptorchidism in adult rats. Within 12 hours after surgery CSCCE activity (expressed as dpm of isocaproic acid-14C formed from cholesterol-26-14C/3 hours/testis) was significantly reduced (P less than 0.01) in cryptorchid testes to approximately 55% of sham-operated control values and remained depressed at less than 50% of control activities 2, 4, 16, and 32 days after surgery. Cryptorchid testis delta5-3beta-HSD activity (measured by a pregnenolone substrate-depletion assay and expressed as mumoles of products/30 minutes/testis) did not differ from controls (P greater than 0.05) 1/2, 2, or 4 days after translocation of testes to the abdominal cavity. By day 8 of cryptorchidism, however, delta5-3beta-HSD activity was reduced to 60% of control values (P less than 0.05) and continued to decline to approximately 30% of controls during the remainder of the experimental period. These observed alterations in enzyme activities suggest an impairment in the ability of cryptorchid rat testes to synthesize androgens and further indicate that testicular CSCCE is more acutely sensitive to the cryptorchid milieu than delta5-3beta-HSD.     

Effect of 5-thio-D-glucose on testosterone biosynthesis in vivo in mice testes

Testicular synthesis of (14C)cholesterol and (14C)testosterone from (14C)acetate were investigated in mice treated with 5-thio-D-glucose at a dose of 33 mg/kg body weight/day for 21 days. The testicular synthesis of free cholesterol as well as steroids were significantly decreased. The steroid synthesizing enzymes, cholesterol esterase, cholesterol side-chain cleaving enzyme, total alpha-hydroxysteroid dehydrogenase and total beta-hydroxysteroid dehydrogenase, were also analysed. Cholesterol esterase and total beta-hydroxysteroid dehydrogenase were significantly reduced whereas total alpha-hydroxysteroid dehydrogenase was unaffected. Hence, a decrease in free cholesterol for steroid synthesis and a decreased activity of the steroidogenic enzyme, beta-hydroxysteroid dehydrogenase, were responsible for the diminished synthesis of testosterone.