Search

A comparison of the effects of perfusion and immersion fixation on localization of delta 53 beta-HSD in adrenocortical and testicular Leydig cells has been made. The results demonstrated that regardless of the mode of fixation, localization of the enzyme (smooth endoplasmic reticulum) and variability in staining intensity were similar in both tissues. Also noted was the uneven distribution of the staining reaction product within the cells. This finding was interpreted as an expression of regional disparity in metabolic activity rather than artefactual since this phenomenon was noted in both perfusion and immersion fixed tissue.

The adrenal homologue of C. batrachus is distributed around the postcardinal vein in the pronephric head kidney. The cortical cells are round or oval in shape. They showed positive reaction for total lipid, glycogen and ascorbic acid. Their intense delta5-3beta HSDH activity indicates their capacity for steroid biosynthesis. In addition, the cortical cells of C. batrachus exhibited strong G-6-PD, NADPH diaphorase, NADH diaphorase, MAO and weak SDH and LDH activity. The presence of MAO suggests the aminergic control of the adrenal in this species and the silver positive fibres seen the cortical cells were hypertrophied, degranulated and the lipid content was also decreased. The chromaffin or medullary cells were distributed in groups among the cortical cells. They are largely oval or angular in shape. They react positively to ferric ferricyanide, chromaffin and argentaffin reactions and ascorbic acid test.

The analysis of bile acids in biological samples has always presented a problem because of their complex nature and low concentration. Recently, newer analytical procedures for bile acids have become available, including enzymatic analysis, radioimmunoassay, thin-layer chromatography (TLC), gas chromatography, high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) with selected ion monitoring (SIM). However, they differ greatly with respect to specificity, sensitivity, accuracy and simplicity. On the other hand, the choice of analytical procedure differs according to the specific aims and the nature of biological samples to be analysed. These newer procedures have been compared in a double-blind fashion by distributing bile, plasma and urine samples to seven participating laboratories. GC-MS-SIM was found to be the most sensitive and reliable, but it requires other procedures for preliminary clean-up and fractionation steps. Enzymatic analysis is simple and gives small analytical errors but tends to over-estimate plasma bile acids. Radioimmunoassay gives variable results but is useful as a screening procedure for large numbers of plasma samples. TLC gives reliable results for biliary bile acids in experienced hands, except for differentiation between conjugated dihydroxycholanoic acids. HPLC, whether using derivatization or with fixed 3 alpha-hydroxy steroid dehydrogenase detection, is suitable for the analysis of major bile acids in normal human serum but not for the identification of unknown minor peaks.

Testes of foetal pigs between 26 to 35 days post coitum (p.c.) were investigated histochemically and ultrastructurally. Diaphorase and delta 5-3 beta-hydroxysteroid dehydrogenase activities were studied using, respectively, NADH and pregnenolone and dihydroxy androsterone as substrates. Ultrastructurally, attention was focused on the development of mesenchymal cells and on the sustentacular cells in the primitive sex cords in an attempt to detect the origin of Leydig cells. Histochemically there is a concentration of activity toward the interstitium with increasing age. Also the reactions increase in intensity. Ultrastructurally no evidence for Leydig cell development from Sertoli cells could be observed. Mesenchymal cells between the sex cords show a development toward Leydig cells. This is absent in mesenchymal cells in the future tunica albuginea. Before 30 days p.c. no 'true' Leydig cells can be observed morphologically. The role of the rough endoplasmic reticulum/mitochondrial complex, which is present in many mesenchymal and sustentacular cells, is discussed.

A sensitive two-step bioluminescent assay for total serum bile acids was developed using commercially available enzymes. In the first step, the bile acids present in 10 microL of alkali-treated serum were oxidised at pH 9.5 by high purity 3 alpha-hydroxysteroid dehydrogenase to form NADH. Then, NADH was quantitated at pH 6.5 under optimal conditions for bioluminescence using FMN:NADH oxidoreductase and luciferase from Photobacterium fischeri. The enzyme/bioluminescent assay correlated well with gas-liquid chromatography and radioimmunoassay methods. Assay of fasting sera in eight healthy subjects revealed cyclic fluctuations in bile acid concentrations which were inversely related to gallbladder volume. These results provide biochemical evidence for interdigestive partial gallbladder emptying as a normal physiological process.

Pre- and postovulatory ovarian follicles of the amago salmon were investigated with special consideration given to the steroid production site. In the preovulatory specimens, the special thecal cells contained abundant mitochondria and smooth endoplasmic reticulum, as in the case of other steroid-producing cells, while the granulosa cells developed rough endoplasmic reticulum like protein-secreting cells. Remarkably ultrastructural changes occurred in the granulosa and the special thecal cells during the oocyte maturation stage; dilation of rough endoplasmic reticulum in the former cell and further development of smooth endoplasmic reticulum in the latter one. Histochemical reactions of delta 5-3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity were only positive in the special thecal cells of the postovulatory follicles. These results strongly suggest that the special thecal cells are the major sites of estrogen precursor synthesis in the amago salmon ovary.

Ovarian development was studied in control and decapitated pig fetuses. Fetuses were decapitated at 42 days postcoitum. At 51, 61, 74, 90 and 112 days postcoitum decapitated and control females were collected. Ovarian weight gradually increased during development in control animals. Deprivation of pituitary hormones as a result of fetal decapitation did not cause a decline in ovarian weight increase. Germ cell maturation in control and decapitated fetuses proceeded in a similar fashion, with secondary follicles being the most advanced stage. Enzyme histochemical activity was present in the primary interstitial gland cells and in granulosa cells and was similar in normal and decapitated fetuses. Both NADH diaphorase activity and 3 beta-hydroxysteroid dehydrogenase activity increased from 51 to 74 days and remained relatively constant thereafter. Since fetal decapitation in the pig hardly influences ovarian development, pituitary dependency of the fetal ovary in the pig is unlikely.

The steroid hormone has an important role in the early stages of reproduction. There has been abundant histochemical evidence that oocytes contain steroid hormones and are able to synthesize these hormones. But there have been few methods of analyzing one oocyte biochemically because it is too small and light. In order to study steroidogenesis in the oocyte, a microassay method sensitive enough to analyze the enzyme activities in one oocyte was developed using enzymatic cyling for amplifying the reaction product to 10,000-fold. An oil-well technique and a microtube method were applied in the assay for achieving the reaction in a medium as small as 1.0 to 5.0 microliters under a stereomicroscope. Immature Wistar rats were superovulated by PMS-hCG administration. Oocytes were collected by puncturing the follicle and flushing the tube. They were freeze-dried after washing to remove cumulus cells. The dry weight of one oocyte was 51.2 +/- 6.2 ng in a quartz fiber fishpole balance. The activity of 3 beta hydroxysteroid dehydrogenase (3 beta HSD) (picomol/oocyte/hr, substrate:pregnenolone) in the PMS-treated oocyte was 2.66 +/- 0.59, which corresponds to 3 times the activity of the ovarian homogenate as control, indicating the high capacity of oocytes to produce progesterone. The activity increased significantly (P less than 0.01) by hCG administration up to 4.17 +/- 0.29 after ovulation, suggesting that gonadotropin regulates steroidogenesis in the oocyte. The activities of G6PD and 6PGD were 8.41 +/- 1.09 picomol/oocyte/min and 3.85 +/- 2.02 picomol/oocyte/hr, respectively. The high activity of G6PD (more than 10 times that of the ovarian homogenate) suggests that the pentose phosphate shunt concerned with steroidogenesis is active in the oocyte. HCG decreased the activities of both G6PD and 6PGD. The present results show that steroidogenesis in the oocyte is very active under the control of gonadotropin, suggesting that steroid hormones may play an important role in oocyte maturation, ovulation and fertilization.

With the potassium ferricyanide method, the localization of the sites of 3 beta-hydroxysteroid dehydrogenase activity has been investigated in the glandular tissue cells, previously fixed, of the Pleurodeles testis. An abundant copper ferrocyanide precipitate, the final reaction product, is observed in the vicinity of the external faces of the membranes of the smooth endoplasmic reticulum, or in contact with them. A very weak reaction occurs in mitochondrial cristae.

3 beta-hydroxysteroid dehydrogenase (3 beta-HDS) activity was demonstrated ultrastructurally in human corpora lutea obtained from the women aged 32-46 who were treated by the hysterectomy for various gynecological indications. The corpora lutea were removed from ovaries and cut into small pieces of 1 mm3. Specimens were incubated at 25 degrees C for 20 min. in the media containing dehydroepiandrosterone 0.6 mg, 0.1 M sodium citrate 0.3 ml, 5 mM potassium ferricyanide 1 ml, 30 mM copper sulfate 1 ml, N.A.D. 3.6 mg, sucrose 1.0 g and 0.1 M phosphate buffer (pH 7.2) 6 ml. For a control study, 1.5 mg cyanoketone (by the courtesy of Dr G.O. Pott), the specific inhibitor for 3 beta-HSD, was added to the media. The materials were fixed in 4% glutaraldhyde and 1% OsO4 and embedded in Epon 812. Electron dense reaction products of copper ferrocyanide were observed in smooth endoplasmic reticulum (sER) and in intercristal and outer space of mitochondria. In control studies, 3 beta-HSD activity were completely abolished. It is concluded that not only sER but also mitochondria has 3 beta-HSD activity in human corpora lutea. Furthermore, mitochondria seems to have capability to synthetize progesterone from cholesterol by itself.

Enzyme histo-cytochemical staining including alkaline phosphatase, acid phosphatase and 3 beta ol dehydrogenase in human fetal adrenal glands (13 to 20 week fetuses) was studied. Enzyme histo-chemical activity of alkaline phosphatase was mainly observed in cell membranes of the fetal cortex. The permanent cortex showed weak or negative activity. Histochemical staining of acid phosphatase was mostly observed in the cytoplasm as large globular structures near the nucleus in the fetal cortex, and intracellular localization of acid phosphatase by electron microscopic enzyme cytochemistry was mainly observed in lysosomes including dense bodies of the fetal cortex. The permanent cortex was only slightly stained for acid phosphatase. The activity of 3 beta ol dehydrogenase was predominantly observed in the cytoplasm of the fetal cortex in the 20 week fetus. Based on these findings, the significance of these enzymes in the fetal cortex is discussed.

An improved digitonin-precipitation radioassay for 3 beta-hydroxy-delta 5-steroid dehydrogenase is presented. It is linear for enzyme protein concentrations up to 0.6 mg and for 37 degrees C incubation times up to 10 min. Sensitivity and reproducibility are improved nine- and sevenfold, respectively, over the method of Philpott and Peron. There is a good correlation with the Philpott and Peron assay (r = 0.958) and with a thin-layer chromatography method (r = 0.997). Beyond being faster, simpler, and more sensitive than other analyses, this radioassay uses one-tenth the quantity of [3H]pregnenolone substrate and is, therefore, a safer and less expensive procedure.

Enzyme histo-cytochemical staining was performed for 3 beta-hydroxysteroid dehydrogenase, alkaline phosphatase and acid phosphatase in the compact and clear cells of the adrenocortical adenomas associated with Cushing's syndrome (Cushing's adenomas). In the compact cells, enzymatic activities of 3 beta-hydroxysteroid dehydrogenase and alkaline phosphatase were stronger than those in the clear cells. Electron microscopic localization of alkaline phosphatase was mostly present on well developed microvilli, while, acid phosphatase was observed in lysosomes near the well developed smooth endoplasmic reticula. On the other hand, in the clear cells, cytochemical localization of alkaline phosphatase was mainly present on the intercellular plasma membrane, while acid phosphatase was mostly observed near the lipid droplets. Based on these data, functional aspects of the compact and clear cells in Cushing's adenomas are discussed.

The ultrastructural localization of the enzyme delta 5-3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) was studied in the goldfish interrenal cells by using the potassium ferricyanide method. Immersion fixation in a mixture of 1% paraformaldehyde and 0.25% glutaraldehyde results in a consistently good ultrastructural preservation and enzyme localization. A precipitate of copper ferrocyanide indicating the localization of 3 beta-HSD activity was observed in close vicinity to the smooth endoplasmic reticulum or in contact with the outer surface of its membrane. A small number of precipitated grains were also found in the lumen of mitochondrial cristae. Addition of phenazine methosulfate increased the intensity of the reaction without changing the localization of the copper ferrocyanide grains. The findings suggest that the outer surface of the smooth endoplasmic reticulum is the major site of 3 beta-HSD activity in goldfish interrenal cells.

Sebaceous glands were isolated by manual dissection using a stereomicroscope from skin specimens of bald scalp of men with male-pattern baldness undergoing hair transplant or scalp reduction surgery and also from specimens taken from hairy and bald areas of scalp at autopsy of adult male victims of accidental death within 3 h post mortem. Homogenates of the isolated glands exhibited activities of delta 5-3 beta-hydroxysteroid dehydrogenase (3 beta HSD), 17 beta-hydroxysteroid dehydrogenase, and testosterone 5 alpha-reductase by the conversion of [3H]dehydroepiandrosterone (DHA) to 3H-delta 4-androstenedione (AD), [3H]testosterone, and [3H]dihydrotestosterone. Homogenates of glands from bald (B) scalp had greater 3 beta HSD activity than homogenates of glands from hairy (H) scalp. After differential centrifugation, 3 beta HSD activity was found mainly in the microsomal and 105,000 X g supernatant fractions. Specific activity of the enzyme based on protein mass was highest in the microsomal fraction; however, the total 3 beta HSD activity in the 105,000 X g supernatent of B glands was significantly (p less than .01) greater than that of H glands. 3 beta HSD activity in sebaceous glands isolated from autopsy specimens did not differ from that of glands isolated from surgical specimens in apparent Km (0.13-0.14 microM), pH optima (8.0), or coenzyme requirement for NAD. Since substantial 3 beta HSD activity was present in the cytosol, and cytosol of B glands showed increased 3 beta HSD activity, the increased conversion of DHA to AD may be a critical step for androgenic action and may be responsible for excessive androgenicity in male-pattern baldness.

The aim of this study was to establish a monolayer culture system for human ovarian thecal cells and to investigate their morphological and functional characteristics. Theca layers were isolated and digested with collagenase-hyaluronidase solution, and dispersed thecal cells were cultured for 10 days in plastic dishes. Histological examination indicated that there were no contaminating granulosa cells in isolated theca layers. Various histochemical studies revealed abundant lipid droplets and 3 beta-hydroxysteroid dehydrogenase activity in cultured cells. The major steroids secreted were delta 4-androstenedione (delta 4) and progesterone(P). delta 4 secretion was very high during the first 2 days (31.6 +/- 1.9 ng/1 X 10(5) cells/2 days) and declined thereafter. P was secreted in moderate amounts throughout the 10 day culture period (9.0-21.3 ng/1 X 10(5) cells/2 days), while estradiol secretion was very low. Subsequently, the responsiveness of cultured thecal cells to gonadotropins and dibutyryl cyclic AMP (Bu2cAMP) was investigated. LH/HCG and Bu2cAMP stimulated delta 4 and P secretion in a dose-related manner. The maximal effective doses of LH and HCG were both 10 ng/ml, and that of Bu2cAMP was 10(-3)M. In conclusion, it was evident that these monolayer-cultured human thecal cells could maintain their morpho-functional characteristics during culture. Therefore, this culture system will provide an excellent model for further studies on the functional properties of thecal cells.

The aim of the present report was to study the course of development of rat Leydig cells from 17 fetal day (f.d.) up to 5 postnatal day (p.n.d.), with the help of enzymhistochemical reaction of delta 5-3 beta Hydroxysteroid-dehydrogenase (3 beta-HSDH). Testes were obtained from 17 to 21 days old embryos and from 1 to 5 days old offsprings. Cryostat sections were cut and processed for enzymhistochemical reaction of 3 beta-HSDH, employing dehydroepiandrosterone as substrate. The areas of the Leydig cells, showing the enzymatic activity, were measured morphometrically. The 3 beta-HSDH positive intertubular Leydig cells appear on 17 f.d., and grow further showing a maximum peak, relative to the size of the testis, on 19 f.d. Thereafter, the percentage of the Leydig cells relative to the size of the testis decreases continuously up to 4 p.n.d.. The Leydig cells did not regress during the period of observation. Postnatally, the large complexes disperse into many small complexes.

Induced zinc deficiency in male albino rats caused a great reduction in the testicular levels of testosterone as compared to control and zinc-supplemented (ZS) rats. Estimation of the testicular levels of follicle-stimulating hormone, luteinizing hormone and prolactin (PRL) in the zinc-deficient (ZD) rats showed higher levels in comparison with both control and zinc-supplemented rats. However, the increase in PRL levels was statistically insignificant. A great reduction in the activity of 3 beta-hydroxysteroid dehydrogenase, an important enzyme involved in testosterone biosynthesis, was demonstrated histochemically in the testes of ZD rats as compared to both control and ZS ones. These results reflect a direct action of zinc deficiency on the testicular steroidogenesis and strongly support the idea that the hypogonadal state associated with zinc deficiency results mainly from some alteration in the testicular steroidogenesis or in other words Leydig cell failure.

Quantitative cytochemistry was used to determine the effect of subjecting pregnant rats to environmental stress on the activity of delta 5-3 beta hydroxysteroid dehydrogenase (3 beta-HSD) in Leydig cells of their fetuses. Enzyme activity was measured by microspectrophotometry in individual Leydig cells in cryostat sections of fetal testes on Days 16-21 postconception. Fetuses of stressed mothers lacked the peak of enzyme activity on Days 18 and 19 of gestation that is characteristic of Leydig cells of normal fetuses at this time. In addition, both before and after these 2 days, 3 beta-HSD activity in Leydig cells of stressed fetuses was significantly higher than normal. The altered developmental pattern of 3 beta-HSD activity in the stressed fetuses largely corresponds to the changes in plasma testosterone found previously in male fetuses of mothers exposed to the same regimen of stress. Thus, in the fetal Leydig cell, the activity of 3 beta-HSD, a key steroidogenic enzyme, can be modified by environmental stress, and provides an index of steroidogenic activity of the fetal testes and of the titers of circulating testosterone.

The ovaries of sexually mature, pregnant mare serum gonadotropin (PMSG) stimulated, 12 week old Mongolian gerbils were investigated morphologically and enzyme histochemically for the appearance of the 3 alpha-hydroxy-steroid and the 3 beta-hydroxysteroid dehydrogenase during the estrous cycle. Up to ovulation, on day 3 of the estrous cycle, the number of vesicular follicles increases continuously. Primarily atretic follicles can be seen on day 4. On day 5 corpora lutea appear, but they degenerate already by day 6. During the entire estrous cycle, 3 alpha-hydroxysteroid dehydrogenase and 3 beta-hydroxysteroid dehydrogenase activity can be found in the theca of tertiary follicles and in the interstitial cells, whereas the theca of secondary follicles and the granulosa of healthy follicles do not exhibit any enzyme activity. The activity decreases from day 1 till day 6. The granulosa of atretic follicles and the cells of corpora lutea show only weak activity. It may be significant that the intensity of enzyme activity in the ovary and the estrogen level in the plasma are differently correlated to the estrous cycle.

In addition to the morphological examination, the measurement of the content of corticosteroids was done in aldosterone-producing adenoma. Histologically, the adenoma consists of four types of cells. The major component of the adenoma was clear-type cells. In this type of cells, the activities of 3 beta-hydroxysteroid dehydrogenase and glucose-6-phosphate dehydrogenase showed weakly positive. The fine structure was characterized by numerous lipid vacuoles and poor organellae. On the contrary, intermediate- and compact-type cells revealed higher than moderate activities of these enzymes. Moreover, marked development of smooth endoplasmic reticulum and mitochondria was noted. Zona glomerulosa-type cells were observed only in a small part. The content of aldosterone and corticosterone in aldosterone-producing adenoma was significantly larger than those of the other type of adenoma. A tendency of positive correlation between the increase of compact-type cells and content of aldosterone was found. The cell origin of this aldosterone-producing adenoma and functional role of clear- and compact-type cells were also discussed.

A method for determining faecal bile acids, suitable for clinical purposes, is introduced. The analysis uses a 0.2-g stool specimen, a simple extraction procedure, and 3 alpha-steroid dehydrogenase determination. The method, which is rapid, has been validated by gas-liquid chromatography and by recovery of internal standards. Stool examination was done in 16 healthy volunteers on free diet and in 25 patients with non-gastrointestinal diseases who were on a fat- and fibre-fixed diet. No difference was found between the two groups, so the data were pooled, and the normal reference interval (mean +/- S.D.) for faecal bile acid output was calculated to be 0-975 mumol/24h.

Enzymatically dispersed testis cells derived from 7-day-old male rats maintained their gonadotropin-stimulated testosterone production for 18 days in culture. Treatment with hCG or LH stimulated androgen production in a dose-dependent manner, with ED50 values of 0.030 +/- 0.007 and 1.0 +/- 0.4 ng/ml for hCG and LH, respectively. Concomitant treatment with a phosphodiesterase inhibitor further enhanced LH action. In contrast, treatment with FSH, GH, or PRL was without effect. Treatment with forskolin, cholera toxin, or 8-bromo-cAMP induced dose-dependent increases in testosterone biosynthesis; this was accompanied by stimulation of 3 beta-hydroxysteroid dehydrogenase activity after treatment with hCG, forskolin, or 8-bromo-cAMP. RIA measurement of different androgens in HPLC-fractionated medium revealed that the main androgen secreted by the neonatal testis cells was testosterone, with lower production of 5 alpha-androstane-3 alpha,17 beta-diol and negligible 5 alpha-dihydrotestosterone, androstenedione, and androsterone. Treatment with epidermal growth factor, GnRH, and arginine vasopressin (AVP) decreased hCG-induced testosterone biosynthesis. Since the inhibitory actions of GnRH and AVP were blocked by concomitant addition of specific hormone antagonists, their inhibitory actions were probably mediated by specific testis receptors. In contrast, treatment with several potent synthetic steroid hormone analogs [diethylstilbestrol (an estrogen), dexamethasone (a glucocorticoid), R5020 (a progestin; 17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione), R1881 (an androgen; 17 beta-hydroxy-17 alpha-methyl-4,9,11-estratrien-3-one), or cyproterone acetate (an antiandrogen; 17 alpha-acetyloxy-6-chloro-1,2-dihydro-(1 beta,2 beta)3'-H-cyclopropa-(1,2) pregna-1,4,6-trien-3,20-dione)] did not affect testosterone biosynthesis in hCG-treated cells. These results demonstrate that testosterone production by neonatal testis cells is maintained by gonadotropins during prolonged culture; the ability of cAMP-generating drugs and a cAMP analog to mimic gonadotropin actions on testosterone biosynthesis and 3 beta-hydroxysteroid dehydrogenase activity suggests a mediatory role of cAMP in gonadotropin action; and AVP, epidermal growth factor, and GnRH, through their putative testis receptors, directly inhibit gonadotropin-stimulated testosterone synthesis, while various steroids (androgens, estrogens, progestins, and glucocorticoids) do not affect Leydig cell function in the neonatal testis. The present culture system offers a unique model for elucidating the hormonal control of Leydig cell androgen biosynthesis during neonatal development.

The localization of 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD) was studied in bovine adrenal glands by light as well as electron microscopic immunocytochemistry, using anti-bovine adrenal 3 beta-HSD antibody. With light microscopy the cytoplasm of the glomerulosa cells was weakly immunostained, while that of the fasciculata-reticularis cells was intensely immunostained though both the capsular connective tissue cells and the medullary cells were entirely negative for this reaction. Electron microscopic immunocytochemistry revealed that the positive reaction products for 3 beta-HSD were present on the membrane of smooth endoplasmic reticulum of the cortical cells, especially that of the fasciculata and reticularis cells. Other cell organelles such as mitochondria and Golgi apparatus were entirely negative. The present results indicate that 3 beta-HSD is present in the membrane of smooth endoplasmic reticulum of bovine adrenal cortical cells.

We investigated the possible secretory capacity of the ovaries of 79 postmenopausal women with and without endometrial carcinoma using chemical, enzymehistochemical and ultrastructural methods. The mean serum levels of estrone, estradiol and total estrogens were higher in ovarian effluent blood than the corresponding values in cubital venous blood. There was a positive correlation between the difference of total estrogens in ovarian and cubital vein sera and, on the other hand, enzymehistochemical and ultrastructural findings in ovarian tissue. No statistical differences were found in the peripheral mean values of estrone and total estrogens between the two groups. The mean level of estradiol, however, seemed to be higher and that of estriol lower in the carcinoma patients than in the control groups.

Detailed histochemical localization of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and 17 beta-HSD was made in the goat testis using both NAD and NADP coenzymes. The substrates used for 3 beta-HSD were dehydroepiandrosterone (DHA) and pregnenolone whereas 17 beta-HSD was localized with testosterone and oestradiol. In general, the activity of the enzymes varied with the cell type, substrate and coenzyme. In seminiferous tubules, DHA and NAD were the preferred substrate and coenzyme respectively for 3 beta-HSD. In addition, in interstitial tissue, NAD was the preferred coenzyme with DHA whereas no such preference existed with pregnenolone. 17 beta-Hydroxysteroid dehydrogenase showed a similar pattern in the two main compartments of the testis, as testosterone and oestradiol were equally utilized and NAD was the preferred coenzyme in both these compartments. The activities of the enzymes increased during the process of spermiogenesis and were higher in seminiferous tubules than in interstitial tissue, especially in elongated spermatids and spermatozoa.

Zinc, magnesium and copper concentrations were measured in homogenates of 20 human prostates with benign hyperplasia and a significant direct relationship between zinc and magnesium contents was found. Activities of the testosterone-5 alpha-reductase in the same homogenates were not significantly changed according to the concentrations of the three metals but activities of the 3 alpha- and 3 beta-hydroxysteroid dehydrogenases were inversely related with the zinc concentrations of the tissue preparations. This inverse relationship may explain the known increased 5 alpha-dihydrotestosterone content in prostatic tissues with high zinc concentrations.

After solubilization of rat adrenal microsomes with sodium cholate, 3 beta-hydroxysteroid dehydrogenase with steroid 5-ene-4-ene isomerase (abbreviated as steroid isomerase) activity was purified to a homogeneous state. The following characteristics of the enzyme were obtained: 3 beta-Hydroxysteroid dehydrogenase together with steroid isomerase was detected as a single protein band in SDS-polyacrylamide gel electrophoresis, where its mol. wt was estimated as 46,500. Either NAD+ or NADH was required for demonstration of steroid isomerase activity. Treatment of the enzyme with 5'-p-fluorosulfonylbenzoyladenosine, an affinity labeling reagent for NAD+-dependent enzyme, diminished both the enzyme activities.

3 alpha-Hydroxyglycyrrhetinate dehydrogenase of Clostridium innocuum, isolated from human intestinal bacteria, was capable of converting 3-ketoglycyrrhetic acid to 3 alpha-hydroxyglycyrrhetic acid. The enzyme was purified to homogeneity by means of butyl-Toyopearl 650M, Sephadex G-150, Matrex Red A, Toyopearl HW-55S, and isoelectric focusing column chromatographies. The purified enzyme showed a specific activity of 156 mumol/min.mg toward 3 alpha-hydroxyglycyrrhetic acid, and showed a single band on SDS-polyacrylamide gel electrophoresis. The apparent molecular weight was 53,000, as estimated by gel filtration, and 30,000, as judged by SDS-polyacrylamide gel electrophoresis. Its isoelectric point was 5.2. The enzyme showed absolute specificity for the 3 alpha-hydroxyl and 3-ketonic groups of 18 alpha- or 18 beta-glycyrrhetic acid and required NADP+ and NADPH as cosubstrates. The enzyme did not act on any 3 alpha-hydroxyl or 3-ketonic group of steroids or bile acids. The enzyme is a novel type of enzyme, defined as 3 alpha-hydroxy-glycyrrhetinate dehydrogenase, being quite different from 3 alpha-hydroxysteroid dehydrogenase [EC 1.1.1.50].

Four isozymes of 3 alpha-hydroxysteroid dehydrogenase (3alpha-HSD) appeared in rat livers to be classified into three categories concerned with the requirement of coenzyme. Two isozymes in the first group had affinity for both NAD and NADP. One of the other isozymes classified in the second was linked with NADP to show specificity for 5beta-androstan-3alpha-ol-17-one (etiocholanolone) as the steroid substrate. An isozyme belonging to the third required only NAD as cofactor. This has the same migration rate of a lactate-dehydrogenase isozyme. In the histochemical observation, the maximal activity of the enzyme was demonstrated with 5-alpha androstan-3alpha-ol-17 one (androsterone) but not with etiocholanolone as a substrate. On the other hand, all 3 alpha-HSD isozymes revealed by electrophoresis showed a higher affinity for etiocholanolone than androsterone. It is worthwhile to note that the zymogram of 3alpha-HSD in the cold acetone-treated section was essentially the same as the zymogram in the intact liver. All isozymes in the section were highest in activity when etiocholanolone was used as a substrate. These findings indicate that in the cold acetone-treated section the enzyme still has affinity for etiocholanolone to resist the histochemical procedure employed.

The histochemical localization of delta 5-3 beta-HSDH in individual follicles isolated from the adult mouse ovary and in ovulated cumulus cell-oocyte masses recovered from the oviduct was examined using a new embedding technique. The procedure employed involves the histochemical staining of such tissue for delta 5-3 beta-HSDH with subsequent embedding in GMA (glycol methacrylate). This method not only permits the acquisition of sections as thin as 3 micron but also preserves the histological detail of the tissue allowing for the specific cellular localization of the enzyme. Results obtained from this technique far surpass those obtained from frozen material. Virgin female mice were injected with PMSG and sacrificed either 10 or 17 h later in order to acquire preovulatory or ovulated oocyte-cumulus cell masses, respectively. The sites of localization of delta 5-3 beta-HSDH corresponded to sites demonstrated by histochemical studies on frozen tissue sections; however, the present study revealed that not all cells of a specific type within the same follicle reacted with the same intensity. Granulosa cells lining the walls of vesicular follicles displayed different degrees of enzyme activity based on their distance from the basement membrane. Intrafollicular tranformed cumulus masses and cumulus cells of ovulated masses within the oviduct did not react uniformly in that some were positive for the enzyme and others were not. Such results indicate that not all cells of a given type in the ovary possess similar delta 5-3 beta-HSDH activity at a particular time. Thus, the cells comprising a specific cellular component of the ovary should be treated as individual entities and not as a homogeneous group with respect to their metabolic activities.

This paper described a modified method for the radiometric determination of hydroxysteroid dehydrogenase activities in rat liver. The principle advantages of this method are the improved precision and a radical reduction in the time involved in performing the assay. The procedure comprises the following steps: incubation of 14C-labelled substrate with coenzyme and cell fraction under optimized conditions; termination of the reaction by addition of organic solvent containing a defined amount of 3H-labelled reaction product; removal of precipitated protein and coenzyme by centrifugation; paper chromatographic isolation of the product; direct quantitation of 14C activity in the product zone of the paper chromatogram. The assay systems have been applied to elucidate and quantitate sex and strain differences in the activities of the above enzymes in Chbb/THOM and Sprague-Dawley rats.

The curve of the specific activity of rat epididymal nuclear delta 4-5 alpha-reductase is bell shaped as a function of age, whereas that of cytoplasmic 3 alpha-hydroxysteroid dehydrogenase does not change significantly with age. The present study examines the subcellular distribution of delta 4-5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase in the caput-corpus and cauda epididymidis during development. A 5-step discontinuous sucrose gradient was developed for fractionation of epididymal homogenates. By using enzyme markers specific for different subcellular organelles, the five different subcellular fractions obtained were shown to be of cytoplasmic, microsomal, mitochondrial, nuclear and spermatozoal origin. 3 alpha-Hydroxysteroid dehydrogenase activity was associated only with the cytoplasmic fraction. The activity of the enzyme did not change significantly with age in either the caput-corpus or cauda epididymidis. delta 4-5 alpha-Reductase activity was found in fractions containing microsomal and nuclear markers. delta 4-5 alpha-Reductase activity in the nuclear fraction of the caput-corpus epididymidis was evident in the youngest age group (Day 25), increased 4-fold and peaked in the next age group (Day 35), and declined with each successive age group: Day 45 (60% of maximum), Day 60 (20% of maximum), Day 75 (15% of maximum) and Day 105 (10% of maximum). In contrast, microsomal delta 4-5 alpha-reductase activity increased successively from Day 25 to Day 105; enzyme activity doubled between these two ages. The ratio of nuclear to microsomal delta 4-5 alpha-reductase activity from the caput-corpus epididymidis thus changed markedly with age: Day 25:1.32; Day 35:3.76; Day 45:2.44; Day 60:1.03; Day 75:0.41; and Day 105:0.21. In the cauda epididymidis nuclear delta 4-5 alpha-reductase activity was only evident at Day 35 and Day 45; in microsomal fractions, activity was first found at Day 35 and did not subsequently change with age. These results demonstrate that: 1) epididymal 3 alpha-hydroxysteroid dehydrogenase activity is found only in the cytoplasmic fraction; 2) delta 4-5 alpha-reductase activity is found in nuclear and microsomal fractions; and 3) the subcellular distribution of delta 4-5 alpha-reductase activity changes markedly with age and epididymal section, suggesting differential regulation of nuclear and microsomal delta 4-5 alpha-reductase activities.

A method for the determination of delta 5 3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta-HSD) activity in intact isolated Leydig cells was established. This method utilizes the conversion of [7-3H]dehydroepiandrosterone (1.04 mumole) to androstenedione and expresses the activity of the enzyme as mumoles of androstenedione produced/microgram DNA/h. The reaction is limited to 0.5 - 4 micrograms DNA of Leydig cells/ml (equivalent to 0.1-0.8 million of Leydig cells/ml) and to 1 h of incubation at 34 degree C. The 3 beta-HSD activity of 44 suspensions of Leydig cells isolated from adult rats was found to be 1.13 +/- 0.03 (SE) mumoles/microgram DNA/h. This new method for direct measurement of 3 beta-HSD activity in intact Leydig cells was found to be rapid, easy to perform and highly reproducible.

Presence of a parahypothalamic-adenohypophyseal way of the influence of the lateral septal nuclei (LSN) lesion on the gonads was suggested on the basis of study of hormonal reserves of the hypothalamo-hypophyseal-testicular system in adult male albino rats in experiments using niftolide (flutamide) after the lesion of the LSN, the medial-basal hypothalamus remaining intact or isolated.

Caffeine, when administered in moderate (30 mg/kg X d) or high (60 mg/kg X d) doses during pregnancy, was shown to cause significant fetal growth retardation of both sexes. Mortality rate at or soon after birth was significantly higher and litter size significantly lower in the litters treated with 60 mg. The subsequent growth rates were also affected. The experimental pups grew more slowly, with growth plateauing at the same age resulting in smaller adults. The male offspring when subjected to short-term stress (one session) in adulthood showed an intact emergency response, demonstrating an adequate ability to react to a sudden environmental change. A significant decrease in 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity, and consequent reduction in testosterone biosynthesis, in the fetal testes at d 18 and 20 of gestation was also found for both doses of caffeine. Low 3 beta-HSD activity persisted to adulthood in the group receiving 60 mg. Lingering effects were observed in a second litter bred 8 wk after the discontinuation of caffeine consumption. In this second breeding, the offspring of both sexes from both caffeine doses were born significantly smaller when compared to the controls. Persistent effects of caffeine were also found in second-generation rats bred from females who were exposed to caffeine in utero. The pups of both sexes were born significantly heavier after a significantly longer gestation. The subsequent growth did not differ from that of the controls. It was suggested that a changed genetic program in the ovarian germ cells of the first generation and/or a changed uterine environment in the second generation may be implicated.

The capacity for steroid production in the corpora lutea (CL) of bank voles was examined by enzyme histochemistry for 3 beta-hydroxysteroid dehydrogenase. The results revealed similar activity in the CL resulting from sterile matings and the CL of pregnancy 2 days after mating. Six days after mating, the CL of pregnancy showed a strong reaction, while there was almost no reaction in the CL from sterile matings, which were clearly regressed at this time. It is suggested that the CL from sterile matings produce progesterone before regression and that this progesterone may prime the luteotrophic complex, increasing fertility of subsequent matings.

The enzymatic activities of thiamine pyrophosphatase (TPPase), acid phosphatases (ACPases), alkaline phosphatases (APases) and steroid-3 beta-ol dehydrogenase (beta-HSDH) in different germ cells and somatic cells in the testis of three cytogenetically determined states of fertility (i.e. normal, impaired fertility and sterility) were studied histochemically in the mouse. The TPPase, ACPases and APases activities showed a characteristic stage dependent pattern when the activities were related to the typical twelve stages of the seminiferous epithelium in the mouse, according to Oakberg (1956). Comparing the enzymatic patterns of the activities in the normal spermatogenic process versus the impaired and sterile conditions, the following conclusions can be drawn: even in impaired and sterile conditions the enzymatic activity patterns retain their characteristic stage dependence; the pattern of beta-HSDH and ACPases is not altered in the impaired and sterile conditions; TPPase and APases patterns are modified in impaired and sterile mice. It is concluded that the kinetics of the enzyme activities can serve as a useful marker for characterizing pathological spermatogenic processes.

3 alpha-Hydroxysteroid dehydrogenase (EC 1.1.1.50) of rat liver cytosol catalyzes the second step in glucocorticoid metabolism, namely the NADPH-dependent reduction of 5 beta-dihydrocortisol to tetrahydrocortisol. The purified enzyme is potently inhibited by the nonsteroidal anti-inflammatory drugs [Penning and Talalay, Proc. natn. Acad. Sci. U.S.A. 80, 4504 (1983)], and the consequences of this inhibition on hepatic glucocorticoid metabolism are now examined. Analyses of the specific activities for the reduction of 5 beta-dihydro-cortisone catalyzed by homogeneous preparations of 3 alpha- and 3 beta-hydroxysteroid dehydrogenases of rat liver cytosol indicated that this steroid was predominantly reduced to the 3 alpha-hydroxysteroid (tetrahydrocortisone). Whether this reaction was catalyzed by the purified 3 alpha-hydroxysteroid dehydrogenase or unprocessed cytosol, it was potently inhibited by indomethacin (IC50 = 0.6 microM). In the presence of 30 microM indomethacin, the rate of reduction of 5 beta-dihydro-glucocorticoids was 16 times smaller than in the absence of drug. Measurement of 5 beta-reductase activity in rat liver cytosol indicated that it was 25-30 times less active than the 3 alpha-hydroxysteroid dehydrogenase. In the presence of 30 microM indomethacin, enough residual dehydrogenase may exist to reduce 5 beta-dihydro-glucocorticoids as they are formed. This was confirmed by incubating the supernatant fraction obtained from the 10,000 g centrifugation of a rat liver homogenate, with [1,2-3H]cortisol plus NADPH in the presence and absence of drug. At the end of the incubation, cortisol metabolites were analyzed by TLC and the results expressed as CL:CM ratio (cortisol left:cortisol metabolized). It was found that no change in this ratio occurred in the presence of the drug. Moreover, the addition of trapping quantities of 5 beta-dihydrocortisol to incubations containing 30 microM indomethacin were insufficient to promote an increase in cortisol levels as reflected by a rather small increase in the CL:CM ratio. Thus, nonsteroidal anti-inflammatory drugs cannot effect a rise in hepatic glucocorticoid levels in vitro, and do not promote a "glucocorticoid saving effect" in this organ.

Purified mouse Leydig cells have been prepared from interstitial cell suspensions using a Percoll gradient procedure. The isolated cells are 88-95% pure as determined by light microscopy. Staining for 3-beta-hydroxysteroid dehydrogenase indicates that over 85% of the Leydig cells recognized by differential interference microscopy are also positive for this enzyme. After separation, the Leydig cells are viable by dye exclusion assays and exhibit normal in situ morphology when examined at the ultrastructural level. Leydig cell suspensions have been used to raise a polyclonal antiserum in rabbits. This antiserum, prior to absorption, reacts in indirect immunofluorescent studies with the surfaces of isolated Leydig cells, with testicular germ cells, with spermatozoa and with somatic cells such as splenocytes. Following absorption with lymphocytes and spermatogenic cells, the antiserum binds only to Leydig cell plasma membranes. Quantitative measurements with 125I-protein A confirm the specific labeling of Leydig cells by the absorbed antiserum. Biochemical identification of the Leydig cell plasma membrane antigen has been accomplished by immunoblotting polyacrylamide gel nitrocellulose transfers. A single major band of Mr approximately 40,000 is detected on one-dimensional transfers; two weakly reactive spots of Mr 43,000 and 45,000 are detected using two-dimensional immunoblots. Blotting experiments conducted using concanavalin A have identified the major Leydig cell constituents reactive with this lectin. The Leydig cell plasma membrane antigen(s) does not bind concanavalin A.

The steroidogenic enzymes 3 beta-hydroxysteroid dehydrogenase and delta 4-5 isomerase (3 beta-HSD) that convert delta 5-hydroxysteroids to delta 4-ketosteroids were measured in human follicular tissue collected during the follicular phase of the menstrual cycle. This study was done in order to determine whether 3 beta-HSD enzyme activity fluctuated during the follicular phase of the menstrual cycle and, if so, whether these changes were reflected by the concentration of steroids in the follicular fluid. A microsomal fraction was prepared from each of 14 follicular-phase follicles, and 3 beta-HSD enzyme activity was estimated by the amount of androstenedione synthesized in the presence of excess substrate (dehydroepiandrosterone) and cofactor (nicotinamide adenine dinucleotide). Androstenedione, dehydroepiandrosterone, and progesterone were measured in the aspirated follicular fluid of each follicle. 3 beta-HSD enzyme activity was undetectable in the smallest (3 to 5 mm) follicles, increased in 5 to 6 mm follicles to 363.2 +/- 60 pmol of androstenedione per milligram per hour, and increased still further in 9 to 11 mm follicles to 5,000 +/- 200 pmol of androstenedione per milligram per hour. The increase in 3 beta-HSD enzyme activity was accompanied by an increase in the concentration of androstenedione in the follicular fluid as well as of progesterone in larger follicles. These data indicate that 3 beta-HSD activity increases during the follicular phase of the menstrual cycle. It is suggested that the primary product of the increased enzyme activity is androstenedione. Since androstenedione is the principal C19 steroid produced by the ovulatory follicle and serves as a substrate for estradiol production, this increase in 3 beta-HSD activity may be important for the associated changes in the late follicular phase that lead to ovulation.

The histochemical reaction for 3 beta-hydroxysteroid dehydrogenase was applied to glutaraldehyde fixed eel testis and successively processed for transmission electron microscopy. The reaction product was found only on the smooth endoplasmic reticulum and in the intermembrane space of mitochondria of the Leydig cells of hCG treated silver eels. The positive cytochemical reaction appears to be coupled with an enlargement of the Leydig cells and an increase of their smooth endoplasmic reticulum.