Immunolocalization of 3 beta-hydroxysteroid dehydrogenase in human ovary

Immunohistochemical localization of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) was performed in 55 cases of morphologically normal human ovaries by using a specific polyclonal antibody against purified human placental 3 beta-HSD. In small developing follicles, immunoreactivity was observed only in the theca interna but also became recognizable in the membrana granulosa with development of the follicle. At a late stage of folliculogenesis, the intensity of the 3 beta-HSD activity in the membrana granulosa was nearly equal to that of theca interna in 2 or 3 large follicles examined. One to several layers of theca interna cells just beneath membrana granulosa did not demonstrate any immunoreactivity of 3 beta-HSD or that of cytochrome P-450 17 alpha-hydroxylase. These unstained theca interna cells did not appear to be directly involved in ovarian steroidogenesis and might be designated as 'enzymically inactive theca interna cells.' Marked immunoreactivity was observed in luteinized theca and granulosa cells of the corpus luteum.     

Changes in mitochondrial and microsomal 3 beta-hydroxysteroid dehydrogenase activity in mouse ovary over the course of the estrous cycle.

3 beta-Hydroxysteroid dehydrogenase (HSD) is located in the endoplasmic reticulum and mitochondria. To determine whether the separate enzymes play different roles in steroidogenesis, the specific activity (SA) of both were measured at four different stages of the mouse estrous cycle. Microsomal HSD activity changed little throughout, averaging 8.7 +/- 0.7 nmol progesterone/min/mg protein. In contrast, mitochondrial HSD activity changed dramatically at diestrus, increasing to 14.4 nmol progesterone/min/mg protein. When measured at proestrus, estrus, and metestrus, mitochondrial HSD activity was 5.5, 7.4, and 4.5 nmol progesterone/min/mg protein, respectively. To ascertain whether the increase in mitochondrial HSD activity at diestrus could be due to a preferential induction of enzyme, its SA and the SA of a mitochondrial inner membrane enzyme, cytochrome C oxidase, were compared to the SA of a mitochondrial outer membrane enzyme, rotenone-insensitive NADH cytochrome C reductase. The SA of all three enzymes changed proportionally at diestrus, suggesting that the increase in mitochondrial HSD activity was not due to its preferential induction. Rather, we believe that the HSD activity in the mitochondrial fraction, as measured at the four stages of the estrous cycle, is a reflection of the combined contributions from an ever changing population of ovarian cells. Mitochondria from luteal cells have the highest HSD activity, and are very likely responsible for the major synthesis of progesterone during the luteal phase.     

3 Alpha-hydroxysteroid dehydrogenase and 3 beta-hydroxysteroid dehydrogenase in the ovary of young Mongolian gerbils

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.     

Activity of 3 beta-hydroxysteroid dehydrogenase/isomerase in the fetal rat ovary

3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD) was examined in rat fetal ovaries. The enzymatic activity was determined by measuring the conversion of radiolabeled pregnenolone to progesterone. 3 beta-HSD, present in 14-day old fetal ovaries showed a regular increase in the course of development. Pretreatment with dcAMP for 48 h enhanced the apparent maximal velocity of the enzyme by about 5-fold without increase in the apparent Km. The increase in 3 beta-HSD activity was not due to the synthesis of pregnenolone observed after dcAMP pretreatment, but it was dependent on protein synthesis. The present results indicate that (1) 3 beta-HSD activity is present in fetal female gonads and the absence of steroid biosynthesis cannot be related to a defect in this enzyme (2) 3 beta-HSD activity is enhanced in the presence of dcAMP. The absence of gonadotropic receptors in the rat ovary before birth could explain the low level of the enzymatic activity measured in fetal ovaries.     

Delta5-3beta-hydroxysteroid dehydrogenase and estradiol-17beta-hydroxysteroid dehydrogenase activity in preimplantation hamster embryos

Preimplantation golden hamster (Mesocricetus auratus) embryos were recovered on days 1 (= day of finding spermatozoa in the vagina) through 4 of pregnancy. Postimplantation embryos were studied in sectioned gestation sacs excised on days 5 and 6. Δ⁵-3β-Hydroxysteroid dehydrogenase (3β-HSD) activity in embryos was determined histochemically. There was no enzyme activity on days 1 and 2. Weak activity was first observed at 08:00–09:00 hr on day 3, the activity then increased, peaked at 01:00–03:00 hr on day 4, considerably declined by 08:00–09:00 hr (day 4), and was absent on days 5 and 6. These results suggest that the preimplantation embryos synthesize steroid hormones. It was previously hypothesized (Dickmann and Dey, 1973, Dickmann and Dey, 1974) that, hormones synthesized by the preimplantation rat embryo participate in the regulation of morula to blastocyst transformation and implantation of the blastocyst. This hypothesis is applicable to the hamster.In addition to 3βHSD, estradiol-17β-hydroxysteroid dehydrogenase activity was observed in day 3 embryos, suggesting that the embryo synthesizes estrogen.     

Histoenzymatic activity of the delta 5-3 beta-hydroxysteroid dehydrogenase of ovary in hibernators (Citellus citellus L)

The ovaries of 24 ground squirrels (Citellus citellus L) were studied in spring (March, April), summer (July), and winter (December). The animals hibernated in a chamber at a temperature of 6-8 degrees C. The activities of the interstitial gland, theca interna, atretic follicles with theca interna and yellow bodies were measured densitometrically and studied by a specially modified histoenzymatic technique. Measurements showed that the endocrine structures were most active in March and least active in July. The atretic follicles had the highest enzymatic activity in April. The quantitative histoenzymatic approach presents an objective base for morphofunctional studies of organs and tissues at different body temperature levels in hibernators.     

Changes in the 17 beta-hydroxysteroid dehydrogenase activity of mouse blastocysts during delayed implantation

The rate of estrone (E1)----estradiol-17 beta (E2) or E2----E1 conversion catalyzed by 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activity was determined for each mouse embryo in modified F-10 medium containing 0.95 microM 3H-E1 or 3H-E2. During delayed implantation, the E1----E2 conversion rate was decreased (p less than 0.005) from 5.69 +/- 0.34 fmol/h/blastocyst on Day 5 to 3.50 +/- 0.46 fmol/h/blastocyst on Day 9, whereas E2----E1 was increased (p less than 0.005) from 7.44 +/- 1.08 to 18.60 +/- 2.04 fmol/h/blastocyst. After estrogen injection, the Day 9 implanting blastocyst showed an increase (p less than 0.005) in E1----E2 conversion to 9.05 +/- 0.64 fmol/h/blastocyst but a slight, insignificant decrease in E2----E1 conversion to 14.2 +/- 1.82 fmol/h/blastocyst. A similar trend was also observed in Day 5 implanting blastocysts when compared to Day 5 delayed blastocysts. Thus, 17 beta-HSD activity in delayed blastocysts favors E2----E1 over E1----E2 conversion in a ratio of 5:1. After estrogen induction of implantation, the E1----E2 conversion rate is stimulated and the ratio of E2----E1 to E1----E2 rate is decreased to 1.5:1. The results suggest that 17 beta-HSD activity may be involved in blastocyst implantation.     

Histochemistry of 3beta-hydroxysteroid dehydrogenase in rat ovary. I. Amethodological study

By recording the incubation time needed for initial appearance of the red and blue formazans the reliability of the histochemical method for 3beta-HSD was investigated: 1. Prefixation of small tissue blocks with 1% W/V methanol-free formaldehyde (pH=7.2) for up to 30 min preserved morphological integrity as well as maximal enzyme activity. Moreover, the substantivity of formazans and lipids was enhanced. 2. Commercial available glutaraldehyde (pH=7.2) induced SH groups in the tissue (even at 0.1% W/V for 5 min) thereby enhancing the Nothing dehydrogenase reaction. 3. Preextraction of lipids with acetone for 20 min at -30 degree C caused no loss of activity and was an inevitable step if a reliable activity pattern had to be achieved (e.g. in interstitial cells). 4. No diffusion of enzyme was noticed within 30 min of preincubation in phosphate buffer (0.2 M, pH=7.2) at 20 degree C. 5. By using the double-section incubation method no diffusion of 3beta-HSD or rediffusion of NADH or PMSH could be noticed withn 45 min of incubation, provided that low concentrations of NAD (0.1 mg/ml) and PMS (0.003 mg/ml) were balanced against the concentration of Nitro BT (0.5 mg/ml) or Tetranitro BT (1.0mg/ml). 6. The utlity of different inhibitors of alkaline phosphomonoesterase was tested and discussed. 7. By inhibiting alkaline phosphomonoesterase with 0.1 mM of L-p-bromotetramisole or 16 mM of beta-glycerophosphate, 3beta-HSD was shown to be exclusively NAD-linked. 8. Levamisole was a potent inhibitor of NADH-tetrazolium reductase as well as 3 beta-HSD, but not of NADPH-tetrazolium reductase. 9. 3beta-HSD possess SH groups requisite for the activity as this enzyme was totally inhibited by N-ethyl maleimide. 10. Whether alcohol dehydrogenases may use steroids as substrate is discussed; It is concluded that preextraction (by acetone) and/or the use of an inhibitor of alcohol dehydrogenase (1,10-phenanthroline) has to be performed. 11. Propylene glycol was a poor solvent for all substrates and was itself an excellent substrate for alcohol dehydrogenase. 12. Specifications for the ideal solvent of steroid substrates in the histochemical practice are proposed. DMSO showed to be promising as a steroid solvent (e.g. extraction of formazans was considerably lower as compared to DMF). 13. The utilization of substrates was descending in the following order (using 1 mM and 0.1 ml/ml of either DMF or DMSO): epiandrosterone, methandriol, dehydroepiandrosterone and pregnenolone. 14. If DMSO was used as solvent for pregnenolone (but not for the other substrates tested) an evident increase of activity was recorded as compared to DMF.     

Follicular fluid stimulation of 3 beta-hydroxysteroid dehydrogenase activity in vitro

Follicular fluid from porcine antral follicles stimulates progesterone secretion by porcine granulosa cells from small antral follicles in vitro. Fluid from large (6-12 mm) follicles has more stimulatory activity than fluid from smaller follicles. In this study, we have examined the action of charcoal-treated and filtered follicular fluid on 3 beta-hydroxysteroid dehydrogenase activity (3 beta-HSD) and the ability of exogenous pregnenolone to increase progesterone secretion. Granulosa cells cultured with 30% follicular fluid in TC 199 (v/v) for 3 days were less dependent on the presence of exogenous pregnenolone to enhance their progesterone secretion and exhibited more 3 beta-HSD activity than control cells incubated in 30% serum in TC 199. The apparent Vmax of 3 beta-HSD was increased 80% in follicular fluid-treated cells over that observed in controls (4.8 vs. 2.6 nM/min/100 mg protein) whereas the apparent Kms for 3 beta-HSD were similar (1.3 +/- 0.34 microM) in both experimental and control cells.     

Oxidative 3alpha-hydroxysteroid dehydrogenase activity of human type 10 17beta-hydroxysteroid dehydrogenase

In vitro enzyme assays have demonstrated that human type 10 17beta-hydroxysteroid dehydrogenase (17beta-HSD10) catalyzes the oxidation of 5alpha-androstane-3alpha,17beta-diol (adiol), an almost inactive androgen, to dihydrotestosterone (DHT) rather than androsterone or androstanedione. To further investigate the role of this steroid-metabolizing enzyme in intact cells, we produced stable transfectants expressing 17beta-HSD10 or its catalytically inactive Y168F mutant in human embryonic kidney (HEK) 293 cells. It was found that DHT levels in HEK 293 cells expressing 17beta-HSD10, but not its catalytically inactive mutant, will dramatically increase if adiol is added to culture media. Moreover, certain malignant prostatic epithelial cells have more 17beta-HSD10 than normal controls, and can generate DHT, the most potent androgen, from adiol. This event might promote prostate cancer growth. Analysis of the 17beta-HSD10 sequence shows that this enzyme does not have any ER retention signal or transmembrane segments and has not originated by divergence from a retinol dehydrogenase. The data suggest that the unique mitochondrial location of this HSD [Eur. J. Biochem. 268 (2001) 4899] does not prevent it from oxidizing the 3alpha-hydroxyl group of a C19 sterol in living cells. The experimental results lead to the conclusion that mitochondrial 17beta-HSD10 plays a significant part in a non-classical androgen synthesis pathway along with microsomal retinol dehydrogenases.     

Kinetic analysis of the placental microsomal 3 beta-hydroxysteroid dehydrogenase activity.

A sensitive accurate assay for the placental microsomal 3 beta-hydroxysteroid dehydrogenase (E.C.1.1.1.51) has been developed using tritiated substrates. Kinetic analysis of the enzyme with 3 beta-hydroxy-5-androsten-17-one and 3 beta-hydroxy-5-pregnen-20-one indicates that the apparent Km values for these substrates are orders of magnitude less than previously described. Analyses were carried out with microsomal preparations from two different placentas. For placenta 1 the apparent Km value for 3 beta-hydroxy-5-androsten-17-one was 14 nM and for 3 beta-hydroxy-5-pregnen-20-one was 36 nM; for placental 2 apparent Km values were 19 nM and 42 nM respectively. The analyses were performed over wide ranges of substrate concentration (about 200 fold), both above and below the Km values and no deviation from linearity of Eadie-Hoftsee plots was observed.     

11 beta-hydroxysteroid dehydrogenase activity in the mammary gland

Levels of 11 beta-hydroxysteroid dehydrogenase activity in mammary gland homogenates from pregnant and lactating Sprague-Dawley rats were determined by incubation with [3H]corticosterone under standard conditions, followed by thin-layer chromatography of incubated media. Enzyme activity was high in virgin and pregnant rats, but fell soon after parturition, suggesting a possible role for this enzyme in the co-ordinate regulation of glucocorticoid effects on milk protein synthesis.     

17 beta-hydroxysteroid dehydrogenase activity in canine pancreas

The mitochondrial fraction of the dog pancreas showed NAD(H)-dependent enzyme activity of 17 beta-hydroxysteroid dehydrogenase. The enzyme catalyzes oxidoreduction between androstenedione and testosterone. The apparent Km value of the enzyme for androstenedione was 9.5 +/- 0.9 microM, the apparent Vmax was determined as 0.4 nmol mg-1 min-1, and the optimal pH was 6.5. In phosphate buffer, pH 7.0, maximal rate of androstenedione reduction was observed at 37 degrees C. The oxidation of testosterone by the enzyme proceeded at the same rate as the reduction of the androstenedione at a pH of 6.8-7.0. The apparent Km value and the optimal pH of the enzyme for testosterone were 3.5 +/- 0.5 microM and 7.5, respectively.     

Isolation of multiple forms of indanol dehydrogenase associated with 17 beta-hydroxysteroid dehydrogenase activity from male rabbit liver

Seven multiforms of indanol dehydrogenase were isolated in a highly purified state from male rabbit liver cytosol. The enzymes were monomeric proteins with similar molecular weights of 30,000-37,000 but with distinct electrophoretic mobilities. All the enzymes oxidized alicyclic alcohols including benzene dihydrodiol and hydroxysteroids at different optimal pH, but showed clear differences in cofactor specificity, steroid specificity, and reversibility of the reaction. Two NADP+-dependent enzymes exhibited both 17 beta-hydroxysteroid dehydrogenase activity for 5 alpha-androstanes and 3 alpha-hydroxysteroid dehydrogenase activity for 5 beta-androstan-3 alpha-ol-17-one. Three of the other enzymes with dual cofactor specificity catalyzed predominantly 5 beta-androstane-3 alpha,17 beta-diol dehydrogenation. The reverse reaction rates of these five enzymes were low, whereas the other two enzymes, which had 3 alpha-hydroxysteroid dehydrogenase activity for 5 alpha-androstanes or 3(17)beta-hydroxysteroid dehydrogenase activity for 5 alpha-androstanes, highly reduced 3-ketosteroids and nonsteroidal aromatic carbonyl compounds with NADPH as a cofactor. All the enzymes exhibited Km values lower for the hydroxysteroids than for the alicyclic alcohols. The results of kinetic analyses with a mixture of 1-indanol and hydroxysteroids, pH and heat stability, and inhibitor sensitivity suggested strongly that, in the seven enzymes, both alicyclic alcohol dehydrogenase and hydroxysteroid dehydrogenase activities reside on a single enzyme protein. On the basis of these data, we suggest that indanol dehydrogenase exists in multiple forms in rabbit liver cytosol and may function in in vivo androgen metabolism.     

Inhibition of rat testis microsomal 3beta-hydroxysteroid dehydrogenase activity by tributyltin

In this study, we have examined the effects of a range of organotin compounds (mono-, di-, tributyltin, mono-, di-, trioctyltin) on the activities of rat testis microsomal 3beta-hydroxysteroid dehydrogenase (3beta-HSD), 17-hydroxylase (17-OHase) and 17beta-hydroxysteroid dehydrogenase (17beta-HSD). 17-OHase activity was inhibited by more than 50% compared with the control rate by 59 microM tributyltin (TBT) but other organotin compounds showed no inhibition. 17beta-HSD activity was unaffected by all organotins tested. 3beta-HSD was inhibited by monooctyltin (81 microM) and by TBT at all concentrations tested in a dose-dependent manner, with almost complete loss of activity at TBT concentrations of 12 microM. The mechanism of inhibition of 3beta-HSD was investigated in kinetic analysis with 0-12 microM TBT. Three rat testis microsomal preparations were incubated with dehydroepiandrosterone as the steroid substrate ranging from 1 to 10,000 nM. Tributyltin was primarily a competitive inhibitor of 3beta-HSD activity, causing an increase in the value of the K(m(app)). However, the mechanism was not entirely competitive as while there was an increase in K(m(app)), a decrease in the V(max(app)) was also observed with increasing concentrations of TBT. Slope and intercept replots demonstrated that the K(i)((app)) from slope replots was around 2.7 microM whereas the K(i)((app)) value from intercept replots was around 30 microM. When compared with the K(m(app)) for 3beta-HSD of around 0.42 microM, TBT could be an effective inhibitor of this enzyme.