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The biosynthesis of C21, C19, and C18 steroids in adrenal, ovary, testis and foeto-placental unit is summarized and the properties of the enzymes involved are described, with particular reference to their substrate specificities and to the possible controlling effects of endogenous steroids. It is concluded that these may exert a considerable regulatory role in vivo in steroid hormone biosynthesis.

The conversion of testosterone to 5 alpha-dihydrotestosterone, catalysed by 4-ene-steroid 5 alpha-reductase (3-oxo-5 alpha-steroid: NADP+ 4-ene-oxidoreductase EC 1.3.1.22) requires NADPH. In the present study, the role of flavins and Co-enzyme Q in this proton transfer was investigated for the first time in any male androgen target tissue. Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) inhibited epididymal nuclear 4-ene-steroid 5 alpha-reductase activity non-competitively with respect to the substrate testosterone. However, neither the oxidized nor reduced forms of Co-enzyme Q affected the Kmapp or the Vmaxapp and the reduced form was unable to support catalytic activity in the absence of NADPH. Further investigation of the effects of flavins revealed that the inhibition was caused by an elevation of NADP+ in the incubations and that the incorporation of a NADPH generating system abolished the inhibition. Therefore, neither flavins nor Co-enzyme Q directly affected the 4-ene-steroid 5 alpha-reductase activity. Further evidence to support this conclusion was obtained when several inhibitors of electron transfer reactions failed to inhibit 4-ene-steroid 5 alpha-reductases from rat epididymides, prostate and seminal vesicles. These findings show that, in male rat androgen target tissues, the conversion of testosterone to 5 alpha-dihydrotestosterone does not require intermediates of electron transfer reactions. We propose that the reduction proceeds by the direct transfer of protons from NADPH to testosterone.

We have investigated the effects of two 4-ene-steroid 5 alpha-reductase inhibitors, diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carboxamide (4-MA) and (4R)-5,10-seco-19-norpregna-4, 5-diene-3,10,20-trione (SECO), on testicular and epididymal androgen biosynthesis. Kinetic analyses revealed that both compounds inhibited epididymal DHT biosynthesis. 4-MA was a competitive inhibitor of epididymal nuclear and microsomal 4-ene-steroid 5 alpha-reductases (3-oxo-5 alpha-steroid: NADP 4-ene-oxidoreductase EC 1.3.1.22) with Kiapp values of 12.8 and 15.1 nmol/l compared to the respective Kmapp values of 185 and 240 nmol/l. Values for the Vmaxapp were always within 70-130% of the control. SECO at 1.0 mumol/l, also inhibited epididymal nuclear and microsomal 4-ene-steroid-5 alpha-reductases, causing respectively 2.9 and 5.2-fold increases in Kmapp. The Vmaxapp values were unchanged. However, SECO concentrations of 5 and 25 mumol/l abolished 4-ene-steroid 5 alpha-reductase activity at all testosterone concentrations. To examine the specificity of these compounds, we investigated their effects on the enzymes that convert pregnenolone to testosterone. Rat testis microsomes converted pregnenolone to testosterone via the 4-ene-3-oxo pathway, with the major metabolites being progesterone, 17-hydroxyprogesterone, 4-androstenedione and testosterone; some 17-hydroxypregnenolone was also formed. Very small amounts of dehydroepiandrosterone (DHA) and 5-androstenediol were detected. SECO, at a concentration that completely inhibited epididymal 4-ene-steroid 5 alpha-reductase activity, did not alter the metabolic profile of pregnenolone metabolism. However, 4-MA prevented the appearance of 4-ene steroids, and large quantities of 17-hydroxypregnenolone and DHA accumulated, suggesting that inhibition of the 3 beta-hydroxysteroid: NAD(P)+ oxidoreductase (EC 1.1.1.51) and 3-oxosteroid 5-ene-4-ene-isomerase (EC 5.3.3.1) [3 beta-hydroxysteroid dehydrogenase-isomerase] was occurring. Optimal conditions for the microsomal conversion of DHA to 4-androstenedione were determined; kinetic analyses of the 3 beta-hydroxysteroid dehydrogenase-isomerase activity revealed that 4-MA inhibited this reaction non-competitively, reducing Vmaxapp values to 25% of the control. The Kiapp determined from the intercept replot, was 121 nmol/l, and the Kmapp was always between 90 and 130% of the control value. It is concluded that SECO is more specific than 4-MA in its effects on androgen biosynthesis in the testis and epididymis and that both these drugs should provide useful tools in assessments of the relative contributions of 5 alpha-reduced androgens to androgen dependent processes.

Epididymal nuclear 4-ene steroid 5 alpha-reductase catalyses the bisubstrate reaction between testosterone and NADPH to produce 5 alpha-dihydrotestosterone (DHT) and NADP+. Previous studies from this laboratory have demonstrated that the 4-ene steroid 5 alpha-reductase reaction proceeds through the direct transfer of protons from NADPH to testosterone, and that while the product DHT does not affect 4-ene steroid 5 alpha-reductase activity, NADP+ is a potent inhibitor of this enzyme. In the present studies we have investigated the mechanism of 4-ene steroid 5 alpha-reductase with respect to the binding of the substrates, testosterone and NADPH. Kinetic analyses revealed that testosterone does not alter the Kmapp for NADPH, and that NADPH does not alter the Kmapp for testosterone. These findings excluded the possibility that the mechanism of 4-ene steroid 5 alpha-reductase is of the ping-pong variety, and that the sequential addition of both substrates is required before any products are released. The lack of change in Kmapp, observed for either substrate, further suggests that both testosterone and NADPH are able to bind to the free enzyme, negating the possibility that substrate addition occurs in an ordered manner. Indeed the kinetic profiles are entirely consistent with the mechanism of 4-ene steroid 5 alpha-reductase being a rapid equilibrium random sequential process in which the binding of the first substrate has no affect on the binding of the second. Mean values for the dissociation constants, Ktestosterone and KNADPH, were 200 nmol/l and 50 nmol/l, respectively. These findings, coupled with those from earlier studies, suggest that the mechanism of epididymal nuclear 4-ene steroid 5 alpha-reductase is a rapid equilibrium random bireactant process, with the possible dead-end complex: testosterone-4-ene steroid 5 alpha-reductase-NADP+.

Sulphotransferase activity has been assayed in porcine liver and testis cytosol using either 3'-phosphoadenosine-5'-phospho [35S]sulphate (PAPS) or unlabelled PAPS as sulphate donors. In porcine liver the sulphotransferase for DHA was linear for up to 10 min, the optimum pH was 7.7 and optimum temperature, 37 degrees C. The apparent Km value was found to be 91 mumol/l and the activity was inhibited non-competitively by 5 alpha-androst-16-en-3 beta-yl sulphate, with all concentrations used (0.02-25 mumol/l) inhibiting the enzyme to the same extent. Time courses for sulphoconjugation of pregnenolone and 5 alpha-androst-16-en-3 beta-ol were linear for up to at least 10 min or up to only 5 min, respectively. The optimum pH values and temperatures were pH 8.0 and 37 degrees C in each case. The porcine testicular sulphotransferase activity for DHA as substrate was linear with time up to 10 min, the apparent Km for the reaction was 2 mumol/l and apparent Vmax 10 nmol/l/mg/min. 5 alpha-Androst-16-en-3 beta-yl sulphate (11.3-45.2 mumol/l) failed to inhibit the enzyme activity. The time-course for the reaction, when pregnenolone was used as substrate, was also linear up to 10 min at the optimum pH 8.0 but, in contrast to the reaction when DHA was the substrate, had an apparent Km of 20 mumol/l and was inhibited by pregnenolone sulphate, 5 alpha-androst-16-en-3 beta-yl sulphate, DHA and 5 alpha-androst-16-en-3 beta-ol, but not by DHA sulphate. 5 alpha-Androst-16-en-3 beta-yl sulphate inhibited the reaction non-competitively and to the same extent at concentrations over the range 11.3-45.2 mumol/l. These data suggest that DHA and pregnenolone may not be sulphoconjugated by the same sulphotransferase. With 5 alpha-androst-16-en-3 beta-ol as substrate, the time-course for its sulphate formation was linear up to 15 min, and this reaction could explain the quantities of 5 alpha-androst-16-en-3 beta-yl sulphate that are found endogenously in porcine testis. Our results further suggest that these quantities could well inhibit the sulphation of pregnenolone in porcine testis in vivo, and the possibility of control of sulphoconjugation in this tissue is discussed. Having regard to the smaller quantities of 5 alpha-androst-16-en-3 beta-yl sulphate present in porcine liver, our results suggest that the sulphation of DHA there may not be so much affected.

Epididymal 4-ene steroid 5 alpha-reductase converts testosterone to 5 alpha-dihydrotestosterone. The enzyme is localized to the nuclear and microsomal fractions, and the activity can be altered by modifying the phospholipids in the membrane environment. To investigate the membrane dependence of 4-ene steroid 5 alpha-reductase, we have treated nuclear and microsomal membranes with combinations of phospholipase A2 and phospholipase C, and examined the effects on 4-ene steroid 5 alpha-reductase activity. Sequential addition of phospholipase A2 and phospholipase C to the nuclear fraction, reduced the 4-ene steroid 5 alpha-reductase activity to approx 25% of the control level. Neither the nature of the phospholipase, nor the sequence of addition altered the inhibition. When both phospholipases were added simultaneously, nuclear 4-ene steroid 5 alpha-reductase activity was inhibited in a linear fashion, and in tests for cooperativity, the effects of phospholipase A2 and phospholipase C were clearly additive. The microsomal enzyme responded differently to sequential phospholipase treatments; if phospholipase A2 was followed by phospholipase C, or phospholipase C followed by phospholipase A2, the 4-ene steroid 5 alpha-reductase activity was, respectively, 13 and 27% of the control. In contrast, sequential addition of the same phospholipase reduced the activity of 4-ene steroid 5 alpha-reductase to approx 40% of the control level. Furthermore, simultaneous addition of phospholipase A2 and phospholipase C to the microsomal fraction, resulted in non-linearity of 4-ene steroid 5 alpha-reductase activity with time, whereas when added individually, linearity of 4-ene steroid 5 alpha-reductase was maintained. Consequently, it was not possible to test for cooperative effects of phospholipases on the microsomal 4-ene steroid 5 alpha-reductase. These findings suggest that for the nuclear 4-ene steroid 5 alpha-reductase, the polar and non-polar regions of the membrane environment have similar functions, which are most likely involved in the maintenance of the structural integrity of the enzyme. For the microsomal enzyme, the polar and non-polar regions of the membrane appear to have different functions, not only for the maintenance of enzyme integrity, but also in the mechanism at the active site.