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The synthetic pathway by which 5{alpha} -androstane-3{alpha} ,17ß-diol (5{alpha} -adiol) is formed in the testes of tammar wallaby pouch young was investigated by incubating testes from d 20–40 males with various radioactive precursors and analyzing the metabolites by thin-layer chromatography and HPLC. [3H]Progesterone was converted to 17-hydroxyprogesterone, which was converted to 5{alpha} -adiol by two pathways: One involves the formation of testosterone and dihydrotestosterone as intermediates, and the other involves formation of 5{alpha} -pregnane-3{alpha} ,17{alpha} -diol-20-one (5{alpha} -pdiol) and androsterone as intermediates. Formation of 5{alpha} -adiol from both [3H]testosterone and [3H]progesterone was blocked by the 5{alpha} -reductase inhibitor 4MA. The addition of nonradioactive 5{alpha} -pdiol blocked the conversion of [3H]progesterone to 5{alpha} -adiol, and [3H]5{alpha} -pdiol was efficiently converted to androsterone and 5{alpha} -adiol. We conclude that expression of steroid 5{alpha} -reductase in the developing wallaby testes allows formation of 5{alpha} -reduced androgens by a pathway that does not involve testosterone as an intermediate.
Introduction
5{alpha} -ANDROSTANE-3{alpha} ,17ß-DIOL (5{alpha} -ADIOL) plays a key role in the formation of the male urogenital tract in the tammar wallaby. It is the predominant androgen formed in the testes of the early tammar pouch young (1), is secreted into plasma at the time of virilization of the urogenital sinus in the male of this species (1), and virilizes the urogenital tract and the external genitalia when administered to female pouch young (2, 3). 5{alpha} -Adiol is also the predominant steroid formed in immature testes of mice (4, 5), rats (6, 7), and golden hamsters (8) and is the principal androgen formed in regenerating Leydig cells of adult rats following treatment with the cytotoxic drug ethane dimethane sulfonate (9) as well as the major androgen formed in adult rat testes after desensitization treatment with an LHRH agonist (10). The capacity to form 5{alpha} -adiol declines as testosterone synthesis increases after d 20 in the mouse (4) and after d 35 in the rat (6), and the shift from formation of 5{alpha} -adiol to testosterone in the immature rat testes is accelerated by the administration of chorionic gonadotropin (11).
The pathway by which 5{alpha} -adiol is synthesized is not entirely clear. We showed that 5{alpha} -adiol can be formed from testosterone in tammar pouch young testes (1), but Eckstein et al. (12) reported that the predominant pathway of 5{alpha} -adiol synthesis in immature rat testes involves 17-hydroxyprogesterone (17-OHP) but not androstenedione as an intermediate. Presumably, 5{alpha} -adiol formation in the immature testes involves one or more 5{alpha} -reduced intermediates because the levels of isoenzymes 1 and 2 of steroid 5{alpha} -reductase are high in rat testes at d 30 and decline rapidly thereafter (13) and because both progesterone and 17-OHP are better substrates for 5{alpha} -reductase than testosterone itself (14). In keeping with this possibility, Tsujimura and Matsumoto identified a variety of 5{alpha} -reduced pregnanes and androgens in immature mouse testes (15). To define the pathway of 5{alpha} -adiol formation in tammar wallaby pouch young, we studied the metabolism of [3H]progestogens and [3H]testosterone in testes removed from pouch young during the period when the male urogenital sinus virilizes (d 20–45 postpartum).
Materials and Methods
Animals
Tammar wallabies (Macropus eugenii) that originated in Kangaroo Island, South Australia, were held in a breeding colony in open yards. Their diet was supplemented with lucerne hay, oats, and fresh vegetables. All experiments followed guidelines of the National Health and Medical Research Council of Australia (1977) and were approved by the Animal Experimentation Ethics Committee of the University of Melbourne. Females were checked regularly for the presence of pouch young, and pouch young ages were determined either from known birth dates or extrapolated from growth curves using head length measurements (16). The sex of each pouch young was identified by the presence of scrotal bulges (males) or pouch and mammary gland primorida (females) (17).
Methods
The pouch young were killed by decapitation (18), and the testes were removed, weighed, dissected free of adjacent structures under a dissecting microscope, and kept in ice-cold normal saline until used for incubations. For the in vitro incubation studies, gonads were blotted, weighed, and added to glass tubes containing radioactive steroids in 50 µl DMEM and some instances either 4 µl ethanol or nonradioactive steroids or 17ß-N,N-diethyl-carbamoyl-4-methyl-4-aza-5{alpha} -androstane-3-one (4MA) dissolved in 4 µl ethanol (19). The tubes were gassed for 30 sec with 95% oxygen-5% carbon dioxide, capped, and incubated with shaking at 37 C for varying periods of time. The reactions were stopped by the addition of 1 ml chloroform/methanol (2:1), and the samples were dried under air at room temperature. The residues were dissolved in 0.2 ml chloroform/methanol (2:1).
For thin-layer chromatography (TLC), 10-µl aliquots were spotted on 20 x 20-cm TLC plastic sheets coated with silica gel 60 (Merck, Darmstadt, Germany) together with 10 µg each of carrier steroids (progesterone, 17-OHP, 5-dihydroprogesterone, 5{alpha} -adiol, dihydrotestosterone, testosterone, androsterone, androstenedione, 5-androstanedione, and in some studies 5{alpha} -pregnane-3{alpha} ,17{alpha} -diol-20-one (5-pdiol). The plates were developed in chloroform:toluene:acetone (50:80:20) for 45 min and air dried; steroids were visualized by spraying with 1% p-anisaldehyde in glacial acetic acid:sulfuric acid (100:2) and heating the plates in an oven at 100 C for 15 min. Each lane was then cut into 10 fractions corresponding to the visualized carrier steroids, and the entire lane was assayed for radioactivity in a liquid scintillation counter.
For separation by HPLC, the residues were dissolved in 100 µl methanol, and aliquots were injected into an 840 instrument (Waters, Woburn, MA) and separated on a 10-µm C18 Bondpak column (3.9 x 300 mm) using a gradient of 60–100% methanol and water. In the standard 45-min run, the initial 23 min was at 64% methanol followed by a 7-min gradient to 100% methanol followed by 15 min at 100% methanol. The column effluent was analyzed with a 757 absorbance detector (Spectraflow, Waters) set at 254 nm and a ß-RAM radioactive flow detector (INUS Systems, Inc., Tampa, FL). Under these conditions the elution times (in minutes) were as follows: androstenedione, 13.7; testosterone, 16.8; 17-OHP, 17.8; dihydrotestosterone, 26.0; progesterone 32.6; androsterone, 33.8; 5-adiol, 34.2; and 5{alpha} -pdiol, 35.4.
Materials
[1,2,6,7-3H]Progesterone [3.8 TBq/mmol] and [7-3H]pregnenolone (0.5 TBq/mmol) were from NEN Life Science Products, Boston MA; 17-hydroxy[1,2,6,7-3H]progesterone (2.40 TBq/mmol) and [1,2,6,7-3H]testosterone (3.70 TBq/mmol) were from Amersham Pharmacia. [1,2,6,7-3H]5{alpha} -pregnane-3{alpha} ,17{alpha} -diol-20-one was prepared as described by hydroxylation of [1,2,6,7-3H]5{alpha} -pregnane-3{alpha} -ol-20-one (3.8 TBq/mmol) (NEN Life Science Products) with microsomes that express a mutant human 17-hydroxylase (CYP 17) that is selectively deficient in 17,20-lyase activity (19). [1,2-3H]5-dihydroprogesterone was prepared by enzymatic reduction of [1,2-3H]progesterone (1.8 TBq/mmol, NEN Life Science Products) using microsomes from yeast that express human steroid 5{alpha} -reductase 2 as described (19). The nonradioactive steroids were from Steraloids, Inc. (Newport, RI) or Sigma (St. Louis, MO). The 5{alpha} -reductase inhibitor 4MA was a gift of Merck (20).
Results
In preliminary experiments d 49 tammar pouch young testes were incubated with 5 µM [3H]pregnenolone for 2 h, and the incubation products were separated by TLC and assayed for radioactivity, and the majority of radioactivity was recovered in progesterone and 17-OHP (46 and 56 nmol/mg testis·2 h, respectively), compared with 17-hydroxypregnenolone (6.4 nmol/mg testis·2 h). We concluded that the predominant system for androgen synthesis in this species is via the {Delta} 4 pathway, and the subsequent experiments used radioactive progesterone and its metabolites.
To investigate the concept that 5{alpha} -adiol could be synthesized by a pathway not involving androstenedione as a substrate, we considered the possibility that 5{alpha} -dihydroprogesterone might be an intermediate in the pathway because progesterone is a better substrate for steroid 5{alpha} -reductase than testosterone or androstenedione (14). However, [3H]progesterone did not appear to be converted in significant amounts to [3H]5{alpha} -dihydroprogesterone (results not shown), and when 5 µM [3H]progesterone and 5 µM [3H]5{alpha} -dihydroprogesterone were compared as substrates for 5{alpha} -adiol formation in pouch young testes in two experiments, the rate of formation from progesterone was almost twice that from dihydroprogesterone (average of 10.0 vs. 5.2 pmol/h per two testes). Thus, 5{alpha} -dihydroprogesterone is probably not a significant intermediate in the conversion of progesterone to 5{alpha} -adiol. However, as illustrated in Fig. 1 (and found consistently), pouch young testes converted [3H]progesterone to a variety of metabolites including 17-OHP, testosterone, 5{alpha} -adiol, androsterone/dihydrotestosterone (which did not separate in this system), and another metabolite that chromatographed in a region between 5{alpha} -adiol and the origin. In time sequence studies, the unknown metabolite was detectable after the appearance of 17-OHP, and its formation was blocked by the addition of the steroid 5{alpha} -reductase inhibitor 4MA (results not shown). We therefore deduced that the metabolite was probably a 5{alpha} -reduced derivative of 17-OHP, which is also an excellent substrate for steroid 5{alpha} -reductase (14).
fig.ommitteed
Figure 1. [3H]Progesterone metabolism in ovaries and testes from tammar wallaby pouch young. Two gonads from tammar wallaby male (average age 26 d) or female (average age 23 d) were added to 50 µl DMEM containing 5 µM [1,2,6,7-3H]progesterone. The tubes were gassed with 95% oxygen-5% carbon dioxide, incubated with shaking at 37 C as indicated, and processed and subjected to TLC as described in the text. O, 17-OHP; , testosterone; , 5-adiol; , 5-pdiol; , dihydrotestosterone.
On the basis of chromatography studies using five different solvent systems for TLC and three different programs for elution of HPLC with methanol and water, we tentatively identified the unknown metabolite not as the predicted metabolite 5-pregnane-17{alpha} -ol-3,20-dione but as its 3{alpha} -reduced derivative 5{alpha} -pdiol. This progesterone metabolite was previously identified in immature mouse testes (15). In contrast, the only metabolite of [3H]progesterone in d 23 tammar pouch ovaries was a small amount of 17-hydroxyprogesterone, indicating that 5-adiol formation does not occur in this tissue (Fig. 1). In all of the TLC systems examined, androsterone cochromatographed with dihydrotestosterone, and the areas for 5-adiol and 5{alpha} -pdiol partially overlapped, so the remainder of the analyses were performed by HPLC.
When testes from d 35 pouch young were incubated with 5 µM [3H]progesterone, [3H]17-OHP, or [3H]5{alpha} -pdiol, 17-OHP was a better substrate than progesterone for the formation of testosterone and 5-adiol, whereas [3H]5-pdiol was converted to androsterone and 5-adiol but not to 4 metabolites (Table 1). The fact that [3H]5-pdiol was converted to androsterone and 5-adiol was in keeping with our interpretation that this compound is an intermediate in 5-adiol synthesis. Furthermore, in trapping experiments in which excess 17-OHP or 5-pdiol was added to incubations of pouch young testes with [3H]progesterone, 17-OHP inhibited the formation of testosterone and 5-adiol similarly, whereas 5-pdiol inhibited the formation of 5-adiol but had little effect on the formation of testosterone (Table 2). Inhibition of steroid 5-reductase with 5 µM 4MA blocks the conversion of [3H]progesterone to 5-adiol and 5-pdiol but does not impair the formation of 17-OHP or testosterone (Fig. 2).
fig.ommitteed
Table 1. Conversion of three [3H]progestogens to 5-adiol by tammar pouch young testes
fig.ommitteed
Table 2. Effect of excess 17-OHP or 5-pdiol on the conversion of [3H]progesterone to 5-adiol by tammar pouch young testes
fig.ommitteed
Figure 2. Effect of inhibition of steroid 5reductase with 4MA on [3H]progesterone metabolism in d 24 tammar wallaby pouch young testes. One testis (0.9–1.6 mg in weight) from tammar wallaby pouch young (average age 24 d) was added to 50 µl DMEM containing 5 µM [1,2,6,7-3H]progesterone and either 4 µl ethanol (A) or 5 µM 4MA in 4 µl ethanol (B). The tubes were gassed with 95% oxygen-5% carbon dioxide, incubated with shaking at 37 C as indicated, and processed by HPLC as described in the text. Symbols are as in Fig. 1.
These various experiments indicated that 17-OHP is metabolized by a 5-reduced pathway to 5-adiol and by a 4 pathway to testosterone, which we had previously shown can also be metabolized to 5-adiol (1). To provide insight into the relative importance of these two pathways of 5-adiol formation, 5 µM [3H]testosterone and 5 µM [3H]progesterone were compared as substrates for the formation of 5-adiol in d 34 pouch young testes (Fig. 3). After 2 h approximately twice as much 5-adiol was formed from progesterone as from testosterone (8.2 vs. 3.8 pmol/mg testis·2 h), and the formation of 5-adiol from both substrates was inhibited by 4MA. These findings are in keeping with the interpretation of Eckstein et al. (12) that the predominant pathway of 5-adiol formation in the immature rat testis does not involve androstenedione as an intermediate.
fig.ommitteed
Figure 3. Comparison of [1,2,6,7-3H]testosterone and [1,2,6,7-3H]progesterone as precursors for 5-adiol formation by d 34 tammar wallaby pouch young testes. One testis (1.8–2.9 mg in weight) from tammar wallaby pouch young (average age 34 d) was added to 50 µl DMEM containing either 5 µM [1,2,6,7-3H]progesterone or 5 µM [1,2,6,7-3H]testosterone and either 4 µl ethanol or 5 µM 4MA in 4 µl ethanol. The tubes were gassed with 95% carbon dioxide, incubated with shaking at 37 C as indicated, and processed by HPLC as described in the text. Symbols are as in Fig. 1.
In immature rat testes, the capacity to form 5-adiol declines (6) in parallel with the decrease in the activity of the steroid 5-reductase isoenzymes (13). In the oldest pouch young testes examined in the present study (d 54), the conversion of [3H]progesterone to 5-adiol and testosterone were similar (Fig. 4), implying that 5-reductase activity had not started to decline by this age.
fig.ommitteed
Figure 4. [3H]Progesterone metabolism by d 54 tammar wallaby pouch young testes. One testis (3.1–3.9 mg in weight) from tammar wallaby pouch young (aged 51 and 57 d) was added to 50 µl DMEM containing 5 µM [1,2,6,7-3H]progesterone. The tubes were gassed with 95% oxygen-5% carbon dioxide, incubated with shaking at 37 C as indicated, and processed by HPLC as described in the text. Symbols are as in Fig. 1.
Discussion
The present studies indicate that 5{alpha} -adiol, the predominant 19-carbon steroid synthesized in testes of wallaby pouch young (1), can be formed by two enzymatic pathways (Fig. 5). Testosterone can be converted to 5-adiol as a result of the action of 5-reductase and 3-hydroxysteroid dehydrogenase, and a second pathway of formation involves 5-pdiol and androsterone as intermediates. Interestingly, we did not identify measurable amounts of 5-pregnane-17{alpha} -ol-3,20-dione as an intermediate. Under ordinary circumstances steroid 5-reductase is a microsomal enzyme, whereas 3-hydroxysteroid dehydrogenase is in the cytosol, but in some tissues the two enzymes appear to colocalize in small organelles (21), and the failure to find significant amounts of 5-pregnane-17-ol-3,20-dione could result from either substrate channeling because of enzyme proximity or a much greater 3-hydroxysteroid dehydrogenase activity in this tissue, compared with 5-reductase. A similar phenomenon was observed by Tsujimura and Matsumoto (15) in immature mouse testes incubated with [3H]progesterone, in which only traces of radioactivity were recovered in 5-pregnane-17-ol-3,20-dione, compared with 5-pdiol. The finding that [3H]5-pdiol is an excellent substrate for 5-adiol formation in tammar pouch young testes is in keeping with the demonstration that this steroid is also an effective substrate for human CYP17 (19). In the immature rabbit testis, 5-androstane-3ß,17ß-diol is the principal 5-reduced androgen (22, 23) and appears to be synthesized via the intermediate 5{alpha} -pregnane-3ß,17-diol-20-one (22). Whether 5-androstane-3ß,17ß-diol is an active androgen in the rabbit is not known. It is of interest that androsterone was identified as a likely intermediate in the present studies only in the experiment using [3H]5-pdiol as substrate and that little radioactivity was recovered in androstenedione following incubation with [3H] progesterone, both phenomena presumably indicating a high level of 17ß-hydroxysteroid dehydrogenase 3 activity in these testes.
fig.ommitteed
Figure 5. Pathways for 5-adiol formation in tammar pouch young testes. CYP17, Steroid 17-hydroxylase; 17HSD3, 17ß-hydroxysteroid dehydrogenase 3; 5-R, steroid 5-reductase; 3-HSD, 3-hydroxysteroid dehydrogenase. Brackets indicate intermediates that are inferred from subsequent metabolism but did not accumulate during the in vitro incubations.
In all mammals studied to date [with the exception of the Australian phalanger (Tricosurus vulpecula) in which large amounts of 5{alpha} -reduced steroids are secreted by the testes of mature animals (24, 25)], the activity of steroid 5-reductase in mature testes is low, and the predominant testicular androgen is testosterone. In contrast, developmental studies in the mouse (4, 5), rat (6, 7, 13), golden hamster (8), and rabbit (21, 22) indicate that 5-reductase activity is high in the immature testis and decreases or becomes undetectable with sexual maturation. We have shown that levels of 5{alpha} -adiol and dihydrotestosterone in tammar wallaby pouch young testes are quite low by d 100 (Wilson, J. D., G. Shaw, M. B. Renfree, and C. Shackleton, unpublished observations), and the finding in the present study that the activity was still high on d 54 indicates that the decline in 5-reductase activity must occur between d 50 and d 100. The fact that 5{alpha} -adiol synthesis in the adult rat is increased following inhibition of gonadotropin secretion (10) and the decline in 5{alpha} -reductase activity in the neonatal rat testes can be accelerated by treatment with chorionic gonadotropin (11) implies that testicular 5-reductase is under negative control by gonadotropins, but the mechanism of this control has not been elucidated. Likewise, the cellular location of 5-reductase in wallaby testes has not been identified, although the fact that in rat testes the enzyme is present in Leydig cells (8, 26) makes it likely that it is similarly located in the wallaby. Pratis et al. (13) reported that the predominant isoenzyme in neonatal rat testis is steroid 5{alpha} -reductase 1, but isoenzyme 2 is also detectable; both activities decline with maturation. The isoenzyme(s) responsible for this activity in tammar wallaby pouch young have not been identified.
Evidence of a variety of types suggests that 5-adiol virilizes tammar wallaby pouch young, at least in part, via oxidation of the 3{alpha} -hydroxyl to form dihydrotestosterone and dihydrotestosterone mediates the androgenic effect by binding to the androgen receptor (2, 3). Under most circumstances dihydrotestosterone is formed in extraglandular tissues by the 5{alpha} -reduction of testosterone, whereas in the tammar pouch young intracellular dihydrotestosterone appears to be formed by the oxidation of 5-adiol (1). It is not known whether circulating 5{alpha} -adiol also has actions in the tammar wallaby that are not mediated exclusively by the androgen receptor, e.g. via a cell surface receptor (27, 28) or by interacting with -aminobutyric acid receptors (29).
It is well established that active androgens can be synthesized by two general pathways, depending on the enzymatic composition of individual species. The 5 pathway involves pregnenolone 17-hydroxypregnenolone dehydroepiandrosterone androstenediol testosterone, and the 4 pathway involves pregnenolone progesterone 17-hydroxyprogesterone androstenedione testosterone. In both pathways the end product is testosterone, which can serve as a precursor for 5-reduced androgens in either the gonads or in extraglandular tissues. The fact that 5-adiol (and dihydrotestosterone) can be formed in the immature gonads of several species by another pathway, apparently determined by the expression of steroid 5-reductase, raises the possibility that 5{alpha} -adiol is in fact a primordial hormone of the testes. Why two different mechanisms evolved for the synthesis of dihydrotestosterone is unclear. We speculate that a high level of 5-reductase in the testes during the time of virilization of the male genitalia causes most 19-carbon steroids to be 5reduced and hence ready dihydrotestosterone precursors. The pathway of 5{alpha} -adiol formation via 5-pdiol circumvents two kinetic barriers to testosterone synthesis, namely 17-OHP is superior to testosterone as a substrate for 5-reductase (13) and 5{alpha} -pdiol appears to be the best substrate for the 17,20-lyase activity of CYP17 (19). The 5-pdiol pathway would thus assure efficient production of 19-carbon steroids even in species such as the human in which 17,20-lyase function of CYP17 is inefficient with 4 steroids (30). In summary, the formation of 5-adiol in fetal and neonatal testes of several species may be the most efficient route to dihydrotestosterone formation as a consequence of inherent properties of the synthetic enzymes.
Acknowledgments
Dr. Byron H. Arison (Merck Sharp & Dohm Research Laboratories, Rahway, NJ) generously performed nuclear magnetic resonance spectroscopy to confirm the assigned structure of 5{alpha} -pregnane-3,20-diol-20-one.
Received July 16, 2002.
Accepted for publication October 23, 2002.
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The synthetic pathway by which 5{alpha} -androstane-3{alpha} ,17ß-diol (5{alpha} -adiol) is formed in the testes of tammar wallaby pouch young was investigated by incubating testes from d 20–40 males with various radioactive precursors and analyzing the metabolites by thin-layer chromatography and HPLC. [3H]Progesterone was converted to 17-hydroxyprogesterone, which was converted to 5{alpha} -adiol by two pathways: One involves the formation of testosterone and dihydrotestosterone as intermediates, and the other involves formation of 5{alpha} -pregnane-3{alpha} ,17{alpha} -diol-20-one (5{alpha} -pdiol) and androsterone as intermediates. Formation of 5{alpha} -adiol from both [3H]testosterone and [3H]progesterone was blocked by the 5{alpha} -reductase inhibitor 4MA. The addition of nonradioactive 5{alpha} -pdiol blocked the conversion of [3H]progesterone to 5{alpha} -adiol, and [3H]5{alpha} -pdiol was efficiently converted to androsterone and 5{alpha} -adiol. We conclude that expression of steroid 5{alpha} -reductase in the developing wallaby testes allows formation of 5{alpha} -reduced androgens by a pathway that does not involve testosterone as an intermediate.
Introduction
5{alpha} -ANDROSTANE-3{alpha} ,17ß-DIOL (5{alpha} -ADIOL) plays a key role in the formation of the male urogenital tract in the tammar wallaby. It is the predominant androgen formed in the testes of the early tammar pouch young (1), is secreted into plasma at the time of virilization of the urogenital sinus in the male of this species (1), and virilizes the urogenital tract and the external genitalia when administered to female pouch young (2, 3). 5{alpha} -Adiol is also the predominant steroid formed in immature testes of mice (4, 5), rats (6, 7), and golden hamsters (8) and is the principal androgen formed in regenerating Leydig cells of adult rats following treatment with the cytotoxic drug ethane dimethane sulfonate (9) as well as the major androgen formed in adult rat testes after desensitization treatment with an LHRH agonist (10). The capacity to form 5{alpha} -adiol declines as testosterone synthesis increases after d 20 in the mouse (4) and after d 35 in the rat (6), and the shift from formation of 5{alpha} -adiol to testosterone in the immature rat testes is accelerated by the administration of chorionic gonadotropin (11).
The pathway by which 5{alpha} -adiol is synthesized is not entirely clear. We showed that 5{alpha} -adiol can be formed from testosterone in tammar pouch young testes (1), but Eckstein et al. (12) reported that the predominant pathway of 5{alpha} -adiol synthesis in immature rat testes involves 17-hydroxyprogesterone (17-OHP) but not androstenedione as an intermediate. Presumably, 5{alpha} -adiol formation in the immature testes involves one or more 5{alpha} -reduced intermediates because the levels of isoenzymes 1 and 2 of steroid 5{alpha} -reductase are high in rat testes at d 30 and decline rapidly thereafter (13) and because both progesterone and 17-OHP are better substrates for 5{alpha} -reductase than testosterone itself (14). In keeping with this possibility, Tsujimura and Matsumoto identified a variety of 5{alpha} -reduced pregnanes and androgens in immature mouse testes (15). To define the pathway of 5{alpha} -adiol formation in tammar wallaby pouch young, we studied the metabolism of [3H]progestogens and [3H]testosterone in testes removed from pouch young during the period when the male urogenital sinus virilizes (d 20–45 postpartum).
Materials and Methods
Animals
Tammar wallabies (Macropus eugenii) that originated in Kangaroo Island, South Australia, were held in a breeding colony in open yards. Their diet was supplemented with lucerne hay, oats, and fresh vegetables. All experiments followed guidelines of the National Health and Medical Research Council of Australia (1977) and were approved by the Animal Experimentation Ethics Committee of the University of Melbourne. Females were checked regularly for the presence of pouch young, and pouch young ages were determined either from known birth dates or extrapolated from growth curves using head length measurements (16). The sex of each pouch young was identified by the presence of scrotal bulges (males) or pouch and mammary gland primorida (females) (17).
Methods
The pouch young were killed by decapitation (18), and the testes were removed, weighed, dissected free of adjacent structures under a dissecting microscope, and kept in ice-cold normal saline until used for incubations. For the in vitro incubation studies, gonads were blotted, weighed, and added to glass tubes containing radioactive steroids in 50 µl DMEM and some instances either 4 µl ethanol or nonradioactive steroids or 17ß-N,N-diethyl-carbamoyl-4-methyl-4-aza-5{alpha} -androstane-3-one (4MA) dissolved in 4 µl ethanol (19). The tubes were gassed for 30 sec with 95% oxygen-5% carbon dioxide, capped, and incubated with shaking at 37 C for varying periods of time. The reactions were stopped by the addition of 1 ml chloroform/methanol (2:1), and the samples were dried under air at room temperature. The residues were dissolved in 0.2 ml chloroform/methanol (2:1).
For thin-layer chromatography (TLC), 10-µl aliquots were spotted on 20 x 20-cm TLC plastic sheets coated with silica gel 60 (Merck, Darmstadt, Germany) together with 10 µg each of carrier steroids (progesterone, 17-OHP, 5-dihydroprogesterone, 5{alpha} -adiol, dihydrotestosterone, testosterone, androsterone, androstenedione, 5-androstanedione, and in some studies 5{alpha} -pregnane-3{alpha} ,17{alpha} -diol-20-one (5-pdiol). The plates were developed in chloroform:toluene:acetone (50:80:20) for 45 min and air dried; steroids were visualized by spraying with 1% p-anisaldehyde in glacial acetic acid:sulfuric acid (100:2) and heating the plates in an oven at 100 C for 15 min. Each lane was then cut into 10 fractions corresponding to the visualized carrier steroids, and the entire lane was assayed for radioactivity in a liquid scintillation counter.
For separation by HPLC, the residues were dissolved in 100 µl methanol, and aliquots were injected into an 840 instrument (Waters, Woburn, MA) and separated on a 10-µm C18 Bondpak column (3.9 x 300 mm) using a gradient of 60–100% methanol and water. In the standard 45-min run, the initial 23 min was at 64% methanol followed by a 7-min gradient to 100% methanol followed by 15 min at 100% methanol. The column effluent was analyzed with a 757 absorbance detector (Spectraflow, Waters) set at 254 nm and a ß-RAM radioactive flow detector (INUS Systems, Inc., Tampa, FL). Under these conditions the elution times (in minutes) were as follows: androstenedione, 13.7; testosterone, 16.8; 17-OHP, 17.8; dihydrotestosterone, 26.0; progesterone 32.6; androsterone, 33.8; 5-adiol, 34.2; and 5{alpha} -pdiol, 35.4.
Materials
[1,2,6,7-3H]Progesterone [3.8 TBq/mmol] and [7-3H]pregnenolone (0.5 TBq/mmol) were from NEN Life Science Products, Boston MA; 17-hydroxy[1,2,6,7-3H]progesterone (2.40 TBq/mmol) and [1,2,6,7-3H]testosterone (3.70 TBq/mmol) were from Amersham Pharmacia. [1,2,6,7-3H]5{alpha} -pregnane-3{alpha} ,17{alpha} -diol-20-one was prepared as described by hydroxylation of [1,2,6,7-3H]5{alpha} -pregnane-3{alpha} -ol-20-one (3.8 TBq/mmol) (NEN Life Science Products) with microsomes that express a mutant human 17-hydroxylase (CYP 17) that is selectively deficient in 17,20-lyase activity (19). [1,2-3H]5-dihydroprogesterone was prepared by enzymatic reduction of [1,2-3H]progesterone (1.8 TBq/mmol, NEN Life Science Products) using microsomes from yeast that express human steroid 5{alpha} -reductase 2 as described (19). The nonradioactive steroids were from Steraloids, Inc. (Newport, RI) or Sigma (St. Louis, MO). The 5{alpha} -reductase inhibitor 4MA was a gift of Merck (20).
Results
In preliminary experiments d 49 tammar pouch young testes were incubated with 5 µM [3H]pregnenolone for 2 h, and the incubation products were separated by TLC and assayed for radioactivity, and the majority of radioactivity was recovered in progesterone and 17-OHP (46 and 56 nmol/mg testis·2 h, respectively), compared with 17-hydroxypregnenolone (6.4 nmol/mg testis·2 h). We concluded that the predominant system for androgen synthesis in this species is via the {Delta} 4 pathway, and the subsequent experiments used radioactive progesterone and its metabolites.
To investigate the concept that 5{alpha} -adiol could be synthesized by a pathway not involving androstenedione as a substrate, we considered the possibility that 5{alpha} -dihydroprogesterone might be an intermediate in the pathway because progesterone is a better substrate for steroid 5{alpha} -reductase than testosterone or androstenedione (14). However, [3H]progesterone did not appear to be converted in significant amounts to [3H]5{alpha} -dihydroprogesterone (results not shown), and when 5 µM [3H]progesterone and 5 µM [3H]5{alpha} -dihydroprogesterone were compared as substrates for 5{alpha} -adiol formation in pouch young testes in two experiments, the rate of formation from progesterone was almost twice that from dihydroprogesterone (average of 10.0 vs. 5.2 pmol/h per two testes). Thus, 5{alpha} -dihydroprogesterone is probably not a significant intermediate in the conversion of progesterone to 5{alpha} -adiol. However, as illustrated in Fig. 1 (and found consistently), pouch young testes converted [3H]progesterone to a variety of metabolites including 17-OHP, testosterone, 5{alpha} -adiol, androsterone/dihydrotestosterone (which did not separate in this system), and another metabolite that chromatographed in a region between 5{alpha} -adiol and the origin. In time sequence studies, the unknown metabolite was detectable after the appearance of 17-OHP, and its formation was blocked by the addition of the steroid 5{alpha} -reductase inhibitor 4MA (results not shown). We therefore deduced that the metabolite was probably a 5{alpha} -reduced derivative of 17-OHP, which is also an excellent substrate for steroid 5{alpha} -reductase (14).
fig.ommitteed
Figure 1. [3H]Progesterone metabolism in ovaries and testes from tammar wallaby pouch young. Two gonads from tammar wallaby male (average age 26 d) or female (average age 23 d) were added to 50 µl DMEM containing 5 µM [1,2,6,7-3H]progesterone. The tubes were gassed with 95% oxygen-5% carbon dioxide, incubated with shaking at 37 C as indicated, and processed and subjected to TLC as described in the text. O, 17-OHP; , testosterone; , 5-adiol; , 5-pdiol; , dihydrotestosterone.
On the basis of chromatography studies using five different solvent systems for TLC and three different programs for elution of HPLC with methanol and water, we tentatively identified the unknown metabolite not as the predicted metabolite 5-pregnane-17{alpha} -ol-3,20-dione but as its 3{alpha} -reduced derivative 5{alpha} -pdiol. This progesterone metabolite was previously identified in immature mouse testes (15). In contrast, the only metabolite of [3H]progesterone in d 23 tammar pouch ovaries was a small amount of 17-hydroxyprogesterone, indicating that 5-adiol formation does not occur in this tissue (Fig. 1). In all of the TLC systems examined, androsterone cochromatographed with dihydrotestosterone, and the areas for 5-adiol and 5{alpha} -pdiol partially overlapped, so the remainder of the analyses were performed by HPLC.
When testes from d 35 pouch young were incubated with 5 µM [3H]progesterone, [3H]17-OHP, or [3H]5{alpha} -pdiol, 17-OHP was a better substrate than progesterone for the formation of testosterone and 5-adiol, whereas [3H]5-pdiol was converted to androsterone and 5-adiol but not to 4 metabolites (Table 1). The fact that [3H]5-pdiol was converted to androsterone and 5-adiol was in keeping with our interpretation that this compound is an intermediate in 5-adiol synthesis. Furthermore, in trapping experiments in which excess 17-OHP or 5-pdiol was added to incubations of pouch young testes with [3H]progesterone, 17-OHP inhibited the formation of testosterone and 5-adiol similarly, whereas 5-pdiol inhibited the formation of 5-adiol but had little effect on the formation of testosterone (Table 2). Inhibition of steroid 5-reductase with 5 µM 4MA blocks the conversion of [3H]progesterone to 5-adiol and 5-pdiol but does not impair the formation of 17-OHP or testosterone (Fig. 2).
fig.ommitteed
Table 1. Conversion of three [3H]progestogens to 5-adiol by tammar pouch young testes
fig.ommitteed
Table 2. Effect of excess 17-OHP or 5-pdiol on the conversion of [3H]progesterone to 5-adiol by tammar pouch young testes
fig.ommitteed
Figure 2. Effect of inhibition of steroid 5reductase with 4MA on [3H]progesterone metabolism in d 24 tammar wallaby pouch young testes. One testis (0.9–1.6 mg in weight) from tammar wallaby pouch young (average age 24 d) was added to 50 µl DMEM containing 5 µM [1,2,6,7-3H]progesterone and either 4 µl ethanol (A) or 5 µM 4MA in 4 µl ethanol (B). The tubes were gassed with 95% oxygen-5% carbon dioxide, incubated with shaking at 37 C as indicated, and processed by HPLC as described in the text. Symbols are as in Fig. 1.
These various experiments indicated that 17-OHP is metabolized by a 5-reduced pathway to 5-adiol and by a 4 pathway to testosterone, which we had previously shown can also be metabolized to 5-adiol (1). To provide insight into the relative importance of these two pathways of 5-adiol formation, 5 µM [3H]testosterone and 5 µM [3H]progesterone were compared as substrates for the formation of 5-adiol in d 34 pouch young testes (Fig. 3). After 2 h approximately twice as much 5-adiol was formed from progesterone as from testosterone (8.2 vs. 3.8 pmol/mg testis·2 h), and the formation of 5-adiol from both substrates was inhibited by 4MA. These findings are in keeping with the interpretation of Eckstein et al. (12) that the predominant pathway of 5-adiol formation in the immature rat testis does not involve androstenedione as an intermediate.
fig.ommitteed
Figure 3. Comparison of [1,2,6,7-3H]testosterone and [1,2,6,7-3H]progesterone as precursors for 5-adiol formation by d 34 tammar wallaby pouch young testes. One testis (1.8–2.9 mg in weight) from tammar wallaby pouch young (average age 34 d) was added to 50 µl DMEM containing either 5 µM [1,2,6,7-3H]progesterone or 5 µM [1,2,6,7-3H]testosterone and either 4 µl ethanol or 5 µM 4MA in 4 µl ethanol. The tubes were gassed with 95% carbon dioxide, incubated with shaking at 37 C as indicated, and processed by HPLC as described in the text. Symbols are as in Fig. 1.
In immature rat testes, the capacity to form 5-adiol declines (6) in parallel with the decrease in the activity of the steroid 5-reductase isoenzymes (13). In the oldest pouch young testes examined in the present study (d 54), the conversion of [3H]progesterone to 5-adiol and testosterone were similar (Fig. 4), implying that 5-reductase activity had not started to decline by this age.
fig.ommitteed
Figure 4. [3H]Progesterone metabolism by d 54 tammar wallaby pouch young testes. One testis (3.1–3.9 mg in weight) from tammar wallaby pouch young (aged 51 and 57 d) was added to 50 µl DMEM containing 5 µM [1,2,6,7-3H]progesterone. The tubes were gassed with 95% oxygen-5% carbon dioxide, incubated with shaking at 37 C as indicated, and processed by HPLC as described in the text. Symbols are as in Fig. 1.
Discussion
The present studies indicate that 5{alpha} -adiol, the predominant 19-carbon steroid synthesized in testes of wallaby pouch young (1), can be formed by two enzymatic pathways (Fig. 5). Testosterone can be converted to 5-adiol as a result of the action of 5-reductase and 3-hydroxysteroid dehydrogenase, and a second pathway of formation involves 5-pdiol and androsterone as intermediates. Interestingly, we did not identify measurable amounts of 5-pregnane-17{alpha} -ol-3,20-dione as an intermediate. Under ordinary circumstances steroid 5-reductase is a microsomal enzyme, whereas 3-hydroxysteroid dehydrogenase is in the cytosol, but in some tissues the two enzymes appear to colocalize in small organelles (21), and the failure to find significant amounts of 5-pregnane-17-ol-3,20-dione could result from either substrate channeling because of enzyme proximity or a much greater 3-hydroxysteroid dehydrogenase activity in this tissue, compared with 5-reductase. A similar phenomenon was observed by Tsujimura and Matsumoto (15) in immature mouse testes incubated with [3H]progesterone, in which only traces of radioactivity were recovered in 5-pregnane-17-ol-3,20-dione, compared with 5-pdiol. The finding that [3H]5-pdiol is an excellent substrate for 5-adiol formation in tammar pouch young testes is in keeping with the demonstration that this steroid is also an effective substrate for human CYP17 (19). In the immature rabbit testis, 5-androstane-3ß,17ß-diol is the principal 5-reduced androgen (22, 23) and appears to be synthesized via the intermediate 5{alpha} -pregnane-3ß,17-diol-20-one (22). Whether 5-androstane-3ß,17ß-diol is an active androgen in the rabbit is not known. It is of interest that androsterone was identified as a likely intermediate in the present studies only in the experiment using [3H]5-pdiol as substrate and that little radioactivity was recovered in androstenedione following incubation with [3H] progesterone, both phenomena presumably indicating a high level of 17ß-hydroxysteroid dehydrogenase 3 activity in these testes.
fig.ommitteed
Figure 5. Pathways for 5-adiol formation in tammar pouch young testes. CYP17, Steroid 17-hydroxylase; 17HSD3, 17ß-hydroxysteroid dehydrogenase 3; 5-R, steroid 5-reductase; 3-HSD, 3-hydroxysteroid dehydrogenase. Brackets indicate intermediates that are inferred from subsequent metabolism but did not accumulate during the in vitro incubations.
In all mammals studied to date [with the exception of the Australian phalanger (Tricosurus vulpecula) in which large amounts of 5{alpha} -reduced steroids are secreted by the testes of mature animals (24, 25)], the activity of steroid 5-reductase in mature testes is low, and the predominant testicular androgen is testosterone. In contrast, developmental studies in the mouse (4, 5), rat (6, 7, 13), golden hamster (8), and rabbit (21, 22) indicate that 5-reductase activity is high in the immature testis and decreases or becomes undetectable with sexual maturation. We have shown that levels of 5{alpha} -adiol and dihydrotestosterone in tammar wallaby pouch young testes are quite low by d 100 (Wilson, J. D., G. Shaw, M. B. Renfree, and C. Shackleton, unpublished observations), and the finding in the present study that the activity was still high on d 54 indicates that the decline in 5-reductase activity must occur between d 50 and d 100. The fact that 5{alpha} -adiol synthesis in the adult rat is increased following inhibition of gonadotropin secretion (10) and the decline in 5{alpha} -reductase activity in the neonatal rat testes can be accelerated by treatment with chorionic gonadotropin (11) implies that testicular 5-reductase is under negative control by gonadotropins, but the mechanism of this control has not been elucidated. Likewise, the cellular location of 5-reductase in wallaby testes has not been identified, although the fact that in rat testes the enzyme is present in Leydig cells (8, 26) makes it likely that it is similarly located in the wallaby. Pratis et al. (13) reported that the predominant isoenzyme in neonatal rat testis is steroid 5{alpha} -reductase 1, but isoenzyme 2 is also detectable; both activities decline with maturation. The isoenzyme(s) responsible for this activity in tammar wallaby pouch young have not been identified.
Evidence of a variety of types suggests that 5-adiol virilizes tammar wallaby pouch young, at least in part, via oxidation of the 3{alpha} -hydroxyl to form dihydrotestosterone and dihydrotestosterone mediates the androgenic effect by binding to the androgen receptor (2, 3). Under most circumstances dihydrotestosterone is formed in extraglandular tissues by the 5{alpha} -reduction of testosterone, whereas in the tammar pouch young intracellular dihydrotestosterone appears to be formed by the oxidation of 5-adiol (1). It is not known whether circulating 5{alpha} -adiol also has actions in the tammar wallaby that are not mediated exclusively by the androgen receptor, e.g. via a cell surface receptor (27, 28) or by interacting with -aminobutyric acid receptors (29).
It is well established that active androgens can be synthesized by two general pathways, depending on the enzymatic composition of individual species. The 5 pathway involves pregnenolone 17-hydroxypregnenolone dehydroepiandrosterone androstenediol testosterone, and the 4 pathway involves pregnenolone progesterone 17-hydroxyprogesterone androstenedione testosterone. In both pathways the end product is testosterone, which can serve as a precursor for 5-reduced androgens in either the gonads or in extraglandular tissues. The fact that 5-adiol (and dihydrotestosterone) can be formed in the immature gonads of several species by another pathway, apparently determined by the expression of steroid 5-reductase, raises the possibility that 5{alpha} -adiol is in fact a primordial hormone of the testes. Why two different mechanisms evolved for the synthesis of dihydrotestosterone is unclear. We speculate that a high level of 5-reductase in the testes during the time of virilization of the male genitalia causes most 19-carbon steroids to be 5reduced and hence ready dihydrotestosterone precursors. The pathway of 5{alpha} -adiol formation via 5-pdiol circumvents two kinetic barriers to testosterone synthesis, namely 17-OHP is superior to testosterone as a substrate for 5-reductase (13) and 5{alpha} -pdiol appears to be the best substrate for the 17,20-lyase activity of CYP17 (19). The 5-pdiol pathway would thus assure efficient production of 19-carbon steroids even in species such as the human in which 17,20-lyase function of CYP17 is inefficient with 4 steroids (30). In summary, the formation of 5-adiol in fetal and neonatal testes of several species may be the most efficient route to dihydrotestosterone formation as a consequence of inherent properties of the synthetic enzymes.
Acknowledgments
Dr. Byron H. Arison (Merck Sharp & Dohm Research Laboratories, Rahway, NJ) generously performed nuclear magnetic resonance spectroscopy to confirm the assigned structure of 5{alpha} -pregnane-3,20-diol-20-one.
Received July 16, 2002.
Accepted for publication October 23, 2002.
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