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Endocrinology, doi:10.1210/en.2005-1251
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Endocrinology Vol. 147, No. 5 2368-2373
Copyright © 2006 by The Endocrine Society

Role of the Alternate Pathway of Dihydrotestosterone Formation in Virilization of the Wolffian Ducts of the Tammar Wallaby, Macropus eugenii

Geoffrey Shaw, Jane Fenelon, Michelle Sichlau, Richard J. Auchus, Jean D. Wilson and Marilyn B. Renfree

Department of Zoology (G.S., J.F., M.S., J.D.W., M.B.R.), University of Melbourne, Victoria 3010, Australia; and Department of Internal Medicine (J.D.W., R.J.A.), University of Texas Southwestern Medical Center, Dallas, Texas 75390-8857

Address all correspondence and requests for reprints to: Geoff Shaw, Department of Zoology, The University of Melbourne, Victoria 3010, Australia. E-mail: g.shaw{at}zoology.unimelb.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Dihydrotestosterone in androgen target tissues is formed under most circumstances by the 5{alpha}-reduction of testosterone, but an alternate pathway involves the oxidation of androstanediol to dihydrotestosterone. To investigate the mechanism by which androgens virilize the Wolffian ducts in the tammar wallaby, [3H]progesterone was incubated with testes from d 10 and 19 pouch young, and radioactivity was recovered in testosterone and androstanediol at both ages. Analysis of the intermediates indicates that androstanediol was formed both from testosterone via 5{alpha}-reduction and 3{alpha}-keto reduction and directly from 5{alpha}-reduced progestogens. 5{alpha}-Reductase activity was high in minces of mesonephros/epididymis from d 6–21 pouch young. When minces of urogenital tract tissues from d 19 pouch young were incubated with [3H]testosterone, [3H]dihydrotestosterone, and [3H]androstanediol, dihydrotestosterone was the principal androgen formed in the mesonephros/epididymis, urogenital sinus, and urogenital tubercle, whereas androstanediol was the principal androgen formed by the testis. In intact pouch young studied between d 10 and 34, administration of the 5{alpha}-reductase inhibitor, 17ß-(N,N-diethyl)carbamoyl-4-methyl-4-aza-5{alpha}-androstan-3-one, blocked virilization of the Wolffian ducts in males, and administration of androstanediol caused virilization of the Wolffian ducts in females. We conclude that dihydrotestosterone, largely formed in the tissue by the oxidation of androstanediol derived from the testes and also the 5{alpha}-reduction of testosterone, is responsible for Wolffian duct virilization in this species.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN MAMMALS, VIRILIZATION of the male urogenital tract during development involves two processes: formation of the epididymides, vasa deferentia, and (in some species) seminal vesicles from the Wolffian ducts and conversion of the urogenital tubercle and urogenital sinus into the phallus, male urethra, and prostate. Both processes are mediated by androgens secreted by the developing testes (1). Development of the urogenital tract is identical in eutherians and marsupials with the exception of the scrotum and mammary glands (2, 3, 4). The scrotum in marsupials forms from a different anlagen, the abdominal skin, rather than from the labioscrotal folds as in eutherian mammals, and its formation is not controlled by androgens (2, 3, 5). Studies of animals with abnormal sex chromosome constitutions indicate that the marsupial scrotum is regulated by the sex chromosome composition independent of hormonal control (2, 5, 6), although as in eutherians differentiation of the male urogenital tract and penis is regulated by testicular hormones.

In eutherians testosterone, the androgen secreted by the fetal testis at the time of formation of the male urogenital tract (7, 8, 9, 10), promotes virilization by two mechanisms. In the urogenital sinus and urogenital tubercle, testosterone is 5{alpha}-reduced to dihydrotestosterone, which acts as the intracellular mediator for formation of the prostate and phallus (1). In contrast, testosterone itself mediates conversion of the Wolffian ducts into the ejaculatory system. Strong evidence for this dual control comes from the study of genetic males with steroid 5{alpha}-reductase 2 deficiency in whom the external genitalia and urethra are female in character, whereas the Wolffian duct system is male (11). In addition, in several species 5{alpha}-reductase is not expressed in Wolffian duct derivatives until male development of the tissues is far advanced (12).

In pouch young of the tammar wallaby, Macropus eugenii, the marsupial in which male differentiation has been studied in greatest detail, dihydrotestosterone is formed by an alternate pathway when the urogenital sinus and urogenital tubercle virilize. 5{alpha}-Androstane-3{alpha},17ß-diol (androstanediol) is the principal androgen secreted by the testis at the time when prostate development is initiated between d 20 and 40 of pouch life (13) and when the phallus begins to differentiate after d 40 (14, 15). In pouch young testes during these phases of development, androstanediol is formed largely by a pathway in which testosterone and androstenedione are not intermediates but in which 17{alpha}-hydroxyprogesterone is converted to 5{alpha}-reduced progestogens, which are metabolized directly to 5{alpha}-androstane-17ß-ol-3-one (androsterone) and androstanediol (16). In target tissues in turn, androstanediol is oxidized to dihydrotestosterone, which, as in eutherians, mediates virilization of the urogenital sinus and urogenital tubercle (13, 15, 17).

The process by which androgens promote formation of the male ejaculatory system in the tammar wallaby has never been defined. The mesonephros is the functional kidney for the first 10 d after birth of the tammar and then gradually regresses to contribute to the epididymis as the metanephic kidney takes over excretory function (18). As in other mammals, virilization of the Wolffian ducts is one of the first processes in development of the male phenotype, commencing around d 10 of pouch life in the wallaby (18), approximately 10 d before the initiation of prostate development. The present study was designed to determine what androgen(s) are synthesized in early pouch young testes, how androgens are metabolized in the mesonephros/Wolffian ducts during this phase, and the effects of the administration of androstanediol and a 5{alpha}-reductase inhibitor on Wolffian duct differentiation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Tammar wallabies (M. eugenii), which originated in Kangaroo Island, South Australia, were held in a breeding colony in open grassy yards. The diet was supplemented with lucerne hay, oats, and fresh vegetables. The experiments followed the 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 the ages of pouch young were either determined from known birth dates or extrapolated from growth curves using head length measurement (19). The sex of each pouch young was identified by the presence of scrotal bulges (males) or pouch and mammary gland primordia (females) (3). The pouch young were killed by decapitation, and the testes and urogenital tracts were removed, dissected free of adjacent structures under a dissecting microscope, and kept in ice-cold normal saline until used for incubations.

Incubation studies
Testes.
For the in vitro measurements of androgen synthesis by testes, individual gonads were blotted, weighed, and added to glass tubes containing 5 µM [3H]progesterone in 50 µl DMEM. The testis weights averaged 0.39 mg in the d 8–13 (n = 5; average 10 d) pouch young and 0.95 mg in the d 18–22 (n = 9; average 19 d) pouch young. The tubes were gassed for 30 sec with 95% oxygen-5% carbon dioxide, capped, and incubated with shaking at 37 C for varying periods. The reactions were stopped by the addition of 1 ml chloroform-methanol (2:1), and the samples were dried under air at room temperature. For separation by HPLC, the residues were dissolved in 0.4 ml methanol, and 40-µl aliquots were injected into a Breeze model 1525 HPLC pump system equipped with model 717 plus autoinjector and a 4.6 x 150 mm, 5-µm C18 symmetry column (Waters Corp., Milford, MA). The column effluent was analyzed with a model 2487 dual-wavelength UV detector set to 254 nm and a ß-RAM model 3 in-line radioactivity detector (IN/US Systems, Inc., Tampa, FL). The samples were separated at 30 C and a flow rate of 1 ml/min with a program that involved a linear gradient of 50–60% methanol in water over 20 min; isocratic 60% methanol for 10 min; a linear gradient to 100% methanol over l5 min; followed by equilibration at 50% methanol for 15 min. The retention times in minutes were as follows: androstenedione, 10.7; testosterone, 13.5; 17{alpha}-hydroxyprogesterone, 15.0; dihydrotestosterone, 20.2; progesterone, 24.3; androstanediol, 25.3; androsterone, 26.6; 5{alpha}-dihydroprogesterone and 5{alpha}-pregnane-3{alpha},17{alpha}-diol-20-one, 36.0; and 5{alpha}-pregnane-3{alpha}-ol-20-one, 39.7. The identity of the radioactivity recovered in areas corresponding to androstanediol, androsterone, and 5{alpha}-pregnane-3{alpha},17{alpha}-diol-20-one was confirmed by thin-layer chromatography (TLC).

Urogenital tract minces.
For in vitro studies of androgen metabolism in various parts of the urogenital tract, minces of dissected tissues (1.8–4.8 mg wet weight) were added to glass tubes containing 100 µl DMEM, 0.1 µM radioactive androgen, and 1% fetal calf serum. All incubations were performed in duplicate. For assessment of the reduction and oxidation of androgens in tissues of the d 19 tammar pouch young, the tubes contained 0.1 µM [3H]testosterone, 0.1 µM [3H]dihydrotestosterone, or 0.1 µM [3H]androstanediol, and for measurement of 5{alpha}-reductase activity in the mesonephros/epididymis at various ages the tubes contained 0.1 µM [3H]testosterone. The studies were performed in duplicate. All tubes were gassed for 30 sec with 95% oxygen-5% carbon dioxide, capped, and incubated with shaking at 37 C for 1 h. The reactions were stopped by the addition of 2 ml chloroform/methanol (2:1), and the samples were dried under air at room temperature and processed by TLC. Namely, residues were dissolved in 0.2 ml chloroform/methanol (2:1), and 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 (dihydrotestosterone, androstanediol, testosterone, androstenedione, and 5{alpha}-androstane-3,17-dione). The plates were developed in dichloromethane/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 at 100 C for 10 min. Each lane was then cut into eight fractions corresponding to the visualized carrier steroids, and each fraction was assayed for radioactivity in a liquid scintillation counter after addition of 5 ml Budget-Solve cocktail.

Treatment of pouch young with a 5{alpha}-reductase inhibitor (4MA) or androstanediol
Ten female pouch young (10 d of age) were randomly assigned to one of two regimens and treated with either 8 µg/g body weight per day of androstanediol dissolved in 10% ethanol in triolein or an equal amount of 10% ethanol in triolein. Ten male pouch young (l0 d of age) were randomly assigned to receive either 25 µg/g body weight per day of the 5{alpha}-reductase inhibitor 4MA dissolved in l0% ethanol in triolein or as a control an equal amount of 10% ethanol in triolein. The animals were treated orally each day from d 10 to 33 by placing the appropriate volume of drug or carrier in a polyethylene tube (0.5 mm inner diameter, 0.8 mm outer diameter), placing the tube in the mouth beside the teat, and allowing the young to suck the contents as described (4). The dose was increased every 7 d to account for the growth in weight of the pouch young, assuming an average weight of 1.8 g from d 10 to 17, 3.9 g from d 18 to 25, and 5.3 g from d 26 to 33.

The pouch young were killed on d 34. The external anatomy was examined, and the lower half of the abdomen containing the reproductive tract was fixed in 10% neutral buffered formalin and embedded in wax. The blocks were serially sectioned at 8 µm and mounted in ribbons of 10 sections on glass slides. Every fourth slide was stained with Harris’s hematoxylin and eosin. The diameters of Wolffian and Mullerian ducts were measured bilaterally in each animal at four different levels along the ducts. The effects of treatment on the averaged diameters of the Wolffian and Mullerian ducts were assessed using t tests.

Materials
[1,2,6,7-3H]progesterone (3.9 TBq/mmol), [1,2,4,5,6,7-3H]dihydrotestosterone (4.6 TBq/mmol), [1,2,6,7-3H]testosterone (2.9 TBq/mmol), and [9,11-3H]5{alpha}-androstane-3{alpha},17ß-diol (1.7 TBq/mmol) were from PerkinElmer Life Sciences, Inc. (Boston MA). The nonradioactive steroids were from Steraloids, Inc. (Newport, RI), and DMEM was from Invitrogen Corp. (Grand Island, NY). Triolein was from Sigma (St. Louis, MO). The 5{alpha}-reductase inhibitor 4MA (17ß-N,N-diethylcarbamoyl-4-methyl-4-aza-5{alpha}-androstan-3-one) was a gift of Merck & Co. (Whitehouse Station, NJ) (20). Budget-Solve cocktail was from Research Products International.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The pattern of metabolism of [3H]progesterone in the testes of d 10 and 19 pouch young (Fig. 1Go) is similar to that previously described in d 24–54 tammar pouch young testes (16). Namely, the metabolites recovered after 90 min included the 19-carbon androgens testosterone, androstanediol, and (at d 19) androsterone. Two other major metabolites were identified: 17{alpha}-hydroxyprogesterone, an intermediate in both pathways of androgen formation, and 5{alpha}-progestane-3{alpha},17{alpha}-diol-20-one (5{alpha}-pdiol), an intermediate in the conversion of 17{alpha}-hydroxyprogesterone to androstanediol and androsterone (16). The change in the profile of metabolites with testis age suggests a progressive increase in activity of both 5{alpha}-reductase and CYP17 so that the transient accumulation of the intermediate 17{alpha}-hydroxyprogesterone seen at d 10 does not occur with d 19 testes because the intermediate is more rapidly metabolized to adiol and testosterone. Consistent with this, in experiments not shown, the only metabolite identified after the incubation of testes from d 6 tammar pouch young with [3H]progesterone was 17{alpha}-hydroxyprogesterone, and the only metabolites identified when d 10 testes were incubated with 5 µM [3H]progesterone and the 5{alpha}-reductase inhibitor 4MA (5 µM) were testosterone and 17{alpha}-hydroxyprogesterone.


Figure 1
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  		FIG. 1. [3H]Progesterone metabolism by testes from d 10 and 19 tammar wallaby pouch young. Individual testes weighing 0.35–0.48 mg from d 9–11 males (average l0.3 d) (A) or weighing 0.9–1.0 mg from d 18–22 males (average 19.1 d) (B) were added to 50 µl DMEM containing 5 µM [1,2,6,7-3H]progesterone, incubated, and processed by HPLC as described in the text. {square}, 17{alpha}-Hydroxyprogesterone; {blacksquare}, 5{alpha}-pdiol; bullet, testosterone; {triangleup}, androsterone; {blacktriangleup}, androstanediol. The values are expressed as picograms per milligram tissue.

 
Because both androstanediol and testosterone are formed by d 10 and 19 pouch young testes, the fate of any testosterone reaching the epididymis was assessed by incubating 0.1 µM [3H]testosterone with minces of mesonephros/epididymis from nine d 6–54 male pouch young and assessing 5{alpha}-reductase activity as the sum of metabolites recovered in dihydrotestosterone, 5{alpha}-androstane-3, 17-dione, and androstanediol after 1 h (Fig. 2Go). 5{alpha}-Reductase activity approximated 150 fmol/mg tissue per hour in d 6, 13, and 21 tissues and decreased to 7–13 fmol/mg tissue per hour by d 30 (P < 0.005; t test).


Figure 2
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  		FIG. 2. 5{alpha}-Reductase in minces of mesonephros/epididymis from male pouch young as a function of age. Tissue minces (1.6–3.0 mg) were added to 0.1 ml DMEM containing 0.1 µM [1,2,6,7-3H]testosterone. The tubes were processed by TLC as described in the text. 5{alpha}-Reductase, expressed as femtomoles per milligram tissue per hour, is the sum of metabolites recovered in the areas of the TLC corresponding to dihydrotestosterone, androstanediol, and 5{alpha}-androstanedione. The points represent average values for individual duplicate assays except that the average for two d 13 pouch young testes is shown.

 
The metabolism of androgen in various parts of the urogenital tract in d 19 male pouch young was assessed by incubating tissue minces with 0.1 µM [3H]testosterone, 0.1 µM [3H]dihydrotestosterone, or 0.1 µM [3H]androstanediol and analyzing the pattern of metabolism at the end of 1 h (Fig. 3Go). In testes, metabolism favors the conversion of testosterone and dihydrotestosterone to androstanediol, whereas in the other tissues examined (epididymis/mesonephros, urogenital sinus, urogenital tubercle, and abdominal skin), the predominant metabolite recovered after incubation with all three androgens is dihydrotestosterone.


Figure 3
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  		FIG. 3. [3H]Androgen metabolism in urogenital tract tissues from a d 19 male tammar wallaby pouch young. Tissue minces were added to 0.1 ml DMEM containing 0.1 µM [1,2,6,7-3H]testosterone, 0.1 µM [1,2,4,5,6,7-3H]dihydrotestosterone, or 0.1 µM [9,11-3H]androstanediol. The tubes were processed by TLC as described in the text. Values are expressed as femtomoles per milligram tissue per hour. T, Testosterone; DHT, dihydrotestosterone; Adiol, androstanediol; 5{alpha}-R, steroid 5{alpha}-reductase; 3{alpha}-HSD, 3{alpha}-hydroxysteroid dehydrogenase.

 
Male pouch young were treated from d 10 to 33 of pouch life with the 5{alpha}-reductase inhibitor 4MA, and female pouch young were similarly treated with androstanediol. The Mullerian and Wolffian ducts in these two groups of animals were examined histologically at the end of the experiment (Fig. 4Go). Wolffian ducts in males that were treated with 4MA were smaller in diameter than those of control males (P < 0.0005) (Fig. 4Go, A, C, and E). In these animals the Mullerian ducts had regressed in the posterior half, but the upper half of the duct remained (Fig. 4Go, A, C, and F). In two animals the Mullerian ducts close to the ovary were distended. In contrast, treatment of females with androstanediol prevented the regression of the Wolffian ducts that occurred in the control females (P < 0.002), with diameters similar to those in males (Fig. 4Go, B, D, and E), but no difference was observed in the diameter of the Mullerian ducts (Figs. 4Go, B, D, and F) in the androstanediol-treated females.


Figure 4
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  		FIG. 4. Representative photomicrographs of Wolffian and Mullerian ducts of control and treated pouch young (A–D) and average duct diameters (E and F). Control males (A) have well-developed Wolffian ducts and only remnants of Mullerian ducts. Treatment of male pouch young with 4MA (C) causes regression of the Wolffian duct and inhibits Mullerian duct regression. In control females (B), the Wolffian duct (arrow) is regressed and Mullerian ducts are large. Treatment with androstanediol from d 10 to 33 of pouch life (D) preserves the Wolffian duct but has no effect on the Mullerian duct. Average diameters (±SEM) of the Wolffian ducts (E) and Mullerian ducts (F) show significant effects of the treatments (asterisks). The scale for all four photomicrographs is the same and is indicated in B.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
5{alpha}-Reductase activity is expressed as early as d 10 in the testes of tammar pouch young, and, as is true in older pouch young (16), androstanediol in these testes is formed by two pathways. The sequence of one pathway is progesterone -> 17{alpha}-hydroxyprogesterone -> 5{alpha}-pdiol -> (androsterone) -> androstanediol, whereas the other pathway is progesterone -> 17{alpha}-hydroxyprogesterone -> (androstenedione) -> testosterone -> dihydrotestosterone -> androstanediol. The failure (or inconsistency) in demonstrating androstenedione and androsterone as intermediates is almost certainly due to the abundant expression of 17ß-hydroxysteroid dehydrogenase-3 in the Leydig cell.

5{alpha}-Reductase is also highly active in the mesonephros/epididymis from d 6 to 21 and declines markedly between d 21 and 30 as the mesonephros regresses and disappears. Consequently, any testosterone that reaches the mesonephros is likely converted to dihydrotestosterone. Similarly, the pattern of androgen metabolism in the d 19 male urogenital tract favors the conversion of any androstanediol reaching the mesonephros/epididymis to dihydrotestosterone. In keeping with these in vitro studies, the administration of 4MA (a potent inhibitor of both isoenzymes of steroid 5{alpha}-reductase) to male pouch young between d 10 and 33 impairs Wolffian duct differentiation, whereas treatment of female pouch young with androstanediol between d 10 and 34 virilizes the Wolffian ducts. These findings indicate that dihydrotestosterone, derived largely from the alternate pathway in which androstanediol from the testes is oxidized to dihydrotestosterone or derived from the 5{alpha}-reduction of any testosterone that reaches the tissue, is responsible for virilization of the Wolffian ducts in the tammar wallaby. In brief, virilization of the Wolffian ducts in tammars occurs by the same process as the virilization of the urogenital sinus and urogenital tubercle in this species (13, 14, 15). This is a novel finding, which contrasts with the situation in the brushtail possum Trichosurus vulpecula (21) and the eutherian species in which the process has been examined (7, 8, 9, 10). In these species testosterone rather than dihydrotestosterone is believed to cause differentiation of the Wolffian duct to form the epididymis, vas deferens, and seminal vesicles.

Although the scrotum has low 5{alpha}-reductase activity (22), it does have androgen receptors (23), so the high production of 5{alpha}-reduced androgens by the early (d 10) testis could potentially influence development of the scrotum of early tammar pouch young. Such an argument has been made in the gray short-tailed opossum Monodelphis domestica (24) in which there are androgen receptors in the scrotum (24, 25) and 3ß-hydroxysteroid-dehydrogenase in the gonads of both sexes 1 d before birth, whereas scrotal anlagen were visible in males only 1 d after birth (24), although in another study, scrotal bulges were readily identifiable on the day of birth in this and the Virginia opossum, Didelphis virginiana (26). However, presence of receptor and hormone does not necessarily mean there is an endocrine interaction, and there is convincing evidence from endocrine manipulations and intersexes that the formation of the scrotum in marsupials is independent of androgens. The scrotum begins to differentiate in male tammar fetuses 4 d before birth at a time when the gonadal primordium consists only of two to three layers of undifferentiated mesenchymal cells (2, 18). Female wallaby and opossum neonates do not develop a scrotum when treated with large doses of exogenous testosterone (4, 27) or grafts of neonatal testes (26), although the reproductive tract is masculinized. Likewise treatment of male pouch young with antiandrogen or 5{alpha}-reductase inhibitor blocks virilization of the reproductive tract but does not affect scrotal growth (27, 28).

In intersex marsupials from a diverse range of marsupial families from both Australia and South America, sexual phenotype of the gonads and internal genitalia is usually at odds with phenotype of scrotum, pouch, and mammary development (2, 5, 6, 29, 30, 31, 32, 33, 34, 35, 36, 37). XO individuals develop phenotypically as females except for the presence of an empty scrotum, and the absence of a pouch and mammary glands. Conversely, XXY intersexes have testes and develop as males except for the absence of a scrotum and the presence of a pouch. Several intersexual marsupials, including macropodids, dasyurids, and opossums, have a hemiscrotum on one side of the midline and a hemipouch and mammary glands on the other side as a result of chromosomal mosaicism (2, 5, 6, 29, 30, 31, 36, 37).

Together these data show that scrotal development in marsupials is directly regulated by a gene or genes on the X chromosome and is independent of gonadal hormones, although, as in eutherians, differentiation of the remainder of the male reproductive tract and penis is regulated by testicular hormones (38, 39, 40).

Mullerian duct regression in developing male tammar young, as in other mammals, is effected by anti-Mullerian hormone (41, 42). The reason for the retention of the Mullerian duct in 4MA-treated males in this study and in estradiol-treated males in a previous study (4) is unexplained. Further study is warranted to explore whether blocking 5{alpha}-reduction of testosterone leads to more aromatization of androgens and consequently more estrogen production, a mechanism that would link the two studies. Estradiol can inhibit anti-Mullerian hormone action (43), and estradiol receptors are present in the developing Mullerian duct (44). Estrogen can also alter anti-Mullerian hormone gene transcription (45), and there is also evidence from humans with androgen insensitivity that full regression of the Mullerian ducts may require androgen action (46) so that inhibiting 5{alpha}-reductase with 4MA may have prevented Mullerian regression through this mechanism.

The alternate pathway of dihydrotestosterone formation comes into play when steroid 5{alpha}-reductase is present in testes; in the mouse the testicular 5{alpha}-reductase is isoenzyme 1 (10), and in rat testes expression of the enzyme appears to be inhibited by chorionic gonadotropin (47) and enhanced by treatment with an LHRH agonist (48). When 5{alpha}-reductase is present in testes, androstanediol is the predominant androgen formed (rather than dihydrotestosterone or androsterone) because of the abundance of 17ß-hydroxysteroid dehydrogenase-3 and 3{alpha}-hydroxysteroid dehydrogenase in the tissue (10, 16).

Exogenous androstanediol is a potent androgen (49, 50), although it binds only weakly to the androgen receptor (51). In the brain androstanediol can alter behavior by modulating {gamma}-aminobutyric acid receptors (52) or through other nongenomic actions (reviewed in Ref.53). Androstanediol may also alter cell function in the reproductive tract via interaction with SHBG and its cell-membrane receptor (54). However, most of its actions can be explained by conversion in target tissues to dihydrotestosterone (17, 55).

The fact that two such different mechanisms exist for dihydrotestosterone formation emphasizes the central role that dihydrotestosterone plays in androgen physiology. To date, however, the alternative route of dihydrotestosterone formation appears to be important in only three circumstances: virilization of the male urogenital tract in the tammar wallaby (13, 14, 15, 16, 17), the neonatal surge of androgen secretion in the rodent (10, 56, 57, 58, 59), and the surge of androgen secretion in the immature brushtail possum (21). It is also possible that this pathway may explain the virilization in human congenital adrenal hyperplasia caused by P450 oxidoreductase deficiency (60). In all other circumstances, dihydrotestosterone appears to be formed by the 5{alpha}-reduction of testosterone in target tissues.


   Acknowledgments
 
We thank Scott Brownlees and Kerry Martin for assistance with the animals.


   Footnotes
 
This work was supported by Grant 350420 from the Australian National Health Medical Research Council and Grant R21DK59942 from the National Institutes of Health.

Disclosure of potential conflicts of interest: The authors declare no conflicts of interest related to this manuscript.

First Published Online February 9, 2006

Abbreviations: Androstanediol, 5{alpha}-Androstane-3{alpha},17ß-diol; androsterone, 5{alpha}-androstan-3{alpha}-ol,17-one; 4MA, 17ß-(N,N-diethyl)carbamoyl-4-methyl-4-aza-5{alpha}-androstan-3-one; 5{alpha}-pdiol, 5{alpha}-pregnane-3{alpha},17{alpha}-diol-20-one; TLC, thin-layer chromatography.

Received October 3, 2005.

Accepted for publication January 30, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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