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-Androstane-3,17ß-Diol Is Secreted by the Testes
Department of Zoology (M.W.L., G.S., J.D.W., M.B.R.), University of Melbourne, Victoria 3010, Australia; and Department of Internal Medicine (J.D.W.), University of Texas Southwestern Medical Center, Dallas, Texas 75390-8857
Address all correspondence and requests for reprints to: Professor Marilyn B. Renfree, Department of Zoology, University of Melbourne, Gate 12, Royal Parade, Victoria 3010, Australia. E-mail: m.renfree{at}unimelb.edu.au.
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Abstract |
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-androstane-3,17ß-diol (5-adiol), but uncertainties exist about the control of penile development. We performed longitudinal studies up to d 150 of pouch life to define normal penile development and the effects of androgen administration and castration. In control animals the male phallus was longer than the female phallus by d 48. Closure of the urethra in males begins around d 60 and continues to at least d 150. Administration of supraphysiological doses of testosterone to females caused penile development equivalent to that of the male and also induced partial closure of the urethral groove by d 150. Castration of male pouch young at d 25 prevented penile development, whereas the penis in males castrated at d 40, 80, or 120 had partial closure of the urethral groove. Administration of 5-adiol to females from d 20–40 also caused partial closure of the urethral groove and some growth of the phallus at d 150, whereas 5-adiol treatment from d 40–80 or 80–120 caused some penile growth but had little effect on urethral development. These findings, together with the fact that we found no sex differences in plasma levels of testosterone, dihydrotestosterone, 5-adiol, dehydroepiandrosterone, or androstenedione from d 51–227, clearly indicate that the action of 5-adiol between d 20 and 40 imprints later differentiation of the male penis. |
Introduction |
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-reductase inhibitor finasteride impair differentiation of the Wolffian ducts, urogenital sinus (into prostate), and phallus (into penis) in all mammalian species studied to date (2, 3, 4, 5, 6). Mutations that impair androgen formation, steroid 5-reductase activity, or the androgen receptor have similar consequences in humans and in other mammals (7). Marsupials provide a unique opportunity to study sexual differentiation because the process occurs largely after birth. Sexual differentiation has been studied in detail in the tammar wallaby, Macropus eugenii, in which differentiation of the testes is first evident with the appearance of seminiferous tubules and onset of hormone production by d 2 post partum (pp) (8, 9, 10). We have previously shown that 4 d before birth genetic males but not genetic females develop scrotal primordia, and females but not males develop mammary primordia (11). Because sexual dimorphism occurs before gonadal differentiation, it presumably arises through direct genetic action dependent on the sex chromosomes (8, 11, 12, 13). Differentiation of the scrotum and pouch and mammary primordia are not affected by testicular hormones after birth (13, 14) so were not assessed in this study. Sexual differentiation of all other parts of the reproductive tract, including the Wolffian and Müllerian ducts, prostate, and phallus are regulated by gonadal hormones. By d 25 pp, Wolffian ducts degenerate in females and the Müllerian ducts degenerate in males (10). Prostatic buds first appear in the urogenital sinus of males by d 25 (4, 12), but sex differences in the phallus have not been demonstrated until after d 60 (10, 15).
As in eutherians, castration of male pouch young (14) or administration of flutamide (4, 16) or finasteride (5) to male tammar young inhibit virilization of the urogenital sinus and phallus. Likewise, administration of androgen (11, 17) or transplantation of testes into female pouch young (14) induces development of a prostate and penis. Testosterone production begins in the testes shortly after birth (18). Testosterone concentrations in the pouch young testes plateau from d 10 to d 40 when the prostate differentiates but decrease by d 60 as testis size increases (18). Testosterone is not detectable in the developing ovaries (18). Despite the sexual dimorphism in gonadal testosterone within pouch young, plasma testosterone and dihydrotestosterone levels are not sexually dimorphic at least up to d 150 (19). Sexually dimorphic development of the wallaby young appears to be mediated by another 5-reduced androgen, 5-androstane-3,17ß-diol (5-adiol). 5-Adiol is secreted by the testes and is sexually dimorphic in the plasma when the prostate begins to differentiate (20). Furthermore, administration of small amounts of 5-adiol to females causes prostatic development (21).
Identification of 5-adiol as a key player in virilization of this species has opened new avenues to understanding the formation of the male reproductive tract. To date, 5-adiol is the only androgen known to be sexually dimorphic in the plasma during the time when the male urogenital tract is forming in any mammalian species (20, 22). Demonstration that 5-adiol is produced by the tammar pouch young testis both from testosterone and directly by side-chain cleavage of 5-reduced pregnanes (23), that it virilizes the urogenital sinus, probably by back conversion to dihydrotestosterone (20), and that it is capable of virilizing the tammar phallus (17) indicates that the traditional model of virilization involving circulating testosterone and conversion to dihydrotestosterone in target tissues (24) does not apply to this species.
Because the tammar phallus differentiates in large part when plasma testosterone and dihydrotestosterone are not sexually dimorphic in the plasma (19), the androgen responsible and the mechanism by which it reaches the phallus in this species are unclear. We therefore designed a variety of longitudinal experiments to define normal phallic differentiation in the two sexes and to investigate the effects of androgen administration for varying periods and of timed castrations on this process.
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Materials and Methods |
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Longitudinal studies of phallic differentiation: effect of androgens or castration
In five control female pouch young and four untreated male pouch young, phallic size was measured at d 20 and then periodically until d 150. Control females received triolein on d 20, 27, and 35 (for volumes and concentrations of control and androgen enanthate regimens, see Ref.17). In addition, five female pouch young were given 10 µg/g body weight/wk testosterone enanthate (Steraloids, Newport, RI) dissolved in triolein (Sigma, St. Louis, MO) from d 20 to d 150, and phallic size was monitored periodically. We have previously shown that a similar regimen using androstanediol enanthate completely virilizes the phallus of females (17). However, that regimen caused supraphysiological growth of the urogenital sinus (17), so we chose to administer testosterone enanthate in this study to see whether the previous effects were due to the potency of 5-adiol enanthate.
To test the effects of critical periods of androgen action on phallic development, three additional groups of female pouch young received 10 µg/g body weight/wk 5-adiol 17-monoenanthate (Steraloids) on d 20, 27, and 34 (d 20–40 group, n = 5), d 40, 47, 54, 61, 68, and 75 (d 40–80 group, n = 4), and d 80, 87, 94, 101, 108, and 115 (d 80–120 group, n = 5). The solutions were administered sc into the abdominal skin through 26-gauge needles. Phallic size was monitored periodically.
For the castration studies, male pouch young were assigned to four treatment groups; d 25 castrate (age range 24–30 d, average age 27 ± 1.1 d; n = 5), d 40 castrate (35–39 d, average 38 ± 0.7 d; n = 5), d 80 castrate (78–83 d, average 81 ± 0.7; n = 6), and d 120 castrate (122–127 d, average 124 ± 1.1; n = 4). Mothers with pouch young were brought into the department on the day of surgery. Pouch young were removed and anesthetized by hypothermia (d 25 and d 40 groups) or with halothane (d 80 and d 120 groups) as previously described (26). The testes were surgically removed, and the incision site was sutured. The pouch young were then revived by hand warming or removal from the gas and reattached to the mother’s teat. Pouch young were checked daily for 3 d after surgery, and phallic size was measured periodically.
Phallic measurements and autopsy procedures
Phalluses were measured with calipers in all groups at the start of treatment and then weekly from d 40 through to d 150. The phallus was everted by gently pressing either side of the genital tubercle, and phallus length was measured from the tip of the glans to the point at which the shaft entered the genital tubercle. Phallus width was measured across the base of the shaft of the phallus at which it entered the genital tubercle. All measurements were taken at least twice and averaged. Animals were killed on d 150 by inhalation of 100% isofluorane. The appearance of the external genitalia was recorded, and the phallus was measured and photographed. The reproductive tract and phallus were then dissected and fixed in 10% neutral buffered formalin, embedded in paraffin wax, serially sectioned (6–8 µm), and stained with hematoxylin and eosin. Transverse or longitudinal sections were taken through the phallus.
In vitro incubations with [1,2-3H]testosterone
Urogenital tracts were dissected from 10 male pouch young aged 32–175 d, and slices of urogenital tubercle/phallus, urogenital sinus/prostate, and abdominal skin (3.1–17 mg in weight) were added to 0.1 ml Dulbecco’s modified Eagle’s MEM containing 0.1 µM [1,2-3H]testosterone (1.57 Bq/mmol) (NEN Life Science Products, PerkinElmer Life Sciences, Boston, MA). Duplicate samples were gassed with 95% oxygen/5% carbon dioxide and incubated at 37 C with shaking for 1 h. The reactions were stopped by the addition of 1 ml chloroform/methanol (2:1), and the samples were dried at room temperature. The residues were dissolved in 0.1 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 (testosterone, dihydrotestosterone, androstenedione, 5-adiol, and 5-androstanedione; Steraloids). The sheets were developed in chloroform/toluene/acetone (80:50:20, vol/vol), and the steroids were visualized by spraying with 1% anisaldehyde in glacial acetic acid/sulfuric acid (100:2) and heating the plates in an oven for 15 min. Each lane was then cut into seven fractions corresponding to the visualized carrier steroids, and each fraction was assayed for radioactivity in a liquid scintillation counter.
Plasma androgen measurements
Plasma was collected from 41 female and 44 male pouch young aged between 51 and 227 d from our colony or from our field site on Kangaroo Island. Pouch young were killed by overdose of sodium pentobarbitone in sterile saline (60 mg/ml, ip) or by cervical dislocation in field samples, and blood was collected by cardiac puncture. To minimize the effects of stress, all pouch young were sampled within 30 min from the time of removal from the pouch to the time of collection. Plasma was frozen and shipped from Melbourne to Dallas on dry ice, where they were pooled into 14 male and 17 female pools of pouch young plasma for analysis to create sufficient volumes for assay (4–14 samples per pool for animals d 100 or less and one to two animals for the older ages) and stored at –20 C before being assayed for the content of total testosterone, dihydrotestosterone, 5-adiol, dehydroepiandrosterone, and androstenedione. Blood samples taken from the caudal tail vein (27) of three adult males were assayed as separate pools. Testosterone, dihydrotestosterone, and 5-adiol were also measured in 33 individual plasma samples from d 91–115 male pouch young that were killed at different times of the day that encompassed a complete 24-h period (but not on the same day).
Direct RIAs were performed by InterScience Institute (Inglewood, CA) using specific antibodies with sensitivities of 0.01 ng/ml for dihydrotestosterone, 0.02 ng/ml for 5-adiol, and 0.02 ng/ml for testosterone. The assays were performed in duplicate, and the results were averaged to provide the value for each pool.
Statistics
Data are reported as mean ± SEM unless otherwise indicated. Phallus length was used as the index of phallus size because width changed little over the time course of this study. Statistical comparisons between groups were made using Student’s t tests, and comparison of growth rates between groups were made by comparing the slopes of linear regressions over 20- or 30-d time windows. Growth curves of control male and female phalluses were smoothed by the LOESS procedure using Systat software (version 10; SPSS Inc., Chicago, IL).
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Results |
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). By d 150, the male phallus was 4 times longer than the female phallus (P < 0.001). The opening of the urethra (urethral meatus) was at the base of the phallus in both sexes until d 60. A urethral groove was present on the ventral surface of the phallus in both sexes at d 60 and started to close in a proximal to distal direction in the male after d 60. The ventral surface of the d 150 female phallus had a large urethral groove up its center, and the urethral meatus was situated at the base of this groove (Fig 2A). The groove was closed over in the male phallus and a seam ran up the ventral surface, but the glandular urethra was incompletely formed at the distal tip (Fig. 2B). The prepuce covered the entire shaft and could be distinguished from the glans. The urethral plate was the dominant histological feature of the female phallus at d 150 (Fig. 3A). The female phallus had a small diverticulum at the urethral meatus, and the erectile tissues were well developed; the corpus spongiosum surrounded the urethral plate, and the paired corpora cavernosa ran dorsolaterally along the phallus into a single structure at its tip. A large keratinized blind-ending duct (diverticulum) that was dorsal to the true urethra was present in the d 150 male phallus (Fig. 3B). Two corpora cavernosa were dorsolateral to these ducts and remained as separate identities at the tip. The corpus spongiosum surrounded both the true urethra and the diverticulum.
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). Little growth occurred in the phalluses of treated females between d 111 and d 150. The urethral groove was closed partway up the shaft, but the prepuce was incompletely formed at the junction between the glans and the shaft at which the groove had failed to close (Fig. 2B). The dorsal prepuce was enlarged and there was profound enlargement of the corpus spongiosum and corpora cavernosum (Fig. 3C).
Oxidation of [3H]testosterone in the urogenital tract
The fact that testosterone enhanced linear growth of the phallus suggests that the amount of androgen available in the cells of the phallus limits early development of the male phallus. Therefore, the metabolism of testosterone was assessed in urogenital sinus/prostate and urogenital tubercle/phallus from male pouch young that varied in age from d 32 to d 175 by incubating tissue slices with 0.1 µM [3H]testosterone for 1 h and then extracting and separating the various metabolites by thin-layer chromatography. The principal metabolites were dihydrotestosterone, androstenedione, and 5-androstanedione. Formation of inactive 17-oxo androgens was assessed as a sum of androstenedione and 5-androstanedione (Table 1). At all ages examined, the rate of 17-oxidation was 3–5 times greater in the urogenital tubercle/phallus than in the urogenital sinus/prostate. This may explain, at least in part, the more rapid virilization of the urogenital sinus/prostate, compared with the urogenital tubercle/phallus.
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). Similarly, dihydrotestosterone levels were equally low in pools of both sexes (Fig. 4C). In three of the female pools, the dihydrotestosterone concentrations were high, and these values were confirmed on a repeat assay. The significance of these elevated values is unknown, but because it does not explain differentiation of the male phallus, it will not be discussed further. The concentration of 5-adiol was also low in all pools, although it was highest in the youngest male pool (0.179 ng/ml; 14 male pouch young aged between 51 and 77 d, average age 66.2 ± 1.69 d). The concentration of 5-adiol was below assay sensitivity in the equivalent female pool (<0.02 ng/ml; six female pouch young aged between 56 d and 83 d, average age 68.8 ± 4.57 d). There were no sex differences in the older pools (Fig. 4A). Concentrations of dehydroepiandrosterone and androstenedione were also low and no different in males than females in these pools (results not shown).
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-adiol (Fig. 4, D–F).
Sequential administration of androgen to females and castration of males
To ascertain the extent to which d 20–40 5-adiol secretions in the male might influence subsequent development of the phallus, two types of experiments were performed. Treatment of females with 5-adiol from d 20–40 caused significant growth of the phallus when examined at d 150, equivalent to approximately half the length of the d 150 male (Fig. 5A). Treatment of females from d 40–80 and d 80–120 had an even greater effect on phallic growth, and these phalluses were not significantly different in length from those of the control males (Fig. 5A). 5-Adiol treatment from d 20–40 caused partial closure of the urethral groove in females (Fig. 2E). Little or no urethral closure occurred in females treated from d 40–80 or d 80–120 (Fig. 2, G and I). The urethral meatus was situated at the base of the phallus in the 5-adiol 40–80 and 80–120 females (Fig. 3, G and I), although major changes occurred in the urethral plate. The corpora cavernosa and corpora spongiosa were enlarged in all 5-adiol-treated females (Fig. 3, E, G, and I).
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). Castration at d 120 yielded variable results; the phallus stopped growing in two pouch young, whereas in the other two, growth continued. There was no difference in size at any time between the phallus of the d 25 and d 40 castrate groups, and they both followed the control female pattern of growth. Similarly, growth was halted in the phalluses of the d 80 castrated males. The ventral surface of the phallus of the d 25 castrates was similar to that of the control females (Fig. 2D). The urethra in all other castrate males appeared to be completely formed to the end of the shaft, and the shaft/prepuce appeared normal, although it was reduced in size in the d 40 and d 80 castrates (Fig. 2, F, H, and J). The glans was severely malformed in the d 40 group, moderately formed in the d 80 group, and similar to that of the untreated males in the d 120 group. The urethral diverticulum in each of the castrated groups was reduced in size, and the urethral meatus moved progressively toward the tip of the phallus Fig. 3, D, F, H, and J). |
Discussion |
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-adiol between d 40 and d 80 or d 80 and d 120 had little or no urethral closure, whereas the urethral groove was closed to the junction between the shaft and the glans in males castrated at d 40 or later and in females treated with 5-adiol between d 20 and d 40.
Because closure of the urethral groove does not occur until after d 60, androgen must imprint later development on the phallus between d 20 and d 40, the period during which 5-adiol levels are elevated in the male circulation (20). The key aspect of the concept of hormonal imprinting is that a critical period of androgen action sensitizes a tissue so that lower concentrations of androgen are required for differentiation (28). The effects of hormonal imprinting from early androgen exposure are believed to be a factor in development of late-life human prostatic hyperplasia (29), and neonatal sex steroids have permanent effects on prostate development in rodents (29, 30). To our knowledge, this is the first evidence that androgen imprinting is involved in phallus differentiation. The change in concentration of circulating androgens in tammar pouch young provides further support for this hypothesis. In accord with our previous work (19), testosterone and dihydrotestosterone concentrations were not sexually dimorphic in any of the plasma pools at ages we assayed. Levels of 5-adiol, which are 2–3 times higher in male than female plasma between d 20 and d 40 (measured by gas chromatography/mass spectroscopy) (20), are low and not sexually dimorphic in pouch young plasma from d 51 to d 227.
Treatment of females with pharmacological doses of testosterone enanthate accelerated phallus growth to the extent that the female phalluses were the same length as those of normal d 150 males by d 100. This suggests that penile growth during this period is limited by the amount of androgen reaching the phallus. The testosterone-treated female phallus grew little after d 111 and was not longer than the male phallus at d 150. Similarly, growth of the phallus of female rhesus monkeys that received testosterone implants at four weekly intervals from postnatal d 5 or 6 was increased, compared with control females, but slowed after wk 8 and ceased when it had reached half the length of the penis, despite ongoing treatment (31). There are two phases of penile growth, an initial phase of differentiation and growth controlled by androgens and a second phase of growth independent of androgens (32). The most likely candidate for the androgen-independent growth phase is growth hormone because humans with growth hormone deficiency (33) or mutations that impair growth hormone receptor (34) develop micropenis despite normal androgen production. Whether this is indeed the case in the tammar will require further investigation.
The length of the phalluses of testosterone enanthate-treated females at d 150 was similar to those of females treated from d 20 to d 150 with equivalent or higher doses of 5-adiol enanthate, but the penile urethra was incompletely formed in testosterone-treated females, whereas females treated for a similar period and with similar doses of 5-adiol had urethras that extended to the tip of the phalluses (17). This difference is almost certainly due to differences in potency between the two hormones. One possible explanation is that the epithelium of the penile urethra might be particularly rich in the oxidative 3-hydroxysteroid dehydrogenase that converts 5-adiol to the active intracellular androgen dihydrotestosterone (20), or it may be relatively deficient in 5-reductase. If either were the case, the level of dihydrotestosterone would be higher in the urethra than in the rest of the penis after 5-adiol treatment and would require a larger dose of testosterone to induce full urethral closure.
Aromatization of testosterone to estradiol could also explain the accelerated growth of the phallus in these females. Estrogens are known to cause hypertrophy of many tissues (35, 36, 37). In a similar study on mice, testosterone caused hypertrophy of the stromal tissue surrounding the urethral seam (38). In the present study, the dorsal aspect of the phallus was enlarged because the urethral folds failed to close. The apparent acceleration in growth of testosterone enanthate-treated phalluses may just reflect the hypertrophic growth of the dorsal hood, but this cannot explain the cessation of growth observed in these phalluses. This issue needs to be resolved. Synthetic estrogens have been linked to an increased rate of hypospadias in boys (39), so perhaps 5-reduction occurs in the testes to protect the fetal tissues from the negative effects of estrogen.
This paper makes two important advances in our understanding of penis development in the tammar wallaby. First, the male phallus starts to grow much earlier than previously recognized, during the period when 5-adiol is sexually dimorphic, and urethral development is imprinted on the phallus during this time. Second, the continuing presence of androgen from d 40 until at least d 120 is essential for complete formation of the male urethra and for full penile growth, but androgen imprinting between d 20 and d 40 accelerates subsequent growth and is essential to initiate urethral closure. These results could have wider implications in the study of hypospadias, the etiology of which remains unresolved.
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Acknowledgments |
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Footnotes |
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Abbreviations: 5-Adiol, 5-Androstane-3,17ß-diol; pp, post partum.
Received February 6, 2004.
Accepted for publication March 25, 2004.
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References |
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-reductase inhibitor-induced abnormalities. Teratology 42:483–496[CrossRef][Medline]
-reductase inhibitor finasteride and the antiandrogen flutamide on prostate and genital differentiation: dose-response studies. Endocrinology 131:1149–1156[Abstract]
-reductase inhibitor, finasteride, on the developing prostate and testis of a marsupial. J Androl 18:123–130-reductase 2 deficiency, testicular feminization, and related disorders. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The metabolic and molecular basis of inherited disease. 8th ed. New York: McGraw-Hill; 4117–4161
-androstane-3,17ß-diol to female tammar wallaby pouch young causes development of a mature prostate and male urethra. Endocrinology 143:2643–2651-androstane-3, 17ß-diol. Proc Natl Acad Sci USA 97:1256–1259
-androstane-3,17ß-diol. Mol Cell Endocrinol 181:111–115[CrossRef][Medline]
-Androstane-3,17ß-diol is formed in tammar wallaby pouch young testes by a pathway involving 5-pregnane-3,17-diol-20-one as a key intermediate. Endocrinology 144:575–580This article has been cited by other articles:
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  G. Shaw, J. Fenelon, M. Sichlau, R. J. Auchus, J. D. Wilson, and M. B. Renfree Role of the Alternate Pathway of Dihydrotestosterone Formation in Virilization of the Wolffian Ducts of the Tammar Wallaby, Macropus eugenii Endocrinology, May 1, 2006; 147(5): 2368 - 2373. [Abstract] [Full Text] [PDF]
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 K-M Fung, E N S Samara, C Wong, A Metwalli, R Krlin, B Bane, C Z Liu, J T Yang, J V Pitha, D J Culkin, et al. Increased expression of type 2 3{alpha}-hydroxysteroid dehydrogenase/type 5 17{beta}-hydroxysteroid dehydrogenase (AKR1C3) and its relationship with androgen receptor in prostate carcinoma. Endocr. Relat. Cancer, March 1, 2006; 13(1): 169 - 180. [Abstract] [Full Text] [PDF]
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