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REPRODUCTION-DEVELOPMENT |
-Androstane-3,17ß-Diol to Female Tammar Wallaby Pouch Young Causes Development of a Mature Prostate and Male Urethra
Department of Zoology, University of Melbourne (M.W.L., G.S., M.B.R., J.D.W.), Victoria 3010, Australia; and Department of Internal Medicine, University of Texas Southwestern Medical Center (J.D.W.), Dallas, Texas 75390-8857
Address all correspondence and requests for reprints to: Michael W. Leihy, Department of Zoology, University of Melbourne, Victoria 3010, Australia. E-mail: ; or to Prof. Marilyn B. Renfree, Department of Zoology, University of Melbourne, 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) by the testes of the tammar wallaby is responsible for initiation of prostatic development after d 20 in male pouch young. To ascertain the role of this hormone in the subsequent growth and differentiation of the prostate and in the development of the male phallus, 5-adiol was administered to tammar female pouch young in two regimens. Administration of the hormone by mouth (8 µg/g body weight·wk) between d 70 and 150 of pouch life caused prostate development equivalent to that in d 150 males and promoted growth and differentiation of the penis, but not masculinization of the urethra. Treatment with a small dose of 5-adiol enanthate (1 µg/g body weight·wk) from d 20–150 produced similar results. However, administration of larger doses of 5-adiol enanthate (10 or 100 µg/g body weight·wk) from d 20–150 caused supraphysiological growth of the prostate, development of a male-type urethra, and penile growth. These results indicate that prostatic development and penile growth can be initiated over a wide time period, but that formation of a male urethra requires androgen action before d 70, when male penile differentiation begins. This further strengthens the hypothesis that 5-adiol is the circulating androgen responsible in this species for virilization during development. |
Introduction |
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-reductase inhibitor finasteride, which impairs the formation of dihydrotestosterone (2, 3, 4). Similarly, in the human, mutations that impair androgen synthesis, steroid 5-reductase, or the androgen receptor prevent formation of the male phenotype (5). The formation of the male phenotype in marsupials, which has been best characterized in the tammar wallaby, Macropus eugenii (6, 7), differs in several regards from the process in eutherian mammals. First, testicular differentiation and hormone production by the testes occur after birth while the young are attached to the mother’s teat. Second, although androgen is formed in the testes of pouch young shortly after birth (8), virilization occurs in distinct phases over a longer period of time than in most eutherian species. For example, although pouch life in the wallaby and pregnancy in humans are both of 9-month duration, the human male phenotype is formed largely between wk 8 and 12 of embryogenesis (2), whereas in the wallaby the epididymis develops between d 10 and 20 postpartum (7), prostatic buds begin to develop between d 20 and 25 (3, 9), and the phallus becomes sexually dimorphic after d 70, beginning with closure of the urethral groove to form the male urethra and continuing with differential growth of the phallus after d 100 (10). The fact that these developmental processes take place in distinct phases when the animal in the pouch is accessible to experimental manipulation has made it possible to define some aspects of male phenotypic differentiation in this marsupial in ways not easily achieved in eutherian mammals.
In eutherians it has not been feasible to measure plasma hormones in the fetus at the time of male phenotypic development, but it was tacitly assumed that the same androgens act during development as in the adult. Thus, it was believed that testosterone is secreted by the testes into plasma and is 5-reduced in the urogenital sinus and urogenital tubercle to dihydrotestosterone, the active androgen for the virilization of these tissues (2). However, in both the tammar wallaby (11) and the gray short-tailed opossum, Monodelphis domestica (12, 13), levels of plasma testosterone and dihydrotestosterone are not sexually dimorphic during the formation of the male phenotype. However, when prostate development is initiated in the tammar wallaby, there is sexual dimorphism in plasma levels of another 5-reduced androgen, 5-androstan-3,17ß-diol (5-adiol), which is synthesized by the testes and secreted into plasma between d 20 and 40 of pouch life (14). 5-Adiol has the capacity to induce the formation of prostate buds in the urogenital sinus of female pouch young when administered between d 20 and 40 of pouch development, but on the basis of studies of the metabolism of 5-adiol in target tissues (14) and comparative studies of the effects of different hormones between d 20 and 40 of pouch life (15), we believe that the hormone acts within target tissues after conversion back to dihydrotestosterone.
The concept that 5-reduced androgen is secreted into the plasma by the testis to initiate prostate development is a radical departure from previous models of androgen action, but it is not known whether 5-adiol has the capacity to virilize the phallus or to promote maturation of the prostate. To provide insight into the later phases of virilization in the tammar pouch young we administered 5-adiol by mouth and 5-adiol monoenanthate by sc injection to female pouch young and characterized the phalluses and prostates on d 150 of pouch life.
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Materials and Methods |
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Animals were killed on d 150 by an overdose of ip sodium pentobarbitone (0.5 ml of 60 mg/ml). The appearance of the external genitalia was recorded, and the phallus was measured. The reproductive tract and phallus were then dissected, fixed in 10% neutral buffered formalin, embedded in paraffin wax, serially sectioned (6–8 µm), and stained with hematoxylin and eosin. Transverse sections were taken through the urogenital sinus, and the phallus was sectioned either transversely or longitudinally. Six untreated male pouch young, aged 150 d, were measured for comparison, and the phalluses from two of these untreated males were processed for histology.
Oral treatment with 5-adiol between d 70 and 150
Female pouch young (70 d of age) were randomly assigned to one of two regimens and received either 56 µg/g body weight·wk (equivalent to 8 µg/g body weight·wk) 5-androstane-3,17ß-diol (Steraloids, Newport, RI; A56, n = 6) or 56 µg/g body weight·wk 5ß-androstane-3,17ß-diol (Steraloids; C56, n = 5) dissolved in peanut oil. 5ß-Androstane-3,17ß-diol was chosen for the controls because it has weak androgenic activity (18). The hormones were administered by mouth (9) through a polyethylene capillary tube (internal diameter, 0.5 mm; external diameter, 0.9 mm) inserted alongside the teat each day from d 70–150 postpartum. The length and width of the phallus and the overall length of the genital eminence (total length) were measured regularly over the course of treatment. The lengths of the head and phallus were also measured in untreated male and female pouch young of the same age span.
Injection of 5-adiol monoenanthate between d 20 and 150
To minimize handling of the animals we devised a treatment regimen that involved weekly, rather than daily, administration. In the second experiment 20-d-old female pouch young were assigned to one of three treatment regimens of 5-androstane-3,17ß-diol-17-monoenanthate (Steraloids) dissolved in triolein (Sigma, St. Louis, MO), namely 1 µg/g body weight·wk (AE1, n = 1), 10 µg/g body weight·wk (AE10, n = 1), or 100 µg/g body weight·wk (AE100, n = 1). One hundred micrograms of 5-adiol enanthate contain 72.3 µg 5-adiol. The enanthate functional group makes the compound more soluble in oil solution so that it is released more slowly into the circulation. The solutions were injected sc in the abdominal wall through 26-gauge needles each week from d 20–150 postpartum. The initial injection volume was 0.05 ml, and the maximal volume injected at any stage of the study was 0.3 ml (Table 1
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Results |
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Administration of 5-adiol caused enlargement of the urogenital sinus in female pouch young in the area where the prostate develops in males. The urogenital sinus region of the A56 females (Fig. 1C) and that of the AE1 female (Fig. 1D) were of similar size and appearance to those of normal d 150 male pouch young (Fig. 1B). The urogenital sinuses of the two animals treated with the larger doses of 5-adiol enanthate (Fig. 1, E and F) were larger than those in the male. Indeed, the prostates in these two animals were considerably larger than the prostates of two juvenile males (not pictured), and the AE100 prostate exuded a white liquid secretion resembling that of adult males. The urogenital sinus of the C56 females was not enlarged (Fig. 1A).
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) resembled those of the 5-adiol-treated females (Fig. 2, C–F). As in the male, prostatic tubules radiated from the prostatic urethra in the A56 females (Fig. 2C) and the AE1 female (Fig. 2D). In the AE10 and AE100 females, the prostates had histological features characteristic of adult tammar prostates with secretion-filled acini (Fig. 2, E and F) and areas of microcyst formation. The C56 females had no prostatic buds in this region (Fig. 2A).
Phallus
The phallus grew in length at a similar rate in males and females until about d 110 (Fig. 3A). At this time, both the normal female phallus and the phalluses of the control females (C56) ceased growth, became smaller relative to body size, and began to assume an adult form. The male phallus continued to grow as before until about d 130, when it exhibited a growth spurt. The phallus in the females treated orally with 5-adiol (A56) followed the male pattern of growth until d 130, when, unlike the male phallus, it stopped growing (Fig. 3A). The width of the phallus did not differ much in males, females, or A56 females (Fig. 3B). The total length of the genital eminence followed a similar pattern of growth to that of the phallus (Fig. 3C). The male and female genital eminences grew at similar rates until about d 110. On approximately d 130, there was an exponential growth spurt in the male. The A56 female genital eminence again followed the male growth pattern until d 130, at which time it stopped growing. Growth of the C56 female phalluses was similar to that of the normal females.
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-adiol (A56) were intermediate in length between the phalluses of the control females (C56) and that of the untreated male (Fig. 4A). The same was true for the measurements of total length (penis length plus that of the genital tubercle; Fig. 4C). The untreated male phalluses were approximately 2.5 times longer than those of the C56 females. The phallus length of the females treated with 5-adiol enanthate increased with dose (Fig. 4), so that the phallus of the animal treated with the lowest dose was similar to those of the orally treated females (A56), and the phalluses of the animals treated with the higher doses were similar to those of the normal males.
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). The ventral surface was grooved, and the urethral meatus (the external opening of the urethra) was situated at the base. The male penis was long and cylindrical (Fig. 5B). The prepuce surrounded the base, and the urethral meatus was situated at the tip. A penile raphe was evident on the ventral surface. The females treated orally with 5-adiol (A56) had a urethral groove on the ventral surface. The phallus of the female in the lowest 5-adiol enanthate group (AE1) had a small prepuce around the base and a urethral groove similar to that in the A56 females (Fig. 5D). The phalluses of the AE10 (Fig. 5E) and AE100 (Fig. 5F) females were male in structure with a well-developed prepuce and a urethral meatus at the distal tip.
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, A–D). The clitoris was surrounded by the genital tubercle and consisted largely of urethral plate epithelium. Approximately one third of the way down the phallus, the plate hollowed out to form a groove. Two corpora cavernosa along the dorsal aspect of the phallus lateral to the urethral plate consisted of long, thin bands of mesenchymal tissue separated by a septum. The right corpora cavernosa was always larger than the left one. A corpus spongiosum was present around the urethra and urethral plate.
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E–H). The erectile bodies of the males were larger than those of the females. Paired corpora cavernosa were prominent from the base to the tip, and the corpus spongiosum was extensively developed around the urethra. A blind-ending duct was identified beginning at the distal end of the urethra and extending proximally about one third of the length of the penis. This duct was also surrounded by corpus spongiosum. The phalluses of the A56 females (Fig. 6, I–L) had a female grooved ventral urethral plate and a blind-ending duct similar to that of the male. The corpora cavernosa were as well developed as in the male.
In the C56 female the phallus and anus were enclosed by the folds of the genital tubercle (Fig. 6M). The urethral meatus opened at the base of the phallus, and the urethral plate extended beneath the corpus cavernosum. The androgenized phalluses of the A56 (Fig. 6N) and AE1 females (Fig. 6O) had prominent blind-ending ducts, small prepuces, and enlarged corpora cavernosa below the dorsal surface.
The phallus of the AE10 female (Fig. 6P) resembled that of the male, with a fully formed male urethra, prominent corpora cavernosa, a blind-ending duct, and prepuce. A corpus spongiosum surrounded the urethra and blind-ending duct. The phallus of the AE100 female (Fig. 6Q) also had a male urethra that was surrounded by a corpus spongiosum and masculine-like corpora cavernosa. The blind-ending duct was not patent, and instead consisted of a solid column of epithelial cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-adiol induces formation of a male urogenital tract, including prostate and phallus, in the d 150 female tammar wallaby. This is a clear demonstration of the potency of this androgen and suggests that it could mediate all androgen-dependent phases of male phenotypic development in this species. The induction of mature prostatic development in the urogenital sinus with both treatment regimens (d 20–150 and d 70–150) indicates that there is a wide window of time in which prostatic development can be initiated in the tammar wallaby.
We have reported that administration of 5-adiol by mouth from d 20–30 of pouch life (14) and of 5-adiol enanthate by injection from d 20–40 (15) induces the development of prostatic buds in the female tammar wallaby. Consequently, it is not surprising then that injection of 1 µg/g body weight·wk 5-adiol enanthate from d 20–150 (AE1) induces prostatic development in a female similar to that in normal d 150 males. This suggests that 1 µg 5-adiol enanthate is close to a physiological dose for prostate development. Administration of the two larger doses of 5-adiol enanthate over the same period caused more extensive growth and maturation of the prostate than in untreated males of the same age. The prostates in these animals resemble the prostatic hyperplasia induced in castrated adult male dogs by administration of 5-adiol (19, 20).
Treatment with the two higher doses of 5-adiol from d 20–150 caused the Mullerian ducts to develop into enlarged uteri distended with fluid. Androgen has a similar effect on Mullerian structures in female pouch young of the American opossum, Didelphis virginiana (21), and in female sheep embryos (22). No Wolffian derivatives were observed in any of the female pouch young, but this is not surprising, as all treatments began after Wolffian ducts atrophy in females (7).
The response of the phallus in the tammar wallaby to the administration of 5-adiol is different from that of the prostate, in that development of a complete male urethra and normal male length only occurred in animals treated from d 20–150. The clitoris of untreated females is flat and wide in contrast to the larger cylindrical structure of the penis (Fig. 7, A and B). An unexpected finding in males was that a blind-ending duct extends dorsal to the urethra from the urethral meatus at the distal tip about a third of the length down the penis. Such a structure has not been reported during organogenesis of the penis in the rabbit (23) or the human (24, 25, 26), but a similar blind-ending duct has recently been observed during normal human penile development (Hutson, J. M., and E. Penington, unpublished observations). Furthermore, duplications of the male urethra, usually termed accessory urethras, are well recognized congenital anomalies in boys, and the most common form of duplication is a sagittal, partial duplication, which may be blind-ending (27, 28, 29).
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The formation of the penile urethra in the tammar follows the classic model of urethral development (10). The urethral plate hollows out to form a groove beginning around d 70 postpartum. The groove then folds over and fuses to form the penile urethra (10). Treatment with 5-adiol from d 70–150 postpartum at a dose of 56 µg/g body weight·wk did not induce formation of a male penile urethra, but did cause formation of the blind-ending duct (Fig. 7C). This may be due to the daily oral dose regimen being insufficient to cause complete urethral development or to the failure to administer hormone before d 70. When the treatment period was begun on d 20, females developed a complete male urethra, but only with the two higher doses of 5-adiol enanthate. Indeed, the females treated with the intermediate (AE10) and high (AE100) doses of 5-adiol enanthate adiol had male phalluses (Fig. 7, B and D), except that the blind-ending duct of the AE100 female was no longer patent and was almost completely fused into a solid column of epithelial cells within the corpus spongiosum.
This study provides the first evidence for a role of 5-adiol in penile development in the tammar. This strengthens our hypothesis that 5-adiol is the circulating androgen that serves as a precursor for the formation of dihydrotestosterone to mediate male sexual differentiation in this species. If this concept is correct, then the amount being secreted by pouch young testes must be somewhere in between 1 µg/g body weight·wk, a dose adequate to virilizes the urogenital sinus, and 10 µg/g body weight·wk, a dose that caused formation of a complete male phallus, but resulted in supraphysiological enlargement of the prostate.
The role of 5-adiol in the development of other mammalian species is not established. However, 5-adiol is the major testicular androgen in the immature rat (33, 34) and mouse (35), and its production declines as testosterone production rises after d 35 in the rat (33) and after d 20 in the mouse (35). 5-Adiol is also the principal androgen formed by the regenerating Leydig cells in adult rats treated with the cytotoxic drug ethane dimethyl sulfonate (36). Furthermore, the administration of 5-adiol to pregnant rats causes profound virilization of the urogenital tracts of female pups (37). Consequently, it is possible that 5-adiol plays a role in male development in other species.
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Acknowledgments |
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Footnotes |
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Abbreviations: 5-adiol, 5-Androstane-3,17ß-diol.
Received January 10, 2002.
Accepted for publication March 26, 2002.
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References |
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-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. Proc Natl Acad Sci USA 97:1256–1259
-androstane-3,17ß-diol. Mol Cell Endocrinol 181:111–115
-androstanediol-induced growth of the dog prostate. Endocrinology 102:1748–1758
-hydroxysteroid dehydrogenase oxidative and reductive activities in rat Leydig cells during pubertal development. Biol Reprod 60:855–860
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) and females (
), C56 females (
), and A56 females (
). A, Phallus length measured from the tip of the phallus to the point of contact with the genital tubercle. B, Phallus width measured at the point of contact with the genital tubercle. C, Total length of genital eminence measured from the tip of the phallus to the point where the genital tubercle makes contact with the body. SE bars indicate three or more measurements for each time.
