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Biology of Reproduction 61, 471-475 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Articles

Virilization of the Male Pouch Young of the Tammar Wallaby Does Not Appear to be Mediated by Plasma Testosterone or Dihydrotestosterone1

Jean D. Wilson2,a, Fredrick W. Georgea, Geoffrey Shawb, and Marilyn B. Renfreeb

a Department of Internal Medicine, The University of Texas Southwestern Medical Center, Dallas, Texas 75235 b Department of Zoology, University of Melbourne, Parkville, Victoria 3052, Australia


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Virilization of the male urogenital tract of all mammals, including marsupials, is mediated by androgenic hormones secreted by the testes. We have previously demonstrated profound sexual dimorphism in the concentrations of gonadal androgens in pouch young of the tammar wallaby Macropus eugenii during the interval when the urogenital sinus virilizes. To provide insight into the mechanisms by which androgens are transported from the testes to the target tissues, we measured testosterone and dihydrotestosterone in plasma pools from tammar pouch young from the day of birth to Day 150. Plasma testosterone levels were measurable (0.5–2 ng/ml) at all times studied, but there were no differences between males and females. These low concentrations of plasma testosterone appear to be derived from the adrenal glands and not the testes. Plasma dihydrotestosterone levels in plasma pools from these animals were also low and not sexually dimorphic.

We conclude that virilization of the male urogenital tract cannot be explained by the usual transport of testosterone or dihydrotestosterone in plasma but may be mediated by the direct delivery of androgens to the urogenital tract via the Wolffian ducts. Alternatively, circulating prohormones may be converted to androgens in target tissues.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In eutherian mammals, development of a male phenotype requires the presence of a testis, whereas female development takes place when ovaries are present or in the absence of functional gonads [1]. Two types of testicular hormones are involved in male development in eutherians. Testosterone and its 5{alpha}-reduced derivative dihydrotestosterone are responsible for virilization of the Wolffian ducts, urogenital sinus, scrotum, and phallus; and Müllerian inhibiting substance (MIS) causes regression of the Müllerian ducts in the male [1–3].

In the marsupial, formation of the scrotum in the male is androgen independent and is determined directly by one or more genes on the X chromosome [4–7]. However, formation of the other components of the male urogenital tract is controlled by mechanisms analogous to those in eutherians; namely, the Müllerian ducts regress under the control of MIS [8, 9], and testicular androgens virilize the Wolffian ducts, the urogenital sinus, and the phallus [1, 2]. The role of testicular androgens in these processes in the marsupial has been established on the basis of a variety of studies in the tammar wallaby Macropus eugenii [10]. Testosterone levels in the testis [11] increase before the onset of virilization of the prostate on Days 20–30 [10, 12–15], and virilization of the male pouch young is prevented by administration of the steroid 5{alpha}-reductase inhibitor finasteride [12] or of the androgen receptor inhibitor flutamide [13]. Furthermore, castration of 10-day-old male pouch young prevents virilization [16], whereas transplantation of testes into 10-day-old females [16] or the administration of testosterone to female pouch young [12, 15] virilizes the urogenital sinus and phallus. Furthermore, the receptor that mediates the actions of testosterone and dihydrotestosterone is expressed in the urogenital tract of both male and female tammar pouch young before the onset of development of the male phenotype [17, 18].

Nevertheless, certain aspects of male phenotypic differentiation in the mammal are incompletely understood. For example, some androgen actions in the developing rabbit embryo appear to be exerted ipsilaterally in that removal of one testis impairs virilization of the Wolffian duct on the same side but not on the opposite side [1], implying that androgen may be transferred directly to the Wolffian ducts. Furthermore, because the development of phenotypic sex takes place early in embryogenesis, it has not been technically feasible to measure plasma androgen levels in any eutherian species during the time when virilization of the male embryo commences.

In the marsupial, in contrast, virilization of the male takes place after attachment of the pouch young to the teat, and as a consequence it is possible to examine the sequence of events in great detail. Fadem and Harder measured plasma androgen levels from Day 1 of postnatal development in the gray short-tailed possum (Monodelphis domestica), but sexual dimorphism was not demonstrated in these levels until after puberty [19]. Xie et al. [20] were unable to measure plasma testosterone in males of the same species until the onset of puberty, despite histological evidence of maturation of the Leydig cells by Day 16 of pouch life. In the present study we report that the levels of plasma testosterone and dihydrotestosterone in the pouch young of the tammar wallaby (M. eugenii) are measurable but that they exhibit no sexual dimorphism during the periods when the prostate and phallus virilize. We also provide evidence that plasma testosterone in the early pouch young of both sexes is probably derived from the adrenal. The demonstration in two marsupial species that virilization of the male urogenital tract cannot be mediated by plasma androgens raises fundamental questions concerning how androgen is transported from the testes to the male urogenital tract during early postnatal development.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Tammar wallabies from Kangaroo Island, South Australia, were kept in open grassy yards and provided with compressed lucerne hay, oats, fresh vegetables, and water ad libitum. Tammar wallabies are seasonal breeders and have a postpartum estrus that results in the presence of a diapausing blastocyst in the uterus while a pouch young is suckled. Removal of pouch young causes reactivation of the quiescent blastocyst, and births occur 26.5 days later on average. Adult female tammars were checked daily for births. In cases in which the day of birth was uncertain, the age of pouch young was assessed by head length [21].

Blood was collected, in heparinized tubes, from adult males and females from the lateral tail vein or by cardiac puncture immediately postmortem as described previously [22]. To assess free testosterone levels, 7 adult males were sampled, in November (n = 1), January (n = 4), and March (n = 2). One hundred and sixty-two pouch young aged between Day 0 (newborn) and Day 150 after birth were removed from the pouch, weighed, measured, and sexed according to the presence or absence of a scrotum or mammary primordia. In pouch young less than 60 days of age, blood was drawn in heparinized capillary pipettes under hypothermic anesthesia, and the animals were killed by decapitation and dissected. Pouch young older than 60 days were killed by overdose of sodium pentobarbitone in sterile saline intraperitoneally, and blood was collected by cardiac puncture. A complete set of samples from 79 pouch young aged Days 0–150 was collected within a single breeding season. A second set was taken between Days 0 and 40 (from a total of 77 pouch young) and Days 50 and 150 (6 pouch young). In a separate experiment, adrenals were collected from 39 pouch young that varied in age from newborn to 75 days. All experiments followed the National Health and Medical Research Council of Australia guidelines (1990) and were approved by the University of Melbourne Animal Ethics Committee.

Plasma and adrenals were frozen and shipped in dry ice from Melbourne to Dallas, where they were stored at -20°C before being assayed for the content of testosterone and dihydrotestosterone. Plasma was combined into pools that included blood from 10 to 12 animals (Days 0–4 and 5–10), 4 to 5 animals (Days 31–40 and 41–50), and 1 to 3 animals (over Day 50). Adrenals from 2–5 pouch young less than 30 days of age were pooled; the remaining assays were performed on a pair of individual adrenals from a single animal.

Hormone Assays

Two methods were used for the assay of testosterone. For the study of adrenal testosterone content and the first series of plasma assays, we used previously described methods for the chromatography and RIA of testosterone and dihydrotestosterone and for the measurement of protein in the tissue samples [11]. In brief, tritiated testosterone (the adrenal samples) or a mixture of tritiated testosterone and dihydrotestosterone (25 µl of plasma in duplicate from each plasma pool) was added for a recovery standard, and the samples were extracted overnight with chloroform:methanol (2:1, v:v); in the case of the adrenal samples, protein content was assayed in the aqueous layer. The chloroform:methanol extracts were dried and chromatographed on small celite-ethylene glycol columns. The fractions containing dihydrotestosterone (plasma samples) were eluted with benzene (7%) in isooctane, and the fractions containing testosterone (plasma and adrenals) were eluted with benzene (20%) in isooctane. The samples were dried under a stream of air and reconstituted in buffer for RIA. The individual RIAs for testosterone [23] and dihydrotestosterone [24] have been described previously. For the second set of plasma pools, testosterone was measured directly, without prior chromatographic separation, by the Mayo Medical Laboratories (Rochester, MN) in 25-µl plasma aliquots using a competitive chemiluminescent immunoassay (the Ciba-Corning ACS testosterone assay) [25]. In all instances plasma assays were performed in duplicate, and these results were averaged to provide the value for each pool. An internal control consisting of measurement of an aliquot of a plasma pool made from three adult male plasma samples was run with each assay.

In one experiment, levels of total and free testosterone were measured in adult male plasma by the Mayo Medical Laboratories using the technique of Vermeulen, Stoica, and Verdonck [26], in which radioactive testosterone is added to the sample prior to dialysis and the radioactivity is measured both inside and outside the tubing after dialysis. In seven plasma samples taken from adult males between November and March, the total testosterone level was 8.3 ± SD 2.0 ng/ml, and the free testosterone level averaged 11.7 ± SD 1.0% of the total level.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Plasma Androgens

Testosterone was measurable but low, in the range of 0.5–2 ng/ml, in most of the plasma pools from Days 0 to 4 until 150 days after birth (Fig. 1a). However, no sex differences were observed in the levels of testosterone in the plasma pools at any time examined, including the periods that encompass the virilization of the prostate (Days 20–30) and virilization of the phallus (Days 100–120) in this species. Because the failure to demonstrate sexual dimorphism in plasma testosterone levels was unexpected, we utilized a different immunoassay procedure to repeat the measurement of testosterone in plasma obtained during subsequent breeding seasons for most of the age groups, and again no sexual dimorphism was observed (Fig. 1a). In three pools of plasma that encompass the interval in which the phallus becomes sexually dimorphic, dihydrotestosterone was barely detectable at less than 0.05–0.1 ng/ml and again exhibited no sexual dimorphism (Fig. 1a).



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  		FIG. 1. Androgen levels in the plasma and adrenal glands of tammar wallaby pouch young as a function of age. a) Testosterone was measured in pools of plasma from male and female pouch young varying in age from newborn (Day 0) to 150 days, and dihydrotestosterone was measured in pools of plasma from male and female pouch young varying in age from 31 to 150 days. Each data point represents the average of duplicate values of assays of different plasma pools. The initial pools were obtained in one breeding season, and the second pools were obtained in subsequent breeding seasons. b) Testosterone was measured in pools of 3–5 adrenal glands (Day 30 or younger) or in individual adrenals at the later ages; for the samples from pouch young < Day 31, the ranges of measurements in 2–3 pools for each age are shown. In the case of the pouch young 31 days or older, individual assays are shown.

Adrenal Androgens

Since testosterone was virtually undetectable in the ovaries of pouch young females at all ages examined [11], we measured testosterone levels in adrenals from pouch young of various ages (Fig. 1b). Testosterone levels in the adrenals of both sexes were almost as high as in testes at comparable ages [11] (Fig. 2), namely, in the range of 0.3–1.0 ng/mg protein, but again exhibited no sexual dimorphism from Day 0 up to Day 40. In the one sample studied in an older animal, the concentration was lower. This finding is similar to that of Catling and Vinson [27], who reported that androgen synthesis in the adrenal of the tammar wallaby fell between Days 19 and 40, as cortisol biosynthesis increased. Since the adrenal appears to be the only source of testosterone in the pouch young females, we conclude that plasma testosterone in the female (and possibly in the male pouch young as well) is probably derived from the adrenal.



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  		FIG. 2. Testosterone content of the gonads of tammar wallaby pouch young as a function of age (redrawn from Renfree et al., Biol Reprod 1992; 47:645, Figure 1 [11]).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study we failed to demonstrate sexual dimorphism in plasma testosterone levels in pouch young of the tammar wallaby during the two periods when virilization of the urogenital sinus and prostate takes place (Fig. 1), including the period when sexual dimorphism in testosterone levels is easily demonstrable in the gonads [11] (Fig. 2). Abundant evidence indicates that virilization of the male tammar pouch young is mediated by testicular androgens [11–17]. Consequently, the findings in the present study, considered together with similar evidence in another marsupial (the gray short-tailed opossum) [19, 20], indicating that virilization of the male marsupial pouch young occurs under circumstances in which there is no sexual dimorphism in plasma testosterone levels, raises profound questions as to how androgenic hormones are delivered from the testis to the developing male urogenital tract. Such a process could occur by at least three mechanisms: 1) direct delivery from the testes down the lumen of the Wolffian duct or via lymphatic transport, 2) secretion by the testis of androgen in the form of a water-soluble derivative or prohormone that is then converted to testosterone and/or dihydrotestosterone within the target tissues, or 3) secretion of free (unbound) androgen into the circulation. Furthermore, different mechanisms of delivery may be involved in the virilization of the different anlage of the male urogenital tract, namely the Wolffian ducts, the urogenital sinus, and the phallus.

As to secretion into the Wolffian ducts, Jost [1] showed that some effects of testicular secretions in virilizing the male rabbit embryo appeared to be exerted ipsilaterally; namely, removal of one testis prevents virilization of the Wolffian duct on that side but does not impair development of the duct on the opposite side or virilization of the urogenital sinus. Veyssiere et al. [28] subsequently showed that the administration of anti-testosterone antibodies to the rabbit embryo prevents virilization of the Wolffian ducts but not the urogenital sinus. Such a phenomenon could be due to a variety of mechanisms, including local diffusion, lymphatic transport, or secretion through the lumen of the Wolffian ducts. Tong et al. [29] provided evidence in favor of the latter mechanism by showing that testosterone-protein conjugates are secreted from embryonic mouse testes into the lumen of the Wolffian ducts in organ culture. It is also interesting in this regard that secretion of testosterone into the rete testes fluid is involved in the maturation of the epididymis at puberty in many species [30]; in this circumstance the hormone in the testicular secretion is bound to the androgen-binding protein (ABP), which is formed in the Sertoli cells of the testes. The ABP-testosterone complex binds to specific receptor sites on cell membranes of epithelial cells in the epididymis and is internalized where the testosterone is cleaved from the protein and undergoes 5{alpha}-reduction to dihydrotestosterone, which in turn mediates the virilization of the tissue (reviewed in [30]). The existence of such a pathway has not been demonstrated to be involved in the formation of the male phenotype in any species and has not been documented to be involved in the virilization of tissues other than the epididymis. However, McDonald and Waring [31] suggested that testosterone could be delivered directly by a similar mechanism down the lumen of the vas deferens to the marsupial prostate, and the blood supply to the prostate of the brush-tailed possum is derived from a network of periurethral arteries and veins from which vessels radiate into the body of the tissue [32]. Although this concept has powerful explanatory potential, particularly for the virilization of the Wolffian ducts, the report by Tyndale-Biscoe and Hinds, indicating that transplantation of testes into 10-day-old female tammar pouch young resulted in virilization of the prostate and penis, suggests that secretion of androgen via the Wolffian ducts cannot explain sexual dimorphism of these tissues [16].

Indeed, demonstration that transplanted testes can virilize the urogenital tract of the female tammar pouch young suggests that virilization is likely mediated by some testis-derived circulating prohormone or hormone derivative that is converted to active androgens in target tissues. There is ample precedent in eutherians and marsupials for the conversion of prohormones to active hormones in target tissues, including estrogen formation in the placenta [33] and brain [34], dihydrotestosterone formation in the male urogenital tract [11, 32, 35], and testosterone synthesis from 19-carbon precursors in multiple extraglandular tissues [36]. Certain aspects of steroid hormone metabolism make the marsupial a prime candidate for extraglandular hormone formation, namely the presence of elevated levels of circulating 17-hydroxyprogesterone and 21-deoxycortisol, indicating a relative deficiency of steroid 21-hydroxylase (CYP21), and higher plasma levels of 11ßOH-androstenedione than in most eutherian mammals [31]. Furthermore, testosterone sulfate can serve as a prohormone for testosterone in some species [37], and aryl sulfatase is known to be present in the tammar ovary [38]. The net consequence is that a variety of potential substrates could serve as circulating prohormones for conversion to androgen.

A third possibility is that virilization is mediated by free (unbound) testosterone secreted into the circulation by the testes. This possibility stems from the fact that androgen transport in the plasma of the tammar wallaby (and a few other marsupial and eutherian species) differs from that in most mammals in that there is no high-affinity transport protein in plasma analogous to sex hormone-binding globulin (SHBG, also termed testosterone-binding globulin, TeBG) [39]. As a consequence, testosterone and dihydrotestosterone in plasma are transported bound to low-affinity, nonsaturable carriers, principally albumin, and albumin is synthesized by the tammar fetus [40]. Consequently, it would be expected that protein-bound as well as free testosterone would be present and that levels would be sexually dimorphic in plasma at the time of virilization of the male tammar pouch young. Interestingly, total plasma testosterone levels in mature tammar males are in the same range as those in sexually mature males in other species, and the concentration of free (dialyzable) testosterone in male tammar plasma during the breeding season averages only about twice the level in sexually mature men, namely about 1.0 ng/ml. If free plasma testosterone plays a role in male phenotypic sexual differentiation, then one must assume that the sexual dimorphism in plasma testosterone levels is obscured by the release of adrenal testosterone into plasma as a part of a general stress reaction. To document that such a mechanism is involved it would be necessary to suppress the secretion of adrenal androgens by inhibiting the secretion of corticotropin and measuring free hormone levels in plasma. It is noteworthy, however, that in mature marsupials of several species the secretion of adrenal androgen does not appear to be under the control of adrenocorticotropic hormone [41–43].

Much additional work will have to be performed to ascertain exactly how androgens are delivered from the testes to the Wolffian ducts, urogenital sinus, and phallus to induce the formation of the male phenotype in pouch young.


   ACKNOWLEDGMENTS
 
We thank Anne Duns and Richard Moyle for assistance with the animals. The wallabies were held under permit numbers RP-92–099 and RP-95–088 from the Department of Conservation and Natural Resources, Victoria, Australia.


   FOOTNOTES
 
1 Aided in part by grant DK03892 from the National Institutes of Health, by grant 980779 from the National Health & Medical Research Council of Australia, and by a grant from the Perot Family Foundation. Back

2 Correspondence: Jean D. Wilson, Department of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas TX 75235–8857. TEL: 214 648 3685; FAX: 214 648 8917;jwils1{at}mednet.swmed.edu Back

Accepted: March 15, 1999.

Received: November 25, 1998.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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G. Shaw, M. B. Renfree, M. W. Leihy, C. H. L. Shackleton, E. Roitman, and J. D. Wilson
Prostate formation in a marsupial is mediated by the testicular androgen 5alpha -androstane-3alpha ,17beta -diol
PNAS, October 12, 2000; (2000) 220412297.
[Abstract] [Full Text]

Home page
 Proc. Natl. Acad. Sci. USAHome page
G. Shaw, M. B. Renfree, M. W. Leihy, C. H. L. Shackleton, E. Roitman, and J. D. Wilson
Prostate formation in a marsupial is mediated by the testicular androgen 5alpha -androstane-3alpha ,17beta -diol
PNAS, October 24, 2000; 97(22): 12256 - 12259.
[Abstract] [Full Text] [PDF]

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