Developmental Biology 334 (2009) 429–436

Contents lists available at ScienceDirect

Developmental Biology
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / d e v e l o p m e n t a l b i o l o g y

Wolffian duct differentiation by physiological concentrations of androgen

delivered systemically
Marilyn B. Renfree a,⁎,1, Jane Fenelon a,1, Gratiana Wijiyanti a, Jean D. Wilson b, Geoffrey Shaw a
a
Department of Zoology, The University of Melbourne, Victoria 3010, Australia
b
Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390-8857, USA

a r t i c l e i n f o a b s t r a c t

Article history: In developing mammalian males, conversion of the Wolffian ducts into the epididymides and vasa deferentia
Received for publication 13 March 2009 depends on androgen secretion by the testes, whereas in females these ducts remain in a vestigial form or
Revised 24 June 2009 regress. However, there is continuing uncertainty whether the androgen needs to be delivered locally, either
Accepted 30 July 2009
by diffusion from the adjacent testis or, by secretion into the lumen of the duct, or whether circulating
Available online 4 August 2009
androgens maintain and virilize the Wolffian ducts. To resolve this uncertainty, we transplanted either day
Keywords:
0–2 or day 8–9 post-partum testes beneath the flank skin of three groups of neonatal (days 0–1) female
Gonadal transplantation tammar wallabies, where they developed and secreted physiological levels of hormones. The Wolffian ducts
Xenotransplantation of all these females were retained and had formed extensive epididymides when examined at days 25, 34
Sexual differentiation and 87 after birth. In the two older groups of females, sampled after the time of prostatic bud formation, the
Marsupial urogenital sinus was virilized and there was extensive prostatic development similar to that of normal males
Genital ducts of the same age, showing that androgen secretion had occurred. Virilization of the Wolffian ducts occurred
Androgen imprinting during an early but short-lived window of sensitivity. This study provides the first clear evidence that under
Testosterone
physiological conditions virilization can be mediated by circulating androgen.
Anti-Müllerian hormone
© 2009 Elsevier Inc. All rights reserved.
Androstanediol
Urogenital system

Introduction
Virilization of the male urogenital tract during mammalian
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. In all
mammalian species—eutherian and marsupial—these processes are
mediated by androgens secreted by the developing testes (George and
Wilson, 1994; Wilson et al., 1995).
In all species studied to date virilization of the urogenital sinus and
external genitalia is mediated by the 5α-reduced androgen dihydro-
testosterone, which is formed by one of two mechanisms in the
tissues of the urogenital tract—either the 5α-reduction of testosterone
of testicular origin to dihydrotestosterone or by the oxidation of
testicular 5α-androstanediol to dihydrotestosterone (George and
Wilson, 1994, Wilson et al, 2003). In both instances circulating
androgens are converted to the more potent dihydrotestosterone
which acts via the androgen receptor to initiate the virilization
process (George and Wilson, 1994; Wilson et al., 2002).
⁎ Corresponding author. Fax: +61 3 93481719.
E-mail address: m.renfree@unimelb.edu.au (M.B. Renfree).
1
These authors contributed equally to the study.
The process by which the Wolffian ducts are converted to the male
ejaculatory system is less well understood in two regards. First, in
eutherians and in some marsupial species testosterone itself is
believed to be the responsible intracellular hormone (George and
Wilson, 1994), whereas in the tammar wallaby, Macropus eugenii the
process appears to be mediated by dihydrotestosterone formed from
circulating androstanediol (Shaw et al. 2006). Two mechanisms have
been considered for delivery of androgen to the developing ducts.
Based on a single rabbit embryo in which a testis was grafted onto the
mesosalpinx of a female fetus, Jost (1953a; 1953b) proposed that
testicular androgen diffused directly down the Wolffian ducts and
promotes virilization of the tissue ipsilaterally. This concept is in
keeping with the fact that in many humans with 46,XY/45,X
mosaicism or with true hermaphroditism, the Wolffian ducts virilize
only on the side with a testis (Donahoe et al., 1978; McKelvie et al.,
1987). Further evidence in favour of this concept was obtained by
Wilson (1973) who showed that the Wolffian ducts of the rabbit can
concentrate radioactive testosterone against a concentration gradient,
by Veyssiere et al. (1982) who demonstrated an increase of
testosterone in rabbit Wolffian ducts during their virilization, and
by Tong et al. (1996) who microinjected embryonic (E) day 14 mouse
urogenital ridges in organ culture with testosterone–albumin–
fluorescein isothiocyanate and found fluorescence distributed
throughout the Wolffian ducts that became maximal in the caudal
end within 48 h. Androgen receptors are localized in the

0012-1606/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.ydbio.2009.07.040
430 M.B. Renfree et al. / Developmental Biology 334 (2009) 429–436

mesenchymal cells at the medial side of the Wolffian duct and only
subsequently in the epithelial cells (Bentvelsen et al, 1995). Thus
these various types of evidence supported the concept that the
Wolffian duct is virilized locally by androgens either passing through
the lumen of the duct or diffusing along the tissue around the duct.
Nevertheless, circulating androgens can virilize the Wolffian ducts:
administration of androgen to pregnant mice (Goldstein and Wilson,
1972) and rats (Schultz and Wilson, 1974) induces formation of
epididymides, vasa deferentia, and seminal vesicles in female
offspring. However, the latter phenomenon are more likely to be a
result of pharmacological rather than physiological effects due to the
doses administered.
The former experiments cannot discount the possibility that
circulating androgens may be involved while the latter experiments
may involve pharmacological rather than physiological effects as well as
the possible metabolism of the administered hormone by the placenta.
We decided to reinvestigate the mechanism of Wolffian duct virilization
by taking advantage of the fact that xenotransplantation well before
maturation of the immune system after days 90–120 post-partum (pp)
(Basden et al., 1997) can be performed in tammar wallaby pouch young
(Tyndale-Biscoe and Hinds, 1989; Whitworth et al, 1996).
The process of sexual differentiation is well documented in the
tammar (O et al., 1988; Renfree and Short, 1988, Shaw et al., 1988;
1990; Renfree et al., 1992; 1995; 1996; Wilson et al., 1995; 2003). The
scrotum, mammary glands, pouch, gubernaculum and processus
vaginalis all differentiate before the gonad is differentiated and are
controlled by a gene or genes on the X chromosome, not steroids (O et
al, 1988; Renfree and Short, 1988; Shaw et al., 1988). The testis is
undifferentiated on the day of birth, and early seminiferous cords do
not develop until 2 days after birth. The ovary remains undifferen-
tiated for longer, and develops cortical and medullary regions by day
8 pp (Renfree et al., 1996). Testicular testosterone is unmeasurable on
the day of birth and contains less than 0.2 ng/mg up to day 4 but
between days 5–10 it has reached 1 ng/mg. This concentration is
sustained until day 40 pp in the testes but there is no measurable
testosterone in ovaries between day of birth and day 70 pp (Renfree
et al., 1992).
The circulating androgen in the tammar is 5α-androstane-3α,17β-
diol (androstanediol), since plasma concentrations of 5α-adiol were
3 times higher in day 20–40 pp male plasma pools (averaging 1.9 ng
ml− 1) than in female pools (averaging 0.6 ng ml− 1) (Shaw et al.,
2000) and there is no sexual dimorphism in the levels of plasma
testosterone or DHT at these ages (Renfree et al., 1992). In the
tammar, dihydrotestosterone is formed by an alternate pathway
during virilization of the urogenital tract (Wilson et al., 2003).
Androstanediol is the principal androgen secreted by the testis at the
time when virilization of the Wolffian ducts commence around days
8–10 of pouch life (Shaw et al., 2006), when prostate development is
initiated between days 20–40 (Shaw et al., 1988; Renfree et al, 1996;
Lucas et al, 1997), and when the phallus differentiates after day 40,
the latter two as a result of a window of sensitivity to androgen
(androgen imprinting) (Leihy et al, 2002, 2004). We therefore
transplanted testes from male young aged less than 2 days old that
had not yet begun to synthesize androgens (Renfree et al., 1992), into
the flank of neonatal female pouch young, a stage at which the
Wolffian ducts are undifferentiated. The recipient females' urogenital
tracts were examined at day 34 and day 87 after birth. In a third group
of recipient females, day 8 pp testes, that are already secreting
androgens (Renfree et al., 1992) were transplanted into females aged
day 0–1, and the recipient urogenital tracts were examined at day 25
post-partum. Our findings demonstrate that virilization of the
Wolffian ducts in this species can be mediated by physiological levels
of systemically circulating androgens.
Materials and methods
Animals
Tammar wallabies (Macropus eugenii) were held in open grassy
yards with shelters provided. Diet was supplemented with com-
pressed lucerne cubes and water ad libitum. Females were checked
daily for births during the breeding season (Tyndale-Biscoe and
Renfree, 1987). On the day of birth (day 0) neonates were sexed by
the presence of scrotal bulges (male) or mammary primordia
(female) (O et al, 1988). Care and treatment of animals conformed
to the National Health and Medical Research Council of Australia
(2004) guidelines and were approved by the University of Melbourne
Animal Experimentation Ethics Committees.
Experimental design
Neonatal young were either given testis grafts or allowed to grow
normally (Table 1). These animals were killed at day 34 or day 87 to
assess the degree of virilization. In a third group, testes from day
8 males when testicular testosterone levels are much higher were
transplanted into neonatal females (Table 1). This group was killed at
day 25 after transplant and compared to young from previous studies
that had been allowed to grow normally (Shaw et al. 1988, Lucas et al.,
1997; Ryhorchuk et al, 1997).
Testicular grafts
Transplantation from male neonate to female neonate
Both testes from males aged 0–2 days pp were transplanted under
the flank skin of neonatal (days 0–1) females as previously described
(Whitworth et al., 1996). Briefly, males were removed from the
pouch, sedated by cooling on ice and then killed by decapitation. The
testes attached to the mesonephroi were removed and placed in
saline while the recipient female was prepared. The recipient female
young was anesthetized by hypothermia (Renfree 2002). A small
incision was made in the abdominal skin on the left hand side. The
two testes with mesonephroi attached were inserted subcutaneously

Table 1
Status of reproductive tissues of female pouch young carrying testicular grafts at autopsy.

Day 25 Day 34 Day 34 Day 34 Day 87 Day 87 Day 87

transp. female control female control male transp. female control female control male transp. female

Number surviving to autopsy 5/6 4/4 5/5 3/5 3/3 3/3 4/5
Testes present and developed 5/5a – 5/5 3/3b – 3/3 2/4
Both Wolffian ducts virilized 5/5 0/4 5/5 3/3 0/3 3/3 4/4
Both Müllerian ducts regressed 0/5 0/4 5/5 0/3 0/3 3/3 0/4
Abnormal ipsilateralc ovary 5/5 0/3 – 2/3 0/3 – 1/4
Abnormal contralateralc ovary 0/5 0/3 – 0/3 0/3 – 1/3
Urogenital sinus virilized – 0/4 5/5 3/3 0/3 3/3 4/4
a
One testicular graft in this group had stopped growing by the time of autopsy.
b
Two testicular grafts in this group had stopped growing by the time of autopsy.
c
Relative position of ovary to testes graft shown.
 M.B. Renfree et al. / Developmental Biology 334 (2009) 429–436
through this incision. All but one female (that was killed at day 87)
received 2 testes with attached mesonephroi, but because of a small
body size of this individual, this remaining female neonate was given
testes without the adjacent mesonephros to reduce the bulk of the graft.
The incisions were closed with 8-0 sutures. The female recipient young
were then warmed by hand and reattached to the teat in the mother's
pouch. Sham operations were not conducted due to limited numbers of
available animals, but our previous studies have shown that surgery of
this type per se has no significant effect on the development of these
animals (Nurse and Renfree, 1994; Whitworth et al, 1996; Rudd et al.,
1999; Renfree, 2002). Effects of grafts in young were assessed by
comparison to unoperated males and females of the same age groups.
The phallus of male tammar young becomes longer than that of the
female only after day 50, though large differences are not obvious until
around day 100 (Leihy et al., 2004). Therefore, in the animals killed at
day 87 the length of the phallus diagonally from the tip to the base was
measured (Leihy et al., 2004) at day 77, 83 and 87. All measurements
were taken at least twice and averaged. At autopsy, graft tissue from
the flank of the recipient females (see Fig. 1) and the entire urogenital
tracts were removed and fixed for histological examination.
Transplantation from males at day 8 into female neonates
Testes were dissected from male pouch young of 8 or 9 days old and
transplanted under the abdominal skin of female recipients aged 0–
1 days pp as above, and the operated recipient females were reattached
to the teat and left to grow in their mothers' pouches for 25 days.
Histology and morphometry
The day 8 testes to the day 0–1 female transplant group were killed
by decapitation at 25 days after transplantation. Females in one of the
day 0–2 testes to day 0–1 female groups were killed at day 34 by
decapitation. The day 87 recipient females were killed by anesthetic
overdose (sodium pentobarbitone, 60 mg/ml i.p., to effect). The external
anatomy and graft position in each recipient female was examined
before the abdominal cavity was opened. The reproductive tracts of the
females and the testicular grafts were removed, fixed in neutral buffered
formalin (10% v/v) and embedded in wax. These blocks were serially
sectioned at 8 μm, and mounted in ribbons of 10 sections on glass slides.
Every 4th slide was stained in Harris's haematoxylin and eosin to
examine the development of the grafted testes, the contra- and
ipsilateral ovaries and their Müllerian and Wolffian ducts.
The development of the testes grafted into the recipient females
was assessed by examining the morphology of the testes and germ
Fig. 1. Female pouch young (wt = 42.3 g) at day 87 with the testes transplant (arrow)
beneath the flank skin. Insert shows a neonate (400 mg) at the same scale. Scale
bar = 2 cm.
431
cells. Gonadal morphometry was assessed using point counts on 6
evenly spaced sections from near the cranial pole to near the caudal
pole. Volume of ovaries and grafted testes were determined using
Cavalieri's principle (Gunderson et al., 1988).
Immunohistochemistry against rabbit polyclonal anti-Müllerian
hormone (AMH) antibody (a kind gift from Dr Nathalie Josso, INSERM,
Paris) with appropriate controls was performed on the testes to assess
the testicular bioactivity for AMH as previously described (Pask et al.,
2004). The development of the ovaries in the female recipients was
assessed by examining the morphology and the presence of mitotic or
meiotic activity of germ cells.
Prostatic development in the female recipients at days 34 and 87
pp was assessed by the presence or absence of prostatic buds and
branching in the urogenital sinus. Such branching is the result of
androgen and is never seen in untreated females. Since prostatic buds
do not develop before day 25 pp in the tammar (Renfree et al., 1996;
Lucas et al, 1997), the day 25 group were not examined for buds.
Wolffian duct and Müllerian duct diameters were measured in
each recipient female on both right and left sides at 4 different levels,
representing proximal, near the gonad, to distal, near the urogenital
sinus. Lumina diameters were averaged.
Statistics
Effects of treatment on continuous variables were assessed using
t-tests or analysis of variance (using repeated measures analysis
where appropriate). T-tests were used to compare volume of gonads
and grafts between treatment groups. Initial general linear models
were used to determine whether a significant difference was present
for the overall effect of treatment on the ducts and to establish a non-
significant effect of side and position. If significant, subsequent
analyses involved repeated measures multivariate analysis. Two
factor ANOVAs were used to compare testes development and phallus
measurements.
Results
Survival of young
One of 6 young from the day 25 recipient group disappeared from
the pouch at day 20. Four of 5 females with grafts remained in their
mother's pouches at day 34 but one disappeared from the pouch
during catching of their mother. Similarly, 4 of 5 female recipients
remained in their mother's pouches at day 87 (Table 1).
Survival of testicular grafts
At day 25 post-transplantation, all of the 5 remaining recipient
females with day 8 testes grafted under the skin of the day 0 female
young developed testes (Table 1). At day 34 and at day 87, all
surviving transplanted female young had visible lumps under the skin
at the site where the testes were grafted (Fig. 1). Three of the 7
females had grafts that were histologically well differentiated as
normal testes whereas the testes in three of the remaining four female
recipients were less well developed. One recipient female at day 34
had a graft that was largely fibrous with only a few seminiferous
tubules identifiable (Fig. 2; Table 1). Remnants of the mesonephros
were present as dark staining condensed tissue in all grafts except the
single female in which only testes were transplanted.
External anatomy of the transplanted female young
Control males had a well developed scrotum. At days 25 and 34 pp
the testes of normal males were visible through the skin, located in
the upper part of the inguinal canal. At day 87 pp, testes were located
in the scrotum. Control females all had a well formed pouch.
432 M.B. Renfree et al. / Developmental Biology 334 (2009) 429–436

Fig. 2. (A) The testis of a control male day 87; (B) the testis at day 87 after transplantation under flank skin of a female; (C) two testes at day 34 after transplantation under flank skin
of a female; (D) ovary of a control female at day 87; (E) an ovary at day 87 of female receiving the testicular graft; and (F) an ovary at day 34 of female receiving the testicular graft. m:
medulla; c: cortex; st: seminiferous tubules; ta: tunica albuginea; sk: flank skin; meso: mesonephros. Scale bar = 500 μm.

Transplantation of testes to recipient females had no obvious
effects on the external genitalia and as expected there was no evidence
of a scrotum. At day 87 pp, the phalluses of control males were larger
than those of control females (4.57 ± 0.13 mm vs 3.35 ± 0.05 mm;
p b 0.005). The phalluses of recipient females were intermediate in
size (4.04 ± 0.13 mm) but were neither significantly larger than
the control females (p = 0.16) nor smaller than control males
(p = 0.25).
Testicular graft development
Histology and morphometry of the grafts
All testicular grafts survived and grew under the flank skin of the
transplanted females (Figs. 1–3). At 25 days post-transplant, the
grafted testes were present in all 5 and still developing in 4 of the 5
young that remained at the end of the experiment. In one female, the
testes appeared to have stopped developing. In some, the two grafted
testes had fused together while in others they remained as two
discrete organs (Fig. 2). In all these testes seminiferous tubules
contained healthy looking germ cells. The Sertoli cells were bounded
by a basement membrane and peritubular myoid cells. The inter-
stitium was comprised of Leydig cells and connective tissue (Fig. 3A).
The Sertoli cells actively produced AMH in 4 of 5 pairs of transplanted
testes as shown by positive immunostaining against rabbit polyclonal
anti AMH (Fig. 3B). One of the 5 testes grafts had stopped developing
but had formed a communal tunica albuginea in addition to individual
tunica, and seminiferous tubules were visible in some sections. This
testis was smaller than the other four testes pairs, and the Sertoli cells
were AMH negative. No AMH staining was observed in the ovaries of
the control females.
Of the testes grafted under the skin in the 3 remaining day 34 pp
female pouch young, one was indistinguishable from a normal
developing testis and had well developed seminiferous cords and a
volume within the range of the control animal's testes (t = 0.943,
df = 4, p = 0.399, ns), one had severely degenerated and had few
remaining seminiferous cords, and one had the two transplanted
testes still visible as two spherical structures with seminiferous cords,
also with a volume range within normal (Fig. 2C). In the 4 remaining
day 87 pp female recipient pouch young, the transplanted testes were
still developing apparently normally in two animals (Fig. 2B), with a
volume within the range of the control males. The other two were
smaller with a somewhat disorganized structure but still obvious
seminiferous cords.
Ovarian development in recipients
The ovaries of the recipient female young that had received day
8 testes differed depending on the proximity of the testicular
transplants at day 25. The ovaries on the side contralateral to the
transplants developed normally with nests of oogonia delineated by
bands of mesenchymal tissue (Fig. 3C, Table 1). They had all reached a
stage of development comparable to that of normal day 25 pp ovaries.
Some oogonia were still dividing mitotically while others had entered
meiotic prophase. In contrast, the ipsilateral ovaries were smaller than
those on the contralateral side, and there was some evidence of early
development of cord-like structures (Fig. 3D). However, there was no
positive immunostaining for AMH detected in any type of cell in these
ovaries (data not shown). These ovaries had fewer oogonia than the
contralateral ovaries, with some of them in mitosis. None of the
surviving oogonia had entered meiotic prophase. The ipsilateral ovary
from the female in which the grafted testis had stopped developing
had germ cells of normal appearance but there were no meiotic germ
cells (Fig. 3D).
In the day 0–1 females that received day 0–2 testes, development
of one ovary was disrupted in 2 of 3 females at day 34 pp and in both
ovaries of one of the day 87 female recipient young with testis
transplants. In these 4 ovaries there were few germ cells and a highly
fibrous medulla containing a few structures resembling seminiferous
 M.B. Renfree et al. / Developmental Biology 334 (2009) 429–436 433

Fig. 3. (A) The day 8 testis at day 25 after transplantation under the flank skin of a neonatal female showing healthy seminiferous tubules with Sertoli cells aligning the tubule and the
gonocytes at the center of the tubule (triangle); (B) AMH protein (brown stain) was detected in the seminiferous tubules of the grafted testes; (C) ovary contralateral to the testicular
graft showing oogonia at the first meiotic prophase (black arrow) and interphase (white arrow); and (D) ovary ipsilateral to the testicular graft was less developed and some
seminiferous cord-like structures were observed (grey arrow). st: seminiferous tubules. Scale bar = 50 μm.

cords (Fig. 2D). In the remaining three female young with testicular
transplants at day 87 pp there was also some development of cord-
like structures in the medulla of the ipsilateral ovaries despite the
presence of healthy looking follicles in the cortex. All the other ovaries
looked normal. At day 34 pp the ovaries of females with testis grafts
were similar in size to the day 34 pp control females. Similarly, there
was no significant difference (t = 2.091, df = 8, p = 0.070, ns) in
ovarian volume at day 87 pp between control females and females
with testis grafts.
Wolffian ducts
Transplantation of testes from either the day 0–2 or day 8 males to
day 0–1 female tammar wallabies caused virilization of the Wolffian
ducts in all animals. All control males had a well developed Wolffian
duct with a columnar epithelium and a lumen patent along its whole
length (Fig. 4). The duct diameter was similar on the left and right
sides (Fig. 5). In all control females, the Wolffian duct had regressed
and only small vestiges remained (Fig. 4). The ipsilateral and

Fig. 4. Wolffian and Müllerian ducts of control and transplanted animals at days 34 (A–C) and 87 (D–F). (A) Control male day 34 with regressed Müllerian duct and persistent
Wolffian duct; (B) mesonephric ridge with retained Wolffian duct of a female at day 34 after testis transplantation; (C) mesonephric ridge with regressed Wolffian duct of control
female at day 34; (D) control male day 87 with virilized Wolffian duct shown as an expanded and convoluted epididymis; (E) mesonephric ridge with enlarged Wolffian and
Müllerian ducts of a female at day 87 after transplantation of testis; (F) mesonephric ridge of control female at day 87 showing the enlarged Müllerian duct. t: testis; Wd: Wolffian
duct; Md: Müllerian duct. Scale bar = 200 μm.
434 M.B. Renfree et al. / Developmental Biology 334 (2009) 429–436
Fig. 5. Wolffian and Müllerian duct lumen diameters in control and transplanted
animals at days 34 and 87. There was a significant difference (p b 0.05; asterisk) at day
34 and day 87 in Wolffian duct diameters between control and testes transplanted
females, whereas there was no significant difference in lumen diameters of females
with transplants and males at either time (p N 0.1). Müllerian ducts of all females were
similar (p N 0.5), whether carrying transplants or not, whereas in males the Müllerian
duct was fully regressed. white bars = day 34 animals; grey bars = day 87 animals,
shaded bars = male animals.
contralateral Wolffian ducts that were retained in females with
testicular transplants at days 34 and 87 pp were similar in diameter to
those of the control males (Figs. 4 and 5), despite the fact that some of
the testis grafts were not still developing by the time of autopsy.
Fig. 6. (A) Urogenital sinus (TS) of a control male at day 87 showing the prostate with extensive branching; (B) urogenital sinus (TS) of a female at day 87 after testis transplantation
showing enlarged branched prostate similar to that of the control male; (C) urogenital sinus of control female at day 87 showing the typical tripartite branching that occurs during
the development of the median and lateral vaginae of the female marsupial urogenital tract. Scale bar = 400 μm.
Müllerian ducts
In the day 25 post-transplant animals, the Müllerian duct
contralateral to the grafts was intact with an average diameter of
30.7 μm (Fig. 4). This duct was comprised of cuboidal epithelial cells
forming a distinct lumen and had an intact basement membrane. A
typical whorl of mesenchyme surrounding the Müllerian duct was
comparable to that of a normal day 10 pp male Müllerian duct
(Whitworth et al., 1996). However, the Müllerian ducts on the
ipsilateral side had begun to regress as indicated by disintegrated
basement membrane and an irregular shape of the duct.
In the day 34 and 87 unoperated females, Müllerian ducts were
well developed and had begun to form the typical complex vaginal
system with two lateral vaginae and a median vaginal strand, but they
were regressed in unoperated males except for a small remnant
where the duct entered the urogenital sinus (Figs. 4 and 5). The duct
in both control and treatment animals varied in diameter along its
length (p b 0.0005; Fig. 4), so 2-way repeated measures analyses of
variance were performed (position along duct, side relative to graft).
There was a significant variation in the diameter of the ducts within
animals between the left and right sides (p = 0.028). Testicular grafts
did not significantly affect the size of the Müllerian duct (Figs. 4 and
5.; p N 0.5) despite the beginnings of ipsilateral regression (see above)
Urogenital sinus
Prostatic buds were visible in the control males at days 34 and day
87 pp, and were absent from control females (Fig. 6). In contrast, all of
the day 34 and day 87 pp females with testis grafts, including the one
where only fibrous remnants of the grafted testis remained, were
clearly virilized and had prostatic buds, demonstrating a response to
androgens (Fig. 6).
Discussion
Transplantation of testes from day 0–2 males to day 0–1 female
recipient tammar young caused virilization of the Wolffian ducts in
animals autopsied at day 34 and at day 87 pp. Similarly,
transplantation of day 8–9 testes (that are known to be synthesizing
greater amounts of testosterone) (Renfree et al., 1992) into female
neonates also caused Wolffian duct retention and development by
25 days post-transplant. Transplantation induced marked virilization
of the urogenital sinus in all recipient females at days 34 and 87 pp, a
clear demonstration of the presence of physiological levels of
androgen. In the tammar wallaby, both these processes of virilization
are mediated by circulating androstanediol which is transformed in
the tissues to the active hormone dihydrotestosterone (Shaw et al.,
 M.B. Renfree et al. / Developmental Biology 334 (2009) 429–436
2000; Shaw et al., 2006). In our previous studies, administration of
supra-physiological doses of androgens (T, DHT and adiol) to female
pouch young caused development of much larger prostates than in
control males by day 150 pp (Lucas et al, 1997; Leihy et al. 2002) and
administration of androgen receptor inhibitor flutamide inhibited bud
formation in males (Ryhorchuk et al, 1997). Consequently, the fact
that the degree of prostate development in this study was similar in
transplanted females and control males at day 87 pp is an excellent
bioassay to indicate that the plasma androgen levels were at a
physiological concentration in the two groups. Our study was
terminated before sexual dimorphism in the phallus is clear-cut and
hence we did not address the issue of whether phallus development
requires local delivery of androgens via the genital tract rather than
delivery in the systemic circulation. However Tyndale-Biscoe and
Hinds (1989) found well developed penises in females 3 years after
day 10 pp testes were transplanted to 10 day old females, so we
conclude that systemic delivery of testicular androgens is also
effective for normal phallus development.
The finding that Wolffian duct development in the transplanted
females was similar to that of the control males at days 34 and 87 pp
was unexpected for two reasons. First, in the long term study of
Tyndale-Biscoe and Hinds (1989), transplantation of testes into
female tammar wallaby pouch young at day 10 pp caused formation
of male urogenital sinus and phallus, but only minor virilization of the
Wolffian ducts, although the Wolffian ducts never completely
regressed. The difference in the findings in the two studies is almost
certainly due to the fact that the transplanted testes in the earlier
study were older: the transplants were between day 10 males and day
10 females (Tyndale-Biscoe and Hinds, 1989). This suggests that there
is a period of sensitivity (or androgen imprinting) of the Wolffian
ducts to androgens earlier than day 10, but after day 8, the age of the
testes grafted into the female neonates of the day 25 post-transplant
group. This is similar to the conclusion of Burns (1961) that the period
of susceptibility to hormones is limited, and once established, the
effects are permanent. There is also a critical window of time between
E15.5 and E17.5 for androgen action in regulating Wolffian duct
differentiation in the rat (Welsh et al., 2007) and in the tammar,
where androgen imprinting is critical for development of the prostate
and phallus (Leihy et al, 2004). Secondly, on the basis of studies of a
single rabbit embryo (Jost 1953a) and mouse embryos (Tong et al.
1996) and of studies of patients with true hermaphroditism and
mixed gonadal dysgenesis (Donahoe et al, 1978; McKelvie et al. 1987)
it is widely believed that virilization of the Wolffian ducts is an
ipsilateral function, depending on diffusion and/or secretion of
testosterone from the testis into the ipsilateral duct lumen. However,
culture of explanted fetal rat reproductive tracts showed that
regression of both Wolffian ducts could be prevented by the presence
of either a single testis or by the replacement of the testis by
testosterone. Interestingly the virilization of the contralateral duct
was prevented if the tract was spread, increasing the distance of the
duct from the intact gonad in the culture (Price and Pannabecker
1956). Wolffian duct differentiation is probably dependent on
androgen mediated signaling from the stroma to the epithelium
(Welsh et al, 2006; 2007) since the androgen receptor is expressed in
the Wolffian duct stroma but not in the epithelium during the early
window of sensitivity in the rat (Bentvelsen et al, 1995; Welsh et al.,
2006; 2007) and in mice the androgen receptor is expressed only in
the Wolffian duct mesenchyme and not the epithelium of the mouse
until after the initial differentiation (stabilization) of the Wolffian
ducts has occurred (Cooke et al, 1991). This distribution of the
androgen receptor supports arguments that virilization of the
Wolffian duct is less likely to be due to androgens flowing down the
duct itself than to those in the circulation, although this possibility
cannot be excluded. The fact that the administration of exogenous
androgens in pharmacological amounts to rodents (Goldstein and
Wilson, 1972; Schultz and Wilson, 1974) virilizes the Wolffian ducts
435
clearly indicates that direct secretion of androgen into the ducts is not
essential. The present findings further demonstrate that under
physiological conditions circulating androgen can virilize the undif-
ferentiated ducts in females. We have previously shown that in this
species the circulating androgen is androstanediol which is converted
to dihydrotestosterone in the target tissues (Shaw et al. 2006).
Ipsilateral androgen secretion as shown by Jost after unilateral
castration or unilateral insertion of a crystal of testosterone after
castration (1953a; 1961; 1970) may be of importance only under
circumstances in which androgen concentrations are less than
optimal. Under most circumstances testosterone is a weaker androgen
than DHT, so this could be a consequence of dependence on
testosterone for Wolffian duct virilization in some species, but in
the tammar the circulating testicular androgen androstanediol is 3α-
oxidized in the Wolffian duct to the more potent androgen
dihydrotestosterone (Shaw et al, 2006). In support of this, Welsh et
al. (2006) suggest that Wolffian duct differentiation is more
susceptible to interference with androgen action than that required
for the initial stabilization of the duct. Thus there appears to be an
absolute requirement for a physiological level of androgens to virilize
both ipsilateral and contralateral ducts that the presence of a single
testis in the earlier experiments could not provide.
It was also unexpected that transplantation of the testes into
females did not cause disappearance of the Müllerian ducts. This
failure might be due to the fact that the secretion of anti-Müllerian
hormone was inadequate for this purpose and that it does work in an
ipsilateral manner as originally formulated by Jost (1953a; 1961;
1970) or that it needs to be present in sufficient quantities during an
early window of sensitivity. Evidence in favor of the latter possibility
is provided by the older (days 8–9 pp) testis transplant animals
whose testes were AMH positive and in which ipsilateral regression of
Müllerian ducts occurred. Müllerian ducts were also retained in four
of the five animals transplanted at day 10 pp but were absent from
males castrated at day 10 pp (Tyndale-Biscoe and Hinds, 1989). In
that study the authors suggested that the 5th animal may have lost its
Müllerian ducts because it was younger (so developmentally less well
developed) when operated (Tyndale-Biscoe and Hinds, 1989). These
data again suggest that there is a small window of sensitivity to AMH
as well as to androgen which must be between day 8 pp (this study)
and day 10 pp (Tyndale-Biscoe and Hinds, 1989) and as discussed
extensively by Burns (1961). Similarly, Wolffian duct development in
the rat is sensitive to a small window of time between E15.5 and E17.5
which coincides with the onset of androgen synthesis by the testis
(Welsh et al., 2007).
Transplantation of testes caused a variable degree of abnormality
in the recipient ovaries, as evidenced by loss of germ cells and
development of seminiferous-like tubules ipsilateral to the testicular
grafts. In the day 25 post-transplant animals that had received day
8 testes that were producing much greater quantities of testosterone
(Renfree et al., 1992), these ipsilateral ovaries were smaller than the
ovaries contralateral to the graft—presumably due to the greater
concentration of androgens (and AMH) produced by the larger testes.
A similar result was obtained when we administered pharmacological
doses of testosterone orally to females for 25 days in which the ovaries
were smaller than controls (Shaw et al. 1988). In one day 87 animal
both the contralateral and ipsilateral ovaries were abnormal.
Coincidentally, this was the only animal in which the mesonephros
was not transplanted with the testes. This animal was smaller on the
day of birth and we conclude that its immaturity made it more even
more susceptible to testicular secretions. This again is consistent with
the idea that there is a narrow, and early, window of sensitivity.
Similar findings were observed in previous transplantation studies
in our laboratory and in ovaries cultured in the presence of anti-
Müllerian hormone (Whitworth et al., 1996). Variable degrees of
dysgenesis also occurred in both ovaries of tammars treated orally
with exogenous, pharmacological doses of testosterone (Shaw et al.
436 M.B. Renfree et al. / Developmental Biology 334 (2009) 429–436
1988). In the present study, the ovarian abnormalities are likely due to
a greater concentration of androgen secretion by the transplanted
testes on the side closest to the transplant during an early, sensitive
period.
Thus grafting testes either from day 0 or day 8 pp males beneath
the skin of day 0–1 pp recipient female tammar wallabies virilizes the
indifferent Wolffian duct when examined at day 25, day 34 and day 87
after birth and induces prostatic development in the urogenital sinus
of these females. This is the first clear-cut evidence that testicular
androgens under physiological conditions do not need to be delivered
locally down the Wolffian duct but can act via the systemic circulation
during an early and narrow window of sensitivity that must be
between day 8 and day 10 post-partum in the tammar wallaby.
Acknowledgments
We thank Kerry Martin and Scott Brownlees for assistance with
the wallabies. This study was supported by the Australian National
Health Medical Research Council grant #350420 and the National
Institutes of Health grant #R21DK59942. MBR was supported by an
Australian Research Council Federation Fellowship.
References
Basden, K., Cooper, D.W., Deane, E.M., 1997. Development of the lymphoid tissues of the
tammar wallaby, Macropus eugenii. Reprod. Fertil. Dev. 9, 243–254.
Bentvelsen, F.M., Brinkmann, A.O., van der Schoot, P., van der Linden, J.E.T.M., van der
Kwast, T.H., Boersma, W.J.A., Schroder, F.H., Nijman, J.M., 1995. Developmental
pattern and regulation by androgens of androgen receptor expression in the
urogenital tract of the rat. Mol. Cell. Endocrinol. 113, 245–253.
Burns, R.K., 1961. In: Young, W.C. (Ed.), Sex and the Internal Secretions, 3rd ed. The
Williams and Wilkins Co., Baltimore, pp. 76–158.
Cooke, P.S., Young, P., Cunha, G.R., 1991. Androgen receptor expression in developing
male reproductive organs. Endocrinology 128, 2867–2873.
Donahoe, P.K., Crawford, J.D., Hendren, W.H., 1978. True hermaphroditism: a clinical
description and a proposed function for the long arm of the Y chromosome.
J. Pediatr. Surg. 13, 293–301.
George, F.W., Wilson, J.D., 1994. Sex determination and differentiation. In: Knobil, E.,
Neill, J.D., Greenwald, G.S., Markert, C.L., Pfaff, D.W. (Eds.), The Physiology of
Reproduction. Raven Press, New York, pp. 3–28.
Goldstein, J.L., Wilson, J.D., 1972. Studies on the pathogenesis of the pseudohermaph-
roditism in the mouse with testicular feminization. J. Clin. Invest. 51, 1647–1658.
Gunderson, H., Bagger, P., Bendtsen, T., Evans, S., Korbo, L., Marcussen, N., Moller, A.,
Nielsen, K., Nyengaard, J., Pakkenberg, F., Verteby, A., MJ, W., 1988. The new
stereological tools: dissector, fractionator, nucleator and point sampled intercepts
and their use in pathological research and diagnosis. Acta Pathol. Microbiol.
Immunol. Scand. 96, 857–881.
Jost, A., 1953a. Fetal intersexuality induced by methylandrostenediol in the rat; with
reference to a probably similar clinical observation. C R Seances Soc. Biol. Fil. 147,
1930–1933.
Jost, A., 1953b. Problems of fetal endocrinology: the gonadal and hypophyseal
hormones. Recent Prog. Horm. Res. 8, 379–418.
Jost, A., 1961. The role of fetal hormones in prenatal development. Harvey Lect. 53,
201–226.
Jost, A., 1970. Hormonal factors in the sex differentiation of the mammalian foetus.
Philos. Trans. R Soc. Lond., B Biol. Sci. 259, 119–130.
Leihy, M.W., Shaw, G., Renfree, M.B., Wilson, J.D., 2002. Administration of 5 α-
androstane-3 α,17 β-diol to female tammar wallaby pouch young causes
development of a mature prostate and male urethra. Endocrinology 143, 2643–2651.
Leihy, M.W., Shaw, G., Wilson, J.D., Renfree, M.B., 2004. Penile development is initiated
in the tammar wallaby pouch young during the period when 5α androstane-
3α,17β-diol is secreted by the testes. Endocrinology 145, 3346–3352.
Lucas, J.C., Renfree, M.B., Shaw, G., Butler, C.M., 1997. The influence of the anti-androgen
flutamide on early sexual differentiation of the marsupial male. J. Reprod. Fertil.
109, 205–212.
McKelvie, P., Jaubert, F., Nezelof, C., 1987. Is true hermaphroditism a primary germ cell
disorder? Pediatr. Pathol. 7, 31–41.
National Health and Medical Research Council, 2004. Australian Code of Practice for the
Care and Use of Animals for Scientific Purposes, 7th ed. Canberra A.C.T, Australian
Government.
Nurse, S.C., Renfree, M.B., 1994. Pubertal development of the pouch and teats in a
marsupial. J. Reprod. Fertil. 101, 279–285.
O, W.S., Short, R.V., Renfree, M.B., Shaw, G., 1988. Primary genetic control of somatic
sexual differentiation in a mammal. Nature 331, 716–717.
Pask, A., Whitworth, D.J., Mao, C.-A., Wei, K.-J., Sankovic, N., Graves, J.A., Shaw, G.,
Renfree, M.B., Behringer, R.R., 2004. Marsupial anti-müllerian hormone gene
structure, regulatory elements, and expression. Biol. Reprod. 70, 160–167.
Price, D., Pannabecker, R., 1956. Organ culture of foetal rat reproductive tracts. Ciba
Found. Colloq. Ageing 2, 3–13.
Renfree, M.B., 2002. Hypothermic anaesthesia of early postnatal marsupial pouch
young. In: Fisher, M., Marbrook, J., Sutherland, G. (Eds.), Learning, Animals and the
Environment: Changing the Face of the Future. Royal Society of New Zealand,
Wellington, pp. 125–127.
Renfree, M.B., Short, R.V., 1988. Sex determination in marsupials: evidence for a
marsupial–eutherian dichotomy. Philos. Trans. R. Soc. Lond., B. 322, 41–54.
Renfree, M.B., Harry, J.L., Shaw, G., 1995. The marsupial male: a role model for sexual
development. Philos. Trans. R. Soc. Lond., Ser. B-Biol. Sci. 350, 243–251.
Renfree, M.B., Wilson, J.D., Short, R.V., Shaw, G., George, F.W., 1992. Steroid hormone
content of the gonads of the tammar wallaby during sexual differentiation. Biol.
Reprod. 47, 644–647.
Renfree, M.B., O, W.S., Short, R.V., Shaw, G., 1996. Sexual differentiation of the
urogenital system of the fetal and neonatal tammar wallaby, Macropus eugenii.
Anat. Embryo. 194, 111–134.
Rudd, C.D., Short, R.V., McFarlane, J.R., Renfree, M.B., 1999. Sexual differentiation of
oestradiol-LH positive feedback in a marsupial. J. Reprod. Fertil. 115, 269–274.
Ryhorchuk, A.R., Shaw, G., Butler, C.M., Renfree, M.B., 1997. Effects of 5α-reductase
inhibitor, Finasteride, on the developing prostate and testis of a marsupial.
J. Androl. 18, 123–130.
Schultz, F.M., Wilson, J.D., 1974. Virilization of the Wolffian duct in the rat fetus by
various androgens. Endocrinology 94, 979–986.
Shaw, G., Renfree, M.B., Leihy, M.W., Shackleton, C.H., Roitman, E., Wilson, J.D., 2000.
Prostate formation in a marsupial is mediated by the testicular androgen 5α-
androstane-3α,17 β-diol. Proc. Natl. Acad. Sci. U. S. A. 97, 12256–12259.
Shaw, G., Renfree, M.B., Short, R.V., O, W.S., 1988. Experimental manipulation of
sexual differentiation in wallaby pouch young treated with exogenous steroids.
Development 104, 689–701.
Shaw, G., Fenelon, J., Sichlau, M., Auchus, R.J., Wilson, J.D., Renfree, M.B., 2006. Role of
the alternate pathway of dihydrotestosterone formation in virilization of the
Wolffian ducts of the tammar wallaby, Macropus eugenii. Endocrinology 147,
2368–2373.
Shaw, G., Renfree, M.B., Short, R.V., 1990. Primary genetic control of sexual
differentiation in marsupials. Aust. J. Zool. 37, 443–450.
Tong, S.Y., Hutson, J.M., Watts, L.M., 1996. Does testosterone diffuse down the Wolffian
duct during sexual differentiation? J. Urol. 155, 2057–2059.
Tyndale-Biscoe, C.H., Hinds, L.A., 1989. Influence of the immature testis on sexual-
differentiation in the tammar wallaby, Macropus eugenii (Macropodidae,
Marsupialia). Reprod. Fertil. Dev. 1, 243–254.
Tyndale-Biscoe, C.H., Renfree, M.B., 1987. Reproductive Physiology of Marsupials.
Cambridge University Press.
Veyssiere, G., Berger, M., Jean-Faucher, C., de Turckheim, M., Jean, C., 1982. Testosterone
and dihydrotestosterone in sexual ducts and genital tubercle of rabbit fetuses
during sexual organogenesis: effects of fetal decapitation. J. Steroid Biochem. 17,
149–154.
Welsh, M., Saunders, P.T.K., Marchetti, N.I., Sharpe, R.M., 2006. Androgen-dependent
mechanisms of Wolffian duct development and their perturbation by flutamide.
Endocrinology 147, 4820–4830.
Welsh, M., Saunders, P.T.K., Sharpe, R.M., 2007. The critical time window for androgen-
dependent development of the Wolffian duct in the rat. Endocringology 148,
3185–3195.
Whitworth, D.J., Shaw, G., Renfree, M.B., 1996. Gonadal sex reversal of the developing
marsupial ovary in vivo and in vitro. Development 122, 4057–4063.
Wilson, J.D., 1973. Testosterone uptake by the urogenital tract of the rabbit embryo.
Endocrinology 92, 1192–1199.
Wilson, J.D., George, F.W., Renfree, M.B., 1995. The endocrine role in mammalian sexual
differentiation. Rec. Prog. Hormone Res. 50, 349–364.
Wilson, J.D., Auchus, R.J., Leihy, M.L., Guryev, O.L., Estabrook, R.W., Osborn, S.M., Shaw,
G., Renfree, M.B., 2003. 5α-Androstane-3α,17β-diol is formed in the tammar
wallaby pouch young testes by a pathway involving 5α-pregnane-3α, 17α-diol-
20-one as a key intermediate. Endocrinology 144, 575–580.
Wilson, J.D., Shaw, G., Leihy, M.L., Renfree, M.B., 2002. The marsupial model for male
phenotypic development. Trends Endocrinol. Metab. 13, 78–83.