Sex Dev 2014;8:273–280
DOI: 10.1159/000363432
Wolffian Duct Development
Geoffrey Shaw Marilyn B. Renfree
Department of Zoology, The University of Melbourne, Melbourne, Vic., Australia
Key Words
Androgens · Epididymis · Growth factors · Marsupial ·
Müllerian ducts · Sexual differentiation · Virilisation ·
Wolffian ducts
Abstract
The Wolffian ducts (WDs) are the progenitors of the epidid-
ymis, vas deferens and seminal vesicles. They form initially
as nephric ducts that acquire connection to the developing
testis as the mesonephros regresses. The development of
the WDs is dependent on androgens. Conventionally, the
active androgen is believed to be testosterone delivered lo-
cally rather than via the systemic circulation. However, re-
cent studies in marsupials show that 5α-reduced steroids
are essential and that these can induce virilisation even
when they are delivered via the systemic circulation. The
development of the WDs involves an interplay between the
duct epithelium and underlying mesenchyme; androgen re-
ceptors in both the epithelium and mesenchyme are need-
ed. The epidermal growth factor and epidermal growth fac-
tor receptor may play a role, possibly via activation of an-
drogen receptor. The formation of the epididymis involves
a complex morphogenetic program to achieve the normal
pattern of coiling, formation of septae, and regional func-
tional differentiation. In part, this process may be mediated
by inhibin beta A as well as by genes from the HOX cluster.
Whilst the development of the WD is androgen dependent,
© 2014 S. Karger AG, Basel
1661–5425/14/0085–0273$39.50/0
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Published online: June 17, 2014
it is clear that there is a complex interplay between andro-
gens, genes and growth factors in the tissues that leads to
the formation of the complex anatomy of the male repro-
ductive duct system in the adult. © 2014 S. Karger AG, Basel
Development of the Wolffian Duct
In mammals, genetic males and females initially have
bipotential gonads and paired Müllerian ducts and Wolff-
ian ducts (WDs), the precursors, respectively, of the fe-
male and male internal reproductive systems. The classic
model for sexual differentiation involves active virilisa-
tion in males, through the action of testicular hormones,
whilst absence of these testicular hormones allows femi-
nisation of these structures by endogenous pathways
[Jost, 1970]. However, there is a growing recognition that
some somatic sexual dimorphisms arise independent of
gonadal hormones and even the testis-determining gene
on the Y chromosome [see Renfree et al., this issue]. The
formation of the Müllerian duct and regulation of its dif-
ferentiation in females and anti-Müllerian hormone-me-
diated regression in males is the subject of another paper
in this issue [see Ipulan and Behringer, this issue]. This
review will focus on differentiation of the WDs and the
mechanisms by which they are virilised in males by an-
drogens.
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Geoffrey Shaw
Department of Zoology
The University of Melbourne
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Melbourne, VIC 3010 (Australia)
E-Mail g.shaw @ zoology.unimelb.edu.au
 The WDs (pronephric and mesonephric ducts) are
named after the German anatomist Caspar Friedrich
Wolff [1774], who first described the mesonephros and
its ducts. This duct plays a fundamental role in fetus, in-
ducing formation of the 3 generations of kidney and act-
ing as the excretory duct of the pro- and mesonephros.
The gonad forms medio-lateral to the mesonephros,
which regresses once the metanephros is formed and
functioning. As this regression occurs, mesonephric tu-
bules differentiate to form efferent ducts connecting the
testis to the WD, and thus, as the duct loses its excretory
function, it acquires a reproductive function. This review
will briefly address the embryology of the duct before
coming to focus on its role as the male reproductive duct
and the mechanisms by which it is virilised.
Embryonic Origins of the WD
The origins of the reproductive tract are intimately in-
terwoven with the development of the embryonic kidneys
and associated ducts. The urogenital ridge arises in the
early embryo as a pair of ridges of intermediate meso-
derm lateral to somites running down the posterior body
wall. The WD arises in the anterior intermediate meso-
derm (the presumptive pronephric region) and elongates
caudally as a cord of cells that eventually undergo mesen-
chymal-epithelial transition (fig. 1A). The migrating epi-
thelial cells of the developing WD actively extend and
withdraw filopodia and lamellipodia, and have cell tails
that extend and retract under control of a RHO/actin-
based mechanism that is essential for normal duct forma-
tion [Atsuta et al., 2013]. The WD sequentially induces
the formation of the pronephros, mesonephros and meta-
nephros. The pronephros forms cranially in the ridge and
is the primary blood filtration and osmoregulatory organ
in fish and amphibian larvae. In mammals, it is transitory
and is never functional as an excretory organ. The WD
induces formation of the mesonephros in the central re-
gion of the nephrogenic mesoderm. This functions as the
kidney in amphibians and embryonic kidney in mam-
mals, though in marsupials, it remains functional for a
period after birth in the altricial young [Wilkes and Jans-
sens, 1986]. Interestingly, the developing elephant has
nephrostomes, openings that directly connect the coelo-
mic cavity and the lumen of the primitive nephric tubule,
in its developing mesonephros [Gaeth et al., 1999]. These
are a characteristic of aquatic vertebrates and may reflect
an ancestral aquatic stage in the elephant lineage [Gaeth
et al., 1999].
274 Sex Dev 2014;8:273–280
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A B C
Fig. 1. Early development of the WD. A The WD arises in the an-
terior intermediate mesoderm (IM) and grows caudally as a cord
of cells before forming a tubule via mesenchymal-epithelial transi-
tion dependent on the Pax2, Pax8 and Lim1 genes. Inductive sig-
nals from the developing WD induce formation of the pronephros
(Pro) and mesonephros (Meso). B The caudal end of the WD con-
nects to the urogenital sinus (UGS), and the WD takes on an excre-
tory role for the mesonephros. The development of the WD re-
quires Emx2 and retinoic acid signalling via retinoic acid receptor
(RAR). Mesoderm adjacent to the caudal WD induces the forma-
tion of the ureteric bud, a process requiring GDNF/RET signalling.
Inductive signals from the ureteric bud initiate the development of
the metanephros (Meta) which migrates cranially. C As develop-
ment proceeds the mesonephros regresses, but the cranial meso-
nephric ducts form into the rete testis/efferent ducts connecting to
the developing testis. Hox genes are differentially expressed cra-
nial to caudal, contributing to regional specification. Androgens
from the testis act on ARs to initiate coiling morphogenesis of the
epididymal region.
The gonads form as epithelial thickenings on the ven-
tromedial surface of the mesonephros (fig.  1B). As the
mesonephros regresses in mammals, remnant meso-
nephric tubules form the rudiments of the rete testis, con-
necting the seminiferous tubules to the mesonephric or
WD (fig. 1C). The definitive adult mammalian kidney is
the metanephros. This originates with dynamic interac-
tions between the caudal WD and the adjacent mesen-
chyme that induces formation of an epithelial bud from
the caudal end of the WD [Renfree et al., 1996], a process
involving GDNF/RET signalling [reviewed in Saxén and
Sariola, 1987; Airik and Kispert, 2007; Menshykau and
Iber, 2013] (fig. 1B, C). The ureteric bud extends crani-
ally and initiates branching morphogenesis to form the
metanephros [Saxén and Sariola, 1987; Airik and Kispert,
2007; Menshykau and Iber, 2013]. The caudal WD also
gives rise, in some species, to the seminal vesicles.
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 The genetic mechanisms underlying initial formation
of the WD have been clarified through investigation of
mutant mice, and the pathways appear similar in both
males and females (fig. 1). Pax2, Pax8 and Lim1 are all
expressed in the developing WD and appear to cooperate
in WD development, probably playing a key role in mes-
enchymal-epithelial transformation which is essential for
formation of the duct and mesonephric tubules. Emx2 is
also needed for WD development, since in Emx2 null
mice the duct initially forms normally but is degenerating
in both males and females by E11.5 [Miyamoto et al.,
1997]. Retinoic acid signalling also appears to be impor-
tant, since double knockout of RARα and RARγ led to
agenesis of the epididymis, vas deferens and seminal ves-
icles [Mendelsohn et al., 1994].
Hormonal Regulation of WD Differentiation
The importance of androgens for WD development
has been well established. Jost’s early experiments dem-
onstrated that castration of fetal male rabbits before the
age when WD regression occurred in females led to a fe-
male ductal phenotype with a regressed WD and the de-
velopment of the Müllerian duct [Jost, 1946, 1970]. Plac-
ing a crystal of testosterone beside the ovary of a fetal
rabbit caused retention of the WD [Jost, 1970]. Testicular
androgens drive differentiation of the seminal vesicles
from the caudal region and the formation of the epididy-
mis from the cranial section. In mice with androgen re-
ceptor knock out (ARKO), WD derivatives, epididymi-
des, vas deferens, and seminal vesicles are absent [Yeh et
al., 2002]. In many humans with complete androgen in-
sensitivity syndrome, WD derivatives are absent, al-
though in many cases some Wolffian derivatives may re-
main, possibly due to some small residual activity of the
androgen receptor (AR) [Hannema et al., 2004]. In fe-
males, remnants of the caudal section of the WD may also
persist in the urogenital sinus tissues lateral to the vaginal
wall as the Gartner’s duct or cyst, and remnants of the
cranial part of the duct may persist in females as the ep-
oophoron and Skene’s glands.
Morphological development of the WD occurs in 2
androgen-dependent phases. The initial phase encom-
passes regression of the duct in females and androgen-
mediated stabilisation and retention of the ducts in males.
This occurs from about E13.5–E16 in mice and rats, by
which stage little remains of the WD in females [Staack et
al., 2003; Welsh et al., 2009]. Following this is a phase of
differentiation in males which includes tubular elonga-
Wolffian Duct Development
tion and marked convolution of the cranial end accom-
panied by cytological and morphological differentiation
of the epididymis and vas deferens [Welsh et al., 2006]. At
the caudal end, the seminal vesicle starts to form. Intrigu-
ingly, there are critical time windows for the hormonal
priming that regulates WD development as well as other
aspects of virilisation [Welsh et al., 2009, 2010; Macleod
et al., 2010]. Blocking androgen action with flutamide af-
ter this critical time window, but before morphological
differentiation, does not prevent formation of a vas and
epididymis [Welsh et al., 2006, 2009; Macleod et al., 2010].
A similar pattern seems to apply also in tammars with re-
spect to WD development [Renfree et al., 2009] (fig. 2).
Neonatal females that received testis transplants had
well-developed WDs at autopsy [Renfree et al., 2009],
whilst when transplants were made at day 10, subsequent
WD development was poor [Tyndale-Biscoe and Hinds,
1989]. Similar time windows where androgen stimulation
is needed to program later morphological differentiation
are also seen in phallus development in wallabies [Leihy
et al., 2004] and in rats [Welsh et al., 2010]. Despite the
importance of androgen exposure in this early time win-
dow, full development and differentiation of the WD and
phallus is also dependent on continuing androgen stimu-
lation.
Two mechanisms have been investigated to define
how the testicular androgen reaches the WD. Jost’s
[1953a, b] early experiments indicated that the effect of
the testis on the WD was unilateral and presumably due
to diffusion from the testis graft or crystal of testosterone
preferentially to the side of the implant. In a single female
rabbit fetus that had a testis grafted adjacent to the ovary
retained the ipsilateral WD, whilst the duct on the con-
tralateral side underwent the regression normally seen in
the female. A number of ‘experiments of nature’ parallel
to Jost’s [1953a, b] observations. For example, in human
cases of disorders of sex development involving unilat-
eral gonadal dysgenesis, the WD associated with the unaf-
fected testis usually develops, whilst on the side of the
dysgenetic gonad, the gonad is commonly retained in the
abdomen and the WD is absent. These data suggest that
androgens from the testis are locally transferred to the
ipsilateral WD. Tong et al. [1996] investigated this ex-
perimentally. They microinjected testosterone-albumin-
FITC into the anterolateral side of testes of mouse 14-day
urogenital ridges in culture. After 17 h, fluorescence was
found all along the WD, and by 24 h, fluorescence was
concentrated at the dilated caudal end. These data are
consistent with the local transfer of androgens from the
developing testis into the adjacent WD.
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 A B
C D
Fig. 2. Experimental manipulation of the WDs in tammars. At day
34 pp in males (A) the WD is developing and the Müllerian duct
(MD) is regressed, whilst in females (B), the MD is developing, and
only a trace of the WD remains (arrow). In males treated with an
inhibitor of 5α-reductase (C), the WD is regressing, whilst in fe-
males treated with androstanediol (D), the WD has not regressed.
An alternative approach to test this hypothesis was
pursued [Renfree et al., 2009] using a marsupial model. As
in eutherians, there are ARs in the WD of the tammar wal-
laby [Butler et al., 1998], and WD development is andro-
gen dependent [Shaw et al., 1988; Ryhorchuk et al., 1997].
However, in marsupials, gonadal differentiation occurs
after birth when the young are accessible for experimental
manipulation [Wilson et al., 2003; Renfree et al., 2006]. In
females, regression of the WD takes place between day 10
and 25 pp [Renfree et al., 1996]. Since the immune system
does not mature until about day 90–120 pp [Basden et al.,
1997], it is possible to xenotransplant early developing go-
nads between animals [Whitworth et al., 1997; Renfree et
al., 2009]. Testes transplanted from neonatal or day 8–9
pp male young to below the flank abdominal skin of a neo-
natal female young developed and secreted physiological
amounts of androgen as assessed by the formation of pros-
tatic tissue in the urogenital sinus (fig. 2). At day 34 pp,
WDs were retained in these females, with a columnar ep-
ithelium and a duct diameter similar to that in control
males, whilst WDs were regressed in the control females.
By day 87 pp, WDs in the females with testis grafts showed
coiling as an epididymis started to form. The development
of the WD in the females with testis grafts was similar on
both sides. Since there was no local association between
the subcutaneous testis grafts and the WDs of the graft
276 Sex Dev 2014;8:273–280
DOI: 10.1159/000363432
E
F
In females that received a testis graft under the skin as neonates
(E), the graft (arrow) has developed, and androgens from it have
virilised the WD (F), which shows characteristic epididymal coils
adjacent to the ovary (Ov). Data from Shaw et al. [2006] and Ren-
free et al. [2009].
recipients, it is clear that testicular androgen delivered via
the systemic circulation was sufficient to prevent WD re-
gression and, indeed, to initiate formation of epididymal
coiling [Renfree et al., 2009]. Thus, at least in the tammar
wallaby, local transfer of androgens from the testis is not
needed for WD development.
The question of which androgen is responsible for vir-
ilisation of the WD has also been considered. In many
androgen target tissues, testosterone is 5α-reduced to
5α-dihydrotestosterone (DHT), which acts as the active
androgen locally. However, there is evidence that testos-
terone may act directly to virilise the WD. Strong evi-
dence for this comes from genetic males with steroid
5α-reductase-2 deficiency, where, although the external
genitalia and urethra are feminised, the WD still virilises
[Wilson et al., 1993]. In addition, in several species, the
WD lacks 5α-reductase during the initial phases of sexual
differentiation, when the duct is stabilised and commenc-
es differentiation. However, in tammar wallabies, there is
evidence that WD differentiation is not mediated by tes-
tosterone. In tammars, the main androgen secreted by the
testis during the early phase of sexual differentiation is
5α-androstane-3α,17β-diol (adiol) rather than testoster-
one or DHT [Shaw et al., 2000; Renfree et al., 2006]. The
tammar WD can readily metabolise the hormonally inac-
tive adiol into DHT [Shaw et al., 2006]. Administering a
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5α-reductase inhibitor, which would suppress testicular
production of adiol and DHT, but not testosterone,
blocked virilisation of the WD in males, showing that, in
tammars, the androgen responsible for WD virilisation is
not testosterone [Shaw et al., 2006]. In addition, treat-
ment of females with adiol, in doses previously shown to
induce prostate development, prevented regression of the
WDs [Shaw et al., 2006].
Androgen Receptors and WD Virilisation
In both male and female mice, ARs are present at E12.5
(the time of onset of testicular androgen production in
males) in the mesenchyme surrounding the cranial WD
and are extended caudally during development from
E14.5 to E18.5 [Cooke et al., 1991; Murashima et al.,
2011]. At E14.5, when the WD is being stabilised in males
but is regressing in females, there are low levels of AR
present in the cranial WD epithelium in both sexes. In
males, the expression of AR increases in both mesen-
chyme and epithelium between E16.5 and E18.5 [Mu-
rashima et al., 2011]. In mice with ARKO, the WD of
males regresses in a manner similar to that in females,
with increased apoptosis of epithelial cells, but no change
in proliferation. In contrast, in male mice with selective
ARKOs in the WD epithelia, the WD was retained, coiled
epididymides were present at E18.5, and normal epididy-
mal morphology was seen in adulthood [Murashima et
al., 2011]. Thus, mesenchymal AR is needed for stabilisa-
tion of the WD and the subsequent elongation and coil-
ing. However, these epithelium-specific ARKO males had
altered expression of functional markers that indicate
that epithelial AR is needed for correct differentiation of
the caput and cauda principal and caput basal cells [Mu-
rashima et al., 2011]. Mice with selective ablation of AR
in the caput epididymis lack an initial segment, have al-
tered epididymal morphology and function, and are in-
fertile [O’Hara et al., 2011].
Whilst these data in mice indicate an anti-apoptotic
effect of androgens, fetal rats treated with the AR blocker
flutamide from E15.5 had reduced cell proliferation in the
WD, but no change in apoptosis [Welsh et al., 2006]. The
failure of flutamide to induce an anti-apoptotic effect may
reflect a low level of residual androgen action in the rats
that is sufficient to prevent the increased apoptosis seen
in ARKO mice. However, the contrast between the reduc-
tion of cell proliferation in flutamide treated rats and the
absence of change in proliferation seen in ARKO mice
[Murashima et al., 2011] remains to be clarified.
Wolffian Duct Development
These data suggest that AR has 2 separate roles in WD
development. Mesenchymal ARs are needed for WD sta-
bilisation, growth and coiling, whilst epithelial ARs are
needed for later development and adult function. How-
ever, the androgen effects depend on interplay between
the mesenchyme and epithelium. Tissue recombination
experiments have clearly indicated that the regionally
specific development of the epithelium is driven by the
underlying mesenchyme. For example, epithelium from
the anterior WD, that will normally form the epididymis,
will adopt a seminal vesicle-like phenotype if placed on
caudal WD mesenchyme [Higgins et al., 1989]. However,
the dialogue is 2-way. Mesenchyme cultured in the ab-
sence of epithelium does not develop normally [Higgins
et al., 1989; Cunha et al., 1991]. Some of this crosstalk may
be mediated by the epidermal growth factor (EGF). Ex-
posure of developing male urogenital systems to antibod-
ies to EGF in culture led to disintegration of the WD in a
dose-dependent manner, even in the presence of exoge-
nous testosterone [Gupta et al., 1991]. Both EGF and the
epidermal growth factor receptor (EGFR) are expressed
in the developing male reproductive tract in a pattern that
correlated with the progression of WD differentiation
[Gupta et al., 1991, 1996; Gupta and Singh, 1996]. EGF is
initially found in both mesenchyme and epithelium, but
by E18 it is principally epithelial [Gupta, 1997], and EGF
can stabilise the developing female WD in vitro [Gupta et
al., 1996]. The action of EGF is not direct, but appears to
involve ARs. At high doses, EGF may activate the AR-
mediated transcription in the absence of androgen, but at
physiological doses, EGF acts to amplify androgen-medi-
ated transcriptional changes via the AR [Gupta, 1999].
This effect may be brought about by activation of MAPK
signalling pathways that might phosphorylate the AR in
ways that enhance gene activation [Gupta, 1999]. Thus,
there is interplay between androgen stimulating EGF,
which, in turn, potentiates androgen’s actions. Hannema
et al. [2004] report that, whilst complete androgen insen-
sitivity syndrome patients with mutations that prevented
AR expression, or mutations in the DNA-binding do-
main that prevented transcriptional activation, always
lacked WD development; 70% of complete androgen in-
sensitivity syndrome patients with substitution muta-
tions in the ligand-binding domain had epididymides
and vas deferens. They hypothesised that there was some
residual AR activity that was activated by locally high tes-
tosterone concentrations in the duct, but it is interesting
to speculate that non-ligand-mediated activation of the
AR via EGF/EGFR may contribute to the development of
epididymis and vas deferens in these patients.
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 Regional Specification
Regional specification of the developing WD into epi-
didymis, vas deferens and seminal vesicle depends on sev-
eral Hox genes [Hsieh-Li et al., 1995; Warot et al., 1997;
Podlasek et al., 1999; Bomgardner et al., 2003]. Hox genes
encode transcription factors, regulate anterior-posterior
patterning, and regulate the regionalisation or patterning
of multiple embryonic tube systems. In male mice, Hoxa9
and Hoxd9 are expressed in the epididymis and vas def-
erens. Hoxa10 and Hoxd10 are expressed in the caudal
epididymis and vas deferens. Hoxa11 is expressed only in
the vas deferens, whilst Hoxa13 and Hoxd13 are expressed
only in the caudal WD [Snyder et al., 2010]. A recent
study implicated miRNA in the final regional maturation
of the epididymis. Conditional knockout of the RNase
III enzyme Dicer1, which is needed for production of
miRNAs from longer precursors, in the proximal 2 seg-
ments of the epididymis from day 12 pp resulted in down-
regulation of epididymis-specific gene markers and pro-
gressive dedifferentiation of the epididymal epithelium
[Björkgren et al., 2012].
Forming the Epididymis
A key event in WD development is the formation of
the epididymis. In mice, the cranial WD expands from
approximately 1 mm in the fetus to around 1 m in the
adult. Clearly, its formation requires both considerable
elongation, and also intricate coiling and packaging,
since the epididymal tube is about 100 times longer than
the body. Elongation of the WD appears to be a result of
cell proliferation and cell rearrangements [reviewed in
Hinton et al., 2011]. This process is clearly mediated by
androgens, since it fails in the absence of AR, or andro-
gen signalling, but the downstream events and molecular
and cellular interactions responsible are still unclear.
Only the cranial part of the WD undergoes this coiling.
The regional variation in coiling is not due to proximity
to the testis. WDs cultured without the testis underwent
dose-dependent androgen-stimulated cell proliferation
(assessed by DNA content) and coiling (assessed by the
number of epididymal curvatures at the end of culture)
[Tsuji et al., 1991]. Rather this appears to be a conse-
quence of regional variation in Hox gene expression. Hox
genes are regionally expressed, and in Hoxa10/Hoxa11
double knockout mice, the developing vas deferens un-
derwent coiling [Branford et al., 2000; Snyder et al.,
2010].
278 Sex Dev 2014;8:273–280
DOI: 10.1159/000363432
The coiling of the elongating epididymal section of the
WD appears to be remarkably well controlled, with a con-
sistent anatomical pattern evolving with time both in vivo
and in explanted urogenital systems in vitro. As with
many aspects of urogenital development, there is a pro-
gressive cranial to caudal progression in the onset of coil-
ing. The pattern of coiling is not generated by differential
rates of cell proliferation along the duct, as the rate of cell
division appears to be uniform along the whole length
[Hinton et al., 2011]. Possibly proliferation and coiling,
whilst occurring at the same time, are under independent
control [Hinton et al., 2011]. As the developing epididy-
mal duct elongates, it is constrained by a fibrous capsule,
so to some extent the coiling may be a consequence of this
constraint. However, the patterning of the coiling and the
formation of septae between sections of coil that later
show segmental gene expression patterns suggests there
is more to the pattern of coiling than simple physical con-
straint.
One factor that has been identified as a regional
paracrine factor necessary for epididymal coiling is in-
hibin beta A [Tomaszewski et al., 2007]. In mice, in-
hibin beta A is present in anterior WD mesenchyme
and mesonephros in both males and females before
E12.5, but later its expression becomes androgen de-
pendent. In male mice with knockout of inhibin beta A,
androgen levels appear to be normal, since several an-
drogen-dependent structures virilised normally. How-
ever, in these mice, the WD remained as a straight duct
in the mesonephros at E17.5 and E19.5, resembling the
duct in control males at E15.5 before coiling normally
starts. These mutant males also had reduced cell prolif-
eration in the WD, but there was no effect on apoptosis.
How inhibin beta A is controlling coiling is unclear, but
the effect may be mediated, at least in part, by upregula-
tion of AR in the epididymal epithelium [Tomaszewski
et al., 2007].
Conclusions
Virilisation of the WDs to form the epididymis, vas
deferens and seminal vesicles is a complex process that
involves complex epithelial-mesenchymal interactions
under the action of androgens. AR activity is modulated
by EGF/EGFR as well as androgens. Development of a
highly coiled epididymis is an astonishing feat of devel-
opmental programming regulated by a number of factors,
including members of the inhibin family, that leads to a
consistent pattern of coiling and regional differentiation
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that is essential for its later function. Whilst the conven-
tional model is that the WD is virilised by testosterone
which is delivered locally from the testis down the duct,
this may not be the only mechanism. Studies in marsupi-
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