REVIEWS

The makings of maleness:

towards an integrated view of
male sexual development
Dagmar Wilhelm* and Peter Koopman*
Abstract | As the mammalian embryo develops, it must engage one of the two distinct
programmes of gene activity, morphogenesis and organogenesis that characterize males
and females. In males, sexual development hinges on testis determination and
differentiation, but also involves many coordinated transcriptional, signalling and endocrine
networks that underpin the masculinization of other organs and tissues, including the brain.
Here we bring together current knowledge about these networks, identify gaps in the overall
picture, and highlight the known defects that lead to disorders of male sexual development.

Hypospadias
Incorrect placement of the
urethral opening in males, not
at the tip of the penis.
*Division of Molecular
Genetics and Development,
Institute for Molecular
Bioscience, The University
of Queensland, Brisbane,
QLD 4072, Australia.
The differences between males and females are a source
of enduring fascination. This is hardly surprising, given
the mysteries surrounding these differences and their
profound effect on our daily lives. For the developmen-
tal biologist, the morphological differences between the
sexes are particularly intriguing given that they arise
through dichotomous differentiation of a common set
of precursor tissues, a unique situation in embryonic
development. For any given species, there are not one
but two developmental biologies.
In simple terms, the development of mammalian
maleness or femaleness hinges on whether testes or
ovaries form in the embryo from the paired, ambipo-
tent structures known as genital ridges1. This decision
depends on the presence and correct function of a male-
determining gene from the Y chromosome, Sry, which
functions in a specific subset of genital ridge cells to
stimulate them to differentiate as Sertoli cells the cells
that interact with and nurture the germ cells. The Sertoli
cells then seem to orchestrate the differentiation of other
cell types required for testis formation, such as the germ
cells and steroid-producing cells. If Sry is absent or does
not function correctly, other regulatory cascades lead to
ovary development and female characteristics.
secondary effects such as infertility and gonadal tumours.
Sexual dysgenesis is often traumatic, stigmatized and
under-recognized as a medical issue in our society,
and identifying responsible genes will be increasingly
important for diagnosing these disorders, counselling
affected patients, and making a prognosis that will inform
gender-assignment options. However, genes responsible
for proper sexual development are often difficult to find
by conventional genetic studies, which require fertility.
The discovery of Sry in 1990 (REFS 2,3) opened the
way for genetic dissection of the cascade of events lead-
ing to male development. There is a growing molecular
and cellular understanding of testis determination and
the early events in testis differentiation, mainly from
analyses of mouse mutants and genotypephenotype
correlations in humans who are affected by disorders
of sexual development. In addition, technologies such
as microarray analysis have identified many genes that
show sex-specific expression during gonad development
and might be important in testis development.
However, key questions still remain. How does SRY
induce Sertoli cell differentiation? How do Sertoli cells
orchestrate the differentiation of the other testicular cell
lineages, including the germ line? What factors, gonadal

ARC Centre of Excellence in

Biotechnology and
Development, Institute for
Molecular Bioscience,
The University of Queensland.
Correspondence to P.K.
e-mail:
p.koopman@imb.uq.edu.au
doi:10.1038/nrg1903
Published online 11 July 2006
In reality, sexual development is vastly more complex,
hinging on delicate networks of molecular signals that
specify sex-specific differentiation, organogenesis and
endocrine function. The fragility of these networks is clear:
disorders of sexual development are among the most com-
mon human birth defects. They range in frequency and also
in severity, from hypospadias to complete sexual ambiguity
and sex reversal (TABLE 1), and are often associated with
or non-gonadal, are necessary to induce secondary sexual
characteristics such as testicular descent and differentia-
tion of the external genitalia? What are the phenotypic
consequences of mutations in genes that are important
in these processes? We focus on these questions in an
effort to summarize the knowns and unknowns of male
sexual development, from testis specification, to the
development of the genital tract, male accessory sex

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Table 1 | Disorders of human sexual development
Syndrome
Hypospadias
Cryptorchidism
XY true hermaphroditism
XY sex reversal
Swyer syndrome
DenysDrash syndrome
Frasier syndrome
WAGR syndrome
Adrenal hypoplasia congenita
Campomelic dysplasia
Persistent Mllerian duct
syndrome
Congenital bilateral aplasia of
vas deferens
X-linked lissencephaly with
abnormal genitalia syndrome
-Thalassaemia/mental
retardation syndrome, X-linked
Congenital adrenal hyperplasia
Androgen insensitivity syndrome Varying from nearly normal male to nearly normal female
Hand-foot-genital syndrome
This table summarizes the sexual disorders mentioned in the text. For references and further reading see REF. 103. AMH, anti-Mllerian hormone; AMHR, anti-
Mllerian hormone receptor; AR, androgen receptor; ARX, aristaless-related homeobox; ATRX, -thalassaemia/mental retardation syndrome, X-linked; CFTR, cystic
fibrosis transmembrane conductance regulator; HOXA13, homeobox A13; INSL3, insulin-like 3; LGR8, the INSL3 receptor; SOX9, SRY-box containing gene 9; WT1,
Wilms tumour 1.
Eutherian mammals
Mammals that have a placenta;
includes all mammals except
monotremes and marsupials.
NATURE REVIEWS | GENETICS
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Phenotype Genes or other alterations
known to be involved
Incorrect placement of the urethral opening in males HOXA13, HOXD13
Failure of testicular descent INSL3, LGR8
Ovotestes, ambiguous genitalia Trisomy of chromosome 22
Ovaries, female genitalia and secondary characteristics SRY, SOX9
XY females with complete gonadal dysgenesis SRY
Pseudohermaphroditism, nephropathy, predisposition to Wilms tumour WT1
Pseudohermaphroditism, progressive glomerulopathy WT1
Wilms tumour, aniridia, genito-urinary malformations, mental retardation WT1
Adrenal hypoplasia, hypogonadotropic hypogonadism DAX1
Skeletal dysmorphology and XY sex reversal SOX9
Normally virilized men with a uterus and fallopian tubes, often with AMH, AMHR
unilateral inguinal hernia or cryptorchidism
Absence of vasa deferentia, infertility CFTR
Microcephaly, lissencephaly, agenesis of the corpus callosum, epilepsy, ARX
poor temperature regulation, chronic diarrhoea, ambiguous genitalia
Severe mental retardation, -thalassaemia, genital abnormalities, facial ATRX
anomalies, lung, kidney and digestive problems
Androgen excess produced by adrenal glands, XX with varying degrees of Enzymes that are necessary
virilization for hormone production
(21-hydroxylase in 95% of patients)
AR
Short first metacarpals and metatarsals, carpal and tarsal fusion, fifth-finger HOXA13, HOXD13
clinodactyly, genital abnormalities in both sexes, hypospadias in the male
organs and external genitalia, to male-specific brain Mutational analyses in mice have shown that several
features (FIG. 1). We aim to move beyond studies of testis transcription-factor genes are required for the early for-
development in order to build up an integrated picture of mation of the indifferent genital ridges, including empty
the breadth of issues in male sexual development. spiracles homologue 2 (Emx2) (REF. 4), GATA-binding
protein 4 (Gata4) (REF. 5), Lim homeobox protein 9
Development of the testes (Lhx9) (REF. 6), steroidogenic factor 1 (Sf1; also known
In eutherian mammals, the first morphological sign of as NR5A1) (REFS 7,8), and Wilms tumour 1 (Wt1) (REF. 4).
male differentiation is the formation of cords in nas- Two of these, WT1 and SF1, are also crucial for the for-
cent testes. Before this, both male and female embryos mation of the genital ridges in humans. Mutations in
develop paired, ambipotent genital ridges that are these genes result in malformed gonads and ambiguous
indistinguishable between sexes. genitalia9,10. Intriguingly, Wt1 and Sf1 have additional,
sex-specific roles later in gonadal development.
The indifferent gonad. The genital ridges form as linear One enigmatic gene, Dax1 (also known as Nr0b1),
swellings on the ventromedial surfaces of the mesone- is expressed in the indifferent gonad of both sexes, and
phroi. Each mesonephros is derived from intermediate gain-of-function and loss-of-function mutations in mice
mesoderm in the aorta-gonad-mesonephros region of affect both testis and ovary determination. It may be that
the trunk, flanking the dorsal aorta and bounded ven- Dax1 levels and thresholds are extremely critical for its
trally by epithelium at its coelomic surface (FIG. 1a). In male versus female function11.
both sexes, mesonephric mesenchyme and thickening
coelomic epithelium contribute to the evagination of the Sry the male determinant. The bifurcation in the
genital ridge from the mesonephros as development pro- development of testes and ovaries is triggered by the
ceeds. In mice, the genital ridges start to appear around expression of Sry. Loss-of-function and gain-of-function
10 days post coitum (dpc), and remain morphologically mutations in mice and humans indicate that this gene
undifferentiated until about 12 dpc, despite different is necessary and sufficient for testis development in
programmes of genetic activity in males and females mammals1214. Its expression is tightly regulated in mice,
beginning around 10.5 dpc (see below). occurring in a wave that starts in the centre of the genital
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a Development of external genitalia b Formation of the aorta-genital-ridge-

mesonephros region
Allantois
Cloacal Hindgut Mesonephros
membrane
Cloaca Urorectal Genital
septum ridge Aorta
10.5 Wolffian duct
dpc 11.5 Coelomic
epithelium Mllerian duct
onwards dpc

Urogenital
Genital sinus c Testis and genital tract differentiation
tubercule
1317.5 Seminal vesicle
dpc Prostatic bud

Testis
Vas Testicular cords
Urethra deferens
Efferent ducts
Distal urethral Rete testes
epithelium
Penile urethra Epididymis

14.518
dpc

e Development of brain dimorphisms d Testicular descent 1st phase 2nd phase

Kidney

Cranial ligament
Scrotum

Gubernaculum Gubernaculum
Gential
tubercle
Figure 1 | Major steps in male sexual differentiation. The external genitalia appear as a mesenchymal swelling between the
two layers of the cloacal membrane at around 10.5 dpc and develop further under the influence of testicular androgens (a).
A transverse section through a mouse embryo at 11.5 days post coitum (dpc) shows the developing urogenital ridge, which
develops out of the intermediate mesoderm on either side of the aorta and is composed of the mesonephros and genital
ridge. Within the mesonephros the Wolffian (male) and Mllerian (female) ducts form. At 11.5 dpc males cannot be
distinguished morphologically from females (b). However, expression of the male-determining gene Sry induces a cascade
of gene expression that results in the differentiation of the genital ridge and the Wolffian duct into the testis and male
genital tract (rete testes, epididymis, vas deferens and seminal vesicle), respectively (c). At 13 dpc the bipotential urogenital
sinus develops and at 17.5 dpc the prostatic bud is formed by the urogenital sinus under the influence of testicular
androgens. Between 14.5 and 18 dpc the testis migrates into the developing scrotum in two phases (d). The first phase is
due to an enlargement of the gubernaculum, whereas during the second phase the gubernaculum migrates into the
scrotum, guided by calcitonin-related peptide that is released by the genito-femoral nerve. The brain develops sexual
dimorphisms, some of which are due to direct genetic effects, although most are caused by sex hormones (e).

Ovotestes
Gonads in which ovarian and
testicular tissue are present
together.
XY true hermaphroditism
This condition comprises the
presence of both ovarian and
testicular tissue either in the
same gonad as an ovotestis, or
an ovary and a testis.
ridges around 10.5 dpc, reaching peak levels and encom-
passing the whole gonad at 11.5 dpc, before declining,
first in the centre then later at the poles, to undetectable
levels around 12.5 dpc (REFS 15,16). However, the molec-
ular mechanism of this regulation remains a mystery.
Several factors have been implicated by virtue of reduced
Sry expression in targeted mouse mutants; these include a
splice variant of WT1, GATA4 and its cofactor FOG2 (also
known as ZFPM2), and the insulin receptor family 5,17,18.
It is not clear in these mutants whether the level of Sry
expression per cell is affected, or the number of cells
that express Sry is reduced, and none of these genes is
expressed in a wave pattern similar to Sry.
Differences between species1921 imply that it is
immaterial how quickly Sry expression is shut down.
However, the time at which peak levels of expression
are attained is crucial. In a phenomenon known as
B6-YDOM sex reversal22, Y chromosomes from several
Mus domesticus variants differ in their ability to induce
testis formation on a C57BL/6J genetic background,
resulting in phenotypes that range from complete XY sex
reversal, to unilateral or bilateral ovotestes, to delayed testis
formation. This has been ascribed to delayed expression
of Sry from these Y chromosomes23,24, adding to previous
evidence that levels of Sry expression are important for
male sex-determining function25. Similar regulatory phe-
nomena might explain the occurrence of ovotestes in some
cases of XY true hermaphroditism in humans. The current
belief is that Sry expression must reach a certain threshold
level within a definite temporal window of competence

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in the precursors of supporting cells, otherwise the the same cell type, the pre-Sertoli cell3840. It is clear that
ovary-determining pathway will initiate in these cells. Sox9 represents an early acting and crucial component
Sry encodes a nuclear, high-mobility group (HMG) of the male sex-determining pathway: loss of function of
domain protein that binds and bends DNA. Outside the SOX9 results in XY gonadal dysgenesis in mice and also in
HMG domain, SRY exhibits little sequence or structural the human disorder campomelic dysplasia, whereas gain
conservation between species, and almost all mutations of function in transgenic mice induces XX maleness4147.
that have a clinical phenotype (for example, XY females Sox9 is present in a wide diversity of metazoans, possibly
with complete gonadal dysgenesis in Swyer syndrome) fulfilling a similar role even in Drosophila melanogaster 48,
reside within this motif, underscoring the importance of suggesting that it is an ancient and conserved effector
this domain26. These mutations generally impair SRY DNA
binding and/or bending, or nuclear translocation2730.
The SRY protein in humans and mice has other
domains that mediate proteinprotein interaction
and transcriptional transactivation in vitro. The sig-
nificance of the former is suggested by some missense
and frameshift mutations causing XY sex reversal in
humans3133. However, it is not clear whether SRY func-
tions as a transcriptional activator in vivo; the frequency
of SRY-negative XX maleness in the human population
has been taken as evidence that SRY might repress a
repressor of the male programme rather than directly
activate a sex-determining cascade34. Either way, it is
curious that no target genes for SRY have been identi-
fied, and the molecular cascade of events triggered by
SRY activity still remains obscure.

Differentiation of Sertoli cells. The genital ridges consist

XY gonadal dysgenesis
This can lead to pure gonadal
dysgenesis, in which patients
have streak gonads
(undeveloped gonadal
structures), Mllerian structures
(owing to insufficient AMH
secretion) and a complete
absence of virilization.
Alternatively, patients can have
dysgenetic testes. In this case,
enough AMH is produced to
regress the Mllerian duct and
there might be enough
testosterone for partial
virilization.
Campomelic dysplasia
A syndrome that is
characterized by skeletal
abnormalities and sex reversal,
caused by mutations in SOX9.
of several cell lineages (FIG. 2), each of which is thought to
be bipotential, generating different cell types in testes and
ovaries depending on the signals received. Among these
is a progenitor known as the supporting-cell precursor
lineage, which gives rise to both the Sertoli cells and the
ovarian granulosa cells that support the development of
germ cells in males and females, respectively. Landmark
experiments by Burgoyne and colleagues35 that involve
mouse XXXY chimaeras showed that all gonadal lineages
were composed of similar numbers of XX and XY cells,
but Sertoli cells were overwhelmingly XY. This indicates
that SRY functions cell autonomously to trigger the differ-
entiation of Sertoli cells, and that pre-Sertoli cells (defined
as supporting cells that express Sry and/or Sox9 (SRY-box
containing gene 9) but are not yet arranged into cords)
can signal to other lineages to induce their male-specific
differentiation and so orchestrate testis development.
However, up to 10% of Sertoli cells in these chimae-
ras were found to be XX. This implies the existence of
a mechanism by which supporting cell precursors that
do not express Sry can be recruited to differentiate into
Sertoli cells through a secreted signal. Recent work has
shown that pre-Sertoli cells produce prostaglandin D2,
which, by binding to its receptor and upregulating Sox9
expression, recruits other cells to the Sertoli cell fate15,36,37.
This might act as a backup mechanism, reinforcing male
sex-determination when Sry expression or function
is impaired.
Downstream of Sry, other factors such as SOX9,
SOX8, DAX1 and FGF9 (fibroblast growth factor 9) have
a role in Sertoli cell differentiation and function. Sox9 is
the best candidate for a direct SRY target gene, although
conclusive proof has yet to be produced. In mice, Sox9 is
expressed shortly after the onset of Sry expression and in
Endothelial cells
Pecam1 Peritubular myoid cells
Ptc
Germ cells
Oct4, Pecam1, E cadherin Leydig cells
Sertoli cells Scc, HSD3B, Ptc
Sox9, Amh, Dhh, Dmrt1
Figure 2 | Histological and gene-expression map of an
early embryonic testis. A haematoxylin and eosin
stained section that illustrates the different cell types of
the testis and the genes that are specifically expressed in
these cells: endothelial cells (red) form the male-specific
vasculature, germ cells (orange) that later develop into
sperm are enclosed by a layer of Sertoli cells (green), which
support and nourish the germ cells, and peritubular myoid
cells (black), which help to maintain cord integrity and are
later responsible for pulsatory contractions that are
required for export of sperm. The steroid-producing Leydig
cells (purple) reside in the interstitium together with other
cells such as macrophages and mesenchymal cells. Amh,
anti-Mllerian hormone; Dhh, desert hedgehog; Dmrt1,
doublesex and mab-3 related transcription factor 1;
Pecam1, platelet/endothelial cell-adhesion molecule 1; Ptc,
patched; Scc, side-chain cleavage (also known as Cyp11a1);
Sox9, SRY-box containing gene 9; HSD3B, hydroxy--5-
steroid dehydrogenase-3.

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Lissencephaly
A brain malformation that
is characterized by the
incomplete development of
the folds of the outer region
of the brain (the cerebral
cortex), which causes the
surface of the brain to appear
abnormally thickened and
unusually smooth.
Ovarian follicle
A cyst in which the oocyte
matures.
624 | AUGUST 2006 | VOLUME 7
of male sex determination, in contrast to Sry, which
is exclusively mammalian. The elusive relationship
between SRY and Sox9, and the probable nature of the
transcriptional cascade downstream of SRY, have been
reviewed extensively elsewhere49,50.
Besides the differentiation of Sertoli cells, testis devel-
opment immediately after the onset of Sry expression
differs from female gonad development in its increased
cell proliferation51 and the induced immigration of
mesonephric cells52,53. Although several factors such
as neurotropin 3 (NT3; also known as NTF3) (REF. 54),
hepatocyte growth factor (HGF)55, and platelet-derived
growth factor- (PDGFA)56 are able to induce this
cell migration in vitro, none has been shown to have
a role in vivo. Presumably, because this migration and
increased proliferation are male-specific events, the fac-
tors that mediate them are regulated either by Sry or one
of its early downstream genes such as Sox9.
The immigrant mesonephric cells are thought to dif-
ferentiate into Leydig, endothelial and peritubular myoid
cells (FIG. 2), depending on interactions with somatic cells
within the gonad. This raises the question of the nature
of these interactions and how these other testicular cell
types differentiate.
Testis differentiation peritubular myoid cells. Peri-
tubular myoid (PM) cells are flat, smooth-muscle-like
cells that ensheath testis cords and are necessary for
cord development and structural integrity53,57. Little is
known about them, partly owing to the lack of a specific
marker 58. One factor that is correlated with differentia-
tion of PM cells is the Sertoli cell-specific, secreted factor
desert hedgehog (DHH). Its receptor patched (PTC; also
known as PTCH1) is expressed on PM and Leydig cells.
Null mutation of Dhh in mice led to impaired differen-
tiation of PM and Leydig cells and subsequently to femi-
nized males5961. Similarly, mutations in human DHH
have been associated with partial and pure XY gonadal
dysgenesis accompanied by impaired cord formation
and reduced testosterone levels6264. Other unknown
Sertoli cell-derived factors might be involved in directing
PM cell differentiation.
Testis differentiation endothelial cells. Although
gonads of both sexes are heavily vascularized from an
early stage65,66, endothelial cells in the testis form a charac-
teristic vasculature with a prominent coelomic vessel on
the anti-mesonephric surface and branches between the
testis cords. The formation of the coelomic vessel, but
not the side branches, is suppressed by the secreted sig-
nalling molecule WNT4. Null mutation of Wnt4 in mice
resulted in the ectopic formation of a coelomic vessel
in XX animals67. Because blood vessels and testis cords
occupy complementary domains, there is likely to be an
interplay between testis cord formation and vascular
patterning, although is not clear which directs which.
Testis differentiation Leydig cells. Fetal Leydig cells
synthesize crucial hormones for male sex differentiation.
The aristaless-related homeobox gene ARX, identified
in X-linked lissencephaly with abnormal genitalia, is
2006 Nature Publishing Group
associated with a block in Leydig cell differentiation68,69.
Additionally, ATRX (-thalassaemia/mental retarda-
tion syndrome, X-linked), also known as XNP or XH2,
is mutated in the ATRX syndrome, which is character-
ized by severe mental retardation, -thalassaemia and
a range of genital abnormalities that suggest that ATRX
could be involved in Leydig cell development70. For
both genes, however, the molecular role during Leydig
cell differentiation is unknown. In addition, signalling
through PDGFs and their receptor PDGFRA have been
identified in knockout mice to be essential for fetal71 and
adult72 Leydig cell differentiation.
Testis differentiation germ cells. Primordial germ cells
originate neither in the gonad nor the mesonephros,
but instead migrate from their origin at the posterior
extremity of the embryo through the hindgut to populate
the genital ridges at around 10.5 dpc in the mouse. Here
they associate with somatic cells to form primitive sex
cords, the precursors of testis cords and ovarian follicles.
Important factors in germ-cell migration include the fra-
gilis proteins IFITM1 and IFITM3 (interferon-induced
transmembrane proteins 1 and 3) (REF. 73), whereas stro-
mal cell-derived factor 1 (SDF1; also known as CXCL12)
and its receptor CXCR4 are involved in the colonization
of the genital ridges74.
Like other cell types in the gonad, germ cells are
influenced by Sertoli cells to differentiate in a male-
specific fashion regardless of their sex-chromosome
genotype. Germ cells in a testis enter a state of mitotic
arrest around 12.5 dpc (REFS 75,76), a state in which they
remain until after birth, whereas germ cells in an ovarian
environment enter meiosis around 13.5 dpc, signalling
the onset of oogenesis77.
Interestingly, the long-standing dogma that germ cells
enter meiosis cell autonomously is questioned by recent
data showing that retinoic acid, produced by the meso-
nephros, induces entry into meiosis. Male germ cells
are protected from the effects of retinoic acid by their
enclosure within the testis cords. Sertoli cells, which sur-
round primordial germ cells in the testis cords, express
CYP26B1, an enzyme that breaks down retinoic acid78,79.
However, the mechanism by which male germ cells arrest
mitotically has not been determined. The fact that after
12.5 dpc male germ cells are committed to develop as
spermatogonia suggests that either mitotic arrest pre-
vents entry into meiosis, or that a temporal window of
competence to respond to retinoic acid signalling exists
within the germ cells.
Germ cells are crucial for the differentiation of ovar-
ian follicles and the maintenance of the ovary, presuma-
bly secreting factors that are required for these processes.
This signalling might be a consequence of entry into
meiosis. By contrast, germ cells are not required for
testicular development or maintenance: fetal testes
in which the germ cells are chemically or genetically
depleted develop normally 80.
Testicular descent. An important aspect of sexual
development is that testes and ovaries end up in different
locations in the body. The testes migrate to their final
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Cryptorchidism
The condition of having
undescended testes.
Persistent Mllerian duct
syndrome
A rare form of male pseudo-
hermaphroditism that is most
commonly characterized by
bilateral fallopian tubes and a
uterus combined with an
otherwise more or less normal
male phenotype.
NATURE REVIEWS | GENETICS
location in two phases. The transabdominal phase of tes-
ticular descent occurs in mice between 14.5 and 18 dpc
(in humans at 8 to 15 weeks of gestation), and is con-
trolled by the enlargement of the caudal genito-inguinal
ligament and the gubernaculum, and by the regression
of the cranial ligament (FIG. 1c). Mutation studies in
mice demonstrated that the insulin-like 3 (INSL3)
hormone, produced by Leydig cells, using its receptor
LGR8 (also named GREAT), is necessary and sufficient
to mediate this phase of testicular descent 81. Mutations
in these genes have been found in male patients with
cryptorchidism , but these account for only a small
proportion of all cases of this common disorder 82.
The second, inguinoscrotal phase is usually completed
by 20 days after birth in mice, and by the thirty-fifth
week in humans. In contrast to the first phase, which
is dependent on the growth of the gubernaculum, this
phase requires the migration of the gubernaculum from
the groin into the scrotum (FIG. 1c). This is guided by the
neurotransmitter calcitonin gene-related peptide (CGRP;
also known as CALCA), which is released by the genito-
femoral nerve under the control of androgens. Mutations
in genes that are involved in androgen signalling and
those that encode transcription factors, such as homeobox
A10 (Hoxa10), Hoxa11 and Desrt (developmentally and
sexually retarded with transient immune abnormalities),
cause disruption of the second stage of descent in mice,
leaving the testes at the level of the bladder, which is in
contrast to Insl3 and Lgr8 mutations that lead to a high
intra-abdominal location83,84. Clearly the regulatory
mechanisms that drive testicular descent are sensitive to
imbalance and malfunction and are often secondary
to other disorders factors that might contribute to the
high frequency of undescended testes in newborn boys.
Development of the male genital tract
Early stages the genital ducts. Both male and female
embryos initially have two pairs of genital ducts. In
males, one pair, the Wolffian (or mesonephric) ducts,
generate the mature genital tract, whereas the other, the
Mllerian (or paramesonephric) ducts, disappear. In
females, the Mllerian ducts survive and the Wolffian
ducts disappear. This represents a different strategy to
that used in gonad development, which involves dichot-
omous differentiation of a bipotential precursor tissue.
However, a bipotential structure, the urogenital sinus,
contributes to the genital tract by forming the prostate in
males and the lower vagina in females (FIG. 1b,d).
In an XY embryo, the Mllerian ducts degenerate
in an active process, involving a TGFB (transform-
ing growth factor-)-family molecule, anti-Mllerian
hormone (AMH; also known as Mllerian-inhibiting
substance or MIS). AMH is secreted by the Sertoli cells
of differentiating testes and binds to its receptor, MISRII
(also known as AMHR2), on the surface of Mllerian
duct mesenchymal cells. This induces a signalling
cascade that results in the production and secretion of
matrix metalloproteinase 2 (MMP2), which induces
apoptosis in the Mllerian duct epithelial cells85. Failure
in these processes in humans can lead to persistent
Mllerian duct syndrome (PMDS)8688.
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Initial
Ros1
Epididymis segment
Interstitium
Caput Bmp7,8
Testis
cords
Bmp4
Corpus
Cauda Bmp7,8
Hoxa9,10,11
Hoxd9,10
Vas deferens
Fgf10
Seminal
vesicle Gdf7
Figure 3 | Schematic representation of the
differentiation of the male genital tract. The Wolffian
duct differentiates under the influence of testicular
androgens into the epididymis, vas deferens and seminal
vesicle. The epididymis can be further divided,
morphologically and functionally, into the initial segment,
caput, corpus and cauda. The sperm, which are produced
in the testes, mature during their passage through the
caput and corpus, whereas the cauda functions
predominantly for storage. The different segments of the
male genital tract are marked by specific gene expression.
Null mutations of these genes result in phenotypes that are
restricted to these segments. Homeobox A10 (Hoxa10) and
Hoxa11 knockout mice are sterile and the epididymis shows
homeotic transformation. In Hoxa10 null mice the cauda
epididymis seems to have transformed into the corpus,
whereas in Hoxa11 knockout mice the vas deferens shows
partial transformation into the cauda. Mutations in bone
morphogenetic protein 4 (Bmp4) result in extensive
degeneration of the epididymal epithelium of the corpus
region, rather than in the caput and cauda regions as for
Bmp7 and Bmp8 knockout mice, whereas mice that are null
for Ros1 show defects in the differentiation of the initial
segment. For the proper development of the seminal
vesicle, fibroblast growth factor 10 (Fgf10) and growth
differentiation factor 7 (Gdf7) are required.
Later stages differentiation of the Wolffian duct. In
males, the Wolffian ducts further develop under the
influence of testosterone, produced by Leydig cells,
into a system of organs the epididymis, vas defer-
ens and seminal vesicle (FIGS 1b,3) that connect the
testes with the urethra. These Wolffian duct derivatives
can be distinguished by their morphologies, specific
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REVIEWS

a differentiation along the Wolffian duct. Knockout

Ventral Outgrowth mouse models of Gdf7, Bmp4, Bmp7, Bmp8a and Bmp8b
(which encode bone morphogenic proteins)8992 and
Genital Bmp4, Hoxa13, the homeobox genes Hoxa10 and Hoxa11 (REFS 93,94)
tubercle Hoxd13, Ptc
result in defects in specific areas of the epididymis and
Cloacal seminal vesicle, confirming the region-specific require-
Shh
membrane ment for signalling molecules and transcription fac-
tors (FIG. 3). Furthermore, the phenotypic effects of a
Urethral fold null mutation of Ros1 implicates the encoded tyrosine
kinase receptor in the regionalization and terminal dif-
Labioscrotal ferentiation of the epididymal epithelium95. Studies of
swellings
mouse mutants have also identified two genes that are
necessary for the proliferation of epithelial cells, and
therefore tube elongation: those that encode the orphan
Dorsal G-protein coupled receptor LGR4 (REF. 96) and relaxin,
a naturally occurring inhibitor of collagen deposition97.
Subsequently, the growth factor PDGFA, expressed
b Hoxa13, Hoxd13
in the epithelium and its receptor PDGFRA in the
surrounding mesenchyme maintain the structural
Apoptosis Bmp7 Fgf8 integrity of the tube98.
Ventral Genital tubercle Proliferation Studies of the human disorder congenital bilateral
Distal urethral epithelium Wnt5a
aplasia of vas deferens (CBAVD) have implicated the
cystic fibrosis transmembrane conductance regulator
Mesenchyme Fgf10 (CFTR) in the development of the male reproductive
Urethral groove tract. A high proportion of males with cystic fibrosis
Shh also display CBAVD and are therefore infertile. However,
many CBAVD patients with CFTR mutations do not
Fusion of
urethral folds show the cystic fibrosis lung phenotype. This is probably
explained by differences in the tissue-specific alterna-
tive splicing of CFTR between the vas deferens and the
lung 99. However, it is not yet known how mutation of this
membrane-bound chloride channel leads to the loss of
vasa deferentia during development.
Dorsal
Development of the external genitalia
Figure 4 | Development of the external genitalia. a | Caudal view of the indifferent A second organ system that differentiates under the
anlage. The cloaca is closed off from the exterior by the cloacal membrane, which is influence of testosterone is the external genitalia. The
bordered anteriorly by the genital tubercle and laterally by the urethral and labioscrotal specialized male and female genital anatomy that has
swellings. Sonic hedgehog (SHH) signalling from the epithelium results in the evolved in mammals provides greatly increased repro-
upregulation of bone morphogenetic protein (Bmp4), homeobox A13 (Hoxa13), Hoxd13 ductive efficiency compared with the system of external
and patched (Ptc) expression in the mesenchyme. This expression pattern exhibits a fertilization found in many birds and fish100. Perhaps
balance of apoptosis that is induced by Bmp4 and proliferation, which is indirectly even more so than the gonads and the duct systems, the
controlled by Hoxa13 and Hoxd13 through the upregulation of fibroblast growth factor 8 vastly different morphologies of the male and female
(Fgf8). This balance is necessary for correct development of the genital tubercle with mis- external genitalia belie their origins from a common set
regulation resulting in hypospadias. b | The genital tubercle elongates to form the penis,
of ambiguous embryonic structures.
while the urethral groove forms on the ventral aspect of the genital tubercle, extending
distally in a solid epithelial plate (distal urethral epithelium). The penile urethra
subsequently forms by proximal to distal fusion of the urethral folds. The labioscrotal The indifferent stage. In mice, at around 10.5 dpc, the
swellings migrate caudally and fuse at the midline to form the scrotum. SHH from the external genitalia first become visible as a small mesen-
epithelium and FGF10 from the mesenchyme maintain their own expression in a positive- chymal swelling between the two layers of the cloacal
feedback loop. HOXA13 and HOXD13 continue to signal back to the distal urethral membrane (FIG. 1d). Subsequently, the ventrally located
epithelium to induce Bmp7 and Fgf8 expression. urethral groove appears, which has a solid plate of epi-
thelial cells at the distal end. This distal urethal epithe-
lium, which is comparable to the apical ectodermal ridge
gene-expression patterns and functions, although they of the limb bud, functions as a signalling centre to stimu-
are contiguous and collaborate with each other in sup- late the outgrowth and differentiation of the genital tuber-
porting male gamete development. Similar to other cle through epithelialmesenchymal interactions. The
testosterone-induced, male-specific organs such as expression and relationship of the key molecules involved
Anlage the prostate and external genitalia, their development in this outgrowth Fgfs, SHH (sonic hedgehog), Wnts,
A group of cells that are
destined to become a specific
depends on epithelialmesenchymal interactions. It is HOXA13 and HOXD13, Bmps and their antagonist
structure or tissue in the adult, believed that region-specific, inductive signals from noggin (FIG. 4; Supplementary information S1 (table))
but have not yet differentiated. the surrounding mesenchyme specify the characteristic are remarkably similar to those involved in limb

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2006 Nature Publishing Group

Branchial arches
A series of paired segmental
structures that are composed
of ectoderm, mesoderm and
neural crest cells that are
positioned on each side of the
developing pharynx. In
mammals, the branchial arches
contribute to pharyngeal
organs and to the connective,
skeletal, neural and vascular
tissues of the head and neck.
Congenital adrenal
hyperplasia
A condition that is in most
cases due to CYP21 deficiency,
and is characterized by the
deficiency in the hormones
cortisol and aldosterone and
an overproduction of
androgens, which results in
ambiguous genitalia in females.
NATURE REVIEWS | GENETICS
REVIEWS
bud development and branchial arch outgrowth during a Initiation AR
craniofacial development101.
?
Differentiation of male external genitalia. After this SHH
initial, bipotential phase, at around 16 dpc in mice and
Urethra
12 weeks of gestation in humans, the development of the
external genitalia becomes sex-specific. 5-Reductase
is expressed in the genital tubercle mesenchyme, and
in males it converts testosterone into 5-dihydrotesto- b Growth HOXA13 SHH
HOXD13
sterone (DHT), the biologically more potent androgen.
DHT signals through the androgen receptor (AR) that NKX3.1
is present in cells of the developing external genitalia,
which leads to a series of morphological changes. The
Urethra
genital tubercle elongates further with the urethral folds
approaching each other, finally fusing, from proximal
to distal, to form the tubular penile urethra (FIG. 4b). The c Branching
morphogenesis FGF10 TGFB1
scrotum is formed by the genital swellings, which move
caudally and fuse in the midline. 5-Reductase and AR FGF7 FGFR2
are also expressed in the female external genitalia, but
do not lead to male differentiation owing to the lack of SHH
testosterone. However, in females with congenital adrenal
hyperplasia, the adrenal glands produce abnormally high Notch
levels of androgens, which lead to varying degrees of BMP4
BMP7
virilization of the female external genitalia102.
In males, mutations resulting in a defective AR,
or low levels of AR, cause androgen insensitivity syn-
drome, the most common form of XY sex reversal. Progression of
This disorder is characterized by deficient or absent d Differentiation and p63 ductal lumen,
maturation FOXA1 differentiation
virilization of 46,XY individuals despite normal or of basal and
even elevated androgen levels. More than 300 muta- luminal cell types,
tions in the X-linked, single-copy AR gene have been and formation
of smooth
described, leading to phenotypes that range from com- muscle cells
plete androgen insensitivity and female phenotype, to
partial insensitivity and ambiguous genitalia, to mild
Urethra
forms with a male phenotype but that are infertile.
The testes do not usually descend; however, there is no
Figure 5 | Morphological and gene-expression changes
uterus because the degeneration of the Mllerian ducts during prostate development. a | Circulating androgens
is androgen-independent. initiate the development of the prostate from the
Mutations in the gene encoding 5-reductase are urogenital sinus; the androgen receptor (AR), which is
another common cause of male pseudohermaphroditism. necessary for budding of the urethral epithelium (green), is
Such individuals are deficient in DHT, which is required expressed in the mesenchyme (pink). An unknown factor,
for the full masculinization of the external genitalia and which could be an activator or repressor, mediates a signal
development of the prostate, but not for the normal dif- to the epithelium, which causes the upregulation of Shh
ferentiation of the Wolffian ducts, epididymides, vasa expression. b | Sonic hedgehog (SHH), at least in part by
deferentia and seminal vesicles103. upregulating the transcription factor NKX3.1, and the
mesenchymal homeobox genes Hoxa13 and Hoxd13
The high prevalence of hypospadias in humans sug-
promote further growth of the prostatic ducts. c | Most of
gests that urethral fusion is a delicate and finely regulated these ducts remain unbranched until birth in rodents but,
process. The cell-surface molecules ephrins, and their subsequently, epithelialmesenchymal interactions result
receptors (Ephs), have been implicated in this process:
in further elongation and branching morphogenesis.
mice in which ephrin B2 and EphB2/EphB3 signalling Ductal branching and budding are inhibited by the
is disrupted show variable levels of incomplete urethral mesenchymal signalling factors BMP4 (bone
tubularization104. In addition, mutations in many of the morphogenetic protein 4) and BMP7, and stimulated by the
above-mentioned genes that control the initial phase antagonist of these Bmps, Notch. FGF7 (fibroblast growth
factor 7) and FGF10, expressed in the mesenchyme, bind to
of genital tubercle outgrowth result in hypospadias,
FGFR2 on epithelial cells, which leads to the maintenance
indicating a requirement for coordinated control of
of SHH expression. This positive regulation is limited by the
urethral fusion and genital tubercle outgrowth. For
negative-feedback loop of downregulation of Fgf
example, mutations in HOXA13 and HOXD13 lead to expression by SHH signalling. d | Finally, in a proximal to
hand-foot-genital syndrome, an autosomal dominant
distal direction, the epithelial cell types differentiate under
disorder, which is characterized by malformation of the control of transcription factors such as p63 and
the distal limbs, accompanied by hypospadias105107. forkhead box A1 (FOXA1), smooth muscle cells form
Furthermore, experiments in rodents have suggested around the epithelium and the ductal lumen advances.
VOLUME 7 | AUGUST 2006 | 627
2006 Nature Publishing Group
REVIEWS

Genital tubercle Penis Brain Wolffian duct Vas deferens,

epididymis, seminal vesicle

Androgen receptor, Shh, Wnts, Androgen receptor, Bmps,

Fgfs, HoxA13, HoxD13, Bmps, Sry? Other genes? Hox, Cftr, Hoxa10, Hoxa11,
noggin, Ephs, ephrins Lgr4, Pdgfa, Pdgfra

Androgens Androgens
External genitalia Androgens Male genital tract

Testicular descent Genital ridge Testis Urogenital sinus Prostate

Androgens
Sry, Sox9, Sox8, Fgf9, Sf1, Androgen receptor, Shh,
Insl3, Lgr8, Hoxa10, Hoxa11,
Dhh, Ptc, Wt1, Dax1, Atrx, Fgfs, Hoxa13, Hoxd13,
Desrt
Arx, Pod1, Pdgfa, Pdgs CD44, follistatin

Figure 6 | Overview of genetic pathways that drive male sexual differentiation in mammals. The Y-linked gene
Sry is the master switch that determines the differentiation of the bipotential genital ridge into a testis (bottom middle
box). Expression of Sry sets in motion a cascade of male-specific gene expression such as Sox9, Sox8 and Fgf9. Later, the
testis has to descend (depicted by a broad arrow) into the scrotum for full functionality. Subsequently, testicular
androgens initiate the differentiation of secondary male sexual characteristics such as the male genital tract, external
genitalia and brain, which involves organ-specific, regulatory gene networks.

that some industrial chemicals, pharmaceuticals,
environmental pollutants and natural products have
anti-androgenic properties that can result in the femi-
nization of male external genitalia, which might explain
the alarming increase in the occurrence of hypospadias
in recent decades108.
(table)). How do the same set of control genes lead to
the formation of these distinct structures? This remains
unknown, but interacting partners might be expressed
differentially in these structures, or different thresholds
or temporal and spatial combinations of expression of
these key genes might occur in different tissues.

Development of the prostate Sexual differences in the brain

The prostate is an essential mammalian-specific, male
accessory sex gland that contributes to the seminal
plasma fluid. This important role in mammalian repro-
duction and the high incidence in humans of prostatic
diseases, including benign and malignant tumours,
make it necessary to understand prostate development
and biology.
The prostate develops from the urogenital sinus,
which is derived from the cloaca that is a caudal extension
of the hindgut (FIG. 1b,d). Interestingly, its development
is similar to that of the external genitalia. The indif-
ferent urogenital sinus forms in both male and female
mice at approximately 13 dpc (7 weeks of gestation in
humans), and remains morphologically indistinguish-
able until 17.5 dpc, when testicular androgens induce
the outgrowth of solid buds from the urogenital sinus
epithelium into the urogenital sinus mesenchyme109,110.
Curiously, the androgen receptor is expressed on the
mesenchyme and induces prostatic epithelial devel-
opment, which implies that there is an unidentified,
secreted, mesenchymal factor that mediates the action
of androgens. After this initial hormone-dependent
stage, the development of the prostate is characterized by
epithelialmesenchymal interactions, resulting in cell dif-
ferentiation and branching morphogenesis, that involve
the same key molecules (FGFs, SHH, BMPs, HOXA13
and HOXD13) as the development of the external
genitalia, in addition to a few others (for example, CD44
and follistatin)111,112 (FIG. 5; Supplementary information S2
Males and females have clear behavioural differences and
are differentially susceptible to many behavioural, emo-
tional and personality disorders. Sex-specific differences
in brain morphology traditionally have been attributed
to the action of steroid hormones that are produced by
the gonads. Although these are still seen as the main fac-
tors, recent data suggest that genetic differences could
also have a direct role.
Microarray analysis identified more than 50 genes
that show a sex-specific expression pattern in the brain
of 10.5 dpc mice, a stage that is too early for any gonadal
hormone influence, which supports the hypothesis that
chromosomal constitution has a role in mammalian
brain differentiation113. These genes encode proteins that
range from those that regulate the cell cycle and con-
trol transcription, to enzymes and structural proteins.
It remains unclear whether the sex-specific expres-
sion of these genes is a cause or consequence of brain
sexual dimorphism, and, if they do contribute to neural
differentiation, what their functions might be.
A more restricted screen was used to test the hypoth-
esis that genes encoded on the sex chromosomes have
a direct role in sexual differentiation of brain and sex-
specific behaviours114. This study used a fully fertile
mouse model in which Sry was moved from the Y chro-
mosome to an autosome. By breeding these mice with
wild-type XX females four genotypes were produced:
XX females, XYSry females, XYSry (Sry+) males, and XX
(Sry+) males, a system in which sex determination is

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2006 Nature Publishing Group
 REVIEWS

Substantia nigra
A region of the ventral
midbrain that contains pigment
and sends afferent dopamine-
releasing neurons to the
striatum.
independent of the Y chromosome. One sexual dimor-
phism, the density of vasopressin-immunoreactive
fibres, was identified that is due to direct effects of sex
chromosomal genes114. However, this mouse model did
not allow the effects of the male-determining gene Sry
to be investigated.
Expression of Sry mRNA has been reported in the
mouse and human brain115118, but the biological sig-
nificance has remained obscure. Recently, Dewing and
co-workers demonstrated that not only the mRNA but
also SRY protein is expressed in tyrosine-hydroxylase-
expressing neurons of the substantia nigra, and that exper-
imentally induced knock-down of Sry expression results
in a decrease of tyrosine hydroxylase, suggesting that the
expression of this enzyme might be directly regulated by
SRY. Tyrosine hydroxylase is the rate-limiting enzyme in
the synthesis of dopamine in the dopaminergic neurons
of the nigrostratial system that controls specific motor
behaviours. Interestingly, experimental, unilateral down-
regulation of Sry has led to quantifiable sensorimotor
deficits in male mice, assessed by akinesia and limb-use
asymmetry tests, which implicates the direct control of
specific motor behaviour by Sry independent of hormo-
nal influences119. Although these experiments provide
evidence that Sry and/or other genes are directly involved
in sexual dimorphism of the brain, more work is needed
to elucidate the precise molecular mechanisms, and how
these are modified by different androgen dosages.
Challenges for the future
The mammalian embryo develops for a significant
period in a sexually ambiguous manner, until the sex-
determining switch gene Sry is activated, testes differ-
entiate and other organs and tissues that differ between
males and females including the brain set off on
their sex-specific developmental trajectories. Therefore,
in both male and female embryos, the complete set of
genes, cell types and precursor structures must exist to
equip the embryo for each of two possible outcomes. A
remarkable set of strategies is used in different parts of
the embryo to achieve this plasticity. For example, cell
lineages inhabit the early genital ridges that are capable
of male-specific or female-specific differentiation,
depending on secreted signals from nascent Sertoli cells.
In the developing duct system there is no bipotential
precursor, but instead both sets of ducts are laid down
initially, with only one surviving at the expense of the
other. The external genitalia, on the other hand, are
constructed from basically the same cell types in males
and females, but are moulded into strikingly different
forms through the action of elaborate endocrine and
paracrine cascades.
Significant progress has been made in identifying
genes and regulatory networks that drive male-specific
differentiation (FIG. 6). The picture that emerges is one
of a complex interplay between transcription factors,
secreted signalling molecules, hormones and recep-
tors that, if disturbed, can result in various phenotypes.
Interestingly, a similar network of hub genes includ-
ing members of the Hox, Fgf, hedgehog and Wnt fami-
lies has an important role in each system (the testes,
prostate and external genitalia). The challenge is to find
the specific members of each family that are unique to
a particular system, and the tissue-specific target genes
that are regulated by this common network.
Despite the advances described above, most cases of
XY sex reversal, SRY-negative XX sex reversal and true
hermaphroditism remain unexplained at the molecular
level. Either a large number of key sex-determining genes
await identification, or mutations outside the coding
regions of crucial known genes such as Sry and Sox9 (for
example, regulatory mutations or mutations that affect
post-transcriptional processing) are more prevalent
than previously suspected or both. Approaches such
as microarray profiling, comparative genomic hybridiza-
tion and mutagenesis screening will no doubt have an
important role in efforts to understand human disorders
of sexual development.
In addition to explaining testis development, these
methods will contribute to the growing picture of how
other parts of the male reproductive system develop.
This, in turn, will provide a clearer picture of how devel-
opment of the seemingly disparate elements of male
ontogeny is coordinated. Until then, we can only wonder
at the complexity of events between the activation of Sry
and the characteristics that make men well, men.

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Acknowledgements
We thank T. Svingen, A. Combes and C. Spiller for helpful
comments on the manuscript, and G. Yamada for help with
Supplementary information S1. DW acknowledges grant
funding by the US National Institutes of Health. PK is an
Australian Professorial Research Fellow of the Australian
Research Council and acknowledges grant funding by the
Australian Research Council and the National Health and
Medical Research Council, Australia.
Competing interests statement
The authors declare no competing financial interests.
DATABASES
The following terms in this article are linked online to:
Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db=gene
ARX | ATRX | CFTR | Dax1 | Emx2 | Gata4 | IFITM1 | IFITM3 |
Lhx9 | Sf1 | Sox9 | Sry | Wt1
OMIM: http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db=OMIM
androgen insensitivity syndrome | ATRX syndrome |
congenital bilateral aplasia of vas deferens | hand-foot-
genital syndrome | Swyer syndrome
UniProtKB: http://ca.expasy.org/sprot
AMH | AR | CGRP | DHH | HGF | INSL3 | MMP2 | NT3 |
PDGFRA | PTC | SDF1 | WNT4
FURTHER INFORMATION
Koopmans homepage:
http://www.imb.uq.edu.au/koopman.html
SUPPLEMENTARY INFORMATION
See online article: S1 (table) | S2 (table)
Access to this links box is available online.
VOLUME 7 | AUGUST 2006 | 631