SEXUAL DIFFERENTIATION

Rodolfo Rey, MD, PhD, Centro de Investigaciones Endocrinológicas “Dr. César Bergadá” (CEDIE),
CONICET-FEI-División de Endocrinología, Hospital de Niños Ricardo Gutiérrez, Gallo 1330, C1425EFD
Buenos Aires; and Departamento de Histología, Biología Celular, Embriología y Genética, Facultad de
Medicina, Universidad de Buenos Aires, C1121ABG Buenos Aires, Argentina. E-mail: [email protected]
Nathalie Josso, MD, PhD, Centre de Recherche Saint-Antoine (CRSA), INSERM UMR_S938, Sorbonne
Université, 75012 Paris, France. E-mail: [email protected]
Chrystèle Racine, PhD, Centre de Recherche Saint-Antoine (CRSA), INSERM UMR_S938, Sorbonne
Université, 75012 Paris, France. E-mail: [email protected]

Updated May 27, 2020

ABSTRACT hormones, or the female pathway in their absence. Since

the presence of the fetal testis plays a determining role in
The chromosomal sex of the embryo is established at the differentiation of the reproductive tract, the term "sex
fertilization. However, 6 weeks elapse in humans before the determination" has been coined to designate the
first signs of sex differentiation are noticed. Sex differentiation of the gonad during early fetal development.
differentiation involves a series of events whereby the
sexually indifferent gonads and genitalia progressively THE BIPOTENTIAL GONAD
acquire male or female characteristics. Believed initially to
be governed entirely by the presence or absence of the SRY No sexual difference can be observed in the gonads until
gene on the Y chromosome, gonadal determination has the 6th week of embryonic life in humans and 11.5 days
proven to rely on a complex network of genes, whose post-coitum (dpc) in mice. Undifferentiated gonads of XX or
balanced expression levels either activate the testis XY individuals are apparently identical and can form either
pathway and simultaneously repress the ovarian pathway ovaries or testes. This period is therefore called indifferent
or vice versa. The presence or absence of primordial germ or bipotential stage of gonadal development.
cells, of extragonadal origin, also has a sexually dimorphic
relevance. Subsequently, internal and external genitalia will
follow the male pathway in the presence of androgens and The Gonadal Ridge
anti-Müllerian hormone (AMH), or the female pathway in
their absence. Here we review the sexually undifferentiated
The urogenital ridges are the common precursors of the
stage of embryonic development, and the anatomic,
histologic, physiologic and molecular aspects of the fetal urinary and genital systems and of the adrenal cortex (1). In
sexual differentiation of the gonads, the internal the human, they develop during the 4 th week post-
reproductive tract and the external genitalia. fertilization at the ventral surface of the cranial
mesonephroi, and are formed by intermediate mesoderm
INTRODUCTION covered by coelomic epithelium. Each urogenital ridge
divides into a urinary and an adreno-gonadal ridge in the 5th
week (Table 1). The adreno-gonadal ridge is the common
Genital sex differentiation involves a series of events
precursor of the gonads and adrenal cortex. The gonadal
whereby the sexually indifferent embryo progressively
acquires male or female characteristics in the gonads, ridge is bipotential and can develop into an ovary or a testis.
genital tract and external genitalia. Sex development Gonads are subsequently colonized by the primordial germ
consists of several sequential stages. Genetic sex, as cells, of extra-gonadal origin. The mesonephroi also give
determined by the chromosome constitution, drives the rise to components of the internal reproductive tract and of
primitive gonad to differentiate into a testis or an ovary. the urinary system.
Subsequently, internal and external genitalia will follow the
male pathway in the presence of specific testicular

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The molecular mechanisms underlying the specific location ingression, thus probably initiating gonadal development
of the gonads on the surface of the mesonephroi begin to (2). However, since there are significant differences in
be unveiled in chicken embryos, where Sonic hedgehog gonadal development between birds and mammals, these
(SHH) signaling mediated by the bone morphogenetic mechanisms need to be explored to establish whether they
protein 4 (BMP4) establishes the dorsoventral patterning of are conserved amongst vertebrates.
the mesoderm and induces coelomic epithelium cell

TABLE 1. Chronology of Human Sex Differentiation*

Age from conception CR length (mm) Event
22 days 2-3 Intermediate mesoderm becomes visible
Primordial germ cells in the yolk sac
24 days 2.5-4.5 Formation of solid Wolffian ducts
Primordial germ cells migrate to the hindgut
26 days 3-5 Wolffian ducts develop a lumen
Primordial germ cells in the hindgut
28 days 4-6 Primordial germ cells migrate to the urogenital ridges
32 days 5-7 Gonadal primordia develop
Growth of Wolffian ducts
33-37 days 7-11 Primordial germ cells reach gonadal ridge
Urogenital sinus is distinguishable
Differentiation of Müllerian ducts
Genital tubercle and urethral folds are visible
41-44 days 11-17 Seminiferous cord differentiation
Differentiation between pelvic and phallic parts of the urogenital sinus
44-50 days 15-20 Seminiferous cords with germ cells
50-60 days 30 Beginning of secretion of AMH
Leydig cell differentiation
Cranial part of Müllerian ducts begins to regress
9 weeks 40 Leydig cells produce testosterone
Beginning of masculinization of urogenital sinus and external genitalia
10 weeks 45-50 Meiotic entry of oocytes in the medulla
Beginning of degeneration of female Wolffian ducts
Male Müllerian ducts have disappeared
Prostatic buds appear
12 weeks 55-60 The vaginal cord is formed
Primordial follicles appear
Seminal vesicles develop
Testis at internal inguinal ring
14 weeks 70 Completion of male urethral organogenesis
16 weeks 100 Primary follicles appear
20 weeks 150 Testosterone serum level is low
Formation of prostatic utricle
22 weeks 180 Vagina reaches perineum
24 weeks 200 Graafian follicles appear
Beginning of penile growth
27-30 weeks 230-265 Inguino-scrotal descent of the testis
36 weeks 300 Secondary and tertiary follicles produce AMH

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* According to O’Rahilly (3).

Several general transcription factors belonging to the large homeobox gene family play an important role in the stabilization
of the intermediate mesoderm and the formation of the urogenital ridges (Table 2). Mice in which Lhx1 (4), Emx2 (5, 6) or
Pax2 (7) have been inactivated fail to develop urogenital derivatives. Most of these ubiquitous factors are essential for the
development of other vital embryonic structures. However, another LIM homeobox gene, Lhx9, seems to be essential only
for the proliferation of somatic cells of the gonadal ridge (8) by interacting with Wt1 to regulate Sf1 (9). LHX9 expression
increases in both XX and XY undifferentiated gonads, and then decreases as Sertoli and granulosa cells differentiate (10,
11). Several other factors are involved in cell proliferation in the gonadal primordium both in XX and XY embryos. For
instance, impairment of the signaling pathway of the insulin/insulin-like growth factor family in mouse knockout models with
disrupted Insr, Igf1r and Insrr leads to a significant reduction of the size of adreno-gonadal ridges in both XX and XY embryos
(12). Also in mice with a knockout of Tcf21, gonads are severely hypoplastic in both XX and XY fetuses (13). GATA4 (14)
and the homeoproteins SIX1 and SIX4 are also essential for early proliferation of gonadal precursor cells and for FOG2-
and SF1-regulated SRY expression (15). The Notch signaling pathway is also involved in somatic cell lineage commitment
during early gonadogenesis in mice. Conditional knockout of Numb and Numbl (antagonists of Notch signaling) in the
undifferentiated gonad results in disruption of the coelomic epithelium and reduction of somatic cell numbers in the gonads
(16). Finally, NRG1 is also required in a dose-dependent manner in order to induce somatic cell proliferation in the gonads
(17). Since cell proliferation is more important in the male than in the female early developing gonad (18, 19), sex-reversal
is often observed in XY embryos with an alteration of gonadal cell proliferation (12). It has been suggested that this is due
to a reduction in the number of SRY-expressing pre-Sertoli cells, resulting in very low levels of SRY expression that are
insufficient to trigger testicular differentiation (discussed in ref. (20).

TABLE 2. Factors Involved in Early Gonadal Ridge Development

Gene Chromosomal Expression Function
localization
ATRX (Alpha- Xq21.1 Widespread Nucleotide excision repair
thalassemia/mental and initiation of
retardation syndrome, transcription
Helicase 2, X-Linked)
CITED2 (CBP/p300- 6q24.1 Widespread WT1 cofactor, regulating
interacting transactivator, SF1 expression in the
with glu/asp-rich c-terminal adrenogonadal primordium
domain, 2)
EMX2 (homolog of empty 10q26.11 Telencephalon Arealization of the
spiracles homeobox gene and epithelial neocortex and induction of
2) components of the the mesenchyme
urogenital system
GATA4 (GATA-binding 8p23.1 Widespread Regulation of coelomic
protein 4) epithelium thickening
INSR (Insulin receptor) 19p13.2 Widespread Metabolic, cell proliferation
IGF1R (Insulin growth 15q26.3
factor 1 receptor)
INSRR (Insulin receptor- 1q23.1
related receptor)
JMJD1A, or KDM3A 2p11.2 Testis, ovary, Demethylases histone H3
(Lysine-Specific kidney, lung, (epigenetic regulation by
Demethylase 3A) heart, brain, liver, modification of chromatin
skeletal muscle, conformation)

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 pancreas, and
spleen
LHX1 (LIM homeobox gene 17q12 Primitive streak, Differentiation and
1) prechordal and development of the head,
intermediate neural and lymphoid
mesoderm, brain, tissues and urogenital
thymus, tonsil structures
LHX9 (LIM homeobox gene 1q31.3 Central nervous Activation of SF1 in
9) system, forelimb gonadal primordia
and hind limb
mesenchyme and
urogenital system
NR5A1 (Nuclear receptor 9q33.3 Gonadal ridges, Stabilization of intermediate
subfamily 5, group A, adrenal gland mesoderm, and
member 1, also SF1: primordia, transcriptional regulation of
Steroidogenic factor 1, or hypothalamus and several genes (StAR,
AD4BP: Adrenal 4 binding pituitary steroid hydroxylases,
protein, or FTZF1: Fushi aromatase, AMH, DAX1
tarazu factor homolog 1) and many other)
NRG1 (Neuregulin 1) 8p12 Widespread, Progenitor cell proliferation
including in the gonads
progenitors of
somatic gonadal
cells
NUMB 14q24.2-q24.3 Widespread, Antagonize NOTCH
and and including coelomic signaling, involved in
NUMBL 19q13.2 epithelium mediating asymmetric
division of cells in the
coelomic epithelium
PAX2 (Paired box gene 2) 10q24.31 Mesonephros, Regulation of WT1
metanephros, expression and of
adrenals, spinal mesenchyme- to-
cord, hindbrain epithelium transition
and optic and otic
vesicles
SIX1 / SIX 4 (Sine oculis 14q23.1 Urogenital ridge Regulation of gonadal
homeobox 1 and 4) derivatives precursor cell proliferation,
and of Fog2 and Sf1
TCF21 (Transcription factor 6q23.2 Epithelium of the Basic helix-loop-helix
21, also POD1: Podocyte- developing transcription factor
expressed 1) gastrointestinal,
genitourinary, and
respiratory
systems
WT1 (Wilms tumor 11p13 Urogenital ridge DNA- and RNA-binding
associated gene 1) derivatives protein with transcriptional
and post-transcriptional
regulating capacity

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The differentiation of the gonadal ridge from the always present. In 46,XX females, SF1 mutations have
intermediate mesoderm requires the expression of sufficient been described in patients with primary ovarian
levels of WT1 and SF1. WT1 was initially isolated from insufficiency (29, 30). SF1 is one of the increasing number
patients with Wilms' tumor, an embryonic kidney tumor of examples of dosage-sensitive mechanisms in human sex
arising from the metanephric blastema. By alternative differentiation, since mutations at the heterozygous state
splicing and alternative translation initiation, WT1 encodes are sufficient to induce sex reversal in XY individuals
more than 20 isoforms of a zinc-finger protein acting as (reviewed in refs. (29, 30).
transcriptional and/or post-transcriptional regulator (20).
The -KTS splicing variant of WT1, lacking the three amino Recent studies using single-cell RNA sequencing (scRNA-
acids lysine (K), threonine (T) and serine (S) at the end of seq) has shed light on the initial steps of lineage trajectories
the third zinc finger, is required for cell survival and and cell fate in the developing gonads (1, 37). A subset of
proliferation in the indifferent gonad, whereas the +KTS cells of the coelomic epithelium expressing GATA4, SF1
variant is involved in the regulation of SRY expression (21). and WT1 are likely to be the precursors of the somatic
The first indication of a role for WT1 in gonadal and renal lineages of the undifferentiated gonads: both the supporting
development was its expression pattern in the urogenital (Sertoli and granulosa) and the steroidogenic (Leydig and
ridges (22). During gonadal differentiation, WT1 is theca) cell populations of the differentiating gonads seem to
expressed in the coelomic epithelium and later in Sertoli and derive from SF1 and WT1-expressing cells present in the
granulosa cells (23). In mice with a knockout of WT1, neither genital ridge (1, 37, 38).
the kidneys nor the gonads develop (24). In humans,
mutations in the WT1 gene do not completely prevent The Germ Cells
urogenital ridge development but may result in gonadal
dysgenesis associated with nephroblastoma (Wilms' tumor)
Initially formed exclusively by somatic cells, the gonads are
and/or nephrotic syndrome owing to glomerular diffuse
subsequently colonized by the primordial germ cells
mesangial sclerosis (25-27).
(PGCs). PGCs derive from pluripotent cells of the posterior
proximal epiblast, which move, at a very early stage of
SF1, also known as Ad4BP or FTZF1 (HGNC approved
embryonic life, through the primitive streak into the extra-
gene symbol: NR5A1), initially described as a regulator of
embryonic region at the base of the allantois (39). Not all of
steroid hydroxylases, is an orphan nuclear receptor
these cells are committed to a germ cell lineage since they
expressed in the hypothalamus, the pituitary, the gonads
also give rise to extra-embryonic mesoderm cells (40).
and the adrenal glands (reviewed in refs. (28-30). In mice
The mechanisms responsible for specification of epiblast
with a knockout of the SF1 gene, the intermediate
cells to become PGCs vary between species (41-43). In
mesoderm is not stabilized and the gonadal and adrenal
mice, PCG specification involves several extraembryonic
primordia soon degenerate (31). SF1 also plays an
ectoderm-derived factors, including bone morphogenetic
important role in spermatogenesis, Leydig cell function,
protein 2 (BMP2) (44), BMP4 (45-47), BMP8B (46) and
ovarian follicle development and ovulation, as
WNT3 (48). Cells of the adjacent epiblast become
demonstrated by a gonad-specific disruption of SF1 (32). A
determined to develop through the germline as they start
recurrent heterozygous p.Arg92Trp variant of the gene is
expressing BLIMP1 (44), encoded by Prdm1. BLIMP1
associated with testicular development in XX subjects (33,
represses somatic fate in the epiblast cells, and together
34). WT1, through interaction with CITED2 (35, 36), and
with PRDM14 and AP2G (encoded by Tfap2c), constitute a
LHX9 (8) regulate the expression of SF1 upstream of the
tripartite genetic network necessary and sufficient for
gonadal development cascade. GATA4 and SOX-family
mouse PGC specification (49). PRDM14 regulates the
factors also regulate SF1 expression in the gonad (28). In
restoration of pluripotency and epigenetic reprogramming in
humans, the phenotype resulting from SF1 mutations does
PGCs, reestablishing the expression of the pluripotency
not exactly match that of Sf1 knockout mice: the clinical
factors OCT3/4 (encoded by Pou5f1), SOX2 and NANOG
spectrum includes severe and partial forms of testicular
(41).
dysgenesis, anorchidism, and even male infertility in
normally virilized individuals; adrenal insufficiency is not

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Instead, embryos of other mammals do not form a structure expression of interferon-induced transmembrane proteins 1
equivalent to the extraembryonic ectoderm, and the origin and 3 (IFITM1 and IFITM3) in the surrounding mesoderm
of the signals that initiate PGC specification remain largely (51). During migration, PGCs proliferate actively but do not
unknown. Notably, in the human embryo, PGC-like cells differentiate (40). Germ cell migration through the dorsal
express very low or no PRDM14, maintain NANOG mesentery to the gonadal ridges and survival/proliferation in
expression, and do not express SOX2. Furthermore, the both XX and XY embryos is driven by signaling between kit
expression of SOX17 is detected before that of BLIMP1 and ligand (KITL, also known as Stem cell factor [SCF], Steel
could be involved in the regulation of PGC specification and factor or mast cell growth factor [MGF]), which is expressed
maintenance of their pluripotency in humans (49, 50). in somatic cells of the gonadal ridge and the hind gut along
the pathway of PGC migration, and its receptor present in
Widespread chromatin modifications are observed: PGCs germ cells, C-KIT (Fig. 1) (52). PGC migration and genital
undergo genome-wide demethylation including erasure of ridge colonization is also dependent on stromal cell-derived
genomic imprints (44), thus reaching a ‘ground state’ in factor 1 (SDF1, also known as CXCL12) and its receptor
terms of epigenetic marks. Re-methylation of germ cell CXCR4 (53) and on interactions with extracellular matrix
genome occurs later during fetal life: in XY germ cells when proteins, like fibronectin and laminin, while proliferation
they have committed to the spermatogenic fate, and in XX and/or survival involve many other factors (39, 40, 52, 54).
germ cells just before ovulation (45).
In the 4thweek, PGCs have migrated and are present in the PGCs are in a bipotential state when they colonize the
yolk sac near the base of the allantois. They can be gonadal ridges, i.e. they still have the capacity to enter
identified by their expression of alkaline phosphatase, either spermatogenesis or oogenesis. Shortly afterwards,
OCT3/4 and the tyrosine kinase receptor C-KIT (Fig. 1A) induced by the gonadal environment, PGCs begin to
(40). Subsequently, PGCs become embedded in the wall of express DAZL, DDX4 (also known as MVH) and low levels
the hind gut, gain motility and migrate through the dorsal of SYCP3 (43), probably owing to promoter demethylation
mesentery to reach the gonadal ridges in the 5 thweek (Fig. (55). DAZL seems to induce PGCs capacity to respond to
1B). Early migration of PGCs is dependent on the specific male or female gonadal signals (56, 57).

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FIGURE 1. REGULATION OF GERM CELL MIGRATION. A: 4-WEEK EMBRYO. DIFFERENTIATION OF PRIMORDIAL
GERM CELLS (PGC) OCCURS FROM EPIBLAST-DERIVED CELLS PRESENT IN THE YOLK SAC NEAR THE BASE
OF THE ALLANTOIS. PGCS EXPRESS PMRD1, THE RECEPTORS C-KIT AND CXCR4, OCT3/4 AND ALKALINE
PHOSPHATASE. FIBRONECTIN AND LAMININ, TOGETHER WITH KITL, SDF1 AND IFITM 1 AND 3 ARE
EXPRESSED IN THE MESODERM ALONG THE PGC PATHWAY. B: 5-WEEK EMBRYO. PGCS MIGRATE ALONG
THE DORSAL MESENTERY OF THE HIND GUT TO THE GONADAL RIDGES.

SEX DETERMINATION pathway. When no testes are present, the genitalia develop
along the female pathway. The existence of ovaries has no
The Determining Role of Testicular effect on fetal differentiation of the genitalia. The paramount
Differentiation importance of testicular differentiation for fetal sex
development has prompted the use of the expression “sex
determination” to refer to the differentiation of the bipotential
The pioneering experiments of fetal sexual differentiation
or primitive gonads into testes.
carried out by Alfred Jost in the 1940’s clearly established
that the existence of the testes determines the sexually
In the next section, we describe the morphological aspects
dimorphic fate of the internal and external genitalia (Fig. 2)
of fetal testicular and ovarian differentiation and the
(58, 59). Irrespective of their chromosomal constitution,
underlying molecular mechanisms, involving genes
when the gonadal primordia differentiate into testes, all
mapping to sex-chromosomes (Fig. 3) and autosomes
internal and external genitalia develop following the male
(Table 3).

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FIGURE 2. Determining role of the testes in fetal sex differentiation. In normal females,
Müllerian ducts are maintained, Wolffian ducts regress. In males, the opposite occurs. In
castrated fetuses, irrespective of genetic or gonadal sex, the reproductive tract differentiates
according to the female pattern.

The Fate of the Undifferentiated Gonadal gonad, the upregulation of SRY induces a destabilization of
Ridge that balance, initiating the testis cascade.

As already mentioned, the gonadal ridges are bipotential THE MALE DETERMINING PATHWAY
until the 6th week after conception in humans, i.e. they have
the capacity to follow the testicular and the ovarian Sex-Determining Region on the Y Chromosome (SRY)
pathways. The discovery of the testis-determining factor
SRY in 1990 was followed by the progressive unveiling of Compelling evidence for the importance of the Y
robust networks of genes, whose balanced expression chromosome for the development of the testes, irrespective
levels either activate the testis pathway and simultaneously of the number of X chromosomes present, has existed since
repress the ovarian pathway or vice versa (Fig. 4). During 1959 (61, 62). However, the identification of the testis-
the formation of the undifferentiated gonadal ridges, a determining factor (TDF) on the Y chromosome did not
common genetic program is established in the supporting- prove easy and several candidates (e.g. HY antigen, ZFY)
cell lineage deriving from the multipotent somatic progenitor were successively proposed and rejected until the SRY
cells in both XX and XY embryos, characterized by a (Sex-determining region on the Y) gene was cloned in 1990
balanced expression of pro-Sertoli (SOX9, FGF9, PGD2) in man (63) and mouse (64). Experimental (65, 66) and
and pregranulosa (WNT4, RSPO1, FST and CTNNB1) clinical (67, 68) evidence clearly established that SRY was
genes (37, 60). Under physiological conditions in the XY the testis-determining factor. Considerable progress has
been made since SRY was identified, and it has become

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clear that sex determination is a far more complex process, that undergo meiotic recombination with homologous
regulated by competing molecular pathways in the sequences of the X chromosome during male
supporting cell lineage of the bipotential gonad. SRY has spermatogenesis. The proximity of SRY to PAR1 makes it
lost much of its prestige because it has a very weak susceptible to translocation to the X chromosome following
transactivation potential, is expressed very transiently in the aberrant recombination and provides an explanation for
mouse, weakly at best in other mammals and not at all in 80% of XX males (73) and for a low proportion of XY
sub-mammalian species (reviewed in ref. (20). Instead, its females. Indeed, mutations and deletions of the SRY locus
target gene encoding the transcription factor SOX9 has only account for 15% of XY females (74, 75).
emerged as the master regulator of testis determination, the
main role of SRY consisting in upregulating the expression While SRY gene exists in almost all mammals as a single
of SOX9 during a very narrow critical time window (69). copy gene, the rat carries 6 copies and the mouse Sry gene
Once time is up, either SOX9 is able to maintain its own has a distinct structure from other mammalian SRY genes
expression with the help of feed-forward enhancing because of the presence of a long-inverted repeat. Also,
mechanisms succeeding in triggering Sertoli cell SRY expression varies between species: in mice a
differentiation or it is silenced by an opposing set of genes functional transcript is present only in pre-Sertoli cells for a
which impose ovarian differentiation. Timing and expression very short period during early gonadogenesis, in goats SRY
level determine which team wins (20, 70, 71) but the battle is expressed in all somatic and germ cells of the gonad
is never over, even after birth, at least in mice (72). during fetal life and restricted to Sertoli cells and
spermatogonia in the adult testis. Human SRY is expressed
SRY is a member of a family of DNA-binding proteins in both Sertoli cells and germ cells at fetal and adult stages
bearing a high mobility group (HMG) box; its gene maps to (reviewed in ref. (20). Proteins that interact with SRY and
the short arm of the Y chromosome (Table 3), very close to could have a relevant function in gonadal differentiation
the pseudoautosomal region 1 (PAR1) (Fig. 3). PAR1 on Yp include SIP-1/NHERF2 (76) and KRAB-O (77).
and PAR2 on Yq are the only regions of the Y chromosome

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FIGURE 3. X and Y chromosome genes involved in sex determination and differentiation.
SRY: Sex-determining region Y chromosome; DAX1: DSS-AHC critical region X chromosome gene 1; AR:
Androgen receptor; and ATRX: Alpha-thalassemia/mental retardation syndrome X-linked are involved in in sex
determination and differentiation. Other genes present in the X and Y chromosomes are: AZF: azoospermia
factor; CSF2RA: Colony-stimulating factor 2 receptor alpha; DAZ: Deleted in azoospermia; FRA-X: Fragile X
syndrome; DMD: Duchenne muscular dystrophy; GK: Glycerol kinase; HY: Histocompatibility antigen Y; IL3RA:
Interleukin 3 receptor alpha; IL9R: Interleukin 9 receptor; Kal1: Kallmann syndrome 1; PAR: Pseudo-autosomal
regions; POLA: DNA polymerase alpha; RBMY: RNA-binding motif protein Y chromosome; SHOX: Short stature
homeo box; USP9Y: Ubiquitin-specific protease 9 Y chromosome; XIST: X inactivation-specific transcript; ZFX:
Zinc finger protein X-linked; ZFY: Zinc finger protein Y-linked.

Owing to its Y-chromosome localization, SRY can only be underlying the initiation of SRY expression begin to be
expressed in the XY gonadal ridge, thus playing a unraveled (Fig. 4). The +KTS splice variant of WT1 (21, 79,
paramount role in tilting the balance between testicular and 80), SF1 (20) and SP1 (81, 82) are able to activate SRY
ovarian promoting genes towards the male pathway. transcription. The transcriptional co-factor CITED2 acts in
the gonad with WT1 and SF1 to increase SRY levels to
A tight regulation of SRY expression is essential for fetal attain a critical threshold to efficiently initiate testis
gonadogenesis: both timing and level of expression are development (35). The +KTS isoform of WT1 might also act
determinant, as revealed by experiments in mouse showing as a posttranscriptional stabilizer of SRY mRNA (70).
that SRY levels must reach a certain threshold at a certain
stage of fetal development to induce testis differentiation The implication of GATA4 on SRY expression is less
(69). SRY expression commences between days 41 and 44 straightforward. The interaction between GATA4 and its
post-fertilization in humans (78). The mechanisms cofactor FOG2 in the gonadal primordium is required for

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normal Sry expression and testicular differentiation in mice activation of Y chromosome genes, amongst which is SRY.
(83). However, whether the effect is specific on Sry ATRX, also known as XH2, is an X-encoded DNA-helicase
transcription or more general on gonadal somatic cell whose mutation results in mental retardation, α-thalassemia
development was not evaluated. Functional GATA-binding and gonadal dysgenesis in XY individuals (89-91). ATRX
sites are present in the mouse and pig Sry promoter but not has a more general effect on chromatin remodeling, which
in the human SRY (84, 85). One possibility is that GATA4 seems to play an important role in the epigenetic regulation
interacts with WT1 (Fig. 4), mainly the +KTS isoform, which of sex determination (92).
binds to the SRY promoter and increases its transcriptional
activity (84). Alternatively, it has been proposed that GATA4 Several other experimental models impairing the
directly acts on the SRY promoter, based on the expression of signaling molecules, which are expressed
experimental observation that GADD45G binds and SRY in the early gonadal ridge in normal conditions, show
activates the mitogen-activated protein kinase kinase reduced or absent SRY expression, develop gonadal
MAP3K4 (also known as MEKK4) to promote agenesis and a female phenotype of the internal and
phosphorylation and activation of the p38 kinase (Table 3), external genitalia. LHX9 (8) is a potential regulator of SRY
which in turn phosphorylates GATA4 thus enhancing its expression. A direct effect of LHX9 on the SRY gene has
binding to the Sry promoter (85, 86) (Fig. 4). These results not been demonstrated but an indirect effect through SF1
are in line with those indicating that MAP3K4 is essential for upregulation has been postulated (20). Loss-of-function
testicular differentiation in mice (87). mutations of the mouse genes encoding the insulin receptor
(Insr), the IGF1 receptor (Igf1r) and the insulin related
SRY expression is also epigenetically regulated: the receptor (Insrr) also result in decreased or absent Sry
demethylase KDM3A, also known as JMJD1A, positively expression (12). However, these factors and signaling
regulates the expression of Sry in mice, as shown by the pathways affect cell proliferation, and decreased SRY
absence of testicular development and consequent sex expression might only reflect the reduced number of cells in
reversal in Jmjd1a-deficient XY mice (88). Histone the gonadal primordium. Indeed, many of these potential
methylation is an important mechanism of epigenetic regulators have not yet been proven to affect SRY
regulation: methylation of lysine 9 of histone H3 (H3K9) is a expression directly.
hallmark of transcriptionally suppressed chromatin.
JMJD1A demethylates H3K9, thus allowing transcriptional
TABLE 3. Factors Involved in Gonadal Differentiation
Gene Chromosomal Expression Function
localization
ATRX (Alpha- Xq21.1 Widespread Nucleotide excision repair and
thalassemia/mental initiation of transcription
retardation syndrome,
Helicase 2, X-Linked)
CBX2 (Chromobox 17q25.3 Widespread Regulation of homeotic genes.
homolog gene 2; or M33 Represses WNT4 signaling
mouse homolog of)
CITED2 (CBP/p300- 6q24.1 Widespread WT1 and SF1 cofactor, regulating
interacting transactivator, SRY expression in the gonad
with glu/asp-rich c-terminal
domain, 2)
COUP-TF2 (Chicken 15q26.2 Widespread Transcription factor (orphan
ovalbumin upstream nuclear receptor) likely involved
promoter transcription in mesenchymal-epithelial
factor 2), or NR2F2 interactions
(Nuclear receptor subfamily
2, group F, member 2)

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 CTNNB1 (β-catenin) 3p22.1 Widespread Upregulates WNT4, FST and
FOXL2
DAX1: DSS-AHC critical Xp21.2 Gonads, Antagonizes SRY, SOX9.
region on the X pituitary, Essential for normal testicular
chromosome 1); or NR0B1 adrenals and ovarian development
(Nuclear receptor subfamily
0, group B, member 1).
DHH (Desert hedgehog) 12q13.12 Sertoli cells Morphogenesis
(testis),
Schwann
cells
(peripheral
nerves)
DKK1 (Dickkopf, xenopus, 10q21.1 Widespread Represses WNT4 binding to the
homolog of, 1) LRP5/6 co-receptor
DMRT1 (Doublesex- and 9p24.3 Gonads and Antagonizes FOXL2
mab3-related transcription several other
factor 1) tissues
FGF9 (Fibroblast growth 13q12.11 Gonads and Upregulation of SOX9 and
factor 9) several other downregulation of WNT4
tissues
FGFR2 (FGF receptor 2) 10q26.13 Gonads and Upregulation of SOX9 and
several other downregulation of WNT4
tissues
FOG2 (Friend of GATA, 8q23.1 Widespread Repression of DKK1
gene 2, or ZFPM2: zinc
finger protein multitype 2)
FOXL2 (Forkhead 3q22.3 Gonads and Antagonizes SOX9. Survival of
transcription factor 2) eyelids meiotic germ cells
FST (Follistatin) 5q11.2 Widespread Antagonizes Activins. Survival of
meiotic germ cells
GADD45G (Growth arrest- 9q22.2 Widespread Phosphorylation of GATA4
and DNA damage-inducible
gene, gamma)
GATA4 (GATA-binding 8p23.1 Widespread Regulation of SRY expression
protein 4)
HHAT (Hedgehog 1q32.2 Gonads Two INHBB subunits form Activin
acyltransferase) B dimer, which induces vascular
endothelial cell migration to the
gonad
INHBB (Inhibin βB, Activin 2q14.2 Gonads Two INHBB subunits form Activin
βB) B dimer, which induces vascular
endothelial cell migration to the
gonad
JMJD1A; or KDM3A 2p11.2 Testis, ovary, Demethylases histone H3
(Lysin-specific demethylase kidney, lung, (epigenetic regulation by
3A) heart, brain,

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 liver, skeletal modification of chromatin
muscle, conformation)
pancreas,
and spleen
MAP3K1 (MAP/ERK 5q11.2 Widespread Phosphorylation of GATA4
Kinase Kinase 1; MEKK1;
MAPKKK1; MEK Kinase)
MAPK14 (Mitogen- 6p21.31 Widespread Phosphorylation of GATA4
activated protein kinase 14;
or p38-MAPK)
NR5A1 (Nuclear receptor 9q33.3 Gonadal Transcriptional regulation of
subfamily 5, group A, ridges, several genes (SRY, SOX9,
member 1, also SF1: adrenal gland STAR, steroid hydroxylases,
Steroidogenic factor 1, or primordia, aromatase, AMH, DAX1 and
AD4BP: Adrenal 4 binding hypothalamus many other)
protein, or FTZF1: Fushi and pituitary
tarazu factor homolog 1)
PDGFB (Platelet-derived 22q13.1 Endothelial Increase in cell proliferation in the
growth factor, beta cells gonadal interstitial tissue
polypeptide)
PDGFRA (PDGF receptor 4q12 Gonadal Increase in cell proliferation in the
α) interstitial gonadal interstitial tissue
cells and
several other
tissues
PTGDS (or PGDS2: 9q34.3 Gonads and Synthesis of prostaglandin D2
Prostaglandin D2 synthase) several other (PGD2), upregulation of SOX9
tissues and its nuclear translocation
RSPO1 (R-spondin family, 1p34.3 Gonads and Upregulates WNT4 by
member 1) skin sequestering the transmembrane
E3 ubiquitin ligases ZNRF3 and
RNF43.
Cooperates with WNT4 signaling,
by antagonizing DKK1, to
stabilize β-catenin and FST
SOX8 (SRY box 8) 16p13.3 Gonads and Transcriptional regulation of
several other SOX9, in cooperation with SF1
tissues
SOX9 (SRY box 9) 17q24.3 Testis, Triggers testis differentiation, and
cartilage regulates several testis-specific
genes
SOX10 (SRY box 10) 22q13.1 Gonads and Transcriptional regulation of
several other SOX9, in cooperation with SF1
tissues
SP1 (Specificity protein 1) 12q.13.13 Widespread Regulation of SRY expression

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 SRY (Sex-determining Yp11.31 Male gonadal Regulates SOX9 and triggers
region on the Y ridge testis differentiation
chromosome)
VEGFA (Vascular 6p21.1 Mesenchymal Induces vascular endothelial cell
endothelial growth factor A) cells of the migration to the gonad
gonadal ridge
and other
organs
WNT4 (Wingless-type 1p36.12 Gonads and Induces β-catenin and silences
MMTV integration site several other FGF9 and SOX9 by binding to
family, member 4) tissues Frizzled receptor
WT1 (Wilms tumor 11p13 Urogenital Transcriptional regulation and
associated gene 1) ridge post-transcriptional stabilization
derivatives of SRY
ZNRF3 (Zinc finger and ring 22q12.1 Widespread Inhibition of WNT signaling by
finger protein 3) targeting Frizzled receptor for
degradation by ubiquitination and
increased membrane turnover

SOX9: A Target of SRY haploinsufficiency resulting in campomelic dysplasia, a

polymalformative syndrome that includes sex-reversal due
SOX9, an autosomal member of the HMG-box protein to gonadal dysgenesis in XY individuals (93, 103), whereas
superfamily mapped to chromosome 17 q24 (93), is the gain-of-function of SOX9 in XX individuals leads to sex
master regulator of Sertoli cell differentiation (94). In the reversal (104).
mouse, SOX9 is expressed at low levels in the bipotential
gonads of both sexes under SF1 regulation (95), but In humans more distant regulatory regions of SOX9 have
persists only in testicular Sertoli cells after SRY expression been identified (105), and confirmed by observations in
has peaked (96-98). SRY and SF1 directly bind to several patients with XY gonadal dysgenesis. No mutation has been
sites within a 3.2-kb testis-specific enhancer (TES) or 1.4- found in the TES sequence (106), instead a 1.9 kb SRY-
kb of its core element (TESCO), present approximately 14 responsive subfragment of a 32.5 kb interval lying 607.1–
kb upstream of the Sox9 promoter and responsible for this 639.6 kb upstream of SOX9 —termed XY SR for XY Sex
expression pattern (95, 99). Together with SF1, SOX9 also Reversal— seems to be the core of the Sertoli-cell
binds and activates TES, thus maintaining its own enhancer of human SOX9. Heterozygous deletions
expression by autoregulation after transient SRY encompassing these sequences were identified in four
expression has ceased in the mouse. families with SRY-positive 46,XY gonadal dysgenesis
without campomelic dysplasia (107) and a deletion of a
SOX9 mimics SRY effects independently of SRY 557–base pair element named enhancer 13 (Enh13),
expression. In fact, overexpression of SOX9 during early reproduced in mice, led to XY sex reversal (108). This
embryogenesis induces testicular differentiation in two region is included in a 1.2-Mb deletion previously described
different models of transgenic XX mice (100, 101). in a case of 46,XY DSD with gonadal dysgenesis and no
Functional analysis of SOX9 during sex determination, by skeletal phenotype (109). Finally, in line with these
conditional gene targeting in mice, has shown that observations, overexpression of SOX9 is supposed to
homozygous deletion of Sox9 in XY gonads interferes with underlie testicular development in familial 46,XX SRY-
sex cord development and with activation of testis specific negative males with a 178-kb duplication or a 96-kb
markers (102). Further evidence for the role of SOX9 in triplication in sequences lying 500–600 kb upstream of
testicular development comes from observations in SOX9 (110, 111).
humans, in whom a double dose of SOX9 expression is SOX9 also affects the differentiation of the reproductive
required. Heterozygous mutations result in tract by upregulating the expression of anti-Müllerian

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hormone (AMH) (112, 113), a Sertoli cell factor involved in gonad (121), and prostaglandin D2 (PGD2) has been
male differentiation of the internal genitalia (see below). shown to upregulate SOX9 in the absence of SRY (122).

SOX8 and SOX10 are two other members of the SOX family SOX9 expression is maintained at high levels in the male
expressed in the gonads and in several other tissues. gonad despite down-regulation of SRY soon after testicular
During mouse embryo development, the expression of determination, at least in the mouse (97, 98). As mentioned,
SOX8 and SOX10 is triggered shortly after that of SOX9, SOX9 is capable of autoregulating its expression (95), and
but at lower level (114-117). SOX8 is regulated by SOX9 other members of the SOX family like SOX3, SOX8 and
(102). Like SOX9 itself, SOX8 and SOX10 can synergize SOX10 are also able to interact with SF1 to maintain SOX9
with SF1 and upregulate SOX9 expression (Fig. 4) upon expression in the male gonad (20, 117).
binding to TESCO (20). SOX8 can bind the canonical target
DNA sequences and activate AMH transcription acting Observations made in XY intersex patients with normal SRY
synergistically with SF1, but with less efficiency than SOX9 together with the discovery of proteins showing a sexually
(114, 118). Later during fetal development, an interaction dimorphic pattern of expression in the gonads following
between SOX9 and SOX8 is required for basal lamina SRY peak have helped to identify other loci, likely to be
integrity of testis cords and for suppression of FOXL2, two involved in testicular differentiation, which are discussed
events essential to the normal development of testis cords below.
(117).
FGF9 and PGD2: Maintaining SOX9 Expression Levels
An X-linked member of the SOX family, SOX3, although not
involved in the normal pathway of fetal gonadal SOX9 upregulates the expression of FGF9 and the
differentiation, is capable of inducing SOX9 expression and synthesis of prostaglandin D2 (PGD2) catalyzed by PGD
testis differentiation when ectopically expressed in the XX synthase. FGF9 interacts with its receptor FGFR2, initiating
gonad (119). It is also possible that indirect mechanisms a feed-forward loop that maintains SOX9 expression and
mediate Sox9 activation, in line with the hypothesis also results in downregulation of WNT4 expression (123-
indicating that SRY might act as a repressor of a negative 126) (Fig. 4). Independently of FGF9, PGD2 interacts with
regulator of the male cascade (120). For instance, targeted its receptor DP to induce SOX9 expression (122, 127) and
disruption of Foxl2 leads to SOX9 upregulation in the XX its nuclear translocation (127, 128), thus increasing its
availability to target genes (80).

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FIGURE 4. Schematic representation of molecular mechanisms involved in determining the fate of the
undifferentiated gonadal ridge. Black arrows indicate a positive regulation; double arrows indicate a positive
feedback loop; red lines indicate a negative regulation; double red lines indicate a mutual antagonism. In the 6th
week of embryonic life, the gonadal ridge is sexually undifferentiated, and various factors are expressed at the
same levels in the XX and the XY gonads. During the 7th week, in the XY gonad, SRY expression is triggered, and
the male pathway prevails driving to the formation of the coelomic vessel. In the XX gonad, the female pathway
prevails, and there is no formation of the coelomic vessel. Reprinted with permission from ref. (129) Freire AV,
Ropelato MG, Rey R. Ovaries and Testes. In: Kovacs C, Deal C, Eds. Maternal-Fetal and Neonatal Endocrinology.
1st Edition. Boston: Academic Press-Elsevier, 2020, pp. 625-641. ISBN 9780128148235. Copyright © 2020 Elsevier
Inc.

As already discussed, somatic cell proliferation is critical for gonads at the same levels: FGF9 near the coelomic surface
early testicular differentiation (18). FGF9 and WNT4 act as and WNT4 near the mesonephric border (130). When SRY
antagonistic signals in the first steps of differentiation of the expression is initiated and upregulates SOX9 in the XY
gonadal ridge (130). FGF9 controls cell proliferation in a gonadal ridge, the balance between FGF9 and WNT4 is
sexually dimorphic fashion: the disruption of FGF9 disrupted: SOX9 enhances FGF9 expression which in turn
expression by targeted deletion in transgenic mice does not maintains high SOX9 levels thus resulting in a feed-forward
affect XX gonads but prevents testicular differentiation and loop that accelerates commitment to the male pathway.
results in sex reversal in XY mice (131). In the mouse, FGF9 WNT4 expression is downregulated when a threshold level
and WNT4 are expressed in the undifferentiated XX and XY of FGF9 is reached (130). FGF9 controls the proliferation of

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a cell population that gives rise to Sertoli progenitors (19). (Dosage Sensitive Sex-reversal) region on Xp21, was the
In Fgf9 knockout mice, initial Sertoli cell differentiation is not first putative testis repressor and/or ovarian determining
hindered: SRY and SOX9 expression is observed but soon gene. A duplication of DSS results in sex-reversal in 46,XY
weakens resulting in an aborted differentiation of Sertoli cell patients (139), and DAX1 overexpression in transgenic XY
precursors (130). Although in experimental conditions, mice impairs testis differentiation by antagonizing the ability
FGF9 is capable of inducing proliferation of coelomic of SF1 to synergize with SRY action on SOX9 (140, 141)
epithelium cells in XX gonadal ridges, this does not result in (Fig. 4). However, the disruption of Dax1 gene in XX mice
Sertoli cell differentiation, clearly indicating that increasing does not prevent ovarian differentiation (142). Furthermore,
cell proliferation is not sufficient to induce testicular DAX1 is essential for normal testicular cord formation (143,
differentiation, and that other pro-testicular signals are also 144). These observations in rodent models, together with
required (131). FGF9 and SOX9 also upregulate AXIN1 and DAX1 expression pattern in the human fetus showing
GSK3β, which promote the destabilization of β-catenin and, persistently low levels in both XX and XY gonads from 33
thus, serve to block ovarian development (132). days post-fertilization (i.e. the bipotential stage) through 15
fetal weeks (78), strongly suggest that low DAX1 levels are
DMRT1, DAX1 and Other Factors Modulating Testis necessary for gonadal development in both sexes.
Versus Ovary Antagonism Abnormally low or high DAX1 expression result in abnormal
gonadal differentiation (145).
DMRT1 is a member of the DM domain transcription factor
family which appears to play a conserved role in vertebrate CBX2, the human homolog of murine M33 (146), does not
male gonad development. In mice, DMRT1 –but not DMRT2 seem to activate SRY expression as initially proposed
or DMRT3– is expressed and required in both germ cells (147), but may act as a stabilizer of SRY action and the
and Sertoli cells of the testis (133). Overexpression of testis pathway by repressing WNT4 downstream target
DMRT1 in XX mice inhibits WNT4 and FOXL2 expression LEF1, involved in ovarian differentiation (148). Interestingly,
and results in partial testicular differentiation and male biallelic mutations in CBX2 were found in a 46,XY girl with
genital development (134), while loss of DMRT1 expression ovarian tissue (149), and XY mice with inactivated Cbx2
activates FOXL2 and reprograms Sertoli cells into developed as female (146).
granulosa cells, even in postnatal life, suggesting that
DMRT1 is essential to maintain mammalian testis MAP3K1, unlike MAP3K4 (87), is not essential for testicular
differentiation life-long in mice (135, 136). differentiation and development in mice (150), but it
modulates the balance between testicular and ovarian male
In humans, deletions of chromosome 9p involving DMRT1, pathways by sequestration of AXIN1 (see “Genetic
DMRT2 and DMRT3 genes are associated with XY male- pathways of ovarian differentiation”). In humans, mutations
to-female sex reversal due to gonadal dysgenesis. Patients in the MAP3K1 gene have been associated with testicular
also present with mental retardation and typical craniofacial dysgenesis (151, 152).
dysmorphia, including trigonocephaly, upward-slanting
palpebral fissures, and less frequently hypertelorism, Similarly, inactivating variants that disrupt ZNRF3 function
epicanthus, flat nasal bridge, low-set ears, microstomia, result in 46,XY DSD in humans and to sex reversal in mice,
micrognathia, short neck, widely spaced nipples, square likely due to gonadal dysgenesis (153).ZNRF3 is an E3
hyperconvex nails, dolichomesophalangy and hypotonia ubiquitin ligase that promotes the degradation by
(137, 138). ubiquitination and the turnover of Frizzled, a WNT receptor
(Fig. 5) (154, 155).
DAX1 (HGNC approved gene symbol: NR0B1), encoding
for an orphan nuclear receptor and mapping to the DSS

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FIGURE 5. WNT and RSPO actions. Under steady state conditions (red dotted arrows), ZNRF3 provokes the
ubiquitination and degradation of Frizzled, receptor of WNT family factors. GSK3 phosphorylates β-catenin,
which is then degraded. R-spondin (RSPO) family members binding to their receptors LGR4/5/6 results in
complex formation with ZNRF3. Consequently, more Frizzled molecules become available for WNT signaling.
Under these conditions (blue full arrows), the complex formed by GSK3, Axin, CKIα and APC is recruited to the
WNT–receptor complex and inactivated, allowing β‑catenin to translocate to the nucleus and regulate target
genes.

COUP-TF2, encoded by NR2F2, is a transcription factor MAMLD1 is expressed in fetal Sertoli and Leydig cells,
likely involved in mesenchymal-epithelial interactions under the control of SF1 (157, 158), and gene variants have
required for organogenesis. In the fetal gonads, COUP-TF2 been associated with a broad phenotypic spectrum of DSD
expression increases as the ovaries develop, and loss-of- (159). However, Mamld1 knockout mice depict a very mild
function mutations in NR2F2 have been described in 46,XX reproductive phenotype (160). The precise role of MAMLD1
ovotesticular SRY-negative DSD (156), indicating that still needs to be established.
COUP-TF2 may be involved in driving the balance towards
ovarian differentiation. Stabilization of Testis Differentiation:
Vascular, Cellular and Molecular Pathways

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In the XY fetus, the initially amorphous cluster of gonadal in which Leydig cells will soon appear. Beyond
cells becomes segregated in two compartments, testicular vascularization, which is necessary to allow efficient export
cords and interstitial tissue, during the 7th week of gestation of testosterone, cell migration from the mesonephros largely
(3). These architectural changes are heralded by gonadal contributes to testicular organogenesis (165, 166) and is
ridge vascularization, a highly dynamic and sexually antagonized by the initiation of meiosis in germ cells (167).
dimorphic process. At variance with the differentiating ovary
that recruits vasculature by typical angiogenesis, the XY The molecular mechanisms underlying sex-specific
gonad recruits and patterns vasculature by a remodeling gonadal vascularization are being progressively unraveled.
mechanism: pre-existing mesonephric vessels disassemble A vascular-mesenchymal cross-talk between VEGFs and
and generate a population of endothelial cells that migrate PDGFs drives gonadal patterning during early fetal life (Fig.
to the gonad, below the coelomic epithelium, where they 4). VEGF-A, expressed in interstitial mesenchymal cells of
reaggregate to form the coelomic vessel, an arterial vessel the undifferentiated gonadal ridge, induces vascular
that runs the length of the testis at its antimesonephric endothelial cell migration to the gonad. In turn, PDGF-B
margin (161, 162). The formation of this vessel is one of the expressed by the endothelial cells is responsible for an
earliest hallmarks of testis development that distinguishes it increase in cell proliferation in the gonadal interstitium, upon
morphologically from the developing ovary (161, 163). binding to its receptor PDGFRα. Disruption of vascular
Evidence now exists for a close spatial relationship between development blocks formation of testis cords (168, 169)
testis vascularization and cord formation (162, 164). while not affecting Sertoli and Leydig cell specification(169).
Furthermore, all of the cells migrating from the In the XX gonadal ridge, WNT4 and its downstream target
mesonephros to the coelomic zone of the differentiating follistatin (FST) repress endothelial cell migration, probably
testis express endothelial markers such as VE-cadherin, an by antagonizing Activin B (Fig. 4). In the XY gonad, the
indication that incoming endothelial, rather than peritubular SRY/SOX9 pathway downregulates WNT4/FST thus
myoid cells, are required for testicular cord formation (164). allowing Activin B, VEGF and other potential as yet
Subsequently, Sertoli cells aggregate and enclose germ unidentified factors to induce male-specific gonadal
cells. The interaction between differentiating peritubular vascularization (170). Genes involved in male sex
myoid cells and Sertoli cells results in the formation of determination are shown in Fig. 6.
basement membrane of the testicular cords. Mesenchymal
cells and matrix and blood vessels fill the interstitial space,

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FIGURE 6. Sex determination and differentiation. Reprinted with permission from ref. (171): Grinspon RP, Rey RA
Molecular Characterization of XX Maleness. International Journal of Molecular Sciences (2019) 20:6089, © 2019 by
the authors. Licensee MDPI, Basel, Switzerland.

Differentiation of Sertoli and Leydig Cells lineage (pre-Sertoli cells) (38, 172-174). SRY-expressing
pre-Sertoli cells lying beneath the coelomic epithelium play
As already mentioned, both the supporting and the a central role in the migration of cells from the mesonephric
steroidogenic cell lineages derive from WT1-positive mesenchyme into the differentiating gonad (175).
somatic progenitors present in the undifferentiated gonadal Experimental work using XX-XY chimeras has shown that
ridges. Wt1-positive cells can express HES1, a Notch not 100% of Sertoli cell precursors need to express SRY to
effector, or not (38). In the subset of WT1-positive and differentiate along the male pathway: in fact, up to 10% of
HES1-negative cells, having delaminated from the coelomic Sertoli cells were XX. However, a threshold number of SRY
epithelium in the central part of the indifferent gonad, SRY expressing –i.e. XY– cells seems to be essential in order for
expression is induced giving rise to the supporting cell

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Sertoli cell differentiation, and thus testicular development, 191, 192). Differentiating Sertoli cells also express growth
to be guaranteed (176). factors, like nerve growth factors (NGFs), which can induce
cell migration from the mesonephros acting through their
Along with SRY, FGF9 might have a role in inducing receptors TRKA (NTRK1) and TRKC (NTRK3) (193, 194).
mesonephric cell migration into the developing fetal testis Sertoli cells aggregate around large, spherical germ cells,
and Sertoli cell differentiation. FGF9 is expressed in Sertoli with a large nucleus and pale cytoplasm, called gonocytes
cells of the fetal testis and Fgf9-null mice have dysgenetic at this stage, which can be observed in the center of
gonads (131, 177) (see below). testicular cord cross-sections (186). The structural basis of
cord formation seems to be dependent on basal lamina
Vanin-1, a cell-surface molecule involved in the regulation deposition between Sertoli and peritubular cells with
of cell migration, might also be responsible for differentiating myofibroblastic characteristics (166). In the interstitial
Sertoli cell association with, and adhesion to, migrating compartment, connective tissue, blood vessels and Leydig
peritubular cells (178). Nexin-1, expressed by early Sertoli cells can be observed. As described above, one particular
cells, could act to maintain the integrity of the basal lamina feature of testicular vasculature is the formation of the
(178). coelomic vessel, a large vessel that appears below the
coelomic epithelium very early in testicular differentiation
Desert hedgehog (DHH) and its receptor PATCHED2 might (161, 195). Surrounding the gonad, the basement
also play a role in Sertoli-peritubular cell interaction and membrane layer underlying the coelomic epithelium
basal lamina deposition (179, 180). DHH is a protein thickens to form the tunica albuginea.
secreted by fetal Sertoli cells, but not by somatic
components of the fetal ovary, immediately after testicular Sertoli and germ cell numbers increase exponentially in the
determination (181). Patched2 is expressed in germ, human fetal testis throughout the second trimester (196) in
peritubular and interstitial cells of the testis (182). Testes response to FSH acting through its receptor in Sertoli cells
develop abnormally during fetal life in Dhh null mice, (197-199), and androgens acting indirectly through the
resulting in XY sex-reversal. Seminiferous cords are peritubular myoid cells (200). This probably explains why
disorganized owing to defects in the basal lamina and newborns with congenital hypogonadotropic hypogonadism
peritubular cells, with germ cells occasionally lying in the have small testes and low serum levels of Sertoli cell
interstitial tissue, and Leydig cells are hypoplastic (179, markers, such as AMH and inhibin B (201, 202). Sertoli cells
180). Homozygous mutations of DHH in 46,XY patients are do not reach a mature state, and meiosis is not initiated in
associated with gonadal dysgenesis (183, 184). the human testis until pubertal age, when all Sertoli cells
reach a high expression level of the androgen receptor
DHH, like other members of the hedgehog family, (203-206). In mice, NRG1 and its receptors ERBB2/3 are
undergoes post-translational modifications including N- also essential for Sertoli cell proliferation, and Nrg1 gene
terminal palmitoylation by HHAT (hedgehog acyl- invalidation leads to Sertoli cell hypoplasia and micro-
transferase), which is essential for efficient signaling. A orchidism (17).
mutation leading to defective HHAT function was found to
cause complete gonadal dysgenesis and female phenotype Morphologically and functionally distinct from testicular
in two 46,XY patients (185). cords, the interstitial compartment contains developing
Leydig cells (Fig. 7), the main androgen producing cells in
Testicular cord formation can be detected in human fetuses the male. The origin of Leydig cells has not been clearly
13-20 mm crown-rump length (43-50 days) beginning in the established: the precursors of fetal Leydig cells have been
central part of the gonad (186). Cord formation is heralded proposed to be either migrating cells from the coelomic
by the development of a new type of cell, the primitive epithelium, the mesonephros or the neural crest or resident
Sertoli cell, characterized by a polarized, large and clear cells present in the adreno-gonadal primordium (reviewed
cytoplasm with abundant rough endoplasmic reticulum and in refs (20, 207, 208). According to the latter hypothesis, a
complex membrane interdigitations (187), a downregulation subset of SF1-expressing cells gives rise to all
of desmin and an upregulation of cytokeratins (188), and the steroidogenic lineages of the gonads and adrenal cortex.
expression of SOX9 (97), AMH (189, 190) and DHH (184, This is supported by the finding of adrenal markers (209)

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and adrenal-like cells in the fetal testis (210, 211) and of after testicular cords have completely formed— and soon
adrenal rests in the testes of male patients with congenital begin to produce testosterone, which plays an essential role
adrenal hyperplasia (212). Mesonephric cells expressing in the stabilization of Wolffian ducts and the masculinization
nestin, a cytoskeletal filament initially characterized in of external genitalia. Leydig cells also produce insulin-like
neural stem cells, are a multipotent progenitor population growth factor 3 (INSL3), a growth factor responsible for the
that gives rise to Leydig cells, pericytes and smooth muscle transabdominal phase of testicular descent (216-218).
cells. However, the first cohort of Leydig cells derive from Although the initial differentiation of fetal Leydig cells
nestin-negative cells, confirming the multiple origins of fetal depends, at least partially, on Sertoli cell-secreted PDGFs
Leydig cells (213). binding to PDGFRα (219) independently of gonadotropin
action (220), further Leydig cell differentiation and
Another particular feature of the mouse testis is that Leydig proliferation depends on placental hCG in the first and
cell populations can be divided into fetal and adult Leydig second trimesters of fetal life and on fetal pituitary LH
cells according to the time they arise. Fetal Leydig cells thereafter acting on the LH/CG receptor (221). At mid-
disappear after birth and are replaced by adult Leydig cells gestation, interstitial tissue is literally packed with Leydig
at puberty (214). Despite their similar functions, fetal and cells; afterwards their number decreases (196, 215).
adult Leydig cells show morphologic and gene expression
differences: some progenitor cells that lose Wt1 expression SF1 action, is suppressed by WNT4-activated DAX1
and are HES1-negative/GLI1-negative become located to expression (222). By counteracting WNT4, and thus
the interstitial tissue, do not express SOX9 and differentiate downregulating DAX1 in interstitial cells of XY gonads, SRY
into fetal Leydig cells, under the effect of the Notch signaling might indirectly enhance SF1 action (223, 224). Finally,
pathway. Another subset of cells that expresses HES1 and ARX is an X-chromosome gene identified in patients with X-
GLI1, under the Hedgehog signaling pathway, are not linked lissencephaly and genital abnormalities probably
initially steroidogenic, but give rise to adult Leydig cells in associated with a block in Leydig cell differentiation (225).
postnatal life (38). FGF9 (131, 177) and DHH (180) are Sertoli cell-secreted
signals involved in Leydig cell differentiation.
In the human fetus, Leydig cells can be identified in the
interstitial tissue by the beginning of the 8th week (215) —

FIGURE 7. Leydig cells accumulate in the testicular interstitial tissue of a 90-mm male human fetus (11th week).
Large eosinophilic Leydig cells with a prominent nucleus are interspersed with mesenchymal cells.

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Timing of Testicular Differentiation inhibition of germ cell entry into meiosis needs to be
elucidated (234, 235).
In order for the fetal testis to adequately differentiate and
secrete masculinizing hormones, not only do all these Chromosomal constitution does not influence sex
factors need to be present at sufficient levels in the right cell differentiation of germ cells: XX germ cells surrounded by
lineage, but their expression must also be initiated within a Sertoli cells differentiate into spermatogonia, whereas XY
narrow time window. In mice, the ability of SRY to induce germ cells in an ovarian context differentiate into oogonia
testis development is limited to a time window of only 6 and then enter meiosis (236). However, germ cells whose
hours after the normal onset of expression in XY gonads. If karyotype is discordant with the somatic lineages fail to
SRY is expressed later, Sox9 gene activation is not progress through gametogenesis and enter apoptosis later
maintained due to failure of FGF9/WNT4 signaling to switch in life.
to a male pattern (69).
The influence of germ cells on the developing gonad is
Germ Cell Interaction with Somatic Cells in the sexually dimorphic: Germ cell progression through meiosis
Developing Testis: Repression of Meiosis is essential for the maintenance of the fetal ovary, otherwise
prospective follicular cells degenerate and streak gonads
Upon arriving in the undifferentiated genital ridge, by the result. In contrast, the development of the testes is not
end of the 5th week, germ cells continue to proliferate by hindered by the lack of germ cells (195).
mitosis and maintain bipotentiality for approximately one
week. Then germ cells in the male gonad become enclosed STABILIZATION OF OVARIAN
in the seminiferous cords and differentiate into the DIFFERENTIATION: CELLULAR AND
spermatogonial lineage, which does not enter meiosis until
MOLECULAR PATHWAYS
the onset of puberty. Gonocyte proliferation in the fetal testis
is inhibited by androgens (226). Prevention of entry into
Genetic Pathways of Ovarian Differentiation
meiosis was first thought to be a specific effect of male
somatic cells since germ cells entering a prospective ovary
The pathway leading to ovarian differentiation and
or those which have failed to enter gonads of either sex
stabilization is far more complex than what was originally
enter meiosis at approximately the same time and develop
hypothesized. In humans, the absence of an active SRY
into oocytes, irrespective of their chromosomal pattern
gene –e.g. SRY mutations or deletions of the Y
(227). Subsequent studies shed light on the sexually
chromosome involving the SRY locus– results in gonadal
dimorphic evolution of gametogenesis in the fetal gonads.
dysgenesis of variable degrees, but is not sufficient to allow
The mesonephros from the indifferent gonad, as well as the
ovarian differentiation: no oocyte meiotic progression or
lung and adrenal gland, synthesize retinoic acid that acts as
follicle development has been described, even during fetal
a meiosis inducer (228, 229). Germ cells embedded in the
life. Recent findings suggest that most probably the
seminiferous cords do not enter meiosis because they are
coordinated action of several factors is needed for the
protected from retinoic acid action: mouse Sertoli cells
differentiation and stabilization of the ovaries (237-239)
express two factors that prevent meiosis onset: FGF9 (230)
(Table 3, Figs. 4, 6 and 8).
and CYP26B1, an enzyme that catabolizes retinoic acid
(231, 232). NANOS2, expressed in germ cells, is also a
WNT4 is a secreted protein that functions as a paracrine
meiosis-preventing protein, since in the fetal testis it
factor to regulate several developmental mechanisms. WNT
represses the expression of STRA8 (233) (for details on
proteins bind to the frizzled (FZ) family of membrane
STRA8, see “Genetic control of oogenesis and
receptors and LRP5/6 co-receptors, leading to the
folliculogenesis“. In human fetal testis, CYP26B1 does not
activation of the phosphoprotein disheveled (DVL) and a
seem to be expressed, and the mechanism underlying the
subsequent increase in cytoplasmic β-catenin levels owing

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to an inhibition of its degradation rate (240). In turn, WNT4
is upregulated by the action of β-catenin, which establishes β-catenin also activates FOXL2 winged helix/forkhead
a positive feedback loop, and also indirectly by the transcription factor, expressed in germ and somatic cells,
GATA4/FOG2 complex, which represses DKK1 (241). more strongly in the female than the male fetal gonad from
DKK1 is capable of binding to the LRP5/6 co-receptor, thus the 8th fetal week (251) and involved in granulosa cell
preventing the formation of the WNT-FZ-LRP5/6 signaling differentiation (252, 253). The high levels of WNT4/β-
complex. WNT4 is expressed at similar levels in the XY and catenin and FOXL2 counteract FGF9 and SOX9, thus
XX bipotential gonads. When SRY upregulates SOX9 in XY leading to the stabilization of the ovarian differentiation
gonads, and the feed-forward loops with FGF9 and PGD2 pathway (238, 239). FOXL2 also represses SF1 expression
are established, WNT4 is silenced (130) (Fig. 4). In XX by antagonizing WT1 in the XX mouse fetus (254). FOXL2
gonads, the absence of SRY releases WNT4 expression, and FST are needed for the survival of meiotic germ cells
which stabilizes β-catenin and silences FGF9 and SOX9 (72, 255, 256). In the XY fetus, SOX9 represses FOXL2
(130). WNT4 also up-regulates DAX1 (222), which expression in the gonad (257). Conversely, inducible
antagonizes SF1 and thereby inhibits steroidogenic deletion of Foxl2 in adult mouse ovarian follicles leads to
enzymes. WNT4-deficient XX mice express the upregulation of Sox9 and reprogramming of adult ovaries to
steroidogenic enzymes 3-hydroxysteroid dehydrogenase testes (72). In goats, XX males develop in the event of a
and 17-hydroxylase, which are required for the production deletion in the autosomal PIS locus (258), where FOXL2
of testosterone and are normally suppressed in the has been identified. In humans, FOXL2 mutations result in
developing female ovary (242). In humans, a duplication of a variety of phenotypes, from streak gonads to adult ovarian
chromosome 1 containing 1p36.12, where human WNT4 failure associated with eyelid abnormalities characterized
maps, causes ambiguous genitalia of XY patients, probably by blepharophimosis, ptosis and epicantus inversus (BPES)
due to low testosterone production (222), whereas (259).
inactivation of both copies of WNT4 in XX human fetuses
results in alterations in gonadal morphology, ranging from Germ cell entry into meiosis is a specific feature of initial
ovotestes to testes, associated with renal agenesis, adrenal ovarian differentiation (Table 3, Figs. 4 and 9). Once
hypoplasia, and pulmonary and cardiac abnormalities stabilized by the cooperative action of WNT4 and RSPO1,
(SERKAL syndrome: Sex reversal with kidney, adrenal and cytoplasmic β-catenin migrates to the nucleus and induces
lung abnormalities) (243). WNT4 is also involved in the the expression of FST. The latter antagonizes Activin B,
development of the internal genital tract (see below). thus repressing endothelial cell migration and the coelomic
vessel formation, one of the earliest testis-specific events
Like WNT4, RSPO1 is expressed in the undifferentiated (170). Wnt4 has a similar effect (256).
gonadal ridge of XY and XX embryos and increases in the
XX gonads in the absence of SRY. RSPO1 binds to G MAP3K1 modulates the balance between female and male
protein–coupled receptors LGR4 and LGR5 (244), pathways. As explained above (see “FGF9 and PGD2:
stimulates the expression of WNT4 and cooperates with it Maintaining SOX9 Expression Levels”), SOX9 and FGF9
to increase cytoplasmic β-catenin (Fig. 5) and FST levels upregulate AXIN1 and GSK3β, which promote the
(245-248). RSPO1 is thought to facilitate WNT-FZ-LRP destabilization of β-catenin, thus blocking ovarian
complex formation through fending off DKK1 and by development. MAP3K1 sequestrates AXIN1; consequently,
sequestering ZNRF3, which promotes FZ degradation by there is a stabilization of β-catenin, which favors the ovarian
ubiquitination and increased turnover (154, 155, 249). The pathway (132). In XY patients with mutations of MAP3K1
increase in WNT4/β-catenin counteracts SOX9, thus that result in increased binding to AXIN1, there is an
leading to the ovarian pathway (170). Loss of function increase of β-catenin leading to defective testicular
mutations in the human RSPO1 gene and Rspo1 gene differentiation and finally resulting in gonadal dysgenesis
ablation in mice result in the formation of ovotestes in the (151).
XX fetus probably owing to SOX9 upregulation (75, 170,
250).

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FIGURE 8. Female sex determination. As in the male, general transcription factors, as LHX1, EMX2 and PAX2, are
necessary for intermediate mesoderm development. The gonadal ridge differentiates from the intermediate
mesoderm following the action of SF1, LHX9 and WT1. WNT4, FST, RSPO1 and β-Catenin should be expressed to
antagonize testis differentiation and promote early ovarian differentiation. Germ cell development (dependent on
BMP family members, KIT ligand and its receptor C-KIT, WNT4, FST, retinoic acid and its receptors, the existence
of two X chromosomes as well as several factors like DAZLA, MSH5, STRA8 and DMC1) are essential for fetal
ovary stabilization. A number of other factors are involved in early folliculogenesis (FOXL2, neurotrophins and
neurotrophin tyrosine kinase receptors, FIGα, NOBOX, SOHLH and members of the TGFβ family like GDF9, AMH
and BMP15).

Ovarian Morphogenesis from the center to the periphery. At week 10, oogonia in the
deepest layers of the ovary enter meiotic prophase, the first
In the XX fetus, the gonad remains histologically unequivocal sign of morphological ovarian differentiation.
undifferentiated after the 7 th week from a histological Subsequently, oogonia become surrounded by a single
standpoint, but a functional differentiation is already layer of follicular (granulosa) cells, they enter meiosis,
detectable: XX gonads become capable of estradiol become oocytes and form primordial follicles (Fig. 9).
production at the same time as XY gonads begin to Initiation of meiosis in the fetal ovary is heralded by the
synthesize testosterone (260). PGCs proliferate by mitosis increase in retinoic acid levels synthesized by retinaldehyde
and differentiate to oogonia. Ovarian maturation proceeds

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dehydrogenase isoform 1 (encoded by ALDH1A1), (263, 270). Furthermore, if germ cells are lost after
expressed in the developing female gonad (261). formation of follicles, these rapidly degenerate (263, 271,
272).
The earliest primary follicles appear at 15-16 weeks and the
first Graafian follicles at 23-24 weeks (262, 263). By the end In XX gonads, very few endothelial cells migrate from the
of the 7th month of gestation, mitotic activity has ceased and mesonephros to the gonad, which suggests that cortical and
almost all germ cells have entered meiotic prophase. medullary domains of the ovary are already established in
Oocytes proceed to the diplotene stage, where they remain early gonadogenesis, although no morphological
until meiosis is completed at the time of ovulation in adult boundaries are evident, consistently with molecular
life. However, not all oocytes undergo meiosis: from 6-7 evidence of discrete gene expression domains specified by
million ovarian follicles at 25 weeks, only 2 million persist at 12.5 dpc in the mouse ovary (255). The coelomic vessel
term (264). Most oocytes undergo apoptosis and follicles formation, characteristic of the differentiating testis, does
become atretic. AMH is produced, albeit in low amounts, not occur in the normal XX gonadal ridge.
after the 23th week of development (265) by granulosa cells
from primary to antral follicles, but not by primordial follicles Granulosa cells, the equivalent of the Sertoli cells of the
(266-268). The dynamics of follicle development and entry testes, originate from 3 possible sources: the ovarian
of germ cells into meiosis is notably different in rodents, in surface epithelium, mesonephric cells from the adjacent
whom meiosis and folliculogenesis only progress after birth rete ovarii, and the existing mesenchymal cells of the genital
(170). ridge (170, 273). Recent evidence in mice shows that many
coelomic epithelial cells ingress to ovarian cortex and give
The involvement of germ cells in the stabilization of the rise to FOXL2-positive granulosa cells (274), confirming that
gonadal structure is one major difference between the ovary other potential granulosa cell precursors are present in the
and the testis, with germ cells being critical only in the gonadal ridge prior to the start of coelomic cell migration
ovaries in terms of maintenance of the somatic component (173, 274). Theca cells, the counterpart of testicular Leydig
of the gonad. In fact, while fetal testis development cells, are thought to derive from fibroblast-like precursors in
progresses normally in the absence of germ cells (269), the ovarian stroma under the control of granulosa cells
ovarian follicles do not develop when germ cells are absent (275).

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FIGURE 9. Developing human fetal ovaries. At 45 days, the ovary is recognizable only because it has not yet
undergone testicular differentiation. In the cortex of the 14-week-old gonad, germ cells are aligned in rows, some
of them have entered the meiotic prophase (arrows). In the medulla, primordial (small arrow head) and primary
(large arrowhead) follicles are visible.

Genetic Control of Oogenesis and Folliculogenesis complex 1 (PRC1), which represses STRA8 and other
factors involved in the differentiation of primordial germ cells
Two major steps mark ovarian development: germ cell and in early meiosis programs until retinoic acid reaches a
migration, proliferation and meiosis onset, followed by threshold (276). Retinoic acid, synthesized by retinaldehyde
folliculogenesis. For a long time, it has been known that two dehydrogenases present in the mesonephros and the
intact X chromosomes are required in the human for ovarian developing ovary (261, 277, 278), binds to the retinoic acid
differentiation and development –in contrast to the mouse, receptor (RAR) present in the germ cells and induces the
in which XY oocytes can occur in experimental conditions expression of STRA8 (229, 234), a transcription factor that
(65)– for ovarian differentiation and development. The lack upregulates DAZL and SYCP3, two proteins involved in the
of two X chromosomes, e.g. in Turner syndrome, results in formation of the synaptonemal complex essential for the
germ cell loss and, subsequently, gonadal dysgenesis (263, onset of meiosis (39). Stabilization of oocytes requires the
271). Therefore, all the factors involved in the proliferation expression of MSH5, a protein involved in DNA mismatch
and migration of PGCs in early embryogenesis (see “The repair (279). In Msh5 null mice, oocytes are lost before the
Germ Cells” section) are essential for ovarian formation. diplotene stage resulting in ovarian dysgenesis. The
In the female gonad, germ cells continue to proliferate by expression of STRA8 takes place in an anterior-to-posterior
mitosis. Meiotic entry is delayed until the 10th week in the wave and is followed by the upregulation of another meiotic
human fetus and the 13th day in the mouse fetus (Table 1), gene Dmc1 (280). For a detailed description of other factors
due to the suppressive effect of the Polycomb repressive involved in oocyte development, see refs. (281) and (282).

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A number of genes are upregulated in the human ovary important for follicle growth beyond the primary stage. An
before and during primordial follicle formation; their increasing number of factors are involved in later steps of
functional implications still need to be elucidated (283). In folliculogenesis (for review, see ref. (39).
mice, neurotrophins (NTs) and their NTRK tyrosine kinase
receptors facilitate follicle assembly and early follicular THE INTERNAL REPRODUCTIVE TRACT
development (284). Factors involved in germ cell meiosis
are also important. Although not essential to ovarian The Indifferent Stage
differentiation, several factors are involved in the
development of ovarian follicles. FIGα is crucial for the
Up to 8 weeks in the human embryo, the internal
formation of primordial follicles (285). AMH regulates the
reproductive tract is similar in both sexes and consists of a
recruitment of primordial follicles into subsequent steps of
set of two unipotential ducts, the Wolffian and Müllerian
folliculogenesis (286, 287), NOBOX, SOHLH1 and
ducts (Fig. 10).
SOHLH2 are critical transcription factors during the
transition from primordial to primary follicles (reviewed in
ref. (39). GDF9 (288, 289) and BMP15 (290, 291) are

FIGURE 10. Undifferentiated reproductive tract. Both Wolffian and Müllerian ducts are present. Müllerian ducts
open in the urogenital sinus at the level of the Müllerian tubercle between the orifices of the Wolffian duct.

Wolffian Ducts

In both the XX and the XY human embryo, Wolffian days old (Table 1) (3). Wolffian ducts elongate caudally and
(mesonephric) ducts originate in the intermediate induce the formation of nephric tubules through a
mesoderm, laterally to somites 8-13 in embryos 24 to 32 mesenchymal‑epithelial transition process. These tubules

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give rise, in a cephalic-to- caudal direction, to the three efferent ducts and the epididymis (298). Epididymal
kidney primordia: pronephros, mesonephros and disjunction from the rete testis reflects a defect in these
metanephros. While the pronephros and mesonephros are processes and can be found in approximately 40 % of
transient structures that soon degenerate, the metanephros patients with cryptorchidism (299).
is one of the main sources of the definitive kidney. Because
Wolffian ducts are crucial for kidney development, abnormal The Wolffian ducts finally reach the caudal part of the
formation of the Wolffian ducts is usually associated with hindgut, the cloaca. A spatiotemporally process of regulated
other malformations in the urinary or genital systems. apoptosis in both the Wolffian ducts and the cloaca is
necessary for Wolffian duct insertion into the cloaca (300).
Several factors have been identified in the induction and The Wolffian ducts become incorporated into the male
development of the Wolffian ducts (292, 293): PAX2 and genital system when renal function is taken over by the
PAX8, acting through GATA3, induce the initial formation, definitive kidney, the metanephros (301).
and LIM1 is required for the extension of the Wolffian ducts
(293). EMX2 is necessary for their maintenance, whereas Müllerian Ducts
FGF8 and its receptors FGFR1 and FGFR2 seem to be
important in the development and maintenance of different Müllerian (paramesonephric) ducts, which give rise to most
segments (cranial or caudal) of the Wolffian ducts (293). of the female reproductive tract, develop after Wolffian
ducts in the urogenital ridges of both XX and XY embryos.
A single ureteric bud evaginates from the Wolffian duct and They arise in 10-mm human embryo (5–6 weeks of
grows dorsally, in response to inductive signals from gestation) as a cleft lined by the coelomic epithelium,
metanephric mesenchyme involving GREMLIN1, BMP4 between the gonadal and mesonephric parts of the
and BMP7 (294). RET signaling is involved in multiple urogenital ridge (3). This coelomic opening will later
aspects of early Wolffian duct development (295). Growing constitute the abdominal ostium of the Fallopian tube. The
caudally, Wolffian ducts undergo extensive elongation and cleft is closed caudally by a solid bud of epithelial cells,
coiling while progressively acquiring a lumen. Factors which burrows in the mesenchyme lateral to the Wolffian
involved in Wolffian duct stabilization, elongation and coiling ducts and then travels caudally inside their basal lamina.
include the SFRP1 and SFRP2, VANGL2, WNT5A and Initially, these cells are mesoepithelial, i.e. they exhibit
PKD1 (293). characteristics of both the epithelium and the mesenchyme;
they will become completely epithelial only in the female, at
As the Wolffian ducts elongate towards the cloaca, they the time male ducts begin to regress (302, 303). At 8 weeks
induce the formation of the mesonephric tubules, most of of development, the growing solid tip of the Müllerian duct,
which finally undergo regression, except close to the testes. now in the pelvis, lies medial to the Wolffian duct, having
There is a number of factors involved in mesonephric tubule crossed it ventrally in its downward course. For a while, the
development, including PAX2, PAX3, PAX8, GATA3, two Müllerian ducts are in intimate contact, then they fuse,
OSR1, WNT9B, WT1, SIX1, FGFR1, FGFR2, FGFR8 and giving rise to the uterovaginal canal (Fig. 11), which makes
SHH (292, 293, 296, 297). The mesonephric tubules give contact with the posterior wall of the urogenital sinus,
rise to the efferent ducts connecting the rete testis with the causing an elevation, the Müllerian tubercle, flanked on both
epididymis. WNT9B knockout male mice fail to develop the sides by the opening of the Wolffian ducts (Fig. 10).

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FIGURE 11. Fused Müllerian ducts flanked by Wolffian ducts in the lower reproductive tract of a 50-mm female
human fetus (10th week).

Development of the Müllerian duct occurs in three phases phase consists in the proliferation and caudal migration of a
(Fig. 12) (302, 303). First, cells of the coelomic epithelium group of cells at the most caudal tip. Müllerian duct
are specified to a Müllerian duct fate. These can be elongation continues in close proximity to the Wolffian duct,
identified by a placode-like thickening of the coelomic then Müllerian ducts cross Wolffian ducts ventrally and fuse
epithelium and by the expression of LHX1 (302, 304) and centrally close to the urogenital sinus.
anti-Müllerian hormone receptor type II (AMHR2) (305,
306). Transcriptional co-factors DACH1 and DACH2 are As could be expected, integrity of protein kinase pathways
required for the formation of Müllerian ducts, possibly by is required for cell proliferation (310). Close contact with the
regulating the expression of LHX1 and WNT7A or other Wolffian duct is also necessary to Müllerian growth; indeed,
factors important for Müllerian duct formation (307, 308). the lack of transcription factors required for Wolffian
During the second phase, these primordial Müllerian cells development, such as LIM1 or PAX2, leads to Müllerian
invaginate from the coelomic epithelium to reach the truncation (see Table 4). Wolffian ducts do not contribute
Wolffian duct. WNT4 expression in the mesonephric cells to the elongating Müllerian tip (302, 311), but act by
mesenchyme is essential for the Müllerian duct progenitor supplying WNT9B, secreted by Wolffian epithelium (298).
cells to begin invagination (304, 309).

The third or elongation phase begins when the invaginating

tip of the Müllerian duct contacts the Wolffian duct. This

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FIGURE 12. Müllerian duct (MD) development can be subdivided into three phases. A. Phase I (initiation): MD
progenitor cells in the mesonephric epithelium (ME) (yellow) are specified and begin to express LHX1. Phase II
(invagination): in response to WNT4 signaling from the mesenchyme, LHX1+ MD progenitor cells invaginate
caudally into the mesonephros towards the WD (blue). Phase III (elongation): the tip of the MD contacts the WD
and elongates caudally in close proximity to the WD requiring structure and WNT9B signaling from the WD. B.
Beginning at ∼ E11.5 in mice, the MD invaginates and extends posteriorly guided by the WD. During elongation,
mesenchymal cells separate the WD and MD anterior to the growing tip (inset I). However, at the MD tip, the MD
and WD are in contact (inset II). At ∼ E12.5, the MD crosses over the WD to be located medially. Elongation is
complete by ∼ E13.5 with the MD reaching the urogenital sinus (UGS). A = anterior (rostral); D = dorsal; P =
posterior (caudal); V = ventral. Reprinted with permission from ref. (303): Mullen RD, Behringer RR. Molecular
Genetics of Müllerian Duct Formation, Regression and Differentiation. Sexual Development 8:281-296 (2014),
Copyright 2014, Karger.

Caudally each Müllerian duct contacts the urogenital sinus portions of the Müllerian ducts lie between the two Wolffian
at the Müllerian tubercle. This is a critical step and its failure ducts near the urogenital sinus. Then during the 8th week,
can lead to lower vaginal agenesis, as it has been observed Müllerian ducts fuse in the midline, leaving temporarily an
in Lhfpl2 mutant mice (312). In weeks 7 and 8, the caudal epithelial septum that disappears one week later giving rise

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to the midline uterovaginal canal. The degree of midline Müllerian duct (316, 317). Extra-cellular matrix is deposited
fusion of Müllerian ducts is extensive in humans, but it is in the peri-Müllerian mesenchyme (318), which
almost inexistent in mice, exhibiting paired oviducts and progressively strangles the Müllerian duct epithelium and
large bilateral uterine horns. Defects in Müllerian duct fusion finally remains the only witness of its former existence.
and retention of the midline septum can lead to various Mesenchymal changes are preceded by the dissolution of
congenital malformations in humans, including separate the basement membrane, which precipitates apoptosis and
hemiuteri, uterus didelphys or unicornis, double vagina or allows extrusion of epithelial cells and their transformation
cervix, vagina with septum etc. (313). into mesenchymal cells (317, 319). Epithelial-mesenchymal
transformation is an important factor of epithelial cell loss
MALE DIFFERENTIATION OF INTERNAL during Müllerian regression.
GENITALIA
Integrity of the WNT/β-catenin pathway is required for
complete Müllerian duct regression in the male, perhaps
Male differentiation of the internal genital tract is
through amplification of the AMH signal (320). β-catenin
characterized by regression of Müllerian ducts and
accumulates in the nucleus (317) upregulating Osterix
differentiation of the Wolffian duct into male accessory
(Osx), also called Sp7, an AMH-induced gene that regulates
organs.
the expression of matrix metallopeptidase 2 (MMP2) (321).
Osx is expressed in male, but not female, Müllerian ducts
Müllerian Duct Regression
before and during regression. Overexpression of human
AMH in female fetuses induces Osx, and Amhr2 knockout
Müllerian regression, the first sign of male differentiation of
males lose Osx expression. Additionally, conditionally
the genital tract, occurs in 55 to 60 day-old human embryos
invalidation of β-catenin in the Müllerian ducts leads to a
(Fig. 13), triggered by anti-Müllerian hormone (AMH) at the
reduction in Osx expression, indicating that OSX is
center of a complex gene regulatory network (reviewed in
downstream of β-catenin in the regression pathway.
ref (314)). Once initiated, the regression of the Müllerian
duct extends caudally as well as cranially, sparing the
Wif1 (WNT inhibitory factor 1) encodes a secreted frizzled-
cranial tip which becomes the Morgagni hydatid, and the
related protein that inhibits WNT signaling. WIF1 shows
caudal end, which participates in the organogenesis of the
many similarities to OSX: it is expressed in the male, but not
prostatic utricle. Müllerian regression of the cranial part of
the female, Müllerian duct and is not detected in Amhr2
the Müllerian duct begins while the duct is still growing
knockout mice. However, Müllerian ducts are absent in Wif1
caudally towards the urogenital sinus (315) and is
knockout male mice, which implies that WIF1 is not
characterized by a wave of apoptosis spreading along the
indispensable for Müllerian duct regression (322).

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FIGURE 13. Regressing Müllerian duct in a 35-mm male human fetus (9th week). Note the fibroblastic ring
surrounding the epithelium of the Müllerian duct (right), the Wolffian duct is visible on the left.

Stabilization and Differentiation of Wolffian Ducts

Testicular Descent
The second aspect of male differentiation of the internal
genital tract is the stabilization and differentiation of the During human fetal development, the testis migrates from
Wolffian ducts (323). After the loss of mesonephric its initial pararenal position to its terminal location in the
functional activity, the mesonephric nephrons and caudal scrotum (Fig. 14). Testicular descent has been subdivided
tubules degenerate but the cranial tubules persist to form into several phases (325). Initially, the upper pole of the
the male efferent ducts. The connections between the testis is connected to the posterior abdominal wall by the
mesonephric tubules and the gonadal primordium are cranial suspensory ligament while a primitive gubernaculum
permanently established in the sixth week; in the male, they extends from the caudal pole to the inner inguinal ring. At
give rise to the rete testis, while in the female, they form the 12 weeks, the cranial suspensory ligament dissolves and
rete ovarii. Between weeks 9 and 13 in the human embryo, the gubernaculum testis swells and pulls the testis down to
the upper part of the Wolffian duct differentiates into the the inguinal ring. After 25 weeks, the gubernaculum bulges
epididymis. Below, it is surrounded by a layer of smooth beyond the external inguinal ring and is hollowed out by a
muscle and becomes the vas deferens, which opens into peritoneal diverticulum called the processus vaginalis. The
the urogenital sinus at the level of Müllerian tubercle. In second –inguinoscrotal– phase of testicular descent occurs
sexually ambiguous individuals, in whom Wolffian and between 27 and 35 weeks after conception. « Physiological
Müllerian ducts coexist, the vas deferens is embedded in » cryptorchidism is frequent in premature infants. In the
the uterine and vaginal walls (reviewed in ref. (324). The female, the cranial ligament holds the ovary in a high
seminal vesicle originates from a dilatation of the terminal position and the gubernaculum, now the round ligament,
portion of the vas deferens in 12-week-old fetuses. remains long and thin.

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FIGURE 14. Testicular descent. Left, Initial phase: the primitive gonad is located near the
kidney, held by the cranial suspensory ligament (CSL) and the gubernaculum testis. Center,
Transabdominal descent: androgen-mediated dissolution of the CSL and insulin like factor 3
(INSL3) mediated swelling of the gubernaculum bring the testis to the internal orifice of the
inguinal canal. Right, Inguino-scrotal migration: the testis passes through the inguinal canal
into the scrotum, this phase is androgen-dependent. Reprinted from ref. (325): Klonisch T,
Fowler PA, Hombach-Klonisch S. Molecular and genetic regulation of testis descent and
external genitalia development. Developmental Biology, 270:1-18 (2004), Copyright 2004, with
permission from Elsevier.
http://www.sciencedirect.com/science/article/pii/S001216060400137X

FEMALE DIFFERENTIATION OF INTERNAL conditionally targeted to the Wolffian mesenchyme, has

GENITALIA shown that the regression of Wolffian ducts in female
embryos is an active process induced by COUP-TF2
through inhibition of the expression of FGFs, which
Female differentiation of the internal genital tract is
otherwise activate the p-ERK pathway in the Wolffian duct
characterized by the disappearance of the Wolffian ducts,
epithelium for its maintenance (326). How androgens
which is complete at 90 days of human fetal development,
interact with this mechanism in males needs to be
except for vestiges such as the Rosenmüller organs or
elucidated.
Gartner canals. Traditionally, the regression of Wolffian
ducts in the female fetus has been ascribed to a passive
Müllerian ducts persist, establish apico-basal
process deriving from the lack of androgen action. Recent
characteristics and develop into an epithelial tube that will
work using a Nr2f2 (encoding COUP-TF2) deletion,
give rise to the endometrium (302), while the surrounding

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mesenchyme differentiates into the myometrium of the uterotubal junction is demarcated by an abrupt increase in
uterus and Fallopian tubes (306). The acquisition of true the diameter of the uterine segment and by the
epithelial characteristics signals the end of the AMH- development of epithelial crypts. The early endometrium is
sensitive window of Müllerian ducts (302). Tubal lined by a closely packed columnar epithelium in which
differentiation involves formation of fimbriae and folds in the gland formation and vacuolated cells can be recognized as
ampullary region (Fig. 15) and acquisition of cilia and gestation advances. The cervix occupies the distal two-
secretory activity by the high columnar epithelium. The thirds of the fetal uterus.

FIGURE 15. MÜLLERIAN DUCTS DEVELOP INTO THE UTERUS AND FALLOPIAN TUBES

THE UROGENITAL SINUS AND EXTERNAL GENITALIA urogenital sinus is individualized in 7-9 mm (~5 week)
human embryos, when a transverse urorectal septum
The Indifferent Stage divides the cloaca into the rectum dorsally and the primitive
urogenital sinus ventrally. The Müllerian tubercle
Up to approximately 9 weeks, the urogenital sinus and demarcates the cranial vesicourethral canal from the caudal
external genitalia remain undifferentiated (Fig. 16). The urogenital sinus.

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FIGURE 16. Sex differentiation of urogenital sinus (left) and external genitalia (right).

The cloaca is closed by the cloacal membrane, formed by thought to invade the genital tubercle to form the midline
ectoderm and endoderm, with no mesoderm in between. In epithelial urethral plate, which lies in the roof of the primary
the 5th week, mesodermal cells spread along the cloacal urethral groove and extends to the tip of the phallus (329,
membrane and give rise to pair of swellings –the cloacal 330). After the corpora cavernosa and glans have
folds–, which form urogenital folds flanking the urogenital differentiated, the ventral surface of the genital tubercle is
sinus and anal folds posteriorly. The urogenital folds fuse depressed by a deep furrow, the urethral groove. The
anteriorly to the cloacal membrane in the midline to form the external genitalia remain undifferentiated up to
genital tubercle. The cloacal membrane is divided by the approximately 9 weeks (327) (Fig. 16).
urorectal septum into the genital membrane anteriorly and
the anal membrane posteriorly. The genital membrane At 12 weeks in males and females alike, the vaginal
disappears in 20-22 mm (~8 week) embryos (327). primordium is formed by the caudal tips of the Müllerian
ducts, and medial and lateral outgrowths of the urogenital
In embryos 8-15 mm long (~6 weeks), the opening of the sinus, the sinovaginal bulbs, which fuse to form the vaginal
urogenital sinus, the ostium, is surrounded by the cord or plate. When the cells of the vaginal plate
labioscrotal swellings, which develop on each side of the desquamate, the vaginal lumen is formed.
urogenital folds. These are connected to the caudal poles
of the genital ridges by fibrous bands which later develop MALE DIFFERENTIATION
into the gubernaculum testis in males and the round
ligament in females.
Urogenital Sinus and Prostate

The genital tubercle, consisting of lateral plate mesoderm

Male orientation of the urogenital sinus is characterized by
and surface ectoderm, emerges as a ventral medial
prostatic development and by the repression of vaginal
outgrowth just cranial to the opening of the ostium (328).
development. Prostatic buds appear at approximately 10
Endodermal epithelial cells from the urogenital sinus are

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weeks at the site of the Müllerian tubercle and grow into system (GH-IGFs) is partly responsible for penile growth,
solid branching cords. Maturation of the prostatic gland is independently of androgens (338-340).
accompanied by development of the prostatic utricle. Two
buds of epithelial cells, called the sino-utricular bulbs in the FEMALE DIFFERENTIATION
male, develop from the urogenital sinus close to the opening
of the Wolffian ducts and grow inwards, fusing with the
Female orientation of the urogenital sinus is characterized
medial Müllerian tubercle, to form the sino-utricular cord,
by lack of prostatic differentiation and the acquisition of a
enclosed within the prostate gland, which canalizes at 18
separate vaginal opening on the surface of the perineum
weeks to form the prostatic utricle, the male equivalent of
(Fig. 16). At the end of the ambisexual stage, the vaginal
the vagina (331).
anlage is located just underneath the bladder neck. In
females, the lower end of the vagina slides down along the
External Genitalia
urethra until the vaginal rudiment opens directly on the
surface of the perineum at 22 weeks. The hymen marks the
Masculinization of the external genitalia begins in human
separation between the vagina and the diminutive
male fetuses 35-40 mm long (~9 weeks) by lengthening of
urogenital sinus, which becomes the vestibule.
the anogenital distance (327) (Fig. 16). Fusion of the
labioscrotal folds, in a dorsal to ventral fashion, forms the
The embryological origin of the vagina is still hotly debated.
epithelial seam (332), which closes the primary urethral
In the generally accepted view, the upper part of the vagina
groove. The literature concerning penile development is
derives from the Müllerian ducts and the lower part from the
controversial. Most textbooks describe it as a two-step
sinovaginal bulbs, which by fusion form the vaginal plate,
process, with the proximal urethra forming by fusion of the
derived from the urogenital sinus (341). It is now thought
urethral folds around the urethral plate and the distal urethra
that the Wolffian ducts do not contribute cells to the
arising from an invagination of the apical ectoderm.
sinovaginal bulbs but they may have a helper function
However, according to Cunha and colleagues (333), the
during downward movement of the vaginal bud in the
entire human male urethra is of endodermal origin, formed
female (342). Atresia of the vagina in the Mayer-Rokitansky-
by the urethral plate dorsally and the fused urethral folds
Küster-Hauser syndrome could be explained by the failure
ventrally. The seam is remodeled into the tubularized
of Wolffian and Müllerian ducts to descend caudally.
urethra without connection to the epidermis. The ventrally
discarded excess epithelial cells migrate into the ventral
Development of female external genitalia is essentially
skin of the penis. Abnormalities of seam formation or
static. The anogenital distance does not increase, the rims
remodeling could explain the vast majority of cases of
of the urethral groove do not fuse, the urethral plate persists
hypospadias in which defects of androgen synthesis or
as an epithelial cord, and the labioscrotal swellings give rise
metabolism cannot be demonstrated (334).
to the labia majora. The dorsal commissure forms at their
junction. The genital folds remain separate and become the
Urethral organogenesis is complete at 14 weeks, apart from
labia minora. When the vagina acquires a separate perineal
a physiological ventral curvature, which can persist up to 6
opening, the diminutive pars pelvina and the pars phallica
months of gestation. However, surprisingly, no size
of the urogenital sinus become the vestibule.
difference exists between penile or clitoral size until 14
weeks (335) despite the fact that serum testosterone levels
CONTROL OF SEX DIFFERENTIATION
peak between 11 to 14 weeks in males (336). The
insensitivity of the male genital tubercle to high levels of
androgens during the second trimester does not correspond Growth Factors
to a low expression of the androgen receptor or of 5α-
reductase type 2 in the corpora cavernosa (337). Maximal GENITAL DUCT FORMATION
phallic growth occurs during the third trimester of fetal life,
at a time when male testosterone levels are declining. The Molecular genetic studies in the mouse have contributed to
action of the growth hormone-insulin-like growth factor the identification of growth factors essential for the
formation of the sexual ducts (Table 4) [see refs. (323) and

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(343) for review]. Since Wolffian ducts are required for the (240, 348). WNT7 is required for the expression of AMHR2;
elongation of Müllerian ducts, absence of growth factors in its absence the Müllerian ducts do not regress in male
necessary to Wolffian development will per se induce fetuses (349). Members of the dachsung gene family,
Müllerian truncation. Many growth factors, such as LIM1, DACH 1 and 2 also play a role by regulating the expression
EMX2, HOXA13, PAX2 and 8 and VANGL2 are essential of LIM1 and WNT7 (307).
also for the development of other organs. In contrast the role
of WNT4A and WNT7A, a subset of the Wnt family Congenital bilateral absence of the vas deferens affects 97-
homologous to the Drosophila wingless gene, is restricted 98%% of patients suffering from cystic fibrosis, a bronchial
to reproductive organs. WNT4 is required in both sexes for and pancreatic disease due to mutations in the cystic
the initial formation of Müllerian ducts (309), mutations of fibrosis transmembrane conductance regulator (CFTR)
WNT4 have been reported in three cases of Müllerian (350). Whether efferent duct maldevelopment is a primary
aplasia associated with hyperandrogenism in girls defect of cystic fibrosis or a secondary degenerative change
(reviewed in refs. (344-347), but have not been detected in resulting from obstruction by mucus is not known at the
classical forms of the Rokitansky-Küster-Mayer syndrome present time.

TABLE 4. Consequences of Null Mutations of Growth Factors on Morphogenesis of Genital Ducts

Growth factors Wolffian ducts Müllerian ducts Gonads References
β-catenin Normal Lack of oviduct coiling. Loss of germ cells in (320, 351,
Lack of regression the ovary. 352)
Testes normal
DACH1/DACH2 Normal Hypoplasia of female Normal (307)
reproductive tract
DICER1 Normal Hypoplasia of female Reduced ovulation (353)
reproductive tract rate
EMX2 Early degeneration Do not form Absent (5)
HOXA13 Rostral ureteral junction Agenesis of caudal Normal (354)
portion
IGF1 Agenesis of caudal portion Infantile uterus No ovulation. (355)
Abnormal Leydig
cells.
LIM1 (LHX1) Do not form Do not form Normal (304)
PI3K/AKT Increased apoptosis Increased apoptosis (310)
PAX2 Early degeneration Early degeneration Normal (356)
PAX8 Normal Endometrium does not Normal (356, 357)
form
Retinoic acid Agenesis of vas deferens Agenesis of uterus and Normal (358)
receptors and seminal vesicles cranial vagina
WNT4 Persist in females Do not form Ovary produces (320)
No regression in males testosterone
WNT7A Normal Persist in males (349, 359,
360)

VAGINA, PROSTATE, URETHRA, AND EXTERNAL vaginal clear-cell adenocarcinoma, vaginal adenosis,
GENITALIA transverse vaginal ridges and structural malformations of
the cervix and uterus, occur in transgenic mice deficient in
Correct vaginal development requires Wnt, Pax and Vangl2 WNT7A, a signaling molecule expressed by the Müllerian
genes (Table 5). Vaginal abnormalities similar to those epithelium, suggesting that DES exposure acts by
elicited by diethylstilbestrol (DES) administration, i.e. deregulating WNT7A during uterine morphogenesis (361).

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WNT7A deficiency could act by interfering with normal genital tubercle outgrowth (368). Sonic hedgehog (SHH)
mesenchymal-epithelial signaling, which is required for signaling regulates many of the mesenchymal genes
correct morphogenesis of the reproductive tract. Vaginal involved (325, 328, 369-371) (Fig. 17). The homeotic genes
opening is regulated by PAX8 (357) and VANGL2 as shown Hoxa13 and Hoxd13 act in a partially redundant manner
in the mutated the loop-tail mouse (362). since double null mutants show more severe urogenital
abnormalities than those with at least one functional allele
SOX9 (363) and FGF10 (364) both play a role in early (372).
prostate bud differentiation.
The secreted frizzled-related proteins (SFRP1 and 2) are Ephrin family factor EFNB2 and receptors EPHB2 and
required for correct gubernaculum development and EPHB3 mediate cell adhesion and patterning events
testicular descent (365). occurring at the midline, including urethral closure and
scrotal fusion, as well as palate fusion (328, 373).
Early patterning of external genitalia is regulated by a Diacylglycerol kinase K (DGKK), an enzyme that
cascade of signaling molecules which orchestrate phosphorylates diacylglycerol, is expressed in the epithelial
interaction between tissue layers and cells of the urethral plate (374). In humans, DGKK is
mesenchymal/epithelial tissues (Table 5). External genitalia strongly associated with hypospadias risk (375, 376).
are appendages emerging from the caudal body trunk, Regulation of urethral tube closure during the androgen-
hence many genes which pattern distal limb development dependent phase of penile development is mediated by
also play a predominant role during genital tubercle FGF10, signaling through the IIIb isoform of fibroblast
formation, for example BMPs (328, 366), Fgf-8 and 10, Hox growth receptor 2 (FGFR2-3b), suggesting that these genes
gene families (for reviews, see refs. (325, 367). β-catenin are downstream targets of the androgen receptor (377).
activates Fgf8 expression in the urethra, required for normal

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FIGURE 17. Growth factors regulating the outgrowth and ambisexual differentiation of the external genitalia. Role
of sonic hedgehog (Shh) in the outgrowth and ambisexual differentiation of the genital tubercle (see table 5 for
references). Most factors, with the exception of Hoxa13, are regulated by sonic hedgehog (Shh), expressed in the
urethral epithelium (light green), and are identical to those regulating limb morphogenesis. Apoptosis is also
affected by Shh. Data obtained from ref. (325): Klonisch T, Fowler PA, Hombach-Klonisch S. Molecular and
genetic regulation of testis descent and external genitalia development. Developmental Biology, 270:1-18 (2004).
http://www.sciencedirect.com/science/article/pii/S001216060400137X.

TABLE 5. Growth Factors in Urogenital Development

Growth factors Role in urogenital development References
BMP4 Restricts prostate ductal budding (378)
BMP7 Closure of the distal urethra (367)
FGF8 Initiation of genital swellings; (379)
Ephrins Urethral closure and scrotal fusion (373)
FGF10 Development of the glans penis and clitoridis, and prostate (364, 369, 379)
FGFR2-IIIB Null mice exhibit severe hypospadias (377)
HOXA10 Atrophic seminal vesicles in null mice (380)
HOXA13 In mice, semi-dominant mutations lead to limb defects, vaginal hypoplasia (354, 381)
and deficiency of the os penis (Hypodactyly syndrome)
In humans, an autosomal dominant mutation produces limb and uterine
abnormalities and urinary tract malformations (Hand-Foot-Genital
syndrome)
HOXD13 Hoxd-13 null mice display decreased ductal branching in the prostate and (382)
seminal vesicle and agenesis of bulbourethral gland
HOXA13/HOXD13 null No genital tubercle, no partition of the cloaca in double mutants (372)
mutants
LTAP Vaginal opening (362)
MSX2 Disruption of vaginal epithelium and lack of caudal Wolffian regression (383)
PAX8 Vaginal opening (357)
SHH/GLI2 Outgrowth and patterning of external genitalia and urogenital sinus (369, 371, 384,
Development of prostatic ducts 385)
Inhibition of apoptosis in penile smooth muscle
Masculinization of external genitalia
SOX9 Lack of ventral prostate development (363)
SFRP1 and 2 Testicular descent (365)
VANGL2 (looptail Imperforate vagina (362, 386)
mouse)
WNT/β-catenin Masculinization of external genitalia (387)

HORMONAL CONTROL OF MALE SEX sex, will develop along female lines provided it is not
DIFFERENTIATION exposed to testicular hormones, the main forces driving
male sex differentiation (Fig. 18).
The classical experiments of Jost (58, 59) (Fig. 2) have
taught us that the reproductive tract, whatever its genetic

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FIGURE 18. Hormonal control of male sex differentiation.

Anti-Müllerian Hormone (AMH)

19) until puberty and at lower levels thereafter (for reviews,
Anti-Müllerian hormone (AMH), a member of the TGFβ see refs. (324, 388)). In the female, AMH begins to be
family, triggers Müllerian regression, the first step of male produced in the second half of fetal life by granulosa cells
sex somatic differentiation. AMH is expressed at high levels of growing follicles (265, 266).
by Sertoli cells from the time of testicular differentiation (Fig.

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FIGURE 19. AMH protein expression by seminiferous tubules of an 11-week-old male human fetus, using an
AMH-specific polyclonal antibody. Note the strong staining of seminiferous tubules.

Low expression of AMH and/or its type II receptor AMHR2 which remain associated by non-covalent bonds (395, 396).
has also been identified in spermatocytes of maturing rat Whether cleavage occurs at the time of secretion or within
testis (389), the endometrium (390), the brain (391), the target tissue is not clear at the present time. This step is
hypothalamus (392), motor neurons (393) and female required for binding of AMH to its primary receptor, at which
pituitary (394). time the AMH complex dissociates, releasing the mature
ligand, the C-terminal homodimer and the N-terminal
TGFβ family ligands are translated as dimeric precursor proregion (396). The homology of AMH to other members
proteins comprising two polypeptide chains, each of the transforming growth factor-β (TGF-β) family is
containing a large N-terminal pro-region and a much smaller restricted to the C-terminus, for which a molecular model
C-terminal mature domain. Processing involves cleavage at has been built, by analogy with crystallized members of the
sites between the two domains and dissociation of the pro- family (397) (Fig. 20). Cleavage and presumably bioactivity
region domain. The AMH molecule is initially synthesized as are enhanced if the endogenous cleavage site RAQR is
a biologically inactive precursor. The precursor is cleaved replaced by a furin/kex2 RARR consensus site (398).
by proteolytic enzymes into C and N terminal fragments

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FIGURE 20. Molecular model of C-terminal AMH. A three-dimensional model of the C-terminal dimer was
generated by comparative modeling using human BMP9 (399) as a template. The wrist epitope, the putative
binding site for the type I receptor, is composed of the prehelix loop and alpha-helix of one monomer together
with the concave side of the fingers of the second monomer (400). A mutation in the prehelix loop of AMH,
Q496H, causes persistent Müllerian duct syndrome (397). Residues in the knuckle epitope of AMH, the putative
binding site for AMHR2, are similar to those present in BMP7 and activin at the interface with ACTR2B (401, 402).
Disulfide bonds (yellow) and Q496 residues (blue) are shown as sticks; residues in the knuckle epitopes are
shown as spheres. Reprinted from ref. (397): Belville C, Van Vlijmen H, Ehrenfels C, Pepinsky RB, Rezaie AR,
Picard J, Josso N, di Clemente N, Cate RL. Mutations of the anti-Müllerian hormone gene in patients with
persistent Müllerian duct syndrome: biosynthesis, secretion and processing of the abnormal proteins and
analysis using a three-dimensional model. Molecular Endocrinology 18:708-721 (2004). Copyright 2004 The
Endocrine Society with permission. http://mend.endojournals.org/content/18/3/708.abstract?sid=22a37d21-69b5-499e-
9996-8b1d4df81215

The human 2.8-kb gene has been cloned (403) and mapped caudate amphibian, Pleurodeles waltl, whose Müllerian
to chromosome 19p13.3 (404). It consists of five exons, the ducts persist in males (414). Even more surprisingly, AMH
last one coding for the C-terminal fragment. The AMH gene orthologs (415, 416) and the AMH type II receptor (417)
has been cloned in many other mammals (405-409), in the have been cloned from the gonads of modern teleost fish,
marsupial tammar wallaby (410), in the chick (411, 412) and which do not possess Müllerian ducts at all. In fish, AMH
American alligator (413), all of which carry Müllerian ducts appears to be involved essentially in germ cell proliferation
which regress in the male. The gene is also present in the and gonadal development (reviewed in ref. (418)), which

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suggests that AMH was initially a regulator of gonadal SOX8 (114)- triggers AMH expression in Sertoli cells by
differentiation which acquired its anti-Müllerian activity binding to a specific response element on the AMH
during the course of evolution without completely promoter. Transcription factors SF1 (112, 113, 422, 424-
relinquishing its former role. Indeed, in higher vertebrates, 431), GATA4 (113, 427, 432-438), WT1 (425, 439)
AMH inhibits Leydig cell differentiation (419) and follicle increase, whereas DAX1 (425) and β-catenin (439) reduce,
maturation (420). SOX9-activated AMH transcription either by binding to
specific response elements or by protein-protein interaction
The ontogeny of AMH expression differs widely between (440, 441). In vivo, genes can affect AMH levels indirectly
males and females. In the human fetal testis, AMH mRNA through their impact on testicular determination instead of
and protein can be detected from the 8th week, when Sertoli acting on gene transcription.
cells begin to form cord-like structures, the future
seminiferous tubules (189) (Fig. 19). In the ovary, AMH Although initially gonadotropin-independent, AMH
production is detectable at 24 week gestation in granulosa production falls under FSH control later in fetal life and after
cells of preantral follicles (265). The timing of the expression birth (113, 197, 442-444). FSH regulates AMH transcription
of AMH is crucial. In the male, high amounts of AMH must through the FSH receptor-Gsα protein-adenylate cyclase-
be expressed before Müllerian ducts lose their cyclic AMP pathway, resulting in a stimulation of protein
responsiveness, i.e. before the end of the 8 th week in the kinase A (PKA) activity. PKA mediates phosphorylation of
human fetus. In the female, to avoid destroying the the transcriptional regulators SOX9, SF1 and AP2, as well
reproductive tract, it must be expressed after the window of as of IκB which releases NFκB. In the nucleus these factors
sensitivity of the Müllerian ducts to its action has closed. activate AMH transcription by binding to their specific
Thus, in both sexes, the initiation of AMH transcription is response elements on the AMH promoter (Fig. 21). LH and
under tight transcriptional control. hCG do not have a direct effect on Sertoli cell AMH
expression, but affect testicular AMH production through
In the mammalian testis, but not in reptiles (413) or birds androgen action, as explained below.
(421), SOX9 (97, 112, 113, 422, 423) -and to a lesser extent

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FIGURE 21. Regulation of testicular AMH production. Left: the onset of AMH expression is gonadotropin-
independent and depends on SOX9 binding to the proximal AMH promoter. Subsequently, SF1, GATA4 and WT1
enhance AMH expression by binding to specific promoter sequences or by interacting with transactivating
factors. DAX1 impairs GATA4 and SF1 binding to the AMH promoters, resulting in lower AMH expression levels.
Right: Later in fetal and postnatal life, FSH regulates AMH production through the FSH receptor-Gsα protein-
adenylate cyclase (AC)-cyclic AMP (cAMP) pathway, resulting in a stimulation of protein kinase A (PKA) activity.
PKA mediates phosphorylation of the transcriptional regulators SOX9, SF1 and AP2, as well as of IκB which
releases NFκB. In the nucleus these factors bind to their specific response elements in proximal (SOX9, SF1) or
distal (AP2 and NFκB) regions of the AMH promoter.
Right figure reprinted from ref. (113): Lasala C, Schteingart HF, Arouche N, Bedecarrás P, Grinspon R, Picard JY,
Josso N, di Clemente N, Rey RA. SOX9 and SF1 are involved in cyclic AMP-mediated upregulation of anti-
Müllerian gene expression in the testicular prepubertal Sertoli cells SMAT1. American Journal of Physiology –
Endocrinology and Metabolism 2011; 301: E539-E547, Copyright 2011 the American Physiological Society.
http://ajpendo.physiology.org/content/301/3/E539.abstract?sid=3829d833-dfdf-4310-bd6f-e7481c62be06

At puberty, FSH stimulation is antagonized by androgens unopposed stimulation by FSH. Androgens act directly on
resulting in a steep fall in AMH secretion by Sertoli cells pubertal Sertoli cells to inhibit AMH promoter activity in the
(445). Androgen action requires the presence of the presence of the androgen receptor (429), even though the
androgen receptor in Sertoli cells. This occurs relatively late AMH promoter does not carry consensus androgen
after birth (Fig. 22) (204, 205, 446) allowing both AMH and response elements (449). For androgens to repress AMH
testosterone to reach high levels in fetuses and neonates. expression, the existence of intact sites for binding of the
In androgen-insensitive patients, affected by mutations of transactivating factor SF1 on the AMH promoter is crucial,
the androgen receptor, AMH levels are abnormally elevated suggesting that the inhibition of AMH promoter activity by
during the perinatal and pubertal stages (447, 448), due to androgens could be due to protein–protein interactions

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between the ligand-bound androgen receptor and SF1 or by cells (450) while estrogens has differential effects according
blockage of SF1 binding to its sites (429). to which estrogen receptor is involved (451), while LH has
no effect in normal cells (452).
Gonadotropins and steroid also regulate AMH in the ovary.
FSH stimulates AMH transcription in cultured granulosa

FIGURE 22. Ontogeny of testicular AMH production. In the mammalian fetal testis, AMH expression is triggered
by the increase of SOX9 levels. It is not prevented by the rise of intratesticular levels of testosterone because
fetal Sertoli cells do not express the androgen receptor (AR). After birth the number of Sertoli cells expressing
the AR progressively increases. At puberty, when testosterone increases again, AR is present and AMH
production is inhibited.

AMH is measurable in human serum by ELISA. Initially, the correlation between the different manual kits after
procedure was used by pediatric endocrinologists to manipulation of standard curves by manufacturers but not
measure testicular AMH in boys, hence the first between manual assays and automated ones, which yield
commercially available kits were suited to the high level of 20-30% lower values (458, 460). It follows that AMH values
AMH concentration of prepubertal males (448). Following obtained with different methods are not interchangeable
the discovery that AMH serum concentration in women (461). Since clinicians are not usually aware of the problem,
mirrors ovarian reserve (453, 454), AMH assay has become serious interpretation errors may arise during patient follow-
a standard procedure in assisted reproduction centers and up. An international standard of human recombinant AMH
more sensitive methods, adapted to the low concentration needs to be developed, particularly since the Immunotech
of AMH in female serum, were developed (455)(456)(457). assay upon which many normative values have been based
In parallel, automated assays, e.g. the (462-464) has been pulled off the market.
electrochemiluminescence Roche Elecsys assay (458) and
the Beckman Coulter Access AMH assay (459), are The uncleaved AMH precursor and non-covalent cleaved
progressively gaining ground, due to increased AMH are both detectable by commercial ELISA kits, but
reproducibility and accelerated turnaround time, only 18 attempts to discriminate between the various AMH forms
minutes for the Roche Elecsys assay. There is reasonable have not proven clinically rewarding (465-468).

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 TGFβ family truncated forms of the AMH primary receptor
AMH is an exceptionally stable biomarker, variations during AMHR2 are not secreted, unless the signal sequence is
the menstrual cycle (469, 470) and diurnal variations in men replaced by the TGFβ one, suggesting that the AMHR2
(471) are minimal. Measurement of AMH in serum has signal sequence is defective (483). A three-dimensional
diagnostic applications in disorders of sex development model of extra- and intracellular domains built by analogy
(324, 472) and as a marker of prepubertal testicular function with crystallized receptors of the TGFβ family (Fig. 23) has
in boys (473-477). In women, AMH levels are a reliable served to analyze structure/activity relationship of the
marker of follicular reserve (453, 454) and may be used with receptor molecule (483, 484).
relative accuracy to predict the onset of menopause (478)
or to follow the evolution of granulosa cell tumors (479, 480). The AMHR2 gene, located on chromosome 12q13.13,
Some AMH mutations with reduced in vitro bioactivity are spans 8 kb pairs and is divided into 11 exons. Exons 1-3
associated with premature ovarian insufficiency (481). In code for the signal sequence and extracellular domain,
contrast, the clinical usefulness of AMH in seminal fluid in exon 4 for most of the transmembrane domain, and exons
men with non-obstructive azoospermia is debatable (482). 5-11 for the intracellular serine/threonine kinase domains
Further discussion of the diagnostic and potentially (485). AMHR2 is expressed in the mesenchymal cells which
therapeutic value of AMH in the adult ovary and testis is surround the Müllerian duct, and also in Sertoli, granulosa
beyond the scope of this review. (486, 487), Leydig (419) and germ cells (389), endometrium
(390), neurons (391, 393) and hypothalamus (392).
AMH Transduction: Type I and II AMH Receptors Expression of the receptor in the peri-Müllerian
mesenchyme requires the presence of the signaling
molecule WNT7A (359). The activity of AMHR2 is enhanced
Like other members of the TGFβ family, AMH signals
by WT1 (488) and by SP600125, an inhibitor of the c-Jun N-
through two distinct membrane-bound receptors, both
terminal kinase (489).
serine/threonine kinases. Unlike other members of the

FIGURE 23. Molecular models of AMHR2 extracellular and intracellular domains. (A) The extracellular domain
exhibits the general three-finger toxin fold of type II receptors and displays five disulfide bridges, four of which
are conserved. Five amino acids (Phe62, Met76, Arg80, Asp81, and Thr108), implicated in binding AMH, are

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shown as spheres. (B) The intracellular domain exhibits the general fold of a two-domain kinase, with an N-lobe
consisting mainly of a five-stranded β-sheet and a C-lobe, which is mainly α-helical. Some of the residues
affected by PMDS mutations (Arg54, His254, Arg406, Asp426, Asp491, and Arg504) are shown as sticks. The inset
shows residues affected by the p.((Gly445_Leu453del) mutation. Reprinted with permission from Elsevier, from
ref. 364 (490): Josso N, Picard JY, Cate RL (2013). The Persistent Müllerian Duct Syndrome. In: New MI, Parsa A,
Yuen TT, O'Malley BW, Hammer GD, eds. Genetic Steroid Disorders. New York, NY (USA): Elsevier

Binding of the receptor to its specific ligand requires The primary AMH receptor, AMHR2, is AMH-specific, a
proteolytic cleavage of the AMH precursor to yield the non- unique example of exclusive ligand-receptor pair within the
covalent complex AMH, but unlike other TGFβ family TGFβ family (492). This specificity may be due to the
members, prior dissociation of this complex is not required. presence of charged residues at the ligand binding interface
Dissociation is triggered by binding to AMHR2 (396) and is (493). In contrast, the downstream elements of the AMH
followed by the assembly of a tetrameric C- transduction pathway are shared with the bone
terminus/receptor complex with two molecules of type I morphogenetic protein family, namely ALK2 (or ACVR1,
receptor. Activated type I receptors then phosphorylate Activin a receptor, type I) ALK3 (or BMPR1A, Bone
receptor-SMADS 1/5/8, which associate with SMAD4 and morphogenetic protein receptor, type IA) and all three BMP
are then shuttled to the nucleus where they regulate receptor SMADS, 1, 5 and 8 (494-496). Another BMP
transcription of target genes. (Fig. 24). receptor, ALK6 (or BMPR1B, Bone morphogenetic protein
The AMH type II receptor is subject to processing (491). receptor, type IB), is engaged by ligand-bound AMHR2
Increased expression of the receptor results in the removal (494) but has an inhibitory effect on AMH activity (497).
of most of its extracellular domain and subsequent retention ALK3 is the more potent AMH type I receptor in the
in the endoplasmic reticulum, resulting in a constitutive Müllerian duct (498), in the Leydig cell (499) and in the
negative regulation. SMAT1 Sertoli cell line (497) but in its absence, ALK2 is
capable of transducing the AMH signal (496, 497).

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FIGURE 24. Model showing processing of AMH, assembly of the AMH receptor signaling complex, and
intracellular signaling. Cleavage of the AMH precursor results in a conformational change in the C-terminal
domain, which allows binding of the AMH non-covalent complex to AMRHII. After dissociation of the N terminal
proregion, the type I receptor is recruited into the complex and phosphorylated by the type II receptor kinase.
The activated type I receptor can then phosphorylate Smads 1/5/8, which associate with Smad 4, translocate to
the nucleus and regulate AMH responsive genes. Courtesy of Dr. Richard Cate. Data obtained from ref. (396): di
Clemente N, Jamin SP, Lugovskoy A, Carmillo P, Ehrenfels C, Picard J-Y, Whitty A, Josso N, Pepinsky RB, Cate
RL. Processing of anti-Müllerian hormone regulates receptor activation by a mechanism distinct from TGF-β.
Molecular Endocrinology 24:2193-2206 (2010). http://mend.endojournals.org/content/24/11/2193.abstract

The Persistent Müllerian Duct Syndrome normally virilized, Müllerian duct derivatives are discovered
incidentally at surgery for either inguinal hernia or
Mutations of human AMH or AMHR2 (324) and gene cryptorchidism (Fig. 25) or following discovery of the
knockout in mice (500, 501) are associated with a rare form condition in a sibling. Older patients may seek medical
of disorder of sex development, the persistent Müllerian attention because of an abdominal tumor, hematuria or
duct syndrome (PMDS). These XY individuals are externally hemospermia.

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FIGURE 25. Operative findings in a patient with PMDS. The Fallopian tubes are tightly attached to the testes,
preventing testicular descent. Note normal male external genitalia.
Reprinted from ref. (484): Abduljabbar M, Taheini K, Picard JY, Cate RL, Josso N. Mutations of the AMH type II
receptor in two extended families with Persistent Mullerian Duct Syndrome: lack of phenotype/genotype
correlation. Hormone Research in Paediatrics 77:291-297 (2012). Copyright 2012 S. Karger AG, Basel, with
permission. http://www.karger.com/Article/FullText/338343.

In patients with PMDS, as in the normal female, the sign of an AMH or AMHR2 mutation in patients with
Müllerian ducts differentiate into Fallopian tubes, uterus, normally regressed Müllerian derivatives (502).
and upper vagina. They retain their close apposition to Approximately half the cases present with bilateral
Wolffian duct derivatives, epididymis, and vas deferens cryptorchidism, the rest with hernia uteri inguinalis or
while remaining tied to the pelvis by the broad ligament (Fig. transverse testicular ectopia in similar proportions. The
25). The clinical features of PMDS are similar in AMH and descended testis is only loosely anchored to the bottom of
AMHR2 mutations and may vary within the same sibship. the processus vaginalis by a thin gubernaculum 4. It is
The mobility of the Müllerian structures determines exposed to an increased risk of torsion and subsequent
testicular location. Bilateral cryptorchidism is observed most degeneration (503). Associated abnormalities such as low
frequently: the uterus remains anchored to the pelvis, and birth weight with or without prematurity or complex
mechanically prevents testicular descent. Alternatively, one metabolic syndromes are suggestive of idiopathic PMDS
or both testes may make it into the inguinal canal or the unrelated to defects in the AMH pathway. Intestinal
scrotum, dragging the uterus along. This may result either malformations have been observed in four cases, consisting
in unilateral cryptorchidism with a hernia containing the of either jejunal atresia or lymphangiectasis. Skeletal
uterus on the opposite side, a condition known as “hernia malformations suggestive of defects in the BMP pathway
uteri inguinalis”. The testis on the opposite side can be have not been reported.
drawn into the same hemiscrotum by gentle traction or may
already be present there; this condition typical of PMDS is Testicular tumors of every denomination, mostly
named “transverse testicular ectopia”. It may be the only seminomas, are a frequent mode of presentation of PMDS

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in older patients, particularly in settings where testicular blood supply is through the internal spermatic and
cryptorchidism has been neglected in childhood. Young the deferential arteries. Often the spermatic vessels are too
patients may be affected by germ cell neoplasia in situ (502, short and must be divided to allow orchidopexy. The viability
504). Early orchidopexy is not necessarily 100% effective to of the testis then becomes wholly dependent upon the
preserve against testicular degeneration (505, 506). deferential artery, which is closely associated with the
Furthermore, the incidence in PMDS adults reaches 33% Müllerian structures and may be severely damaged by
(502) compared to 18% for simple cryptorchidism (507), attempts to remove them (514). Most authors recommend
suggesting that misplacement of the testis may not be the partial hysterectomy, limited to the fundus and proximal
only factor driving testicular cancer. Evolution depends Fallopian tubes, or the simple division of Müllerian
upon the histological type of the tumor; choriocarcinomas structures in the midline. If the length of the gonadal vessels
and mixed germ cell tumors share a dim prognosis. is the limiting factor; a Fowler–Stephens orchidopexy or
microvascular autotransplantation (515) may produce good
Uterine tumors occur less frequently, hematuria is usually results. Intracytoplasmic sperm injection may be helpful in
the presenting symptom (508). They should not be the case of ejaculatory duct defects. Orchidectomy is
confused with degeneration of the prostatic utricle often inevitable if the testes cannot be brought down.
mistakenly called “Müllerian” cysts (509). Farikullah et al
(510) reported 11 cases of Müllerian degeneration in males, The serum level of AMH in prepubertal patients depends on
but only 3 qualified as PMDS. Exceptionally, in an elderly the molecular origin of the syndrome. Before puberty, the
PMDS patient, hematospermia may be due to endocrine level of serum AMH allows easy discrimination between
imbalance with low testosterone and high estrogen AMH and AMHR2 mutations. In nearly all patients with AMH
secretion (511). mutations, AMH levels are extremely low or undetectable.
AMH gene mutations with a normal AMH serum level are
Infertility is the most common complication of PMDS. very unusual and should be regarded with suspicion. We
Pubertal development is normal, spermatogenesis is not have documented only one such case, a Gln 496 His
unheard of (512), yet few patients actually father children mutation, which is thought to affect binding of AMH to its
and stringent evidence of paternity is lacking. In all cases at type 1 receptor ALK3 (397). Menabo et al (516) reported a
least one testis was in a scrotal position (reviewed in ref. case of PMDS attributed to AMH variants with a normal
(502). There are several causes of infertility: the excretory AMH level but they did not rule out an AMHR2 mutation.
ducts may not be properly connected to the testis or the AMH levels are relatively low in normal infants shortly after
germinal epithelium may be damaged by longstanding birth, but then repeat determinations show a progressive
cryptorchidism. Paradoxically, all fertile PMDS patients rise with increasing age.
fathered children before their condition was diagnosed and
surgically addressed. Surgery may compromise the Serum AMH levels are within normal limits for age in
testicular blood supply or the vasa deferentia, particularly if AMHR2 mutations and in idiopathic PMDS, unrelated to
hysterectomy is undertaken without proper dissection of the defects in known components of the AMH pathway.
male excretory ducts included in the uterine wall. The Obviously, serum AMH is not detectable, whatever the
prognosis may improve with modern surgical and assisted genotype, in the case of anorchia (503) and may be
reproduction techniques. In inbred populations where abnormally low in cryptorchid patients. Testosterone and
fertility is a crucial issue, as in the Middle East (513), genetic gonadotropin levels are normal for age. After pubertal
counseling is recommended and molecular screening maturation, serum AMH declines physiologically, and it may
should be carried out if a consanguineous union is be difficult to discriminate between AMH and AMHR2.
contemplated.
Approximately 80% of PMDS cases are due to AMH or
Treatment should aim primarily towards the prevention of AMHR2 mutations, in about equal proportions. The first
the two main complications of PMDS, cancer and infertility. AMH mutation was reported in 1991 (517) in a Moroccan
Both goals are served by replacing the testes in the scrotum family. At the time of writing, early 2020, a total of 84 families
but excising the uterus to allow abdominal testes to descend affected by AMH mutations have been published in the
into the scrotum carries significant risks. The primary world literature. All exons are affected (Fig 26). Exon 1, the

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site of most recurrent mutations (Table 6) is hit hardest, is the runner up. Although it is shorter, the 3’ end of exon 5
exon 5 is next but when the number of base pairs is taken that codes for the bioactive C-terminal domain of AMH is
into account, the relatively short exon 2 with its 13 mutations targeted nearly twice as often as the 5’ end.

FIGURE 26. Mutations of the AMH and AMHR2 genes in the Persistent Müllerian Duct Syndrome (PMDS).
Mutations of the AMH (top) and AMHR2 genes (bottom) in the Persistent Müllerian Duct Syndrome (PMDS). The 3'
end of the AMH gene (picture in red) codes for the C-terminal domain, responsible for bioactivity, yet mutations
are spread along the whole length of the gene. Similarly, mutations of the AMHR2 affect intracellular and
extracellular domains alike.

Altogether, 67 different AMH alleles bearing all types of destruction of the GATA site adjacent to SF1-102 results in
mutations have been described in PMDS. Missense and inactivation of the AMH promoter (430).
stop mutations are the most frequent, insertions are rare
(see details in ref. (502). One deletion is of particular A few AMH mutations have been reproduced by site-
interest, because it disrupts the SF1 response element directed mutagenesis, cloned into an expression vector and
located at -228 in the AMH promoter. Inactivation of the - transfected into COS cells to allow study of the secretion of
102 site does not prevent Müllerian regression in transgenic the mutant protein into the culture medium (397). These
mice. The greater impact of the -228 deletion detected in studies confirm that most single nucleotide variations of the
the PMDS patient may be due to the vicinity of the -102 SF1 AMH gene act by affecting the stability and secretion of the
site to a GATA site, to which SF1 can indirectly bind through hormone, explaining why nearly all patients with AMH
protein/protein interaction with GATA4. This hypothesis is mutations, regardless of the site of the mutation, have a very
supported by transactivation experiments showing that low level of circulating AMH.

TABLE 6. Recurrent (n≥4) Mutations of AMH in PMDS

Exon cDNA Protein Families (n)
1 c.35T>G p.(Val12Gly) 4
1 c.283C>T p.(Arg95*) 6
1 c.301G>A p.(Gly101Arg) 4
1 c.343_344delCT p.(Leu115Thrfs*58) 5
1 c.367C>T p.(Arg123Trp) 7
2 c.500A>G p.(Tyr167Cys) 5

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Up to now, 90 families with various AMHR2 mutations have such as β-catenin (320) or patterning genes not specifically
been published, the first in 1995 (485). Since AMHR2 involved in reproductive development. Studies with next
mutations lead to PMDS by blocking response to AMH, the generation sequencing are underway to resolve this issue.
level of circulating AMH is normal for age, in contrast to Women homozygous for AMH or AMHR2 mutations are
PMDS due to AMH mutations. normally fertile but it is too early to know whether, similar to
“AMH-null” mice (286), they will experience premature
A total of 75 independent mutant alleles have been ovarian failure due to follicular depletion
described, targeting all 11 exons and 5 introns; their location
within the gene are shown in Fig.26B. Most are missense or Androgens
stop mutations. Two cases of classical PMDS due to a
microdeletion of the chromosomal region 12q13.13, the Testosterone or dihydrotestosterone (DHT), binding to the
locus of the gene for AMHR2, have been reported. One same androgen receptor (AR), are the main factors involved
case involved a homozygous microdeletion of five exons of in maintenance of the Wolffian duct and differentiation of
the AMHR2 gene. In the second case, the whole AMHR2 male sex accessory organs and external genitalia.
gene was deleted from the maternally inherited
chromosome. The patient’s paternal allele carried a stop Testosterone Biosynthesis
mutation, which was initially thought to be homozygous by
Sanger sequencing (518).
Beginning at 9 weeks, testosterone is produced from
cholesterol by chorionic gonadotropin stimulation of fetal
The most prevalent mutation, a 27-base deletion in exon 10
Leydig cells through the coordinated action of steroidogenic
(c.1332_1358del) pictured on Fig. 23 results in the deletion
enzymes (Fig. 27 and Table 7), most of which are also
of 9 amino acids from an alpha helix within the kinase
expressed in the adrenal gland, explaining why many
domain and affects 37% of families with receptor mutations.
steroidogenic disorders are common to the testis and
The proportion reaches 62% of Northern European families,
adrenal. Most steroidogenic enzymes are either
where it probably represents a founder effect. This mutation
hydroxysteroid dehydrogenases or cytochromes P450,
is easily detected by PCR, without the need for sequencing.
residing either on the mitochondrial membrane (type I) or in
the endoplasmic reticulum (type II) (524). The initial step in
Not all PMDS cases have benefited from molecular study.
steroidogenesis, conversion of cholesterol into
In many countries, genetic studies are not readily available
pregnenolone, is mediated by the P450 side-chain cleavage
for PMDS, and cases have been published with only clinical
enzyme (P450scc), a type I cytochrome located at the inner
data (519, 520). Owing to lack of molecular
mitochondrial membrane. However, the inner mitochondrial
characterization, it is difficult to interpret the unusual sex-
membrane contains relatively little cholesterol, so the rate-
linked familial transmission of PMDS reported in two
limiting step of steroidogenesis is the transfer of cholesterol
families (521, 522).
from the outer to the inner mitochondrial membrane. This
step is dependent on steroidogenic acute regulatory protein
In approximately 20% of PMDS patients, careful
(StAR) regulated essentially by a trophic hormone
sequencing of AMH and AMHR2 exons and adjacent
stimulated cAMP/PKA pathway (525). The exact
portions of introns have failed to yield an explanation. Either
mechanism of StAR-mediated cholesterol transport into the
a mutation has escaped detection or other genes are
mitochondria is not completely understood.
involved. The AMH and BMP families share type I receptors
and cytoplasmic effectors, which could be implicated in
Pregnenolone is subsequently metabolized into 17α-
PMDS. Initially, BMP receptors were considered unlikely
hydroxypregnenolone and dehydroepiandrosterone
candidates because an intact BMP pathway is required for
(DHEA) by P450c17. This type II cytochrome bears two
survival beyond the embryonic stage. However, this might
distinct activities: a 17α-hydroxylase activity responsible for
not hold for mild missense mutations (523). Alternatively,
the conversion of pregnenolone to 17α-
idiopathic PMDS could be caused by mutations in other
hydroxypregnenolone and a 17-20 lyase activity, capable of
genes involved in Müllerian duct development/regression
converting 17α-hydroxypregnenolone to DHEA. P450c17

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receives electrons from NADPH via the flavoprotein P450 compound progesterone into 17α-hydroxyprogesterone and
oxidoreductase (POR) (526, 527). Cytochrome b5 is Δ4-androstenedione.
required for optimal 17,20 lyase activity (528, 529).
P450c17 and its partner proteins also convert the Δ 4

FIGURE 27. Steroidogenesis. Steroidogenesis: the “classic” and “backdoor” pathways for dihydrotestosterone
(DHT) synthesis. See Table 7 for enzyme nomenclature. DHEA: dehydroepiandrosterone, DHP:
dihydroprogesterone. Reprinted from ref. (530): Fluck CE, Meyer-Boni M, Pandey AV, Kempna P, Miller WL,
Schoenle EJ, Biason-Lauber A. Why boys will be boys: two pathways of fetal testicular androgen biosynthesis
are needed for male sexual differentiation. American Journal of Human Genetics 89:201-218 (2011). Copyright
2011, with permission from Elsevier. http://www.cell.com/AJHG/abstract/S0002-9297(11)00262-X (top figure), and
ref. (531): Wilson JD, Shaw G, Leihy ML, Renfree MB. The marsupial model for male phenotypic development.
Trends in Endocrinology and Metabolism, 13:78-83 (2002), Copyright 2002, with permission from Elsevier.
http://www.sciencedirect.com/science/article/pii/S1043276001005252 (bottom figure).

Two additional enzymes, 3β- and 17β-hydroxysteroid dehydrogenases have been identified: 3ß-HSD type 1,
dehydrogenases are required for the synthesis of expressed mainly in the placenta, mammary gland and skin,
testosterone. Two isoforms of 3ß-hydroxysteroid and 3ß-HSD type 2, expressed in the gonads and adrenal

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glands. Only mutations in the type 2 gene result in the testis and is the only one involved in fetal male sexual
congenital adrenal hyperplasia and/or DSD (532, 533). differentiation (534). XY patients with impaired HSD17B3
usually develop with female or ambiguous external
The final testicular enzyme in testosterone biosynthesis is genitalia; however, Wolffian ducts derivatives are present in
17ß-hydroxysteroid dehydrogenase (17β-HSD), formerly most, probably due to accumulation of the weak androgens
known as 17-ketosteroid reductase, which reduces 17- Δ4-androstenedione and Δ5-DHEA. The type 2, HSD17B2,
ketosteroids to 17β-hydroxysteroids, i.e. Δ4- is expressed in the liver and has the capacity for
androstenedione to testosterone and the Δ5 steroid DHEA testosterone synthesis. This could explain the virilization
to androstenediol. Three isoforms of 17ß-HSD have been observed at puberty in XY patients with HSD17B3
identified. The type 3 isoform, HSD17B3, is expressed in deficiency (534, 535).

TABLE 7. Proteins Involved in Androgen Production

Protein Main Role Gene Chromosome
Steroidogenic acute Regulatory Cholesterol trafficking STAR 8p11.2
Protein (StAR)
P450scc (P450 side chain Cholesterol side-chain CYP11A1 15q24.1
cleavage enzyme) cleavage
Cytochrome P450, family 11,
subfamily A, polypeptide 1
P450c17 (17α- Metabolizes pregnenolone CYP17A1 10q24.32
hydroxylase/17,20-lyase)
Cytochrome P450, family 17,
subfamily A, polypeptide 1
P450 oxidoreductase (POR) Electron donor to P450c17 POR 7q11.23
Cytochrome b5, type A Regulation of 17,20-lyase CYB5A 18q22.3
activity
3β-hydroxysteroid Conversion of Δ5 to Δ4 HSD3B2 1p12
dehydrogenenase 2 (3β-HSD 2) steroids
17β-hydroxysteroid Reduction of 17 keto to 17β- HSD17B3 9q22.32
dehydrogenenase 3 (17β-HSD hydroxysteroids
3)
5α-reductase type 2 Reduction of T to DHT SRD5A2 2p23.1
5α-reductase type 1 Reduction of progesterone SRD5A1 5p15.31
and 17OH-progesterone
Aldo-keto reductase family 1, Oxidoreduction of 3α- AKR1C2 10p15.1
member C2 androstanediol/ DHT *
3α-hydroxysteroid
dehydrogenase, type III (3α-
HSD)
Aldo-keto reductase family 1, id but less efficient AKR1C4 10p15.1
member C4
3α-hydroxysteroid
dehydrogenase, type I
17β-hydroxysteroid Oxidizes 3α-androstanediol to HSD17B6 12q13.3
dehydrogenase 6 (17β-HSD 6) DHT RODH
Retinol dehydrogenase 1
3α-hydroxysteroid epimerase

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 P450aro (aromatase) Aromatizes androgens to CYP19A1 15.q21.2
Cytochrome P450, family 19, estrogens
subfamily A, polypeptide 1
Steroidogenic factor 1 (SF1) Regulates several NR5A1 9q33.3
Nuclear receptor subfamily 5, steroidogenic enzymes
group A, member 1
Adrenal-4 binding protein
(AD4BP)
Fushi tarazu factor 1 (FTZF1)
* The direction of the reaction depends on cofactor availability (530). The four last enzymes act exclusively in the alternate
pathway of DHT synthesis.

DHT Production: Classic and Alternative

(Backdoor) Pathways Testosterone, however, is not an obligatory precursor of
DHT (Fig. 27, bottom). Observations in a marsupial, the
tammar wallaby (531), have shown that the testis itself
Testosterone itself is not a very active androgen; its
produces biologically significant amounts of DHT through
metabolite DHT is the main virilizing agent during male
an alternate or “backdoor” pathway without using
reproductive development. The conversion of testosterone
testosterone, DHEA or androstenediol as intermediates.
to DHT amplifies the androgenic signal through several
Additional enzymes not part of the classic pathway can
mechanisms. DHT cannot be aromatized to estrogen, and
mediate the direct oxidation of 5α-androstanediol to DHT
thus its effects are purely androgenic. Testosterone and
(524, 530). This "backdoor" pathway contributes to
DHT bind to the same androgen receptor but DHT does so
virilization in the human fetus as demonstrated by the
with greater affinity which results in a stabilization of the
genetic studies of Flück and her colleagues (530) in two
hormone-receptor complex for a longer period of time (536).
families with 46,XY DSD. After they failed to demonstrate
mutations in known steroidogenic enzymes, they explored
In the classic pathway of DHT production (Fig. 27, top),
genes acting in the alternate pathway of androgen
testosterone is converted to DHT inside the target cell by
synthesis. This led to discovery of mutations in the genes
the enzyme 5α-reductase type 2 coded by the SRD5A2
AKR1C2 and AKR1C4, alias 3α-hydroxysteroid
gene expressed in fetal genital skin, in male accessory sex
dehydrogenase (or reductase) type I and type III. In the
glands and in the prostate (537). In tissues equipped with
alternate pathway these enzymes catalyze the reduction of
5α-reductase at the time of sex differentiation, such as the
dihydroprogesterone and 17OH-dihydroprogesterone to
urogenital sinus and external genitalia, DHT is the active
allopregnanolone and 17OH-allopregnanolone, the
androgen (538). During embryogenesis, 5α-steroid
precursor of androsterone and androstanediol. Their role in
reductase type-2 encoded by the SRD5A2 gene plays a
the oxidation of the latter to testosterone is hypothetical
central role in the differentiation of the male phenotype.
because they have very high affinity for NADP(H), which
Patients with 5α-reductase deficiency virilize very poorly at
favors reductive reactions and low affinity for NAD(H) which
these levels (539, 540). Another functional isoenzymes of
favors the opposite, thus they are expected to function
5α-reductase, with a different pH optimum, has been
primarily as a reductase (541). AKR1C2 is expressed in the
characterized (537): 5α-reductase type 1, transiently active
fetal, but not the adult testis, AKR1C4 is expressed at low
in newborn skin and scalp and permanently expressed in
levels in both tissues. The deleterious effect of AKR1C2/4
liver after birth and in skin from the time of puberty, is not
mutations proves that testicular DHT synthesis through the
expressed in the fetus. Tissue distribution and ontogeny of
alternate pathway is required for normal fetal sex
both isoforms as well as mutation studies in humans with
differentiation.
46,XY DSD clearly indicate that type 2 plays the major role
in sexual differentiation but the emergence of type 1
Gonadotropin Control of Testosterone
probably accounts for the pubertal virilization of the type 2-
deficient patients. Production

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Testosterone production by the human fetal testis is differentiation, with the exception of penile growth, occur at
detectable at 9 weeks, peaks between 14 and 17 weeks and the time Leydig cells are controlled by hCG.
then falls sharply, so that in late pregnancy the serum
concentrations of testosterone overlap in males and In contrast, mutations in the LH/CG receptor of Leydig cells
females. Gonadotropin stimulation is not required for the result in severe virilization defects (544). LH and hCG signal
initiation of steroid synthesis (220) but is necessary to through a common seven-transmembrane domain receptor
maintain Leydig cell function subsequently. Testicular and coupled to G proteins present on testicular Leydig cells. The
serum levels of testosterone are closely correlated with human gene located on chromosome 2p21, contains 11
human chorionic gonadotropin (hCG) concentration; the exons. The first ten encode a long N-terminal extracellular
peak of fetal testicular steroidogenic activity coincides with domain responsible for hormone binding, while the 11th
the acme of concentrations of hCG in the circulation. In adult exon encodes the whole transmembrane domain, involved
Leydig cells, the capacity to respond to sustained in the cAMP/PKA signal transduction pathway. A
gonadotropic stimulation by increased androgen production functioning LH/CG receptor is absolutely necessary to
is curtailed by the development of a refractory state, due to achieve a normal development of the fetal Leydig cell
receptor down-regulation (542). Fetal Leydig cells population and androgen production. Loss of function
apparently escape desensitization, allowing them to mutations lead to 46,XY DSD (reviewed in ref. (545), with
maintain a high testosterone output during the several the exception of the deletion of exon 10, which was
weeks necessary to male differentiation of the genital tract. identified in a patient with normal male phenotype but lack
The fetal pituitary takes over when chorionic gonadotropin of pubertal development (546, 547). This suggests that
declines in the 3rd trimester (reviewed in ref. (543) (Fig. 28). exon 10 is required for signal transduction of pituitary LH but
Impaired LH secretion in 46,XY fetuses does not result in not hCG.
DSD because the most important steps of sexual

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FIGURE 28. Control of testosterone production in the human fetus. Note the low testosterone
concentration during the last trimester, at the time that hCG production by the placenta has abated.
Data obtained from ref. (548): Winter JSD, Faiman C, Reyes F (1981). Sexual endocrinology of fetal
and perinatal life. In: Austin CR, ed. Mechanisms of Sex Differentiation in Animals and Man. London:
Academic Press; p.205-253.

The Androgen Receptor kb comprises 8 exons. Exon 1 is the longest and codes for
the amino-terminal transactivation domain. A highly
Testosterone and DHT exert their action on androgen- polymorphic CAG triplet containing 14-35 repeats towards
dependent tissues by binding to the androgen receptor, a the 5’-end of exon 1, is useful as a genetic marker for
member of the steroid receptor family (Fig. 29). Mutations inheritance of X chromosomes. Interestingly, expansion of
of this receptor lead to the androgen insensitivity syndrome, the trinucleotide repeat which encodes this long tract of
a relatively common disorder of sex development typically glutamine residues segregates with X-linked spinal and
characterized by a female external genital appearance in bulbar atrophy a degenerative neuropathy characterized by
XY patients despite a normal or excessive production of the accumulation of the mutated receptor in the nucleus and
testicular hormones (see ref. (549) for review). The cytoplasm of motor neurons (reviewed in ref. (551). Exons
androgen receptor is encoded by a single-copy gene 2 and 3 code for sequences containing two zinc fingers
located on the long arm of the X chromosome, locus Xq12 implicated in DNA binding. Most mutations occur in exons 4
(550). It spans 75-90 kb and its open reading frame of 2.75 to 8, which encode the steroid hormone binding domain.
The 5’-portion of exon 4 codes for the hinge region between

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the DNA- and steroid-binding domains, and plays a The androgen receptor binds to specific DNA motifs, the
regulatory role (552). A complete database of androgen androgen response elements (ARE), present in the
receptor mutations is available from McGill University in promoter regions of androgen-activated genes. The
Montreal (553). consensus or classic ARE consists of two palindromic half
sites spaced by three base pairs
In contrast to receptors for other steroid sex hormones, (AGAACAnnnTGTTCT).while the so-called "selective"
which reside in the nucleus even in the absence of ligand AREs, such as the one in intron 1 of the SRD5A2 gene (556)
binding, the androgen receptor resides mainly in the resemble direct repeats of the same hexamer (557). After
cytoplasm, associated with heat-shock and other binding to AREs on the promoters of androgen-responsive
chaperone proteins, in the absence of hormone and genes, the androgen receptor regulates their transcriptional
translocates into the nucleus in the presence of ligand activity. It is aided in this task by co-regulators, partner
(554). Nuclear localization is controlled by a nuclear proteins that facilitate assembly of the preinitiation complex
localization signal spanning the second zinc finger and the through chromatin remodeling. These include the p160
hinge region competing with an androgen-regulated nuclear family of coactivators, which interact selectively with the
export signal in the ligand binding domain (555). The agonist-bound form of AR (558-560). Attempts at blocking
androgen/AR complex can also signal through non-DNA the androgen receptor by preventing its interaction with co-
binding-dependent pathways. However, the physiological activators are part of the therapeutic strategy in prostate
relevance of these actions remaining largely unknown cancer (554).
(554).

FIGURE 29. Androgen receptor protein, cDNA and gene.

The Case of the Wolffian Ducts: The Role of Local degeneration (537, 538). Because of its close proximity to
Testosterone the testis, the Wolffian duct is exposed to a very high local
concentration of testosterone, a source of androgen not
In fetal Wolffian ducts, 5α-reductase is expressed only after available to organs receiving testosterone only via the
the ambisexual, critical, stage of male sex differentiation, peripheral circulation (Fig. 30) (297). Patients with androgen
thus testosterone itself, not DHT, saves them from insensitivity whose androgen receptor retains very low but

www.EndoText.org 59
significant residual activity have a female phenotype but inguinoscrotal phase of testicular descent. The mechanism
retain an epididymis or vas deferens (561). Wolffian duct of androgenic action on the gubernaculum is controversial.
differentiation is programmed during a critical time window, Androgens could act through the genitofemoral nerve and
between 15.5 and 17.5 dpc in the rat fetus. Because the the neuropeptide calcitonin gene-related peptide (565,
androgen receptor is expressed in the Wolffian duct stroma 566). Thus, any condition associated with decrease of fetal
but not in the epithelium during this time, Wolffian duct testicular production or action may impair testicular descent.
differentiation is likely to be dependent on androgen-
mediated signaling from the stroma to the epithelium (562). The first, transabdominal, phase of testicular descent is
controlled by Insulin-like factor 3 (INSL3), a member of the
Two phase can be described in the development of the insulin/relaxin hormone superfamily secreted by Leydig
Wolffian ducts (297). In the first phase, testosterone induces cells, signaling through its G protein-coupled receptor
the stabilization of the ducts (in rodents, this occurs LGR8, now known as relaxin family peptide receptor 2
between embryonic days 13 and 16). Afterwards the (RXFP2) (217, 567). INSL3 acts by inducing male
Wolffian ducts undergo elongation and convolution of the development of the gubernaculum testis. Mutations of
cranial end, where the epididymis and vas deferens INSL3 have been detected in cryptorchid patients (568),
differentiate, and the seminal vesicles form at the caudal similarly deletion of Rxfp2 targeted to mesenchymal
end. gubernacular cells leads to high cryptorchidism in mice
(569). Prenatal DES treatment, which is associated with
Control of Testicular Descent cryptorchidism, impairs Insl3 expression in mouse testis
and interferes with gubernacular development (570).
Androgens are required to mediate the disappearance of
the cranial suspensory ligament (563, 564) and later for the

FIGURE 30. Respective roles of testosterone(T) and dihydrotestosterone (DHT) in sex differentiation. Normal
androgen physiology in mammals. Testosterone and dihydrotestosterone are assumed to work by binding to the
same receptor protein and forming hormone–receptor complexes of different allosteric configurations.
Abbreviations: AR, androgen receptor; 17β-HSD3, 17β-hydroxysteroid dehydrogenase type 3; LHR, luteinizing

www.EndoText.org 60
hormone receptor; 5α-R2, steroid 5α-reductase type 2. Reprinted from ref. (531): Wilson JD, Shaw G, Leihy ML,
Renfree MB. The marsupial model for male phenotypic development. Trends in Endocrinology and Metabolism,
13:78-83 (2002), Copyright 2002, with permission from Elsevier.
http://www.sciencedirect.com/science/article/pii/S1043276001005252.

HORMONAL CONTROL OF FEMALE 575). Phthalates also adversely affect male differentiation
DIFFERENTIATION by increasing the expression of COUP-TF2, a transcription
factor which represses steroidogenic enzymes (576).
Evidence from animal studies show that environmental
Estrogens, Diethylstilbestrol, Xenoestrogens
exposure to endocrine disrupting chemicals is at least
partially responsible (reviewed in (577, 578). Phthalates
The conclusion that ovarian hormones are not necessary to
may act as pseudo-estrogens (biphenol A, alias BPA) or as
female development of the female reproductive tract (58,
antiandrogens (diethylhexylphthalate, alias DEHP) (579);
59) is supported by the female phenotypic development of
however caution is required for interpretation of animal
45,X or 46,XY subjects with bilateral gonadal aplasia and of
studies because of species differences. In human testes,
aromatase knockout mice unable to synthesize estrogens.
germ cells appear the most susceptible to damage by
Yet, inappropriate estrogen exposure is clearly detrimental.
phthalates (580). Atrazine, a herbicide widely used in the
The most tragic illustration of estrogen toxicity is the « DES
United States, demasculinizes male gonads and reduces
story ». Diethylstilbestrol (DES), a synthetic estrogen, was
sperm count by interfering with phosphodiesterase
widely administered to pregnant women in the early 1940s
enzymes and SF1 (581).
in the hope of preventing abortion. It was later discovered
that female progeny exhibited severe abnormalities of the
CONCLUSION
reproductive tract: vaginal clear-cell adenocarcinoma,
vaginal adenosis and squamous metaplasia, transverse
A bewildering number of hormones and growth factors is
vaginal ridges and structural malformations of the cervix
involved in sex determination and differentiation, making it
and uterus (571, 572).
one of the best studied developmental processes. The
uncovering of an active genetic pathway towards ovarian
Environmental chemicals that exert deleterious effects upon
development has overturned the dogma of a default
the endocrine axis are called endocrine disruptors. By
pathway towards female gonadal differentiation. For the
binding to nuclear hormone receptors, they may affect
moment, testicular hormones retain their primacy in
sexual differentiation. Unregulated exposure to
modeling the reproductive tract but who knows what
xenoestrogens such as bisphenol A is now incriminated in
surprises the future holds in store?
the occurrence of cryptorchidism and hypospadias (573-

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