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Sex determination and sex reversal
Giovanna Camerino, Pietro Parma, Orietta Radi and Stella Valentini
Sex determination in mammals is based on a genetic cascade
that controls the fate of the gonads. Gonads will then direct the
establishment of phenotypic sex through the production of
hormones. Different types of sex reversal are expected to occur
if mutations disrupt one of the three steps of gonadal
differentiation: formation of the gonadal primordia, sex
determination, and testis or ovary development.
Dipartimento di Patologia Umana ed Ereditaria, Università di Pavia,
Via Forlanini 14, 27100 Pavia, Italy
Corresponding author: Camerino, Giovanna ([email protected])
Current Opinion in Genetics & Development 2006, 16:289–292
This review comes from a themed issue on
Genetics of disease
Edited by Andrea Ballabio, David Nelson and Steve Rozen
Available online 2nd May 2006
0959-437X/$ – see front matter
# 2006 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.gde.2006.04.014
Gonadal sex determination and sex reversal:
the general design
In mammals, sex is genetically determined and is based
on the chromosomal constitution of the embryo. Presence
or absence of the Y chromosome, or of the Y-encoded SRY
(sex-determining region on the Y chromosome) gene, determines the sex of the gonads: XY embryos develop testes,
whereas XX embryos develop ovaries. In turn, the phenotypic sex of the embryo (i.e. the development of
secondary sex characteristics, including internal and
external genitalia) depends on the sex of the gonads.
However, there is not a perfect evenness in the two
pathways. Whereas functional testes are necessary for
male differentiation of the embryo, female differentiation
does not require the presence of functional ovaries.
Testes produce hormones that are necessary and sufficient to bring about male differentiation in the embryo:
these include the anti-Müllerian hormone (AMH), which
induces regression of the female internal genitalia; the
androgens testosterone and dihydrotestosterone, which
direct the differentiation of the internal and external male
genitalia; and insulin-like factor 3 (INSL3), which,
together with testosterone, directs the descent of the
testis into the scrotum. Conversely, sex hormones are
not required for the ‘default’ female differentiation
pathway. Thus, a phenotypic female will be formed if
the gonads develop into ovaries but also if the gonads do
not develop correctly (i.e. gonadal dysgenesis or gonadal
agenesis) and if AMH and androgens do not reach the
threshold level. Accordingly, XY sex-reversal (genetic
males develop as females) in humans is relatively frequent (approximately 1 in 3000 newborns) and genetically heterogeneous, whereas XX sex-reversal (genetic
females develop as males) is more rare (1 in 20000 newborn) and is usually caused by translocations of SRY to the
X chromosome or to an autosome.
Excellent reviews on sex determination and gonadal
differentiation have been published [1,2,3,4]. Far
from being comprehensive, this review only focuses on
the process of gonadal sex determination and on the
effects of its disruption on the establishment of the
phenotypic sex of the embryo. The review is also very
schematic; for example, we do not discuss ambiguous
genitalia, generally caused by inadequate levels of testicular hormones.
Formation of the gonadal primordia
In the mouse, gonadal development begins at embryonic
day 10.0 (E10), with the thickening of the coelomic
epithelium adjacent to the ventro-medial surface of the
mesonephros. Gonadal primordia are morphologically
indistinguishable in male and female embryos. Primordial
germ cells are specified in the epiblast and migrate to the
developing gonads between E10 and E11 [5].
Loss-of-function mutations in a number of transcription
factor genes (including Emx2, Lhx9, Sf1, Wt1–KTS, Pod1
and M33) result in the degeneration of gonads before the
period of sex determination, suggesting that these proteins are required for the specification and maintenance
of the gonadal primordia [1,2,3]. However, many of
these genes have been shown to play important roles both
in the development of other organs and in later stages of
gonadal differentiation. If compatible with life, these
mutations cause gonadal dysgenesis (or agenesis), and
embryos develop a female phenotype irrespective of their
chromosomal sex (i.e. XY sex-reversal).
To date, only two of these transcription factors have been
linked to an abnormal sexual development in humans.
Heterozygous mutations in SF1 (Online Mendelian
Inheritance in Man [OMIM] database, 184757) cause
XY sex-reversal associated with adrenal failure. Different
types of mutations in WT1 (OMIM, 607102) have been
associated with various syndromes: WAGR, Denys–
Drash, and Frasier, each characterized by different
Current Opinion in Genetics & Development 2006, 16:289–292
290 Genetics and disease
degrees of gonadal dysgenesis associated with kidney
anomalies or predisposition to kidney tumors.
Sex determination in XY gonads
Testes begin to differentiate earlier than ovaries, suggesting that the initiation of the male pathway is an active
process diverting the gonadal primordia towards testis
fates before ovarian commitment. Although the male
and female gonads are morphologically identical until
around E12, the two differentiation pathways have previously started to branch out at the molecular level. In
mice, Sry is switched on in the XY gonad a few hours after
E10.5. Its expression peaks around E11.5 and is turned
off shortly before E12.5. Little is known about the
regulation of SRY transcription, although three factors,
Gata4, Fog2 and Wt1(+KTS) have been implicated
Sertoli cells, the supporting cell lineage of the testis, are
responsible for AMH production and for supporting the
proliferation and differentiation of germ cells. They are
the first somatic cells of the gonads to be committed to a
testicular fate and they play a fundamental role in the first
stages of testis differentiation. Sry is expressed in Sertoli
cell precursors, and — at least in mouse — must act
during a very crucial time-window in order to properly
activate male testis determination [6,7].
One of the first cellular events triggered by Sry expression
is cellular proliferation in the XY coelomic epithelium,
possibly to produce sufficient Sertoli cell precursors to
initiate testis development [8]. Fgf9 and possibly IGF
have been shown to play a role in this process [9,10].
At the molecular level, the molecular target(s) of SRY are
still elusive, although several indirect observations suggest that the transcription factor Sox9 is one of these
[6,11]. Low levels of Sox9 are expressed in XX and XY
gonadal primordia. Once the SRY protein product reaches
a threshold concentration level, Sox9 is up-regulated in
XY gonads and, at around the same period, is downregulated in XX gonads [12]. Moreover, the Sox9 protein,
which is cytoplasmic in both sexes prior to the onset of Sry
expression, is translocated to the nucleus of XY preSertoli cells [13]. Sry, a protein involved in chromatin
remodelling and transcription regulation, might act cellautonomously to trigger differentiation of Sertoli cells and
expression of Sox9. However, historical XX $ XY chimera studies and more recent data revealed that not all
Sox9-expressing Sertoli cell have experienced Sry expression, suggesting the existence of a paracrine signal —
possibly prostaglandin D2 (PGD2) — driving the Sox9
expression in these cells [14,15]. This mode of Sertoli
cells’ recruitment might serve to increase the number of
supporting cells during testis development. PGD2 has
also been shown to induce SOX9 nuclear translocation in
NT2–D1 Sertoli-like cells [16].
Current Opinion in Genetics & Development 2006, 16:289–292
Sex determination in XX gonads
In contrast to the situation in the male gonad, where
dramatic morphological changes follow Sry expression, in
the female gonad the first cellular event occurs at E13.5,
when germ cells enter into meiosis. However, the femalespecific molecular program is activated as early as it is in
the male gonad [17–19]. In a recent large-scale transcriptional analysis, 2306 genes were found to be expressed in
a sex-specific manner in the somatic compartment of the
gonads between E10.5 and E13.5: 1223 were overexpressed in XX embryos, and 1083 in XY embryos [17].
Mechanisms for ovarian commitment and early development remain elusive. Three genes, upregulated in XY
gonads during the period of sex determination, have been
proposed as candidate ovary-determining. Overexpression of DAX1 in the male gonads results in XY sex-reversal
in both humans and mice. However, knockout and transgenic studies in mice indicate that Dax1 is not required for
normal ovarian development but is crucial for testicular
development [4]. Wnt4-null XX mice are masculinized as
a result of the ectopic presence of androgen-producing
cells and the lack of a Müllerian duct. Nevertheless, Wnt4
has been shown to be directly required for Müllerian duct
development, and the androgen-producing cells express
markers specific to adrenal steroidogenic cells. However,
Wnt4 possesses some ’anti-male’ properties, because its
expression appears to inhibit the migration of endothelial
and steroidogenic cells into the developing ovary (see
below) [4]. Finally, Pailhoux et al. [20] demonstrated that
the deletion of an 11.7-kb DNA element is responsible for
PIS syndrome, a XX sex-reversal condition in the goat.
The deletion was shown to affect the transcription of at
least 2 genes: PISRT1, encoding a 1.5-kb mRNA devoid
of open reading frames; and FOXL2. Mutations in FOXL2
in humans cause BPES syndrome (blepharophimosis
ptosis epicanthus inversus syndrome), which is associated
with premature ovarian failure [21]. Again, null mutations
of Foxl2 in mice do not affect initial ovary formation, but
lead to failure in primary follicle formation [22,23].
Sex determination and sex reversal
The gonadal primordium is unique among all organs
because it can develop in two different organs, a testis
or an ovary. Defects in the establishment of the gonadal
primordia or in the differentiation of the gonads after sex
determination generally result in defective organ formation. By contrast, mutations affecting the sex determination process can result in real sex reversal (i.e. in redirecting a genetically female gonad to a testicular fate or
vice versa).
In theory, XX sex-reversal can be produced by the
expression of testis-determining genes in XX gonads
(i.e. gain-of-function mutations) or by loss-of-function
mutations in ovary-determining genes. Approximately
85% of the cases of XX sex-reversal in man are due to
Sex determination and sex reversal Camerino, Parma, Radi and Valentini 291
the translocation of the SRY gene to the X chromosome or
to an autosome. SRY-independent XX sex-reversal is
extremely rare and its molecular basis in man unknown.
In the mouse model, XX sex-reversal has been artificially
produced only by the ectopic expression of the testisdetermining genes Sry and Sox9 in the developing XX
gonads [24,25,26,27]. As previously discussed, loss-offunction mutations in the three candidate ovary-determining genes, Dax1, Foxl2 and Wnt4, do not result in
complete XX sex-reversal in mouse [22,23,28–33]. A
heterozygous mutation in WNT4 has been associated with
Müllerian duct regression and virilization in an XX
patient [34].
Loss-of-function mutations in human SRY are characterized by gonadal dysgenesis after birth, and only account
for approximately 10–15% of XY sex-reversal in man.
Haploinsufficiency for SOX9 causes campomelic dysplasia associated with XY sex-reversal. To our knowledge, no
gain-of-function mutations have been associated with XY
ambiguous genitalia. Conversely, mutations in genes
required for ovarian development might cause ovarian
dysgenesis [4].
More than fifteen years after the SRY gene was identified,
a large proportion of the genes involved in the genetic
pathway for gonadal sex determination and differentiation still remain unidentified. Accordingly, mutational
screenings in sex-reversed patients have demonstrated
that only a small subset of patients carry mutations in
known genes. The integration of new and old approaches
is likely to reveal many new players in the field. Positional
cloning efforts and large-scale mutagenesis screens in
families and in sporadic cases with subtle genomic rearrangements are in progress. Large-scale transcriptional
analyses for genes expressed in a sex-specific manner
have identified genes that are dimorphically expressed in
the first stages of gonadal differentiation. Functional
analysis of many of these genes is underway.
Gonadal differentiation
Following sex determination and Sertoli cell differentiation, several cellular events take place in the XY gonad.
Sertoli cells polarize and aggregate around germ cells,
thereby causing the reorganization of the gonad into
two compartments: the testis cords, composed of Sertoli
and germ cells; and the interstitial space between the
cords. Migration of mesonephric cells into the XY gonad,
mainly consisting of endothelial, perivascular and peritubular myoid cells, is also a male-specific event. Peritubular
myoid cells surround Sertoli cells and cooperate to deposit
the basal lamina at the periphery of testis cords. Migrating
endothelial cells associate to establish the coelomic vessel
that promotes the efficient export of testosterone from the
early testis. Finally, fetal Leydig cells, the androgen-producing cells, differentiate between E12.5 and E13.5 in the
interstitial space between cords (reviewed in [1,2]).
In the female gonad, the first cellular event occurs at
around E13.5, when germ cells enter into meiosis and will
arrest at prophase 1 at birth. Germ cells play an essential
role in the development of the ovary, because XX gonads
depleted of germ cells fail to form ovarian follicles and
degenerate [5,35]. In the presence of meiotic germ cells,
the somatic cells of the gonad differentiate into follicles
that surround the oocytes. Several of the genes required
for follicle formation have been identified, including the
two transcription factor genes Figa and Foxl2, whereas
Wnt4 and follistatin are required during early gonad
development to repress aspects of testis differentiation
in XX gonads [4].
Loss-of-function mutations in several of the genes
involved in the differentiation of the testis (e.g. Dhh,
Arx and Pod1 [1,2,3]), cause XY sex-reversal or
Work in our laboratory was supported by grants from Coperativa Est
Ticino, Telethon, European Community, MIUR and CNR.
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