Control of Sex Development PDF

Best Practice & Research Clinical Endocrinology & Metabolism 24 (2010) 163–186
Contents lists available at ScienceDirect
Best Practice & Research Clinical

Endocrinology & Metabolism
journal homepage: www.elsevier.com/locate/beem
Control of sex development

Anna Biason-Lauber, MD, Assistant Professor of Pediatric Endocrinology *
Division of Endocrinology/Diabetology, University Children’s Hospital, Steinwiesstrasse 75, CH-8032 Zurich, Switzerland
Keywords:
The process of sexual differentiation is central for reproduction of
sex development almost all metazoan, and therefore, for maintenance of practically
sex determination all multicellular organisms. In sex development, we can distinguish
sex differentiation two different processes, sex determination, that is the develop-
ovary mental decision that directs the undifferentiated embryo into
testis a sexually dimorphic individual. In mammals, sex determination
disorders of sex development (DSD) equals gonadal development. The second process known as sex
bipotential state
differentiation takes place once the sex determination decision has
been made through factors produced by the gonads that determine
the development of the phenotypic sex. Most of the knowledge on
the factors involved in sexual development came from animal
models and from studies of cases in whom the genetic or the
gonadal sex does not match the phenotypical sex, that is, patients
affected by disorders of sex development (DSDs).Generally
speaking, factors influencing sex determination are transcriptional
regulators, whereas factors important for sex differentiation are
secreted hormones and their receptors.
This review focusses on these factors and whenever possible,
references regarding the ‘prismatic’ clinical cases are given.
Ó 2009 Elsevier Ltd. All rights reserved.
Abbreviations: DSD, Disorders of sex development; Pax2, Paired Homeobox 2; Emx2, Empty spiracle homolog 2; Lhx9, Lim
Homeobox protein 9; WT1, Wilms Tumour 1; GATA4, GATA-binding protein 4; SRY, Sex determining Region Y; SOX9, SRY- box 9; SF1/
NR5A1, Steroidogenic Factor 1/Nuclear Receptor subfamily 5, group A, member 1; DAX1, Dosage sensitive sex reversal, Adrenal
hypoplasia congenital critical region on the X chromosome gene 1; DMRT1, Doublesex and MAB3-Related Transcription factor 1;
DHH, Desert Hedgehog; ATRX, Alpha Thalassemia, mental Retardation syndrome, X-linked; CBX2, Chromobox homolog 2; TSPYL1,
Testis-specific protein Y-like-1; MAMLD1, Mastermind-Like Domain-Containing Protein 1; PGD2S, Prostaglandin D2 synthase; RSPO1,
Root-plate specific Spondin 1; WNT4/FST, Wingless Type MMTV intergration site family, member 4/Follicostatin; FOXL2, Forkhead
transcription factor; AMH/AMHR ¼ MIS, Anti-Müllerian Hormone/Receptor ¼ Müllerian Inhibiting Substance; HOXA, Homeobox A;
Lim1/LHX1, Lim Homeobox protein 1; LH/CG, Luteinising Hormone/Corionic Gonadotropin; POF, Premature ovarian failure; ODG,
Ovarian dysgenesis; FMR1, Fragile X Mental Retardation 1; DIAPH2, Diaphanous (dia) homolog 2; GDF9, Growth Differentiation
Factor 9; BMP15, Bone Morphogenic Protein 15; NOBOX, Newborn Ovary Homeobox; FIGLA, Factor in Germ Line Alpha.
* Tel.: þ 41 44 266 7623; Fax:þ 41 44 266 7169.
E-mail address: Anna.Lauber@kispi.uzh.ch
1521-690X/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.beem.2009.12.002
164 A. Biason-Lauber / Best Practice & Research Clinical Endocrinology & Metabolism 24 (2010) 163–186
Physiology of sex development
In sex development, we can distinguish two different processes, ‘sex determination’, that is, the
developmental decision that directs the undifferentiated embryo into a sexually dimorphic individual
and ‘sex differentiation’ that takes place once the sex determination decision has been made through
factors produced by the gonads that determine the development of the phenotypic sex. At the
beginning of gestation (1st and 2nd week), embryos of the two sexes differ only by their karyotypes.
Starting at week 3, specific genes lead to the differentiation of the gonads, which in turn, produce
hormones inducing anatomical and psychological differences, leading to behavioural differences that
are ultimately influenced by the social environment. As very nicely put by Sekido and Lovell-Badge:
‘‘Overall, sex determination is a story of opposing forces and crucial alliances but, although the winning
team takes all, its rule can be surprisingly tenuous.’’1
The main steps of intrauterine sex differentiation are depicted in Fig. 1. At gestational weeks 6–7, the
paramesonephric duct (Müllerian duct) develops next to the mesonephric duct. If testes develop and
secrete testosterone, the mesonephric (Wolffian) duct increases in size and differentiates into
epididymis, vas deferens and prostate. A glycoprotein secreted from the testicular Sertoli cells known
as anti-Müllerian hormone (AMH) or Müllerian inhibiting substance (MIS) results in Müllerian duct
regression. If testes do not develop, the mesonephric duct does not grow and eventually degenerates,
whereas the paramesonephric duct proliferates and the fallopian tube, uterus and the upper third of
the vagina develop (Fig. 2).
In mammals, including humans, the differentiation of the gonads is the turning point of this whole
process. The classical textbook theory says that in the presence of the sex-determining region on the
Y chromosome (SRY), the ‘default’ ovarian pathway of sex determination will be inhibited and
therefore, testes will be formed. In the XX individual, due to the absence of SRY, no inhibition of the
‘default’ programme will take place and ovaries will develop. Ovarian-determining factors might
help the process of differentiation. However, these factors are yet to be determined. The second
model called the ‘Z-factor theory’ was proposed to explain the cases where XX individuals develop
testes in the absence of SRY. According to this theory, the XX gonad expresses a factor that has both
anti-testis and pro-ovary functions. SRY in XY individuals acts as an inhibitor of the Z-factor to lift the
block on the male pathway. In this case, the bipotential gonad will differentiate into a testis.2 The
Z-factor remains unknown.
It is important to notice that most of the knowledge on the factors involved in sexual development
came from studies of cases in whom the genetic or the gonadal sex does not match the phenotypical
sex, that is, patients affected by defects of sex development3 and from animal models. This review
focusses on these factors and whenever possible, references on the ‘prismatic’ clinical cases are given.
Fig. 3 and Table 1 summarise the role of such factors and the effects of their mutations in mice and
men.
Factors involved in sex determination
Indifferentiated state (bipotential)
Pax2
Pax-2 is a transcriptional regulator of the paired-box family and is widely expressed during the
development of both ductal and mesenchymal components of the urogenital system. Pax-2 homozy-
gous mutant newborn mice lack kidneys, ureters and genital tracts. These defects might be attributed
to dysgenesis of both ductal and mesenchymal components of the developing urogenital system.4
In humans, genetic defects in PAX2 are identified in renal-coloboma syndrome, without genito-
urinary abnormalities.
Emx2
The homeobox gene Emx2 is a mouse homologue of a Drosophila head gap gene empty spiracles
(ems) and is essential for the development of dorsal telencephalon.5 At the same time, Emx2 is
A. Biason-Lauber / Best Practice & Research Clinical Endocrinology & Metabolism 24 (2010) 163–186 165
GW
0
Development of Wolffian ducts 4
Migration of germ cells 5 Migration of germ cells

6 Development of Müllerian ducts
Differentiation of seminiferous tubules 7
Regression of Müllerian ducts. 8

Appearance of Leydig cells
9 1st meiotic prophase in oogonia
Masculinization ofexternal
Masculinizationof externalgenitalia
genitaliabegins
begins 10 Wolffian ducts regression
Fetal testes in the internal inguinal rings

12-14
Penile urethra completed
Appearance of first spermatogonia 14
16 Appearance of first ovarian follicles
Max Leydig cells. Testosterone peak 17
Regression Leydig
Leydigcells.
cells.Testosterone
Testosteronelower
lower 20
1st multilayer ovarian follicles

24
Canalisation of the vagina
Descent of testes 28 Cessation of oogonia multiplication

//
38
Fig. 1. Embryonic development. GW: Gestational Week. Data derived from PC Sizonenko in Pediatric Endocrinology, edited by
J. Bertrand, R. Rappaport, and PC Sizonenko, (Baltimore: Williams & Wilkins, 1993), pp. 88–99.
expressed in the epithelial components of the developing urogenital system and, in Emx2 mutant mice,
the kidneys, ureters, gonads and genital tracts were completely missing. Degeneration of the Wolffian
duct and mesonephric tubules was also abnormally accelerated in these mice without the formation of
the Müllerian duct.6
Mutations in EMX2 are a rare cause of schizencephaly and somatic mutations in EMX2 have been
found in endometrial carcinomas,7 but, again, no human sex development defect due to EMX2
mutations has been described to date.
Fig. 2. The undifferentiated sexual system at 6–7 week of gestation. Precursor structure and their mature counterparts have the
same color. Fossum G. Atlas of Clinical Gynecology: Pediatric and Adolescent Gynecology. Edited by Morton Stenchever
(series editor), Alvin F. Goldfarb.
Lhx9
Transcripts of the LIM homeobox gene Lhx9 are present in urogenital ridges of mice at embryonic
day 9.5; later, they localise to the interstitial region as morphological differentiation occurs. In mice
lacking Lhx9 function, germ cells migrate normally, but somatic cells of the genital ridge fail to
proliferate and a discrete gonad fails to form. In the absence of testosterone and anti-Müllerian
hormone, genetically male mice are phenotypically female. The expression of steroidogenic factor 1
(Sf1), a nuclear receptor essential for gonadogenesis, is reduced to minimal levels in the Lhx9-deficient
genital ridge, indicating that Lhx9 may lie upstream of Sf1 in a developmental cascade. Lhx9 mutants do
not exhibit additional major developmental defects.8
Fig. 3. Sexual Development cascade. Mutations in various genes can lead to a variety of syndromes of dysgenesis involving the
müllerian or wolffian ducts, gonads, kidneys, and adrenal glands as a result of a deficiency or excess of the proteins shown. For
abbreviations see title page. Modified from McLaughlin and Donahoe, 2004.155
Although LHX9 mutations may underlie certain forms of isolated gonadal agenesis in humans, none
of such mutations has yet been found.
WT1
The WT1 gene encodes a zinc finger DNA-binding protein that is inactivated in some Wilms’
tumours. Mutation analysis in human patients and genetic experiments in mice revealed that WT1 has
a role in development as well as in tumour suppression. WT1 has at least four splicing variants
depending on the presence of exon 5 (17 amino acids in the mature protein) and/or nine nucleotides
coding for the three amino acids KTS (KTS). Post-transcriptional modifications of the Wt1 pre-mRNA
lead to the production of up to 24 isoforms, which increase the complexity of Wt1 action. WT1 is
already expressed within the undifferentiated gonad, and its importance there has been demonstrated
in Wt1/ mice, in which the gonads undergo apoptosis. Since the gonads and adrenal cortex share
a common primordium,9 it is not a surprise that adrenal glands are also affected in these mice.10
Expression of Sf1 and expression of WT1 in the gonad begin about simultaneously and might be
dependent. Wilhelm and Englert demonstrated that WT1, and more specifically, its DNA-binding form
WT1(–KTS), can activate the Sf1 promoter in vitro and in vivo.11 The apoptotic phenotype in Wt1-
knockout mice might therefore be caused by a lack of Sf1 expression. Alternatively, WT1 might activate
an Sf1-independent pathway required for adrenogenital survival. This could include the anti-apoptotic
factor Bcl-2, which might be directly activated by WT1.12 A second role for WT1 during gonad
formation occurs at the level of sex determination, through its control over Sry expression. Other
potential targets during gonad formation include Wnt-4,13 Dax114 and AMH, all of which seem to be
activated by WT1(–KTS) variants. Dax1, a gene that can interfere with male sexual development in
mouse and man was proposed to be a repressor of the AMH promoter that could displace WT1 from its
complex with Sf1. For more details see Wagner et al.15
Mutations in the WT1 gene have been identified in patients with renal disease, such as Wilms tumour
and isolated diffuse mesangial sclerosis (IDMS), and complex diseases such as Wilms tumour, aniridia,
Table 1
Human sex determination: effect of mutation
Chromosome Molecular Phenotype

(Human) defect
Human Mouse
Sexual development Other organs Sexual development Other organs
XY XX Both sexes XY XX Both sexes
Bipotential State
PAX2 10q24.3- LOF Normal Normal Renal-coloboma No genital tract No genital tract No kidneys
q25.1 syndrome No ureters
EMX2 10q26.1 LOF Normal (?) Normal (?) Schizencephaly No urogenital tract No genital tract No kidneys
No gonads No gonads No ureters
Neural abnormalities
Lhx9 1q31.q32 LOF NR NR NR No gonads No gonads Neural abnormalities
Müllerian ducts
WT1 11p3 LOF Ambiguous or Normal No urogenital tract No urogenital No kidneys
female (Frasier) Nephropathy/ No gonads tract Retina abnormalities
external genitalia anyridia: No gonads
Dysgenetic gonads - Wilms tumor
- WAGR syndrome
- Denys–Drash
syndrome
- Frasier
syndrome
- Isolated diffuse
mesangial
sclerosis
- Meacham
syndrome.
GATA4 8p23.1-p22 LOF Normal Normal Atrial septal defects Normal Dysgenetic Cardial defects
ovaries (?)
Testes
SRY Yp11.3 LOF - Dysgenetic testes n.a. Normal - Dysgenetic testes n.a. Normal
- Müllerian + - Müllerian +
- Female or ambiguous
external genitalia
SOX9 17q24.3- LOF - Dysgenetic testes Normal (?) Camptomelic No urogenital tract No genital tract No kidneys
q25.1 - Müllerian +/ dysplasia No gonads No gonads No ureters
Neural abnormalities
SF1/NR5A1 9q33 LOF Dysgenetic or Premature Adrenal Absent gonads Absent gonads Absent adrenals
absent testes ovarian failure insufficiency
- Female or ambiguous
external genitalia
- Müllerian +
DAX1/NROB1 Xp21.3-p21.2 LOF Normal Normal - Adrenal hypoplasia Normal (impaired Normal - Adrenal
Delayed puberty congenita (X-linked) spermatogenesis) hypoplasia
- Hypogonadotropic congenita
hypogonadims. (X-linked)
- Hypogonadotropic
hypogonadims.
Duplication - Dysgenetic or Normal Cleft palate
absent testes Mental retardation
- Female, ambiguous
or male external genitalia
- Müllerian +/
DMRT1 9q24.3 LOF - Dysgenetic testes Normal - Facial abnormalities NR NR NR
- Female or ambiguous - Mental retardation
external genitalia - Microcephaly
- Müllerian +/
DHH 12q13.1 LOF - Dysgenetic testes Normal Minifascicular Impaired NR Normal
- Ambiguous genitalia polineuropathy spermatogenesis
ATRX Xq13 LOF - Dysgenetic testes - Severe NR NR Embryonic
- Ambiguous or male psycho-motor lethality due to
external genitalia retardation, neural abnormalities
- Müllerian NR - Dysmorphic face,
- Thalassemia
(X-linked)
CBX2 17q25 LOF - Ovaries with oocytes NR Normal
- Female external genitalia Gonads: Gonads
- Müllerian + Testes 0% Testes 0%
Ovaries 50% Ovaries (smaller) 87.5%
Testis +1x Ovary Testis + 1x Ovary 0%
(NOT ovotestis) 25% Ovary/1x Null 12.5%
Undefined 25% Internal genitalia
Internal genitalia Female (Bipartite Uterus)
Female (Bipartite Uterus) 87.5% Female + Male
75% Female + Male (Monopartite Uterus +
(Monopartite Uterus + Deferens) 12.5%
Deferens) 25% Male (Deferentes) 0%
Male (Deferentes) 0% External genitalia
External genitalia Male 0%
Male 16.6% Female 75%
Female 33.3% Intersex 25%
Intersex 50% Fertility
Fertility All animals are sterile
All animals are sterile
(continued on next page)
Table 1 (continued)
Chromosome Molecular Phenotype

(Human) defect Human Mouse
Sexual development Other organs Sexual development Other organs
XY XX Both sexes XY XX Both sexes
WNT4 1p35 Duplication - Dysgenetic testes NR - Cleft lips and palate, Disruption normal Normal NR
- Ambiguous - Tetralogy of Fallot, testicular vasculature
external - Intra uterine and function, but
genitalia growth retardation, does not lead to an
XY sex-reversed
- Müllerian + - Microcephaly
TSPYL1 6q22-q23 LOF - Dysgenetic NR Sudden death in infancy NR NR NR
testes
- Ambiguous
genitalia
MAMLD1 Xq28 LOF/GOF Hypospadia NR Myotubular myopathy NR NR NR
H-PDGS2 4q21-q22 LOF cryptorchidism NR None Normal Normal More severe
inflammatory
response
Ovaries
RSPO1 1p34.2 LOF Normal - Testes Palmoplantar
- Müllerian hyperkeratosis with
- Ambiguous squamous cell
external genitalia carcinoma of skin
WNT4 1p35 LOF (heterozygote) NR - Ovarian Renal agenesis Normal - Masculinized Renal
LOF (homozygote) hyperandrogenism - Renal agenesis ovaries agenesis
- Müllerian aplasia - Cardial and - Müllerian
- Ovotestis/testis pulmonary agenesis
- Müllerian aplasia abnormalities
- Ambiguous external - Adrenal agenesis
genitalia (SERKAL
syndrome)
FOXL2 3q23 LOF Normal Primary ovarian Blefaroptosis/ Normal Primary - Small size
failure and sterility epichantus inversus ovarian - Absent
syndrome insufficiency upper eyelid
and sterility
LOF, Loss-of-function; NR, Not reported; n.a., not applicable; see text for abbreviation.
genito-urinary anomalies and mental retardation syndrome (WAGR syndrome), Frasier syndrome,
Denys–Drash syndrome (DDS) and Meacham syndrome. The association of aniridia, hemihypertrophy
and other congenital anomalies with Wilms tumour was first described by Miller et al. in 1964.16 The
syndrome subsequently became known as the WAGR syndrome. Aniridia is due to the PAX6 gene,
whereas the other features are due to WT1 gene defects.17 Frasier syndrome is a rare disorder defined by
46, XY disorders of sex development (DSD) and progressive glomerulopathy.18 Patients present with
normal female external genitalia, streak gonads and XY karyotype and frequently develop gonado-
blastoma. Wilms tumour is not a feature of the syndrome. Frasier syndrome is caused by a mutation in
the donor splice site in intron 9 of WT1, with the predicted loss of the þKTS isoform.19 In contrast to
Frasier syndrome, most individuals with Denys–Drash syndrome20,21 have ambiguous genitalia or
a female phenotype, an XY karyotype and dysgenetic gonads. Renal symptoms are characterised by
diffuse mesangial sclerosis, usually before the age of 1 year, and the patients frequently develop Wilms
tumour. Pelletier identified mutations of various types in WT1 as the molecular cause of DDS.22 Mea-
cham described two 46, XY individuals with a novel constellation of genital, cardiac and pulmonary
malformations. The genital abnormalities consisted of a true double vagina, retention of Müllerian
structures and undervirilisation of the external genitalia.23 Although phenotype and sometimes
genotype overlap with that of DDS patients,24 the Meacham syndrome patients do not have renal disease.
GATA4
GATA-4, a member of the GATA family of transcription factors, is required for proper development of
the heart. It is also present in the gonads and may be a regulator of gonadal gene expression. In mice of
both sexes, GATA-4 specifically marked the developing somatic cell lineages (Sertoli in testis and
granulosa in ovary) but not primordial germ cells. Interestingly, abundant GATA-4 expression was
maintained in Sertoli cells throughout embryonic development but was markedly down-regulated
shortly after the histological differentiation of the ovary on embryonic day 13.5. This pattern of
expression suggested that GATA-4 might be involved in early gonadal development and possibly sexual
dimorphism.25
In humans, mutations in GATA-4 have been related to atrial septal heart defects and genital
ambiguity in some 46,XY individuals.
Testes
SRY
The discovery of Sry, the mammalian Y-chromosomal sex-determining gene, promised to answer
many questions relating to the genetics and developmental biology of sex determination.26,27 The simple
transgenesis experiment of adding Sry function to an XX mouse to induce male phenotype confirmed the
master switch role of this gene.28 However, since then, little else about this gene has proven to be simple
or straightforward. The fact that Sry resides on the Y chromosome makes it vulnerable to degradation. As
a result, it is minimally conserved and shows some functional flaws, surprising indeed for a gene on
which the survival and propagation of mammalian species depends so much.
In the mouse embryo, Sry exhibits a tightly controlled and limited spatiotemporal profile of
expression in the precursors of Sertoli cells of the XY gonad.29–31 Early studies revealed that Sry is first
expressed around 10.5 days post coitum (dpc), shortly after the emergence of the genital ridges, reaches
peak levels of expression at 11.5 dpc, and is extinguished shortly after 12.5 dpc in mouse.32–34 SRY
clearly throws a molecular switch to engage a male-specific cascade of molecular events, but continued
expression of SRY is not required for these events to unfold.
Despite almost 20 years of work since the discovery of SRY, little is known about its biochemical
function. The HMG domain, a DNA-binding and DNA-bending motif, plays a central role, being the
only region conserved between species and the site of almost all clinical mutations causing XY
gonadal dysgenesis. It seems that SRY acts on a single gene, Sox9, the expression of which is then
rapidly reinforced by positive regulatory loops. SOX9 then drives Sertoli cell formation and, therefore,
testis differentiation. If SRY is absent or fails to act in time, Sox9 is silenced and development of the
follicle cell and ovary ensues, with b-catenin being one of the crucial components driving this
process.
Mutations in SRY are the cause of 46, XY DSD due to pure gonadal dysgenesis, originally described as
XY, female with gonadal dysgenesis.35
SOX9
Several lines of evidence indicate that Sox9 is the best candidate for a direct SRY target gene.30,31,36
First, Sox9 expression is strongly up-regulated soon after the expression of SRY begins, whereas it is
down-regulated in the ovary. Second, cell-fate mapping experiments show that SRY-positive cells
exclusively become SOX9-positive Sertoli cells. Third, heterozygous mutations in SOX9 are responsible
for the human skeletal malformation syndrome – campomelic dysplasia in which most XY patients
have male-to-female sex reversal. Similarly, targeted Sox9 ablation in mice also leads to ovary devel-
opment in XY embryos.37,38
However, the expression of Sox9 in the genital ridge is not entirely dependent on SRY and the
normal gonadal Sox9 transcriptional regulation consists of a SRY-independent initiation; a SRY-
dependent up-regulation; and SRY-independent maintenance in adult life.
SF1 is a good candidate for initiating or sensitising Sox9 expression because early sex-independent
expression of Sox9 is abolished in Sf1 null mutant gonads.30,39
SF1 sensitises Sox9, initiating a low level of expression in the genital ridge of both sexes at 10.5 dpc.
In the male, SF1 (probably with other factors such as WT1KTS) also activates Sry expression. Sox9
expression is up-regulated by the action of SRY together with SF1, whereas it is down-regulated in the
female. This down-regulation is unlikely to be passive, implying the presence of one or more currently
unknown repressors. After the transient expression of Sry has ceased, high levels of SOX9 are main-
tained by its direct autoregulation and via FGF9 signalling (Fig. 4).
As mentioned, mutations in SOX9 lead to 46, XY DSD and skeletal abnormalities known as camp-
tomelic dysplasia.
SF1/NR5A1
Human SF-1/NR5A1 is a 461-amino-acid protein that shares structural homology with other
members of the nuclear receptor superfamily (www.nursa.org). Critical functional domains of this
protein include: an amino-terminal 2 zinc finger DNA-binding domain (DBD), an accessory DNA-
binding region, a hinge region and a ligand-binding domain (LBD) that forms an AF-2 structure.
The expression pattern of SF-1 is consistent with its central role in regulating adrenal development,
gonad determination and differentiation and in the hypothalamic–pituitary control of reproduction
and metabolism.
In the mouse, Sf-1 is expressed in the early adrenogonadal primordium from 9 dpc and, thereafter,
in the developing adrenal gland and gonad.40–42 In humans, SF-1 expression has been shown in the
developing adrenal gland and bipotential gonad at 32–33 days post conception.43,44 Following testis
determination (from approximately 42 days onwards in humans), SF-1 expression is consistently
maintained in the somatic cells of the early testis, where it may play a crucial role together with SRY in
supporting SOX9 expression.36 In Sertoli cells, SF-1 activates expression of AMH from around 7 weeks
gestation, which leads to the regression of Müllerian structures in the developing male foetus. In Leydig
cells, SF-1 activates the expression of steroidogenic enzyme systems from 8 weeks gestation, which
results in androgenisation of the external genitalia. In females, persistent expression of SF-1 has been
reported in early ovarian development in humans,44 whereas Sf-1 expression may decline in the
mouse. However, SF-1 is detectable in somatic cells (granulosa and theca cells) of the adult ovary.45 The
exact role SF-1 plays in the ovary is unclear, as the related nuclear receptor LRH-1 (NR5A2) may also
have a role in regulating aromatisation and oestrogen synthesis.
In addition to its expression in the adrenal gland and gonad, SF-1 is also expressed in the developing
ventromedial hypothalamus (VMH), pituitary gonadotropes and spleen.46–48
Deletion of the gene (Nr5a1) encoding Sf-1 in XY mice results in impaired adrenal development,
complete testicular dysgenesis with Müllerian structures and female external genitalia.
These studies in mice prompted the search for the human counterpart. Initial studies to identify
SF1/NR5A1 mutations in humans focussed on patients with primary adrenal failure, 46, XY gonadal
dysgenesis and Müllerian structures. The first prismatic case was identified by Achermann et al. in 1999
in a patient with such a phenotype.49
Mutation in the human SF1/NR5A1 gene has a phenotypic spectrum that ranges from complete
testicular dysgenesis with Müllerian structures, through individuals with mild clitoromegaly or genital
ambiguity, to severe penoscrotal hypospadias or even anorchia.50
Although not essential for ovarian determination,51 heterozygote mutations in NR5A1 have recently
been linked to ovarian insufficiency.52
DAX1/NR0B1
DAX1 encodes a member of the orphan nuclear hormone receptor (NHR) family of transcriptional
regulators. The C-terminal domain shows homology to the ligand-binding domain (LBD) of related
NHRs, although a ligand remains unknown. The N-terminal domain represents a novel domain
comprising 3.5 repeats of a 65- to 67-amino-acid motif containing two putative zinc fingers, and
possibly defining a nucleic acid-binding domain.
DAX1 has a transcriptional silencing domain, which has been proposed to interact directly with
corepressors to mediate repression.53 At least two corepressors, Alien and NCoR, interact in vitro with
the DAX1 C-terminus,54,55 one of which (Alien) is expressed in the testis.56
In cultured cells, DAX1 represses Sf1-mediated transcriptional activation of steroidogenic genes,
such as Cyp11A, Cyp17 and Cyp19, essential for the synthesis of androgens and oestrogens; it interacts
with the AF2 domain of ligand-bound oestrogen receptors, ERa and ERb and repress ER-mediated
activation of target reporter genes. It is probable that DAX1 and SF1 interact during gonadogenesis in
a similar manner to that seen during steroidogenesis. Together, these studies suggest a common
function for DAX1 as a repressor of SF1 ‘trans’-activation in both steroidal and gonadal tissues.
Inactivating mutations in DAX1 (NR0B1) can lead to adrenal insufficiency with glucocorticoid and
mineralocorticoid deficiency.57 These mutations are responsible for many, but not all, patients with the
cytomegalic form of adrenal hypoplasia congenita (AHC). These mutations also cause hypogonadotropic
hypogonadism (HH), which is consistent with the expression of DAX1 in the hypothalamus and pituitary.
Duplications of the region of the X chromosome containing DAX1 cause dosage-sensitive sex
reversal.57,58 Transgenic XY mice with additional copies of the mouse DAX1 orthologue expressed at
high levels do not have male-to-female sex reversal, but do show delayed testicular development.59
DMRT1
Raymond and co-workers60 isolated the male sexual regulatory gene mab361 from the nematode
Caenorhabditis elegans and found that it is related to the Drosophila melanogaster sexual regulatory
gene ‘doublesex’ (dsx).62 Both genes encode proteins with a DNA-binding motif that Raymond et al.
referred to as the ‘DM domain’. The same authors identified a human gene, which they designated
DMT1 that encodes a protein with a DM domain and found that DMT1 is expressed only in the testis.
Muroya et al.63 reported clinical and molecular findings in five 46, XY and one 46, XX patients with
distal 9 p monosomy. Some of the XY and the XX subjects had female external genitalia, one case
showed ambiguous external genitalia, and another exhibited male external genitalia with left crypt-
orchidism and right intrascrotal testis. Gonadal explorations at gonadectomy revealed the presence of
various morphologies, from streak gonad and right agonadism to hypoplastic testes.
DHH
In an animal model, Bitgood and collaborators64 found that male Desert Hedgehog (Dhh)-null mice
were sterile and failed to produce mature spermatozoa, and that the peripheral nerves of Dhh-null
mutant mice were highly abnormal. The perineurial sheaths surrounding the nerve fascicles were
abnormally thin, and extensive minifascicles consisting of perineurial-like cells were formed within
the endoneurium.
In a patient with 46 XY partial gonadal dysgenesis (PGD) associated with minifascicular poly-
neuropathy, Umehara et al.65 found a homozygous missense mutation at the initiating codon in exon 1
of the DHH gene, which predicted a failure of translation.
ATRX
The protein encoded by this gene contains an ATPase/helicase domain, and thus it belongs to the
SWI/SNF family of chromatin-remodelling proteins.
XY patients with deletions or mutations in the alpha thalassemia, mental retardation syndrome,
X-linked (ATRX) gene display varying degrees of sex reversal, implicating ATRX in the development of
the human testis.66 To explore further the role of ATRX in mammalian sex differentiation, Pask et al.67
cloned and characterised the homologous gene in a marsupial. To their surprise, active homologues of
ATRX were detected on the marsupial Y as well as the X chromosome. The Y-borne copy (ATRY) dis-
played testis-specific expression. This, as well as the sex reversal of ATRX patients, suggested that ATRY
is involved in testis development in marsupials and may represent an ancestral testis-determining
mechanism that predated the evolution of SRY as the primary mammalian male sex-determining gene.
The authors found no evidence for a Y-borne ATRX homologue in mouse or human, implying that this
gene has been lost in eutherians and its role supplanted by the evolution of SRY from SOX3 as the
dominant determiner of male differentiation.
The range of genital phenotypes caused by ATRX mutations, together with the observation that
ATRX patients always have some testicular tissue, suggests that ATRX functions in sex differentiation
rather than sex determination in humans.68
CBX2/M33
When Katoh-Fukui and co-workers ablated the M33 gene in mice, they observed sex reversal in XY
animals.69 M33 is a member of the Polycomb(PcG)/Trithorax (TrxG) proteins. Polycomb group (PcG)
proteins are highly conserved regulatory factors that were initially discovered in Drosophila. PcG genes
are best known for their role in maintaining silent expression states of Hox genes during development,
while trithorax group (trxG) proteins maintain expression of these genes. They act by regulating
chromatin structure and chromosome architecture at their target loci (for more information http://
www.igh.cnrs.fr/equip/cavalli/link.PolycombTeaching.html).
M33-deficient mice have male-to-female sex reversal with perfectly normal female gonads and
genitalia in 50% of the cases, but are invariably sterile. Similarly, we made the intriguing discovery of
a loss-of-function double heterozygote mutation state in a 46, XY girl with normal ovaries at histology,
normal uterus and external female genitalia, accidentally diagnosed because of a discrepancy between
prenatal karyotype and phenotype at birth. Functional studies demonstrated that the mutated CBX2
does not properly bind to and does not adequately regulate the expression of target genes such as SF1/
NR5A1, which are essential for sex development. Our data identify CBX2 as essential for normal human
male gonadal development, suggest that it lies upstream of SRY in the human sex development cascade
and identify a novel autosomal recessive cause of defect of sex development.70
TSPYL1
Testis-specific protein Y-like-1 (TSPYL1) is a member of the TSPY-SET-NAP1L1 family of chromatin
modifiers71 that are involved in nucleosome assembly.72 Besides being linked to beta globin regulation,73
mutations in TSPYL1 were found by Puffenberger and co-workers in a previously unrecognised
syndrome called Sudden Infant Death with Dysgenesis of the Testes (SIDDT). Genotypic males with
SIDDT had foetal testicular dysgenesis and ambiguous genitalia, with findings such as intra-abdominal
dysplastic testes, deficient foetal testosterone production, fusion and rugation of the gonadal sac and
partial development of the penile shaft. The 46, XX sexual development was normal. Affected infants
appeared normal at birth, but developed signs of viscero-autonomic dysfunction early in life, followed by
death before age 12 months due to abrupt cardiorespiratory distress.74,75 Interestingly, cases of sudden
infant death without testicular dysgenesis do not have mutations in TSPYL1,75 suggesting the testis-
specific role of this protein.
MAMLD1/CXorf6
In the course of identifying the gene responsible for X-linked myotubular myopathy (MTM1), which
maps to proximal Xq28, Laporte et al. (1997) identified a novel gene, Mastermind-Like Domain-Con-
taining Protein 1 (MAMLD1, also called Chromosome X Open Reading Frame 6: CXORF6, F18),76
expressed in several tissues including foetal Sertoli and Leydig cells around the critical period for sex
development (E12.5–E14.5) . Most patients with deletion of this region of chromosome X also had
ambiguous genitalia. The proof of the importance of MAMLD1 came when Fukami et al. performed
direct sequencing of the coding exons 3–6 of the CXORF6 gene and their flanking splice sites in 166
CBX2 DAX1
FGF9R
Sry PGD2
Sf1
Sf1
Sertoli cells
Tes
Testis
esttis
Sox9 Sox9 Amh
XY Sry
WT1 -cat
SF1 Foxl2
Lhx9
GATA4
Rspo1
Wnt4
XX
Sox9
Ovary
Ovary Follicles
Sox9
-cat Foxl2
ER
Wnt4 Aromatase
Rspo1
TIME
Fig. 4. A time-line of events in mammalian sex determination (simplified). After the establishment of the bipotential genital ridge by
several genes, the early expression of Sf1 and Wt1 initiates also that of Sox9 in both sexes. b-catenin also begin to accumulate as
a response to Rspo1–Wnt4. In XX cells, b-catenin levels accumulate to repress SOX9 activity. In XY cells, with the contribuition of
CBX2, increasing levels of SF1 activate Sry expression and then SRY, together with SF1, increase Sox9 expression. CBX2 influences
directly SF1 expression and might do so indirectly by removing the DAX1 repression on SF1. Once SOX9 levels reach a critical
threshold, several positive regulatory loops are initiated, including autoregulation of its own expression and formation of feed-
forward loops via FGF9 or PGD2 signaling. If SRY activity is weak, low or late, it fails to boost Sox9 expression before b-catenin levels
accumulate sufficiently to shut it down. At later stages, in XX gonads, FOXL2 increases and can maintain granulosa (follicle) cell
differentiation by repressing Sox9 expression. In the testis, SOX9 promotes the testis pathway, including Amh activation, and it also
probably represses ovarian genes, including Wnt4 and Foxl2. Solid lines: validated influence; broken line: possible influence.
individuals with various types of 46, XY disorders of sex development. They identified three nonsense
mutations in four Japanese individuals with penoscrotal hypospadias as the most evident phenotype.77
Two years later, Fukumi et al. again demonstrated that MAMLD1 acts as a transcription factor by
transactivating the promoter of a non-canonical Notch target gene hairy/enhancer of split 3 (Hes3),
augments testosterone production, most likely under the regulation of SF1.78
More recently, a gain-of-function mutation in MAMDL1 was described in a 46, XX DSD woman with
virilisation (clitoromegaly, streak gonads and small uterus),79 suggesting a role of this protein in
ovarian development.
PGD2S
Lipocalin-type prostaglandin D2 synthase (L-PGDS) and haematopoietic prostaglandin D2 synthase
(H-PGDS) are two enzymes expressed in the testes and involved in the nuclear translocation and
expression of SOX9, a central factor in testis determination during foetal life. Knockout of L- and H-PGDS
in male mice induces delayed testicular organisation and a uni- or bilateral testis migration defect.
Wilhelm et al. found that Pgds was expressed in embryonic mouse Sertoli cells immediately after
the onset of Sry and Sox9 expression. Pgds up-regulation was mediated by Sox9, but not Sry, and
required the binding of dimeric Sox9 to a paired SOX recognition site within the Pgds 5-prime flanking
region.80
The process of testicular descent is not fully understood, but several factors, such as Hoxa-10,
epidermal growth factor (EGF) and calcitonin gene-related peptide (CGRP), together with androgens
and insulin-like hormone 3 (Insl3), have been suggested to regulate it. An H-PGDS gene (but not
L-PGDS) abnormality was identified in one boy referred to our paediatric endocrinology clinic at 15
months with bilateral cryptorchidism and severe growth retardation (–4DS). Functional analysis of the
mutations showed about a 40% decrease in glutathione transferase activity (indispensable for PGDS
activity) and total prostaglandin D2 synthase abolition. Taken together, these data suggested that
H-PGDS defect can be responsible for cryptorchidism in humans.81
Ovaries
RSPO1
Kamata et al.82 identified the mouse R-spondin gene, which encodes a 265-amino-acid sequence
with a calculated molecular mass of 29 kD. Unlike other secretory proteins of the thrombospondin
family, R-spondin contains no apparent secretion cleavage site, but has a putative N-terminal nuclear
localisation signal. R-spondin transcripts were observed mainly in central nervous system (CNS)
tissues. Outside the CNS, R-spondin transcripts were detected in forelimb buds after 10 dpc, especially
in the body trunk and the proximal posterior part of the anterior limb bud. Kim and co-workers found
that human R-spondin is expressed in enteroendocrine cells as well as in epithelial cells from various
tissues.83
In mice, the ovarian phenotype of XX, Rspo1/ is striklingly similar to that of Wnt4/ female
mice, especially for the formation of coelomic vessels and the presence of functional steroidogenic
cells. These animal models suggest that the ovarian phenotype in Rspo1/ mice is at least in part due
to failing overexpression of Wnt4 in XX gonads. By contrast, Rspo1/ female mice do not show
adrenal or uterine abnormalities, as Wnt4/ mice do, in agreement to the fact that Rspo1 is not
expressed in adrenals or mesonephros, indicating that activation of Wnt4 in these organs is inde-
pendent of Rspo1 (For a review, see Chassot et al.84).
The essential role of R-spondin 1 in human ovarian development was demonstrated by studying
individuals with palmoplantar hyperkeratosis with squamous cell carcinoma of skin and 46 XX, DSD in
whom Parma et al. demonstrated mutations in the RSPO1 gene.85 The authors concluded that RSPO1 is
produced and secreted by fibroblasts and regulates keratinocyte proliferation and differentiation. The
presence of ‘functional’ testes in the sex-reversed individuals was confirmed by the absence of
Müllerian derivatives and by the masculinisation of the internal and external genitalia, presumably
induced by functioning Sertoli and Leydig cells, respectively. All sex-reversed individuals were sterile.
Notably, the data suggested that normal RSPO1 is not required for testis differentiation and function.
Parma et al. speculated that RSPO1 may synergise with WNT4 in XX gonads to stabilise beta-catenin.85
WNT4
WNT4 is a member of the WNT family of secreted molecules that function in a paracrine manner to
affect a number of developmental changes. WNT proteins bind to members of the Frizzled (FZ) family
of cell-surface receptors and possibly to the single-pass transmembrane protein LDL-receptor-related
proteins 5 and 6 (LRP5 and LRP6).86 The binding of WNT to FZ ultimately leads to reduced degradation
of b-catenin with consequent b-catenin-dependent activation of T-cell factor/lymphocyte enhancer
factor transcription factors, and induction of Wnt-responsive genes.87 Wnt proteins can also signal via
a b-catenin independent non-canonical pathway involving protein kinase C (PKC) and c-jun
NH2-terminal kinase (JNK).88
(Readers interested in learning more about WNT proteins should visit the informative site http://
www.stanford.edu/wrnusse/wntwindow.html).
Wnt4 is produced in ovarian somatic cells (pre-granulosa cells). Wnt4 up-regulates Dax1,89 a gene
known to antagonise SF1, and thereby inhibits steroidogenic enzymes. Vainio and collaborators90
observed in Wnt4-deficient mice that gonadal development and steroidogenic function were affected
exclusively in female Wnt4-knockout mice, whereas both male and female mice had similar defects in
kidney development and adrenal function. Wnt4-knockout female mice were masculinised, as
demonstrated by the absence of Müllerian ducts and the presence of Wolffian ducts, and expressed the
steroidogenic enzymes 3b-hydroxysteroid dehydrogenase and 17a-hydroxylase, which are required for
the production of testosterone and are normally suppressed in the developing female ovary. The
ovaries of these mice also had fewer oocytes, suggesting a role of Wnt4 in the life of female germ cells.
This function is crucial for the organisation of ovarian structure, since female germ cells have a central
role in this process and in maintenance of the ovary, as demonstrated by the fact that when oocytes are
either absent91 or lost after follicle formation92 ovarian follicles never form or degenerate subse-
quently. In contrast, testis development proceeds in the absence of germ cells. WNT4 and follicostatin93
appear to maintain oocyte viability once germ cells have reached their final destination in the gonad.
In humans, more copies of WNT4, due to duplication of chromosome 1p31-p35, were found in
a patient with ambiguous genitalia, severe hypospadia, fibrous gonads and remnants of both Müllerian
and Wolffian ducts,89 that is, male-to-female sex reversal. On the other end of the spectrum, when both
copies of the gene are inactive, as in the case of homozygote mutations, a severe clinical entity, called
the SERKAL syndrome results.94 The syndrome was described in three 46, XX foetuses and is charac-
terised by female-to-male sex reversal with ambiguous genitalia, gonadal morphology ranging from
ovotestis to normal testis, renal agenesis, adrenal hypoplasia and pulmonary and cardiac abnormalities.
In the middle of such a spectrum, one might expect to find patients with intermediate defects of sex
development. Searches for clinically relevant WNT4 mutations in large cohorts of these patients were
sometimes unsuccessful.95 We described three women with absent Müllerian structures (uterine and
fallopian tubes) and clinical signs of androgen excess. Their phenotype resembles that of patients with
the Mayer–Rokitansky–Küster–Hauser (MRKH) syndrome and is also strikingly similar to that of
Wnt4-knockout female mice. This constellation of findings prompted us to search for and allowed us to
find mutations in the WNT4 gene in these patients.96–99
From a more clinical point of view, we agree with Clement-Ziza100 that the WNT4 mutation should
not be looked for in the MRKH syndrome. However, an MRKH-like syndrome associated with hyper-
androgenism should be investigated for mutations in this gene, and may classify for a distinct clinical
entity.
FOXL2
FOXL2 is a single-exon gene encoding a forkhead/winged helix (fkh) transcription factor.101 FOXL2 is
a nuclear protein. In vertebrates, FOXL2 is one of the earliest known markers of ovarian differentia-
tion.102 Thus, it may play a role in the early stage of development of the ovarian somatic compartment.
As it is still strongly expressed in postnatal and adult follicular cells, it may also play a role in follicle
development and/or maintenance during fertile life.
Despite the importance of FOXL2 in ovarian development and maintenance, only a few transcrip-
tional targets have been described so far.103,104 In the context of the pituitary, FOXL2 seems to stimulate
the expression of the gonadotropin-releasing hormone (GnRH) receptor. Foxl2 influence the expres-
sion of aGSU, StAR and several steroidogenic enzymes.
Mutations in FOXL2 are responsible for blepharophimosis–ptosis–epicanthus inversus syndrome
(BPES), which is a genetic disease leading to complex eyelid malformation and other mild craniofacial
abnormalities and can be associated with premature ovarian failure.101,105
Genes and proteins responsible for the maintanance of ovarian function, such as BMP15, GDF9 and
NOBOX, whose mutations cause premature ovarian insufficiency, will be discussed later in this article.
Müllerian ducts
AMH
AMH produced by foetal Sertoli cells is responsible for regression of Müllerian ducts and is the
anlage for uterus and Fallopian tubes during male sex differentiation. A member of the transforming
growth factor-beta superfamily, AMH signals through two transmembrane receptors, type II (which is
specific) and type I receptors, shared with the bone morphogenetic protein family.
Mutations of the AMH and AMH receptor type II (AMHR-II) genes lead to persistence of the uterus
and Fallopian tubes in males.106–109
HOXA
HOX genes are a family of regulatory molecules that encode conserved transcription factors
controlling aspects of morphogenesis and cell differentiation during normal embryonic development.
Hox genes are also expressed in the adult uterus. Hox genes are essential both for the development of
Müllerian tract in the embryonic period and adult function. Sex steroids regulate Hox gene expression
during embryonic and endometrial development in the menstrual cycle. EMX2 and b3-integrin acting
downstream of Hoxa10 gene are likely involved in both these developmental processes.110,111
The role of WNT4 and EMX2 in sex development has been discussed above.
Lim1/LHX1
Lim1 encodes a LIM-class homeodomain transcription factor that is essential for head and kidney
development. In the developing urogenital system, Lim1 expression has been documented in the
Wolffian (mesonephric) duct, the mesonephros, metanephros and foetal gonads. Using a Lim1 lacZ
knock-in allele in mice, we identified a previously unreported urogenital tissue for Lim1 expression, the
epithelium of the developing Müllerian duct that gives rise to the oviduct, uterus and upper region of
the vagina of the female reproductive tract. Lim1 expression in the Müllerian duct is dynamic, corre-
sponding to its formation and differentiation in females and regression in males. Although female
Lim1-null neonates had ovaries, they lacked a uterus and oviducts. A novel female mouse chimaera
assay was developed and revealed that Lim1 is required cell-autonomously for Müllerian duct
epithelium formation. These studies demonstrate an essential role for Lim1 in female reproductive
tract development.112
Gonadal dysgenesis
Gonadal dysgenesis is characterised by a progressive loss of primordial germ cells on the developing
gonads of an embryo. This loss leads to extremely hypoplastic and dysfunctioning gonads mainly
composed of fibrous tissue; hence the name ‘streak gonads’.
Although most aetiologies of gonadal dysgenesis have been already described in previous sections
of this article, some causes merit special mention.
Chromosomal abnormalities
Turner syndrome, pure gonadal dysgenesis. 45, X0, variants and mosaics
The syndrome is named after Henry Turner, an Oklahoma endocrinologist, who described it in 1938.113
It is a chromosomal disorder in which all or part of one of the X chromosome is absent. In some cases, the
missing chromosome is present in some cells but not others, a condition referred to as mosaicism. There
are characteristic physical abnormalities, such as short stature, swelling, broad chest, low hairline, low-set
ears and webbed necks. Girls with Turner syndrome typically experience gonadal dysgenesis, which
results in amenorrhoea and sterility. Concurrent health concerns are also frequently present, including
congenital heart disease, hypothyroidism, diabetes, vision problems, hearing concerns and many other
auto-immune diseases. Finally, a specific pattern of cognitive deficits is often observed, with particular
difficulties in visuospatial, mathematical and memory areas. A detailed review of such a complex disease
is beyond the purpose of this article and can be read, for instance, in the article by Bondy.114
Klinefelter syndrome, seminiferous tubules dysgenesis. 47, XXY, variants and mosaics
The syndrome was named after Harry Klinefelter, an endocrinologist at Massachusetts General
Hospital in Boston, Massachusetts, who first described it in 1942.115 The principal effects are devel-
opment of small testicles and reduced fertility. Some degree of language learning impairment may be
present, and neuropsychological testing often reveals deficits in executive functions. In adults, possible
characteristics vary widely and include little to no signs of affectedness, a lanky, youthful build and
facial appearance or a rounded body type with some degree of gynaecomastia. Some variations, for
example, XXYY and mosaicism are possible with different severity of symptoms. For more details see
Bojesen and Gravholt, Lee et al. and Paduch et al.116–118
Mixed gonadal dysgenesis and ovostesticular DSD (true hermaphroditism)
Mixed gonadal dysgenesis. Some authors reserve the term ‘mixed gonadal dysgenesis’ (MGD) for indi-
viduals who present a 45, X/46, XY karyotype with a testis on one side and a streak on the other side
(seeKim et al.119 and references therein); other authors apply the term to all patients who have varying
degrees of asymmetrical gonadal dysgenesis with testicular differentiation on either side, bilateral
streak testis or bilateral dysgenetic testis.
Ovotesticular DSD (OT-DSD or true hermaphroditism). This is the rarest form of intersexuality in humans,
and the term is applied to an individual who has both well-developed ovarian and testicular tissues.
The differentiation of OT-DSD from MGD is important for three reasons. First, a bilateral gonadectomy
is recommended as soon as possible in all individuals with MGD containing Y-chromosome material.
There is no benefit in delaying surgery because approximately one-third of patients with MGD develop
gonadoblastoma during the 1st to 4th decades of life and because 30% of gonadoblastomas are over-
grown by more malignant germ cell tumours, such as germinoma, endodermal sinus tumour, imma-
ture teratoma, embryonal carcinoma or choriocarcinoma. In OT-DSD, however, only the removal of the
opposite gonad from the assigned gender and a biopsy of remaining gonadal tissue for histological
evaluation may be appropriate. Second, gender assignment is critical for the treatment of patients of
OT-DSD because these patients usually do not have any other developmental malformations, and thus
normal sexual and reproductive functions can be achieved by proper management and sex assignment
at a young age. After the removal of the testicular portion of an ovotestis, about 38% of these patients
with a 46, XX karyotype menstruate by age 14 years120; ovulation121 and successful childbearing have
also been described.122,123 By contrast, it is important that bilateral gonadectomies be performed in
patients with MGD before the patient reaches puberty, not only to prevent the development of
malignant tumours but also to avoid virilisation if the patient is to be raised as female. Third, certain
medical problems, such as deficient immunoglobulin levels, aberrant bony development of inner ear
structures and cardiovascular and renal anomalies, are more common in MGD patients than in OT-DSD
patients; therefore, patients with MGD should receive more medical attention.
For a differential diagnosis, chromosomal constitutions are not helpful. Various types of chromo-
somal abnormalities have been described in cases of OT-DSD, such as 46, XX; 46, XY; 46, XX/46, XY;
45X/46, XY; and others. MGD cases also have heterogeneous chromosomal changes but are usually
characterised by a 45, X/46, XY; 45, X/47, XYY; or 46, XY karyotype.124 The pathogenesis of OT-DSD and
MGD still remains unclear. In cases of 46, XX true hermaphroditism, mosaicism with a Y-bearing cell
line at low level125 or partial inactivation of the Y-bearing X chromosome that was formed during
paternal meiosis126,127 have been suggested to explain the mechanism of abnormal sexual develop-
ment. In cases of MGD, mutations in SRY genes were usually absent128; however, cytogenetic mosai-
cism, mutation in the genes downstream of SRY and unpaired or incompletely paired X chromosome
have been proposed as a mechanism to cause defective formation of the follicular mantle and
degeneration of oocytes.
Histopathologically, there are no major differences in the testicular part of the gonads between the
two entities, on the one hand. On the other hand, a rise of testosterone levels after human chorionic
gonadotropin (hCG) is observed exclusively in cases of OT-DSD and will not occur in MGD patients. The
presence of numerous primordial follicles containing primary oocytes with or without maturing
follicles is considered well-developed ovarian tissue when making a definitive diagnosis of OT-DSD
after birth.
For a more extensive review see Kim et al.119
Single gene defects
46, XX
Besides those described in Section 2.3 and Table 1, an additional form of gonadal dysgenesis in
women is premature ovarian failure (POF), defined as menopause at age less than 40 years.
POF1 is associated to premutations (mutations that do not cause symptoms and needs a second
mutation to become ‘full’) in Fragile X Mental Retardation 1 (FMR1) gene (Xq26-q28).129 An interna-
tional collaborative study of 760 women from fragile X families130 found that 395 carried a pre-
mutation, 128 carried a full mutation and 237 were non-carriers. In 63 (16%) of the premutation
carriers, menopause occurred before the age of 40, compared with none of the full-mutation carriers
and one (0.4%) of the controls, indicating a significant association between premature menopause and
premutation carrier status.
POF2A is caused by mutations in the Drosophila Diaphanous (dia) homolog 2 (DIAPH2, Xq22). In the
fruit fly, mutant alleles of dia affect spermatogenesis or oogenesis and lead to sterility. Bione et al.131
identified DIAPH2, which they called DIA, as the gene disrupted by a breakpoint in a family with
premature ovarian failure and stated that DIAPH2 is the first human member of the FH1/FH2 protein
family. Members of this family affect cytokinesis and other actin-mediated morphogenetic processes
that are required in the early steps of development.
POF2B. Bione et al.131considered the POF1B gene (Xq21) a candidate for premature ovarian failure
because it is located within the critical region for normal ovarian function, escapes X inactivation and
a breakpoint in the third intron was identified in a POF patient with an X;1 translocation132; however,
they could not demonstrate any mutation in POF1B in their POF patients. In the same work, they
determined that Pof1B expression is absent from adult mouse ovary but is present between embryonic
day 16.5 and postnatal day 5, suggesting that the gene may be critical for the steps required for early
ovary development leading to the final number of germ cells. Based on its expression pattern in mouse
development, Bione et al. also hypothesised that POF1B could play a role in the pairing of meiotic
chromosomes and that alteration in its function could lead to cell death and a drastic reduction in the
final number of oocytes created during ovarian development. Alternatively, POF1B may act as an anti-
apoptosis factor, to slow down the process of germ cell loss; as a consequence, loss of function of
POF1B could lead to exaggerated germ cell apoptosis and POF. In 2006, Lacombe et al.133 identified a G-
to-A transition at nucleotide 1132 in exon 10 of the POF1B gene that resulted in an arg329-to-gln
(R329Q) mutation in a Lebanese family in which premature ovarian failure mapped to the Xq21.1-
q21.33. Affected family members were homozygous. The R329Q variant altered the arginine within an
amino acid triplet, serine–leucine–arginine, that is part of a protein kinase C phosphorylation
recognition site.
POF3 is caused by mutations in FOXL2 (see above).
POF4 is associated with defects of bone morphogenic protein 15 (BMP15; Xp11.2). This entity is also
known as ovarian dysgenesis 2 (ODG2). The bone morphogenetic protein (BMP) family is part of the
transforming growth factor-beta superfamily, which includes large families of growth and differenti-
ation factors. By Northern blot analysis, Dube et al.134 showed that mouse Bmp15 is expressed only in
the ovary and precisely in the oocyte soon after primordial follicles are recruited, and that expression is
maintained until after ovulation. Studies by Aaltonen and co-workers135 showed that in human ovary
BMP15 (which they called GDF9B) is expressed in the oocytes of late primary follicle. Otsuka et al.136
showed BMP15 expression in oocytes throughout follicular development. The importance of BMP15 for
human disease was first proven by Di Pasquale et al.,137 who reported an Italian family in which two
sisters had hypergonadotropic ovarian failure due to ovarian dysgenesis and a dominant negative
mutation in BMP15. Later reports confirmed the role of BMP15 in POF.138,139
POF5. Newborn ovary homeobox (7q35) NOBOX is a homeobox gene that is preferentially expressed
in oocytes. In mice, it is essential for folliculogenesis and regulation of oocyte-specific genes.140 In
a 35-year-old Caucasian woman with premature ovarian failure, Qin et al.141 identified heterozygosity
for a 1064G–A transition in exon 6 of the NOBOX gene, resulting in an arg355-to-his (R355H) substi-
tution within the highly conserved homeodomain region.
POF6. Factor in germ line alpha (FIGLA, 2p12) is a helix–loop–helix transcription factor expressed in
all ovarian follicular stages and in mature oocytes, with less frequent expression in preimplantation
embryos. Zhao and co-workers142 analysed the FIGLA gene in 100 Chinese women with premature
ovarian failure and identified a 22-bp deletion and a 3-bp in-frame deletion in two patients, respec-
tively, that were not found in controls.
POF7 is related to mutations in SF1/NR5A1 (see above).
Ovarian dysgenesis (ODG1). Hypergonadotropic ovarian dysgenesis with normal karyotype can be
caused by mutations in the gene encoding follicle-stimulating hormone receptor (FSHR, 2p21–p26).
Elliott et al.143 reported the condition in three sisters who had normal stature and sex chromatin but
had never menstruated and had severe osteoporosis. Somatic features of Turner syndrome were not
found. In the Finnish population in which ovarian dysgenesis with normal XX karyotype is relatively
common (approximately 1 in 8,300 females), Aittomaki et al.144 demonstrated that affected females are
homozygous for a C-to-T transition in exon 7 of the FSHR gene, predicting an ala189-to-val substitution
in the extracellular ligand-binding domain of the protein.
FSHR mutations also appear to play a role in ovarian hyperstimulation syndrome. Greb and
collaborators145 showed that the FSH receptor ser680/ser680 genotype was associated with higher
ovarian threshold to FSH, decreased negative feedback of luteal secretion to the pituitary during the
intercycle transition and longer menstrual cycles.
Syndrome-associated ODG
Perrault syndrome. This syndrome is characterised by ovarian dysgenesis and sensorineural deaf-
ness.146,147 Although the exact genetic cause of the disease remains unknown, there is evidence for an
autosomal recessive mode of inheritance.147
Immunodeficiency, pulmonary fibrosis, ODG. Somech et al.148described two sisters, born of first-cousin
parents of Sri Lankan descent, who presented in infancy with immunodeficiency, gonadal dysgenesis
and fatal pulmonary fibrosis. The infants displayed no dysmorphic features. Immune studies
demonstrated combined humoral and cellular abnormalities including reduced immunoglobulin
production, absence of lymphoid tissue, markedly reduced T lymphocyte numbers and function and
reduced newly thymus-derived T cells. Both infants succumbed to rapidly progressive pulmonary
fibrosis; autopsy revealed streak ovaries bearing no discernible oocytes. The molecular basis of the
disease is not known.
Mitochondrial dysfunction. Two sisters were described by Hisama et al.149 with hypergonadotropic
hypogonadism, marked short stature and recurrent episodes of dehydration with metabolic acidosis.
Both patients had normal intelligence and normal 46, XX karyotypes. Laboratory studies suggested
mitochondrial dysfunction; however, results of mitochondrial DNA studies and of biochemical analysis
of electron transport chain activity in skeletal muscle were normal.
46, XY
Besides the discussion in Section 2.2. and Table 1 Testes, an entity called alopecia universalis, XY
gonadal dysgenesis with ambiguous genitalia with an XY karyotype and SRY positivity and lar-
yngomalacia has been described. El-Shanti et al.150 reported five individuals from two unrelated
families from Jordan with alopecia universalis congenita, gonadal dysgenesis and laryngomalacia that
persisted beyond infancy. An additional feature of this entity was growth delay due to growth hormone
deficiency.
Environmental
Testicular dysgenesis syndrome

The term ‘testicular dysgenesis syndrome’ (TDS) was first coined in 2001.151 It was hypothesised
that many cases of abnormal spermatogenesis, cryptorchidism, hypospadias and testicular cancer may
have a common aetiology, and that all these clinical problems may result from an irreversible devel-
opmental disorder originating in early foetal life. Aetiological factors that explain how TDS may arise
include genetic defects (mosaicisms for sex chromosome aneuploidy) and polymorphism (still
unclear), environmental and lifestyle factors and intrauterine growth disorders, which may act sepa-
rately or in concert. Exposure to endocrine-disrupting compounds such as phthalates (mainly used as
plasticisers), which act as anti-androgens, seem to be central risk factors. Lifestyle such as maternal
smoking during pregnancy152 and increasing body mass index in young men also negatively influence
testicular function.153 These factors lead to testicular dysgenesis with decreased Leydig cell and Sertoli
cells function. Leydig cell deficiency causes androgen insufficiency and INSL3 decrease explaining
cryptorchidism, hypospadia and impaired spermatogenesis. Sertoli cells dysfuntion contributes to
infertility and to the onset of testicular cancer (for a review see Joensen et al.154). Article 7 discusses this
problem in greater detail.
Factors involved in sex differentiation
LH/CG receptor, steroidogenic enzymes, steroid receptors
The role of these factors in sex development will be discussed in great detail in articles 4–6.
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Best Practice & Research Clinical Endocrinology & Metabolism 24 (2010) 163–186

Contents lists available at ScienceDirect

Best Practice & Research Clinical

Control of sex development

Physiology of sex development

Factors involved in sex determination

Indifferentiated state (bipotential)

Development of Wolffian ducts 4

Migration of germ cells 5 Migration of germ cells

Regression of Müllerian ducts. 8

Fetal testes in the internal inguinal rings

Appearance of first spermatogonia 14

16 Appearance of first ovarian follicles

Max Leydig cells. Testosterone peak 17

1st multilayer ovarian follicles

Descent of testes 28 Cessation of oogonia multiplication

Chromosome Molecular Phenotype

Sexual development Other organs Sexual development Other organs

XY XX Both sexes XY XX Both sexes

Chromosome Molecular Phenotype

Mixed gonadal dysgenesis and ovostesticular DSD (true hermaphroditism)

Single gene defects

Testicular dysgenesis syndrome

Factors involved in sex differentiation

LH/CG receptor, steroidogenic enzymes, steroid receptors

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