The Chromosomal Basis of Sex
• Soon after cytogenetic analysis became feasible, the fundamental basis of the XX/XY
system of sex determination became apparent.
• Males with Klinefelter syndrome were found to have 47 chromosomes with two X
chromosomes as well as a Y chromosome (karyotype 47,XXY), whereas most Turner
syndrome females were found to have only 45 chromosomes with a single X
chromosome (karyotype 45,X).
• The sex chromosomes play a determining role in specifying primary (gonadal) sex, a
number of genes located on both the sex chromosomes and the autosomes are
involved in sex determination and subsequent sexual differentiation.
The Y Chromosome
• In male meiosis, the X and Y chromosomes normally pair by segments at the ends of
their short arms and undergo recombination in that region.
• The pairing segment includes the pseudoautosomal region of the X and Y
chromosomes (A second, smaller pseudoautosomal segment is located at the distal
ends of Xq and Yq.).
• By comparison with autosomes and the X chromosome, the Y chromosome is
relatively gene poor and contains only about 50 genes.
• Notably, however, the functions of a high proportion of these genes are related to
gonadal and genital development.
Embryology of the Reproductive System
• The current concept is that development into an ovary or a testis is determined by the
coordinated action of a sequence of genes that leads normally to ovarian
development when no Y chromosome is present or to testicular development when a
Y is present.
• The ovarian pathway is followed unless a Y-linked gene, designated testis-
determining factor (TDF ), acts as a switch, diverting development into the male
• In the presence of a Y chromosome (with the TDF gene), the medullary tissue forms
typical testes with seminiferous tubules and Leydig cells that, under the stimulation of
chorionic gonadotropin from the placenta, become capable of androgen secretion.
• If no Y chromosome is present, the gonad begins to differentiate to form an ovary,
beginning as early as the eighth week of gestation and continuing for several weeks; the
cortex develops, the medulla regresses, and oogonia begin to develop within follicles.
• Beginning at about the third month, the oogonia enter meiosis I, but this process is
arrested at dictyotene until ovulation occurs many years later.
The Testis-Determining Gene, SRY
• The earliest cytogenetic studies established the testis-determining function of the Y
• In the ensuing three decades, different deletions of the pseudoautosomal region and
of the sex-specific region of the Y chromosome in sex-reversed individuals were used
to map the precise location of the primary testis-determining region on Yp.
• Whereas the X and Y chromosomes normally exchange in meiosis I within the Xp/Yp
pseudoautosomal region, in rare instances, genetic recombination occurs outside of
the pseudoautosomal region, leading to two rare but highly informative abnormalities:
XX males and XY females.
• Each of these sex-reversal disorders occurs with an incidence of approximately 1 in
• XX males are phenotypic males with a 46,XX karyotype who usually possess some Y
chromosomal sequences translocated to the short arm of the X.
• Similarly, a proportion of phenotypic females with a 46,XY karyotype have lost theTDF.
• The SRY gene (sex-determining region on the Y ) lies near the pseudoautosomal
boundary on the Y chromosome, is present in many 46,XX males, and is deleted or
mutated in a proportion of female 46,XY patients, thus strongly implicating SRY in male
SRY is expressed only briefly early in development in cells of the germinal ridge just
before differentiation of the testis.
SRY encodes a DNA-binding protein that is likely to be a transcription factor, although
the specific genes that it regulates are unknown.
Thus, by all available genetic and developmental criteria, SRY is equivalent to the TDF
gene on the Y chromosome.
However, the presence or absence of SRY does not explain all cases of abnormal sex
SRY is not present in about 10% of unambiguous XX males and in most cases of XX
true hermaphrodites or XX males with ambiguous genitalia.
Further, mutations in the SRY gene account for only about 15% of 46,XY females.
Y-Linked Genes in Spermatogenesis
• Interstitial deletions in Yq have been associated with at least 10% of cases of
nonobstructive azoospermia (no sperm detectable in semen) and with approximately
6% of cases of severe oligospermia (low sperm count).
• These findings suggest that one or more genes, termed azoospermia factors (AZF),
are located on the Y chromosome, and three nonoverlapping regions on Yq (AZFa,
AZFb, and AZFc) have been defined.
• Molecular analysis of these deletions has led to identification of a series of genes that
may be important in spermatogenesis.
• For example, the AZFc deletion region contains several families of genes expressed
in the testis, including the DAZ genes (deleted in azoospermia) that encode RNA-
binding proteins expressed only in the premeiotic germ cells of the testis.
•De novo deletions of AZFc arise in about 1 in 4000 males and are mediated by
recombination between long repeated sequences.
• AZFa and AZFb deletions, although less common, also involve recombination.
• The prevalence of AZF mutations, deletions, and sequence variants in the general
male population as well as their contribution to spermatogenic failure remain to be fully
•Approximately 2% of otherwise healthy males are infertile because of severe defects in
sperm production, and it appears likely that de novo deletions or mutations account for at
least a proportion of these.
• Thus, men with idiopathic infertility should be karyotyped, and Y chromosome
molecular testing and genetic counseling may be appropriate before the initiation of
assisted reproduction for such couples.
• Not all cases of male infertility are due to chromosomal deletions.
• For example, a de novo point mutation has been described in one Y-linked gene,
USP9Y, the function of which is unknown but which must be required for normal
The X Chromosome
• Aneuploidy for the X chromosome is among the most common of cytogenetic
• The relative tolerance of the human karyotype for X chromosome abnormalities can
be explained in terms of X chromosome inactivation, the process by which most
genes on one of the two X chromosomes in females are silenced epigenetically and
fail to produce any product.
X Chromosome Inactivation
The theory of X inactivation is that in somatic cells in normal females (but not in
normal males), one X chromosome is inactivated early in development, thus
equalizing the expression of X-linked genes in the two sexes.
In normal female cells, the choice of which X chromosome is to be inactivated is a
random one that is then maintained in each clonal lineage.
Thus, females are mosaic with respect to X-linked gene expression; some cells
express alleles on the paternally inherited X but not the maternally inherited X,
whereas other cells do the opposite.
This pattern of gene expression distinguishes most X-linked genes from imprinted
genes (which are also expressed from only one allele but determined by parental
origin, not randomly) as well as from the majority of autosomal genes that are
expressed from both alleles.
Although the inactive X chromosome was first identified cytologically by the presence
of a heterochromatic mass (called the Barr body) in interphase cells, there are many
epigenetic features that distinguish the active and inactive X chromosomes.
As well as providing insight into the mechanisms of X inactivation, these features can
be useful diagnostically for identifying the inactive X chromosome in clinical material.
The promoter region of many genes on the inactive X chromosome is extensively
modified by addition of a methyl group to cytosine by the enzyme DNA
DNA methylation is restricted to CpG dinucleotides and contributes to formation of an
inactive chromatin state.
Additional differences between the active and inactive X chromosomes involve the
histone code and appear to be an essential part of the X inactivation mechanism.
For example, the histone variant macroH2A is highly enriched in inactive X chromatin
and distinguishes the two X's in female cells.
In patients with extra X chromosomes, any X chromosome in excess of one is
Thus, all diploid somatic cells in both males and females have a single active X
chromosome, regardless of the total number of X or Y chromosomes present.
Although X chromosome inactivation is clearly a chromosomal phenomenon, not all
genes on the X chromosome are subject to inactivation.
Extensive analysis of expression of nearly all X-linked genes has demonstrated that
at least 15% of the genes escape inactivation and are expressed from both active
and inactive X chromosomes.
In addition, another 10% show variable X inactivation; that is, they escape
inactivation in some females but not in others.
Notably, these genes are not distributed randomly along the X; many more genes
escape inactivation on distal Xp (as many as 50%) than on Xq (just a few percent).
This finding has important implications for genetic counseling in cases of partial X
chromosome aneuploidy, as imbalance for genes on Xp may have greater clinical
significance than imbalance of Xq.
Chromosomal Features of X
• Inactivation of most X-linked genes on the
• Random choice of one of two X chromosomes in
• Inactive X:
– Heterochromatic (Barr body)
– Late-replicating in S phase
– Expresses XIST RNA
– Associated with macroH2A histone modifications in
Sex Chromosomes and X Inactivation
From the study of structurally abnormal, inactivated X chromosomes, the X
inactivation center has been mapped to proximal Xq, in band Xq13.
The X inactivation center contains an unusual gene, XIST, that appears to be a key
master regulatory locus for X inactivation.
XIST, an acronym for inactive X (Xi)-specific transcripts, has the novel feature that it
is expressed only from the allele on the inactive X; it is transcriptionally silent on the
active X in both male and female cells.
Although the exact mode of action of XIST is unknown, X inactivation cannot occur in
The product of XIST is a noncoding RNA that stays in the nucleus in close
association with the inactive X chromosome and the Barr body.
Nonrandom X Chromosome Inactivation
X inactivation is normally random in female somatic cells and leads to mosaicism for
two cell populations expressing alleles from one or the other X.
However, there are exceptions to this when the karyotype involves a structurally
For example, in almost all patients with unbalanced structural abnormalities of an X
chromosome (including deletions, duplications, and isochromosomes), the
structurally abnormal chromosome is always the inactive X, probably reflecting
secondary selection against genetically unbalanced cells that could lead to significant
Because of this preferential inactivation of the abnormal X, such X chromosome
anomalies have less of an impact on phenotype than similar abnormalities of
autosomes and consequently are more frequently observed.
Nonrandom inactivation is also observed in most cases of X;autosome translocations.
If such a translocation is balanced, the normal X chromosome is preferentially
inactivated, and the two parts of the translocated chromosome remain active, again
probably reflecting selection against cells in which autosomal genes have been
In the unbalanced offspring of a balanced carrier, however, only the translocation
product carrying the X inactivation center is present, and this chromosome is invariably
inactivated; the normal X is always active.
These nonrandom patterns of inactivation have the general effect of minimizing, but
not always eliminating, the clinical consequences of the particular chromosomal defect.
Because patterns of X inactivation are strongly correlated with clinical outcome,
determination of an individual's X inactivation pattern by cytogenetic or molecular
analysis is indicated in all cases involving X; autosome translocations.
• One consequence sometimes observed in balanced carriers of X;autosome
translocations is that the break itself may cause a mutation by disrupting a gene on
the X chromosome at the site of the translocation.
• The only normal copy of the particular gene is inactivated in most or all cells because
of nonrandom X inactivation of the normal X, thus allowing expression in a female of
an X-linked trait normally observed only in hemizygous males.
• Several X-linked genes have been identified when a typical X-linked phenotype has
been found in a female who then proved to have an X;autosome translocation.
• The general clinical message of these findings is that if a female patient manifests an
X-linked phenotype normally seen only in males, high-resolution chromosome
analysis is indicated.
• The finding of a balanced translocation can explain the phenotypic expression and
show the gene's probable map position on the X chromosome.
X-Linked Mental Retardation
• An additional feature of the X chromosome is the high frequency of mutations,
microdeletions, or duplications that cause X-linked mental retardation.
• The collective incidence of X-linked mental retardation has been estimated to be 1 in
500 to 1000 live births.
• In many instances, mental retardation is but one of several abnormal phenotypic
features that together define an X-linked syndrome, and more than 50 X-linked genes
have been identified in families with such disorders.
• However, there are many other genes at which mutations lead to isolated or
nonsyndromic X-linked mental retardation, often of the severe to profound kind.
• The number of such genes is consistent with the finding in many large-scale surveys
that there is a 20% to 40% excess of males among persons with mental retardation.
• Detailed chromosome analysis is indicated as an initial evaluation to rule out an
obvious cytogenetic abnormality, such as a deletion.
In biology, a hermaphrodite is an organism that has reproductive organs normally
associated with both male and female sexes.
Historically, the term hermaphrodite has also been used to describe
ambiguous genitalia and gonadal mosaicism in individuals of gonochoristic species,
especially human beings.
The word hermaphrodite entered the English lexicon in the 15th century, derived
from the Greek Hermaphroditos a combination of the names of the gods Hermes
(male) and Aphrodite (female).
Recently, the word intersex has come into preferred usage for humans, since the
word hermaphrodite is considered to be misleading and stigmatizing,as well as
"scientifically specious and clinically problematic."
True hermaphroditism in humans differs from pseudohermaphroditism in which the
person has both X and Y chromosomes (not to be confused with the normal XY
chromosome of males), having both testicular and ovarian tissue, and having
ambiguous-looking external genitalia.
One possible pathophysiologic explanation of this rare phenomenon is a
parthenogentic division of a haploid ovum into two haploid ova.
Upon fertilization of the two ova by two sperm cells (one carrying an X and the other
carrying a Y chromosome), the two fertilized ova are then fused together resulting in a
person having dual genitalial, gonadal and genetic sex.
The XY Female, the XX Male, and the True
The most conserved gene in sex differentiation is DMRT1, mapping to 9p24.3.
DMRT1 is expressed in greater amount in the male embryonic gonads than in the
female, and expression is localized to the sex cords, which will give rise to the Sertoli
cells of the testis.
The DAX1 gene located at Xp21.3 is a repressor of male gene expression.
The sequence whereby germ cells acquire a testicular or an ovarian destiny is
exquisitely dose-sensitive: too much DAX1 product displaces WT1 from its association
with SF1, and the testis-inducing influences of SRY and SOX9 are thwarted.
More genes along this pathway will, no doubt, be discovered.
WT1 comes under the influence of WNT4, on chromosome 1p35, and this gene is
Two 46,XY women having an unbalanced translocation involving the same 6p25
breakpoint, t(X;6) in one and t(6;13) in the other, may point to the existence in this region
of a further sex-determining locus.
XY Pure Gonadal Dysgenesis (Swyer Syndrome)
The rare familial form provides a unique example of a Mendelian condition that can be
inherited in an X-linked recessive, Y-linked, or sex-limited autosomal dominant mode.
In the X-linked forms or autosomal dominant forms, the XY female has a perfectly
normal Y chromosome, with a normal SRY testis-determin-ing gene; presumably, there
is a mutation in a gene (whether this be X-linked or autosomal) controlling a later event
in the testicular developmental pathway.
In the Y-linked form, there is a mutation in the SRY gene.
In some Y-hemizygotes, the mutant gene has nevertheless been able to reach a
threshold of operation and to induce testis development, while in others with the same
mutation it has not.
Thus, for example, an XY male with a mutation in SRY may be a normal fertile man,
while his XY child may be a daughter.
The threshold is apparently all-or-nothing: partial expression, that is to say, an intersex
state, does not result .
A man may be a gonadal mosaic for an SRY deletion, as presumably was the father in
Barbosa et al. (1995).
Two sisters with XY gonadal dysgenesis (one with gonadoblastoma) had a deletion of
SRY, but their father showed a normal SRY result; there were three other normal sisters
and six normal brothers.
Complete Androgen Insensitivity Syndrome (Testicular
Here, the defect lies further down the developmental path.
The gonad becomes a testis, and produces testicular secretions, but the genital tract,
internal and external, is resistant to the effects of androgen.
The inheritance is X-linked recessive, and the locus is the androgen receptor gene at
One example is on record in which, in a sense, the X-linkage was directly visible to
the cytogeneticist: that is to say, the X chromosome was abnormal, including the region
containing the androgen receptor locus.
An affected aunt and niece had the karyotype 46,Y,inv(X)(q11.2q27) and the
connecting mother 46,X,inv(X) (q11.2q27).
The individual appears externally very much as a female, but there is amenorrhea and
pubic and axillary hair is absent.
Internally, the vagina is short, and the uterus and tubes are represented only by
remnants; the testes may be in the inguinal canal.
XY Female with Extragonadal Defects
A number of rare conditions exist in which sex reversal coexists with physical,
metabolic, and/or mental defect.
By way of example, one of these is campomelic dysplasia (campomelia refers to long
bone bowing) with sex reversal, a syndrome of skeletal dysplasia with female genital
The usual cause is a mutation within the SOX9 gene (at 17q24.3-q25.1), one of the
genes operating on the sexual differentiation pathway and which also influences limb
bud mesenchymal development .
Another cause of campomelic dysplasia is deletion in an apparently balanced 17q
translocation with haploinsufficiency at the locus.
A significant recurrence risk applies, on the order of 5%, which may include the
etiologies of mildly affected transmitting parent, parental gonadal mosaicism, recessive
forms, and familial rearrangement involving 17q .
Most XX males arise from the presence of Yp material (rarely visible cytogenetically)
on one of the X chromosomes, from occult XXY/XX mosaicism, or from the inappropriate
activity of a gene that is normally switched on only in response to a Y-originating genetic
In about three-quarters of cases the SRY gene is present, typically the consequence
of an abnormal exchange between the X and Y during meiosis I in gametogenesis in the
father, and thus clearly a sporadic event.
These are referred to as SRY+
The phenotype in the SRY+
XX male is similar to that of Klinefelter syndrome,
presumably reflecting the similar basic genotypes of active X + inactive X + SRY in the
two conditions; however, the XX male differs in being of normal height and of unimpaired
Margarit et al. (1998) describe six SRY+
cases due to translocation of Yp material to
Xp22.3, in whom different Y breakpoints could be identified, but whose clinical
phenotypes were very similar: normal intelligence, normal stature, and testicular atrophy
In these SRY+
XX males, a more accurate designation would be 46,X,der(X)t(X;Y), or
more fully, 46,X,der(X)t(X;Y)(p22.3;p11.2), although the exchange is not usually visible
on standard cytogenetics.
Thus this entity is also discussed in the section on the X;Y translocation.
True hermaphroditism generally presents as a problem in determining the sex of a
newborn infant- in other words, genital ambiguity.
The formal definition of true hermaphroditism is that the gonads comprise both
ovarian and testicular elements: there may be a testis and an ovary, or one or both may
be an ovotestis.
The most common karyotype is 46,XX, seen in 60%.
A third have mosaicism with one cell line which includes Y chromosomal sequences,
mostly 46,XX/46,XY, and a few are 46,XY.
Most of the 46,XX cases test negative on peripheral blood analysis for the SRY gene,
and in some of these the basis of the defect may be sporadic inappropriate activation of
the testicular developmental cascade in part of the gonadal tissue during its embryonic
XX true hermaphroditism is unusually prevalent among certain indigenous Black
populations in Southern Africa, and Spurdle et al. (1995) excluded the presence of SRY
and of uniparental disomy X in all of 16 individuals studied.
Alternatively, an apparent XX karyotype may harbor Y material, as Margarit et al.
(2000) show in a woman reared as a boy with hypospadias who went on to have gender
change surgery after testing 46,XX.
Several years later, reanalysis revealed a tiny segment of Yp translocated on to the X
long arm, 46,X,der(X),t(X;Y)(q28;p11.31).
A more common explanation in the 46,XX case may be cryptic mosaicism within the
gonad itself, with an island or islands of tissue containing the SRY gene.
One 46,XY case had a postzygotic mutation in SRY with SRY+
Presumably the SRY+
line was responsible for the testicular elements in the gonad
and the SRY-
line, for the ovarian elements.
The different sex chromosome constitution of normal human male and female cells has been appreciated for more than 50 years. Soon after cytogenetic analysis became feasible, the fundamental basis of the XX/XY system of sex determination became apparent. Males with Klinefelter syndrome were found to have 47 chromosomes with two X chromosomes as well as a Y chromosome (karyotype 47,XXY), whereas most Turner syndrome females were found to have only 45 chromosomes with a single X chromosome (karyotype 45,X). These findings promptly and unambiguously established the crucial role of the Y chromosome in normal male development. Furthermore, compared with the dramatic consequences of autosomal aneuploidy, these karyotypes underscored the relatively modest effect of varying the number of X chromosomes in either males or females. The basis for both observations is now understood in terms of the unique biology of the Y and X chromosomes. Whereas the sex chromosomes play a determining role in specifying primary (gonadal) sex, a number of genes located on both the sex chromosomes and the autosomes are involved in sex determination and subsequent sexual differentiation. In most instances, the role of these genes has come to light as a result of patients with abnormalities in sexual development, whether cytogenetic, mendelian, or sporadic, and many of these are discussed in a section later in this chapter
The structure of the Y chromosome and its role in sexual development have been determined at both the molecular and genomic levels. In male meiosis, the X and Y chromosomes normally pair by segments at the ends of their short arms and undergo recombination in that region. The pairing segment includes the pseudoautosomal region of the X and Y chromosomes, so called because the X- and Y-linked copies of this region are essentially identical to one another and undergo homologous recombination in meiosis I, like pairs of autosomes. (A second, smaller pseudoautosomal segment is located at the distal ends of Xq and Yq.) By comparison with autosomes and the X chromosome, the Y chromosome is relatively gene poor and contains only about 50 genes. Notably, however, the functions of a high proportion of these genes are related to gonadal and genital development.
By the sixth week of development in both sexes, the primordial germ cells have migrated from their earlier extraembryonic location to the gonadal ridges, where they are surrounded by the sex cords to form a pair of primitive gonads. Up to this time, the developing gonad, whether chromosomally XX or XY, is bipotential and is often referred to as indifferent. The current concept is that development into an ovary or a testis is determined by the coordinated action of a sequence of genes that leads normally to ovarian development when no Y chromosome is present or to testicular development when a Y is present. The ovarian pathway is followed unless a Y-linked gene, designated testis-determining factor (TDF), acts as a switch, diverting development into the male pathway. In the presence of a Y chromosome (with the TDF gene), the medullary tissue forms typical testes with seminiferous tubules and Leydig cells that, under the stimulation of chorionic gonadotropin from the placenta, become capable of androgen secretion. The spermatogonia, derived from the primordial germ cells by successive mitoses, line the walls of the seminiferous tubules, where they reside together with supporting Sertoli cells. If no Y chromosome is present, the gonad begins to differentiate to form an ovary, beginning as early as the eighth week of gestation and continuing for several weeks; the cortex develops, the medulla regresses, and oogonia begin to develop within follicles. Beginning at about the third month, the oogonia enter meiosis I, but this process is arrested at dictyotene until ovulation occurs many years later. While the primordial germ cells are migrating to the genital ridges, thickenings in the ridges indicate the developing genital ducts, the mesonephric (formerly called wolffian) and paramesonephric (formerly called müllerian) ducts. In the male, the Leydig cells of the fetal testes produce androgen, which stimulates the mesonephric ducts to form the male genital ducts. The Sertoli cells produce a hormone (müllerian inhibitory substance) that suppresses formation of the paramesonephric ducts. In the female (or in an embryo with no gonads), the mesonephric ducts regress, and the para-mesonephric ducts develop into the female duct system. Duct formation is usually completed by the third month of gestation. In the early embryo, the external genitalia consist of a genital tubercle, paired labioscrotal swellings, and paired urethral folds. From this undifferentiated state, male external genitalia develop under the influence of androgens. In the absence of a testis, female external genitalia are formed regardless of whether an ovary is present.