d. tnher. Metab. Dis.

15 (1992) 518-525
© SSIEM and KluwerAcademicPublishers. Printed in the Netherlands

Abnor alities of Hu an Sex Deter ination

M. A. FERGUSON-SMITH
Cambridge University, Department of Pathology, Tennis Court Road, Cambridge
CBV2 IQP, UK

Summary: Cytogenetic and molecular studies in patients with abnormalities

of sex determination have been the key to the isolation and investigation of
candidates for the primary testis determining factor (TDF). A gene, SRY,
isolated from the sex determining region of the Y chromosome within 5 kilobases
of the pairing segment boundary, has been characterized recently which fulfils
the expectations of TDF. It is expressed in the embryonic gonads at the critical
time of differentiation; it is highly conserved among mammals; it has the
structure of a transcription regulator; and mutations within its conserved
domain are found in 10% of sex-reversed XY females. The murine homologue
of this gene has been shown to cause sex reversal in XX embryos following
injection of a 14 kb DNA fragment containing SRY into fertilized eggs. However,
most XX true hermaphrodites and a proportion of XX sex-reversed males lack
SRY despite the presence of testicular differentiation. It is postulated that the
constitutive activation of an X-linked gene, TDF-2, normally regulated by SRY,
is responsible for male differentiation in these cases. The female phenotype of
XY individuals with duplications of Xp may be the result or deletion of
disruption of TDF-2.

Sex is determined by the Y chromosome. Males usually have a Y chromosome and

females usually do not. In mice and men XXY individuals are male and X0 individuals
are female. In Drosophila, on the other hand, XXY individuals are female and X0
individuals are male. This tells us that the primary male determining signal or testis
determining factor (TDF) is carried by the Y chromosome in our species, while in
Drosophila the primary signal is different and is the ratio between the number of X
chromosomes and autosomes. The sex determining system is an important system
to study in both species because it provides a marvellous model for the molecular
basis of tissue differentiation. It is a particularly good model in man because the sex
chromosome dimorphism provides such an obvious starting point.
This review therefore starts appropriately with consideration of Figure 1, which
shows the human X and Y chromosomes at the pachytene stage of the prophase of
the first meiotic division. This reveals that the sex chromosomes pair together only
at one end, their short arms. This is important because it allows the two sex
chromosomes to segregate evenly into different gametes. If they did not pair, random
assortment into different gametes would result in 50% of zygotes having XXY or X0

518
Human S e x Determination 519

Figure 1 The XY sex bivalent at the pachytene stage of first meiosis. The pairing segment is
seen at the top with the differential parts of the X (left) and Y (right) presenting a more diffuse
appearance. The testis determining factors (TDF) are located on the differential part of the Y
chromosome close to the boundary of the pairing segment. TDF is therefore at risk of being
transferred to the X, should an abnormal recombination event occur outside the pairing
segment. Electron micrograph by A. Chandley, reproduced by permission of the publishers
from Essential Medical Genetics (Connor and Ferguson-Smith 1990)

sex chromosomes, leading to offspring with the Klinefelter and Turner syndromes,
respectively. It is also important that the sex determining region of the Y is in the
non-pairing or differential segment so that crossing over does not occur. Crossing
over might, of course, transfer male determinants from the Y to the X, leading to
apparent sex reversal in XX and XY individuals.
The first step towards the discovery of the nature of the sex determining mechanism
was to locate the site of the male determining genes on the Y. Fortunately, karyotype-
phenotype studies in patients with Y-chromosome aberrations are very helpful. In
short, those individuals who have retained the distal part of the Y short arm are
male and those who have lost it are female. The key individuals are those with
pseudodicentric isochromosomes for the long arm of the Y chromosome. These Y
chromosome aberrations have breakpoints in the short arm of the Y and are
essentially duplication-deficiency aberrations in which there is variable loss of the
terminal part of the short arm. Most of these patients are female, without any
evidence of masculinization. However, a few patients are male, and in these the
breakpoints occur in the pairing segment distal to the male determining region
(Ferguson-Smith et al 1987). The boundary between the pairing and non-pairing
region of the Y (the pseudoautosomal boundary) therefore marks the distal limit of

J. Inher. Metab. Dis. 15 (1992)

520 Ferguson-Smith

the male determining region, while the most distal breakpoint in the non-pairing
region found in female patients with long-arm isochromosomes of the Y marks the
proximal limit of the male determining region.
The critical region must contain testis-determining factors. Developmental studies
in mouse and rabbit embryos show that the first sign of testis determination occurs
in the supporting cell lineage which surround the incoming germ cells (McLaren
1988). Changes in the undifferentiated gonad occur first in the male, with the
transformation of the supporting cells into Sertoli cells. The Sertoli cells secrete anti-
Mullerian hormone which causes regression of the female internal genitalia. They
also stimulate the interstitial cells to produce testosterone, which is vital for the
differentiation of the Wolffian ducts and male external genitalia. The Sertoli cells
form tubular structures around the incoming germ cells, inhibit meiosis, and nurture
the spermatogonia. Jost (1947) showed that if the undifferentiated fetal gonad were
removed before a critical stage of development,.none of this occurred and the internal
and external genitalia ducts developed by default along female lines; unilateral
castration in male embryos resulted in female differentiation on the same side. The
Sertoli cells appear to be the important component in male differentiation.
Without a Y chromosome, the supporting cell lineage of the undifferentiated gonad
becomes the pre-follicular cells of the ovary. They surround the incoming germ cells
which enter meiosis and become primordial follicles. As no anti-Mullerian hormone
is produced, the Mullerian ducts develop into uterus tubes and upper vagina. The
interstitial cells are unstimulated and Wolllian ducts do not develop.

SEX REVERSAL
There are some rare and apparent exceptions to this series of events, and these
exceptions have proved important in the search for the primary sex determining
genes. A few patients with Klinefelter syndrome and a few patients with pure gonadal
dysgenesis have what seem to be normal female and male karyotypes, respectively.
They appear to be examples of sex reversal.
The XX males are not quite typical of XXY Klinefelter syndrome, in that their
height is below average and within the normal female range. They seldom show
much intellectual impairment and gynaecomastia seems more common. They are
azoospermic and have all the endocrinological features of Klinefelter syndrome,
including small testes and characteristic testicular histology.
It was noted 25 years ago that a few of these XX males had failed to inherit an
X-linked blood group gene from their father. This suggested the hypothesis that the
Xg blood group locus on the X might have been exchanged for testis determinants
on the Y during an abnormal crossover event in male meiosis (Ferguson-Smith 1966).
Once recombinant DNA techniques were applied to test this idea, it became clear
that the majority of XX males had Y-specific sequences in their DNA (Guellaen et
al 1984) and that these sequences came from the short arm of the Y. In situ
hybridization using some of these sequences soon showed that part of the tip of the
short arm of the Y had been transferred to the X.
Could XY females result from toss of male determinants by the same mechanism?
XY females tend to be taller than average women, and their sterility and sexual

J. lnher. Metab. Dis. 15 (1992)

Human Sex Determination 521

infertilism is due to ovarian failure. The ovaries are represented by thin streaks of
ovarian stroma without follicles, and these remnants are liable to malignancy through
the formation of unusual tumours (gonadoblastoma) which often develop into
dysgerminomas. Some believe that these tumours develop from XY germ cells in an
abnormal environment. DNA studies in these cases only rarely show loss of Y-
specific sequences and so it is concluded that most XY females result from mechanisms
other than abnormal X - Y interchange. More importantly, no XY female has been
shown to carry paternal X-chromosome-specific sequences (such as the Xg blood
group locus), which might be expected if there had been abnormal X Y interchange
during paternal meiosis.

THE SEARCH FOR THE TESTIS DETERMINING FACTOR

Studies in sex-reversed XX males and the rare XY females who have loss of Y-specific
sequences have been important in efforts to clone candidates for TDF. Early
candidates such as the Bkm sequence, which is sex-specific in some reptiles (Jones
and Singh 1981) and the H-Y male specific histocompatibility antigen (Wachtel et al
1975) were soon excluded. Bkm sequences are rare and not sex-specific in man, and
the H-Y antigen locus maps to the long arm of the human Y chromosome (Simpson
et al t987). The first plausible candidate for T D F was described by Page and
colleagues (1987). They identified an expressed gene on the short arm of the Y which
coded for a zinc finger transcriptional activator gene. This gene, termed ZFY, was
present in an XX male and absent in an XY female who had about t40 kilobases
deleted from her Y chromosome as a result of an apparently balanced X-autosomal
translocation. ZFY was found to have a homologue on the short arm of the X which
escaped X-inactivation, apparently excluding the possibility of a gene dosage model
for sex determination.
Most XX males were shown to have the ZFY gene, which was also found to be
highly conserved on the Y chromosome of other mammals. However, almost
immediately a Y-positive XX male was identified who lacked ZFY (Ferguson-Smith
and Affara 1988). In addition, many XX true hermaphrodites and XX males were
found who appeared to have no Y sequences whatsoever. Moreover, ZFY proved to
be autosomal among marsupial species that have an X - Y sex determining system.
ZFY was finally excluded as a candidate for T D F by the demonstration that its
expression is confined to the germ-cell lineage in the mouse and could not be
demonstrated in embryonic testes which lacked germ cells. The possibility remains
that ZFY has an important rote in germ cell maturation.
The search for a better candidate for T D F then concentrated on those XX males
that lacked ZFY yet had other more distal Y-specific sequences. Palmer et al (1989)
described three XX males with less than 60 kb of the distal end of the differential
segment of the Y~ The sex determining region in these patients was further reduced
to 35 kb and characterization of cloned sequences from within this region revealed
a new gene, SRY (for sex determining region of the Y), which was conserved and Y-
specific among a wide range of mammals (Sinclair et al 1990). The SRY gene was
found to map within the sex determining region of the mouse Y chromosome. This
routine homologue (Sty) was shown to be deleted in an XY female mouse and

J. Inher. Metab. Dis. 15 (t992)

522 Ferguson-Smith

expressed in the developing gonads of male mouse embryos at the critical stage of
testis differentiation. SRY appears to code for a transcription factor which shares a
DNA-binding motif with the yeast mating-type protein, Mc. The evidence that SRY
is the testis determining factor appeared to be clinched by the subsequent observation
of de novo mutations in SRY in two XY females with gonadal dysgenesis (Berta et
al 1990; Jager et al 1990a). At the time of writing, it now seems that about 10% of
XY females may have mutations in the SRY gene.
The latest and most compelling finding to confirm the testes determining nature
of SRY comes from transgenic studies in the mouse. Koopman and colleagues (1991)
have transferred a 14kb fragment including Sry from the male determining region
of the mouse into fertilized mouse eggs. Sex-reversed XX male embryos and adult
mice with sterile testes and male external genitalia were identified as chromosomally
female by the presence of sex chromatin in amnion and the absence of Zfy sequences
in their DNA.

SEX REVERSAL IN THE ABSENCE OF SRY

By analogy with the elegant studies of sex differentiation in Drosophila species and
in Caenorhabditis elegans (Hodgkin 1990), the genetic control of sex differentiation
in mammals is likely to result from a cascade of genes, each gene switching on the
next gene in the series. The primary switch in Drosophila and C. elegans is the X
chromosome: autosome ratio, whereas in mammals it is clearly a Y-linked factor and
almost certainly the transcription factor coded by SRY. It follows that in individuals
who lack SRY and in whom testicular differentiation occurs none the less, there must
be a gain-of-function mutation later in the sex determination pathway. In fact,
testicular differentiation could well result from the constitutive activation of the gene
regulated by SRY. Several questions can now be asked: What is the nature of this
gene? Where is it expressed? And where is it likely to be located?
It seems most likely that the product of this gene may be found in the Sertoli cells
of the testes. However, there is no need for the gene to be Y-linked. In fact there are
some observations which suggest that it might map to the X chromosome. No doubt
the answer wilt come from DNA-binding studies using the product of the SRY gene.
Meanwhile, we can look for clues among other abnormalities of human sex
determination, in particular the XX true hermaphrodites and Y-negative XX males.
The phenotype of XX true hermaphrodites is characteristic. All have stature within
the normal female range and the diagnosis may be apparent in infancy or childhood
because of ambiguity of the external genitalia. Those cases missed in childhood will
usually be apparent in adolescence because of virilization of the external genitalia
and development of the mammary glands. There is usually a phallus with perineal
hypospadias, a vaginal pouch and variable development of the uterus and Fallopian
tubes depending on the nature of the ipsilateral gonad. The most frequent finding is
an ovary on one side and a testis on the other, with suppression of the Mullerian
ducts on the side of the testis. Bilateral ovotestes are usually associated with uterus
and bilateral tubes and in these patients virilization is less than in those with a
unilateral testis.

J. lnher, Metab. Dis. 15 (1992)

Human Sex Determination 523

True hermaphrodites seem to be of average intelligence and the condition is not

usually associated with other developmental malformations. Almost all cases have
proved to have a Y-negative XX genotype when tested by PCR for the presence of
the Y-pseudoautosomal boundary sequences. There are, however, two important
exceptions in the patients described by Sinclair and colleagues (1990) and Jager and
colleagues (1990b). Both have Y-specific sequences at the autosomal boundary which
include SRY. One case has bilateral ovotestes and the other has an ovary on one
side and a testis on the other. It is possible that mosaicism for testis determining and
ovary determining gonadal cells has been the result of non-random inactivation of
the X chromosome carrying the SRY gene as postulated previously (Ferguson-Smith
1966). The exact breakpoint on the X chromosome may be critical in determining
whether or not the Y-specific sequences are subject to inactivation, and this may
explain why most XX males are not hermaphrodites. Alternatively, only a small
proportion of cells with an active SRY locus may be sufficient to ensure complete
male differentiation.
Mosaicism involving the Y chromosome has long been known in association with
true hermaphroditism. XX/XY chimeras have been described in man (Gartler et al
1962) and artificial XX/XY chimeras have been constructed in the mouse (Tarkowski
1964), both with similar sex differentiation. In addition, true hermaphrodites are
known with XX/XXY mosaicism. However, in both XX/XY chimeras and XX/XXY
mosaics, it is more usual to find unambiguous male differentiation, presumably
because only a small proportion of Y-bearing cells are required to ensure differentiation
of a testis.
Y-negative XX males, i.e. those that do not have the pseudoautosomal Y boundary
sequence, show interesting phenotypic differences when compared to the majority of
Y-positive XX males. Most show varying degrees of hypospadias and some have
cryptorchidism (Ferguson-Smith et al 1990)- findings which suggest an aetiological
reIationship with XX true hermaphrodites rather than with Y-positive XX males.
Furthermore, there are a number of rare families in which there are sibships containing
Y-negative true hermaphrodites, Y-negative XX males and normal males and females.
The pattern of inheritance in these families is consistent with that of an X-linked
mutation transmitted either by normal males or by non-manifesting females in which
the mutation is subject to X-inactivation.
The occurrence of these familial cases is the most important evidence in favour of
an X-linked gene for sex determination, TDF-2, normally regulated by SRY.
Constitutive activation of the TDF-2 gene will have no effect on an XY individual
but may result in the sex reversal of an XX individual. If TDF-2 is subject to X-
inactivation, a constitutive mutation in one X chromosome may lead to mosaicism
for testis determining and ovary determining cells and, in cases with the appropriate
proportions in the gonadal anlage, to true hermaphroditism.
Perhaps the only justification for considering such a hypothesis is that it may
suggest studies which might lead to an improved understanding of sex determination
and differentiation. One consequence of the existence of the postulated TDF-2 is that
its deletion might be expected to result in sex reversal in an XY individual. Thus a
search for X-linked deletion in XY females could be rewarding. In this respect it is

J. Inher. Metab. Dis. 15 (t992)

524 Ferguson-Smith

noteworthy that there are several cases reported in which a chromosome aberration
resulting in the duplication of part of the distal end of the X chromosome resulted
in female sex differentiation despite the presence of an apparently normal Y
chromosome (Bernstein et al 1980; Scherer et al 1989). These aberrations could
conceivably disrupt the T D F - 2 locus and thus interfere with the pathway to normal
testis differentiation.

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J. Inher. Metab. Dis. 15 (1992)