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OCR for page 28
Every Ceil Has a Sex
ABSTRACT
The biological differences between the sexes have long been recognized at
the biochemical and cellular levels. Rapid advances in molecular biology
have revealed the genetic and molecular bases of a number of sex-based
differences in health and human disease, some of which are attributed to
sexual genotype XX in the female and XY in the male. Genes on the sex
chromosomes can be expressed differently between males and females be-
cause of the presence of either single or double copies of the gene and
because of the phenomena of different meiotic effects, X inactivation, and
genetic imprinting. The inheritance of either a male or a female genotype is
further influenced by the source (maternal or paternal) of the X chromo-
some. The relative roles of the sex chromosome genes and their expression
explains X-chromosome-linked disease and is likely to illuminate the rea-
sons for heterogeneous expression of some diseases within and between the
sexes.
The notion that there are biological differences between the sexes is
most evident and comfortable when it is applied to the reproductive sys-
tem. However, sex differences have been identified or suggested at many
levels of biological organization, from biochemical to behavioral. For the
majority of the population, as well as a substantial fraction of scientists,
not all known differences are obvious, and not all of those that have been
suggested or suspected are easily explainable in biological terms.
In terms of genetic mechanisms, two general models attempt to ex-
28
OCR for page 29
EVERY CELL HAS A SEX
29
plain how an individual's genes give rise to sex differences (Figure 2-1~.
In the first model, a series of critical hormone-responsive genes, shared by
both males and females, are influenced differently in the alternative hor-
monal milieus of the male or female throughout their life spans, thus
leading to or contributing to the many differences observed between the
sexes. In the second model (which is not necessarily exclusive of the first
one), one or more genes, located on the sex chromosomes and thus ex-
pressed differently in the two sexes, encode proteins involved in rate-
limiting or rate-influencing steps in biochemical or physiological path-
ways that are critical to establishing differences between the sexes.
The purpose of this chapter is twofold: (1) to describe those differ-
ences that exist between males and females at the biochemical and cellu-
lar levels and that result directly from the defining genotypic difference
between male and female mammals, namely, an XY (male) sex chromo-
some constitution versus an XX (female) sex chromosome constitution,
and (2) to describe how males and females may transmit to their offspring
genetic information that is the same but that is transmitted at different
observed phenotypic or genotypic ratios. This information will then serve
as a foundation for consideration of the onset of sex differences during
development and throughout life in response to both intrinsic and extrin-
SiC exposures.
SEX AND THE HUMAN GENOME
Males and females have partially different genomes. Viewed from a
purely reductionist standpoint, many differences between the male and
female sexes are predicted to be rooted in differences between the genetic
contents of male and female cells and differences in the expression of
those genetic contents. As the complete DNA sequence of the human
genome has now been determined, it is important to place the discussions
of this chapter into the context of the human genome.
The human genome contains, by current measurements, a little more
than 3 billion base pairs of DNA (Lander, 1996; National Human Genome
Research Institute, 2000~. Earlier estimates predicted an estimated 50,000
to 100,000 different genes (National Human Genome Research Institute,
2000~. The most recent estimates, based on the current drafts of the hu-
man genome sequence, suggest that there are approximately 30,000 hu-
man genes (International Human Genome Sequencing Consortium, 2001;
Venter et al., 2001~. However, this lower figure may be a minimum esti-
mate because it is derived using an algorithm that identifies genes on the
basis of their similarity to a modest sized panel of already characterized
human genes.
The hallmark of human biology is variation, and much of the ob-
served variation both within and between the sexes is encoded within the
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30
EXPLORING THE BIOLOGICAL CONTRIBUTIONS TO HUMAN HEALTH
Model I
Hormones
Model Ha
Model rib
Gene Expression in:
Male Female
Gene 1
Gene 2
Gene 3
Gene 'N'
Male A ~ B
Female A ~ B
Male A
Female A
tt
t
| X-linked gene product |
C ~ D
C ~ D
I Y-linked gene product
B
B
C ~ D
C ~ D
FIGURE 2-1 Schematic representation of two general models used to explain
sex differences in gene expression. In Model I, hormones in males and females
differentially influence the level of expression of different genes (Gene 1 to Gene
N) in the genome. Arrows indicate the direction and the magnitude of the effect.
In Model II, a rate-limiting step in various pathways (e.g., metabolic or signal
transduction pathways) is encoded by or influenced by an X- or Y-chromosome-
linked gene that shows dosage differences between males and females. Thus, the
net amounts of Products C and D differ between the sexes.
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EVERY CELL HAS A SEX
31
human genome. At the DNA level, an estimated 1 of every 1,300 bases on
the autosomes (non-sex-determining chromosomes) differs between any
two individuals (International SNP Map Working Group, 2001; Nickerson
et al., 1998; Venter et al., 2001~. In other words, the genomes of individuals
may differ at some 4 to 6 million base positions. Some of these differences
will lead to gene products that are functionally distinct, for example,
receptors that differ in their affinity or rate of turnover, enzymes that
differ in their steady-state levels, and genes that differ in their degree of
hormone responsiveness. Although ongoing studies of human DNA
variation will soon provide a more robust estimate, one can calculate
from previous studies of enzyme variation and more recent investiga-
tions of gene variation (Zwick et al., 2000) that the precise composition
and functioning of thousands of proteins will differ between any two
individuals.
Notwithstanding this degree of population-level variation in the DNA
sequence, most of the genes in the genome are thought to not differ in
either sequence or level of expression as a simple consequence of the sex
of the individual. However, as will be illustrated more fully in the follow-
ing sections, there are three types of genes (see also Box 2-1) in which an
individual's sex per se is likely to play a role.
First, genes on the Y chromosome are expressed only in males, and
many of these have no counterpart on the X chromosome or autosomes;
thus, expression of these genes will be limited to males.
Second, some genes on the X chromosome are expressed at higher
levels in females than in males. Although the process of X-chromosome
inactivation equalizes the effective dosage of most X-chromosome genes
between male and female cells by inactivating one of the two X chromo-
somes in female cells, not all genes on the inactivated X chromosome
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32
EXPLORING THE BIOLOGICAL CONTRIBUTIONS TO HUMAN HEALTH
respond to this mechanism. The relatively few genes that are not equal-
ized can have significant effects on the phenotypes of cells.
Third, the expression of many genes is likely to be influenced by
hormonal differences between the two sexes. For example, some of these
may be genes whose expression is limited to sexually dimorphic tissues
or cell types (e.g., the ovary, testis, prostate, and breast), whereas others
may be globally expressed but subject to hormonal regulation in different
tissues or at different times during development (see Chapter 3~.
Although only a limited number of genes have been examined to
date, from the standpoint of sexual dimorphism, new approaches to quan-
tification of the expression of genes in different samples on a genome-
wide basis promise to change this. DNA arrays, or "gene chips," contain-
ing tens of thousands of human genes can be queried to compare their
levels of expression between different tissues or different sexes under a
variety of physiological or hormonal conditions (Lander, 1996; Lockhart
and Winzeler, 2000~. Such studies will yield a large database of gene
expression data. More difficult will be determination of the relative ef-
fects of differences in gene expression on the characteristic phenotypic
differences seen between males and females. Nonetheless, this new tech-
nology with DNA arrays promises to provide a comprehensive functional
view of the genome in different cellular states, and studies that address
differences in expression throughout the male and female genomes should
reap a rich harvest.
BASIC MOLECULAR GENETICS:
WHAT IS THE POTENTIAL FOR DIFFERENCES
BETWEEN THE SEXES?
The issue of whether there should be genetic differences in basic cel-
lular biochemistry between female and male cells (as a direct result of sex
chromosome constitution rather than hormonal influences) (see Figure 2-
1 and Box 2-1) is often approached from two opposing perspectives. Ge-
neticist lacques Monod's famous adage that "What's true of Escherichia
cold is true of an elephant" represents the point of view that genes have
been conserved over time and among species. This view has had extraor-
dinary staying power in molecular biology and genetics, and if "yeast"
was substituted for "E. coli," the statement would have even greater vital-
ity. If the basic biochemistries of organisms separated by a billion years of
evolution are so similar, then (so goes the logic) why should one expect
that males and females within the same species should exhibit important
differences in their basic biochemistries? An opposing perspective ac-
knowledges that the majority of human disease-causing mutations ex-
hibit dominant or semidominant effects (McKusick, 2000~. Thus, a change
in the activity of a single gene can have a large effect on the organism that
OCR for page 33
EVERY CELL HAS A SEX
X
1,000-2,000 genes
Most subject to
X inactivation
At least 10%
escape
X inactivation
\1
RPS4X
XIST
.
,1
l
l
l
l
33
y
<50 genes
o
Involved in:
~ Sex determination
O Spermatogenesis
X-Y Homologous
regions
I Pseudoautosomal
regions
· X-Y homologous
genes, regions
FIGURE 2-2 Comparison of gene contents and gene organizations on the X and
Y chromosomes (see text for details).
carries that gene. Because the sex chromosomes comprise approximately
5 percent of the total human genome (Figure 2-2), there is the potential for
1 in 20 biochemical reactions to be differentially affected in male versus
female cells. From this standpoint, it is difficult to imagine that male and
female cells will not differ in at least some aspects of basic biochemistry,
given the complexity of most biological pathways.
Males Have a Y Chromosome, Females Do Not
The male genome differs from the female genome in the number of X
chromosomes that it contains, as well as by the presence of a Y chromo-
some. It is the overriding presence of a gene on the Y chromosome (SPY)
that results in development of the male gonadal phenotype. However,
apart from causing the dramatic divergence from the female develop-
mental pathway (which the indeterminate gonad would otherwise follow
and which has been discussed in a number of reviews [Hiort and
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34
EXPLORING THE BIOLOGICAL CONTRIBUTIONS TO HUMAN HEALTH
Holterhus, 2000, Sinclair, 1998; Vilain and McCabe, 1998~), it was long
considered a valid biological question to ask whether the Y chromosome
carried any genes of "importance." The paucity and nature of traits that
were thought, by genetic criteria, to segregate with the Y chromosome
("hairy ears," for example [Dronamraju, 1964~) tended to reinforce the
notion that the Y chromosome encoded the male gonadal phenotype
(Koopman et al., 1991), one or more genes involved in male fertility (Lahn
and Page, 1997), the HY male transplantation antigen (Wachtel et al.,
1974), and not much else. Surprisingly, recent studies show that the Y
chromosome carries some genes that are involved in basic cellular func-
tions and that are expressed in many tissues (Lahn and Page, 1997~.
Cytologically, the Y chromosome consists of two genetically distinct
parts (Figure 2-2~. The most distal portion of the Y-chromosome short arm
(Yp) is shared with the most distal portion of the X-chromosome short
arm (Xp) and normally recombines with its X-chromosome counterpart
during meiosis in males. This region is called the "pseudoautosomal re-
gion" because loci in this region undergo pairing and exchange between
the two sex chromosomes during spermatogenesis, just as genes on auto-
somes exchange between homologues. There is also a second pseudo-
autosomal region involving sequences on the distal long arms of the sex
chromosomes (Watson et al., 1992) (Figure 2-2~. The remainder of the Y
chromosome (the Y-chromosome-specific portion) does not recombine
with the X chromosome and strictly comprises "Y-chromosome-linked
DNA" (although some of the nonrecombining part of the Y chromosome
retains residual homology to X-chromosome-linked genes, reflecting the
shared evolutionary history of the two sex chromosomes [see billowy.
The pseudoautosomal regions reflects the role of the Y chromosome as
an essential pairing homologue of the X chromosome during meiosis in
males (Rappold, 1993), whereas the Y-chromosome-specific region, in-
cluding the testis-determining factor gene, SPY, provides the chromo-
somal basis of sex determination.
The Y chromosome is one of the smallest human chromosomes, with
an estimated average size of 60 million base pairs, which is less than half
the size of the X chromosome. Cytologically, much of the long arm (Yq) is
heterochromatic and variable in size within populations, consisting
largely of several families of repetitive DNA sequences that have no obvi-
ous function. A significant proportion of the Y-chromosome-specific se-
quences on both Yp and Yq are, in fact, homologous (but not identical) to
sequences on the X chromosome. These sequences, although homologous,
should not be confused with the pseudoautosomal regions. Pseudoauto-
somal sequences may be identical on the X and Y chromosomes, reflecting
their frequent meiotic exchange, whereas the sequences on Yp and Yq
homologous with the Y and X chromosomes are more distantly related to
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EVERY CELL HAS A SEX
35
each other, reflecting their divergence from a common ancestral chromo-
some (Lahn and Page, 1999~.
Only about two dozen different genes are encoded on the Y chromo-
some (although some are present in multiple copies). Unlike collections of
genes that are located on the autosomes and the X chromosome and that
reflect a broad sampling of different functions without any obvious chro-
mosomal coherence, Y-chromosome-linked genes demonstrate functional
clustering and can be categorized into only two distinct classes (Lahn and
Page, 1997~. One class consists of genes that are homologous to X-chromo-
some-linked genes and that are, for the most part, expressed ubiquitously
in different tissues. Some of these genes are involved in basic cellular
functions, thus providing a basis for functional differences between male
and female cells. For example, the ribosomal protein S4 genes on the X
and Y chromosomes encode slightly different protein isoforms (Watanabe
et al., 1993~; thus, ribosomes in male cells will differ characteristically
from ribosomes in female cells, setting up the potential for widespread
biochemical differences between the sexes. The second class of Y-chromo-
some-linked genes consists of Y-chromosome-specific genes that are ex-
pressed specifically in the testis and that may be involved in spermatogen-
esis (Figure 2-2~. Deletion or mutation of some of these genes has been
implicated in cases of male infertility, but otherwise, these genes have no
obvious phenotypic effects (Kent-First et al., 1999; McDonough, 1998~.
Females Have Two X Chromosomes, Males Have One
Male and female genomes also differ in the other sex chromosome,
the X chromosome, in that females have twice the dose of X-chromosome-
linked genes that males have. The X chromosome consists of approxi-
mately 160 million base pairs of DNA (about 5 percent of the total haploid
genome) and encodes an estimated 1,000 to 2,000 genes (Figure 2-2~. By
the nature of X-chromosome-linked patterns of inheritance, females can
be either homozygous or heterozygous for X-chromosome-linked traits,
whereas males, because they have only a single X chromosome, are hem-
izygous. Of those X-chromosome-linked genes known to date, most are X
chromosome specific; only pseudoautosomal genes and a few genes that
map outside of the pseudoautosomal region have been demonstrated to
have functionally equivalent Y-chromosome homologues (Willard, 2000~.
Products of X-chromosome-linked genes, like those on the autosomes,
are involved in virtually all aspects of cellular function, intermediary
metabolism, development, and growth control. Although many are re-
sponsible for general cellular functions and are expressed widely in dif-
ferent tissues, others are specific to particular tissues or particular time
points during development, and several are known to be responsible for
steps in gonadal differentiation (Pinsky et al., 1999~.
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36
EXPLORING THE BIOLOGICAL CONTRIBUTIONS TO HUMAN HEALTH
X-Chromosome Inactivation Compensates for
Differences in Gene Dosage
The twofold difference between males and females in the dosage of
genes on the X chromosome is negated at many loci by the process of X-
chromosome inactivation (Figure 2-3~. X-chromosome inactivation is, on
~3
Random X
Inactivation
X with mutant allele active
X with normal allele active
I'
~J
.
Variability of Mosaicism
Increasing clinical expression or severity in heterozygotes
FIGURE 2-3 Schematic representation of X-chromosome inactivation in female
somatic cells. Inactivation early in development is believed to be random, with
an equal probability a priori that the maternal or paternal X chromosome will be
active or inactive. Females are therefore epigenetic mosaics. However, the distri-
bution of cells that express alleles from one or the other X chromosome can vary
widely in different tissues or individuals. Inter- or intraindividual variation in
the expression of one allele at X-chromosome-linked loci may result from selec-
tion against cells that express a particular allele (such as a mutant G6PD [glucose-
6-phosphate dehydrogenase] allele) or stochastic variation in the proliferation of
cells, in which one or the other X chromosome has been inactivated. The pheno-
type observed in females heterozygous for X-chromosome-linked traits can also
vary widely, with an increasing level of clinical expression or increasing severity
correlating with the proportion of cells expressing a mutant allele from the active
X chromosome. Variations between females may also occur as the result of differ-
ences in the levels at which particular X-chromosome-linked genes may escape
inactivation or become reactivated as cells age (Wareham et al., 1987~.
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EVERY CELL HAS A SEX
37
a cytological level, a large-scale process in which one of the two X chro-
mosomes becomes heterochromatic. The end result of this process can be
seen under the microscope as the Barr chromatin body in the nucleus of
the female cells. X-chromosome inactivation is associated with extensive
silencing of genes on the affected X chromosome and occurs in almost
every cell of XX females but does not occur in XY males. The one docu-
mented exception to this rule occurs, reciprocally, in reproductive cells;
the single X chromosome of males becomes heterochromatic in spermato-
cytes, whereas both X chromosomes are thought to be active in primary
oocytes. This unusual characteristic in which both X chromosomes are
active in a single cell also occurs very early in the development of female
embryos. Because the process of X-chromosome inactivation is not com-
pleted until near the time of implantation (reviewed by Willard [2000~),
there is a preimplantation developmental window during which there
may be basic differences in cellular chemistry between female and male
embryos. It is unknown whether the differences in gene expression that
have been shown to occur (Gutierrez-Adan et al., 2000; Latham et al.,
2000) or that may occur during this period influence the establishment of
additional differences between the sexes during the postimplantation or
postnatal periods.
In any case, the simple fact of X-chromosome inactivation leads to
two levels of difference between males and females. The first is that XX
cells must operate whatever cellular machinery is required to initiate and
establish the inactivation of an X chromosome in all mitotically active
cells and also (perhaps) to actively maintain the inactive state of one X
chromosome in terminally differentiated cells first. The second level of
difference is superimposed on the first and is a property of populations of
XX cells: females, by virtue of not inactivating the same X chromosome in
every cell, are "epigenetic mosaics."
X-Chromosome-Based Differences Between Cells
There has been substantial recent progress in understanding the bio-
chemistry and molecular biology of the X-chromosome inactivation pro-
cess. These advances have been described in detail in several recent
reviews (Heard et al., 1997; Willard, 2000), but the overall conclusion
relevant to this report is that genes involved in the initiation, establish-
ment, or maintenance of the X-chromosome inactivation process are or
have been expressed in every somatic cell of females. Although some of
the genes in the X-chromosome inactivation pathway may be expressed
at some level or at some time in males (Daniels et al., 1997; Ray et al.,
1997), the overall process that results in the cytologically visible hetero-
chromatization of an entire chromosome is a fundamentally "female"
characteristic, whether considered in viva or in vitro. Here, then, is a basic
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38
EXPLORING THE BIOLOGICAL CONTRIBUTIONS TO HUMAN HEALTH
biochemical process that is a fundamental consequence of having two X
chromosomes. The biochemical results of the process can be measured
and quantified in the tissues of individual females or in cells in culture
dishes. The process affects genes that are involved in many important
metabolic processes as well as genes that are known to be important in the
regulation of expression of other genes (Amir et al., 1999; Melcher et al.,
2000).
Because there is a stochastic or random component in the choice of
which of the two X chromosomes is inactivated (Puck and Willard, 1998),
individual females have two epigenetically distinct populations of cells:
those in which the maternally derived X chromosome remains active and
those in which the paternally derived X chromosome remains active (Fig-
ure 2-3~. By contrast, males have only an active maternally derived X
chromosome in all of their cells.
This X-chromosome-based, female-specific mosaicism is often in-
voked as the reason for much of the dramatic sex differences observed in
the severities of recessive X-chromosome-linked disease phenotypes
(McKusick, 2000~. All cells of XY males must suffer the consequences of a
mutation in an X-chromosome-linked gene, but only that fraction of a
female's cells that carry the mutation on the active X chromosome will be
affected. Such situations have resulted, in some cases, in strong somatic
selection against cells that bear the mutation on the active X chromosome
and thus avoidance (or minimization) of the disease phenotype (Belmont,
1996; Willard, 2000~.
It should be noted that the stochastic nature of the initial choice of
which X chromosome to inactivate can be influenced by many factors.
Environmental, epigenetic, and genetic factors have all been demonstrated
to influence the X-chromosome inactivation pattern (the proportion of a
female's cells with a designated active X chromosome) of individual fe-
males (Puck and Willard, 1998~. The relative importance of each may be
different in different individuals, to the extent that all sisters within an
individual family may show nearly identical patterns of X-chromosome
inactivation, whereas identical twins in other families may exhibit wide
variations in the proportions of their cells that have one or the other active
X chromosome.
Not all of the genes on the X chromosome respond to the inactivation
process by transcriptional silencing (Willard, 2000~. This fact may lead
secondarily to biochemical differences between XX cells and XY cells. As
many as 10 to 15 percent of X-chromosome-linked genes have been iden-
tified as being expressed from the inactive X chromosome, at least in cells
in culture, and are therefore said to "escape" X-chromosome inactivation
(Carrel et al., 1999~. Some of these are transcribed from both the active and
the inactive X chromosome at similar levels, whereas others appear to be
transcribed from the inactive X chromosome at reduced, but still signifi-
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EVERY CELL HAS A SEX
39
cant, levels (Carrel and Willard, 1999; Fisher et al., 1990~. Regardless of the
level at which such genes escape X-chromosome inactivation, it is likely
that XX cells will produce higher levels of gene product from some of
these loci than XY cells will. It has been suggested that some of these
differences may lead to sex-specific levels of risk for certain diseases, such
as the suspected relationship between gastrin-releasing peptide receptor
and smoking-related lung cancer (Shriver et al., 2000~. Gastrin-releasing
peptide is expressed by both the active and the inactive X chromosomes,
and elevated levels of gastrin-releasing peptide are hypothesized to be
associated with an elevated risk of lung cancer in women who smoke.
An interesting genomic consideration resulting from the study of
genes that escape X-chromosome inactivation is that their distribution
along the X chromosome is not random. A higher proportion of the genes
on the short arm of the X chromosome than on the long arm of the X
chromosome escape X-chromosome inactivation (Carrel et al., 1999~. This
issue may reflect the different evolutionary histories of the X-chromo-
some arms (Lahn and Page, 1999) but may also be related to whether
particular X-chromosome-linked genes have homologues on the Y chro-
mosome (legalian and Page, 1998~. It is of some interest that the particular
genes that escape inactivation appear to differ among different females
(Carrel and Willard, 1999), thus providing additional avenues for differ-
ences between and within the sexes. It is unknown whether there are
significant population variations in patterns of inactivation and thus X-
chromosome-linked gene expression.
The X-Chromosome Dosage Matters
In general, the possible effects of any variant in an X-chromosome-
linked gene may differ between the sexes for a variety of reasons, as
outlined below.
· Gene dosage. For genes that are specific to the X chromosome and
that escape X-chromosome inactivation, female cells (with two X chromo-
somes) may contain higher levels of the gene product than male cells,
which have only a single X chromosome. Depending on the cellular role
of the particular gene product, this dosage difference may have more
pervasive effects on the expression of other genes in the genome. For
example, a twofold change in the level of an X-chromosome-linked tran-
scription factor might lead to dramatic effects on the levels of genes regu-
lated by that transcription factor.
· Mosaicism. For genes that are subject to X-chromosome inactiva-
tion, most females are mosaics of two cell populations, one expressing
alleles on the paternally inherited X chromosome and one expressing
alleles on the maternally inherited X chromosome (see below). Thus, ex-
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40
EXPLORING THE BIOLOGICAL CONTRIBUTIONS TO HUMAN HEALTH
pression of an X-chromosome-linked phenotype is often much more vari-
able in females than in males.
· Hemizygosity. Because males have only a single X chromosome,
functional variants cannot be "masked" by a second X chromosome. Thus,
males often demonstrate a clearer, more common, or more extreme ver-
sion of the variant phenotype than females do.
· X-chromosome-linked dominant traits. A dramatic example of the
effect of male hemizygosity for X-chromosome-linked traits involves X-
chromosome-linked dominant mutations that are lethal in males in utero
and that are therefore evident only in females. For example, X-chromo-
some-linked incontinentia pigment) is a relatively benign dermatological
condition in females, but it is lethal in males who inherit a mutant allele
(Smahi et al., 2000~.
Differences Between Male and Female Cells That Have Not
Been Linked to Sex Chromosomes
The incidence of a number of diseases whose etiologies cannot be
traced to the sex chromosomes differ dramatically between males and
females (McKusick, 2000~. Although the basis for these differences in inci-
dence is most often ascribed to hormonal influences, the possibility that
other genetic differences are at fault cannot be discounted.
EFFECTS OF PARENTAL IMPRINTING ON THE
EXPRESSION OF GENETIC INFORMATION
The discovery that some genes are expressed only from the maternal
allele and that others are expressed only from the paternal allele, a phe-
nomenon called "genomic imprinting" (reviewed by Tilghman [1999~),
reinforces the concept that there are multiple biochemical differences be-
tween the gametogenic cells of males and females and that these differ-
ences may affect the expression of genetic information in the next genera-
tion.
Because autosomes are transmitted equally to both sexes, it is not
predicted that inheritance of imprinted genetic information on the auto-
somes should have a differential effect on male versus female offspring.
The situation is different for sex chromosomes. Imprinting-related differ-
ences between the sexes do exist for the X chromosome. Males have only
a maternal X chromosome, but females have both a maternal X chromo-
some and a paternal X chromosome; therefore, X-chromosome-linked
genes that pass through the paternal germ line have the potential to affect
the phenotype of female offspring but not that of male offspring. In this
regard, there is direct evidence that the imprinting process affects the
expression of alleles in females at the Xist locus (a gene that is critical to
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EVERY CELL HAS A SEX
41
the process of X-chromosome inactivation and that is expressed primarily
from the paternal allele in some extraembryonic cells) in females (re-
viewed by Lyon [1999~.
There is also indirect evidence that imprinting affects the expression
of a locus on Xp that has female-specific effects on cognitive and behav-
ioral phenotypes. The latter evidence is derived from studies of patients
with Turner syndrome, who have inherited only one X chromosome (XO)
from either the mother or the father (Skuse et al., 1997~. These findings
may have broader implications for cognitive function or behavior in males
and females because males inherit only a maternal X chromosome,
whereas females inherit both a maternal X chromosome and a paternal X
chromosome.
UNEXPECTED OR NONOBVIOUS SEX DIFFERENCES
Sex-Specific Meiotic Effects
Although the basic mechanism of meiosis, the creation of haploid
gametes from diploid precursors, is universal, there are both quantitative
and qualitative differences between males and females in the production
of gametes. These differences have characteristic effects on the ways that
males and females drive the evolutionary process, as well as the mecha-
nisms by which diseases that result from genetic defects are manifest.
The three most important differences between males and females in
the gametogenic process are as follows: (1) the number of stem cell divi-
sions that occur to give rise to the germ cell population, (2) the timing of
the first and second meiotic divisions, and (3) the number of gametes
produced from each primary germ cell.
The male produces billions of sperm from a population of stem cells
that continue to divide throughout the entire adult life. In contrast, the
female produces a relatively small number of ova (~500) from a limited
population of oocytes that arise early in embryogenesis. These oocytes are
arrested at the meiotic prophase from fetal life until ovulation, which may
occur as many as 50 years after the initiation of meiosis. This simple
numerical difference in the number of stem cell divisions between the two
sexes dictates that most mutations resulting from errors in DNA replica-
tion take place in the male germ line (Haldane, 1935), although the magni-
tude of this difference and whether additional factors may contribute are
subjects of debate (Hurst and Ellegren, 1998~. On the other hand, the
protracted length of time that an individual ovum may be arrested at the
meiotic prophase is correlated with the fact that aneuploidy (gain or loss
of one or more chromosomes) resulting from nondisjunction (improper
separation of chromosomes at nuclear division) occurs much more fre-
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42
EXPLORING THE BIOLOGICAL CONTRIBUTIONS TO HUMAN HEALTH
quently through the female germ line than through the male germ line
(Hassold et al., 2000~.
Although all four products of meiosis in the male have the potential
to become functional sperm, each primary oocyte gives rise to only a
single ovum. Additional differences in the meiotic process are found in
the observed rate of recombination and the consequent length of the hu-
man genetic map obtained by measurement with chromosomes from fe-
males compared with those obtained by measurement with chromosomes
from males. In general, there is more recombination over the autosomes
during female meiosis than during male meiosis (Broman et al., 1998~.
Only the comparatively small, "pseudoautosomal" portion of the X and Y
chromosomes recombine during male meiosis, but the rate of recombina-
tion in this region is approximately 10 times greater than the rate of re-
combination in this region during female meiosis (Hunt and LeMaire,
1992; Rappold, 1993~.
Sex-of-Offspring-Specific Transmission Ratio Distortion
In a number of instances the inheritance of alleles from heterozygous
parents does not appear to be equal between male and female offspring
(Naumova et al., 1998; Pardo-Manuel de Villena et al., 2000; Siracusa et
al., 1991; see also Sapienza [1994] for a review). Such sex-specific biases in
the inheritance of genetic information are not expected per se (especially
in the case of autosomal loci) but may be due to a number of causes,
including meiotic drive, preferential cosegregation of sex chromosomes
with one of a pair of homologous chromosomes, preferential fertilization,
and preferential death of the embryo of one sex. These biases that are
specific to the sex of the offspring have been observed as a result of
transmission through both male and female parents (reviewed by
Sapienza [1994~. As a practical matter, it is important to consider the
source and magnitude of any observed inheritance biases because they
may affect the mapping and identification of genetic traits that are more
prevalent in one sex than the other. An interesting side effect of sex-of-
offspring-specific transmission ratio distortion is that the observed fre-
quencies of alleles at some loci may differ between the sexes.
GENETICS AS A TOOL
Genetics has long been an important tool for the dissection of biologi-
cal mechanisms. Its use, however, has been limited by the investigator's
ability to find appropriate mutant phenotypes and then to identify the
gene and gene product responsible for producing the phenotype. Re-
cently, an array of genetic techniques has greatly expanded the power of
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EVERY CELL HAS A SEX
43
genetics. These techniques exploit the ability to clone specific genes and
modify their sequences to destroy or modify the gene product. This modi-
fied sequence is then inserted into the genome of an intact animal or
cultured cell to determine the effect on the phenotype. The details of
introducing transgenes vary with species, but in the most successful cases,
the transgene can exactly replace the resident gene. In other cases,
transgenes cannot be specifically targeted and the resident gene may still
be present; thus, the types of questions that can be asked are more limited.
Transgenic techniques have overcome several problems of conven-
tional genetics. They greatly speed study because there is a direct link
between the gene used in the experiment and the phenotype. The investi-
gator can precisely specify the gene modification rather than depend on
random mutagenesis, making it possible to focus not only on a specific
gene but also on a particular feature of that gene. For example, by remov-
ing the domain responsible for phosphorylation, one can study its role in
the parent protein.
Two classes of important genes are difficult to study by conventional
genetics. The first class is redundant genes, such as families of genes that
all fulfill the same function. It is unlikely that random mutagenesis will
knock out all members of even a very small family of genes, but targeted
transgenes can easily achieve this to allow study of the action of this gene
family. A second class of important genes includes those that produce
lethal phenotypes or that have effects in one developmental stage that
preclude study of their activity in a later stage. Techniques that allow the
transgene to be specifically inactivated in a tissue of choice offer ways to
bypass these problems because the gene can be expressed normally ex-
cept in the tissue where it is being studied. Similar techniques can be used
to drive inappropriate expression of a gene in specific tissues where the
inappropriate expression can be informative.
Transgenic techniques can also be used to construct model systems to
meet specific requirements. For example, mouse transgenic systems are
being used in many laboratories as models for human genetic diseases
and for cancer studies. The models need not be restricted to mouse genes
but can also contain transgenes of human origin to study specific interac-
tions. The increasing number of mice being bred to carry transgenes of
interest makes it possible to rapidly test gene interactions by breeding
different transgenic animals. Despite the power of these new techniques,
however, interpretation of experimental results is not always straightfor-
ward. For example, the manipulations involved with introducing the new
DNA sequence can sometimes introduce unexpected genetic changes, ei-
ther at the locus under study or at unrelated loci. In addition, identical
transgene or knockout models may have variable phenotypes, depending
on the strain's background (just as for "normal" mutant alleles).
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44
EXPLORING THE BIOLOGICAL CONTRIBUTIONS TO HUMAN HEALTH
FINDINGS AND RECOMMENDATIONS
Findings
Males and females have partially different genomes:
· The Y chromosome carries genes that are involved in basic cellular
functions and that are expressed in many different tissues.
· In females, the majority of genes on one of the two X chromosomes
are silenced in every cell. This inactivation makes each female a func-
tional mosaic because some cells express one X chromosome and others
cells express the other one. The advantages of heterozygosity can be am-
plified by selection against cells in which the active X chromosome carries
a detrimental allele.
· Some genes on the inactive X chromosome are not silenced, lead-
ing to higher levels of their products in female cells.
· Female cells must have cellular machinery to establish and main-
tain the inactivation of the X chromosome.
· Male and female germ cells differentially imprint the genetic infor-
mation to be transmitted to their progeny.
These findings argue that there are multiple, ubiquitous differences
in the basic cellular biochemistry of males and females that can affect an
individual's health. Many of these differences do not necessarily arise as a
result of differences in the hormonal environment of the male and female
but are a direct result of the genetic differences between the two sexes.
Recommendation
RECOMMENDATION 1: Promote research on sex at the cellular level.
The committee recommends that research be conducted to
· determine the functions and effects of X-chromosome- and Y-
chromosome-linked genes in somatic cells as well as germ-line cells,
· determine how genetic sex differences influence other levels of
biological organization (cell, organ, organ system, organism), including
susceptibility to disease, and
· develop systems that can identify and distinguish between the
effects of genes and the effects of hormones.
The phenotypic differences between males and females are deter-
mined, initially, by genes on the sex chromosomes. Sex chromosome-
linked genes can be expressed in both germ-line and somatic cells and
could influence an individual's phenotype, including disease susceptibil-
ity, at many levels.
Representative terms from entire chapter:
x chromosomes
