Download Animal models for Klinefelter`s syndrome and their relevance for the

Molecular Human Reproduction, Vol.16, No.6 pp. 375– 385, 2010
Advanced Access publication on March 21, 2010 doi:10.1093/molehr/gaq024
NEW RESEARCH HORIZON Review
Animal models for Klinefelter’s
syndrome and their relevance
for the clinic
Joachim Wistuba *
Institute of Reproductive and Regenerative Biology, Centre of Reproductive Medicine and Andrology, University Clinics, Domagkstrasse 11,
48149 Münster, Germany
*Correspondence address. Tel: +49-251-83-52043; Fax: +49-251-83-54800; E-mail: [email protected]
Submitted on December 15, 2009; resubmitted on March 4, 2010; accepted on March 17, 2010
abstract: In mammals, the contribution of the Y chromosome is paramount for male sexual determination; however, the presence of a
single functional X chromosome is also of importance. In contrast to females where X inactivation is seen; the X chromosome of the male
stays active. When, due to meiotic non-disjunction events, males are born with a supernumerary X chromosome, the resulting 47, XXY
karyotype is referred to as Klinefelter’s syndrome. This frequent genetic condition is most commonly associated with infertility, hypogonadism, gynecomastia and cognitive impairments. The condition has also been associated with a reduced life expectancy, insulin resistance, dyslipidemia, increased body fat mass and reduced bone mineral content. In a variety of species, male animals with karyotypes resembling
Klinefelter’s syndrome arise and develop a subset of features similar to those seen in humans. The availability of these animals is driving
efforts to experimentally address the pathophysiology of the condition. To date, two models, 41, XXY and 41, XXY* (mutated Y chromosome) male mice, have been established which resemble aspects of the pathophysiology of Klinefelter’s syndrome. Experiments performed in
these models confirm that the presence of a supernumerary X chromosome causes germ cell loss, cognitive deficits, Leydig cell hyperplasia,
and that their Sertoli cells are capable of supporting germ cells of normal karyotype. This review summarizes the generation and characterization of the animal models for Klinefelter’s syndrome and suggests experimental strategies to improve our understanding of the mechanisms underlying the pathophysiology of Klinefelter’s syndrome.
Key words: Klinefelter’s syndrome / 46, XX men / mouse models / X chromosome imbalance / infertility
Introduction
Klinefelter’s syndrome, the chromosomal aberration of 47, XXY,
results from a segregation failure of the sex chromosomes during
meiosis (Lanfranco et al., 2004). Meiotic non-disjunction of chromosomes can occur during the differentiation of gametes and results in
aberrant karyotypes leading to various diseases, as long as the extra
chromosomes are not causing lethal alterations. Among such disorders, Klinefelter’s syndrome, which severely affects the male phenotype, is the most frequent (Lanfranco et al., 2004; Aksglaede et al.,
2006; Wikström and Dunkel, 2008). It is often diagnosed late in life,
mostly after puberty, when the phenotypic features become
obvious. Although known for more than six decades and addressed
in numerous studies, our understanding of the genetic and molecular
mechanisms underlying the complex pathophysiology is still poor.
The elucidation of the detrimental mechanisms which are driven by
the supernumerary X chromosome requires experimental approaches
that are limited in humans because of ethical implications. Therefore,
there is a need for the generation and characterization of animal
models which closely resemble the manifestations of the syndrome
(Lue et al., 2001, 2005; Lewejohann et al., 2009). Although aberrant karyotypes which cause similar phenotypes have been reported in other
mammalian species, none of these animals can adequately serve as an
experimental model, primarily due to simple practical concerns.
Consequently, it was a breakthrough when a mouse line with a
mutated Y chromosome was discovered (Eicher et al., 1991) and
after the application of a staggered breeding scheme (Fig. 1) resulted
in the generation of male mice with a supernumerary X chromosome
which showed a phenotype comparable to Klinefelter’s syndrome. To
date, from the initial strain, two mouse models, the 41, XXY (Lue
et al., 2005, 2009) and the 41, XXY* mouse (Lewejohann et al.,
2009), have been established and characterized. All features thus far
examined in these animals show, as good as possible in an animal
model, a close resemblance to those of the human disorder. Although
further studies are still needed to determine whether these animals
have comparable metabolic changes to those seen in patients (e.g.
bone physiology), these mice are to date the most promising option
available to investigate Klinefelter’s syndrome. They also present the
opportunity to employ novel experimental approaches which have
thus far not been possible.
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376
Wistuba
Figure 1 Scheme of the staggered breeding protocol providing male mice with a supernumerary X chromosome serving as animal models
for Klinefelter’s syndrome (Eicher et al., 1991; Hunt and Eicher 1991).
(A) Animals were obtained from the B6Ei.Lt-Y* strain. The spontaneously mutated male mice carried a Y chromosome, designated Y*, which acquired a new centromere
distally and lost the normal centromere. (B) Breeding such phenotypically normal and fertile 40, XY* males to 40, XX females provoked sex chromosomal non-disjunction
events during meiosis and result in 25% of the male offspring exhibiting the 41, XXY* karyotype. These males have been demonstrated to resemble many features of
Klinefelter’s syndrome, although the Y chromosome is not completely separated but in close association with one of the X chromosomes (Lewejohann et al., 2009;
Wistuba et al., in press). Female XY*X littermates of those infertile males are fertile and can be mated to XY wild-type males giving raise to offspring of many different
karyotypes; of which, some are apparently lethal (e.g. Y0, YY*X). From these, breeding males of the XYY*X karyotype were bred further with female wild-type mice and
from these breedings finally 41, XXY males derive, the second mouse model for Klinefelter’s syndrome (Lue et al., 2005, 2009).
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Animal models for Klinefelter’s syndrome
Heterosomes and the male
phenotype
In mammals, the presence of a Y chromosome is the main signal for male
sexual determination. The fetal activation of the gene sry (sex-determining
region on the Y chromosome) is responsible for the further sexual differentiation of the undifferentiated gonad into a testis (for review, see McElreavey and Fellous, 1999; Wistuba et al., 2007). In addition, the male
phenotype depends on the presence of a single X chromosome which
needs to be always active. The situation in females is different; two X
chromosomes are present and their expression activity has to be
balanced. In mammals, X chromosome inactivation (XCI) controls the
genetic imbalance arising from the X: autosome ratio, i.e. the different
number of X chromosomes between the sexes. In female cells, dosage
compensation is achieved by transcriptional silencing of one X chromosome (Lyon, 1961); the single X chromosome of the male cells undergoes
no such inactivation. Two different ways of XCI are possible: by imprinting and by random XCI. Imprinted XCI leads to paternal X chromosome
silencing in the extraembryonic tissue, whereas random XCI takes place
in the epiblast where either the maternally or paternally inherited X
chromosome is inactivated (Ng et al., 2007). In random XCI, inactivation
of the X chromosome (Xi) is initiated in somatic cells at a X inactivation
center (Xic) which contains regulatory elements responsible for detection of the number of X chromosomes, and the selection of one X
chromosome to remain active (Xa) and the other to be inactivated
(Plath et al., 2002). The Xi-specific gene transcript (Xist) is located in
the Xic, (Chow et al., 2005) and encodes an untranslated RNA that
initiates silencing in the X chromosome. Xist repression on the activated
X chromosome is achieved by methylation of CpG-sites in the Xist promoter region. Conversely, when the Xist promoter region is not methylated, it is transcriptionally active and as a consequence, the X
chromosome is silenced. The possession of a single, active X chromosome can therefore be seen as a prerequisite for the correct formation
of a male phenotype and is as important as the presence of a Y
chromosome.
The supernumerary X
chromosome in male mammals
Male mammals normally exhibit a karyotype containing one active Y
chromosome and one active X chromosome. However, during
meiotic division, the separation of chromosomes can fail, leading to
non-disjunction events and resulting in males with a supernumerary
X chromosome. The ensuing karyotype is neither lethal nor exclusively human, as it occurs in many mammalian species. In men, two
possible phenotypes containing a single supernumerary X exist:
46, XX men and 47, XXY Klinefelter patients (de la Chapelle,
1972; Lanfranco et al., 2004; Yencilek and Baykal, 2005; Vorona
et al., 2007). Karyotypic aberrations resembling the later condition
have been reported in a variety of mammals such as the shrew
(Searle and Jones, 2002), cat (Centerwall and Benirschke, 1975),
boar (Hancock and Daker, 1981; Mäkinen et al., 1998), bull (Dunn
et al., 1980; Molteni et al., 1999; Joerg et al., 2003; Słota et al.,
2003), horse (Kubień et al., 1993; Mäkinen et al., 2000) and dog
(Reimann-Berg et al., 2008). Even a male Siberian tiger exhibiting a karyotype of 39, XXY has been described (Suedmeyer et al., 2003).
Furthermore, the Klinefelter karyotype is not homogeneous and can
be seen in the form of a XY/XXY mosaic (Lanfranco et al., 2004).
Not surprisingly karyotypically mosaic males (XX/XY or XXY/XY,
respectively) have also been found in rams (Bunch et al., 1991) and
goats (Bongso et al., 1982), where the mosaicism can result in intersexuality or true hermaphroditism. A case of sex chromosomal mosaicism very similar to that of mosaic Klinefelter patients has also been
reported in the baboon (Papio hamadryas, Dudley et al., 2006).
However, in contrast to the human, in the mosaic animal males, the
phenotype appears not to be milder, but rather more pronounced
in the ‘female’ direction.
When summarized, all studies on supernumerary X male mammals
describe symptomatic features that, to a certain extent, resemble the
aspects of human Klinefelter’s syndrome, namely hampered androgenization, loss of germ cells, Leydig cell hyperplasia and infertility. In the
dog, a correlation between the disturbed karyotype and the early
onset of testicular cancer has also been suggested (Reimann-Berg
et al., 2008).
Therefore, it could be surmised that the principles and mechanisms
by which the supernumerary X chromosome influences the male phenotype are in general terms similar, and likely to be related to an
inability to handle the X-linked gene expression in a male environment. Evidence that similar principles do in fact underlie the disorder
opened the route for the generation and utilization of animal models
to address the pathophysiology of Klinefelter’s syndrome.
Klinefelter’s syndrome: a
rationale for the generation of
animal models
The 47, XXY karyotype which defines Klinefelter’s syndrome is not a
rare event. With a prevalence of 0.2% in the male population, it is
the most common chromosomal disorder causing male infertility
(Lanfranco et al., 2004). As roughly 10% of X chromosome-encoded
genes are specifically expressed in the testis (Ross et al., 2006) with
some of these expressed in spermatogonia only (Wang et al., 2001),
it is not surprising that the presence of an aberrant X would detrimentally affect fertility status. However, the infertility caused by loss of
germ cells during childhood, the subsequent germ cell-depleted
testis and the degenerated seminiferous tubules seen at the onset of
puberty (Aksglaede et al., 2006), is only one aspect of the condition.
The imbalance of the sex chromosomes also disturbs the male endocrine milieu causing hypergonadotropic hypogonadism (De Braekeleer
and Dao, 1991; Nielsen and Wohlert, 1991; Yoshida et al., 1997;
Christiansen et al., 2003; Kamischke et al., 2003; Simpson et al.,
2003; Lanfranco et al., 2004) and is associated with decreased life
expectancy (Bojesen et al., 2004, 2006; Swerdlow et al., 2005) due
to alterations in metabolic functions (e.g. insulin resistance, gynecomastia and dyslipidemia). Increased body fat mass is often found in
prepubertal Klinefelter’s patients which in turn may cause an unfavorable ratio of fat to muscle mass provoking the impaired metabolic
profile observed in adult patients and changed body proportions
such as tall stature (Lanfranco et al., 2004; Kamischke et al., 2003;
Aksglaede et al., 2006). Metabolic perturbations may also explain
the lower bone mineral density of Klinefelter’s patients, and their
increased risk to develop osteoporosis (Aksglaede et al., 2008).
378
There is evidence that Klinefelter’s patients also have elevated risk for
breast cancer, non-Hodgkin lymphoma (Swerdlow et al., 2005) and
some extragonadal germ cell tumors (Aizenstein et al., 1997;
Aguirre et al., 2006).
Mild cognitive and behavioral defects, which deteriorate when more
than one extra X are present, are also associated with the condition and
indicate that the unbalanced X chromosome-linked gene expression
also interferes with brain function (Temple and Sanfilippo, 2003; van
Rijn et al., 2006; Itti et al., 2006). It has been shown that adults score
significantly lower in cognitive exercises (DeLisi et al., 2005) while
young boys have impaired executive skills of social processing supposed
to be linked to X chromosome-dependent effects in areas of cognitive
strength (Temple and Sanfilippo, 2003; van Rijn et al., 2006). To date, it
is not clear whether the hypogonadal endocrine milieu resulting from
Klinefelter’s may also induce changes in the subcortical pathways
which contribute to these cognitive deficits (Itti et al., 2006).
It is typical of Klinefelter’s syndrome that there are only minor physical anomalies prior to puberty, leading very often to a late diagnosis.
As the onset of puberty is also accompanied by accelerated germ cell
depletion which often results in the complete loss of germ cells
(Wikström et al., 2006a), delayed detection of the condition is particularly harmful to the future fertility potential of these patients as it limits
the therapeutic options: namely, the preservation of the few differentiated germ cells that may remain in these patients.
Despite the insights provided by the numerous investigations on the
clinical consequences of Klinefelter’s syndrome performed in the past,
our understanding of the syndrome on the molecular and cellular level
remained limited due to the lack of in-depth mechanistic studies, only
possible with the use of experimental models.
Mouse models for
Klinefelter’s syndrome
The observation that the sex chromosome aberration resulting in a
supernumerary X chromosome in male mammals exists in many
species and always results in a similar disturbance of the phenotype
leads to the idea that the generation of animal models for Klinefelter’s
syndrome could offer a route to investigate the pathophysiology of the
syndrome experimentally. However, as this aberrant karyotype is rare
and occurs randomly, few such animals are available for study. Furthermore, the condition is almost always accompanied by infertility,
meaning that generation of similar animals is extremely difficult, if
not entirely unlikely. As a consequence, large systematic investigations
of the general mechanisms underlying the syndrome cannot be conducted using these animals, and their usefulness has been limited to
small, mainly descriptive studies of the condition.
Over the last decades, attempts have been made to generate chimeric mouse models for Klinefelter’s syndrome by injecting embryonic
stem cells with an extra X chromosome into mouse blastocysts. Offspring with XXY karyotype from these chimeric mice have been
obtained (Lue et al., 2001) and showed alterations of the male phenotype comparable to those of Klinefelter’s syndrome. However, maintaining this mouse line has proven to be quite difficult as the XXY mice
could not be established as a permanent line and therefore required
the repeated use of chimeric females; thus, becoming a sort of ‘transient’ model which is difficult to handle in experimental settings.
Wistuba
Fortunately, male mice with mutated Y chromosomes were discovered some years ago, allowing several groups to establish males with
aberrant karyotypes using a staggered four-generation breeding
scheme from the B6Ei.Lt-Y* strain (Fig. 1; Eicher et al., 1991; Hunt
and Eicher, 1991; Lue et al., 2005).
The spontaneously mutated male mice carry a Y chromosome,
designated Y*, which acquired a new centromere distally and lost
the normal centromere. Breeding such phenotypically normal and
fertile 40, XY* males to 40, XX females provoked sex chromosomal
non-disjunction events during meiosis, resulting from a changed structure of the Y* chromosome and produced animals with various chromosomal aberrations such as XXY, XXY*, XXY*Y, XY*Y, XYY*X,
XYY*, XY*X (Hunt and Eicher, 1991). Two of these models (male
41, XXY and the 41, XXY*) have been analyzed in depth and reveal
a phenotype that closely resembles the features of Klinefelter’s
patients (Lue et al., 2005, 2009; Lewejohann et al., 2009).
Initially, these mice were assessed solely for their suitability as a
model organism for Klinefelter’s syndrome. However, their major disadvantage was that a definitive identification of the karyotype was only
possible post-mortem, thus severely impairing experimental work and
efficient breeding of those animals. As a consequence, beyond the
descriptive analysis of certain physiological, histological and morphological features, no further experiments were possible. Although
experimental settings for further studies have been suggested, such
as transplantation experiments analyzing the interaction between
germ line and somatic testicular environment (Mroz et al., 1999),
these were never performed due to the lack of appropriate tools
and techniques.
Early studies on juvenile XXY mice showed that spermatogonia
were present and morphologically normal, but that a progressive
loss of germ cells began within the first 10 days of life (Hunt et al.,
1998), indicating that the early proliferation and migration of primordial germ cells was normal in XXY mice but that mitotic proliferation
declined from post-coital day 12.5 leading to germ cells being progressively lost in early post-natal life (Hunt et al., 1998; Lue et al.,
2001).
However, when testicular cells were propagated in vitro, the testicular differentiation and morphology of both germ cells and Sertoli cells
were normal, suggesting that an abnormality in the interaction
between somatic and the germ line components could be responsible
for the germ cell loss (Hunt et al., 1998; Wang et al., 2001). Furthermore, Mroz et al. (1998) observed that in XXY mice, spermatogonial
stem cells with a second X chromosome disappeared via apoptotic
pathways while mosaic stem cells showing a XY karyotype though
able to survive gave rise to a higher number of aneuploid daughter
cells. This observation indicates that while only XY spermatogonia
survive in the XXY testis, when they undergo meiosis, they are
more prone to meiotic abnormalities. This raises the possibility that
increased meiotic errors are not due to the XXY testis per se, but
rather a more generalized event in the severely oligozoospermic
testis (Mroz et al., 1998) which in turn brings into question the influence and regulatory control of the Sertoli cells that support ‘normal’
germ cells.
Over the last decade, new techniques have become available which
enable the karyotyping of living animals. Fluorescence in situ hybridization (FISH) allows the detection of sex chromosomes in metaphase
fibroblast cultures obtained from tail tip biopsies (Lue et al., 2005,
379
Animal models for Klinefelter’s syndrome
2009) or from interphase chromosomes of leukocytes from peripheral
blood samples (Lewejohann et al., 2009). This methodological
advancement has allowed for improvements in breeding resulting in
novel experimental approaches and a ‘re-launch’ of the mouse
models for the exploration of mechanisms underlying the pathophysiology of Klinefelter’s syndrome.
Confirmation of the models:
male mice resembling
Klinefelter’s syndrome
Lue et al. (2005, 2009) provided the most convincing evidence that
their 41, XXY mouse males were a suitable model for the characteristic disturbances seen in the karyotypically aberrant male phenotype.
They showed that adult XXY mice had small testes with seminiferous
tubules of decreased diameter containing Sertoli cells only and an
interstitium with a higher number of Leydig cells, a testicular histology
which reflected the situation in adult Klinefelter’s patients. Furthermore, these Sertoli cells showed reduced immunolocalization for
the androgen receptor. The endocrine profile of the animals was
also similar to that of patients, namely hypergonadotropic hypogonadism with decreased plasma testosterone and increased FSH levels. The
loss of germ cell in this mouse model also became significant 7 days
after birth (Table I).
Lue et al. (2005, 2009) succeeded in obtaining substantial numbers
of 41, XXY male mice. However, keeping in mind the complex fourgeneration breeding scheme necessary to achieve XXY males and
the impaired fertility of mice with aberrant karyotypes, the model
proved to be extremely time-consuming and difficult, especially the
ability to obtain sufficient animal numbers needed for complex
experimental designs. Using this breeding scheme, we could not
obtain 41, XXY males exhibiting a complete Y chromosome from
our colony. Although we obtained B6Ei.Lt-Y* females of the karyotype XY*X and by further breeding some male offspring with a 42,
XYY*X karyotype, these animals did not give rise to 41, XXY
male offspring, as they either died before puberty or were infertile
never achieving pregnancies when mated with females. An explanation may be that in contrast to the other model (Lue et al.,
2005), the sex chromosomal aberrations in the female XY*X and
in XYY*X males in our colony severely influence meiotic progression
and therefore result in infertile animals (Lewejohann et al., 2009;
Wistuba et al., in press).
However, employing the B6Ei.Lt-Y* strain, an alternative male
mouse model carrying a supernumerary X chromosome has been
generated which had not been characterized in detail thus far
(Lewejohann et al., 2009). The mice, derived from breeding XY*
males with XX females, display a 41, XXY* karyotype and constitute
25% of the total male offspring. When analyzed with FISH probes
which identify the sex chromosomes, these animals were found to
have a Y-chromosomal signal in close proximity with one of the X
chromosomes while their XY* littermates and the male C57/Bl 6
wild-type animals show two distinct and separated signals.
The investigation of these 41, XXY* mice first aimed at answering
the question of whether these mice could best serve as a model of
Klinefelter’s syndrome or a model for 46, XX males for which no
animal model is available so far (Vorona et al., 2007).
Characterization of the male 41, XXY* mice revealed that reduction
of the Y chromosome and its junction to a X chromosome presented
a pathophysiology (Lewejohann et al., 2009; Wistuba et al., in press;
Table I) comparable to the 41, XXY mice which closely resembled
all the features of Klinefelter’s syndrome. Namely, small testis due
to the complete loss of germ cells (Fig. 2) that occurred between
the second week of life and the onset of puberty (slightly delayed
Table I Comparison of patients with a supernumerary X chromosome and the mouse models available.
Feature
Mouse models
..............................................................
41, XXY
Y
41, XX *
Patients
...............................................................
Klinefelter’s syndrome
47, XXY
References
Men 46, XX
.............................................................................................................................................................................................
Body proportions
Not addressed
Heavier, bigger
Bigger, changed proportions
Smaller
[1– 4]
Androgenization
Impaired
Impaired
Impaired
Impaired
[1, 2, 4– 6]
Testosterone
Reduced (not significant)
Reduced (not significant)
Reduced (lower limit)
Reduced (lower limit)
[1, 2, 4– 6]
Serum LH
Elevated
Elevated
Elevated
Elevated
[1, 2, 4, 6]
Serum FSH
Elevated
Elevated
Elevated
Elevated
[1, 2, 4, 5]
Cognition
Impaired
Impaired
Impaired
Not known
[1, 5, 7, 8, 9, 10]
Germ cell loss
Significant from d7 pp
Between d 14 and 21 pp
Peripubertal
Prepubertal
[1, 2, 4– 6]
Leydig cell number
Hyperplasia
Hyperplasia
Hyperplasia
Hyperplasia
[4– 6, 11]
Leydig cell steroidogenesis
in vitro
Not addressed
Elevated
Not addressed
Not addressed
[6]
Leydig cell marker gene
expression
Not addressed
Elevated
Not addressed
Not addressed
[6]
Germ cell maturation?
Not found
Not found
Focally in some patients
Not found
[2, 12]
X inactivation
Not addressed
50%
50%
20%
[6, 13]
References: 1, Lewejohann et al. (2009); 2, Lanfranco et al. (2004); 3, Kamischke et al. (2003); 4, Vorona et al. (2007); 5, Lue et al. (2005); 6, Wistuba et al. (in press), 7, van Rijn et al.
(2006); 8, Temple and Sanfilippo (2003), 9, Itti et al. (2006); 10, DeLisi et al. (2005); 11, Aksglaede et al. (2006); 12, Sciurano et al. (2009); 13, Poplinski et al. (2010).
380
Wistuba
Figure 2 Characterization of 41, XXY* male mice. (A) Metaphase FISH identification of heterosomes in a 41, XXY* male nucleus counterstained by DAPI.
(A) The green probe detects the X chromosomes in the cell arrested in metaphase spread. (B) The red probe detects the Y chromosome. (C) The overlay shows the Y
chromosome signals in close association with one X chromosome. Testicular histology of adult 41, XXY* versus 40, XY* littermate, hematoxylin staining. (D) All seminiferous
tubules of the XY* controls exhibited complete spermatogenesis and apparently normal distribution and number of Leydig cells (LC). (E) In contrast, 41, XXY* males presented SCO tubules of decreased diameter and LC hyperplasia. The bar represents 10 mm. (F) The loss of germ cells in adult 41, XXY* males is reflected in significantly
reduced testis weight compared with both control groups (P , 0.05, SIGMASTAT 2.03 (SPSS Inc., Chicago, IL, USA; ANOVA; XXY*: n ¼ 72, XY*: n ¼ 69, C57Bl/6:
n ¼ 36). These data summarize results from previous publications in which also the methodology was described in detail (Lewejohann et al., 2009; Wistuba et al., in
press). Hypergonadotropic hypogonadism results in (G) reduced testosterone (XY* littermate controls versus XXY*: not significantly different: P ¼ 0.621, C57Bl/6 XY
versus XXY*: significantly different: P ¼ 0.040; XXY*: n ¼ 45, XY*: n ¼ 45, C57Bl/6: n ¼ 33) but (H) significantly elevated FSH and (I) LH serum levels in XXY* mice compared with both control groups (P , 0.05; ANOVA with the Holm– Sidak method; SIGMASTAT 2.03, XXY*: n ¼ 39, XY*: n ¼ 38, C57Bl/6: n ¼ 29). None of the
parameters measured significantly differed between the both control groups. (F –I) All measurements were performed as previously described in Lewejohann et al. (2009).
Animal models for Klinefelter’s syndrome
381
Figure 3 X inactivation in mouse models, Klinefelter’s patients and 46, XX men: comparison of the percentages of the Xist promoter region methylation degree for the mouse 41, XXY* model (left side) and men with a supernumerary X chromosome, i.e. 47, XXY Klinefelter’s patients, 46, XX men
(right side).
Normal men and women as well as female and male littermates and C57Bl/6 male mice served as controls (modified figure from data reported in Wistuba et al., in press
and Poplinski et al., 2010).
when compared with XXY mice but in the same developmental
period as it occurs in patients), hyperplasia of the Leydig cells
and hypergonadotropic hypogonadism (Fig. 2), the hallmarks of
Klinefelter’s patients.
The final validation of the male 41, XXY* mice as a suitable model was
obtained when X inactivation was studied in these animals and compared with Klinefelter’s patients and 46, XX men (Poplinski et al.,
2010; Wistuba et al., in press; Fig. 3). Recently, it has been shown
that the epigenetic pattern of X inactivation in Klinefelter’s patients is
similar to that of healthy women and remarkably different from
healthy men and 46, XX males, in that it shows distinct hypomethylation
of the XIST promoter region, findings that are in agreement with the
XIST mRNA expression levels seen in Klinefelter’s patients (Stabile
et al., 2008). Therefore, the finding that male 41, XXY* mice exhibited
a Xist methylation pattern (50% methylated) very similar to that of
female littermates and distinctly different from that seen in XY* and
XY male mice (85% methylated) indicated a normal inactivation of
the second X chromosome and supported the conclusion that these
XXY* males truly mimicked Klinefelter’s syndrome (Fig. 3).
Mouse models resembling
Klinefelter’s syndrome: what are
they good for?
In summary, to date, two mouse models, the 41, XXY (Lue et al.,
2005, 2009) and 41, XXY* (Lewejohann et al., 2009; Wistuba et al.,
in press), are available and have been demonstrated to resemble
the pathophysiology related to the supernumerary X chromosome
of Klinefelter’s syndrome.
How important the availability of such models is can be derived from
the various ways in which the mouse models confirmed the relation
between the presence of a supernumerary X chromosome and
certain features seen in patients. For example, it has long been
debated whether the mild cognitive defects observed in Klinefelter’s
men were due to chromosomal imbalance and therefore directly
caused by disturbances of brain function or whether cognition and
behavior in these patients was a secondary effect resulting from sociopsychological problems experienced because of the changed male phenotype. Both mouse models were tested experimentally and confirmed
delayed conditional learning in a Pavlovian association (Lue et al., 2005),
in which the 41, XXY mice achieved the task but over a significant longer
period, and 41, XXY* mice exposed to a non-conditional Novel Object
Task testing for memory recognition were not able to solve the task at
all (Lewejohann et al., 2009). As Klinefelter’s patients develop mild cognitive and behavioral defects, perform cognitive exercises and executive
skills poorly (Temple and Sanfilippo, 2003; DeLisi et al., 2005; Itti et al.,
2006; van Rijn et al., 2006), the results of the animal experimental work
suggest that future analysis of the cognitive features of the condition
should address the physiological and metabolic changes of the brain
dependent on X-linked gene expression and/or metabolic and endocrine changes. Such analyses require the direct correlation of the dysfunction with changes of structures and organ-specific gene
expression, an avenue only possible when animal models are employed,
382
and which will provide an understanding of what the supernumerary X
chromosome does in the brain and how this can be addressed therapeutically, e.g. by the administration of testosterone (i.e. as a link
between testosterone and spatial cognition, activation of the cortical
network, and the dendritic spine formation has been shown in patients
and rodent models; Zitzmann et al., 2001; Lue et al., 2005; Zitzmann,
2006; Leonard et al., 2007; Leranth et al., 2007).
Male mice with an extra X chromosome have also played a key role
in the analysis of the main symptoms of Klinefelter’s syndrome: hypergonadotropic hypogonadism and testicular failure. So far, unbiased
microscopical quantification of testicular cell types was impossible
due to the small size of biopsies obtained from patients. However,
stereological techniques analyzing the entire testis of the experimental
animals confirmed, what was only an assumption thus far, that in the
testis a massive Leydig cell hyperplasia was present (41, XXY; Lue
et al., 2005, 41, XXY*; Wistuba et al., in press). Furthermore, although
the Leydig cells were mature and had a functional LH receptor, they
were hyperactive as shown by their steroidogenic response in vitro
with elevated transcriptional activity, despite intratesticular testosterone levels being similar to controls (Wistuba et al., in press). Interestingly, while decreasing levels of serum INSL3 are found in 47, XXY
boys from midpuberty onwards (Wikström et al., 2006b) which culminate in significantly low levels in adult Klinefelter’s syndrome patients
(Bay et al., 2005), the Leydig cells of 41, XXY* mice revealed a significantly higher expression of the homologous gene Rlf (and of all other
Leydig cell marker genes analyzed). Despite this seeming discrepancy,
as Rlf levels in serum have not been measured in the mouse nor INSL3
transcription in the Leydig cells of patients, it is possible that the findings are not irreconcilable and that the mouse also exhibits decreased
Rlf serum protein levels and INSL3 transcription is increased in
patients’ Leydig cells, but that this increase is not reflected in serum
values because transportation of Rlf is hampered in a similar way to
that we suggested for testosterone (Wistuba et al., in press). Hypergonadotropic hypogonadism in these animals cannot be explained
by dysfunctional Leydig cells but may be due to interactions within
the changed testicular environment that influence the endocrine function of the cells (Wistuba et al., in press). This observation may have
important implications for the re-assessment of the androgenizationrelated pathophysiological features of Klinefelter’s syndrome and
might result in new strategies of treatment (Wistuba et al., in press).
Previous studies (Guttenbach et al., 1997) found a low proportion
(3%) of ‘hyperhaploid’ sperm from a small number of Klinefelter’s
patients with focal spermatogenesis. On the basis of this indirect evidence, the authors suggest that the few sperm obtained from Klinefelter’s patients must be derived from 47, XXY spermatogonia. In
contrast, a recent elegant study which analyzed testicular biopsies
from six Klinefelter’s patients with foci of ongoing spermatogenesis
employed FISH to determine the chromosomal content of differentiating germ cells undergoing meiosis (Sciurano et al., 2009) and found
that all progressing germ cells were euploid but embedded in 47,
XXY Sertoli cells. The authors concluded that the focal spermatogenesis observed in some patients is driven by such 46, XY germ cells
which have undergone random expulsion of the supernumerary X
chromosome during mitotic propagation. A finding which is consistent
with and supported by Lue et al.(2009) who demonstrated the ability
of XXY mouse testes depleted of germ cells to support complete
spermatogenic differentiation of transplanted 46, XY spermatogonia.
Wistuba
The confirmation of several previously held assumptions and the
highlighting of new aspects of the syndrome that probably would be
still debated without experimental data have shown the importance
and utility of the animal models for Klinefelter’s syndrome.
Perspectives offered by the
Klinefelter mouse models
After the successful establishment and characterization of the animal
models, numerous applications utilizing these male mice for the
more in-depth exploration of sex chromosomal imbalance in the
male have now become possible.
The demonstration of normal inactivation of the supernumerary X
chromosome leads to the suggestion that the aberrant phenotype
observed in the 41, XXY* males might be due to X-linked genes
that ‘escape’ silencing (Wistuba et al., in press). In contrast to
female cells, male cell metabolism is not adapted to the expression
of X-linked genes from two alleles. These ‘escapees’ could act
either directly or influence autosomal gene over expression via regulatory elements. Thus, future experiments should identify candidate
genes that are functionally involved in features related to Klinefelter’s
syndrome such as impaired recognition, hypergonadotropic hypogonadism, Leydig cell function and germ cell differentiation. To establish
a proof of principle, this hypothesis needs to be tested on both the
transcriptional and translational levels. If the hypothesis holds,
human homologues could be identified that would act as targets for
the development of novel therapies.
We have found that hypergonadotropic hypogonadism is evident in
41, XXY* male mice, but Leydig cell function is not hampered
(Wistuba et al., in press), neither on the mRNA expression level
nor in regard to steroidogenic activity. The question thereby arises
why serum testosterone levels are not at normal values in males
with a supernumerary X chromosome? Although intratesticular testosterone levels appear not different from those of littermate controls,
serum androgen values are lower. Various mechanisms could be
responsible for this seeming inconsistency: first, although Sertoli cells
of XXY mice are capable of supporting germ cell maturation (Lue
et al., 2009), they could have disturbed endocrine function and overproduce androgen-binding protein (ABP). Although sex-hormone
binding globulin (SHBG) and ABP are products of homologous
genes in mouse and humans, unlike SHBG, ABP secretion into
serum is minimal and has almost exclusively tissue-specific function;
namely, regulating the androgen availability in the extracellular
spaces of the testis (Reventos et al., 1993; Larriba et al., 1995). An
overexpression of ABP, therefore, would bind the testosterone produced by the Leydig cells in the seminiferous epithelium and thereby
reduce its availability in the circulation, thus explaining the situation
seen in 41, XXY* mice (Wistuba et al., in press). This may be
caused by an increased number of Sertoli cells; however, as yet no
such increase has been described. Secondly, the blood flow in the
testis might be disturbed, possibly by fewer or smaller blood
vessels. Alternatively, a failure of the regulation of the conversion of
testosterone into estrogen might contribute to lowered serum testosterone values. Presently, this remains in the area of speculation, as no
information exists on estrogen levels in these mice so far. The mouse
models offer the possibility to address these questions making use of a
383
Animal models for Klinefelter’s syndrome
comprehensive analysis combining morphometry and stereology to
assess blood vessel proportion and absolute Sertoli cell number in
XXY* testes. To date, both questions have not been examined in Klinefelter’s patients due to the quantity of testicular tissues necessary.
In adult Klinefelter’s patients, while the disturbed hypogonadal
endocrine milieu can be treated by the administration of exogenous
testosterone, for the two other problematic features of the syndrome
(infertility due to germ cell loss and cognitive deficits), no therapies are
available. The possible rescue of cognitive deficits by hormonal treatment has not been systematically examined so far, but initial studies do
provide encouragement. Assessment of the models’ responses to the
non-conditional Novel Object task showed a clear positive correlation
between serum testosterone levels and learning performance in
normal males but not in the 41, XXY* littermates (Lewejohann
et al., 2009), suggesting that the learning failure might be associated
with a disturbance of androgenization. There are numerous possible
reasons that could explain this observation. For example, testosterone
levels did not pass a certain threshold; hormone reception via the
androgen receptor (AR) of the brain is disturbed; conversion of androgens into estrogens in the brain is altered or X-linked genes are not
normally expressed in the brain. To elucidate which mechanism(s) is
responsible for the cognitive defects, studies need to be undertaken
into the phenotypical changes after early (pre-/peripubertal) testosterone administration, expression patterns of the AR and of the estrogen receptor system (ERb/Era) in the brain, the conversion of
androgens to estrogens in the brain by analysis of enzymatic activity
and chip experiments on up or down-regulation of X-linked candidate
genes. All these experiments are only possible using the animal
models.
As mentioned previously, in some Klinefelter’s patients, foci of spermatogenesis persist in the testis after puberty and during adulthood,
possibly due to random propulsion of the extra X chromosome
during mitotic progression of spermatogonia (Sciurano et al., 2009).
The majority of patients unfortunately have complete loss of their
spermatogenic germ cells leaving the testis with the characteristic features of Sertoli cell-only syndrome. To address this issue, innovative
approaches are needed to the preservation of fertility and the germ
line. One such approach could be correction of the cells’ karyotype
which would allow them to differentiate with the support of the
Sertoli cells. For the technical and procedural details of such an undertaking to be developed, mouse models will be crucial. Recently, a
method for in vitro differentiation of male mouse germ cells was
reported making use of novel three-dimensional matrices in cell cultures (Stukenborg et al., 2008, 2009). By differentiating immature spermatogonia from juvenile mouse testes in vitro into morphologically
mature spermatozoa, this approach showed that, at the experimental
level at least, in vitro differentiation is possible. In the future, a similar
approach could be used clinically to preserve fertility and generate
gametes from patients. A possible strategy could be the isolation of
spermatogonia from Klinefelter’s patients before puberty and culturing
these early germ cells under conditions that induce high mitotic
activity. If the hypothesis that due to a higher mitotic activity, the
number of germ cells losing the supernumerary X chromosome
should increase, then germ cell culture might result in sufficient
numbers of 46, XY spermatogonia being produced (Sciurano et al.,
2009). Subsequent culture in three-dimensional matrices or
re-transplantation into a hosts’ testis (Lue et al., 2009) would allow
the germ cells with a normal karyotype to differentiate. Once established and proven in the mouse models, the approach could be
tested on human germ cells and if successful would offer a therapeutic
option to patients who currently have no alternatives other than
donation or adoption.
Conclusion
Studies performed on the two mouse models have demonstrated that
they possess many of the characteristic features of Klinefelter’s syndrome. Similarities have been found in perturbations of the endocrine
and testicular environments as well as the cognitive deficits associated
with the condition. However, certain questions remain and further
investigations are necessary to fully characterize all the features of
the mice and thus their validity as true models of Klinefelter’s syndrome. In particular, the evaluation of their metabolic profile and its
similarity to that of the human would be of considerable interest, in
view of the altered bone physiology found in patients,
One of the most important questions regarding Klinefelter’s syndrome is whether the phenotype is due to chromosomal imbalance,
endocrine changes, altered gene expression or a mixture of all. To
date, neither human nor animal studies have been able to provide adequate answers. However, the availability of these new models provides the opportunity to conduct previously impossible investigations
which will contribute to our understanding of the molecular and cellular mechanisms of sex chromosome imbalance in the male and, may in
time, provide the basis for the development of novel therapeutic
strategies which are needed to meet the challenges presented by
Klinefelter’s syndrome.
Acknowledgements
The author would like to thank Martin Heuermann, Günter Stelke,
Reinhild Sandhowe-Klaverkamp, Petra Köckemann and Jutta Salzig
for excellent technical assistance and to Con Mallidis for language
editing. Oliver S. Damm, Andreas Poplinski, Tuula Hämäläinen, Steffi
Werler, Matthias Dittmann, Lars Lewejohann, Eberhard Nieschlag,
C. Marc Luetjens, Jörg Gromoll and Manuela Simoni are gratefully
acknowledged for the support of the work reviewed and fruitful
discussions.
Funding
Studies from J.W.’s lab at the Centre of Reproductive Medicine and
Andrology discussed herein and in the preparation of this article
were supported by the Deutsche Forschungs Gemeinschaft (DFG
No. WI 27-23/1-1).
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