doi:10.1093/humrep/den112
Human Reproduction Vol.23, No.6 pp. 1263–1270, 2008
Advance Access publication on April 16, 2008
Nuclear organization in human sperm: preliminary evidence
for altered sex chromosome centromere position
in infertile males
K.A. Finch1,2, K.G.L. Fonseka1, A. Abogrein1, D. Ioannou1, A.H. Handyside2, A.R. Thornhill2,
N. Hickson1 and D.K. Griffin1,3
1
Department of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, UK; 2The London Bridge Fertility, Gynaecology
and Genetics Centre, One St Thomas Street, London SE1 9RY, UK
3
Correspondence address. Tel: þ44-1227-823022; Fax: þ44-1227-763912; E-mail: [email protected]
BACKGROUND: Many genetic defects with a chromosomal basis affect male reproduction via a range of different
mechanisms. Chromosome position is a well-known marker of nuclear organization, and alterations in standard patterns can lead to disease phenotypes such as cancer, laminopathies and epilepsy. It has been demonstrated that normal
mammalian sperm adopt a pattern with the centromeres aligning towards the nuclear centre. The purpose of this
study was to test the hypothesis that altered chromosome position in the sperm head is associated with male infertility.
METHODS: The average nuclear positions of fluorescence in-situ hybridization signals for three centromeric probes
(for chromosomes X, Y and 18) were compared in normoozoospermic men and in men with compromised semen parameters. RESULTS: In controls, the centromeres of chromosomes X, Y and 18 all occupied a central nuclear location.
In infertile men the sex chromosomes appeared more likely to be distributed in a pattern not distinguishable from a
random model. CONCLUSIONS: Our findings cast doubt on the reliability of centromeric probes for aneuploidy
screening. The analysis of chromosome position in sperm heads should be further investigated for the screening of
infertile men.
Keywords: male infertility; sperm; centromere; FISH
Introduction
A range of chromosomally related genetic defects has been
associated with male infertility phenotypes (Chandley et al.,
1979; de Braekeleer and Dao, 1991; Shah et al., 2003;
Griffin and Finch, 2005). Constitutional structural abnormalities, e.g. inversions and balanced translocations (Chandley
et al., 1979), Y chromosome deletions (Affara and Mitchell,
2000) and constitutional trisomy (mosaic or full blown) (e.g.
Skakkebaek et al., 1973; Sheridan et al., 1989) are the most
cited. While these different types of chromosome abnormalities lead to infertility via various different etiologies
(e.g. some involve asynapsis and altered recombination
patterns while others do not) collectively they highlight the
continuing importance of cytogenetically based genotype–
phenotype correlations in studies of male reproduction. More
recently, the association between compromised semen
parameters and increased sperm disomy (presumably as a
result of aberrant meiotic behaviour) has become well documented with many authors reporting a 10– 30-fold increase in
the most extreme cases (Tempest and Griffin, 2004).
However, to date a correlation between altered chromosome
position in sperm head nuclei and male infertility has not yet
been established.
Chromosome territory position in the interphase nucleus is
commonly regarded as an indicator of nuclear organization in
a range of cell types and developmental stages (Foster and
Bridger, 2005). Spermatogenesis is a developmental process
in which nuclear organization and architecture have been
studied extensively (Zalensky et al., 1993, 1995; Tilgen
et al., 2001; Sbracia et al., 2002; Mudrak et al., 2005).
Sperm nuclear architecture may be important for spatial chromatin differentiation and may help to direct normal development of the fertilized egg. Such architecture is thought to
have evolved alongside mammal-specific regulatory systems
such as X inactivation and genomic imprinting (Greaves
et al., 2003). During pachytene, the sex chromosomes are transcriptionally silenced and condensed to form the sex body,
which is located towards the periphery of the nucleus
(reviewed in Turner, 2007). Moreover, Foster et al. (2005)
established in porcine testes that the sex chromosomes then
reposition from the nuclear periphery to the nuclear centre,
occuring between the secondary spermatocyte and round
# The Author 2008. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.
All rights reserved. For Permissions, please email: [email protected]
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Finch et al.
spermatid stages. During spermiogenesis, remodelling of the
chromatin takes place as the histones are replaced, first by transition proteins, and then by protamines, leading to highly compacted chromatin (Kierszenbaum and Tres, 1975; Loir and
Courtens, 1979; Meisrich et al., 1979, 2003; Kistler et al.,
1996).
In the sperm of monotreme mammals, chromosomes are
arranged in a specified order. Work by Greaves et al. (2003)
suggested that the X chromosome lies in the region that
makes first contact with the egg, and this position may be
related to its predisposition for inactivation. Greaves et al.
(2001) focused on the positions of chromosomes in marsupial
sperm, demonstrating that chromosomes occupy fixed positions in both immature and mature sperm; this may be important in establishing patterns of gene activity in the developing
embryo. In rats (Rattus rattus) telomeres in sperm heads are
located peripherally, whereas peri-centromeres are more
interior (Meyer-Ficca et al., 1998). Human sperm chromosome
territories are organized into loop domains that are attached at
specific sites to the sperm nuclear matrix (Kalandadze et al.,
1990; Nadel et al., 1995; Kramer and Krawetz, 1996).
Nuclear architecture in the mammalian sperm is thought to
be characterized by the clustering of the 23 centromeres into
a ‘chromo-centre’ which is positioned well inside the nucleus
(Zalensky et al., 1993, 1995; Tilgen et al., 2001; Sbracia
et al., 2002; Mudrak et al., 2005; Zalensky and Zalenskaya,
2007), although the nature and compactness of the chromocentre may vary (Zalensky and Zalenskaya, 2007). Moreover
the human X chromosome territory shows preference for a
position in the anterior half of the nucleus (Luetjens et al.,
1999; Hazzouri et al., 2000; Zalensky and Zalenskaya,
2007). Patterns of organization of the chromosome territory
and its associated relative centromere and telomere positioning
suggest that DNA loop domains may be mediated by the
nuclear matrix (Ward and Zalensky, 1996); it has been
further hypothesized that the spatial organization of the malehaploid genome is important in sperm function and early development (Sotolongo and Ward, 2000). Moreover, functionally
abnormal sperm, in which the nuclear matrix has been chemically disrupted (and thus presumably the nuclear organization
perturbed), are thought to be unable to produce viable offspring
(Ward et al., 1999). However it has yet to be determined
whether individual infertile men have compromised nuclear
organization as measured by assays of chromosome territory
position. The purpose of this study was to test the hypotheses
(i) that three centromeric loci occupy a central position in a
population of normozoospermic males and, (ii) if so, whether
this pattern is altered in men with compromised semen
parameters.
Materials and Methods
Source of samples
Control sperm nuclei preparations were made from nine chromosomally normal men (controls 1–9, Table I) with normal semen parameters (according to WHO criteria 1999). The infertile cohort
comprised sperm samples from 15 men undergoing male factor IVF
treatment (Patients 10 –24). These patients were classified as
1264
oligozoospermic (O) if sperm concentration was ,20 million/ml,
severe oligozoospermic (sO) if ,5 million/ml, asthenozoospermic
(A) if ,25% forward motility and teratozoospermic (T) if .85%
abnormal forms. Thus, Patients 10–13 were classified ‘O’, Patient
14 as ‘A’, Patients 15–17 as ‘AT’, 18 –19 were ‘OA’, 20 and 21
were ‘sO’, 22 was ‘sOA’ and 23 –24 ‘sOAT’ (Table I). All samples
were from men participating in sperm donation or under treatment
for infertility at The London Bridge Fertility, Gynaecology and
Genetics Centre in London, or at the National Infertility Centre,
Abumeliana Fertility Clinic, Farah Fertility Centre or Misurata Infertility Centre in Libya. All men were chromosomally normal according
to standard G-banded karyotyping, but none was tested for submicroscopic Y chromosome deletions. All participants gave informed
consent for the use of their sperm for research purposes and this
work was approved under the auspices of the treatment licence
awarded by the HFEA to the London Bridge Fertility Centre, the
Libyan Ministry of Health and the Local Research and Ethics committee of the University of Kent.
Preparation of samples
Sperm samples were prepared from fresh ejaculate, washed in 10 mM
NaCl + 10 mM Tris pH 7.0 sperm buffer solution, re-suspended and
centrifuged at 300g for 5 min. The supernatant was removed to
leave 1 ml remaining, the pellet re-suspended and the process
repeated up to five times. Samples were fixed drop-wise to a final
volume of 5 ml using 3:1 methanol:acetic acid. The solution was
re-suspended and centrifuged at 300g for 5 min. The supernatant
was removed to leave 1 ml remaining, the pellet re-suspended and
the process repeated three times.
Fluorescence in-situ hybridization
Briefly, 5 ml of sample was spread over an area 20 20 mm on a
Poly-L-lysine slide (VWR International, Leicestershire, UK)
allowed to dry at room temperature, the optimal density checked
and the sample area marked on the underside of the slide using a
diamond pen. The slide was incubated on a hot plate at 658C for
1.5 h. Sperm cells were swollen by exposure to 0.1 M DTT for
20 min, treated with 1% pepsin/0.01 M HCl for 20 min (398C),
fixed in 4% paraformaldehyde, dehydrated and air-dried. To increase
the level of sperm swelling DTT incubations of 40 and 60 min were
used. Either the pan-centromeric probe set or the pre-prepared triple
colour Aneuvysion# probe set for the centromeres of chromosomes
18 (aqua), X (green) and Y (red) (Vysis, Abbott laboratories, UK)
were applied to the nuclear DNA under a coverslip. This was sealed
using rubber cement and both nuclear and probe DNA were denatured
simultaneously using a ‘HYBrite’ (Vysis) hotplate for 5 min at 758C.
Hybridization at 378C continued for 16 –24 h and post-hybridization
washes consisted of 0.4 standard saline citrate (SSC)/0.3%
Tween-20 (Sigma) at 738C for 3 min and 1 min in 2 SSC/0.1%
Tween-20 at room temperature before dehydration in an alcohol
series. Slides were then air-dried, mounted in Vectashield anti-fade
medium (Vector Laboratories, CA, USA). FISH signals were evaluated using a BX61 (Olympus, Hertfordshire, UK) microscope
equipped for epifluorescence; images were captured using either
Cytovysion Software (Applied Imaging/Genetix, Hampshire, UK)
or SmartCapture software (Digital Scientific UK, Cambridge, UK)
and exported as JPEG and/or TIFF files to Paint Shop Pro 9.1 for
aneuploidy and locus position analysis.
Scoring and interpretation
Signals were classified as representing separate hybridization events if
two or more similarly sized fluorescent foci could be identified that
Infertility and centromere position in sperm
Table I. Summary of location of three loci (18c, Xc and Yc) in controls (1– 9) and infertile men (Patients 10–24).
Diagnosis
18c
Xc
Yc
Control
1
2
3
4
5
6
7
8
9
Pool
Normozoospermic (N)
Normozoospermic (N)
Normozoospermic (N)
Normozoospermic (N)
Normozoospermic (N)
Normozoospermic (N)
Normozoospermic (N)
Normozoospermic (N)
Normozoospermic (N)
Pooled normozoospermic (N)
Central/Medial*
Central*
Medial*
Central*
Central**
Central/Medial*
Central*
Central/Medial**
Central*
Central*
Not significant
Central*
Central*
Central/Medial*
Central*
Central*
Central*
Central*
Central*
Central*
Central/Medial**
Central*
Central*
Central*
Central*
Central*
Central*
Not significant
Central/Medial*
Central*
Patient
10
11
12
13
Pool
14
‘Pool’
15
16
17
Pool
18
19
Pool
20
21
Pool
22
23
24
Pool
Oligozoospermic (O)
Oligozoospermic (O)
Oligozoospermic (O)
Oligozoospermic (O)
Pooled oligozoospermic (N)
Asthenozoospermic (A)
Pooled Asthenozoospermic (N)
Asthenoterotozoospermic (AT)
Asthenoterotozoospermic (AT)
Asthenoterotozoospermic (AT)
Pooled asthenoteratoozoospermic (AT)
Oligoasthenozoospermic (OA)
Oligoasthenozoospermic (OA)
Pooled oligoasthenoozoospermic (OA)
Severe oligozoospermic (sO)
Severe oligozoospermic (sO)
Pooled severe oligozoospermic (sO)
Severe oligoasthenozoospermic (sOA)
Severe oligoasthenoteratozoospermic (sOAT)
Severe oligoasthenoteratozoospermic (sOAT)
Pooled severe oligoasthenoteratozoospermic
Medial*
Central*
Central/Medial**
Central/Medial*
Central/Medial*
Central/Medial*
Central/Medial*
Central**
Central*
Central*
Central*
Central*
Central/Medial*
Central*
Medial*
–
Medial*
Central*
Central/Medial*
Central *
Central*
Central/Medial*
Central/Medial*
–
–
Central/Medial*
Not significant
Not significant
–
Central/Medial*
–
Central/Medial*
Not significant
Central**
Not significant
Not significant
–
Not significant
Central/Medial*
Not significant
Central/Medial*
Central/Medial*
–
Central/Medial*
Not significant
Central*
Central/Medial*
Not significant
Not significant
Not significant
Central/Medial*
–
Not significant
–
–
–
–
Central/Medial**
Central/Medial**
Central*
–
–
Central*
Data representing individual men and defined pools of semen are given. *P , 0.01, **P , 0.05, not significant P 0.05. Medial, predominantly in shell 3, 2/3
or 3/4; Central, predominantly in shell 5 or 4/5; Central/Medial, predominantly in shell 4 or 3-; – , experiment unsuccessful or less than 50 cells could be
scored reliably.
were greater than one signal’s diameter apart. If the loci were less than
one signal’s diameter apart, the signal was classed as a ‘split signal’
and thus scored as a single hybridization event. Only haploid, nondisomic nuclei were scored.
Analysis of locus position in interphase nuclei
Adaptations of previously published approaches for three-dimensional
extrapolations from two-dimensional data were used. An approach
very similar to that of Croft et al. (1999) and Boyle et al. (2001)
was employed to assess chromosome location using two-dimensional
images. A transparent five-circle template (Fig. 1) was used consisting
of five concentric circles derived from concentric spheres of equal
volume. Five spheres of linearly increasing volume were considered
(from V = 1 to V = 5). The radius of each sphere was calculated from
the equation: V = 4/3 p r3.
Hence,
rffiffiffiffiffiffiffiffiffiffiffiffi
0:75V
3
:
p
The template was overlaid on the nucleus image using the rotation,
vertical and horizontal size editing functions to best fit the nucleus
(Fig. 1). Chromosome loci signals were scored according to which of
the five rings they appeared in: area (shell) one was defined as the outermost ring and area (shell) five as the innermost. If a probe spanned more
than one shell it was scored depending upon where the majority of that
signal lay. Signal scores were compiled for each nucleus using
Microsoftw Excel. Sperm heads were not of sufficient quality (particularly in infertile patients) to determine the polarity of the cell and hence
the relative anterior/posterior chromosome territory position.
Statistical analysis
In testing the null hypothesis that distributions were not significantly
different from what would be observed from a random pattern, our
‘expected’ values for each shell were derived from the number of
signals scored divided by the number of rings (five). In essence, if a
random pattern were present, we would expect all bars in the histogram to be roughly of the same height. For each locus, in each cell
type our raw data (i.e. the number of signals that were actually
scored) constituted our ‘observed’ values. From there, a chi-squared
test was used to determine the presence any of non-random distributions. Given that there were five individual values (one for each
shell) that made up the subsequent chi-squared total, P-values were
determined at four degrees of freedom. Distributions were considered
significantly different when compared with the nuclear counterstain at
P 0.05 and ‘highly’ significantly different when P 0.01. This
approach represents only a minor modification from previously
described methodology (e.g. Croft et al., 1999; Bridger et al., 2000;
Boyle et al., 2001; Meaburn et al., 2005a, b). Results were included
only when at least 50 sperm could be scored.
Histogram presentation of results
Relative positions were presented by ‘% relative observed frequency’
in order to allow direct comparisons between graphs (Fig. 2).
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Finch et al.
Figure 1: Sperm nuclei with FISH signals and 5 shell template overlaid.
The nuclear counterstain is DAPI (blue), the chromosome 18 centromeric probes is aqua, the X chromosome centromeric probe is green
and the Y chromosome probe red.
In addition to the chi-squared data therefore, the shape of the
graphical distributions was taken into account including analysis of
standard error of the mean (as depicted by the standard error bars in
the histograms—Fig. 2). In order to determine whether the highest
bar in the histogram was significantly greater than its neighbour(s),
individual chi-square tests (1 df) were performed on the raw data.
This (and visual inspection of the shape of the graph) allowed us to
determine the ‘directivity’ of our distributions, i.e. whether the
signal was distributed peripherally, medially or centrally. For the
purposes of this study, a ‘central’ location was defined as a nonrandom distribution where the bar representing shell 5 (or 2 bars representing shells 4 and 5) was (were) significantly higher that its (their)
nearest neighbour by chi-squared test. A central/medial location
was defined as a non-random distribution where the bar representing
shell 4 (or 3 bars representing shells 3–5) was (were) significantly
higher that its (their) nearest neighbour by chi-squared test and so
on. Therefore, in order to determine the mean nuclear position of
each centromere we considered (i) whether the distribution was significantly different from that predicted by a random pattern and (ii)
if so, in which shell (or shells) was (were) the signal seen most often.
Results
Position of centromeric loci in the sperm head using
a pan-centromeric probe
A probe that recognizes all human chromosomes was applied
to control human sperm heads and revealed that, unlike in
mice, a clear and fixed chromocentre was not as easily discernable. Numerous spots appeared in the sperm head and visual
1266
Figure 2: Distribution of loci (18c, Xc, Yc) in controls.
One example for each locus is given.
inspection indicated a general tendency to the nuclear centre.
However meaningful ‘shell’ analysis was not practicable due
to our inability to discern overlapping signals with any accuracy. For this reason, shell/histogram analysis was continued
only on individual centromeres.
Identification of the three individual centromeric loci in
sperm nuclei of men with normal semen parameters
Our results provide evidence for a chromo-centre arrangement
with all three loci occupying a central or central/medial
location in the majority of normozoospermic control males
(Table I). Figure 2 shows representative examples of the
central positions for each of the centromeric loci in control
patients. In addition our findings show that this central position
of the centromeric array is maintained in a range of sperm head
sizes under a range of swelling conditions (results not shown).
Only 3 out of 27 analyses (11.1%) displayed a pattern for centromeric loci that differed from a non-random, central (or
central/medial) pattern. Specifically: in control 1 the distribution of locus Xc was not significantly different from that
Infertility and centromere position in sperm
predicted by a random distribution (Table I), control 3
displayed a significant ‘medial’ location (shell 3) for 18c
and, for control 8, the Y chromosome centromere distribution
observed was not significantly different from that predicted
by a random pattern (Table I). All nine controls therefore
showed a non-significant, central (or central-medial) pattern
for at least two out of three centromeric loci (Table I).
Investigation of the position of three centromeric loci
(X, Y and 18) in sperm nuclei of men with impaired
semen parameters compared to the controls
Given that these men had fewer sperm and that hybridization
efficiencies were less successful than in controls, a larger proportion of experiments were not successful despite repeated
attempts. We were able to score all three signals in Patients
11, 14 and 22 and two signals in all other patients with the
exception of Patient 17 where only the 18c signal was analysable (Table I). On average, hybridization efficiencies (i.e. the
proportion of nuclei that displayed a discernable signal) were
96% for control samples (range 75– 100%) and 66% for
patients (range 0 – 98%).
The distribution of locus 18c (where sufficient numbers
of cells could be analysed) was significantly non-random
(P , 0.05) in all infertile men (Patients 10– 24), the same as
it was in all controls (1 – 9). In the control group, 18c deviated
from the ‘usual’ central (or central/medial) position in only
one male (11.1% - control 3); similarly among infertile men
(Patients 10– 24), the same locus deviated from the ‘usual’
pattern in 2 out of 14 patients (14.3%). For the centromere of
chromosome 18, therefore, patterns observed in the patient
group were not significantly different from those in the
control group. However, among the infertile males (Patients
10 –24) the pattern of signal distribution for the X chromosome
centromere was not significantly different from that predicted
by a random model in 4 out of 10 (40%) measurements and
similarly for the Y chromosome centromere in three out of
eight (37.5%) measurements. Therefore an apparently
random pattern of signal distribution for the sex chromosome
centromeres was seen in 7 out of 18 measurements (38.9%)
(Table I). This ‘apparently random’ distribution among the
sex chromosomes (both individually and collectively) was
observed significantly more often (in fact over three times
more frequently) (P , 0.01 by t-test) compared with the
control group.
Significant non-random, central distributions of loci 18c, Xc
and Yc were observed in all oligozoospermic men (Patients
10 –13) with the exception of Yc for Patient 12 and 18c for
Patient 10. The distribution of 18c was non-random and
central/medial but not significantly different from that predicted by a random distribution for Xc and Yc in a single asthenozoospermic male (Patient 14). In asthenoteratozoospermic
patients (15 – 17), all but one displayed a non-random distribution for the loci studied (see Fig. 3), while in oligoasthenozoospermic patients (18 and 19) significant non-random,
central distributions for 18c were largely observed. Severe
oligozoospermic patients (20 and 21) showed significant nonrandom distributions for loci 18c and Yc, appearing most frequently in medial and central/medial positions, respectively,
Figure 3: Distribution of loci (18c, Xc, Yc) in patient 17
(asthenoterotozoospermic).
though locus Xc displayed a pattern not significantly different
from that predicted by a random distribution in Patient 20. In a
single severe oligoasthenozoospermic patient (22), all three
loci (18c, Xc and Yc) displayed significant non-random,
central (or central/medial) distributions, and in severe oligoasthenoteratozoospermic (OAT) patients (23 and 24), loci
18c and Xc showed a significant non-random distribution at
central/medial positions (see Fig. 4) with one (apparently
random) exception. All of the above results are expressed in
more detail in Table I.
Discussion
Our results provide evidence that the three centromeric loci
analysed preferentially occupy a central position as predicted
by Zalensky et al. (1993, 1995) and Zalenskaya and Zalensky
(2004) with very little but nonetheless some inter-individual
variation. However, the use of the pan-centromeric probe
revealed that this pattern was not nearly as clear-cut as the
defined and punctate chromo-centre seen in murine sperm.
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Finch et al.
Figure 4: Distribution of loci (18c, Xc, Yc) in patient 23 (severe
oligoasthenoteratozoospermic).
One obvious initial practical implication of our data is that
probes which more commonly occupy the nuclear periphery
are more likely to detect a higher proportion of disomies than
probes which occupy the nuclear centre (such as the sex
chromosome probes and the centromeric probes) because of
the increased likelihood of signals at the nuclear centre overlapping. For the most part, the probes of choice for studies of
male meiotic non-disjunction (for review see Tempest and
Griffin, 2004) are those that recognize centromeric arrays,
largely because of the bright signals that they produce.
However, the observation that these probes are found more
often at the nuclear centre calls into question their utility
when drawing conclusions about the absolute levels of aneuploidy in the male gamete. Similarly although studies comparing data from the same chromosome between patients may well
be valid, inter-chromosomal comparisons may not, e.g. if a
peripherally located locus is compared with centrally located
one. On another technical point it was also noteworthy that
hybridization efficiencies were lower for infertile patients compared with normal controls. While there is no easy explanation
for this observation it did mirror the overall quality of the
semen samples used in this study.
Our results also provide some preliminary evidence in
support of the hypothesis that, at least in certain men, altered
nuclear organization for the sex chromosome is associated
1268
with male factor infertility. While a failure to establish significant evidence for a central or central/medial location of the
X and Y chromosome probes (both individually and collectively) was not common in the control group, it was a regular
occurrence in the cohort of infertile men, happening in nearly
half of the cases (where the signals could be analysed) and
three and a half to four times more frequently than in the
control group. Altered chromosome position has been associated with a range of cell disorders including epilepsy, certain
types of laminopathies and cancer (Borden and Manuelidis,
1988; Misteli, 2004; Meaburn et al., 2005a, b). To the best of
our knowledge this is the first time that alterations in nuclear
organization have been related to a human reproductive
phenotype.
Our findings may suggest a relationship between impaired
spermatogenic regulation and altered nuclear organization in
certain men. Foster et al. (2005) first reported the migration
of the sex chromosomes from the nuclear periphery to the
nuclear centre in porcine spermatogenesis by the round spermatid stage. In parallel, the organization of chromosome territories in a ‘chromo-centre’ (also by the round spermatid stage)
is now an accepted model in a number of species including
mouse and human (Zalenskaya and Zalensky, 2004, 2007;
Namekawa et al., 2006). Recently there has been clear evidence for the juxtaposition of the post-meiotic sex chromatin
(PMSC) with the chromo-center in murine spermatogenesis
(Namekawa et al., 2006). Migration to the nuclear centre at
the round spermatid stage is accompanied by a reactivation
of 33 X chromosome genes that had previously been inactivated by meiotic sex chromosome inactivation (MSCI) including Pctk1, Ube2a and Pdk3 (Namekawa et al., 2006). Turner
et al. (2006) noted that MSCI is an inevitable consequence of
asynapsis (e.g. of the sex chromosome bivalent) and is an
example of the general mechanism of meiotic silencing of
unsynapsed chromatin (MSUC). Turner (2007) goes on to
argue that MSCI/MSUC is involved in protecting the gamete
from generating excessive levels of aneuploidy (which also
can be generated by synaptic errors). Compromised semen
parameters are associated with increased levels of sperm aneuploidy (reviewed in Tempest and Griffin, 2004) and, according
to our results, increased failure of migration of the PMSC to
the nuclear centre by the round spermatid stage is also possible.
It therefore prompts us to speculate that mechanisms involved
in the genesis of male infertility and its association with
increased levels of aneuploidy may well be related to MSCI/
MSUC. A closer examination of chromosome position and
its relationship between sperm aneuploidy and compromised
semen parameters in a larger group of patients may enable us
to determine this.
In infertility clinics, males are routinely screened for standard semen parameters such as concentration, motility and
morphology. Increased sperm aneuploidy is associated with
compromised semen parameters and was first reported in the
late 1990s (Pang et al., 1999). Some infertility clinics are
now adopting FISH aneuploidy screening in sperm as a standard protocol. Indeed we have argued that sperm aneuploidy
levels should constitute another semen parameter (Griffin
et al., 2003). If FISH experiments are already being performed
Infertility and centromere position in sperm
to assess chromosome copy number, the additional time also to
assess nuclear organization would be minimal. It should be
stressed however that our results, as they stand, although
showing a significant association between sex chromosome
position in sperm heads and infertility, do not provide evidence
that sex chromosome centromere position analysis constitutes
an accurate diagnostic test for infertility. It remains to be
seen whether assays for chromosome position will ultimately
constitute yet another semen parameter. However, in the
meantime, studies such as those involving three-dimensional
analyses, would help to indicate whether altered nuclear organization is a general phenomenon, or whether it applies to a
specific sub-set of patients.
Authors’ contributions
K.A.F., K.G.L.F., N.H. and D.K.G. performed the experiments,
data collection and analysis. A.A., A.H.H. and A.R.T. provided
patient material and critically appraised the manuscript. D.I.
performed the experiments to address the reviewer comments.
D.K.G. (with assistance from A.H.H. and A.R.T.) conceived
and managed the project.
Funding
K.A.F was funded by a BBSRC quota studentship awarded to
the University of Kent. D.I. is funded by a BBSRC industrial
CASE studentship awarded to Digital Scientific UK (Cambridge, UK). K.G.L.F., A.A. and N.H. are/were self-funded
postgraduate students at the University of Kent.
References
Affara NA, Mitchell MJ. The role of human and mouse Y chromosome genes in
male infertility. J Endocrinol Invest 2000;23:630– 645.
Borden J, Manuelidis L. Movement of the X-Chromosome in Epilepsy. Science
1988;242:1687– 1691.
Boyle S, Gilchrist S, Bridger JM, Mahy NL, Ellis JA, Bickmore WA. The
spatial organization of human chromosomes within the nuclei of normal
and emerin-mutant cells. Hum Mol Genet 2001;10:211–219.
Bridger JM, Boyle S, Kill IR, Bickmore WA. Re-modelling of nuclear
architecture in quiescent and senescent human fibroblasts. Curr Biol
2000;10:149–152.
Chandley AC. The chromosomal basis of human infertility. Br Med Bull
1979;35:181–186.
Croft JA, Bridger JM, Boyle S, Perry P, Teague P, Bickmore WA. Differences
in the localization and morphology of chromosomes in the human nucleus.
J Cell Biol 1999;145:1119– 1131.
De Braekeleer M, Dao TN. Cytogenetic studies in male infertility: a review.
Hum Reprod 1991;6:245– 250.
Foster HA, Bridger JM. The genome and the nucleus: a marriage made by
evolution. Genome organisation and nuclear architecture. Chromosoma
2005;114:212–229.
Foster HA, Abeydeera LR, Griffin DK, Bridger JM. Non-random chromosome
positioning in mammalian sperm nuclei, with migration of the sex
chromosomes during late spermatogenesis. J Cell Sci 2005;118:1811– 1820.
Greaves IK, Svartman M, Wakefield M, Taggart D, De Leo A, Ferguson-Smith
MA, Rens W, O’Brien PC, Voullaire L, Westerman M et al. Chromosomal
painting detects non-random chromosome arrangement in dasyurid
marsupial sperm. Chromosome Res 2001;9:251– 259.
Greaves IK, Rens W, Ferguson-Smith MA, Griffin D, Marshall Graves JA.
Conservation of chromosome arrangement and position of the X in
mammalian sperm suggests functional significance. Chromosome Res
2003;11:503–512.
Griffin DK, Finch KA. The genetic and cytogenetic basis of male infertility.
Hum Fertil (Camb) 2005;8:19– 26.
Griffin DK, Hyland P, Tempest HG, Homa ST. Safety issues in assisted
reproduction technology - Should men undergoing ICSI be screened
for chromosome abnormalities in their sperm? Hum Reprod 2003;18:
229– 235.
Hazzouri M, Rousseaux S, Mongelard F, Usson Y, Pelletier R, Faure AK,
Vourc’h C, Sele B. Genome organization in the human sperm nucleus
studied by FISH and confocal microscopy. Mol Reprod Dev 2000;55:
307– 315.
Kalandadze AG, Bushara SA, Vassetzky YS, Jr, Razin SV. Characterization of
DNA pattern in the site of permanent attachment to the nuclear matrix
located in the vicinity of replication origin. Biochem Biophys Res Commun
1990;168:9–15.
Kierszenbaum AL, Tres LL. Structural and transcriptional features of the
mouse spermatid genome. J Cell Biol 1975;65:258–270.
Kistler WS, Henriksen K, Mali P, Parvinen M. Sequential expression
of nucleoproteins during rat spermiogenesis. Exp Cell Res 1996;225:
374– 381.
Kramer JA, Krawetz SA. Nuclear matrix interactions within the sperm genome.
J Biol Chem 1996;271:11619– 11622.
Loir M, Courtens JL. Nuclear reorganization in ram spermatids. J Ultrastruct
Res 1979;67:309– 324.
Luetjens CM, Payne C, Schatten G. Non-random chromosome positioning in
human sperm and sex chromosome anomalies following intracytoplasmic
sperm injection. Lancet 1999;353:1240.
Meaburn KJ, Levy N, Toniolo D, Bridger JM. Chromosome positioning is
largely unaffected in lymphoblastoid cell lines containing emerin or
A-type lamin mutations. Biochem Soc Trans 2005a;33:1438– 1440.
Meaburn KJ, Parris CN, Bridger JM. The manipulation of chromosomes by
mankind: the uses of microcell-mediated chromosome transfer.
Chromosoma 2005b;114:263– 274.
Meistrich ML, Göhde W, White RA, Longtin JL. ‘Cytogenetic’ studies of
spermatids of mice carrying Cattanach’s translocation by flow cytometry.
Chromosoma. 1979;74:141–151.
Meistrich ML, Mohapatra B, Shirley CR, Zhao M. Roles of transition nuclear
proteins in spermiogenesis. Chromosoma 2003;111:483– 488.
Meyer-Ficca M, Muller-Navia J, Scherthan H. Clustering of pericentromeres
initiates in step 9 of spermiogenesis of the rat (Rattus norvegicus) and
contributes to a well defined genome architecture in the sperm nucleus.
J Cell Sci 1998;111:1363–1370.
Misteli T. Spatial positioning: A new dimension in genome function. Cell
2004;119:153–156.
Mudrak O, Tomilin N, Zalensky A. Chromosome architecture in the
decondensing human sperm nucleus. J Cell Sci 2005;118:4541– 4550.
Nadel B, De Lara J, Ward WS. Structure of the rRNA genes in the hamster
sperm nucleus. J Androl 1995;16:517–522.
Namekawa SH, Park PJ, Zhang LF, Shima JE, McCarrey JR, Griswold MD,
Lee JT. Postmeiotic sex chromatin in the male germline of mice. Curr
Biol 2006;16:660– 667.
Pang MG, Hoegerman SF, Cuticchia AJ, Moon SY, Doncel GF, Acosta AA,
Kearns WG. Detection of aneuploidy for chromosomes 4, 6, 7, 8, 9, 10,
11, 12, 13, 17, 18, 21, X and Y by fluorescence in-situ hybridization in
spermatozoa from nine patients with oligoasthenoteratozoospermia
undergoing intracytoplasmic sperm injection. Hum Reprod 1999;14:
1266– 1273.
Sbracia M, Baldi M, Cao D, Sandrelli A, Chiandetti A, Poverini R, Aragona C.
Preferential location of sex chromosomes, their aneuploidy in human sperm,
and their role in determining sex chromosome aneuploidy in embryos after
ICSI. Hum Reprod 2002;17:320–324.
Shah K, Sivapalan G, Gibbons N, Tempest H, Griffin DK. The genetic basis of
infertility. Reproduction 2003;126:13 –25.
Sheridan R, Llerena J, Jr, Matkins S, Debenham P, Cawood A, Bobrow M.
Fertility in a male with trisomy 21. J Med Genet 1989;26:294– 298.
Skakkebaek NE, Hulten M, Philip J. Quantification of human seminiferous
epithelium. IV. Histological studies in 17 men with numerical and structural
autosomal aberrations. Acta Pathol Microbiol Scand [A] 1973;81:112–124.
Sotolongo B, Ward WS. DNA loop domain organization: the three-dimensional
genomic code. J Cell Biochem Suppl 2000;35:23 –26.
Tempest HG, Griffin DK. The relationship between male infertility and
increased levels of sperm disomy. Cytogenet Genome Res 2004;107:83 –94.
Tilgen N, Guttenbach M, Schmid M. Heterochromatin is not an adequate
explanation for close proximity of interphase chromosomes 1 –Y, 9 –Y,
and 16–Y in human spermatozoa. Exp Cell Res 2001;265:283– 287.
Turner JMA. Meiotic sex chromosome inactivation. Development
2007;134:1823–1831.
1269
Finch et al.
Turner JMA, Mahadevaiah SK, Ellis PJI, Mitchell MJ, Burgoyne PS.
Pachytene asynapsis drives meiotic sex chromosome inactivation and
leads to substantial postmeiotic repression in spermatids. Dev Cell
2006;10:521– 529.
Ward WS, Zalensky AO. The unique, complex organization of the
transcriptionally silent sperm chromatin. Crit Rev Eukaryot Gene Expr
1996;6:139– 147.
Ward WS, Kimura Y, Yanagimachi R. An intact sperm nuclear matrix may be
necessary for the mouse paternal genome to participate in embryonic
development. Biol Reprod 1999;60:702–706.
Zalenskaya IA, Zalensky AO. Non-random positioning of chromosomes in
human sperm nuclei. Chromosome Res 2004;12:163–173.
1270
Zalensky A, Zalenskaya I. Organization of chromosomes in spermatozoa: an
additional layer of epigenetic information? Biochem Soc Trans
2007;35:609–611.
Zalensky AO, Allen MJ, Kobayashi A, Zalenskaya IA, Balhorn R, Bradbury
EM. Well-defined genome architecture in the human sperm nucleus.
Chromosoma 1995;103:577–590.
Zalensky AO, Breneman J, Zalenskaya IA, Brinkley BR, Bradbury EM.
Organization of centromeres in the decondensed nuclei of mature human
sperm. Chromosoma 1993;102:509–518.
Submitted on January 3, 2008; resubmitted on February 28, 2008; accepted on
March 13, 2008