Molecular Human Reproduction vol.2 no.7 pp. 485-488, 1996
Analysis of the sex chromosomal equipment in spermatozoa of
a 47,XYY male using two-colour fluorescence in-situ hybridization
S.Mercier1, F.Morel, C.Roux, M.C.CIavequin and J.L.Bresson
Service de Cytog6n6tique, Immunocytologie, Biologie du DeVeloppement et de la Reproduction, Hdpital Saint Jacques
and URA CNRS 561, Faculte de Medecine, Place Saint Jacques, F25030 Besangon, France
1
To whom correspondence should be addressed
The sex chromosomes in spermatozoa of a 47,XYY fertile male were analysed simultaneously by dual
fluorescence in-situ hybridization (FISH), with two probes (pHY2.1 and pXBR). Of the 100 000 cells analysed,
95 179 spermatozoa (95.18%) exhibited one or more hybridization signals. Of the hybridized nuclei, 85.37%
showed a normal sex chromosome constitution (37.37% X-bearing cells and 48.00% Y-bearing cells), with an
X:Y ratio of 0.78:1. A total of 14.63% of the hybridized nuclei exhibited sex chromosome aneuploidy with a
majority of XY- and YY-bearing spermatozoa (9.37 and 4.65% respectively). Even if the majority of spermatozoa
have chromosomal haploidy, a large proportion of them exhibits numerical errors for the sex chromosomes.
These observations raise questions about the commonly-admitted notions concerning the absence of
chromosomal risk for XYY male offspring.
Key words: fluorescence interphase in-situ hybridization/sex chromosome/spermatozoa/XYY male
Introduction
Over the past 20 years, contradictory opinions have been
reported on the behaviour and the distribution of both Y
chromosomes during the spermatogenesis of 47.XYY males.
On the one hand, meiotic analyses of testicular biopsies were
performed, but these studies were limited by the difficulty in
obtaining testicular samples. On the other hand, Y-specific
stainings were carried out on either testicular sections or
ejaculate spermatozoa smears, but the non-X-chromosome
identification gave incomplete results. In some cases, these
studies showed disturbances in the complete spermatogenesis
process, and generally resulted in a precocious loss of the
second Y chromosome in XYY germ cells (Chandley et al,
1976; Faed et al, 1976; Speed et al, 1991). However, Hulten
and Pearson (1971) reported an XYY childless patient with
YY bivalents in 45% of first division meiotic metaphase cells.
After meiotic studies in an XYY male, Luciani et al. (1973)
observed spermatocytes with X + Y + Y diakinetic univalent
associations.
The data concerning XYY male fertility showed that some
patients have either primary or secondary clinical infertility
(Court Brown et al, 1968; Chandley et al, 1975), while others
may have several children (Thompson et al, 1967). The notion
of normal offspring of 47.XYY males is usually admitted, but
data on these offspring remain very subjective.
This article reports the results of a simultaneous identification, by dual fluorescence in-situ hybridization (FISH), of both
X and Y chromosomes in 100 000 spermatozoa of the ejaculate
of a 47.XYY karyotype male donor.
Materials and methods
This study concerns one sample of frozen semen of 47.XYY male
spermatozoa provided by the Spermotheque de la F6d6ration Francaise
© European Society for Human Reproduction and Embryology
de Centre d'Etude et de Conservation des Oeufs et du Sperme
humains (CECOS), with values for conventional semen parameters
falling within normal ranges. This man had already had children and
was volunteering to become a sperm donor, but the cytogenetic
analysis of blood peripheric lymphocytes showed a 47.XYY homogeneous karyotype.
A total of 57 sperm samples, belonging to another prospective
study on aneuploidy detection by dual FISH (data not shown), were
used as a control population (control 1). These samples were selected
at random and submitted for FISH analysis without previous freezing.
The karyotype of these men was unknown. One frozen sperm sample
of a donor with a 46.XY normal karyotype and normal semen
parameters, also provided by Spermotheque was used as a second
control (control 2).
Preparation and decondensation of sperm nuclei
After thawing, in order to eliminate the preservation solution, each
frozen sample was cleared of glycerol and 'egg yolk' by three
washings using a Tyrode buffer. All frozen or fresh semen samples
were washed three times in phosphate-buffered saline (PBS), and
then carefully resuspended at a concentration of 25 X106 spermatozoa/
ml in the same buffer. Washed suspension (200-250 (il) was dropped
onto clean and dry glass slides, air dried, and incubated overnight
in a fixative (methanol:acetic acid, 3:1) at room temperature. The
slides were treated for 10 min with a solution of 0.2 M Tris HC1
(pH 8.6) containing 1.25% papain, 0.16% dithioerythritol and 0.5%
dimethylsulphoxide. The fixed sperm heads were decondensed to
approximately twice their original size, then air dried and fixed again
in 95% ethanol for 15 min (Schwerin et al, 1991).
Preparation of the molecular DNA probes
Four DNA probes were used for this study: a Yq heterochromatinspecific probe (pHY2.1; Cooke et al, 1982), an X-specific a-satellite
probe (pXBR; Willard et al., 1983), and two autosomal a-satellite
probes, an 18-specific probe (L1.84) and an 8-specific probe (pJM 128),
both provided by the American Type Culture Collection (ATCC;
485
S.Mercier et al.
spemMtogonia
Statistical analysis
An independent x 2 test was used to compare the results obtained for
the 47,XYY male with those observed in each control population.
Results
AAAA
in
ii ii
Figure 1. Theoretical distribution of the meiotic segregation of the
three chromosomes in a 47,XYY male.
Rockville, Maryland, USA). The probes were labelled by nick
translation according to a kit (Boehringer Mannheim, Meylan, France)
either with biotin-7-dATP (Gibco BRL, Cergy Pontoise, France)
(pXBR and L1.84 probes) or with digoxigenin-11-dUTP (Boehringer
Mannheim) (pHY2.1 and pJM128 probes).
Two-colour in-shu hybridization
To determine the frequencies of X- and Y-bearing cells, and the
frequencies of disomic cells for X and/or Y, two-colour FISH was
performed, according to the classical method described by Lichter
et al. (1988). A mixture of X- and Y-specific probes was applied to
two slides of 47,XYY male spermatozoa smears, to one slide of each
control 1 sperm smear, and to two slides for control 2. The hybridization was performed overnight at 37°C. After post-hybridization
washings, the hybrids were detected by aminomethylcoumarin acetic
acid (AMCA)-labelled avidin D (Vector Laboratories, Biosys,
Compiegne, France), followed by a mix of biotinylated anti-avidin
D and fluorescein isothiocyanate (hllC)-labelled anti-digoxigenin
(Boehringer Mannheim), and finally AMCA-avidin D. The slides
were finally mounted with a mix of glycerol, anti-fade reagent (Vector
Laboratories) and propidium iodide (PI; Sigma Aldrich, Saint Quentin
Fallavier, France). Two-colour FISH with the probes LI.84 and
pJM128 was performed on one slide of the 47.XYY male semen
sample in order to identify the frequency of diploid cells.
Criteria for hybridized nuclei selection and analysis
The slides were examined with a Zeiss Axiophot epifluorescence
microscope. A spermatozoon was considered to be monosomic for
the tested chromosome when one fluorescent signal was seen and
disomic when two fluorescent signals of the same colour were clearly
positioned within the sperm head, comparable in brightness and size,
and at least one signal apart. These strict scoring criteria may lead
to a slight underestimation of the frequency of disomy. However,
without these criteria, there would undoubtedly be a risk of overestimating the frequencies of disomy, as described by Martin and
Rademaker (1995). The non-fluorescent nuclei, which could be either
nullisomic nuclei for the chromosomes or non-hybridized nuclei due
to a technical failure, could not be interpreted. Certain populations
of sperm nuclei were also eliminated from scoring and analysis:
those with overlapping sperm heads, disrupted nuclei with indistinct
margins, heads without tails, and nuclei which were non-swollen or
swollen to more than three times their original size due to excessive
decondensation.
Concerning chromosomal identification, the frequency of each
hybridization pattern was compared with that observed in the control
populations.
486
A total of 100 000 nuclei for the tested sperm sample, 31 260
nuclei for the control 1 population and 49 855 for the control
2 population (Table I) were scored after hybridization with
pXBR and pHY2.1. The hybridization efficiency rates were
95.2, 96.4 and 97.1% respectively. The non-fluorescent nuclei
were excluded; corrected values presented in Table II were
used in the following data analyses.
The frequency of X- and Y-bearing spermatozoa was 37.37
and 48.0% respectively for the tested sample. There was an
excess of Y-bearing cells with a X:Y ratio of 0.78:1. Both
control populations showed frequencies of X- and Y-bearing
cells close to 50% (49.94% X-bearing cells and 49.81%
Y-bearing cells for control 1; 50.16% X-bearing cells and
49.77% Y-bearing cells for control 2) with a sex ratio close
to that expected (1:1).
In the tested sample, 85.37% of hybridized nuclei (81 259
out of 95 179) exhibited chromosomal haploidy in comparison
with 99.75% (30 046 out of 30 122) for control 1 and 99.93%
(48 366 out of 48 399) for control 2. The difference in haploid
chromosomal nuclei frequencies between the tested sample
and control 1 was significant (%2 = 350, P < 0.001). A
significant difference was also obtained for the haploid chromosomal nuclei frequencies of the tested sample compared to
those of control 2 (x2 = 502, P < 0.001).
In the 47,XYY sample, the frequency of disomic spermatozoa was 4.65% for the Y chromosome and 0.34% for the X
chromosome. The proportion of XY-bearing spermatozoa was
9.37%. Other patterns of chromosomal aneuploidies were found
with 0.21% XXY-nuclei and 0.06% XYY-nuclei. Therefore, the
total frequency of hybridized cells with numerical errors for
sex chromosomes was 14.63%. In the control populations,
nuclei with a numerical error for sex chromosomes were found
with frequencies <0.25 and 0.07% for control 1 and 2
respectively, with significantly higher values for control 1
(X2 = 32, P < 0.001). Whatever control population used, the
percentage of nuclei with chromosomal aneuploidies in the
tested sample (14.63%) was always higher than that of control
1 (0.25%, x2 = 4764, P < 0.001) and that of control 2
(0.066%, x2 = 7750, P < 0.001).
Relating to the evaluation of diploid cell number using
8 and 18 chromosome probes, the hybridization onto one
spermatozoa smear of the 47.XYY male sperm sample showed
that 98% of nuclei exhibited a signal of hybridization. Among
the 48 663 scored nuclei, 56 nuclei (0.11%) showed four spots
which corresponded to two 8-signals and two 18-signals; 87
nuclei (0.18%) showed two 8-spots and one 18-spot, and 83
nuclei (0.17%) exhibited one 8-spot and two 18-spots. These
results indicated that the frequency of double-disomic nuclei
for both autosomes could be estimated at 0.0003%
(0.18X0.17%), and thus the frequency of diploid nuclei
included in our results can be considered as identical to the
frequency of nuclei with two 8 and two 18-signals (0.11%).
FISH analysis of a 47.XYY male
Table I. Percentage of scored nuclei after two-colour fluorescence in-situ hybridization (FISH) with pXBR and pHY2.1 on sperm samples of the 47XYY
male, of the control 1 population (n = 57). and of the control 2 (46XY male). Figures in parentheses are number of scored nuclei
47.XYY
(n = 100 000)
control 1
(n = 31 260)
control 2
(n = 49 855)
35.57
(35.570)
48.12
(15 042)
48.69
(24 275)
45.69
(45 689)
48.00
(15 004)
48.32
(24 091)
XY
YY
XX
XXY
XYY
Non-fluorescent
8.91
(8914)
0.15
(47)
0.05
(25)
4.43
(4427)
0.05
(16)
0.01
(5)
0.32
(323)
0.04
(13)
0.004
(2)
0.20
(204)
0
(0)
0
(0)
0.05
(52)
0
(0)
0.002
(1)
4.82
(4821)
3.64
(1138)
2.92
(1456)
Table II. Corrected percentage of hybridized nuclei scored after two-colour fluorescence in-situ hybridization (FISH) with pXBR and pHY2.1 on sperm
samples of the 47,XYY male and of both control populations. Figures in parentheses are number of scored nuclei
47.XYY
(n = 95 179)
control 1
(n = 30 122)
control 2
(n = 48 399)
37.37
(35 570)
49.94
(15 042)
50.16
(24,275)
48.00
(45 689)
49.81
(15 004)
49.77
(24 091)
XY
YY
XX
XXY
XYY
9.37
(8914)
0.16
(47)
0.05
(25)
4.65
(4427)
0.05
(16)
0.01
(5)
0.34
(323)
0.04
(13)
0.004
(2)
0.21
(204)
0
(0)
0
(0)
0.06
(52)
0
(0)
0.002
(1)
An identical study of 8 and 18 chromosome identification was
performed on one slide of each sperm sample of the control
1 population. This study was part of an evaluation of aneuploidy
(data not shown). The value of diploid nuclei in this sperm
population was estimated at 0.07% for the scored nuclei.
Discussion
The aim of this study was to identify the X and Y chromosomes
in one frozen sperm sample from a 47.XYY fertile male. Twocolour FISH with specific chromosomal probes allowed us to
screen a large sample of sperm nuclei for numerical errors.
Results were compared with those obtained in two different
control populations: the first made up of 57 fresh semen
samples of males with unknown karyotypes, and the second,
a frozen sperm sample of a fertile male donor with a normal
46,XY karyotype.
The X: Y ratios found in the two types of control populations
were similar, but differences were observed in the frequencies
of chromosomal aneuploidies found in both controls, possibly
reflecting inter-sample variations. These results confirmed
those obtained by other research teams (Spriggs et al, 1995).
The use of two types of control populations should reduce the
risk of error interpretation.
The nuclei with no hybridization signal are difficult to
interpret. They may occur as a result of a hybridization
technique failure, linked with the quality of spermatozoa
chromatin decondensation for example. However, the decondensation methods have been well tested for several years
(Roux et al, 1988), and our hybridization efficiency rates are
similar to those in the literature, even if values of 99% have
been reported (Rousseaux and Chevret, 1995). These nuclei
may be nullisomic nuclei for the chromosomes after nondisjunction during meiosis I or meiosis n. In the sperm sample
of a male with a normal 46,XY karyotype, the percentage of
nullisomic spermatozoa as a result of meiotic non-disjunction
can easily be deduced from the percentage of disomic sperm-
atozoa. In the present case of XYY male spermatozoa, such
an evaluation is more difficult. Therefore, in our study, the
number of spermatozoa with chromosomal abnormalities does
not include the number of chromosomal nullisomic spermatozoa.
The result of this work, concerning the sex chromosome
aneuploidies of spermatozoa in a 47.XYY male, indicates: (i)
among normal spermatozoa, an excess of Y-bearing spermatozoa, with a very disparate sex ratio; (ii) a significantly
higher frequency of abnormal spermatozoa with two or three
chromosomes, especially chromosomal disomic spermatozoa;
(iii) among these disomic spermatozoa, a few XX-bearing
spermatozoa (0.34%) and a large majority of YY- and XYbearing spermatozoa (about 14%) with twice as many XY(9.37%) as YY-bearing cells (4.65%). The XX- and XXYbearing cells are necessarily the result of segregation errors,
but the XYY-cells may also correspond to diploid cells.
However the percentage of XYY-cells (0.06%) seems low,
clearly below the estimated percentage of diploid cells in our
sample. Perhaps the XYY-bearing sperm frequency has been
slightly underestimated because of our scoring criteria. Alternatively, concerning the cells with diploidy of meiosis I origin,
it may reflect the phenomenon of one Y elimination as
suggested by some authors (Chandley et al., 1976; Faed et al.,
1976). None of these results agree with other reported data
(Chandley et al, 1976; Faed et al, 1976).
In our study, the diploid cell frequency was assessed by
double-colour FISH with two autosomal probes. Some authors
evaluate the frequencies of diploid cells in sperm samples by
triple-colour FISH (Schattman et al, 1993; Chevret et al.,
1995; Spriggs et al, 1995, 1996). These studies involved
normal sperm samples with, among the abnormal chromosomal
cells, a majority of XY-bearing cells. In the case of a 47.XYY
male sperm sample, we feel that two-colour FISH is an
adequate method because the number of XYY-bearing cells
represents a very small portion of scored abnormal chromosomal cells.
487
S.Mercier et al.
A theoretical meiotic distribution of the chromosomes in
XYY spermatogonia, irrespective of any hypothetical correction by one Y chromosome elimination and of any added nonsegregation (Figure 1), result, in the same number of normal
and abnormal spermatozoa with, among the normal spermatozoa, twice as many Y- as X-bearing cells and, among the
abnormal spermatozoa, twice as many XY- as YY-bearing
spermatozoa. The abnormal sperm values reported here are
much lower than expected as shown in this theoretical distribution. However, the patterns of such a distribution suggest either
an excess of Y in the normal spermatozoa, or an excess of
XY in the abnormal spermatozoa, as if some germ cells had
distributed an X and two Y during meiotic chromosomal
segregation (Luciani et al., 1973), and that a larger number of
germ cells had distributed an X and only one Y, perhaps by
eliminating one Y.
In conclusion, although this study was based on only one
case, it suggests that, in a male with a 47.XYY karyotype, a
relatively large proportion of spermatozoa may have sex
chromosome aneuploidy, in particular XY or YY, which could
lead to 47.XXY or 47.XYY conceptuses. However, it would
be interesting to pursue this study by evaluating larger numbers
of sperm samples obtained from different 47.XYY males.
Nevertherless, our results raise questions about the commonly
admitted notions concerning the absence of chromosomal risk
for 47,XYY male offspring.
Acknowledgements
The authors thank the 'Spermotheque de la F6d6ration Francaise de
CECOS', the 'Centre d'Etude et de Conservation des Oeufs et du
Sperme humains (CECOS) de 1'Ouest' (Professor Lc Lannou) and
the 'CECOS de BesancxHi-Franche Comte''. They are grateful to
Professor Cooke of Western General Hospital of Edinburgh, UK, and
to Professor Willard of Case Western Reserve University of Ohio,
USA, for providing the respective probes pHY2.1 and pXBR. They
would also like to thank N.Maitret for her technical assistance.
This work was supported by the 'Association Rdgionale pour le
De'veloppement des Etudes biologiques en Ge'ndtique et Reproduction
humaine'.
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Received on January 26, 1996; accepted on April I, 1996