Human Reproduction, Vol.27, No.5 pp. 1518– 1524, 2012
Advanced Access publication on March 1, 2012 doi:10.1093/humrep/des051
ORIGINAL ARTICLE Reproductive genetics
Meiotic non-disjunction mechanisms
in human fertile males
L. Uroz 1,2 and C. Templado 1,*
1
Departament de Biologia Cel lular, Fisiologia i Immunologia, Facultat de Medicina, Universitat Autònoma de Barcelona, Bellaterra, Barcelona
08193, Spain 2Gravida, Centre Internacional de Reproducció Humana Assistida de Barcelona, Barcelona 08034, Spain
*Correspondence address. Tel: +34-935811905; Fax: +34-935811025; E-mail: [email protected]
Submitted on December 12, 2011; resubmitted on January 18, 2012; accepted on February 2, 2012
background: In humans, little is known about the mechanisms of non-disjunction working in male meiosis, although considerable attention has been given to these mechanisms in female meiosis. The present study explores the origin of meiotic non-disjunction during human
spermatogenesis and the chromosomes most commonly involved in this process.
methods: We used Multiplex fluorescence in situ hybridization to carry out meiotic analyses in metaphase I (MI) and metaphase II (MII)
spermatocytes from three fertile donors. Testicular biopsy was obtained during a vasectomy procedure.
results: We examined a total of 317 MI and 248 MII spermatocytes. The frequency of numerical chromosome abnormalities at MII
(14.5%) was 5.5 times higher than at MI (2.5%). We observed 88 (27.7%) spermatocytes I with chromosome bivalents with a low
chiasma count, usually small chromosomes displaying two separated univalents. Chromosomes X, Y and 21 were the most commonly
found as achiasmate chromosomes at MI and the most frequently involved in disomy at MII. Hyperploidy frequency in spermatocytes II
(disomy) was significantly higher (P , 0.001) than that found in spermatocytes I (trisomy).
conclusions: Achiasmate non-disjunction and premature separation of sister chromatids appear to be the two main non-disjunction
mechanisms during the first meiotic division in human spermatogenesis, and both mechanisms contribute equally to the genesis of aneuploidy. The elevated frequencies of disomy detected in spermatocytes II are significantly higher than those previously described in human
spermatozoa, suggesting the existence of a postmeiotic checkpoint monitoring numerical abnormalities.
Key words: chromosome abnormalities / meiosis / meiotic segregation / non-disjunction / spermatogenesis
Introduction
In humans, trisomy is the most common chromosome abnormality
and it is the leading cause of miscarriage and mental retardation.
Around 90% of trisomies originate during paternal or maternal
meiosis (Hassold et al., 2007). Paternal origin of autosomal trisomies
is low (0–30% of cases, depending on the chromosome affected)
(reviewed by Hall et al., 2007), whereas the paternal contribution to
sex chromosome trisomies is more clinically relevant (10% in
47,XXX conditions, 50% in 47,XXY cases and 100% in 47,XYY individuals) (Hall et al., 2006). Trisomy is the result of chromosome missegregation, basically through meiotic non-disjunction (Márquez et al.,
1996). Three main non-disjunction mechanisms have been proposed
on the basis of data obtained from human oocytes II (Hassold and
Hunt, 2001). First, the failure to resolve chiasmata between homologous chromosomes at anaphase I, known as ‘true’ non-disjunction.
Second, the achiasmate non-disjunction, characterized by the
absence of chiasmata between a pair of homologs followed by their
migration to the same pole of the meiotic spindle. The third
mechanism, called premature separation of sister chromatids
(PSSC), consists of chromatid segregation during anaphase instead of
whole chromosome segregation (Angell, 1991). Direct studies on
metaphase II (MII) oocytes report achiasmate non-disjunction and
PSSC as the main mechanisms generating aneuploidy in female
meiosis I (reviewed by Pellestor et al., 2005, 2006; Rosenbusch,
2006). However, little is known about the relevance of each individual
non-disjunction mechanism in human male meiosis I and its contribution to the production of aneuploid spermatozoa.
Basically, non-disjunction in human male meiosis I may result either
from improper pairing, abnormal synapsis or anomalous recombination during prophase I, or from abnormal chromosome orientation
and premature loss of cohesion between chromatids at anaphase I.
Studies in trisomic conceptuses (reviewed by Lamb et al., 2005) and
in pachytene spermatocytes and spermatozoa from infertile men (Ferguson et al., 2007; Sun et al., 2008) found an association between abnormal meiotic recombination and aneuploidy. However, these
previous works were not able to determine the causative mechanisms
of meiotic non-disjunction because the analysis of such mechanisms
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Meiotic non-disjunction mechanisms in fertile males
requires the combined cytogenetic analysis of spermatocytes at metaphase I (MI) and MII. Studies at these meiotic stages are scarce, owing
to the difficulty of obtaining testicular tissue. Meiotic analyses carried
out on fertile and infertile men described abnormal chiasma count
and numerical chromosome abnormalities (Skakkebaek et al., 1973;
Chandley et al., 1976; Koulischer et al., 1982; Egozcue et al., 1983;
Laurie et al., 1985; Guichaoua et al., 1986; Uroz et al., 2009, 2011)
but they were not focused on studying non-disjunction mechanisms.
Multiplex fluorescence in situ hybridization (M-FISH), first described
in somatic cells (Speicher et al., 1996), allows the simultaneous identification of each human chromosome with a specific combination of
five fluorochromes. This technique has been recently applied to metaphase spermatocytes in order to identify meiotic abnormalities
(Sarrate et al., 2004) or to analyze non-disjunction in one infertile
man (Uroz et al., 2008).
The aims of the present work are (a) to detect the mechanisms
causing non-disjunction in human male meiosis I and (b) to identify
the chromosomes most commonly involved in aneuploid spermatocytes. We have analyzed spermatocytes at MI and MII stages from
three fertile men by M-FISH.
Materials and Methods
Subjects
Data collection and meiotic analysis
Spermatocyte images were captured under an epifluorescence microscope
(Olympus Bx60, Hamburg, Germany) using a Power Macintosh. The
M-FISH technique required the capture of each fluorochrome in separate
images and metaphase analysis was carried out in the merged image. MI
and MII were analyzed with the SpectraVysion software (Digital Scientific,
Cambridge, UK) by two different observers, and chromosome abnormalities were classified according to the International System for Human
Cytogenetic Nomenclature (ISCN) (Schaffer et al., 2009). Conservative
estimates of aneuploidy were calculated by doubling the hyperploidy frequency to avoid technical artifacts. Hypoploid metaphases were included
in this study only when showing other chromosome abnormalities. Only
spermatocyte II metaphases with 46 chromosomes distributed randomly
in a nucleated metaphase were considered diploid.
Statistical analyses
The two-tailed Fisher’s exact test was performed to determine whether
there were significant differences in chromosome abnormalities between
MI and MII spermatocytes or whether the frequencies of X- and
Y-bearing MII spermatocytes differed from the expected 1:1 ratio. Statistical analyses were performed using the Statistical Package for the Social
Sciences, and P-values ,0.05 were considered significant.
Results
Testicular samples were collected during a vasectomy procedure from
three healthy fertile males, aged 28, 36 and 41 years. Spermatocyte metaphases I from all three donors were previously analyzed by uniform stain in
a larger study (Uroz et al., 2011) (donors 16, 8 and 13, respectively), and
they were selected on the basis of the number and quality of their meiotic
cells. All three subjects produced at least one naturally conceived offspring.
One of the donors was a smoker (case 1) and none of them had been
exposed to known mutagens or radiation. Semen samples from the individuals analyzed in our study were not available. All donors signed an
informed consent form prior to the surgery and the study was approved
by our University’s and Institutional Ethics Committees.
A total of 565 spermatocytes I (317) and II (248) from three fertile
donors were karyotyped by M-FISH. Synaptic or recombination
errors were detected by counting the chiasma number in each bivalent. Both chromosome segregation disorders and premature loss
of chromatid cohesion were analyzed in MII spermatocytes.
Meiotic analysis was carried out in metaphases, simultaneously
stained with DAPI and hybridized by M-FISH (Fig. 1). DAPI counterstain enabled (a) the detection of meiotic chromosome abnormalities
and (b) the differentiation between the p and q arm in most chromosomes by marking centromeres as darker spots. Meanwhile, M-FISH
colored images allowed the identification of the chromosomes
involved in meiotic chromosome abnormalities.
Spermatocyte spreading
Chiasma number in MI spermatocytes
Testicular biopsy was obtained under local anesthesia during a vasectomy
procedure and meiotic cells were fixed according to the protocol previously described by our group (Uroz et al., 2008). Meiotic chromosome
preparations were preserved at 2208C until M-FISH hybridization.
In MI, sex chromosomes had one chiasma in 73.5% of cases (lineal bivalent configuration) (Fig. 1A and B) and two chiasmata in 1.3% (circular bivalent configuration). The remaining 25.2% of spermatocytes
I showed dissociated sex chromosomes. We also observed the presence of two univalents for autosomes 10, 13, 21 and 22 in 1.9% of MI
cells, and monochiasmate medium-sized bivalents for chromosome
pairs 7 and 8 in 0.6% (Fig. 1C and D).
The M-FISH technique
The M-FISH technique employs whole-chromosome probes labeled with
different combinations of 5 fluorochromes for the simultaneous identification of the 24 human chromosomes (Speicher et al., 1996). Meiotic
chromosome spreads were hybridized by M-FISH following the manufacturer’s instructions (SpectraVysion Assay, Vysis Inc., Downers Grove, IL,
USA), with minor modifications. Briefly, slides were washed in 2× standard saline citrate/0.1% NP-40 for 5 min, dehydrated in an ethanol series
and air-dried. Meiotic preparations were pretreated with 0.005% pepsin
for 5 min at 378C, post-fixed in 1% formaldehyde for 10 min and denatured in 70% formamide for 1.5 min at 738C. Meiotic chromosome
spreads were later counterstained with 4′ ,6-diamidino-2-phenylindole
(DAPI) in antifade solution (DAPI II, Vysis Inc., Downers Grove, IL, USA).
Numerical chromosome abnormalities in
MI and MII spermatocytes
The frequencies of numerical chromosome abnormalities in MI and
MII spermatocytes are shown in Table I. Hyperploidy frequency in
spermatocytes II (disomy) was significantly higher (P , 0.001) than
that found in spermatocytes I (trisomy). Four trisomic spermatocytes
I (1.3%) contained an extra chromosome (univalent) 18, 19, 22 or
Y. Disomy at MII (6.9%) was observed as either an extra chromosome
(3.6%) or an extra chromatid (3.2%) (Fig. 1G and H). It involved
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Uroz and Templado
Discussion
The use of M-FISH for karyotyping MI and MII spermatocytes from
fertile men provides a good approach to study the mechanisms of
non-disjunction in human male meiosis I. M-FISH is essentially useful
in (a) identifying chromosomes involved in aneuploidies, (b) increasing
the number of analyzable spermatocytes, especially at the MII stage,
where there is a high level of overlapping and touching chromosomes
and (c) distinguishing diploidy at MII from two juxtaposed metaphases.
Although M-FISH is too expensive and time-consuming to be used in
clinical routine screening, it is a powerful technique to identify
chromosome abnormalities, and it could be suitable for those cases
requiring an accurate meiotic analysis.
Low chiasma count in human
spermatocytes I
The main abnormality found in meiosis I is the presence of bivalents
with a decreased number or absence of chiasmata. Chromosome
21, the smallest one in the karyotype, and the sex chromosomes,
whose pairing is limited to the pseudoautosomal region, appear frequently as two separated univalents. In accordance with our results,
other meiotic studies in fertile male donors reported G-group and
sex chromosomes as the most susceptible to having no chiasmata at
MI (Skakkebaek et al., 1973; Uroz et al., 2011) or no recombination
foci at pachytene (Sun et al., 2006) (reviewed by Tempest, 2011).
The absence of chiasmata has been correlated with an abnormal
chromosome segregation in meiosis I both in trisomic 21 (Savage
et al., 1998; Oliver et al., 2009) and 47,XXY conceptuses (Hassold
et al., 1991; Lorda-Sanchez et al., 1992; Thomas et al., 2000). In the
present work, chromosomes X, Y and 21 were the most commonly
implicated in disomy in spermatocytes II, corroborating the results
reported in spermatozoa from healthy men using FISH (reviewed by
Templado et al., 2005, 2011).
Origin of disomy and diploidy in human
spermatocytes
Figure 1 Human spermatocytes at metaphase stage. Metaphase
spermatocytes were analyzed sequentially by DAPI counterstain (A,
C, E, G) and by M-FISH (B, D, F, H). Normal MI (A, B). MI with
a bivalent seven showing one single distal chiasma (C, D). Normal
MII (E, F). MII with an extra X chromatid (G, H).
essentially sex chromosomes (3.2%) and chromosome 21 (1.2%)
(Table I). Diploidy was found in 0.8% of the MII spermatocytes.
Chromosome cohesion abnormalities in
MII spermatocytes
Separated sister chromatids at MII were seen in 6.9% of the cells.
The 5.5-fold increase in the frequency of aneuploidy in spermatocytes
II versus spermatocytes I suggests that aneuploidy originates mainly by
non-disjunction during anaphase I, and is probably caused by missegregation of bivalents showing no chiasmata or decreased chiasma count
(27.7%). The occurrence of trisomy in spermatocytes I (2.5%) implies
that some aneuploidy in human spermatozoa originates by pre-existing
aneuploidy in the germ cells.
The presence of diploidy at MII and its absence in MI spermatocytes
shows that this abnormality arises principally during the first meiotic
division. Some authors (reviewed by Egozcue et al., 2000) have suggested that spermatocytes I with univalents generate diploid spermatocytes II, since spermatocytes I bypass the spindle assembly
checkpoint without carrying out cytokinesis in meiosis I.
On the other hand, the detection of spermatocytes II with separated sister chromatids provides the first evidence that balanced
PSSC (or balanced pre-division), described in fresh oocytes II
(Sandalinas et al., 2002; Garcia-Cruz et al., 2010), also occurs in
human male meiosis. Separated sister chromatids may be at risk of improper segregation at anaphase II, leading to aneuploid spermatozoa.
However, it is not possible to rule out that some of the spermatocytes
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Meiotic non-disjunction mechanisms in fertile males
Table I Meiotic chromosome abnormalities in 317 MI and 248 MII spermatocytes from three human fertile donors
analyzed by M-FISHa
MI spermatocytes
No. (%)
MII spermatocytes
No. (%)
.............................................................................................................................................................................................
Abnormal chiasma count
Sex chromosomes
88 (27.8)
Chomatid separation
17 (6.9)
80 (25.2)
Sex chromosomes
3 (1.2)
24,X,Y (75 cells)
23,2X,+Xcht,+Xcht,+21
24,X,Y,2II(21),+I(21)
22,2X,+Xcht
24,X,Y,+ace(1)
21,+Xcht,2Y,+Ycht,+Ycht,214,+14cht,+14cht
23,X,Y,2II(6),+ace(10),2II(21),+I(21)
Autosomes
24,X,Y,+ace(22)
25,X,Y,+der(7)
Autosomes
14 (5.7)
21,+Xcht,2Y,+Ycht,+Ycht,214,+14cht,+14cht
22,X,21,+1cht,+1cht
8 (2.5)
22,Y,22,+2cht,+2cht
23,XY,II(7,7)(xma ¼ 1)
22,X,23,+3cht,+3cht
23,XY,II(8,8)(xma ¼ 1)
22,X,23,+3cht
23,XY,2II(10),+I(10)
22,X,26,+6cht,+6cht
23,XY,chrb(3),2II(13),+I(13),+I(13)
20,X,26,+6cht,+6cht,27,+7cht,+7cht,213,+13cht,+13cht
23,XY,2II(21),+I(21)
22,Y,27,+7cht,+7cht
24,X,Y,2II(21),+I(21)
22,Y,chtb(7),28,+8cht,+8cht,chtb(8)
23,X,Y,2II(6),+ace(10),2II(21),+I(21)
22,Y,213,+13cht,+13cht
24,XY,2II(22),+I(22),+I(22)
22,Y,222,+22cht
22,Y,+ace(7),222,+22cht,+22cht
Numerical abnormalities
4 (1.3)
Trisomy
Numerical abnormalities
Disomy
24,X,II(Y)
17 (6.9)
Extra whole chromosome
23,XY,2II(4),+?I(8)
24,XY (3 cells)
24,XY,+?I(19)
24,XY,chtb(X)(q?)
24,XY,+I(22)
Conservative aneuploidy
36 (14.5)
9 (3.6)
24,XY,26,+7,211,+12
8 (2.5)
23,2X or –Y,+9
23,2X,+Xcht,+Xcht,+21
Extra chromatid
8 (3.2)
23,Y,+Xcht
21,+Xcht,2Y,+Ycht,+Ycht,214,+14cht,+14cht
23,X,+Xcht
23,X,+4cht
23,Y,+15cht
23,X,+16cht
23,X,+21cht
23,Y,+21cht
Conservative aneuploidy
Diploidy
34 (13.7)
2 (0.8)
46,XY (2 cells)
M-FISH, multiplex fluorescence in situ hybridization.
a
Spermatocytes showing more than one meiotic chromosome abnormality were listed in each category.
with separated sister chromatids could correspond in fact to early anaphase II.
Non-disjunction mechanisms in meiosis I
We have detected three different non-disjunction mechanisms in
fertile men leading to aneuploidy during the first meiotic
division: PSSC, achiasmate non-disjunction and, less frequently, ‘true’
non-disjunction (Fig. 2).
PSSC, characterized by the presence of an extra (or missing) chromatid at MII (Angell, 1991), was detected in around 50% of disomies
observed in spermatocytes II. This non-disjunction mechanism is
common in human oocytes (Pellestor et al., 2006; Jones, 2008;
Garcia-Cruz et al., 2010), and has been recently described by our
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Uroz and Templado
Figure 2 Non-disjunction mechanisms in human male meiosis I producing aneuploid spermatozoa. (A) Normal segregation of homologous chromosomes during meiosis I. (B) PSSC involves segregation of sister chromatids from an univalent in meiosis I. (C) Achiasmate non-disjunction (ND) originates by random segregation of two separated homologous chromosomes. (D) ‘True’ ND, probably caused by abnormal segregation of lineal
medium-sized bivalents with a single distal chiasma.
group in human male meiosis in an infertile man (Uroz et al., 2008).
According to our data, chromosomes X, Y and 21 are the most commonly affected by PSSC in MII spermatocytes and correspond to those
frequently found as separated univalents at MI. A cytogenetic study of
mouse oocytes proposed that extra or missing chromatids in oocytes
II arise by bi-orientation of univalents and their mitotic-like segregation
during meiosis I (Kouznetsova et al., 2007).
The remaining 50% of disomies found in spermatocytes II had an
extra whole chromosome that could originate from achiasmate nondisjunction. This is based on the high incidence both of sex chromosome univalency at MI and of XY disomy at MII. Accordingly, high
levels of sex chromosomes with no recombination focus in pachytene
spermatocytes have been related to increased XY disomy in spermatozoa (Sun et al., 2008).
The medium-sized monochiasmate bivalents observed in this study,
previously described in infertile males (Templado et al., 1981), could
be at risk of missegregation. The open configuration of these bivalents
may favor an interlocking with other bivalents aligned in the metaphase
plate, resulting in non-disjunction during meiosis I (McDermott, 1966).
A recent study in yeast has demonstrated that bivalents with a single
distal chiasma are prone to mono-orientation (Lacefield and Murray,
2007). This mechanism could probably correspond to ‘true’ nondisjunction. Alternatively, bivalents having a single distal chiasma
could undergo a premature chiasma resolution resulting in achiasmate
non-disjunction of the two separated homologous chromosomes
(McDougall et al., 2005).
Meiotic non-disjunction in humans: few
differences between male and female
As observed in the current work and in human female meiosis
(reviewed by Pellestor et al., 2006; Jones, 2008; Fragouli et al.,
2011), spermatogenesis and oogenesis show resemblances with
regard to meiosis I non-disjunction events, such as the similar contribution of PSSC and achiasmate non-disjunction to the genesis of aneuploidy and the predominant involvement of chromosomes of
smaller size.
Unexpectedly, the level of numerical abnormalities in MII spermatocytes is similar to that described in fresh donated oocytes II from
young women (Sandalinas et al., 2002) and three times higher than
that reported in spermatozoa from healthy donors (Templado et al.,
2005, 2011). This decrease of numerical abnormalities during spermatogenesis would imply the existence of a postmeiotic checkpoint at
the spermatid stage (de Rooij and de Boer, 2003; Guichaoua et al.,
2005) or in spermatozoa (Rodrigo et al., 2004), arresting specifically
those germ cells with aneuploidy and diploidy. This postmeiotic checkpoint would also be responsible for the lower paternal origin of trisomies in humans (reviewed by Hassold et al., 2007), instead of
differences in the incidence of meiotic chromosome segregation
errors between males and females. Confirmation of the existence of
this male checkpoint and the determination of the spermatogenesis
stage in which it occurs has clinical implications in reproductive techniques, especially in ICSI of epididymal and testicular spermatozoa.
Meiotic non-disjunction mechanisms in fertile males
In conclusion, aneuploidy and diploidy in spermatocytes II could be
caused mainly by the absence of chiasmata at MI and, to a lesser
extent, by errors in segregation of bivalents with a low chiasma
number. As in female meiosis, the two main non-disjunction mechanisms during the first meiotic division in human spermatogenesis are
PSSC and achiasmate non-disjunction, principally affecting chromosome 21 and sex chromosomes. The levels of numerical chromosome
abnormalities in spermatocytes II and oocytes II from young women
are similar, whereas spermatozoa show lower levels. This decrease
would indicate the existence of a checkpoint during human spermatogenesis, which monitors and arrests those spermatocytes II with numerical abnormalities.
Acknowledgements
The authors wish to thank Raquel Garcia-Cruz for her technical assistance and Dr Anna Estop for her careful reading of the manuscript.
Authors’ roles
Both authors, L.U. and C.T., have participated in study design, execution, analysis, manuscript drafting and critical discussion.
Funding
This work was supported by the Generalitat de Catalunya, Spain
[2009SGR-01107, 2005FI00399 to L.U.].
Conflict of interest
None declared.
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