Hum. Reprod. Advance Access originally published online on February 11, 2008
Human Reproduction 2008 23(4):982-987; doi:10.1093/humrep/dem427
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Premature separation of sister chromatids in human male meiosis
1 Departament de Biologia Cellular, Fisiologia i Immunologia, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain 2 Servei d'Andrologia, Fundació Puigvert, 08025 Barcelona, Spain
3 Correspondence address. E-mail: cristina.templado{at}uab.es
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Abstract |
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BACKGROUND: Meiotic disorders result in meiotic arrest and aneuploid spermatozoa, leading to male infertility, spontaneous abortions or affected offspring. We carried out meiotic studies in an infertile male to detect meiotic nondisjunction mechanisms leading to aneuploidy in spermatogenesis.
METHODS AND RESULTS: Meiotic studies were performed in testicular and semen samples from a 38-year-old teratozoospermic male with normal somatic karyotype and a history of spontaneous abortions. We analysed 262 spermatocytes (69 pachytene cells, 106 metaphases I (MI), 87 metaphases II (MII)) by multiplex-fluorescence in situ hybridization and 20 193 spermatozoa by multicolour-FISH with probes for chromosomes 9, 10, 15, 21, X and Y. The results indicate high increase of 21 and XY disomies, as well as diploidy in both spermatocytes at MII and spermatozoa (P < 0.0001). Achiasmate segregation of sex chromosomes was found in 3.4% of spermatocytes II, preceded by early-dissociated XY bivalent at MI (41.5% of cells). We also detected premature separation of sister chromatids (PSSC) in 4.6% of MII.
CONCLUSIONS: This individual presents high levels of numerical abnormalities in germ cells, caused by two different nondisjunction mechanisms during meiosis I. To our knowledge, this work represents the first time that PSSC has been demonstrated in human male germ cells.
Key words: aneuploidy/male infertility/meiosis/nondisjunction/premature separation of sister chromatids
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Introduction |
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Meiotic chromosome disorders causing human male infertility and sterility are abnormalities present only in germ cells and not detectable in somatic karyotype. Meiotic chromosome disorders lead to meiotic arrest, resulting in oligozoospermia and azoospermia and spermatozoa with numerical chromosomal abnormalities, resulting in spontaneous abortions or affected offspring.
The most common chromosome abnormalities in spontaneous abortions are sex-chromosome monosomy (45,X) and trisomies 16, 21 and 22; whereas among newborns, trisomy 21 and sex-chromosome trisomies account for the great majority of additional chromosomes (Hassold and Hunt, 2001
). Although automosomal aneuploidies are preferentially of maternal origin, paternal errors account for nearly 50% of 47,XXYs (Arnedo et al., 2006), 75–80% of 45,Xs (Hassold et al., 1992; Martínez-Pasarell et al., 1999) and 100% of 47,XYYs. Most of the aneuploidies in humans originate at meiosis and the main mechanism leading to aneuploidy in spermatozoa is considered nondisjunction (Márquez et al., 1996). Several possible patterns of abnormal segregation have been described in meiosis (Hassold and Hunt, 2001), including ‘true' nondisjunction, achiasmate nondisjunction and premature separation of sister chromatids (PSSC) (Angell, 1991). The relevance of each nondisjunctional mechanism varies depending on the affected chromosomes (Hassold et al., 2007).
Most meiotic studies in humans have been carried out on metaphase I (MI) spermatocytes to elucidate the etiology of male infertility (Skakkebaek et al., 1973; Chandley et al., 1976; Templado et al., 1976; Egozcue et al., 1983, 2000). These classical cytogenetic studies indicated that 18.5% of infertile patients have meiotic arrest (Egozcue et al., 1983) and 6–8% show meiotic abnormalities in spermatocytes I (Egozcue et al., 2000), which can represent up to 17.5% in oligoasthenozoospermic males (Vendrell et al., 1999). In accordance with these results, fluorescent in situ hybridization (FISH) studies in decondensed sperm nuclei have shown increased levels of aneuploidy in infertile patients with abnormal sperm parameters (Egozcue et al., 2000) and meiotic abnormalities in spermatocyte I (Aran et al., 1999), and in couples with recurrent abortions (Rubio et al., 1999).
Cytogenetic studies in MI and MII spermatocytes provide a useful tool for detecting meiotic chromosome abnormalities (numerical and structural) and synaptic errors, as well as meiotic chromosome missegregation. This approach additionally facilitates analysis of recombination levels by counting chiasmata at MI. Recently, the application of multiplex-FISH (M-FISH) (Speicher et al., 1996) to meiotic chromosomes (Sarrate et al., 2004) has allowed the identification of individual chromosomes, characterization of structural chromosome abnormalities and analysis of chiasmata pattern for individual bivalents. Moreover, this technique increases the number of analysable spermatogenic cells, particularly those at MII stage.
Here, we report on meiotic abnormalities analysed using M-FISH in spermatocytes I and spermatocytes II and multicolour FISH in spermatozoa from an infertile male with normal somatic karyotype and a history of spontaneous abortions, with special emphasis on the detection of meiotic nondisjunction mechanisms leading to aneuploidy.
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Case report
Testicular tissue and a sperm sample were obtained from a 38-year-old infertile male with normal somatic karyotype and normal FSH profile. The patient was referred to our laboratory as his wife had had two spontaneous abortions that had not been karyotyped. His semen analysis showed teratozoospermia (1% of morphologically normal forms, according to the strict criteria) (Kruger et al., 1988
). Sperm count (28 x 106spermatozoa/ml) and progressive motility (15.4 x 106spermatozoa/ml of forward motility) were within the normal parameters (WHO, 1999). The donor had no history of exposure to any known mutagens, clastogens, radiation or drugs. The current study was approved by our university's Ethics Committee, and informed consent was obtained from the patient before undergoing this study.
Control donors
FISH analysis of sperm aneuploidies for chromosomes 6, 21, X and Y in 18 healthy donors aged 24–74 years (control group) has already been reported by our group (Bosch et al., 2001).
Meiotic preparations
Testicular biopsy was obtained under local anesthesia and processed using a modification of the technique described by Evans et al. (1964). Briefly, testicular tissue was treated with hypotonic solution (1% sodium citrate) for 30 min and minced carefully for 15 min. After sedimentation of the seminiferous tubules, the cells were fixed three times in methanol:glacial acetic acid (3:1) and smeared onto clean slides.
M-FISH in spermatocytes
Slides were treated with 0.005% pepsin for 2 min and subjected to M-FISH protocol in accordance with manufacturer's instructions (SpectraVysion Assay, Vysis Inc; Downers Grove, IL, USA). Spermatogenic cells were counterstained with 4,6-diamino-2-phenylindole in antifade solution (DAPI III, Vysis Inc; Downers Grove, IL, USA). An epifluorescence microscope (Olympus Bx60, Hamburg, Germany) and a Power Macintosh G3 with SpectraVysion software (Digital Scientific, Cambridge, UK) were used for cell evaluation and image capturing. Each spermatocyte was previously analysed using DAPI counterstain to count chiasmata and to detect most of the meiotic abnormalities, given the good preservation of chromatin. M-FISH analysis of the same cells allowed chromosome identification. Chromosomal abnormalities were karyotyped as described by the International System for Human Cytogenetic Nomenclature (ISCN) (Schaffer LG, 2005).
Sperm preparations
The semen sample was washed three times in hypotonic solution (0.075 M KCl), fixed three times with methanol:glacial acetic acid (3:1), smeared onto clean slides and preserved at –20°C until in situ hybridization.
Probes
FISH analysis was performed using centromeric probes for chromosomes 9 (CEP9, Spectrum Green), 10 (CEP10, Spectrum Aqua), 15 (CEP15, Spectrum Green), X (CEPX, Spectrum Aqua) and Y (CEPY SatIII, Spectrum Orange), and a locus-specific probe for chromosome 21 (LSI21, Spectrum Orange) (Vysis Inc; Downers Grove, IL, USA).
FISH in spermatozoa
Slides were treated with 0.005% pepsin for 5 min, washed twice with sodium standard citrate (SSC), and post-fixed with 1% formaldehyde for 2 min, washed twice in phosphate-buffered saline and dehydrated in an ethanol series. Sperm nuclei were decondensed in a solution of 5 mM dithiothreitol, 0.1 M Tris (pH 7.4) and 1% Triton X-100 at 37°C for 15–16 min, washed with 2xSSC and dehydrated in an ethanol series. Sperm DNA was denatured with 70% formamide at 74°C for 5 min and hybridized in a moist chamber at 37°C for 72 h, with probes for chromosomes 15, X and Y on one slide, and probes for chromosomes 9, 10 and 21 on another. Post-hybridization washes followed manufacturer's instructions. Sperm nuclei were counterstained with DAPI in antifade solution (DAPI II, Vysis Inc; Downers Grove, IL, USA). An epifluorescence microscope (Olympus Bx60, Hamburg, Germany) was used for signal counting and a Power Macintosh G3 with Smartcapture software (Digital Scientific, Cambridge, UK) was used for image capturing. A minimum of 10 000 spermatozoa was scored per assay, using strict criteria that we have previously described (Bosch et al., 2001). All ambiguous signals were examined by a second observer.
Statistical analysis
To determine whether there were significant differences in the mean number of chiasmata between normal spermatocytes and those with dissociated sex chromosomes, a Student's t-test was applied.
To compare disomy and diploidy frequencies in spermatozoa found in our patient with those obtained in the control group, we used the Chi-square test with Yates correction.
To investigate whether there were significant differences in the frequencies of numerical abnormalities between MII spermatocytes and spermatozoa, we used the Chi-square test with Bonferroni correction.
A value of P < 0.05 was considered significant.
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A total of 262 spermatocytes were analysed by M-FISH at different meiotic stages: 69 at pachytene, 106 at MI and 87 at MII. FISH analysis using probes for chromosomes 9, 10, 15, 21, X and Y was carried out in 20 193 decondensed sperm nuclei.
Chromosome abnormalities in MI, MII and spermatozoa
Table I summarizes the numerical and structural chromosome abnormalities observed in spermatocytes I and II at metaphase. The frequency of structural abnormalities was 7.5% at MI and 4.6% at MII. The most common type of structural aberration observed was chromosome breaks, followed by deletions and acentric fragments (Fig. 1A–C).
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Since hypoploidy may be due to technical artefacts, incidence of aneuploidy was considered as twice that of hyperploidy (Márquez et al., 1996
). The percentage of hyperploidy was 0% at MI and 6.9% at MII, with a frequency of conservative aneuploidy of 0% and 13.8% at MI and MII, respectively. Nondisjunction during the first meiotic division was detected for chromosome 21 and sex chromosomes as single extra or missing chromatid in 4.6% of MII cells (Fig. 1D–F), or as one whole extra chromosome in 3.4% of MII cells. The percentage of diploidy was 0% and 8% in spermatocyte I and II, respectively. All diploid spermatocytes II were 46,XY.
FISH analysis in spermatozoa (Table II) showed significantly higher levels (P < 0.0001) of disomy 21, disomy XY and diploidy compared with our control group (Bosch et al., 2001). There was a significant decrease (P < 0.0001) in the frequencies of disomy XY and diploidy in spermatozoa compared to those found in MII spermatocytes.
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Synaptic errors observed in MI
All pachytene spermatocytes analysed had condensed sex chromosomes with apparently normal pairing, but the presence of crossing-over between them could not be determined in this meiotic stage using this approach. Achiasmate sex chromosomes were detected in 41.5% of spermatocytes at MI (Fig. 1A–C), and autosomal desynapsis was found in 7.5% of cells, in all cases affecting one autosomal bivalent of C group (bivalents 6, 8, 8, 9, 10, 11, 11 and 12) showing one single chiasma. Circular sex chromosomes with two crossovers were observed in two spermatocytes I (1.9%).
The chiasmata counts were carried out in 30 well-spread spermatocytes I: 15 of these with a normal XY bivalent and the remaining 15 with dissociated sex chromosomes (Table III). The mean number of chiasmata per spermatocyte was 51.3, this being significantly lower in MI with dissociated XY pair (mean count/cell 49.0; range 44–55) than in normal MI (mean count/cell 53.7; range 51–58) (P < 0.0001). This decrease in the number of chiasmata was generalized for all chromosomes, but was statistically significant only for chromosome 4 (P = 0.02), chromosome 9 (P = 0.03), chromosome 10 (P = 0.02) and chromosome 12 (P = 0.04).
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Discussion |
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In this study, we carried out meiotic analysis in spermatocytes I, spermatocytes II and spermatozoa on an infertile patient with severe teratozoospermia (1% of normal forms) and a history of recurrent abortions, in order to detect meiotic errors. The use of M-FISH technique allowed us to identify each chromosome and to increase the number of analysable spermatocyte metaphases.
Structural and numerical chromosomal abnormalities
The most common types of structural abnormalities in both MI and MII (chromosome breaks, deletions and acrocentric fragments) were also the most frequent types found in sperm karyotypes (Estop et al., 1995). The frequency of structural chromosomal aberrations found here at MII was similar to that described both for infertile males at the same meiotic stage (5.5%) (Laurie et al., 1985), and for control donors analysed in sperm complements (6.6%) (Templado et al., 2005). None of the MI spermatocytes was hyperploid or diploid in this patient, indicating that numerical chromosome abnormalities in later meiotic stages must have originated after spermatogonial divisions.
The only chromosomes involved in disomies at MII were 21 and sex chromosomes, which usually only present a single chiasma in meiosis I (Laurie and Hultén, 1985; Sun et al., 2004). FISH analysis of these chromosomes in spermatozoa showed significantly increased levels of disomy 21 and disomy XY (a frequency six times higher than XX and YY disomies). This increase of disomy has been described in other FISH studies carried out in spermatozoa from infertile patients with teratozoospermia (Templado et al., 2002) and in couples with repetitive abortions (Rubio et al., 1999).
The frequencies of disomy XY and diploidy in spermatozoa was lower than that observed at MII, probably due to: 1) a light spermatogenic arrest between spermatocyte II and spermatozoa, or 2) hybridization failures in FISH on spermatozoa. In our case, the low sperm count (28 x 106 spermatozoa/ml), slightly higher than that established as normal (
20 x 106 spermatozoa/ml) (WHO, 1999), would support the first hypothesis.
Only one similar study (Ma et al., 2006) has been carried out on an infertile male with severe oligoasthenoteratozoospermia and an abortus 45,X of paternal origin, using both immunofluorescent studies of synaptonemal complexes and FISH in spermatozoa. The meiotic results obtained from this individual are similar to those that we have observed: an apparently normal sex body with no XY recombination and high levels for 21 and XY disomies and diploidy in spermatozoa. Unfortunately, no available data on nondisjunctional mechanisms can be obtained with the approaches used by Ma et al. (2006).
Early dissociation of the XY pair and chiasmata counts
In our patient, frequency of early dissociated sex chromosomes (41.5%) is higher than that found in other infertile males analysed by classical meiotic studies (range 6.7–28%) (Hultén et al., 1966; Chandley et al., 1976; Laurie and Hultén, 1985). Despite this high frequency of unpaired XY bivalent at MI, all spermatocytes at pachytene stage had an apparently normal sex body. These data seem to reflect that early XY dissociation occurs after their pairing (desynapsis) by the lack of chiasmata between the two homologous chromosomes.
The mean number of chiasmata per cell in our subject is similar to that previously described in infertile (51.3) (Laurie and Hultén, 1985) and control males (51.2) (Skakkebaek et al., 1973) in MI spermatocytes. However, spermatocytes I with dissociated sex chromosomes showed a significant decrease in the total number of chiasmata, this even being significant for some medium-sized autosomal bivalents (pairs 4, 9, 10 and 12). Desynapsis of individual bivalents was described previously by our group in infertile males (Templado et al., 1981). Desynaptic bivalents could migrate to the same pole because of mono-orientation in the metaphase plate (‘true' nondisjunction), leading to disomic and nullisomic gametes. In fact, a recent study in yeast has demonstrated that chromosomes lacking crossovers near the centromere are more prone to mono-orient (Lacefield and Murray, 2007). Existence of specific chromosomes that are more sensitive to meiotic nondisjunction was described by FISH studies in spermatozoa from fathers of aneuploid children of paternal origin (Soares et al., 2001a,b).
Nondisjunctional mechanisms in human male meiosis
The analysis of numerical chromosome abnormalities in MII spermatocytes and spermatozoa allowed us to study chromosome missegregation in the first and second meiotic division, respectively. We observed two of the three types of nondisjunction proposed by Hassold and Hunt (2001), since ‘true’ nondisjunction was not detected in this study. Both nondisjunctional mechanisms found in this individual (achiasmate nondisjunction and PSSC) originated during the first meiotic division.
Achiasmate nondisjunction was observed as increased levels for both dissociated sex chromosomes at MI and for disomy XY in MII and spermatozoa. The relationship between disomic XY spermatozoa and the absence of recombination has been demonstrated by DNA polymorphism studies in paternally derived 47,XXY individuals and their fathers (Hassold et al., 1991), and by recombination studies carried out in 24,XY spermatozoa (Shi et al., 2001).
Early XY dissociation could also explain the high levels of diploidy present in spermatocyte II and spermatozoa. In fact, infertile males with meiotic abnormalities such as the individual studied in our case, also present an increased frequency of diploid spermatozoa (Aran et al., 1999). In agreement with this, Egozcue et al. (2000) have suggested that erratic chromosomes may be unable to migrate to the poles at anaphase and thus a single, diploid spermatocyte II is produced, giving rise to diploid spermatozoa.
Surprisingly, the most common type of nondisjunction observed was PSSC, detected in MII as one extra or missing chromatid affecting chromosomes 21, X and Y. These observations were corroborated by increased levels of disomy 21 and disomy XY in spermatozoa. The presence of achiasmate sex chromosomes and the PSSC for these chromosomes described in this work have been reported in a recent study on mouse oocytes (Kouznetsova et al., 2007). The authors proposed that achiasmatic chromosomes (univalents) are bi-oriented in a mitotic manner in MI and evade the spindle assembly checkpoint, contributing to aneuploidy in oocytes.
Chromatid predivision was first described by Angell (1991) at female meiosis II, and is considered to be as important as whole chromosome nondisjunction in the genesis of aneuploidies in human oocytes (Pellestor et al., 2006). The only reference to premature separation of chromatids in male meiosis was described at pachytene stage in 11% of spermatocytes in an infertile male with a high incidence of desynapsis (86%) and absence of sex body (87%) (Guichaoua et al., 2005); however, no evidence that this abnormality results in nondisjunction can be demonstrated by studies at early prophase I. To our knowledge, the present study provides the first evidence that PSSC occurs in human male meiosis. This segregation error has probably not previously been detected in spermatogenesis because of the low number of analysable MII in classical meiotic preparations. The use of M-FISH increases the number of analysable MII, even if these are not well-spread, and allows chromosome identification.
In conclusion, our subject has an increased risk of recurrent spontaneous abortion and affected offspring due to the presence of high levels of diploidy and aneuploidy observed in his germ cells. The presence of univalents in meiosis I could result in both aneuploid spermatozoa through PSSC or achiasmate nondisjunction, and diploid spermatozoa. In our study, PSSC was observed directly for first time in human spermatocytes at MII, using M-FISH technique. Future meiotic studies on nondisjunctional mechanisms will clarify whether PSSC in human male meiosis is as common as in female germ cells.
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Ministerio de Ciencia y Tecnología (BFI2002-01193); Generalitat de Catalunya (2005SGR-00495, 2005FI00399 to L.U.).
Authors Roles
L.U., experiment performing, analysis of data, manuscript writing; O.R., samples collection, analysis of data, manuscript writing; C.T., experiment design, analysis of data, manuscript writing.
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Submitted on October 18, 2007; resubmitted on November 20, 2007; accepted on December 17, 2007.
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