1 Service de Médecine et de Biologie de la Reproduction B, Hôpital Arnaud de Villeneuve, 34295 Montpellier Cedex 5 2 CNRS-UPR 1142, Institut de Génétique Humaine, 141 rue de la Cardonille, F-34396 Montpellier Cedex 5 3 Service de Gynécologie Obstétrique, Hôpital Arnaud de Villeneuve, 34295 Montpellier Cedex 5 4 Service de Génétique Moléculaire et Chromosomique, Hôpital Arnaud de Villeneuve, 34295 Montpellier Cedex 5 and 5 Service de Génétique Clinique, Hôpital Arnaud de Villeneuve, 34295 Montpellier Cedex 5, France
6 To whom correspondence should be addressed. Email: franck.pellestor{at}igh.cnrs.fr
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Abstract |
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Key words: FISH/meiotic segregation/Robertsonian translocation/sperm
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Introduction |
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Men carrying Robertsonian translocation have a normal phenotype. However, they may have spermatogenesis alterations expressed by oligozoospermia or azoospermia, and they may be affected by reproductive failure owing to imbalances in chromosome meiotic segregation (Scriven et al., 2001).
The direct analysis of sperm chromosomal constitution can be used to determine meiotic segregation patterns of translocated chromosomes, and to predict the risk of having unbalanced conceptuses. Direct data were first obtained using the human spermhamster egg fertilization system. To date, six men heterozygous for Robertsonian translocation have been investigated using this method (Balkan and Martin, 1983; Pellestor et al., 1987
; Martin, 1988
; Pellestor, 1990
; Martin et al., 1992
; Syme and Martin, 1992
). Although this approach allowed the direct karyotyping of individual human spermatozoa, the procedure remained time-consuming, labour intensive and did not result in the chromosome analysis of large numbers of sperm nuclei (from 24 to 149) (Guttenbach et al., 1997
).
Fluorescence in-situ hybridization (FISH) with chromosome-specific DNA probes has offered a new strategy for investigating the meiotic segregation of translocations. Several studies have been carried out, essentially focusing on the most frequent Robertsonian translocations, i.e. the t(13;14) and t(14;21) (Rousseaux et al., 1995; Escudero et al., 2000
; Honda et al., 2000
; Morel et al., 2001
; Frydman et al., 2001
; Anton et al., 2004
). Only one case of Robertsonian translocation between two homologous chromosomes, a t(21q;21q), has been investigated by sperm FISH analysis (Acar et al., 2002
).
In this report we present the first analysis of sperm chromosome segregation in a man heterozygous for an uncommon (13;22) Robertsonian translocation. Both locus-specific probes and whole chromosome painting probes were used in parallel, in order to compare the efficiency and the accuracy of the two procedures, and to provide complementary data on the male meiotic segregation of this rare chromosomal rearrangement.
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Materials and methods |
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The patient gave his informed consent prior to participation in the study, which was approved by the Ethical Board of the Montpellier Hospital.
Sperm from a fertile, 35-year-old man with normal sperm parameters and a normal karyotype was used as control.
Sperm preparation
Sperm samples were collected in sterile containers after 3 days of sexual abstinence. After liquifying at room temperature, the samples were washed three times in 1 x phosphate-buffered saline by centrifugation (5 min at 2000 rpm). The final pellets were fixed for 1 h in fresh fixative (methanol:glacial acetic acid 3:1) at 20°C. The sperm suspensions were then dropped onto clean microscopic slides and air-dried. Slides were kept for 2 days at room temperature before use in FISH reactions.
Before in-situ labelling reactions, the sperm nuclei decondensation was performed by slide incubation in 25 mmol/l dithiothreitol (Sigma, St Louis, MO, USA), in 1 mol/l TrisHCl solution at room temperature for 5 min. Slides were then washed twice in 2 x SSC, dehydrated through an ethanol series and air-dried. The decondensation of sperm nuclei was checked under a phase-contrast microscope.
FISH procedure
Two distinct probe mixtures were used in this study. The first mixture consisted of Vysis locus-specific probes (LSI), for chromosome 13 (LSI 13, spanning the 13q14 region and labelled in SpectrumOrange) and chromosome 22 (LSI 22, spanning the 22q13.3 region and labelled in SpectrumGreen). The second probe mixture was composed of two Vysis whole chromosome painting (WCP) probes, i.e. a chromosome 13 painting probe (WCP 13) labelled in SpectrumGreen, and a chromosome 22 painting probe (WCP 22) labelled in SpectrumOrange (Vysis, Downers Grove, IL, USA). The probes were prepared according to the manufacturer's instructions.
The labelling efficiency of both LSI probes and WCP probes was determined by scoring the proportion of labelled nuclei on samples of 200 metaphasic and interphasic lymphocytes from the patient and the control subject, in order to assess the efficiency of probe binding and characterisation of the derivative chromosomes. There was no significant difference (P>0.05) in hybridization efficiency (from 99.60% to 99.88%) between the patient and the control, and the detection of segregation patterns was efficient in both metaphases and interphase nuclei.
Before hybridization, both probe and slide preparations were denatured separately. The probe mixture were denatured for 5 min at 73°C in a water bath, whereas the slides were denatured by immersion in 70% formamide/2 x SSC at 72°C for 3 min, dehydrated again and air-dried.
Each hybridization mixture was applied to the denatured sperm nuclei preparations and slides were covered with coverslips, sealed with rubber cement and hybridized overnight in a dark, moist chamber. Coverslips were then gently removed and the slides were washed for 10 min in a 50% formamide/50% 2 x SSC solution at 46°C, followed by a 10 min wash in 2 x SSC at 46°C, and a 5 min wash in 2 x SSC/0.5% Tween 20 solution, and finally mounted with DAPI (100 ng/ml) in antifade solution.
The slides were examined by two independent observers using a Leitz fluorescence microscope DMRB (Leica SA, Rueil-Malmaison, France), equipped with a DAPI single band-pass, a fluorescein single band-pass filter, a rhodamine single band-pass filter, a fluorescein/rhodamine double band-pass filter, and a triple filter set for simultaneous observation of fluorescein, rhodamine and DAPI signals. Only individual and well-delineated sperm nuclei were scored. Previously described standard assessment criteria were followed for the analysis of in-situ sperm labelling (Pellestor et al., 2001). The scoring criteria were similar for LSI and WCP probes. Briefly, overlapping sperm nuclei, disrupted nuclei or large nuclei with diffuse signals were not considered. Sperm nuclei were scored as having two identical signals when the two spots were of equal size and intensity and were separated by at least the diameter of one hybridization domain. In painting assays, nuclei with two signals of different colours clearly coupled one with the other, were considered as displaying a balanced chromosomal pattern.
Data analysis
The 2-test was used to statistically analyse the segregation patterns observed in the patient and compare the fluorescent phenotypes between the translocation carrier and the control subject. Comparison of data between the two procedures was also performed using
2-test. Differences were considered to be significant when P<0.05.
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Results |
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Among the 11 787 sperm nuclei analysed in the translocation carrier, 4735 nuclei were scored using the WCP probe set (Figure 1) and 7052 nuclei using the LSI probe set (Figure 2). The results are summarized in Table I. In the two assays, similar frequencies of normal and balanced spermatozoa resulting from alternate segregation were found (86.70% and 85.19%, respectively). Unbalanced fluorescent patterns, resulting from adjacent segregation modes, were observed in 12.79% of nuclei scored with LSI probes and 14.36% of nuclei from the WCP assay. There was no significant difference (P>0.05) between these values. In the two assays, the distribution of the different unbalanced patterns (nullisomies and disomies 13 or 22) was similar, with rates of imbalances ranging from 2.66% to 4.24% (Table I).
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Discussion |
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To carry out the present FISH study, we utilized two different procedures, i.e. LSI and WCP probes. The LSI method has been used in the majority of previous FISH sperm studies performed in Robertsonian translocation carriers (Rousseaux et al., 1995; Escudero et al., 2000
; Honda et al., 2000
; Frydman et al., 2001
; Acar et al., 2002
; Anton et al., 2004
), whereas WCPs have been used only once for sperm analysis in three Robertsonian translocation (13;14) carriers (Morel et al., 2001
). The use of LSI on sperm can be limited by the DNA compaction and inefficient decondensation of sperm nuclei, or the small size of fluorescent signals usually obtained with these probes. Signals generated by WCP appeared easier to detect in situ on sperm nuclei because of their size and intensity. Previous uses of painting probes on human sperm have demonstrated that this type of probe possessed both the specificity and the sensitivity required for the in-situ scoring of chromosomal imbalances in human sperm (Rives et al., 1998
; 1999
; Morel et al., 2001
). In addition, the use of WCP requires only a moderate sperm decondensation to provide a high hybridization efficiency (Rives et al., 1998
).
The combined use of these two labelling procedures allowed us to compare the efficiency of the two techniques on human sperm, and provided an internal control for the analysis of meiotic segregation. Both procedures gave similar results in terms of balanced and unbalanced nuclei. This indicates that FISH with WCP is an accurate approach for sperm study in Robertsonian translocation carriers, and so can be used to complement the LSI technique. Morel et al. (2001) reported that the WCP method allowed the in-situ differentiation of both normal spermatozoa (with two signals of different colour separated by at least one diameter) and balanced spermatozoa (with two signals of different colour coupled one with the other). In our study, although such a distinction was possible in numerous sperm nuclei, a significant proportion of nuclei also displayed partially overlapping signals. This observation lead us to consider that the WCP approach was not efficient enough to precisely estimate the proportion of normal and balanced nuclei resulting from the alternate segregation mode. Consequently, no distinct classification was made between normal and balanced sperm nuclei.
The observed high frequency (89%) of sperm nuclei resulting from alternate segregation is in good agreement with results from previous analysis of sperm in Robertsonian translocation carriers, using either the humanhamster fertilization system or FISH procedure (Pellestor, 1990; Honda et al., 2000
; Anton et al., 2004
). Similar high incidences of alternate meiotic segregation in sperm of Robertsonian translocation carriers were also observed in other mamalian species such as mice and bulls (Gropp and Winking, 1981
; Tateno et al., 1994
). All these data support the existence of a similar meiotic behaviour of rearranged chromosomes in Robertsonian translocations. The predominance of alternate segregation over other segregation types does not appear to be influenced by the difference in chromosomes involved in the translocation. Meiotic analyses of trivalent synaptonemal complexes in Robertsonian translocations have shown the predominant pairing of the acrocentric elements in cis-configuration. Such configuration favours alternate meiotic segregation (Vidal et al., 1982
; Luciani et al., 1984
; Navarro et al., 1991
).
The overall frequency of unbalanced spermatozoa resulting from adjacent segregation modes is 12%. This is also consistent with the results of previous FISH studies (results ranging from 10.8% to 22.6%). The similarity of meiotic configurations in Robertsonian translocation could explain the relatively homogeneous rates of imbalances. In addition, both the number and the location of chiasmata could contribute to produce similar proportions of imbalances in all Robertsonian translocations. Because of the correlation between the line of chromosomal segregation and the chiasmata line, there is a strong prevalence of alternate segregation, resulting in a low rate of unbalanced spermatozoa, and consequently in a low risk of imbalance in progeny of male carriers. However, the incidences of imbalances in sperm of Robertsonian translocation carriers are always higher than the incidences of imbalances drawn from studies of fetuses (Boué and Gallano, 1984) or newborns (Daniel et al., 1989
). This finding indicates that there is a strong in-utero selection against unbalanced conceptuses. In this way, it is interesting to note that studies of female carriers of Robertsonian translocation performed by polar body FISH analysis (Munne et al., 2000
; Durban et al., 2001
) reported significantly higher rates of unbalanced oocytes (3236%). Such variations emphasize the difference in the outcome of adjacent segregation in male and female meiosis, probably linked to the weakness of the female meiosis checkpoint mechanisms (LeMaire-Adkins et al., 1997
). Also, some reports of preimplantation genetic diagnosis for Robertsonian translocation carriers have indicated elevated rates of imbalanced chromosomal constitution (Conn et al., 1998
; Iwarsson et al., 2000
; Alves et al., 2002
). The observed abnormalities were essentially mosaicism and chaotic chromosomal constitution. However, these data are still in discussion, since other PGD reports with no evidence for high frequency of unbalanced embryo do not support the contention that Robertsonian translocations could predispose to malsegregation, and suggest that the observed chromosomal abnormalities could essentially result from culture conditions (Scriven et al., 2001
). Post-zygotic events affecting the chromosomal segregation during early cleavage stages could also influence the occurrence of unbalanced conceptuses, and this could be a patient-related phenomenon.
The production of unbalanced gametes renders difficult the investigation of meiotic segregation in Robertsonian translocation based on data from live birth or prenatal diagnoses. The sperm analysis of Robertsonian translocation carriers constitutes a unique and efficient approach for predicting the meiotic behaviour of these chromosomal rearrangements and estimating the risk of occurrence of unbalanced embryo in translocation carrier couples. The present study has shown that the rare Robertsonian translocation (13;22) displayed a similar distribution of balanced and unbalanced sperm patterns as the common Robertsonian translocations previously studied, thus suggesting that the behaviour of acrocentric chromosomes was similar in all cases of centric fusion. Further investigations of other rare Robertsonian translocations are now required to confirm these data.
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Acknowledgements |
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References |
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Submitted on November 5, 2004; resubmitted on January 5, 2005; accepted on January 21, 2005.