Hum. Reprod. Advance Access originally published online on June 3, 2006
Human Reproduction 2006 21(9):2335-2339; doi:10.1093/humrep/del190
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Analysis of non-crossover bivalents in pachytene cells from 10 normal men
1 Department of Medical Genetics, University of Calgary 2 Department of Genetics, Alberta Children’s Hospital, Calgary, Alberta, Canada 3 Institute of Human Genetics and Anthropology, Jena, Germany 4 Department of Urology, Department of Obstetrics and Department of Gynecology and Reproductive Sciences, University of California San Francisco, San Francisco, CA and 5 Department of Preventive Medicine, Northwestern University Medical School, Chicago, IL, USA
6 To whom correspondence should be addressed at: Department of Genetics, Alberta Children’s Hospital, 1820 Richmond Road S.W., Calgary, Alberta, Canada T2T 5C7. E-mail: rhmartin{at}ucalgary.ca
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Abstract |
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BACKGROUND: Bivalents with no recombination foci (possible achiasmates) are unable to orient properly on the metaphase plate or to segregate chromosomes to daughter cells. Non-crossover bivalents are known to cause meiotic arrest in various organisms. METHODS: Individual non-crossover bivalents were identified in 886 pachytene cells (19 492 bivalents) from testicular biopsies of 10 normal men. Fluorescence staining combined with centromere-specific multicolour fluorescence in situ hybridization (cenM-FISH) was used to identify mismatch repair gene mutation of human mutL homologue 1 (MLH1) recombination foci along each bivalent synaptonemal complex (SC). RESULTS: A total of 60 autosomal non-crossovers (SCs without an MLH1 focus) were found, and of these, chromosomes 21 (2.1%) and 22 (1.7%) had a significantly higher proportion than chromosomes 11, 12, 19 (each 0.1%), 13 (0.2%), 14 (0.6%), 16 (0.5%) and 15, 17, 18, 20 (each 0.3%) (P < 0.05). Sex chromosome univalents had a frequency of 27%, higher than that observed in any autosomal bivalent (P < 0.0001). CONCLUSIONS: These results suggest that G-group chromosomes and sex chromosomes are most susceptible to having no recombination foci and thus would be more susceptible to non-disjunction during spermatogenesis. This is consistent with previous observations from sperm karyotyping and FISH analysis, which demonstrate that chromosomes 21 and 22 and the sex chromosomes have a significantly increased frequency of aneuploidy compared with other autosomes.
Key words: aneuploidy/MLH1/non-crossover bivalent/pachytene spermatocytes/synaptonemal complex
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionAcknowledgementsReferences |
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During prophase I of meiosis, homologous chromosomes pair, synapse and recombine. Recombination not only involves the exchange of genetic material between homologous chromosomes but plays another equally crucial role in tethering the homologues together and thereby facilitating proper segregation (i.e. disjunction) (Hassold et al., 2000
). The improper segregation of homologous chromosomes (i.e. non-disjunction) leads to chromosomally unbalanced oocytes or sperm (Hassold and Hunt, 2001). If these gametes participate in fertilization, the outcome is an aneuploid embryo, with either trisomy or monosomy. Aneuploidy is the leading cause of fetal loss, mental impairment and developmental disabilities.
Despite the obvious clinical importance of non-disjunction, the predisposing genetic and environmental factors still remain a mystery. Altered recombination formation may be a major contributor to non-disjunction (Lynn et al., 2004). It has been recognized that the absence or reduced numbers of crossovers can increase the likelihood of non-disjunction in model organisms (e.g. flies and yeast; Hassold et al., 2000), and many recent studies have linked reduced or absent recombination with aneuploidy and infertility in humans (Hassold et al., 1991, 1995; Lamb et al., 1997; Savage et al., 1998; Thomas et al., 2000; Shi et al., 2001). These observations suggest that reduced recombination frequency is a risk factor for non-disjunction and meiotic arrest (reviewed in Egozcue et al., 2005). However, we have almost no information about the recombination frequency in individual chromosomes and the frequency of bivalents without recombination foci.
It is crucial to understand a chromosome-specific effect on recombination events over the genome in fertile men, because it not only provides the information about the regulation of recombination formation but can identify the chromosome-to-chromosome variation in recombination and thus predict the incidence of aneuploidies for different chromosomes. This will provide clues to identify the source of clinically observed trisomies as well. Fortunately, recent advances in immunofluorescence methods make it possible to directly characterize recombination events, by identifying the mismatch repair gene mutation of human mutL homologue 1 (MLH1) recombination foci along the synaptonemal complexes (SCs; the proteinaceous structure linking homologous chromosomes in prophase of meiosis I) (Baker et al., 1996; Barlow and Hultén, 1998; Anderson et al., 1999; Lynn et al., 2002; Tease et al., 2002; Sun et al., 2004a). These methods, in combination with centromere-specific multicolour fluorescence in situ hybridization (cenM-FISH) (Nietzel et al., 2001; Oliver-Bonet et al., 2003; Sun et al., 2004a), allow the analysis of the recombination distributions of individual chromosomes in human germ cells both across the whole genome and at the chromosomal level (Oliver-Bonet et al., 2003; Sun et al., 2004a, 2005).
This study identified non-crossover bivalents from 10 fertile men using the immunocytogenetic assay and cenM-FISH. The potential clinical implications are discussed. To our knowledge, this is the first study to perform a whole-genome analysis of non-crossover bivalents in a large population of normal men.
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Materials and methods |
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Sample collection
Testicular samples were obtained from patients undergoing orchiectomy for testicular cancer (n = 3; Calgary, Alberta, Canada) and vasovasostomy for fertility (n = 7; University of California at San Francisco, CA, USA). These patients were ascertained for reasons unrelated to meiotic defects or infertility. Histological examination showed normal spermatogenesis in these 10 patients (ages 47–81 years). Testicular tissues were kept in phosphate-buffered saline (PBS; pH 7.4) until use and transferred on ice to Calgary by air courier where appropriate. We have previously demonstrated that the cold storage of testicular tissue for 2 days does not affect recombination frequencies (Sun et al., 2004b
). Patients gave informed consent, and this study received ethical approval from the institutional review boards at the University of Calgary and at the University of California at San Francisco.
Fluorescence immunostaining and cenM-FISH
Slides with chromosome spreads were subjected to immunofluorescence staining, as described previously (Sun et al., 2004a). Primary antibodies against the following proteins were used: SCP1 (transverse filament proteins of SC; 1:1000 dilution; a gift from P. Moens, York University), SCP3 (lateral element proteins of SC; 1:250 dilution; a gift from T. Ashley, Yale University), MLH1 (marks the site of recombination foci; 1:100 dilution; Oncogene, San Diego, CA, USA) and CREST (calcinosis, Raynaud’s phenomenon, esophageal dysfunction, sclerodactyly, telangiectasia; marks the centromere; 1:100 dilution; a gift from M. Fritzler, University of Calgary). These primary antibodies were detected using a cocktail of secondary antibodies (donkey antisera) conjugated with different fluorochromes: 1-amino-4-methylcoumarin-3 acetic acid (AMCA) and Cy3 (1:100 dilution; Jackson Immunoresearch, West Grove, PA, USA) and Alexa 488 and Alexa 555 (1:125 dilution; Molecular Probes, Eugene, OR, USA). Primary and secondary antibodies were incubated overnight at 37°C and for 90 min at 37°C, respectively. Slides were examined on a Zeiss Axiophot epifluorescence microscope equipped with propidium iodide (PI), fluorescein isothiocyanate (FITC) and 4',6-diamidino-2-phenylindole (DAPI) filters and a cooled charged coupled device (CCD) camera. Three fluorescent images (red, green and blue) for the SC, MLH1 sites and CREST locations, respectively, were captured using Applied Imaging Cytovision 3.1 software (Applied Imaging, Santa Clara, CA, USA). Spreads were localized using a gridded finder slide.
Each pachytene-stage nucleus used for analysis met the following criteria: (i) the correct numbers of bivalents (i.e. 22 autosomes and 1 sex body) were present; (ii) the SCs were not overlapped with other SCs or bent back on themselves, allowing all foci to be scored and (iii) background was fairly low, allowing the SCs to be distinguished from background noise and from each other. MLH1 signals were scored if they were distinct and localized on an SC. SCs were classified as normally synapsed if they were completely linear, without any obvious bubbles, forks, loops or irregularities. Only cells with an identifiable XY pair were analysed from each man. SCs with 0 MLH1 foci were noted.
After the analysis of the captured immunofluorescence images, cenM-FISH was carried out on the same spermatocytes. This technique allows simultaneous identification of each autosome. Techniques developed by Nietzel et al. (2001) and Oliver-Bonet et al. (2003) were modified to make use of the microwave-decondensed/codenatured FISH technique (Ko et al., 2001). Cells were decondensed for 5 s in dithiothreitol (DTT) and 30 s in 3,5-diiodosalicylic acid, lithium salt (LIS)/DTT at medium power (550 W). Hybridization buffer [10% dextran sulphate, 2x saline sodium citrate (SSC), 55% formamide] was pre-warmed to 50°C, added to the cenM-FISH probes and warmed at 50°C until all probes were dissolved. Probes were applied to the slide, a glass cover slip was sealed in place with rubber cement, the probes and cells were microwave codenatured for 80 s at 1100 W, and the slide was incubated in a humid chamber at 37°C for 
24 h. A post-hybridization wash (0.4x SSC, 0.1% NP-40, 70°C) was carried out, streptavidin-Alexa 647 (1:58 dilution; Molecular Probes) solution was applied under a plastic cover slip, and the slide was incubated at 37°C for 40 min in a humid chamber. The slide was washed, with constant agitation, for 10 min in 4x SSC, air-dried and mounted in DAPI. Cells previously analysed by antibody immunostaining were relocated, and six fluorescent images (blue, aqua, green, gold, red and far-red) were captured for each cell using Applied Imaging Cytovision 3.1 software (Applied Imaging).
Analysis of non-crossover bivalents
After cenM-FISH identification of each pachytene bivalent, the images of corresponding SC spreads were analysed for MLH1 focus frequency along each SC. Because there is no significant difference in the mean MLH1 focus frequency between the two groups—cancer patients and the vasectomy patients or patients from either locale, Calgary or San Francisco [P = 0.68, nested analysis of variance (ANOVA) with person nested within group]—data were pooled from the 10 men.
The numbers of MLH1 foci per autosomal bivalent were scored, and the presence of an MLH1 focus in the XY pair was evaluated. Bivalents with no MLH1 foci were then assigned to individual chromosomes. Fisher’s exact test was applied to compare the frequency of non-crossover bivalents among normal men. The frequency of non-crossover bivalent 21 was compared with that of all other autosomes, the frequency bivalent 22 was compared with that of all other autosomes, and the frequency the sex chromosomes was compared with that of all autosomal chromosomes.
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Results and discussion |
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A total of 1000 pachytene-stage spermatocytes were analysed to determine the MLH1 focus frequency in 10 normal men (100 cells per man). The overall mean frequency of MLH1 foci was 49.7 (±5.0 SD), with significant inter-individual variability among the men (Table I; P < 0.0001, one-way ANOVA). The men were 47–81 years of age, and we found a significant age effect on the frequency of MLH1 foci (P < 0.01, Spearman’s correlation analysis). Clear cenM-FISH signals could be seen in 886 pachytene-stage spreads to identify individual bivalents. An example of pachytene SCs, with the identification of individual bivalents and cenM-FISH signals in the same cell, is shown in Figure 1. On average, there were 1, 3, 12, 5, 1 and 0 autosomal SCs, respectively, with 0, 1, 2, 3, 4 and 5 MLH1 foci. Overall, 5% of cells had one or more autosomal SCs without an MLH1 focus. The frequency of sex bodies without MLH1 foci was 27%. A total of 19 492 autosomal bivalents from the 886 cells were scored to determine the recombination frequency. Only 60 of the 19 492 autosomal SCs (0.3%) lacked an MLH1 focus. Thus, non-crossover autosomal chromosome pairs are rare in normal control men. However, this was more frequent for the sex chromosome bivalent, as 239 of them were missing an MLH1 focus.
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We then identified the individual SCs with no MLH1 foci. SCs 1–10 did not have any bivalents without MLH1 foci. Of the 60 autosomal SCs without MLH1 foci, 19 were identified as chromosome 21; 15 were chromosome 22; 5 were chromosome 14; 4 were chromosome 16; 3 were for each of chromosomes 15, 17, 18 and 20; 2 were chromosome 13; and 1 was for each of chromosomes 11, 12 and 19. Individual non-crossover bivalents in individual donors are presented in Table I. The overall frequency of these bivalents in different chromosomes from 10 men is presented in Figure 2. There was no evidence from these data for a correlation between the mean MLH1 focus frequency per cell and the frequency of cells with at least one SC missing an MLH1 focus (P > 0.05, Pearson’s correlation analysis). Also, there was no correlation between the frequency of MLH1 foci per cell and the frequency of cells without MLH1 foci in the sex body (P = 0.35, Spearman’s correlation analysis).
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A significant difference in the frequency of non-crossover bivalents in chromosome 21 (P < 0.006, Fisher’s exact test), chromosome 22 (P < 0.05, Fisher’s exact test) and the sex chromosome (P < 0.0001, Fisher’s exact test) was found compared with other chromosomes. There was no significant difference between the frequency of non-crossover bivalent 21 and that of bivalent 22 (P = 0.60). The highest frequency of autosomal SCs lacking an MLH1 focus was observed in chromosome 21. These results suggest that G-group chromosomes and sex chromosomes are most susceptible to having no recombination foci; in addition, the smaller chromosomes are more likely to be achiasmate than larger ones.
Recently, we reported the first recombination maps for each individual autosome from the normal man (Sun et al., 2004a). This report detailed the frequency and distribution of each MLH1 focus on each meiotic chromosome and ranked the length of bivalent SCs. We found a very strong correlation between SC length and the number of MLH1 foci per SC (Sun et al., 2004a). Chromosomes 21 and 22 are the smallest chromosomes in the whole genome. The pairing region of X and Y chromosomes is restricted to the pseudoautosomal region. Chromosomes 21 and 22 and the sex chromosome pairs generally have only one single crossover during meiosis I (Sun et al., 2004a). The length of SC for chromosome 14 is shorter than that of SCs for chromosomes 15, 16 and 17, and correspondingly, there is a higher frequency of bivalents without MLH1 foci in chromosome 14. Because SC length is strongly correlated with recombination frequency (Lynn et al., 2002; Sun et al., 2004a), it is likely that the chromosomes with the shortest SCs are more likely to be achiasmate than chromosomes with long SCs.
The presence of non-crossover bivalents is of interest because it is possible that the lack of recombination or subsequent univalents will trigger a meiotic checkpoint and interfere with the completion of meiosis. Achiasmate bivalents are known to lead to meiotic arrest in various organisms (Egozcue et al., 1983; Bascom-Slack et al., 1997; Roeder and Bailis, 2000). However, non-recombinant chromosomes can sometimes escape the pachytene checkpoint, and then they are at increased risk of undergoing non-disjunction (Koehler et al., 1996). Significant reductions in recombination have been observed in paternally derived cases of trisomy 21 (resulting in Down syndrome) (Savage et al., 1998). Direct PCR analysis of human sperm (single sperm typing) indicated that the lack of recombination in the pseudoautosomal region was a significant cause of XY non-disjunction. Also, the studies of liveborn paternally derived cases of Klinefelter’s syndrome (47,XXY) are associated with a lack of recombination between the X and the Y chromosomes (Lorda-Sanchez et al., 1992; MacDonald et al., 1994). In the human female, a decreased frequency of recombination or an alteration in the position of exchanges is also associated with non-disjunction (Hassold and Hunt, 2001). Thus, reduced or absent recombination is a molecular risk factor for chromosome non-disjunction in humans.
Although we do not know the frequency of aneuploidy for these bivalents in the 10 men in this study, the frequency and distribution of aneuploidy in spermatozoa from normal men have been intensively studied. The disomy frequencies for these chromosomes (Shi and Martin, 2000a) are presented alongside the frequencies of non-crossover bivalents in Figure 2. Compared with other autosomes, chromosomes 21 and 22 showed significantly higher disomy frequencies in spermatozoa from normal men obtained by the human–hamster system and by multicolour-FISH analysis on decondensed sperm nuclei (Shi and Martin, 2000a; Templado et al., 2005). Significantly higher frequencies of aneuploidy for the sex chromosomes compared with autosomes have been consistently found in normal and infertile men (Martin et al., 1995; Shi and Martin, 2000b). Also, most sex chromosomal aneuploidy is of paternal origin (Hassold et al., 1991; Hassold and Hunt, 2001). These studies suggest that chromosomes 21 and 22 and the sex chromosomes have a greater tendency to suffer segregation errors during spermatogenesis than the rest of the autosomes. It has been noted that the frequencies of these non-crossover SCs (especially SC 21 and SC 22) were higher than the frequencies of their corresponding aneuploidies in human sperm. These results imply that more non-crossover bivalents may cause meiotic arrest before proceeding to the first meiotic division and less of them escape from the meiotic checkpoints to produce aneuploidy in sperm.
Recently, Codina-Pascual et al. (2006) published the only other study in which bivalents without MLH1 foci were identified from two fertile men, using the similar methodology. Non-crossover bivalents were found in SCs 8, 13, 14 and 18–22 in their study, and among these SCs, SC 21 had the highest frequency of SCs with no MLH1 foci. We did not observe any non-crossover SC 8 from our 10 men, but both studies found that the absence of an MLH1 focus occurred more frequently in smaller chromosomes. The mean frequency of the absence of an MLH1 focus in the sex chromosomes was found to be 31% in two men, similar to our results in 10 men. Our results corroborate the observation that the sex chromosomes and bivalents 21 and 22 were the most prone to become achiasmate.
Studies of recombination and non-disjunction will provide important insights into the aetiology of human trisomies. In this study, failure in recombination was markedly increased for chromosomes 21 and 22 and the sex chromosomes, the same chromosomes that are most frequently observed as aneuploid sperm.
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We thank T. Ashley, M. Fritzler and P. Moens for the generous gift of antibodies and the patients for their participation in the study. R.H.M. holds the Canada Research Chair in Genetics, and the research was funded by the Canadian Institutes of Health Research (CIHR) grant MA7961. F.S. and M.O.-B. are the recipients of a CIHR Strategic Training Fellowship in Genetics, Child Development and Health. T.L. is supported in part by the EU (ICA2-CT-2000-10012).
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Submitted on March 23, 2006; resubmitted on April 13, 2006; accepted on May 3, 2006.
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