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Human Molecular Genetics Advance Access originally published online on September 9, 2005
Human Molecular Genetics 2005 14(20):3013-3018; doi:10.1093/hmg/ddi332
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Discontinuities and unsynapsed regions in meiotic chromosomes have a cis effect on meiotic recombination patterns in normal human males

Fei Sun1,2, Maria Oliver-Bonet1,2, Thomas Liehr3, Heike Starke3, Kiril Trpkov4, Evelyn Ko2, Alfred Rademaker5 and Renée H. Martin1,2,*

1Department of Medical Genetics, University of Calgary, Calgary, Alberta, Canada T2N 4N1, 2Department of Genetics, Alberta Children's Hospital, Calgary, Alberta, Canada T2T 5C7, 3Institute of Human Genetics and Anthropology, 07743 Jena, Germany, 4Department of Pathology, Rockyview Hospital, Calgary, Alberta, Canada T2V 1P9 and 5Cancer Center Biometry Section, Northwestern University Medical School, Chicago, IL 60611-4402, USA

* To whom correspondence should be addressed at: Department of Genetics, Alberta Children's Hospital, 1820 Richmond Road SW, Calgary, Alberta, Canada T2T 5C7. Tel: +1 4039437369; Fax: +1 4035439100; Email: rhmartin{at}ucalgary.ca

Received August 3, 2005; Accepted August 26, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
During meiosis, homologous chromosome pairing is essential for subsequent meiotic recombination (crossover). Discontinuous chromosome regions (gaps) or unsynapsed chromosome regions (splits) in the synaptonemal complex (SC) indicate anomalies in chromosome synapsis. Recently developed immunofluorescence techniques (using antibodies against SC proteins and the crossover-associated MLH1 protein) were combined with fluorescence in situ hybridization (using centromere-specific DNA probes) to identify bivalents with gaps/splits and to examine the effect of gaps/splits on meiotic recombination patterns during the pachytene stage of meiotic prophase from three normal human males. Gaps were observed only in the heterochromatic regions of chromosomes 9 and 1, with 9q gaps accounting for 90% of these events. Most splits were also found in chromosomes 9 and 1, with 58% of splits occurring on 9q. Gaps and splits significantly altered the distribution of MLH1 foci on the SC. On gapped SC 9q, the frequency of MLH1 foci was decreased compared with controls, and single 9q crossovers tended toward a more distal distribution. Furthermore, the larger the gap the more distal the location of the MLH1 focus closest to the q arm's telomere. MLH1 foci on split SC 9 had distributions similar to those of gapped SC 9; however, splits did not change the frequencies of MLH1 foci on SC 9. This is the first demonstration that gaps and splits have an effect on meiotic recombination in humans.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Meiotic recombination (crossover) frequency and positioning are under tight genetic control and recombination is a crucial pre-requisite for proper meiotic chromosome segregation (1Go). The first identified molecular correlation between human non-disjunction and altered placement of recombination events was that an enrichment of single crossovers in the distal chromosome 21q was associated with an increased probability of non-disjunction at meiosis I (2Go). These types of crossover configurations are also known to be susceptible to non-disjunction in the fruit fly and in yeast (3Go,4Go). Thus, exploring the factors affecting crossover distribution is needed in order to understand the basis of the association between recombination and human non-disjunction.

The synaptonemal complex (SC), a tripartite structure with two parallel lateral elements and a central element held together by transverse filaments (5Go), mediates homologous chromosome pairing, synapsis and recombination during meiotic prophase I (6Go). A DNA mismatch repair protein, MLH1, has been identified as a marker of meiotic recombination sites (reviewed in 7Go). The patterns of genetic recombination in males with normal and abnormal spermatogenesis were characterized utilizing immunofluorescence methodologies, using antibodies against lateral element proteins (SCP3) and transverse filament proteins (SCP1) to visualize SCs (8Go) and antibodies against MLH1 to identify recombination sites (9Go–15Go). These studies suggest that the number of crossovers and their cytological correlates, chiasmata, vary in striking correlation to the length of the prophase chromosome axes at the SC stage (10Go,12Go,16Go). Therefore, any perturbation altering SC length has the potential to change the distribution of recombination foci on that SC, so incomplete SC formation may be an important factor in the distribution of crossovers. Although some researchers have shown that SC defects lead to abnormalities in recombination, which in turn lead to non-disjunction in model organisms (reviewed in 17Go), there has been no investigation to date into whether defects in chromosome synapsis can alter the pattern of recombination events in humans.

Discontinuities in the visible SC (gaps) and unsynapsed chromosome regions (splits) along the SC have been observed in pachytene cells from normal human males (9Go,11Go,14Go,15Go). In this study, the effect of these perturbations on MLH1 focus distributions in individual SCs from three normal human males were studied, in order to investigate the potential relationship between incomplete SC formation and recombination patterns at the pachytene stage. This was accomplished using immunofluorescence in spermatocytes, followed by centromere-specific multicolour fluorescence in situ hybridization (cenM-FISH) (18Go,19Go).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
An example of pachytene SCs, with identification of individual bivalents and cenM-FISH signals in the same cell, is shown in Figure 1. A total of 261 pachytene stage spreads with clear cenM-FISH signals was analyzed from the 300 spreads already analyzed by immunofluorescence in three normal men. Gaps (expressed as unstained segments in the solid-stained chromosomal bivalents, Fig. 1) or splits in the SCs were observed in some pachytene nuclei. Most gaps (71%) and nearly all splits were restricted to the early substages of pachytene (as classified by 9). The uniform, narrow, pale lateral elements of localized splits in early pachytene SCs could be definitively distinguished from pre-synaptic zygotene bivalent regions, as the latter have characteristic irregularities in thickness and brightness.



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  		Figure 1. Human pachytene spermatocyte with SCs shown in red, centromeres in blue and MLH1 foci in yellow. Note that SC 9 has a gap (g) (upper). Subsequent cenM-FISH analysis allows identification of individual chromosomes so that recombination (MLH1) foci can be analyzed for each SC (lower).

 
Gaps/splits were localized to specific autosomal SCs using cenM-FISH: gaps were restricted to pericentromeric regions on the q arm of bivalent 9 and bivalent 1 (and therefore unlikely due to technical artefact); most splits were also observed in the pericentromeric regions on the q arm of bivalent 9 (58% of 44 cells with splits) and bivalent 1 (11%). Less commonly observed were splits in bivalents 13 (9%), 21 (7%), 15 (5%) and 3, 4, 6, 14 and 20 (2% each). Bivalent 9 had the largest and most frequently observed gaps (91% of 72 cells with gaps, mean length=4.31 µm) and splits (mean length=4.95 µm). The mean lengths of gaps and splits in bivalent 1 were 2.89 µm (seven cells analyzed) and 4.63 µm (five cells analyzed), respectively.

Overall, the mean number of gaps in the 2200 autosomal SCs analyzed per individual was 46 (range: 23–66). The mean proportion of cells with gaps in 100 spermatocytes was 34%, and the mean proportion of cells containing splits, less common in occurrence than gaps, was 4% (mean number: 7; range: 0–14). Individual data on the frequency of gaps/splits from the three patients are presented in Table 1. As SC 9 had the most frequently observed gaps/splits in pachytene stage cells and the largest number of spreads with gaps/splits, the effect of gaps/splits on the number and distribution of MLH1 foci in SC 9 was examined in this study.


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  		Table 1. Individual frequencies of gaps/splits in autosomal SCs in three men
 
The mean numbers of MLH1 foci in the p arm, q arm and entire bivalent for normal and gapped/split SC 9 were assessed in the 261 cells that were analyzed by cenM-FISH (Table 2). There was a significant difference in the numbers of MLH1 foci in the q arm and in the whole bivalent between normal and gapped SC 9 (P=0.024, P=0.029, respectively), indicating that gaps were associated with a decreased frequency of MLH1 foci in SC 9. MLH1 focus frequencies were not significantly different between split and normally-synapsed SC 9.


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  		Table 2. MLH1 focus frequencies in normally-synapsed, gapped and split SC 9
 
Positions of MLH1 foci on normally-synapsed and gapped/split SC 9 are expressed as a distance from the MLH1 focus to the telomere in Table 3. When gapped/split SC 9 had a single MLH1 focus on each arm, the distance from the q arm telomere to its MLH1 focus was significantly less than in normal SC 9 (for gapped SC 9, P<0.001; for split SC 9, P=0.02). The distance for double crossovers in the q arm of gapped/split SC 9, however, roughly approximated those in normal controls (Table 3).


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  		Table 3. Distance (µm, mean±SE) from MLH1 foci to the telomeres of normally-synapsed, gapped/split SC 9
 
To examine the influence of the length of gaps/splits on recombination focus position in the SC, correlation analysis between the length of gaps/splits and the distance from the most distal MLH1 focus to the telomeres was performed (Fig. 2). There was a significant inverse relationship between the length of gaps/splits and the distance from the telomere to the distal MLH1 focus on the q arm (gapped SC 9: r=–0.4964, P<0.001; split SC 9: r=–0.5460, P<0.01, Pearson's correlation coefficient). Thus, as the length of gaps/splits increased, the most distal recombination focus on the q arm was located closer to the telomere.



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  		Figure 2. The relationship in SC 9 between (A) the length of gap and the distance from the p arm's MLH1 focus to its telomere, (B) the length of gap and the distance from the q arm's distal MLH1 focus to its telomere, (C) the length of split and the distance from the p arm's MLH1 focus to its telomere and (D) the length of split and the distance from the q arm's distal MLH1 focus to its telomere.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In the present study, SC configurations showed that during the pachytene stage of meiotic prophase I, bivalent 9 had the most structural variability: gaps/splits were very common in the heterochromatic region of the q arm. The presence of gaps/splits had a profound effect on the location of MLH1 foci: on gapped/split SC 9q with a single recombination site, there was a significant likelihood that MLH1 foci were more distally located. A reduction in recombination frequency was found in SC 9 with gaps, but not in those with splits.

The pericentromeric region on bivalents 9 and 1, where gaps/splits were typically found, corresponds to the location of C-heterochromatin in the proximal segment of the q arm of mitotic chromosomes 9 and 1 (20Go). These segments represent the heterochromatic blocks 9qh and 1qh, as confirmed by the simultaneous hybridization of a whole chromosome paint probe and a qh-specific probe for chromosome 9 and chromosome 1 in spermatocytes at the pachytene stage (21Go). Heterochromatin is characterized by repetitive DNA, reduced recombination rates, few protein-coding regions and gene silencing (22Go–24Go). Moreover, in translocation carriers, heterochromatin exerted transcriptional repression on the nearby translocated euchromatic genes (25Go,26Go). In the Drosophila female, heterochromatic SC elements are less rigid and structured than their euchromatic counterparts and lack recombination nodules, and are often structurally abnormal or even absent in some cases (27Go–30Go). Finally, recent studies indicate that unsynapsed chromosome regions are transcriptionally silenced relative to synapsed regions during the pachytene stage of meiosis in the mouse in both males and females (31Go).

Among autosomes, chromosome 9 contains the largest block of heterochromatin, and in 6–8% of humans the structure of this heterochromatin region is highly polymorphic (32Go–35Go). This large heterochromatin block may explain why this region of SC 9 is more susceptible to SC irregularities than are other bivalents: the gaps (lack of visible SC elements) and splits (incomplete SC elements) in 9qh may be attributable to heterochromatic gene silencing of SC elements and a paucity of protein-coding for SC elements.

Although the molecular mechanisms of how gaps/splits act on crossover distributions are not known, the association between altered recombination patterns and meiotic non-disjunction has been studied (reviewed in 36Go). The absence of chiasmata or inappropriately located chiasmata was linked to sporadic non-disjunction in Drosophila melanogaster oocytes (4Go). Genetic maps based on meiosis I non-disjunctional errors were generated for human trisomies of chromosomes 15, 16, 18 and 21 and the sex chromosomes, and in each instance the trisomic map was significantly shorter than the control map (reviewed in 36Go). Direct analysis of human sperm (single sperm typing) indicated that lack of recombination in the pseudoautosomal region was a significant cause of XY non-disjunction (37Go). Thus, reduced recombination may play a significant role in the genesis of non-disjunction in humans.

Alteration in the positioning of crossovers in human non-disjunctional meiosis has also been reported. Hassold et al. (38Go) found that in trisomy 16 patients, crossovers were much more distally located than expected and Lamb et al. (2Go) observed that trisomy 21 patients had an enrichment of crossovers in the distal 21q. Thus, at least for some trisomies, distal crossovers are a risk factor for non-disjunction.

In this study, both gapped and split SC 9 had an alteration in the position of recombination events, whereas only gapped SC 9 had a decreased recombination frequency (suggesting that gaps and splits may be inherently different in nature). Although no data on sperm aneuploidies for chromosomes 9 or 1 are available for these three men, a tendency for these chromosomes to have higher disomy frequencies than other autosomes has been reported in human sperm (39Go). Whether the altered recombination patterns caused by synaptic anomalies in SC 9 contribute to chromosome 9 non-disjunction in sperm awaits further study.

In summary, in human meiosis, bivalent 9 is susceptible to synaptic anomalies, which in turn alter the distribution of recombination foci in the SC. This is the first demonstration that a chromosome-specific structural variation has an effect on meiotic recombination patterns in normal humans. Further research will be needed in order to explore the molecular nature of the gaps/splits in SC 9 and possible clinical consequences.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Testicular samples were obtained from three cancer patients ascertained for reasons unrelated to meiotic defects or infertility. Histological examination showed normal spermatogenesis in these three patients (aged 47–81). Testicular tissue was transported in phosphate buffered saline (PBS, pH 7.4) and processed using methods described elsewhere (12Go). This study received ethical approval from the institutional review board of the University of Calgary.

Slides with chromosome spreads were subjected to immunofluorescence staining as described previously (12Go). Primary antibodies against the following proteins were used: SCP1 (1:1000 dilution, a gift from P. Moens, York University), SCP3 (1:250 dilution, a gift from T. Ashley, Yale University), MLH1 (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), 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 and three fluorescent images (red, green, blue) were captured using Applied Imaging Cytovision 3.1 software (Applied Imaging Corporation, Santa Clara, CA, USA). Spreads were localized using a gridded finder slide.

Each pachytene-stage nucleus used for analysis met the following criteria: (1) the correct numbers of bivalents (i.e. 22 autosomes and 1 sex body) were present, (2) the SCs were not overlapped with other SCs or bent back on themselves, allowing all foci to be scored and (3) background was fairly low, allowing the SCs to be distinguished from background noise and from each other, with the telomeres of the SC arms clearly visible. MLH1 signals were scored if they were distinct and localized on an SC. The XY bivalent was excluded from MLH1 scoring, as it desynapses earlier than the autosomes. SCs were classified as normally synapsed if they were completely linear, without any obvious bubbles, forks, loops or irregularities. One hundred pachytene-stage cells were analyzed from each male, the number of MLH1 foci per autosomal bivalent was scored and the number and location of gaps/splits in the SCs were noted in these spreads.

After analysis of the captured immunofluorescence images, centromere-specific multicolour FISH (cenM-FISH) was carried out on the same spermatocytes. This technique allows simultaneous identification of each autosome. Techniques developed by Nietzel et al. (19Go) and Oliver-Bonet et al. (18Go) were modified to make use of the microwave-decondensed/codenatured FISH technique (40Go). 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 sulfate, 2x standard 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 1% NP-40, 70°C) was carried out, streptavidin–Alexa 647 (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 4',6-diamidino-2-phenylindole (DAPI). Cells previously analyzed 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 Corporation, Santa Clara, CA, USA).

After cenM-FISH identification of each pachytene bivalent, the images of corresponding SC spreads were analyzed using MicroMeasure 3.3 (available from the web site: http://www.colostate.edu/Depts/Biology/MicroMeasure) to measure the lengths of gaps and splits. MicroMeasure is an image analysis application that allows collection of data for a wide variety of chromosome parameters from electronically captured images (41Go). The gap/split length was measured inline: the gap or split was measured in a straight line from where the SC ended on one side of the gap or split to where the SC began again on the other side. In addition, the curved length of the splits was measured following the actual path of the split from one end to the other. As there was no significant difference between inline and curved split lengths (P=0.22, Student's t-test), inline lengths have been used for all gap and split lengths in this study.

The number and position of each MLH1 focus on normally-synapsed, gapped and split SCs were scored and analyzed on individually identified bivalents in SC spreads using MicroMeasure 3.3. As the nature of gaps is unknown (it is known only that nothing is visible in that portion of the SC; it is not known if there are structures that are not visible with current technologies or if there is an actual void in the region), it was important to measure the absolute distance from the telomere to the MLH1 foci (a distance that does not include a gap or split) to more accurately reflect the position of the MLH1 foci on the SC.

Fisher's exact test was used to examine the difference in the mean MLH1 focus numbers in the SCs. The independent sample t-test was used to compare the distance from MLH1 foci to the telomeres in normally-synapsed, gapped and split SC 9. The relationship between the sizes of gaps/splits and the positions of MLH1 foci in the SCs was analyzed using Pearson's correlation test.


   ACKNOWLEDGEMENTS
 
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).

Conflict of Interest statement. None declared.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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