Synapsis and meiotic recombination analyses: MLH1 focus in the XY pair as an indicator

Montserrat Codina-Pascual1,5, Maria Oliver-Bonet1, Joaquima Navarro1, Mercè Campillo2, Ferran García3, Susana Egozcue3, Carlos Abad4, Josep Egozcue1 and Jordi Benet1,5

1 Unitat de Biologia i Genètica Mèdica, Departament de Biologia Cel·lular, Fisiologia i Immunologia, 2 Laboratori de Medicina Computacional, Unitat de Bioestadística, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, 3 Unitat Andrologia, Institut Marquès, 08024 Barcelona and 4 Servei d'Urologia, Consorci Hospitalari Parc Taulí, 08208 Sabadell, Spain

5 To whom correspondence should be adressed. Email: jordi.benet{at}uab.es or montserrat.codina{at}uab.es


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Anomalies in meiotic prophase I have been related to partial or total meiotic arrest. These anomalies include an abnormal synaptic process, resulting in disorders in meiotic recombination. METHODS: In the present study, we analyse primary spermatocytes from 12 infertile men (four with non-obstructive azoospermia, six with oligoastenoteratozoospermia, one with astenoteratozoospermia and one normozoospermic) and five control fertile donors using immunocytological techniques for synaptonemal complex, meiotic recombination and centromeric proteins. RESULTS: Mean numbers of MLH1 foci per cell, frequencies of cells presenting an MLH1 focus in the XY pair and percentages of cells affected by abnormal synaptic patterns (gaps and splits) are reported for each of the infertile patients and control men. A positive correlation between the frequency of cells showing a recombination focus in the XY pair and the number of autosomal recombination foci per cell is found. CONCLUSIONS: Reduced recombination in the XY pair and an increased number of cells affected by gaps may explain some idiopathic male infertility cases. The results suggest that recombination in the XY pair could be an indicator for general recombination frequency and for a successful meiotic process.

Key words: male infertility/meiotic recombination/pachytene/synaptonemal complexes/XY pair


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Meiosis is the process by which the whole genome of a cell is reduced to half to produce genetically different haploid cells for sexual reproduction. During meiotic prophase I, homologous chromosomes pair, synapse and recombine. These three events are essential for a successful meiotic process; any alteration of the meiotic process results in a reduction of the quality and quantity of gametes and, in the most severe cases, it can cause a complete meiotic arrest. Several meiotic checkpoints have been proposed to control this process (Roeder and Bailis, 2000Go). When any of the meiotic sequential events does not occur properly, the checkpoint switches on and prevents the cell from proceeding.

The incidence of infertility in human males is ~10%. Synaptic anomalies in meiotic chromosomes have been described and associated with male infertility. Indeed, up to 8% of the general infertile population show meiotic defects, and 74% of them correspond to an abnormal synapsis (revised in Egozcue et al., 2005Go). A correlation between a higher number of meiotic abnormalities and more severely affected semenograms has also been reported (Vendrell et al., 1999Go).

When chromosomes synapse, a tri-axial proteinaceous core, the synaptonemal complex (SC), is built along the pairing axis. At pachytene, homologous chromosomes are fully synapsed. Some structural meiosis-specific proteins of the SC, SCP1, SCP2 and SCP3, have been identified in the last few years (Meuwissen et al., 1992Go; Lammers et al., 1994Go; Schalk et al., 1998Go). Mutations of the Scp3 gene in mice (Yuan et al., 2000Go) and in human males (Miyamoto et al., 2003Go) demonstrated that defects in synapsis of homologous chromosomes at pachytene arrests the meiotic process and leads to infertility. In the past, studies of the SC were performed using unspecific protein staining (AgNO3). In a series of infertile men, abnormal synaptic processes were reported (Navarro et al., 1986Go). Since the development of immunolabelling methods, the interest in SC analysis has been renewed. Recently, abnormal synaptic processes have been reported in two azoospermic men by immunocytogenetic analysis of their spermatocytes (Judis et al., 2004Go; Sun et al., 2004aGo).

Disorders in meiotic recombination have also been described as a possible cause of meiotic arrest (revised in Egozcue et al., 2005Go). A DNA mismatch repair protein MLH1 (Baker et al., 1996Go; Barlow and Hultén, 1998Go; Anderson et al., 1999Go) and a cyclin-dependent kinase Cdk2 (Ashley et al., 2001Go) co-localize in late recombination nodules. Both of them have been shown to be involved in reciprocal recombination. A reduction in the MLH1 foci number per cell has also been reported in some azoospermic men (Gonsalves et al., 2004Go; Sun et al., 2004aGo).

The aim of this study was to analyse synapsis and meiotic recombination patterns in infertile and control men. Spermatocyte spreads were immunolabelled by using antibodies against synaptonemal complex proteins (SCP3 and SCP1), a late recombination nodule protein (MLH1) and centromeric proteins (CENP).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Testicular biopsies from 12 infertile patients and from five control donors, of proven fertility, were obtained under local anaesthesia. Infertility patients were diagnosed after 2 years of pregnancy failure. Written consent was given by all patients, and the study was approved by the Institutional Ethics Committee.

Four infertile patients were azoospermic (AZO 1–4), six oligoastenoteratozoospermic (OTA 1–6), one astenoteratozoospermic (AST) and another normozoospermic (NOR). Semenograms were classified according to the WHO parameters (World Health Organization, 1999Go). In the control group, the testicular biopsies of patients C1, C2 and C3 were obtained while they were undergoing vasectomy. Testicular biopsies of patients C4 and C5 were obtained while they were undergoing a vasectomy reversal.

Sample treatment
The testicular tissue was processed for meiotic chromosome analysis (Evans et al., 1964Go) and for SC immunocytogenetic analysis (Codina-Pascual et al., 2004Go). For the immunocytology of spermatocytes, the primary antibodies used were rabbit anti-SCP3 (Lammers et al., 1994Go) and rabbit anti-SCP1 (Meuwissen et al., 1992Go) (both gifts from Dr Christa Heyting, University of Wageningen, The Netherlands), anti-CENP (CREST serum given by Dr William Earnshaw, University of Edinburgh, UK) and mouse anti-MLH1 (Pharmingen, San Diego, CA). The secondary antibodies applied were tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit IgG antibody and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG antibody (both from Sigma; Madrid, Spain). The Pacific Blue-conjugated rabbit anti-human IgG (from Sigma; Madrid, Spain) labelled with Zenon Reaction (Molecular Probes, Spain) was applied in a third round. Finally, slides were counterstained with antifade (Vector lab Inc., Burlingame, CA). A fluorescent photomicroscope (Olympus Bx60) and Power Macintosh G3 with Smartcapture software (Digital Scientific, Cambridge, UK) were used for cell evaluation and image capture.

Cell analysis
Pachytene cells immunolabelled for SCP3, SCP1, MLH1 and CENP were captured and analysed. We considered as pachytene only the cells in which the XY pair was identifiable. Only pachytene nuclei with clear MLH1 labelling were included in the study. Cells were analysed according to three main variables: pachytene stage, meiotic recombination and synapsis.

According to the XY pair stages (Solari, 1980Go), nuclei were classified as early pachytene (stages 1 and 2) or late pachytene (stages 3, 4 and 5) (Figure 1a).



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Figure 1. Immunolabelled pachytene cells with synaptonemal complexes (SCs) in red, MLH1 in green and the centromere in blue. XY is identified in each cell. (a) XY pair morphologies observed during pachytene. XY pair stages 1 and 2 correspond to early pachytene (EP) and stages 3, 4 and 5 to late pachytene (LP). (b) Normal pachytene cell of a control donor. (c) Pachytene cell of an infertile patient showing multiple splits (large arrows). (d) Pachytene cell of an infertile patient showing multiple gaps (small arrows). (e) Pachytene cell of an infertile patient showing a generalized fragmentation of the SCs.

 
For meiotic recombination analysis, the number of MLH1 foci per cell and the presence of an MLH1 focus in the XY pair were evaluated.

To analyse synapsis, the existence of discontinuities in the SCs (gaps) and of splits in bivalents (unpaired lateral elements) was analysed. Based on the number of gaps present in the cell, nuclei were initially classified into four groups: NA, nuclei not affected by gaps; SA, nuclei slightly affected having from one to two SCs with gaps; MA, nuclei moderately affected with three or more SCs with gaps; and HA, nuclei highly affected with gaps in all the SCs. However, for data analysis the NA+SA and MA+HA groups were joined, respectively, into normal and gap-affected nuclei groups.

Data analysis
The {chi}2 test and Fisher test were applied when needed for qualitative data analysis. The Student test and Mann–Whitney test were applied to quantitative data comparisons between two groups. For other quantitative comparisons, analysis of variance (ANOVA) was used. Pearson's correlation coefficient was calculated for correlation analysis.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Classic meiotic chromosome analysis results are displayed in Table I.


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Table I. MLH1 immunocytogenetic results in spermatocytes of infertile and control men

 
For immunocytogenetic analysis, a total of 846 pachytene nuclei, 224 for the control group and 622 for the infertile men, were studied. Table I shows the number of cells analysed and the frequency of early pachytenes (complementary to that of late pachytenes) for the infertile cases, for each semenogram group and for controls. In the control group, 35.5% of pachytene cells were classified as early pachytene and the rest (64.5%) as late (Figure 1a). Similar results were found for the infertile group in which 34.4% were early and 65.6% were late pachytenes. Interindividual differences in the early and late pachytene percentages are detected in both control and infertile groups (P<0.0005). Early pachytene ranged from 18.8 to 52.8% for the control group and from 17.9 to 61.7% for the infertile group.

MLH1 foci analysis
The number of MLH1 foci was scored per each cell. Table I also displays the mean number of MLH1 foci per pachytene and the frequency of cells presenting an MLH1 focus in the XY pair for the analysed cases. The mean number of MLH1 foci per cell observed in controls is 48.8±2.3 and ranges from 36 to 63 foci per cell. In the infertile group, a mean of 47.3±3.3 foci per cell is found, ranging from 34 to 66, which is not different from the control results. A significant interindividual variation in the average number of MLH1 foci per cell in all cases is detected (P<0.0005), ranging from 42.9±2.1 to 52.3±4.2 MLH1 foci (Figure 2). However, two infertile cases showing univalents in metaphase I chromosome spreads (OTA3 and OTA4) and the NOR patient present a significant reduction in the number of MLH1 foci per cell when compared with the mean (P<0.0001) and minimum (P<0.003) control values (Table I). Pachytene nuclei were also classified regarding the presence or absence of an MLH1 focus in the XY pair. In the infertile group, the mean average of pachytene cells having an MLH1 focus in the XY pair (59.2%, range 40–76.2%) is not significantly lower than that seen in the control group (69.9%, range 59.7–80.5%). In both control and infertile groups, the average frequencies of cells showing an MLH1 focus in the XY pair in early pachytene cells (72.7 and 61.7%, respectively) are not significantly different from the average frequencies observed in late pachytenes (67.9 and 58.2%, respectively). Two cases, OTA6 and AZO4, displayed a significantly lower number of cells presenting an MLH1 focus in the XY pair (40 and 45%, respectively) when compared with the control mean value (69.9%) (P<0.006). However, when individually comparing the above results with the lower control value (59.7%), the difference is not significant (Table I).



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Figure 2. Mean (±SD) number of MLH1 foci per cell is drawn for the 17 individuals.

 
The mean number of autosomal MLH1 foci per cell was compared between the nuclei with an MLH1 focus in the XY pair and the nuclei without an MLH1 focus. Interestingly, the number of autosomal MLH1 foci per cell displayed a relationship with the presence or absence of the MLH1 focus in the XY pair (P=0.001). The mean value of autosomal MLH1 foci per cell was significantly higher in nuclei with an MLH1 focus in the XY pair. This increase was found for both control (P=0.022) and infertile (P=0.004) groups. A significant correlation between the frequency of cells with an MLH1 focus in the XY pair and the mean number of autosomal MLH1 foci per nuclei was observed for the 17 cases (P=0.013, r2=0.34) (Figure 3).



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Figure 3. Relationship between the frequency of cells with an MLH1 focus in the XY pair and the mean number of MLH1 foci per cell in autosomal SCs (y=2.37x–49.5; P=0.013, r2=0.34).

 
Synapsis analysis
Unsynapsed bivalent regions (splits) were seen in both control (11.7%) and infertile (11.2%) groups (Figure 1c). Table II shows the frequencies of pachytene nuclei showing a single split and multiple splits in SC regions in infertile patients and in control men. High interindividual variation in the presence of splits is detected in the control (P=0.002) and in the infertile groups (P<0.0005). Observed ranges are from 2.4 to 29.2% in control donors and from 0 to 26.2% in infertile patients. In general, the presence of a single small split is much more frequent in pachytene nuclei (80.4% of splits) than two or more splits. For almost all cases analysed, splits were seen more frequently in early nuclei than in late ones. No variation in the number of MLH1 foci per cell or in the frequency of MLH1 foci in the XY pair was detected in cells with splits.


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Table II. Frequencies of pachytene nuclei showing single (1S) and multiple splits (≥2S) in infertile patients and in control men

 
Some pachytene cells had discontinuities (gaps) in their SCs. Table III shows frequencies of pachytene nuclei showing SCP3 and SCP1 gaps in infertile and control men. The results are indicated according to the degree to which the bivalent set is affected. A significantly higher frequency of nuclei affected by gaps is observed in infertile cases (31.3%) when compared with control donors (20.2%) (P=0.046) (Figure 1d and e). Patients AST and AZO1 have more gap-affected cells (60 and 55.3%, respectively) than control donors (P<0.0001).


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Table III. Frequencies of pachytene nuclei having SCP3 and SCP1 gaps in infertile and control men

 
About 3% of cells presented a general fragmentation of their SCs (Figure 1e). MLH1 foci have always been seen in regions where the SC is structured. The number of MLH1 foci in the cell and the presence of MLH1 in the XY pair do not vary significantly whatever the degree to which cells were affected by gaps.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This work reports the results of synapsis and meiotic recombination analysis by the immunocytogenetic labelling technique of pachytene spermatocytes of infertile and control men. In control donors, 35.5% of the pachytenes cells correspond to the early pachytene stage. In the infertile group, a close average frequency is found (34.4%). A previous study showed similar frequencies of early (37%) and late (63%) nuclei in 46 pachytene cells of a control donor (Barlow and Hultén, 1998Go).

Recombination levels in spermatocytes
In this study, no significant difference in the number of MLH1 foci at early or at late pachytene has been detected, corroborating previous observations in which no relationship between MLH1 foci count and early–late pachytene stage was found (Barlow and Hultén, 1998Go; Lynn et al., 2002Go). These results indicate that, once the cell has acquired the full MLH1 complement by early pachytene, the mean number of MLH1 foci per cell remains the same throughout the pachytene stages.

Significant interindividual variation in the mean number of MLH1 foci per cell was encountered, which ranged from 42.9±2.1 to 52.3±4.2 MLH1 foci. All reported studies of MLH1 foci count in men with normal spermatogenesis showed interindividual variations in the range described here (Barlow and Hultén, 1998Go; Lynn et al., 2002Go; Gonsalves et al., 2004Go; Hassold et al., 2004Go; Sun et al., 2004bGo,cGo). The presence of an interindividual variation in the MLH1 foci frequency suggests the idea that the number of MLH1 foci would be more related to an individual behaviour than to a specific fertility group.

A significant reduction in MLH1 foci number has been found in three patients when compared with our controls. However, this reduction would not be significant if compared with control donors of other reported studies (Gonsalves et al., 2004Go; Sun et al., 2004cGo). This discordance between the results could be due to the interindividual variation in the number of MLH1 foci per cell. In cases presenting a strong reduction in the MLH1 foci counts, as reported in some azoospermic males (Gonsalves et al., 2004Go; Sun et al., 2004aGo), no discordances in the results could be found when compared with any control series.

In this study, we have obtained ranges of MLH1 foci per cell from 36 to 63 for the control group, and from 34 to 66 for the infertile group. Similar ranges are also observed in previous studies (Barlow and Hultén, 1998Go; Lynn et al., 2002Go; Gonsalves et al., 2004Go; Hassold et al., 2004Go; Sun et al., 2004bGo,cGo). It has been proposed recently that the minimum number of expected MLH1 foci is 39, one for each of the 39 autosomal arms (excluding the short arms of acrocentric chromosomes) (Lynn et al., 2002Go). Recently, the MLH1 foci pattern of each single bivalent has been analysed (Sun et al., 2004bGo). Taking into account ranges of MLH1 foci per bivalent, the theoretically expected range of recombination events in a single nucleus would be from 33 to 74. In this study, minimum values of meiotic recombination ranges in a cell are similar to the theoretical ones, but maximum values differ by ~10 units. This indicates the existence of a positive interference in the number of MLH1 foci in the cell. This interference would ensure a minimum of crossing-overs, but would reduce the maximum potential of recombination events in human males. Sexual differences in this interference may exist. This interference would be stronger in human males, as spermatocytes show fewer recombination events than oocytes (Tease et al., 2002Go). Differences between individuals and between sexes in epigenetic factors controlling this interference might explain interindividual and intersex variation in interference intensity.

Frequencies of cells with an MLH1 focus in the XY pair observed in the present study are similar to the 56.5 and 73% previously reported (Barlow and Hultén, 1998Go; Sun et al., 2004bGo). Nevertheless, patients AZO4 and OTA6 have a frequency of cells with an MLH1 focus in the XY pair lower than 45%, indicating a low recombination frequency between sex chromosomes. A reduction in the recombination in the PAR1 region of the XY pair can lead to an abnormal disjunction of the sex chromosomes. Consequently, it may increase the sex chromosome aneuploidy (Shi et al., 2001Go) or even cause a meiotic arrest (Hale, 1994Go). In patients AZO4 and OTA6, partial meiotic arrest and meiotic arrest, respectively, were detected by meiotic chromosomes analysis, which is in agreement with the XY recombination results.

The present study describes for the first time a correlation between the percentage of XY pairs showing an MLH1 focus and the mean number of autosomal MLH1 foci per cell. The presence of an MLH1 focus at the XY pair was strongly correlated with a higher autosomal recombination frequency. Similarly, levels of XY and autosomal pairing were described to correlate positively (Mittwoch and Mahadevaiah, 1992Go). Also, the rate of XY bivalents has been reported as an indicator for successful spermatogenesis in azoospermic men (Yogev et al., 2002Go).

The results obtained in our study suggest that the frequency of an MLH1 focus in the XY pair could be a marker for general recombination frequency and for the meiotic process to proceed.

Synaptic behaviour: split and gap incidence
Pachytene cells of infertile men presented splits in a mean of 11.2% cells, not different from those observed in control patients. However, considerable differences among cases have been seen in both infertile and control groups. The fact that 80.4% of the splits seen are small and unique in the pachytene nuclei suggests that these splits correspond to the heterochromatic regions of bivalents 1 and 9. These splits have been observed more frequently in early than in late pachytene nuclei, indicating that they may result from a delay of the heterochromatin to synapse. Polymorphisms in these heterochromatic regions may explain the high interindividual variation observed in the frequency of splits. Discontinuities (gaps) in the SCs of a cell are seen in ~50% of pachytene nuclei in control patients. According to a study of a fertile man in which SCs of chromosomes 1 and 9 frequently (>50%) showed gaps in their heterochromatic regions (Barlow and Hultén, 1996Go), pachytene nuclei presenting a gap in one or two SCs have been considered as normal nuclei.

The observed pachytene cells showing a general fragmentation of their SCs (Figure 1e) could correspond to nuclei of degenerative cells in apoptosis.

The AST and AZO1 infertile cases show a significant increase of nuclei (>55%) affected by gaps. If more nuclei are affected, more cells may fail to go through meiotic checkpoints, leading to a reduction in cell counts or to cells with an abnormal morphology and motility.

AZO1 has the highest frequency of early pachytene nuclei (61.7%), some of them with general SC fragmentation, and also shows <45% of normal pachytene cells. These results suggest an early pachytene partial arrest caused by synaptic defects, in agreement with the few post-pachytene meiotic stages observed in meiotic chromosome analysis.

In summary, we report the results of an immunocytogenetic analysis of synapsis and meiotic recombination in a series of infertile and control men. We have demonstrated that reduced recombination in the XY pair and increased number of cells affected by gaps may explain male infertility in certain cases. Therefore, this cytological analysis is a useful tool to better understand some idiopathic male infertility. However, the great interindividual variability in meiotic recombination and in the synaptic process, seen in this and in other studies, makes comparisons between groups and individuals still complicated. To better understand the effects of meiotic recombination and synaptic abnormalities on fertility and to mark out limits between pathological and normal incidence values, further analysis in infertile patients will be required. Also, the application of centromere or subtelomere multiplex-fluorescence in situ hybridization (FISH) methodologies for the identification of all SCs (Oliver-Bonet et al., 2003Go; Codina-Pascual et al., 2004Go) should provide information about the bivalents most affected by synaptic and recombination disorders. Finally, the relationship between frequency of MLH1 foci in the XY pair and the average number of MLH1 foci per cell found in the present study suggests that the meiotic recombination frequency in the XY pair could be an indicator for general recombination frequency and for a successful meiotic process.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We thank Dr C.Heyting and Professor W.Earnshaw for SCP1, SCP3 antibodies and CREST serum, respectively. M.C.P. is the recipient of a grant of the Generalitat de Catalunya (2001FI00468). This study was supported by Fondo Investigacion Sanitaria (Madrid) (project PI020258) and the Generalitat de Catalunya (project 2001 SGR 00201).


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on September 16, 2004; resubmitted on February 14, 2005; accepted on March 14, 2005.