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RESEARCH |
1 Departament de Biologia Cellular, Fisiologia i Immunologia, Universitat Autònoma de Barcelona, 08193-Bellaterra, Spain, 2 Servei de Ginecologia i Obstetrícia, Hospital Materno-infantil de la Vall d’Hebron, Barcelona, Spain, 3 Max-Planck-Institute for Molecular Genetics, Ihnestrasse 73, D-14195 Berlin, Germany and 4 Institute of Radiation Biology Bw Neuherbergstr. 1180937, München, Germany
Correspondence should be addressed to M Garcia, Unitat de Biologia, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193-Bellaterra, Spain; Email: montserrat.garcia.caldes{at}uab.es
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Abstract |
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Introduction |
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At the leptotene stage, SYCP3 and SYCP2 form an axial element (AE) along each chromosome (Lammers et al. 1994, Offenberg et al. 1998). During this stage, initiation of recombination can be monitored by immunostaining of the phosphorylated version of histone H2AX (
H2AX) (Mahadevaiah et al. 2001, Roig et al. 2004, Lenzi et al. 2005). At the zygotene stage, homolog pairing starts and SYCP1 organizes the central element of the SC by developing transverse filaments between closely aligned regions of AEs (Meuwissen et al. 1992); from this time on, AEs are called lateral elements (LEs). In order to promote homologous synapsis, chromosome ends (telomeres) cluster at a limited portion of the nuclear envelope, known as bouquet topology (for reviews see Scherthan 2001 and Harper et al. 2004). Studies in cattle and human have revealed that bouquet topology lasts longer in the mammalian female than in the male (Bojko 1983, Pfeifer et al. 2003, Roig et al. 2004). At pachytene, all homologs are completely paired (bivalents). Finally, at diplotene, SCs almost completely dissolve. The substages of prophase I are characterized by the initiation and progression of recombination which can be followed by the dynamic turnover of repair proteins (reviewed in Cohen & Pollard 2001, Moens et al. 2001).
In mammalian males meiosis is a continuous process, while in the female meiotic prophase occurs during early fetal life, and arrests before birth at a specialized diplotene–dictyotene stage. The meiotic division is blocked until the female enters puberty. Despite the difficulties associated with the study of human female meiotic prophase, some aneuploid ovaries have been studied using classical staining techniques (Luciani et al. 1976, Wallace & Hulten 1983, Speed 1984), fluorescent in situ hybridization (FISH) (Cheng et al. 1995, 1998) or immunofluorescent (IF) (Barlow et al. 2002) techniques.
In these studies, pairing anomalies implying the presence of the extra chromosome are described. Nevertheless, in all cases, at least two homologs seem to pair normally forming a bivalent, and even pairing of the three homologs (trivalent formation) has been described in all studied cases, with different efficiency (Luciani et al. 1976, Wallace & Hulten 1983, Speed 1984, Cheng et al. 1995, 1998, Barlow et al. 2002). In some of these papers a disturbing effect of the extra chromosome on the pairing process of other homologs is suggested (Cheng et al. 1998).
In those studies in which the pairing process is analyzed in silver-stained preparations (Wallace & Hulten 1983, Speed 1984), a thickening of the unpaired axial element (univalent) of the extra chromosome is seen, suggesting a different proteinaceous composition. This phenomenon is not restricted to humans, and has also been observed in rat meiocytes (Martínez-Flores et al. 2001). However, recent studies in which IF techniques have been applied to stain SYCP3 in human trisomic 21 oocytes have not observed any univalent thickening, suggesting that SYCP3 is not implicated in this phenomenon (Barlow et al. 2002).
Trisomy 18 syndrome, also known as Edwards’ syndrome, has a meiotic maternal origin in 89% of cases (Hassold & Hunt 2001) and causes major physical abnormalities, growth delay and severe mental retardation. Very few affected children survive beyond their first year.
The aim of the present study was to analyze the pairing process of homologs in ovaries from three trisomic 18 fetuses in order to assess the effect of the presence of an extra chromosome 18 on the progress of chromosomal events in the female meiotic prophase I. To this end, immunofluorescent staining (IF) to SYCP1 and SYCP3 proteins, chromosome painting, and centromere and telomere FISH were performed. As it has been recently described that human SCs are also formed of a cohesin axis (see above), analysis of a specific meiotic cohesin REC8 (Prieto et al. 2004) was performed in order to check whether cohesins are responsible for the unpaired univalent thickening observed in previous studies (Wallace & Hulten 1983, Speed 1984, Martínez-Flores et al. 2001).
Furthermore, as ataxia telangiectasia and Rad3 related kinase (ATR) has been described as a marker for late-pairing chromosomes in mouse pachytene spermatocytes (Moens et al. 1999, Baart et al. 2000), IF to ATR was performed to investigate its distribution in aneuploid oocytes. Due to the absence of any published data about ATR in humans, ATR distribution in human euploid spermatocytes and oocytes is also described.
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Materials and Methods |
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) were used after legal interruption of pregnancy according to the Ethics Rules Committee of the Hospital de la Vall d’Hebron of Barcelona, Spain. The age of each case was calculated from the last menstrual period and from echography.
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DNA probes and labeling
Centromeres of all human chromosomes were detected by FISH with alpha satellite-DNA sequences (pan-centromeric probe), as obtained by alphoid-specific PCR from genomic human DNA (Weier et al. 1991) and were labeled with Cy3-dUTP by random priming. All chromosome telomeres were detected after applying FISH with an Fluorescien (FITC)-conjugated (CCCTAA3) peptide nucleic acid (PNA) telomere probe (DAKO, Glostrup, Denmark; pan-telomeric probe).
Commercial Whole Chromosome 18 Probe (WCP) Cy3-conjugated and Whole Chromosome 13 Probe Cy3-conjugated (Cambio, Cambridge, UK), as well as a dual locus-specific identification (LSI) probe 13/21 (Oncor, Qbiogene, Irvine, CA, USA) and a locus-specific biotinylated probe for the 18q subtelomeric region (YAC 932Q10) were used to analyze the homolog-pairing process.
DNA was counterstained applying an antifade solution (Vector Laboratories) containing 0.1 µg/ml DAPI (4',6'-diamidino-2-phenylindole; Sigma).
Fluorescent in situ hybridization (FISH) on methanol:acetic acid spread oocytes
In order to study the homolog chromosome 18 pairing process, FISH was performed on methanol:acetic acid spread oocytes as described before with slight modifications (Barlow & Hultén 1997). Slides were post-fixed in a formaldehyde solution and denatured in 70% formamide in 2 x SSC at 69 °C for 4 min. Probe denaturation was performed as described by the manufacturer and the denatured probe was applied after slide dehydration. Three post-hybridization washes were performed in 50% formamide in 2 x SSC, 2 x SSC and 0.4 x SSC 0.05% Tween 20 at 45 °C. LSI 18 probe detection was performed applying Avidin FITC-conjugated antibody (1/150) for 1 h at 37 °C.
In this part of the study 719 oocytes were analyzed. Oocyte staging was performed according to the morphological criteria previously described (Garcia et al. 1987).
Immunofluorescent (IF) staining
The axial elements of the SCs were identified with a rabbit polyclonal serum against SYCP3 (Meuwissen et al. 1992). Homolog synapsis was tracked by marking the central element of the SC with a rabbit polyclonal serum against SYCP1 (Lammers et al. 1994) (both were kind gifts of Christa Heyting, Wageningen, The Netherlands). The cohesin axis was detected using a mouse polyclonal serum against cohesin REC8 (Prieto et al. 2004, a kind gift of José Luís Barbero, DIO/CNB, Trescantos, Madrid, Spain). Staining of all chromosome telomeres was performed applying a mouse monoclonal antibody (Imgenex, San Diego, CA, USA) against telomeric protein TIN2 (Kim et al. 1999). Finally, ATR dynamics were described using a rabbit polyclonal antibody against ATR (Abcam, Cambridge, UK).
IF staining was performed as described by Roig et al.(2004). Primary antibodies were diluted in PTBG (PBS, 0.2% BSA, 0.2% gelatin, 0.05% Tween 20) and incubated overnight at 4 °C in a humid chamber.
After washing away unattached antibodies with PBTG, detection was performed using some of the following fluorochrome-conjugated secondary antibodies (diluted in PTBG; all Jackson ImmunoResearch Laboratories, West Grove, PA, USA): goat anti-rabbit Cy3, goat anti-rabbit FITC antibody, goat anti-rabbit Cy5, a goat anti-mouse Cy3 antibody, a goat anti-mouse FITC antibody and a goat anti-mouse Cy5 antibody. Secondary antibodies were incubated for one hour at 37 °C in a humid chamber. Later, excess secondary antibodies were washed off with PBTG and the fluorescent signals were fixated with 1% formaldehyde in PBS. DNA was counterstained as mentioned above.
FISH on immunostained preparations
IF stained preparations were hybridized with an 18q sub-telomeric probe as described by Scherthan et al.(1996) with modifications. DAPI staining was removed by washing for 5 min in water. A first denaturation of the slide was performed in 70% formamide in a 2 x SSC solution for 5 min at 70 °C. The slide was then washed in water and a 3-h NaSCN (Sodium Thiocyanate) 1 M treatment was applied at 65 °C. A second denaturation was then performed as mentioned before. Dehydration of the slide was carried out before applying the denatured probe to the slide. Hybridization was performed at 37 °C in a humid chamber for at least 48 h. Slide washes and probe detection were performed as described above.
Microscopy and image analysis
Preparations were evaluated using an Olympus BX70 fluorescence microscope (Olympus Optical Co.). Images were captured and produced by Smart Capture software. (Vysis, Grove, IL, USA) Images were further processed using Adobe Photoshop (Adobe, San Jose, USA) to match the fluorescent intensity seen in the microscope.
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). The karyotype of V54 obtained from the somatic culture was 46, XX, thus validating this sample as a control case (Table 1).
Chromosomal dynamics in trisomy 18 oocytes
IF staining for SYCPs, telomeric proteins, as well as a dual-color FISH applying pan-centromeric and pan-telomeric probes performed on structurally preserved oocyte preparations and ovary sections respectively, were carried out in order to check for pairing progression in trisomy 18 oocytes. These experiments revealed the same chromosome and protein dynamics as described in euploid oocytes (Fig. 1) (Roig et al. 2004).
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). However, tight telomeric clustering was not found until the zygotene stage (Fig. 1B). At this stage, synapsis was initiated and the first SYCP1 fibers were seen mainly near the bouquet base, although some interstitial pairing was also seen (Fig. 1C). At the pachytene stage nuclear polarization was resolved and complete pairing existed and SYCP1 was found along the entire length of the synaptonemal complex. At the diplotene/dictyotene stage, SYCP1 was found as little dots and SYCP3 as small threads, telomeres were found inside the nuclei and centromeres were sometimes seen in a line-like organization, as was reported in the euploid human oocytes (Roig et al. 2004).
Bouquet topology is increased in trisomic oocytes
In order to compare meiotic prophase progression in aneuploid and euploid cases, the percentage of oocytes displaying a bouquet topology in each case was analyzed. In the trisomic 18 cases, 47%, 56% and 55% of the oocytes (Table 1) had their telomeres clustered in a limited sector of the nuclear periphery (bouquet topology; Fig. 1). In euploid cases of a similar developmental stage, the bouquet proportion was 36%, 28%, 34%, 36% and 38% (Table 1). Thus, a statistically significant (P < 0.0001) increase in the proportion of the bouquet stage oocytes per ovary in trisomy 18 cases was observed.
As noted before in euploid oocytes (Table 1 and Roig et al. 2004), the trisomy 18 oocyte bouquet frequencies were statistically significantly increased (P < 0.0001) over those observed in human spermatogenesis.
Cohesin core and SC organization during aneuploid meiotic prophase
Silver-stained preparations of trisomic oocytes revealed a significant thickening of the unpaired univalent (Wallace & Hulten 1983, Speed 1984). Thus, REC8 distribution throughout the meiotic prophase in trisomic 18 oocytes was analyzed to see whether this phenomenon was caused by cohesin proteins and whether aneuploidy could affect organization of cohesin cores. In order to properly follow homolog synapsis, IF staining was also performed on the major component of the central element of the SC (SYCP1).
REC8 first appeared at the preleptotene stage as a diffuse signal throughout the nucleus (not shown). At the leptotene stage, REC8 was organized as thin, curly threads (Fig. 2A). At the zygotene stage, REC8 was present as thin threads in unsynapsed regions and as thicker threads which colocalize with SYCP1 corresponding to synapsed regions between homologs (Fig. 2B). At the pachytene stage, complete colocalization of REC8 and SYCP1 was seen in the majority of the oocytes (Fig. 2C, see below). At the diplotene stage, SYCP1 started to disappear and REC8 persisted as long, thin threads in SYCP1-devoid regions (Fig. 2D).
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, Table 2). In total, 193 oocytes were analyzed.
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), most oocytes (71.43%) displayed three independent signals for the paint and the subtelomeric region. The remaining oocytes displayed two signals, one corresponding to two joint subtelomeric signals colocalizing with one paint 18 signal (consistent with an already paired bivalent 18) and the other was the unpaired third chromosome 18 (univalent 18). During the zygotene stage (Fig. 4C,D), 22% of oocytes had three signals (unpaired chromosome 18). Two signals (bivalent plus univalent) were seen in 37% of oocytes. Finally, one single signal comprising all three chromosome 18 paired (trivalent) was seen in 42% of oocytes. At the pachytene stage, the vast majority of the oocytes analyzed (92%) displayed a trivalent (Fig. 4E). The remaining 8% of oocytes showed two separate signals corresponding to a bivalent plus a univalent. Diplotene-stage oocytes generally displayed only one signal (83.33%) (Fig. 4F), but oocytes with two separate chromosome 18 signals were also observed.
The high frequency of single signals observed at the pachytene stage contrasted with previous studies performed on trisomic 21 oocytes (Luciani et al. 1976, Wallace & Hulten 1983, Speed 1984, Barlow et al. 2002). Single signals observed in methanol:acetic acid preparations at the pachytene stage could either come from synapsed chromosomes or from aligned homologs. Thus, to determine if pairing or alignment of the extra chromosome existed, FISH, using a subtelomeric 18q probe on REC8 and SYCP1 IF preparations, was performed (Fig. 5). All the pachytene oocytes analyzed (n = 10) showed a single 18q subtelomeric signal matching with a SYCP1 thread (Fig. 5C). Despite the small number of nuclei studied, this value is almost the same as that observed by FISH in the previous experiment on methanol:acetic acid spread preparations (Table 2). Similarly, all the leptotene oocytes (n = 5) presented three unsynapsed chromosome 18s (Fig. 5A). Fifty percent of the zygotene oocytes analyzed (n = 40) displayed a bivalent 18 plus a univalent 18 (Fig. 5B). Only two diplotene oocytes could be analyzed –one showed a bivalent plus a univalent configuration, the other presented a desynapsed trivalent (Fig. 5D).
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) showed that chromosome 13s were not paired at leptotene – 100% of the studied oocytes had two univalents. Chromosome 13s paired during the zygotene stage, where we observed 70% of the oocytes displaying one bivalent signal. At the pachytene stage, all nuclei analyzed displayed one chromosome 13 bivalent.
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). The results showed no significant differences to those obtained in trisomic 18 oocytes. Almost all leptotene oocytes analyzed displayed two chromosome 13 univalents (91%). Seventy percent of the zygotene oocytes showed one chromosome 13 bivalent. All pachytene and diplotene oocytes showed only one chromosome 13 bivalent.
ATR location in human meiosis
It has been proposed that ATR controls the correct pairing of the homologs in the male mouse, as has been found preferentially in late-pairing chromosomes in pachytene-stage spermatocytes (Keegan et al. 1996, Moens et al. 1999). Therefore, and given the delayed formation of the trivalent 18 (Fig. 3), the ATR dynamics in trisomic 18 oocytes were studied.
ATR distribution in euploid human spermatocytes
As ATR detection has only been performed on mouse spermatocytes, ATR distribution in human spermatocytes was first analyzed in order to characterize the location of this protein during male meiotic prophase (Fig. 6). Spermatocyte staging was performed according to parameters described previously (Solari 1980).
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), where ATR was seen to decorate almost the entire length of the SC (Fig. 6B). At late pachytene, ATR was predominantly located at the sex body and some ATR foci persisted on SCs, some of them marking telomeres (Fig. 6C). At the diplotene stage, a few ATR foci were present on the SCs.
ATR distribution in human oocytes
When ATR distribution in euploid oocytes was analyzed, it appeared that ATR distribution was similar to that in human spermatocytes described above. In euploid oocytes, ATR first appeared at leptotene as a few foci. At the zygotene stage, ATR was present at the unsynapsed and synapsed regions of the chromosomes. No remarkable differences in ATR staining were observed between ‘late pairing’ and ‘synapsed’ homologs at the late zygotene stage/early pachytene stage, mirroring the situation found in human spermatocytes. At early pachytene, ATR foci were present along the whole length of the SCs. At late pachytene, ATR foci remarkably decreased; some of them were located at the telomeres. Finally, oocytes at the diplotene stage had few ATR foci.
The same ATR distribution was found in trisomic 18 oocytes (Fig. 6D) and apparently no differential ATR staining was observed for the trivalent 18 at late zygotene/early pachytene.
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Discussion |
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According to previous observations made in human euploid oocytes (Prieto et al. 2004), development of REC8 chromosome cores and synapsis progression was not clearly found to be overtly altered in the presence of an extra copy of human chromosome 18, thus suggesting that cohesin core organization is not affected by the presence of an extra chromosome. The sequence of chromosomal events also mirrored those of euploid human oocytes (Roig et al. 2004). Therefore, it seems aneuploidy does not grossly affect chromosome core and SC formation, which agrees with previous reports (Luciani et al. 1976, Wallace & Hulten 1983, Speed 1984, Cheng et al. 1995, 1998, Barlow et al. 2002).
The results of trivalent formation are similar to those described earlier for trisomic cases in human trisomy 21 oocytes (Wallace & Hulten 1983, Speed 1984, Barlow et al. 2002) and in the rat (Martínez-Flores et al. 2001). However, thickening of the asynapsed chromosome core was not observed in preparations stained with REC8 and SYCP1, contrasting with previous observations made with electron microscopy (Wallace & Hulten 1983, Speed 1984, Martínez-Flores et al. 2001). These results indicate that the different proteinaceous composition of the unpaired univalent is not related to meiotic cohesin REC8. IF data also corroborate the absence of SYCP3 and SYCP1 implication in this phenomenon, agreeing with previous observations (Barlow et al. 2002).
An extra chromosome 18 does not alter pairing of other chromosomes
Studies performed on Down’s syndrome oocytes suggest that an extra chromosome may alter the pairing of other homologs (Cheng et al. 1998). When a comparison was made of the chromosome 13 pairing process found in Edwards’ syndrome oocytes with control oocytes from a case at the same developmental stage, no obvious differences were found. Moreover, similar timing was presented. In contrast, Cheng et al.(1998) reported that chromosome 13 suffered a pairing delay in trisomy 21 oocytes. The differences in chromosome 13 pairing found between trisomy 21 and trisomy 18 oocytes may be caused by the frequent association existing between acrocentric NOR-bearing chromosomes 13 and 21, which is commonly observed both in euploid and aneuploid oocytes (I Roig and M Garcia, data not published).
Moreover, normal SYCP1 dynamics obtained in SYCP1 IF experiments also provide evidence that general chromosome synapsis progresses with no obvious differences compared with euploid oocytes (Roig et al. 2004).
Cheng et al.(1998) suggested that the possible interference caused by the extra chromosome 21 was diminished by the formation of a trivalent 21, mirroring observations made by other authors (Speed 1984, Rodriguez & Burgoyne 2000). Thus, the high trivalent-18 formation efficiency observed in this study could explain the absence of an interfering effect of the extra chromosome in the other chromosome-pairing processes.
Presence of an extra chromosome 18 delays bouquet resolution in trisomic oocytes
The bouquet has been implied as a mediator of chromosome pairing (Loidl 1990, Scherthan 2001). A prolonged bouquet stage has been found between species with a similar genome size, but different chromosome complement (mouse vs cattle, where the latter has more chromosomes and thus a longer bouquet stage; see Pfeifer et al. 2001 for discussion) and disomic yeast strains (Rockmill & Roeder 1998). The significant increase in bouquet frequency in trisomy 18 oocytes as compared with that of euploid cases of similar stage (Table 1) suggests that the oocyte modulates the bouquet stage in response to an extra chromosome.
Results obtained in methanol:acetic acid spreads suggest that chromosome-18 pairing is an efficient process to achieve the trivalent condition. Trivalent formation is preceded in leptotene and zygotene by the formation of a bivalent plus a univalent, since the portion of oocytes with bivalent plus univalent was higher at leptotene and zygotene than in later stages (Table 2). Moreover, late-pairing trivalent 18s shown in Fig. 3 reflect the fact that normally a trivalent is formed from a bivalent plus a univalent. In all of the images, there seems to be an already-paired bivalent (black) to which the univalent (gray) tries to pair. This observation may reflect the requirement to form a trivalent due to the cell being allowed to pass a pachytene-pairing checkpoint (Roeder & Bailis 2000). It may be that the time required for bringing about the encounter of surplus chromosomes takes longer, and therefore bouquet topology lasts longer in aneuploid cells compared with euploid oocytes. It may also be that trivalent formation is a late event in prophase I and that the cells spend more time until pairing is finally achieved.
REC8-SYCP1 IF studies have revealed that the three chromosome 18 cores were not perfectly aligned in some early pachytene oocytes (Fig. 3), thus mirroring the findings of Barlow et al.(2002). Therefore, it could be that the bouquet also serves a role in mediating and/or maintaining homolog alignment. Alternatively, the presence of proteins involved in the recombination process, such as Replication Protein A (RPA) at the trivalent 18 which is formed at early pachytene (not shown) may reflect the fact that delayed recombination may have the potential to elicit a bouquet prolongation (Scherthan 2003, Roig et al. 2004).
Nevertheless, Edwards’ syndrome is characterized by a general developmental delay, which could not be excluded as playing a role in the origin of the delayed bouquet resolution found in trisomic 18 oocytes. However, this seems unlikely since prophase sub-stage frequencies found in trisomy 18 (V70) and euploid (V54) cases were similar (Table 3). In the same way, preliminary results obtained in Down’s syndrome oocytes (Robles P, Roig I, Garcia R, Egozcue J and Garcia M, unpublished data) seem to support the hypothesis that the extended bouquet could be due mainly to the presence of an extra chromosome.
ATR in aneuploid oocytes
The specific ATR decoration of the late-pairing bivalents found in mouse spermatocytes (Keegan et al. 1996, Moens et al. 1999, Baart et al. 2000) was not observed in human spermatocytes or oocytes in this study. Moreover, in the cells with late-pairing bivalents, ATR was most often located at the synapsed regions rather than at the asynapsed regions (Fig. 6A1 and A2). In this case, late zygotene spermatocytes showed that ATR was located mostly at the paired region of the XY bivalent (Fig. 6A3). This is in contrast to what has been reported for the male mouse where ATR was largely seen in asynapsed regions at the late zygotene stage and no ATR staining was observed at the pachytene stage except for the sex body (Keegan et al. 1996, Moens et al. 1999, Baart et al. 2000). A recent study performed on male mouse spermatocytes suggests a pachytene checkpoint function for ATR, which could be related to recombination rather than to synapsis (Perera et al. 2004). Our results seem to be in agreement with the latter hypothesis.
This is the first study in which a combination of FISH and IF techniques have been used in order to analyze homolog pairing in human trisomic oocytes. The usage of a dual approach enables the study of a large number of oocytes with accurate precision. Our results show that even in the presence of an extra chromosome the pairing process in human oocytes is generally completed, yielding evidence of the reliability of the homolog-chromosome pairing process in human female meiosis. However, further analysis in human euploid and aneuploid oocytes and their peculiarities in homolog pairing is required in order to gain deeper insights into the occurrence of this phenomenon in the human species.
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Acknowledgements |
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The English of this manuscript has been revised by a native English speaking instructor of English of this University. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
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Footnotes |
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References |
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