Abstract
Only little is known about the meiotic prophase events in human oocytes, although some of them are involved in the origin of aneuploidies. Here, a broad study of the pairing and synaptic processes in 3263 human euploid and 2613 aneuploid oocytes (47,XX, +21 and 47,XX, +13), using different techniques and methods, is presented in order to elucidate the characteristics of this essential meiotic process. Our results reaffirm the existence of a common high efficiency in the pairing process leading to the obtainment of a bivalent for all chromosomes studied in euploid and aneuploid cases. Nevertheless, this high efficiency was insufficient to consistently produce trivalents in aneuploid oocytes. Trivalent 21 was only observed in 48.8% of the 47,XX, +21 pachytene-stage oocytes studied, and trivalent 13 was found in 68.7% of the 47,XX, +13 pachytene-stage oocytes analyzed. Our data confirm the hypothesis which suggests that in human oocytes the presence of an extra chromosome could interfere in bouquet dynamics. In addition, the pairing process of the X chromosome is altered in trisomic 21 oocytes, providing evidence of the influence that an extra chromosome 21 may cause meiotic progression.
Introduction
Meiosis is the special cell division that provides haploid cells, called gametes, which ensure species diploidy after fertilization. In order to complete this process, the nucleus undergoes substantial reorganizations, which mainly promote three major events, all of which occur during the first meiotic division: first, the encounter of the homologue chromosomes (alignment and pairing) and the establishment of a physical interaction between them (synapsis), mediated by a proteinaceous structure called the synaptonemal complex (SC; Fawcett 1956, Moses 1956). Secondly, the exchange of genetic material between homologue chromosomes (for a review see Marcon & Moens 2005). Finally, at the anaphase of the first meiotic division, each homologue is segregated to one pole of the cell.
In humans, aneuploidy is mainly caused by errors produced during the initial stages of the female meiotic process (Hassold & Hunt 2001). However, human female meiosis is still poorly understood because it is initiated during the fetal time period, and the obtainment of samples for the analysis is difficult. Nevertheless, some papers have studied human oocyte I using classical approaches mainly focusing on the establishment of the time frame in which meiotic prophase takes place in the human female, but also analyzing how homologues synapse (Ohno et al. 1962, Baker 1963, Blandau 1969, Kurilo 1981, Bojko 1983, Speed 1985, Garcia et al. 1987, 1989). Fluorescence in situ hybridization (FISH) has enabled the analysis of particular homologue pairing processes of euploid and aneuploid oocytes using whole chromosome probes (WCP) alone or combined with locus identification probes (Cheng & Gartler 1994, Cheng et al. 1995, 1998, 1999, Cheng & Naluai-Cecchini 2004, Roig et al. 2005a) or particular sets of probes to study oocytes with rearranged genomes (Cheng et al. 1999, Cheng & Naluai-Cecchini 2004). More recently, studies have used immunofluorescence (IF) to primarily analyze meiotic recombination (Barlow & Hultén 1997, Hartshorne et al. 1999, Tease et al. 2002, Roig et al. 2004, 2005b, Lenzi et al. 2005). In most of these studies, important differences concerning homologue synapsis and the recombination progression process between male and female meiosis are described both in humans (Bojko 1983, Rasmussen & Holm 1984, Roig et al. 2004, Lenzi et al. 2005, Oliver-Bonet et al. 2005) and mice (Morelli & Cohen 2005), suggesting that these may be involved in the differences observed in the origin of human aneuploidy.
Because most of the recent studies performed in mammalian meiosis have focused on studying meiotic recombination, only little is known about the homologue pairing process and synapsis, and this is also true for human oocytes (Tease et al. 2002, Cheng & Naluai-Cecchini 2004, Roig et al. 2004, 2005a, 2005b, Lenzi et al. 2005). On the other hand, chromosomes 13, 21, and X are involved in some of the most common human trisomies, and in the aneuploidies shown by oocytes at metaphase II (Cupisti et al. 2003, Pujol et al. 2003). In this paper, an extensive analysis of the pairing process and synapsis of homologues 13, 21, and X in 5876 oocytes is performed. Different technical approaches have been used combining FISH and IF techniques to analyze both the pairing process and synapsis. Analysis has been performed in spread and 3D preserved preparations from euploid and aneuploid cases in order to improve our knowledge about homologue pairing and synapsis implication in the origin of human aneuploidy.
Materials and Methods
Biological material
In this study, a total of 13 fetuses (Table 1) were used after legal interruption of pregnancy according to the Ethics Rules Committee of the Hospital de la Vall d’Hebron, Barcelona, Spain. Of these, seven were euploid fetuses, five were prenatally diagnosed for Down syndrome, and one was prenatally diagnosed for Patau syndrome. The age of each case was calculated from the last menstrual period and echogram. The results presented in this paper are based on 12 samples that, all of them, were used for the first time in this study, and sample V94 was used in a previous study with different goals (Table 1; Roig et al. 2005b). Nevertheless, in order to deepen the knowledge of the pairing and synaptic processes, our results obtained in this study were compared with previously published data (Roig et al. 2004, 2005a, 2005b).
Processing of the sample
In those cases in which sample karyotype was not known, the ovaries were processed as described previously (Roig et al. 2003) in order to obtain a somatic chromosome complement of the sample.
Samples were also processed to obtain structurally preserved nuclei preparations (Roig et al. 2004), which enabled a 3D study of the nucleus, and oocyte spreads for IF and FISH purposes, as described recently (Martínez-Flores et al. 2003, Roig et al. 2004, 2005b), were used to characterize homologue chromosome pairing and synapsis in detail.
Fluorescence in situ hybridization (FISH)
In order to study the homologue chromosome 21, 13, and X pairing processes, FISH was performed in euploid and aneuploid methanol:acetic acid spread oocytes as described previously (Roig et al. 2005b). Different commercial (Vysis, Downers Grove, IL, USA, and Cambio, Cambridge, UK) and non-commercial (provided by Dr R Stanyon, National Cancer Institute-Frederick, Frederick, MD, USA) WCP, as well as a 13q14/21q22 locus-specific identification (LSI) probe (Oncor, Gaithersburg, MD, USA), were used. DNA was counter-stained by applying an antifade solution (Vector Laboratories, Peterborough, UK) containing 0.1 μg/ml 4′,6′-diamidino-2-phenylindole (Sigma).
Oocyte staging was performed according to the morphological criteria previously described (Garcia et al. 1987). As a brief summary, leptotene-stage oocytes are characterized by the start of homologue chromosome condensation and individualization. At the zygo-tene stage, the synaptic process starts, and thickening of the bivalents is visible where it is completed. At the pachytene stage, bivalents are completely synapsed, thus they appear thicker than that in the rest of the stages. Finally, at the diplotene stage, homologues separate, but they remain close, joined only by the chiasmata.
Immunostained preparations used to analyze synapsis of the homologue chromosomes were also FISHed with LSI probe 13q14/21q22 (Oncor) with the aim to detect the homologue 21s, following the protocol described elsewhere (Roig et al. 2005b).
IF staining
The IF techniques were used to analyze the evolution of synapsis of the homologue chromosomes in spread and 3D nuclear-preserved preparations of euploid and aneuploid oocytes.
Synapsis was followed using a mouse polyclonal serum against meiotic-specific cohesin REC8 (Prieto et al. 2004) provided by Dr José Luís Barbero, DIO/CNB (Spain), as well as a rabbit polyclonal serum against the SC central element-specific protein synaptonemal complex protein (SYCP1) (Lammers et al. 1994), which was provided by Dr Christa Heyting (Wageningen, the Netherlands).
Meiotic prophase evolution was also followed by analyzing the presence of bouquet topology in 3D preserved cells using a rabbit polyclonal serum against the axial element of SC, the SYCP3 protein (Meuwissen et al. 1992), provided by Dr Christa Heyting (Wageningen) and a mouse monoclonal antibody (MAB; Imgenex, San Diego, CA, USA) against telomeric protein TRF1 interacting protein 2 (TIN2) (Kim et al. 1999).
IF staining was performed as described previously (Roig et al. 2004). Primary antibodies were diluted in PBGT (PBS, 0.2% BSA, 0.2% gelatin, and 0.05% Tween 20) and incubated overnight at 4 °C in a humid chamber.
After washing away unreacted antibodies with PBGT, detection was performed by the following fluorochrome-conjugated secondary antibodies (all from Jackson ImmunoResearch Laboratories, diluted in PBGT): goat anti-rabbit Cy3, a goat anti-rabbit fluorescein isothiocyanate (FITC) antibody, a goat anti-mouse Cy3 antibody, and a goat anti-mouse FITC antibody. Secondary antibodies were incubated for 1 h at 37 °C in a humid chamber. Later, washing off of the secondary antibodies excess with PBGT and fixation of the fluorescent signals with 1% formaldehyde in PBS were performed. DNA was counterstained as mentioned above.
Microscopy and image analysis
The preparations were evaluated using an Olympus BX70 fluorescence microscope (Olympus Optical Co). Images were taken and produced by SmartCapture software (Digital Scientific Ltd, Cambridge, UK), and further processed using Adobe Photoshop to match the fluorescent intensity seen in the microscope.
Statistical analysis
The Fisher F-test, lineal-by-lineal analysis, and logistic regression were applied in this study, as detailed below in each section, using SPSS 13.0 software (SPSS Inc., Chicago, IL, USA). The degree of significance applied in each test is also indicated.
Results
Pairing of homologues 21 and X in human euploid fetuses
To analyze the pairing of chromosomes 21 and X, FISH was performed in spread oocytes with WCP 21 or X. A total of 2867 euploid oocytes were analyzed.
The results presented in Table 2 show that chromosome pairing, as defined by the presence of a single WCP signal in the oocyte, in human euploid oocytes starts at the leptotene stage for both homologue pairs, as described previously for chromosomes 13, 18, and 21 (Cheng et al. 1995, 1998, Roig et al. 2005a, 2005b). Although the pairing process of chromosome 21 progresses faster than that of the X chromosome, it shows a higher error rate (0.3% of the oocytes presented two separate chromosome 21s at the pachytene stage), whereas this was not observed for the X chromosome in any oocyte (Fisher F-test, P = 0.507), these differences between two chromosomes being not statistically significant. As it has been reported for chromosome 18 (Roig et al. 2005a), the presence of leptotene-stage oocytes with three chromosome 21s from euploid fetuses has been observed, demonstrating the existence of pre-meiotic non-disjunction events. In contrast, this was not observed for the X chromosome.
Pairing of homologous chromosomes 13 and 21 in oocytes from fetuses with trisomies 13 and 21
To analyze the pairing process of the homologue chromosomes involved in trisomy, FISH was performed in oocytes from 47,XX, +13 and 47,XX, +21 fetuses (Tables 3 and 4) by applying WCP 13 and WCP 21, as well as a dual 13q14/21q22 LSI. A total of 296 oocytes from 47,XX, +21 (n = 132) and 47,XX, +13 (n = 164) were analyzed.
In both trisomic cases, presence of a total trivalent configuration at the pachytene stage was observed. However, results suggest that chromosome 13 presents greater efficiency than chromosome 21 does in obtaining a complete pairing along the whole chromosome length of the three homologues: trivalent 13 is found in 68.6% of the trisomic 13 pachytene-stage oocytes, while trivalent 21 is only observed in 48.8% of the trisomic 21 pachytene-stage oocytes (Fig. 1; Tables 3 and 4). Our results also indicate that the trivalent configuration is maintained until the diplotene stage for both 13 and 21 homologues, with the same proportion found at the pachytene stage (lineal-by-lineal analysis, P = 0.863 and P = 0.624 for homologues 13 and 21 respectively). Other differences have been observed between these two homologues: chromosome pairing process analysis at the leptotene stage shows that homologue pairing starts earlier in trisomic 13 oocytes (Tables 3 and 4), although statistical analysis has concluded that these differences are not significant (Fisher F-test, P = 0.274). In addition one trisomic 21 pachytene-stage oocyte with three univalents was found, while this has not been observed in trisomic 13 oocytes.
Synapsis of chromosome 21 homologues in oocytes from fetuses with trisomy 21
Analysis of the synaptic process of the three homologue 21s found in trisomic 21 oocytes was performed. In order to assess whether the pairing process efficiency found was sufficient to progress throughout prophase or SC formation among the three homologues was needed, SC formation was analyzed by IF following meiotic-specific cohesin REC8 and the SC central element protein SYCP1. Identification of chromosome 21s was performed by FISH using a LSI 13q14/21q22 probe.
At the pachytene stage, 35.7% of the oocytes analyzed (n = 42) presented a complete trivalent (Fig. 2A), 28.6% displayed a partial trivalent (Fig. 2B), and 35.7% of the oocytes showed a bivalent plus a univalent (Fig. 2C). No pachytene-stage oocyte with three univalent 21s was observed. These results are similar to those obtained by FISH analysis (Table 4, lineal-by-lineal analysis, P = 0.294).
Presence of an extra chromosome delays bouquet resolution
During meiotic prophase, telomeres appear clustered along the nuclear membrane displaying bouquet topology, which promotes homologue encounters and consequently the pairing process (for a recent review see Scherthan 2006). Recent investigations have demonstrated that the bouquet lasts longer in mammalian females when compared with the males (Pfeifer et al. 2003, Roig et al. 2004), and the presence of an extra chromosome may delay bouquet resolution in human oocytes (Roig et al. 2005b). To investigate this delay in trisomic 21 oocytes, an analysis of the percentage of oocytes displaying telomeric clustering was performed in six trisomic 21 cases. Three-dimensional nuclear-preserved preparations were immunostained against telomeric protein TIN2 and SC component proteins SYCP3 and SYCP1. A total of 1231 oocytes were analyzed.
Oocytes from trisomy 21 fetuses showed a significantly higher bouquet frequency than oocytes from euploid fetuses (Fig. 3A and Table 5; logistic regression, P ≤ 0.001).
In order to study the magnitude of the delay in bouquet formation in oocytes from trisomy 21 fetuses, the proportion of oocytes with telomeric clustering corresponding to the late stages of bouquet formation at the pachytene stage was compared in oocytes from euploid and trisomy 21 fetuses. The proportion was higher in oocytes from trisomy 21 fetuses but not significantly different (Fig. 3B and Table 5; logistic regression, P ≥ 0.005).
Presence of an extra chromosome 21 affects the chromosome X pairing process
As previous studies have suggested that the presence of an extra chromosome 21 affects the autosomal 13 pairing process (Cheng et al. 1998), it was decided to investigate whether the extra chromosome 21 could also affect the X on oocytes from female fetuses with trisomy 21. A complete analysis of the X homologue pairing process was done for 1418 oocytes from fetuses with trisomy 21 and 1457 oocytes from fetuses with a euploid karyotype (Table 6).
Comparing the leptotene and zygotene stages, there was no significant difference between oocytes from euploid or aneuploid oocytes (Fisher F-test, P = 0.187). However, nine pachytene-stage oocytes from trisomic 21 fetuses had two univalent X chromosomes, which indicated a significantly higher error rate in the X pairing process (Fisher F, P = 0.02). This is consistent with the hypothesis that an extra chromosome 21 affects X chromosome pairing.
Discussion
In this paper, an extensive analysis of the pairing process, followed by FISH, and synapsis, analyzed by the detection of the SC central element protein SYCP1, of homologue chromosomes 13 and 21 is described in 3263 oocytes from fetuses with a euploid chromosome complement and 2613 oocytes from fetuses with chromosome aneuploidy.
Homologue pairing process in euploid fetuses
Analysis of chromosome 21 and X pairing has revealed that this is a very efficient process, as has been proposed previously (Roig et al. 2005a, 2005b), which ensures the encounter of the homologues achieving a low pairing error rate. Pachytene-stage bivalent formation efficiency obtained for chromosomes 21 and X do not significantly differ from data recently published concerning the chromosome 13 and 18 pairing processes (Roig et al. 2005a; lineal-by-lineal analysis, P = 0.977). Thus, taking into consideration the data from all of the chromosomes analyzed in both papers, an overall pairing error rate of 0.14% can be obtained. This rate is very low when compared with the high rate of unbalanced oocytes II found in humans, which is ~25% as recently reviewed by Morelli & Cohen (2005). These data agree with the hypothesis that implication of the pairing process in the origin of the high number of human unbalanced oocytes is minimal (Roig et al. 2005a). Additionally, these data could suggest that pairing process efficiency may be similar in all chromosomes, independent of chromosomal size, morphology or DNA content, but more studies should be performed analyzing other chromosomes to assess this hypothesis.
As has been reported in earlier studies (Cheng et al. 1995, 1998, Roig et al. 2005a, 2005b) for chromosomes 13, 18, and 21, we have observed that pairing of the 21 and X homologues starts during the late leptotene stage. Since the first piece of evidence of telomeric clustering has been detected during the leptotene/zygotene transition in human oocytes (Roig et al. 2004, 2005b), as was already proposed in humans (Bojko 1983) and demonstrated in other species (Scherthan 2003), these results seem to support the hypothesis that bouquet topology promotes homologue encounter and facilitates the synaptic process during meiotic prophase.
In this study, pre-meiotic non-disjunction events for chromosome 21 have been identified. We suggest that these pre-meiotic non-disjunction events are probably due to a precocious separation of sister chromatids, which occurred previously in prophase stages, and would increment the rate of unbalanced oocytes and the risk of aneuploidies, as was proposed by Cupisti et al.(2003) in a previous study with oocytes at metaphase II. Our study showed leptotene-stage oocytes from euploid fetuses with three chromosome 21s (0.2%, Table 2), which has also been described previously for chromosome 18 (0.6%; Roig et al. 2005a). The difference between the two studies (likelihood ratio χ2 test, P ≤ 0.001) and the absence of three univalents for the X chromosome or chromosome 13 indicates that the rate of pre-meiotic non-disjunction may be different for different chromosomes.
Homologue pairing process in aneuploid oocytes
The presence of an extra chromosome may alter other homologue pairing processes
Some authors have suggested that the presence of an extra chromosome may alter the pairing process of other chromosomes (Cheng et al. 1998). An interchromosomal effect has been principally suggested to occur in individuals affected with chromosomal translocations (Rousseaux et al. 1995, Oliver-Bonet et al. 2001, Blanco et al. 2003, Morel et al. 2004). In addition, studies performed in trisomic 21 oocytes suggested the existence of an interference for the homologue pairing process in chromosome 13 due to the presence of an extra chromosome 21 (Cheng et al. 1998). Nevertheless, this effect does not seem to be universal, because studies performed in trisomic 18 oocytes have not found any alteration of the chromosome 13 pairing process (Roig et al. 2005b). Since chromosomal interference in trisomic 21 oocytes has only been observed to occur between autosomes (Cheng et al. 1998), we investigated whether this effect could also occur in the X chromosome. Our study suggests that the presence of an extra chromosome 21 interferes withd the X chromosome pairing process (Fisher F, P = 0.02). However, the mechanism by which these homologue pairs interact is not clear. The present study and previous studies (Cheng et al. 1998) may indicate that the extra chromosome 21 non-specifically interferes with a particular chromosome during prophase. On the contrary, this interference of the extra chromosome has not been observed in trisomic 18 oocytes, most likely because in almost all of the pachytene-stage oocytes, the three chromosome 18s are found forming a trivalent (Roig et al. 2005b). In this sense, the interference found in trisomic 21 oocytes would be produced by the univalent 21 present at the pachytene stage, which may affect the pairing process of the rest of the homologues.
Bouquet topology in aneuploid oocytes
It has been reported previously that the proportion of oocytes with bouquet formation was significantly increased in trisomy 18 fetuses when compared with euploid fetuses (P < 0.001; Roig et al. 2005b). Results obtained in trisomic 21 oocytes corroborate this first observation found in aneuploid oocytes. The bouquet delay observed for trisomic 21 is not significantly different from the one previously found for trisomic 18 oocytes (Roig et al. 2005b). Therefore, taking into account that the trivalent formation efficiency for chromosome 21 is significantly lower than that observed for chromosome 18, it is our hypothesis that the bouquet delay would be necessary for the pairing of the three homologues, but it would not be sufficient to complete trivalent conformation. However, we cannot rule out that this bouquet delay could indicate a disturbed recombination process due to the presence of an extra chromosome in these oocytes.
Trivalent efficiency
Chromosome size may influence the pairing process and, as shown in this paper and by other experiments conducted in our laboratory, homologue pairing seems to progress more quickly in shorter chromosomes than in longer ones during human female meiosis (Table 2; Garcia et al. data not published). This fact was also observed in male meiosis of Rattus norvegicus (Scherthan & Schonborn 2001). Nevertheless, some authors have proposed that the double-strand breaks (DSBs) originated at the leptotene stage may help in the homologue pairing process (Moens et al. 1997, Tarsounas et al. 1999) and, as it has been published, longer chromosomes have more DSBs than the shorter ones. Alternatively, DNA content may also influence the homologue pairing process. Chromosomes 13 and 21 are nucleolar organization region (NOR)-bearing chromosomes; thus, as shown to occur during the meiotic prophase of R. norvegicus (Martínez-Flores et al. 2003), they may interact with the nucleolus during meiotic prophase. This event, which cannot occur in chromosome 18, may make the pairing process of the 13 and 21 homologues more difficult and therefore decrease the efficiency of trivalent 13 and 21 formation when compared with that of trivalent 18. Surprisingly, these pairing efficiency differences have not been detected in euploid oocytes (Table 2, and Roig et al. 2005a), implying that the pairing process for chromosomes 13, 18, and 21 is efficient enough to guarantee the formation of at least a bivalent at the pachytene stage.
Results found in trisomic 13 and 21 oocytes, as well as those already published for trisomic 18 oocytes, indicate that the trivalent formed at the pachytene stage is maintained until the diplotene stage. This finding is linked to the fact that the three homologues recombine among themselves, most likely two by two. Moreover, this process implies the existence of at least two recombination events among the three homologues in order to keep them together until the diplotene stage. Finding two recombination points in a trivalent 21 is a significant increase in the recombination rate observed for bivalent 21 in euploid oocytes by Tease et al.(2002) and agrees with the preliminary observations performed in our laboratory (Robles and Garcia). Thus, it seems that recombination events may adapt to the number of chromosomes synapsed. Alternatively, as proposed recently (Borner et al. 2004), crossover designation is established early in meiotic prophase when homologues start pairing; thus, we believe, in the case of the trisomic 21 oocytes, the three homologue 21s may contact only two by two; therefore, two crossovers are designated for trivalent 21.
Nonhomologous synapsis has been reported to occur in trisomic 21 oocytes (Barlow et al. 2002). Nevertheless, we have not observed any indication of this happening in our samples. Similarly, one of the first cytological analyses of trisomic 21 oocytes (Speed 1984) reported that only a small fraction of the pachytene-stage oocytes presented a full triple synapsis of the chromosome 21s. These data are in disagreement with our findings, possibly related to different factors. First, the technical approaches used in the mentioned studies are different from those used here. Moreover, in this study, the number of oocytes analyzed is significantly higher than those reported in the other studies. Secondly, a high variability between samples concerning synaptic configurations achieved by the three chromosome 21s has been observed in previous studies (Barlow et al. 2002) and here (data not shown). This fact would be in agreement with other studies focused in the recombination process, suggesting the existence of a high variability between samples in humans (Lenzi et al. 2005).
In summary, we conclude that the present study agrees with previous studies performed in euploid oocytes in which a high pairing process efficiency was described (Cheng & Gartler 1994, Cheng et al. 1995, Roig et al. 2005a). Our study presents the first analysis of the pairing process for chromosome 13 in oocytes from fetuses with trisomy 13. This study also presents indirect evidence of the existence of recombination in the trivalent, which would ensure maintenance of the three homologues jointly until the diplotene stage. Nevertheless, more studies should be performed in order to test the hypothesis proposed here regarding pre-meiotic non-disjunction, existence of an interchromosomal effect in human oocytes, identifying the regulators that control bouquet resolution in meiocytes, and finally, discovering the controlling factors governing the increased recombination rate observed in trivalent 21.
Biological material, gestational age, referral indication, and karyotype.
| Case | GA | Referral indication | Karyotype |
|---|---|---|---|
| GA, gestational age. | |||
| aKaryotype obtained from prenatal diagnosis. bKaryotype obtained from fetal ovarian culture. cThis sample was used in Roig et al. (2005b). | |||
| V17 | 16 | Down syndrome | 47,XX, +21a |
| V19 | 21 | Down syndrome | 47,XX, +21a |
| V26 | 20 | Down syndrome | 47,XX, +21a |
| V81 | 22 | Down syndrome | 47,XX, +21b |
| V83 | 22 | Down syndrome | 47,XX, +21b |
| V99 | 20 | Down syndrome | 47,XX, +21a |
| V113 | 17 | Down syndrome | 47,XX, +21a |
| V114 | 16 | Down syndrome | 47,XX, +21a |
| V116 | 22 | Down syndrome | 47,XX, +21a |
| V88 | 20 | Patau syndrome | 47, XX, +13a |
| V9 | 22 | Neural tube malformation | 46,XXa |
| V10 | 22 | Ventricular hypoplasy | 46,XXa |
| V11 | 22 | Cardiopathology | 46,XXa |
| V85 | 22 | Cardiopathology | 46,XXa |
| V89 | 22 | Social reason | 46,XXa |
| V94c | 20 | Neural tube malformation | 46,XXa |
| V109 | 18 | Anencephaly | 46,XXa |
Distribution of chromosome 21 and X signals throughout the meiotic prophase in euploid cases.
| Chromosome 21 | Chromosome X | |||||
|---|---|---|---|---|---|---|
| 1 Bivalent % (n) | 2 Univalents % (n) | 3 Univalents % (n) | 1 Bivalent % (n) | 2 Univalents % (n) | 3 Univalents % (n) | |
| Leptotene | 48.7 (207) | 51.1 (217) | 0.2 (1) | 36.2 (187) | 63.8 (329) | 0.0 (0) |
| Zygotene | 64.3 (155) | 35.7 (86) | 0.0 (0) | 42.4 (150) | 57.6 (204) | 0.0 (0) |
| Pachytene | 99.7 (742) | 0.3 (2) | 0.0 (0) | 100.0 (587) | 0.0 (0) | 0.0 (0) |
Distribution of chromosome 13 signals throughout the meiotic prophase in a trisomic 13 case.
| Trivalent % (n) | Partial trivalent % (n) | Bivalent plus univalent % (n) | 3 Univalents % (n) | |
|---|---|---|---|---|
| aDue to the desynapsis existing at the diplotene stage, all trivalent configurations found have been classified as ‘trivalent’. | ||||
| Leptotene | 0.0 (0) | 0.0 (0) | 18.4 (7) | 81.6 (31) |
| Zygotene | 30.9 (21) | 8.8 (6) | 42.6 (29) | 17.6 (12) |
| Pachytene | 68.6 (35) | 5.9 (3) | 25.5 (13) | 0.0 (0) |
| Diplotene | 71.4 (5)a | – | 28.6 (2) | 0.0 (0) |
Distribution of chromosome 21 signals throughout the meiotic prophase in a trisomic 21 case.
| Trivalent % (n) | Partial trivalent % (n) | Bivalent plus univalent % (n) | 3 Univalents % (n) | |
|---|---|---|---|---|
| aDue to the desynapsis existing at the diplotene stage, all trivalent configurations found have been classified as ‘trivalent’. | ||||
| Leptotene | 0.0 (0) | 0.0 (0) | 0.0 (0) | 100.0 (7) |
| Zygotene | 22.2 (6) | 18.5 (5) | 33.3 (9) | 25.9 (7) |
| Pachytene | 48.8 (41) | 16.7 (14) | 33.3 (28) | 1.2 (1) |
| Diplotene | 57.1 (8)a | – | 42.9 (6) | 0.0 (0) |
The proportion of bouquet and bouquet pachytene formations in oocytes from fetuses of different gestational ages with a euploid chromosome complement or with trisomy 21.
| Case | GA | Karyotype | Bouquet % (n) | Pachytene bouquet % (n) |
|---|---|---|---|---|
| n.a., Not analyzed. | ||||
| aPercentages obtained from Roig et al. (2005b). | ||||
| V81 | 22 | 47,XX, +21 | 45.0 (160) | 37.7 (98) |
| V83 | 22 | 47,XX, +21 | 60.0 (90) | 40.5 (79) |
| V99 | 20 | 47,XX, +21 | 44.3 (88) | 38.4 (78) |
| V113 | 17.3 | 47,XX, +21 | 48.7 (78) | 39.3 (61) |
| V114 | 16 | 47,XX, +21 | 47.0 (44) | 47.0 (23) |
| V116 | 22 | 47,XX, +21 | 47.0 (100) | n.a |
| V85 | 22 | 46,XX | n.a | 27.3 (84) |
| V89 | 22 | 46,XX | n.a | 26.0 (100) |
| V94 | 20 | 46,XX | 28.0a | 30.0 (100) |
| V109 | 18 | 46,XX | 31.2 (64) | 31.2 (48) |
X chromosome pairing process in euploid and trisomy 21 cases.
Chromosome 13 pairing process in oocytes from a trisomy 13 fetus. Pachytene oocytes (A) displaying pairing of the three chromosome 13s. Pachytene-stage oocyte (B) with a bivalent and a univalent 13. Pachytene-stage oocyte (C) with a partial trivalent 13.
Citation: Reproduction 133, 5; 10.1530/REP-06-0243
Pachytene-stage oocytes from a fetus with trisomy 21 showing different types of synapsis. (A) Total trivalent: all homologue 21s paired and synapsed. REC8 (red) and SYCP1 (green) colocalize along the homologue 21s with a single FISH signal (blue diffused signal) in the nucleus. (B) Bivalent plus univalent: two homologue 21s synapsed displaying a bivalent where REC8 (red) and SYCP1 (green) colocalize, whereas the third homologue is asynapsed with only REC8 (red) staining. (C) Partial trivalent: two homologue 21s completely synapsed and the other homologue is synapsed with them in certain regions; REC8 (red) and SYCP1 (green) staining are present in these regions.
Citation: Reproduction 133, 5; 10.1530/REP-06-0243
(A) The proportion of oocytes with bouquet formation from fetuses with a euploid chromosome complement (Roig et al. 2005b) or trisomy 21. (B) The proportion of oocytes with pachytene-stage bouquet formation. (C) A trisomy 21 oocyte at the zygotene stage with bouquet formation; dual IF was used with SYCP3 (green) and TIN2 (red). (D) A trisomy 21 oocyte with bouquet formation in late prophase; dual IF was used with SYCP1 (green) and TIN2 (red).
Citation: Reproduction 133, 5; 10.1530/REP-06-0243
In memoriam of Prof. J Egozcue Cuixart, our exceptional teacher, enthusiastic colleague and beloved friend. The authors wish to thank Mr Miguel Ángel Brieño and Ms Aïda Casanovas for their critical comments of the first draft of this manuscript, and all of the staff of the Hospital de la Vall d’Hebron, in particular Ms Núria Camats, for helping in sample collection. We also thank Dr Miguel Martínez, of the Statistical Laboratory of the School of Medicine (UAB, Spain), for his assistance with the analysis of the results. The English of this manuscript has been corrected by a native English-speaking instructor of our university. This work has been funded by a grant from the Spanish Ministerio de Sanidad (FIS 02/0297) and a grant from Universitat Autònoma de Barcelona (PRP2006-02). PR received a fellowship from a Ministerio de Sanidad grant (FIS 02/0297) and RG a fellowship from the Generalitat de Catalunya (2004FI 00953). The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
References
Baker TG1963 A quantitative and cytological study of germ cells in human ovaries. Proceedings of the Royal Society of London. Series B. Biological Sciences 158 417–433.
Barlow AL & Hultén MA1997 Combined immunocytogenetic and molecular cytogenetic analysis of meiosis I oocytes from normal human females. Zygote 6 27–38.
Barlow AL, Tease C & Hultén MA2002 Meiotic chromosome pairing in fetal oocytes of trisomy 21 human females. Cytogenetic and Genome Research 96 45–51.
Blanco J, Farreras A, Egozcue J & Vidal F2003 Meiotic behavior of the sex chromosomes in a 45,X/46,X,r(Y)/46,X,dic r(Y) patient whose semen was assessed by fluorescence in situ hybridization. Fertility and Sterility 79 913–918.
Blandau RJ1969 Observations on living oogonia and oocytes from human embryonic and fetal ovaries. American Journal of Obstetrics and Gynecology 104 310–319.
Bojko M1983 Human meiosis VIII. Chromosome pairing and formation of the synaptonemal complex in oocytes. Carlsberg Research Communications 48 457–483.
Borner GV, Kleckner N & Hunter N2004 Crossover/noncrossover differentiation, synaptonemal complex formation, and regulatory surveillance at the leptotene/zygotene transition of meiosis. Cell 117 29–45.
Cheng EY & Gartler SM1994 A fluorescent in situ hybridization analysis of X chromosome pairing in early human female meiosis. Human Genetics 94 389–394.
Cheng EY & Naluai-Cecchini T2004 FISHing for acrocentric associations between chromosomes 14 and 21 in human oogenesis. American Journal of Obstetrics and Gynecology 190 1781–1785.
Cheng EY, Chen YJ & Gartler SM1995 Chromosome painting analysis of early oogenesis in human trisomy 18. Cytogenetics and Cell Genetics 70 205–210.
Cheng EY, Chen YJ, Bonnet G & Gartler SM1998 An analysis of meiotic pairing in trisomy 21 oocytes using fluorescent in situ hybridization. Cytogenetics and Cell Genetics 80 48–53.
Cheng EY, Chen YJ, Disteche CM & Gartler SM1999 Analysis of a paracentric inversion in human oocytes: nonhomologous pairing in pachytene. Human Genetics 105 191–196.
Cupisti S, Conn C, Fragouli E, Whalley K, Millis J, Faed M & Delhanty J2003 Sequential FISH analysis of oocytes and polar bodies reveals aneuploidy mechanisms. Prenatal Diagnosis 23 663–668.
Fawcett DW1956 The fine structure of chromosomes in the meiotic prophase of vertebrate spermatocytes. Journal of Biophysical and Biochemical Cytology 2 403–406.
Garcia M, Dietrich M, Freixa L, Vink ACG, Ponsa M & Egozcue J1987 Development of the first meiotic prophase stages in human fetal oocytes observed by light microscopy. Human Genetics 77 223–232.
Garcia M, Dietrich M, Pujol R & Egozcue J1989 Nucleolar structures in chromosome and SC preparations from human oocytes at 1st meiotic prophase. Human Genetics 82 147–153.
Hartshorne GM, Barlow AL, Child TJ, Barlow DH & Hulten MA1999 Immunocytogenetic detection of normal and abnormal oocytes in human fetal ovarian tissue in culture. Human Reproduction 14 172–182.
Hassold T & Hunt P2001 To err (meiotically) is human: the genesis of human aneuploidy. Nature Reviews. Genetics 2 280–291.
Kim S, Kaminker P & Campisi J1999 TIN2, a new regulator of telomere length in human cells. Nature Genetics 23 405–412.
Kurilo LF1981 Oogenesis in antenatal development in man. Human Genetics 57 86–92.
Lammers JH, Offenberg HH, van Aalderen M, Vink AC, Dietrich AJ & Heyting C1994 The gene encoding a major component of the lateral elements of synaptonemal complexes of the rat is related to X-linked lymphocyte-regulated genes. Molecular and Cellular Biology 14 1137–1146.
Lenzi ML, Smith J, Snowden T, Kim M, Fishel R, Poulos BK & Cohen PE2005 Extreme heterogeneity in the molecular events leading to the establishment of chiasmata during meiosis I in human oocytes. American Journal of Human Genetics 76 112–127.
Marcon E & Moens PB2005 The evolution of meiosis: recruitment and modification of somatic DNA-repair proteins. BioEssays 27 795–808.
Martínez-Flores I, Egozcue J & García M2003 Synaptic process in rat (Rattus norvegicus): influence of the methodology on the results. Microscopy Research and Technique 60 450–457.
Meuwissen RL, Offenberg HH, Dietrich AJ, Riesewijk A, van Iersel M & Heyting C1992 A coiled-coil related protein specific for synapsed regions of meiotic prophase chromosomes. EMBO Journal 11 5091–5100.
Moens PB, Chen DJ, Shen Z, Kolas N, Tarsounas M, Heng HH & Spyropoulos B1997 Rad51 immunocytology in rat and mouse spermatocytes and oocytes. Chromosoma 106 207–215.
Morel F, Douet-Guilbert N, Roux C, Tripogney C, Le Bris MJ, De Braekeleer M & Bresson JL2004 Meiotic segregation of a t(7;8)(q11.21;cen) translocation in two carrier brothers. Fertility and Sterility 81 682–685.
Morelli MA & Cohen PE2005 Not all germ cells are created equal: aspects of sexual dimorphism in mammalian meiosis. Reproduction 130 761–781.
Moses MJ1956 Chromosomal structures in crayfish spermatocytes. Journal of Biophysical and Biochemical Cytology 2 215–218.
Ohno S, Klinger HB & Atkin NB1962 Human oogenesis. Cytogenetics 1 42–52.
Oliver-Bonet M, Navarro J, Codina-Pascual M, Carrera M, Egozcue J & Benet J2001 Meiotic segregation analysis in a t(4;8) carrier: comparison of FISH methods on sperm chromosome metaphases and interphase sperm nuclei. European Journal of Human Genetics 9 395–403.
Oliver-Bonet M, Turek P, Sun F, Ko E & Martin R2005 Temporal progression of recombination in human males. Molecular Human Reproduction 11 517–522.
Pfeifer C, Scherthan H & Thomsen PD2003 Sex-specific telomere redistribution and synapsis initiation in cattle oogenesis. Developmental Biology 255 206–215.
Prieto I, Tease C, Pezzi N, Buesa JM, Ortega S, Kremer L, Martínez A, Martínez-A C, Hultén MA & Barbero JL2004 Cohesin component dynamics during meiotic prophase I in mammalian oocytes. Chromosome Research 12 197–213.
Pujol A, Boiso I, Benet J, Veiga A, Durban M, Campillo M, Egozcue J & Navarro J2003 Analysis of nine chromosome probes in first polar bodies and metaphase II oocytes for the detection of aneuploidies. European Journal of Human Genetics 11 325–336.
Rasmussen SW & Holm PB1984 The synaptonemal complex, recombination nodules and chiasmata in human spermatocytes. Symposia of the Society for Experimental Biology 18 331–413.
Roig I, Vanrell I, Ortega A, Cabero L, Egozcue J & Garcia M2003 The use of foetal ovarian stromal cell culture for cytogenetic diagnosis. Cytotechnology 41 45–49.
Roig I, Liebe B, Egozcue J, Cabero L, Garcia M & Scherthan H2004 Female-specific features of recombinational double-stranded DNA repair in relation to synapsis and telomere dynamics in human oocytes. Chromosoma 113 22–33.
Roig I, Robles P, Garcia R, Martin M, Egozcue J, Cabero L, Barambio S & Garcia M2005a Evolution of the meiotic prophase and of the chromosome pairing process during human fetal ovarian development. Human Reproduction 20 2463–2469.
Roig I, Robles P, Garcia R, Martínez-Flores I, Egozcue J, Liebe B, Scherthan H & Garcia M2005b Chromosome 18 behaviour in human trisomic oocytes. Presence of an extra chromosome extends bouquet stage. Reproduction 129 565–575.
Rousseaux S, Chevret E, Monteil M, Cozzi J, Pelletier R, Devillard F, Lespinasse J & Sele B1995 Meiotic segregation in males heterozygote for reciprocal translocations: analysis of sperm nuclei by two and three colour fluorescence in situ hybridization. Cytogenetics and Cell Genetics 71 240–246.
Scherthan H2003 Knockout mice provide novel insights into meiotic chromosome and telomere dynamics. Cytogenetic and Genome Research 103 235–244.
Scherthan H2006 Factors directing telomere dynamics in synaptic meiosis. Biochemical Society Transactions 34 550–553.
Scherthan H & Schonborn I2001 Asynchronous chromosome pairing in male meiosis of the rat (Rattus norvegicus). Chromosome Research 9 273–282.
Speed RM1984 Meiotic configurations in female trisomy 21 foetuses. Human Genetics 66 176–180.
Speed RM1985 The prophase stages in human foetal oocytes studied by light and electron microscopy. Human Genetics 69 69–75.
Tarsounas M, Morita T, Pearlman RE & Moens PB1999 RAD51 and DMC1 form mixed complexes associated with mouse meiotic chromosome cores and synaptonemal complexes. Journal of Cell Biology 147 207–220.
Tease C, Hartshorne GM & Hultén MA2002 Patterns of meiotic recombination in human oocytes. American Journal of Human Genetics 70 1469–1479.
