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ATR, BRCA1 and γH2AX localize to unsynapsed chromosomes at the pachytene stage in human oocytes
RBMOnline - Vol 18 No 1. 2009 37-44 Reproductive BioMedicine Online; www.rbmonline.com/Article/3543 on web 3 November 2008
Article ATR, BRCA1 and GH2AX localize to unsynapsed chromosomes at the pachytene stage in human oocytes Dr Montserrat Garcia Caldés is a full professor of Medical Genetics at the Medicine School of the Universitat Autònoma de Barcelona, Spain. Her research focuses on the biology of reproduction, in particular human female meiosis, the origin of human aneuploidy and the effects of genotoxic agents in the maternal germ line.
Dr Montserrat Garcia Caldés R Garcia-Cruz1, I Roig1,2, P Robles1, H Scherthan3, M Garcia Caldés1,4 Unitat de Biologia Cellular i Genètica Mèdica, Departament de Biologia Cellular, Fisiologia i Immunologia, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain; 2Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA; 3Ins. Für Radiobiologie der Bundeswehr, Neuherbergstr.11, D-80937 München, FRG 4 Correspondence: Tel: +34 935811905; Fax: +34 935811025; e-mail: [email protected]
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Abstract Asynapsis of homologous chromosomes at the pachytene stage has been associated with gametogenic failure and infertility, but the cellular mechanisms involved are currently unknown in human meiocytes. In mice, the protein encoded by the breast-cancer susceptibility gene Brca1 has been described to direct kinase ATR (ataxia telangiectasia and Rad3 related) to any unpaired DNA at the pachytene stage, where ATR triggers H2AX phosphorylation, resulting in the silencing of those chromosomes. In this study, the distribution of ATR, BRCA1 and the phosphorylated histone GH2AX is assessed by immunofluorescence in human oocytes and it is found that they localize at unpaired chromosomes at the pachytene stage. Evidence is shown to propose that BRCA1, ATR and GH2AX in the human may be part of a system such as the one previously described in mouse, which signals unsynapsed chromosomes at pachytene and may lead to their silencing. Keywords: asynapsis, ATR, BRCA1, GH2AX, meiosis, oocyte
Introduction During the meiotic prophase, homologous chromosomes synapse, through the formation of a tripartite proteinaceous structure, the synaptonemal complex, and recombine. These two unique features are crucial for the segregation of the homologues and for the achievement of balanced haploid gametes. To date, several studies have reported increased rates of synaptic defects in patients with azoospermia and oligozoospermia, which lead to spermatogenesis arrest and infertility, either because of the synaptic defect per se or because of associated reduced recombination (Chaganti et al., 1980; Vidal et al., 1982; Navarro et al., 1986; Judis et al., 2004; Guichaoua et al., 2005; Sun et al., 2005; Topping et al., 2006). In the human female, meiotic studies linking synaptic defects and sterility are nonexistent, because the particular timing of oogenesis makes it impracticable to associate a meiotic prophase event (occurring in the fetal ovary), with a fertility phenotype (occurring in the
adult woman). However, several studies have described the synaptic process in euploid and aneuploid oocytes, the latter showing synaptic anomalies derived from their missing or extra chromosomes (Luciani et al., 1976; Wallace and Hulten, 1983, 1985; Speed, 1984, 1985, 1986, 1988; Garcia et al., 1987; Jagiello et al., 1987; Cheng and Gartler, 1994; Cheng et al., 1995, 1998, 1999; Barlow et al., 2002; Roig et al., 2005a,b; Robles et al., 2007). In any case, these studies are descriptive and, currently, little is known about the cellular mechanism underlying asynapsis or how synaptic errors may lead to meiotic arrest in human meiocytes. In higher eukaryotes, data on this subject come mainly from mice where synaptic defects have long been related to meiotic failure (Miklos, 1974; Burgoyne and Baker, 1984; Burgoyne et al., 1992; Odorisio et al., 1998).
© 2009 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB23 8DB, UK
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Article - ATR, BRCA1 and GH2AX localization in human oocytes - R Garcia-Cruz et al.
Recently, kinase ATR (ataxia telangiectasia and Rad3 related), the protein encoded by the breast-cancer susceptibility gene Brca1, and phosphorylated histone GH2AX have been shown to participate in the recognition of asynapsed chromosomes. The co-localization of ATR, BRCA1 and GH2AX at unpaired sites in wild-type, but not in Brca1-mutant mice meiocytes, suggests a role for BRCA1 in the recruitment of ATR at the sex body and any unsynapsed chromosome at the pachytene stage leading to the phosphorylation of H2AX by ATR. In turn, this event leads to the inactivation of these chromosomes (Turner et al., 2004, 2005), most likely by replacement of nucleosomes within the unsynapsed chromatin (van der Heijden et al., 2007). Other histone modifications, such as ubiquitination of histone H2A, are also linked to the silencing of unpaired chromosomes in the mouse (Baarends et al., 2005). This phenomenon, called meiotic silencing of unsynapsed chromatin (MSUC) (Schimenti, 2005), may underlie the more specialized process, known as meiotic sex chromosome inactivation (MSCI) (Monesi, 1965), which leads to the silencing of the XY body in mammalian spermatocytes. The aim of the present study was to further analyse the distribution of ATR, BRCA1 and GH2AX in human oocytes and to elucidate whether they associate with asynaptic chromosomes and could therefore be implicated in the synapsis checkpoint in the human female. To this end, co-localization studies of ATR and GH2AX and of BRCA1 distribution were performed in human oocytes exhibiting pairing defects due to the presence of an extra chromosome 21. Finally, immunofluorescence with an antibody against the active form of RNA polymerase II was performed to analyse whether unpaired pachytene chromosomes are also transcriptionally silenced in the human.
Materials and methods Biological material For this study, four 47,XX+21 fetuses (V113, V116, V125 and V152) were used. Gestational ages were 17, 22, 22 and 21 weeks respectively. Ovaries were obtained from female fetuses after legal interruption of pregnancy, according to the Ethics Rules Committee of the Hospital de la Vall d’Hebron, Barcelona, Spain. Ovaries were removed within 1 h after delivery and transported in 1% penicillin–streptomycin phosphate-buffered saline. Human testes material used in the RNA polymerase experiment was obtained from an adult male with proven fertility.
Meiotic preparations Samples were processed as described elsewhere (MartinezFlores et al., 2003; Roig et al., 2004) to obtain oocyte spreads for immunofluorescence purposes. Human testes material was processed as previously described (Scherthan et al., 1996).
Immunofluorescence
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A standard protocol was used for immunostaining of meiocytes. The following primary antibodies were used: mouse polyclonal serum against cohesin REC8 (Prieto et al., 2004) (courtesy of JL Barbero, Madrid, Spain), rabbit polyclonal serum against
synaptonemal complex central element protein 1 (SYCP1; Meuwissen et al., 1992) (courtesy of C Heyting, Wageningen, The Netherlands), goat polyclonal antibody against kinase ATR (Santa Cruz Biotechnology, Santa Cruz, USA), rabbit polyclonal antibody against histone GH2AX (Upstate, Lake Placid, USA), rabbit polyclonal antibody against BRCA1 (Abcam, Cambridge, UK) and mouse monoclonal antibody against the phosphorylated form of RNA Polymerase II (Covance, Berkeley, USA). Fluorochromeconjugated secondary antibodies were used for detection (Jackson ImmunoResearch Laboratories, West Grove, USA, and Molecular Probes, Carlsbad, USA). DNA was counterstained by applying an antifade solution (Vector Laboratories, Burlingame, USA) containing 0.1 Mg/ml 4',6'-diamidino-2-phenylindole (DAPI; Sigma, Munich, Germany).
Fluorescent in-situ hybridization (FISH) on immunostained preparations Immunofluorescence-stained preparations were hybridized with a locus-specific probe for 21q22 (Qbiogene, Morgan Irvine, USA) or with a whole chromosome 21 paint probe (Metasystems, Altlussheim, Germany). The hybridization protocol used has been previously described (Roig et al., 2005b).
Microscopy and image analysis Preparations were visualized on an Olympus BX70 (Olympus Optical Co., Hamburg, Germany) fluorescence microscope. Images were captured and produced by Smart Capture software (Digital Scientific, Cambridge, UK) and further processed using Adobe Photoshop (Adobe Systems Inc., USA) to match the fluorescence intensity seen in the microscope. Micromeasure 3.3 software (available at http://www.biology.colostate.edu/ MicroMeasure) was used for analysis of synaptonemal complex length.
Results ATR localizes to unpaired axial elements at pachytene To elucidate whether ATR, BRCA1 and GH2AX associate with unsynapsed chromosomes in human oocytes, 47,XX+21 oocytes were used as a model because they exhibit a high percentage of unpaired chromosomes at pachytene (Luciani et al., 1976; Wallace and Hulten, 1983; Speed, 1984; Jagiello et al., 1987; Cheng et al., 1998; Barlow et al., 2002), which is substantially higher than in other trisomic cases (Robles et al., 2007). Therefore, Down syndrome oocyte preparations were stained using antibodies against ATR and antibodies against lateral element cohesin REC8 and central element protein SYCP1 to ascertain the presence of synapsed/ unsynapsed chromosomes. Pachytene oocytes were divided into different groups depending on the synaptic status of the three chromosomes 21. A first group (n = 29) showed two chromosomes 21 synapsed in a normal bivalent configuration while the third chromosome 21 remained completely unpaired as revealed by the absence of SYCP1 staining. Both bivalent and univalent were confirmed by
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Article - ATR, BRCA1 and GH2AX localization in human oocytes - R Garcia-Cruz et al.
posterior FISH analysis using a specific probe for chromosome 21. ATR marked the asynapsed chromosome 21 in 79.3% (n = 23/29) of the oocytes, either as an intense linear signal (37.9%, n = 11/29) (Figure 1A) or as a linear track of bright foci (41.4%, n = 12/29) (Figure 1B), while bivalent 21 did not show such staining. Such ATR staining on unsynapsed chromosome axes has also been observed in euploid oocytes showing pairing defects (data not shown). However, in 20.7% (n = 6/29) of the trisomic 21 oocytes analysed, the unsynapsed chromosome 21 did not show differential ATR staining (Figure 1C). A second group (n = 8) of oocytes showed the three chromosomes 21 completely aligned and engaged in a synaptic-like structure of the three axial elements. ATR did not associate with any of these trivalents (Figure 1D). A third group of oocytes (n = 27) displayed the three chromosomes 21 partially synapsed, meaning that the three homologues only paired in some portions thus creating a partial trivalent configuration. In this group, ATR staining pattern was variable: some of them showed differential ATR staining on the unpaired portion of the partial trivalent, either as a continuous signal (14.81%, n = 4/27) or as ATR bright foci (33.33%, n = 9/27), while other partial trivalent configurations did not show differential staining on the unpaired portion of the trivalent (51.85%, n = 14/27). It is worth mentioning that isolated ATR bright foci were frequently observed on other chromosomes other than the unsynapsed, but never as a linear track on the same chromosome, a feature only observed on unsynapsed axes. This feature was also observed in euploid pachytene oocytes (data not shown).
BRCA1 associates with any unpaired axial elements at pachytene The staining of human 47,XX+21 oocytes using antibodies against BRCA1 and REC8 revealed that BRCA1 foci were present at paired and unpaired axes at zygotene, but the signal became more intense at unsynapsed areas and pairing forks from mid-zygotene onwards (Figure 1E), a feature that matches the situation in euploid oocytes (data not shown). In addition, BRCA1 distribution was analysed in a total of 29 trisomic pachytene oocytes. In 100% of the oocytes showing a bivalent plus univalent configuration (n = 20), BRCA1 specifically marked the unsynapsed chromosome 21 (Figure 1F), either as a continuous signal in the majority of cases or as a linear track of bright foci in the few others. In those oocytes that showed the three chromosomes 21 forming a partial trivalent (n = 6), BRCA1 always marked the unsynapsed portions of all of them. Finally, a third group of oocytes was found where the three chromosomes 21 seemed to be totally synapsed, forming a total trivalent (n = 3). In these cells, BRCA1 did not mark any of these trivalents, similar to the ATR findings.
GH2AX accumulates at the unpaired chromosomes marked by ATR The analysis of trisomic 21 oocytes stained for ATR, GH2AX and REC8 showed that an intense GH2AX signal appeared on the chromatin encompassing those univalents recognized by
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ATR at pachytene (Figure 2), either as a linear signal (Figure 2A) or as a linear track of bright foci (Figure 2B). Therefore, in such pachytene cells, two different kinds of GH2AX signal coexist in the nuclei (Figure 2C). One type of focus is associated with the synaptonemal complexes, as has been observed in euploid oocytes where it has been related to the signalling of double-strand breaks generated during the meiotic process (Roig et al., 2004; Lenzi et al., 2005). This kind of focus frequently co-localizes with a focus of ATR (Figure 2A and 2B, arrows), a feature that has also been observed in euploid oocytes (data not shown). In addition, in trisomic 21 oocytes, a second type of GH2AX signal was observed, which was widely spread on the chromatin and surrounding the axial elements and that appeared only on those unpaired chromosomes that were stained by ATR, as mentioned above. However, such GH2AX on the univalent did not appear on all pachytene cells displaying univalents 21, and it was suspected that such a signal appeared as the pachytene stage progressed. Taking into account that chromosomes compact as pachytene steps forward, the oocytes were divided into three subgroups according to the length of the univalent (above 10 Mm, between 10 and 7.5 Mm, and less than 7.5 Mm) in order to assess the evolution of GH2AX signal on unsynapsed chromosome 21 with pachytene stage progression (Figure 3). On univalents, the GH2AX signal was seen either as a massive signal, totally or partially covering the axial element, or as isolated foci of an intensity and shape equivalent to the GH2AX recombinational foci. Results on GH2AX distribution in each of the three subgroups presented in Figure 3 show how the recombinational GH2AX signal disappears in favour of the spread GH2AX signal as the axial element reduces in length. On the other hand, the massive signal covering the whole length of the unsynapsed chromosome axis appears as the axial elements of chromosome 21 compacts. Therefore, it can be concluded that H2AX on the chromatin of the whole univalent 21 becomes progressively phosphorylated as pachytene stage progresses.
MSUC occurs during human female meiosis The transcriptional status of human meiocytes was analysed using an antibody against the active form of RNA polymerase II. As this approach has not been used in human meiocytes before, the RNA polymerase signal was first analysed on human spermatocytes. The RNA polymerase signal becomes more intense as prophase progresses from leptotene to pachytene (data not shown). As expected, the presence of RNA polymerase at the pachytene stage was evidently reduced or absent in the defined area of the nuclei covering the XY body of the spermatocytes (Figure 4A), showing the silencing of the unpaired regions of chromosomes X and Y. The transcriptional status of univalents 21 in human 47,XX+21 oocytes was analysed in pachytene and diplotene cells. A decrease in the RNA polymerase signal was observed at GH2AX-positive areas, revealing that the chromosomes that remain unpaired are at least partially transcriptionally silenced (n = 26). The decrease in the RNA polymerase signal was hardly visible in early pachytene nuclei, but became more perceptible as pachytene progressed (Figure 4B) and even more evident in diplotene cells. However, there was always a residual RNA
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Figure 1. Localization of ataxia telangiectasia and Rad3 related (ATR) and BRCA1 in 47,XX+21 oocytes. Cohesin REC8 labels the axial elements and the synaptonemal complex central element protein 1 (SYCP1). Locus-specific identification probe for 21q22 (LSI 21) shows the position of chromosome 21 (stained after immunofluorescence). (A–C) Pachytene-stage oocytes where two chromosomes 21 are synapsed (arrow) and the third chromosome 21 remains unpaired (arrowhead). ATR marks the unpaired chromosome, either as a continuous signal (A) or by a bright foci pattern (B). (C) An oocyte is shown with an unpaired chromosome 21 not marked by ATR. (D) Pachytene-stage oocyte where the three chromosomes 21 have engaged in total synapsis (arrowhead). ATR does not label the trivalent. (E) Zygotene-stage oocyte stained for BRCA1. BRCA1 remains at unpaired axial elements and pairing forks (enlarged image details). (F) Pachytene-stage oocyte with a univalent 21 (arrowhead) and a bivalent 21 (arrow). BRCA1 covers the unpaired chromosome 21. Bar in right panel of E represents 4.5 Mm. Bar in A represents 10 Mm and applies to all other panels in Figure 1.
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Figure 2. Localization of the phosphorylated histone GH2AX in 47,XX+21 pachytene oocytes. Cohesin REC8 labels the axial elements. (A, B) Oocytes where the unpaired chromosomes 21 are marked by ataxia telangiectasia and Rad3 related (ATR) either as a linear staining (A) or as a track of bright foci (B). GH2AX is distributed throughout the chromatin of the univalent in both situations. Arrows show co-localization between ATR and GH2AX foci on synaptonemal complex. (C) Enlarged image of the GH2AX staining from the same oocyte as in (A). GH2AX shows two different kinds of signal: small clouds of GH2AX on the synaptonemal complex (arrows) and a large signal throughout the chromatin of the univalent (arrowhead). Bar in A represents 10 Mm and also applies to panel B. RBMOnline®
Article - ATR, BRCA1 and GH2AX localization in human oocytes - R Garcia-Cruz et al.
GH2AX foci GH2AX spread signal partially covering the univalent 21 GH2AX spread signal covering the whole univalent 21
100
Percentage
80 60 40 20 0 4.79–7.5
7.5–10
>10
Univalent length (Mm)
Figure 3. Phosphorylated histone GH2AX signal on the unpaired chromosome 21 in pachytene 47,XX+21 oocytes according to univalent length.
Figure 4. RNA synthesis as disclosed by RNA polymerase II immunostaining in human pachytene spermatocytes and in 47,XX+21 pachytene oocytes. Cohesin REC8 labels the axial elements. (A) The RNA polymerase signal is absent in the XY body on human spermatocytes. (B) Pachytene oocyte with an unpaired chromosome 21. Phosphorylated histone GH2AX marks the chromatin of the univalent and the RNA polymerase II signal is lower. Whole chromosome 21 paint probe (WCP21) shows the position of chromosome 21 in the same nucleus (stained after immunofluorescence). DAPI = 4a,6a-diamidino-2-phenylindole. Bar represents 10 Mm and applies to all panels.
polymerase signal and the decrease was never comparable to the one observed in the XY domain in human spermatocytes. During this analysis, a diminished RNA polymerase signal was also detected in areas of some pachytene and diplotene nuclei with GH2AX-positive domains that resulted from pairing errors at regions different from chromosome 21 univalents. It has to be mentioned that both in oocytes and spermatocytes the expression of RNA polymerase was not homogeneous in the rest of the nuclei and that other domains showed lower RNA polymerase levels, most probably due to constitutional silent regions such as pericentromeric and other heterochromatic regions.
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Discussion Human oogenesis is an extremely error-prone process that progresses with an elevated rate of atresia (Hassold and Hunt, 2001; Hunt and Hassold, 2008; Martin, 2008). The identification of the mechanisms involved in this major hitch has been one of the fundamental subjects of the scientific and medical community involved in reproductive biology, yet the exact biological basis is still poorly understood. However, different meiotic and cellular processes have been claimed to be involved in the high ratio of atresia and aneuploidy: abnormal patterns of meiotic recombination (for a review see Lamb et al., 2005), pairing and
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Article - ATR, BRCA1 and GH2AX localization in human oocytes - R Garcia-Cruz et al.
synapsis defects (Speed, 1985, 1988; Tease et al., 2006), loss of chromosome cohesion (Hodges et al., 2005), spindle aberrations and congression failure (Volarcik et al., 1998; Hodges et al., 2002), the existence of a chromosome aggregation phase during oocyte maturation (Otsuki and Nagai, 2007), lack of checkpoint at the anaphase/metaphase transition (LeMaire-Adkins et al., 1997), environmental factors and others. The present study has attempted to shed some light on the current knowledge of the mechanisms functioning in the human oocyte when synapsis defects occur. Selective association of ATR, BRCA1 and GH2AX with unpaired chromosome axes was observed, lending itself to supportive evidence that indicates that BRCA1, ATR and GH2AX may be part of a mechanism in the human similar to the one previously described in mouse (Turner et al., 2005), where it has been described that BRCA1 guides ATR to regions that remain unpaired at pachytene, followed by H2AX phosphorylation by ATR. This study’s findings show that the unpaired chromosomes showed an extensive GH2AX signal on the chromatin of the univalent, which has been related to the silencing of unpaired chromosomes (Baarends et al., 2005; Turner et al., 2005) different from the much more focused signal that appears on the rest of the synaptonemal complexes in euploid and aneuploid oocytes and related to the recombination process (Roig et al., 2004; Lenzi et al., 2005). Therefore, these findings highlight the importance of the change in the chromatin state produced by H2AX phosphorylation, which may contribute to different processes during meiotic prophase, such as facilitating DNA repair and as part of the meiotic checkpoint apparatus, possibly inhibiting access of the transcriptional machinery to chromatin and thus silencing unpaired chromosomes. This is especially significant in the human female, when both processes may be operational at the same time during the pachytene stage. The finding that a proportion of univalents were not recognized by ATR substantially differs from what has been described in the mouse (Turner et al., 2005). However, the study shows that any unpaired DNA at the pachytene stage is recognized by BRCA1, a protein whose role in the recruitment of ATR to unsynapsed DNA has been reported (Turner et al., 2005). The fact that all unpaired DNA is recognized by BRCA1, but not by ATR, indicates that, in the human female, BRCA1 also seems to act upstream of ATR in the synapsis checkpoint and could have a role in recruiting ATR to the unsynapsed DNA. Therefore, the absence of ATR in some univalents may just be a question of timing, rather than a decreased efficiency in detecting asynapsed chromosomes. This hypothesis is supported by the finding that the GH2AX signal encompassing the univalent, which is a feature linked to the ATR signalling on the univalent, also appears as pachytene progresses.
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During the progression of this study, different meiotic configurations between the three chromosomes 21 were found, as others have previously reported (Luciani et al., 1976; Wallace and Hulten, 1983; Speed, 1984; Jagiello et al., 1987; Barlow et al., 2002; Robles et al., 2007): bivalent plus univalent, partially synapsed trivalents and totally synapsed trivalents were found. Of these nuclei, only the ones showing totally synapsed trivalents escape from ATR-BRCA1-GH2AX signalling at pachytene. This observation leads us to believe that, in trivalent configurations, not only pairing but also complete synapsis is achieved between
the three homologues in all their length, creating a unique synaptonemal complex configuration. Similarly, in 47,XYY men, spermatocytes achieving full synapsis between the three PAR regions are able to escape the pachytene checkpoint and progress through meiosis (Milazzo et al., 2006). The observation that RNA polymerase II signal is diminished on GH2AX-positive chromatin corroborates that unpaired chromosomes are at least partially silenced in the human female, similar to what has been reported in the mouse, where it is speculated that this fact could lead to the apoptosis of those nuclei. However, in the present study, the finding of a considerable amount of diplotene cells with spread GH2AX signals leads us to speculate that MSUC and the ATR checkpoint function is not sufficient to prevent such nuclei from progressing into diplotene. Sciurano et al. (2006) reported the presence of spermatocytes at the division stage showing GH2AX patches in an azoospermic man with severe pairing defects and extensive BRCA1 staining in prophase spermatocytes, also suggesting that asynapsis does not necessarily abort meiocytes at the prophase stage. In the study samples, the presence of a normally transcribing bivalent 21 and a silenced third 21 copy could create a normal level of chromosome 21 transcripts, which could explain why these nuclei progress beyond pachytene. The impossibility of studying human trisomic 21 oocytes beyond diplotene leaves further aspects and potential consequences of this phenomenon at later stages in the dark. Therefore, it seems that MSUC does not necessarily have detrimental consequences for human trisomic oocytes. However, the existence of this phenomenon could have devastating consequences for monosomic oocytes since the transcription of a whole chromosome would be null from pachytene. For that reason, the assessment of the consequences of MSUC in monosomic oocytes would be of great interest. Nevertheless, contrary to the situation in mice, the proportion of XO human oocytes that reach pachytene is almost nil (Speed, 1986; R Garcia-Cruz and M Garcia, unpublished) and, on the other hand, the chance of obtaining samples from other monosomic fetuses, old enough to enable meiotic studies to be performed, is highly improbable. This makes the analysis of the effect of MSUC in monosomic oocytes impracticable at the moment. Nevertheless, the effects of MSUC in the human could have an enormous implication not only in monosomic oocytes but also, and therefore its significance, in euploid oocytes. A high rate of pairing defects is known to occur in human oocytes (Speed, 1988; Hartshorne et al., 1999; Tease et al., 2006) and, according to the results of this study, this may lead to the silencing of such unpaired DNA sequences. Although not reported here, during this study a considerable number of pachytene and diplotene oocytes with GH2AX-positive domains were found, and lower RNA polymerase expression in chromosomes other than univalent 21. The effects of the inactivation of fragments of chromosomes could have an enormous impact on human oogenesis and could partially contribute to the elevated rate of atresia that usually occurs in the oocyte population of the developing human ovary.
Acknowledgements We wish to thank Dr JL Barbero and Dr C Heyting for kindly providing REC8 and SYCP1 antibodies, respectively. We also thank Dr James Turner, Dr Paul Burgoyne, Dr Scott Keeney and RBMOnline®
Article - ATR, BRCA1 and GH2AX localization in human oocytes - R Garcia-Cruz et al.
all the members of the Keeney laboratory, as well as MA Brieño and A Casanovas for their critical reading of the manuscript. The English of this manuscript has been read and corrected by Mr Chuck Simmons, a native English-speaking instructor of English of the Universitat Autònoma de Barcelona. RGC is recipient of a grant from Agència de Gestió d’Ajuts Universitaris i de Recerca de la Generalitat de Catalunya (2004FI00953). This work was carried out with financial support from: Fondo de Investigación Sanitaria (FIS 02/0297); Universtitat Autònoma de Barcelona (PRP2006–02); Ministerio de Ciencia y Tecnología (BFU2006–1295).
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Speed RM 1984 Meiotic configurations in female trisomy 21 foetuses. Human Genetics 66, 176–180. Sun F, Greene C, Turek PJ et al. 2005 Immunofluorescent synaptonemal complex analysis in azoospermic men. Cytogenetic and Genome Research 111, 366–370. Tease C, Hartshorne G, Hulten M 2006 Altered patterns of meiotic recombination in human fetal oocytes with asynapsis and/or synaptonemal complex fragmentation at pachytene. Reproductive BioMedicine Online 13, 88–95. Topping D, Brown P, Judis L et al. 2006 Synaptic defects at meiosis I and non-obstructive azoospermia. Human Reproduction 21, 3171–3177. Turner JM, Mahadevaiah SK, Fernandez-Capetillo O et al. 2005 Silencing of unsynapsed meiotic chromosomes in the mouse. Nature Genetics 37, 41–47. Turner JM, Aprelikova O, Xu X et al. 2004 BRCA1, histone H2AX phosphorylation, and male meiotic sex chromosome inactivation. Current Biology 14, 2135–2142. van der Heijden GW, Derijck AA, Posfai E et al. 2007 Chromosomewide nucleosome replacement and H3.3 incorporation during mammalian meiotic sex chromosome inactivation. Nature Genetics 39, 251–258.
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Declaration: The authors report no financial or commercial conflicts of interest. Received 17 March 2008; refereed 2 April 2008; accepted 18 July 2008.
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Article ATR, BRCA1 and GH2AX localize to unsynapsed chromosomes at the pachytene stage in human oocytes Dr Montserrat Garcia Caldés is a full professor of Medical Genetics at the Medicine School of the Universitat Autònoma de Barcelona, Spain. Her research focuses on the biology of reproduction, in particular human female meiosis, the origin of human aneuploidy and the effects of genotoxic agents in the maternal germ line.
Dr Montserrat Garcia Caldés R Garcia-Cruz1, I Roig1,2, P Robles1, H Scherthan3, M Garcia Caldés1,4 Unitat de Biologia Cellular i Genètica Mèdica, Departament de Biologia Cellular, Fisiologia i Immunologia, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain; 2Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA; 3Ins. Für Radiobiologie der Bundeswehr, Neuherbergstr.11, D-80937 München, FRG 4 Correspondence: Tel: +34 935811905; Fax: +34 935811025; e-mail: [email protected]
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Abstract Asynapsis of homologous chromosomes at the pachytene stage has been associated with gametogenic failure and infertility, but the cellular mechanisms involved are currently unknown in human meiocytes. In mice, the protein encoded by the breast-cancer susceptibility gene Brca1 has been described to direct kinase ATR (ataxia telangiectasia and Rad3 related) to any unpaired DNA at the pachytene stage, where ATR triggers H2AX phosphorylation, resulting in the silencing of those chromosomes. In this study, the distribution of ATR, BRCA1 and the phosphorylated histone GH2AX is assessed by immunofluorescence in human oocytes and it is found that they localize at unpaired chromosomes at the pachytene stage. Evidence is shown to propose that BRCA1, ATR and GH2AX in the human may be part of a system such as the one previously described in mouse, which signals unsynapsed chromosomes at pachytene and may lead to their silencing. Keywords: asynapsis, ATR, BRCA1, GH2AX, meiosis, oocyte
Introduction During the meiotic prophase, homologous chromosomes synapse, through the formation of a tripartite proteinaceous structure, the synaptonemal complex, and recombine. These two unique features are crucial for the segregation of the homologues and for the achievement of balanced haploid gametes. To date, several studies have reported increased rates of synaptic defects in patients with azoospermia and oligozoospermia, which lead to spermatogenesis arrest and infertility, either because of the synaptic defect per se or because of associated reduced recombination (Chaganti et al., 1980; Vidal et al., 1982; Navarro et al., 1986; Judis et al., 2004; Guichaoua et al., 2005; Sun et al., 2005; Topping et al., 2006). In the human female, meiotic studies linking synaptic defects and sterility are nonexistent, because the particular timing of oogenesis makes it impracticable to associate a meiotic prophase event (occurring in the fetal ovary), with a fertility phenotype (occurring in the
adult woman). However, several studies have described the synaptic process in euploid and aneuploid oocytes, the latter showing synaptic anomalies derived from their missing or extra chromosomes (Luciani et al., 1976; Wallace and Hulten, 1983, 1985; Speed, 1984, 1985, 1986, 1988; Garcia et al., 1987; Jagiello et al., 1987; Cheng and Gartler, 1994; Cheng et al., 1995, 1998, 1999; Barlow et al., 2002; Roig et al., 2005a,b; Robles et al., 2007). In any case, these studies are descriptive and, currently, little is known about the cellular mechanism underlying asynapsis or how synaptic errors may lead to meiotic arrest in human meiocytes. In higher eukaryotes, data on this subject come mainly from mice where synaptic defects have long been related to meiotic failure (Miklos, 1974; Burgoyne and Baker, 1984; Burgoyne et al., 1992; Odorisio et al., 1998).
© 2009 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB23 8DB, UK
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Recently, kinase ATR (ataxia telangiectasia and Rad3 related), the protein encoded by the breast-cancer susceptibility gene Brca1, and phosphorylated histone GH2AX have been shown to participate in the recognition of asynapsed chromosomes. The co-localization of ATR, BRCA1 and GH2AX at unpaired sites in wild-type, but not in Brca1-mutant mice meiocytes, suggests a role for BRCA1 in the recruitment of ATR at the sex body and any unsynapsed chromosome at the pachytene stage leading to the phosphorylation of H2AX by ATR. In turn, this event leads to the inactivation of these chromosomes (Turner et al., 2004, 2005), most likely by replacement of nucleosomes within the unsynapsed chromatin (van der Heijden et al., 2007). Other histone modifications, such as ubiquitination of histone H2A, are also linked to the silencing of unpaired chromosomes in the mouse (Baarends et al., 2005). This phenomenon, called meiotic silencing of unsynapsed chromatin (MSUC) (Schimenti, 2005), may underlie the more specialized process, known as meiotic sex chromosome inactivation (MSCI) (Monesi, 1965), which leads to the silencing of the XY body in mammalian spermatocytes. The aim of the present study was to further analyse the distribution of ATR, BRCA1 and GH2AX in human oocytes and to elucidate whether they associate with asynaptic chromosomes and could therefore be implicated in the synapsis checkpoint in the human female. To this end, co-localization studies of ATR and GH2AX and of BRCA1 distribution were performed in human oocytes exhibiting pairing defects due to the presence of an extra chromosome 21. Finally, immunofluorescence with an antibody against the active form of RNA polymerase II was performed to analyse whether unpaired pachytene chromosomes are also transcriptionally silenced in the human.
Materials and methods Biological material For this study, four 47,XX+21 fetuses (V113, V116, V125 and V152) were used. Gestational ages were 17, 22, 22 and 21 weeks respectively. Ovaries were obtained from female fetuses after legal interruption of pregnancy, according to the Ethics Rules Committee of the Hospital de la Vall d’Hebron, Barcelona, Spain. Ovaries were removed within 1 h after delivery and transported in 1% penicillin–streptomycin phosphate-buffered saline. Human testes material used in the RNA polymerase experiment was obtained from an adult male with proven fertility.
Meiotic preparations Samples were processed as described elsewhere (MartinezFlores et al., 2003; Roig et al., 2004) to obtain oocyte spreads for immunofluorescence purposes. Human testes material was processed as previously described (Scherthan et al., 1996).
Immunofluorescence
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A standard protocol was used for immunostaining of meiocytes. The following primary antibodies were used: mouse polyclonal serum against cohesin REC8 (Prieto et al., 2004) (courtesy of JL Barbero, Madrid, Spain), rabbit polyclonal serum against
synaptonemal complex central element protein 1 (SYCP1; Meuwissen et al., 1992) (courtesy of C Heyting, Wageningen, The Netherlands), goat polyclonal antibody against kinase ATR (Santa Cruz Biotechnology, Santa Cruz, USA), rabbit polyclonal antibody against histone GH2AX (Upstate, Lake Placid, USA), rabbit polyclonal antibody against BRCA1 (Abcam, Cambridge, UK) and mouse monoclonal antibody against the phosphorylated form of RNA Polymerase II (Covance, Berkeley, USA). Fluorochromeconjugated secondary antibodies were used for detection (Jackson ImmunoResearch Laboratories, West Grove, USA, and Molecular Probes, Carlsbad, USA). DNA was counterstained by applying an antifade solution (Vector Laboratories, Burlingame, USA) containing 0.1 Mg/ml 4',6'-diamidino-2-phenylindole (DAPI; Sigma, Munich, Germany).
Fluorescent in-situ hybridization (FISH) on immunostained preparations Immunofluorescence-stained preparations were hybridized with a locus-specific probe for 21q22 (Qbiogene, Morgan Irvine, USA) or with a whole chromosome 21 paint probe (Metasystems, Altlussheim, Germany). The hybridization protocol used has been previously described (Roig et al., 2005b).
Microscopy and image analysis Preparations were visualized on an Olympus BX70 (Olympus Optical Co., Hamburg, Germany) fluorescence microscope. Images were captured and produced by Smart Capture software (Digital Scientific, Cambridge, UK) and further processed using Adobe Photoshop (Adobe Systems Inc., USA) to match the fluorescence intensity seen in the microscope. Micromeasure 3.3 software (available at http://www.biology.colostate.edu/ MicroMeasure) was used for analysis of synaptonemal complex length.
Results ATR localizes to unpaired axial elements at pachytene To elucidate whether ATR, BRCA1 and GH2AX associate with unsynapsed chromosomes in human oocytes, 47,XX+21 oocytes were used as a model because they exhibit a high percentage of unpaired chromosomes at pachytene (Luciani et al., 1976; Wallace and Hulten, 1983; Speed, 1984; Jagiello et al., 1987; Cheng et al., 1998; Barlow et al., 2002), which is substantially higher than in other trisomic cases (Robles et al., 2007). Therefore, Down syndrome oocyte preparations were stained using antibodies against ATR and antibodies against lateral element cohesin REC8 and central element protein SYCP1 to ascertain the presence of synapsed/ unsynapsed chromosomes. Pachytene oocytes were divided into different groups depending on the synaptic status of the three chromosomes 21. A first group (n = 29) showed two chromosomes 21 synapsed in a normal bivalent configuration while the third chromosome 21 remained completely unpaired as revealed by the absence of SYCP1 staining. Both bivalent and univalent were confirmed by
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Article - ATR, BRCA1 and GH2AX localization in human oocytes - R Garcia-Cruz et al.
posterior FISH analysis using a specific probe for chromosome 21. ATR marked the asynapsed chromosome 21 in 79.3% (n = 23/29) of the oocytes, either as an intense linear signal (37.9%, n = 11/29) (Figure 1A) or as a linear track of bright foci (41.4%, n = 12/29) (Figure 1B), while bivalent 21 did not show such staining. Such ATR staining on unsynapsed chromosome axes has also been observed in euploid oocytes showing pairing defects (data not shown). However, in 20.7% (n = 6/29) of the trisomic 21 oocytes analysed, the unsynapsed chromosome 21 did not show differential ATR staining (Figure 1C). A second group (n = 8) of oocytes showed the three chromosomes 21 completely aligned and engaged in a synaptic-like structure of the three axial elements. ATR did not associate with any of these trivalents (Figure 1D). A third group of oocytes (n = 27) displayed the three chromosomes 21 partially synapsed, meaning that the three homologues only paired in some portions thus creating a partial trivalent configuration. In this group, ATR staining pattern was variable: some of them showed differential ATR staining on the unpaired portion of the partial trivalent, either as a continuous signal (14.81%, n = 4/27) or as ATR bright foci (33.33%, n = 9/27), while other partial trivalent configurations did not show differential staining on the unpaired portion of the trivalent (51.85%, n = 14/27). It is worth mentioning that isolated ATR bright foci were frequently observed on other chromosomes other than the unsynapsed, but never as a linear track on the same chromosome, a feature only observed on unsynapsed axes. This feature was also observed in euploid pachytene oocytes (data not shown).
BRCA1 associates with any unpaired axial elements at pachytene The staining of human 47,XX+21 oocytes using antibodies against BRCA1 and REC8 revealed that BRCA1 foci were present at paired and unpaired axes at zygotene, but the signal became more intense at unsynapsed areas and pairing forks from mid-zygotene onwards (Figure 1E), a feature that matches the situation in euploid oocytes (data not shown). In addition, BRCA1 distribution was analysed in a total of 29 trisomic pachytene oocytes. In 100% of the oocytes showing a bivalent plus univalent configuration (n = 20), BRCA1 specifically marked the unsynapsed chromosome 21 (Figure 1F), either as a continuous signal in the majority of cases or as a linear track of bright foci in the few others. In those oocytes that showed the three chromosomes 21 forming a partial trivalent (n = 6), BRCA1 always marked the unsynapsed portions of all of them. Finally, a third group of oocytes was found where the three chromosomes 21 seemed to be totally synapsed, forming a total trivalent (n = 3). In these cells, BRCA1 did not mark any of these trivalents, similar to the ATR findings.
GH2AX accumulates at the unpaired chromosomes marked by ATR The analysis of trisomic 21 oocytes stained for ATR, GH2AX and REC8 showed that an intense GH2AX signal appeared on the chromatin encompassing those univalents recognized by
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ATR at pachytene (Figure 2), either as a linear signal (Figure 2A) or as a linear track of bright foci (Figure 2B). Therefore, in such pachytene cells, two different kinds of GH2AX signal coexist in the nuclei (Figure 2C). One type of focus is associated with the synaptonemal complexes, as has been observed in euploid oocytes where it has been related to the signalling of double-strand breaks generated during the meiotic process (Roig et al., 2004; Lenzi et al., 2005). This kind of focus frequently co-localizes with a focus of ATR (Figure 2A and 2B, arrows), a feature that has also been observed in euploid oocytes (data not shown). In addition, in trisomic 21 oocytes, a second type of GH2AX signal was observed, which was widely spread on the chromatin and surrounding the axial elements and that appeared only on those unpaired chromosomes that were stained by ATR, as mentioned above. However, such GH2AX on the univalent did not appear on all pachytene cells displaying univalents 21, and it was suspected that such a signal appeared as the pachytene stage progressed. Taking into account that chromosomes compact as pachytene steps forward, the oocytes were divided into three subgroups according to the length of the univalent (above 10 Mm, between 10 and 7.5 Mm, and less than 7.5 Mm) in order to assess the evolution of GH2AX signal on unsynapsed chromosome 21 with pachytene stage progression (Figure 3). On univalents, the GH2AX signal was seen either as a massive signal, totally or partially covering the axial element, or as isolated foci of an intensity and shape equivalent to the GH2AX recombinational foci. Results on GH2AX distribution in each of the three subgroups presented in Figure 3 show how the recombinational GH2AX signal disappears in favour of the spread GH2AX signal as the axial element reduces in length. On the other hand, the massive signal covering the whole length of the unsynapsed chromosome axis appears as the axial elements of chromosome 21 compacts. Therefore, it can be concluded that H2AX on the chromatin of the whole univalent 21 becomes progressively phosphorylated as pachytene stage progresses.
MSUC occurs during human female meiosis The transcriptional status of human meiocytes was analysed using an antibody against the active form of RNA polymerase II. As this approach has not been used in human meiocytes before, the RNA polymerase signal was first analysed on human spermatocytes. The RNA polymerase signal becomes more intense as prophase progresses from leptotene to pachytene (data not shown). As expected, the presence of RNA polymerase at the pachytene stage was evidently reduced or absent in the defined area of the nuclei covering the XY body of the spermatocytes (Figure 4A), showing the silencing of the unpaired regions of chromosomes X and Y. The transcriptional status of univalents 21 in human 47,XX+21 oocytes was analysed in pachytene and diplotene cells. A decrease in the RNA polymerase signal was observed at GH2AX-positive areas, revealing that the chromosomes that remain unpaired are at least partially transcriptionally silenced (n = 26). The decrease in the RNA polymerase signal was hardly visible in early pachytene nuclei, but became more perceptible as pachytene progressed (Figure 4B) and even more evident in diplotene cells. However, there was always a residual RNA
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Figure 1. Localization of ataxia telangiectasia and Rad3 related (ATR) and BRCA1 in 47,XX+21 oocytes. Cohesin REC8 labels the axial elements and the synaptonemal complex central element protein 1 (SYCP1). Locus-specific identification probe for 21q22 (LSI 21) shows the position of chromosome 21 (stained after immunofluorescence). (A–C) Pachytene-stage oocytes where two chromosomes 21 are synapsed (arrow) and the third chromosome 21 remains unpaired (arrowhead). ATR marks the unpaired chromosome, either as a continuous signal (A) or by a bright foci pattern (B). (C) An oocyte is shown with an unpaired chromosome 21 not marked by ATR. (D) Pachytene-stage oocyte where the three chromosomes 21 have engaged in total synapsis (arrowhead). ATR does not label the trivalent. (E) Zygotene-stage oocyte stained for BRCA1. BRCA1 remains at unpaired axial elements and pairing forks (enlarged image details). (F) Pachytene-stage oocyte with a univalent 21 (arrowhead) and a bivalent 21 (arrow). BRCA1 covers the unpaired chromosome 21. Bar in right panel of E represents 4.5 Mm. Bar in A represents 10 Mm and applies to all other panels in Figure 1.
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Figure 2. Localization of the phosphorylated histone GH2AX in 47,XX+21 pachytene oocytes. Cohesin REC8 labels the axial elements. (A, B) Oocytes where the unpaired chromosomes 21 are marked by ataxia telangiectasia and Rad3 related (ATR) either as a linear staining (A) or as a track of bright foci (B). GH2AX is distributed throughout the chromatin of the univalent in both situations. Arrows show co-localization between ATR and GH2AX foci on synaptonemal complex. (C) Enlarged image of the GH2AX staining from the same oocyte as in (A). GH2AX shows two different kinds of signal: small clouds of GH2AX on the synaptonemal complex (arrows) and a large signal throughout the chromatin of the univalent (arrowhead). Bar in A represents 10 Mm and also applies to panel B. RBMOnline®
Article - ATR, BRCA1 and GH2AX localization in human oocytes - R Garcia-Cruz et al.
GH2AX foci GH2AX spread signal partially covering the univalent 21 GH2AX spread signal covering the whole univalent 21
100
Percentage
80 60 40 20 0 4.79–7.5
7.5–10
>10
Univalent length (Mm)
Figure 3. Phosphorylated histone GH2AX signal on the unpaired chromosome 21 in pachytene 47,XX+21 oocytes according to univalent length.
Figure 4. RNA synthesis as disclosed by RNA polymerase II immunostaining in human pachytene spermatocytes and in 47,XX+21 pachytene oocytes. Cohesin REC8 labels the axial elements. (A) The RNA polymerase signal is absent in the XY body on human spermatocytes. (B) Pachytene oocyte with an unpaired chromosome 21. Phosphorylated histone GH2AX marks the chromatin of the univalent and the RNA polymerase II signal is lower. Whole chromosome 21 paint probe (WCP21) shows the position of chromosome 21 in the same nucleus (stained after immunofluorescence). DAPI = 4a,6a-diamidino-2-phenylindole. Bar represents 10 Mm and applies to all panels.
polymerase signal and the decrease was never comparable to the one observed in the XY domain in human spermatocytes. During this analysis, a diminished RNA polymerase signal was also detected in areas of some pachytene and diplotene nuclei with GH2AX-positive domains that resulted from pairing errors at regions different from chromosome 21 univalents. It has to be mentioned that both in oocytes and spermatocytes the expression of RNA polymerase was not homogeneous in the rest of the nuclei and that other domains showed lower RNA polymerase levels, most probably due to constitutional silent regions such as pericentromeric and other heterochromatic regions.
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Discussion Human oogenesis is an extremely error-prone process that progresses with an elevated rate of atresia (Hassold and Hunt, 2001; Hunt and Hassold, 2008; Martin, 2008). The identification of the mechanisms involved in this major hitch has been one of the fundamental subjects of the scientific and medical community involved in reproductive biology, yet the exact biological basis is still poorly understood. However, different meiotic and cellular processes have been claimed to be involved in the high ratio of atresia and aneuploidy: abnormal patterns of meiotic recombination (for a review see Lamb et al., 2005), pairing and
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synapsis defects (Speed, 1985, 1988; Tease et al., 2006), loss of chromosome cohesion (Hodges et al., 2005), spindle aberrations and congression failure (Volarcik et al., 1998; Hodges et al., 2002), the existence of a chromosome aggregation phase during oocyte maturation (Otsuki and Nagai, 2007), lack of checkpoint at the anaphase/metaphase transition (LeMaire-Adkins et al., 1997), environmental factors and others. The present study has attempted to shed some light on the current knowledge of the mechanisms functioning in the human oocyte when synapsis defects occur. Selective association of ATR, BRCA1 and GH2AX with unpaired chromosome axes was observed, lending itself to supportive evidence that indicates that BRCA1, ATR and GH2AX may be part of a mechanism in the human similar to the one previously described in mouse (Turner et al., 2005), where it has been described that BRCA1 guides ATR to regions that remain unpaired at pachytene, followed by H2AX phosphorylation by ATR. This study’s findings show that the unpaired chromosomes showed an extensive GH2AX signal on the chromatin of the univalent, which has been related to the silencing of unpaired chromosomes (Baarends et al., 2005; Turner et al., 2005) different from the much more focused signal that appears on the rest of the synaptonemal complexes in euploid and aneuploid oocytes and related to the recombination process (Roig et al., 2004; Lenzi et al., 2005). Therefore, these findings highlight the importance of the change in the chromatin state produced by H2AX phosphorylation, which may contribute to different processes during meiotic prophase, such as facilitating DNA repair and as part of the meiotic checkpoint apparatus, possibly inhibiting access of the transcriptional machinery to chromatin and thus silencing unpaired chromosomes. This is especially significant in the human female, when both processes may be operational at the same time during the pachytene stage. The finding that a proportion of univalents were not recognized by ATR substantially differs from what has been described in the mouse (Turner et al., 2005). However, the study shows that any unpaired DNA at the pachytene stage is recognized by BRCA1, a protein whose role in the recruitment of ATR to unsynapsed DNA has been reported (Turner et al., 2005). The fact that all unpaired DNA is recognized by BRCA1, but not by ATR, indicates that, in the human female, BRCA1 also seems to act upstream of ATR in the synapsis checkpoint and could have a role in recruiting ATR to the unsynapsed DNA. Therefore, the absence of ATR in some univalents may just be a question of timing, rather than a decreased efficiency in detecting asynapsed chromosomes. This hypothesis is supported by the finding that the GH2AX signal encompassing the univalent, which is a feature linked to the ATR signalling on the univalent, also appears as pachytene progresses.
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During the progression of this study, different meiotic configurations between the three chromosomes 21 were found, as others have previously reported (Luciani et al., 1976; Wallace and Hulten, 1983; Speed, 1984; Jagiello et al., 1987; Barlow et al., 2002; Robles et al., 2007): bivalent plus univalent, partially synapsed trivalents and totally synapsed trivalents were found. Of these nuclei, only the ones showing totally synapsed trivalents escape from ATR-BRCA1-GH2AX signalling at pachytene. This observation leads us to believe that, in trivalent configurations, not only pairing but also complete synapsis is achieved between
the three homologues in all their length, creating a unique synaptonemal complex configuration. Similarly, in 47,XYY men, spermatocytes achieving full synapsis between the three PAR regions are able to escape the pachytene checkpoint and progress through meiosis (Milazzo et al., 2006). The observation that RNA polymerase II signal is diminished on GH2AX-positive chromatin corroborates that unpaired chromosomes are at least partially silenced in the human female, similar to what has been reported in the mouse, where it is speculated that this fact could lead to the apoptosis of those nuclei. However, in the present study, the finding of a considerable amount of diplotene cells with spread GH2AX signals leads us to speculate that MSUC and the ATR checkpoint function is not sufficient to prevent such nuclei from progressing into diplotene. Sciurano et al. (2006) reported the presence of spermatocytes at the division stage showing GH2AX patches in an azoospermic man with severe pairing defects and extensive BRCA1 staining in prophase spermatocytes, also suggesting that asynapsis does not necessarily abort meiocytes at the prophase stage. In the study samples, the presence of a normally transcribing bivalent 21 and a silenced third 21 copy could create a normal level of chromosome 21 transcripts, which could explain why these nuclei progress beyond pachytene. The impossibility of studying human trisomic 21 oocytes beyond diplotene leaves further aspects and potential consequences of this phenomenon at later stages in the dark. Therefore, it seems that MSUC does not necessarily have detrimental consequences for human trisomic oocytes. However, the existence of this phenomenon could have devastating consequences for monosomic oocytes since the transcription of a whole chromosome would be null from pachytene. For that reason, the assessment of the consequences of MSUC in monosomic oocytes would be of great interest. Nevertheless, contrary to the situation in mice, the proportion of XO human oocytes that reach pachytene is almost nil (Speed, 1986; R Garcia-Cruz and M Garcia, unpublished) and, on the other hand, the chance of obtaining samples from other monosomic fetuses, old enough to enable meiotic studies to be performed, is highly improbable. This makes the analysis of the effect of MSUC in monosomic oocytes impracticable at the moment. Nevertheless, the effects of MSUC in the human could have an enormous implication not only in monosomic oocytes but also, and therefore its significance, in euploid oocytes. A high rate of pairing defects is known to occur in human oocytes (Speed, 1988; Hartshorne et al., 1999; Tease et al., 2006) and, according to the results of this study, this may lead to the silencing of such unpaired DNA sequences. Although not reported here, during this study a considerable number of pachytene and diplotene oocytes with GH2AX-positive domains were found, and lower RNA polymerase expression in chromosomes other than univalent 21. The effects of the inactivation of fragments of chromosomes could have an enormous impact on human oogenesis and could partially contribute to the elevated rate of atresia that usually occurs in the oocyte population of the developing human ovary.
Acknowledgements We wish to thank Dr JL Barbero and Dr C Heyting for kindly providing REC8 and SYCP1 antibodies, respectively. We also thank Dr James Turner, Dr Paul Burgoyne, Dr Scott Keeney and RBMOnline®
Article - ATR, BRCA1 and GH2AX localization in human oocytes - R Garcia-Cruz et al.
all the members of the Keeney laboratory, as well as MA Brieño and A Casanovas for their critical reading of the manuscript. The English of this manuscript has been read and corrected by Mr Chuck Simmons, a native English-speaking instructor of English of the Universitat Autònoma de Barcelona. RGC is recipient of a grant from Agència de Gestió d’Ajuts Universitaris i de Recerca de la Generalitat de Catalunya (2004FI00953). This work was carried out with financial support from: Fondo de Investigación Sanitaria (FIS 02/0297); Universtitat Autònoma de Barcelona (PRP2006–02); Ministerio de Ciencia y Tecnología (BFU2006–1295).
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Declaration: The authors report no financial or commercial conflicts of interest. Received 17 March 2008; refereed 2 April 2008; accepted 18 July 2008.
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