ORIGINAL ARTICLECraigA.Hodges· PatriciaA.Hunt
Simultaneous analysis ofchromosomes andchromosome-associatedproteins inmammalian oocytes andembryos
Received: 19 February 2002 / Revised: 3 April 2002 / Accepted: 3 April 2002 / Published online: 30 May 2002©Springer-Verlag 2002
AbstractCytogenetic analyses of mammalian eggs andpreimplantation embryos have been limited by the diffi-cult and tedious task of preparing chromosomes fromsingle cells or small numbers of cells. In this report wedescribe a new technique that is both reliable and com-paratively simple. Further, since the technique does notuse the conventional 3:1 methanol:acetic acid fixative, ithas the advantage of producing high-resolution chromo-some preparations without destroying chromosome-asso-ciated proteins. Thus, this method provides a sensitivemeans of conducting studies of a heretofore inaccessibleperiod of mammalian development, and of studying pro-teins thought to mediate both meiotic chromosome seg-regation and chromatin modifications in the preimplanta-tion embryo.
Introduction
Cytogenetic studies of oocytes and embryos have beenhampered by the difficulty of obtaining suitable prepara-tions from single cells. An air-drying technique devel-oped by Tarkowski (1966) has been used extensively, butthis procedure involves the in situ fixation of oocytesand embryos to microscope slides and results in variablequality of the cytogenetic preparations and artifactualloss of chromosomes. While a modification developed
by Kamiguchi et al. (1976) improved reliability, the la-Materials andmethodsborious nature of the technique precludes the analysis of
large numbers of oocytes or embryos. Thus, althoughMouse oocytes or preimplantation embryos are collected and cul-tured using standard protocols (Wassarman and DePamphilis
slight modifications of these techniques have been used1993). Collection and culture of germinal vesicle stage oocytesextensively in studies of humans and mice to evaluate re-can be used to obtain oocytes at various stages of the first meioticcombination frequencies (e.g., Jagiello et al. 1976; division (e.g., prometaphase, metaphase, and anaphase/telophase,)Jagiello and Fang 1979; Lawrie et al. 1995), determineor cells that have completed the first division and are arrested at
metaphase II (e.g., as described previously in LeMaire-Adkins et
nondisjunction frequencies (reviewed in Bond and al. 1997). Similarly, mouse embryos can be collected from the ovi-Edited by: T. Hassold
C.A.Hodges· P.A.Hunt (✉)
DepartmentofGenetics, CaseWesternReserveUniversity, Cleveland, Ohio, USA
e-mail: pah13@po.cwru.edu
Chandley 1983), and assess the meiotic segregation be-havior of structurally abnormal chromosomes (e.g.,Tease 1998), the methodology remains difficult, error-prone, and tedious.
In the past decade our understanding of different class-es of chromosome-associated proteins that mediate thesegregation of chromosomes during both meiotic and mi-totic cell division has increased dramatically (reviewed inManey et al. 2000; Lee and Orr-Weaver 2001). Unfortu-nately, most such proteins are lost from the chromosomesduring the fixation process in the conventional cytogenet-ic procedure. Thus, studies of these proteins have gener-ally relied on whole cell fixation methods (e.g., Simerlyand Schatten 1993) or the use of a cytospin procedure(e.g., Saffery et al. 2000), both of which limit chromo-some resolution. The problem is particularly pronouncedfor oocytes and preimplantation embryos, where the largecytoplasmic volume presents special problems with re-spect to background staining and/or antibody accessibility(Simerly and Schatten 1993). To circumvent these prob-lems, we developed a modified fixation technique as de-tailed below. This method not only preserves chromo-some-associated proteins but provides a simplified meth-od of producing analyzable chromosome preparationsfrom oocytes and early cleavage stage embryos.
ducts 6–72h after mating to obtain preimplantation embryos rang-ing from the one-cell to the blastocyst stage (Wassarman and DeP-amphilis 1993). For both oocytes and embryos the zona pellucidais removed prior to fixation by brief exposure to 1% pronase (CalBiochem) in culture medium. Zona-free oocytes or embryosare washed in medium and transferred to a petri dish coated in 1%agar to prevent attachment.
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Fig.1A–FProtein localization and fluorescence in situ hybridiza-tion (FISH) in oocytes and preimplantation embryos. Chromo-some preparations from diakinesis/metaphase I stage mouse oo-cytes immunostained with antibodies to CENP-E (A), CREST an-tiserum (B), BUB1 (C), and phophorylated histone H3 (D). A sin-gle cell from a four-cell mouse embryo immunostained with anantibody to CENP-E (E) illustrates the use of this technique tostudy the early cleavage divisions. Subsequent FISH with a Y-spe-cific probe of the same cell (F). Note that although the fixationtechnique preserves chromosome-associated proteins, the morpho-logical detail is sufficient to allow the resolution of individual sis-ter chromatids (e.g., arrowheadsin A) and the identification of theunivalent X chromosome in oocytes from an XO female mouse(e.g., arrowin A). Barrepresents 10µm
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(2)For specific antibodies it may be necessary to modify the pHof the paraformaldehyde solution; however, we have observedinconsistent spreading as the pH is lowered and find that opti-mal chromosome morphology is achieved with a basic pH of~9.2.
(3)The amount of paraformaldehyde on the slide can be modifiedto achieve optimal chromosome morphology; too much willcause excessive spreading and chromosome loss, while too lit-tle results in inefficient spreading of chromosomes.
(4)Oocytes and embryos should be delivered in a minimal amountof medium; excessive medium can affect both chromosomemorphology and protein stabilization.As detailed below, we have successfully used standard immuno-histochemical staining procedures on these preparations with anti-bodies to CENP-E, BUB1 (gifts from T. Yen), phosphorylated his-tone H3 (Upstate Biotechnology), MAD2 (Covance) and CRESTserum detected with appropriate rhodamine- or fluorescein iso-thiocyanate-conjugated secondary antibodies.
Fig.2A–CAnaphase I and metaphase II chromosomes frommouse oocytes. AChromosomes from a mouse oocyte captured ata transient stage of anaphase when chiasmata between most ho-mologous chromosomes have been resolved but chromosomeshave not yet segregated. BA late anaphase I/telophase I oocyte il-lustrating the preservation of chromosome orientation that is pos-sible with this protocol. CA metaphase II-arrested oocyte. Itshould be noted that although this fixation method reduces the ar-tifactual loss of chromosomes, removal of the zona pellucida re-sults in loss of the polar body in a proportion of cells. Barrepre-sents 10µm
For fixation, a clean microscope slide is dipped in a solution of1% paraformaldehyde in distilled H2O (pH9.2) containing 0.15%Triton X-100 and 3mM dithiothreitol. With a finely drawn pipet,up to 20 oocytes or embryos are carefully pipetted along thelength of the slide. The oocytes or embryos will burst within sec-onds of exposure to the fixation and slowly “melt” onto the slide.Optimal spreading of the chromosomes is achieved when the cellsare expelled evenly across the slide and the slide is allowed to dryslowly in a humid chamber for several hours before being washedin 0.4% Photoflo (Kodak) in distilled H2O and dried at room tem-perature. Slides can be stored at –20°C prior to staining.
As with other techniques, the morphology and spreading of thechromosomes is variable, but this can be minimized as follows:(1)Ensure that the Triton X-100 is completely dissolved beforeusing the paraformaldehyde solution.
Results anddiscussion
Advantages of the technique
As shown in Fig.1, this technique produces excellentquality chromosome preparations from both mouse oo-cytes and early embryos, and preliminary studies suggestthat comparable results can be obtained with human oo-cytes (data not shown). Furthermore, in our hands, awide variety of chromosome-associated proteins (e.g.,CENP-E, BUB1, MAD2, phosphorylated histone H3,and centromere-associated proteins recognized byCREST serum) are preserved and can be detected byroutine immunostaining procedures. The examples pro-vided in Fig.1 include both proteins that associate spe-cifically with the centromere/kinetochore (Fig.1A, B, C,E) and with the chromosome arms (Fig.1D), demon-strating the preservation of chromosome-associated pro-teins along the length of the chromosome. Further, toidentify specific chromosomes, standard fluorescence insitu hybridization (FISH) procedures can be used(Fig.1F); however, because chromosome denaturation
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Fig.3A–DA comparison ofmitotic and meiotic chromo-somes. Chromosome prepara-tions were fixed using the newtechnique and immunostainedwith an antibody to CENP-E(red) to identify the kineto-chores. These examples illus-trate the utility of the techniquein detecting subtle differencesin chromosome-associated pro-teins. AA pair of homologouschromosomes from an oocytefixed at metaphase I. Note thata single CENP-E focus is ob-served in association with thesister kinetochores of each ho-molog. BA metaphase II chro-mosome exhibiting the splayedsister chromatids typical of thisstage. Note that sister chromat-ids are clearly separated andthat there are two distinctCENP-E foci. CA metaphasechromosome from a four-cellembryo. Note that sister chro-matids remain tightly joinedbut two distinct CENP-E fociare evident. DA univalent Xchromosome from a metaphaseI oocyte from an XO female.Note that, even in the absenceof a homolog, a single CENP-Efocus is observed in associationwith the sister kinetochores ofthis metaphase I chromosome.Barrepresents 10µm
procedures may weaken or destroy immunofluorescencestaining, images should be captured prior to FISH.Application of the technique to study factors affectingchromosome segregation
This fixation technique not only provides a means of an-alyzing chromosome-associated proteins but also pro-vides access to meiotic stages that have traditionallybeen difficult to study. For example, preparation of chro-mosomes of metaphase II-arrested oocytes is notoriouslydifficult since cells at this stage are fragile and, duringthe harsh fixation procedure, subject to both chromo-some loss and physical separation of sister chromatids.However, in our experience, the comparably milder fixa-tion process used in this technique alleviates productionof these artifacts (e.g., Fig.2C). Further, unlike previousmethods that involve both hypotonic pretreatment ofcells and a harsh fixation procedure, both of which serveto disrupt chromosome orientation, this slow fixationmethod allows chromosomes to melt onto the slide, pre-serving the orientation of cells fixed at anaphase and te-lophase (e.g., Fig.2A, B).The ability to produce high-quality chromosomepreparations while retaining chromosome-associatedproteins has obvious applications in the study of chro-mosome segregation. To illustrate the utility of the tech-nique, a comparative study of meiotic and mitotic chro-mosomes fixed via this procedure and stained with anantibody to the kinetochore-associated motor protein,CENP-E, is shown in Fig.3. Two large protein complex-es function to ensure proper segregation during mitoticcell division: Cohesion between sister chromatids is es-tablished during S-phase and maintains a physical con-nection between sisters until anaphase, and a functionalkinetochore established at the centromere of each sisterfacilitates the attachment and movement of chromatidson the spindle (reviewed in Maney et al. 2000; Lee andOrr-Weaver 2001). Meiotic cell division requires modifi-cation of both protein complexes: successful completionof the first meiotic division necessitates the establish-ment of cohesion between homologous chromosomes,which is accomplished by the process of recombinationand the formation of chiasmata. In addition, to ensurethat homologs rather than sisters segregate, coordinatedbehavior of sister kinetochores is required so that attach-ments to the same rather than opposite spindle poles are
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established. Further, at anaphase I, cohesion between homologs must be resolved while retaining that betweensister centromeres to allow segregation of sister chro-matids at the second division (reviewed in Lee and References
BondDJ, ChandleyAC (1983) Aneuploidy. (Oxford monographs
on medical genetics, vol 11) Oxford University Press, NewOrr-Weaver 2001).
As illustrated in Fig.3, the differences between mitot-ic, first meiotic, and second meiotic chromosomes withrespect to both cohesion and kinetochore behavior can bevisualized in chromosomes prepared with the new tech-nique. Immunostaining with an antibody to the kineto-chore-associated motor protein CENP-E reveals clearlyseparated sister kinetochores on mitotic (Fig.3C) andsecond meiotic (Fig.3B) metaphase chromosomes, whilethe sister kinetochores of a pair of homologous chromo-somes (Fig.3A) or a univalent chromosome (Fig.3D) atthe first meiotic division are indistinguishable. In addi-tion, differences in cohesion between mitotic and secondmeiotic chromosomes are evident (i.e., compare Fig.3Band C; chromosomes at second meiotic metaphase dis-play a characteristic splaying of sister chromatids that re-sults from the loss of arm cohesion at anaphase I). Theability to discern subtle differences in the localization ofcentromere-associated proteins on high-resolution chro-mosome preparations illustrates the utility of this methodof chromosome preparation.
In addition to providing new methodology for thestudy of proteins thought to mediate chromosome segre-gation, this technique has other potential applications.For example, the preimplantation period is inherentlydifficult to study, and the ability to obtain high-resolu-tion chromosome preparations of early cleavage stageembryos (e.g., Fig.1E) may provide important temporalinsight regarding post-fertilization changes in chromatinstructure; e.g., the complex process of genome activationoccurs during the early cleavage divisions and is thoughtto be regulated by changes in chromatin protein content(reviewed in Latham 1999), with maternally and pater-nally inherited chromosomes exhibiting methylation dif-ferences (Mayer et al. 2000) and differences in gene ex-pression (reviewed in Latham 1999). Further, it seemslikely that the technique can be modified to provide asimplified method of polar body biopsy or preimplanta-tion diagnosis (reviewed in Verlinsky and Kuliev 1996),thus aiding diagnostic procedures associated with assist-ed human reproduction.
Acknowledgementsgifts of antibodies used in these studies. We also thank C. Bean forWe would like to thank T. Yen for generousproviding the mouse embryos and T. Hassold for helpful discus-sion. This work was supported by National Institutes of Healthgrant R01 HD31866 to P.A. Hunt; C.A. Hodges was supported byNational Institutes of Health training grant GM 08613.
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