Abstract Cytogenetic analys of mammalian eggs and preimplantation embryos have been limited by the diffi-cult and tedious task of preparing chromosomes from single cells or small numbers of cells. In this report we describe a new technique that is both reliable and com-paratively simple. Further, since the technique does not u the conventional 3:1 methanol:acetic acid fixative, it has the advantage of producing high-resolution chromo-some preparations without destroying chromosome-asso-ciated proteins. Thus, this method provides a nsitive means of conducting studies of a heretofore inaccessible period of mammalian development, and of studying pro-teins thought to mediate both meiotic chromosome g-regation and chromatin modifications in the preimplanta-tion embryo.
Introduction
Cytogenetic studies of oocytes and embryos have been hampered by the difficulty of obtaining suitable prepara-tions from single cells. An air-drying technique devel-oped by Tarkowski (1966) has been ud extensively, but this procedure involves the in situ fixation of oocytes and embryos to microscope slides and results in variable quality of the cytogenetic preparations and artifactual loss of chromosomes. While a modification developed
by Kamiguchi et al. (1976) improved reliability, the la-borious nature of the technique precludes the analysis of
large numbers of oocytes or embryos. Thus, although slight modifications of the techniques have been ud extensively in studies of humans and mice to evaluate re-combination frequencies (e.g., Jagiello et al. 1976; Jagiello and Fang 1979; Lawrie et al. 1995), determine nondisjunction frequencies (reviewed in Bond and Chandley 1983), and asss the meiotic gregation be-havior of structurally abnormal chromosomes (e.g.,Tea 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 the gregation of chromosomes during both meiotic and mi-totic cell division has incread dramatically (reviewed in Maney et al. 2000; Lee and Orr-Weaver 2001). Unfortu-nately, most such proteins are lost from the chromosomes during the fixation process in the conventional cytogenet-ic procedure. Thus, studies of the proteins have gener-ally relied on whole cell fixation methods (e.g., Simerly and Schatten 1993) or the u of a cytospin procedure (e.g., Saffery et al. 2000), both of which limit chromo-some resolution. The problem is particularly pronounced for oocytes and preimplantation embryos, where the large cytoplasmic volume prents special problems with re-spect to background staining and/or antibody accessibility (Simerly and Schatten 1993). To circumvent the prob-lems, we developed a modified fixation technique as de-tailed below. This me
thod not only prerves chromo-some-associated proteins but provides a simplified meth-od of producing analyzable chromosome preparations from oocytes and early cleavage stage embryos.
Materials and methods Mou oocytes or preimplantation embryos are collected and cul-tured using standard protocols (Wassarman and DePamphilis
1993). Collection and culture of germinal vesicle stage oocytes can be ud to obtain oocytes at various stages of the first meiotic division (e.g., prometapha, metapha, and anapha/telopha,)or cells that have completed the first division and are arrested at
化妆的正确步骤metapha II (e.g., as described previously in LeMaire-Adkins et
wojtekal. 1997). Similarly, mou embryos can be collected from the ovi-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 pellucida is removed prior to fixation by brief exposure to 1% prona (CalBiochem) in culture medium. Zona-free oocytes or embryos are washed in medium and transferred to a petri dish coated in 1%agar to prevent attachment.
Edited by : T. Hassold
C.A.Hodges · P.A.Hunt (✉)
Department of Genetics, Ca Western Rerve University, Cleveland, Ohio, USA
e-mail: pah13@po.cwru.edu
Chromosoma (2002) 111:165–169DOI 10.1007/s00412-002-0195-3
mountain topCraig A.Hodges · Patricia A.Hunt
Simultaneous analysis of chromosomes and chromosome-associated proteins in mammalian oocytes and embryos
Received: 19 February 2002 / Revid: 3 April 2002 / Accepted: 3 April 2002 / Published online: 30 May 2002©Springer-Verlag 2002
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Fig.1A–F Protein localization and fluorescence in situ hybridiza-tion (FISH) in oocytes and preimplan
tation embryos. Chromo-some preparations from diakinesis/metapha I stage mou oo-cytes immunostained with antibodies to CENP-E (A), CREST an-tirum (B), BUB1 (C), and phophorylated histone H3 (D). A sin-gle cell from a four-cell mou embryo immunostained with an antibody to CENP-E (E) illustrates the u of this technique to study the early cleavage divisions. Subquent FISH with a Y-spe-cific probe of the same cell (F). Note that although the fixation technique prerves chromosome-associated proteins, the morpho-logical detail is sufficient to allow the resolution of individual sis-ter chromatids (e.g., arrowheads in A) and the identification of the univalent X chromosome in oocytes from an XO female mou (e.g., arrow in A). Bar reprents 10µm
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For fixation, a clean microscope slide is dipped in a solution of 1% paraformaldehyde in distilled H 2O (pH 9.2) containing 0.15%Triton X-100 and 3mM dithiothreitol. With a finely drawn pipet,up to 20 oocytes or embryos are carefully pipetted along the length of the slide. The oocytes or embryos will burst within c-onds of exposure to the fixation and slowly “melt” onto the slide.Optimal spreading of the chromosomes is achieved when the cells are expelled evenly across the slide and the slide is allowed to dry slowly in a humid chamber for veral hours before being washed in 0.4% Photoflo (Kodak) in distilled H 2O 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 the chromosomes is variable, but this can be minimized as follows:(1)Ensure that the Triton X-100 is completely dissolved before using the paraformaldehyde solution.
Fig.2A–C Anapha I and metapha II chromosomes from mou oocytes. A Chromosomes from a mou oocyte captured at a transient stage of anapha when chiasmata between most ho-mologous chromosomes have been resolved but chromosomes have not yet gregated. B A late an
adaptation
apha I/telopha I oocyte il-lustrating the prervation of chromosome orientation that is pos-sible with this protocol. C A metapha II-arrested oocyte. It should 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. Bar repre-nts 10µm
(2)For specific antibodies it may be necessary to modify the pH of the paraformaldehyde solution; however, we have obrved inconsistent 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 modified to achieve optimal chromosome morphology; too much will cau 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 amount of medium; excessive medium can affect both chromosome morphology and protein stabilization.As detailed below, we have successfully ud standard immuno-histochemical staining procedures on the preparations with anti-bodies to CENP-E, BUB1 (gifts from T. Yen), phosphorylated his-tone H3 (Upstate Biotechnology), MAD2 (Covance) and CREST rum detected with appropriate rhodamine- or fluorescein iso-thiocyanate-conjugated condary antibodies.
kuleResults and discussion
Advantages of the technique
As shown in Fig.1, this technique produces excellent quality chromosome preparations from both mou oo-cytes and early embryos, and preliminary studies suggest that comparable results can be obtained with human oo-cytes (data not shown). Furthermore, in our hands, a wide variety of chromosome-associated proteins (e.g.,CENP-E, BUB1, MAD2, phosphorylated histone H3,and centromere-associated proteins recognized by CREST rum) are prerved and can be detected by routine 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 prervation of chromosome-associated pro-teins along the length of the chromosome. Further, to identify specific chromosomes, standard fluorescence in situ hybridization (FISH) procedures can be ud (Fig.1F); however, becau chromosome denaturation
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procedures may weaken or destroy immunofluorescence staining, images should be captured prior to FISH.Application of the technique to study factors affecting chromosome gregation
This fixation technique not only provides a means of an-alyzing chromosome-associated proteins but also pro-vides access to meiotic stages that have traditionally been difficult to study. For example, preparation of chro-mosomes of metapha II-arrested oocytes is notoriously difficult since cells at this stage are fragile and, during the harsh fixation procedure, subject to both chromo-some loss and physical paration of sister chromatids.However, in our experience, the comparably milder fixa-tion process ud in this technique alleviates production of the artifacts (e.g., Fig.2C). Further, unlike previous methods that involve both hypotonic pretreatment of cells and a harsh fixation procedure, both of which rve to disrupt chromosome orientation, this slow fixation method allows chromosomes to melt onto the slide, pre-rving the orientation of cells fixed at anapha and te-lopha (e.g., Fig.2A, B).The ability to produce high-quality chromosome preparations while retaining chromosome-associated proteins has obvious applications in the study of chro-mosome gregation. To illustrate the utility of the tech-nique, a comparative study of meiotic and mitotic chro-mosomes fixed via this procedure and stained with an antibody to the kinetochore-associated motor protein,CENP-E, is shown in Fig.3. Two large protein complex-es function to ensure proper gregati二级建造师教材下载
on during mitotic cell division: Cohesion between sister chromatids is es-tablished during S-pha and maintains a physical con-nection between sisters until anapha, and a functional kinetochore established at the centromere of each sister facilitates the attachment and movement of chromatids on the spindle (reviewed in Maney et al. 2000; Lee and Orr-Weaver 2001). Meiotic cell division requires modifi-cation of both protein complexes: successful completion of the first meiotic division necessitates the establish-ment of cohesion between homologous chromosomes,which is accomplished by the process of recombination and the formation of chiasmata. In addition, to ensure that homologs rather than sisters gregate, coordinated behavior of sister kinetochores is required so that attach-
ments to the same rather than opposite spindle poles are
Fig.3A–D A comparison of mitotic and meiotic chromo-somes. Chromosome prepara-tions were fixed using the new technique and immunostained with an antibody to CENP-E (red ) to identify the kineto-chores. The examples illus-trate the utility of the technique in detecting subtle differences in chromosome-associated pro-teins. A A pair of homologous chromosomes from an oocyte fixed at metapha I. Note that a single CENP-E focus is ob-rved in association with the sister kinetochores of each ho-molog. B A metapha II chro-mosome exhibiting the splayed sister chroma
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tids typical of this stage. Note that sister chromat-ids are clearly parated and that there are two distinct CENP-E foci. C A metapha chromosome from a four-cell embryo. Note that sister chro-matids remain tightly joined but two distinct CENP-E foci are evident. D A univalent X chromosome from a metapha I oocyte from an XO female.Note that, even in the abnce of a homolog, a single CENP-E focus is obrved in association with the sister kinetochores of this metapha I chromosome.Bar reprents 10µm
established. Further, at anapha I, cohesion between homologs must be resolved while retaining that between sister centromeres to allow gregation of sister chro-matids at the cond division (reviewed in Lee and Orr-Weaver 2001).
As illustrated in Fig.3, the differences between mitot-ic, first meiotic, and cond meiotic chromosomes with respect to both cohesion and kinetochore behavior can be visualized in chromosomes prepared with the new tech-nique. Immunostaining with an antibody to the kineto-chore-associated motor protein CENP-E reveals clearly parated sister kinetochores on mitotic (Fig.3C) and cond meiotic (Fig.3B) metapha chromosomes, while the sister kinetochores of a pair of homologous chromo-somes (Fig.3A) or a univalent chromosome (Fig.3D) at the first meiotic division are indistinguishable. In addi-tion, differences in cohesion between mitotic and cond meiot
ic chromosomes are evident (i.e., compare Fig.3B and C; chromosomes at cond meiotic metapha dis-play a characteristic splaying of sister chromatids that re-sults from the loss of arm cohesion at anapha I). The ability to discern subtle differences in the localization of centromere-associated proteins on high-resolution chro-mosome preparations illustrates the utility of this method of chromosome preparation.
In addition to providing new methodology for the study of proteins thought to mediate chromosome gre-gation, this technique has other potential applications. For example, the preimplantation period is inherently difficult to study, and the ability to obtain high-resolu-tion chromosome preparations of early cleavage stage embryos (e.g., Fig.1E) may provide important temporal insight regarding post-fertilization changes in chromatin structure; e.g., the complex process of genome activation occurs during the early cleavage divisions and is thought to 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 ems likely that the technique can be modified to provide a simplified 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.
Acknowledgements We would like to thank T. Yen for generous gifts of antibodies ud in the studies. We also thank C. Bean for providing the mou embryos and T. Hassold for helpful discus-sion. This work was supported by National Institutes of Health grant R01 HD31866 to P.A. Hunt; C.A. Hodges was supported by National Institutes of Health training grant GM 08613.References
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