Abstract Cytogenetic analyses 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 use 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 sensitive means of conducting studies of a heretofore inaccessible period 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 been hampered by the difficulty of obtaining suitable prepara-tions from single cells. An air-drying technique devel-oped by Tarkowski (1966) has been used 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 these techniques have been used 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 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 the segregation of chromosomes during both meiotic and mi-totic cell division has increased 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 these proteins have gener-ally relied on whole cell fixation methods (e.g., Simerly and 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 pronounced for oocytes and preimplantation embryos, where the large cytoplasmic 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 preparations from oocytes and early cleavage stage embryos.
Materials and methods Mouse 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 used to obtain oocytes at various stages of the first meiotic division (e.g., prometaphase, metaphase, and anaphase/telophase,)or cells that have completed the first division and are arrested at
metaphase II (e.g., as described previously in LeMaire-Adkins et
al. 1997). Similarly, mouse 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% pronase (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, Case Western Reserve University, Cleveland, Ohio, USA
e-mail: pah13@
Chromosoma (2002) 111:165–169DOI 10.1007/s00412-002-0195-3
Craig A.Hodges · Patricia A.Hunt
Simultaneous analysis of chromosomes and chromosome-associated proteins in mammalian oocytes and embryos
Received: 19 February 2002 / Revised: 3 April 2002 / Accepted: 3 April 2002 / Published online: 30 May 2002©Springer-Verlag 2002
