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Telophase enucleation: An improved method to prepare recipient cytoplasts for use in bovine nuclear transfer

1998; Wiley; Volume: 49; Issue: 1 Linguagem: Inglês

10.1002/(sici)1098-2795(199801)49

1098-2795

Vilceu Bordignon, Lawrence C. Smith,

Animal Genetics and Reproduction

Molecular Reproduction and DevelopmentVolume 49, Issue 1 p. 29-36 Embryo Development Telophase enucleation: An improved method to prepare recipient cytoplasts for use in bovine nuclear transfer Vilceu Bordignon, Vilceu Bordignon Centre de recherche en reproduction animale, Faculte de médecine vétérinaire, Université de Montréal, Saint-Hyacinthe, Quebec, CanadaSearch for more papers by this authorLawrence C. Smith, Corresponding Author Lawrence C. Smith [email protected] Centre de recherche en reproduction animale, Faculte de médecine vétérinaire, Université de Montréal, Saint-Hyacinthe, Quebec, CanadaCentre de recherche en reproduction animale, Faculte de médecine vétérinaire, Université de Montréal, Saint-Hyacinthe, PQ, J25 7C6 CanadaSearch for more papers by this author Vilceu Bordignon, Vilceu Bordignon Centre de recherche en reproduction animale, Faculte de médecine vétérinaire, Université de Montréal, Saint-Hyacinthe, Quebec, CanadaSearch for more papers by this authorLawrence C. Smith, Corresponding Author Lawrence C. Smith [email protected] Centre de recherche en reproduction animale, Faculte de médecine vétérinaire, Université de Montréal, Saint-Hyacinthe, Quebec, CanadaCentre de recherche en reproduction animale, Faculte de médecine vétérinaire, Université de Montréal, Saint-Hyacinthe, PQ, J25 7C6 CanadaSearch for more papers by this author First published: 07 December 1998 https://doi.org/10.1002/(SICI)1098-2795(199801)49:1 3.0.CO;2-QCitations: 65AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat Abstract The enucleation of oocytes to be used as host cytoplasts for embryo reconstruction by nuclear transfer is an important limiting step when cloning mammals. We propose an enucleation technique based on the removal of chromatin after oocyte activation, at the telophase stage, by aspirating the second polar body and surrounding cytoplasm. In a preliminary experiment to determine an optimal activation protocol, oocytes were matured for 26 and 30 hr and exposed for 5 min to 7% ethanol and/or for 3 hr at either 25 or 4°C. Relative to most activation treatments tested, oocytes matured for 30 hr and exposed to ethanol alone showed highest activation rates, as determined by low levels of H1 kinase activity within 90 min from exposure and high pronuclear formation (82%) after 12 hr of culture. No synergistic effect on activation rates was observed when oocytes also were exposed to reduced temperature after ethanol treatment. Microsurgical removal of the telophase-stage chromatin in a small volume of cytoplasm adjacent to the second polar body was significantly more effective in enucleating than aspiration of a larger cytoplasm volume surrounding the first polar body of metaphase-arrested oocytes (98% versus 59%; P < 0.01). Moreover, compared with a nuclear transfer protocol based on enucleation of metaphase-arrested oocytes followed by aging and cooling, more (38% versus 16%; P < 0.001) and better-quality blastocytes (126 versus 84 nuclei per blastocyst; P < 0.02) were obtained from embryos reconstructed using the telophase procedure. Higher development potential of embryos reconstructed by the telophase procedure may be attributed to (1) the selection of oocytes that activate and respond by extruding the second polar body, (2) avoiding the use of DNA dyes and ultraviolet irradiation, and (3) the limited removal of cytoplasm during enucleation. The ease with which telophase enucleation can be performed is likely to render this technique widely useful for research and practice on mammalian cloning. Mol. Reprod. Dev. 49:29–36, 1998. © 1998 Wiley-Liss, Inc. References Barnes FL, Collas P, Powell R, King WA, Westhusin M, Shepherd D (1993): Influence of recipient oocyte cell cycle stage on DNA synthesis, nuclear envelope breakdown, chromosome constitution, and development in nuclear transplant bovine embryos. Mol Reprod Dev 36: 33–41. 10.1002/mrd.1080360106 PubMedWeb of Science®Google Scholar Ben-Yosef D, Oron Y, Shalgi R (1995): Low-temperature and fertilization-induced Ca2+ changes in rat eggs. 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