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A reliable, noninvasive technique for spindle imaging and enucleation of mammalian oocytes

Nature Biotechnology volume 18, pages 223–225 (2000)Cite this article

Abstract

Factors affecting the efficiency of animal cloning remain to be elucidated1,2. Enucleation of recipient oocytes is a critical step in cloning procedures and typically is performed by aspirating a portion of the cytoplasm underlying the first polar body. Enucleation is evaluated using epifluorescence after Hoechst staining for DNA3,4,5,6, which may disrupt functions of the cytoplast, especially mitochondria7. Mitochondrial DNA in Dolly and other cloned sheep has been shown to derive exclusively from recipient oocytes8. Not only might evaluation of the aspirated karyoplast portion5 inadequately reflect the state of the cytoplast, it is also time consuming. Here we report a reliable, noninvasive technique for spindle imaging and enucleation of oocytes using a new microscope, the Pol-Scope9. The efficiency of enucleation was 100%, and only 5.5% of the oocytes' mitochondria entered the karyoplast upon Pol-Scope-directed removal of the spindle. Moreover, Pol-Scope imaging of spindles and micromanipulation did not compromise the developmental competence of reconstituted oocytes and cytoplasts.

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Figure 1: The use of the Pol-Scope for spindle imaging, spindle removal-directed enucleation, and reconstitution of metaphase II oocytes.
Figure 2: Pol-Scope imaging of metaphase II spindles (indicated by arrowheads) of a bovine oocyte (A) free of zona pellucida and (B) with zona pellucida intact.

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Acknowledgements

This work was supported in part by the National Institute of Health (NIH K081099) and Women and Infants Hospital/ Brown Faculty Research Fund. We thank Dr. Peter Smith and Shinya Inoue for encouraging advice.

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Authors and Affiliations

  1. Department of Ob/Gyn, Women and Infants Hospital, Brown University, Providence, 02905, RI

    Lin Liu, James R. Trimarchi & David L. Keefe

  2. Marine Biological Laboratory, Woods Hole, 02543, MA

    Lin Liu, Rudolf Oldenbourg, James R. Trimarchi & David L. Keefe

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  1. Lin Liu
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  2. Rudolf Oldenbourg
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  4. David L. Keefe
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Correspondence to David L. Keefe.

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Liu, L., Oldenbourg, R., Trimarchi, J. et al. A reliable, noninvasive technique for spindle imaging and enucleation of mammalian oocytes. Nat Biotechnol 18, 223–225 (2000). https://doi.org/10.1038/72692

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