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Related Experiment Videos

Immunoprobe Localization by Correlative Microscopy.

Calarco1

  • 1Department of Anatomy, Box 0452, University of California, San Francisco, CA 94143

Microscopy and Microanalysis : the Official Journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada
|May 3, 2000
PubMed
Summary

Light microscopy can misidentify organelles in mammalian oocytes. This study shows gamma-tubulin and centrosome antigens localize to multivesicular aggregates, not standard microtubule organizing centers, in eggs.

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Area of Science:

  • Cell Biology
  • Microscopy Techniques
  • Oocyte Biology

Background:

  • Mammalian oocytes are difficult to study with electron microscopy (EM) due to hydration and size.
  • Light microscopy (LM) techniques like immunofluorescence are often used, but can lead to misinterpretations.
  • Previous studies presumed standard microtubule organizing centers (MTOCs) house centrosome antigens.

Purpose of the Study:

  • To investigate the accuracy of LM for predicting organelle ultrastructure in mammalian oocytes.
  • To correlate light and electron microscopy data for centrosome and MTOC antigen localization.
  • To clarify the identity and function of organelles containing these antigens.

Main Methods:

  • Correlative light and electron microscopy (LM-EM) was employed.

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  • Immunofluorescence and post-embedding immuno-EM were used for antigen detection.
  • Antibodies against 5051 and gamma-tubulin (a conserved MTOC element) were utilized, with Lowicryl embedding for optimal EM results.

    • Main Results:

    • Prior to nuclear breakdown, 5051 and gamma-tubulin antigens were found exclusively in multivesicular aggregates (MVAs).
    • MVAs bear no ultrastructural resemblance to standard MTOCs.
    • Gamma-tubulin's presence suggests MVAs are precursors to centrosomes with a unique ultrastructure.

    Conclusions:

    • Light microscopy alone can lead to erroneous conclusions about organelle identity and function in mammalian oocytes.
    • Multivesicular aggregates, not standard MTOCs, contain key centrosome/MTOC antigens before nuclear breakdown.
    • MVAs represent a distinct organelle type serving as centrosomal precursors.