Elena Rebollo and Cayetano Gonzlez 1 Introduction

Meiosis is one of the key stages of spermatogenesis during which two rounds of chromosome segregation follow a single doubling of the DNA, thus reducing the chromosome number to produce haploid cells. Drosophila spermatocytes have been used extensively to study the basic mechanisms that govern cell division and meiosis. They are well suited for this purpose because they are easily accessible, readily identifiable, and relatively large and abundant. Moreover, large collections of mutants that disrupt many aspects of cell division and green fluorescent protein (GFP)-fusion proteins that label different components of the cell division machinery, are available in Drosophila and can be studied in these cells (1-12).

Classically, studies in Drosophila male meiosis have been performed by direct observation of unfixed and unstained cysts under phase-contrast optics (3,8,9,13). Detailed analyses of chromosome behavior have required staining with aceto-orcein or Hoechst dye (reviewed in ref. 14). In the mid-1990s, a reliable fixation protocol was developed that allows immunofluorescence staining (15), thus providing a better basis to study the organization of the major cell structures and organelles, as well as the localization of proteins of interest. Although very informative, these methods based on either short-lived or fixed cells do not allow for the observation of meiosis progression, which is essential to follow the dynamic events that take place during cell division. The first description of a method for generating primary cultures of primary Drosophila spermatocytes that could be visualized by time-lapse video microscopy was published by Church and Lin (16). Using this method in combination with

From: Methods in Molecular Biology, vol. 247: Drosophila Cytogenetics Protocols Edited by: D. S. Henderson © Humana Press Inc., Totowa, NJ

micromanipulation and electron microscopy, they provided beautiful descriptions of kinetochore and chromosome behavior during the first meiotic division in Drosophila testes (16,17). However, this approach has not been exploited at all. It has not been until very recently that time-lapse video microscopy of cultured Drosophila spermatocytes has been used again to study meiosis in Drosophila males (18-21).

To some extent, Drosophila spermatocytes are actually well suited for this technique: They remain flat, thus producing sharp phase-contrast images during cell division; they have large spindles and centrosomes beautifully delineated by phase dark spindle and aster-associated membranes; and the small number of chromosomes, only four, makes it possible to follow each of them in detail. However, the tolerance of Drosophila spermatocytes to culture is very limited. They can break easily if the manipulations required to set up the cultures are not carried out with extreme care, and they are very sensitive to light, thus imposing some limitation in terms of the recording conditions. We have introduced some minor, yet useful, modifications in the original protocol described by Church and Lin (16) that ameliorate these problems and allow for relatively long recording sessions, including observations of such critical steps as cytokinesis, interkinesis, and the second meiotic division, which have not been reported previously (Rebollo and González, unpublished data). We have also set up the conditions for two-channel imaging to record both phase-contrast as well as fluorescence images from the same cell, so that the localization of GFP-fusion proteins can be followed. Special attention is paid to the culturing steps. Although the technique looks unbelievably simple, it requires some practice and expertise until viable cells can be followed under the microscope.

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