Live-cell Imaging and Quantitative Analysis of Meiotic Divisions in Males.

Live-imaging of meiotic cell division has been performed in extracted spermatocytes of a number of species using phase-contrast microscopy. For the nematode , removal of spermatocytes from gonads has damaging effects, as most of the extracted spermatocytes show a high variability in the timing of meiotic divisions or simply arrest during the experiment. Therefore, we developed a live-cell imaging approach for filming of spermatocyte meiosis in whole immobilized males, thus allowing an observation of male germ cells within an unperturbed environment. For this, we make use of strains with fluorescently labeled chromosomes and centrosomes. Here we describe how to immobilize male worms for live-imaging. Further, we describe the workflow for the acquisition and processing of data to obtain quantitative information about the dynamics of chromosome segregation in spermatocyte meiosis I and II. In addition, our newly developed approach allows us to re-orient filmed spindles , regardless of the initial 3D orientation in the worm, and analyze spindle dynamics in living worms in a statistically robust manner. Our live-imaging approach is also applicable to hermaphrodites and should be expandable to other fluorescently labelled nematodes or other fully transparent small model organisms.