Abstract
Live-imaging of meiotic cell division has been performed in extracted spermatocytes of a number of species using phase-contrast microscopy. For the nematode Caenorhabditis elegans, 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 in situ filming of spermatocyte meiosis in whole immobilized C. elegans 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 in silico, 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 C. elegans hermaphrodites and should be expandable to other fluorescently labelled nematodes or other fully transparent small model organisms.
Highlights
[Background] In the past, studies on male meiosis have been performed on isolated and stained specimens to image meiotic spindles, with obvious limitations of using fixed specimens (Boring, 1909; Boveri, 1909; Goldstein, 1977)
Extracted insect spermatocytes were observed with phase-contrast and polarized light microscopy and very elegant experiments were performed on spindles or single chromosomes using micromanipulators and laser microsurgery (King and Nicklas, 2000; Nicklas et al, 2001; Forer et al, 2013; Sheykhani et al, 2017)
In the nematode Caenorhabditis elegans, microscopic investigation of spermatocyte meiosis was previously achieved by labelling of isolated and fixed gonads with antibodies (Howe et al, 2001; Shakes et al, 2009; Peters et al, 2010; Schvarzstein et al, 2013)
Summary
Platinum wire with a diameter of 0.2 mm (Agar Scientific, catalog number: AGE404-2). 8. High-precision cover slips (18 mm x 18 mm x 0.17 μm, Carl Roth, catalog number: LH22.1) 9. C. elegans strain N2 (reference strain; Brenner, 1974) 12. Agarose (Carl Roth, catalog number: 3810.2) 15. NaCl (Carl Roth, catalog number: 9265.1) 17. Cholesterol (Carl Roth, catalog number: 8866.1) 18. CaCl2 (Carl Roth, catalog number: CN93.1) 19. MgCl2 (Carl Roth, catalog number: 2189.1) 20. NaOCl (Carl Roth, catalog number: 9062.3) 21. KH2PO4 (Carl Roth, catalog number: P749.2) 22. Na2HPO4 (Carl Roth, catalog number: P030.1) 23. MgSO4 (Carl Roth, catalog number: P027.1) 24. Spinning disc confocal microscope (Olympus, model: IX83) 8.
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