Meiosis is a central feature of sexually reproducing eukaryotes that results in haploid gametes. The spindle assembly checkpoint (SAC) is essential for precise cell division as it monitors connections between kinetochores and spindle microtubules. Inhibition of the SAC may result in chromosome segregation failure and aneuploidy, which can lead to cell death, or defective progeny. Despite the critical function of the SAC to meiotic cell division, we still do not understand how it fully functions, particularly in male meiosis. This study aims to characterize the SAC's response to erroneous kinetochore attachment and faulty positioning of chromosomes in male meiosis, using C. elegans as a model. To perturb meiotic chromosome segregation, we treated wild-type and
mad-2 (a SAC component) mutant worms with colchicine, a drug that depolymerizes microtubules, affecting all microtubule-kinetochore interactions. Live imaging revealed a metaphase delay in spermatocytes following release from colchicine (metaphase length WT: 9.6 plus or minus 1.6 mins, n=11; WT + colchicine=22.5 plus or minus 6.0 mins, n=9). The metaphase delay was SAC-dependent as no delay was observed in the SAC mutant (
mad-2: 9.6 plus or minus 1.8 mins, n=8;
mad-2 + colchicine: 10.7 plus or minus 1.6 mins, n=10). To date, our results suggest that the SAC is activated and induces a cell cycle delay in response to microtubule depolymerization at metaphase I of spermatogenesis. However, MAD2, which normally accumulates to high levels on unattached kinetochores, was not detected on unattached kinetochores following colchicine treatment. This preliminary data suggests that the SAC is operating differently in spermatogenesis than in mitotically-dividing germ cells or embryos. To further examine SAC sensitivity and function, we are analyzing the
zim-2 and
zim-1 mutants, which cause defects in pairing, synapsis, and chromosome segregation of a single pair or two chromosome pairs, respectively. Using CRISPR-Cas9, we generated both
zim-2 and
zim-1 mutants in a C. elegans strain expressing GFP::Tubulin and mCherry::Histone H2B to allow for analysis of chromosome segregation in vivo. We are currently examining the timing of metaphase I in
zim-2 and
zim-1 mutants in the presence and absence of the SAC to determine the sensitivity of the SAC in male meiosis.