Investigating a role for CHTF18 in meiotic crossover formation

Formation of DNA crossovers is a key process in meiosis that increases genetic diversity and ensures accurate chromosome segregation in gametes. Each pair of homologs must possess at least one crossover, called the obligate crossover, to properly segregate during meiosis I. Previously, we revealed a role for the DNA replication protein, CHTF18 (Chromosome Fidelity Factor 18), in mammalian meiosis. In Chtf18-/- mice, meiotic DSB repair is defective, homologs separate prematurely, and spermatocytes and oocytes possess fewer crossovers than wild-type mice. In addition, some homologs completely lack crossovers. These findings indicate a function for CHTF18 in regulating crossover number and ensuring the obligate crossover. Currently, we utilized Chtf18-/- male meiotic chromosome spreads to investigate a role for CHTF18 in crossover formation. We performed immunofluorescence microscopy and evaluated intermediates in the main crossover pathway (Class I). We found that MSH4, an early marker of crossover intermediates, is decreased during the pachytene stage of prophase I in Chtf18-/- spermatocytes compared to wild-type controls. We confirmed that MLH1 foci, a marker of final crossovers, are also decreased. Since MSH4 recruitment is important for crossover formation, these results suggest that CHTF18 may play a role in crossover formation. Finally, we optimized our method of preparing male meiotic spreads for imaging with structured illumination microscopy (SIM). We were able to visualize complete and incomplete synapsis of homologs in zygotene cells at a higher resolution with SIM than conventional microscopy. Collectively, our results provide a foundation to investigate further a role for CHTF18 in crossover formation.