For assessment of meiotic progression, sporulating cells were fixed with ethanol, stained with 4′,6-diamidino-2-phenylindole, and photographed under wet mount using a Nikon Optiphot equipped for epifluorescence as previously described (Krisak et al. 1994 (link)). Sporulation efficiency was analyzed by phase-contrast microscopy, with cells containing two or more spores per ascus scored as positive. For live-cell imaging, it was necessary to use multicopy plasmids to detect the Smk1-GFP and Ssp2-GFP signals. Cells containing an integrated nuclear HTB2-mCherry marker (Table 1) carrying 2µ-based SMK1-GFP or SSP2-GFP plasmids (Table 2) were inoculated from selective medium into YEPA for overnight growth and transferred to sporulation medium as previously described (Tio et al., 2015 (link)). A Leica DM-RXA with oil immersion was used to image 8 μl of sporulating cells, using a 60× lens, at the indicated times. Completion of MII was monitored using the HTB2-mCherry marker and further assayed for GFP fluorescence. The fraction of postmeiotic cells that showed GFP fluorescence varied between trials from 8% to 20% due to plasmid loss. All Smk1-GFP and Ssp2-GFP experiments described in this study were repeated independently at least three times with a minimum of 100 fluorescent cells examined.