Preparation of spermatozoa for FISH analysis
Slides were fixed in Carnoy’s fixative (3:1 methanol:acetic acid) at room temperature for 15 minutes and placed on a 37°C slide moat overnight. Sperm decondensation was achieved by placing slides into a Coplin jar containing 10 mmol/l dithiothreitol (DTT; Sigma Chemical Co., St. Louis, MO, USA) in 100 mmol/L tris(hydroxymetyl) aminomethane (Trizma HCl; Sigma Chemical Co.) at 22°C for 3 minutes. Slides were then washed for 1 minute in 2x standard saline citrate (SSC; Vysis, Downers Grove, IL, USA) and hybridized with fluorescent probes. Negative controls were included in each assessment and processed identically, except for the probe hybridization step [