2.5 Electron microscopy and concentrated Papanicolaou staining
For scanning electron microscopy (SEM), the sperm samples were centrifuged at 400×g for 10 min at room temperature. The supernatants were carefully aspirated, and the pellets were suspended and fixed in 2.5% glutaraldehyde for 30 min at 4°C. Next, the samples were evenly spread onto 20-mm-diameter slides and fixed in 2.5% glutaraldehyde overnight at 4°C. Following primary fixation, the slides were washed three times in 1 ×PBS and postfixed in 1% osmic acid for 1 h at 4°C. Then, gradient dehydration was performed sequentially with 30%, 50%, 75%, 95%, and 100% ethanol for 10 min. Subsequently, the slides were dried to temperature with a CO2 critical-point dryer (Eiko HCP-2, Hitachi). Finally, all of the dried specimens were mounted on aluminum stubs, sputter-coated by an ionic sprayer meter (Eiko E-1020, Hitachi), and analyzed by SEM (Hitachi S3400).
For transmission electron microscopy (TEM), 1.0 mL of the sperm samples was washed routinely and centrifuged at 400 × g for 15 min. Then, the seminal plasma was removed, and the sperm pellets were fixed in 3% glutaraldehyde. Next, the samples were postfixed in 1% buffered OsO4, dehydrated through gradient acetone solutions, and embedded in Epon 812. It was necessary to make the samples into half-thin slices (800 nm) to localize the sperm under the light microscope. Finally, the ultrathin sections (80 nm) were double-stained with lead citrate and uranyl acetate before being observed and photographed via TEM (TECNAI G2 F20, Philips).