Sperm Preparation for Transmission Electron Microscopy

Sperm was prepared for transmission electron microscopy (TEM) (JEM-1400; JEOL, Tokyo, Japan) as previously described [39 (link)]. Briefly, sperm was washed in PBS and centrifuged at 300 g for 5 min; this process was repeated twice. The sperm pellet was extremely gently mixed with 0.1% glutaraldehyde at 4 °C overnight. The fixed sperm pellet was rinsed with 0.1 M phosphate buffer (pH 7.2) and then treated with 1% osmium tetroxide at room temperature for 2 h. It was then rinsed again with phosphate buffer and progressively dehydrated through a series of ethanol treatments at increasing concentrations. The Spurr’s resin kit (cat-14300; Electron Microscopy Sciences, PA, USA) was used to embed the sperm pellet at room temperature overnight. Finally, 75-nm thin sections of the embedded samples were cut using an ultramicrotome (EM-UC7, Leica microsystems, Wetzlar, Germany) and were mounted on copper grids. Counter-staining was performed with uranyl acetate and lead citrate. An ultramicrograph was acquired using TEM (JEM-1400; JEOL, Tokyo, Japan) at 100 Kva.