Osmium tetraoxide

Following overnight fixation in 2.5% EM grade glutaraldehyde at 4 °C, samples were washed (3 × 5 min) in 0.1 M sodium cacodylate buffer prior to OTO (osmium tetraoxide–thiocarbohydrazide–osmium) processing. Briefly, this method included post fixation in equal volumes of 4% osmium tetraoxide (Agar Scientific Ltd. Essex, UK) and 3% potassium ferrocyanide (Sigma-Aldrich, St. Louis, MO, USA) for 60 min on ice. Following post fixation, samples were rinsed (3 × 5 min) in dH₂O before incubating with thiocarbohydrazide (Sigma-Aldrich, St. Louis, MO, USA) for 20 min. Additional dH₂O washing steps (3 × 5 min) were applied before incubation in 2% aqueous osmium for 30 min at room temperature. Following OTO processing, bacterial samples were stained in 1% aqueous uranyl acetate (1 h at 4 °C) followed by lead aspartate (200 µM) for 30 min in the dark. Between these steps, washing with dH₂O was performed. After the final washing step, bacterial samples were dehydrated and dried as previously outlined.

To better characterize the impact of the reported candidate variant on sperm ultrastructure, sperm from P0504 (an individual carrying this variant) were subjected to transmission electron microscopy. Sperm were collected from fresh ejaculate and washed with M2 medium (Sigma-Aldrich Co. Ltd., Irvine, UK) by centrifugation at 300 g for 10 min. Sperm cells were fixed in 0.1 M phosphate buffer, pH 7, containing 3% glutaraldehyde (Grade I; Sigma-Aldrich Co., St. Louis, MO, USA) (room temperature, 90 min). After two washes in PBS + 1% sucrose, samples were resuspended in 0.2 M sodium cacodylate buffer. Secondary fixation was performed with 1% osmium tetra-oxide (Agar Scientific Ltd., Essex, UK) and was followed by dehydration in graded alcohol and embedding in Epon resin (Polysciences Inc., Warrington, PA, USA). After staining with toluidine blue-Azur II, semi-thin sections were examined on a Zeiss Axioscope photomicroscope (Carl Zeiss, GmbH, Jena, Germany). The use of a Reichert OmU2 ultramicrotome (Reichert-Jung AG, Wien, Austria) allowed us to obtain ultra-thin sections (90 nm), which were then stained with uranyl acetate and lead citrate. These sections were examined with a JEOL JEM 100CX II electron microscope (Jeol Ltd., Tokyo, Japan). All images were acquired with Digital Micrograph software coupled to a Gatan Erlangshen CCD (charge-coupled device) camera.