Sydney ivf sperm medium

Aliquots of freshly collected ejaculates (2 to 5 x 10⁶ spermatozoa) from 5 patients were mixed with 1 ml of Sydney IVF Sperm Medium (COOK medical, Brisbane, Australia) and centrifuged for 7 minutes at 400 g. Then, the pellets were suspended in 1 ml of PBS 1x with 20 μl of PI 30 μg/ml for 30 minutes. Before sorting, the stained samples were filtered with CellTrics 100 μm-filters (Partec, Görlitz, Germany). PI-dimmer or PI-brighter populations were isolated using the BD Influx cell sorter (BD Biosciences, San Jose, CA, USA) and the Sortware software (BD Biosciences, San Jose, USA). The following settings were used for sorting: 80 μm nozzle, sample pressure: 14–15 psi, event rate: 3,000–4,000 events/second. Sterile PBS 1x solution was used as sheath fluid and the sorted spermatozoa were collected in 14 ml polystyrene round-bottom tubes (BD Falcon tubes, Biosciences, Durham, NC, USA), containing 1 ml of the Sydney IVF-medium. The sorting was aborted after 1x10⁶ spermatozoa were obtained. The sorted samples were centrifuged at 400 g for 7 minutes and resuspended in 1 ml Sydney IVF Sperm Medium.

Unless otherwise stated, chemicals and reagents were purchased from Sigma-Aldrich Quimica S.A. (Madrid, Spain). Sydney IVF Sperm Medium was provided by Cook Medical (Barcelona, Spain). 4–15% polyacrylamide gels were supplied by Bio-Rad (Madrid, Spain) and PVDF membranes by Merck (Madrid, Spain). The primary antibodies were: anti-phospho-PKA substrates (PKAs-P) (Cell Signaling Technology, Beverly, USA, #9624), anti-phosphotyrosine (Tyr-P) (Abcam, Cambridge, UK, #ab10321) and anti-β-tubulin (β-TUB) (Sigma-Aldrich Quimica S.A., Madrid, Spain, #T0198)^{78 (link),79 (link)}. Horseradish peroxidase-conjugated anti-rabbit and anti-mouse IgGs were obtained from Santa Cruz Biotechnology (Heidelberg, Germany, #sc2004) and Bio-Rad (Madrid, Spain, #1706516), respectively. PageBlue was provided by Thermo Scientific (Rockford, IL, USA), whereas acetonitrile, trifluoroacetic acid and formic acid by Fisher Scientific (UK). Trypsin Gold Proteomics Grade and ProteaseMax surfactant were purchased from Promega Corporation (Madison, MI, USA).

Spermatozoa gained from ejaculation were processed using the swim-up method (13 (link)) and incubated at a temperature of 37°C with 5% O₂, 6% CO₂ and 89% N₂ in Sydney IVF Sperm Medium (Cook Medical, Brisbane, Australia).