Donated human spermatozoa with normal semen characteristics (concentration, motility; WHO 2010) from healthy volunteers with no known fertility problems were used in this study. Samples were obtained by masturbation after ≥ 48 h of sexual abstinence, liquefied for 30 min at 37°C, and processed using density gradient centrifugation, to separate cells using 40/80% Percoll (Sigma Aldrich, UK) fractions. Every experiment has been performed on pooled spermatozoa from ≥ 3 donors each.
For experiments in non-capacitating conditions, we used a buffer system slightly modified from [23 (link)] using Minimal Essential Medium Eagle (Sigma), supplemented with HEPES (1 M solution, Gibco), sodium lactate (DL-solution, Sigma), sodium pyruvate (100 mM solution, Gibco) and bovine serum albumin (7.5% solution, Sigma).
For experiments in capacitating conditions, we used a buffer system slightly modified from the HTF from [63 (link)] to a final composition of 97.8 mM NaCl, 4.69 mM KCl, 0.2 mM MgSO4, 0.37 mM KH2PO4, 2.04 mM CaCl2, 0.33 mM Na-pyruvate, 12.4 mM Na-lactate, 2.78 mM glucose, 21 mM HEPES, 25 mM NaHCO3 and 3 mg/mL BSA. All buffer components were supplied by Sigma-Aldrich unless otherwise stated and all buffer systems were adjusted to pH 7.4.
For experiments in non-capacitating conditions, we used a buffer system slightly modified from [23 (link)] using Minimal Essential Medium Eagle (Sigma), supplemented with HEPES (1 M solution, Gibco), sodium lactate (DL-solution, Sigma), sodium pyruvate (100 mM solution, Gibco) and bovine serum albumin (7.5% solution, Sigma).
For experiments in capacitating conditions, we used a buffer system slightly modified from the HTF from [63 (link)] to a final composition of 97.8 mM NaCl, 4.69 mM KCl, 0.2 mM MgSO4, 0.37 mM KH2PO4, 2.04 mM CaCl2, 0.33 mM Na-pyruvate, 12.4 mM Na-lactate, 2.78 mM glucose, 21 mM HEPES, 25 mM NaHCO3 and 3 mg/mL BSA. All buffer components were supplied by Sigma-Aldrich unless otherwise stated and all buffer systems were adjusted to pH 7.4.
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