2D-DIGE

Two independent 2D-DIGE analyses were performed to compare the protein profiles of spermatozoa and EM before and after cryopreservation with different cryoprotectants (DMSO or MeOH; n = 7 for each group). Before analysis, the EM was concentrated using an Amicon ultracentrifuge filter with a cutoff of 3 kDa (Millipore). Aliquots containing approximately 800 μg of spermatozoa and EM proteins were processed using a Clean-Up Kit (GE Healthcare) according to the manufacturer's protocol. For each biological replicate, 50 μg of protein extract of each sample type (spermatozoa and EM from fresh and cryopreserved samples with MeOH or DMSO) was labeled with 400 pmol of Cy3 or Cy5 (GE Healthcare), with dye swaps to exclude dye bias. An internal standard was created by mixing equal amounts of each sample within the experiment and was labeled with Cy2. After incubation on ice and in the dark for 30 min, the reaction was terminated by the addition of 10 mM lysine. The three labeled samples were then combined within each experiment and diluted with rehydration buffer (8 M urea, 2% CHAPS, 18 mM dithiothreitol (DTT), 0.5% carrier ampholyte, pH 3–10 NL) to 340 μL. The combined samples were loaded on a pH gradient strip (24 cm, pH 3–10 NL) for isoelectric focusing (IEF) on an Ettan IPGphor system (Amersham Biosciences) as described by Dietrich et al.²⁸. After IEF, the strips were first equilibrated in equilibration solution (50 mM Tris–HCl (pH 8.8), 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, traces of bromophenol blue, and 1% (w/v) DTT) for 15 min and later in the same solution except that DTT was replaced by 4% (w/v) iodoacetamide for an additional 15 min. IPG strips were transferred onto 12.5% vertical polyacrylamide gels (with a gel size of 25.5 × 19.6 cm and a thickness of 1 mm) cast on low-fluorescence glass plates using an Ettan DALTsix system (GE Healthcare, Uppsala, Sweden). Electrophoresis was conducted overnight at a constant current of 1.5 W per gel. The Cy2-, Cy3-, and Cy5-labeled images were acquired on a Typhoon 9400 scanner (Amersham Biosciences) at excitation and emission wavelengths of 488/520, 532/580, and 633/670 nm, respectively. Intragel spot detection and quantification and Intergel matching and quantification were performed using differential in-gel analysis (DIA) and biological variation analysis (BVA) modules of DeCyder software version 6.5 (Amersham Biosciences) according to Dietrich et al.¹⁵.