Polytron vdi 12

Daily testicular spermatid production was measured as described previously in Maurice et al. 2018 [33 (link)]. Briefly, the thawed testis without the tunica, one for each mated male, was homogenized in 10% DMSO/0.9% NaCl (Sigma-Aldrich, Oakville, ON, Canada) using a Polytron VDI 12 (VWR International, Radnor, PA, USA) twice for 15 s with a 30 s pause between. Samples were sonicated at 40% power for 1 min and 0.1% trypan blue (Sigma-Aldrich, Oakville, ON, Canada) was added to colour the spermatid heads, which were counted using a hemacytometer. Daily testicular spermatid production was calculated according to the equation: ((Mean count of hemacytometer/0.00004 μL volume of secondary square in hemacytometer) × 100.5 mL of total number of rat testis suspension)/6.10 days for spermatogenesis cycle.

Right atrial samples were pulverized in liquid nitrogen congelation and sonicated (Brandson Sonifier 250, ICN Hubber S.A.) in 500 μL of ice-cold 10 mM Tris HCl, pH 7.4 buffer containing a protease inhibitor cocktail (Roche Molecular Systems, Belmont, CA, USA). The sonicated tissue was further homogenized using a Polytron (VDI 12, VWR, Barcelona, Spain) for three periods of 10 s each. The homogenate was centrifuged at 12,000 × g at 4 °C for 30 min. The membranes were dispersed in 50 mM Tris HCl (pH 7.4) and 10 mM MgCl₂, washed, and resuspended in the same medium as described previously [34 (link)]. Protein concentration was determined using the BCA protein assay kit (Thermo Fisher Scientific Inc., Rockford, IL, USA) and 10 μg of protein was used for immunoblotting.