Optical tweezers and non-ratiometric fluorescent-dye-based studies of respiration in sperm mitochondria

The purpose of this study is to investigate how the mitochondrial membrane potential affects sperm motility using laser tweezers and a non-ratiometric fluorescent probe, DiOC(6)(3). A 1064 nm Nd:YVO4 continuous wave laser was used to trap motile sperm at a power of 450 mW in the trap spot. Using customized tracking software, the curvilinear velocity (VCL) and the escape force from the laser tweezers were measured. Human (Homo sapiens), dog (Canis lupis familiaris) and drill (Mandrillus leucophaeus) sperm were treated with DiOC(6)(3) to measure the membrane potential in the mitochondria-rich sperm midpieces. Sperm from all three species exhibited an increase in fluorescence when treated with the DiOC(6)(3). When a cyanide inhibitor (CCCP) of aerobic respiration was applied, sperm of all three species exhibited a reduction in fluorescence to pre-dye levels. With respect to VCL and escape force, the CCCP had no effect on dog or human sperm, suggesting a major reliance upon anaerobic respiration (glycolysis) for ATP in these two species. Based on the preliminary study on drill sperm, CCCP caused a drop in the VCL, suggesting potential reliance on both glycolysis and aerobic respiration for motility. The results demonstrate that optical trapping in combination with DiOC(6)(3) is an effective way to study sperm motility and energetics.