Laser tweezers to Investigate sperm motility and mitochondrial respiration in three vertebrate species

The purpose of this study is to investigate how the mitochondrial membrane potential affects sperm motility using laser tweezers and the fluorescent probe, DiOC6(3). A 1064 nm Nd:YVO4 continuous wave laser was used to trap motile sperm at a power of about 450 mW in the focused trap spot. Using customized tracking software, the curvilinear velocity (VCL) and the escape force (Pesc) from the laser tweezers were measured. Human (Homo sapiens), dog (Canis lupis familiaris), and drill (Mandrillus lecuophaeus) sperm were labeled with the fluorescent dye DiOC6(3) to measure membrane potential in the mitochondria-rich sperm midpiece. The VCL was measured frame by frame before trapping, and the fluorescence was measured before and during trapping. Fluorescence intensity of the dye-treated sperm was compared to unlabeled sperm. Controls were performed to verify that the DiOC6(3) does not affect sperm motility. Carbonyl cyanide m-chlorophenylhydrazone (CCCP) was used to inhibit mitochondrial respiration and cause a loss of fluorescence. The results show that DiOC6(3) has no effect on the VCL and Pesc of the sperm even though there was a marked reduction in dye fluorescence when the CCCP was applied. Drill sperm exhibited a drop in VCL upon addition of CCCP, indicating a potential reliance on both glycolysis and aerobic respiration for motility. These results confirm that aerobic mitochondrial respiration is not the primary driving force for motility in human and dog sperm, but may be important for some primate species. DiOC6(3) was also proven to be an effective dye to study sperm mitochondrial energetic