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

Abstract

Optical macro-tweezers: trapping of highly motile micro-organisms G Thalhammer, R Steiger, S Bernet et al. AbstractThe 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.

Effects of viscosity on sperm motility studied with optical tweezers

“…11 The DiOC 6 ð3Þ-treated sperm were tested in viscosities of 1, 6, and 9 cP. The fluorescence data were aggregated and graphed in Fig.…”

“…11 Briefly, a Zeiss fluor arc lamp provides the excitation light. A red filter above the image plane allows for separation of phase contrast (670 nm) and fluorescence (500 to 570 nm) images.…”

“…The final sperm dilutions of 30,000 sperm per mL were loaded into cover slide dishes (about 0.5 μL of sperm in 2.5 mL methylcellulose þ mHTFþ SSS media) and mounted on the microscope stage. 11 For fluorescent studies 3,3 0 -dihexyloxacarbocyanine iodide [DiOC 6 ð3Þ], a nonratiometric carbocyanine dye supplied by Invitrogen (Carlsbad, CA), was added to the prepared sperm: 40-nM stock DiOC 6 ð3Þ dye was added into 250 μL of prepared human sperm and was incubated at 37°C for 20 min. 11 The sperm sample was loaded into the optical system to measure midpiece mitochondrial fluorescence.…”

“…11 For fluorescent studies 3,3 0 -dihexyloxacarbocyanine iodide [DiOC 6 ð3Þ], a nonratiometric carbocyanine dye supplied by Invitrogen (Carlsbad, CA), was added to the prepared sperm: 40-nM stock DiOC 6 ð3Þ dye was added into 250 μL of prepared human sperm and was incubated at 37°C for 20 min. 11 The sperm sample was loaded into the optical system to measure midpiece mitochondrial fluorescence. Stock solutions of DiOC 6 ð3Þ dye were prepared with dimethyl sulfoxide (DMSO).…”

“…DiOC 6 ð3Þ is used in this study because it has been demonstrated to be an effective dye for measuring membrane potentials in sperm. 11 …”