73 APPLICATION OF QUANTUM DOT CONJUGATES FOR INVESTIGATING MAMMALIAN SPERMATOZOA

Abstract

Self-illuminating quantum dots are nanoparticles that are less than 100 nm in diameter. Their coating with the light-emitting protein Renilla luciferase forms complexes that have promising applications in in vivo imaging. These complexes can be further combined to specific tags such as antibodies or peptides for various in vitro studies. Especially in reproduction, these conjugates may contribute to a better comprehension of molecular events associated with fertilization and beyond. To this end, we evaluated the ability of mammalian spermatozoa to harmlessly incorporate nanoparticles. Motile spermatozoa of freshly collected boar and stallion semen were purified, washed in PBS/polyvinylpyrrolidone (PVP) (1 mg mL^–1) and adjusted at desired concentrations according to experiments. Spermatozoa were fixed at 10⁷ in Experiment 1 and incubated for 30 min at 37°C with 0, 1, or 5 nM quantum dots conjugated with the bioluminescence resonance energy transfer and R9 cell internalization peptide (BRET-Qdot-R9). In Experiment 2, different amounts of spermatozoa (25 × 10⁶, 50 × 10⁶ and 100 × 10⁶) were incubated as in Experiment 1 with 0 or 1 nM BRET-Qdot-R9. After incubation, aliquots of BRET-Qdot-R9-loaded spermatozoa in both experiments were set aside for motility analysis using the computer-assisted sperm analyzer (only boar data shown). Remaining spermatozoa were centrifuged to eliminate the excess of BRET-Qdot-R9 and washed twice with PBS/PVP at 22°C. Resulting pellets were re-suspended with 50 μL of PBS/PVP and aliquots were mounted on slides for confocal fluorescence microscopic evaluation. Remaining cells and supernatants were mixed with 2 μg of coelenterazine and immediately imaged for bioluminescence using the IVIS-100 Imaging System. Experiments were repeated 3 times and analysed (Student's t-test; P < 0.05 for threshold of significance). Higher light emissions were detected in tubes containing both spermatozoa and BRET-Qdot-R9 as compared to the control (0 nM BRET-Qdot-R9) and last wash-derived supernatants. Low background signals of coelenterazine were observed in tubes (± sperm) without BRET-Qdot-R9. Experiment 1 revealed a dose-dependent response of BRET-Qdot-R9, whereas experiment 2 indicated a potential decrease of light emission with the higher sperm quantity. Interestingly, the presence of BRET-Qdot-R9 did not compromise sperm motility; however, the sperm/BRET-Qdot-R9 ratio appears essential to maintain comparable proportions of motile and rapid spermatozoa to the control group (86 ± 6 and 74 ± 5% for control vs 63 ± 17 and 46 ± 15%, 81 ± 8 and 68 ± 11% and 93 ± 1 and 85 ± 1% for 25 × 10⁶, 50 × 10⁶ and 100 × 10⁶ sperm/1 nM ratios, respectively; P ≥ 0.05; mean ± standard error of the means). The BRET-Qdot-R9 fluorescence signal was mostly detected in the sperm head and intermediate piece with 1 nM BRET-Qdot-R9, whereas the entire spermatozoa fluoresced with 5 nM BRET-Qdot-R9. This study indicates that the incorporation of BRET-Qdot-R9 by boar and stallion spermatozoa does not impair their motility. Further investigations are needed to evaluate the proportion of cells incorporating BRET-Qdot-R9.

Supported by the USDA-ARS Biophotonics Initiative #58-6402-3-0120.