Background: The fertility performance of animals is still a mystery and the full comprehension of mammalian gametes maturation and early embryonic development remains to be elucidated. The recent development in nanotechnology offers a new opportunity for real-time study of reproductive cells in their physiological environments. As a first step toward that goal, we evaluated the effectiveness of a fluorescent and luminescent nanoparticle for in vitro and ex vivo imaging of porcine gametes.
Methods: Freshly harvested boar sperm were labeled with red-shifted (655 nm) quantum dot nanoparticles conjugated (QD+) or not (QD-) with plasminogen antibody and evaluated. Subsets of labeled spermatozoa were loaded into straws and placed within the lumen of gilt reproductive tracts for ex vivo intra-uterine imaging. Porcine cumulus-oocyte complexes (COCs) were matured in the presence of QD- or QD+. Ovarian follicles were microinjected with QD- or QD+ and placed in culture for up to 4 days. After labeling, all samples were supplemented with coelenterazine, the luciferase substrate, and immediately submitted to bioluminescence analysis, followed by fluorescence and hyperspectral imaging. Data were analyzed with ANOVA and P < 0.05 indicated significant differences.
Results: All labeled-samples revealed bioluminescence emission that was confirmed by fluorescence and hyperspectral imaging of the QD localization within the cells and tissues. Over 76% of spermatozoa and both immature and mature COCs were successfully labeled with QD- or QD+. The QD- fluorescence appeared homogenously distributed in the oocytes, while found in the entire sperm length with a higher accumulation within the mid-piece. Labeled-follicles exhibited a progressive migration of QD nanoparticles within the follicle wall during culture. In contrast, QD+ fluorescence signals appeared condensed and stronger in the follicle cells, sperm head, and sub-plasma membrane area of mature oocytes. Weaker QD+ signals were detected in the cumulus cells. Fluorescence and hyperspectral microscope imaging showed comparable intracellular QD localization. Ex-vivo intra-uterine bioluminescence imaging of labeled spermatozoa revealed stronger signals captured over the oviducts, with uterine body allowing the lowest signal detection.
Conclusion: Findings indicate that conjugated and non-conjugated fluorescent nanoparticles can be used for effective labeling of mammalian gametes for in vitro monitoring and potential in vivo targeted-imaging.