Self-illuminating quantum dots for non-invasive bioluminescence imaging of mammalian gametes

Abstract

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 cumulusoocyte 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.

Current and future prospects for nanotechnology in animal production

“…These self-illuminating, inorganic nanoparticles are of interest to the field of theriogenology as they are biocompatible, photo-stable, and have a greater signal intensity than organic fluorescent molecules previously used to image gametes and other cell types in vivo [ 68 – 70 ]. Feugang et al [ 68 , 71 ] have demonstrated the real-time tracking ability of bioluminescent resonance energy transfer-conjugated quantum dot (BRET-QD) nanoparticles in vitro, in situ, and ex vivo using pig male gametes ( Sus scrofa domesticus ). Quantum dots can provide targeted or non-targeted imaging as a function of their size, emitted wavelengths, and conjugation possibilities [ 68 , 71 ].…”

“…Feugang et al [ 68 , 71 ] have demonstrated the real-time tracking ability of bioluminescent resonance energy transfer-conjugated quantum dot (BRET-QD) nanoparticles in vitro, in situ, and ex vivo using pig male gametes ( Sus scrofa domesticus ). Quantum dots can provide targeted or non-targeted imaging as a function of their size, emitted wavelengths, and conjugation possibilities [ 68 , 71 ]. This engineered nano particle provides a new mean to visualize the molecular and cellular events during fertilization, in a similar way to fluorescent proteins, but at greater tissue depths [ 71 , 72 ].…”

“…Quantum dots can provide targeted or non-targeted imaging as a function of their size, emitted wavelengths, and conjugation possibilities [ 68 , 71 ]. This engineered nano particle provides a new mean to visualize the molecular and cellular events during fertilization, in a similar way to fluorescent proteins, but at greater tissue depths [ 71 , 72 ]. Signal strength of quantum dots are dose-dependent and a higher concentration may be required for in vivo imaging on larger animals.…”

Fluorescent nanoparticles as tools in ecology and physiology

“…Since the complex did not hinder the movement of spermatozoa, it could be used for in vivo tracking of fertilisation success. This application also demonstrates the usefulness of QD technologies for deep tissue imaging of large animals, which is challenging with more traditional methods that are less specific and bright (Feugang et al, 2015). CdSe/ZnS QDs capped with dihydrolipoic acid (DHLA) were used to track a protein that regulates the cell cycle in African clawed frog (Xenopus laevis) embryos in vivo (Brandt et al, 2015).…”

“…Streptavidin-conjugated CdSe/ZnS QDs injected into a zebrafish blastomere at the two-cell stage were transferred from mother to daughter cells through cell division via cytoplasmic bridges and exhibited bright fluorescence even in late developmental stages such as organogenesis (Rieger et al, 2005). A CdSe/ZnS QD complex involving a fluorescent dye, a cellpenetrating peptide and an antibody related to fertilisation regulation was utilised for tracking the movement of domestic pig (Sus scrofa domesticus) spermatozoa in female reproductive tracts (Feugang et al, 2015). The QD complexes specifically attached to male gametes, indicating that plasminogen is present in sperm cells.…”