147 POTENTIAL USE OF CONJUGATED QUANTUM DOT NANOPARTICLES FOR BIO-LABELLING OF MAMMALIAN GAMETES

Quantum dot nanoparticles (QD) have unique optical properties and high photo-stability. In our previous study, we reported the ability of boar spermatozoa and ovarian follicles to incorporate QD coated with light emitting protein luciferase (QD-BRET) for successful bioluminescence and fluorescence imaging. These reports indicated that co-incubation of QD-BRET with spermatozoa before IVF of pig oocytes does not affect their fertilizing potential, and neither microinjected QD-BRET into cultured porcine ovarian follicles affected their development (Feugang et al. 2012 Reprod. Fertil. Dev.). In the current study, we tested whether the conjugation of QD-BRET with antibody (anti-plasminogen) allows noninvasive molecular labelling in living spermatozoa and ovarian follicular cells. Gilt reproductive tracts were collected at a local abattoir and washed in a pre-warmed 0.9% NaCl (37°C). In experiment 1 (n = 3), ovaries were dissected and antral follicles of 4 to 8 mm in diameter were transferred to NCSU-23+ supplements. Follicles were incubated (37°C, 5% CO2), in a humidified environment (Day 0). The following day, 60 pmol of QD-BRET alone (QD–) or QD-BRET+ antibody (QD+) was microinjected into intact follicles and half of the culture medium was changed. Follicles were subsequently returned to 48 h of incubation (Day 3). In parallel (experiment 2; n = 3), cumulus-oocytes complexes were aspirated from antral follicles (4–8 mm) and matured (0 or 48 h at 38.5°C, 20% O2) in the presence or absence of 1 nM QD conjugates (QD– or QD+). In experiment 3 (n = 4), motile boar spermatozoa (108) were incubated (0.5 h, 37°C) with or without 1 nM QD conjugates (QD– or QD+). For bioluminescence imaging (IVIS 100), coelenterazine was microinjected into follicles or mixed with spermatozoa or oocytes, imaged, and data collected (photons/sec). Labelled spermatozoa were evaluated through flow cytometry and confocal and hyperspectral microscopes were used for QD fluorescence evaluation. Results indicate that follicle morphology and structure were not affected by the presence of QD– or QD+ during culture. Bioluminescence imaging showed that all samples were able to pick up both QD-BRET conjugates. The flow cytometery indicated a significantly higher proportion (90 ± 2%) of sperm labelled with QD+ than those without antibody (QD–: 75 ± 6%; P < 0.05). This latter group had significantly less fluorescence intensity compared to QD+ (P < 0.05). The motility of labelled spermatozoa were not affected and were comparable to the control (untreated) group. Hyperspectral imaging revealed predominance, stronger, and more localised fluorescence signals of QD+ than QD– in sperm heads and follicular cells. The QD+ signal was detected as 9 times higher in cumulus-oocytes complexes, whereas a redistribution of plasminogen towards the subcortical region was observed. In conclusion, the results indicate that QD conjugates are potential tools for noninvasive molecular labelling and tracking of mammalian gametes. However, further studies are still needed to confirm the usefulness of QD conjugates during early embryo development and to minimize their possible toxicity. Supported by the USDA-ARS Grant #58-6402-3-0120.