113 EX VIVO IMAGING OF OVARIAN FOLLICLES AND INTRAUTERINE SPERMATOZOA USING QUANTUM DOTS

The rapid progress in nanotechnology has permitted the production of biocompatible quantum dots (QD). These particles of less than 100 nm in diameter have unique optical properties and high photo-stability, and their possible attachment to biomolecules offers a great potential for noninvasive and ultrasensitive imaging of molecular targets in living cells and animals. Here, we used a QD coated with light-emitting protein luciferase (QD-BRET) to assess their passive cellular uptake in developing ovarian follicles, and to conduct photonic imaging of spermatozoa within the excised uterus. Pig ovaries (experiment 1) and reproductive tracts (RT) were collected at a local abattoir and washed in a pre-warmed (37°C) 0.9% NaCl (n = 3). Ovaries were dissected, and antral follicles of 4 to 8 mm in diameter were transferred to NCSU-23 medium supplemented with 3 mg mL–1 BSA, 1% insulin-transferrin-selenium (ITS), 1.5 ng mL–1 porcine FSH, 30 ng mL–1 human LH, and 7.5% porcine serum. Follicles were incubated at 37oC under 5% CO2, in a humidified environment (day 0). The following day, 60 pM QD-BRET was microinjected into intact follicles that were subsequently returned to the incubator. Half of the medium was changed every 2 days. In experiment 2 (n = 3), motile boar spermatozoa (5 × 107) were incubated (30 minutes at 37°C) with 0 or 1 nM QD-BRET. Later, spermatozoa were centrifuged and pellets resuspended in PBS. For QD-BRET analyses, follicles were collected on Day 2, 4, and 6, and injected with 750 ng ofcoelenterazine, while 2 µg were mixed with spermatozoa. Spermatozoa were then transferred into 0.5-mL plastic straws and placed inside of warmed RT sections (oviduct, uterine horn, and body). A subset of filled straws kept outside the tissues served to evaluate the proportion of light transmitted across tissue sections. All samples were immediately imaged for bioluminescence emission after addition of coelenterazine and data were recorded (photons/sec). In parallel, aliquots of spermatozoa and prepared follicle sections (4 µm) were submitted to confocal laser and/or transmission electron microscopy (TEM) evaluation of QD-BRET. Observed morphology and structure of follicles were not affected by the presence of QD-BRET during culture. At each evaluation day, injected follicles emitted light, which intensities appeared inversely proportional to the follicle size. Fluorescence signals indicated the incorporation of QD-BRET in granulosa cells, while lesser or no signals were detected in the theca cells or the antrum, respectively. Ex vivo tissue imaging revealed the highest transmission light (22%) across the oviduct, which was 3× and 20× greater than the signals captured over the uterine horn and body, respectively (P ≤ 0.05, ANOVA). The QD-BRET fluorescence in spermatozoa was mainly located in the head and mid-piece areas, which was confirmed by TEM. These results indicate that QD conjugates are potential tools for noninvasive functional analyses in growing ovarian follicles and for tracking sperm migration within the reproductive tract. Further studies are still needed to optimise the use of QD and minimise their possible toxicity. USDA-ARS Grant #58-6402-3-0120.