149 DEVELOPMENT OF AN IN VITRO BIOLUMINESCENT SPERM BINDING ASSAY: PRELIMINARY DATA

Quantum dot technology has enabled researchers to incorporate the intrinsic properties of such nanoparticles into physiological exploration. Previous work from our laboratory has demonstrated that quantum dots can be incorporated into spermatozoa without deleterious effects to physiological parameters such as motility, viability, and fertilizing potential (Feugang et al. 2012). However, the journey of spermatozoa within the female reproductive tract is met with many physicochemical obstacles and checkpoints that include the binding of spermatozoa to utero-oviducal epithelial cells. Moreover, the binding ability/affinity of quantum dot-labelled spermatozoa has not been tested and therefore, the objective of this study is to test the binding semblance of quantum dot-labelled spermatozoa to uterine epithelial cells compared to normal sperm, and the subsequent use of the technology to develop a bioluminescent sperm binding assay. Porcine uterine epithelial (PUE) cells were seeded into 96-well clear-bottomed plates (20 000 cells/well) and allowed to grow to 95% confluency. Motile spermatozoa were selected from fresh pooled semen of fertile boars and labelled with quantum dot nanoparticles to form quantum sperm, as previously described (Feugang et al. 2012). Final concentrations of 107 labelled (QD+) and non-labelled (QD–) spermatozoa were added to monolayers of PUE cells and co-incubated in PBS/polyvinylpyrrolidone (PVP) at 37°C, 5% CO2. The control consisted of PUE cells alone in the PBS/PVP medium. Each treatment was performed in triplicate and experiments were repeated 3 times. After 1 h of co-incubation, the supernatant from each well was transferred to the adjacent three wells. The co-incubated wells containing expected PUE-sperm binding were then washed 3 times with PBS/PVP to eliminate any unbound sperm. PUE-quantum sperm (QD+) and PUE-non-labelled sperm (QD–) complexes were verified using bright field microscopy, followed by measurement of photonic emission from each well (GloMax Multi Detection System, Promega, Madison, WI, USA). Data was analysed by ANOVA with the threshold of significance fixed at P < 0.05. There were no visual differences in binding patterns between QD+ and QD–, which appeared similar under the microscope. However, the photonic signals (relative luminescent units; RLU) from QD+ wells were significantly higher than both the control and QD– wells (2534.84 ± 639.91 v. 542.46 ± 639.91 and 806.48 ± 639.91 RLU; P < 0.05). Supernatants collected from the QD+ wells, representing unbound quantum sperm, had the highest photonic emissions when compared to all other wells, with or without spermatozoa (19 948.23 ± 639.91 RLU; P < 0.05). Results demonstrate that quantum dot nanoparticles can be incorporated into boar spermatozoa without affecting their binding affinity to uterine epithelial cells, and their subsequent use in a biophotonic sperm binding assay. Further optimization and experimentations are ongoing to establish whether bioluminescent quantum sperm could serve to develop sensitive in vitro binding assays to better characterise sperm viability. Support was provided by U.S. Department of Agriculture Agricultural Research Service (USDA-ARS) grant number 58-6402-3-0120