The use of two fluorescent dyes to identify sperm in a competitive bindingassay to oocytes

The relationship of most sperm laboratory assays to male fertility is incon sistent. Assays that measure traits required to fertilize oocytes are expec ted to have the most predictive value. A new assay that measures the compet itive ability oi two sperm samples to bind to oocytes was developed. Two po pulations of sperm were labeled using a pair of lipophilic dyes. A concentr ation of 75 mu M of the two dyes, DiQ (4-[4-(dihexadecylamino)styryl]-N-met hylquinolinium iodide; an orange-red dye) and DiOC(16) (3,3'-dihexadecyloxa carbocyanine perchlorate; a yellow-green dye), intensely stained 66 and 73% of sperm, respectively, without affecting sperm motility or oocyte-binding ability. Because sperm could be stained with fluorescent dyes, sperm from two semen samples were mixed together in a droplet, and oocytes were added to allow sperm to bind oocytes competitively. Oocyte-bound sperm from each sample were counted. Binding was specific; nonspecific sperm binding was es timated by sperm bound to two-cell mouse embryos and averaged one to three sperm per embryo. Staining with DiQ or DiOC(16) did not affect oocyte-bindi ng ability since more than 80% of the sperm bound were stained with either dye. Furthermore, ii different ratios of DiQ- or DiOC(16)-stained sperm fro m the same ejaculate were prepared in droplets and oocytes were added, the percentage of sperm bound to the oocytes reflected the percentage of sperm in the droplet; there was no differential effect of either dye. This assay used fixed oocytes because sperm bound equally to fixed and fresh bovine oo cytes. This competitive oocyte-binding assay allows one to make a series of pairwise comparisons between a group of males or to include an internal co ntrol sample in sperm-oocyte binding assays. This assay may allow more accu rate prediction of the oocyte-binding ability of sperm.