The Use of Two Fluorescent Dyes to Identify Sperm in a Competitive Binding Assay to Oocytes

Abstract

The relationship of most sperm laboratory assays to male fertility is inconsistent. Assays that measure traits required to fertilize oocytes are expected to have the most predictive value. A new assay that measures the competitive ability of two sperm samples to bind to oocytes was developed. Two populations of sperm were labeled using a pair of lipophilic dyes. A concentration of 75 μM of the two dyes, DiQ (4‐[4‐(dihexadecylamino)styryl]‐N‐methylquinolinium iodide; an orangered dye) and DiOC16 (3,3′‐dihexadecyloxacar‐bocyanine 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. Oocytebound sperm from each sample were counted. Binding was specific; nonspecific sperm binding was estimated by sperm bound to two‐cell mouse embryos and averaged one to three sperm per embryo. Staining with DiQ or DiOC16 did not affect oocyte‐binding ability since more than 80% of the sperm bound were stained with either dye. Furthermore, if different ratios of DiQ‐ or DiOC16‐stained sperm from 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 oocytes. This competitive oocyte‐binding assay allows one to make a series of pairwise comparisons between a group of males or to include an internal control sample in spermoocyte binding assays. This assay may allow more accurate prediction of the oocyte‐binding ability of sperm.

Fluorescent labelling of boar spermatozoa for quantitative studies on competitive sperm–oviduct binding

“…Despite their limited ability to stain spermatozoa, lipophilic fluorescent dyes such as 4-[4-(dihexadecylamino)styryl]-Nmethylquinolinium iodide (DiQ) and 3,3 0 -dihexadecyloxacarbocyanine perchlorate (DIOC 16 ) have been used in heterospermic in vitro approaches to rank boars according to their sperm-zona binding capacity (Miller et al 1998;Braundmeier et al 2004). Staining of almost all spermatozoa was achieved only at the expense of inhibiting sperm motility.…”