Miller D J, Demers J M, Braundmeier A G, Behrens M L
Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana 61801, USA.
J Androl. 1998 Nov-Dec;19(6):650-6.
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 microM of the two dyes, DiQ (4-[4-(dihexadecylamino)styryl]-N-methylquinolinium iodide; an orange-red dye) and DiOC16 (3,3'-dihexadecyloxacarbocyanine 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 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 sperm-oocyte binding assays. This assay may allow more accurate prediction of the oocyte-binding ability of sperm.
大多数精子实验室检测与男性生育能力之间的关系并不一致。预计那些测量使卵母细胞受精所需特性的检测具有最大的预测价值。一种新的检测方法被开发出来，用于测量两个精子样本与卵母细胞结合的竞争能力。使用一对亲脂性染料对两群精子进行标记。75微摩尔浓度的两种染料，即DiQ（4-[4-(二十六烷基氨基)苯乙烯基]-N-甲基喹啉碘化物；一种橙红色染料）和DiOC16（3,3'-二十六烷基氧杂羰花青高氯酸盐；一种黄绿色染料），分别使66%和73%的精子强烈染色，且不影响精子活力或与卵母细胞结合的能力。由于精子可用荧光染料染色，将来自两个精液样本的精子在一滴中混合，然后加入卵母细胞，使精子竞争性地结合卵母细胞。对每个样本中与卵母细胞结合的精子进行计数。结合是特异性的；非特异性精子结合通过与二细胞小鼠胚胎结合的精子来估计，每个胚胎平均有一到三个精子。用DiQ或DiOC16染色不影响与卵母细胞结合的能力，因为超过80%结合的精子被其中任何一种染料染色。此外，如果在液滴中制备来自同一射精样本的不同比例的DiQ或DiOC16染色精子，并加入卵母细胞，与卵母细胞结合的精子百分比反映了液滴中精子的百分比；两种染料均无差异效应。该检测使用固定的卵母细胞，因为精子与固定的和新鲜的牛卵母细胞结合程度相同。这种竞争性卵母细胞结合检测方法允许在一组雄性之间进行一系列成对比较，或在精子-卵母细胞结合检测中纳入内部对照样本。这种检测方法可能会更准确地预测精子与卵母细胞结合的能力。
