Livestock Improvement Corporation, Hamilton, New Zealand.
Theriogenology. 2012 Oct 15;78(7):1476-86. doi: 10.1016/j.theriogenology.2012.06.021. Epub 2012 Aug 24.
Head-to-head agglutination of bull sperm occurs when semen is highly diluted in an egg yolk-citrate diluent without streptomycin. The objectives were to investigate causes of sperm agglutination and the underlying mechanism. Aliquots of bull semen were diluted in a base diluent (BD) supplemented with various test components and the percentage of agglutinated sperm (% AggSp) was quantified at 1, 5, 24, 48, and 72 h of incubation. When sperm were incubated at 22 °C, no agglutination was observed in BD for up to 72 h, whereas the % AggSp was 5.0, 41.7, 72.2, 91.1, and 92.8% in BD + 5% egg yolk (BD + EY) at 1, 5, 24, 48 and 72 h, respectively. However, no sperm agglutination was observed in BD + EY if incubation temperature was 37 °C. Addition of 5 or 10 mM ethylenebis (oxyethyleneni-trilo) tetra-acetic acid to BD + EY reduced the % AggSp from 95% to <5% at 72 h (P < 0.001), but addition of 5 mM CaCl(2) to BD failed to induce sperm agglutination in the absence of egg yolk, implicating calcium and other factors in egg yolk. Addition of the citrate-soluble fraction (CSF) of egg yolk to BD induced sperm agglutination similar to whole egg yolk, whereas water- and saline-soluble fractions of egg yolk were ineffective. The sperm-agglutinating efficacy of CSF (the % AggSp = 95% at 72 h) was reduced by dialysis (20%; P < 0.05), partially restored by addition of 5 mM CaCl2 (70%; P < 0.05), but the calcium effect was neutralized by addition of 5 mM ethylenebis (oxyethyleneni-trilo) tetra-acetic acid (1.7%; P < 0.05), again implicating calcium. Addition of 30 μM of a protein kinase A inhibitor (H-89) to an agglutinating diluent failed to inhibit sperm agglutination, whereas addition of 2 mM of a cAMP analogue, dbcAMP, to a nonagglutinating diluent failed to induce sperm agglutination. Agglutination status had no effect on sperm plasma membrane/acrosome status and mitochondrial membrane potential. In conclusion, calcium and other component(s) in the CSF of egg yolk induced head-to-head agglutination of bull sperm in a time- and temperature-dependent manner. Although the mechanism of agglutination was not determined, the cAMP- protein kinase A signaling pathway was not involved.
当精液在没有链霉素的蛋黄-柠檬酸盐稀释液中高度稀释时，会发生公牛精子的头对头聚集。目的是研究精子聚集的原因和潜在机制。将公牛精液等分试样稀释在基础稀释液（BD）中，添加各种测试成分，并在孵育 1、5、24、48 和 72 小时时定量测定聚集的精子百分比（% AggSp）。当精子在 22°C 下孵育时，BD 在 72 小时内不会发生聚集，而在 BD+5%蛋黄（BD+EY）中，1、5、24、48 和 72 小时的% AggSp 分别为 5.0、41.7、72.2、91.1 和 92.8%。然而，如果孵育温度为 37°C，则在 BD+EY 中不会观察到精子聚集。向 BD+EY 中添加 5 或 10 mM 乙二胺四乙酸（EDTA）可将 72 小时时的% AggSp 从 95%降低至<5%（P<0.001），但在没有蛋黄的情况下，向 BD 中添加 5 mM CaCl2 不会诱导精子聚集，这表明钙和蛋黄中的其他因素参与了精子聚集。向 BD 中添加蛋黄的可溶柠檬酸部分（CSF）可诱导类似于全蛋黄的精子聚集，而蛋黄的水溶和盐溶部分则无效。CSF（72 小时时的% AggSp=95%）的精子聚集效力通过透析（20%；P<0.05）降低，部分通过添加 5 mM CaCl2 恢复（70%；P<0.05），但添加 5 mM EDTA 会中和钙的作用（1.7%；P<0.05），再次表明钙的作用。向聚集稀释液中添加 30 μM 蛋白激酶 A 抑制剂（H-89）不能抑制精子聚集，而向非聚集稀释液中添加 2 mM cAMP 类似物 dbcAMP 不能诱导精子聚集。聚集状态对精子质膜/顶体状态和线粒体膜电位没有影响。总之，蛋黄 CSF 中的钙和其他成分以时间和温度依赖的方式诱导公牛精子的头对头聚集。虽然聚集的机制尚未确定，但 cAMP-蛋白激酶 A 信号通路不参与其中。
