Simpson A M, White I G
Department of Veterinary Physiology, University of Sydney, Australia.
Cell Calcium. 1988 Feb;9(1):45-56. doi: 10.1016/0143-4160(88)90037-1.
The highly selective fluorescent Ca2+ indicator 'quin 2' has been loaded into ram and boar spermatozoa as the acetoxymethyl ester, 'quin 2/AM', which is hydrolysed and trapped in the cytoplasm. Loadings of several mM were not toxic to spermatozoa as judged by motility. Fluorescence measurements (mean +/- S.E.M.) indicated a normal cytoplasmic free-calcium concentration, [Ca2+]i, of 193 nM +/- 0.2 (n = 10) for ejaculated ram sperm, 175 nM +/- 3.9 (n = 10) for cauda epididymal boar sperm and 105 nM +/- 10 (n = 10) for the caput sperm. After cold shock ejaculated ram and cauda epididymal boar sperm did not retain quin 2, due presumably to structural damage. However, cold shocked caput boar sperm could be readily loaded with quin 2 and had a [Ca2+]i similar to control sperm. Sodium azide, propranolol and caffeine did not affect the [Ca2+]i of ram and boar sperm, however theophylline, dibutyryl c-AMP and La3+ significantly reduced it. The inhibitors rotenone and antimycin A, and the uncouplers 2,4-DNP and CCCP caused a transient elevation of [Ca2+]i, most likely resulting from release of mitochondrial calcium. The increased [Ca2+]i following addition of the ionophore A23187, was highly pH dependent in ram spermatozoa and it was critical to increase the pH of the medium above 7.5; the increase in [Ca2+]i was apparently not dependent on the oxidative metabolism of the sperm as addition of the uncouplers 2,4-DNP and CCCP had no effect on [Ca2+ )i. Addition of filipin to ram and boar sperm resulted in a large increase in [Ca2+]i but addition of filipin to ionophore-treated sperm caused [Ca2+]i to fall well below control levels.
高选择性荧光钙离子指示剂“喹啉 2”以乙酰氧基甲酯形式（“喹啉 2/AM”）被加载到公羊和公猪精子中，它在细胞质中水解并被捕获。通过活力判断，几毫摩尔的加载量对精子无毒。荧光测量（平均值±标准误）表明，射精公羊精子的正常细胞质游离钙浓度[Ca2+]i 为 193 nM±0.2（n = 10），附睾尾部公猪精子为 175 nM±3.9（n = 10），附睾头部精子为 105 nM±10（n = 10）。冷休克后，射精公羊和附睾尾部公猪精子无法保留喹啉 2，推测是由于结构损伤。然而，冷休克的附睾头部公猪精子可以很容易地加载喹啉 2，且其[Ca2+]i 与对照精子相似。叠氮化钠、普萘洛尔和咖啡因不影响公羊和公猪精子的[Ca2+]i，但茶碱、二丁酰 c - AMP 和 La3+显著降低了它。抑制剂鱼藤酮和抗霉素 A，以及解偶联剂 2,4 - DNP 和 CCCP 导致[Ca2+]i 短暂升高，最可能是由于线粒体钙释放。添加离子载体 A23187 后[Ca2+]i 的增加在公羊精子中高度依赖于 pH，将培养基 pH 提高到 7.5 以上至关重要；[Ca2+]i 的增加显然不依赖于精子的氧化代谢，因为添加解偶联剂 2,4 - DNP 和 CCCP 对[Ca2+]i 没有影响。向公羊和公猪精子中添加制霉菌素会导致[Ca2+]i 大幅增加，但向离子载体处理的精子中添加制霉菌素会使[Ca2+]i 降至对照水平以下。
