Department of Botany, University of Alberta, Edmonton, Alberta, T6G 2E9 Canada.
Plant Physiol. 1992 Sep;100(1):47-53. doi: 10.1104/pp.100.1.47.
Our previous studies showed that the common maize (Zea mays L.) sperm isolation medium (Brewbaker and Kwack salts in 0.44 m sucrose without buffering) caused cell lysis in vitro. In an attempt to remedy this situation, 6 sugars, 10 buffers, 5 pH values, and 3 membrane protective agents were screened to improve longevity and viability of isolated Zea mays sperm cells as estimated by hemacytometry and flow cytometry. Use of 0.55 m galactose in the isolation solution increased sperm yield by 2.5-fold compared with sucrose, and suspension of isolated sperm cells in the galactose solution gave the best longevity among the six sugars. Buffering the galactose solution with 2 mm 2-(N-morpholino)ethanesulfonic acid significantly improved longevity, whereas other buffers had no effect or decreased the longevity and/or viability. Among the five pH values tested (5.0, 6.0, 6.7, 7.0, and 8.0), pH 6.7 appeared to be optimal for maintenance of both longevity and viability. Screening of membrane protectants showed that cysteine caused a rapid decrease in cell viability and increased lysis, whereas dithiothreitol increased the cell numbers but lowered their viability. Addition of 0.1% bovine serum albumin increased cell numbers and viability, and about 70% of the cells remained viable after 72 h of suspension. Cell longevity and viability were also improved in 0.44 m sucrose when the solution was conditioned with 2-(N-morpholino)ethanesulfonic acid and bovine serum albumin. Use of 2-(N-morpholino)ethanesulfonic acid and bovine serum albumin inthe isolation and suspension medium significantly improved the viability and longevity of sperm cells isolated from Zea mays pollen.
我们之前的研究表明，常用的玉米（Zea mays L.）精子分离介质（Brewbaker 和 Kwack 盐在 0.44 m 蔗糖中，无缓冲液）在体外会导致细胞裂解。为了改善这种情况，我们筛选了 6 种糖、10 种缓冲液、5 种 pH 值和 3 种膜保护剂，以通过血细胞计数和流式细胞术来提高分离的玉米精子细胞的活力和存活率。与蔗糖相比，在分离溶液中使用 0.55 m 半乳糖可使精子产量增加 2.5 倍，并且将分离的精子细胞悬浮在半乳糖溶液中可获得六种糖中最长的寿命。用 2 mM 2-（N-吗啉基）乙磺酸缓冲半乳糖溶液可显著提高精子活力，而其他缓冲液则没有效果或降低了精子活力和/或存活率。在测试的 5 个 pH 值（5.0、6.0、6.7、7.0 和 8.0）中，pH 6.7 似乎最有利于维持精子的活力和存活率。膜保护剂的筛选表明，半胱氨酸会导致细胞活力迅速下降和细胞裂解增加，而二硫苏糖醇则增加了细胞数量，但降低了其活力。添加 0.1%牛血清白蛋白可增加细胞数量和活力，并且在悬浮 72 小时后，约 70%的细胞仍保持活力。在 0.44 m 蔗糖溶液中添加 2-（N-吗啉基）乙磺酸和牛血清白蛋白也可改善溶液的条件，从而提高精子活力和存活率。在分离和悬浮介质中使用 2-（N-吗啉基）乙磺酸和牛血清白蛋白可显著提高从玉米花粉中分离出的精子细胞的活力和寿命。
