METHOD FOR FRACTIONATION AND PURIFICATION OF SEA-URCHIN SPERM
METHOD FOR FRACTIONATION AND PURIFICATION OF SEA-URCHIN SPERM
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DOI：
10.1111/j.1440-169x.1973.00255.x
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发表时间：
1973-01-01
影响因子：
2.5
通讯作者：
HARRIS, FL
中科院分区：
文献类型：
--
作者：
ATHERTON, RW;HARRIS, FL
研究了海胆精子亚基的分离纯化方法。当使用经典的超声处理或均质化技术时，观察到细胞的碎片化而不是细胞亚基的分级分离。酒精溶液中的钙冲击能有效去除精子纤毛，但对精子鞭毛的去除效果不明显。发现酒精诱导整个精子之间的凝聚力，从而不可能通过离心分离孤立的亚基。无酒精的钙休克不能有效分离精子亚基，通过在0.22 μ m过滤的无钙培养基中应用逐步涡旋剪切作用，成功分离精子亚基。Nomarski光学分析表明细胞碎片很少或没有。用各种不连续的蔗糖-海水溶液进行纯化被证明是一种成功的纯化分级分离的头部、中段和尾部的技术。本文报道的程序可用于获得分级分离和纯化的精子头部、中段和尾部。这些方法不需要对材料进行酶、均质化或超声处理。
Procedures for fractionation and purification of sea urchin sperm subunits were studied. Fragmentation of cells was observed rather than fractionation of cell subunits when classic sonication or homogenization techniques were used. Calcium shock in alcohol solution which successfully removes Protozoa cilia was not effective in removing sperm flagella. Alcohol was found to induce cohesion among whole sperm rendering separation of isolated subunits by centrifugation impossible. Calcium shock without alcohol in the medium was not effective in achieving dissociation of sperm subunits.Successful fractionation of sperm subunits was achieved by application of a stepwise vortex‐shearing action in a 0.22 micron filtered calcium‐free medium. Analysis by Nomarski optics indicated little or no cellular fragmentation. Centrifugation with various discontinuous sucrose‐sea water solutions proved to be a successful technique to purify the fractionated heads, midpieces, and tails.The procedures reported here can be used to obtain fractionated and purified sperm cell heads, midpieces, and tails. These procedures do not require enzyme, homogenization, or sonication treatment of the material.