FLOW CYTOMETRIC STUDIES OF BICARBONATE-MEDIATED CA-2+ INFLUX IN BOAR SPERM POPULATIONS
FLOW CYTOMETRIC STUDIES OF BICARBONATE-MEDIATED CA-2+ INFLUX IN BOAR SPERM POPULATIONS
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DOI：
10.1002/mrd.1080350214
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发表时间：
1993-06-01
影响因子：
2.5
通讯作者：
MILLER, NGA
中科院分区：
文献类型：
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作者：
HARRISON, RAP;MAIRET, B;MILLER, NGA
将载有Ca ~（2+）探针Fluo-3的猪精子在类似于体外受精（IVF）所用的各种台氏培养基中孵育，然后通过双色流式细胞术分析样品;在培养基中加入碘化丙啶以检测膜损伤如果培养基含有碳酸氢盐/CO2，（一种被认为促进获能的成分），部分活精子群体经历了相当大的Ca 2+流入头部和尾部隔室。反应细胞的百分比在约30分钟后达到最大值，但在此期间和之后，死细胞的数量也稳定增加。这种碳酸氢盐介导的细胞死亡增加发生在外部Ca 2+的情况下。有证据表明，碘化丙啶的进入之前的质膜的渗透性的变化，检测泄漏的羧基二氯荧光素，因此推断，钙离子内流检测荧光-3是由于质膜的不稳定。咖啡因和罂粟碱（最著名的磷酸二酯酶抑制剂）都能产生类似的反应，但环腺苷酸和腺苷酸环化酶的激活剂都没有任何作用。底物对该过程没有影响，但是，与聚乙烯醇相比，血清白蛋白增强了该过程。这种去稳定化与获能的确切相关性尚不清楚，但似乎重要的是，该过程由IVF培养基中经常特别包括的组分介导或增强，并且不同的个体细胞在不同时间后响应。
Boar spermatozoa loaded with the Ca2+ probe fluo-3 were incubated in various Tyrode's-based media similar to those used for in vitro fertilization (IVF), and samples were then analysed by two-colour flow cytometry; propidium iodide was included in the media to detect membrane-damaged (''dead'') cells.If media contained bicarbonate/CO2 (a component thought to promote capacitation), part of the live sperm population experienced a considerable influx of Ca2+ into both head and tail compartments. The percentage of responding cells reached a maximum after about 30 min, but both during and after this period there was also a steady increase in the number of dead cells. This bicarbonate-mediated increase in cell death took place in the absence of external Ca2+ . Evidence was obtained that the entry of propidium iodide was preceded by a change in permeability of the plasma membrane, detectable by leakage of carboxydichlorofluorescein, and it was therefore deduced that the Ca2+ influx detected by fluo-3 was due to destabilization of the plasma membrane. A similar response could be produced by both caffeine and papaverine (best known as phosphodiesterase inhibitors), but neither cyclic AMP nor activators of adenylate cyclase had any effect. There was no influence of substrate on the process, but, in comparison to poly(vinyl alcohol), serum albumin enhanced it.The precise relevance of this destabilization to capacitation is not yet clear, but it seems significant that the process is mediated or enhanced by components often specifically included in IVF media, and that different individual cells respond after different times.