Capacitation-like alterations in cooled boar spermatozoa: assessment by the chlortetracycline staining assay and immunodetection of tyrosine-phosphorylated sperm proteins
Capacitation-like alterations in cooled boar spermatozoa: assessment by the chlortetracycline staining assay and immunodetection of tyrosine-phosphorylated sperm proteins
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DOI：
10.1016/s0378-4320(02)00128-8
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发表时间：
2002-10-16
影响因子：
2.2
通讯作者：
Kato, S
中科院分区：
文献类型：
--
作者：
Kaneto, M;Harayama, H;Kato, S
本研究的目的是通过金霉素（CTC）染色试验和酪氨酸磷酸化精子蛋白的免疫检测来表征冷却公猪精子中发生的变化。从10头成年公猪采集精子，洗涤后重悬于Tris-柠檬酸-葡萄糖（TCG）溶液中。将精子悬浮液在5小时内缓慢冷却至4 ℃，并保持2天。在冷却处理前后回收精子悬液的等分试样，然后用于CTC染色测定和酪氨酸磷酸化精子蛋白的免疫检测。在冷却处理之前，几乎所有用CTC染色的精子的特征在于整个头部上的均匀荧光（F模式：未获能的精子）。然而，在冷却处理后，显著更高百分比的精子表现出B模式，在顶体后区域有一条荧光减弱的暗带，而在顶体区域有一条相对明亮的荧光（获能精子的模式）。与此同时，在精子中出现了一个32 kDa的酪氨酸磷酸化蛋白。然而，通过向精子悬浮液中补充精浆（20%（v/v）），可减弱冷却精子中发生的这些变化。此外，在获能支持培养基（改良的Krebs-Ringer碳酸氢盐; mKRB）中孵育5小时的精子中也观察到了相同的变化。这些结果表明，冷却可以诱导获能样改变与32 kDa的精子蛋白的酪氨酸磷酸化的猪精子。(C)2002 Elsevier Science B. V.保留所有权利。
This study was undertaken in order to characterize alterations occurring in cooled boar spermatozoa by a chlortetracycline (CTC) staining assay and immunodetection of tyrosine-phosphorylated sperm proteins. Spermatozoa were collected from 10 mature boars, washed and then resuspended in a Tris-citric acid-glucose (TCG) solution. The sperm suspensions were slowly cooled to 4 degreesC over 5 h and held for 2 days. Aliquots of the sperm suspensions were recovered before and after the cooling treatment and then used for the CTC staining assay and immunodetection of tyrosine-phosphorylated sperm proteins. Before the cooling treatment, almost all of the spermatozoa stained with CTC were characterized by uniform fluorescence over the whole head (an F pattern: uncapacitated spermatozoa). After the cooling treatment, however, significant higher percentages of spermatozoa exhibited a B pattern with a dark band of diminished fluorescence in the post acrosomal region and a relatively bright fluorescence in the acrosomal region (the pattern of capacitated spermatozoa). Coincidently, a 32 kDa tyrosine-phosphorylated protein appeared in the spermatozoa. However, these alterations occurring in the cooled spermatozoa were attenuated by the supplementation to the sperm suspensions with seminal plasma (20% (v/v)). Additionally, the same alterations were observed in the spermatozoa incubated in a capacitation-supporting medium (a modified Krebs-Ringer bicarbonate; mKRB) for 5 h. These results suggest that cooling could induce capacitation-like alterations in boar spermatozoa that were associated with the tyrosine phosphorylation of the 32 kDa sperm protein. (C) 2002 Elsevier Science B.V. All rights reserved.