Relationship of Protein Tyrosine Phosphorylation State With Tolerance to Frozen Storage and the Potential to Undergo Cyclic AMP-Dependent Hyperactivation in the Spermatozoa of Japanese Black Bulls
Relationship of Protein Tyrosine Phosphorylation State With Tolerance to Frozen Storage and the Potential to Undergo Cyclic AMP-Dependent Hyperactivation in the Spermatozoa of Japanese Black Bulls
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DOI：
10.1002/mrd.21233
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发表时间：
2010-10-01
影响因子：
2.5
通讯作者：
Fukushima, Moriyuki
中科院分区：
文献类型：
--
作者：
Harayama, Hiroshi;Nishijima, Kazuhiro;Fukushima, Moriyuki
本研究的目的是阐明蛋白酪氨酸磷酸化状态与冷冻保存的日本黑牛精子的精子特性之间的关系。用含有聚乙烯醇的PBS洗涤精子，然后与细胞可渗透的cAMP类似物cBiMPS孵育以诱导鞭毛超活化。孵育前后，精子用于酪氨酸磷酸化蛋白的免疫检测，顶体形态学状态的评估和运动的评估。在公牛的冷冻储存的精子被归类为具有高品位的顶体条件孵育前，精子酪氨酸磷酸化蛋白，包括33 kDa的酪氨酸磷酸化SPACA 1蛋白，被定位在顶体和赤道亚段的前部区域。41-和33-kDa的精子酪氨酸磷酸化蛋白质的免疫检测水平的蛋白质印迹和酪氨酸磷酸化蛋白质和SPACA 1蛋白质的免疫荧光在精子顶体的前部区域的公牛，其冷冻储存的精子被归类为具有低级顶体条件较低。另一方面，在与cBiMPS孵育后，至少10个酪氨酸磷酸化蛋白的免疫检测水平在精子的连接和主要部分中增加，与鞭毛过度激活的诱导一致。许多精子也表现出类似的检测模式，公猪超活化精子。这些结果是一致的建议，酪氨酸磷酸化蛋白的免疫检测水平是有效的标志物，可以预测的水平的耐受性冷冻储存和潜在的经历cAMP依赖性超活化的精子的个人日本黑牛。
The aim of this study was to elucidate the relationship between protein tyrosine phosphorylation state and sperm characteristics in frozen-stored spermatozoa of Japanese Black bulls. The spermatozoa were washed with PBS containing polyvinyl alcohol and then incubated with cell-permeable cAMP analog cBiMPS to induce flagellar hyperactivation. Before and after incubation, the spermatozoa were used for immunodetection of tyrosine-phosphorylated proteins, assessment of morphological acrosome condition and evaluation of motility. In bulls whose frozen-stored spermatozoa were classified as having a high-grade acrosome condition before incubation, sperm tyrosine-phosphorylated proteins, including the 33-kDa tyrosine-phosphorylated SPACA1 protein, were localized in the anterior region of the acrosome and equatorial subsegment. The immunodetection level of the 41- and 33-kDa sperm tyrosine-phosphorylated proteins in the Western blots and the immunofluorescence of tyrosine-phosphorylated proteins and SPACA1 proteins in the anterior region of the sperm acrosome were lower in bulls whose frozen-stored sperm were classified as having a low-grade acrosome condition. On the other hand, after incubation with cBiMPS, immunodetection levels of at least 10 tyrosine-phosphorylated proteins increased in the connecting and principal pieces of spermatozoa, coincident with the induction of flagellar hyperactivation. Many of the spermatozoa also exhibited detection patterns similar to those of boar hyperactivated spermatozoa. These results are consistent with the suggestion that immunodetection levels of tyrosine-phosphorylated proteins are valid markers that can predict the level of tolerance to frozen storage and the potential to undergo cAMP-dependent hyperactivation for the spermatozoa of individual Japanese Black bulls.