Laboratory of Reproductive Biology, Graduate School of Agricultural Science, Kobe University, Kobe, Japan.
Mol Reprod Dev. 2010 Oct;77(10):910-21. doi: 10.1002/mrd.21233.
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.
本研究旨在阐明日本黑牛冷冻储存精子中蛋白酪氨酸磷酸化状态与精子特征之间的关系。精子先用含聚乙烯醇的 PBS 洗涤，然后用细胞通透的 cAMP 类似物 cBiMPS 孵育以诱导鞭毛超激活。孵育前后，使用免疫检测法检测酪氨酸磷酸化蛋白、评估顶体形态和评估运动性。在冷冻储存精子在孵育前被分类为具有高等级顶体状态的公牛中，包括 33 kDa 酪氨酸磷酸化 SPACA1 蛋白在内的精子酪氨酸磷酸化蛋白定位于顶体的前区和赤道亚段。Western blot 中 41 kDa 和 33 kDa 精子酪氨酸磷酸化蛋白的免疫检测水平以及精子顶体前区酪氨酸磷酸化蛋白和 SPACA1 蛋白的免疫荧光在冷冻储存精子被分类为具有低等级顶体状态的公牛中较低。另一方面，用 cBiMPS 孵育后，连接部和主部的至少 10 种酪氨酸磷酸化蛋白的免疫检测水平增加，与鞭毛超激活的诱导一致。许多精子还表现出与猪超激活精子相似的检测模式。这些结果与以下观点一致，即酪氨酸磷酸化蛋白的免疫检测水平是预测个体日本黑牛精子对冷冻储存的耐受水平和对 cAMP 依赖性超激活的潜在能力的有效标志物。
