Kumaresan A, Siqueira A P, Hossain M S, Johannisson A, Eriksson I, Wallgren M, Bergqvist A S
Division of Reproduction, Department of Clinical Sciences, Swedish University of Agricultural Sciences, SLU, PO Box 7054, Uppsala SE-750 07, Sweden.
Reprod Fertil Dev. 2012;24(4):531-42. doi: 10.1071/RD11074.
Protein tyrosine phosphorylation in sperm is associated with capacitation in several mammalian species. Although tyrosine phosphorylated proteins have been demonstrated in cryopreserved sperm, indicating capacitation-like changes during cryopreservation, these changes have not yet been quantified objectively. We monitored tyrosine phosphorylation, intracellular calcium and sperm kinematics throughout the cryopreservation process, and studied the relationships among them in boar spermatozoa. Sperm kinetics changed significantly during cryopreservation: curvilinear velocity, average path velocity and straight line velocity all decreased significantly (P < 0.05). While the percentage of sperm with high intracellular calcium declined (P < 0.05), global phosphorylation increased significantly (P < 0.01). Specifically, cooling to 5 °C induced phosphorylation in the spermatozoa. After cooling, a 32-kDa protein not observed in fresh semen appeared and was consistently present throughout the cryopreservation process. While the level of expression of this phosphoprotein decreased after addition of the second extender, frozen-thawed spermatozoa showed an increased expression. The proportion of sperm cells with phosphorylation in the acrosomal area also increased significantly (P < 0.05) during cryopreservation, indicating that phosphorylation might be associated with capacitation-like changes. These results provide the first quantitative evidence of dynamic changes in the subpopulation of boar spermatozoa undergoing tyrosine phosphorylation during cryopreservation.
在几种哺乳动物中，精子中的蛋白质酪氨酸磷酸化与获能相关。尽管在冷冻保存的精子中已证实存在酪氨酸磷酸化蛋白，这表明在冷冻保存过程中发生了类似获能的变化，但这些变化尚未得到客观量化。我们在整个冷冻保存过程中监测了公猪精子的酪氨酸磷酸化、细胞内钙和精子运动学，并研究了它们之间的关系。在冷冻保存过程中，精子运动学发生了显著变化：曲线速度、平均路径速度和直线速度均显著降低（P < 0.05）。虽然细胞内钙含量高的精子百分比下降（P < 0.05），但整体磷酸化显著增加（P < 0.01）。具体而言，冷却至5°C会诱导精子发生磷酸化。冷却后，在新鲜精液中未观察到的一种32 kDa蛋白质出现，并在整个冷冻保存过程中持续存在。虽然在添加第二种稀释液后这种磷蛋白的表达水平下降，但冻融精子的表达增加。在冷冻保存过程中，顶体区域发生磷酸化的精子细胞比例也显著增加（P < 0.05），这表明磷酸化可能与类似获能的变化有关。这些结果首次提供了公猪精子在冷冻保存过程中酪氨酸磷酸化亚群动态变化的定量证据。
