Zilli L, Schiavone R, Storelli C, Vilella S
Department of Biological and Environmental Sciences and Technologies, Laboratory of Comparative Physiology, University of Salento, Via Provinciale Lecce-Monteroni, 73100 Lecce, Italy.
Cryobiology. 2008 Oct;57(2):150-5. doi: 10.1016/j.cryobiol.2008.07.006. Epub 2008 Jul 25.
We have previously demonstrated that in sea bream Sparus aurata motility initiation determined changes in the phosphorylation state of some proteins. This paper describes an investigation of the effect of a freezing-thawing procedure on the protein phosphorylation/dephosphorylation pattern. Proteins extracted from fresh and cryopreserved spermatozoa (before and after motility activation) were separated on SDS-PAGE, blotted on nitrocellulose membrane and treated with anti-phosphotyrosine, anti-phosphothreonine, or anti-phosphoserine antibodies. The results obtained demonstrate that the cryopreservation protocol has a strong effect on the phosphorylation state of proteins. In general, compared to fresh sperm, phosphorylated proteins are most numerous in both activated and non-activated cryopreserved sperm, and in particular we observed a dramatic increase in threonine phosphorylation. However, frozen-thawed sperm showed a minor number of proteins that changed their phosphorylation state after motility activation. Among these, we identified the acetyl-coenzyme A synthetase that plays a role in sperm motility initiation in both fresh and cryopreserved sperm.
我们之前已经证明，在金头鲷中，运动启动决定了一些蛋白质磷酸化状态的变化。本文描述了一项关于冻融程序对蛋白质磷酸化/去磷酸化模式影响的研究。从新鲜和冷冻保存的精子（运动激活前后）中提取的蛋白质在SDS-PAGE上分离，印迹到硝酸纤维素膜上，并用抗磷酸酪氨酸、抗磷酸苏氨酸或抗磷酸丝氨酸抗体处理。获得的结果表明，冷冻保存方案对蛋白质的磷酸化状态有很强的影响。一般来说，与新鲜精子相比，在激活和未激活的冷冻保存精子中磷酸化蛋白质数量最多，特别是我们观察到苏氨酸磷酸化显著增加。然而，冻融精子在运动激活后显示出少量改变其磷酸化状态的蛋白质。其中，我们鉴定出了在新鲜和冷冻保存精子的运动启动中都起作用的乙酰辅酶A合成酶。
