McClean R, MacCallum C, Blyde D, Holt W, Johnston S
School of Animal Studies, University of Queensland, Gatton, Australia.
Cryo Letters. 2006 Jul-Aug;27(4):253-60.
This study examined the hypothesis that filamentous actin associated with the complex cytoskeleton of the kangaroo sperm head and tail may be contributing to lack of plasma membrane plasticity and a consequent loss of membrane integrity during cryopreservation. In the first study, the distribution of G and F actin within Eastern Grey Kangaroo (EGK, Macropus giganteus) cauda epididymidal spermatozoa was successfully detected using DNAse-FITC and a monoclonal F-actin antibody (ab205, Abcam), respectively. G-actin staining was most intense in the acrosome but was also observed with less intensity over the nucleus and mid-piece. F-actin was located in the sperm nucleus but was not discernable in the acrosome or sperm tail. To investigate whether cytochalasin D (a known F-actin depolymerising agent) was capable of improving the osmotic tolerance of EGK cauda epididymal spermatozoa, sperm were incubated in hypo-osmotic media (61 and 104 mOsm) containing a range of cytochalasin D concentrations (0-200 microM). Cytochalasin D had no beneficial effect on plasma membrane integrity of sperm incubated in hypo-osmotic media. However, when EGK cauda epididymidal sperm were incubated in isosmotic media, there was a progressive loss of sperm motility with increasing cytochalasin D concentration. The results of this study indicated that the F-actin distribution in cauda epididymidal spermatozoa of the EGK was surprisingly different from that of the Tammar Wallaby (M. eugenii) and that cytochalasin-D does not appear to improve the tolerance of EGK cauda epididymidal sperm to osmotically induced injury.
本研究检验了一个假设，即与袋鼠精子头部和尾部复杂细胞骨架相关的丝状肌动蛋白可能导致冷冻保存过程中质膜可塑性的缺乏以及随之而来的膜完整性丧失。在第一项研究中，分别使用DNA酶-FITC和单克隆F-肌动蛋白抗体（ab205，Abcam）成功检测了东部灰袋鼠（EGK，大袋鼠）附睾尾精子中G-肌动蛋白和F-肌动蛋白的分布。G-肌动蛋白染色在顶体中最为强烈，但在细胞核和中段也有较弱的染色。F-肌动蛋白位于精子细胞核中，但在顶体或精子尾部无法辨别。为了研究细胞松弛素D（一种已知的F-肌动蛋白解聚剂）是否能够提高EGK附睾尾精子的渗透耐受性，将精子在含有一系列细胞松弛素D浓度（0-200 microM）的低渗培养基（61和104 mOsm）中孵育。细胞松弛素D对在低渗培养基中孵育的精子的质膜完整性没有有益影响。然而，当EGK附睾尾精子在等渗培养基中孵育时，随着细胞松弛素D浓度的增加，精子活力逐渐丧失。本研究结果表明，EGK附睾尾精子中F-肌动蛋白的分布与帚尾袋貂（M. eugenii）的分布惊人地不同，并且细胞松弛素D似乎不能提高EGK附睾尾精子对渗透压诱导损伤的耐受性。
