Petrunkina A M, Jebe E, Töpfer-Petersen E
Institute for Reproductive Medicine, School of Veterinary Medicine Hannover, Foundation, Hannover, Germany.
J Cell Physiol. 2005 Aug;204(2):508-21. doi: 10.1002/jcp.20317.
Spermatozoa of many species initially respond to hypotonicity as perfect osmometers. Thereafter they undergo a regulatory process resulting in a decrease in cell volume, similar to that reported for somatic cells. Regulatory volume increase (RVI), a complementary process which is assumed to occur following initial shrinkage of sperm volume after exposure to a hypertonic medium, has not yet been described in detail for spermatozoa. In this study, we investigated whether spermatozoa are able to regulate their volume after hypertonic stress and whether this ability is maintained in preserved sperm. Cell volume changes were recorded using electronic cell sizing. Sperm response to the ion channels blockers quinidine, tamoxifen, and dydeoxyforskolin, and to protein kinase/phosphatase inhibitors lavendustin, staurosporine, and vanadate was studied to investigate possible mechanisms of RVI. Annexin V staining was used in combination with propidium iodide to determine whether hypertonic stress may induce apoptosis. Overall protein tyrosine phosphorylation under hypertonic conditions was measured via flow cytometry using antiphosphotyrosine antibody. Spermatozoa exposed to hypertonic stress initially responded with an abundant subpopulation according to the perfect osmometer model and recovered their volume from this shrinkage after 20 min. RVI was inhibited by quinidine and tamoxifen, which indicates the involvement of the important cellular ions sodium and chloride in this process. Volume regulatory ability was essentially maintained during storage of liquid semen. However, the response of the sperm population was heterogeneous. A second population raised, containing spermatozoa with larger volumes, which demonstrated irregularities in the volume response with respect to osmotic challenge, ion channel blockers, and storage. Under hypertonic conditions, both protein kinase inhibitors (PKI) led to increased isotonic volumes and to elevated initial relative volumes and subsequent volume decrease. RVI was inhibited by the vanadate. Hypertonic stress did not result in an increase in early apoptotic cells, but produced a shift toward late necrotic cells. Substitution of sodium and chloride by choline and sulfate resulted in decreased isotonic volume of sperm treated with lavendustin. Tyrosine phosphorylation levels were reduced after 20 min under hypertonic conditions. It was concluded that RVI is regulated via a protein tyrosine kinase-dependent pathway, and that dephosphorylation occurs when volume regulation is required. The necrotic volume increase (NVI) is associated with the accumulation of sodium and chloride following uncontrolled opening of the channels. The ability to regulate volume after exposure to hypertonic conditions is important for sperm functionality and can have practical applications in spermatological diagnostics and cryopreservation.
许多物种的精子最初作为完美的渗透计对低渗性作出反应。此后，它们会经历一个调节过程，导致细胞体积减小，这与体细胞的情况类似。调节性容积增加（RVI）是一个补充过程，假定在精子暴露于高渗介质后体积最初收缩之后发生，目前尚未针对精子进行详细描述。在本研究中，我们调查了精子在高渗应激后是否能够调节其体积，以及这种能力在保存的精子中是否得以维持。使用电子细胞大小测量记录细胞体积变化。研究了精子对离子通道阻滞剂奎尼丁、他莫昔芬和双脱氧佛司可林，以及对蛋白激酶/磷酸酶抑制剂拉文达ustin、星形孢菌素和钒酸盐的反应，以研究RVI的可能机制。膜联蛋白V染色与碘化丙啶联合使用，以确定高渗应激是否可能诱导细胞凋亡。使用抗磷酸酪氨酸抗体通过流式细胞术测量高渗条件下的总体蛋白酪氨酸磷酸化。暴露于高渗应激的精子最初根据完美渗透计模型以大量亚群作出反应，并在20分钟后从这种收缩中恢复其体积。RVI被奎尼丁和他莫昔芬抑制，这表明重要的细胞离子钠和氯参与了这一过程。在液体精液储存期间，容积调节能力基本得以维持。然而，精子群体的反应是异质性的。出现了第二个群体，其中包含体积较大的精子，这些精子在体积对渗透压挑战、离子通道阻滞剂和储存的反应方面表现出不规则性。在高渗条件下，两种蛋白激酶抑制剂（PKI）均导致等渗体积增加以及初始相对体积升高和随后的体积减小。RVI被钒酸盐抑制。高渗应激并未导致早期凋亡细胞增加，但导致向晚期坏死细胞的转变。用胆碱和硫酸盐替代钠和氯导致用拉文达ustin处理的精子等渗体积减小。在高渗条件下20分钟后酪氨酸磷酸化水平降低。得出的结论是，RVI通过蛋白酪氨酸激酶依赖性途径调节，并且在需要体积调节时发生去磷酸化。坏死性容积增加（NVI）与通道不受控制地开放后钠和氯的积累有关。暴露于高渗条件后调节体积的能力对精子功能很重要，并且在精子学诊断和冷冻保存中可能具有实际应用价值。
