Mohan R, Atreja S K
Reproductive Biochemistry Laboratory, Animal Biochemistry Division, National Dairy Research Institute, Karnal, Haryana 132001, India.
Cryobiology. 2015 Jun;70(3):253-61. doi: 10.1016/j.cryobiol.2015.03.007. Epub 2015 Mar 30.
Considering the importance of cytochrome c in both life and death, it was of significant interest to investigate the expression of cytochrome c, its tyrosine phosphorylation status and immunolocalization patterns in a frozen-thawed buffalo sperm cell in comparison to in vitro capacitated [heparin (10 μg/ml) induced, for 4h] and stress [apoptotic (10 μM staurosporine), oxidative (25 μM H2O2) and osmotic (180 mM NaCl) for 4h] induced conditions. Proteins were subjected to immunoblotting and probed by using monoclonal anti-phosphotyrosine antibodies. A significant (p<0.05) increase in expression of tyrosine phosphorylated cytochrome c was observed in capacitated buffalo sperm in comparison to frozen-thawed samples. cAMP protein kinase-A dependent and extracellular signal-regulated kinase independent tyrosine phosphorylation of cytochrome c was found during in vitro capacitation of buffalo spermatozoa. Localized increase in cytochrome c and tyrosine phosphorylated proteins were observed in frozen thawed and capacitated sperm. The information generated in this study can be used to understand the molecular mechanism of regulation of an apoptotic protein (cytochrome c) by tyrosine phosphorylation (a capacitation marker) in a frozen thawed sperm cell which could be a good target to combat apoptosis.
鉴于细胞色素c在生死过程中的重要性，相较于体外获能（肝素（10μg/ml）诱导，4小时）以及应激（凋亡（10μM星形孢菌素）、氧化（25μM H2O2）和渗透（180mM NaCl）处理4小时）诱导条件下的水牛精子细胞，研究冻融水牛精子细胞中细胞色素c的表达、其酪氨酸磷酸化状态及免疫定位模式具有重要意义。蛋白质经免疫印迹处理，并用单克隆抗磷酸酪氨酸抗体进行检测。与冻融样本相比，在获能的水牛精子中观察到酪氨酸磷酸化细胞色素c的表达显著（p<0.05）增加。在水牛精子体外获能过程中发现细胞色素c的环磷酸腺苷蛋白激酶 -A依赖性和细胞外信号调节激酶非依赖性酪氨酸磷酸化。在冻融和获能的精子中观察到细胞色素c和酪氨酸磷酸化蛋白的局部增加。本研究中产生的信息可用于理解冻融精子细胞中酪氨酸磷酸化（一种获能标记）对凋亡蛋白（细胞色素c）的调节分子机制，冻融精子细胞可能是对抗细胞凋亡的良好靶点。
