Expression and localization of five members of the testis-specific serine kinase (Tssk) family in mouse and human sperm and testis
Expression and localization of five members of the testis-specific serine kinase (Tssk) family in mouse and human sperm and testis
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DOI：
10.1093/molehr/gaq071
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发表时间：
2011-01-01
影响因子：
4
通讯作者：
Salicioni, Ana M.
中科院分区：
文献类型：
--
作者：
Li, Yahui;Sosnik, Julian;Salicioni, Ana M.
睾丸特异性丝氨酸/苏氨酸激酶(TSSK)家族的成员可能在睾丸中的精子分化和/或受精中起作用。为了深入了解这些激酶的功能相关性，我们在mRNA和蛋白质水平上检测了它们的表达。定量聚合酶链式反应分析证实，所有5个tssk的mRNAs几乎都是在减数分裂后的睾丸中表达。克隆了重组鼠和人的Tssk，并用于验证一系列商用和定制的Tssk抗体。免疫组织化学结果表明，Tssk1、Tssk2、Tssk4和Tssk6存在于小鼠精子和小鼠睾丸生殖细胞中，而Tssk3不存在。在人类精子中也检测到TSSK1、TSSK2和TSSK6，而TSSK3缺失。在小鼠和人类精子中，Tssk1是部分可溶的，而Tssk2、Tssk4和Tssk6在非离子洗涤剂中不溶。体外重组TSSK2活性测定表明，在5 mM的镁离子浓度和Km为10mM时，Tssk2的活性最高。结合Tssk1/Tssk2双基因敲除小鼠和Tssk6基因缺失的小鼠不育的结果，表明这些酶可以作为男性避孕的靶标。
Members of the testis-specific serine/threonine kinases (Tssk) family may have a role in sperm differentiation in the testis and/or fertilization. To gain insight into the functional relevance of these kinases, their expression was examined both at the mRNA and protein levels. Quantitative PCR analysis confirmed that all five Tssk mRNAs are almost exclusively expressed postmeiotically in the testis. Recombinant mouse and human Tssks were cloned and used for validation of an array of commercial and custom-made antibodies against Tssks. Immunolocalization in mouse testis, and in mouse and human sperm, showed that Tssk1, Tssk2, Tssk4 and Tssk6, but not Tssk3, were present in mouse sperm and in germ cells from mouse testis. TSSK1, TSSK2 and TSSK6 were also detected in human sperm, while TSSK3 was absent. In both mouse and human sperm, Tssk1 was partially soluble, while Tssk2, Tssk4 and Tssk6 were insoluble in non-ionic detergents. In vitro recombinant TSSK2 activity assays showed maximum enzymatic activity at 5 mM Mg2+ and a Km for ATP of similar to 10 mu M. These, observations together with findings that the Tssk1/Tssk2 double knock-out as well as the Tssk6 null mice are sterile without presenting other detectable defects, suggest that these kinases could be used as targets for male contraception.