Cloning and Functional Analysis of Histones H3 and H4 in Nuclear Shaping during Spermatogenesis of the Chinese Mitten Crab, Eriocheir sinensis

During spermatogenesis in most animals, the basic proteins associated with DNA are continuously changing and somatic-typed histones are partly replaced by sperm-specific histones, which are then successively replaced by transition proteins and protamines. With the replacement of sperm nuclear basic proteins, nuclei progressively undergo chromatin condensation. The Chinese Mitten Crab (Eriocheir sinensis) is also known as the hairy crab or river crab (phylum Arthropoda, subphylum Crustacea, order Decapoda, and family Grapsidae). The spermatozoa of this species are aflagellate, and each has a spherical acrosome surrounded by a cup-shaped nucleus, peculiar to brachyurans. An interesting characteristic of the E. sinensis sperm nucleus is its lack of electron-dense chromatin. However, its formation is not clear. In this study, sequences encoding histones H3 and H4 were cloned by polymerase chain reaction amplification. Western blotting indicated that H3 and H4 existed in the sperm nuclei. Immunofluorescence and ultrastructural immunocytochemistry demonstrated that histones H3 and H4 were both present in the nuclei of spermatogonia, spermatocytes, spermatids and mature spermatozoa. The nuclear labeling density of histone H4 decreased in sperm nuclei, while histone H3 labeling was not changed significantly. Quantitative real-time PCR showed that the mRNA expression levels of histones H3 and H4 were higher at mitotic and meiotic stages than in later spermiogenesis. Our study demonstrates that the mature sperm nuclei of E. sinensis contain histones H3 and H4. This is the first report that the mature sperm nucleus of E. sinensis contains histones H3 and H4. This finding extends the study of sperm histones of E. sinensis and provides some basic data for exploring how decapod crustaceans form uncondensed sperm chromatin.在多数动物的精子发生（spermatogenesis）过程中，与DNA结合的碱性蛋白持续发生动态变化：体细胞型组蛋白（somatic-typed histones）首先被精子特异性组蛋白（sperm-specific histones）部分替代，随后过渡蛋白（transition proteins）与鱼精蛋白（protamines）依次取代上述组蛋白。随着精子核碱性蛋白的替换，细胞核逐步发生染色质浓缩（chromatin condensation）。中华绒螯蟹（Eriocheir sinensis）又称毛蟹、河蟹，隶属于节肢动物门（Arthropoda）、甲壳亚门（Crustacea）、十足目（Decapoda）、方蟹科（Grapsidae）。该物种的精子无鞭毛（aflagellate），每个精子均具有球形顶体（acrosome），外被杯状细胞核，此为短尾类（brachyurans）所特有的结构。中华绒螯蟹精子细胞核的一个有趣特征是缺乏电子致密染色质（electron-dense chromatin），但其形成机制尚不清楚。本研究通过聚合酶链式反应（polymerase chain reaction, PCR）扩增克隆得到组蛋白H3与H4的编码序列。蛋白质免疫印迹（Western blotting）实验结果显示，精子细胞核中存在组蛋白H3和H4。免疫荧光（immunofluorescence）与超微结构免疫细胞化学（ultrastructural immunocytochemistry）实验证实，精原细胞（spermatogonia）、精母细胞（spermatocytes）、精子细胞（spermatids）以及成熟精子的细胞核中均存在组蛋白H3和H4；其中组蛋白H4在精子细胞核中的核标记密度有所下降，而组蛋白H3的标记密度无显著变化。实时荧光定量聚合酶链式反应（quantitative real-time PCR, qRT-PCR）结果显示，组蛋白H3和H4的mRNA表达水平在有丝分裂与减数分裂阶段高于精子形成（spermiogenesis）后期。本研究证实，中华绒螯蟹的成熟精子细胞核中存在组蛋白H3和H4，这是首次报道中华绒螯蟹成熟精子细胞核中含有组蛋白H3和H4。该发现拓展了中华绒螯蟹精子组蛋白的研究范畴，同时为探索十足目甲壳动物如何形成未浓缩的精子染色质提供了基础数据。