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Volume 43 Issue 11
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Cloning and distribution of histone H2A during spermatogenesis of the Chinese mitten crab (Eriocheir sinensis)

  • Corresponding author: Xianjiang KANG, xjkang@hbu.edu.cn
  • Received Date: 2019-03-13
    Accepted Date: 2019-05-28
  • Spermatogenesis is the process in which spermatogonia develop into mature spermatozoa. In most animals, this process involves changes in the basic protein associated with DNA. Somatic-type histones are partially or totally replaced by transition proteins, which in turn are replaced by protamines with strong alkalinity. With the replacement of basic proteins, nuclei progressively undergo chromatin condensation. The Chinese mitten crab (Eriocheir sinensis) sperm nucleus chromatin arrange loosely, and its formation is not clear. In this research, DNA sequence encoding histone H2A was cloned by PCR method. Thereafter, polyclonal antibody against the histone H2A was generated in rabbit, histone H2A distribution during E. sinensis spermatogenesis was detected through immunofluorescence. The encoding sequence of E. sinensis H2A contains 369 bp that encodes a protein of 123 amino acids. The predicted molecular weight of H2A was 13.1 ku. Protein alignment studies demonstrated that this histone H2A had a high identity with its counterparts in Penaeus vannmei and P.monodon, and the homology both are 99.19%. Immunofluorescence demonstrated that histone H2A was present through the spermatogenesis of E.sinensis. In spermatogonia and spermatocytes, H2A was mainly distributed in the nuclei and cytoplasm, while in spermatids and spermatozoa, H2A was mainly in the nuclei. The persistence of H2A in the mature sperm nucleus of E. sinensis might have some association with the uncondensed chromatin.
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Cloning and distribution of histone H2A during spermatogenesis of the Chinese mitten crab (Eriocheir sinensis)

    Corresponding author: Xianjiang KANG, xjkang@hbu.edu.cn
  • 1. College of Life Science, Hebei University, Baoding    071002, China
  • 2. Science and Technology Department, Hebei University of Chinese Medicine, Shijiazhuang    050200, China

Abstract: Spermatogenesis is the process in which spermatogonia develop into mature spermatozoa. In most animals, this process involves changes in the basic protein associated with DNA. Somatic-type histones are partially or totally replaced by transition proteins, which in turn are replaced by protamines with strong alkalinity. With the replacement of basic proteins, nuclei progressively undergo chromatin condensation. The Chinese mitten crab (Eriocheir sinensis) sperm nucleus chromatin arrange loosely, and its formation is not clear. In this research, DNA sequence encoding histone H2A was cloned by PCR method. Thereafter, polyclonal antibody against the histone H2A was generated in rabbit, histone H2A distribution during E. sinensis spermatogenesis was detected through immunofluorescence. The encoding sequence of E. sinensis H2A contains 369 bp that encodes a protein of 123 amino acids. The predicted molecular weight of H2A was 13.1 ku. Protein alignment studies demonstrated that this histone H2A had a high identity with its counterparts in Penaeus vannmei and P.monodon, and the homology both are 99.19%. Immunofluorescence demonstrated that histone H2A was present through the spermatogenesis of E.sinensis. In spermatogonia and spermatocytes, H2A was mainly distributed in the nuclei and cytoplasm, while in spermatids and spermatozoa, H2A was mainly in the nuclei. The persistence of H2A in the mature sperm nucleus of E. sinensis might have some association with the uncondensed chromatin.

  • 在鸟类、哺乳类等多数动物的精子发生过程中,与DNA结合的碱性蛋白会逐渐发生变化,组蛋白首先被精子特异的组蛋白替代,随后被一种过渡蛋白替代,最后被碱性更强的鱼精蛋白替代,随着替代蛋白碱性的增强,精子核一步步浓缩,最终,成熟的精子核是浓缩的,染色质高度聚集[1-7]。但是有一类十足目(Decapoda)甲壳动物,其精子核不同于绝大多数动物,为非浓缩核,核内遗传物质排列疏松,呈均匀的细网状[8]。早期研究认为,此类动物成熟精子中不含组蛋白等碱性蛋白[9-10],所以其精子核为非浓缩核。但是最近的研究发现蜘蛛蟹(Maja brachydactyla)和黄道蟹(Cancer pagurus)的成熟精子核内存在4种核心组蛋白[11-12],远海梭子蟹(Portunus pelagicus)的成熟精子中含有组蛋白H2B和H3[13],这些研究结果与早期有关蟹类组蛋白的研究结果不同,使学者们对十足目甲壳动物精子的碱性蛋白及其非浓缩核又有了新的认识。

    中华绒螯蟹(Eriocheir sinensis),隶属于十足目、方蟹科(Grapsidae)、绒螯蟹属。其精子无鞭毛、呈不规则的球形结构,主要由一个球状的顶体和包裹在外的细胞核组成[14-15]。核内染色质排列疏松,但是其非浓缩机制尚不清楚。本课题组前期研究发现,中华绒螯蟹的成熟精子核中含有组蛋白H2B、H3和H4[16-18],且与其非浓缩核有一定的关系。为了进一步探究组蛋白H2A在精子发生中的变化和功能,本实验克隆了中华绒螯蟹组蛋白H2A基因,并制备相应抗体,通过免疫荧光对其进行了定位,为进一步探讨中华绒螯蟹精子非浓缩核的成因提供参考。

1.   材料与方法
  • 大肠杆菌(Escherichia coli)DH5α、Rosetta(DE3)感受态细胞购自北京博迈德科技发展有限公司;pET-30a(+)表达载体为本实验室保存。

  • 雄性中华绒螯蟹,体质量15~100 g,购自河北保定白洋淀,雄性新西兰大白兔(Oryctolagus cuniculus)由河北大学实验中心提供。

  • DNA提取试剂盒,pMD-19T Vector,限制性内切酶EcoRI、HindⅢ,T4 DNA连接酶购自宝生物工程(大连)有限公司,辣根过氧化物酶(HRP)标记的羊抗兔IgG抗体购自北京索莱宝科技有限公司,Alexa Fluor488标记亲和纯化山羊抗兔IgG(H+L)二抗购自Abcam公司。

  • 从GenBank上查询到了十足目的凡纳滨对虾(Litopenaeus vannamei)(Q6PV61.3)的H2A全长氨基酸序列,斑节对虾(Penaeus monodon)(ABX84387.1)的H2A部分氨基酸序列,将其进行比对发现,相似度高达98.37%,考虑到组蛋白的保守性,根据其比对结果,使用Primer Premier 5.0 设计一对引物H2AF和H2AR (表1)。

    引物
    primers
    引物序列(5′–3′)
    primer sequence (5′–3′)
    用途
    usage
    H2AF CCTGGATGGTTACAACAGCCTTA 扩增H2A基因
    H2AR ATTCTTCAGCAGCACGACGAG 扩增H2A基因
    BDF CCGGAATTCATGTCCGGTCGCGGAAA 构建表达载体
    BDR CCCAAGCTTTTACTTCTTCTCGGTCTTCTTGGGC 构建表达载体
    注: 下划线碱基分别为EcoRI和HindⅢ酶切位点
    Notes: underlined bases are EcoRI and HindⅢ restriction enzyme cutting site, respectively

    Table 1.  Information of used primers

  • 用DNA提取试剂盒提取中华绒螯蟹精巢基因组DNA,以其为模板,扩增条件为94 °C预变性5 min,随后94 °C变性30 s,67 °C退火30 s,72 °C延伸1.5 min,循环30次,每次循环降低0.5 °C,之后再94 °C变性30 s,52 °C退火30 s,72 °C延伸1.5 min,循环15次,最后72 °C延伸10 min。PCR产物经胶回收试剂盒回收,与pMD-19T载体连接,转化大肠杆菌DH5α感受态细胞,挑取单菌落放大培养,进行菌液PCR验证,将得到的阳性克隆送上海生工生物工程股份有限公司测序,并保种命名为pMD-19T-H2A

  • 设计分别带有酶切位点EcoRI和HindⅢ的原核表达引物BDF和BDR,引物序列见表1。对扩增片段和pET-30a(+)载体分别进行双酶切,切胶纯化,以T4 DNA连接酶连接,构建重组质粒pET-30a-H2A,转化至DH5α感受态细胞,筛选阳性克隆进行菌液PCR验证并送去测序。

  • 将上述鉴定正确的pET-30a-H2A转化至E. coil Rosetta (DE3)感受态细胞中,挑取单克隆接种到Kana抗性LB液体培养基,至菌液A600为0.6~0.8时,加入终浓度为0.2 mmol/L的IPTG诱导,并在诱导4 、8 、18 h时,分别取菌液离心,用PBS重悬菌体,进行超声破碎,将破碎后的菌液离心,上清液和沉淀分别进行SDS-PAGE分析。参照Ni-NTA Agarose柱试剂盒的方法对上清液和包涵体蛋白进行纯化。

  • 将过夜诱导的菌液离心,收集菌体,用含有尿素的Buffer B溶液悬浮,随后进行超声破碎,将破碎后的菌液离心,取上清液用镍柱进行纯化。

  • 免疫之前,分离部分血清作为阴性对照。以纯化的重组蛋白为抗原,首次免疫将蛋白与等体积的完全弗氏佐剂充分乳化,在兔脊柱两旁多点皮下注射。以后间隔7~10 d注射1次,第4次免疫后取血,检测抗体的效价,之后进行加强免疫,7 d后心脏采血分离血清。

  • 取中华绒螯蟹的精巢,加入一定量的PBS匀浆,将匀浆液4 °C,5 000 r/min,离心5 min,取上清液,再次离心(13 000 r/min,4 °C)10 min,收集上清液保存于−80 °C冰箱备用。取纯化的重组蛋白和提取的精巢蛋白,进行SDS-PAGE电泳。电泳结束后将胶上蛋白转移至PVDF膜,5%脱脂奶粉室温封闭2 h,以制备的H2A多抗作为一抗,采用HRP标记的羊抗兔IgG作为二抗,ECL化学发光法显影并拍照。

  • 取不同发育时期的中华绒螯蟹精巢、输精管、储精囊,立即放入4%多聚甲醛中固定48 h,经30%蔗糖脱水,OCT包埋,徕卡冰冻切片机切片(厚5 μm)。

  • 免疫荧光具体操作步骤参考文献[14]。对于精子切片,在进行抗原修复之前,用2 mol/L HCl于37 °C酸化1 h。

2.   结果
  • PCR产物经1.0%的琼脂糖凝胶电泳检测,在约369 bp处有特异性条带出现,与预期大小相符(图1)。对测序结果进行分析,去掉载体序列后得到369 bp的核苷酸序列,编码123个氨基酸,预测分子量为13.1 ku(图2)。

    Figure 1.  Amplification of histone H2A

    Figure 2.  Nucleotide and deduced amino acid sequences of coding region of E. sinensis histone H2A gene

    用ClustalX软件,将中华绒螯蟹的H2A氨基酸序列与人(Homo sapiens,AAN59974.1),小鼠(Mus musculus,NP-835489.1)、非洲爪蟾(Xenopus laevis,AAA49769.1)、凡纳滨对虾和斑节对虾的H2A氨基酸序列进行了比对(图3),它们的相似度分别为84.62%、83.85%、83.85%、99.19%和99.19%,比对结果证实了组蛋白H2A进化上的保守性,同时也表明本实验成功克隆到中华绒螯蟹H2A基因的编码区。

    Figure 3.  Multiple alignment of H2A amino acid sequences

  • SDS-PAGE分析显示,重组菌株pET-30a-H2A/Rosetta经IPTG诱导,能成功表达重组蛋白,且蛋白在上清液和包涵体中均有表达(图4)。经Ni-NTA Agarose纯化后可获得高纯度的目的蛋白(图5)。

    Figure 4.  SDS-PAGE analysis of recombinant protein induced by IPTG

    Figure 5.  SDS-PAGE analysis of the purified recombinant protein

  • 经过Western blot验证,过镍柱纯化的重组蛋白在其大小位置(约22 ku)出现了一条特异性的条带,说明制备的抗体可以很好的与重组蛋白识别。而在与精巢蛋白的反应中,除了在蛋白预期位置(约13.1 ku)出现一条阳性反应条带之外,在上方还出现了一条非特异性条带。而对照组(免疫前血清)没有阳性反应(图6)。

    Figure 6.  Result of Western blot

  • 精原细胞染色质比较分散,部分聚集成小团块,免疫荧光结果显示H2A在精原细胞的细胞核和细胞质中均有分布(图版-1~3);精母细胞期,染色质聚集成较大团块,H2A在细胞核和细胞质中均有分布(图版-4~6);精细胞为圆形或者长圆形,细胞核直径约3.8 μm,H2A主要位于精细胞的细胞核中(图版-7~9);中华绒螯蟹成熟的精子由中央一个圆的顶体囊和外面包裹的一层薄薄的核杯组成,由于2 mol/L HCl酸化破坏了DNA结构,故DAPI染色显示不出核的结构,免疫荧光结果显示H2A主要存在于成熟精子的细胞核中(图版-10~11)。

    Figure 图版.  Location of histone H2A in spermatogenesis of E. sinensis

3.   讨论
  • 组蛋白的编码基因不含内含子,转录产物没有polyA尾巴[19-20],所以本实验提取基因组DNA来克隆H2A基因。组蛋白在进化上非常保守,即使在亲缘关系较远的种属中,4种组蛋白的氨基酸序列都非常相似。如海胆组织与小牛胸腺的H3仅有1个氨基酸的差异。小牛的H4序列与豌豆(Pisum sativum)相比,只有2个氨基酸的差异。中华绒螯蟹H2A的氨基酸序列与凡纳滨对虾和斑节对虾也仅有1个氨基酸的差异,进一步验证核心组蛋白在进化上是高度保守的。

    Western blot检测发现,在与精巢蛋白的反应中,除了在预期位置(13.1 ku)出现条带外,在其上方还出现了1条非特异性条带。组蛋白H2A的变体很多,目前已经发现的有H2A.X、H2A.Z、H2A.V、H2ABbd(Barr body deficient)、macroH2A和精巢特异的TH2A等[21-22]。可能是本研究制备的抗体同时识别了H2A及其变体,抑或是有部分蛋白形成了二聚体,或者蛋白发生了修饰,导致其分子量变大。

    有关十足目甲壳动物精子中组蛋白的研究,不同学者有不同的发现,早期研究主要采用一些组织化学的染色方法,研究者认为蟹类的精子核之所以是非浓缩是因为核内不含组蛋白等碱性蛋白。Vaughn等[23]发现在沙蟹(Emerita analoga)的精细胞时期,大量的核蛋白逐渐减少,最终成熟的精子核中不含有组蛋白或者鱼精蛋白。在刀额新对虾(Metapenaeus ensis)和锯缘青蟹(Scylla serrata)中也有类似的报道[24]。Chevaill[25]认为以往的研究大多缺乏定量测定,细微的染色很有可能被忽略[25],即使精子核存在碱性蛋白,可能由于技术问题而检测不到。最近的研究发现,一些蟹类(蜘蛛蟹、黄道蟹)的成熟精子中含有组蛋白[11-12]。本研究发现H2A存在于精子发生的整个时期,在精原细胞和精母细胞的细胞核和细胞质中均有分布,精原细胞和精母细胞不断进行减数分裂,需要在细胞质中大量合成蛋白质。精细胞时期,不需要大量合成蛋白质,此时组蛋白主要存在于细胞核中。在成熟的精子核内依旧存在组蛋白H2A,据此推测,H2A在中华绒螯蟹成熟精子核内的保留可能与其非浓缩的染色质有关,因为在鼠[26]、牛[27]、鱼[28]等物种中,此时正是组蛋白被鱼精蛋白和过渡蛋白等碱性更强的蛋白替代而导致核进一步浓缩的过程。而中华绒螯蟹成熟精子核内组蛋白的保留,说明其核内较少或者没有发生组蛋白的替代,导致中华绒螯蟹的精子核不能进一步浓缩。免疫荧光检测到精子核内组蛋白H2A是在经HCl酸化之后,HCl酸化能够破坏DNA的双螺旋结构、进而暴露H2A抗原,这说明H2A位于组蛋白八聚体的位置可能比较特殊,也暗示中华绒螯蟹精子核内虽然存在H2A、H2B、H3和H4 4种组蛋白,但组蛋白和DNA够成的核小体八聚体可能不同于一般的在人和鼠等这些具有浓缩精子核的动物当中的核小体结构。

    大多数动物的精子核为浓缩核,染色质高度聚集,这种高度浓缩的染色质是精子在受精之前对遗传物质DNA免受环境刺激的一种保护[29]。而中华绒螯蟹成熟的精子在受精之前聚集在精囊[14-15],这也是一个类似的保护机制,有了这种保护,中华绒螯蟹的精子可以拥有包装疏松的染色质结构而其DNA又不会被外界环境所破坏。此外,中华绒螯蟹非浓缩的精子染色质也可能与其特殊的受精方式有关,当精子接触到卵母细胞的外膜时,顶体外翻,精子核被牵引着通过一个相对窄的通道进入卵质[30-32],这一过程导致了细胞核的变形。如果精子染色质包装紧密,其灵活性受到限制,这一过程便不会发生。甲壳动物非浓缩的精子核可能是其染色质对于这种特殊受精方式的一种适应。

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