基于5羟甲基胞嘧啶的中华绒螯蟹非浓缩核精子发生机制

Eriocheir sinensis is an economically important crustacean in Decapoda. The mechanism of spermatogenesis with uncondensed nucleus in Decapoda is still unclear although the mechanisms of spermatogenesis with condensed nuclei in mammals and many other animals have been better understanded. 5-hydroxymethylcytosine (5hmC) distributes in diverse animal tissues and plays several epigenetic roles, such as participating in DNA demethylation, regulation of gene expression and reprogramming of imprinting. Presently, we discovered that 5hmC also distributed during the spermatogenesis in E. sinensis. Therefore, we supposed that 5hmC was likely to play very important roles in the regulation of the spermatogenesis with uncondensed nucleus, which has not been reported. Accordingly, we should explore the relationship between 5hmC and the loose state of nucleus material, the hypothetical role that 5hmC mediates the expression of spermatogenesis-relevant genes, as well as the possible function that the active hydroxymethylated genes at the given phases of spermatogenesis directly regulate the process of spermatogenesis, in the levels ranging from gene to genome to nucleus genetic material to cell, using male germ cells at the diverse phases of spermatogenesis in E. sinensis as materials, utilizing the biological techniques (such as PCR, improved Southern blot, DNA sequencing, and two-dimensional electrophoresis) and the Blast in databanks, in order to illustrate the mechanism of this species spermatogenesis with uncondensed nucleus based on 5hmC. The results may help to better the understandings of mechanism of protecting genetic material in the uncondensed nucleus spermatozoa.

中华绒螯蟹是一种重要的十足目经济甲壳动物。虽然已知哺乳动物等的浓缩核精子发生机制，但十足目动物的非浓缩核精子发生机制还不清楚。5羟甲基胞嘧啶存在于多种动物组织，具有DNA去甲基化、调控基因表达、参与印记重编程等多种表观遗传学功能。近来，我们发现中华绒螯蟹精子发生过程中也存在5羟甲基胞嘧啶， 因此推测5羟甲基胞嘧啶可能在调控非浓缩核精子发生过程中起着十分重要的作用，而这还未见报到。据此，本立项以中华绒螯蟹各种雄性生殖细胞为研究对象，利用PCR、改良的DNA印迹、DNA测序和二维电泳等技术及数据库检索，在基因、基因组、核遗传物质、细胞等层次探讨5羟甲基胞嘧啶与细胞核松散状态的相关性、5羟甲基胞嘧啶对基因表达的调控作用及精子发生各阶段特异表达的羟甲基化基因对精子发生的直接调控作用，以期阐明基于5羟甲基胞嘧啶的中华绒螯蟹非浓缩核精子发生机制，为更好地理解非浓缩核精子的遗传物质保护机制提供帮助。

中华绒螯蟹是重要的十足目经济甲壳动物，其非浓缩核精子的发生机制还不清楚。5羟甲基胞嘧啶（5hmC）与DNA的去甲基化和结构松弛及基因激活等相关。本项目通过5hmC定位和定量及基因组甲基化和羟甲基化图谱及基因表达谱分析，筛选相关羟甲基化DNA片段和表达基因，从5hmC与细胞核松散状态的相关性、5hmC对基因表达的调控作用及相关基因对精子发生的调控作用三个方面，阐释了中华绒螯蟹非浓缩核精子的发生机制。另外我们还分析了基因组修饰与该物种性早熟的相关性。以该物种为材料，我们建立了分离各种雄性生殖细胞的胰酶分步消化法，发现精细胞内5hmC含量最高；获得了精子发生中的基因组甲基化和羟甲基化图谱及基因表达谱，筛选了大量精子发生相关DNA片段和表达基因；发现1975个与雄性生殖细胞结构相关基因（MSG）及583个参与精子核脱浓缩的遗传和表观遗传因子基因（NDG）；构建了单分子分辩率的精子基因组甲基化草图，其中mCG、mCHG和mCHH形式的mC所占比例分别为99.93%、0.05%和0.02%。结果表明，基因组整体的高羟甲基化和低甲基化使该物种精子核内的遗传物质在结构上相对松散，基因组非编码区DNA序列如微卫星序列的高羟甲基化通过松弛染色质结构或引起基因转座等激活脱浓缩相关基因的表达，精巢rRNA基因、蛋白编码基因及其他相关功能基因（尤其MSG及NDG）通过高羟甲基化和去甲基化而被激活，这些因素共同参与精子发生、精子核脱浓缩及稳定和保护精子核。另外，我们还构建了长江水系该物种线粒体基因组，并发现其精巢线粒体基因组的甲基化和羟甲基化现象。我们还发现，精巢基因组总体低羟甲基化参与调控早熟过程，精巢基因组内一些特定DNA序列，如部分微卫星序列、18S和28S rRNA基因、一些蛋白编码基因及预测基因的羟甲基化或甲基化可以作为鉴别性早熟的重要分子指标及具有经济意义的生长性状和育种参考的评价指标。