鲟鱼生殖细胞移植研究

The Chinese sturgeon (Acipenser sinensis) is a large anadromous bony fish, which is currently endangered due to the deteriorated natural habitat caused by anthropogenic activities. Although the artificial propagation of the Chinese sturgeon has already been successfully conducted, many problems exist, such as the rather tough and long period of sexual maturation of the breeding sturgeons. Therefore new methods are badly needed for shortering their reproduction period.. Spermatogonial stem cell transplantation has already been applied to many fishes, such as zebrafish, rainbow trout, Nile tilapia and pejerrey. Considering its mature artificial propagation technique and short period of first sexual maturation, Siberian sturgeon is the appropriate recipient of germ cell transplantation. In the present study, the primordial gonad cell\development-related genes such as dnd, vasa and nanos will be cloned, and the temporal and spatial expression patterns of the acquired genes during embryo development will be studied. In addition, gene specific Morpholino (MO) knockdown and mRNA rescue experiments will be used to explore their biological functions. Finally, MO of suitable germ cell genes will be injected into fertilized eggs of Siberian sturgeon to get the sterile recipients. Mosaic individuals will be obtained by tranplatation of Chinese sturgeon spermatogonial stem cells into the sterile Siberian sturgeon recipients. Bioinformatic analysis like transcriptome sequencing will be adopted for further investigating the developing mechanism of the donor-derived stem cell in the allogeneic recipient. Through this study, spermatogonial stem cell transplantation technique in sturgeons will be established, which should provide the basis for species conservation and sustained resource exploitation of Acipenser sinensis.

中华鲟为大型江海洄游性鱼类，由于人类活动的影响，其生存环境恶化，物种处于濒危状态。中华鲟全人工繁殖已获成功，但人工养殖中华鲟性成熟晚且性腺很难发育成熟，迫切需要引进新方法来缩短其繁殖周期。鱼类生殖细胞移植研究进展很快，已在虹鳟、罗非鱼和银汉鱼中获得成功。达氏鲟性成熟时间短，人工繁殖技术成熟，是中华鲟生殖细胞移植的合适受体。本研究拟克隆达氏鲟vasa、dnd和nanos等生殖细胞发育调控基因，研究其在胚胎发育过程中的时空表达特征；进行基因敲降和拯救实验，确定其在达氏鲟生殖细胞发育中的功能；敲降上述所得关键调控基因，制备生殖细胞缺失的达氏鲟作为受体。随后，分离中华鲟精原细胞并移植到不育受体中获得生殖细胞嵌合体；对供体在受体内的发育状况进行监测并采用转录组分析的方法探讨供体生殖细胞在受体内的发育机制。本研究将建立鲟鱼生殖细胞移植技术体系，为中华鲟等濒危动物的物种保护及其资源的可持续利用提供基础。

中华鲟为大型江海洄游性鱼类，由于水利水电工程建设、航运、污染和过度捕捞等人类活动的影响，其生存环境恶化，2017、2018连续两年自然繁殖活动中断，物种处于极度濒危状态。中华鲟全人工繁殖虽已获成功，但人工养殖中华鲟性成熟时间很长且性腺很难发育成熟，迫切需要引进新方法来缩短其繁殖周期，加强其种质资源保存及物种保护。鱼类生殖干细胞具有发育为两性配子的潜能，生殖干细胞移植技术已被证明是濒危物种保护的有效手段之一。达氏鲟与中华鲟亲缘关系很近，性成熟时间相对较短，人工繁殖技术成熟，是中华鲟生殖细胞移植的优良受体。本研究首先克隆得到达氏鲟、中华鲟生殖细胞标记基因vasa、dnd、nanos的全长cDNA 序列，获得其序列和系统进化特征。随后，采用实时荧光定量PCR、RNA 原位杂交和免疫组织化学等方法，在mRNA 水平和蛋白水平研究了vasa、dnd 和nanos 基因在达氏鲟胚胎和出膜幼鱼中的表达特征和亚细胞定位，发现它们均为生殖细胞特异表达基因。mRNA定点表达实验结果表明，vasa、dnd、nanos均可以作为鲟鱼原始生殖细胞的标记基因。接着，采用dnd 基因Mopholino显微注射方法探讨了dnd基因敲降对达氏鲟原始生殖细胞迁移及形成等性腺早期发育的影响；采用紫外线照射达氏鲟受精卵的方法，探讨了鲟鱼不育受体制备技术。最后，我们以达氏鲟精巢细胞和中华鲟卵巢细胞作为供体，达氏鲟出膜8天幼鱼作为移植受体，在国内首次成功建立了鲟鱼生殖细胞鉴定、分离、纯化、显微注射等移植技术体系。并且，以匙吻鲟作为生殖细胞供体，达氏鲟作为移植受体，成功实现了鲟鱼科间生殖细胞高效移植。我们还研究了供体生殖细胞在受体内的发育规律，对匙吻鲟移植嵌合体的性腺与作为对照的正常达氏鲟性腺进行了转录组比较分析，初步发现二者在基因表达上无显著差异。本研究开展的鲟鱼生殖细胞早期发育机理解析及生殖细胞移植技术体系的建立，为中华鲟等濒危鱼类的种质资源保存和物种保护提供了新的手段，也为鱼类种质资源保存及利用研究提供了一定理论基础及有效技术支撑。