鱼类生殖细胞移植的研究进展及应用前景

生殖细胞移植是指将供体的生殖细胞移植到同种或异种受体体内,供体生殖细胞嵌合到受体性腺,经过增殖、分化并最终发育为功能性配子的过程。作为辅助生殖技术,它不仅为珍稀濒危动物的繁育和保护提供了新途径,同时也为生殖干细胞的功能研究提供了有效手段。鱼类生殖细胞移植研究首先在模式鱼类斑马鱼中开展,经过十多年的发展,取得了一系列突破性的进展:主要包括先后建立了以胚胎、仔鱼和成鱼为受体的生殖细胞移植体系,精原和卵原干细胞的发现拓宽了供体生殖细胞的选择,受体的选择与制备方法的完善。该技术在缩短鱼类性成熟周期、性控育种、珍稀濒危鱼类保护等方面具有巨大的应用前景,已成功在多种淡水和海水鱼类中开展了研究和应用。本文结合作者的研究实践和经验,系统地梳理和总结了鱼类生殖细胞移植的研究进展,指出了该技术实践应用的关键问题,并探讨了其应用前景。     

鱼类两性嵌合体及其在种质保存中的应用前景

本文对鱼类两性嵌合体的概念、产生以及在科研和生产上的意义作一综述,并首次提出鱼类两性嵌合体在鱼类种质资源保存中的应用价值。两性嵌合体（ａｍｐｈｉｇｅｎｅｔｉｃｍｏｓａｉｃｓ）是指在组织学成分中同时混杂存在着雌雄两类生殖细胞的生殖腺（性腺）,是鱼类中一类特殊的生殖腺。两性嵌合体根据其中两类生殖细胞数量的相对比例以及特定的遗传和生理条件,有的是不育的（即不能产生具有受精能力的精子或卵子）,有的是能育的（如正常排卵受精）;有的两性嵌合体则是性转变过程中的中间过渡形式。判别鱼类的一种生殖腺是不是两性嵌合…     

鱼类原始生殖细胞发育与生殖操作技术研究进展

原始生殖细胞是胚胎发育过程中最早建立的一群生殖干细胞,是有性生殖动物生殖发育的基础。鱼类原始生殖细胞特化遵循“先成论”的模式,早期胚胎发育过程中获得母源生殖质组分的细胞特化为原始生殖细胞,特化形成的原始生殖细胞需要维持其生殖干细胞命运,并经过长距离迁移最终到达性腺原基的位置。原始生殖细胞特化、迁移和命运维持过程受到多种基因和信号通路的综合调控。研究鱼类原始生殖细胞发育不仅有助于深入理解脊椎动物细胞特化、迁移和命运维持等基本生物学过程的调控机理,而且是开发新的养殖鱼类生殖控制和生殖干细胞移植技术的重要基础。本文概述了鱼类原始生殖细胞发育的基础理论以及生殖操作技术研究进展,并展望了未来的发展趋势,以期为开发重要养殖鱼类优良种质创制新技术提供参考。     

显微注射技术在制备鱼类嵌合体和转基因海水鱼上的应用

以鱼类胚胎细胞和胚胎干细胞为核供体进行细胞移植构建鱼类嵌合体研究方面,现有的成功报道均采用显微注射方法;在转基因海水鱼类研究中,显微注射也是最为常用的技术,本实验室在花鲈胚胎干细胞嵌合体构建和外源基因向花鲈胚胎的转移研究中取得的结果也充分证实,显微注射技术是开展海水鱼类细胞移植和转基因研究的首选技术。     

基于dnd基因标记的大黄鱼原始生殖细胞发生发育的初步研究

在鱼类的早期胚胎发育中生殖细胞便与体细胞分离,形成所谓的原始生殖细胞(Primordial germ cells,PGCs)。母源性基因Dead end(dnd)在胚胎发育中特异地表达于原始生殖细胞,编码一种进化上保守的RNA结合蛋白,对生殖细胞发育具有重要作用。本研究通过PCR获得大黄鱼(Larimichthys crocea)dnd基因(Lcdnd)的部分cDNA序列,结合实时定量PCR、整胚原位杂交等技术,研究Lcdnd在各组织和胚胎发育中的表达。实时定量PCR结果显示：dnd在性腺中特异表达,且其在卵巢中的表达量高于精巢;检测的各期胚胎中都有dnd的表达,随着胚胎的发育其表达量呈下降的趋势。整胚原位杂交结果显示：Lcdnd在早期卵裂阶段主要分布于分裂沟,到囊胚期至原肠早期则开始定位于细胞内,原肠早期时定位在胚环中的中内胚层,且阳性信号增多,暗示此时原始生殖细胞的形成。接着原始生殖细胞沿胚环向胚体形成处(胚盾)迁移;随着胚胎的发育,发现胚体两侧的阳性信号增多;到体节发生期,阳性信号细胞分布于胚体两侧的侧板中胚层,随着体节的发生这些细胞沿背腹轴向腹部运动;发育到孵出期时,到达了发育中的原始生殖嵴。本研究首次使用dnd作为一种较为可靠的大黄鱼生殖细胞标记,揭示了大黄鱼原始细胞的起源与迁移,为大黄鱼生殖细胞发生发育及养殖大黄鱼的育性控制的研究奠定了一定的基础。     

温度和白消安对星点东方鲀成鱼生殖细胞枯竭的影响

为了解高温和白消安注射对星点东方鲀(Takifugu niphobles)成鱼生殖细胞发生的影响,设置32℃高温组、32℃高温-白消安组和空白对照组,比较3组的生长、存活率以及生殖细胞凋亡情况。结果显示,高温-白消安组的体重、肥满度显著低于其他组(P<0.05),而全长与其他各组差异不显著(P>0.05),各组的存活率均在90.9%以上。高温-白消安组对生殖细胞的凋亡作用比高温组更加明显,卵巢基本看不到正常的生殖细胞,且有大量的吞噬作用造成的黄褐色沉积,生殖细胞凋亡造成的空白面积比例要显著地高于高温组(P<0.05);精巢中生殖细胞凋亡的精小囊占总的精小囊的比例高达100.0%±0.0%,也显著高于高温组空泡精小囊占总的精小囊的比例(P<0.05)。得出结论:高温-白消安组较高温组更易诱导星点东方鲀内源生殖细胞凋亡,从而可制备出生殖细胞移植的适宜受体。     

鱼类遗传改良研究综述

本文对全世界范围内鱼类遗传改良研究的概况及其有关问题进行了全面的介绍和评述,从选择育种、杂交育种、性别控制、多倍体诱导、细胞核移植和细胞融合、低温保存技术、DNA分子标记育种、转基因技术等方面系统介绍了鱼类遗传改良的方法、在水产养殖中的应用和已取得的成就,并对鱼类遗传改良今后的发展趋势进行了展望,旨在为今后进一步开展鱼类遗传改良研究提供参考。     

利用鳍条组织进行流式细胞实验的可行性研究

流式细胞术能快速检测生物细胞的DNA含量和细胞周期,在鱼类遗传育种方面已得到广泛运用。目前的研究多基于鱼类血液和生殖细胞开展实验,存在周期性和操作难度的限制。该研究利用草鱼(Ctenopharyngodon idellus)不同组织进行流式分析,在鳍条组织中得到了明显的峰图,并在4种鱼类的重复实验中得到了稳定结果,且不同鱼类的鳍条组织均存在细胞周期,大部分细胞停留在G_0/G_1期。利用斑马鱼(Danio rerio)作为内标测定出团头鲂(Megalobrama amblycephala)鳍条组织的相对DNA含量(C值)为(1.25±0.07)pg,与相关研究结果基本吻合,认为利用鳍条组织作为血液和生殖细胞的替代材料进行流式细胞实验是科学可行的。     

亲鱼卵球成熟的规律与亲鱼培育的关系

探讨鱼类性腺成熟过程以及影响性腺成熟过程的主要因素，这对生产有很大意义。鱼类性腺的成熟程度取决于生殖细胞的发育程度，而生殖细胞的生长发育主要分三期，即卵原细胞的增殖期、卵母细胞的生长期和成熟期。从人工繁殖的角度看，最有实际意义的是生长期及成熟期。     

鱼类原生殖细胞的研究进展

除单细胞动物外,动物体由两部分细胞组成:体细胞和生殖细胞,二者均由同一个受精卵发育而成.现在学术界通常认为:对于大多数动物来说,生殖细胞并非是在胚胎发育的后期才在生殖腺中产生的,而是在胚胎发育的一开始就与体细胞分开了,那时它被称为原生殖细胞(PGCs).随着胚胎发育的进行,PGCs通过不同的途径逐渐迁入正在发育的生殖腺中,随后与其共同分化成为精巢或卵巢[1].     

Germ cell transplantation as a potential biotechnological approach to fish reproduction

Although the use of germ cell transplantation has been relatively well established in mammals, the technique has only been adapted for use in fish after entering the 2000s. During the last decade, several different approaches have been developed for germ cell transplantation in fish using recipients of various ages and life stages, such as blastula-stage embryos, newly hatched larvae and sexually mature specimens. As germ cells can develop into live organisms through maturation and fertilization processes, germ cell transplantation in fish has opened up new avenues of research in reproductive biotechnology and aquaculture. For instance, the use of xenotransplantation in fish has lead to advances in the conservation of endangered species and the production of commercially valuable fish using surrogated recipients. Further, this could also facilitate the engineering of transgenic fish. However, as is the case with mammals, knowledge regarding the basic biology and physiology of germline stem cells in fish remains incomplete, imposing a considerable limitation on the application of germ cell transplantation in fish. Furthering our understanding of germline stem cells would contribute significantly to advances regarding germ cell transplantation in fish.     

Interspecific germ cell transplantation: a new light in the conservation of valuable Balkan trout genetic resources?

Interspecific transplantation of germ cells from the brown trout Salmo trutta m. fario and the European grayling Thymallus thymallus into rainbow trout Oncorhynchus mykiss recipients was carried out in order to improve current practices in conservation of genetic resources of endangered salmonid species in the Balkan Peninsula. Current conservation methods mainly include in situ efforts such as the maintenance of purebred individuals in isolated streams and restocking with purebred fingerlings; however, additional ex situ strategies such as surrogate production are needed. Steps required for transplantation such as isolation of high number of viable germ cells and fluorescent labeling of germ cells which are to be transplanted have been optimized. Isolated and labeled brown trout and grayling germ cells were intraperitoneally transplanted into 3 to 5 days post hatch rainbow trout larvae. Survival of the injected larvae was comparable to the controls. Sixty days after transplantation, fluorescently labeled donor cells were detected within the recipient gonads indicating successful incorporation of germ cells (brown trout spermatogonia and oogonia—27%; grayling spermatogonia—28%; grayling oogonia—23%). PCR amplification of donor mtDNA CR fragments within the recipient gonads additionally corroborated the success of incorporation. Overall, the transplantation method demonstrated in this study presents the first step and a possible onset of the application of the germ cell transplantation technology in conservation and revitalization of genetic resources of endangered and endemic species or populations of salmonid fish and thus give rise to new or improved management strategies for such species.     

Biotechnology applied to fish reproduction: tools for conservation

This review discusses the new biotechnological tools that are arising and promising for conservation and enhancement of fish production, mainly regarding the endangered and the most economically important species. Two main techniques, in particular, are available to avoid extinction of endangered fish species and to improve the production of commercial species. Germ cell transplantation technology includes a number of approaches that have been studied, such as the transplantation of embryo-to-embryo blastomere, embryo-to-embryo differentiated PGC, larvae to larvae and embryo differentiated PGC, transplantation of spermatogonia from adult to larvae or between adults, and oogonia transplantation. However, the success of germ cell transplantation relies on the prior sterilization of fish, which can be performed at different stages of fish species development by means of several protocols that have been tested in order to achieve the best approach to produce a sterile fish. Among them, fish hybridization and triploidization, germline gene knockdown, hyperthermia, and chemical treatment deserve attention based on important results achieved thus far. This review currently used technologies and knowledge about surrogate technology and fish sterilization, discussing the stronger and the weaker points of each approach.     

石鲽仔、幼鱼性腺发育的组织学观察

利用组织学方法对人工培育的石鲽(Kareius bicoloratusBasiewsky)仔、幼鱼性腺发育进行研究。结果表明,性腺的发育与体长密切相关。刚孵化石鲽的原生殖细胞数目为2个,孵化3 d数目增至8个,之后经过迁移,至孵化9 d到达生殖嵴。在全长为7.2～8.5 mm(孵化9～11 d)的仔鱼中,性腺原基中的体细胞迅速增殖并包围原生殖细胞,后者在全长10～15 mm(孵化后10～35 d)的仔幼鱼中增殖成为生殖干细胞。原始性腺在全长15～30 mm(孵化40～60 d)的幼鱼中逐渐发育完善,呈细线状,位于腹腔后部中肾管下方紧贴体壁。雌性性腺最早在全长32.5 mm(孵化66 d)的个体中出现分化特征,至全长89～102 mm时雌性性腺特征完全分化。雄性性腺的分化较雌性性腺晚,最早在全长为91 mm的幼鱼中开始,至全长为114～118 mm时雄性性腺分化特征已经十分明显。     

Colonization,proliferation, and survival of intraperitoneally transplanted yellowtail <Emphasis Type="Italic">Seriola quinqueradiata</Emphasis> spermatogonia in nibe croaker <Emphasis Type="Italic">Nibea mitsukurii</Emphasis> recipient

Recently, we developed an intraspecies spermatogonial transplantation technique in a pelagic egg spawning marine teleost, nibe croaker Nibea mitsukurii. Nibe croaker is an ideal candidate recipient for spermatogonial transplantation since it has a short generation time and small body size. In the present study, yellowtail Seriola quinqueradiata spermatogonia were transplanted into nibe croaker larvae, and the behavior of transplanted spermatogonia in recipient gonads was observed. Three weeks post-transplantation, yellowtail spermatogonia were incorporated into the gonads of 72 out of 88 recipients. An antiproliferating cell nuclear antigen was detected in incorporated yellowtail spermatogonia, suggesting that the xenogenic germ cells were proliferating in recipient gonads. Yellowtail vasa-positive spermatogonia survived for 11 months after transplantation in the gonads of recipient fish. Thus, we showed that the microenvironment in nibe croaker gonads can support the colonization, proliferation, and survival of germ cells derived from a different taxonomic family.     

黑脊倒刺鲃vasa同源基因的克隆及表达分析

为了从分子水平上研究鱼类生殖细胞发育的机制,首次从重要的经济养殖鱼类——黑脊倒刺鲃中克隆了斑马鱼vasa的同源基因ScVHG。该cDNA长1962bp,编码654个氨基酸,氨基酸序列中含有DEAD蛋白家族特有的9个保守结构域。经比对发现,其编码的蛋白序列与斑马鱼VASA蛋白等具有高度的同源性,与斑马鱼、罗非鱼、虹鳟、银鲫、黄鳝、金鱼、金枪鱼、草鱼的VASA蛋白相似度分别为77%,77%,79%,90%,74%,89%,79%和86%。RT-PCR结果显示:在成鱼不同的组织中,ScVHG仅在精巢和卵巢中表达。RNA原位杂交结果进一步表明:在精子发生中,ScVHG只在精原细胞中表达,而在后期的精母细胞、精子细胞中均未发现明显的表达;在卵子发生中,ScVHG在卵原细胞和卵母细胞的各个时相中均有表达,在卵原细胞中表达最为强烈,而在随后各时相的卵母细胞中,阳性信号逐渐减弱。研究认为,该基因中含有的保守序列、序列的相似性以及该基因在生殖细胞中的特异性表达等结果均表明,克隆得到的ScVHG是斑马鱼vasa的同源基因,此基因可作为一种有效的分子标记,用于黑脊倒刺鲃以及其他鱼类生殖细胞发育机制的研究。