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。     

鱼类生殖细胞移植技术的发展及应用前景

鱼类生殖细胞移植技术是通过诱导不同物种之间生殖系嵌合体来实现。原始生殖细胞和精原细胞(或卵原细胞)是诱导生殖细胞系嵌合体的关键材料。在过去的十多年中,通过采用不同的供体生殖细胞和不同发育阶段(囊胚期胚胎、初孵仔鱼和成鱼)的鱼为受体,开发出多种鱼类生殖细胞移植技术。这些成果的取得,为生殖细胞移植技术应用于诸多水产养殖新兴领域打下了坚实基础。该技术可以应用于:(1)生殖细胞生物学和转基因鱼等基础研究;(2)遗传资源和濒危鱼种的保护;(3)鱼类性别选择育种;(4)有效提高放流鱼苗的遗传多样性。     

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.     

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.     

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.     

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。     

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。     

The implications for aquaculture practice of epigenomic programming of components of the endocrine system of teleostean embryos: lessons learned from mammalian studies

Epigenetics is the study of changes in gene expression patterns that occur without any modification of the underlying nucleotide sequence of the DNA. Modifications of the so‐called epigenome include complex transient or permanent chemical changes of the DNA or histone proteins resulting in the suppression or enhancement of gene expression, together with an array of post‐translational events that modify the translational products. Epigenomic programming (EP) of the genome is an essential component of embryonic development in animals from the totipotent fertilized egg to the pluripotent stem cells, stem cell differentiation and final tissue and organ formation. Many of these EP processes are influenced transiently and some permanently by environmental influences. In eutherian mammals, environmentally related EP of embryos is linked to permanent changes in the phenotype of the progeny, some of which have been associated with adult onset metabolic disorders. Moreover, because some of the epigenetic remodelling occurs in both the soma and germ line, the resultant phenotypic characteristics (some of which are linked to disease states) may be heritable. Although far less is known about the effects of environmentally linked EP on the ontogeny of fishes, the available information suggests that the EP processes are similar amongst all vertebrates, and there are clear parallels between fish and mammals that are discussed in this paper. Our perspective takes the well‐established findings in mammals and uses them to proactively extrapolate to the as yet under‐recognized implications of EP for fish biology and for fish production in intensive aquaculture.     

Sertoli cell proliferation and FSH signalling in African catfish, Clarias gariepinus

Sertoli cell proliferation occurs mainly during the phase of rapid spermatogonial proliferation, allowing the cyst-forming Sertoli cells to form an increasingly large space for housing the growing germ cell clone. There is no information in fish on the regulation of Sertoli cell proliferation; follicle-stimulating hormone (FSH) stimulates Sertoli cell proliferation in mammals. Increasing or decreasing FSH and FSH receptor expression experimentally in male African catfish was associated with respective changes in Sertoli cell proliferation or testis growth, suggesting that also in fish, one role of FSH may be to regulate Sertoli cell numbers.     

Fin cell cryopreservation and fish reconstruction by nuclear transfer stand as promising technologies for preservation of finfish genetic resources

Domestication is a long-term process during which wild fish are acclimated to farming conditions and hopefully are reproduced over several generations, possibly using selective breeding. Preservation of the genetic diversity of the original population, together with that of the ongoing selection steps, is important for ecological and economical purposes. Cryobanking of reproductive cells is one answer to meet this need. In fish, however, only sperm can be cryopreserved as neither oocytes nor embryos are capable of handling the freeze-thawing stress. In this review, we explore to what extent diploid cells obtained from fin pieces can be used for the preservation of both parental genomes. The main parameters, which should be under control to ensure proper production of fin cells in culture and to enable cryopreservation of the material are described. After cryobanking of such non-reproductive cells, fish can be reconstructed using the nuclear transfer technology whose potentials and difficulties are discussed. The gametes produced by the fish reconstructed after somatic cells nuclear transfer are different to some extent from the gametes obtained after the direct transplantation of primordial germ cell or spermatogonial germ cells into host embryos or larvae. However, in some cases, only somatic cells can be obtained in quantities which would be compatible with strain restoration purposes. From the knowledge available today, it is reasonable to expect that nuclear transfer becomes available for fish reconstruction, even if restricted to high-tech biotechnology facilities. Therefore, cryobanking of fin somatic cells can be farsightedly considered for high-throughput diploid genome conservation.     

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。     

Cell junctions in fish seminiferous epithelium

Similar to mammals, in fish the cellular interactions between Sertoli cells (SC) and germ cells (GC) in the seminiferous epithelium have important structural and functional roles. In this review, we give a brief summary of these interactions, in particular those on the cell junctions. Despite the scarcity of detailed empirical data, it appears that both basic types of adhesive junctions (actin- and intermediate filaments-related) are present between SC. However, the actin-related multifunctional junction known as the “ectoplasmic specialization” is seemingly present only in some cartilaginous fish. Conversely, SC in other fish species are joined by actin-related junctions similar to typical zonulae or puncta adherens found in other epithelia. Adhesive junctions are also found between SC and GC and between GC and GC, and due to their particular characteristics these junctions are known as “desmosome-like junctions”. In terms of intercellular communication, connexins and gap junctions have been shown to occur between SC in fish, and they may be involved in the coordination of the synchronous development of GC within the cysts. It is also possible that gap junctions may form an interconnected network between SC and GC within a cyst. Concerning the SC barrier, tight junctions between fish SC apparently form a functional barrier only in cysts containing haploid GC, and different from mammals, meiotic GC in fish are not shielded from the vascular system. In summary, although still not well investigated, cell–cell interactions in the seminiferous epithelium of fish seem to be crucial for GC development, and their disturbance, for example by changing environmental conditions, will probably affect GC survival and fertility.     

中华鳖Gper基因克隆、表达及其在精巢中的功能

G蛋白偶联雌激素受体（Gper）是一种膜雌激素受体,介导雌激素的非基因组途径。为研究G蛋白偶联雌激素受体基因（ cDNA全长序列,利用qRT-PCR分析其在不同组织及胚胎不同发育阶段的性腺中的表达模式,并通过来曲唑（letrozole）和Gper抑制剂G-15处理雄鳖初步分析Gper在精巢中的作用。结果显示,中华鳖 cDNA序列全长2023 bp,包含705 bp 5''非编码区、241 bp 3''非编码区和1077 bp开放阅读框,编码358个氨基酸,其氨基酸序列上有7个跨膜结构域和Asp-Arg-Tyr（DRY）三联体结构,基因编码蛋白分子量为41.084 kD,等电点为6.844。表达量变化呈相同趋势：16期表达量最高,随着性腺分化过程表达量显著降低。Letrozole处理组中2表达量明显升高;G-15处理组精巢中,精子发生与促细胞凋亡相关基因表达量显著升高。结果表明,可能参与中华鳖性腺分化早期过程,并调控雄性生殖细胞增殖。     

Spermatogenesis and its endocrine regulation

Three major phases compose spermatogenesis: mitotic proliferation of spermatogonia, meiosis of spermatocytes, and spermiogenesis, the restructuring of spermatids into flagellated spermatozoa. The process is fuelled by stem cells that, when dividing, either self-renew or produce spermatogonia that are committed to proliferation, meiosis, and spermiogenesis. During all phases, germ cells are in close contact with and require the structural and functional support of Sertoli cells. In contrast to germ cells, these somatic cells express receptors for sex steroids and follicle-stimulating hormone (FSH), the most important hormones that regulate spermatogenesis. A typical Sertoli cell response to an endocrine stimulus would be to change the release of a growth factor that would then mediate the hormone's effect to the germ cells. Recent studies in the Japanese eel have shown, for example, that in the absence of gonadotropin Sertoli cells produce a growth factor (an orthologue of anti-Müllerian hormone) that restricts stem cell divisions to the self-renewal pathway; also estrogens stimulate stem cell renewal divisions but not spermatogonial proliferation. Gonadotropin or 11-ketotestosterone (11-KT) stimulation, however, induces spermatogonial proliferation, which is in part mimicked by another Sertoli cell-derived growth factor (activin B). Since FSH (besides luteinizing hormone, LH) stimulates steroidogenesis in fish, and since FSH is the only gonadotropin detected in the plasma of sexually immature salmonids, increased FSH signalling may be sufficient to initiate spermatogenesis by activating both Sertoli cell functions and 11-KT production. Another important androgen is testosterone (T), which seems to act via feedback mechanisms that can compromise FSH-dependent signalling or steroidogenesis. The testicular production of T and 11-KT therefore needs to be balanced adequately. Further research is required to elucidate in what way(s) 11-KT stimulates later stages of development, such as entry into meiosis and spermiogenesis. At this period, LH becomes increasingly important for the regulation of androgen production. Results from mammalian models suggest that during the later phases, the control of germ cell apoptosis via Sertoli cell factors is an important regulatory mechanism. In many species, sperm cells cannot fertilize eggs until having passed a maturation process known as capacitation, which includes the acquisition of motility. Progestins that are produced under the influence of LH appear to play an important role in this context, which involves the control of the composition of the seminal plasma (e.g., pH values). This revised version was published online in July 2006 with corrections to the Cover Date.     

胚胎干细胞的研究进展及应用前景

干细胞(Stem Cell)是一类具有分化潜能和自我复制的早期未分化细胞。胚胎干细胞(Embryonic stem cells,ES细胞)是一种早期胚胎内细胞团(inner cell mass,ICM)或原始生殖细胞(primordial germ cell,PGC)经体外分化抑制培养,分离和克隆得到的具有发育全能性的高度未分化细胞。本文综述了胚胎干细胞的形态学、生长特性、免疫学鉴定方法,ES细胞抑制分化和诱导分化的机理以及饲养层、血清和细胞因子等影响胚胎干细胞分离克隆的因素,并进一步阐述了胚胎干细胞的应用前景以及存在的问题。     

Embryonic stem cells in fish: current status and perspectives

Totipotent embryonic stem (ES) cells represent a bridge that links in vitro and in vivo manipulations of animal genomes and have enormous potential for genetic engineering of livestock. We have recently established feeder cell-free conditions for culturing cells of midblastula embryos (MBE) of the medaka (Oryzias latipes) and obtained several stable cell lines that show all features of mouse ES cells in vitro. One of these lines, MES1, has been demonstrated to retain a diploid karyotype and can be induced to differentiate into various cell types in vitro. Upon microinjection into albino host blastulae, MES1 cells are able to form pigmented chimeras. Genotype-specific PCR analysis revealed that 90% of host blastulae transplanted with MES1 cells developed into chimeric fry. This high frequency was not compromised by cryostorage or DNA transfection of the donor cells. Transplantation of genetically labelled MES1 cells revealed a wide contribution to numerous organs derived from all three germ layers and differentiation into various types of functional cells. These ES properties of MES1 line was not abolished by stable gene transfer and long-term selection. Thus MES1 cells may represent a first promising cellular vehicle for the production of genetically modified fish. The genetic background has been found to have a profound effect on the efficacy of ES cell derivation and of chimera formation.     

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。