核移植与线粒体

线粒体是哺乳动物的产能、供能细胞器,与生长、发育、衰老和凋亡等多种细胞事件及疾病有关。哺乳动物核移植可能导致克隆胚胎及后代中线粒体的杂合性,从而影响到个体的表型甚至导致线粒体疾病。文章阐明了哺乳动物中线粒体的生物学功能及遗传特性,并分析了核移植中供体细胞和受体卵胞质两种来源的线粒体在同种胚胎细胞核移植、同种及异种体细胞核移植重构胚发育进程中的变化以及可能影响线粒体杂合性的一些因素,对其可能导致的线粒体疾病及解决方法进行了简单的阐述。

     

重构胚中线粒体命运的研究进展

线粒体是哺乳动物细胞中最为重要的细胞器之一,是除细胞核外惟一含有功能性基因组的细胞器,通过氧化磷酸化产生维持细胞正常生理功能的ATP。重构胚中线粒体的命运及线粒体与供体核间的互作关系已越来越成为研究的焦点,综述了同种、异种核移植,ICSI,卵胞质移植及异源线粒体注射后,重构胚中线粒体的命运。     

动物异种细胞核移植技术研究进展

作为研究动物胚胎发育过程中核质关系重要手段的细胞核移植技术正在逐步完善。异种细胞核移植技术不仅可用于保护濒危野生动物和珍贵的动物遗传资源,而且在研究核质相互作用和物种之间的进化关系上有着独特的用途。尽管目前同种核移植在许多哺乳动物上都获得成功,但异种核移植研究还刚刚开始,有许多问题亟待解决。本仅就现已发表的少量献,简要综述了动物异种核移植研究的历史与现状、影响因素、面临问题和应用前景。     

哺乳动物体细胞核移植中供体细胞的研究进展

在哺乳动物体细胞核移植中,供体细胞是影响其效率的主要因素之一。供体细胞的类型、细胞周期、细胞的培养代数、冷藏与冷冻处理,以及供体动物的性别、年龄等都可能影响核移植胚胎的发育。根据现有资料,简要综述了在哺乳动物体细胞核移植中有关供体细胞的研究进展。     

动物种间核移植研究概况

近年来,世界各地研究者正在努力完善异种体细胞核移植的技术环节和基础研究,本文就国外种间核移植的研究、我国哺乳动物种间核移植的研究情况进行整理分析。     

兔核移植胚胎起始发育的超微结构研究

对兔核移植胚胎起始发育的超微结构变化进行电镜观察,并与供体桑椹胚细胞,受体卵母细胞及同期正常受精胚胎的超微结构进行比较,“原核”期兔核移植胚胎的超微结构明显不同于供体桑椹胚细胞及受体卵母细胞的超微结构,而与同期正常受精胚胎相似,但有些核移植胚胎中皮质反应,及核仁和线粒体中出电子致密的网眼结构,与正常受精卵存在差别,分裂至２－细胞期时,与正常２－细胞胚超微结构更相似,结果提示,兔胚胎细胞核移植后,供     

哺乳动物线粒体遗传和在体细胞克隆胚胎中的命运

线粒体是哺乳动物重要的细胞器之一,为细胞的生命活动提供能量.线粒体是除细胞核外唯一含有功能性基因组DNA的细胞器.由于线粒体在哺乳动物早期胚胎的发育中有多方面重要的作用,因此线粒体对体细胞克隆胚胎发育的影响成为体细胞克隆动物研究的热点.就线粒体的结构特点和遗传特性及其在同种、异种动物克隆早期胚胎发育过程中的命运以及可能的遗传机制进行综述.同时,也将比较注射异源线粒体后,线粒体在注射胚胎中的发育命运.     

核重编程机制的影响因素

哺乳动物体细胞核移植技术在农业、生物技术、医药生产和濒危动物保护等方面具有很大的潜力和应用价值,已成为目前发育生物学研究的重要方法。但是核重编程仍是核移植技术的关键因素,制约了重构胚胎干细胞的研究。只有供核发生完全重编程,重构胚胎才能正常发育。核重编程与供核者的年龄,供核细胞的组织来源、分化状态、细胞周期、传代次数,供核细胞的表观遗传标记以及供卵者的年龄、卵子的成熟度等因素有关。创造各种适于核重编程的条件有利于从更高的起点开展核移植胚胎干细胞的研究,提高重枸胚胎干细胞建系效率。     

哺乳动物体细胞异种核移植胚胎早期发育的研究进展

体细胞异种核移植是指将一个物种的体细胞移植到另一物种的去核卵母细胞中,移入的体细胞核在受体胞质中重编程并发育成新个体的实验方法.该方法为拯救濒危物种和获取灵长类胚胎干细胞提供了可能的途径.但这方面的研究目前还只获得初步的进展,核重编程不完全以及异种胚胎的囊胚率低仍是其面临的主要难点.本文从基因表达、表观重编程、线粒体异质性、核重塑和核移植体系优化等方面入手,介绍近年来哺乳动物体细胞异种核移植的研究进展,并探讨异种重构胚重编程所面临的关键问题和可能获得成功的方法.     

供核细胞对体细胞核移植效率的影响

利用体细胞核移植技术克隆动物、生产转基因家畜具有极大的应用潜力。然而,核移植效率低下、克隆后代形态异常等问题仍然制约着体细胞核移植技术的产业化进展。影响体细胞核移植效率的因素很多,该文着重从供核细胞的类型、细胞体外培养、细胞凋亡及转基因操作等方面阐述其对体细胞核移植效率的影响。     

In vitro development of reconstructed bovine embryos and fate of donor mitochondria following nuclear injection of cumulus cells

In this study we examined the developmental potential of reconstructed embryos and the fate of donor mitochondria during preimplantation development after nuclear transfer in cattle. Isolated cumulus cells were used as donor cells in nuclear transfer. Cumulus cells labelled with MitoTracker Green FM fluorochrome were injected into enucleated bovine MII oocytes and cultured in vitro. MitoTracker labelling on donor cells did not have a detrimental effect on blastocyst formation following nuclear transfer. Cleavage rate was about 69% (56/81) and blastocyst formation rate was 6.2% (5/81) at 7 days after nuclear transfer. The labelled mitochondria dispersed to the cytoplasm and became distributed between blastomeres and could be identified up to the 8- to 15-cell stage. Small patches of mitochondria were detected in some 8- to 15-cell stage embryos (5/20). However, donor mitochondria were not detected in embryos at the 16-cell stage and subsequent developmental stages. In the control group, mitochondria could be identified in arrested 1-cell embryos up to 7 days after nuclear transfer. These results suggest that disappearance of the labelled donor mitochondria in nuclear transfer bovine embryos is not due to fading of the fluorochrome marker, but is rather an as yet undefined cytoplasmic event.     

Microinjection of serum-starved mitochondria derived from somatic cells affects parthenogenetic development of bovine and murine oocytes

Microinjection of isolated mitochondria into oocytes is an effective method to introduce exogenous mitochondrial DNA. In nuclear transfer procedures in which donor cell mitochondria are transferred with nuclei into recipient oocytes; development and survival rates of reconstructed embryos may be also directly influenced by mitochondrial viability. Mitochondrial viability is dramatically affected by cell culture conditions, such as serum starvation prior to nuclear transfer. This study was conducted to examine the influence of exogenous mitochondria using bovine and mouse parthenogenetic models. Mitochondria were isolated from primary cells at confluency and after serum starvation. The bovine oocytes injected with serum-starved mitochondria showed lower rates of morula and blastocyst formation when compared to uninjected controls (P < 0.05). However, the developmental rates between non-starved mitochondria injection and controls were not different (P > 0.05). The murine oocytes injected with serum-starved mitochondria showed lower rates of development when compared with non-starved mitochondria and controls (P < 0.01). In contrast to mitochondria transfer, ooplasm transfer did not affect murine or bovine parthenogenetic development (P > 0.05). The overall results showed that injection of serum-starved mitochondria influenced parthenogenetic development of both bovine and murine oocytes. Our results illustrate that the somatic mitochondria introduction accompanying nuclei has the capacity to affect reconstructed embryo development; particularly when using serum-starved cells as donor cells.     

Complete replacement of the mitochondrial genotype in a Bos indicus calf reconstructed by nuclear transfer to a Bos taurus oocyte

Due to the exclusively maternal inheritance of mitochondria, mitochondrial genotypes can be coupled to a particular nuclear genotype by continuous mating of founder females and their female offspring to males of the desired nuclear genotype. However, backcrossing is a gradual procedure that, apart from being lengthy, cannot ascertain that genetic and epigenetic changes will modify the original nuclear genotype. Animal cloning by nuclear transfer using host ooplasm carrying polymorphic mitochondrial genomes allows, among other biotechnology applications, the coupling of nuclear and mitochondrial genotypes of diverse origin within a single generation. Previous attempts to use Bos taurus oocytes as hosts to transfer nuclei from unrelated species led to the development to the blastocyst stage but none supported gestation to term. Our aim in this study was to determine whether B. taurus oocytes support development of nuclei from the closely related B. indicus cattle and to examine the fate of their mitochondrial genotypes throughout development. We show that indicus:taurus reconstructed oocytes develop to the blastocyst stage and produce live offspring after transfer to surrogate cows. We also demonstrate that, in reconstructed embryos, donor cell-derived mitochondria undergo a stringent genetic drift during early development leading, in most cases, to a reduction or complete elimination of B. indicus mtDNA. These results demonstrate that cross-subspecies animal cloning is a viable approach both for matching diverse nuclear and cytoplasmic genes to create novel breeds of cattle and for rescuing closely related endangered cattle.     

Microinjection of cytoplasm or mitochondria derived from somatic cells affects parthenogenetic development of murine oocytes

Cloned mammals are readily obtained by nuclear transfer using cultured somatic cells; however, the rate of generating live offspring from the reconstructed embryos remains low. In nuclear transfer procedures, varying quantities of donor cell mitochondria are transferred with nuclei into recipient oocytes, and mitochondrial heteroplasmy has been observed. A mouse model was used to examine whether transferred mitochondria affect the development of the reconstructed oocytes. Cytoplasm or purified mitochondria from somatic cells derived from the external ear, skeletal muscle, and testis of Mus spretus mice or cumulus cells of Mus musculus domesticus mice were transferred into M. m. domesticus (B6SJLF1 and B6D2F1) oocytes to observe parthenogenetic development through the morula stage. All B6D2F1 oocytes injected with somatic cytoplasm or mitochondria showed delayed development when compared to oocytes injected with buffer. The developmental rates were not different among injected cell sources, with the exception of testis-derived donor cells injected into B6SJLF1 oocytes (P < 0.01). The developmental rate of B6D2F1 oocytes injected with buffer alone (98.8% survival) was different from those injected with somatic cytoplasm (60.8% survival) or somatic mitochondria (56.5% survival) (P < 0.01). Conversely, injection of ooplasm into B6D2F1 oocytes did not affect parthenogenetic development (100% survival). Our results indicate that injection of somatic cytoplasm or mitochondria affected parthenogenetic development of murine oocytes. These results have further implications for in vitro fertilization protocols employing ooplasmic transfer where primary oocyte failure is not confirmed.     

Temperature-sensitive yeast mutants defective in mitochondrial inheritance

The distribution of mitochondria to daughter cells is an essential feature of mitotic cell growth, yet the molecular mechanisms facilitating this mitochondrial inheritance are unknown. We have isolated mutants of Saccharomyces cerevisiae that are temperature-sensitive for the transfer of mitochondria into a growing bud. Two of these mutants contain single, recessive, nuclear mutations, mdm1 and mdm2, that cause temperature-sensitive growth and aberrant mitochondrial distribution at the nonpermissive temperature. The absence of mitochondria from the buds of mutant cells was confirmed by indirect immunofluorescence microscopy and by transmission electron microscopy. The mdm1 lesion also retards nuclear division and prevents the transfer of nuclei into the buds. Cells containing the mdm2 mutation grown at the nonpermissive temperature sequentially form multiple buds, each receiving a nucleus but no mitochondria. Neither mdm1 or mdm2 affects the transfer of vacuolar material into the buds or causes apparent changes in the tubulin- or actin-based cytoskeletons. The mdm1 and mdm2 mutations are cell-cycle specific, displaying an execution point in late G1 or early S phase.     

Rate of gene transfer from mitochondria to nucleus: effects of cytoplasmic inheritance system and intensity of intracellular competition

Endosymbiotic theory states that mitochondria originated as bacterial intracellular symbionts, the size of the mitochondrial genome gradually reducing over a long period owing to, among other things, gene transfer from the mitochondria to the nucleus. Such gene transfer was observed in more genes in animals than in plants, implying a higher transfer rate of animals. The evolution of gene transfer may have been affected by an intensity of intracellular competition among organelle strains and the organelle inheritance system of the organism concerned. This article reveals a relationship between those factors and the gene transfer rate from organelle to nuclear genomes, using a mathematical model. Mutant mitochondria that lose a certain gene by deletion are considered to replicate more rapidly than normal ones, resulting in an advantage in intracellular competition. If the competition is intense, heteroplasmic individuals possessing both types of mitochondria change to homoplasmic individuals including mutant mitochondria only, with high probability. According to the mathematical model, it was revealed that the rate of gene transfer from mitochondria to the nucleus can be affected by three factors, the intensity of intracellular competition, the probability of paternal organelle transmission, and the effective population size. The gene transfer rate tends to increase with decreasing intracellular competition, increasing paternal organelle transmission, and decreasing effective population size. Intense intracellular competition tends to suppress gene transfer because it is likely to exclude mutant mitochondria that lose the essential gene due to the production of lethal individuals.     

The influence of enucleation on the ultrastructure of in vitro matured and enucleated cattle oocytes

The enucleation of recipient oocytes in nuclear transfer experiments is generally carried out by aspirating one third of the ooplasm adjacent to the first polar body. It was supposed that this enucleation step affects the ultrastructure of the remaining cytoplast, resulting in a decline or destruction of its cellular compartments. Even if the transferred nucleus had the potential to support the development of a single-cell nucleus transfer embryo to the blastocyst stage, meiotic division could be stopped at any stage if the destruction of the ultrastructure of host cytoplasm resulted in a limited metabolism. The present study was conducted to investigate the influence of the enucleation procedure on the ultrastructure of the remaining ooplast. In vitro matured oocytes; in vitro matured and enucleated oocytes; and in vitro matured and enucleated oocytes that were subsequently cultivated in vitro for additional 4 h were prepared for transmission electron microscopy (TEM). An examination of ultra-thin sections showed that the arrangement of organelles in all matured oocytes was in accordance with that already described for normal oocyte development. Immediately after enucleation no major differences in the arrangement of cortical granules, mitochondria, smooth endoplasmic reticulum (SER), lipid droplets and vacuoles were found compared with nonmanipulated oocytes. After enucleation and 4 h of culture, 24- and 36-h matured oocytes differed from each other in the arrangement of large aggregates of SER surrounded by a wall of mitochondria and lipid droplets. These complexes were still found in the 24-h but not in 36-h matured, enucleated and cultivated oocytes. Clusters of SER, mitochondria and lipid droplets were described by different authors as having metabolic activity. The results of this study in connection with results from nuclear transfer experiments suggest that these aggregates and their metabolic activity can be transferred with cytoplasm from 24- but not 36-h matured oocytes. Only cytoplasm from the 24-h matured oocytes showed a development-supporting effect when fused to enucleated recipient cells before nuclear transfer.     

Why mitochondrial genes are most often found in nuclei

A very small fraction of the proteins required for the propagation and function of mitochondria are coded by their genomes, while nuclear genes code the vast majority. We studied the migration of genes between the two genomes when transfer mechanisms mediate this exchange. We could calculate the influence of differential mutation rates, as well as that of biased transfer rates, on the partitioning of genes between the two genomes. We observe no significant difference in partitioning for haploid and diploid cell populations, but the effective size of cell populations is important. For infinitely large effective populations, higher mutation rates in mitochondria than in nuclear genomes are required to drive mitochondrial genes to the nuclear genome. In the more realistic case of finite populations, gene transfer favoring the nucleus and/or higher mutation rates in the mitochondrion will drive mitochondrial genes to the nucleus. We summarize experimental data that identify a gene transfer process mediated by vacuoles that favors the accumulation of mitochondrial genes in the nuclei of modern cells. Finally, we compare the behavior of mitochondrial genes for which transfer to the nucleus is neutral or influenced by purifying selection.