人-牛异种克隆胚构建及其线粒体来源

通过人-牛异种核移植技术获得异种克隆囊胚, 便于在不消耗人类卵母细胞的情况下从异种克隆胚中分离出人类干细胞。通过透明带下注射法将人胎儿成纤维细胞和牛耳成纤维细胞分别注入去核牛卵母细胞中构建异种和同种胚胎, 并比较两者之间的融合率、卵裂率、8-细胞发育率以及囊胚率。并对处于2-细胞、4-细胞、8-细胞、桑椹胚、囊胚阶段的异种克隆胚的线粒体DNA来源进行检测。结果表明, 异种克隆胚体外各个阶段的发育率均低于同种克隆胚, 尤其是8-细胞到囊胚阶段的发育率, 以及囊胚率都显著低于同种克隆胚(P<0.05)。异种克隆胚在2-细胞到桑椹胚阶段检测到人、牛线粒体DNA共存, 囊胚阶段只检测到牛线粒体DNA。结果表明: 牛卵母细胞可以重编程人胎儿成纤维细胞, 完成异种克隆胚植入前的胚胎发育, 异种克隆胚由于核质相互作用的不谐调, 影响其发育能力, 使其囊胚率显著低于同种克隆胚。牛线粒体DNA存在于植入前异种胚胎发育的各个阶段。异种克隆胚胎用于人类胚胎干细胞分离具有可行性。     

Limited demethylation leaves mosaic-type methylation states in cloned bovine pre-implantation embryos

Cloning by nuclear transfer (NT) has been riddled with difficulties: most clones die before birth and survivors frequently display growth abnormalities. The cross-species similarity in abnormalities observed in cloned fetuses/animals leads us to suspect the fidelity of epigenetic reprogramming of the donor genome. Here, we found that single-copy sequences, unlike satellite sequences, are demethylated in pre-implantation NT embryos. The differential demethylation pattern between genomic sequences was confirmed by analyzing single blastocysts. It suggests selective demethylation of other developmentally important genes in NT embryos. We also observed a reverse relationship between methylation levels and inner cell mass versus trophectoderm (ICM/TE) ratios, which was found to be a result of another type of differential demethylation occurring in NT blastocysts where unequal methylation was maintained between ICM and TE regions. TE-localized methylation aberrancy suggests a widespread gene dysregulation in an extra-embryonic region, thereby resulting in placental dysfunction familiar to cloned fetuses/animals. These differential demethylations among genomic sequences and between differently allocated cells produce varied overall, but specified, methylation patterns, demonstrating that epigenetic reprogramming occurs in a limited fashion in NT embryos.     

Fate of maternal mtDNA following 60Co inactivation of maternal nuclear DNA in unfertilized salmonid eggs.

The effects of gamma irradiation on nuclear DNA and mitochondrial DNA (mtDNA) were examined by exposing unfertilized salmonid eggs to a 60Co source. Brown trout (Salmo trutta) eggs exposed to 60Co were fertilized with sperm from brook trout (Salvelinus fontinalis), and brook trout eggs exposed to 60Co were fertilized with sperm from splake males (S. namaycush x S. fontinalis). In both types of matings only paternal allozymes were found in embryos, confirming the inactivation of the nuclear genome in the eggs. Analysis of mtDNA in these same embryos showed exclusively maternal mtDNA. The absence of paternal mtDNA among any of the embryos supports the predominance of maternal inheritance of mtDNA in vertebrates and suggests that mtDNAs are more resistant to cobalt inactivation than nuclear DNAs based on structure or numerical superiority to maternal nuclear DNA. Inactivation of maternal nuclear DNA, fertilization, and an induced return to the diploid state provide a means for producing an inbred organism having the nuclear genome of the paternal parent (androgenetic) and the mitochondrial genome of the female.     

Development of bovine-ovine interspecies cloned embryos and mitochondria segregation in blastomeres during preimplantation

The objective of the study was to investigate interspecies somatic cell nuclear transfer (iSCNT) embryonic potential and mitochondrial DNA (mtDNA) segregation during preimplantation development. We generated bovine-ovine reconstructed embryos via iSCNT using bovine oocytes as recipient cytoplasm and ovine fetal fibroblast as donor cells. Chromosome composition, the total cell number of blastocyst and embryonic morphology were analyzed. In addition, mtDNA copy numbers both from donor cell and recipient cytoplasm were assessed by real-time PCR in individual blastocysts and blastomeres from 1- to 16-cell stage embryos. The results indicated the following: (1) cell nuclei of ovine fetal fibroblasts can dedifferentiate in enucleated bovine ooplasm, and the reconstructed embryos can develop to blastocysts. (2) 66% of iSCNT embryos had the same number of chromosome as that of donor cell, and the total cell number of iSCNT blastocysts was comparable to that of sheep parthenogenetic blastocysts. (3) RT-PCR analysis in individual blastomeres revealed that the ratio of donor cell mtDNA: recipient cytoplasm mtDNA remained constant (1%) from the one- to eight-cell stage. However, the ratio decreased from 0.6% at the 16-cell stage to 0.1% at the blastocyst stage. (4) Both donor cell- and recipient cytoplasm-derived mitochondria distributed unequally in blastomeres with progression of cell mitotic division. Considerable unequal mitochondrial segregation occurred between blastomeres from the same iSCNT embryos.     

Fate of donor mitochondrial DNA in cloned bovine embryos produced by microinjection of cumulus cells

This study examined the fate of donor mitochondrial DNA during preimplantation development after nuclear transfer (NT) in cattle. Frozen-thawed cumulus cells were used as donor cells in the nuclear transfer. Mitochondrial DNA heteroplasmy in the nuclear transfer embryos was analyzed by allele-specific PCR (AS-PCR), direct DNA sequencing, and DNA chromatography. AS-PCR analysis for the detection of donor mitochondrial DNA was performed at the 1-, 2-, 4-, 8-, 16-cell, morula, and blastocyst stages of the embryos. The mitochondrial DNA from donor cells was detected at all developmental stages of the nuclear transfer embryos. However, mitochondrial DNA heteroplasmy was not observed in direct DNA sequencing of displacement-loop sequence from nuclear-transfer-derived blastocyst embryos. To confirm the mtDNA heteroplasmy in cloned embryos, the AS-PCR product from NT-derived blastocysts was analyzed by DNA sequencing and DNA chromatography. The nucleotides of NT-derived blastocysts were in accordance with the nucleotides from donor cells. These results indicate that the foreign cytoplasmic genome from donor cells was not destroyed by cytoplasmic events during preimplantation development that followed nuclear transfer.     

Trophoblast-specific DNA methylation occurs after the segregation of the trophectoderm and inner cell mass in the mouse periimplantation embryo

The first cell differentiation in the mammalian development separates the trophoblast and embryonic cell lineages, resulting in the formation of the trophectoderm (TE) and inner cell mass (ICM) in blastocysts. Although a lower level of global DNA methylation in the genome of the TE compared with ICM has been suggested, the dynamics of the DNA methylation profile during TE/ICM differentiation has not been elucidated. To address this issue, first we identified tissue-dependent and differentially methylated regions (T-DMRs) between trophoblast stem (TS) and embryonic stem (ES) cells. Most of these TS–ES T-DMRs were also methylated differentially between trophoblast and embryonic tissues of embryonic day (E) 6.5 mouse embryos. Furthermore, we found that the human genomic regions homologous to mouse TS–ES T-DMRs were methylated differentially between human placental tissues and ES cells. Collectively, we defined them as cell-lineage-based T-DMRs between trophoblast and embryonic cell lineages (T–E T-DMRs). Then, we examined TE and ICM cells isolated from mouse E3.5 blastocysts. Interestingly, all T-DMRs examined, including the Elf5, Pou5f1 and Nanog loci, were in the nearly unmethylated status in both TE and ICM and exhibited no differences. The present results suggest that the establishment of DNA methylation profiles specific to each cell lineage follows the first morphological specification. Together with previous reports on asymmetry of histone modifications between TE and ICM, the results of the current study imply that histone modifications function as landmarks for setting up cell-lineage-specific differential DNA methylation profiles.     

动物线粒体基因组研究进展

对动物线粒体分子生物学的最新研究进展进行了较详细的阐述.从线粒体基因组(mtDNA)的研究背景出发,重点介绍了动物线粒体基因组的组成和结构特点,以及目前动物mtDNA与核基因组的关系、线粒体基因的遗传、起源和进化研究中的热点问题.     

Inheritance of organelle DNA sequences in a citrus-poncirus intergeneric cross

Many land plants deviate from the maternal pattern of organelle inheritance. In this study, heterologous mitochondrial and chloroplast probes were used to investigate the inheritance of organelle genomes in the progeny of an intergeneric cross. The seed parent was LB 1-18 (a hybrid of Citrus reticulata Blanco cv. Clementine x C. paradisi Macf. cv. Duncan) and the pollen parent was the cross-compatible species Poncirus trifoliata (L.) Raf. All 26 progeny examined exhibited maternal inheritance of plastid petA and petD loci. However, 17 of the 26 progeny exhibited an apparent biparental inheritance of mitochondrial atpA, cob, coxII, and coxIII restriction fragment length polymorphisms (RFLPs) and maternal inheritance of mitochondrial rrn26 and coxI RFLPs. The remaining nine progeny inherited only maternal mitochondrial DNA (mtDNA) configurations. Investigations of plant mitochondrial genome inheritance are complicated by the multipartite structure of this genome, nuclear gene control over mitochondrial genome organization, and transfer of mitochondrial sequences to the nucleus. In this study, paternal mtDNA configurations were not detected in purified mtDNA of progeny plants, but were present in progeny DNA preparations enriched for nuclear genome sequences. MtDNA sequences in the nuclear genome therefore produced an inheritance pattern that mimics biparental inheritance of mtDNA.     

Altered Levels of Mitochondrial DNA Are Associated with Female Age,Aneuploidy, and Provide an Independent Measure of Embryonic Implantation Potential

Mitochondria play a vital role in embryo development. They are the principal site of energy production and have various other critical cellular functions. Despite the importance of this organelle, little is known about the extent of variation in mitochondrial DNA (mtDNA) between individual human embryos prior to implantation. This study investigated the biological and clinical relevance of the quantity of mtDNA in 379 embryos. These were examined via a combination of microarray comparative genomic hybridisation (aCGH), quantitative PCR and next generation sequencing (NGS), providing information on chromosomal status, amount of mtDNA, and presence of mutations in the mitochondrial genome. The quantity of mtDNA was significantly higher in embryos from older women (P=0.003). Additionally, mtDNA levels were elevated in aneuploid embryos, independent of age (P=0.025). Assessment of clinical outcomes after transfer of euploid embryos to the uterus revealed that blastocysts that successfully implanted tended to contain lower mtDNA quantities than those failing to implant (P=0.007). Importantly, an mtDNA quantity threshold was established, above which implantation was never observed. Subsequently, the predictive value of this threshold was confirmed in an independent blinded prospective study, indicating that abnormal mtDNA levels are present in 30% of non-implanting euploid embryos, but are not seen in embryos forming a viable pregnancy. NGS did not reveal any increase in mutation in blastocysts with elevated mtDNA levels. The results of this study suggest that increased mtDNA may be related to elevated metabolism and are associated with reduced viability, a possibility consistent with the ‘quiet embryo’ hypothesis. Importantly, the findings suggest a potential role for mitochondria in female reproductive aging and the genesis of aneuploidy. Of clinical significance, we propose that mtDNA content represents a novel biomarker with potential value for in vitro fertilisation (IVF) treatment, revealing chromosomally normal blastocysts incapable of producing a viable pregnancy.     

The FANCM family Mph1 helicase localizes to the mitochondria and contributes to mtDNA stability

Mitochondria are membrane-bound organelles found in eukaryotic cells where they generate energy through the respiratory chain. They contain their own genome that encodes genes critical to the mitochondrial function, but most of their protein content is synthetized from nuclear encoded genes. Damages to the mtDNA can cause mutations and rearrangements with an impact on the respiratory functions of the cell. DNA repair factors are able to localize to mitochondria to restore mtDNA integrity and ensure its proper inheritance. We describe in this article the mitochondrial localization of the Mph1/FANCM helicase that serves critical roles in nuclear DNA repair processes. Mph1 localizes to mitochondria and its functions contribute to the mtDNA integrity under mtDNA damaging conditions.     

Normal specification of the extraembryonic lineage after somatic nuclear transfer

To examine the establishment and maintenance of trophectoderm (TE) lineage in somatic cloned blastocysts, the expression of Cdx2, a key molecule for specification of TE fate, was immunohistochemically examined simultaneously with Oct4 expression. Cloned mouse embryos were made by nuclear transfer using cumulus cells, tail-tip fibroblasts, and embryonic stem cells. After 96 h of culture, the rates of Oct4-expressing blastocysts were as low as 50% and 60% for cumulus and fibroblast clones, respectively. However, regardless of Oct4 expression, the majority of those cloned blastocysts (> 90%) normally expressed Cdx2. Thus, even though somatic cloned embryos have reduced potential to produce the inner cell mass lineage, the TE lineage can be established and maintained.     

Replicative advantage and tissue-specific segregation of RR mitochondrial DNA between C57BL/6 and RR heteroplasmic mice

To investigate the interactions between mtDNA and nuclear genomes, we produced heteroplasmic maternal lineages by transferring the cytoplasts between the embryos of two mouse strains, C57BL/6 (B6) and RR. A total of 43 different nucleotides exist in the displacement-loop (D-loop) region of mtDNA between B6 and RR. Heteroplasmic embryos were reconstructed by electrofusion using a blastomere from a two-cell stage embryo of one strain and an enucleated blastomere from a two-cell stage embryo of the other strain. Equivalent volumes of both types of mtDNAs were detected in blastocyst stage embryos. However, the mtDNA from the RR strain became biased in the progeny, regardless of the source of the nuclear genome. The RR mtDNA population was very high in most of the tissues examined but was relatively low in the brain and the heart. An age-related increase of RR mtDNA was also observed in the blood. The RR mtDNAs in the reconstructed embryos and in the embryos collected from heteroplasmic mice showed a different segregation pattern during early embryonic development. These results suggest that the RR mtDNA has a replicative advantage over B6 mtDNA during embryonic development and differentiation, regardless of the type of nuclear genome.     

Epigenetic asymmetry in the mammalian zygote and early embryo: relationship to lineage commitment?

Epigenetic asymmetry between parental genomes and embryonic lineages exists at the earliest stages of mammalian development. The maternal genome in the zygote is highly methylated in both its DNA and its histones and most imprinted genes have maternal germline methylation imprints. The paternal genome is rapidly remodelled with protamine removal, addition of acetylated histones, and rapid demethylation of DNA before replication. A minority of imprinted genes have paternal germline methylation imprints. Methylation and chromatin reprogramming continues during cleavage divisions, but at the blastocyst stage lineage commitment to inner cell mass (ICM) or trophectoderm (TE) fate is accompanied by a dramatic increase in DNA and histone methylation, predominantly in the ICM. This may set up major epigenetic differences between embryonic and extraembryonic tissues, including in X-chromosome inactivation and perhaps imprinting. Maintaining epigenetic asymmetry appears important for development as asymmetry is lost in cloned embryos, most of which have developmental defects, and in particular an imbalance between extraembryonic and embryonic tissue development.     

Blastocysts derived from adult fibroblasts of a rhesus monkey ( Macaca mulatta) using interspecies somatic cell nuclear transfer

In non-human primates, it is difficult to collect sufficient numbers of oocytes for producing identical embryos by somatic cell nuclear transfer (SCNT). Because of this factor, inter-species SCNT (iSCNT) using heterospecific oocytes is an attractive alternative approach. The objective of this study was to produce iSCNT-derived blastocysts using enucleated cow (Bos taurus) metaphase II oocytes and adult rhesus monkey (Macaca mulatta) fibroblasts. Ear skin tissue from a 6-year-old male rhesus monkey was collected by biopsy and fibroblasts were isolated. Immature cumulus-oocyte complexes from cow ovaries were collected and matured in vitro in Medium 199. The enucleated oocytes were reconstructed with rhesus monkey fibroblasts and iSCNT embryos were cultured in modified synthetic oviduct fluid in an atmosphere of 5-5.5% CO2 under various conditions (37-39 °C and 5-20% O2) to examine the effects of in vitro culture conditions. Most embryos were arrested at the 8- or 16-cell stage and only three blastocysts were derived in this way using iSCNT from a total of 1153 cultured activated embryos (0.26% production rate). Two of the three blastocysts were used for counting nuclear numbers using bisbenzimide staining, which were 51 and 24. The other iSCNT-derived blastocyst was used to analyse mitochondrial DNA (mtDNA) by PCR, and both rhesus monkey and cow mtDNA were detected. Although the development rate was extremely low, this study established that iSCNT using two phylogenetically distant species, including a primate, could produce blastocysts. With improvements in the development rate, it may be possible to produce rhesus monkey iSCNT-derived embryonic stem cell lines for studies on primate nucleus and cow mitochondria interaction mechanisms.     

Should I stay or should I go: Protection and maintenance of DNA methylation at imprinted genes

Recent findings shed light on the coordination of two fundamental, yet mechanistically opposing, processes in the early mammalian embryo. During the oocyte-to-embryo transition and early preimplantation development nuclear reprogramming occurs. This resetting of the epigenome in maternal and paternal pronuclei to a ground state is the essential step ensuring totipotency in the zygote, the first embryonic stage. Radical, global DNA demethylation, which occurs actively in the paternal and passively in the maternal genome, is a prominent feature of nuclear reprogramming; yet, this process poses a danger to a subset of methylated sequences that must be preserved for their germline to soma inheritance. Genomic imprinting and its importance were demonstrated three decades ago by a series of experiments generating non-viable mammalian uniparental embryos. Indeed, imprinted loci, gene clusters with parent-of-origin specific gene expression patterns, must retain their differential methylation status acquired during gametogenesis throughout embryogenesis and in adult tissues. It is just recently that the molecular players that protect/maintain imprinting marks during reprogramming in preimplantation embryos have been identified, in particular, an epigenetic modifier complex formed by ZFP57 and TRIM28/KAP1. The interaction of these and other molecules with the newly formed embryonic chromatin and imprinted genes is discussed and highlighted herein.     

Should I stay or should I go: Protection and maintenance of DNA methylation at imprinted genes

Recent findings shed light on the coordination of two fundamental, yet mechanistically opposing, processes in the early mammalian embryo. During the oocyte-to-embryo transition and early preimplantation development nuclear reprogramming occurs. This resetting of the epigenome in maternal and paternal pronuclei to a ground state is the essential step ensuring totipotency in the zygote, the first embryonic stage. Radical, global DNA demethylation, which occurs actively in the paternal and passively in the maternal genome, is a prominent feature of nuclear reprogramming; yet, this process poses a danger to a subset of methylated sequences that must be preserved for their germline to soma inheritance. Genomic imprinting and its importance were demonstrated three decades ago by a series of experiments generating non-viable mammalian uniparental embryos. Indeed, imprinted loci, gene clusters with parent-of-origin specific gene expression patterns, must retain their differential methylation status acquired during gametogenesis throughout embryogenesis and in adult tissues. It is just recently that the molecular players that protect/maintain imprinting marks during reprogramming in preimplantation embryos have been identified, in particular, an epigenetic modifier complex formed by ZFP57 and TRIM28/KAP1. The interaction of these and other molecules with the newly formed embryonic chromatin and imprinted genes is discussed and highlighted herein.     

Methylation reprogramming and chromosomal aneuploidy in in vivo fertilized and cloned rabbit preimplantation embryos

Active demethylation of the paternal genome but not of the maternal genome occurs in fertilized mouse, rat, pig, and bovine zygotes. To study whether this early demethylation wave is important for embryonic development, we have analyzed the global methylation patterns of both in vivo-fertilized and cloned rabbit embryos. Anti-5-methylcytosine immunofluorescence of in vivo-fertilized rabbit embryos revealed that the equally high methylation levels of the paternal and maternal genomes are largely maintained from the zygote up to the 16-cell stage. The lack of detectable methylation changes in rabbit preimplantation embryos suggests that genome-wide demethylation is not an obligatory requirement for epigenetic reprogramming. The methylation patterns of embryos derived from fibroblast and cumulus cell nuclear transfer were similar to those of in vivo-fertilized rabbit embryos. Fluorescence in situ hybridization with chromosome-specific BACs demonstrated significantly increased chromosomal aneuploidy rates in cumulus cell nuclear transfer rabbit embryos and embryos derived from nuclear transfer of rabbit fibroblasts into bovine oocytes compared with in vivo-fertilized rabbit embryos. The incidence of chromosomal abnormalities was correlated with subsequent developmental failure. We propose that postzygotic mitotic errors are one important explanation of why mammalian cloning often fails.