Factors affecting early embryonic development in cattle: relevance for bovine cloning

Female infertility represents a major challenge for improving the production efficiency in the dairy industry. Historically, fertility has declined whereas milk yield has increased tremendously due to intensive genetic selection. In vivo evidence reveals about 60% pregnancy loss takes place during the first month following fertilization. Meanwhile, early embryo development is significant for somatic cell nuclear transfer in cattle as a large proportion of cloned embryos fail to develop beyond peri-implantation stage. Oocyte quality is of utmost importance for the early embryo to develop to term for both fertilized and cloned embryos. Epigenetic reprogramming is a key process occurring after fertilization and critical roles of epigenetic modifiers during preimplantation development are now clear. Incomplete epigenetic reprogramming is believed to be a major limitation to cloning efficiency. Treatment of cloned embryos with epigenetic modifying drugs (e.g., Trichostatin A) could greatly improve cloning efficiency in both mice and cattle. Recently, the rapid progress in high-throughput sequencing technologies has enabled detailed deciphering of the molecular mechanisms underlying these events. The robust efficiency of genomic editing tools also presents an alternative approach to the functional annotation of genes critical to early development.     

Epigenetic dynamics of imprinted X inactivation during early mouse development

The initiation of X-chromosome inactivation is thought to be tightly correlated with early differentiation events during mouse development. Here, we show that although initially active, the paternal X chromosome undergoes imprinted inactivation from the cleavage stages, well before cellular differentiation. A reversal of the inactive state, with a loss of epigenetic marks such as histone modifications and polycomb proteins, subsequently occurs in cells of the inner cell mass (ICM), which give rise to the embryo-proper in which random X inactivation is known to occur. This reveals the remarkable plasticity of the X-inactivation process during preimplantation development and underlines the importance of the ICM in global reprogramming of epigenetic marks in the early embryo.     

Genetics. Reprogramming X inactivation

Inactivation of one of the two X chromosomes occurs in all cells of female adult mice so that genes are expressed from only one X chromosome. In a Perspective, Clerc and Avner describe an elegant series of experiments in mouse embryos cloned from adult and embryonic female cell nuclei (Eggan et al.) that reveal how the inactivation state of the X chromosomes is reprogrammed.     

拟水狼蛛染色体的初步观察

以附肢形成之前的蜘蛛胚胎细胞为材料,经秋水仙素处理、纸渗、固定、制片及染色,对拟水狼蛛的体细胞染色体进行了初步观察．结果表明：拟水狼蛛的染色体数为：雄性２ｎ＝２６,雌性２ｎ＝２８,性别决定机制为Ｘ１Ｘ２Ｏ．Ｃ－带分析表明,所有染色体都为端着丝粒染色体,端部有特异性染色质存在．     

胚胎移植方法和受体母猪因素对克隆猪生产效率的影响

【目的】建立高效的胚胎移植技术体系,以提高克隆猪的生产效率。【方法】比较不同移植方法和不同受体母猪状况的胚胎移植分娩率和克隆猪的出生效率,利用体外发育能力相似的不同品种和发育阶段的克隆胚胎混合移植确定最佳的受体母猪发情同期时间。【结果】克隆胚胎经由输卵管伞移植比输卵管打孔移植具有更高的移植分娩率和克隆猪效率（2.2% 和0.4%,P＜0.01）,而胚胎移植后辅助人工授精会降低克隆效率（0.6% 和 2.2%,P＜0.01）。利用经产母猪作为移植受体比青年猪受体具有更高的克隆效率（3.0%和0.8%,P＜0.01）和平均窝产仔数（（5.5±0.7）头和（2.7±0.3）头,P＜0.05）。受体母猪发情时间比胚胎激活时间晚12-36 h组的克隆效率分别为显著（P＜0.05）和极显著（P＜0.01）,高于晚0 h和早12-24 h 组的克隆效率（2.0%、0.5%和0%）。最佳的受体母猪发情时间为晚于克隆胚胎激活后24 h,克隆效率达3.0%。【结论】选择自然发情时间晚于克隆胚激活后24 h的经产母猪为受体,通过输卵管伞端移植胚胎,能够获得较高的克隆效率,是猪克隆胚胎移植的较理想方法。     

延边黄牛体细胞克隆囊胚染色体标本制备的研究

[目的]探讨适合延边黄牛体细胞克隆胚胎染色体标本制备的方法。[方法]利用延边黄牛体细胞克隆产生的囊胚制备染色体标本,采用传统的细胞遗传学方法,将延边黄牛体细胞克隆囊胚分别在含有0.11、.0和10.0μg/ml浓度秋水仙素的CR1aa培养液中继续培养4或6 h,以使细胞分裂停留在中期。处理后的囊胚移入柠檬酸钠低渗液中,室温下处理15~20 min。将低渗后的单个胚胎转移至载玻片上,固定并用体积分数为25%的Giemsa溶液染色1~5 h,蒸馏水冲洗脱色,于室温干燥。显微镜下计算胚胎细胞数和有丝分裂指数以及标本制备质量。[结果]结果表明,1.0μg/ml秋水仙素处理6 h后的染色体标本制备效果较为理想。[结论]该研究为制备延边黄牛体细胞克隆囊胚染色体标本提供适宜的试验参数。     

马-牛无透明带卵异种克隆的研究

 【目的】开展异种克隆,为濒危动物的保护、细胞核重编程与核质互作研究提供新途径。【方法】以马耳成纤维细胞为供体,牛卵母细胞为受体,采用无透明带手工克隆方法构建异种胚胎,比较不同的激活液、培养液、体细胞同期方式、核移植方法对马-牛异种克隆胚胎体外发育的影响。【结果】（1）A23187 + 6-DMAP联合激活获得的克隆胚胎发育较好,囊胚率2.60%;（2）克隆胚胎于mCR1aa中的卵裂率和桑葚率显著高于CR1aa和SOF（83.65%vs 77.49%,74.87%;22.36%vs19.37%,18.98%,P＜0.05）,且发育到囊胚（2.72%）,CR1aa与SOF间无显著差异;（3）供体细胞经血清饥饿或接触抑制处理后,获得的异种克隆胚的融合率、卵裂率、桑葚率和囊胚率无显著差异;（4）无透明带手工克隆法的融合率显著高于显微注射法（90.33% vs 72.94%）,卵裂率、桑葚率和囊胚率无显著差异;（5）马-牛异种克隆胚与牛同种克隆胚比较,融合率和卵裂率无差别,桑葚率和囊胚率有显著差异（21.78%vs63.52%;2.71%vs37.48%）。【结论】牛卵母细胞能支持马体细胞去分化,进行核重编程。但由于二者的物种距离较远,异种克隆胚胎的发育能力远低于同种克隆胚胎。     

Clonal propagation of primate offspring by embryo splitting

Primates that are identical in both nuclear and cytoplasmic components have not been produced by current cloning strategies, yet such identicals represent the ideal model for investigations of human diseases. Here, genetically identical nonhuman embryos were produced as twin and larger sets by separation and reaggregation of blastomeres of cleavage-stage embryos. A total of 368 multiples were created by the splitting of 107 rhesus embryos with four pregnancies established after 13 embryo transfers (31% versus 53% in vitro fertilization controls). The birth of Tetra, a healthy female cloned from a quarter of an embryo, proves that this approach can result in live offspring.     

不同培养液对牛体细胞核移植胚胎发育的影响

旨在为牛体细胞克隆胚胎体外培养体系的建立奠定基础。利用体细胞核移植技术生产牛重构胚,将重构胚随机分成2组,分别用mSOF和G1.5/G2.5培养,统计2种培养液对牛核移植克隆胚胎发育的影响,用TUNEL荧光检测系统检测2个培养组胚胎细胞凋亡情况,同时用Real time RT-PCR检测热应激蛋白基因Hsp70和凋亡相关基因Bax在2个培养组的相对表达水平。两组的卵裂率分别为85.6%和87.0%、囊胚率分别为22.9%和27.1%,差异都不显著(P>0.05),但G1.5/G2.5组的8-细胞形成率显著高于mSOF组(P<0.05);mSOF组凋亡率为12.1%±1.9%,显著高于G1.5/G2.5组(P<0.05),G1.5/G2.5组的Hsp70和Bax相对表达量显著低于mSOF组(P<0.05)。结果表明,G1.5/G2.5培养液更有利于牛体细胞核移植胚胎的体外发育。     

Gene body-specific methylation on the active X chromosome

Differential DNA methylation is important for the epigenetic regulation of gene expression. Allele-specific methylation of the inactive X chromosome has been demonstrated at promoter CpG islands, but the overall pattern of methylation on the active X(Xa) and inactive X (Xi) chromosomes is unknown. We performed allele-specific analysis of more than 1000 informative loci along the human X chromosome. The Xa displays more than two times as much allele-specific methylation as Xi. This methylation is concentrated at gene bodies, affecting multiple neighboring CpGs. Before X inactivation, all of these Xa gene body-methylated sites are biallelically methylated. Thus, a bipartite methylation-demethylation program results in Xa-specific hypomethylation at gene promoters and hypermethylation at gene bodies. These results suggest a relationship between global methylation and expression potentiality.     

延边黄牛核移植胚胎的活性氧含量的测定

为了研究延边黄牛体细胞克隆胚胎内部的活性氧水平,以延边黄牛孤雌激活胚胎为对照,用2,′7′-二氯荧光素二乙酸酯和二氢乙锭两种染料分别对体细胞克隆胚胎进行染色,测定其内部超氧阴离子和过氧化氢含量。结果表明,2细胞期处理组间超氧阴离子水平差异不显著(P0.05),其它细胞期体细胞克隆组均显著高于孤雌激活组(P0.05),过氧化氢水平各细胞期体细胞克隆胚胎均显著高于孤雌激活胚胎(P0.05)。因此,延边黄牛体细胞克隆胚胎氧化损伤水平显著高于孤雌激活胚胎。     

In vivo and in vitro development of Tibetan antelope (Pantholops hodgsonii) interspecific cloned embryos

The Tibetan antelope is endemic to the Tibetan Plateau, China, and is now considered an endangered species. As a possible rescue strategy, the development of embryos constructed by interspecies somatic cell nuclear transfer (iSCNT) was examined. Tibetan antelope fibroblast cells were transferred into enucleated bovine, ovine and caprine oocytes. These cloned embryos were then cultured in vitro or in the oviducts of intermediate animals. Less than 0.5% of the reconstructed antelope-bovine embryos cultured in vitro developed to the blastocyst stage. However, when the cloned antelope-bovine embryos were transferred to caprine oviducts, about 1.6% of the embryos developed to the blastocyst stage. In contrast, only 0.7% of the antelope-ovine embryos developed to the morula stage and none developed to blastocysts in ovine oviducts. The treatment of donor cells and bovine oocytes with trichostatin A did not improve the embryo development even when cultured in the oviducts of ovine and caprine. When the antelope-bovine embryos, constructed from oocytes treated with roscovitine or trichostatin A, were cultured in rabbit oviducts 2.3% and 14.3% developed to blastocysts, respectively. It is concluded that although some success was achieved with the protocols used, interspecies cloning of Tibetan antelope presents difficulties still to be overcome. The mechanisms resulting in the low embryo development need investigation and progress might require a deeper understanding of cellular reprogramming.     

Mammalian ZFY sequences exist in reptiles regardless of sex-determining mechanism

In some reptiles, egg incubation temperature determines whether the embryo hatches as male or female; in others, sex chromosomes determine sex. A cloned gene (ZFY) representing the putative testis-determining factor in mammals was hybridized to genomic DNA of reptiles with sex chromosomes and to DNA of reptiles with temperature-dependent sex determination. No sex differences in hybridization patterns were observed. Hybridization of ZFY to polyadenylated RNA indicates that reptilian versions of this gene are expressed in embryos of both sexes during the temperature-sensitive period. If these highly conserved sequences are important in reptilian sex determination, then temperature-dependent and genotypic sex determination may have a similar molecular basis. For reptiles with XX/XY or ZZ/ZW systems, the absence of sex differences in hybridization patterns raises the question of whether the ZFY sequences reside on their sex chromosomes.     

牛-山羊异种体细胞克隆胚胎线粒体形态超微结构观察#br#

 【目的】通过对正常受精胚胎、山羊同克隆胚胎(山羊去核卵与布尔山羊耳皮肤成纤维细胞重组)以及牛-山羊异种克隆胚胎(山羊去核卵与布尔山羊耳皮肤成纤维细胞重组)中线粒体超微结构的观察与比对,从亚细胞水平揭示异种克隆胚胎出现异常的原因,为异种克隆技术的进一步研究提供新思路。【方法】利用透射电镜分别对山羊正常受精胚胎、山羊同种克隆胚胎及牛-山羊异种克隆胚胎的原核、2-细胞、4-细胞、8-细胞及桑椹胚阶段的早期胚胎进行线粒体超微结构观察。【结果】山羊自然受精胚胎、山羊同种克隆胚胎的线粒体均为帽状,随着胚胎的发育,线粒体由电子密度高、嵴少的未成熟状态发育为电子密度低、嵴丰富的成熟线粒体,而牛-山羊异种克隆胚胎从2-细胞胚胎至桑椹胚都存在一种具有多个分叶的线粒体,但这种形态异常的线粒体同样也能够形成电子密度低、嵴丰富的成熟线粒体。【结论】牛卵母细胞与山羊体细胞之间不能有效的进行核-质互作,本研究中具体表现为出现多分叶形线粒体,从而影响牛-山羊异种克隆胚胎的发育。     

Expression of the mammalian X chromosome before and after fertilization

The activity of hypoxanthine-guanine phosphoribosyltransferase in unfertilized mouse ova and in mouse embryos at the two-cell stage is proportional to the number of X chromosomes present during oogenesis. This indicates that the enzyme is X-linked in the mouse and that inactivation of the X chromosome does not occur during oogenesis. However, the genetic dosage effect of the X chromosomes is not present after the increase in hypoxanthine-guanine phosphoribosyltransferase activity in the late morula and the blastocyst stages. These results indicate that the X-linked enzyme lacuts is expressed sometimne after fertilization but before the morula stage.     

小型猪体细胞克隆试验

[目的]建立体细胞克隆小型猪的技术平台并了解克隆效率。[方法]运用体细胞核移植技术,以小型猪胎儿成纤维细胞作为核移植供体细胞,以体外成熟卵母细胞作为核移植受体细胞进行体细胞克隆猪生产,并利用STR序列对所出生的克隆猪进行个体识别。[结果]将重构胚胎移植到5头代孕母猪体内,其中一头代孕母猪成功产下2头克隆小型猪。经过个体识别试验证实克隆小型猪来自供核细胞,与代孕母猪无直接亲缘关系。[结论]克隆小型猪的成功出生表明所建立的技术平台适于小型猪的克隆。     

Reactivation of the paternal X chromosome in early mouse embryos

It is generally accepted that paternally imprinted X inactivation occurs exclusively in extraembryonic lineages of mouse embryos, whereas cells of the embryo proper, derived from the inner cell mass (ICM), undergo only random X inactivation. Here we show that imprinted X inactivation, in fact, occurs in all cells of early embryos and that the paternal X is then selectively reactivated in cells allocated to the ICM. This contrasts with more differentiated cell types where X inactivation is highly stable and generally irreversible. Our observations illustrate that an important component of genome plasticity in early development is the capacity to reverse heritable gene silencing decisions.     

Production of early monozygotic twin bovine embryos in vitro by the blastomere separation and coculture technique

The objective of this study was to establish an efficient system of producing early monozygotic twin bovine embryos in vitro using the blastomere separation and coculture technique. In this study, early eight-cell embryos were chosen to optimize the separation method, and multi-coculture tactics were applied to improve the efficiency of this production system. Bovine embryo blastomeres(groups of at least 30 at the eight-cell stage) were separated into eight segments(to regard an eight-cell embryo as a tangerine, a blastomere as one segment) and one, two and four segments(blastomeres) were cultured respectively in microwells on the bottom of the four-well dish(Nunc, Denmark) with 400 μL of culture medium under paraffin oil. Four different types of coculture tactics(cocultured with nothing, intact embryos, bovine cumulus cells(b CCs), intact embryos b CCs) were applied to the group of four segments(blastomeres). Finally, diameter and inner cell mass(ICM):trophectoderm(TE) cell ratio was measured as a criterion to assess the quality of the twin embryos which were derived from bovine separated blastomeres. Our results showed that rate of blastocyst formation of the four segments group was significantly greater than one or two group(P0.05). In addition, rate of blastocyst formation was significantly increased when the four segments were cocultured with intact embryo b CCs(P0.05). Although the ICM, TE and total cells of blastocysts derived from separated blastomeres was less than the control group from intact embryo(P0.05), more important quality indicator of the blastocyst diameter and ICM:TE cell ratio was similar between our experimental group and the control group(P0.05). Thus, these results suggest that combined with intact embryos b CCs coculture system, culturing four isolated segments(blastomeres) per microwell is an efficient system of producing early monozygotic twin bovine embryos. Furthermore, our results also indicate that the quality of blastocysts derived from separated blastomere may be similar to those derived from intact eight-cell embryos.     

Microinjected DNA from the X chromosome affects sex determination in Caenorhabditis elegans

The signal for sex determination in the nematode Caenorhabditis elegans is the ratio of the number of X chromosomes to the number of sets of autosomes (X/A ratio). By previous genetic tests, elements that feminized chromosomal males appeared to be widespread on the X chromosome, but the nature of these elements was not determined. In experiments to define a feminizing element molecularly, cloned sequences were added to chromosomally male embryos by microinjection into the mother. Three different X-chromosome clones, including part of an actin gene, part of a myosin heavy chain gene, and all of two myosin light chain genes, feminize chromosomal males. Both somatic and germline aspects of sex determination are affected. In contrast, about 40 kilobases of nematode autosomal DNA, phage lambda DNA, and plasmid pBR322 DNA do not affect sex determination. A feminizing region was localized to a maximum of 131 base pairs within an intron of the X-linked actin gene; a part of the gene that does not have this region is not feminizing. The results suggest that short, discrete elements found associated with many X-linked genes may act as signals for sex determination in C. elegans.