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.     

Role of histone H3 lysine 27 methylation in X inactivation

The Polycomb group (PcG) protein Eed is implicated in regulation of imprinted X-chromosome inactivation in extraembryonic cells but not of random X inactivation in embryonic cells. The Drosophila homolog of the Eed-Ezh2 PcG protein complex achieves gene silencing through methylation of histone H3 on lysine 27 (H3-K27), which suggests a role for H3-K27 methylation in imprinted X inactivation. Here we demonstrate that transient recruitment of the Eed-Ezh2 complex to the inactive X chromosome (Xi) occurs during initiation of X inactivation in both extraembryonic and embryonic cells and is accompanied by H3-K27 methylation. Recruitment of the complex and methylation on the Xi depend on Xist RNA but are independent of its silencing function. Together, our results suggest a role for Eed-Ezh2-mediated H3-K27 methylation during initiation of both imprinted and random X inactivation and demonstrate that H3-K27 methylation is not sufficient for silencing of the Xi.     

X-Chromosome inactivation in cloned mouse embryos

To study whether cloning resets the epigenetic differences between the two X chromosomes of a somatic female nucleus, we monitored X inactivation in cloned mouse embryos. Both X chromosomes were active during cleavage of cloned embryos, followed by random X inactivation in the embryo proper. In the trophectoderm (TE), X inactivation was nonrandom with the inactivated X of the somatic donor being chosen for inactivation. When female embryonic stem cells with two active X chromosomes were used as donors, random X inactivation was seen in the TE and embryo. These results demonstrate that epigenetic marks can be removed and reestablished on either X chromosome during cloning. Our results also suggest that the epigenetic marks imposed on the X chromosomes during gametogenesis, responsible for normal imprinted X inactivation in the TE, are functionally equivalent to the marks imposed on the chromosomes during somatic X inactivation.     

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.     

机械分割的小鼠桑椹胚和囊胚的发育能力

利用机械方法二等份分割小鼠的桑椹胚、早期囊胚和晚期囊胚,并在体外培养分割的半胚到晚期囊胚。将这些囊胚移植到假孕受体鼠子宫内以评价他们附植后发育的能力。在体外培养中发现,囊胚期分割的胚胎比桑椹胚期分割的胚胎有较高的成活率。通过附植前胚胎细胞数量分析得知,囊胚期半胚的细胞数量大约是对照组的一半,但内细胞团细胞与整个胚胎细胞的数量比不变。虽然从半胚发育到囊胚期的胚胎附植率仅略低于对照组,但半胚形成胎儿的能力却显著低于全胚。组织学分析表明,附植后的这种低成活率现象是由于缺乏卵柱发育造成的,而此卵柱则与半胚中的内细胞团的细胞数量减少有关。移植到第3天假孕受体鼠中的半胚,附植后的发育情况明显地比移植到第4天受体中的好。     

Research on Growth Behavior of Embryos for Bovine and Murine on Primary Murine Embryos Fibroblast Cell Feeder Layer

The difference in growth behavior between bovine embryos and murine embryos was studied on PMEF(primary murine embryos fibroblast)feeder layer. The results showed as follows: With embryos having attached, bovine embryonic trophoblast formed a transparent membranous structure covering on inner cell mass (ICM), however, murine embryonic trophoblast formed disc structure. Bovine embryos formed four kinds of ICM colonies with different morphology including the mass-like, the net-like, the stream-like and the mixture-like colonies. Compared with Murine ICM, the bovine ICM grew more fast. So, the bovine ICM was passaged at first after a culture of approximately 5 - 6 days in vitro, but murine ICM was passaged at first after an attachment of 3 - 4 days on PMEF feeder layer. The mixture colonies of bovine ICM differentiated very early, while the others differentiated very late. Most ICM-like mass of Bovine grew in a defined spot, but bovine ICMs like stream and ICMs like net proliferated fast and dispersed quickly. We found that the single blastomeres derived from late bovine morula and late murine morula formed sub-blastophere; moreover, the bovine ICM cell would differentiate rapidly if the trophoblast was removed.     

牛胚胎在小鼠胎儿成纤维细胞饲养层的生长行为

 比较了牛胚胎与小鼠胚胎在体外培养过程中的生长行为，结果表明，牛滋养层细胞与内细胞团（ｉｎｎｅｒ ｃｅｌｌｍａｓｓ，简称ＩＣＭ）连接不紧密，其迅速增殖，形成半透明囊腔结构；小鼠胚胎滋养层与ＩＣＭ密切相连，形成盘状结构，并推移原代小鼠胎儿成纤维细胞（Ｐｒｉｍａｒｙ ｍｕｒｉｎｅ ｅｍｂｒｙｏｓｆｉｂｒｏｂｌａｓｔ，ＰＭＥＦ）饲养层。牛ＩＣＭ形态呈现多样性，表现为团状、条状、网状和混合型；小鼠ＩＣＭ形态单一，多呈圆盘状。体外培养的牛胚胎发育速度比小鼠胚胎迟缓，牛ＩＣＭ初次传代时间为胚胎体外培养开始后５～６ｄ，小鼠ＩＣＭ初次传代时间为胚胎体外培养后３～４ｄ。牛ＩＣＭ分化程度由小到大依次为团状ＩＣＭ、条状ＩＣＭ、混合型正 和网状ＩＣＭ。牛和小鼠胚胎附着率及ＩＣＭ克隆率由大到小均依次为孵化胚、囊胚和桑褡胚，但桑褡胚和囊胚附着率和ＩＣＭ克隆率均无显著差异（Ｐ＞０．０５）。与全胚相比较，牛和小鼠裸胚（无透明带或透明带不完整的胚胎）贴壁时间提前，但ＩＣＭ形成率和ＩＣＭ的生长行为均无显著差异；小鼠和牛桑椹胚卵裂球（中、晚期）体外分化抑制培养，形成亚囊胚而不能获得 ＥＳ细胞；剥离牛胚胎滋胚层细胞，ＩＣＭ细胞更易分化。     

Molecular coupling of Xist regulation and pluripotency

During mouse embryogenesis, reversion of imprinted X chromosome inactivation in the pluripotent inner cell mass of the female blastocyst is initiated by the repression of Xist from the paternal X chromosome. Here we report that key factors supporting pluripotency-Nanog, Oct3/4, and Sox2-bind within Xist intron 1 in undifferentiated embryonic stem (ES) cells. Whereas Nanog null ES cells display a reversible and moderate up-regulation of Xist in the absence of any apparent modification of Oct3/4 and Sox2 binding, the drastic release of all three factors from Xist intron 1 triggers rapid ectopic accumulation of Xist RNA. We conclude that the three main genetic factors underlying pluripotency cooperate to repress Xist and thus couple X inactivation reprogramming to the control of pluripotency during embryogenesis.     

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.     

Research on Isolation and Clone of Embryonic Stem Cell-Like in Bovine

Bovine embryonic stem cell would be invaluable for researching the aspect of animal cloning, production transgenic animal and discussion of gene function in vitro. With the object of establishing an effective culture system for isolation and clone of bovine pluripotent stem cell, we cultured bovine embryos and mouse embryos including morula blastula and hatached blastula and obtained animal ICM on Primary marine embryonic fibroblast (Primary murine embryonic fibroblast, PMEF) feeder layer with tissue medium(DMEM supplemented with 15ml/100ml NBS ,0.1μmol/L Na2SeO3, 0. 1mmol/L β-mercaptoethanol, 1 000ng/ml LIF,10 ng/ml IGF, 1mmol/L necessary amino acid and 1mmol/L L-glutamine), then, we obtained mouse ICM and bovine ICM. Moreover, we isolated and cloned the 6 passage bovine ES like cells(12 cell lines) and 9 passage marine ES like cells (52 cell lines) deriving from bovine ICM and murine ICM respectively on the feeder layer of PMEF by disaggregating ICM and ES cell clones of bovine and murine into smaller clumps through digesting with 0. 125g/100ml trypsin and 0.02g/100ml EDTA and scattering with a glass needle. The pluripotency of both murine and bovine ES like cells was identified with morphological character, histochemistry identification, karyotype analysis and differentiation of ES cells in vitro or in vivo. This result showed that bovine embryonic stem cell and murine embryonic stem cell had developmental pluripotency.     

巨噬细胞集落刺激因子(GM-CSF)对小鼠胚胎体外发育的影响

以小鼠自然受精与体外受精胚胎为模型,探索鼠源巨噬细胞集落刺激因子(GM-CSF)在小鼠胚胎早期发育过程中的生理功能。分别使用添加不同剂量(对照组:0 ng.mL-1;试验组:0.5、2和10 ng.mL-1)GM-CSF的化学限定培养基连续培养小鼠自然受精、体外受精原核期与2细胞期胚胎,检测其胚胎着床前发育效率(卵裂率、囊胚率)及囊胚的质量(囊胚总细胞数、ICM/总细胞数的比率、凋亡指数)。结果发现,对自然受精胚胎而言,原核时期添加不同剂量的GM-CSF,其卵裂率、囊胚总细胞数、ICM/总细胞数的比率在各组之间均差异不显著,但10 ng.mL-1试验组的囊胚率显著低于对照组(61.6±5.1)%(P0.05);2细胞时期添加不同剂量的GM-CSF,其囊胚率、囊胚总细胞数、ICM/总细胞数的比率在各组之间均差异不显著,但试验组中的囊胚凋亡指数均显著低于对照组(P0.05)),并且试验组中2 ng.mL-1的囊胚凋亡指数最低,显著低于0.5 ng.mL-1(P0.05),其他试验组之间的囊胚凋亡指数没有统计学上的差异性。对体外受精胚胎来说,原核时期添加GM-CSF,其卵裂率、囊胚率、囊胚总细胞数、ICM/总细胞数的比率在各组之间均无显著性差异;在2细胞时期,10 ng.mL-1组的囊胚率显著低于对照组及其他2个试验组(P0.05),但各组之间的囊胚总细胞数、ICM/总细胞数的比率均无显著性差异。本研究结果表明,在化学限定培养基中添加一定剂量的GM-CSF有利于改善小鼠自然受精与体外受精囊胚的质量,但剂量过高可能不利于小鼠受精胚胎的早期发育。     

Autosomal dominant mutations affecting X inactivation choice in the mouse

X chromosome inactivation is the silencing mechanism eutherian mammals use to equalize the expression of X-linked genes between males and females early in embryonic development. In the mouse, genetic control of inactivation requires elements within the X inactivation center (Xic) on the X chromosome that influence the choice of which X chromosome is to be inactivated in individual cells. It has long been posited that unidentified autosomal factors are essential to the process. We have used chemical mutagenesis in the mouse to identify specific factors involved in X inactivation and report two genetically distinct autosomal mutations with dominant effects on X chromosome choice early in embryogenesis.     

Germ cell specification in mice

Specification of germ cells in mice occurs relatively late in embryonic development. It is initiated by signals that induce expression of Blimp1, a key regulator of the germ cell, in a few epiblast cells of early postimplantation embryos. Blimp1 represses the incipient somatic program in these cells and promotes progression toward the germ cell fate. Blimp1 may also have a role in the maintenance of early germ cell characteristics by ensuring their escape from the somatic fate as well as possible reversion to pluripotent stem cells.     

无血清培养山羊内细胞团的研究

对32只关中奶山羊进行超排处理,获得胚胎263枚,其中囊胚122枚。采用机械法、酶消化法和免疫外科法分离囊胚83枚,得到内细胞团(ICM)65个。将ICM培养于小鼠胎儿成纤维细胞饲养层上,研究含血清和无血清条件下ICM的增殖规律,并比较添加不同生长因子对ICM增殖的影响。结果表明,无血清培养时,ICM细胞增殖相对较慢,分化减少;添加LIF、bFGF、两种同时添加或均不添加对原代ESC样集落形成率的影响不显著(P>0.05),但添加10 ng/mL bFGF稍微有助于ICM增殖。山羊ICM适宜于无血清培养。     

牛与小鼠胚胎在分化抑制培养系统中的行为比较

在自制培养基中 ,以原代小鼠胎儿成纤维细胞为饲养层 ,观察比较了牛与小鼠胚胎在体外分化抑制培养体系中的生长行为。结果表明 ,牛囊胚一般培养 36～ 6 0 h后孵化脱带 ,96～ 12 0 h后贴壁 ,12 0～ 144 h为ICM传代的最佳时刻。小鼠囊胚一般培养 12～ 2 4h孵化脱带 ,2 4～ 48h贴壁 ,72～ 96 h为传代的最佳时刻。牛胚胎贴附于饲养层上生长 ,极易从饲养层上剥离 ;小鼠胚胎镶嵌在饲养层中 ,滋养层细胞与饲养层细胞间连接紧密。牛ICM色深发黑 ,集落隆起程度较低 ;小鼠 ICM呈暗黄色 ,有呈柱状增殖的趋势。牛滋养层呈网状 ,小鼠滋养层为较致密的单层薄膜     

Epigenetic reprogramming in plant and animal development

Epigenetic modifications of the genome are generally stable in somatic cells of multicellular organisms. In germ cells and early embryos, however, epigenetic reprogramming occurs on a genome-wide scale, which includes demethylation of DNA and remodeling of histones and their modifications. The mechanisms of genome-wide erasure of DNA methylation, which involve modifications to 5-methylcytosine and DNA repair, are being unraveled. Epigenetic reprogramming has important roles in imprinting, the natural as well as experimental acquisition of totipotency and pluripotency, control of transposons, and epigenetic inheritance across generations. Small RNAs and the inheritance of histone marks may also contribute to epigenetic inheritance and reprogramming. Reprogramming occurs in flowering plants and in mammals, and the similarities and differences illuminate developmental and reproductive strategies.     

LIF与心肌条件液在小鼠ES细胞分离中的差异比较

通过比较LIF和大鼠心肌条件液在小鼠ES细胞分离培养过程中的差异,从而选择较为合适的培养液用于小鼠ES细胞的分离培养与深入研究.取怀孕3.5 d小鼠囊胚,培养于小鼠胎儿成纤维细胞饲养层上,然后根据ES细胞培养液的不同分成2组,一组添加LIF的ES细胞培养液,另一组添加由大鼠心肌条件液组成的ES细胞培养液.结果显示,小鼠ICM的孵出率在心肌条件液中为76.30%,LIF条件液中为59.35%,两者差异极显著(p＜0.01);在LIF条件液中比心肌条件液中能较早地分离出ICM,时间差分别为11、11、12、10和12 h,平均为11.2 h;对传代的ES细胞集落,培养48 h时周边出现分化现象的ES细胞集落所占的比例在心肌条件液中为51.55%,LIF条件液中为31.69%,两者差异显著(p＜0.05),第五代小鼠ES细胞核型正常率在心肌细胞条件液中为78.6%,稍高于LIF条件液中的76%,差异不显著.     

Transient homologous chromosome pairing marks the onset of X inactivation

Mammalian X inactivation turns off one female X chromosome to enact dosage compensation between XX and XY individuals. X inactivation is known to be regulated in cis by Xite, Tsix, and Xist, but in principle the two Xs must also be regulated in trans to ensure mutually exclusive silencing. Here, we demonstrate that interchromosomal pairing mediates this communication. Pairing occurs transiently at the onset of X inactivation and is specific to the X-inactivation center. Deleting Xite and Tsix perturbs pairing and counting/choice, whereas their autosomal insertion induces de novo X-autosome pairing. Ectopic X-autosome interactions inhibit endogenous X-X pairing and block the initiation of X-chromosome inactivation. Thus, Tsix and Xite function both in cis and in trans. We propose that Tsix and Xite regulate counting and mutually exclusive choice through X-X pairing.