Involvement of LIMK1/2 in actin assembly during mouse embryo development

LIMKs (LIMK1 and LIMK2) are serine/threonine protein kinases that involve in various cellular activities such as cell migration, morphogenesis and cytokinesis. However, its roles during mammalian early embryo development are still unclear. In the present study, we disrupted LIMK1/2 activity to explore the functions of LIMK1/2 during mouse early embryo development. We found that p-LIMK1/2 mainly located at the cortex of each blastomeres from 2-cell to 8-cell stage, and p-LIMK1/2 also expressed at morula and blastocyst stage in mouse embryos. Inhibition of LIMK1/2 activity by LIMKi 3 (BMS-5) at the zygote stage caused the failure of embryo early cleavage, and the disruption of LIMK1/2 activity at 8-cell stage caused the defects of embryo compaction and blastocyst formation. Fluorescence staining and intensity analysis results demonstrated that the inhibition of LIMK1/2 activity caused aberrant cortex actin expression and the decrease of phosphorylated cofilin in mouse embryos. Taken together, we identified LIMK1/2 as an important regulator for cofilin phosphorylation and actin assembly during mouse early embryo development.     

Derivation of Human Trophoblast Stem Cells

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家兔胚泡ICM在体外培养系统中的行为

一、用显微外科方法从兔早期胚泡分离来的ICM在所用的体外培养系统中的行为,因ICM的生长状态而不同。呈带状生长的ICM从近端向远处延伸,而细胞的分化则从远端逐渐向近端进行,它具有明显的极性。细胞的分化秩序井然,层次分明。因此呈带状生长的ICM适用于进行细胞分化和细胞谱系的研究。二、呈团块状生长的ICM,没有明显的极性,细胞分化开始于团块的外表面,逐渐向中央进行。细胞分化开始稍晚一些,速度也较慢,这种生长状态的ICM适用于分离胚胎干细胞。三、兔子胚外内胚层从ICM分化来,它经历了二次分化的过程。第一次发生于培养后第三天(相当于交配后第七天),形成第一胚外内胚层-胚外体壁内胚层,它向远处迁移,远离原始外胚层。培养后四天(相当于交配后第八天),形成第二种胚外内胚层-胚外脏壁内胚层,它尾随体壁内胚层向远处迁移,其后缘的部分细胞向原始外胚层附近的滋胚层下面侵润。四、胚外体壁内胚层和脏壁内胚层的细胞是由少数决定了的和离开ICM的细胞分化和增殖而来。五、文中讨论了早期胚胎细胞分化的谱系问题。     

兔胚泡肽合成片段及其与着床有关的生理功能

本研究测定了兔胚泡肽（ＲＢＰ２）的氨基酸序列,它由５４个氨基酸组成,其序列的第４位氨基酸至第３４位氨基酸与兔子宫珠蛋白Ｎ端氨基酸序列完全相同。用计算机软件系统模拟分析,从５４个氨基酸中选取了抗原性最高的一段氨基酸（ａａ２０－ａａ３４）,然后人工合成了含有１５个氨基酸的小肽片段（ＳＰＦ）。研究表明：用溴脱氧尿嘧啶掺入淋巴细胞的方法测定了ＳＰＦ对细胞增殖作用的影响,当浓度范围在８０μｇ／ｍｌ至３２０μ     

双炔失碳酯对兔移植胚泡着床的影响

本文用胚泡移植技术验证了双炔失碳酯对子宫内膜的影响是胚泡不能着床的重要原因。将 29只胚龄4d的兔胚泡移植到给药后假孕4d的子宫,结果没有一只胚泡着床;而对照组的5只受卵兔,移植了41只胚泡,有26只着床成功。 为了了解双炔失碳酯对黄体的影响,在5只妊娠兔上,从交配后1—7d与对照组比较血清孕酮浓度的变化,结果表明:对照组的血清孕酮浓度随着妊娠天数显著升高,妊娠第7天时达 17.35±2.12ng/ml,而给药组升高不明显,第7天仅 1.83±1.03ng/ml,为对照组的1/9。已知足够的孕酮浓度对妊娠的维持和胚泡的成功着床具有十分重要的作用。由此推测,双炔失碳酯抑制孕酮的分泌势必影响子宫内膜的发育,从而阻碍胚泡的着床。     

BMP4 plays a role in apoptosis during human preimplantation development

Comprehensive understanding of lineage differentiation and apoptosis processes is important to increase our knowledge of human preimplantation development in vitro. We know that BMP signaling is important for different processes during mammalian development. In mouse preimplantation embryos, BMP signaling has been shown to play a role in the differentiation into extra‐embryonic trophectoderm (TE) and primitive endoderm (PE). In this study, we aimed to investigate the effect of bone morphogenetic protein 4 (BMP4) supplementation on human preimplantation embryos cultured in vitro. The BMP4 treatment impaired human blastocyst formation. No differences in the expression of the early lineage markers NANOG, CDX2, GATA3, and GATA6 were found between BMP4‐treated embryos and controls. Instead, BMP4 supplementation triggered apoptosis in the human blastocyst. We focused on P53, which is known to play a major role in the apoptosis. In BMP4‐treated embryos, the P53 responsive gene expression was not altered; however, the P53 deacetylase SIRT1 was downregulated and acetylated P53 was increased in mitochondria. Altogether, our findings suggest that BMP4 plays a role in the apoptosis during human preimplantation development.     

Improvement of in vitro and early in utero porcine clone development after somatic donor cells are cultured under hypoxia

Genetically engineered pigs serve as excellent biomedical and agricultural models. To date, the most reliable way to generate genetically engineered pigs is via somatic cell nuclear transfer (SCNT), however, the efficiency of cloning in pigs is low (1–3%). Somatic cells such as fibroblasts frequently used in nuclear transfer utilize the tricarboxylic acid cycle and mitochondrial oxidative phosphorylation for efficient energy production. The metabolism of somatic cells contrasts with cells within the early embryo, which predominately use glycolysis. We hypothesized that fibroblast cells could become blastomere‐like if mitochondrial oxidative phosphorylation was inhibited by hypoxia and that this would result in improved in vitro embryonic development after SCNT. In a previous study, we demonstrated that fibroblasts cultured under hypoxic conditions had changes in gene expression consistent with increased glycolytic/gluconeogenic metabolism. The goal of this pilot study was to determine if subsequent in vitro embryo development is impacted by cloning porcine embryonic fibroblasts cultured in hypoxia. Here we demonstrate that in vitro measures such as early cleavage, blastocyst development, and blastocyst cell number are improved (4.4%, 5.5%, and 17.6 cells, respectively) when donor cells are cultured in hypoxia before nuclear transfer. Survival probability was increased in clones from hypoxic cultured donors compared to controls (8.5 vs. 4.0 ± 0.2). These results suggest that the clones from donor cells cultured in hypoxia are more developmentally competent and this may be due to improved nuclear reprogramming during somatic cell nuclear transfer.