小鼠植入前胚胎紧密化相关基因Crg1的克隆

从已获得的运用抑制消减杂交技术(Suppression Subtractive Hybridization,SSH)分离、克隆和筛选代表8-细胞早期胚胎和紧密化8-细胞胚胎差别表达基因的ESTs片段(GenBank登录号：BQ740263、BQ740251)入手,经比较二者的同源性发现这两个EST末端反向互补,拼接成一个cDNA片段,经分析此序列包含一个完整的阅读框,提交给GenBank,登录号为AY134859。根据此序列设计引物从小鼠8-细胞紧密化胚胎cDNA中经PCR扩增出目的片段,克隆入pUCm—T载体后测序而获得全长cDNA,为小鼠植入前胚胎紧密化相关基因Crg1,分析比较证明Crg1基因与AY134859基本吻合。Crg1基因的cDNA全长为810bp,只有一个外显子,编码由150个氨基酸组成,分子量理论值为17．67kD的蛋白质。与最新的小鼠基因组工作草图进行电子杂交,该基因被定位在小鼠的14号染色体上。RT—PCR实验证明在小鼠植入前各个时期的胚胎、小鼠胚胎干细胞中均有表达,在小鼠胚胎成纤维细胞中没有表达。半定量RT—PCR实验证明Crg1基因在紧密化胚胎中表达较8—细胞胚胎高。采用Northern—blot手段分析Crg1基因在成年小鼠的8种组织中的表达情况,结果表明该基因只在小鼠卵巢中有微弱的表达,转录本大小为1．2kh,而在成年小鼠的脑、心脏、肾、睾丸、肝脏、肺、脾等中没有表达。研究表明,Crg1基因可能与小鼠胚胎紧密化及保持细胞的全能性相关。     

小鼠着床前胚胎发育中调控因子的时序表达及功能

哺乳动物着床前胚胎发育最基本的问题之一是高度分化的卵母细胞如何过渡到全能性的卵裂球。这一转换过程受到了多种调控因子正或负的调控作用,这些特定调控因子在着床前胚胎发育过程中呈现出较为显著的时空表达特征,它们对早期胚胎的进一步正常发育有重要的作用。研究这些调控因子在着床前胚胎发育中的表达模式和调控机制,将有助于我们对早期胚胎发育机制的进一步了解。作者主要对近些年来有关生长因子与细胞因子（如：PAF、IL－1、IGF、MIF）以及特定转录因子（如：SP1、TBP、mTEF、eIF、myc、c-jun等）在小鼠着床前胚胎发育中的时空表达及其相应功能的研究做一简要介绍。     

Response of preimplantation murine embryos to heat shock as modified by developmental stage and glutathione status

Summary Objectives were to characterize developmental changes in response to heat shock in the preimplantation mouse embryo and to evaluate whether ability to synthesize glutathione is important for thermal resistance in mouse embryos. Heat shock (41° C for 1 or 2 h) was most effective at disrupting development to the blastocyst stage when applied to embryos at the 2-cell stage that were delayed in development. Effects of heat shock on ability of embryos to undergo hatching were similar for 2-cell, 4-cell, and morula stage embryos. The phenomenon of induced thermotolerance, for which exposure to a mild heat shock increases resistance to a more severe heat shock, depended upon stage of development and whether embryos developed in vitro or in vivo. In particular, induced thermotolerance was observed for morulae derived from development in vivo but not for 2-cell embryos or morulae that developed in culture. Administration of buthionine sulfoximine to inhibit glutathione synthesis did not increase thermal sensitivity of 2-cell embryos or morulae but did reduce subsequent development of 2-cell embryos at both 37° and 41° C. In summary, changes in the ability of 2-cell through morula stages to continue to develop following a single heat shock were generally minimal. However, 2-cell embryos delayed in development had reduced thermal resistance, and therefore, maternal heat stress may be more likely to cause mortality of embryos that are already compromised in development. There were also developmental changes in the capacity of embryos to undergo induced thermotolerance. Glutathione synthesis was important for development of embryos but inhibition of glutathione synthesis did not make embryos more susceptible to heat shock.     

Comparative ultrastructure of the yolk material in preimplantation stages of the hamster,mouse, and rat embryos

Yolk material of preimplanation stages of embryos of the hamster, mouse, and rat were examined by a standardized electron microscopical procedure. The material was encountered as fibrils, scattered more or less densely in the cytoplasm. In the hamster, the material was present in large masses and the fibrils had a chain-like appearance when cut longitudinally. The ultrastructure of the fibrils was compatible with a helical pattern. The fibrils had a width of about 40 nm and the pitch (the axial distance of the repeating unit) was about 30 nm. In the mouse, the yolk material was dispersed in the cytoplasm forming small plaque-like groups. Also, in this species the fibrils were chain-like but smaller than in the hamster. The fibrils were often closely situated, resulting in images with varying crystalline appearances. In the rat, the yolk appeared as light areas occupying a substantial part of the cytoplasm. The fibrils in the yolk plaques were sparse and diffusely outlined. They were thinner than the fibrils of the mouse-yolk material, did not display any helical pattern at the resolution used, but showed a periodicity.     

Analysis of chromatin structure in mouse preimplantation embryos by fluorescent recovery after photobleaching

Zygotes are totipotent cells that have the ability to differentiate into all cell types. It is believed that this ability is lost gradually and differentiation occurs along with the progression of preimplantation development. Here, we hypothesized that the loose chromatin structure is involved in the totipotency of one-cell stage embryos and that the change from loose to tight chromatin structure is associated with the loss of totipotency. To address this hypothesis, we investigated the mobility of eGFP-tagged histone H2B (eGFP-H2B), which is an index for the looseness of chromatin, during preimplantation development based on fluorescent recovery after photobleaching (FRAP) analysis. The highest mobility of eGFP-H2B was observed in pronuclei in 1-cell stage embryos and mobility gradually decreased during preimplantation development. The decrease in mobility between the 1- and 2-cell stages depended on DNA synthesis in 2-cell stage embryos. In nuclear transferred embryos, chromatin in the pseudopronuclei loosened to a level comparable to the pronuclei in 1-cell stage embryos. These results indicated that the mobility of eGFP-H2B is negatively correlated with the degree of differentiation of preimplantation embryos. Therefore, we suggest that highly loosened chromatin is involved in totipotency of 1-cell embryos and the loss of looseness is associated with differentiation during preimplantation development.     

Quantitative analysis of retromer complex-related genes during embryo development in the mouse

The retromer complex is a heteropentameric protein unit associated with retrograde transport of cargo proteins from endosomes to the trans-Golgi network. Functional silencing study of the Vps26a gene indicated the important role of the retromer complex during early developmental stages in the mouse. However, individual expression patterns and quantitative analysis of individual members of the retromer complex during the early developmental stages has not been investigated. In this study, we conducted quantitative expression analysis of six retromer complex genes (Vps26a, Vps26b, Vps29, Vps35, Snx1, and Snx2) and one related receptor gene (Ci-mpr) during the eleven embryonic stages with normal MEF (mouse embryonic fibroblast) and Vps26a(-/-) MEF cells. Remarkably, except for Vps26a (maternal expression pattern), all tested genes showed maternal-zygotic expression patterns. And five genes (Vps26b, Vps29, Vps35, Snx2, and Ci-mpr) showed a pattern of decreased expression in Vps26a(-/-) MEF cells by comparative analysis between normal MEF and Vps26a(-/-) MEF cells. However, the Snx1 gene showed a pattern of increased expression in Vps26a(-/-) MEF cells. From our results, we could assume that retromer complex-related genes have important roles during oocyte development. However, in the preimplantation stage, they did not have significant roles.     

New member of the Snf1/AMPK kinase family,Melk, is expressed in the mouse egg and preimplantation embryo

The initial phase of mammalian preimplantation development is directed by stored maternal mRNAs and their encoded proteins, yet most of the molecules controlling this process have not been described. We have used differential display analysis of cDNA libraries prepared from unfertilized eggs and preimplantation embryos to isolate three maternal cDNAs that represent novel genes exhibiting different patterns of expression during this developmental period. One of these, Melk, encodes a protein with a kinase catalytic domain and a leucine zipper motif, a new member of the Snf1/AMPK family of kinases. This gene product may play a role in the signal transduction events in the egg and early embryo. Mol. Reprod. Dev. 47:148–156, 1997. © 1997 Wiley-Liss, Inc.