Studies on cell cycle and gene expression of mammalian preimplantation embryos cultured in an oviductal environment

第一年测序结果表明，输卵管环境下培养的胚胎中ATP合酶（ATPase 6）、NADH-泛醌氧化还原酶链2（ND2）、核糖体蛋白S6（S6）、S-腺苷甲硫氨酸脱羧酶（S-AMDC）和核自身抗原精子蛋白基因（NASP）存在差异表达。另一方面，RGS2（G蛋白信号传导调节2）和Tcl1基因在无输卵管组织培养的胚胎中强烈表达。 ATPase 6 和 ND2 基因由线粒体 DNA 编码，对于 ATP 的产生至关重要。 S6 是参与蛋白质翻译的主要蛋白质之一。这表明ATP合成相关基因的表达和2细胞阶段跨国活性的增加可能是维持体外正常发育所必需的。从第二年的结果来看，我们发现了几个在不同培养条件下差异表达的基因。在几个被测序的基因中，有几个未知的序列，其中一个（称为c-1）在没有输卵管环境培养的胚胎中强烈表达。利用EST-Walk和5'RACE，我们获得了大约1600bp的新基因序列。测序后，使用 RT-PCR 检查该基因在各种体细胞组织和卵中的表达模式。该基因在肝、肾、脾、心、肺和脑中未观察到表达，但在卵母细胞、胚胎和卵巢中观察到表达。Western blotting分析结果显示，Cyclin B的量在细胞周期中逐渐增加，并在细胞周期后期G2期达到峰值，但在有或无输卵管组织培养的胚胎中其量没有显着差异。这表明有或无输卵管组织培养的胚胎中Cdc2激酶活性的差异并不取决于Cyclin B表达量的差异，并且Cyclin B的量与2细胞阶段的发育停滞没有直接关系。此外，对2细胞胚胎中Cyclin B的免疫染色分析表明，在有输卵管组织培养的胚胎中，Cyclin B在向M期过渡期间在细胞核中积累，而在没有输卵管组织培养的胚胎中，Cyclin B持续定位在细胞质中。此外，还证明 Cyclin B 保留在 2 细胞封闭胚胎的细胞质中。结论表明该新基因可能参与胚胎的发育，特别是在胚胎发生的较早期阶段；然而，该基因的详细功能仍有待确定。此外，蛋白质分析数据表明，无输卵管组织培养的2细胞胚胎在G2/M转变过程中Cyclin B的核积累存在缺陷，该缺陷可能导致体外培养的2细胞阻断。较少的

The results of the sequencing in the first year indicated that ATP synthase (ATPase 6), NADH-ubiquinone oxidoreductase chain 2 (ND2), ribosomal protein S6 (S6), S-adenosylmethionine decarboxylase (S-AMDC) and nuclear autoantigenic sperm protein genes (NASP) were differentially expressed in the embryos cultured in the oviductal environment. On the other hand, RGS2 (regulation of G-protein signaling 2) and Tcl1 genes were strongly expressed in the embryos cultured without oviductal tissue. The ATPase 6 and ND2 genes are encoded by mitochondrial DNA and are essential for the production of ATP. S6 is one of the major proteins involved in protein translation. This indicates that the expression of ATP synthesis-related genes and an increase of transnational activity at the 2-cell stage may be required to maintain normal development in vitro. From the results of second year, we found several genes that are differentially expressed under different culture conditions. Of several genes that were … More sequenced, there were several unknown sequences, one (named c-1) of which was strongly expressed in embryos cultured without oviductal environment. Using EST-Walk and 5' RACE, we obtained about 1600bp sequence of the novel gene. After sequencing, the expression pattern of the gene in various somatic tissues and eggs is examined using RT-PCR. The expression of the gene was not observed in liver, kidney, spleen, heart, lung and brain but was observed in oocyte, embryo and ovary.From the result of western blotting analysis, the amount of Cyclin B gradually increased during cell cycle and peaked at the late G2 phase of cell cycle, however, the amount does not differ significantly in both embryos cultured with or without oviductal tissue. This indicates that the difference of Cdc2 kinase activity in embryos cultured with or without oviductal tissue does not depend on the difference of the amount of Cyclin B expression, and that the amount of Cyclin B is not directly in relation to the developmental arrest at the 2-cell stage. Furthermore, immunostaining analysis of Cyclin B in the 2-cell embryos revealed that the Cyclin B in embryos cultured with oviductal tissue accumulates in the nucleus during the transition to M phase, while that in embryos cultured without oviductal tissue localizes in the cytoplasm continuously. In addition, it is demonstrated that Cyclin B remains in the cytoplasm in 2-cell blocked embryos.In. conclusion, it is indicated that the novel gene may involve in the development of embryos, especially in relatively early stage of embryogenesis; however, the detail function of the gene remains to be determined. In addition, data obtained from protein analysis indicate that the 2-cell embryos cultured without oviductal tissue have defects in nuclear accumulation of Cyclin B during the G2/M transition and the defects may cause the 2-cell block of in vitro culture. Less