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，在输卵管环境培养的胚胎中，ATP合成酶（ATPase 6）、nadh -泛醌氧化还原酶链2 （ND2）、核糖体蛋白S6 （S6）、s -腺苷蛋氨酸脱羧酶（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（调节g蛋白信号2）和Tcl1基因在无输卵管组织培养的胚胎中强烈表达。ATP酶6和ND2基因由线粒体DNA编码，对ATP的产生至关重要。S6是参与蛋白质翻译的主要蛋白之一。这表明ATP合成相关基因的表达和跨国活动的增加可能需要在2细胞阶段维持体外正常发育。从第二年的结果来看，我们发现了几个在不同培养条件下表达差异的基因。在被测序的几个基因中，有几个未知的序列，其中一个（命名为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，我们获得了该新基因约1600bp的序列。测序后，利用RT-PCR检测该基因在各种体细胞组织和卵子中的表达模式。该基因在肝、肾、脾、心、肺和脑中均未表达，但在卵母细胞、胚胎和卵巢中均有表达。western blotting分析结果显示，细胞周期中Cyclin B的含量逐渐增加，在细胞周期的G2期后期达到峰值，但在有无输卵管组织培养的胚胎中，Cyclin B的含量差异不显著。这表明在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的表达量与2细胞期发育停滞没有直接关系。此外，对2细胞胚胎细胞周期蛋白B的免疫染色分析表明，有输卵管组织培养的胚胎细胞周期蛋白B在向M期过渡期间在细胞核中积累，而无输卵管组织培养的胚胎细胞周期蛋白B则持续定位于细胞质中。此外，在2细胞阻断的胚胎中，细胞周期蛋白B仍然存在于细胞质中。结论：该基因可能参与了胚胎的发育，特别是在胚胎发生的较早阶段；然而，该基因的详细功能仍有待确定。此外，蛋白分析数据表明，无输卵管组织培养的2细胞胚胎在G2/M过渡过程中存在Cyclin B核积累缺陷，这种缺陷可能导致离体培养的2细胞阻滞。少

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