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

输卵管环境下培养的哺乳动物植入前胚胎的细胞周期和基因表达研究

• 批准号: 10660270
• 负责人: MINAMI Naojiro
• 金额: $ 2.24万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10660270/
• 关键词: mouse embryo; gene expression; cell cycle; oviductal environment; differential display; cyclin B; mouse embryo; 2-cell stage; gene expression; oviductal environment

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

第一年测序结果表明，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-腺苷甲硫氨酸脱羧酶（S-AMDC）和核自身抗原精子蛋白基因(NASP) 在输卵管环境中培养的胚胎中差异表达。 G 蛋白信号传导 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 S6 的产生至关重要，S6 是参与蛋白质的主要蛋白质之一。这表明，在体外维持正常发育可能需要ATP合成相关基因的表达和跨国活性的增加。在不同培养条件下差异表达的几个基因中，有几个非常未知的序列，其中一个（称为 c-1）在使用 EST-Walk 和 5' RACE 培养的胚胎中表达。我们获得了大约1600bp的新基因序列，通过RT-PCR检测了该基因在各种体组织和卵中的表达模式，在肝脏、肾脏、脾脏等组织中未观察到该基因的表达。心脏、肺和脑中也有观察到，但在卵母细胞、胚胎和卵巢中也有观察到。从western blotting分析的结果来看，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，但其量并没有增加。在有或没有输卵管组织培养的胚胎中，Cdc2 激酶活性的差异不依赖于 Cyclin B 表达量的差异。 Cyclin B 的量与 2 细胞阶段的发育停滞没有直接关系。此外，2 细胞胚胎中 Cyclin B 的免疫染色分析表明，与输卵管组织一起培养的胚胎中的 Cyclin B 在细胞核中积累。在向M期过渡期间，而在没有输卵管组织培养的胚胎中，Cyclin B持续定位于细胞质中。此外，证明Cyclin B在2-细胞中保留在细胞质中。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相对早期阶段，但该基因的详细功能仍有待确定。分析表明，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细胞阻滞。

项目成果

N.Minami: "Reproductive Biology Update-Novel Tools for Assessment of Environmental Toxicity"Shoukadoh Booksellers Company. 498 (1998)

N.Minami：“生殖生物学更新-评估环境毒性的新工具”Shoukadoh 书商公司。

A.Ohashi: "Effect of an oviductal environment on the expression and cellular localization of cell cycle regulatory proteins in mouse 2-cell embryos"Biol.Reprod.. 60. 131 (1999)

A.Ohashi：“输卵管环境对小鼠 2 细胞胚胎中细胞周期调节蛋白的表达和细胞定位的影响”Biol.Reprod.. 60. 131 (1999)

N.Minami: "Gene expression in mouse 2-cell embryos developed in an oviductal environment in vitro"Biol.Reprod.. 60. 131 (1999)

N.Minami：“在体外输卵管环境中发育的小鼠 2 细胞胚胎中的基因表达”Biol.Reprod.. 60. 131 (1999)

N.Minami: "Gene expression in mouse 2-sell embryos developed in an oviductal environment in vitro"Biol.Reprod.. 60. 159 (1999)

N.Minami：“在体外输卵管环境中发育的小鼠 2-sell 胚胎中的基因表达”Biol.Reprod.. 60. 159 (1999)

S.Hashimoto: "Ultrasound-guided follicle aspiration : effects of the frequency of a linear transvaginal probe on the collection of bovine oocytes"Theriogenology. 52. 131-138 (1999)

S.Hashimoto：“超声引导的卵泡抽吸：线性经阴道探针的频率对牛卵母细胞收集的影响”动物发生学。