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

第一年的测序结果表明，ATP合成酶（ATPase 6），NADH-泛醌氧化还原酶链2（ND 2），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（NASP）基因在输卵管环境中培养的胚胎中差异表达。另一方面，RGS 2（G蛋白信号调节2）和Tcl 1基因在无输卵管组织培养的胚胎中强烈表达。ATP酶6和ND 2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质之一。这表明ATP合成相关基因的表达和在2-细胞阶段的跨国活动的增加可能是维持体外正常发育所必需的。从第二年的结果中，我们发现了几个在不同培养条件下差异表达的基因。几个基因， ...更多信息 测序结果表明，有几个未知序列，其中一个（命名为c-1）在无输卵管环境培养的胚胎中强表达。利用EST-Walk和5' RACE技术获得了该基因的全长约1600 bp的序列。测序后，使用RT-PCR检测该基因在各种体细胞组织和卵中的表达模式。Western blotting分析结果表明，Cyclin B的表达量在细胞周期中逐渐增加，在细胞周期的G2期晚期达到高峰，而在有输卵管组织和无输卵管组织培养的胚胎中，Cyclin B的表达量无显著性差异。这表明在有或无输卵管组织培养的胚胎中Cdc 2激酶活性的差异不依赖于Cyclin B表达量的差异，并且Cyclin B的量与2-细胞期的发育停滞没有直接关系。免疫组化分析表明，在有输卵管组织的2-细胞胚胎中，Cyclin B在向M期过渡的过程中主要集中在细胞核中，而在无输卵管组织的2-细胞胚胎中，Cyclin B主要集中在细胞质中。此外，还证实了Cyclin B在2-细胞阻滞的胚胎中仍然存在于细胞质中。结论：该基因可能参与胚胎发育，尤其是胚胎发育的早期阶段，但其具体功能尚不清楚。此外，蛋白质分析结果表明，在无输卵管组织的2-细胞胚胎培养中，细胞周期蛋白B在G2/M期的核内积累存在缺陷，这种缺陷可能导致体外培养的2-细胞阻滞。少

项目成果

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)

S. Hashimoto, N. Minami, M. Yamada and H. Imai: "An excessive concentration of glucose during in vitro maturation impairs the developmental competence of bovine oocytes after in vitro fertilization : relevance to intracellular reactive oxygen species and

S. Hashimoto、N. Minami、M. Yamada 和 H. Imai：“体外成熟过程中葡萄糖浓度过高会损害体外受精后牛卵母细胞的发育能力：与细胞内活性氧和

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 Booksellers Company，498 (1998)

H.Iwata, S.Akamatsu, N.Minami and M.Yamada: "Effects of antioxidants on the development of bovine IVM/IVF embryos in various concentration of glucose." Theriogenology. 50. 365-375 (1998)

H.Iwata、S.Akamatsu、N.Minami 和 M.Yamada：“不同浓度葡萄糖下抗氧化剂对牛 IVM/IVF 胚胎发育的影响”。

N. Minami, K. Sasaki, A. Aizawa, M. Miyamoto, A. Ohashi and H. Imai: "Gene expression in mouse 2-cell embryos developed in an oviductal environment in vitro."Biol. Reprod. 60. 131 (1999)

N. Minami、K. Sasaki、A. Aizawa、M. Miyamoto、A. Ohashi 和 H. Imai：“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。