|Budget Amount *help
¥3,500,000 (Direct Cost : ¥3,500,000)
Fiscal Year 1999 : ¥1,800,000 (Direct Cost : ¥1,800,000)
Fiscal Year 1998 : ¥1,700,000 (Direct Cost : ¥1,700,000)
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
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