Submission Number: NAO-4-25-6

Abstract Number: 370

A NOVEL GENE OF MOUSE EARLY PREIMPLANTATION EMBRYOS EXPRESSED IN AN ENVIRONMENT DEPENDENT MANNER.

Naojiro Minami* ¹, Masakazu Miyamoto ¹, Kana Sasaki ¹, Akira Aizawa ², Akihiro Ohashi* ¹ and Hiroshi Imai ¹

Laboratory of Reproductive Physiology, Graduate School of Agriculture, Kyoto University Kyoto, Japan ¹
Maebashi Institute of Animal Science, Livestock Improvement Association Japan, Inc. Maebashi, Japan ²

Abstract:
When mouse embryos are cultured from 1-cell stage in vitro, the development of embryos with the exception of some inbred strain and their F1 hybrids, is arrested specifically at the G2 phase of the second cell cycle, a phenomenon that has been referred to as the "2-cell block". The developmental arrest at the 2-cell stage, however, is overcome by culturing embryos with oviductal tissue. In the present study, to examine the difference of gene expression in 2-cell embryos cultured with or without oviductal tissue, we performed fluorescein differential display (FDD-RT-PCR). Oocytes were recovered from female CD-1 mice to produce IVF embryos. Fertilized embryos were cultured with or without oviductal tissue for 32-34 h. RT-PCR was performed using 5'-AAgCTTgATTgCC-3' and fluorescein-labeled oligo dT primers. The fluorescein-labeled products were resolved on a 6% denaturing gel. The gel on its glass plate was directly subjected to a fluorescein image scanner to detect the expressed genes, and the differentially expressed bands were excised and sequenced. From the results of FDD-RT-PCR, 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 5f 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. In addition, the difference of the expression in 2-cell embryos was already observed at 1 h after first cleavage, and no expression was observed after the 4-cell stage. This indicates that the 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, as it is reported that the transcription from zygotic gene is mainly activated at the 2-cell stage and the translational activity increases at this stage, it is inferred that there is a relationship between zygotic gene activation and developmental arrest at the 2-cell stage. .

Keywords: mouse embryo, gene expression, 2-cell block, differential display

This abstract is being presented at: 8:00 AM in session:
Preimplantation Embryo/Fetus