228 EXPRESSION OF zag1 IN MOUSE PRE-IMPLANTATION EMBRYOS

Abstract

Embryonic gene activation (EGA) first occurs during the second half of the mouse 1-cell embryo (Latham KE 1999 Int. Rev. Cytol. 193, 71–124). Moreover, precise regulation of EGA is considered to be essential for normal embryo development. To understand the molecular basis for the regulation of EGA, we have focused on the identification and functional characterization of genes activated at the late 1-cell stage of the mouse embryo. Recently, we have identified and isolated a novel gene, termed zag1 (zygotic activating gene 1), transcribed specifically at the EGA, using a fluoro-differential display method with oocytes and embryos at 15 h post-insemination. Messenger RNA of zag1 expressed at lower level in the oocyte than that in the embryo at 15 h post-imsemination. In this study, we investigated the potential function of zag1 by analysis of mRNA expression and protein distribution in mouse tissues and pre-implantation embryos. Nucleotide sequence analysis of zag1 cDNA revealed that the open reading frame of 1726 bps encodes a protein of 575 amino acids with a predicted molecular mass of 66 kDa. The deduced amino acid sequence indicated that zag1 protein might be a soluble protein with a bipartite nuclear targeting sequence, a NACHT NTP domain, and an APT/GTP binding site motif as a predicted functional domain. Two μg of Poly(A)⁺ RNA from various tissues of adult mice were subjected to Northern blot analysis using the mouse zag1 cDNA probe. We detected this gene abundantly expressed in mouse testis and ovary by approximately 2- to 3-fold compared with one in other mouse tissues (heart, liver, kidney, lung, brain, skeletal muscle, and spleen). zag1 transcript and protein, as assessed by RT-PCR and immunoblotting, respectively, were slightly present in ovulated oocytes, gradually decreased in the early 1-cell embryos, but re-expressed in the late 1-cell and early 2-cell stage embryos which coincided with the mouse EGA. Subsequent to microinjection of an expression vector encoding zag1-enhanced green fluorescent protein (EGFP), fused protein into male pronucleus of 1-cell embryos was detected in the nuclei of 2-cell embryos. These findings suggest that zag1 may be functionally associated with early embryonic development.

This study was supported by a Grant-in-Aid for the 21st Century COE Program of the Japan MEXT, and by a grant from the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence of the JST.