136 FUNCTIONAL ANALYSIS OF RHOPHILIN-2 GENE IN PRE-IMPLANTATION MOUSE EMBRYO

Abstract

Zygotic gene activation (ZGA), which starts at the G2 phase at the 1-cell stage (Latham 1999), promotes the reprogramming of gene expression and is critical for the subsequent development of pre-implantation embryos. We have investigated the molecule function of many gene clusters, DD clones obtained by Differential-Display assays for ovulated eggs at the M II stage, and 1-cell embryos at the G2 phase. The differential expression of rhophilin-2 shown in DD assays was also confirmed by 3 independent real-time PCR analyses (P < 0.05). For these reasons, in this study, we focused on the rhophilin-2 gene, which regulates cytoskeletal organization (Peck et al. 2002). At first, we identified a protein that interacts with the Rhophilin-2 protein by a yeast 2-hybrid system. To confirm the interaction between Rhophilin-2 and the putative protein obtained by a yeast two-hybrid system, we used a co-immunoprecipitaion assay. We also investigated the expression profiles of rhophilin-2 and the transcripts of the identified protein in ovary and pre-implantation embryos using real-time PCR and immunofluorescence (IF) analysis. The ICR mice at 48 h after PMSG priming were primed with hCG, and ovaries were collected at 7 h after hCG priming. Pre-implantation embryos were collected at 1-cell, 2-cell, and 4-cell stages, and cDNA was produced by mRNA isolated from 10 oocytes or embryos in each group and was subjected to real-time PCR using a TaqMan Probe system (ABI). Sectioned ovaries and pre-implantation embryos were analyzed by IF analysis using antibody of Rhophilin-2 and the identified protein. This is the first report that GABA receptor-association protein (GABARAP) was identified as a protein that interacts with Rhophilin-2, as a result of using the yeast 2-hybrid system and subsequent co-immunoprecipitation assay. After fertilization, transcript levels of rhophilin-2 significantly decreased from the 1-cell stage to the 2-cell stage (P < 0.05), but transcript levels of GABARAP significantly increased from the 1-cell stage to the 2-cell stage (P < 0.05). The IF analysis revealed localization of Rhophilin-2 and GABARAP at the nucleolus of all follicle stage in the ovary. Moreover, Rhophiln-2 and GABARAP were found to be localized on the microtubules of 1-cell and 2-cell embryos, but no signal of Rhophilin-2 was detected in 4-cell embryos. These results suggest that Rhophilin-2 protein regulates the cytoskeletal organization in 1-cell to 2-cell embryos and is involved in the molecular mechanism of cell division by coupling with GABARAP. This study was supported by a Grant-in-Aid for the 21st Century COE Program of the Japan Mext and by a grant for the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technology Excellence of the JST.