Tokoro, Mikiko, Kawasumi, Miyuri, Nagai, Kohei, Ikegami, Haruka, Amano, Tomoko, Saeki, Kazuhiro, Hosoi, Yoshihiko, Iritani, Akira, and Matsumoto, Kazuya
Mammalian embryos undergo major change in their gene expression patterns throughout developmental stages of preimplantation. Following fertilization, the first change is the maternal transcripts to zygotic transition, in which transcripts expressed from the zygotic genome replace maternal transcripts that direct early development of fertilized embryos. The second change of major gene activation, named mid-preimplantation gene activation, precedes the dramatic morphological and functional changes from the morula to blastocyst stage. Currently, genome-wide expression and regulation of genes in preimplantation mouse embryos are explored using several methods, including mRNA differential display and microarray analysis. However, from the aspects of many molecular biological studies, mRNA and protein expressions are not necessarily correlated, as well as little is known regarding the proteome of preimplantation embryos. Here, we studied to obtain detailed information about protein profiles of mouse preimplantation embryos at each developmental stage. Oocytes and zygotes were collected from superovulated ICR female mice by conventional method. In brief, oocytes were collected 48h after PMSG injection, and the following stages collected at all stages (unfertilized egg, 1-cell, 2-cell, 4-cell, 8-cell, morula, and blastocyst stages). All samples were washed with PBS(-) and transferred into 1.5mL tubes and stored at 80°C until protein extracts. The eggs were extracted in lysis buffer containing 7M urea, 2M Thiourea, 4% CHAPS, 0.5% IPG buffer 0.05% TBP, BPB, and protease inhibitors. Isoelectric focusing of the egg extracts was performed using the Immobiline DryStrip of pH 3-10 (GE healthcare). The strip gels were placed on 10% SDS-polyacrylamide gels, stained with SYPRO Ruby (invitrogen). After gel image analysis, gel spots of unfertilized eggs were cut out for MS and MS/MS analysis. The peptide mass fingerprint data generated by the MALDI-TOF/TOF experiments were interpreted using MASCOT. Individual protein profiles during developmental stages were analyzed using TT900 (PerkinElmer). 2D gel electrophoresis of whole egg extract from 600, 10000, and 15000 unfertilized eggs was performed in order to identify as many proteins of the egg proteome as possible. 323 spots excised from the gel of unfertilized eggs were subjected to MALDI-TOF/TOF analysis. 105 spots were identified. The identified proteins were classified into 12 groups. Proteins concerning the protein metabolism and modification were present in the highest numbers. Other proteins were classified into factors such as nucleoside, nucleotide and nucleic acid metabolisms, carbohydrate metabolism, immunity and defense. In addition, we obtained the expression profiles of eggs during developmental stages. In conclusion, this proteome analysis of mouse embryos provides an opportunity to expand our knowledge of the molecular physiology of preimplantation embryos. Detailed comparison of the results from transcriptomics and proteimics are required to understand the molecular mechanism of the embryo development.