Expression Profiling of Tetraploid Mouse Embryos in the Developmental Stages Using a cDNA Microarray Analysis

Abstract

Abstract. Tetraploid embryos normally develop to the blastocyst stage before implantation, but fail to survive after implantation. To better understand these characteristics of the tetraploid embryo, we produced tetraploid embryos by electrofusion and analyzed expressed genes that participated in mammalian embryogenesis using a DNA microarray analysis and a publicly available bioinformatics analysis of hatched tetraploid and diploid blastocysts. Transcriptome analysis with the DNA microarray revealed that the expression level of most genes was almost the same between diploid and tetraploid blastocysts. We found that the expression levels 2,800 genes were increased, but the expression levels over 1,600 genes were decreased in tetraploid blastocysts, which have a genomic composition identical to that of diploid blastocysts. In tetraploid blastocysts, the levels of 15 genes were decreased more than two-fold compared with the levels in diploid blastocysts. Among these downregulated genes, Ccnb1 (cyclin B1), which was decreased by 3-fold, seemed to play a particularly important role in the cellular organization of the tetraploid blastocyst. To classify the major functional classes of all the genes differentially expressed between diploid and tetraploid blastocysts, we employed a publicly available bioinformatics database, the VisuaL Annotation Display (VLAD). VLAD revealed several altered pathways in tetraploid blastocysts. Some of the enhanced biological processes were moderately involved with chromosome organization, while the suppressed processes were significantly involved with cell division and the mitotic cell cycle, metabolic processes and protein localization and transport. Taken together, our results revealed a large population of downregulated genes in tetraploid hatched blastocysts, and our convergent data suggest that the downregulation might primarily individualize the tetraploid phenotype in hatched blastocysts. Key words: Blastocyst, Microarray, Mouse, Tetraploid (J. Reprod. Dev. 55: [670][671][672][673][674][675] 2009) pontaneous duplication of the mammalian genome happens in less than 1% of fertilizations [1,2]. Tetraploids can be produced by either inhibition of cleavage or blastomere fusion [3][4][5][6]. Currently, the most commonly used method of producing tetraploid embryos in mice is electrofusion by electrical stimulation of 2-cellstage embryos [7,8].After blastomere fusion, tetraploid embryos can be cultured to the blastocyst stage by using a standard preimplantation embryo culture medium, which implies that tetraploid cells might have the normal function of many cells in the early embryo before implantation. Polyploids abort or die at various developmental stages after implantation [9,10]. Mouse tetraploid embryos have been reported to develop to midgestation, but abort spontaneously [11][12][13][14]. Although full-term tetraploid embryos have been reported by Snow et al. [3,15,16], Tarkowski et al. (1977) were unable to produce embryos beyond the tenth day of development [4].So far, ther...

Altered Gene Expression Profiles in Mouse Tetraploid Blastocysts

“…However, in this study, we compared gene expression profiles of the tetraploid blastocyst with those of parthenote-and diploid-derived blastocysts [19]. As a result of differences in the methods of statistical analysis, Kawaguchi's group may have found that genes involved in cell division and the cell cycle in tetraploid blastocysts were mainly downregulated, whereas the present study showed that the main differential expressions among tetraploid-, parthenote-and diploidderived blastocysts involved apoptosis-related genes.…”

“…Previously, Kawaguchi et al (2009) reported the profiling of global gene expression using microarray analysis to identify molecular and cellular pathways that were affected by tetraploid and diploid hatched blastocysts of mice. However, in this study, we compared gene expression profiles of the tetraploid blastocyst with those of parthenote-and diploid-derived blastocysts [19].…”

“…Although these modifications are caused by changes in Oct4/Cdx2 expression profiles, a major question is how the correct pluripotencyrelated genes kept active in the ICM are selectively inactivated upon switching to the tetraploid state through fusion at the 2-cell stage. In hatched tetraploid blastocysts, there were alterations in the metabolic processes and in protein localization and transport according to a cDNA microarray analysis [19]. To understand the mechanisms underlying early differentiation of tetraploid embryos during preimplantation development, we set out to compare the expression of multiple genes in tetraploid-, haploid-and diploidderived blastocysts.…”

Tetraploid Complementation Assay

“…11 DNA microarray comparison of tetraploid and diploid blastocysts revealed differences in expression levels, with genes involved in cell division and cell cycle significantly suppressed in the tetraploid embryos. 24 Another microarray analysis demonstrated that Oct4, Sox2, and Klf4 in tetraploid blastocysts were expressed at 10% of the level observed in diploid embryos, 25 which could contribute to the observed defects in development of tetraploid embryos.…”

A novel hypothesis for Alzheimer disease based on neuronal tetraploidy induced by p75NTR

“…Genome multiplication in mammals has been associated with alterations in a wide range of cellular processes, including enhanced protein turnover and transcriptional rate, shift from aerobic to anaerobic metabolism, and stress response. 130,131 Interestingly, neurons expressing Swedish mutant APP, known to trigger re-entering into the cell cycle, 37,38 show extensive alterations in gene expression including those involved in energy metabolism, nucleotide and protein synthesis, and oxidative stress, [132][133][134] suggesting that these changes in gene expression may be linked to tetraploidization. Transcription may also be altered at advanced stages of AD, when neurons show active cdc2/cyclin B1, since RNA polymerase II becomes phosphorylated by this kinase.…”