72 HISTONE METHYLATION AND GENE EXPRESSION PATTERNS IN PRE-IMPLANTATION EMBRYOS DERIVED FROM INTRA- AND INTER-SPECIES NUCLEAR TRANSFER

Abstract

The differential epigenetic changes in embryos from different species provide a model to study how the nucleus from one species interacts with cytoplasm from another species. In this study we examined histone methylation at lysine 9 of histone 3 (K9H3) and lysine 20 of histone H4 (K20H4) and the expression levels of three early development-related genes (Oct-4, Hsp 70.1 and Hprt) in individual intra- and inter-species cloned and control embryos at the 1-, 2-, 4- and 8-cell stages. Mouse fetal fibroblast (MFF) nuclei were transferred into mouse, bovine, or rabbit oocytes. As control, we used in vivo derived (mouse and rabbit) or in vitro-produced (bovine) embryos. Histone methylation was detected by anti-MeK9H3 and anti-MeK20H4 antibodies. Gene expression analysis was performed using a quantitative RT-PCR technique (Daniels et al. 2000 Biol. Reprod. 63, 1034-1040). Data were analyzed by Student's t-test. No embryos from inter-species cloning (MFF-bovine and MFF-rabbit) survived beyond the 8-12 cell stage. MFF-mouse and MFF-bovine embryos exhibited demethylation of K9H3 and K20H4 at the 2-cell stage and the methylation level was increased at the 4-cell stage, but no demethylation was observed at the 2-cell stage of MFF-rabbit embryos and the methylation level in these embryos was significantly higher than that of in vivo rabbit embryos. The level of Oct-4 mRNA was low at the 1- and 2-cell stages of in vivo mouse embryos and increased at the 8-cell stage. No significant increase in Oct-4 transcript was detected at the 8-cell stage of inter-species cloned embryos. The expression of Hsp 70.1 in in vivo mouse embryos was increased at the 2-cell stage and decreased to a level similar to that in the zygote at the 8-cell stage. In cloned embryos, Hsp 70.1 transcripts were also increased at the 2-cell stage, but there was no significant decrease of Hsp70.1 mRNA abundance at the 8-cell stage of inter-species embryos as compared to the corresponding 2-cell stage. For MFF-mouse embryos, Hsp 70.1 expression was increased at the 2-cell stage, but at the 8-cell stage the transcript level was at the level similar to that in inter-species clones. Hprt expression was increased at the 8-cell stage of in vivo mouse embryos. The dynamic change of Hprt transcript in MFF-mouse embryos was not significantly different from that of in vivo mouse embryos, but no significant change of Hprt expression occurred in the development of MFF-bovine and MFF-rabbit embryos. Differential epigenetic characteristics of mouse somatic nucleus after transfer into oocytes from different species suggest the existence of incompatibilities of nuclear-cytoplasm interaction between distantly related species. This abnormal interaction at the time of genome activation may affect normal development.

This work was supported by the Bayerische Forschungsstiftung and by Therapeutic Human Polyclonals, Inc.