61 BOVINE OOPLASM PARTIALLY REMODELS CHIMPANZEE SOMATIC NUCLEI FOLLOWING SOMATIC CELL NUCLEAR TRANSFER

Abstract

Interspecies somatic cell nuclear transfer (iSCNT) is a tool to address basic questions related to nucleus–cytoplasm interactions between different species. This approach may have practical applications, such as endangered species preservation. To date, live offspring have been obtained only when closely related species were used, such as cow/gaur, cow/buffalo, and domestic cat/wild cat. Several groups have reported preimplantation development of iSCNT embryos using a diverse collection of species; however, further development of these embryos either failed or was not reported. The aim of this study was to investigate developmental events that take place during reprogramming of a somatic nucleus in an enucleated oocyte from a distant species. Our experimental model was cow/chimpanzee iSCNT. We confirmed the donor cell genome origin in SCNT embryos by karyotyping. Embryonic genome activation (EGA) was monitored by Br-UTP incorporation assay and RT-PCR analysis of 6 genes. Chromatin remodeling events such as histone acetylation, histone methylation, and DNA methylation were observed by immunocytochemistry along with examination of Oct4 and Nanog promoter methylation by bisulfite sequencing. As an indicator of embryonic metabolism, ATP levels and apoptotic status of embryonic nuclei were determined by ATP detection and TUNEL assays, respectively. Development of iSCNT embryos did not progress beyond the 8- to 16-cell stage, whereas their cow/cow SCNT counterparts readily developed to the blastocyst stage. Karyotype analysis showed a donor-specific, diploid genomic content in most of the iSCNT nuclei (46 chromosomes). Major EGA takes place at the 16- and 8-cell stages in cow and chimpanzee embryos, respectively. However, our analyses showed a partial EGA pattern in iSCNT embryos at the 8-cell stage, as indicated by lower levels of Br-UTP incorporation and irregular expression of certain embryonic genes compared to that in cow/cow SCNT embryos. GAPDH, Oct4, and Stella transcripts were detected, while Nanog, Glut1, and DSC2 genes failed to be expressed in chimpanzee somatic nuclei, as opposed to robust expression observed for all 6 genes in bovine SCNT embryos. Dynamic chromatin remodeling events as monitored by H3K27 methylation and H4K5 acetylation-specific antibodies were similar in both intra- and interspecies SCNT embryos from 1-cell to 8–16-cell stages. A global demethylation pattern was observed in iSCNT embryos from 1-cell to 8–16-cell stages, which was not significantly different from that of bovine SCNT embryos by immunocytochemistry. However, bisulfite sequencing indicated incomplete demethylaton of Oct4 and Nanog promoters in 8-cell iSCNT embryos, unlike the complete demethylation of the same promoter regions observed in their bovine intraspecific counterparts. ATP levels were significantly higher in bovine SCNT embryos than in iSCNT embryos; TUNEL assay did not reveal any difference in apoptotic status of the nuclei from both types of embryos. Collectively, our results suggest that bovine ooplasm can partially remodel chimpanzee somatic nuclei. However, the remodeling activity was not sufficient to drive embryo development past the 8–16-cell stage.