Donor-host mitochondrial compatibility improves efficiency of bovine somatic cell nuclear transfer

Abstract

Background: The interaction between the karyoplast and cytoplast plays an important role in the efficiency of somatic cell nuclear transfer (SCNT), but the underlying mechanism remains unclear. It is generally accepted that in nuclear transfer embryos, the reprogramming of gene expression is induced by epigenetic mechanisms and does not involve modifications of DNA sequences. In cattle, oocytes with various mitochondrial DNA (mtDNA) haplotypes usually have different ATP content and can further affect the efficiency of in vitro production of embryos. As mtDNA comes from the recipient oocyte during SCNT and is regulated by genes in the donor nucleus, it is a perfect model to investigate the interaction between donor nuclei and host oocytes in SCNT.

Results: We investigated whether the in vitro development of reconstructed bovine embryos produced by SCNT would be influenced by mtDNA haplotype compatibility between the oocytes and donor cells. Embryos from homotype A-A or B-B showed significantly higher developmental ability at blastocyst stages than the heterotype A-B or B-A combinations. Post-implantation development ability, pregnancy rate up to day 90 of gestation, as well as percent of term births were higher in the homotype SCNT groups than in the heterotype groups. In addition, homotype and heterotype SCNT embryos showed different methylation patterns of histone 3-lysine 9 (H3K9) genome-wide and at pluripotency-related genes (Oct-4, Sox-2, Nanog).

Conclusion: Both histone and DNA methylation show that homotype SCNT blastocysts have a more successful epigenetic asymmetry pattern than heterotype SCNT blastocysts, which indicates more complete nuclear reprogramming. This may result from variability in their epigenetic patterns and responses to nuclear reprogramming. This suggests that the compatibility of mtDNA haplotypes between donor cells and host oocytes can significantly affect the developmental competence of reconstructed embryos in SCNT, and may include an epigenetic mechanism.

Figure 1

mtDNA haplotypes determined by different restriction sites and frequencies of A and B types are noted (n > 500).

Figure 2

Epigenetic profiles of homotype SCNT (A-A and B-B), heterotype SCNT (A-B and B-A), and IVF embryos. The bovine embryos were double stained for histone H3K9 methylation (green) and DNA (blue, stained with DAPI to identify the nuclear compartment). The right column shows representative images of the anti-histone H3-dimethylK9 (H3K9) immunofluorescence from IVF embryos (a, n = 35), A-A SCNT embryos (b, n = 11), B-B SCNT embryos (c, n = 17), A-B SCNT embryos (d, n = 12), and B-A SCNT embryos (e, n = 13). Blastocysts from IVF (a) and homotype SCNT (A-A and B-B) treatments (b, c) showed hypomethylated trophectoderm and hypermethylated ICM, whereas heterotype SCNT (A-B and B-A) blastocysts (d, e) had a more homogeneous pattern between trophectoderm and ICM.

Figure 3

DNA methylation patterns of pluripotency gene promoters in various embryos. The results were obtained from three independent DNA samples. Each horizontal row of circles represents an individual sequencing result from one amplicon. Open and filled circles indicate unmethylated and methylated CpG dinucleotides, respectively.