The Kinetics of Donor Cell mtDNA in Embryonic and Somatic Donor Cell-Derived Bovine Embryos

Abstract

The mechanisms controlling the outcome of donor cell-derived mitochondrial DNA (mtDNA) in cloned animals remain largely unknown. This research was designed to investigate the kinetics of somatic and embryonic mtDNA in reconstructed bovine embryos during preimplantation development, as well as in cloned animals. The experiment involved two different procedures of embryo reconstruction and their evaluation at five distinct phases of embryo development to measure the proportion of donor cell mtDNA (Bos indicus), as well as the segregation of this mtDNA during cleavage. The ratio of donor cell (B. indicus) to host oocyte (B. taurus) mtDNA (heteroplasmy) from blastomere-(NT-B) and fibroblast-(NT-F) reconstructed embryos was estimated using an allele-specific PCR with fluorochrome-stained specific primers in each sampled blastomere, in whole blastocysts, and in the tissues of a fibroblast-derived newborn clone. NT-B zygotes and blastocysts show similar levels of heteroplasmy (11.0% and 14.0%, respectively), despite a significant decrease at the 9-16 cell stage (5.8%; p Ͻ 0.05). Heteroplasmy levels in NT-F reconstructed zygotes, however, increased from an initial low level (4.7%), to 12.9% (p Ͻ 0.05) at the 9-16 cell stage. The NT-F blastocysts contained low levels of heteroplasmy (2.2%) and no somatic-derived mtDNA was detected in the gametes or the tissues of the newborn calf cloned. These results suggest that, in contrast to the mtDNA of blastomeres, that of somatic cells either undergoes replication or escapes degradation during cleavage, although it is degraded later after the blastocyst stage or lost during somatic development, as revealed by the lack of donor cell mtDNA at birth.

Xenooplasmic Transfer between Buffalo and Bovine Enables Development of Homoplasmic Offspring

“…In vitro maturation (IVM), in vitro fertilization (IVF), ovum pickup (OPU), parthenogenetic activation, enucleation, electrofusion, in vitro culture (IVC) of embryos, and embryo transfer were performed as described in Ferreira et al (2007). Briefly, oocytes obtained postmortem from the ovaries of crossbred cows (B. indicus nDNA and B. taurus mtDNA) were IVM for 24 h and after 12 h of IVF were denuded of cumulus cells by pipetting in 0.5% hyluronidase, and selected by the presence of the second polar body (2nd PB), which were used as recipient zygotes for XOT.…”

“…Briefly, sperm and blood (from offspring) samples had total genomic DNA extracted following a standard procedure (Ferreira et al, 2007). The genetic identity between the calves and the sperm was determined by microsatellite analysis using 11 microsatellite markers (Genoa Biotecnologia AS, Sã o Paulo, SP, Brazil).…”

“…For molecular analysis, oocyte and embryo samples were treated as described by Chiaratti et al (2010), whereas tissue samples had the genomic DNA extracted following a standard procedure (Ferreira et al, 2007). The proportion of buffalo mtDNA was determined by quantitative polymerase chain reaction (qPCR) using a discriminating (buffalo mtDNA) assay and normalization for a nondiscriminating (buffalo þ bovine mtDNAs) assay .…”

“…However, development failure is common after SCNT, especially after interspecies SCNT (iSCNT), and its causes remain widely unknown. SCNT generates reconstructed oocytes with a mixture of mitochondria from different origins (e.g., heteroplasmy), that often disappear to generate offspring that are in most cases homoplasmic for mitochondria donated by the host ooplast (Ferreira et al, 2007;Hiendleder et al 2003Hiendleder et al , 2005Meirelles et al, 2001;Steinborn et al, 1998).…”

“…In this regard, it is plausible to propose that the procedure of iSCNT leads to nuclear-mitochondrial incompatibilities that prevent embryo development and birth of healthy offspring (Chen et al, 2002;Gó mez et al, 2009;Hua et al, 2007;Kitiyanant et al, 2001;Lanza et al, 2000;Lu et al, 2005;Mastromonaco et al, 2007;Saikhun et al, 2002). The issue of a nuclear-mitochondrial incompatibility has been studied using xenomitochondrial cybrid models produced by the fusion of a somatic cell with a cytoplast (e.g., a cell previously enucleated) of another species (Kenyon and Moraes, 1997;Trounce and Pinkert, 2007) and using SCNT to analyze effects during embryo=fetal development in both inter- (Chen et al, 2002;Gó mez et al, 2009;Lanza et al, 2000;Loi et al, 2001;Mastromonaco et al, 2007) or intraspecific (Bowles et al, 2008;Ferreira et al, 2007;Hiendleder et al 2003Hiendleder et al , 2005Meirelles et al, 2001;Steinborn et al, 1998) animal models. However, various factors involved in SCNT success (e.g., ooplasmic factors involved in somatic nucleus dedifferentiation) could prevent proper conclusions.…”