No abstract

Mitochondrial DNA (mtDNA) mutations occur naturally in skeletal muscle fibers from aged rhesus macaques. In addition, mtDNA mutations have been observed in germinal vesicle oocytes from fertile monkeys. The goal of this study was to determine whether the rhesus macaque mitochondrial common deletion was present in oocytes and embryos generated by in-vitro embryo production (IVP), as well as in rhesus adult and embryonic stem cell lines. The rhesus common deletion was detected in IVP-generated embryos, three IVP-derived embryonic stem cell lines (ORMES 1, 2 and 7), one in-vivo-derived embryonic stem cell line (R4) and multiple passages of an adult bone marrow stromal cell (BMSC) line. Mitochondrial DNA from an adult adipose stromal cell (ATSC) line was compared with mtDNA from an immortalized line transfected with a retroviral vector expressing telomerase, ATSC-TERT. Multiple passages of the ATSC line harboured a dramatically higher level of the rhesus common deletion than the immortalized ATSC-TERT line. Accumulation of mtDNA mutations in oocytes, embryos and subsequent embryonic stem cell lines, as well as adult stem cell lines, may contribute to mitochondrial dysfunction, and thereby impair ATP production. The authors believe this information establishes a compelling argument for the parallel development of embryonic stem cell technology in non-human primates and humans.

Mitochondria are the most abundant organelles in mammalian oocytes and early embryos. Previous data have shown that mitochondrial DNA (mtDNA) deletions are present both in human oocytes and in embryos from in vitro fertilization (IVF) patients and suggest that accumulation of these deletions may contribute to mitochondrial dysfunction and impaired ATP production. In addition, high levels of mitochondrial mutations are present in skeletal muscle fibers from aged rhesus macaques. The specific aims of this study were to determine whether the mitochondrial common deletion is present in non-human primate oocytes and embryos generated by IVF and to determine whether mtDNA mutations are already present in immature oocytes from rhesus ovaries. Using a nested primer polymerase chain reaction (PCR) strategy, we determined the frequency of the rhesus common deletion in immature oocytes compared with stimulated oocytes and embryos. There was a low incidence (21%) of the rhesus common deletion present in immature, unstimulated oocytes derived from necropsied ovaries of 2 to 10-yr-old rhesus macaques. However, there was >3-fold increase (71.4%) in the frequency of deleted mtDNA in stimulated oocytes and IVF embryos from age-matched fertile monkeys. We postulated that, in addition to skeletal muscle, a similar time-dependent accumulation of mtDNA deletions occurs in fertile rhesus macaque oocytes and embryos. We are now investigating the effects of culture and passage number on mtDNA deletions in primate adult and embryonic stem cells. We propose the rhesus monkey to be an excellent model to assess the quality of gametes and embryos, as well as stem cells, and their developmental competence in human and non-human primates. This study was supported by National Institutes of Health grants RR15395 and HD045966.