PARENT SESSION
SLIDE SESSION 7: REPRODUCTIVE TECHNOLOGIES
Chairs: Nicola Dean, Jay Baltz, Jamie Cowell (Trainee)
Univ Ottawa-Lamoureaux 122
2:30 PM-4:30 PM
48
DISTRIBUTION OF MITOCHONDRIAL DNA IN HETEROPLASMIC MOUSE OOCYTES AND THEIR POLAR BODIES.
Dean, Nicola1, Battersby, Brendan3, Gosden, Roger 2, Tan, Seang Lin 1,2, Ao, Asangla 2,3, Shoubridge, Eric3, 1 3 2
ABSTRACT- This study is to determine if preimplantation genetic diagnosis (PGD) for women with mitochondrial DNA (mtDNA) mutations could be performed on the polar body of an oocyte giving an accurate indication of the mutant mtDNA level in the resulting embryo. MtDNA is found in multiple copies in the cytoplasm of cells and is inherited maternally. Usually, all copies of mtDNA are homoplasmic (have the same sequence) but mutations produce heteroplasmy. Pathogenic mutations show a phenotype when the mutant load reaches a threshold affecting energy production. Extensive mtDNA replication occurs during the development of an oogonium to primary oocyte (from around 2 X 102 to around 103 copies), leading to a wide variation in the degree of heteroplasmy between oocytes. In a heteroplasmic mouse model, the percentage distribution of types of mtDNA between the oocyte and first polar body was elucidated. Oocyte and polar body samples were collected from mice with known levels of heteroplasmy. Duplicate aliquots for each sample underwent a radioactive polymerase chain reaction to amplify a region of mtDNA encoding a complex I subunit, and digested products were separated on a polyacrylamide gel. The relative proportions of bands specific to each genotype were compared to give the level of heteroplasmy for each sample. Five mice (with 30 -70% heteroplasmy) gave complete duplicate results for oocytes and polar bodies. Even though a wide variation in the levels of heteroplasmy was found in the gametes compared with the maternal genotype, the level in each oocyte was consistent with that of the polar body (range 5 - 80%). This suggests the segregation of mtDNA during the first meiotic division is random. Initial conclusions from this data are that PGD could be offered for mtDNA diseases on the polar body of unfertilized oocytes, as long as such transmission is reflective of human mtDNA diseases.
KEY WORDS: mitochondrial DNA, heteroplasmy, oocyte, preimplantation genetic diagnosis