104 ASYMMETRICAL DISTRIBUTION OF MITOCHONDRIAL DNA INTO BLASTOMERES OF TWO-AND FOUR-CELL MOUSE EMBRYOS

Abstract

Mammalian embryos are thought to cleave equally during early development. Therefore, the contents including organelles such as mitochondria (mt) in oocytes are supposed to be delivered symmetrically into each blastomere of the resulting embryo. However, a recent report showed higher oxygen consumption, ATP production, and mt distribution in trophectoderms from mouse blastocysts than that in inner cell masses (Houghton 2006 Differentiation 74, 11–18). We hypothesized that this phenomenon could be preceded by the asymmetrical mt distribution into blastomeres at earlier stages. Oocytes, 2-cell embryos, and 4-cell embryos from BDF1 were measured to determine volumes, ATP contents, and mtDNA copy numbers in the whole egg and individual blastomeres that had been separated in Ca/Mg-free KSOM with added 0.02% EDTA. The volumes of blastomeres were calculated by the sphere volume formula. The ATP contents were determined using a luminometer and commercial kits (BacTiter-Glo, Promega, Madison, WI, USA). The mtDNA copy numbers were quantified absolutely using real-time PCR. The data was analyzed by 1-way ANOVA followed by Fisher's least significant difference test. We found about a 10% difference in volume between the largest and the smallest blastomeres from a 4-cell embryo. At the 4-cell stage, the largest blastomeres were significantly larger than the middle-sized and smallest blastomeres, and the next largest blastomeres were significantly larger than the smallest blastomeres (largest: 41 093 µm³, next largest: 39 671 µm³, middle-sized: 37 725 µm³, smallest: 36 518 µm³; n = 25). The procedure for separation of blastomeres did not significantly reduce theATP contents of embryos. We observed the same pattern of significant differences in theATP contents among the 4 blastomeres at the 4-cell stage (largest: 0.141 pmol, next largest: 0.124 pmol, middle-sized: 0.112 pmol, smallest: 0.098 pmol, n = 27). The largest blastomeres from 4-cell embryos contained about 1.4-fold higherATP than the smallest blastomeres. There was no significant difference in the mtDNA copy numbers in oocytes (n = 7), whole 2-cell embryos (n = 6), whole 4-cell embryos (n = 13), the sum of 2 blastomeres from the same 2-cell embryo (n = 6), and the sum of 4 blastomeres from the same 4-cell embryo (n = 6; 245 071 ± 22 696, 267 567 ± 30 989, 262 931 ± 12 952, 239 717 ± 16 813, 247 012 ± 16 166, respectively; ± SEM). However, the larger blastomere of 2-cell embryo had a higher mtDNA copy number than the smaller blastomeres (largest: 137 100 ± 11 493, smallest: 102 617 ± 6205; P < 0.05). The largest blastomeres of 4-cell embryo had about 1.4 times (P < 0.05) higher mtDNA copy numbers than the smallest blastomeres (largest: 72 072 ± 4112, next largest: 66 198 ± 5767, middle-sized: 57 596 ± 3922, smallest: 51 146 ± 3081). We found differences in volumes, ATP contents, and mtDNA copy numbers among blastomeres from the same embryos at the 4-cell stage. These qualitative differences could be related to embryonic metabolism in mouse early development.