Mitochondria have a broad range of functions that affect reproduction, and structural as well as quantitative variation in mtDNA has been associated with gamete quality and reproductive success. To investigate the mitochondria effect on in vitro embryo production, we collected oocytes by ultrasound-guided follicular aspiration from donor cows known to differ in the developmental capacity, measured by the blastocyst formation rate, of their oocytes. To evaluate the potential effects of mtDNA and mitochondrial function on oocyte quality, the donor cows' mtDNA control region was sequenced and, after pairwise comparisons of polymorphisms, animals were grouped into two major haplogroups. The number of mtDNA molecules per oocyte was quantified by real-time PCR, and the adenosine triphosphate (ATP) content was measured in each oocyte to identify variations between haplogroups. Overall, ATP stocks in oocytes of the two haplogroups differed significantly (P < 0.05; means +/- SEM) both at the germinal vesicle and metaphase II stages (2.8 +/- 0.06 pmol vs. 2.6 +/- 0.07 pmol and 2.9 +/- 0.1 pmol vs. 2.3 +/- 0.06 pmol, respectively). The proportion of development to blastocyst was significantly different between haplogroups (22.3 +/- 2.1 % vs. 36.7 +/- 2.9 %). The number of mtDNA molecules per oocyte was highly variable (377 327 +/- 14 104, ranging from 2.0 x 10(3) to 1.2 x 10(6)) but not significantly different between the two haplogroups; significant differences were observed between animals without any apparent relationship to blastocyst production. These data suggest that mitochondria and mtDNA haplogroup affect the developmental capacity of bovine oocytes in vitro.

The aim of this study was to characterize the respective influences of the paternal and the maternal components on the timing of the first S-phase in the bovine zygote. In vitro-matured oocytes were fertilized in vitro with sperm conferring a high blastocyst rate (embryos of group 1) or a low blastocyst rate (embryos of group 2). Resulting zygotes were either allowed to develop in vitro to the blastocyst stage or exposed to 5'-bromo-2'-deoxyuridine in order to characterize the timing of their first S-phases. Timing of pronuclear formation was similar in the two groups, but the onset of S-phase and the first cleavage occurred earlier in group 1 than in group 2. We also showed that the length of the S-phase represented 30% of the first cell cycle in group 1 and 20% in group 2. Differences in times of onset of the first S-phase observed between embryo groups concerned both male and female pronuclei in a similar manner and were not dependent on the maternal component of the zygote. Our data demonstrated that the precocity of the onset of the first S-phase stemmed from a paternal control exerted during a transient period of the G1-phase.

NBD-phallacidin revealed a polymerized actin distribution in the cortical region of the rabbit egg and along junctional feet. Staining with anti-alpha-tubulin antibody showed that the microtubule distribution was restricted to the barrel-shaped spindle. After cryoprotective treatment in the presence of propanediol, cortical polymerized actin was no longer visible within the egg and along junctional feet but filamentous actin was still present after treatment with dimethylsulphoxide. However, exposure to dimethylsulphoxide or propanediol led to the appearance of microtubules in the cytoplasm and to a disassembly of the spindle often associated with anomalies in chromosome position. Cytoplasmic microtubules formed by the action of propanediol were still present after freezing, thawing, and removal of the cryoprotectant, but after recovery of eggs in culture, they disappeared and barrel-shaped spindles were able to reform. When the effect of propanediol addition on in-vivo fertilization and development of frozen oocytes was examined, 39% (79/200) of frozen oocytes were fertilized and 9% (9/105) developed to normal fetuses, compared to 81% (38/47) and 32% (12/38) respectively for unfrozen control oocytes.

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