| Abstract: | Mitochondria are reported to be critical in in vitro maturation ofoocytes and subsequent embryo development after fertilization, but their contributionfor fertilization has not been investigated in detail. In the present study, weinvestigate the contribution of mitochondria to fertilization using reconstructedporcine oocytes by fusion of ooplasmic fragments produced by serial centrifugations(centri-fusion). Firstly, we evaluated the characteristics of ooplasmic fragments.Three types of fragments were obtained by centrifugation of porcine oocytes maturedin vitro for 46 h: brownish (B), transparent (T) and large (L)fragments containing both B and T parts in a fragment. The production efficiencies ofthese types of fragments were 71.7, 91.0 and 17.8 fragments/100 oocytes,respectively. In experiments, L fragments were excluded because they contained bothbrownish and transparent components that were apparently intermediate between B and Tfragments. Observations by confocal microscopy after staining with MitoTracker RedCMXRos® and transmission electron microscopy revealed highly condensedactive mitochondria in B fragments in contrast to T fragments that contained onlysparse organelles. We reconstructed oocytes by fusion of a karyoplast and twocytoplasts from B and T fragments (B and T oocytes, respectively). The B oocytesshowed higher sperm penetration (95.8%) and male pronuclear formation rates (94.2%)by in vitro fertilization than T oocytes (66.7% and 50.0%,respectively). These results suggest that the active mitochondria in oocytes may berelated to their ability for fertilization. |
