359 THE EFFICIENT MAINTENANCE OF OXYGEN CONSUMPTION BY PORCINE OOCYTES RELATES TO THE HIGH COMPETENCE OF OOCYTE MATURATION

Abstract

We succeeded in noninvasively and quantitatively determining the oxygen consumption rate of single bovine oocytes and embryos by scanning electrochemical microscopy (SECM). Results from our earlier studies with bovine embryos established a positive relationship between oxygen consumption and embryo quality by using SECM (Abe et al. 2004 J. Mamm. Ova Res. 21, 22–30). However, the oxygen consumption of porcine oocytes and the relationship between oxygen consumption and oocytes quality were still unclear. The aims of this study were: (1) to assess the oxygen consumption of single porcine cumulus–oocyte complexes (COCs) and oocytes; and (2) to examine the change of oxygen consumption of oocytes during IVM. The COCs collected from 2–5 mm follicles of a porcine ovary were classified into four grades according to morphological criteria (A, compact multilayered cumulus; B, 3 to 5 layers of compact cumulus; C, less than 3 layers of cumulus, including small areas of denuded zonae pellucidae; and D, completely or mostly denuded). Oxygen consumption of these COCs was measured using by the SECM system (HV-403; Research Institute for Functional Peptides, Yamagata, Japan). After the measurement, cumulus cells were removed from COCs mechanically, and oxygen consumption of the oocytes was evaluated. The COCs were cultured in TCM-199 supplemented with 10% porcine follicular fluid and 10 IU mL^-1 eCG. After IVM, oxygen consumption and ATP content of the oocytes were determined. ATP content was measured using a commercial assay kit (Promega K.K. Japan, Chuo-Ku, Tokyo, Japan) based on the luciferin-luciferase reaction. Moreover, distribution of active mitochondria in the oocytes during IVM was investigated using MitoTracker Orange staining (Invitrogen Japan K.K., Tokyo, Japan). The rates of oxygen consumption and oocyte maturation of higher-graded COCs and oocytes tended to be higher. In matured oocytes with a first polar body, oxygen consumption (F × 10^-14 mol s^-1) was maintained at a high level during IVM (GV oocytes: 0.60 ± 0.03; matured oocytes: 0.57 ± 0.01). ATP content (pmol/oocyte) of matured oocytes was significantly increased by IVM culture (GV oocytes: 1.16 ± 0.11; matured oocytes: 2.06 ± 0.06). On the other hand, in non-matured oocytes without a first polar body, oxygen consumption (0.31 ± 0.04) and ATP content (1.51 ± 0.21) were significantly lower than in matured oocytes. The mitochondrial distribution pattern of the oocytes during IVM was changed into an aggregated type (mitochondria distributed to the inner region of the cytoplasm with aggregation of larger fluorescent areas) from a peripheral type (mitochondria evenly distributed to the periphery of the cytoplasm) through a semi-peripheral type (mitochondria distributed to the periphery of the cytoplasm with small spots of fluorescence intensity). However, most of non-matured oocytes showed a semi-peripheral type. These results demonstrated that the oocytes with high oxygen consumption and ATP levels showed higher competence of oocyte maturation. Furthermore, these findings suggest that mitochondrial reorganization may be partly related to the oxygen consumption and consequently the quality of porcine oocytes.