After the 2nd centrifugation at 6,000 × g for 4 sec, three
types of fragments – brownish (B), transparent (T) and large (L) – (A, B and
C, respectively) were obtained. The distribution after staining with
MitoTracker Red CMXRos (MTR) (D, B fragment; E, T fragment) and relative
fluorescence intensities of active mitochondria in the B and T fragments are
shown (F). Data for relative fluorescence intensity of active mitochondria
are given as means ± SEM. a,b A significant difference
(P<0.05) in relative fluorescence intensity of active mitochondria was
evident between the two groups. Three trials were performed. Total numbers
of oocytes measured in each group are given in parentheses. Transmission
electron microscopy demonstrated that B fragments contained condensed
mitochondria (G), similar to the lower layer of oocytes after the first
centrifugation (see Fig. 1I),
whereas T fragments had few organelles (H), suggesting that they originated
from the middle layer (see Fig.
1H). Scale bars indicate 50 µm, 10 µm and 1 µm for plates A−C, D and
F and G and I, respectively.