The cover image shows epifluorescent images of differential mitochondrial polarity reported by JC-1. Epifluorescent images of high-polarized mitochondria (HPM) images reported by JC-1 J-aggregate fluorescence and detected in the FITC channel in zona-free MII stage mouse oocytes (A) are shown in (B-D). (E) shows the typical level of sperm attachment to zona-free oocytes after insemination in vitro. After vigorous passages though several rinses followed by fixation and DAPI staining, the number of persistently attached sperm was determined by counting fluorescent sperm heads (SP, see F). In the presence of the proton ionophore FCCP (G), mitochondrial depolarization results in the loss of J-aggregate fluorescence while green cytoplasmic fluorescence indicates the presence of the JC-1 monomer (H). DAPI staining of FCCP-treated oocytes showed the absence of sperm attachment and the presence of an intact metaphase II spindle (MII, see I). After removal of FCCP, subplasmalemmal J-aggregate fluorescence progressively returned (J, L) and when inseminated, sperm attached (SP) to the oolemma where the subjacent mitochondria were J-aggregate positive (arrows, J, K). After FCCP-treatment, J-aggregate fluorescence in the subplasmalemmal cytoplasm was often discontinuous (arrows, L), but levels of persistent sperm binding were normal (M). Culture of intact (N) and zona-free oocytes (O) at 25°C precluded normal levels of JC-1, J-aggregate formation (FITC channel, O; RITC channel P). However, sperm attachment at normal levels occurred at this reduced temperature and unlike oocyte mitochondria in the subplasmalemmal (asterisk, Q), those in the midpiece of sperm showed J-aggregate fluorescence (Q, MP) (See Van Blerkom and Davis, pp. 759-770).
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