Figure 1.
Altered mitochondrial morphology in cumulus cells of diabetic mice.
Cumulus-oocyte complexes collected from control and diabetic mice were subjected to transmission electron microscopy analysis and mitochondrial structure was compared. (A) Representative electron micrograph of mitochondria from control cumulus cells, showing bean-shaped structures with numerous transversely orientated cristae enveloped by an intact outer membrane; (B–C) Representative electron micrographs show alterations in mitochondrial morphology of diabetic cumulus cells: (B) small spherical mitochondria with fewer and disarrayed cristae and a decreased electron density of the matrix (arrow), and (C) mitochondria with membrane rupture or large vacuoles (arrow). Higher magnification views of boxed regions are also presented (right panel). (D) Quantification analyses of abnormal mitochondria in cumulus cells from control and diabetic mice. Data are presented as mean percentage of abnormal mitochondria ± SD in total examined mitochondria. * p<0.05 vs control.
Figure 2.
Decreased mitochondrial membrane potential in cumulus cells of diabetic mice.
Cumulus-oocyte complexes from control and diabetic mice were stained with JC-1 to evaluate mitochondrial membrane potential (Δψm) by fluorescence microscopy. Representative images of cumulus cells are shown. (A) Generally, mitochondria in cumulus cells of control mice were predominantly in red form (arrows), indicating the high Δψm. In contrast, the loss of red fluorescence and thus increased green mitochondria were observed in cumulus cells of diabetic mice (arrowheads), indicating the low Δψm. (B) Histogram shows the ratio of red to green fluorescence intensity calculated to characterize Δψm. Note the decreased Δψm in cumulus cells of diabetic mice. Error bars indicate ± SD. * p<0.05 vs controls. Scale bar: 20 µm.
Figure 3.
Mitochondrial distribution was disrupted in cumulus cells of diabetic mice.
Cumulus-oocyte complexes collected from control and diabetic mice were labeled with MitoTracker Red to visualize mitochondrial localization and costained with DAPI to visualize nuclei. Representative confocal sections of cumulus cells are shown. (A) Mitochondria of most cumulus cells from control mice show perinuclear distribution pattern (a–d). Notably, in cumulus cells from diabetic mice, aggregating distribution pattern of mitochondria was readily observed (e–h; arrowhead). (B) Quantification of cumulus cells with each mitochondrial distribution pattern from control and diabetic mice. Data are expressed as mean percentage ± SD from three independent experiments in which at least 200 cells were analyzed. * p<0.05 vs control. Scale bar: 20 µm.
Figure 4.
Increased mitochondrial biogenesis in cumulus cells of diabetic mice.
Cumulus cells removed from cumulus-oocyte complexes were collected for analysis. (A) mtDNA content was calculated using quantitative real-time PCR by measuring the ratio of cytochrome b (mitochondrial gene) to β-actin (nuclear gene) DNA levels in cumulus cells of control and diabetic mice. (B–D) mRNA levels of genes implicated in mitochondrial biogenesis determined by real-time RT-PCR in cumulus cells from control and diabetic mice. At least three experiments were performed and data are presented as the mean ± SD of the fold changes. * p<0.05 vs control.
Figure 5.
Reduced ATP and citrate content in cumulus cells of diabetic mice.
Cumulus cells removed from cumulus-oocyte complexes were collected to determine the levels of ATP and citrate. Values are expressed as pmoles per µg DNA. (A–B) Histogram shows the average ATP and citrate content in cumulus cells from control and diabetic mice. All measurements were performed in triplicate. Error bars indicate ± SD. * p<0.05 vs controls.
Figure 6.
Increased apoptosis in cumulus cells of diabetic mice.
(A) Cumulus-oocyte complexes from control and diabetic mice were stained with TUNEL to visualize apoptotic cells (green) and counterstained with DAPI to confirm nuclear status (blue). Arrows indicate the condensed nuclei of apoptotic cells. Representative confocal sections of cumulus cells are shown. (B) Frequency of TUNEL-positive nuclei in cumulus cells from control and diabetic mice. Data represent mean ± SD of three independent experiments in which at least 30 COCs were analyzed. * p<0.05 vs controls. Scale bar: 20 µm.
Figure 7.
Cytochrome c translocation and caspase-3 activation in apoptotic cumulus cells of diabetic mice.
(A) Cumulus-oocyte complexes collected from control mice were stained with cytochrome c antibody and MitoTracker to determine the subcellular localization of cytochrome c (green) and mitochondria (red), and counterstained with DAPI to visualize nuclei (blue). Representative confocal sections of cumulus cells are shown. Cytochrome c shows a punctuate distribution pattern and co-localizes with the mitochondria (yellow) in control cumulus cells. (B) Cumulus-oocyte complexes collected from diabetic mice were stained with cytochrome c antibody (green), activated caspase-3 antibody (red) and DAPI (blue). All apoptotic cumulus cells of diabetic mice (arrows), as evidenced by positive staining of active caspase-3 antibody (red; arrows) and condensed chromatin (blue; arrows), always display diffuse staining of cytochrome c (green; arrows), indicating its translocation from mitochondria to cytoplasm. In contrast, those non-apoptotic cumulus cells of diabetic mice (green, lower panel; arrowheads) stained negatively with active caspase-3 antibody still show mitochondria-localized cytochrome c. Scale bars: 20 µm.