Fig. 2
Expression of GSK3α and CypD in sperm mitochondria. A Representative western blot images of GSK3α and CypD expression in mitochondrial and cytosolic fractions after incubation in TCM for 1 h. GSK3α protein was found in both cytosol (Cyto) and mitochondrial (Mito) fractions, and p-GSK3α(Ser21) was higher in the cytosol fraction than in the mitochondrial fraction. In addition, CypD was only found in the mitochondria fraction. At least three independent experiments were subjected to western blot analysis. B Representative western blot images of changes in cellular amount of CypD and GSK3α in sperm cytosol and mitochondria during capacitation. CypD and GSK3α were markedly decreased after incubation in TCM for 1 h. ATP5A and GAPDH are mitochondria and cytosol markers, respectively. At least three independent experiments were subjected to western blot analysis. C Representative western blot images of change in p-GSK3α(Ser21) level in cytosol and mitochondria during capacitation. The p-GSK3α(Ser21) levels in cytosol and mitochondria were markedly increased during capacitation. At least three independent experiments were subjected to western blot analysis. D Representative images of immunolocalization of GSK3α/β and CypD in cauda epididymal spermatozoa. Immunoreactivity of GSK3α/β was found in the head, midpiece, and principal piece of the tail. Immunoreactivity of CypD was co-localized in the midpiece with GSK3. Immunoreactivity of both GSK3α/β and CypD was decreased after incubation in TCM for 1 h. Rabbit and mouse IgG were used as a negative control of GSK3α/β and CypD antibodies, respectively. Sperm head, midpiece, and principal piece of the tail are annotated by asterisk, arrows, and arrowheads, respectively. Nuclei were stained blue by DAPI. At least three independent experiments were subjected to immunocytochemistry. AU, arbitrary units. Bar, 10 μm