Long-term apoptosis-related protein expression in the diabetic mouse ovary

Abstract

Emerging evidence has shown that oocytes from diabetic ovaries exhibit delayed maturation, mitochondrial dysfunction and meiotic defects, which are related increased apoptosis. The main objective of the present study was to analyze the apoptosis pathways activated during follicular loss at multiple time points in a diabetic mouse model. Twenty BALB/c mice were used in this study, and diabetes mellitus was induced by streptozotocin injection. Three diabetic and two control animals were sacrificed on days 15, 20, 70 and 80 posttreatment. The ovaries were then removed; one was used for follicular counting, TUNEL, immunohistochemistry and immunofluorescence, while the other was used for Western blot analysis. The proteins studied were BAX, BCL2, t-BID, FAS, FASL, active caspase 8, active caspase 9 and active caspase 3. Follicular apoptosis decreased over time, with the highest values observed at 15 days posttreatment. Granulosa cells were positive for active caspase 3, which showed constant expression levels at all time points. FAS, FASL, t-BID and active caspase 8 showed strong cytoplasmic immunostaining in the oocytes and granulosa cells of the diabetic mice, with significant increases observed at 15, 20 and 70 days posttreatment. BAX expression was slightly higher in the diabetic mouse ovaries than in the control ovaries at 15, 20 and 70 days posttreatment, whereas the highest active caspase 9 expression was at observed 20 days posttreatment. Low BCL2 protein levels were detected in the diabetic mouse ovaries at all time points. This study describes for the first time the behavior of apoptosis-related proteins in the diabetic mouse ovary and shows not only that the FAS/FASL pathway contributes to follicular loss but also that antral follicles are the most affected.

Fig 1. Effects of diabetes on follicular count.

Total follicular count at the different time points. Bar graphs are plotted as the mean ± SD. * Significant differences, Wilcoxon-Mann-Whitney test, p < 0.05.

Fig 2. General histology and follicular apoptosis in diabetic mouse ovaries.

(A) The diabetic mouse ovaries showed strong follicular degeneration associated with follicular apoptosis at different stages of maturation at all time points. (B) The number of apoptotic follicles was significantly increased in the diabetic ovaries at 15, 20 and 70 days posttreatment compared to the control group. (C) Antral follicles were the most affected at 15, 20 and 70 days posttreatment. PF, primary follicle; APF, apoptotic primary follicle; SF, secondary follicle; ASF, apoptotic secondary follicle; TF, tertiary follicle; ATF, apoptotic tertiary follicle; AF, antral follicle; AAF, apoptotic antral follicle. Bars in A represent 100 μm. Bar graphs in B are plotted as the mean ± SD. * Significant differences, Wilcoxon-Mann-Whitney test, p < 0.05.

Fig 3. Follicular apoptosis at different time points in diabetic mouse ovaries detected by TUNEL assay.

The diabetic ovaries showed positive TUNEL signals in granulosa cells (insets). Positive signal (white arrow). PF, primary follicle; APF, apoptotic primary follicle; SF, secondary follicle; ASF, apoptotic secondary follicle; TF, tertiary follicle; ATF, apoptotic tertiary follicle; AF, antral follicle; AAF, apoptotic antral follicle. Bars represent 100 μm.

Fig 4. Western immunoblotting of apoptosis-related proteins.

The protein levels of FAS, FASL, t-BID, C8A, C3A, and BAX were higher in the diabetic mouse ovaries than in the control mouse ovaries at 15, 20 and 70 days posttreatment. C9A expression was significantly higher in the diabetic mouse ovaries than in the control mouse ovaries at 20 days posttreatment. BCL2 showed low expression levels in the diabetic mouse ovaries. Bar graphs are plotted as the mean ± SD. * Significant differences, Wilcoxon-Mann-Whitney test, p < 0.05.

Fig 5. Immunohistochemical and immunofluorescence analysis of apoptosis-related proteins in diabetic mouse ovaries.

(A) The diabetic mouse ovaries showed strong C3A, FAS, FASL t-BID and BAX expression in the granulosa cells and oocytes of follicles from the primary to antral stage. BCL2 showed weak immunoreactivity in the diabetic mouse ovaries compared to the control ovaries. (B) C8A and C9A expression levels were higher in the diabetic mouse ovaries than in the control mouse ovaries. Positive cells (black arrow). PF, primary follicle; APF, apoptotic primary follicle; SF, secondary follicle; ASF, apoptotic secondary follicle; TF, tertiary follicle; ATF, apoptotic tertiary follicle; AF, antral follicle; AAF, apoptotic antral follicle. Bars in A and B represent 50 μm.