Diabetes Induces Abnormal Ovarian Function via Triggering Apoptosis of Granulosa Cells and Suppressing Ovarian Angiogenesis

Abstract

Diabetes triggers abnormal ovarian follicular development and consequently leads to infertility. Here, we established a type 2 diabetes mouse model by feeding with high fat diet (HFD) for 15/20 weeks and assessed the effect of diabetes on follicular development and ovarian angiogenesis. After fed with HFD for 15 weeks, mice had the characteristics of type 2 diabetes, which was much more serious after 20 weeks on HFD. After 20 weeks on HFD, the mice had shown abnormal ovarian morphology with hyaline appearance, much less blood vessel, follicular development arrest and less of granulosa cells (GCs) in mature follicles, but not in ovaries from 15 weeks on HFD. Elevated makers of DNA damage, ER stress and apoptosis of GCs were observed in ovaries from HFD for 20 weeks. Additionally, diabetes significantly suppressed ovarian angiogenesis with the evidence of down-regulation of CD31 via inhibiting HIF1α-VEGF signaling pathway in time-dependent. We concluded that diabetes triggers abnormal ovarian function via inducing GCs apoptosis and suppressing ovarian angiogenesis.

Keywords: DNA damage.; angiogenesis; apoptosis; diabetes; granulosa cells (GCs).

Figure 1

Diet induced-obese (DIO) type 2 diabetes caused abnormal ovarian follicular development. (A) Body weight of mice during fed with different diet (n=8); (B) Body mass index (BMI) of mice after 15/20 weeks fed with normal diet and HFD (n=8); Blood glucose level during GTT after 15 weeks(C) and 20 weeks(D) fed with different diet(n=6); Blood glucose level during ITT after 15 weeks(E) and 20 weeks(F) fed with different diet (n=6). (G) Blood glucose level of mice after 15/20 weeks fed with normal diet and HFD (n=8); (H-O) Representative images of H&E staining shown morphology of ovaries from mice after 15/20 weeks fed with different diet, in H, I, J, K and M, scale bars= 150 μm; in L and N, scale bars = 15 μm; in O, scale bars = 30 μm. *P<0.05 vs. 15W-NC group; **P<0.01 vs. 15W-NC group; ***P<0.001 vs. 15W-NC group; ^#P<0.05 vs. 20 W-NC group; ^## P<0.01 vs. 20 W-NC group; ^### P<0.001 vs. 20 W-NC group. ***P<0.001 vs. 15W-NC group; ^### P<0.001 vs. 20 W-NC group; ✝ P<0.001 vs. 15W-HFD group. NC: normal control diet group; HFD: high fat diet group; CL: corpus luteum; OC: oocyte; GC: granulosa cell; W: weeks.

Figure 1

Diet induced-obese (DIO) type 2 diabetes caused abnormal ovarian follicular development. (A) Body weight of mice during fed with different diet (n=8); (B) Body mass index (BMI) of mice after 15/20 weeks fed with normal diet and HFD (n=8); Blood glucose level during GTT after 15 weeks(C) and 20 weeks(D) fed with different diet(n=6); Blood glucose level during ITT after 15 weeks(E) and 20 weeks(F) fed with different diet (n=6). (G) Blood glucose level of mice after 15/20 weeks fed with normal diet and HFD (n=8); (H-O) Representative images of H&E staining shown morphology of ovaries from mice after 15/20 weeks fed with different diet, in H, I, J, K and M, scale bars= 150 μm; in L and N, scale bars = 15 μm; in O, scale bars = 30 μm. *P<0.05 vs. 15W-NC group; **P<0.01 vs. 15W-NC group; ***P<0.001 vs. 15W-NC group; ^#P<0.05 vs. 20 W-NC group; ^## P<0.01 vs. 20 W-NC group; ^### P<0.001 vs. 20 W-NC group. ***P<0.001 vs. 15W-NC group; ^### P<0.001 vs. 20 W-NC group; ✝ P<0.001 vs. 15W-HFD group. NC: normal control diet group; HFD: high fat diet group; CL: corpus luteum; OC: oocyte; GC: granulosa cell; W: weeks.

Figure 2

DIO-type 2 diabetes triggered oxidative stress and DNA damages of ovary. (A) LPO levels in ovaries. (B) The protein levels of p-H2AX, p-CHK1, p-CHK2, p-cdc25 and P53 in ovaries from mice after 15/20 weeks fed with different diet. (C-G) Intensities of p-H2AX, p-CHK1, p-CHK2, p-cdc25 and P53 normalized to β-actin. Data are presented as the mean ± standard deviation (n=3); *P<0.05 vs. 15W-NC group; ^#P<0.05 vs. 20W-NC group. p‑:phosphorylated; NC: normal control group; HFD: high‑fat diet group; W: weeks.

Figure 3

DIO-type 2 diabetes induced ER stress of ovary. (A) The protein levels of p-PERK, p-IRE1α, p-eIF2α, CHOP and BiP in ovaries from mice after fed with different diet for 20 weeks. (B) Intensities of p-PERK normalized to PERK, p-IRE1α normalized to IRE1α, p-eIF2α normalized to eIF2α, CHOP and BiP normalized to β-actin. (C) Immunohistochemical staining of CHOP in ovaries from mice after fed with different diet for 20 weeks, scale bars= 30 μm. Data are presented as the mean ± standard deviation (n=3). ^#P<0.05 vs. 20W-NC group. NC: normal control diet group; HFD: high fat diet group; CL: corpus luteum; OC: oocyte; GC: granulosa cell; W: weeks.

Figure 4

DIO-type 2 diabetes triggered caspase activation and subsequent apoptosis in ovary. (A) Representative images of TUNEL staining shown apoptotic cells (red signal) in ovaries from mice after fed with different diet for 20 weeks, in A and E, scale bars= 30 μm; in B, D, F and H, scale bars= 15 μm; in C and G, scale bars= 150 μm. (B) The protein levels of Caspase8 and Caspase 3 in ovaries from mice after fed with different diet for 20 weeks. (C) Intensities of cleaved-Caspase8 and cleaved-Caspase 3 normalized to β-actin. Data are presented as the mean ± standard deviation (n=3). ^# P<0.05 vs. 20W-NC group. NC: normal control diet group; HFD: high fat diet group; CL: corpus luteum; OC: oocyte; GC: granulosa cell; W: weeks.

Figure 5

DIO-type 2 diabetes suppressed angiogenesis of ovary. (A-D) Morphological appearance of ovaries from mice after 15/20 weeks fed with different mice. (E) Immunohistochemical staining of CD31 in ovaries from mice after 15/20 weeks fed with different diet, scale bars= 30 μm. Yellow arrow: blood vessel. NC: normal control diet group; HFD: high fat diet group; CL: corpus luteum; OC: oocyte; GC: granulosa cell; W: weeks.

Figure 6

DIO-type 2 diabetes inhibited HIF1α-VEGF signaling pathway in ovary. (A) The mRNA levels of HIF1α and VEGF in ovaries from mice after 15/20 weeks fed with different diet. (B) The protein levels of HIF1α and VEGF in ovaries from mice after 15/20 weeks fed with different diet. (C-D) Immunohistochemical staining of VEGF in ovaries from mice after 15/20 weeks fed with different diet, scale bars= 30 μm. Data were presented as the mean ± standard deviation (n=3). *P<0.05 vs. 15W-NC group; ^#P<0.05 vs. 20 W NC groups. NC: normal control diet group; HFD: high fat diet group; CL: corpus luteum; OC: oocyte; GC: granulosa cell; W: weeks.

Figure 7

A schematic diagram illustrating the effect of diabetes on apoptosis of granulosa cells and ovarian angiogenesis in ovary. Type 2 diabetes not only induced oxidative stress, then triggered DNA damage and ER stress, thereby led to GCs apoptosis, but also inhibited HIF1α-VEGF signaling pathway and subsequently suppressed ovarian angiogenesis, consequently induced abnormal ovarian function.