Effects of Vitamin D Deficiency on Proliferation and Autophagy of Ovarian and Liver Tissues in a Rat Model of Polycystic Ovary Syndrome

Abstract

Aim: We aimed to examine the alterations of the insulin signaling pathway, autophagy, nitrative stress and the effect of vitamin D supplementation in the liver and ovaries of vitamin D deficient hyperandrogenic rats.

Methods: Female Wistar rats received eight weeks of transdermal testosterone treatment and lived on a low vitamin D diet (D-T+). Vitamin D supplementation was achieved by oral administration of vitamin D3 (D+T+). Sham-treated (D+T-) and vitamin D deficient animals (D-T-) served as controls. (N = 10-12 per group).

Results: D-T+ animals showed decreased LC3 II levels in the liver and increased p-Akt/Akt and p-eNOS/eNOS ratios with decreased insulin receptor staining in the ovaries. Vitamin D supplementation prevented the increase of Akt phosphorylation in the ovaries. Vitamin D deficiency itself also led to decreased LC3 II levels in the liver and decreased insulin receptor staining in the ovaries. D-T+ group showed no increase in nitrotyrosine staining; however, the ovaries of D-T- rats and the liver of D+T+ animals showed increased staining intensity.

Conclusion: Vitamin D deficiency itself might lead to disrupted ovarian maturation and autophagy malfunction in the liver. Preventing Akt phosphorylation may contribute to the beneficial effect of vitamin D treatment on ovarian function in hyperandrogenism.

Keywords: autophagy; hyperandrogenism; insulin resistance; oxidative stress; polycystic ovary syndrome (PCOS); vitamin D.

Figure 1

Timeline of chronic treatment in different treatment groups. Adolescent Wistar rats were divided into four groups—half of the animals received transdermal testosterone treatment (0.0333 mg/body weight grams 5 times weekly) for 8 weeks, while another half did not receive androgens. Half of the animals in each group were fed low vitamin D chow while the other half received adequate vitamin D supplementation (weekly 1.4 NE/body weight grams per os on the 3rd, 4th, 5th, 6th, and 7th week after a 500 NE saturation on the 2nd week). On the 6th treatment week, an oral glucose tolerance test (OGTT) was performed. On the 8th treatment week, the animals were sacrificed, and their tissues were kept at −80 °C until further studies.

Figure 2

Alterations in insulin signaling pathway and autophagy in the liver. (A) Akt phosphorylation in liver tissue. (B) eNOS phosphorylation in liver tissue. (C) Protein S6 phosphorylation in the liver. (D) LC3 II formation in the liver. LC3 II levels normalized to GAPDH. Data are presented as mean ± SEM. Two-way (factor 1—vitamin D supplementation vs. vitamin D deficiency; factor 2—control vs. testosterone treatment) ANOVA; t: p < 0.05 control vs. testosterone treatment; Tukey’s post hoc test (comparison of experimental groups), * p < 0.05 vs. D+T– group, ** p < 0.01 vs. D+T– group; n = 5–8 in each group.

Figure 3

Alterations in insulin signaling pathway and autophagy in ovarian tissue. (A) Akt phosphorylation in ovarian tissue. (B) eNOS phosphorylation in ovarian tissue. (C) Immunohistochemical staining of ovarian tissue with anti-IRβ antibody. (D) Protein S6 phosphorylation in the ovary. (E) LC3 II formation in ovarian tissue. For Panel A–B and D–E: Data are presented as mean ± SEM. Two-way (factor 1—vitamin D supplementation vs. vitamin D deficiency, factor 2—control vs. testosterone treatment) ANOVA, tt: p < 0.01 control vs. testosterone treatment, ttt: p < 0,001 control vs. testosterone treatment; Tukey’s post hoc test (comparison of experimental groups) * p < 0.05 vs. D+T– group, ^ p < 0.01 vs. D–T– group; n = 5–8 in each group. For Panel D: Data are presented as mean ± SEM. Two-way (factor 1—follicle size, factor 2—experimental group) ANOVA; Tukey’s post hoc test (comparison of experimental groups) * p < 0.05 vs. D+T– group among large follicles, ** p < 0.01 vs. D+T– group among large follicles; n = 5–8 in each group.

Figure 4

Nitrative stress in the liver and in the ovaries. (A) Immunohistochemical staining of liver tissue with anti-NT antibody. (B) Immunohistochemical staining of ovarian tissue with anti-NT antibody. Data are presented as mean ± SEM. One-way ANOVA with Tukey’s post hoc test. * p < 0.05 vs. D+T– group, # p < 0.05 vs. D–T+ group, ^ p < 0.05 vs. D–T– group; n = 5–8 in each group.

Figure 4

Nitrative stress in the liver and in the ovaries. (A) Immunohistochemical staining of liver tissue with anti-NT antibody. (B) Immunohistochemical staining of ovarian tissue with anti-NT antibody. Data are presented as mean ± SEM. One-way ANOVA with Tukey’s post hoc test. * p < 0.05 vs. D+T– group, # p < 0.05 vs. D–T+ group, ^ p < 0.05 vs. D–T– group; n = 5–8 in each group.

Figure 5

Vitamin D receptor density in the liver and in the ovaries. (A) Immunohistochemical staining of liver tissue with anti-VDR antibody. (B) Immunohistochemical staining of ovarian tissue with anti-VDR antibody. Data are presented as mean ± SEM. Two-way (factor 1—vitamin D supplementation vs. vitamin D deficiency, factor 2—control vs. testosterone treatment) ANOVA, t: p < 0.05 control vs. testosterone treatment, ttt: p < 0,001 control vs. testosterone treatment, d: p < 0.05 vitamin D supplementation vs. vitamin D deficiency, dd: p < 0.01 vitamin D deficiency vs. vitamin D deficiency; Tukey’s post hoc test (comparison of experimental groups), ** p < 0.01 vs. D+T– group,*** p < 0.001 vs. D+T– group, ^ p < 0.05 vs. D–T– group; n = 5–8 in each group.

Figure 6

Summary of the changes in receptor density, insulin signaling pathway and autophagy in different treatment groups.