Establishment of a mouse model of ovarian oxidative stress induced by hydrogen peroxide

Abstract

Introduction: Oxidative stress, resulting from environmental changes, significantly affects female fertility. Developing a mouse model to study oxidative stress lays the groundwork for research into human reproductive health and livestock fertility.

Materials and methods: In this study, we established and evaluated an oxidative stress model by administering hydrogen peroxide (H₂O₂) to mice. ICR mice of similar age (7-8 weeks old) and average body weight (31.58 ± 1.12 g) were randomly assigned to four groups (A, B, C, and D). Group A served as the control and was injected with a saline solution, while groups B, C, and D received saline solutions containing 0.75%, 1.50%, and 3.0% H₂O₂, respectively, over one week. We measured the body weights of all mice before and after the experimental period.

Results and discussion: Our findings showed that the average body weight of mice in groups A and B increased, while groups C and D experienced weight loss. Group C showed a significantly lower average weight gain compared to groups A and B, and group D exhibited an even more pronounced reduction in weight gain. Although group D had a high mortality rate, there was no significant difference in mortality rates among groups B, C, and D. Serum malondialdehyde (MDA) content increased with higher concentrations of H₂O₂, with a significant difference noted between groups C and A. Catalase (CAT) activity in group B was significantly higher than in group A, while superoxide dismutase (SOD) activity in group C was notably elevated compared to groups A and B. Conversely, glutathione peroxidase (GSH-Px) activity in group C was significantly lower than in both group A and group B. Hematoxylin and eosin (HE) staining revealed changes in ovarian morphology and follicle dynamics. The percentage of atretic follicles in group C was significantly higher than in the control group, and group D had a significantly lower total number of healthy follicles compared to the untreated group. Increased H₂O₂ content resulted in a reduction of ovary size and an irregular appearance in group D.

Conclusion: Based on our findings, treatment with 1.50% H₂O₂ effectively established an oxidative stress model in mice within 1 week. This model serves as a valuable reference for future clinical studies on oxidative stress and reproductive disorders in female animals and humans.

Keywords: antioxidant enzymes; hydrogen peroxide; mouse model; ovary; oxidative stress.

Figure 1

The body weight progression and mortality of mice under different H₂O₂ treatments in 1 week. (A) The average weight gain of every mouse in different groups. (B)The mortality ratio of mice in the different treatment groups. Group A was injected with 0.9% saline as the control; group B was injected with 0.75% H₂O₂ solution; group C was injected with 1.5% H₂O₂ solution; group D was injected with 3.0% H₂O₂ solution. Bars represent mean ± SEM. The asterisks indicated statistically significant differences, * represented P < 0.05 and ** represented P < 0.01.

Figure 2

Changes of serum oxidation and antioxidant levels in mice treated with different H₂O₂. The letters indicated statistically significant differences, a, b represented P < 0.05 and A, B represented P < 0.01. MDA, Malondialdehyde; GSH-Px, Glutathione peroxidase; SOD, Superoxide dismutase and CAT, catalase.

Figure 3

The ovary sections of mice stained by HE (Hematoxylin-Eosin). (A–D) indicated mice were treated with 0%, 0.75%, 1.5%, and 3.0% H₂O₂. (E). The labels of different follicles at morphology and classification.

Figure 4

(A) The total number of follicles in four different H₂O₂-treated groups with control, 0.75%, 1.50%, and 3.0% H₂O₂. (B) The number of healthy follicles in four treated groups. (C) The ratio of atretic follicles to total number of follicles in four treated with control, 0.75%, 1.50%, and 3.0% H₂O₂. (D) The number of follicles in each category in four treated groups. (E) The proportion of each level follicle to total follicles in four treated groups. P, Primary follicles; S, Secondary follicles; T, Tertiary follicles; Prf, Preovulatory follicles; TF, The total number of follicles. Letters indicate statistically significant differences, a and b indicate P < 0.05, and A indicate P < 0.01.