Thymoquinone improves folliculogenesis, sexual hormones, gene expression of apoptotic markers and antioxidant enzymes in polycystic ovary syndrome rat model

Abstract

Background: Nowadays, polycystic ovary syndrome (PCOS) is a prevalent cause of infertility affecting women of reproductive age around the world. Thymoquinone is a natural antioxidant, derived from Nigella sativa.

Objectives: The current study aimed to evaluate the protective effects of thymoquinone on the detrimental effects of PCOS rats induced with letrozole.

Methods: Thirty-two female rats were randomly divided into four groups: (1) Control, (2) PCOS, (3) PCOS+5 mg/kg thymoquinone and (4) PCOS+10 mg/kg thymoquinone. Thymoquinone was administered every 3 days for 30 days. Ovaries were histopathologically and stereologically examined, and antioxidant and apoptotic enzymes gene expression in ovaries and sex hormones in serum were measured.

Results: The number of unilaminar, multilaminar, antral, and graffian follicles, volume density of corpus luteum (p < 0.01), and GPx1 gene expression in ovaries and level of FSH in the blood increased in both thymoquinone groups when compared to untreated PCOS (p < 0.05). Ovaries in thymoquinone groups showed a significant reduction in the number of atretic follicles, ovary weight and volume, volume density of cortex and ovarian cysts, Bax gene expression (p < 0.01) and Bax/Bcl2 ratio as well as levels of luteinizing hormone (LH), LH/FSH ratio and testosterone (p < 0.05) in the blood of female rats when compared to PCOS group. Administration of thymoquinone restored the most detrimental effects of PCOS on ovaries (p < 0.01) and sexual hormones (p < 0.05) in rats.

Conclusions: These data suggest that thymoquinone has improved effects on ovarian function in the PCOS rat model. Therefore, thymoquinone might be useful as a protective agent and adjunct treatment in PCOS patients.

Keywords: Bax; Bcl2; FSH; LH; testosterone.

FIGURE 1

Approximation of the ovarian volume and volume density of the cortex, medulla, corpus luteum and ovarian cysts using the point‐counting method. The countable points are hitting the cortex, medulla, corpus luteum and ovarian cysts at the right upper corner of the cross (arrow).

FIGURE 2

Estimation of the number of follicles using the counting frame; arrows indicate follicles types.

FIGURE 3

Comparison of the microscopic images of the ovaries in different steps of intermediation. Control group (a): The different developmental stages of folliculogenesis were normal in the healthy control groups, PCOS group (b): The PCOS group showed a significant increase in the number of ovarian cyst and atretic follicles, PCOS+ 5 mg/kg thymoquinone group (c) and PCOS+ 10 mg/kg thymoquinone group (d): In these groups, a significant reduction in the number of ovarian cysts and atretic follicle were observed along with the higher volume of the corpus luteum and healthy follicles. a–d: H&E staining with magnification at × 40. The arrow indicates normal follicles (antral and preantral follicles). Abbreviations: A.F, atretic follicles; C.F, cystic follicles; C.L, corpus luteum; G.F, graafian follicles.

FIGURE 4

The number of follicles was determined on 20 μm thickness sections (n = 8). The results are shown as the Mean ± SD. Abbreviations: Con, control; H.D, PCOS + 10 mg/kg thymoquinone; L.D, PCOS + 5 mg/kg thymoquinone; PCOS, polycystic ovary syndrome. *p < 0.05 and **p < 0.01, Con group vs. PCOS, L.D and H.D. ††p < 0.01, PCOS vs. L.D and H.D groups.

FIGURE 5

The volume density of cortex, medulla, corpus luteum and ovarian cysts was estimated on 5 μm thickness sections using the point‐counting method (n = 8). The results are shown as the Mean ± SD. Abbreviations: Con, control; H.D, PCOS + 10 mg/kg thymoquinone; L.D, PCOS + 5 mg/kg thymoquinone; PCOS, polycystic ovary syndrome. *p < 0.05 and **p < 0.01, Con group vs. PCOS, L.D and H.D. †p < 0.05 and ††p < 0.01, PCOS vs. L.D and H.D groups.

FIGURE 6

Levels of sexual hormones in blood samples of treatment groups (n = 8). The results are shown as the Mean ± SD. Abbreviations: Con, control; H.D, PCOS + 10 mg/kg thymoquinone; L.D, PCOS + 5 mg/kg thymoquinone; PCOS, polycystic ovary syndrome. *p < 0.05 and **p < 0.01, Con group vs. PCOS, L.D and H.D. †p < 0.05 and ††p < 0.01, PCOS vs. L.D and H.D groups.

FIGURE 7

Gene expression of apoptotic markers and antioxidant enzymes. Expression pattern of Bax (a), Bcl2 (b), Bax/Bcl2 ratio (c), glutathione peroxidase 1 (GPx1) (d), superoxide dismutase 1 (Sod1) (e) and catalase (Cat) (f) in ovarian tissue of different groups. The results are shown as the Mean ± SD. Abbreviations: Con, control; H.D, PCOS + 10 mg/kg thymoquinone; L.D, PCOS + 5 mg/kg thymoquinone; PCOS, polycystic ovary syndrome. *p < 0.05 and **p < 0.01, Con group vs. PCOS, L.D and H.D. †p < 0.05 and ††p < 0.01, PCOS vs. L.D and H.D groups.