Consecutive Low Doses of Streptozotocin Induce Polycystic Ovary Syndrome Features in Mice

Abstract

Polycystic ovarian syndrome (PCOS) is a common reproductive endocrine disorder in reproductive-age women. Due to its various pathophysiological properties and clinical heterophenotypes, the mechanism of PCOS pathogenesis is still unclear. Several animal models have been used to study PCOS and allow the exploration of the specific mechanism underlying PCOS. We focused on streptozotocin (STZ) to develop a non-steroidal and non-diabetic PCOS model. We administered multiple STZ injections to female C57BL/6 mice (3-4 weeks old) at different concentrations: STZ-15 (15 mg/kg), STZ-30 (30 mg/kg), and STZ-60 (60 mg/kg) treatments. During the experimental period, we analyzed body weight, blood glucose levels, and estrous cycle pattern. Furthermore, five weeks after STZ administration, we examined hormone levels and the morphology of ovarian tissues. Mice in the STZ-15 group did not show differences in body weights, blood glucose level, insulin level, and insulin tolerance compared to wild-type and control groups whereas those in the STZ-60 group presented a typical diabetes phenotype. In the case of the STZ-30 group, only increased blood glucose level was observed. Total testosterone levels were significantly elevated in STZ-15 and STZ-30 groups. Luteinizing hormone (LH) and estradiol levels were not significantly changed in the STZ-treated groups. The number of ovarian antral follicles and atretic follicles significantly increased in the ovary of mice in the STZ-15 and STZ-30 groups. All STZ-treated groups manifested irregular estrus cycles. However, the patterns of estrous cycles were different between mice treated with different STZ concentrations. We found that PI3K-AKT and IRS-1 signaling in the ovary was enhanced by low doses of STZ treatment. Taken together, our finding indicates that multiple injections of STZ at low doses induce PCOS features in mice without induction of diabetes features.

Keywords: animal models; ovary; polycystic ovary syndrome; streptozotocin; testosterone.

Figure 1

Analyses of bodyweight and glucose level in streptozotocin (STZ)-treated mice. (A) Bodyweight of mice in each group during the experimental period: wild-type (WT, diamonds with black dotted line); control (CON, diamonds with a black line); 15 mg/kg streptozotocin (STZ-15, rectangles with bright orange line); 30 mg/kg streptozotocin (STZ-30, triangles with dark orange line); and 60 mg/kg streptozotocin (STZ-60, circles with red line) groups. (B) Blood glucose levels in each group. The injection of 60 mg/kg STZ induced a typical diabetes condition whereas lower doses of STZ injections did not. (C) Plasma insulin levels: WT, white diamonds; CON, black diamonds; STZ-15, bright orange rectangles; STZ-30, dark orange triangles; and STZ-60, red circles. Treatment with STZ injections did not significantly suppress insulin levels in STZ-15 and STZ-30 groups. (D) Insulin tolerance tests (ITT) were performed at 5 weeks after STZ treatment (left) and expressed as a ratio compared to initial values (right). Mice in the STZ-60 showed signs of insulin resistance. (E) Calculated homeostasis model assessment of insulin resistance (HOMA-IR) index: WT, white bar; CON, black bar; STZ-15, orange bar; STZ-30, dashed orange bar; and STZ-60, red bar. Insulin resistance was not observed in STZ-15 and STZ-30 groups. (a) significant differences compared with the WT group; (b) significant differences compared with the CON group; (c) significant differences compared with the STZ-15 group; (d) significant differences compared with the STZ-30 group; n.s., not significant.

Figure 2

Hormonal assay for testosterone. (A) Plasma total testosterone levels in wild-type (WT, white diamonds); control (CON, black diamonds); 15 mg/kg streptozotocin (STZ-15, bright orange rectangles); 30 mg/kg streptozotocin (STZ-30, dark orange triangles); and 60 mg/kg streptozotocin (STZ-60, red circles) groups. Mice in the STZ-15 and STZ-30 groups developed hyperandrogenism. (B) Plasma luteinizing hormone (LH) levels. Treatment with STZ injections did not elevate circulating LH levels. (C) Estradiol (E2) levels. Treatment with STZ injections did not suppress E2 synthesis. (a) significant differences compared with the WT group; (b) significant differences compared with the CON group. n.s., not significant.

Figure 3

Polycystic ovary syndrome (PCOS) phenotype of STZ-treated mice ovary. (A) Ovary weight in wild-type (WT, white diamonds); control (CON, black diamonds); 15 mg/kg streptozotocin (STZ-15, bright orange rectangles); 30 mg/kg streptozotocin (STZ-30, dark orange triangles); and 60 mg/kg streptozotocin (STZ-60, red circles) groups. (B) Representative microscopic images of ovary sections (hematoxylin and eosin staining; scale bar = 1000 μm). (C) The number of antral follicles per ovary indicating multi follicular formation in the ovaries of mice in the STZ-15 and STZ-30 groups. (D) The number of atretic follicles per ovary indicating that STZ injections compromised ovarian functions. (E) The number of corpora lutea per ovary showing the decreased abundance of corpora lutea in the STZ-60 group. (a) significant differences compared with the WT group; (b) significant differences compared with the CON group; (c) significant differences compared with the STZ-15 group; (d) significant differences compared with the STZ-30 group; n.s., not significant.

Figure 4

PCOS phenotype of STZ-treated mice ovarian follicles. (A) Diameter of ovaries in wild-type (WT, white diamonds); control (CON, black diamonds); 15 mg/kg streptozotocin (STZ-15, bright orange rectangles); 30 mg/kg streptozotocin (STZ-30, dark orange triangles); and 60 mg/kg streptozotocin (STZ-60, red circles) groups. (B) The average diameter of mature follicles. (C) Average corpus luteum (CL) diameter. Corpora lutea were enlarged in the STZ-15 group. (D) Representative microscopic images showing the theca cell layer of antral follicles in each group (hematoxylin and eosin staining; scale bar = 100 μm). (E) Theca cell layer thickness indicating significant changes induced by STZ injections. (a) significant differences compared with the WT group; (b) significant differences compared with the CON group; (e) significant differences compared with the STZ-60 group; n.s., not significant.

Figure 5

Alterations of estrous cycles in STZ-treated mice. (A) Estrous cycle stage classification of each group during 14 days in wild-type (WT, white diamonds); control (CON, black diamonds); 15 mg/kg streptozotocin (STZ-15, bright orange rectangles); 30 mg/kg streptozotocin (STZ-30, dark orange triangles); and 60 mg/kg streptozotocin (STZ-60, red circles) groups. Treatment with STZ injections disrupted ovarian functions. (B) The number of normal estrous cycles in each group during 2 weeks in CON, black bar; STZ-15, orange bar; STZ-30, dashed orange bar; and STZ-60, red bar, groups. (C) The number of each estrous cycle (%) stage during 14 days indicating that STZ injections alter regular ovary functions in mice. E, Estrus; P, Proestrus; M/D, Metestrus or diestrus.

Figure 6

Changes in the IRS-1 and PI3K-AKT pathway in STZ-treated ovary. The expression of specific proteins in STZ-treated ovary. IRS-1 and pAKT were up-regulated along with the STZ treatment dose in the ovary.