The effect of androgens on ovarian follicle maturation: Dihydrotestosterone suppress FSH-stimulated granulosa cell proliferation by upregulating PPARγ-dependent PTEN expression

Abstract

Intraovarian hyperandrogenism is one of the determining factors of follicular arrest in women with polycystic ovary syndrome (PCOS). Using androgenized rat models, we investigated the effects of androgens on metabolism, as well as on factors involved in follicular arrest and the reduced number of estrus cycles. The dihydrotestosterone (DHT)-treated rats had fewer estrus cycles, higher numbers of large arrested follicles and an increased in body weight gain compared with the dehydroepiandrostenedione (DHEA)- and placebo-treated rats. In cultured rat granulosa cells, DHT suppressed follicle stimulating hormone (FSH)-induced granulosa cell proliferation and increased the accumulation of cells in the G2/M phase. DHT decreased phosphorylated Akt (p-Akt) and cyclin D1 levels through increasing PTEN. DHT-promoted PTEN expression was regulated by peroxisome proliferator-activated receptor gamma (PPARγ) in granulosa cells. Meanwhile, in the large follicles of the DHT-treated rats, the expressions of PPARγ and PTEN were higher, but the expression of p-Akt and proliferating cell nuclear antigen (PCNA) were lower. Conclusively, DHT and DHEA produced differential effects on metabolism in prepubertal female rats like clinical manifestations of women with PCOS. DHT treatment may affect ovarian follicular maturation by altering granulosa cell proliferation through the regulation of enhancing PPARγ dependent PTEN/p-Akt expression in the granulosa cells.

Figure 1. Body weight and food intake in androgenized rats.

Female SD rats were randomly divided into three experimental groups (control, DHT, and DHEA) and subcutaneously implanted with 90-day continuous-release pellets containing 7.5 mg DHT (daily dose, 83 μg), 200 mg DHEA (daily dose, 2.2 mg), or 7.5 mg placebo. The data are shown as means ± SD. (A) Weekly body weight per rat. Numbers of rats = 22, 19, and 21 for the DHT, DHEA and placebo groups, respectively. (B) Weekly food intake amount per rat. Number of rats = 9 in each group. In both (A,B), the levels of the DHT group were significantly higher than those of the other 2 groups, as determined by repeated measures ANOVA. *P < 0.05.

Figure 2. Effects of DHT and DHEA on estrous cycles and follicular maturation.

The data are shown as means ± SD. (A) The interestrus intervals of the rats were determined by vaginal smears. The interestrus interval of the DHT group was significantly higher than that of the other two groups. Number of rats = 16, 14, and 14 in the DHT, DHEA and placebo groups, respectively. (B) At the end of the study, the rat ovaries were fixed for histology. Representative HE staining is shown (40×), bar = 300 μm. (C) Quantitative results of total antral follicle count, including small and large antral follicles, among the 3 groups. The large follicle count of the DHT group was significantly higher than that of the other 2 groups. (D) Quantitative results of the mature and ovulated follicle count, including the Graafian follicles and corpus lutea, among the 3 groups. The mature and ovulated follicle count of the DHT group was significantly lower than that of the other 2 groups. For the antral follicle measurements in (C,D), number of rats = 8, 7, and 8 in the DHT, DHEA and placebo groups, respectively. Between-group comparisons are indicated. *P < 0.05.

Figure 3. DHT inhibited in vitro FSH-induced granulosa cell proliferation through interfering cell cycle.

(A) Fifty μg of total protein form rat granulosa cells and human granulosa cells (hGC) were used to determine the specificity of the antibody to FSHR of rat granulosa cells (rGC) by immunoblotting (left panel). Ratio of FSHR positive cells were analyzed by quantifying the FSHR-positive cells by flow cytometry using a FACScan and the Cell Quest software. The white histograms are of isotype controls, whereas the orange overlays were of FSHR-positive cells (right panel). (B) Rat granulosa cells were treated with FSH and different doses of DHT or DHEA, and after 72 hours, cell growth was determined by the MTT assay. The data are shown as means ± SD of 3 independent experiments. Granulosa cell growth was significantly suppressed by co-treatment with FSH and DHT (exceeding 25 uM) in comparison with FSH treatment only. (C) Rat granulosa cells were pretreated with DHT (50 μM), bicalutamide (50 μM), or insulin (500 nM) for one hour prior to FSH (10 ng/ml) treatment. After 24 hours, the cell cycle phase was determined by propidium iodide staining and FACScan analysis. Populations in the subG1, G1, S, and G2/M phases are shaded M1 to M4, respectively. Representative cell cycle histograms of each group are shown. N = 3. (D) The percentage of cells in each cell cycle phase was analyzed and quantified by Cell Quest software, and the data represent the mean ± SD of 3 independent experiments. The percentage of cells in the G2/M phase significantly decreased following treatment with FSH. Compared with cells treated with FSH alone, the number of cells in G2/M phase increased significantly following treatment with DHT and FSH. The percentage of cells in G2/M phase significantly decreased following treatment with DHT/FSH/Bicalutamide, in comparison with cells treated with DHT and FSH. Between-group comparisons were performed as indicated *P < 0.05.

Figure 4. DHT inhibited cell proliferation through regulating p-Akt expression by enhancing PTEN and suppressing resultant cyclin D1 expression.

The data are means ± SD. (A) Rat granulosa cells were pre-treated with different doses of DHT for 1 hour prior to FSH treatment, and after 24 hours, cell lysates were used to measure the cyclin protein levels by western blot. Expression of tubulin was used as loading control. Representative immunoblottings of protein are shown. N = 3. Relative density (RD) of each lane was determined by ImageJ, lane 1 was defined as 1. Statistical comparisons were done between lane 1 with the other lane. *P < 0.05. (B) Rat granulosa cells were pre-treated with DHT (50 μM) or insulin (500 nM) for 1 hour prior to FSH (10 ng/ml) treatment, and after 24 hours, cell lysates were used to measure the indicated protein level by western blot. Expression of β-actin was used as loading control. Representative immunoblottings of protein are shown. N = 3. ImageJ determined RD and lane 1 was defined as 1. Statistical comparisons were done between lane 1 with the other lane. *P < 0.05. (C) Rat granulosa cells were pre-treated with DHT or DHEA, and after 24 hours, cell lysates were used to measure the PTEN level by western blot. Expression of tubulin was used as loading control. Representative immunoblottings of protein are shown. N = 3. ImageJ determined RD and lane 1 was defined as 1. Statistical comparisons were done between lane 1 with the other lane. *P < 0.05. (D) Rat granulosa cells were pretreated with PTEN siRNA(si) or control siRNA(25 μM) for 24 hours and following with or without FSH(10ng/ml) and DHT(50 μM) treatment. After 3 days, cell growth was determined by the MTT assay, N=3. Between-group comparisons were performed as indicated *P < 0.05.

Figure 5. DHT promoted PTEN expression by enhancing PPARγ expression in ovarian granulosa cells.

(A) Rat granulosa cells were pretreated with siRNAs of p53, Egr-1, and PPARγ for 24 hours, then the protein levels of p53, Egr-1, PPARγ were determined by western blot and significantly decreased after corresponding siRNAs treatment. *P < 0.05. (B) Rat granulosa cells were pretreated with siRNAs of p53, Egr-1 and PPARγ respecitvely for 24 hours prior DHT (25uM) treatment for another 24 hours, then PTEN mRNA expression was determined by Q-RT-PCR. PPARγ siRNA significantly suppressed the DHT-upregulated PTEN expression in ovarian granulosa cells. *P < 0.05. (C) PPARγ protein levels were determined by western blot after treatment of different doses of DHT for 24 hours in rat granulosa cells. The levels of PPARγ were significantly increased with incremental dosage of DHT treatment. Statistical comparisons were done between lane 1 with the other lane, *P < 0.05, N = 3. (D) PPARγ mRNA levels were determine by Q-RT-PCR after treatment of different doses of DHT for 24 hours in rat granulosa cells. The mRNA levels of PPARγ were significantly increased with incremental dosage of DHT treatment in rat granulosa cells, but decreased with bicalutamide treatment. Between-group comparisons were performed as indicated *P < 0.05, N = 3.

Figure 6. Expression of PCNA, p-Akt, PTEN, PPARγ and apoptosis marker in large antral follicles of placebo-, DHEA- and DHT-treated rats.

(A) The expression of PCNA, p-Akt, PTEN, PPARγ and TUNEL assay. The representative immunohistochemical staining image of PCNA, p-Akt, PTEN, PPARγ and the TUNEL assay results using ovaries from placebo-, DHEA-, and DHT-treated rats are presented at 400x magnification. (B) Quantitative results of immunohistochemistry analysis. A total of 5 follicles per ovary were counted for analysis of granulosa cells with positive staining, and 3 ovaries per group and 1 ovary per rat were collected for analysis. The data are shown as means ± SD and represent comparisons with the control group *P   < 0.05. (C) Western blot analysis of PTEN and PPAR-γ proteins in ovarian tissue (N = 3/group). Each ovarian tissue was homogenized and extracted, the equal amount of protein extracts were subjected to immunoblotting as described in the materials and methods. Actin was used as an internal control.