The mechanism of mTOR (mammalian target of rapamycin) in a mouse model of polycystic ovary syndrome (PCOS)

Abstract

Polycystic ovary syndrome (PCOS) is a common and complex endocrine disorder affecting 5-10% of women in reproductive age that is characterized by hyperandrogenism, oligo- or anovulation and infertility. However the pathophysiology of PCOS still remains unknown. The mammalian target of rapamycin (mTOR) is a central component that regulates various processes including cell growth, proliferation, metabolism, and angiogenesis. mTOR signaling cascade has recently been examined in ovarian follicles where it regulates granulosa cell proliferation and differentiation. mTOR functions as two complexes, mTOR complex 1 and 2. Therefore, we hypothesized that mTORC1 and/or 2 may have important role in proliferation of theca and granulosa cells in PCOS. In the present study, we sought to determine the mTOR signaling pathway in PCOS mouse ovary. We designed 3 groups: Control (C, no treatment), PCOS (P, The injection of DHEA (6 mg/100 g BW in 0.1 ml of sesame oil) (s.c) for 20 consecutive days), Vehicle (V, daily (s.c) sesame oil alone injection). Our results showed that mTORC1 and mTORC2-mediated signaling may play a role in PCOS mouse ovary. These findings provide evidence that mTORC1 and mTORC2 may have responsibility in increased ovarian follicular cell proliferation and growth in PCOS. Consequently, these results suggest that the mTOR signaling pathways (mTORC1 and mTORC 2) may create new clinical strategies to optimize developmental competence of PCOS should target correction of the entire follicle growth, oocyte development process and anovulatory infertility in PCOS.

Figure 1

Morphological comparison of control and DHEA-treated PCOS mouse ovary.A, control ovary; B, Corpus Luteum (CL) structure from control ovary. C, DHEA-treated PCOS mouse ovary; showed different stage developing follicles but an increase in the number of cysts, preantral follicle and atretic follicles. D, Follicular cyst (FC) structure from DHEA-treated PCOS mouse ovary. The morphology of cysts is characterized by a thin layer of theca cells and a compacted formation of granulosa cells.

Figure 2

Atretic follicles from different stage of follicular development in DHEA-treated PCOS mouse ovary. Unhealthy oocytes have seen with degenerated cytoplasm and zona pellucida (A–D). The arrows show early preantral follicles with compact oocytes and zona pellucida (Figure B and D).

Figure 3

Serum Estradiol (E2) (A) and Progesterone (P) (B) levels were evaluated by ELISA. Female mice of 25 day old (before treatment postnatal 25-day old, PN25), 45 day old (25 day old BalbC mice + 20 day treatment (DHEA-treated/sesame oil-treated) or no treatment (control group) = 45 day old). The levels of estradiol and progesterone examined increased after treatment with DHEA. C: Control group, DHEA: DHEA-treated PCOS group, V: Vehicle group.

Figure 4

Expression of mTOR and serine 2448–phosphorylated form of mTOR (P-mTOR) double staining by immunohistochemistry in DHEA-treated mouse ovary. mTOR expression was shown mainly cytoplasmic nature of total within granulosa cells in various stages of follicular development in control, DHEA-treated and vehicle groups. P-mTOR expression was highly enriched in mitotic granulosa cells in all stage of developing follicles in three groups (arrow, A–D). Negative control has no staining (D, insert).

Figure 5

mTORC1 and mTORC2 protein expression in control (C), DHEA-treated PCOS (DHEA) and vehicle (V) groups. (P < 0.05).

Figure 6

mTORC1 and mTORC2 protein expression levels in control (C), DHEA-treated PCOS (DHEA) and vehicle (V) groups. (P < 0.05).

Figure 7

P70S6K (A and B) and P-P70S6K (C and D) protein expression in control (C), DHEA-treated PCOS (DHEA) and vehicle (V) groups. (P < 0.05).

Figure 8

PKCalpha (A and B) and P-PKCα/beta (Thr638/641 phosphorylation) (C and D) protein expression in control (C), DHEA-treated PCOS (DHEA) and vehicle (V) groups. (P < 0.05).

Figure 9

PCNA protein expression in control (C), DHEA-treated PCOS (DHEA) and vehicle (V) groups. (P < 0.05).