In the present study we mimicked the human anovulatory PCOS using DHEA-treated mouse model. The population of antral follicles increased in PCOS mouse ovary, but there is evidence that disordered folliculogenesis also involves the smaller, preantral follicles
. Beside of increased number of growing immature follicle, communication in between oocyte and granulosa cells is disturbed. Therefore, there is a failure of selection of dominant follicle, and the number of arrested and atretic follicles is significantly increased in polycystic ovaries
. In our study, we observed excess immature and atretic follicles at the different stage of folliculogenesis. Ovarian morphology and hormone status were investigated in female rats given daily androstenedion injections and Okutsu et al. showed that androstenedione administration enhances apoptosis in the inner part of granulosa cell layers of antral follicles, which subsequently leads to the formation of ovarian follicular cysts and exposure to excess androstenedione stimulates premature luteinization of granulosa cells, which is most likely due to the loss of oocyte-granulosa cell communication
. The findings suggested here show that mice from the DHEA group exhibited increased levels of both serum estradiol (E2) and progesterone (P). We suggested that after daily injection of DHEA, hyperandrogenized environment occur the increased concentration of serum E2 would result in unfavorable conditions for producing follicles destined for ovulation. Previous studies work with the same animal model, have reported a similar hormonal regulation
. This hormonal profile suggests an increased steroidogenic activity, which is widely described in PCOS
[1, 36]. The follicles from anovulatory women with PCOS hypersecrete E2 when compared with size-matched follicles from normal ovaries or polycystic ovaries from ovulatory women
[36–38]. Additionally, absent of corpora lutea structure in PCOS ovary showed an anovulatory infertility model.
A characteristic of the ovarian morphology that was seen in DHEA-treated mice was the presence of follicular cysts. These results demonstrate that ovarian follicular cysts are formed from antral follicles and that androstenedione treatments selectively disrupt the later stages of ovarian follicle development (follicle maturation) and subsequent ovulation. Thus, the current results demonstrate that enhanced E2 and P level is the cause of the follicular cyst formation, which may prevent the development of dominant follicles. Moreover, we showed that exposure to excess DHEA stimulated increased estradiol and progesterone levels. Therefore, excess DHEA stimulation may cause to the loss of oocyte-granulosa cell communication and degeneration of healthy oocytes and follicle
[39, 40]. Additionally, DHEA induces increased steroidogenesis in the proliferating and differentiative granulosa cells of early antral follicles as they develop into cysts.
In our present study, mTOR and its downstream effectors were elevated in DHEA-treated PCOS mouse model. The mTORC1 and mTORC2 have crucial roles in different pathways as energy and nutrient sensing, metabolism, cell growth, and differentiation
[41–46]. We showed previously that mTOR is ubiquitously expressed in mouse ovary with predominantly cytoplasmic and perinuclear expression in granulosa cells. However, the P-mTOR (serine 2448) is strongly enriched within mitotic granulosa cells and localizes in the region of the mitotic spindle and also near actin filament–containing structures, including the contractile ring of cytokinesis
. Here we showed mTOR signal proteins in DHEA-treated PCOS mouse model. mTOR and P-mTOR (Serine-2448) showed more protein expression in P group than C and V group. Raptor and GβL (LST8) are two major components of mTORC1 have role in cell proliferation and differentiation. There is no difference about Raptor and GβL (LST8) expression between three groups. Therefore, we suggested that Raptor and GβL (LST8) proteins have important role in activation of mTORC1 and then stimulation of downstream proteins. In this situation, we suggested that Serine-2448 phosphorylated form of mTOR may be responsible from increased granulosa and theca cell proliferation in PCOS mouse ovary. Our PCNA western blot results confirmed that DHEA-treated PCOS mouse ovary showed increased proliferation compared to other groups as shown by increased PCNA expression. P70S6K is well defined downstream signal protein of mTORC1. mTOR regulates cell growth and proliferation functions through cytoplasmic targets such as P70S6K. Bachmann et al. showed that mTOR shuttles between the nucleus and cytoplasm, and nucleocytoplasmic shuttling of mTOR is required for the maximal activation of S6K1
. mTOR stimulates translation by phosphorylating P70S6 kinase and, consequently, the 40S ribosomal protein S6
. Activation of this pathway is required for FSH-mediated induction of several follicular differentiation markers, including luteinizing-hormone receptor (LHR), inhibin-α, microtubule-associated protein 2D, and the PKA type IIβ regulatory subunit
. Based on our results we suggested that activation of mTORC1 has stimulatory effect on P70S6K downstream signal protein. Therefore, P-P70S6K protein level showed difference than P70S6K. Interestingly in our study we detected decreased P-P70S6K protein expression in PCOS group and suggested that P-P70S6K might decrease in DHEA-treated PCOS mouse ovary because of dysfunctional folliculogenesis. Namely, follicular differentiation signals with FSH stimulation cannot show enough effect through decreased level of FSH in PCOS. Thence, we suggested that insufficient P70S6K activation causes the arrest of follicular development. Thus, P-P70S6K showed decreased level in DHEA-treated PCOS mouse ovary.
mTORC2 is composed of mTOR, Rictor, mSIN1, and GβL (LST8), and this branch of mTOR action is resistant to acute inhibition by rapamycin. Our study is the first to show showed weak P-mTOR (Serine-2481) protein level showed very weak expression in all groups, but increased in DHEA-treated PCOS mouse ovary. Moreover, Rictor protein level showed increased expression in PCOS mouse ovary when we compare with control and vehicle groups. We suggested that mTORC2 signal pathway may have important role in PCOS. PKCalpha is an important protein which has role in cell proliferation, differentiation and apoptosis. mTORC2 involved in the phosphorylation of PKCalpha and in post-translational processing
. Ablation of mTORC2 components (Rictor, Sin1 or mTOR) abolished phosphorylation of the PKCalpha
. In our study PKCalpha and its phosphorylated form P-PKCα/beta (Thr638/641) showed same intensity for protein level in three experimental groups. At the same time, there is no difference between PCOS and control group for PKCalpha and P-PKCα/beta (Thr638/641) protein levels. Therefore we suggested that mTORC2 may use also different downstream signal proteins beside PKCalpha in DHEA-treated PCOS mouse ovary. We therefore suggested that Rho and Akt downstream proteins of mTORC2 may also have roles in PCOS mouse ovary.
The characteristic morphological feature of polycystic ovaries in anovulatory women is accumulation of antral follicles in the range of 2–8 mm in diameter. Therefore follicular maturation is disturbed, resulting in premature arrest of follicular growth. Additionally oocyte-granulosa cells communication is also disrupted and apparent failure to select a dominant follicle
. The results presented here indicate that mTORC1 and mTORC2 together may have important regulatory function in PCOS mouse model. Different abnormalities may appear depending on stress or nutrition and they may effect directly follicular growth. As it is known that changes in lipid metabolism disorder in PCOS causes women to gain weight excessively. These findings in PCOS suggest a direct relationship between the energy metabolism of cells and the mTOR signaling mechanisms. Both stress and eating disorders have seen in PCOS (as commonly observed in patients with PCOS suffer from obesity) as well as hormonal imbalance. The mechanism of mTOR by affecting the development of cystic structures, and can cause anovulation. Therefore we suggested that the increase in mTOR activation (mTORC1 and mTORC2) causes increased granulosa cell proliferation. In another study, blockade of central mTOR signaling by rapamycin caused decreased LH secretion
. Consequently, we suggested that Rapamycin (the inhibitor of mTOR) may be a compensatory mechanism attempting to increase protein synthesis and regulate stimulation of luteinized hormone secretion for preventing or treating anovulatory PCOS.
PCOS patients have high levels of LH and show anovulatory follicles in their ovary. This might be due either to abnormalities in LH secretion or to an augmentation of the LH stimulus through hyperinsulinemia and/or hyperandrogenemia
. Phosphorylation of mTOR in PCOS mouse model may be potentially due to estrogens or other derivatives. So it is necessary to further investigate how DHEA is related, directly and indirectly mTOR signal mechanisms effect to folliculogenesis and ovulation process.
In summary, we have found that DHEA increases mTORC1 and mTORC2 expression in mouse ovary. It appears, since DHEA increased mTOR expression in proliferative and differentiative-stage cells (premature luteinization of granulosa cells), mTOR signal pathways in DHEA metabolism might play important roles in the PCOS mouse ovary that results in disturbance of the dominant follicle selection and leads to abnormal follicular development and cystogenesis.