The mTOR System Can Affect Basic Ovarian Cell Functions and Mediate the Effect of Ovarian Hormonal Regulators

Both reproductive hormones and the mammalian target of rapamycin (mTOR) intracellular signalling system, including mTOR complex 1 (mTORC1), mTOR complex 2 (mTORC2) and its key enzyme sirtuin 1 (SIRT 1) are involved in the control of ovarian processes but the interrelationship between hormones and mTOR has been studied insufficiently. The aim of our in vitro experiments was to elucidate the involvement of mTOR in the control of basic ovarian cell functions and in mediating the action of upstream hormonal stimulators. In the first series of experiments, we examined the effect of the known hormonal regulators of ovarian functions, Follicle-Stimulating Hormone (FSH), oxytocin (OT) and insulin-like growth factor I (IGF-I) (all at 0, 1, 10 and 100 ng mL-1 doses), on the accumulation of SIRT1 in porcine ovarian granulosa cells. In the second series of experiments, we examined the effects of mTOR blockers, PF 046 (an inhibitor of mTORC1) and WYE 687 (inhibitor of both mTORC1 and mTORC2) (both at a dose of 1 μg mL-1) on both basal and FSH-induced (0, 1, 10 and 100 ng FSH mL-1 doses) basic ovarian functions (proliferation, apoptosis and steroidogenesis) of cultured porcine granulosa cells. The accumulation of SIRT1, PCNA (a proliferation-related peptide) and Bax (an apoptosis-related peptide) was detected by immunocytochemistry. The release of progesterone (P4) and testosterone (T) was analysed by EIA. It was observed that either FSH or OT additions increased the SIRT1 accumulation in ovarian cells, whilst IGF-I addition decreased it. The PF 046, when given alone, inhibited ovarian cell proliferation but did not affect apoptosis or the release of P4 and T. The WYE 687, when added alone did not affect proliferation and apoptosis but inhibited the P4 and T release by ovarian cells. The FSH, when given alone, stimulated proliferation did not affect apoptosis and increased the release of both P4 and T. In the presence of PF 046, FSH did not significantly alter proliferation, induced apoptosis and suppressed the P4 and T release, i.e., this inhibitor of mTORC1 prevented the proliferation-stimulating effect induced by the pro-apoptotic action of FSH and inverted the stimulatory action of FSH on steroidogenesis. The WYE 687 prevented the effect of FSH on both proliferation and apoptosis, promoted the FSH-induced P4 release but did not affect the FSH action on the T output, i.e., the inhibition of both mTORC1 and mTORC2 inverted the FSH action on ovarian cell proliferation and apoptosis but not the action on steroidogenesis. Our observations suggest the following: (1) mTORC1 can be involved in the up-regulation of ovarian cell proliferation but not that of apoptosis and steroidogenesis (2) mTORC2 can be involved in the up-regulation of the release of both P4 and T but not the up-regulation of ovarian cell proliferation and apoptosis, (3) FSH is involved in the stimulation of ovarian cell proliferation and P4 and T release and (4) hormones (FSH, OT and IGF-I) can regulate ovarian mTOR and the ability of mTOR to modify FSH effects suggests that mTOR can mediate the actions of hormone on ovarian cells. It is hypothesized that both mTORC1 and mTORC2 may be mediators of the FSH action on ovarian cell proliferation, apoptosis and steroidogenesis.