Role of mTOR Signaling in Female Reproduction

mTOR signaling is essential for embryonic development both in vivo and in vitro . In Drosophila melanogaster , cell size and embryonic lethality are reduced when TOR or S6 kinase is silenced ( 124 , 125 ). In mice, disruption of mTOR completely results in the death of embryos shortly after implantation, which was attributed to impaired cell proliferation in both embryonic and extraembryonic compartments. This was proven by in vitro experiments in which both the inner cell mass (ICM) and trophoblasts fail to proliferate in mTOR-deleted mouse blastocysts ( 19 ). When mTOR is disrupted partly, mutant embryos die at midgestation coitum, which is accompanied by failed telencephalon cell growth and rotation around the embryonic body axis ( 126 , 127 ). Different from that with mTOR, mice deficient in S6K1 are viable and smaller in body size, which could be explained by the fact that S6K1 only represents one part of mTOR signaling ( 128 ). Drugs that inhibit mTOR signaling impair blastocysts growth, but the effects are reversible. When treated with rapamycin (200 nM), growth of the ICM is not inhibited, but trophoblast outgrowth of blastocysts is impeded ( 19 ). Interestingly, the proliferation of embryonic stem cells is also refractory to rapamycin. An insufficient number of rapamycin receptors or the existence of mTORC2 could be the reason for this ( 19 ). Another exciting discovery is that the inhibition of both mTORC1 and mTORC2 complexes using INK128 or RapaLink-1 results in a diapause state of blastocysts. Diapause is a reversible pausing state triggered by unfavorable conditions ( 129 , 130 ). When the inhibition is relieved, blastocysts can give rise to live, fertile mice ( 131 ). mTOR signaling also takes part in mammalian embryonic differentiation, during which embryonic stem cells and pluripotent stem cells grow into the endoderm, ectoderm and mesoderm ( 132 ). Interestingly, mTOR signaling presents different functions in mouse embryonic stem cells and human pluripotent stem cells. In mice, the activation mTOR induces mouse embryonic stem cell differentiation. Increasing mTOR activity via withdrawal of the cytokine leukemia inhibitory factor from mouse embryonic stem cell culture can induce mouse embryonic stem cell differentiation ( 133 , 134 ). Further, a decrease in mTORC2 activity, resulting from the knockdown of calcineurin, impairs mesoderm differentiation ( 135 ). In contrast to that in mouse embryonic stem cells, mTOR inhibition promotes human pluripotent stem cell differentiation. The knockdown of Raptor in human pluripotent stem cells induces mesendoderm differentiation ( 136 ), and the same effect occurs when treating human pluripotent stem cells with rapamycin ( 137 ). The mechanism underlying this difference between mice and humans is not understood, and additional studies are needed to explore this, as the cell populations are similar ( 138 ).

mTOR forms the catalytic subunit of two different multi-molecular complexes known as mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) ( 20 ). mTORC1, characterized by its sensitivity to rapamycin treatment, consists of mTOR, Raptor, mLST8/GβL, PRAS40, and DEPTOR. mTORC2, which is insensitive to acute rapamycin treatment but is inhibited by prolonged usage ( 21 ), is composed of mTOR, Rictor, mLST8/GβL, DEPTOR, mSin1, and protor1/2 ( 22 ). mTORC1 plays a vital role in coordinating several cellular processes to help cells grow and divide. These processes include protein synthesis, lipid and nucleotide biogenesis, and autophagy ( 22 ). The critical effectors downstream of mTORC1 involving protein synthesis are p70S6 Kinase 1 (S6K1) and eIF4E Binding Protein 1 (4EBP1) ( 23 ), which deserve special emphasis for their important roles in female reproduction as described in the following sections. The cellular pathways upstream of mTORC1 include growth factors, stress, and amino acids ( 22 ). Briefly, numerous growth factor pathways inhibit tuberous sclerosis complex (TSC) ( 24 , 25 ), which is composed of TSC1, TSC2, and TBC1D7 ( 26 ), relieve the inactivation of Ras homolog enriched in brain (Rheb) ( 27 – 30 ), and stimulate mTORC1 kinase activity. Certain incompatible stresses such as hypoxia, low ATP levels, or DNA damage, activate AMP-activated protein kinase (AMPK) pathway, which inhibits mTORC1 via the phosphorylation of Raptor ( 31 ) or TSC2 ( 32 – 34 ). For amino acids, there are two distinct pathways that stimulate mTORC1, and these are dependent or independent of the Rag GTPases ( 35 – 37 ). The most important function of mTORC2 is the phosphorylation of Akt, which subsequently affects cell growth and proliferation ( 38 ). Moreover, mTORC2 activates several members of the AGC family, regulating cytoskeletal remodeling and cell migration ( 39 – 41 ). The cellular pathways upstream of mTORC2 are insulin/PI3K and mTORC1 ( 42 , 43 ).

Rapamycin is a macrocyclic lactone produced by the bacterium Streptomyces hygroscopicus that was previously used as an antifungal agent but abandoned soon after because of its immunosuppressive effect ( 1 , 2 ). The protein target of rapamycin (TOR) was originally discovered in genetic mutation studies using Saccharomyces cerevisiae ( 3 ) and subsequently found to be the target of the rapamycin–FKBP12 complex in mammalian cells, which is now referred to as the mammalian target of rapamycin (mTOR) ( 4 – 6 ). mTOR is sensitive to diverse environmental inputs including nutrients and growth factors, and regulates various fundamental cell processes including cell growth, metabolism, differentiation, and autophagy ( 7 ). The dysregulation of mTOR has been observed in many diseases like cancer ( 8 ), diabetes ( 9 ), neurodegenerative disorders ( 10 ), and aging ( 10 ). Further, targeting mTOR is one of the most promising fields for the efficient treatment of these diseases. Cellular metabolism comprises the foundation of all biological activities including female reproduction ( 11 , 12 ). Recently, accumulating lines of evidence have shown that mTOR-regulated processes are important for folliculogenesis ( 13 ), oocyte meiotic maturation ( 14 ), ovarian somatic cell proliferation and steroidogenesis ( 15 ), puberty onset ( 16 ), ovarian aging ( 17 ), endometrium changes ( 18 ), and embryonic development ( 19 ). In this review, the role of mTOR signaling in female reproduction will be discussed, and the data describing alterations to this pathway under pathological conditions will be summarized.

As mTOR signaling affects many processes associated with reproduction, manipulating this pathway to preserve fertility has been explored in preclinical studies. The pharmacologic downregulation of mTOR protects the ovarian reserve in the presence of ovarian toxic drugs in animal models ( 139 ), as mTOR is related to primordial follicle activation. In fact, anticancer therapies such as genotoxic or antimitotic agents hardly impair most oocytes in primordial follicles. However, when the ovary is repeatedly exposed to chemotherapy, primordial follicles are activated and grow to replace damaged growing follicles ( 140 , 141 ). Thus, regimens to counteract follicle activation could prevent premature ovarian failure induced by toxicity. It was also confirmed that mTOR inhibitors can promote follicular quiescence and prevent premature ovarian failure when used in cyclophosphamide-induced and cisplatin-induced ovarian dysfunctional animal models ( 139 , 142 , 143 ). Activating mTOR signaling is a vital mechanism used in primordial follicle growth activation (PFGA) ( 144 ), which has great potential to assist reproduction. PFGA is an explored technology that is expected to provide benefits for patients with diminished ovarian reserve or malignant tumors. One problem with hormonal stimulation strategies used clinically is that they cannot fully utilize most follicles remaining in the ovary such as primordial follicles but largely rely on the population of growing follicles. Thus, a critical step to fully exploit the ovarian follicle is to overcome the mechanism resulting in the growth-arrest of primordial follicles ( 144 ). mTOR activators (phosphatidic acid, propranolol) can induce the awakening of primordial follicles in the human ovarian cortex such that more mature follicles can be obtained in combination with other primordial follicle activators ( 145 , 146 ). Polycystic ovarian syndrome (PCOS) is a multifactorial endocrinopathy that affects reproduction and metabolism ( 147 , 148 ). Many conclusions involving the relationship between PCOS and mTOR signaling are paradoxical and complex. For example, in a DHEA-induced PCOS mouse model, the expression of mTOR and p-mTOR (serine-2448) in the ovary was found to be higher than that in normal mice, but S6K1 is decreased in the DHEA-treated PCOS mouse ovary. With respect to this, the authors speculated that insufficient S6K1 activation causes an arrest in follicular development but they could not explain the discrepancy in expression between mTOR and downstream signaling ( 149 ). In another study, mTOR protein levels in luteal GCs of PCOS patients were the same as those in healthy women. However, less mTOR protein expression is observed in luteal GCs with PCOS compared to that in healthy patients upon stimulation with insulin ( 150 ). Therefore, further experiments are needed to delineate the accurate expression of mTOR in PCOS. Another characteristic linking PCOS and mTOR is metabolic disorder during PCOS. Rapamycin was found to enhance insulin sensitivity and improve serum lipid profiles after long term treatment ( 151 ). Thus, rapamycin is expected to be used to eliminate metabolic syndrome with PCOS despite potential adverse effects on fertility and short-term metabolism. Endometriosis, a gynecological disease caused by the dislocation of endometrial cells, is characterized by inflammation and to progesterone resistance ( 152 ). Inadequate responses to progesterone in eutopic and ectopic endometrial cells and tissue contribute to increased cell proliferation ( 153 ), in which mTOR is involved. The ectopic endometrium of patients with endometriosis exhibits increased phosphorylation of mTOR compared to that in the eutopic endometrium ( 154 – 156 ). Moreover, the inhibition of mTOR can suppress endometriotic foci in a rat/mouse model of endometriosis ( 157 , 158 ) and promote human endometriotic cell apoptosis via autophagy induction ( 113 , 159 ). In addition to the ectopic endometrium, aberrant decidualization in the eutopic endometrium of women with endometriosis impairs implantation partially through the activation of PI3K/AKT ( 160 , 161 ). As mTORC2 is downstream of AKT, it can be postulated that mTOR is activated in the eutopic endometrium, but this requires experiments for confirmation.

ZG collected the information, designed the figures, wrote the manuscript, and submitted the manuscript. QY contributed to manuscript conception, design, and critical discussion. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

mTOR signaling plays a vital role in regulating female reproduction, which has been demonstrated based on data from genetic, pharmacological, and clinical studies ( Table 2 , Figure 1 ). mTOR signaling participates in various process that occur in the ovary, including ovarian reserve, follicle development, oocyte meiotic maturation, ovarian aging, and proliferation and steroidogenesis of ovarian somatic cells, among others. There are two impressive functions of mTOR signaling in the ovary. First, elevated mTOR expression in both oocytes and primordial follicle GCs activates the primordial follicle, which has great potential for applications of ovarian reserve protection and PFGA. Second, suppressing mTOR hinders oocyte meiotic maturation, which could limit the use of mTOR-suppressing drugs for fertility-related diseases. In addition to that in the ovary, mTOR appears to be crucial for hypothalamus functions, endometrium changes, and embryo development. Finally, preclinical evidence suggests the possibility of applying mTOR modulators to ameliorate fertility issues associated with POF, PCOS, and endometriosis. Role of mTOR signaling in female reproductive cells and organs. Involvement of mTOR in several processes linked to female reproduction. The impressive events are presented involving mTOR in primordial follicle activation, oocyte meiotic maturation, somatic cells, endometrium, and post-fertilization events. AKT, Protein kinase B; E2, Estradiol-17β; 4EBP1, eIF4E Binding Protein 1; ERK, Extracellular signal regulated kinase; KITL, KIT ligand; mTOR, Mammalian target of rapamycin; mTORC1, mTOR complex 1; P4, Progesterone; PI3K, Phosphoinositide 3-kinase; PKA, Protein kinase A; PKC, Protein kinase C; S6K1, p70S6 Kinase 1. Although rapamycin and catalytic mTOR inhibitors are already successfully used to prevent the rejection of transplants or treat some types of cancer, many studies are needed to translate experimental results to clinical use for female reproductive diseases. On one hand, mTOR seems to be much more than a simple positive or negative trigger of female reproduction. Under physiological conditions, mTOR is regulated by different factors in a joint effort to determine the outcome of several processes including folliculogenesis, ovulation, endometrium changes, or embryonic development. On the other hand, the complete inhibition of mTOR would cause severe dose-limiting toxicities based on the indispensable role of mTOR in most human tissues. Thus, it has yet to be determined if it is worthwhile to cure non-life threatening diseases with an mTOR inhibitor ( 162 , 163 ). Future work should focus on the development of tissue-specific therapeutics to avoid drawbacks associated with effects on unrelated tissues. Overall, mTOR clearly plays an important role in female reproduction. However, much work is needed to fully understand the mechanisms underlying the role of mTOR in female reproduction and to completely unlock the therapeutic potential of this signaling pathway.