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Ovarian Kaleidoscope Database (OKdb)



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Hsueh lab


since 01/2001:

mechanistic target of rapamycin OKDB#: 2406
 Symbols: MTOR Species: human
 Synonyms: SKS, FRAP, FRAP1, FRAP2, RAFT1, RAPT1  Locus: 1p36.22 in Homo sapiens

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General Comment Inhibition of mTOR induces a paused pluripotent state. Bulut-Karslioglu A et al. (2016) Cultured pluripotent stem cells are a cornerstone of regenerative medicine owing to their ability to give rise to all cell types of the body. Although pluripotent stem cells can be propagated indefinitely in vitro, pluripotency is paradoxically a transient state in vivo, lasting 2-3 days around the time of blastocyst implantation. The exception to this rule is embryonic diapause, a reversible state of suspended development triggered by unfavourable conditions. Diapause is a physiological reproductive strategy widely employed across the animal kingdom, including in mammals, but its regulation remains poorly understood. Here we report that the partial inhibition of mechanistic target of rapamycin (mTOR), a major nutrient sensor and promoter of growth, induces reversible pausing of mouse blastocyst development and allows their prolonged culture ex vivo. Paused blastocysts remain pluripotent and competent-able to give rise to embryonic stem (ES) cells and live, fertile mice. We show that both naturally diapaused blastocysts in vivo and paused blastocysts ex vivo display pronounced reductions in mTOR activity, translation, histone modifications associated with gene activity and transcription. Pausing can be induced directly in cultured ES cells and sustained for weeks without appreciable cell death or deviations from cell cycle distributions. We show that paused ES cells display a remarkable global suppression of transcription, maintain a gene expression signature of diapaused blastocysts and remain pluripotent. These results uncover a new pluripotent stem cell state corresponding to the epiblast of the diapaused blastocyst and indicate that mTOR regulates developmental timing at the peri-implantation stage. Our findings have implications in the fields of assisted reproduction, regenerative medicine, cancer, metabolic disorders and ageing.//////////////////

NCBI Summary: The protein encoded by this gene belongs to a family of phosphatidylinositol kinase-related kinases. These kinases mediate cellular responses to stresses such as DNA damage and nutrient deprivation. This protein acts as the target for the cell-cycle arrest and immunosuppressive effects of the FKBP12-rapamycin complex. The ANGPTL7 gene is located in an intron of this gene. [provided by RefSeq, Sep 2008]
General function Cell cycle regulation
Comment Pharmacological Inhibition of mTORC1 Prevents Over-Activation of the Primordial Follicle Pool in Response to Elevated PI3K Signaling. Adhikari D et al. The majority of ovarian primordial follicles must be preserved in a quiescent state to allow for the regular production of gametes over the female reproductive lifespan. However, the molecular mechanism that maintains the long quiescence of primordial follicles is poorly understood. Under certain pathological conditions, the entire pool of primordial follicles matures simultaneously leading to an accelerated loss of primordial follicles and to premature ovarian failure (POF). We have previously shown that loss of Pten (phosphatase and tensin homolog deleted on chromosome ten) in mouse oocytes leads to premature activation of the entire pool of primordial follicles, subsequent follicular depletion in early adulthood, and the onset of POF. Lack of PTEN leads to increased phosphatidylinositol 3-kinase (PI3K)-Akt and mammalian target of rapamycin complex 1 (mTORC1) signaling in the oocytes. To study the functional and pathological roles of elevated mTORC1 signaling in the oocytes, we treated the Pten-mutant mice with the specific mTORC1 inhibitor rapamycin. When administered to Pten-deficient mice prior to the activation of the primordial follicles, rapamycin effectively prevented global follicular activation and preserved the ovarian reserve. These results provide a rationale for exploring the possible use of rapamycin as a drug for the preservation of the primordial follicle pool, and the possible prevention of POF. mTOR kinase inhibition results in oocyte loss characterized by empty follicles in human ovarian cortical strips cultured in?vitro. McLaughlin M 2011 et al. OBJECTIVE To determine whether oocyte loss is induced by mTOR kinase inhibition in human cortical strips as seen in model organisms in?vivo and in?vitro. DESIGN Ovarian cortex was collected at two centers and cut into small strips. Strips were cultured for 6 days with or without the mTOR inhibitor rapamycin (RAP; 100 nM). Strips were then embedded in paraffin, and serial sections were prepared. SETTING Samples were collected in general obstetric (Edinburgh), gynecologic surgery (New Haven), and fertility preservation assisted reproductive technology (ART) (New Haven) practices. PATIENT(S) Ovarian cortex collected from patients (15-34 years of age) during cesarean section (donated tissue) was removed for the purposes of fertility preservation or was prepared after oophorectomy. INTERVENTION(S) Tissue was used for research purposes only, with no subsequent patient intervention. MAIN OUTCOME MEASURE(S) Follicles were counted and assessed in each serial section. Caspase activity was monitored to determine whether mTOR inhibition activated apoptosis. RESULT(S) The RAP inclusion in cultures results in significantly fewer follicles compared with ethanol vehicle-treated controls. Furthermore, RAP treatment resulted in the induction of follicles that lacked an oocyte in any serial section (30/161 follicles vs. 1/347 ethanol vehicle-treated follicles). Caspase activity was not elevated by RAP treatment. CONCLUSION(S) mTOR inhibition results in a conserved destruction of the oocyte by adjacent granulosa cells (GC) in the absence of increased caspase activity. This model of oocyte loss is not consistent with classic apoptosis/atresia. /////////////////////////
Cellular localization Cytoplasmic
Ovarian function Follicle development, Initiation of primordial follicle growth, Antral follicle growth, Follicle atresia, Steroid metabolism, Oocyte maturation
Comment Short-term rapamycin treatment increases ovarian lifespan in young and middle-aged female mice. Dou X et al. (2017) Although age-related ovarian failure in female mammals cannot be reversed, recent strategies have focused on improving reproductive capacity with age, and rapamycin is one such intervention that has shown a potential for preserving the ovarian follicle pool and preventing premature ovarian failure. However, the application is limited because of its detrimental effects on follicular development and ovulation during long-term treatment. Herein, we shortened the rapamycin administration to 2 weeks and applied the protocol to both young (8 weeks) and middle-aged (8 months) mouse models. Results showed disturbances in ovarian function during and shortly after treatment; however, all the treated animals returned to normal fertility 2 months later. Following natural mating, we observed prolongation of ovarian lifespan in both mouse models, with the most prominent effect occurring in mice older than 12 months. The effects of transient rapamycin treatment on ovarian lifespan were reflected in the preservation of primordial follicles, increases in oocyte quality, and improvement in the ovarian microenvironment. These data indicate that short-term rapamycin treatment exhibits persistent effects on prolonging ovarian lifespan no matter the age at initiation of treatment. In order not to disturb fertility in young adults, investigators should in the future consider applying the protocol later in life so as to delay menopause in women, and at the same time increase ovarian lifespan.////////////////// Everolimus, an mTOR pathway inhibitor, is highly successful on ovarian hyperstimulation syndrome by reducing ovarian weight and progesterone levels: a preclinical experimental randomized controlled study. Kosmas IP et al. (2015) The usefulness of various pathways inhibitors, Everolimus, an inhibitor of mammalian target of rapamycin (mTOR), Infliximab, a monoclonal antibody which blocks the tumor necrosis factor-a (TNF-a), Erlotinib, a tyrosine protein kinase inhibitor of the epidermal growth factor receptor (EGFR), Metformin, an activator of AMP-activated protein kinase enzyme (AMPK) and vascular permeability reducers were explored in an ovarian hyperstimulation syndrome (OHSS) rat model. Sixty-three female Wistar rats were randomly divided in seven groups. The control group received saline, while the OHSS group received recombinant -- follicle-stimulating hormone (rec-FSH) for four consecutive days. The other five groups received rec-FSH for 4 d and Everolimus daily, Infliximab once, Erlotinib daily, Metformin daily and Vitamin C daily, respectively. All groups received human chorionic gonadotropin (hCG) at the fifth day. The efficacy of Everolimus administration for various intervals was also explored. Significantly reduced ovarian weight was observed in the Everolimus group (rec-FSH + hCG + mTOR inhibitor) compared to the OHSS group (p < 0.001). The Everolimus group also showed the lowest progesterone (PRG) concentration (p = 0.007). The Erlotinib group (rec-FSH + hCG + EGFR inhibitor) presented with the lowest graafian follicle number, while the Everolimus group was characterized by the lowest corpus luteum number. The vascular permeability and the estradiol levels did not differ between groups. Finally, the Everolimus intra-comparison showed no difference in all measured outcomes. Studying the different pathways linked to vascular endothelial growth factor (VEGF) pathway, we conclude that targeting mTOR pathways is beneficial for reducing ovarian weight and PRG levels in an OHSS animal model.////////////////// New strategy for in vitro activation of primordial follicles with mTOR and PI3K stimulators. Sun X et al. (2015) Abstract It had been known for decades that primordial follicles in mammalian ovaries are assembled with definite numbers and represent the ovarian reserve throughout the reproductive life. Intra-oocyte PI3K/mTOR pathways have been indicated to play a central role on the activation of primordial follicles. Genetic modified mouse models with chronic activation of PI3K/mTOR signals in primordial oocytes showed premature activation of all primordial follicles and eventually their exhaustion. On the other hand, this may suggest that, unlike chronic activation of PI3K/mTOR, its acute activation in infertility would activate primordial follicles, permitting fertility during the treatment. Previously, PI3K stimulators were reported as a temporary measure to accelerate primordial follicle activation and follicular development in both mouse and human, and were applied in the treatment of infertility in premature ovarian failure (POF) patients. To address whether mTOR stimulators could play similar role in the process, we transiently treated neonatal and aged mouse ovaries with mTOR stimulators-phosphatidic acid (PA) and propranolol. Our results demonstrated the stimulators increased activation of primordial follicles and the production of progeny. Human ovarian cortex cubes were also treated with mTOR or/and PI3K stimulators in vitro. When they were used separately, both of them showed similar promotive effects on primordial follicles. Surprisingly, after joint-treatment with the two kinds of stimulators together, synergistic effects on follicular development were observed. Based on increased efficiency of follicular activation in humans, here we propose in vitro transient treatment with mTOR and PI3K stimulators as an optimized protocol for the application in different clinical conditions with limited follicle reserve.////////////////// Rapamycin rescues the poor developmental capacity of aged porcine oocytes. Lee SE 2014 et al. Unfertilized oocytes age inevitably after ovulation, which limits their fertilizable life span and embryonic development. Rapamycin affects mammalian target of rapamycin (mTOR) expression and cytoskeleton reorganization during oocyte meiotic maturation. The goal of this study was to examine the effects of rapamycin treatment on aged porcine oocytes and their in vitro development. Rapamycin treatment of aged oocytes for 24 h (68 h in vitro maturation ; 44 h+10 ?M rapamycin/24 h, 47.52?5.68) or control oocytes (44 h IVM; 42.14?4.40) significantly increased the development rate and total cell number compared with untreated aged oocytes (68 h IVM, 22.04?5.68) (p<0.05). Rapamycin treatment of aged IVM oocytes for 24 h also rescued aberrant spindle organization and chromosomal misalignment, blocked the decrease in the level of phosphorylated-p44/42 mitogen-activated protein kinase (MAPK), and increased the mRNA expression of cytoplasmic maturation factor genes (MOS, BMP15, GDF9, and CCNB1) compared with untreated, 24 h-aged IVM oocytes (p<0.05). Furthermore, rapamycin treatment of aged oocytes decreased reactive oxygen species (ROS) activity and DNA fragmentation (p<0.05), and downregulated the mRNA expression of mTOR compared with control or untreated aged oocytes. By contrast, rapamycin treatment of aged oocytes increased mitochondrial localization (p<0.05) and upregulated the mRNA expression of autophagy (BECN1, ATG7, MAP1LC3B, ATG12, GABARAP, and GABARAPL1), anti-apoptosis (BCL2L1 and BIRC5; p<0.05), and development (NANOG and SOX2; p<0.05) genes, but it did not affect the mRNA expression of pro-apoptosis genes (FAS and CASP3) compared with the control. This study demonstrates that rapamycin treatment can rescue the poor developmental capacity of aged porcine oocytes. ///////////////////////// Caloric Restriction Promotes the Reserve of Follicle Pool in Adult Female Rats by Inhibiting the Activation of Mammalian Target of Rapamycin Signaling. Li L 2014 et al. Caloric restriction (CR) is known to increase the number of primordial follicles and prolong the reproductive life span. However, how CR modulates follicular development is not well understood. In the present study, we examined the effects of CR on follicular development in rats and investigated the underlying mechanism. After 10 weeks of CR or high-fat diet, ovarian follicles at different developmental stages were examined by histological analysis. Plasma levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and estrogen (ESG) were measured, and the levels of mammalian target of rapamycin (mTOR), p70S6 kinase (p70S6K), and phosphorylated p70S6K in the ovary were detected by Western blot. The results showed that the reserve of follicle pool in CR rats was increased, accompanied by decreased level of phosphorylated p70S6K in the ovary, and decreased serum LH, FSH, and ESG levels. Taken together, these results suggest that CR may suppress ovarian follicular development and enhance the follicle pool reserve by inhibiting mTOR signaling. ///////////////////////// Rapamycin-sensitive mTORC1 signaling is involved in physiological primordial follicle activation in mouse ovary. Tong Y 2013 et al. In mammals, resting female oocytes reside in primordial ovarian follicles. An individual primordial follicle may stay quiescent for a protracted period of time before initiating follicular growth, also termed 'activation'. Female reproductive capacity is sustained by the gradual, streamlined activation of the entire population of primordial follicles, but this process also results in reproductive senescence in older animals. Based on the recent findings that genetically triggered, excessive mTORC1 (mammalian target of rapamycin complex 1) activation in mouse oocytes leads to accelerated primordial follicle activation, we examined the necessity of mTORC1 signaling in physiological primordial follicle activation. We found that induction of oocyte mTORC1 activity is associated with early follicular growth in neonatal mouse ovaries. Pharmacological inhibition of mTORC1 activity in vivo by rapamycin treatment leads to a marked, but partial, suppression of primordial follicle activation. The suppressive effect of rapamycin on primordial follicle activation was reproduced in cultured ovaries. While rapamycin did not apparently affect several plausible cellular targets in neonatal mouse ovaries, such as mTORC2, AKT, or cyclin-dependent kinase (CDK) inhibitor p27-KIP1, its inhibitory effect on Cyclin A2 gene expression implies that mTORC1 signaling in oocytes may engage a Cyclin A/CDK regulatory network that promotes primordial follicle activation. The current work strengthens the concept that mTORC1-dependent events in the oocytes of primordial follicles may represent potential targets for intervention in humans to slow the depletion of the ovarian reserve. Mol. Reprod. Dev. ? 2013 Wiley Periodicals, Inc. /////////////////////////Inactivation of mTor arrests bovine oocytes in M I stage, despite reversible inhibition of 4E-BP1 phosphorylation. Mayer S 2014 et al. The mammalian target of rapamycin (mTor), a Ser/Thr protein kinase, is implicated in the phosphorylation-triggered inactivation of translation repressors, the so called eukaryotic initiation factor 4E (eIF4E)-binding proteins (4E-BPs). Previous observations in porcine and bovine oocytes revealed an increasing phosphorylation of 4E-BP1 during meiotic maturation. The factor is hypophosphorylated in the germinal vesicle (GV) stage and highest phosphorylated in the metaphase II (M II). In the present approach we intended to block 4E-BP1 phosphorylation specifically to impair initiation of translation and elucidate effects on resumption of meiosis. Torin2, which acts as an active-site mTor inhibitor, reduces 4E-BP1 phosphorylation without any effect on eIF4E and arrests up to 60% of the oocytes in the M I stage. Effects of Torin2 treatment, analyzed by site-specific substrate phosphorylation, were also observed at protein kinase B (Akt, PKB) and cyclin dependent kinases (CDKs). However, if at all, only minor side effects were found at protein kinase A, C (PKA, PKC), ATM/ATR (Ataxia telangiectasia mutated/AT and Rad3-related protein) and the mitogen activated protein kinases (MAPK) ERK1, 2. The inhibition of 4E-BP1 phosphorylation by Torin2 is reversible when cultivating oocytes for additional 24?h in Torin2-free medium. However, even so, oocytes persist in the M I stage. This may indicate the necessity of spatiotemporally regulated translation during meiosis, which cannot be restored later. In conclusion, Torin2 enables an effective and specific inhibition of 4E-BP1 phosphorylation and such an approach may be valuable to investigate maturation specific protein synthesis in more detail. Mol. Reprod. Dev. ? 2014 Wiley Periodicals, Inc. ///////////////////////// Luteinizing Hormone/Human Chorionic Gonadotropin-Mediated Activation of mTORC1 Signaling Is Required for Androgen Synthesis by Theca-Interstitial Cells. Palaniappan M et al. LH triggers the biosynthesis of androgens in the theca-interstitial (T-I) cells of ovary through the activation of a cAMP-dependent pathway. We have previously shown that LH/human chorionic gonadotropin (hCG) activates mammalian target of rapamycin complex 1 (mTORC1) signaling network, leading to cell proliferation. In the present study, we provide evidence that the LH/hCG-mediated activation of the mTORC1 signaling cascade is involved in the regulation of steroidogenic enzymes in androgen biosynthesis. Treatment with LH/hCG increased the expression of downstream targets of mTORC1, ribosomal protein S6 kinase 1, and eukaryotic initiation factor 4E as well as steroidogenic enzymes. LH/hCG-mediated stimulation of the steroidogenic enzyme mRNA was blocked by the mTORC1 inhibitor, rapamycin. This inhibitory effect was selective because rapamycin failed to block hCG-mediated increase in the expression of Star mRNA levels. Furthermore, pharmacological targeting of mTORC1 with rapamycin also blocked LH/hCG- or forskolin-induced expression of cAMP response element-binding protein (CREB) and steroidogenic enzymes (P450 side-chain cleavage enzyme, 3?hydroxysteroid dehydrogenase type 1, and 17a-hydroxylase/17,20 lyase) but produced no effect on steroidogenic acute regulatory protein levels. These results were further confirmed by demonstrating that the knockdown of mTOR using small interfering RNA selectively abrogated the LH/hCG-induced increase in steroidogenic enzyme expression, without affecting steroidogenic acute regulatory protein expression. LH/hCG-stimulated androgen production was also blocked by rapamycin. Furthermore, the pharmacological inhibition of mTORC1 or ribosomal protein S6 kinase 1 signaling prevented the LH/hCG-induced phosphorylation of CREB. Chromatin immunoprecipitation assays revealed the association of CREB with the proximal promoter of the Cyp17a1 gene in response to hCG, and this association was reduced by rapamycin treatment. Taken together, our findings show for the first time that LH/hCG-mediated activation of androgen biosynthesis is regulated by the mTORC1 signaling pathway in T-I cells. mTOR Controls Ovarian Follicle Growth by Regulating Granulosa Cell Proliferation. Yu J et al. We have shown that inhibition of mTOR in granulosa cells and ovarian follicles results in compromised granulosa proliferation and reduced follicle growth. Further analysis here using spontaneously immortalized rat granulosa cells has revealed that mTOR pathway activity is enhanced during M-phase of the cell cycle. mTOR specific phosphorylation of p70S6 kinase and 4E-BP, and expression of Raptor are all enhanced during M-phase. The predominant effect of mTOR inhibition by the specific inhibitor Rapamycin (RAP) was a dose-responsive arrest in the G1 cell cycle stage. The fraction of granulosa cells that continued to divide in the presence of RAP exhibited a dose-dependent increase in aberrant mitotic figures known as anaphase bridges. Strikingly, estradiol consistently decreased the incidence of aberrant mitotic figures. In mice treated with RAP, the mitotic index was reduced compared to controls, and a similar increase in aberrant mitotic events was noted. RAP injected during a superovulation regime resulted in a dose-dependent reduction in the numbers of eggs ovulated. Implications for the real-time regulation of follicle growth and dominance, including the consequences of increased numbers of aneuploid granulosa cells, are discussed. Cross Talk Between Estradiol and mTOR Kinase in the Regulation of Ovarian Granulosa Proliferation. Yu J et al. Treatment of ovarian granulosa cells and follicles with the mammalian target of rapamycin (mTOR) kinase inhibitor results in biphasic effects where nanomolar rapamycin (RAP) results in reduced proliferation, mitotic anomalies, and attenuated follicle growth, while the picomolar RAP results in accelerated follicle growth. Here, we tested whether such effects are specific to RAP or could be mimicked by 2 alternative mTOR inhibitors, everolimus (EV) and temsirolimus (TEM), and whether these effects were dependent on the presence of estradiol (E2). Spontaneously immortalized rat granulosa cells (SIGCs) were cultured in dose curves of RAP, EV, TEM, or vehicle with or without E2. Proliferation and phosphorylation of mTOR targets p70S6 kinase and 4E-binding protein (BP) were determined. Cell cycle gene array analysis and confirmatory quantitative reverse transcriptase polymerase chain reaction were performed upon cells treated with picomolar RAP versus controls. Nanomolar RAP, EV, and TEM reduced SIGC proliferation and decreased phospho-p70 and 4E-BP. Picomolar concentrations accelerated proliferation without affecting mTOR substrate phosphorylation. Acceleration of growth by picomolar inhibitor required E2. Picomolar drug treatment altered the transcription of cell cycle regulators, increasing Integrin beta 1 and calcineurin expression, and decreasing inhibin alpha, Chek1, p16ARF, p27/Kip1, and Sestrin2 expression. At nanomolar concentrations, mTOR inhibitors attenuated granulosa proliferation. Accelerated growth and alterations in cell cycle gene transcription found with picomolar concentrations required E2 within the intrafollicular concentration range. The low concentrations of inhibitors required to increase granulosa proliferation suggest a novel use to support the growth of ovarian follicles. A putative mitotic checkpoint dependent on mTOR function controls cell proliferation and survival in ovarian granulosa cells. Yaba A et al. The conserved target of rapamycin (TOR) proteins are involved in sensing nutrient levels and/or stress and the resultant control of cell growth, size, and survival. The authors assess mammalian TOR (mTOR) kinase expression in the mouse ovary and also the expression of its cofactors, Raptor, Rictor, and LST8. In granulosa cells, mTOR demonstrates high cytoplasmic/perinuclear expression. The kinase-active serine 2448-phosphorylated form of mTOR (P-mTOR) is present at very high levels during the M-phase. P-mTOR was enriched on or near the mitotic spindle and also near the contractile ring during cytokinesis. Rapamycin inhibition of mTOR resulted in both reduced granulosa cell proliferation and reduced follicle growth in vitro, each in a dose-dependent fashion. Follicles cultured in rapamycin did not undergo atresia. mTOR inhibition results in a reduction in granulosa cell proliferation, supporting a model in which stress and nutritional cues may directly influence ovarian follicle growth. /////////////////Fertilization-Induced Autophagy in Mouse Embryos Is Independent of mTORC1. Yamamoto A 2014 et al. Autophagy is a dynamically regulated intracellular degradation system that is important for cellular processes such as amino acid production during starvation and intracellular quality control. Previously, we reported that autophagy is suppressed in oocytes, but is rapidly up-regulated after fertilization. During this period, autophagy is thought to be important for the generation of amino acids from the bulk degradation of maternal proteins that have accumulated during oogenesis. However, the mechanism of autophagy induction after fertilization is presently unknown. In most cell types, autophagy is negatively controlled by mammalian target of rapamycin complex 1 (mTORC1), which is typically regulated by amino acids and insulin or related growth factors. In this study, we determined the role of mTORC1 in fertilization-induced autophagy. On the basis of the phosphorylation status of mTORC1 substrates, we found that mTORC1 activity was relatively high in metaphase II (MII) oocytes, but was rapidly decreased within 3 h of fertilization. However, chemical inhibition of mTORC1 by Torin1 or PP242 in MII oocytes or fertilized embryos did not induce autophagy. In addition, activation of mTORC1 by cycloheximide did not inhibit fertilization-induced autophagy in fertilized embryos. By contrast, the phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 effectively suppressed autophagy in these embryos. These data suggest that, even though autophagy induction and post-fertilization mTORC1 activity are inversely correlated with each other, as observed in other cell types, mTORC1 suppression is neither essential nor sufficient for fertilization-induced autophagy, highlighting a unique feature of the regulation mechanism of autophagy-mediated intracellular turnover in early embryos. /////////////////////////
Expression regulated by FSH, LH
Comment HCG-mediated activation of mTORC1 signaling plays a crucial role in steroidogenesis in human granulosa lutein cells. Moravek MB et al. (2016) Luteinizing hormone/human chorionic gonadotropin stimulates progesterone biosynthesis in the corpus luteum by activating cyclic adenosine monophosphate/protein kinase A cascade. Recent studies have shown that cyclic adenosine monophosphate-mediated activation of protein kinase A interacts with the mammalian target of rapamycin signaling pathways. Furthermore, the use of mammalian target of rapamycin inhibitors for immunosuppression in transplant patients has shown adverse effects in reproductive functions. This study examined whether the mammalian target of rapamycin pathway plays any role in luteinizing hormone-mediated regulation of progesterone production. Human granulosa lutein cells were isolated from follicular aspirates of women undergoing in vitro fertilization. Cells were cultured for 72 h and treated with human chorionic gonadotropin (50 ng/ml) for different time periods with or without pretreatment with mammalian target of rapamycin complex 1 inhibitor, rapamycin, (20 nM) for 1 h. Expression of steroidogenic enzymes, including steroidogenic acute regulatory protein, cholesterol side chain cleavage enzyme, and 3β-hydroxysteroid dehydrogenase type 1 messenger RNA, were examined by real-time polymerase chain reaction after 6 h of human chorionic gonadotropin treatment. Expressions of phospho-ribosomal protein S6 kinase and cholesterol side chain cleavage enzyme were analyzed after 15 min and 24 h of human chorionic gonadotropin treatment, respectively. Progesterone production was analyzed by an enzyme immunoassay kit after human chorionic gonadotropin (50 ng/ml) or forskolin (10 μM) treatment for 24 h. Treatment with human chorionic gonadotropin increased the expression of downstream targets of mammalian target of rapamycin complex 1, as well as cholesterol side chain cleavage enzyme, 3β-hydroxysteroid dehydrogenase type 1 and steroidogenic acute regulatory protein messenger RNAs. These increases were inhibited by rapamycin pretreatment. Increased progesterone production in response to treatment with human chorionic gonadotropin or forskolin was also blocked by rapamycin pretreatment. Our findings support a role for mammalian target of rapamycin complex 1 in regulating steroidogenesis in human granulosa lutein cells.////////////////// AKT is involved in granulosa cell autophagy regulation via mTOR signaling during rat follicular development and atresia. Choi J 2013 et al. In this study, we examined whether granulosa cell autophagy during follicular development and atresia was regulated by the class I phosphoinositide-3 kinase/protein kinase B (AKT) pathway, which is known to control the activity of mammalian target of rapamycin (mTOR), a major negative regulator of autophagy. Ovaries and granulosa cells were obtained using an established gonadotropin-primed immature rat model that induces follicular development and atresia. Autophagy was evaluated by measuring the expression level of microtubule-associated protein light chain 3-II (LC3-II) using western blots and immunohistochemistry. The activity of AKT and mTOR was also examined by observing the phosphorylation of AKT and ribosomal protein S6 kinase (S6K) respectively. After gonadotropin injection, LC3-II expression was suppressed and phosphorylation of AKT and S6K increased in rat granulosa cells. By contrast, gonadotropin withdrawal by metabolic clearance promoted LC3-II expression and decreased phosphorylation of AKT and S6K. In addition, in-vitro FSH treatment of rat granulosa cells also indicated inhibition of LC3-II expression accompanied by a marked increase in phosphorylation of AKT and S6K. Inhibition of AKT phosphorylation using AKT inhibitor VIII suppressed FSH-mediated phosphorylation of S6K, followed by an increase in LC3-II expression. Furthermore, co-treatment with FSH and AKT inhibitor increased the levels of apoptosis and cell death of granulosa cells compared with the single treatment with FSH. Taken together, our findings indicated that AKT-mediated activation of mTOR suppresses granulosa cell autophagy during follicular development and is involved in the regulation of apoptotic cell death. ///////////////////////// Hormone Stimulates Mammalian Target of Rapamycin Signaling in Bovine Luteal Cells via Pathways Independent of AKT and Mitogen-Activated Protein Kinase: Modulation of Glycogen Synthase Kinase 3 and AMP-Activated Protein Kinase. Hou X et al. LH stimulates the production of cAMP in luteal cells, which leads to the production of progesterone, a hormone critical for the maintenance of pregnancy. The mammalian target of rapamycin (MTOR) signaling cascade has recently been examined in ovarian follicles where it regulates granulosa cell proliferation and differentiation. This study examined the actions of LH on the regulation and possible role of the MTOR signaling pathway in primary cultures of bovine corpus luteum cells. Herein, we demonstrate that activation of the LH receptor stimulates the phosphorylation of the MTOR substrates ribosomal protein S6 kinase 1 (S6K1) and eukaryotic translation initiation factor 4E binding protein 1. The actions of LH were mimicked by forskolin and 8-bromo-cAMP. LH did not increase AKT or MAPK1/3 phosphorylation. Studies with pathway-specific inhibitors demonstrated that the MAPK kinase 1 (MAP2K1)/MAPK or phosphatidylinositol 3-kinase/AKT signaling pathways were not required for LH-stimulated MTOR/S6K1 activity. However, LH decreased the activity of glycogen synthase kinase 3B (GSK3B) and AMP-activated protein kinase (AMPK). The actions of LH on MTOR/S6K1 were mimicked by agents that modulated GSK3B and AMPK activity. The ability of LH to stimulate progesterone secretion was not prevented by rapamycin, a MTOR inhibitor. In contrast, activation of AMPK inhibited LH-stimulated MTOR/S6K1 signaling and progesterone secretion. In summary, the LH receptor stimulates a unique series of intracellular signals to activate MTOR/S6K1 signaling. Furthermore, LH-directed changes in AMPK and GSK3B phosphorylation appear to exert a greater impact on progesterone synthesis in the corpus luteum than rapamycin-sensitive MTOR-mediated events.
Ovarian localization Oocyte, Cumulus, Granulosa, Theca, Luteal cells
Comment Mechanistic target of rapamycin (MTOR) signaling during ovulation in mice. Siddappa D 2014 et al. A complex network of endocrine/paracrine signals regulates granulosa cell function in ovarian follicles. Mechanistic target of rapamycin (MTOR) has recently emerged as a master intracellular integrator of extracellular signals and nutrient availability. The objectives of the present study were to characterize the expression pattern and kinase activity of MTOR during follicular and corpus luteum development, and to examine how inhibition of MTOR kinase activity affects preovulatory maturation of ovarian follicles. MTOR expression was constitutive throughout follicular and corpus luteum development. Gonadotropins induced MTOR kinase activity in the ovary, which was inhibited by rapamycin treatment (10??g/g body weight, intraperitoneal injection). Inhibition of human chorionic gonadotropin (hCG)-induced MTOR activity during preovulatory follicle maturation did not change key events of ovulation. Granulosa cells of rapamycin-treated mice showed reduced MTOR kinase activity at 1h and 4h post-hCG and overexpression of hCG-induced ovulation genes at 4h post-hCG. Overexpression of these ovulatory genes was associated with hyper-activation of extracellular signal regulated kinase 1/2 (ERK1/2), which occurred in response inhibition of MTOR with rapamycin and suggested that MTOR may function as a negative regulator of the mitogen-activated protein kinase (MAPK) pathway. Indeed, simultaneous inhibition of MTOR and ERK1/2 activities during preovulatory follicle maturation caused anovulation. Inhibition of hCG-induced ERK1/2 activity alone suppressed MTOR kinase activity, indicating that MAPK pathway is upstream of MTOR. Thus, normal ovulation appears to be a result of complex interactions between MTOR and MAPK signaling pathways in granulosa cells of ovulating follicles in mice. Mol. Reprod. Dev. ? 2014 Wiley Periodicals, Inc. ///////////////////////// Distribution and association of mTOR with its cofactors, raptor and rictor, in cumulus cells and oocytes during meiotic maturation in mice. Kogasaka Y et al. Mammalian target of rapamycin (mTOR), a Ser/Thr protein kinase, is the catalytic component of 2 distinct signaling complexes, mTOR-raptor complex (mTORC1) and mTOR-rictor complex (mTORC2). Recently, studies have demonstrated mitosis-specific roles for mTORC1, but the functions and expression dynamics of mTOR complexes during meiotic maturation remain unclear. In the present study, to evaluate the roles of respective mTOR complexes in maternal meiosis and compare them with those in mitosis, we sought to elucidate the spatiotemporal immunolocalization of mTOR, the kinase-active Ser2448- and Ser2481-phosphorylated mTOR, and raptor and rictor during cumulus-cell mitosis and oocyte meiotic maturation in mice. mTOR principally accumulated around the chromosomes and on the spindle. Phosphorylated mTOR (Ser2448 and Ser2481) exhibited elevated fluorescence intensities in the cytoplasm and punctate localization adjacent to the chromosomes, on the spindle poles, and on the midbody during mitotic and meiotic maturation, suggesting functional homology of mTOR between the 2 cell-division systems, despite their mechanistically distinctive spindles. Raptor colocalized with mTOR during both types of cell division, indicating that mTORC1 is predominantly associated with these events. Mitotic rictor uniformly distributed through the cytoplasm, and meiotic rictor localized around the spindle poles of metaphase-I oocytes, suggesting functional divergence of mTORC2 between mitosis and female meiosis. Based on the general function of mTORC2 in the organization of the actin cytoskeleton, we propose that mTORC1 controls spindle function during mitosis and meiosis, while mTORC2 contributes to actin-dependent asymmetric division during meiotic maturation in mice. Mol. Reprod. Dev. ? 2013 Wiley Periodicals, Inc. FSH activation of HIF-1 by the PI3-kinase/AKT/Rheb/mTOR pathway is necessary for induction of select protein markers of follicular differentiation. Alam H, et al sought to elucidate the role of AKT in follicle-stimulating hormone (FSH)-mediated granulosa cell (GC) differentiation. Their results define a signaling pathway in GCs whereby the inactivating phosphorylation of tuberin downstream of phosphatidylinositol-3 (PI3)-kinase/AKT activity leads to ras homolog enriched in brain (Rheb) and subsequent mammalian target of rapamycin (mTOR) activation. mTOR then stimulates translation by phosphorylating p70 S6 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-alpha, microtubule-associated protein 2D, and the PKA type IIbeta regulatory subunit. FSH also promotes activation of the transcription factor hypoxia-inducible factor-1 (HIF-1). FSH-stimulated HIF-1 activity is inhibited by the PI3-kinase inhibitor LY294002, the Rheb inhibitor FTI-277, and the mTOR inhibitor rapamycin. Finally, the authors find that the FSH-mediated up-regulation of reporter activities for LHR, inhibin-alpha, and vascular endothelial growth factor is dependent upon HIF-1 activity as a dominant negative form of HIF-1alphainterferes with the up-regulation of these genes. These results show that FSH enhances HIF-1 activity downstream of the PI3-kinase/AKT/Rheb/mTOR pathway in GCs and that HIF-1 activity is necessary for FSH to induce multiple follicular differentiation markers. Role of the PI3-Kinase and ERK Pathways in the Induction of HIF-1 Activity and the HIF-1 Target VEGF in Ovarian Granulosa Cells in response to Follicle Stimulating Hormone. Alam H et al. FSH stimulation of granulosa cells (GCs) results in increased hypoxia-inducible factor (HIF)-1alpha protein levels and HIF-1 activity that is necessary for up-regulation of certain FSH target genes including vascular endothelial growth factor (VEGF). We report that the role of the phosphatidylinositol (PI)-3-kinase/AKT pathway in increasing HIF-1alpha protein in FSH-stimulated GCs extends beyond an increase in mammalian target of rapamycin (mTOR)-stimulated translation. FSH increases phosphorylation of the AKT target mouse double minute 2 (MDM2); a phosphomimetic mutation of MDM2 is sufficient to induce HIF-1 activity. The PI3-kinase/AKT target forkhead box-containing protein O subfamily 1 (FOXO1) also effects the accumulation of HIF-1alpha as evidenced by the ability of a constitutively active FOXO1 mutant to inhibit the induction by FSH of HIF-1alpha protein and HIF-1 activity. Activation of the PI3-kinase/AKT pathway in GCs by IGF-1 is sufficient to induce HIF-1alpha protein but surprisingly not HIF-1 activity. HIF-1 activity also appears to require a PD98059-sensitive protein (kinase) activity stimulated by FSH that is both distinct from mitogen-activated extracellular signal-regulated kinase (ERK) kinase1/2 or 5 and independent of the PI3-kinase/AKT pathway. These results indicate that FSH-stimulated HIF-1 activation leading to up-regulation of targets such as VEGF requires not only PI3-kinase/AKT-mediated activation of mTOR as well as phosphorylation of FOXO1 and possibly MDM2 but also a protein (kinase) activity that is inhibited by the classic ERK kinase inhibitor PD98059 but is not ERK1/2 or 5. Thus, regulation of HIF-1 activity in GCs by FSH under normoxic conditions is complex and requires input from multiple signaling pathways.
Follicle stages Primordial, Antral
Comment Arraztoa JA, et al 2005 reported the identification of genes expressed in primate primordial oocytes. Rapamycin preserves the follicle pool reserve and prolongs the ovarian lifespan of female rats via modulating mTOR activation and sirtuin expression. Zhang XM et al. To maintain the normal length of female reproductive life, the majority of primordial follicles must be maintained in a quiescent state for later use. In this study, we aimed to study the effects of rapamycin on primordial follicle development and investigate the role of mTOR and sirtuin signaling. Rats were treated every other day with an intraperitoneal injection of rapamycin (5mg/kg) or vehicle. After 10weeks of treatment, ovaries were harvested for hematoxylin and eosin (HE) staining, and analysis by immunohistochemistry and Western blotting. HE staining showed that the number and percentage of primordial follicles in the rapamycin-treated group were twice the control group (P<0.001). Immunohistochemical analysis showed that mTOR and phosphorylated-p70S6K were extensively expressed in surviving follicles with strong staining observed in the cytoplasm of the oocyte. Western blotting showed decreased expression of phosphorylated mTOR and phosphorylated p70S6K in the rapamycin-treated group, and increased the expression of both SIRT1 and SIRT6 compared to the control group (P<0.05). Taken together, these results suggest that rapamycin may inhibit the transition from primordial to developing follicles and preserve the follicle pool reserve, thus extending the ovarian lifespan of female rats via the modulation of mTOR and sirtuin signalings.
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created: March 3, 2004, 9:20 a.m. by: hsueh   email:
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