Inhibitory Effect of mTOR Activator MHY1485 on Autophagy: Suppression of Lysosomal Fusion

Autophagy is a major degradative process responsible for the disposal of cytoplasmic proteins and dysfunctional organelles via the lysosomal pathway. During the autophagic process, cells form double-membraned vesicles called autophagosomes that sequester disposable materials in the cytoplasm and finally fuse with lysosomes. In the present study, we investigated the inhibition of autophagy by a synthesized compound, MHY1485, in a culture system by using Ac2F rat hepatocytes. Autophagic flux was measured to evaluate the autophagic activity. Autophagosomes were visualized in Ac2F cells transfected with AdGFP-LC3 by live-cell confocal microscopy. In addition, activity of mTOR, a major regulatory protein of autophagy, was assessed by western blot and docking simulation using AutoDock 4.2. In the result, treatment with MHY1485 suppressed the basal autophagic flux, and this inhibitory effect was clearly confirmed in cells under starvation, a strong physiological inducer of autophagy. The levels of p62 and beclin-1 did not show significant change after treatment with MHY1485. Decreased co-localization of autophagosomes and lysosomes in confocal microscopic images revealed the inhibitory effect of MHY1485 on lysosomal fusion during starvation-induced autophagy. These effects of MHY1485 led to the accumulation of LC3II and enlargement of the autophagosomes in a dose- and time- dependent manner. Furthermore, MHY1485 induced mTOR activation and correspondingly showed a higher docking score than PP242, a well-known ATP-competitive mTOR inhibitor, in docking simulation. In conclusion, MHY1485 has an inhibitory effect on the autophagic process by inhibition of fusion between autophagosomes and lysosomes leading to the accumulation of LC3II protein and enlarged autophagosomes. MHY1485 also induces mTOR activity, providing a possibility for another regulatory mechanism of autophagy by the MHY compound. The significance of this study is the finding of a novel inhibitor of autophagy with an mTOR activating effect.     

ERK and Akt signaling pathways function through parallel mechanisms to promote mTORC1 signaling

The mammalian target of rapamycin (mTOR) is a protein kinase that, when present in a complex referred to as mTOR complex 1 (mTORC1), acts as an important regulator of growth and metabolism. The activity of the complex is regulated through multiple upstream signaling pathways, including those involving Akt and the extracellular-regulated kinase (ERK). Previous studies have shown that, in part, Akt and ERK promote mTORC1 signaling through phosphorylation of a GTPase activator protein (GAP), referred to as tuberous sclerosis complex 2 (TSC2), that acts as an upstream inhibitor of mTORC1. In the present study we extend the earlier studies to show that activation of the Akt and ERK pathways acts in a synergistic manner to promote mTORC1 signaling. Moreover, we provide evidence that the Akt and ERK signaling pathways converge on TSC2, and that Akt phosphorylates residues on TSC2 distinct from those phosphorylated by ERK. The results also suggest that leucine-induced stimulation of mTORC1 signaling occurs through a mechanism distinct from TSC2 and the Akt and ERK signaling pathways. Overall, the results are consistent with a model in which Akt and ERK phosphorylate distinct sites on TSC2, leading to greater repression of its GAP activity, and consequently a magnified stimulation of mTORC1 signaling, when compared with either input alone. The results further suggest that leucine acts through a mechanism distinct from TSC2 to stimulate mTORC1 signaling.     

SIRT1 Negatively Regulates the Mammalian Target of Rapamycin

The IGF/mTOR pathway, which is modulated by nutrients, growth factors, energy status and cellular stress regulates aging in various organisms. SIRT1 is a NAD+ dependent deacetylase that is known to regulate caloric restriction mediated longevity in model organisms, and has also been linked to the insulin/IGF signaling pathway. Here we investigated the potential regulation of mTOR signaling by SIRT1 in response to nutrients and cellular stress. We demonstrate that SIRT1 deficiency results in elevated mTOR signaling, which is not abolished by stress conditions. The SIRT1 activator resveratrol reduces, whereas SIRT1 inhibitor nicotinamide enhances mTOR activity in a SIRT1 dependent manner. Furthermore, we demonstrate that SIRT1 interacts with TSC2, a component of the mTOR inhibitory-complex upstream to mTORC1, and regulates mTOR signaling in a TSC2 dependent manner. These results demonstrate that SIRT1 negatively regulates mTOR signaling potentially through the TSC1/2 complex.     

Efficacy of combined inhibition of mTOR and ERK/MAPK pathways in treating a tuberous sclerosis complex cell model

Tuberous sclerosis complex （TSC） is an autosomal dominant tumor syndrome which afflicts multiple organs and for which there is no cure, such that TSC patients may develop severe mental retardation and succumb to renal or respiratory failure. TSC derives from inacti- vating mutations of either the TSC1 or TSC2 tumor suppressor gene, and the resulting inactivation of the TSC1/TSC2 protein complex causes hyperactivation of the mammalian target of rapamyein （mTOR）, leading to uncontrolled cell growth and proliferation. Recent clinical trials of targeted suppression of mTOR have yielded only modest success in TSC patients. It was proposed that abrogation of a newly identified mTOR-mediated negative feedback regulation on extracellular signal-regulated kinase/mitogen-activated protein kinase （ERK/MAPK） signaling pathway and on the well-documented RTK-PI3K-AKT signaling cascade could limit the efficacy of mTOR inhibitors in the treatment of TSC patients. Therefore, we speculate that dual inhibition of mTOR and ERK/MAPK pathways may overcome the disadvantage of single agent therapies and boost the efficacy of mTOR targeted therapies for TSC patients. Investigation of this hypothesis in a TSC cell model revealed that mTOR suppression with an mTOR inhibitor, rapamycin （sirolimus）, led to up-regulation of ERK/MAPK signaling in mouse Tsc2 knockout cells and that this augmented signaling was attenuated by concurrent administration of a MEK1/2 inhibitor, PD98059. When compared with monotherapy, combinatorial application of rapamycin and PD98059 had greater inhibitory effects on Tsc2 deficient cell proliferation, suggesting that combined suppression of mTOR and ERK/MAPK signaling pathways may have advantages over single mTOR inhibition in the treatment of TSC patients.     

DNMT1, a Novel Regulator Mediating mTORC1/mTORC2 Pathway-Induced NGF Expression in Schwann Cells

Schwann cells play an important role in maintaining the normal function of peripheral nerves via the secretion of nerve growth factor (NGF). The mTOR signaling pathway is known as a kind of Ser/Thr protein kinase that regulates various cell functions. DNA methyltransferase 1 (DNMT1) is an epigenetic regulator and downstream target of the mTOR pathway. In the present study, we explored the relationship between NGF expression and the mTOR pathway/DNMT1 in RSC96 cells. The results showed that both rapamycin and Torin 1 downregulated NGF expression via the inhibition of phospho-mTOR (Ser 2448) and phospho-S6K1 (Thr 389). Similarly, the silencing of RAPTOR and RICTOR decreased NGF expression by 56.7% and 52.4%, respectively, in RSC96 cells compared with the control siRNA treatment, which was accompanied by reduced phospho-S6K1 (Thr 389). The mTOR/S6K1 activator MHY1485 increased NGF expression by 28.7% and 17.1% 1 day and 2 day after stimulation, respectively, compared to the corresponding control group in RSC96 cells. Furthermore, DNMT1 was enhanced by 94.5% and 42.5% with mTOR pathway inhibitor (rapamycin and Torin 1, respectively) treatment for 3 day compared with the control group. Additionally, the inhibition of DNMT1 with a chemical inhibitor or a specific shRNA plasmid upregulated NGF in RSC96 cells. In summary, our findings suggest that DNMT1 is the downstream target of the mTOR pathway and mediates the mTOR pathway inhibition-induced reduction in NGF expression in Schwann cells. Activation of the mTOR signaling pathway and/or inhibition of DNMT1 increased NGF expression, which may benefit patients suffering from NGF deficiencies, such as diabetic peripheral neuropathy.     

Ecdysteroid regulation of ovarian growth and oocyte maturation in the red flour beetle,Tribolium castaneum

Previous studies from our laboratory showed the involvement of juvenile hormone (JH) and ecdysteroid signaling in the regulation of female reproduction in the red flour beetle, Tribolium castaneum. JH regulates vitellogenin (Vg) synthesis in the fat body but the role of ecdysteroid signaling is not known. Here, we report on ecdysteroid regulation of ovarian growth and oocyte maturation. Microarray analysis of RNA isolated from ovaries showed the up-regulation of several genes coding for proteins involved in ecdysteroid signaling on the 4th day after female adult eclosion. The functional analyses of genes coding for proteins involved in ecdysteroid and JH signaling pathways by RNA interference (RNAi) revealed that ecdysteroids but not JH regulate ovarian growth and primary oocyte maturation. Ultrastructural studies showed the temporal sequences of key events in oogenesis including the development of primary oocytes, the differentiation and development of follicle epithelial cells, and the formation of intercellular spaces to facilitate uptake of Vg protein. RNAi studies showed that ecdysone receptor (EcR) and ultraspiracle (USP) are required for the ovarian growth, primary oocyte maturation and the growth and migration of the follicle cells. These studies suggest important roles for ecdysteroids in the regulation of oocyte maturation in the beetle ovaries.     

New strategy for in vitro activation of primordial follicles with mTOR and PI3K stimulators

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 2 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.     

Loss of Tuberous Sclerosis Complex 2 (TSC2) as a Predictive Biomarker of Response to mTOR Inhibitor Treatment in Patients with Hepatocellular Carcinoma

BACKGROUND: Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death globally. Mechanistic target of rapamycin (mTOR) is frequently up-regulated in HCC and plays an important role in HCC tumorigenesis. Tumors with loss of tuberous sclerosis complex 2 (TSC2), a negative regulator of mTOR signaling, tend to respond well to mTOR inhibitors. We analyzed TSC2 expression status in Korean patients with HCC and evaluated the correlation between TSC2 loss and response to the mTOR inhibitor, everolimus. METHODS: We retrospectively assessed 36 patients with advanced HCC who had received sorafenib at a single center in Korea between 2008 and 2014, and for whom tumor specimens were available for TSC2 immunohistochemical analysis (IHC). Three patient-derived tumor cell lines (PDCs) were analyzed by western blotting to determine TSC2 expression and drug sensitivity to mTOR. RESULTS: Twelve of 36 patients (33.3%) showed low to undetectable levels of TSC2 expression. No significant differences were observed in progression-free survival (PFS) or overall survival with sorafenib treatment based on TSC2 expression status. Two patients were treated with everolimus after sorafenib failure; one patient, with moderate TSC2 expression, experienced stable disease with a PFS of 5.8 months; the other, with high TSC2 expression, experienced rapid progression. PDC models demonstrated that the TSC2-low HCC PDC line was significantly more sensitive to everolimus than the TSC2-high HCC PDC lines. CONCLUSION: Loss of TSC2 may predict improved response to everolimus in HCC patients, but further studies are needed to confirm the predictive role of TSC2 expression for everolimus treatment.     

The proto-oncogene c-src is involved in primordial follicle activation through the PI3K, PKC and MAPK signaling pathways

ABSTRACT: BACKGROUND: C-src is an evolutionarily conserved proto-oncogene that regulates cell proliferation, differentiation and apoptosis. In our previous studies, we have reported that another proto-oncogene, c-erbB2, plays an important role in primordial follicle activation and development. We also found that c-src was expressed in mammalian ovaries, but its functions in primordial follicle activation remain unclear. The objective of this study is to investigate the role and mechanism of c-src during the growth of primordial follicles. METHODS: Ovaries from 2-day-old rats were cultured in vitro for 8 days. Three c-src-targeting and one negative control siRNA were designed and used in the present study. PCR, Western blotting and primordial follicle development were assessed for the silencing efficiency of the lentivirus c-src siRNA and its effect on primordial follicle onset. The expression of c-src mRNA and protein in primordial follicle growth were examined using the PCR method and immunohistochemical staining. Furthermore, the MAPK inhibitor PD98059, the PKC inhibitor Calphostin and the PI3K inhibitor LY294002 were used to explore the possible signaling pathways of c-src in primordial folliculogenesis. RESULTS: The results showed that Src protein was distributed in the ooplasmic membrane and the granulosa cell membrane in the primordial follicles, and c-src expression level increased with the growth of primordial follicle. The c-src -targeting lentivirus siRNAs had a silencing effect on c-src mRNA and protein expression. Eight days after transfection of rat ovaries with c-src siRNA, the GFP fluorescence in frozen ovarian sections was clearly discernible under a fluorescence microscope, and its relative expression level was 5-fold higher than that in the control group. Furthermore, the c-src-targeting lentivirus siRNAs lowered its relative expression level 1.96 times. We also found that the development of cultured primordial follicles was completely arrested after c-src siRNA knockdown of c-src expression. Furthermore, our studies demonstrated that folliculogenesis onset was inhibited by Calphostin, PD98059 or LY294002 treatment,but none of them down-regulated c-src expression. In contrast, the expression levels of p-PKC, p-ERK1/2 and p-PI3K in the follicles were clearly decreased by c-src siRNA transfection. Correspondingly, both Calphostin and LY294002 treatment resulted in a decrease in the p-PKC level in follicles, but no change was observed in the PD98059 group. Finally, LY294002 treatment decreased the p-PI3K expression level in the follicles, but no changes were observed in the PD98059 and Calphostin groups. CONCLUSIONS: C-src plays an important role in regulating primordial follicle activation and growth via the PI3K-PKC- ERK1/2 pathway.     

The tuberous sclerosis protein TSC2 is not required for the regulation of the mammalian target of rapamycin by amino acids and certain cellular stresses

Amino acids positively regulate signaling through the mammalian target of rapamycin (mTOR). Recent work demonstrated the importance of the tuberous sclerosis protein TSC2 for regulation of mTOR by insulin. TSC2 contains a GTPase-activator domain that promotes hydrolysis of GTP bound to Rheb, which positively regulates mTOR signaling. Some studies have suggested that TSC2 also mediates the control of mTOR by amino acids. In cells lacking TSC2, amino acid withdrawal still results in dephosphorylation of S6K1, ribosomal protein S6, the eukaryotic initiation factor 4E-binding protein, and elongation factor-2 kinase. The effects of amino acid withdrawal are diminished by inhibiting protein synthesis or adding back amino acids. These studies demonstrate that amino acid signaling to mTOR occurs independently of TSC2 and involves additional unidentified inputs. Although TSC2 is not required for amino acid control of mTOR, amino acid withdrawal does decrease the proportion of Rheb in the active GTP-bound state. Here we also show that Rheb and mTOR form stable complexes, which are not, however, disrupted by amino acid withdrawal. Mutants of Rheb that cannot bind GTP or GDP can interact with mTOR complexes. We also show that the effects of hydrogen peroxide and sorbitol, cell stresses that impair mTOR signaling, are independent of TSC2. Finally, we show that the ability of energy depletion (which impairs mTOR signaling in TSC2+/+ cells) to increase the phosphorylation of eukaryotic elongation factor 2 is also independent of TSC2. This likely involves the phosphorylation of the elongation factor-2 kinase by the AMP-activated protein kinase.     

mTOR: A pathogenic signaling pathway in developmental brain malformations

The mTOR signaling network functions as a pivotal regulatory cascade during the development of the cerebral cortex. Aberrant hyperactivation of mTOR as a consequence of loss-of-function gene mutations encoding mTOR inhibitor proteins such as TSC1, TSC2, PTEN and STRADα has been recently linked to developmental cortical malformations associated with epilepsy and neurobehavioral disabilities. Investigation of mTOR signaling in these disorders provides for the first time exciting future avenues for assessment of biomarkers, patient stratification and prognostic measures as well as the opportunity for targeted therapy to regulate mTOR activity across all age groups. As we learn more about mTOR and its activity in the developing brain, many challenges will arise that must be overcome before widespread clinical therapeutics can be implemented.     

The p53 target Plk2 interacts with TSC proteins impacting mTOR signaling,tumor growth,and chemosensitivity under hypoxic conditions

Tuberous sclerosis complex 1 (TSC1) inhibits mammalian target of rapamycin (mTOR), a central promotor of cell growth and proliferation. The protein product of the TSC1 gene, hamartin (referred to as TSC1) is known to interact with Polo-like kinase 1 (Plk1) in a cell cycle regulated, phosphorylation-dependent manner. We hypothesized that the p53 target gene, Plk2, is a tumor suppressor, mediating its tumor suppressor function through interactions with TSC1 that facilitate TSC1/2 restraint of mTOR under hypoxic stress. We found that human lung tumor cells deficient in Plk2 grew larger than control tumors, and that Plk2 interacts with endogenous TSC1 protein. Additionally, C-terminal Plk2-GST fusion protein bound both TSC1 and TSC2 proteins. TSC1 levels were elevated in response to Adriamycin and cells transiently over-expressing Plk2 demonstrated decreased phosphorylation of the downstream target of mTOR, ribosomal protein p70S6 kinase during hypoxia. Plk2 levels were inversely correlated with cytoplasmic p70S6K phosphorylation. Plk2 levels did not increase in response to DNA damage (Adriamycin, CPT-11) when HCT 116 and H460 cells were exposed to hypoxia. TSC1-deficient mouse embryonic fibroblasts with TSC1 added back demonstrated decreased S6K phosphorylation, which was further decreased when Plk2 was transiently over-expressed. Interestingly, under normoxia, Plk2 deficient tumor cells demonstrated increased apoptosis in response to various chemotherapeutic agents including CPT-11 but increased resistance to apoptotic death after CPT-11 treatment under hypoxia, and tumor xenografts comprised of these Plk2-deficient cells were resistant to CPT-11. Our results point to a novel Plk2-TSC1 interaction with effects on mTOR signaling during hypoxia, and tumor growth that may enable targeting Plk2 signaling in cancer therapy.     

GLUT1 enhances mTOR activity independently of TSC2 and AMPK

Enhanced GLUT1 expression in mesangial cells plays an important role in the development of diabetic nephropathy by stimulating signaling through several pathways resulting in increased glomerular matrix accumulation. Similarly, enhanced mammalian target of rapamycin (mTOR) activation has been implicated in mesangial matrix expansion and glomerular hypertrophy in diabetes. We sought to examine whether enhanced GLUT1 expression increased mTOR activity and, if so, to identify the mechanism. We found that levels of GLUT1 expression and mTOR activation, as evidenced by S6 kinase (S6K) and 4E-BP-1 phosphorylation, changed in tandem in cell lines exposed to elevated levels of extracellular glucose. We then showed that increased GLUT1 expression enhanced S6K phosphorylation by 1.7- to 2.9-fold in cultured mesangial cells and in glomeruli from GLUT1 transgenic mice. Treatment with the mTOR inhibitor, rapamycin, eliminated the GLUT1 effect on S6K phosphorylation. In cells lacking functional tuberous sclerosis complex (TSC) 2, GLUT1 effects on mTOR activity persisted, indicating that GLUT1 effects were not mediated by TSC. Similarly, AMP kinase activity was not altered by enhanced GLUT1 expression. Conversely, enhanced GLUT1 expression led to a 2.4-fold increase in binding of mTOR to its activator, Rheb, and a commensurate 2.1-fold decrease in binding of Rheb to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) consistent with mediation of GLUT1 effects by a metabolic effect on GAPDH. Thus, GLUT1 expression appears to augment mesangial cell growth and matrix protein accumulation via effects on glycolysis and decreased GAPDH interaction with Rheb.     

Tuberous sclerosis complex gene products,Tuberin and Hamartin,control mTOR signaling by acting as a GTPase-activating protein complex toward Rheb

BACKGROUND: Tuberous Sclerosis Complex (TSC) is a genetic disorder that occurs through the loss of heterozygosity of either TSC1 or TSC2, which encode Hamartin or Tuberin, respectively. Tuberin and Hamartin form a tumor suppressor heterodimer that inhibits the mammalian target of rapamycin (mTOR) nutrient signaling input, but how this occurs is unclear. RESULTS: We show that the small G protein Rheb (Ras homolog enriched in brain) is a molecular target of TSC1/TSC2 that regulates mTOR signaling. Overexpression of Rheb activates 40S ribosomal protein S6 kinase 1 (S6K1) but not p90 ribosomal S6 kinase 1 (RSK1) or Akt. Furthermore, Rheb induces phosphorylation of eukaryotic initiation factor 4E binding protein 1 (4E-BP1) and causes 4E-BP1 to dissociate from eIF4E. This dissociation is completely sensitive to rapamycin (an mTOR inhibitor) but not wortmannin (a phosphoinositide 3-kinase [PI3K] inhibitor). Rheb also activates S6K1 during amino acid insufficiency via a rapamycin-sensitive mechanism, suggesting that Rheb participates in nutrient signaling through mTOR. Moreover, Rheb does not activate a S6K1 mutant that is unresponsive to mTOR-mediated signals, confirming that Rheb functions upstream of mTOR. Overexpression of the Tuberin-Hamartin heterodimer inhibits Rheb-mediated S6K1 activation, suggesting that Tuberin functions as a Rheb GTPase activating protein (GAP). Supporting this notion, TSC patient-derived Tuberin GAP domain mutants were unable to inactivate Rheb in vivo. Moreover, in vitro studies reveal that Tuberin, when associated with Hamartin, acts as a Rheb GTPase-activating protein. Finally, we show that membrane localization of Rheb is important for its biological activity because a farnesylation-defective mutant of Rheb stimulated S6K1 activation less efficiently. CONCLUSIONS: We show that Rheb acts as a novel mediator of the nutrient signaling input to mTOR and is the molecular target of TSC1 and TSC2 within mammalian cells.     

Evidence for a role for anti-Mullerian hormone in the suppression of follicle activation in mouse ovaries and bovine ovarian cortex grafted beneath the chick chorioallantoic membrane

The first critical transition in follicular development, the activation of primordial follicles to leave the pool of resting follicles and begin growth, is poorly understood, but it appears that the balance between inhibitory and stimulatory factors is important in regulating the exodus of follicles from the resting pool. There is evidence that anti-Mullerian hormone (AMH; also known as MIS) inhibits follicle activation in mice, but whether it plays a similar role in non rodent species is not known. When pieces of bovine ovarian cortex, rich in primordial follicles, are cultured in serum-free medium, most follicles initiate growth, but when cortical pieces are grafted beneath the chorioallantoic membrane (CAM) of chick embryos, follicle activation does not occur. Since embryonic chick gonads of both sexes produce and secrete high levels of AMH, the hypothesis that the AMH in the chick circulation inhibits follicle activation was tested. In Experiment 1, whole newborn mouse ovaries were grafted beneath the CAM (placed "in ovo") or cultured in vitro for 8 days. In vitro (or after 8 days in vivo) follicles activated and proceeded to the primary or secondary stage, but activation was suppressed in ovo. This inhibition was reversed if ovaries were removed from beneath the CAM and cultured in vitro. In contrast, when ovaries from mice null mutant for the AMH type II receptor were CAM-grafted in Experiment 2, follicle activation occurred in a similar fashion to activation in vitro. This finding strongly implicates AMH as the inhibitor of follicle activation in ovo. Since chick embryonic gonads are the source of circulating AMH, chicks were gonadectomized in Experiment 3, prior to grafting of pieces of bovine ovarian cortex beneath their CAMs. Bovine primordial follicles activated in the gonadectomized chicks, similar to the results for mice lacking the AMH type II receptor. Taken together these experiments provide strong evidence that AMH is the inhibitor of mouse follicle activation present in the circulation of embryonic chicks and provide indirect, and hence more tentative, evidence for AMH as an inhibitor of bovine follicle activation.     

Cryopreservation of the mouse ovary inhibits the onset of primordial follicle development

The cryopreservation of ovarian tissue has been reported to affect the development of preantral follicles. However, the effect of cryopreservation of ovarian tissue on the development of primordial follicles remains to be elucidated. This study was conducted to evaluate the effect of cryopreservation on the development of frozen-thawed mouse primordial follicles. One-day-old mouse ovaries were cryopreserved by either slow-freezing or a vitrification method. The development of primordial follicles was evaluated histologically and also with markers for follicle development such as: GDF-9, inhibin-alpha subunit and ZP3 in fresh and frozen-thawed ovaries cultured for five days. The proportion of apoptotic and necrotic areas was analyzed in fresh and frozen-thawed ovaries at one and five days after culture, in order to examine the viability of ovarian cells that influence primordial follicle development. The development rate of primordial follicles was significantly lower in slow-frozen and vitrified ovaries than the fresh controls after five days of in vitro culture (P<0.05). The mRNA expression for all developmental markers was slightly decreased in the frozen-thawed ovaries; this difference was not significant. The proportion of apoptosis was significantly increased in the slow-frozen and vitrified ovaries compared to the fresh ovaries at one day (P<0.05); however, there was no difference at five days after culture. The proportion of the area of necrosis was significantly higher in slow-frozen and vitrified ovaries compared to the fresh ovaries at one and five days after culture (P<0.05). Our preliminary data suggest that ovarian tissue cryopreservation using slow-freezing and vitrification methods inhibits development of primordial follicles. This may be caused by the death of ovarian cells through apoptosis and necrosis after cryopreservation.