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Promotion of ovarian follicle growth following mTOR activation: synergistic effects of AKT stimulators.

Cheng Y, Kim J, Li XX, Hsueh AJ - PLoS ONE (2015)

Bottom Line: Mature oocytes derived from MHY1485-activated ovarian grafts could be successfully fertilized, leading the delivery of healthy pups.We further treated ovaries with the mTOR activator together with AKT activators (PTEN inhibitor and phosphoinositol-3-kinase stimulator) before grafting and found additive enhancement of follicle growth.Our studies demonstrate the ability of an mTOR activator in promoting follicle growth, leading to a potential strategy to stimulate preantral follicle growth in infertile patients.

View Article: PubMed Central - PubMed

Affiliation: Program of Reproductive and Stem Cell Biology, Department of Ob/Gyn, Stanford University School of Medicine, Stanford, CA, United States of America.

ABSTRACT
Mammalian target of rapamycin (mTOR) is a serine/threonine kinase and mTOR signaling is important in regulating cell growth and proliferation. Recent studies using oocyte- and granulosa cell-specific deletion of mTOR inhibitor genes TSC1 or TSC2 demonstrated the important role of mTOR signaling in the promotion of ovarian follicle development. We now report that treatment of ovaries from juvenile mice with an mTOR activator MHY1485 stimulated mTOR, S6K1 and rpS6 phosphorylation. Culturing ovaries for 4 days with MHY1485 increased ovarian explant weights and follicle development. In vivo studies further demonstrated that pre-incubation of these ovaries with MHY1485 for 2 days, followed by allo-grafting into kidney capsules of adult ovariectomized hosts for 5 days, led to marked increases in graft weights and promotion of follicle development. Mature oocytes derived from MHY1485-activated ovarian grafts could be successfully fertilized, leading the delivery of healthy pups. We further treated ovaries with the mTOR activator together with AKT activators (PTEN inhibitor and phosphoinositol-3-kinase stimulator) before grafting and found additive enhancement of follicle growth. Our studies demonstrate the ability of an mTOR activator in promoting follicle growth, leading to a potential strategy to stimulate preantral follicle growth in infertile patients.

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Treatment of ovaries with MHY1485 increased phosphorylation of mTOR pathway proteins and promoted secondary follicle development in vitro.A) Treatment of ovaries with MHY1485 increased phosphorylation of mTOR as well as S6K1 and rpS6. Ovaries from day 10 mice were treated with MHY1485 for 3h before immunoblotting. Blotting figures for each immunoblotting test were cropped from full-length blots that are presented in S1 Fig. B) Ovarian weight changes. Paired ovaries from day 10 mice were incubated with MHY1485 with media changes at day 2 of culture. At the end of 4 days of incubation, ovaries were fixed before weighing, followed by histological analyses. Numbers in parentheses denote number of ovaries used. * P<0.05. C) Ovarian histology; bars: 100 um. D) Follicle dynamics. N = 5. * P<0.05.
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pone.0117769.g001: Treatment of ovaries with MHY1485 increased phosphorylation of mTOR pathway proteins and promoted secondary follicle development in vitro.A) Treatment of ovaries with MHY1485 increased phosphorylation of mTOR as well as S6K1 and rpS6. Ovaries from day 10 mice were treated with MHY1485 for 3h before immunoblotting. Blotting figures for each immunoblotting test were cropped from full-length blots that are presented in S1 Fig. B) Ovarian weight changes. Paired ovaries from day 10 mice were incubated with MHY1485 with media changes at day 2 of culture. At the end of 4 days of incubation, ovaries were fixed before weighing, followed by histological analyses. Numbers in parentheses denote number of ovaries used. * P<0.05. C) Ovarian histology; bars: 100 um. D) Follicle dynamics. N = 5. * P<0.05.

Mentions: Based on recent studies showing the ability of MHY1485 to activate the mTOR pathway in rat hepatocyte and PC3 cell line [12,14], we investigated ovarian phosphorylation of mTOR and downstream proteins in this signaling pathway after MHY1485 treatment. Ovaries from day 10 mice were incubated for 3h with 10 uM of MHY1485 before immunoblotting analyses. As shown in Fig. 1A, treatment with MHY1485 increased phospho-mTOR levels without affecting total mTOR content. Activated mTORC1 complex phosphorylates Thr389 in ribosomal S6 kinase (S6K), thereby activating it to subsequently phosphorylate ribosomal protein S6 (rpS6) and promote ribosome biogenesis [15]. We further monitored S6K1 and rpS6 phosphorylation in ovarian tissues. As shown in Fig. 1A, MHY1485 treatment also increased the phosphorylation of downstream S6K1 and rpS6 proteins without affecting total S6K1 and rpS6 levels. These findings demonstrate the ability of MHY1485 to stimulate the mTOR signaling pathway in the ovary.


Promotion of ovarian follicle growth following mTOR activation: synergistic effects of AKT stimulators.

Cheng Y, Kim J, Li XX, Hsueh AJ - PLoS ONE (2015)

Treatment of ovaries with MHY1485 increased phosphorylation of mTOR pathway proteins and promoted secondary follicle development in vitro.A) Treatment of ovaries with MHY1485 increased phosphorylation of mTOR as well as S6K1 and rpS6. Ovaries from day 10 mice were treated with MHY1485 for 3h before immunoblotting. Blotting figures for each immunoblotting test were cropped from full-length blots that are presented in S1 Fig. B) Ovarian weight changes. Paired ovaries from day 10 mice were incubated with MHY1485 with media changes at day 2 of culture. At the end of 4 days of incubation, ovaries were fixed before weighing, followed by histological analyses. Numbers in parentheses denote number of ovaries used. * P<0.05. C) Ovarian histology; bars: 100 um. D) Follicle dynamics. N = 5. * P<0.05.
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pone.0117769.g001: Treatment of ovaries with MHY1485 increased phosphorylation of mTOR pathway proteins and promoted secondary follicle development in vitro.A) Treatment of ovaries with MHY1485 increased phosphorylation of mTOR as well as S6K1 and rpS6. Ovaries from day 10 mice were treated with MHY1485 for 3h before immunoblotting. Blotting figures for each immunoblotting test were cropped from full-length blots that are presented in S1 Fig. B) Ovarian weight changes. Paired ovaries from day 10 mice were incubated with MHY1485 with media changes at day 2 of culture. At the end of 4 days of incubation, ovaries were fixed before weighing, followed by histological analyses. Numbers in parentheses denote number of ovaries used. * P<0.05. C) Ovarian histology; bars: 100 um. D) Follicle dynamics. N = 5. * P<0.05.
Mentions: Based on recent studies showing the ability of MHY1485 to activate the mTOR pathway in rat hepatocyte and PC3 cell line [12,14], we investigated ovarian phosphorylation of mTOR and downstream proteins in this signaling pathway after MHY1485 treatment. Ovaries from day 10 mice were incubated for 3h with 10 uM of MHY1485 before immunoblotting analyses. As shown in Fig. 1A, treatment with MHY1485 increased phospho-mTOR levels without affecting total mTOR content. Activated mTORC1 complex phosphorylates Thr389 in ribosomal S6 kinase (S6K), thereby activating it to subsequently phosphorylate ribosomal protein S6 (rpS6) and promote ribosome biogenesis [15]. We further monitored S6K1 and rpS6 phosphorylation in ovarian tissues. As shown in Fig. 1A, MHY1485 treatment also increased the phosphorylation of downstream S6K1 and rpS6 proteins without affecting total S6K1 and rpS6 levels. These findings demonstrate the ability of MHY1485 to stimulate the mTOR signaling pathway in the ovary.

Bottom Line: Mature oocytes derived from MHY1485-activated ovarian grafts could be successfully fertilized, leading the delivery of healthy pups.We further treated ovaries with the mTOR activator together with AKT activators (PTEN inhibitor and phosphoinositol-3-kinase stimulator) before grafting and found additive enhancement of follicle growth.Our studies demonstrate the ability of an mTOR activator in promoting follicle growth, leading to a potential strategy to stimulate preantral follicle growth in infertile patients.

View Article: PubMed Central - PubMed

Affiliation: Program of Reproductive and Stem Cell Biology, Department of Ob/Gyn, Stanford University School of Medicine, Stanford, CA, United States of America.

ABSTRACT
Mammalian target of rapamycin (mTOR) is a serine/threonine kinase and mTOR signaling is important in regulating cell growth and proliferation. Recent studies using oocyte- and granulosa cell-specific deletion of mTOR inhibitor genes TSC1 or TSC2 demonstrated the important role of mTOR signaling in the promotion of ovarian follicle development. We now report that treatment of ovaries from juvenile mice with an mTOR activator MHY1485 stimulated mTOR, S6K1 and rpS6 phosphorylation. Culturing ovaries for 4 days with MHY1485 increased ovarian explant weights and follicle development. In vivo studies further demonstrated that pre-incubation of these ovaries with MHY1485 for 2 days, followed by allo-grafting into kidney capsules of adult ovariectomized hosts for 5 days, led to marked increases in graft weights and promotion of follicle development. Mature oocytes derived from MHY1485-activated ovarian grafts could be successfully fertilized, leading the delivery of healthy pups. We further treated ovaries with the mTOR activator together with AKT activators (PTEN inhibitor and phosphoinositol-3-kinase stimulator) before grafting and found additive enhancement of follicle growth. Our studies demonstrate the ability of an mTOR activator in promoting follicle growth, leading to a potential strategy to stimulate preantral follicle growth in infertile patients.

Show MeSH
Related in: MedlinePlus