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Effects of metformin on fertilisation of bovine oocytes and early embryo development: possible involvement of AMPK3-mediated TSC2 activation

  • Olympia Pikiou (a1), Anna Vasilaki (a2), George Leondaritis (a2), Nikos Vamvakopoulos (a3) and Ioannis E. Messinis (a4)...


Studies on bovine oocytes have revealed that the activation of adenosine monophosphate activated protein kinase (AMPK) by millimolar concentrations of metformin controls nuclear maturation. Tuberous sclerosis complex 2 (TSC2) has been identified as a downstream target of AMPK. The objective of this study was to investigate the effects of addition of low concentrations of metformin (1 nM to 10 μM) on the percentage of cultured cumulus–oocyte complexes (COC) giving rise to cleavage-stage embryos and AMPK-mediated TSC2 activation. Metformin was supplemented either throughout in vitro embryo production (IVP) or only during in vitro fertilization (IVF). COC were matured in vitro, inseminated, and presumptive zygotes cultured for a further 72 h post insemination before the percentage of COC that gave rise to zygotes and early embryo development was assessed. The presence of TSC2 in bovine embryos and its possible AMPK-induced activation were assessed by immunocytochemistry. Metformin had a dose-dependent effect on the numbers of cultured COC that gave rise to embryos. Drug treatment either throughout IVP or only during IVF decreased the percentage of ≥8-cell embryos (1 μM, P < 0.05; 10 μM, P < 0.01; and 0.1 μM, 10 μM, P < 0.01, respectively) and increased the percentage of 2-cell embryos (10 μM, P < 0.01 and P < 0.05 respectively). The percentage of cultured COC that gave rise to zygotes was not affected by metformin. TSC2 is expressed in early embryos. Metformin (10 μM) either throughout IVP or during IVF only, increased AMPK-induced PhosphoS1387-TSC2 immunoreactivity (P < 0.01) and this increase corresponded to the total TSC2 protein levels expressed in cells. Our results suggest that there is a dose-dependent negative effect of metformin on the ability of oocytes to cleave following insemination, possibly mediated through an AMPK-induced activation of TSC2.


Corresponding author

All correspondence to: Ioannis E. Messinis. Department of Obstetrics and GynecologyUniversity of Thessaly Medical School 10 Larissa, Greece Tel: +30 2413502795. Fax: +30 2413501019. e-mail:


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Adamiak, S.J., Powell, K., Rooke, J.A., Webb, R. & Sinclair, K.D. (2006). Body composition, dietary carbohydrates and fatty acids determine post-fertilisation development of bovine oocytes in vitro. Reproduction 131, 247–58.
Adhikari, D., Flohr, G., Gorre, N., Shen, Y., Yang, H., Lundin, E., Lan, Z., Gambello, M.J. & Liu, K. (2009). Disruption of Tsc2 in oocytes leads to overactivation of the entire pool of primordial follicles. Mol. Hum. Reprod. 15, 765–70.
Bai, X. & Jiang, Y. (2010). Key factors in mTOR regulation. Cell. Mol. Life Sci. 67, 239402.
Bailey, C.J. (1997). Metformin and its role in the management of type II diabetes. Curr. Opin. Endocrinol. Diabetes 4, 40–7.
Bilodeau-Goeseels, S., Sasseville, M., Guillemette, C. & Richard, F.J. (2007). Effects of adenosine monophosphate-activated kinase activators on bovine oocyte nuclear maturation in vitro. Mol. Reprod. Dev. 74, 1021–34.
Corradetti, M.N., Inoki, K., Bardeesy, N., DePinho, R.A. & Guan, K.L. (2004). Regulation of the TSC pathway by LKB1: evidence of a molecular link between tuberous sclerosis complex and Peutz–Jeghers syndrome. Genes Dev. 18, 1533–8.
Downs, S.M., Hudson, E.R. & Hardie, D.G. (2002). A potential role for AMP-activated protein kinase in meiotic induction in mouse oocytes. Dev. Biol. 245, 200–12.
Eng, G.S., Sheridan, R.A., Wyman, A., Chi, M.M.Y., Bibee, K.P., Jungheim, E.S. & Moley, K.H. (2007). AMP kinase activation increases glucose uptake, decreases apoptosis and improves pregnancy outcome in embryos exposed to high IGF-I concentrations. Diabetes 56, 2228–34.
Gao, X. & Pan, D. (2001). TSC1 and TSC2 tumor suppressors antagonize insulin signalling in cell growth. Genes Dev. 15, 1383–92.
Goossens, K., Vandaele, L., Wydooghe, E., Thys, M., Dewulf, J., Peelman, L.J. & Van Soom, A. (2011). The importance of adequate fixation for immunofluorescent staining of bovine embryos. Reprod. Domest. Anim. 46, 1098–103.
Guertin, D.A. & Sabatini, D.M. (2007). Defining the role of mTOR in cancer. Cancer Cell 12, 922.
Gwinn, DM., Shackelford, D.B., Egan, D.F., Mihaylova, MM., Mery, A., Vasquez, D.S., Turk, B.E. & Shaw, R.J. (2008). AMPK phosphorylation of Raptor mediates a metabolic checkpoint. Cell 30, 214–26.
Hardie, D.G. & Carling, D. (1997). The AMP-activated protein kinase: fuel gauge of the mammalian cell? Eur. J. Biochem. 246, 259–73.
Hatziefthimiou, A., Kiritsi, M., Kiropoulou, C., Vasilaki, A., Sakellaridis, N. & Molyvdas, P.A. (2009). Regional differences in the modulatory role of the epithelium in sheep airway. Clin. Exp. Pharmacol. Physiol. 36, 668–74.
Hazeleger, N.L., Hill, DJ., Stubbings, R.B. & Walton, J.S. (1995). Relationship of morphology and follicular fluid environment of bovine oocytes to their developmental potential in vitro. Theriogenology 43, 509–22.
Hong, S.G., Jang, G., Oh, H.J., Koo, O.J., Park, J.E., Park, H.J., Kang, S.K. & Lee, B.C. (2009). The effects of brain-derived neurotrophic factor and metformin on in vitro developmental competence of bovine oocytes. Zygote 17, 187–93.
Hundal, R.S. & Inzucchi, S.E. (2003). Metformin: new understandings, new uses. Drugs 63, 1879–94.
Inoki, K., Zhu, T. & Guan, K. (2003). TSC2 mediates cellular energy response to control cell growth and survival. Cell 115, 577–90.
Ito, N. & Rubin, G.M. (1999). Gigas, a Drosophila homolog of tuberous sclerosis gene product-2, regulates the cell cycle. Cell 96, 529–39.
Karttunen, P., Uusitupa, M. & Lamminsivu, U. (1983). The pharmacokinetics of metformin: a comparison of the properties of a rapid-release and a sustained release preparation. Int. J. Clin. Pharmacol. 21, 31–6.
Kwong, W.Y., Adamiak, SJ., Gwynn, A., Singh, R. & Sinclair, K.D. (2010). Endogenous folates and single-carbon metabolism in the ovarian follicle, oocyte and pre implantation embryo. Reproduction 139, 112.
Leverve, X.M., Guigas, B., Detaille, D., Batandier, C., Koceir, EA., Chauvin, C., Fontaine, E. & Wiernsperger, N.F. (2003). Mitochondrial metabolism and type 2 diabetes: a specific target for metformin. Diabetes Metab. 29, 6588–94.
Lord, J.M., Flight, I.H.K. & Norman, R.J. (2003). Metformin in polycystic ovary syndrome: systematic review and meta-analysis. BMJ 327, 951–5.
Luo, Z., Zang, M. & Guo, W. (2010). AMPK as a metabolic tumor suppressor: control of metabolism and cell growth. Future Oncol. 6, 457–70.
Manning, B.D. & Cantley, L.C. (2003). Rheb fills a GAP between TSC and TOR. Trends Biochem. Sci. 28, 573–6.
Matthaei, S., Hamann, A., Klein, H.H., Benecke, H., Kreymann, G., Flier, JS. & Greten, H. (1991). Association of metformin's effect to increase insulin-stimulated glucose transport with potentiation of insulin-induced translocation of glucose transporters from intracellular pool to plasma membrane in rat adipocytes. Diabetes 40, 850–7.
Mayes, M.A., Laforest, M.F., Guillemette, C., Gilchrist, R.B. & Richard, F.J. (2007). Adenosine 5’ monophosphate kinase-activated protein kinase (PRKA). activators delay meiotic resumption in porcine oocytes. Biol. Reprod. 76, 589–97.
Meric-Bernstam, F. & Gonzalez-Angulo, A. (2009). Targeting the mTOR signaling network for cancer therapy. J. Clin. Oncol. 27, 2278–87.
Musi, N., Hirshman, M.F., Nygren, J., Svanfeldt, M., Bavenholm, P., Rooyackers, O., Zhou, G., Williamson, J.M., Ljunqvist, O., Efendic, S., Moller, D.E., Thorell, A. & Goodyear, L.J. (2002). Metformin increases AMP-activated protein kinase activity in skeletal muscle of subjects with type 2 diabetes. Diabetes 51, 2074–81.
Nardo, L.G. & Rai, R. (2001). Metformin therapy in the management of polycystic ovary syndrome: endocrine, metabolic and reproductive effects. Gynecol. Endocrinol. 15, 373–80.
Potter, C.J. & Xu, T. (2001). Mechanisms of size control. Curr Opin Genet Dev 11, 279–477.
Sabatini, D.M. (2006). mTOR and cancer: insights into a complex relationship. Nat. Rev. Cancer 6, 729–34.
Sancak, Y., Thoreen, C.C., Peterson, T.R., Lindquist, R.A., Kang, S.A., Spooner, E., Carr, S.A. & Sabatini, D.M. (2007). PRAS40 is an insulin-regulated inhibitor of the mTORC1 protein kinase. Mol. Cell 25, 903–15.
Sancak, Y., Peterson, T.R., Shaul, Y.D., Lindquist, R.A., Thoreen, C.C., Bar-Peled, L. & Sabatini, D.M. (2008). The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1. Science 320, 1496–501.
Shackelford, D.B. & Shaw, R.J. (2009). The LKB1–AMPK pathway: metabolism and growth control in tumour suppression. Nat. Rev. Cancer 9, 563–75.
Shaw, R.J., Bardeesy, N., Manning, B.D., Lopez, L., Kosmatka, M., DePinho, R.A. & Cantley, L.C. (2004). The LKB1 tumor suppressor negatively regulates mTOR signalling. Cancer Cell 6, 91–9.
Shaw, R.J., Lamia, K.A., Vasquez, D., Koo, S.H., Bardeesy, N., DePinho, R.A., Montminy, M. & Cantley, L.C. (2005). The kinase LKB1 mediates glucose homeostasis in liver and therapeutic effects of metformin. Science 310, 1642–6.
Stapleton, D., Mitchelhill, KI., Gao, G., Widmer, J., Michell, B.J., Teh, T., House, C.M., Fernandez, S., Cox, T., Witters, L.A. & Kemp, B.E. (1996). Mammalian AMP-activated protein kinase subfamily. J. Biol. Chem. 271, 611–4.
Stojkovic, M., Machado, SA., Stojkovic, P., Zakhartchenko, V., Hutzler, P., Goncalves, P.B. & Eckhard, W. (2001). Mitochondrial distribution and adenosine triphosphate content of bovine oocytes before and after in vitro maturation: correlation with morphological criteria and developmental capacity after in vitro fertilization and culture. Biol. Reprod. 64, 904–9.
Tapon, N., Ito, N., Dickson, B.J., Treisman, J.E. & Hariharan, I.K. (2001). The Drosophila tuberous sclerosis gene homologs restrict cell growth and cell proliferation. Cell 105, 345–55.
Tosca, L., Froment, P., Solnais, P., Ferre, P., Foufelle, F. & Dupont, J. (2005). Adenosine 5’ monophosphate-activated protein kinase regulates progesterone secretion in rat granulosa cells. Endocrinology 146, 4500–13.
Tosca, L., Solnais, P., Ferre, P., Foufelle, F. & Dupont, J. (2006a). Metformin-induced stimulation of adenosine 5′ monophosphate-activated protein kinase (PRKA) impairs progesterone secretion in rat granulosa cells. Biol. Reprod. 75, 342–51.
Tosca, L., Crochet, S., Ferre, P., Foufelle, F., Tesseraud, S. & Dupont, J. (2006b). AMP activated protein kinase activation modulates progesterone secretion in granulosa cells from hen preovulatory follicles. J. Endocrinol. 190, 8597
Tosca, L., Uzbekova, S., Chabrolle, C. & Dupont, J. (2007). Possible role of 5′ AMP activated protein kinase in the metformin-mediated arrest of bovine oocytes at the germinal vesicle stage during in vitro maturation. Biol. Reprod. 77, 452–65.
Towler, M.C. & Hardie, D.G. (2007). AMP-activated protein kinase in metabolic control and insulin signalling. Circ. Res. 100, 328–41.
Vasilaki, A., Papadaki, T., Notas, G., Kolios, G., Mastrodimou, N., Hoyer, D., Tsilimbaris, M., Kouroumalis, E., Pallikaris, I. & Thermos, K. (2004). Effect of somatostatin on nitric oxide production in human retinal pigment epithelium cell cultures. Invest. Ophthalmol. Vis. Sci. 45, 1499–506.
Wullschleger, S., Loewith, R. & Hall, M.N. (2006). TOR signalling in growth and metabolism. Cell 124, 471–84.
Zhou, G., Myers, R., Li, Y., Chen, Y., Shen, X., Fenyk-Melody, J., Wu, M., Ventre, J., Doebber, T., Fujii, N., Musi, N., Hirshman, M.F., Goodyear, L.J. & Moller, D.E. (2001). Role of AMP-activated protein kinase in mechanism of metformin action. J. Clin. Invest. 108, 1167–74.



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