H2O2-induced mild stress in relation with in vitro ovine oocyte developmental competence: implications for blastocyst apoptosis and related genes expression

K. Nikdel; M. Aminafshar; A. Mohammadi-Sangcheshmeh; N. EmamJomeh-Kashan; E. Seyedjafari

doi:10.14715/cmb/2017.63.5.9

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Vol. 63 No. 5: Issue 5

Issue Published : July 9, 2017

The undersigned hereby assign all rights, included but not limited to copyright, for this manuscript to CMB Association upon its submission for consideration to publication on Cellular and Molecular Biology. The rights assigned include, but are not limited to, the sole and exclusive rights to license, sell, subsequently assign, derive, distribute, display and reproduce this manuscript, in whole or in part, in any format, electronic or otherwise, including those in existence at the time this agreement was signed. The authors hereby warrant that they have not granted or assigned, and shall not grant or assign, the aforementioned rights to any other person, firm, organization, or other entity. All rights are automatically restored to authors if this manuscript is not accepted for publication.

H2O2-induced mild stress in relation with in vitro ovine oocyte developmental competence: implications for blastocyst apoptosis and related genes expression

https://doi.org/10.14715/cmb/2017.63.5.9

K. Nikdel

Department of Animal Science, Faculty of Agriculture and Natural Resources, Science and Research Branch, Islamic Azad University, Tehran, Iran

M. Aminafshar

Department of Animal Science, Faculty of Agriculture and Natural Resources, Science and Research Branch, Islamic Azad University, Tehran, Iran

A. Mohammadi-Sangcheshmeh

Department of Animal and Poultry Science, College of Aburaihan, University of Tehran, Pakdasht, Tehran, Iran

N. EmamJomeh-Kashan

Department of Animal Science, Faculty of Agriculture and Natural Resources, Science and Research Branch, Islamic Azad University, Tehran, Iran

E. Seyedjafari

Department of Biotechnology, College of Science, University of Tehran, Tehran, Iran

Corresponding Author(s) : A. Mohammadi-Sangcheshmeh

amohammadis@gmail.com

Cellular and Molecular Biology, Vol. 63 No. 5: Issue 5
Article Published : May 20, 2017

Abstract

In this study, in vitro maturation was performed in presence of various concentrations (0, 10, 100, or 1000 µM) of H2O2. The intracellular glutathione (GSH) level, fertilization, cleavage, and blastocyst rates, total cell number, and apoptotic cell number and expression of Bax, Bcl-2, and p53 genes in blastocyst-stage embryos were studied. At 10 Î¼M H2O2 concentration, a higher GSH level was detected in comparison to the other groups while oocytes exposed to 1000 Î¼M H2O2 had the lowest GSH level. Treatment of oocytes with 1000 Î¼M H2O2 decreased the rate of two pronuclei formation as compared with other groups. A higher rate of blastocyst formation was seen in 100 Î¼M H2O2 group as compared with the control group. However, exogenous H2O2 in maturation medium did not affect total cell numbers and apoptotic cell ratio at the blastocyst stage. Moreover, mRNA transcript abundance of Bax, Bcl-2, and p53 genes was similar between blastocysts derived from H2O2-induced oocytes and control blastocysts. Treatment of oocytes with H2O2 at mild level during in vitro maturation had a positive effect on GSH level and this, in turn, may lead to improvement in preimplantation embryonic development.

Keywords

Blastocyst GSH level Hydrogen peroxide Oocyte Apoptosis.

Nikdel, K., Aminafshar, M., Mohammadi-Sangcheshmeh, A., EmamJomeh-Kashan, N., & Seyedjafari, E. (2017). H2O2-induced mild stress in relation with in vitro ovine oocyte developmental competence: implications for blastocyst apoptosis and related genes expression. Cellular and Molecular Biology, 63(5), 43–49. https://doi.org/10.14715/cmb/2017.63.5.9

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References

Enright BP, Lonergan P, Dinnyes A, Fair T, Ward FA, Yang X, et al. Culture of in vitro produced bovine zygotes in vitro vs in vivo: implications for early embryo development and quality. Theriogenology. 2000;54(5):659-73.
Izquierdo D, Villamediana P, Lopez-Bejar M, Paramio MT. Effect of in vitro and in vivo culture on embryo development from prepubertal goat IVM-IVF oocytes. Theriogenology. 2002;57(5):1431-41.
Lonergan P, Rizos D, Gutierrez-Adan A, Moreira PM, Pintado B, de la Fuente J, et al. Temporal divergence in the pattern of messenger RNA expression in bovine embryos cultured from the zygote to blastocyst stage in vitro or in vivo. Biol Reprod. 2003;69(4):1424-31.
Rizos D, Ward F, Duffy P, Boland MP, Lonergan P. Consequences of bovine oocyte maturation, fertilization or early embryo development in vitro versus in vivo: implications for blastocyst yield and blastocyst quality. Mol Reprod Dev. 2002;61(2):234-48.
Chakravarthi VP, Kona S, Kumar AS, Bhaskar M, Rao V. Quantitative patterns of expression of gap junction genes during in vivo or in vitro development of ovarian follicles in sheep. Small Ruminant Research. 2016;143:35-42.
Chakravarthi VP, Kona S, Kumar AS, Bhaskara M, Rao V. Stage specific expression of cell cycle genes during in vivo or in vitro development of ovarian follicles in sheep. Small Ruminant Research. 2016;143:1-7.
Paramio M. In vivo and in vitro embryo production in goats. Small Ruminant Research. 2010;89(2):144-8.
Wani N. In vitro maturation and in vitro fertilization of sheep oocytes. Small Ruminant Research. 2002;44(2):89-95.
Kattal N, Cohen J, Barmat LI. Role of coculture in human in vitro fertilization: a meta-analysis. Fertil Steril. 2008;90(4):1069-76.
Lindgren KE, Hreinsson J, Helmestam M, Wanggren K, Poromaa IS, Karehed K, et al. Histidine-rich glycoprotein derived peptides affect endometrial angiogenesis in vitro but has no effect on embryo development. Syst Biol Reprod Med. 2016;62(3):192-200.
Valiollahpoor Amiri M, Deldar H, Ansari Pirsaraei Z. Impact of supplementary royal jelly on in vitro maturation of sheep oocytes: genes involved in apoptosis and embryonic development. Syst Biol Reprod Med. 2016;62(1):31-8.
Xu L, Wang Y, Zhou P, Cao YX, Huang TH, Chian RC. Cytogenetic analysis of in vivo and in vitro matured oocytes derived from naturally cycling and stimulated mice. Syst Biol Reprod Med. 2008;54(3):155-62.
Zhang YL, Liu FJ, Chen XL, Zhang ZQ, Shu RZ, Yu XL, et al. Dual effects of molybdenum on mouse oocyte quality and ovarian oxidative stress. Syst Biol Reprod Med. 2013;59(6):312-8.
Yang HW, Hwang KJ, Kwon HC, Kim HS, Choi KW, Oh KS. Detection of reactive oxygen species (ROS) and apoptosis in human fragmented embryos. Hum Reprod. 1998;13(4):998-1002.
Dumollard R, Carroll J, Duchen MR, Campbell K, Swann K. Mitochondrial function and redox state in mammalian embryos. Semin Cell Dev Biol. 2009;20(3):346-53.
Tatone C. Oocyte senescence: a firm link to age-related female subfertility. Gynecol Endocrinol. 2008;24(2):59-63.
Barnham KJ, Masters CL, Bush AI. Neurodegenerative diseases and oxidative stress. Nat Rev Drug Discov. 2004;3(3):205-14.
Kim MJ, Oh HJ, Park JE, Kim GA, Park EJ, Jang G, et al. Effects of mineral supplements on ovulation and maturation of dog oocytes. Theriogenology. 2012;78(1):110-5.
Bosman E, Esterhuizen AD, Rodrigues FA, Becker PJ, Hoffmann WA. Effect of metformin therapy and dietary supplements on semen parameters in hyperinsulinaemic males. Andrologia. 2015;47(9):974-9.
Vandaele L, Thys M, Bijttebier J, Van Langendonckt A, Donnay I, Maes D, et al. Short-term exposure to hydrogen peroxide during oocyte maturation improves bovine embryo development. Reproduction. 2010;139(3):505-11.
Chirino-Galindo G, Mejia-Zepeda R, Palomar-Morales M. Change in lipoperoxidation but not in scavenging enzymes activity during polyamine embryoprotection in rat embryo cultured in hyperglycemic media. In Vitro Cell Dev-An. 2012;48(9):570-6.
DeNunzio M, Gomez G. Extracellular glutathione promotes migration of hydrogen peroxide-stressed cultured chick embryonic skin cells. In Vitro Cell Dev-An. 2014;50(4):350-7.
Jang BC, Paik JH, Kim SP, Bae JH, Mun KC, Song DK, et al. Catalase induces the expression of inducible nitric oxide synthase through activation of NF-KB and PI3K signaling pathway in Raw 264.7 cells. Biochem Pharmacol. 2004;68(11):2167-76.
Johnson CS, Blanton MR, Hunter ES. Effects of ethanol and hydrogen peroxide on mouse limb bud mesenchyme differentiation and cell death. In Vitro Cell Dev-An. 2004;40(3-4):108-12.
Yuh HS, Yu DH, Shin MJ, Kim HJ, Bae KB, Lee DS, et al. The effects of various antioxidants on the development of parthenogenetic porcine embryos. In Vitro Cell Dev-An. 2010;46(2):148-54.
Reth M. Hydrogen peroxide as second messenger in lymphocyte activation. Nat Immunol. 2002;3(12):1129-34.
Chaube SK, Prasad PV, Thakur SC, Shrivastav TG. Hydrogen peroxide modulates meiotic cell cycle and induces morphological features characteristic of apoptosis in rat oocytes cultured in vitro. Apoptosis. 2005;10(4):863-74.
Kemal Duru N, Morshedi M, Oehninger S. Effects of hydrogen peroxide on DNA and plasma membrane integrity of human spermatozoa. Fertil Steril. 2000;74(6):1200-7.
Yazaki T, Hiradate Y, Hoshino Y, Tanemura K, Sato E. L-carnitine improves hydrogen peroxide-induced impairment of nuclear maturation in porcine oocytes. Anim Sci J. 2013;84(5):395-402.
Morales H, Tilquin P, Rees JF, Massip A, Dessy F, Van Langendonckt A. Pyruvate prevents peroxide-induced injury of in vitro preimplantation bovine embryos. Mol Reprod Dev. 1999;52(2):149-57.
Donnay I, Knoops B. Peroxiredoxins in gametogenesis and embryo development. Subcell Biochem. 2007;44:345-55.
Finkel T. Oxidant signals and oxidative stress. Curr Opin Cell Biol. 2003;15(2):247-54.
Veshkini A, Asadi H, Khadem AA, Mohammadi-Sangcheshmeh A, Khazabi S, Aminafshar M, et al. Effect of Linolenic acid during in vitro maturation of ovine oocytes: embryonic developmental potential and mRNA abundances of genes involved in apoptosis. J Assist Reprod Genet. 2015;32(4):653-9.
Mohammadi-Sangcheshmeh A, Soleimani M, Deldar H, Salehi M, Soudi S, Hashemi SM, et al. Prediction of oocyte developmental competence in ovine using glucose-6-phosphate dehydrogenase (G6PDH) activity determined at retrieval time. J Assist Reprod Genet. 2012;29(2):153-8.
Abazari-Kia AH, Mohammadi-Sangcheshmeh A, Dehghani-Mohammadabadi M, Jamshidi-Adegani F, Veshkini A, Zhandi M, et al. Intracellular glutathione content, developmental competence and expression of apoptosis-related genes associated with G6PDH-activity in goat oocyte. J Assist Reprod Genet. 2014;31(3):313-21.
Abazari-Kia AH, Dehghani-Mohammadabadi M, Mohammadi-Sangcheshmeh A, Zhandi M, Salehi M. Regulation of embryonic development and apoptotic-related gene expression by brain-derived neurotrophic factor in two different culture conditions in ovine. Theriogenology. 2015;84(1):62-9.
Veshkini A, Khadem AA, Mohammadi-Sangcheshmeh A, Alamouti AA, Soleimani M, Gastal EL. Linolenic acid improves oocyte developmental competence and decreases apoptosis of in vitro-produced blastocysts in goat. Zygote. 2015:1-12.
Chesney JA, Eaton JW, Mahoney JR, Jr. Bacterial glutathione: a sacrificial defense against chlorine compounds. J Bacteriol. 1996;178(7):2131-5.
Greenberg JT, Demple B. Glutathione in Escherichia-Coli Is Dispensable for Resistance to H2o2 and Gamma-Radiation. Journal of Bacteriology. 1986;168(2):1026-9.
Sutton-McDowall ML, Purdey M, Brown HM, Abell AD, Mottershead DG, Cetica PD, et al. Redox and Anti-Oxidant State Within Cattle Oocytes Following In Vitro Maturation With Bone Morphogenetic Protein 15 and Follicle Stimulating Hormone. Molecular Reproduction and Development. 2015;82(4):281-94.
White AR, Collins SJ, Maher F, Jobling MF, Stewart LR, Thyer JM, et al. Prion protein-deficient neurons reveal lower glutathione reductase activity and increased susceptibility to hydrogen peroxide toxicity. Am J Pathol. 1999;155(5):1723-30.
Tatone C, Di Emidio G, Barbaro R, Vento M, Ciriminna R, Artini PG. Effects of reproductive aging and postovulatory aging on the maintenance of biological competence after oocyte vitrification: insights from the mouse model. Theriogenology. 2011;76(5):864-73.
Pribenszky C, Molnar M, Cseh S, Solti L. Improving post-thaw survival of cryopreserved mouse blastocysts by hydrostatic pressure challenge. Anim Reprod Sci. 2005;87(1-2):143-50.
Pribenszky C, Pribenszky C, Siqueira FE, Molnar M, Harnos A, Rumpf R. Improved post-warming developmental competence of open pulled straw-vitrified in vitro-produced bovine blastocysts by sublethal hydrostatic pressure pretreatment. Reprod Fert Develop. 2008;20(1):125-.
Du Y, Pribenszky CS, Molnar M, Zhang X, Yang H, Kuwayama M, et al. High hydrostatic pressure: a new way to improve in vitro developmental competence of porcine matured oocytes after vitrification. Reproduction. 2008;135(1):13-7.
Du YT, Lin L, Schmidt M, Bogh IB, Kragh PM, Sorensen CB, et al. High hydrostatic pressure treatment of porcine oocytes before handmade cloning improves developmental competence and cryosurvival. Cloning Stem Cells. 2008;10(3):325-30.
Lin L, Kragh PM, Purup S, Kuwayama M, Du Y, Zhang X, et al. Osmotic stress induced by sodium chloride, sucrose or trehalose improves cryotolerance and developmental competence of porcine oocytes. Reprod Fertil Dev. 2009;21(2):338-44.
Kaarniranta K, Elo M, Sironen R, Lammi MJ, Goldring MB, Eriksson JE, et al. Hsp70 accumulation in chondrocytic cells exposed to high continuous hydrostatic pressure coincides with mRNA stabilization rather than transcriptional activation. Proc Natl Acad Sci U S A. 1998;95(5):2319-24.
Hochhauser E, Kivity S, Offen D, Maulik N, Otani H, Barhum Y, et al. Bax ablation protects against myocardial ischemia-reperfusion injury in transgenic mice. Am J Physiol Heart Circ Physiol. 2003;284(6):H2351-9.
Knudson CM, Korsmeyer SJ. Bcl-2 and Bax function independently to regulate cell death. Nat Genet. 1997;16(4):358-63.

References

Enright BP, Lonergan P, Dinnyes A, Fair T, Ward FA, Yang X, et al. Culture of in vitro produced bovine zygotes in vitro vs in vivo: implications for early embryo development and quality. Theriogenology. 2000;54(5):659-73.

Izquierdo D, Villamediana P, Lopez-Bejar M, Paramio MT. Effect of in vitro and in vivo culture on embryo development from prepubertal goat IVM-IVF oocytes. Theriogenology. 2002;57(5):1431-41.

Lonergan P, Rizos D, Gutierrez-Adan A, Moreira PM, Pintado B, de la Fuente J, et al. Temporal divergence in the pattern of messenger RNA expression in bovine embryos cultured from the zygote to blastocyst stage in vitro or in vivo. Biol Reprod. 2003;69(4):1424-31.

Rizos D, Ward F, Duffy P, Boland MP, Lonergan P. Consequences of bovine oocyte maturation, fertilization or early embryo development in vitro versus in vivo: implications for blastocyst yield and blastocyst quality. Mol Reprod Dev. 2002;61(2):234-48.

Chakravarthi VP, Kona S, Kumar AS, Bhaskar M, Rao V. Quantitative patterns of expression of gap junction genes during in vivo or in vitro development of ovarian follicles in sheep. Small Ruminant Research. 2016;143:35-42.

Chakravarthi VP, Kona S, Kumar AS, Bhaskara M, Rao V. Stage specific expression of cell cycle genes during in vivo or in vitro development of ovarian follicles in sheep. Small Ruminant Research. 2016;143:1-7.

Paramio M. In vivo and in vitro embryo production in goats. Small Ruminant Research. 2010;89(2):144-8.

Wani N. In vitro maturation and in vitro fertilization of sheep oocytes. Small Ruminant Research. 2002;44(2):89-95.

Kattal N, Cohen J, Barmat LI. Role of coculture in human in vitro fertilization: a meta-analysis. Fertil Steril. 2008;90(4):1069-76.

Lindgren KE, Hreinsson J, Helmestam M, Wanggren K, Poromaa IS, Karehed K, et al. Histidine-rich glycoprotein derived peptides affect endometrial angiogenesis in vitro but has no effect on embryo development. Syst Biol Reprod Med. 2016;62(3):192-200.

Valiollahpoor Amiri M, Deldar H, Ansari Pirsaraei Z. Impact of supplementary royal jelly on in vitro maturation of sheep oocytes: genes involved in apoptosis and embryonic development. Syst Biol Reprod Med. 2016;62(1):31-8.

Xu L, Wang Y, Zhou P, Cao YX, Huang TH, Chian RC. Cytogenetic analysis of in vivo and in vitro matured oocytes derived from naturally cycling and stimulated mice. Syst Biol Reprod Med. 2008;54(3):155-62.

Zhang YL, Liu FJ, Chen XL, Zhang ZQ, Shu RZ, Yu XL, et al. Dual effects of molybdenum on mouse oocyte quality and ovarian oxidative stress. Syst Biol Reprod Med. 2013;59(6):312-8.

Yang HW, Hwang KJ, Kwon HC, Kim HS, Choi KW, Oh KS. Detection of reactive oxygen species (ROS) and apoptosis in human fragmented embryos. Hum Reprod. 1998;13(4):998-1002.

Dumollard R, Carroll J, Duchen MR, Campbell K, Swann K. Mitochondrial function and redox state in mammalian embryos. Semin Cell Dev Biol. 2009;20(3):346-53.

Tatone C. Oocyte senescence: a firm link to age-related female subfertility. Gynecol Endocrinol. 2008;24(2):59-63.

Barnham KJ, Masters CL, Bush AI. Neurodegenerative diseases and oxidative stress. Nat Rev Drug Discov. 2004;3(3):205-14.

Kim MJ, Oh HJ, Park JE, Kim GA, Park EJ, Jang G, et al. Effects of mineral supplements on ovulation and maturation of dog oocytes. Theriogenology. 2012;78(1):110-5.

Bosman E, Esterhuizen AD, Rodrigues FA, Becker PJ, Hoffmann WA. Effect of metformin therapy and dietary supplements on semen parameters in hyperinsulinaemic males. Andrologia. 2015;47(9):974-9.

Vandaele L, Thys M, Bijttebier J, Van Langendonckt A, Donnay I, Maes D, et al. Short-term exposure to hydrogen peroxide during oocyte maturation improves bovine embryo development. Reproduction. 2010;139(3):505-11.

Chirino-Galindo G, Mejia-Zepeda R, Palomar-Morales M. Change in lipoperoxidation but not in scavenging enzymes activity during polyamine embryoprotection in rat embryo cultured in hyperglycemic media. In Vitro Cell Dev-An. 2012;48(9):570-6.

DeNunzio M, Gomez G. Extracellular glutathione promotes migration of hydrogen peroxide-stressed cultured chick embryonic skin cells. In Vitro Cell Dev-An. 2014;50(4):350-7.

Jang BC, Paik JH, Kim SP, Bae JH, Mun KC, Song DK, et al. Catalase induces the expression of inducible nitric oxide synthase through activation of NF-KB and PI3K signaling pathway in Raw 264.7 cells. Biochem Pharmacol. 2004;68(11):2167-76.

Johnson CS, Blanton MR, Hunter ES. Effects of ethanol and hydrogen peroxide on mouse limb bud mesenchyme differentiation and cell death. In Vitro Cell Dev-An. 2004;40(3-4):108-12.

Yuh HS, Yu DH, Shin MJ, Kim HJ, Bae KB, Lee DS, et al. The effects of various antioxidants on the development of parthenogenetic porcine embryos. In Vitro Cell Dev-An. 2010;46(2):148-54.

Reth M. Hydrogen peroxide as second messenger in lymphocyte activation. Nat Immunol. 2002;3(12):1129-34.

Chaube SK, Prasad PV, Thakur SC, Shrivastav TG. Hydrogen peroxide modulates meiotic cell cycle and induces morphological features characteristic of apoptosis in rat oocytes cultured in vitro. Apoptosis. 2005;10(4):863-74.

Kemal Duru N, Morshedi M, Oehninger S. Effects of hydrogen peroxide on DNA and plasma membrane integrity of human spermatozoa. Fertil Steril. 2000;74(6):1200-7.

Yazaki T, Hiradate Y, Hoshino Y, Tanemura K, Sato E. L-carnitine improves hydrogen peroxide-induced impairment of nuclear maturation in porcine oocytes. Anim Sci J. 2013;84(5):395-402.

Morales H, Tilquin P, Rees JF, Massip A, Dessy F, Van Langendonckt A. Pyruvate prevents peroxide-induced injury of in vitro preimplantation bovine embryos. Mol Reprod Dev. 1999;52(2):149-57.

Donnay I, Knoops B. Peroxiredoxins in gametogenesis and embryo development. Subcell Biochem. 2007;44:345-55.

Finkel T. Oxidant signals and oxidative stress. Curr Opin Cell Biol. 2003;15(2):247-54.

Veshkini A, Asadi H, Khadem AA, Mohammadi-Sangcheshmeh A, Khazabi S, Aminafshar M, et al. Effect of Linolenic acid during in vitro maturation of ovine oocytes: embryonic developmental potential and mRNA abundances of genes involved in apoptosis. J Assist Reprod Genet. 2015;32(4):653-9.

Mohammadi-Sangcheshmeh A, Soleimani M, Deldar H, Salehi M, Soudi S, Hashemi SM, et al. Prediction of oocyte developmental competence in ovine using glucose-6-phosphate dehydrogenase (G6PDH) activity determined at retrieval time. J Assist Reprod Genet. 2012;29(2):153-8.

Abazari-Kia AH, Mohammadi-Sangcheshmeh A, Dehghani-Mohammadabadi M, Jamshidi-Adegani F, Veshkini A, Zhandi M, et al. Intracellular glutathione content, developmental competence and expression of apoptosis-related genes associated with G6PDH-activity in goat oocyte. J Assist Reprod Genet. 2014;31(3):313-21.

Abazari-Kia AH, Dehghani-Mohammadabadi M, Mohammadi-Sangcheshmeh A, Zhandi M, Salehi M. Regulation of embryonic development and apoptotic-related gene expression by brain-derived neurotrophic factor in two different culture conditions in ovine. Theriogenology. 2015;84(1):62-9.

Veshkini A, Khadem AA, Mohammadi-Sangcheshmeh A, Alamouti AA, Soleimani M, Gastal EL. Linolenic acid improves oocyte developmental competence and decreases apoptosis of in vitro-produced blastocysts in goat. Zygote. 2015:1-12.

Chesney JA, Eaton JW, Mahoney JR, Jr. Bacterial glutathione: a sacrificial defense against chlorine compounds. J Bacteriol. 1996;178(7):2131-5.

Greenberg JT, Demple B. Glutathione in Escherichia-Coli Is Dispensable for Resistance to H2o2 and Gamma-Radiation. Journal of Bacteriology. 1986;168(2):1026-9.

Sutton-McDowall ML, Purdey M, Brown HM, Abell AD, Mottershead DG, Cetica PD, et al. Redox and Anti-Oxidant State Within Cattle Oocytes Following In Vitro Maturation With Bone Morphogenetic Protein 15 and Follicle Stimulating Hormone. Molecular Reproduction and Development. 2015;82(4):281-94.

White AR, Collins SJ, Maher F, Jobling MF, Stewart LR, Thyer JM, et al. Prion protein-deficient neurons reveal lower glutathione reductase activity and increased susceptibility to hydrogen peroxide toxicity. Am J Pathol. 1999;155(5):1723-30.

Tatone C, Di Emidio G, Barbaro R, Vento M, Ciriminna R, Artini PG. Effects of reproductive aging and postovulatory aging on the maintenance of biological competence after oocyte vitrification: insights from the mouse model. Theriogenology. 2011;76(5):864-73.

Pribenszky C, Molnar M, Cseh S, Solti L. Improving post-thaw survival of cryopreserved mouse blastocysts by hydrostatic pressure challenge. Anim Reprod Sci. 2005;87(1-2):143-50.

Pribenszky C, Pribenszky C, Siqueira FE, Molnar M, Harnos A, Rumpf R. Improved post-warming developmental competence of open pulled straw-vitrified in vitro-produced bovine blastocysts by sublethal hydrostatic pressure pretreatment. Reprod Fert Develop. 2008;20(1):125-.

Du Y, Pribenszky CS, Molnar M, Zhang X, Yang H, Kuwayama M, et al. High hydrostatic pressure: a new way to improve in vitro developmental competence of porcine matured oocytes after vitrification. Reproduction. 2008;135(1):13-7.

Du YT, Lin L, Schmidt M, Bogh IB, Kragh PM, Sorensen CB, et al. High hydrostatic pressure treatment of porcine oocytes before handmade cloning improves developmental competence and cryosurvival. Cloning Stem Cells. 2008;10(3):325-30.

Lin L, Kragh PM, Purup S, Kuwayama M, Du Y, Zhang X, et al. Osmotic stress induced by sodium chloride, sucrose or trehalose improves cryotolerance and developmental competence of porcine oocytes. Reprod Fertil Dev. 2009;21(2):338-44.

Kaarniranta K, Elo M, Sironen R, Lammi MJ, Goldring MB, Eriksson JE, et al. Hsp70 accumulation in chondrocytic cells exposed to high continuous hydrostatic pressure coincides with mRNA stabilization rather than transcriptional activation. Proc Natl Acad Sci U S A. 1998;95(5):2319-24.

Hochhauser E, Kivity S, Offen D, Maulik N, Otani H, Barhum Y, et al. Bax ablation protects against myocardial ischemia-reperfusion injury in transgenic mice. Am J Physiol Heart Circ Physiol. 2003;284(6):H2351-9.

Knudson CM, Korsmeyer SJ. Bcl-2 and Bax function independently to regulate cell death. Nat Genet. 1997;16(4):358-63.

Author biographies is not available.