- Abbas Ahmadi
 - - Department of Embryology, Faculty of Veterinary, Urmia University, Urmia, Iran
- Rajabali Sadrkhanlou
- - Department of Embryology, Faculty of Veterinary, Urmia University, Urmia, Iran
Received: 8/14/2010 Accepted: 12/7/2010 - Publisher : Avicenna Research Institute |
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Abstract
Background: Etiologically, oxidative stress can be considered as one of the reasons for defective embryonic development which leads to developmental arrest due to necrosis or apoptosis. Under in vivo conditions, multiple mechanisms act to protect the embryo against reactive oxygen species (ROS), but under in vitro conditions most of these mechanisms are absent leading to higher levels of ROS in the culture medium. The objective of this study was to compare the antioxidant effects of Tempol, 4-hydroxy-2, 2, 6, 6-tetramethylpiperidine-1-oxyl, a permeable synthetic antioxidant, on mouse pre-implantation embryonic development in vitro conditions in the presence or absence of oxidative stress. Methods: Mature oocytes from mouse were retrieved following ovarian stimulation by the administration of Pregnant Mare Serum Gonadotropin (PMSG) and hCG. Upon in vitro fertilization, the zygotes were cultured in different groups in HTF medium containing 4 mg/ml BSA. To study the effects of oxidative stress on embryo development, the zygotes were cultured for an hour in a medium containing different concentrations of H2O2. After washing, the zygotes were transferred to the culture plate. The zygotes were later placed in the media containing different concentrations of Tempol following their culture in 10 µM H2O2 for one hour to study the effects of different concentrations of the substance in the absence of other oxidative stresses. The data were later compared and statistically analyzed. Results: The pre-implantation embryonic development decreased significantly in the case group, compared to the control group after a short exposure to H2O2, – the effect being more noticeable in higher concentrations. Tempol reduced the impairments resulting from the oxidative stress to some extent. Under in vitro conditions and a concentration of 0.5 µM, Tempol improved embryonic development quality, quantitatively and morphologically. Tempol increased the percentage of two-cell embryos from 91.78% in the control group to 96.99% (p < 0.05), blastocysts from 67.80% in the controls to 81.33% (p < 0.05) in the cases, and significantly decreased embryonic arrest from 32.19% in the controls to 18.67% in the cases (p < 0.05). Conclusion: ROS has a major role in embryonic arrest, witnessed in embryo cultures in vitro conditions. The present study showed that supplementation of embryo cultures with Tempol improved the embryonic development. It seems that addition of permeable synthetic antioxidants, such as Tempol, to embryo cultures could protect embryos from oxidative damage and improve embryonic development.
Keywords:
Developmental arrest, Embryonic development, Mouse, Oxidative stress, Reactive oxygen species, Synthetic Antioxidant, Tempol, Zygote To cite this article:References
- Gonzalez-Flecha B, Reides C, Cutrin JC, Llesuy SF, Boveris A. Oxidative stress produced by suprahep-atic occlusion and reperfusion. Hepatology. 1993;18 (4):881-9. [PubMed]
- Sikka SC. Relative impact of oxidative stress on male reproductive function. Curr Med Chem. 2001;8 (7):851-62. [PubMed]
- Sarafian TA, Bredesen DE. Is apoptosis mediated by reactive oxygen species? Free Radic Res. 1994;21 (1):1-8. [PubMed]
- 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. [PubMed]
- Luvoni GC, Keskintepe L, Brackett BG. Improve-ment in bovine embryo production in vitro by glutathione-containing culture media. Mol Reprod Dev. 1996;43(4):437-43. [PubMed]
- Bedaiwy MA, Falcone T, Mohamed MS, Aleem AA, Sharma RK, Worley SE, et al. Differential growth of human embryos in vitro: role of reactive oxygen species. Fertil Steril. 2004;82(3):593-600. [PubMed]
- Goto Y, Noda Y, Narimoto K, Umaoka Y, Mori T. Oxidative stress on mouse embryo development in vitro. Free Radic Biol Med. 1992;13(1):47-53. [PubMed]
- Goyanes VJ, Ron-Corzo A, Costas E, Maneiro E. Morphometric categorization of the human oocyte and early conceptus. Hum Reprod. 1990;5(5):613-8. [PubMed]
- Yoneda A, Suzuki K, Mori T, Ueda J, Watanabe T. Effects of delipidation and oxygen concentration on in vitro development of porcine embryos. J Reprod Dev. 2004;50(3):287-95. [PubMed]
- Carbone MC, Tatone C, Delle Monache S, Marci R, Caserta D, Colonna R, et al. Antioxidant enzymatic defences in human follicular fluid: charac-terization and age-dependent changes. Mol Hum Reprod. 2003;9(11):639-43. [PubMed]
- Catt JW, Henman M. Toxic effects of oxygen on human embryo development. Hum Reprod. 2000; 15 Suppl 2:199-206. [PubMed]
- Wang X, Falcone T, Attaran M, Goldberg JM, Agarwal A, Sharma RK. Vitamin C and vitamin E supplementation reduce oxidative stress-induced embryo toxicity and improve the blastocyst devel-opment rate. Fertil Steril. 2002;78(6):1272-7. [PubMed]
- Guérin P, El Mouatassim S, Ménézo Y. Oxidative stress and protection against reactive oxygen spe-cies in the pre-implantation embryo and its sur-roundings. Hum Reprod Update. 2001;7(2):175-89. [PubMed]
- Ali AA, Bilodeau JF, Sirard MA. Antioxidant re-quirements for bovine oocytes varies during in vitro maturation, fertilization and development. Theriogenology. 2003;59(3-4):939-49. [PubMed]
- Meirelles FV, Caetano AR, Watanabe YF, Ripa-monte P, Carambula SF, Merighe GK, et al. Genome activation and developmental block in bovine embryos. Anim Reprod Sci. 2004;82-83:13-20. [PubMed]
- Nagy A, Gestsenstion M, Vintersten K, Behringer R. Manipulating the mouse embryo. 3nd ed. New York: Cold Spring Harbor Laboratory; 2000; p. 565-98.
- Cebral E, Carrasco I, Vantman D, Smith R. Preim-plantation embryotoxicity after mouse embryo ex-position to reactive oxygen species. Biocell. 2007; 31(1):51-9. [PubMed]
- Tarkowski AK. An Air-Drying Method for Chromosome Preparations from Mouse Eggs. Cytogenetics. 1966;5(6):394-400. [PubMed]
- Chaudière J, Ferrari-Iliou R. Intracellular anti-oxidants: from chemical to biochemical mechanisms. Food Chem Toxicol. 1999;37(9-10):949-62. [PubMed]
- Szeto HH. Cell-permeable, mitochondrial-targeted, peptide antioxidants. AAPS J. 2006;8(2):E277-83. [PubMed]
- Forrest VJ, Kang YH, McClain DE, Robinson DH, Ramakrishnan N. Oxidative stress-induced apoptosis prevented by Trolox. Free Radic Biol Med. 1994;16(6):675-84. [PubMed]
- Tada-Oikawa S, Oikawa S, Kawanishi M, Yamada M, Kawanishi S. Generation of hydrogen peroxide precedes loss of mitochondrial membrane potential during DNA alkylation-induced apoptosis. FEBS Lett. 1999;442(1):65-9. [PubMed]
- Mitchell JB, Samuni A, Krishna MC, DeGraff WG, Ahn MS, Samuni U, et al. Biologically active metal-independent superoxide dismutase mimics. Biochemistry. 1990;29(11):2802-7. [PubMed]
- Garner DL, Thomas CA, Gravance CG, Marshall CE, DeJarnette JM, Allen CH. Seminal plasma addition attenuates the dilution effect in bovine sperm. Theriogenology. 2001;56(1):31-40. [PubMed]
- Lindemann CB, Kanous K. The cyclic nidroxide free radical, 4-hidroxy-2,2,6,6,tetramethylpiperidine-1-oxyl (TEMPOL), is effective in prolonging the motility of bull sperm in vitro. Biol Reprod. 1991;44(suppl 1):117.
- Donoghue AM, Donoghue DJ. Effects of water- and lipid-soluble antioxidants on turkey sperm viability, membrane integrity, and motility during liquid storage. Poult Sci. 1997;76(10):1440-5. [PubMed]
- Mara L, Accardo C, Pilichi S, Dattena M, Chessa F, Chessa B, et al. Benefits of TEMPOL on ram semen motility and in vitro fertility: a preliminary study. Theriogenology. 2005;63(8):2243-53. [PubMed]
- Li J, Foote RH, Liu Z, Giles JR. Development of rabbit zygotes into blastocysts in defined protein-free medium and offspring born following culture and embryo transfer. Theriogenology. 1997;47(5): 1103-13. [PubMed]