- Farzad Rajaei
 - - Cell Culture Laboratory, Department of Anatomy, Qazvin University of Medical Sciences, Ghazvin, Iran
- Takshij Otoi
- - Department of Reproduction, Faculty of Veterinary, Yamaguchi University, Yamaguchi, Japan
Received: 4/1/2006 Accepted: 4/1/2006 - Publisher : Avicenna Research Institute |
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Abstract
Introduction: The aim of the present study was to investigate the effects of different cryoprotectants (CPs) on DNA fragmentation of in vitro produced blastocysts to determine an appropriate cryoprotectant for embryo cryopreservation. Therefore, the precise aims of the study were to assess the toxic effects of different cryoprotectants in terms of survival rate and to evaluate the effects of different CPs on DNA fragmentation in in vitro produced porcine blastocysts. Ethylene glycol, 1,2 propanediol and glycerol are common cryoprotectants widely used for embryo cryopreservation in different animals as well as humans. Materials & Methods: 197 porcine blastocysts were produced in vitro and 160 blastocysts were random-ly selected and divided into 4 groups. 40 blastocysts were placed in phosphate buffer solution (PBS) with-out any cryoprotectants for 1 hour in room temperature (23-25C) as the control group. The rest of the blastocysts were exposed to 3 different cryoprotectants (10% solutions) ethylene glycol (EG), 1, 2 pro-panediol (PG) and glycerol (Gly) for 1 hour in a 3- step method in room temperature. The survival rate was assessed after culture in NCSU-37 medium for 24 hours as the proportion of recovered embryos with the reformation of blastocele observed by stereomicroscopy at 40 magnifications. The apoptotic indices were evaluated after staining by TUNEL technique to label apoptotic nuclei and later were counter-stained by propidium iodide (PI) to label all nuclei and were analyzed by fluorescent microscopy. Then, the sur-vival rate was compared with the data obtained from the control group. Through ANOVA and Fishers exact test the data were analyzed while employing StatView software and the level of significance was considered as 0.05%. Results: Exposing porcine blastocysts to different cryoprotectants results in an increase in DNA fragmentation, although the apoptotic index in blastocysts with blastocele compared to those without them were lower in the study, disregard of the kind of cryoprotectant. Conclusion: It is concluded that CPs can decrease the survival rate of porcine blastocysts by increasing the percentage of DNA fragmentation but EG has the least effect on DNA fragmentation.
Keywords:
Cryoprotectants, Cytotoxicity, Apoptosis, Pig, DNA fragmentation, Blastocyst To cite this article:References
- Hardy K. Cell death in the mammalian blastocyst. Mol Hum Reprod.1997;3:919-925.
- Jurisicova A., Rogers I., Fasciani A., Casper R.F., Varmuza S. Effect of maternal age and conditions of fertilization on programmed cell death during murine preimplantation embryo development. Mol Hum Reprod.1998;4:139-145.
- Levy R.R., Cordonier H., Czyba J.C., Guerin J.F. Apoptosis in preimplantation mammalian embryo and genetics. Ital J Anat Embryol.2001;106:101-108.
- Hochachka P.W. Defense strategies against hypoxia and hypothermia. Science.1986;231:234-241.
- Gavrieli Y., Sherman Y., Ben-Sasson S.A. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol. 1992;119(3):493-501.
- Yamadori I., Yoshino T., Kondo E., Cao L., Akagi T., Matsuo Y., Minowada J. Comparison of two methods of staining apoptotic cells of leukemia cell lines. Terminal deoxynucleotidyl transferase and DNA polymerase I reactions. J Histochem Cytochem. 1998; 46(1):85-90.
- Neuber E., Luetjens C.M., Chan A.W., Schatten G.P. Analysis of DNA fragmentation of in vitro cultured bovine blastocysts using TUNEL. Theriogenology. 2002;57(9):2193-202.
- Pampfer S., Vanderheyden I., McCracken J.E., Vesela J., De Hertogh R. Increased cell death in rat blasto-cysts exposed to maternal diabetes in utero and to high glucose or tumor necrosis factor-alpha in vitro. Development.1997;124(23):4827-36.
- Emiliani S., Van den Bergh M., Vannin A.S., Bira-mane J., Englert Y. Comparison of ethylene glycol, 1, 2- propanediol and glycerol for cryopreservation of slow-cooled mouse zygotes, 4-cell embryos and blas-tocysts. Hum Reprod.2000;15(4):905-10.
- Fahning M.L., Garcia M.A. Status of cryopreservation of embryos from domestic animals. Cryobiology.1992; 29:1-18.
- Friedler S., Giudice L.C., Lamb E.J. Cryopreserva-tion of embryos and ova. Fertil Steril.1988;49:743-64.
- Marquez-Alvarado Y.C., Galina C.S., Castilla B., Leon H., Moreno-Mendoza N. Evidence of damage in cryopreserved and fresh bovine embryos using the Tunel technique. Reprod Domest Anim.2004;39(3): 141-5.
- رجايي فرزاد، سليمانيراد جعفر، نيكنفس بهروز، غفاري معرفت. اثرات انجماد شيشهاي بر ميزان آپوپتوز در مرحله بلاستوسيست. فصلنامه پزشكي باروري و ناباروري، شماره اول، سال پنجم، زمستان 1382، صفحات: 22-14.
- Kikuchi K., Kashiwazaki N., Noguchi J., Shimada A., Takahashi R., et al. Developmental competence after transfer to recipients of porcine oocytes Matured, fertilized, and cultured in vitro. Biol Reprod.1999;60: 336-40.
- Yuge M., Otoi T., Nii M., Murakami M., Karja N.W., et al. Effects of cooling ovaries before oocyte aspira- tion on meiotic competence of porcine oocytes and of exposing in vitro matured oocytes to ambient tempera-ture on in vitro fertilization and development of the oocytes. Cryobiology.2003;47(2):102-8.
- Wyllie A.H., Kerr J.F., Currie A.R. Cell death: the significance of apoptosis. Int Rev Cytol.1980;68:251-306.
- Leese H.J., Donnay I., Thompson J.G. Human assis-ted conception: a cautionary tale. Lessons from domes-tic animals. Hum Reprod.1998;4(Suppl):184-202.
- Wyllie A.H. Apoptosis: an overview. Br Med Bull. 1997;53:451-465.
- Jurisicova A., Varmuza S., Casper R.F. Programmed cell death and human embryo fragmentation. Mol Hum Reprod.1996;2:93-98.
- Men H., Monson R.L., Parrish J.J., Rutledge J.J. Detection of DNA damage in bovine metaphase II oocytes resulting from cryopreservation. Mol Reprod Dev.2003;64:245-250.
- Oda K., Gibbons W.E., Leibo S.P. Osmotic shock of fertilized mouse ova. J Reprod Fertil.1992;95:737-747.
- Pedro P.B., Zhu S.E., Makino N., Sakurai T., Edashige K., Kasai M. Effects of hypotonic stress on the survival of mouse oocytes and embryos at various stages. Cryobiology.1997;35:150-158.
- Brison D.R., Schultz R.M. Apoptosis during mouse blastocyst formation: evidence for a role for survival factors including transforming growth factor alpha. Biol Reprod.1997;56:1088-1096.
- Kasai M. Simple and efficient methods for vitrifica-tion of mammalian embryos. Anim Reprod Sci.1996; 42:67-75.