Reproductive and Developmental Biology

The Korean Society of Animal Reproduction (한국동물번식학회)
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Developmental Characteristics of SCNT Pig Embryos Knocked-out of Alpha-1,3-Galactosyltransferase Gene

  • Published : 2009.09.30
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Abstract

This study was performed to comprehend the developmental characteristics of cloned embryos knocked out (KO) of $\alpha$-1,3-galactosyltransferase (GalT) gene. Immature oocytes were collected and cultured for 40 hrs (1-step) or 20hrs (with hormone) + 20hrs (without hormone) (2-step). The embryos transferred with miniature pig ear fibroblast cell were used as control. The reconstructed embryos were cultured in PZM-3 with 5% $CO_2$ in air at $38.5^{\circ}C$ for 6 days. To determine the quality of the blstocysts, TUNEL and quantitative realtime RT-PCR were performed. The embryos were transferred to a surrogate (Landrace) at an earlier stage of the estrus cycle. The maturation rate was significantly higher in 2-step method than that of 1-step (p<0.05). The blastocyst development of GalT KO embryos was significantly lower than that of normal cloned embryos (p<0.05). The total and apoptotic cell number of GalT KO blastocysts was not different statistically from control. The relative abundance of Bax-$\alpha$/Bcl-xl ratio was significantly higher in both cloned blastocysts than that of in vivo blastocysts (p<0.05). Taken together, it can be postulated that the lower developmental potential and higher expression of apoptosis related genes in GalT KO SCNT embryos might be a cause of a low efficiency of GalT KO cloned miniature pig production.

Keywords

References

  1. Brenner CA, Exley GE, Alikani M, Cohen J, McElhinny AS, Stachecki JJ, Scott RT, Warner CM (1997): Apoptosis and human embryo survival. Fertility and Sterility Supplement 1: 86 https://doi.org/10.1016/S0015-0282(97)84891-1
  2. Byrne AT, Southgate J, Brison DR, Leese HJ (1999): Analysis of apoptosis in the preimplantation bovine embryo using TUNEL. J Reprod Fertil 117:97-105 https://doi.org/10.1530/jrf.0.1170097
  3. Cheong HT, Ikeda K, Martinez MA, Katagiri S, Takahashi Y (2000): Development of reconstituted pig embryos by nuclear transfer of cultured cumulus cells. Reprod Fertil Dev 12:15-20 https://doi.org/10.1071/RD00051
  4. Cooper DK, Koren E, Oriol R (1993): Genetically engineered pigs. Lancet 342:682-683 https://doi.org/10.1016/0140-6736(93)91791-J
  5. Dai Y, Vauqht TD, Boone J, Chen SH, Phelps CJ, Ball S, Monahan JA, Jobst PM, McCreath KJ, Lamborn AE, Cowell-Lucero JL, Wells KD, Colman A, Polejaeva IA, Ayares DL (2002): Targeted disruption of the alpha 1,3-galactosyltransferase gene in cloned pigs. Nat Biotechnol 20:251-255 https://doi.org/10.1038/nbt0302-251
  6. Forsberg EJ, Strelchenko NS, Augenstein ML, Betthauser JM, Childs LA, Eilertsen KJ, Enos JM, Forsythe TM, Golueke PJ, Koppang RW, Lange G, Lesmeister TL, Malon KS, Mell GD, Misica PM, Pace MM, Pfister-Genskow M, Voelker GR, Watt SR, Bishop MD (2002): Production of cloned cattle from in vitro systems. Biol Reprod 67:327-333 https://doi.org/10.1095/biolreprod67.1.327
  7. Fujimura T, Murakami H, Kurome M, Takahagi Y, Shigehisa T, Nagashima H (2008): Effects of recloning on the efficiency of production of alpha 1,3 galactosyltransferase knockout pigs. J Reprod Dev 54: 58-62 https://doi.org/10.1262/jrd.19110
  8. Funahashi H, Day BN (1993): Effects of the duration of exposure to hormone supplements on cytoplasmic maturation of pig oocytes in vitro. J Reprod Fertil 98:179-185 https://doi.org/10.1530/jrf.0.0980179
  9. Galili U (2001): The alpha-gal epitope (Gal alpha 1-3Gal beta 1-4GlcNAc-R) in xenotransplantation. Biochemie 83:557-563 https://doi.org/10.1016/S0300-9084(01)01294-9
  10. Hao Y, Lai L, Mao J, Im GS, Bonk A, Prather RS (2003): Apoptosis and in vitro development of preimplantation porcine embryos derived in vitro or by nuclear transfer. Biol Reprod 69:501-507 https://doi.org/10.1095/biolreprod.103.016170
  11. Hara A, Hirose Y, Wang A, Yoshimi N, Tanaka T, Mori H (1996): Localization of Bax and Bcl-2 proteins, regulators of programmed cell death, in the human central nervous system. Virchows Arch 429: 249-253 https://doi.org/10.1007/BF00198341
  12. Hwang IS, Park MR, Moon HJ, Shim JH, Kim DH, Yang BC, Ko YG, Yang BS, Cheong HT, Im GS (2008): Osmolarity at early culture stage affects development and expression of apoptosis related genes (Bax-α and Bcl-xl) in pre-implantation porcine NT embryos. Mol Reprod Dev 75:464-471 https://doi.org/10.1002/mrd.20785
  13. Iguma LT, Lisauskas SF, Melo EO, Franco MM, Pivato I, Vianna GR, Sousa RV, Dode MA, Aragao FJ, Rech EL, Rumpf R (2005): Development of bovine embryos reconstructed by nuclear transfer of transfected and non-transfected adult fibroblast cells. Genet Mol Res 4:55-66
  14. Im GS, Yang BH, Lai L, Liu Z, Hao Y, Prather RS (2005): Fragmentation and development of preimplantation porcine embryos derived by parthenogenetic activation and nuclear transfer. Mol Reprod Dev 71:159-165 https://doi.org/10.1002/mrd.20258
  15. Joziasse DH, Oriol R (1999): Xenotransplantation: the importance of the Galalpha1,3Gal epitope in hyperacute vascular rejection. Biochim Biophys Acta 1455:403-418 https://doi.org/10.1016/S0925-4439(99)00056-3
  16. Kobayashi M, Asakuma S, Fukui Y (2007): Blastocyst production by in vitro maturation and development of porcine oocytes in defined media following intracytoplasmic sperm injection. Zygote 15: 93-102 https://doi.org/10.1017/S0967199406004035
  17. Kolbe T, Holts W (1999): Intracytoplasmic injection (ICSI) of in vivo or in vitro matured oocytes with fresh ejaculated or frozen-thawed epididymal spermatozoa and additional calcium ionophore activation in the pig. Theriogenology 52:671-682 https://doi.org/10.1016/S0093-691X(99)00161-2
  18. Kolber-Simonds D, Lai L, Watt SR, Denaro M, Arn S, Augenstein ML, Betthauser J, Carter DB, Greenstein JL, Hao Y, Im GS, Liu Z, Mell GD, Murphy CN, Park KW, Rieke A, Ryan DJ, Sachs DH, Forsberg EJ, Prather RS, Hawley RJ (2004): Production of alpha-1,3-galactosyltransferase null pigs by means of nuclear transfer with fibroblasts bearing loss of heterozygosity mutations. Proc Natl Acad Sci USA 101:7335-7340 https://doi.org/10.1073/pnas.0307819101
  19. Lenton EA, King H, Thomas EJ, Smith SK, McLachlan RI, MacNeil S, Cooke ID (1988): The endocrine environment of the human oocyte. J Reprod Fertil 82:827-841 https://doi.org/10.1530/jrf.0.0820827
  20. Levy RR, Cordonier H, Czyba JC, Goerin JF (2001): Apoptosis in preimplantation mammalian embryo and genetics. Ital J Anat Embryol 106:101-108
  21. Logan JS (2000): Prospects for xenotransplantation. Curr Opin Immunol 12:563-568 https://doi.org/10.1016/S0952-7915(00)00139-4
  22. Moon HJ, Shim JH, Hwang IS, Park MR, Kim DH, Ko YG, Park CK, Im GS (2009): Effect of FBS (fetal bovine serum) and pFF(porcine follicular fluid) on in vitro maturation development of porcine parthenogenetic and nuclear transfer embryos. Reprod Dev Biol 33: 85-91
  23. Nottle MB, Beebe LF, Harrison SJ, McIlfatrick SM, Ashman RJ, O'Connell PJ, Salvaris EJ, Fisicaro N, Pommey S, Cowan PJ, d'Apice AJ (2007): Production of homozygous $\alpha$-1,3-galactosyltransferase knockout pigs by breeding and somatic cell nuclear transfer. Xenotransplantation 14:339-344 https://doi.org/10.1111/j.1399-3089.2007.00417.x
  24. Oltvai ZN, Milliman CL, Korsmeyer SJ (1993): Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 74:609-619 https://doi.org/10.1016/0092-8674(93)90509-O
  25. Park JH, Kwon DJ, Lee BK, Hwang IS, Park CK, Yang BK, Cheong HT (2009): Comparison of microtubule distributions between somatic cell nuclear transfer and parthenogenetic porcine embryos Reprod Dev Biol 33:13-18
  26. Phelps CJ, Koike C, Vaught TD, Boone J, Wells KD, Chen SH, Ball S, Specht SM, Polejaeva IA, Monahan JA, Jobst PM, Sharma SB, Lamborn AE, Garst AS, Moore M, Demetris AJ, Rudert WA, Bottino R, Bertera S, Trucco M, Starzl TE, Dai Y, Ayares DL (2003): Production of 1,3-galactosyltransferase-deficient pigs. Science 299:411-414 https://doi.org/10.1126/science.1078942
  27. Prather RS, Hawley RJ, Carter DB, Lai L, Greenstein JL (2003): Transgenic swine for biomedicine and agriculture. Theriogenology 59:115-123 https://doi.org/10.1016/S0093-691X(02)01263-3
  28. Qiao S, Nagasaka T, Harada T, Nakashima N (1998): p53, Bax and Bcl-2 expression, and apoptosis in gestational trophoblast of complete hydatidiform mole. Placenta 19:361-369
  29. Viet Linh N, Dang-Nquyen TQ, Nquyen BX, Manabe N, Nagai T (2009): Effects of cysteine during in vitro maturation of porcine oocytes under low oxygen tension on their subsequent in vitro fertilization and development. J Reprod Dev Epub ahead of print https://doi.org/10.1262/jrd.09-075H
  30. Xiao D, Zhang L (2008): Upregulation of Bax and Bcl-2 following prenatal cocaine exposure induces apoptosis in fetal rat brain. Int J Med Sci 5:295-302
  31. Wang WH, Abeydeera LR, Han U-M, Prather RS, Day BN (1999): Morphologic evaluation and actin filament distribution in porcine embryos produced in vitro and in vivo. Biol Reprod 60:1020-1028 https://doi.org/10.1095/biolreprod60.4.1020
  32. Yin XM, Oltvai ZN, Veis-Novack DJ, Linette GP, Korsmeyer SJ (1994): Bcl-2 gene family and the regulation of programmed cell death. Cold Spring Harb Symp Quant Biol 59:387-393
  33. Zakhartchenko V, Mueller S, Alberio R, Schernthaner W, Stojkovic M, Wenigerkind H, Wanke R, Lassnig C, Mueller M, Wolf E, Brem G. (2001): Nuclear transfer in cattle with non-transfected and transfected fetal or cloned transgenic fetal and postnatal fibroblasts. Mol Reprod Dev 60:362-369 https://doi.org/10.1002/mrd.1098
  34. Zhao J, Ross JW, Hao Y, Spate LD, Walters EM, Samuel MS, Rieke A, Murphy CN, Prather RS (2009): Significant improvement in cloning efficiency of an inbred miniature pig by histone deacetylase inhibitor treatment after somatic cell nuclear transfer. Biol Reprod 81:525-530 https://doi.org/10.1095/biolreprod.109.077016