DOI QR코드

DOI QR Code

Targeted Suppression of Connexin 43 in Ovine Preimplantation Embryos by RNA Interference Using Long Double-stranded RNA

  • Yan, Zhen (Key Laboratory of Ministry of Education of China for Mammal Reproduction Biology and Biotechnology of Inner Mongolia University) ;
  • Ma, Yu Zhen (Key Laboratory of Ministry of Education of China for Mammal Reproduction Biology and Biotechnology of Inner Mongolia University) ;
  • Liu, Dong jun (Key Laboratory of Ministry of Education of China for Mammal Reproduction Biology and Biotechnology of Inner Mongolia University) ;
  • Cang, Ming (Key Laboratory of Ministry of Education of China for Mammal Reproduction Biology and Biotechnology of Inner Mongolia University) ;
  • Wang, Rui (Key Laboratory of Ministry of Education of China for Mammal Reproduction Biology and Biotechnology of Inner Mongolia University) ;
  • Bao, Shorgan (Key Laboratory of Ministry of Education of China for Mammal Reproduction Biology and Biotechnology of Inner Mongolia University)
  • Received : 2009.05.13
  • Accepted : 2009.09.29
  • Published : 2010.04.01

Abstract

RNA interference (RNAi) is an acknowledged useful and effective tool to study gene function in various cells. Here, we suppressed the Connexin 43 (Cx 43) gene expression during in vitro development of ovine pre-implantation embryos using the RNAi method. The 353 bp Cx 43 double-stranded RNA was microinjected into in vitro fertilized ovine zygotes, and the levels of target mRNA and protein were investigated. Control groups included uninjected zygotes or those injected with RNase-free water. The dsRNA injection resulted in the specific reduction of Cx 43 transcripts as analyzed by quantitative real-time RT-PCR and decreased protein levels as shown by Western blot analysis at the blastocyst stage. Microinjection of Cx 43 dsRNA led to 20.3%, 21.7% and 34.5% blastocyst rates and 19.2%, 37.5% and 41.3% hatched blastocyst rates in Cx 43 dsRNA-injected, water-injected and uninjected groups, respectively. Then the RNAi could not significantly affect cell number and cell death rates of blastocysts. Therefore, suppression of Cx 43 dsRNA and proteins did not apparently affect the development potential of ovine pre-implantation embryos but may play a role in embryo quality. RNAi technology is a promising approach to study gene function in early ovine embryogenesis.

Keywords

Connexin 43;RNA Interference;Ovine Embryo

Acknowledgement

Supported by : National Natural Science Foundation of China, Natural Science Foundation of Inner Mongolia of China

References

  1. Betteridge, K. J., C. Smith, R. B. Stubbings, K. P. Xu and W. A. King. 1989. Potential genetic improvement of cattle by fertilization of fetal oocytes in vitro. J. Reprod. Fertil. Suppl. 38:87-98
  2. Bevilacqua, A., R. Loch-Caruso and R. P. Erickson. 1989. Abnormal development and dye coupling produced by antisense RNA to gap junction protein in mouse preimplantation embryos. Proc. Natl. Acad. Sci. USA. 86:5444-5448 https://doi.org/10.1073/pnas.86.14.5444
  3. Cabot, R. A., M. Hannink and R. S. Prather. 2002. CRM1-mediated nuclear export is present during porcine embryogenesis, but is not required for early cleavage. Biol. Reprod. 67:814-819 https://doi.org/10.1095/biolreprod.102.004960
  4. Cabot, R. A. and R. S. Prather. 2003. Cleavage stage porcine embryos may have differing developmental requirements for karyopherins alpha2 and alpha3. Mol. Reprod. Dev. 64:292-301 https://doi.org/10.1002/mrd.10238
  5. Enright, B. P., P. Lonergan, A. Dinnyes, T. Fair, F. A. Ward, X. Yang and M. P. Boland. 2000. Culture of in vitro produced bovine zygotes in vitro vs. in vivo: implications for early embryo development and quality. Theriogenology 54:659-673 https://doi.org/10.1016/S0093-691X(00)00381-2
  6. Fire, A., S. Xu, M. K. Montgomery, S. A. Kostas, S. E. Driver and C. C. Mello. 1998. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806-811 https://doi.org/10.1038/35888
  7. Ghassemifar, M. R., J. J. Eckert, F. D. Houghton, H. M. Picton, H. J. Leese and T. P. Fleming. 2003. Gene expression regulating epithelial intercellular junction biogenesis during human blastocyst development in vitro. Mol. Hum. Reprod. 9:245-252 https://doi.org/10.1093/molehr/gag033
  8. MacIntyre, D. M., H. C. Lim, K. Ryan, S. Kimmins, J. A. Small and L. A. MacLaren. 2002. Implantation-associated changes in bovine uterine expression of integrins and extracellular matrix. Biol. Reprod. 66:1430-1436 https://doi.org/10.1095/biolreprod66.5.1430
  9. Reaume, A. G., P. A. de Sousa, S. Kulkarni, B. L. Langille, D. Zhu, T. C. Davies, S. C. Juneja, G. M. Kidder and J. Rossant. 1995. Cardiac malformation in neonatal mice lacking connexin43. Science 267:1831-1834 https://doi.org/10.1126/science.7892609
  10. Rizos, D., P. Lonergan, M. P. Boland, R. Arroyo-Garcia, B. Pintado, J. de la Fuente and A. Gutierrez-Adan. 2002a. Analysis of differential messenger RNA expression between bovine blastocysts produced in different culture systems: implications for blastocyst quality. Biol. Reprod. 66:589-595 https://doi.org/10.1095/biolreprod66.3.589
  11. Rizos, D., F. Ward, P. Duffy, M. P. Boland and P. Lonergan. 2002b. 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. 61:234-248 https://doi.org/10.1002/mrd.1153
  12. Wrenzycki, C., D. Herrmann, J. W. Carnwath and H. Niemann. 1996. Expression of the gap junction gene connexin43 (Cx43) in preimplantation bovine embryos derived in vitro or in vivo. J. Reprod. Fertil. 108:17-24 https://doi.org/10.1530/jrf.0.1080017
  13. Wrenzycki, C., D. Herrmann, J. W. Carnwath and H. Niemann. 1999. Alterations in the relative abundance of gene transcripts in preimplantation bovine embryos cultured in medium supplemented with either serum or PVA. Mol. Reprod. Dev. 53:8-18 https://doi.org/10.1002/(SICI)1098-2795(199905)53:1<8::AID-MRD2>3.0.CO;2-K
  14. Wrenzycki, C., D. Herrmann, L. Keskintepe, A. Martins, Jr., S. Sirisathien, B. Brackett and H. Niemann. 2001. Effects of culture system and protein supplementation on mRNA expression in pre-implantation bovine embryos. Hum. Reprod. 16:893-901 https://doi.org/10.1093/humrep/16.5.893
  15. Wianny, F. and M. Zernicka-Goetz. 2000. Specific interference with gene function by double-stranded RNA in early mouse development. Nat. Cell Biol. 2:70-75 https://doi.org/10.1038/35000016
  16. Hardy, K. 1997. Cell death in the mammalian blastocyst. Mol. Hum. Reprod. 3:919-925 https://doi.org/10.1093/molehr/3.10.919
  17. Livak, K. J. and T. D. Schmittgen. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402-408 https://doi.org/10.1006/meth.2001.1262
  18. Tesfaye, D., P. Lonergan, M. Hoelker, F. Rings, K. Nganvongpanit, V. Havlicek, U. Besenfelder, D. Jennen, E. Tholen and K. Schellander. 2007. Suppression of connexin 43 and E-cadherin transcripts in in vitro derived bovine embryos following culture in vitro or in vivo in the homologous bovine oviduct. Mol. Reprod. Dev. 74:978-988 https://doi.org/10.1002/mrd.20678
  19. Lee, S., N. B. Gilula and A. E. Warner. 1987. Gap junctional communication and compaction during preimplantation stages of mouse development. Cell 51:851-860 https://doi.org/10.1016/0092-8674(87)90108-5
  20. Larue, L., M. Ohsugi, J. Hirchenhain and R. Kemler. 1994. Ecadherin null mutant embryos fail to form a trophectoderm epithelium. Proc. Natl. Acad Sci. USA. 91:8263-8267 https://doi.org/10.1073/pnas.91.17.8263
  21. Paradis, F., C. Vigneault, C. Robert and M. A. Sirard. 2005. RNA interference as a tool to study gene function in bovine oocytes. Mol. Reprod. Dev. 70:111-121 https://doi.org/10.1002/mrd.20193
  22. Schmittgen, T. D., B. A. Zakrajsek, A. G. Mills, V. Gorn, M. J. Singer and M. W. Reed. 2000. Quantitative reverse transcription-polymerase chain reaction to study mRNA decay: comparison of endpoint and real-time methods: Anal. Biochem. 285:194-204 https://doi.org/10.1006/abio.2000.4753
  23. Stein, P., F. Zeng, H. Pan and R. M. Schultz. 2005. Absence of non-specific effects of RNA interference triggered by long 471
  24. Ewart, J. L., M. F. Cohen, R. A. Meyer, G. Y. Huang, A. Wessels, R. G. Gourdie, A. J. Chin, S. M. Park, B. O. Lazatin, S. Villabon and C. W. Lo. 1997. Heart and neural tube defects in transgenic mice overexpressing the Cx43 gap junction gene. Development 124:1281-1292
  25. Hu, X., S. Hipolito, R. Lynn, V. Abraham, S. Ramos and F. Wong-Staal. 2004. Relative gene-silencing efficiencies of small interfering RNAs targeting sense and antisense transcripts from the same genetic locus. Nucleic Acids Res. 32:4609-4617 https://doi.org/10.1093/nar/gkh790
  26. Lonergan, P., D. Rizos, A. Gutierrez-Adan, P. M. Moreira, B. Pintado, J. de la Fuente and M. P. Boland. 2003. 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. 69:1424-1431 https://doi.org/10.1095/biolreprod.103.018168
  27. Riethmacher, D., V. Brinkmann and C. Birchmeier. 1995. A targeted mutation in the mouse E-cadherin gene results in defective preimplantation development. Proc. Natl. Acad Sci. USA. 92:855-859 https://doi.org/10.1073/pnas.92.3.855
  28. Lykke-Andersen, K., M. J. Gilchrist, J. B. Grabarek, P. Das, E. Miska and M. Zernicka-Goetz. 2008. Maternal Argonaute 2 is essential for early mouse development at the maternal-zygotic transition. Mol. Biol. Cell 19:4383-4392 https://doi.org/10.1091/mbc.E08-02-0219
  29. Adona, P. R., P. R. Pires, M. D. Quetglas, K. R. Schwarz and C. L. Leal. 2008. Nuclear maturation kinetics and in vitro embryo development of cattle oocytes prematured with butyrolactone I combined or not combined with roscovitine. Anim. Reprod. Sci. 104:389-397 https://doi.org/10.1016/j.anireprosci.2007.06.013
  30. Svoboda, P., P. Stein, H. Hayashi and R. M. Schultz. 2000. Selective reduction of dormant maternal mRNAs in mouse oocytes by RNA interference: Development 127:4147-4156
  31. Nganvongpanit, K., H. Muller, F. Rings, M. Gilles, D. Jennen, M. Holker, E. Tholen, K. Schellander and D. Tesfaye. 2006. Targeted suppression of E-cadherin gene expression in bovine preimplantation embryo by RNA interference technology using double-stranded RNA. Mol. Reprod. Dev. 73:153-163 https://doi.org/10.1002/mrd.20406
  32. Winer, J., C. K. Jung, I. Shackel and P. M. Williams. 1999. Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro. Anal. Biochem. 270:41-49 https://doi.org/10.1006/abio.1999.4085
  33. Rizos, D., A. Gutierrez-Adan, S. Perez-Garnelo, J. De La Fuente, M. P. Boland and P. Lonergan. 2003. Bovine embryo culture in the presence or absence of serum: implications for blastocyst development, cryotolerance, and messenger RNA expression. Biol. Reprod. 68:236-243
  34. Prather, R. S. and N. L. First. 1993. Cell-to-cell coupling in earlystage bovine embryos: A preliminary report. Theriogenology 39:561-567 https://doi.org/10.1016/0093-691X(93)90243-X
  35. Wang, S., Y. Liu, G. R. Holyoak, R. C. Evans and T. D. Bunch. 1998. A protocol for in vitro maturation and fertilization of sheep oocytes. Small Rumin. Res. 29:83-88 https://doi.org/10.1016/S0921-4488(97)00098-9

Cited by

  1. Production of small RNAs by mammalian Dicer vol.468, pp.6, 2016, https://doi.org/10.1007/s00424-016-1817-6