mRNA expression of Bcl-2, Bax, caspase-3 and -7 cannot be used as a marker for apoptosis in bovine blastocysts

Anim Reprod Sci. 2008 Jun;106(1-2):168-73. doi: 10.1016/j.anireprosci.2007.12.016. Epub 2008 Jan 3.

Abstract

At present the most widely used technique for apoptosis detection in embryos remains the in situ visualization of DNA fragmentation by terminal deoxynucleotidyl transferase-dUTP nick end labelling (TUNEL) assay although this technique may be prone to artefacts. The aim of the present study was to investigate if the mRNA expression of a set of genes involved in apoptosis (Bax, Bcl-2, caspase-3 and -7) at an earlier point in the apoptotic cascade could be a good marker for apoptosis in in vitro produced bovine embryos. After normalization to the geometric mean of three reference genes, GAPD, YWHAZ and SDHA, mRNA expression levels of Bax, Bcl-2, caspase-3 and -7 were compared in embryos treated with an apoptosis inducer, staurosporine and in non-treated embryos. None of the genes were differently expressed in treated in comparison with non-treated embryos. In conclusion, mRNA expression of Bax, Bcl-2, caspase-3 and-7 cannot be used as a reliable apoptosis detection method. Immunofluorescent staining of caspase-3 and -7 is a better choice where as for Bcl-2 no reliable and practicable alternative is available at the moment.

Publication types

  • Comparative Study
  • Evaluation Study

MeSH terms

  • Animals
  • Apoptosis / drug effects
  • Apoptosis / genetics*
  • Blastocyst / metabolism*
  • Caspase 3 / genetics*
  • Caspase 3 / metabolism
  • Caspase 7 / genetics*
  • Caspase 7 / metabolism
  • Cattle*
  • Enzyme Inhibitors / pharmacology
  • Female
  • Gene Expression Regulation, Developmental / drug effects
  • Genes, bcl-2*
  • Genetic Markers
  • Genetic Techniques
  • RNA, Messenger / metabolism
  • Reproducibility of Results
  • Staurosporine / pharmacology
  • bcl-2-Associated X Protein / genetics*
  • bcl-2-Associated X Protein / metabolism

Substances

  • Enzyme Inhibitors
  • Genetic Markers
  • RNA, Messenger
  • bcl-2-Associated X Protein
  • Caspase 3
  • Caspase 7
  • Staurosporine