mRNA expression of Bcl-2, Bax, caspase-3 and -7 cannot be used as a marker for apoptosis in bovine blastocysts.
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.
Vandaele
L
L
Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, Merelbeke, Belgium. leen.vandaele@ugent.be
Goossens
K
K
Peelman
L
L
Van Soom
A
A
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics*; D001755:Blastocyst / Q000378:metabolism*; D053148:Caspase 3 / Q000235:genetics* / Q000378:metabolism; D053179:Caspase 7 / Q000235:genetics* / Q000378:metabolism; D002417:Cattle; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D019254:Genes, bcl-2; D005819:Genetic Markers; D005821:Genetic Techniques; D012333:RNA, Messenger / Q000378:metabolism; D015203:Reproducibility of Results; D019311:Staurosporine / Q000494:pharmacology; D051028:bcl-2-Associated X Protein / Q000235:genetics* / Q000378:metabolism
D004791:Enzyme Inhibitors; D005819:Genetic Markers; D012333:RNA, Messenger; D051028:bcl-2-Associated X Protein; D053148:Caspase 3; D053179:Caspase 7; D019311:Staurosporine