Interactions among activity of glucose-6-phosphate dehydrogenase in immature oocytes, expression of apoptosis-related genes Bcl-2 and Bax, and developmental competence following IVP in cattle.
This study was conducted to understand whether the level of G6PDH activity assessed in immature bovine oocytes by means of BCB test was correlated with the level of expression of apoptosis-related genes such as Bcl-2 and Bax in immature and mature oocytes. This information should support previous findings suggesting that G6PDH activity is a useful marker for determining oocyte quality, thereby increasing the validity of BCB test in oocyte selection. Up to now, there are no data estimating the relation between G6PDH activity and the expression of apoptosis-related genes in oocytes. The expression of Bcl-2 and Bax genes was estimated on the mRNA and protein levels, respectively using real-time PCR and Western-blotting. To evaluate developmental competence of these oocytes, cumulus-oocyte complexes classified as BCB+ (low activity of G6PDH), BCB- (high activity of G6PDH) and Control were used for in vitro embryo production. In immature oocytes, the Bax transcript level in BCB- oocytes was significantly higher (P<0.001) in comparison to Control. In mature oocytes, the Bcl-2 transcript level was significantly lower in BCB+ oocytes (P<0.01) and in BCB- oocytes (P<0.05) in comparison to Control. However, no relation was found between the activity of G6PDH and the expression of the Bcl-2 or Bax proteins, both in immature and mature oocytes. Our results on the transcript level seem to indicate that oocytes subjected to BCB staining show tendency towards apoptosis. However, results obtained at the protein level did not confirm this conclusion. The usefulness of the BCB test as the indirect marker of apoptosis seems to be questionable. The lack of significant differences in the blastocyst rates developed from BCB+ and Control oocytes decreases the validity of BCB test in IVP technology.
Opiela
J
J
National Research Institute of Animal Production, Department of Biotechnology of Animal Reproduction, Krakowska 1 st, Balice/Kraków, 32-083 Balice, Poland. jopiela@izoo.krakow.pl
Katska-Ksiazkiewicz
L
L
Lipiński
D
D
Słomski
R
R
Bzowska
M
M
Ryńska
B
B
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D015153:Blotting, Western / Q000662:veterinary; D002417:Cattle / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D004396:Coloring Agents / Q000737:chemistry; D047108:Embryonic Development / Q000502:physiology; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary; D005786:Gene Expression Regulation; D019254:Genes, bcl-2; D005954:Glucosephosphate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D009865:Oocytes / Q000201:enzymology / Q000502:physiology*; D010053:Ovary / Q000201:enzymology / Q000502:physiology*; D010078:Oxazines / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000662:veterinary; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051028:bcl-2-Associated X Protein / Q000096:biosynthesis* / Q000235:genetics
D004396:Coloring Agents; D010078:Oxazines; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D051028:bcl-2-Associated X Protein; C006797:Brilliant Cresyl Blue; D005954:Glucosephosphate Dehydrogenase