Changes in MPF and MAPK activities in porcine oocytes activated by different methods.
The effect of different oocyte activation methods on the dynamics of M-phase promoting factor (MPF) and mitogen-activated protein kinase (MAPK) activity in porcine oocytes were examined. Three activativation methods were tested: (1) electroporation (EP); (2) electroporation combined with butyrolactone I (BL), an inhibitor of cdc2 and cdk2 kinases; (3) electroporation followed by a treatment with cycloheximide (CHX), a protein synthesis blocker. The activity of cdc2 in MII oocytes was 0.067+/-0.011pmol/oocyte/min (mean+/-S.E.M.), which by 1h decreased in every treatment group (P<0.05) and stayed at low levels until 6h post-activation, approximately the time of pronuclear formation. The initial MAPK activity (0.123+/-0.017pmol/oocyte/min) also decreased 1h after each type of activation treatment (P<0.005). However, in the electroporation only group, activity reached its lowest level at 3h; thereafter, it started to recover and at later time points, MAPK activity did not differ from that in non-treated oocytes (P>0.1). In contrast, oocytes where electroporation was followed by protein kinase or protein synthesis inhibition had low MAPK activity by the time pronuclei were to be formed. Pronuclear formation in these groups (86.3+/-3.3% for EP+BL and 87.6+/-3.7% for EP+CHX) was higher compared to that found in the EP-only oocytes (69.4+/-3.3%; P<0.05). These findings demonstrated that electroporation alone efficiently triggered the inactivation of MPF but not that of MAPK. In order to achieve low MAPK activity to allow high frequency of pronuclear formation, electroporation should be followed by a treatment that inhibits protein synthesis or specific protein kinases. The combined activation methods provided stimuli that efficiently induced both MPF and MAPK inactivation and triggered pronuclear formation with high frequencies.
Nanassy
L
L
Department of Animal Breeding Science, Center of Agricultural Sciences, University of Debrecen, Böszörményi Street 138, Debrecen, Hungary.
Lee
K
K
Javor
A
A
Machaty
Z
Z
D015107:4-Butyrolactone / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D003513:Cycloheximide / Q000494:pharmacology; D018274:Electroporation; D005260:Female; D016200:Maturation-Promoting Factor / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009865:Oocytes / Q000187:drug effects / Q000201:enzymology*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D013552:Swine / Q000378:metabolism*
D047428:Protein Kinase Inhibitors; D011500:Protein Synthesis Inhibitors; C082868:butyrolactone I; D003513:Cycloheximide; D016200:Maturation-Promoting Factor; D020928:Mitogen-Activated Protein Kinases; D015107:4-Butyrolactone