Effect of 6-dimethylaminopurine on electrically activated in vitro matured porcine oocytes.
The effect of the protein kinase inhibitor, 6-dimethylaminopurine (6-DMAP), on the maturation promoting factor (MPF) activity, pronuclear formation, and parthenogenetic development of electrically activated in vitro matured (IVM) porcine oocytes was investigated. Oocytes were activated by exposure to two DC pulses, each of 1.5 kV/cm field strength and 60 microsec duration, applied 1 sec apart. In the first experiment, subsequent incubation with 2 or 5 mM 6-DMAP for 3 hr increased the incidence of blastocyst formation compared with no treatment, whereas incubation with 2 or 5 mM 6-DMAP for 5 hr did not. In the proceeding experiments, oocytes exposed to 6-DMAP were incubated with 2 mM of the reagent for 3 hr. Assaying histone H1 kinase activity in the second experiment revealed that the levels of active MPF in electrically activated oocytes treated with 6-DMAP were depleted more rapidly and remained depleted for longer compared with electrical activation alone. The kinetics of MPF activity following 6-DMAP treatment were similar to that found in inseminated oocytes in the third experiment. The effect of 6-DMAP was correlated with an increased incidence of parthenogenetic blastocyst formation. A fourth experiment was undertaken to examine the diploidizing effect of 6-DMAP. Electrically activated oocytes treated with 6-DMAP and cytochalasin B, either alone or in combination, displayed a higher incidence of second polar body retention compared with those that were untreated or treated with cycloheximide alone. After 6 days of culture in vitro, parthenotes exposed to 6-DMAP, either alone or in combination with cytochalasin B, formed blastocysts at a greater rate compared with those exposed to cytochalasin B alone, cycloheximide alone or no treatment. The combined 6-DMAP and cytochalasin B treatment induced the highest rate of blastocyst formation (47%), but the numbers of trophectoderm and total cells in these blastocysts were lower compared with those obtained following exposure to 6-DMAP alone. These results suggest that the increased developmental potential of 6-DMAP-treated parthenotes may be attributable to the MPF-inactivating effect of 6-DMAP, rather than the diploidizing effect of 6-DMAP.
Grupen
Christopher G
CG
Reproductive Biotechnology Division, BresaGen Limited, Adelaide, South Australia, Australia. chris.grupen@adelaide.edu.au
Mau
James C
JC
McIlfatrick
Stephen M
SM
Maddocks
Simon
S
Nottle
Mark B
MB
D000225:Adenine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D000818:Animals; D001755:Blastocyst / Q000378:metabolism; D004558:Electric Stimulation; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D066298:In Vitro Techniques; D009028:Morula / Q000378:metabolism; D009865:Oocytes / Q000187:drug effects* / Q000378:metabolism; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases; D013552:Swine
D004791:Enzyme Inhibitors; D047428:Protein Kinase Inhibitors; C001020:N(6),N(6)-dimethyladenine; D011494:Protein Kinases; C102333:histone H1 kinase; D000225:Adenine