Activation of Pig Oocytes using Calcium Ionophore: Effect of the Protein Kinase Inhibitor 6‐dimethyl aminopurine

Abstract

The aim of our study was to investigate the parthenogenetic activation of in vitro matured pig oocytes after their combined treatment with calcium ionophore A 23187 and the inhibitor of protein kinases, 6-dimethylaminopurine (6-DMAP) and to study the further embryonic development of oocytes activated using this treatment. The oocytes were exposed to ionophore (10, 25 or 50 microM) for 0.5, 1, 3, 5 or 7 min and then cultured with 6-DMAP (0 or 2 microM) The highest activation rate (up to 88% of the activated eggs reached the pronuclear stage) was observed after combined treatment of the oocytes with 50 microM ionophore and 6-DMAP. The highest rate of embryonic development was observed after treatment with 25 microM ionophore without 6-DMAP, when up to 51% of the eggs developed beyond two-cell stage, 2% of the eggs developed up to the stage of morula and up to 3% of the eggs reached the stage of blastocyst. When 50 microM ionophore was used, the embryonic development of the activated eggs was arrested before the morula and blastocyst stage. After treatment of the activated eggs with 6-DMAP, we did not observe any development beyond the stage of 16 blastomeres. We can conclude that combined treatment with calcium ionophore A 23187 and 6-DMAP increases the activation rate in pig oocytes matured in vitro, but this combined treatment exerts a detrimental effect on further embryonic development of the activated eggs.

The role of nitric oxide in parthenogenetic activation of pig oocytes: A review

“…In this respect, the NO-dependent oocyte activation differs from activating protocols based on the elevation of intracellular levels of free calcium ions, which is successful even after a short-term treatment (several minutes) with calcium-enhancing drugs (e.g. Machatý et al, 1997;Jílek et al, 2000Jílek et al, , 2001. On the other hand, several hours of treatment are needed when the activation of pig oocytes is induced using cyclopiazonic acid, which elevates the intracellular calcium levels through the inhibition of calcium-dependent ATPases acting as intracellular sarco(endo)plasmic Ca 2+ .…”

An insufficient increase of cytosolic free calcium level results postovulatory aging-induced abortive spontaneous egg activation in rat

“…The inositol 1,4,5-triphosphate-sensitive stores are the primary source for an increase of cytosolic free Ca 2+ level during EA [3][4][5]. Calcium ionophore A23187 (CI) increases cytosolic free Ca 2+ level and induces EA in vitro [6][7][8][9].…”

Activation of protein kinase C suppresses fragmentation of pig oocytes aged in vitro

“…This capability was not significantly changed even when the oocytes were aged for 24 h in the presence of PKC activators. Parthenogenetic activation by standard activation protocol based on the combined effect of calcium ionophore and DMAP (Jílek et al, 2001) was similarly effective in oocytes aged under the effect of PMA and in oocytes aged in a PMAfree culture medium. The ratio of parthenogenetically activated oocytes was similar in oocytes aged 1 day with PMA and 'fresh' oocytes exposed to parthenogenetic activation immediately after finishing their maturation.…”

“…Oocyte activation and culture of embryos Oocytes were activated using the method described by Jílek et al (2001). Briefly, oocytes matured in vitro were denuded of their cumulus cells and were subjected to a 5-min treatment with 25 mM calcium ionophore A23185.…”