title: The effect of protein kinase C activator and nitric oxide donor on oocyte activation and cortical granule exocytosis in porcine eggs.
abstract: Nitric oxide (NO) and protein kinase C (PKC) are involved in the activation of mammalian oocytes, although their role in the exit from the metaphase II stage and cortical granule (CG) exocytosis is still not fully understood. The aim of this study was to verify whether the NO-donor together with specific PKC-activators induce the complete activation of porcine oocytes assessed as meiosis resumption and a cortical reaction. Pig maturated oocytes were treated with the NO-donor S-nitroso-N-acetylpenicillamine (SNAP, 2 mM) or PKC-activators such as phorbol-12-myristate-13-acetate (PMA, 100 nM), 1-oleoyl-2-acetyl-sn-glycerol (OAG, 400 μM) and l-α-phosphatidylinositol-3,4,5-trisphosphate dipalmitoyl heptaammonium salt (DPAM, 2 μM). To study the combined effect of NO-donor and PKC-activators, aliquots of oocytes were also incubated with SNAP (0.5 mM) together with PKC-activators at the same concentration as above (SNAP-DPAM, SNAP-OAG and SNAP-PMA groups). After in                vitro maturation, an aliquot of oocytes was placed in a fresh medium without NO-donor or PKC-activators (Control group). Another aliquot of oocytes was activated by calcium ionophore A23187 (25 μM, 5 min). The results showed that 0% of the control oocytes reassumed meiosis. However, both the PKC-activators (DPAM 44.0 ± 10.0%, OAG 63.3 ± 1.0% and PMA 45.0 ± 16.5%) as well as the NO-donor alone (48.7 ± 21.0%) significantly induced exit from MII. Interestingly, the combination of PKC-activators and SNAP mainly restrained to the meiosis resumption (SNAP-OAG 0, SNAP-DPAM 17.4 ± 2.5% and SNAP-PMA 38.4 ± 8.5%). Control oocytes did not show a cortical reaction and the area occupied by CG reached 25.9 ± 1.7%, whereas CGs were partially released after Ca2+ ionophore treatment (13.0 ± 3.2%). Treatment with PKC-activators induced a cortical reaction compared with the control group (8.6 ± 2.5, 6.7 ± 1.9 and 0.7 ± 0.4%, respectively, for DPAM, OAG and PMA groups). However, treatment with the NO-donor alone (SNAP group 17.2 ± 2.2%) or combined with any PKC-activator prevented cortical reaction (SNAP-DPAM 20.7 ± 2.6%, SNAP-OAG 16.7 ± 2.9% or SNAP-PMA 20.0 ± 2.4%). Besides, meiosis resumption was not always accompanied by a cortical reaction, indicating that these two activation events are independent. In conclusion, PKC-activators alone induce CG exocytosis to the same degree as calcium ionophore. However, an NO-donor alone or combined with PKC-activators is not able to induce a cortical reaction in pig oocytes.
authors.affiliation: Department of Veterinary Sciences, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences in Prague, 165 21 Prague 6-Suchdol, Czech Republic. tumoval@af.czu.cz
authors.forename: L
authors.initials: L
authors.lastname: Tůmová
authors.forename: R
authors.initials: R
authors.lastname: Romar
authors.forename: J
authors.initials: J
authors.lastname: Petr
authors.forename: M
authors.initials: M
authors.lastname: Sedmíková
mesh_terms: D000818:Animals; D004075:Diglycerides / Q000494:pharmacology; D020536:Enzyme Activators / Q000494:pharmacology*; D005089:Exocytosis / Q000187:drug effects*; D005260:Female; D008540:Meiosis / Q000187:drug effects; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology*; D009865:Oocytes / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D018129:Phosphatidylinositol Phosphates / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D026423:S-Nitroso-N-Acetylpenicillamine / Q000494:pharmacology; D034421:Sus scrofa; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D004075:Diglycerides; D020536:Enzyme Activators; D020030:Nitric Oxide Donors; D018129:Phosphatidylinositol Phosphates; C060974:phosphatidylinositol 3,4,5-triphosphate; D009569:Nitric Oxide; D026423:S-Nitroso-N-Acetylpenicillamine; C038237:1-oleoyl-2-acetylglycerol; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate