Research Article

Nitric oxide synthase isoforms and the effect of their inhibition on meiotic maturation of porcine oocytes

Eva Chmelíková^a2, Michal Ješeta^a3, Markéta Sedmíková^a1 c1, Jaroslav Petr^a4, Lenka Tůmová^a2, Tomáš Kott^a4, Petra Lipovová^a5 and František Jílek^a2

^a1 Department of Veterinary Sciences, Czech University of Life Sciences in Prague, Kamýcká 129, 165 21 Prague 6 – Suchdol, Czech Republic.

^a2 Czech University of Life Sciences in Prague, Department of Veterinary Sciences, Kamýcká 129, 165 21 Prague 6 – Suchdol, Czech Republic.

^a3 Veterinary Research Institute, Hudcova 70, Brno, 621 00, Czech Republic.

^a4 Research Institute of Animal Production, Přátelství 815, 104 01 Prague 10 – Uhříněves, Czech Republic.

^a5 Institute of Chemical Technology, Department of Biochemistry and Microbiology, Technická 5, 166 28 Praha 6 Prague, Czech Republic.

Summary

In this paper we assessed: (i) the change in nitric oxide synthase (NOS) isoforms' expression and intracellular localization and in NOS mRNA in porcine oocytes during meiotic maturation; (ii) the effect of NOS inhibition by N^ω-nitro-l-arginine methyl ester (l-NAME) and aminoguanidine (AG) on meiotic maturation of cumulus–oocyte complexes (COC) as well as denuded oocytes (DO); and (iii) nitric oxide (NO) formation in COC. All three NOS isoforms (eNOS, iNOS and nNOS) and NOS mRNA (eNOS mRNA, iNOS mRNA and nNOS mRNA) were found in both porcine oocytes and their cumulus cells except for nNOS mRNA, which was not detected in the cumulus cells. NOS isoforms differed in their intracellular localization in the oocyte: while iNOS protein was dispersed in the oocyte cytoplasm, nNOS was localized in the oocyte cytoplasm and in germinal vesicles (GV) and eNOS was present in dots in the cytoplasm, GV and was associated with meiotic spindles. l-NAME inhibitor significantly suppressed metaphase (M)I to MII transition (5.0 mM experimental group: 34.9% MI, control group: 9.5% MI) and at the highest concentration (10.0 mM) also affected GV breakdown (GVBD); in contrast also AG inhibited primarily GVBD. The majority of the oocytes (10.0 mM experimental group: 60.8%, control group: 1.2%) was not able to resume meiosis. AG significantly inhibited GVBD in DO, but l-NAME had no significant effect on the GVBD of these cells. During meiotic maturation, NO is formed in COC and the NO formed by cumulus cells is necessary for the process of GVBD.

(Received June 10 2009)

(Accepted October 07 2009)

(Online publication January 29 2010)

Keywords:

• Cumulus cells;
• Meiotic maturation;
• NOS;
• Oocyte;
• Pig

Correspondence:

^c1 All correspondence to: Markéta Sedmíková. Department of Veterinary Sciences, Czech University of Life Sciences in Prague, Kamýcká 129, 165 21 Prague 6 – Suchdol, Czech Republic. Tel: +420 224382933. Fax: +420 234381841. e-mail: sedmikova@af.czu.cz