Platelet-activating factor-stimulated production of reactive oxygen species in ovarian granulosa cells from periovulatory follicles

Abstract

The platelet-activating factor (PAF) is a proinflammatory lipid present in the fluid of ovarian Graafian follicle. Ovarian blockage of the PAF receptor (PAFr) reduces ovulations in the rat whereas underlying mechanism is poorly understood. Mural granulosa cells (MGC) were mechanically isolated from the theca interna of bovine periovulatory follicle. The mRNA abundance for PAFr, progesterone receptor and cyclooxygenase-2 were measured by real-time PCR. Cytosolic calcium (Ca2+) concentration was assayed by microscopy using Fura-2 AM as indicator, 8-isoprostaglandin F2α (8-isoPGF2α) by an ELISA kit. Fluorescent products arising from intracellular oxidation of hydroethidine (HE) and dihydrorhodamine (DHR) were quantified by flow cytometry. The cells expressed PAFr mRNA and PAFr protein and responded to cPAF (nonhydrolyzable form of PAF) with a pulsating increase in Ca2+, demonstrating functional PAFr. Elevation of Ca2+ was reversed by WEB-2086, an inverse PAFr agonist. cPAF elevated the level of 8-isoPGF2α in the medium of MGC cultured with luteinizing hormone (LH). cPAF alone had no significant influence on the oxidation of HE and DHR, or 8-iso-PGF2α level. In MGC from vital periovulatory follicle, PAF and LH signaling plays an important role in regulating the production of excessive oxidants. Blockage of PAFr seems to interfere with these regulatory processes essential for ovulation. © 2009 International Society for Advancement of Cytometry.