Platelet-activating factor-stimulated production of reactive oxygen species in ovarian granulosa cells from periovulatory follicles.
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 F(2alpha) (8-isoPGF(2alpha)) 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-isoPGF(2alpha) 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-isoPGF(2alpha) 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.
Xu
Jinxian
J
Unit of Reproductive Biology, Research Institute for the Biology of Farm Animals, Dummerstorf, Germany.
Krüger
Burkhard
B
Vernunft
Andreas
A
Löhrke
Berthold
B
Viergutz
Torsten
T
D000818:Animals; D020013:Calcium Signaling / Q000187:drug effects; D002417:Cattle; D015232:Dinoprostone / Q000494:pharmacology; D005260:Female; D005456:Fluorescent Dyes / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D006107:Granulosa Cells / Q000187:drug effects / Q000378:metabolism*; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D010060:Ovulation / Q000187:drug effects / Q000502:physiology*; D010084:Oxidation-Reduction / Q000187:drug effects; D010972:Platelet Activating Factor / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*
D005456:Fluorescent Dyes; D010972:Platelet Activating Factor; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D015232:Dinoprostone