BIOLOGY OF REPRODUCTION 46, 630-635 (1992) 630 Multifactorial Regulation of Prostaglandin Synthesis in Preovulatory Goldfish Ovarian Follicles1 RONALD G. KELLNER and GLEN VAN DER KRAAK2 Department of Zoology, University of Guelph, Guelph, Ontario, Canada NIG 2W1 ABSTRACT Goldfish preovulatory ovarian follicles (prior to germinal vesicle breakdown) were utilized for studies investigating the actions of activators of different signal transduction pathways on prostaglandin (P6) production. The protein kinase C (P1(C) activators phorbol 1 2-myristate 13-acetate (PMA; 100-400 nM), 1-oleoyl-2-acetylglycerol (5 and 25 g/ml), and 1,2-dioctanoylglycerol (10 and 50 g/ml) stimulated PGE production; the inactive phorbol 4s-phorbol didecanoate, which does not activate PKC, had no effect. Calcium ionophore A23 187 (0.25-4.0 saM) stimulated P611 production and acted in a synergistic manner with activators of PKC. Although produced in lower amounts than PGE, PGF was stimulated by PMA and A23 187. The direct activator of phos- pholipase A2, melittin (0.1-1.0 saM), stimulated a dose-related increase in PGE production, whereas chloroquine (100 PM), a putative inhibitor of phospholipase A,, blocked basal and PMA + A23 187-stimulated PGE production. Several drugs known to elevate intracellular levels of cAMP including the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.1-1.0 mM), for- skolin (10 .iM), and dibutyryl cAMP (dbcAMP; 5 mM) attenuate PMA + A23 187-stimulated PGE production. Meittin-stianulated production of P611 was inhibited by dbcAMP, suggesting that the action of cAMP was distal to the activation of phospholipase A2. In summary, these studies demonstrate that activation of PKC and elevation of intracellular calcium levels stimulate PG production, in part, through activation of phospholipase A2. The adenylate cyclase/cAMP signalling pathway is inhibitory to PG production by goldfish ovarian fofficles. INTRODUCTION The adenylate cyclase/cAMP and inositol trisphosphate/ diacylglycerol (1P3/DAG) signalling pathways participate in the regulation of ovarian eicosanoid production in mam- mals. LH and FSH, which are known activators of adenylate cyclase, as well as drugs that elevate intracellular cAMP lev- els (cholera toxin, dibutyryl cAMP [dbcAMP]) stimulate ovarian prostaglandin (PG) production [1-5]. Calcium ionophore A23 187 and either phorbol esters or synthetic diacylglycerols, which mimic the actions of 1P3 and DAG, respectively, also stimulate the production of ovarian PGs and prostacyclins in several mammalian species [5-9]. Although the actions of the adenylate cyclase/cAMP and 1P3/DAG pathways on the regulation of ovarian steroido- genesis [10-12] and ovulation [13] have been well defined in teleost species such as the goldfish, the factors control- ling ovarian PG production are poorly understood. Injec- tion of exogenous gonadotropins stimulate ovarian PGE and PGF production [14, 15], which supports the concept that gonadotropins mediate the periovulatory PG increase de- scribed in several species [15-19]. However, drugs that el- evate cAMP levels inhibit PGE2 and PGF2 production by postpartum follicles from the guppy [20]. On the basis of a preliminary report [21], there is evidence that the 1P3/DAG pathway exerts a stimulatory effect on PGE and PGF pro- duction by brook trout ovarian follicles. The present study investigates the putative role of different signal transduc- Accepted November 15, 1991. Received July 8, 1991. ‘This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada to G.V.D.I( ‘Correspondence. FAX: (519) 767-1656. tion pathways in the regulation of PGE and PGF production by preovulatory ovarian follicles from the goldfish. Animals MATERIALS AND METHODS Goldfish, common or comet variety, were purchased from Grassyforks Fisheries Co., Martinsville, IN. Fish were held in 1.2-m diameter circular tanks with flow-through water at 14-16#{176}Cunder a constant photoperiod (14L: 1OD). Fish were fed a commercial trout diet once a day to satiation. Follicle Incubations The protocol for follicle incubations followed proce- dures previously described for studies on the regulation of ovarian steroidogenesis [10,22]. Preovulatory fish were killed by spinal transection and ovaries were placed in modified Cortland’s saline [22]. Each experiment used full-grown ovarian follicles (0.9-1.1 mm in diameter) obtained from a single fish; the oocytes had not undergone final matura- tion as the germinal vesicle occupied a central position. Fully grown follicles were separated from smaller vitellogenic follicles under a dissecting microscope. Follicles were added in groups of 20 to each well of polystyrene tissue culture plates (Falcon 3047; Fisher Scientific Co., Toronto, ON, Can- ada). Immediately before addition of test compounds, the medium was replaced with fresh Cortland’s saline. Rou- tinely, follicles were incubated with test compounds in air for 4 h at 18#{176}C; in preliminary studies, follicles were in- cubated for various times up to 21 h. The incubation vol- ume was 1 ml. The medium was then removed and stored