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The Rat Ovarian Phospholipase A₂ System: Gene Expression, Cellular Localization, Activity Characterization, and Interleukin-1 Dependence¹

Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, University of Maryland School of Medicine, Baltimore, Maryland 21201

Address all correspondence and requests for reprints to: Dr. Eli Y. Adashi, Departments of Obstetrics, Gynecology, and Physiology, University of Maryland School of Medicine, 11–013 BRB, 655 West Baltimore Street, Baltimore, Maryland 21201. E-mail: eadashi{at}umabnet.ab.umd.edu

We have previously demonstrated that interleukin-1ß (IL-1ß), a putative intermediary in the ovulatory process, is a potent stimulator of ovarian PG biosynthesis. In this communication, we examine the possibility that this IL-1 effect reflects in part the induction of arachidonic acid mobilization by phospholipase A₂ (PLA₂). Molecular probing of whole ovarian material revealed the immature rat ovary to be a site of modest secretory PLA₂ (sPLA₂) gene expression. However, no change in ovarian sPLA₂ gene expression was noted during the periovulatory period. Comparable probing for cytosolic PLA₂ (cPLA₂) failed to disclose a quantifiable signal. However, in situ hybridization localized both sPLA₂ and cPLA₂ (sPLA₂ > cPLA₂) transcripts to the granulosa cell layer of the ovarian follicle. Treatment of cultured whole ovarian dispersates with IL-1ß produced significant (P < 0.01) increments in the steady state levels of transcripts corresponding to both sPLA₂ (1.7-fold increase) and cPLA₂ (5-fold increase), an effect reversed by an IL-1 receptor antagonist, suggesting mediation via a specific IL-1 receptor. Treatment with cycloheximide, a protein synthesis inhibitor, resulted in significant attenuation of the ability of IL-1ß to up-regulate sPLA₂ and cPLA₂ gene expression as well as medium PLA₂ activity. Treatment with aminoguanidine, an inhibitor of inducible nitric oxide synthase, led to augmentation of the ability of IL-1ß to up-regulate sPLA₂ and cPLA₂ gene expression as well as medium PLA₂ activity. Total cellular PLA₂ activity proved time, cell density, and calcium dependent, with an optimal pH of 8.0–9.0 and K_m values in the low micromolar range (2–5 µM). Our observations 1) establish the rat ovary as a site of sPLA₂ and cPLA₂ gene expression, 2) localize the corresponding transcripts to the granulosa cell layer, and 3) establish IL-1ß as an up-regulatory agent for ovarian sPLA₂ and cPLA₂ gene expression as well as for ovarian PLA₂ activity. These findings also indicate that the IL-1 effect is 1) receptor mediated, 2) contingent in part upon de novo protein biosynthesis, and 3) inhibited by nitric oxide. These observations support the proposition that PLA₂ may be a key component in the IL-1-stimulated biosynthesis of ovarian PGs.

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