Role and regulation of prostaglandin production in granulosa cell DNA synthesis during ovarian follicular development.

Abstract

The aim of this study was to examine the role and regulation of eicosanoids in the mitogenic actions of TGF$\alpha$ on granulosa cells during follicular development, as well as its involvement in the regulation of urokinase plasminogen activator, an enzyme playing an important role in tissue remodelling. Granulosa cells from the first (F1), fifth, and sixth (F5-6) largest preovulatory follicles were cultured in the presence of TGF$\alpha ,$ and/or TGF$\beta ,$ or TGF$\alpha$ together with inhibitors of phospholipase A$\sb2,$ cyclooxygenase or lipoxygenase, leukotrienes (LTs), lysophosphatidyl choline (LPC), lysophosphatidic acid (LPA) and/or prostaglandins (PGs). TGF$\alpha$ stimulated PG secretion in a concentration-dependent manner. This stimulation was suppressed concentration-dependently by hydroxyurea (1.5-6 mM). TGF$\alpha$-induced DNA synthesis in F1 and F5-6 granulosa cells was suppressed by inhibitors of phospholipase A$\sb2$ and cyclooxygenase, while an inhibitor of lipoxygenase was ineffective. The inhibition was concentration-dependent and could be attenuated by exogenous PGE$\sb2.$ Likewise, PGF and PGE production was suppressed by ONO-RS-82, ibuprofen, naproxen, and indomethacin. Moreover, PGE$\sb2$ and, to a lesser extent, PGF$\sb{2\alpha}$ increased basal ($\sp3$H) thymidine incorporation and enhanced DNA synthesis induced by a submaximal stimulatory concentration of TGF$\alpha .$ The mitogenic effect of PGs was more evident in granulosa cells from F5-6 than from the F1 follicle. In contrast, leukotrienes (+)5(s)-hydroxy-(6E, 8Z, 11Z, 14Z)-eicosatetraenoic acid and lysophospholipids had no effect on granulosa cell DNA synthesis. Cyclooxygenase (COX) and cytosolic phospholipase A$\sb2$ (cPLA$\sb2)$ protein and mRNA levels were also determined. The increase in PG secretion produced by TGF$\alpha$ was accompanied by an elevation of COX II content which was concentration- and time-dependent. Maximal increase in COX II mRNA abundance was evident at 4 and 8 h in cells from F1 and F5-6, respectively. While TGF$\alpha$-stimulated PG secretion was higher in cells from a mature follicle (F1), the magnitude of change in COX II mRNA abundance and protein content was not dependent on follicular maturation. TGF$\beta$ significantly suppressed basal and TGF$\alpha$-induced COX II transcript levels. COX I transcript, however, was undetectable. Treatment with TGF$\alpha$ caused a shift from an electrophoretically fast migrating protein to a slow form, a phenomenon sensitive to inhibitors of serine/threonine kinase as well as MAP kinase pathways. In contrast, TGF$\beta$ suppressed cPLA$\sb2$ expression, but failed to prevent the mobility shift of cPLA$\sb2$ induced by TGF$\alpha .$ The inhibition of cPLA$\sb2$ by TGF$\beta$ is more pronounced in granulosa cells at the early stage of follicular development. Mothers Against dpp (MAD) and its related proteins (MADR) are believed to be important components of the cell signalling pathway for the transforming growth factor beta (TGF$\beta )$ superfamily. We have examined the presence of MADR2 and MADR1 in granulosa cells at different stages of follicular development, and the influence of TGF$\beta$ in vitro on their expression. We have demonstrated the presence of MADR2 and MADR1 in hen granulosa cells at different stages of follicular development. The expression of MADR2, but not of MADR1, was up-regulated by TGF$\beta$ in vitro in a concentration- and time-dependent manner. Granulosa cell MADR2 expression was maximal during early stages of follicular development. The changes in MADR2 expression were accompanied by reciprocal alterations in the expression of cPLA$\sb2 .$ (Abstract shortened by UMI.)