Inhibitory effect of retinoic acid on the development of immature porcine granulosa cells to mature cells

The present study investigated the effect of retinoic acid (RA) on the diff erentiation of granulosa cells prepared from porcine ovaries. The granulosa cells were precultured for 15 h, then cultured for 48 h with FSH and furth er treated for 24 h with LH in order to induce their transformation into lu teal cells. After the cells had been exposed to 1 mu M retinoids (RA, retin al and retinol) for 87 h, analysis of the LH receptor mRNA expression, an i ndicator of granulosa cell differentiation, was carried out by using semiqu antitative RT-PCR. The results showed that there was a decrease in LH recep tor mRNA levels, and that RA had a more potent effect on these levels than the other two retinoids. When cells were exposed to RA in the immature stag e (before the addition of FSH) or the early stage of development (0-24 h af ter the addition of FSH), expression of LH receptor mRNA was greatly dimini shed. When the immature cells were cultured for 15 h with RA, then washed a nd cultured for 48 h with FSH and for 24 h with LH, the expression of LH re ceptor mRNA was not reversed. In the differentiated cells (24 h after the a ddition of FSH), however, RA no longer had any inhibitory effect. When the immature cells were exposed to IA, FSH-induced expression of c-fos mRNA was markedly decreased. In contrast, expression of c-jun and activating transc ription factor-4 mRNAs remained constant. However, the expression of c-fos mRNA was not decreased by forskolin. The results indicate that RA is a pote nt inhibitor in the immature stage of porcine granulosa cell differentiatio n, probably through decreased expression of FSH receptor, but that RA does not inhibit differentiation in the mature stage of the cells.