Evidence that PACAP and GnRH down-regulate follicle-stimulating hormone-β mRNA levels by stimulating follistatin gene expression: effects on folliculostellate cells, gonadotrophs and LβT2 gonadotroph cells

Abstract

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates α-subunit transcription and lengthens LH-β mRNA transcripts, but reduces FSH-β mRNA levels in rat pituitary cell cultures. PACAP also stimulates follistatin transcription, an effect which may explain the decrease in FSH-β mRNA. To begin to investigate the cells in which PACAP activates the follistatin gene, quantitative in situ hybridization for follistatin mRNA combined with immunostaining for LHβ and S100 protein was performed. In control cultures, follistatin mRNA was expressed in 70% of gonadotrophs and in 47% of folliculostellate cells (S-100+). PACAP increased ( P<0.001) both the number of follistatin-expressing cells as well as the number of grains per cell in both gonadotrophs and folliculostellate cells, while GnRH only affected ( P=0.01) gonadotrophs. Follistatin and FSH-β gene expression in rat pituitary cultures were also measured by competitive quantitative RT-PCR and northern analysis, respectively. Both PACAP and GnRH increased ( P<0.05) follistatin gene expression and suppressed ( P<0.05) FSH-β mRNA, and the effect of PACAP together with GnRH on follistatin exceeded that of GnRH alone. PACAP regulation of follistatin and FSH-β gene expression was studied further in LβT2 cells that were found to express receptors for the specific PACAP receptor, PAC 1. Follistatin mRNA was undetectable in cultures exposed to control media, or stimulated with PACAP, GnRH or rh-activin-A. In contrast to the results in primary pituitary cultures, PACAP increased FSH-β mRNA in these follistatin-deficient cells. Moreover, using transient transfection, PACAP stimulated transcription of ovine-FSH-β-luciferase. GnRH likewise increased FSH-β mRNA and stimulated FSH-β gene transcription in LβT2 cells. Activin-A increased FSH-β gene expression dose-dependently, and activin induction of FSH-β mRNA was blocked completely by 3-fold excess follistatin. These results indicate that PACAP stimulates follistatin gene expression in both gonadotrophs and folliculostellate cells, and provide further evidence that follistatin is required for PACAP or continuous GnRH to down-regulate FSH-β mRNA. These experiments suggest a mechanism by which PACAP influences FSH production selectively by an autocrine effect on gonadotrophs and by a paracrine mechanism through folliculostellate cells that involves follistatin.