Transcriptional regulation of the ovine follicle-stimulating hormone-β gene by activin and gonadotropin-releasing hormone (GnRH):: Involvement of two proximal activator protein-1 sites for GnRH stimulation

Previous studies from our laboratory demonstrated that a transgene consisting of the promoter for the ovine FSH P-subunit gene and a luciferase reporter (wt-oFSH beta Luc) was expressed and regulated like the FSH beta gene in vivo and in vitro. In the present study pituitary cultures were prepared from these transgenic mice as well as mice carrying mutated oFSH beta Luc lacking two functional activator protein-1 (AP-1) sites at -120 and -83 bp (mut-oFSH beta Luc). These AP-1 sites were reported necessary for induction of oFSH beta Luc by GnRH in a HeLa cell system. To examine the importance of the two AP-1 sites in mediating GnRH and activin effects in primary gonadotropes, pituitary cultures derived from transgenic mice were pretreated with follistatin to remove activin or activin-like factors present in the cultures. Follistatin lowered luciferase expression in cultures carrying both wt-oFSH beta Luc and mut-oFSH beta Luc transgenes by 74-86%, and subsequent addition of activin induced luciferase expression of both wt- and mut-transgenes by 4- to 14-fold within 4 h, suggesting that these AP-1 sites are not involved in activin stimulation of FSH beta gene transcription. When GnRH was added along with activin, the wt-oFSH beta Luc transgene was induced 200% compared with activin alone, but this effect was not observed with the mut-oFSH beta Luc transgene. These data confirmed the HeLa cell studies showing that GnRH signals through two AP-1 sites to increase oFSH beta transcription in gonadotropes. However, as the mutation of both AP-1 sites had no apparent effect on the expression and regulation of the transgene in vivo (basal, castration, GnRH down-regulation, cycle stage, and GnRH immunoneutralization), it appears that these AP-1 sites have little influence over the major effect of GnRH observed in vivo. These data also showed that activin plays a major role in transcriptional regulation of the FSH beta gene, and the oFSH beta promoter contains the activin response element(s) that is as yet undefined.