FOLLICLE-STIMULATING-HORMONE (FSH) INCREASES FSH RECEPTOR MESSENGER-RIBONUCLEIC-ACID WHILE DECREASING FSH BINDING IN CULTURED PORCINE GRANULOSA-CELLS

The goal of the present studies was to compare direct effects of porcine FSH (pFSH) on [I-125]pFSH-binding sites with effects of pFSH on FSH receptor mRNA in cultured porcine granulosa cells. Cells from immature follicles were cultured on laminin-coated plates in serum-free medium for up to 6 days in the absence or presence of pFSH (1-100 ng/ml) or cholera toxin (0.04-400 ng/ml), which activates adenylyl cyclase independently of the FSH receptor. RRA indicated that [I-125] pFSH binding to cells cultured without stimulator increased more than 10-fold with time in culture. Addition of pFSH to cultures resulted in a dose-dependent decrease in binding, assessed after removal of bound PFSH. Equilibrium saturation binding analysis indicated that pFSH (10 ng/ml) caused a 39% decrease in binding sites in cells cultured for 6 days. At the same time, pFSH increased progesterone production 9.5-fold. Cholera toxin (4 ng/ml) increased [I-125]pFSH binding 110% and progesterone production 8.9-fold. Northern hybridization analysis of cultured granulosa cell mRNA using a porcine FSH receptor cDNA revealed three transcripts for the FSH receptor [2.2, 3.5, and 4.2 kilobases (kb)], with the major transcript being 4.2 kb in length. Addition of either pFSH (10 ng/ml) or cholera toxin (10 ng/ml) to cultures of granulosa cells increased the intensity of pFSH receptor transcripts compared with control values, with the 4.2-kb message remaining predominant. Hybridization with a porcine LH receptor cDNA revealed different transcripts (2.4, 4.0, 4.7, 7.0, and 11.0 kb), with the major transcript being 4.7 kb in length. Addition of either pFSH or cholera toxin to the cultures increased the intensity of all LH receptor transcripts; however, cholera toxin was more effective than pFSH. pFSH and cholera toxin increased the intensity of each species to different extents, although the 4.7-kb transcript remained predominant. These results indicate that exposure to FSH in culture results in down-regulation of the FSH receptor. Down-regulation is accompanied by increased FSH receptor mRNA levels, suggesting that FSH enhances FSH receptor synthesis.