REGULATION OF FOLLISTATIN GENE-EXPRESSION IN THE OVARY AND IN PRIMARYCULTURES OF PORCINE GRANULOSA-CELLS

Experiments were designed to test the hypotheses that (1) follistatin gene expression in granulosa cells is regulated during follicular grow th, and (2) that alteration of follistatin mRNA concentration can be h ormonally induced in primary cultures of porcine granulosa cells. RNA isolated from granulosa cells from small (1-3 mm diameter), medium (3- 5 mm) and large (> 5 mm) follicles of prepubertal and postpubertal sow s was analysed by hybridization to a porcine follistatin cDNA probe. A mounts of follistatin mRNA increased with follicular diameter, but no differences in follicular follistatin mRNA were detected between prepu bertal and postpubertal sows. Treatment of cultured porcine granulosa cells with FSH or LH for 20 h stimulated follistatin mRNA concentratio n by a factor of two (100 ng FSH ml(-1)) and a factor of 1.5 (10 ng LH ml(-1)), respectively, over untreated controls. Treatment of cultured granulosa cells with 200 ng FSH ml(-1), 200 ng LH ml(-1), 10 mu mol d ibutyryl cAMP l(-1), 30 mu mol forskolin l(-1) and 100 ng cholera toxi n ml(-1) stimulated follistatin mRNA accumulation in granulosa cells b y factors of 4.9, 3.7, 1.6, 13.7 and 3.5, respectively, compared with control cultures. Stimulation of follistatin mRNA accumulation in cult ured,oranulosa cells by dibutyryl cAMP (30, 100 and 300 mu mol l(-1)) and forskolin (3, 10 and 100 mu mol l(-1)) was dose dependent. FSH and forskolin induced time-dependent increases in follistatin mRNA concen tration in cultured granulosa cells, with maximal induction occurring 72 h after treatment (a factor of 4.5 for FSH and 15.5 for forskolin). These results demonstrate that (1) increased follistatin mRNA in gran ulosa cells is associated with increased follicular diameter, (2) incr eased follistatin mRNA in cultured porcine granulosa cells can be indu ced by gonadotrophins, and (3) the gonadotrophin-induced increase in f ollistatin mRNA accumulation in cultured granulosa cells can be mimick ed by agents that increase intracellular cAMP concentrations.