Effects of metabolic hormones and growth factors on forskolin and dibutyryl adenosine 3′,5′-cyclic monophosphate induced steroidogenic responses by porcine granulosa cells in vitro

Abstract

The present experiments were conducted to examine the influence of metabolic hormones and growth factors on progesterone and estradiol secretion by porcine granulosa cells in culture and to determine the possible site(s) of action of these agents on steroidogenesis. Porcine granulosa cells, obtained from medium-sized (4–6 mm) nonatretic follicles of prepubertal gilts slaughtered at a commercial abattoir, were cultured at a density of 2 × 106 viable cells well−1 in serum-free Eagle's Minumum Essential Medium for 72 h with 0, 10 or 100 ng mL−1 of GH, insulin or IGF-I or IGF-II. At 72 h, each treatment group was divided into three subgroups, one subgroup remained untreated (basal) while either 25 μM FK or 3 mM dbcAMP was added to the other two subgroups. Additionally, androstenedione (10−7 M) was provided to all cultures as a substrate for estradiol biosynthesis. Media were collected at 96 h for determination of progesterone and estradiol content. Both IGF-I and IGF-II increased (P < 0.01) basal progesterone and estradiol secretion and enhanced the FK- and dbcAMP-induced stimulation of progesterone and estradiol secretion (P < 0.01). Insulin, at 100 ng mL−1, had a modest but significant positive effect on both FK- and dbcAMP-stimulated steroidogenic responses. In contrast, GH inhibited basal, FK- and dbcAMP-induced progesterone and estradiol production. Based on the stimulatory effects of both IGFs above those seen with dbcAMP alone and the inhibitory effects of GH in the face of dbcAMP stimulation, we conclude that their effects are likely mediated by non-cAMP dependent pathways. Key words: Steroidogenesis, granulosa cells, growth hormone, IGF, insulin, pigs