Recent studies have implicated insulin-like growth factor I (IGF-I) as an intraovarian regulator of follicular growth and differentiation. Therefore, we investigated the possibility that cattle selected for twin births may have increased concentrations of IGF-I within the ovarian follicle and(or) in peripheral blood. The estrous cycles of 14 cows with histories of producing twins and 12 control monotocous cows were synchronized with 35 mg of prostaglandin F2 alpha (PGF2 alpha). Blood and follicular fluid were collected 48-50 h post-administration of PGF2 alpha (follicular phase of the estrous cycle). Concentrations of IGF-I were measured by RIA after acid-ethanol treatment of serum or follicular fluid. Twin-producing cows had a greater (p less than 0.05) number of large (greater than or equal to 4 mm) follicles and 47% greater (p less than 0.05) concentrations of IGF-I in peripheral blood than control cows. Cattle selected for high twinning frequency also had greater (p less than 0.05) concentrations of IGF-I (+/- SE) in the two largest follicles than control (unselected) cows (327 +/- 28 vs. 243 +/- 29 ng/ml). IGF-I concentrations in pooled small (1-3.9 mm) follicles were less (p less than 0.05) than in large follicles but did not differ between control and twin-producing cattle. In addition, the percentage of IGF-I concentrations measured in follicular fluid to that of serum was lower (p less than 0.05) in small follicles than in large follicles, and was greater (p less than 0.05) in large follicles of control (93.2 +/- 5.3%) than twin-producing (76.2 +/- 4.4%) cattle.(ABSTRACT TRUNCATED AT 250 WORDS)
The observation that GH deficiency delays the onset of puberty has raised the question of the effect of GH on gonadal development. In addition, recent studies in the rat have indicated that GH is able to elevate ovarian levels of immunoreactive insulin-like growth factor I (iIGF-I) in vivo and enhance FSH-induced granulosa cell differentiation in vitro. To evaluate further the possibility of direct effects of GH on ovarian function, we have studied the action of GH on the secretion of iIGF-I and progesterone by cultured porcine granulosa cells from immature follicles. The effects of GH were compared with those of estradiol (E2) and FSH, hormones previously shown to stimulate steroidogenesis and iIGF-I production in this system. GH-stimulated cultures secreted 7.8 times as much iIGF-I per cell as control cultures, while cultures treated with E2 plus FSH secreted 4.5 times as much, and the combination of all three hormones produced an additional increment. The GH-dependent immunoreactivity was localized to two peaks on gel filtration which coeluted with authentic IGF-I and with an IGF-binding protein. In contrast to the results with iIGF-I secretion, GH was a relatively ineffective stimulator of progesterone secretion, resulting in levels 2.6 times the control value, compared to levels 7.4-fold the control value in cultures treated with E2 plus FSH. However, when the three agonists were combined, a synergistic interaction was observed which resulted in progesterone values 33.3 times the control value. In parallel studies, PRL was unable to mimic the effects of GH on iIGF-I or progesterone secretion. In summary, GH has direct stimulatory actions on porcine granulosa cells. Compared to E2 and FSH, established stimulators of these cells, GH is at least comparable in effectiveness with regard to iIGF-I secretion, but less effective as a stimulator of steroidogenesis. However, GH dramatically enhances the effects of E2 and FSH on progesterone secretion. These effects of GH could be important during the onset of puberty, when GH levels in plasma are elevated.
Previous studies have established the ovarian granulosa cell as a site of insulin-like growth factor-I (IGF-I) secretion and action, suggesting an autocrine function for this peptide in the ovary. To better understand how this putative autocrine system is regulated and its interface with the classic ovarian trophic hormones FSH, LH, and estradiol (E2), we have studied the effects of these hormones on the secretion of immunoreactive IGF-I (iIGF-I) by cultured porcine granulosa cells. Immature granulosa cells were cultured under serum-free conditions which were optimized to allow maximal iIGF-I production and hormonal responsivity. Measurements of iIGF-I were made after minimizing the influence of IGF-binding proteins by either acid gel filtration or reverse phase chromatography. Since the two preparative procedures gave roughly comparable results, the more expeditious reverse phase procedure was chosen for most samples. Cycloheximide virtually eliminated measurable iIGF-I in culture, suggesting that the peptide measured was newly synthesized, and degradation of IGF-I by cultured granulosa cells was negligible. Consequently, the medium levels provided an accurate indication of cellular secretion over the collection period. Under optimal culture conditions, iIGF-I was readily measurable and responsive to treatment with ovarian trophic hormones. The iIGF-I levels in several experiments with these hormones were as follows: FSH treatment, 1.58 +/- 0.21 times the control value (n = 5 experiments); E2 treatment, 1.26 +/- 0.12 times the control value (n = 5); E2 plus FSH, 3.12 X 0.31 times the control value (n = 8); LH, 1.33 +/- 0.12 times the control value (n = 3); LH plus FSH, 1.78 +/- 0.2 times the control value (n = 1). To assess the role of cAMP in the mediation of gonadotropin effects in this system, granulosa cells were treated with a phosphodiesterase inhibitor (methylisobutylxanthine), which resulted in iIGF-I levels 1.61 +/- 0.7 times the control level. In the presence of FSH, a further stimulatory effect was demonstrated (3.76 +/- 0.29 times control). In addition, the cAMP analog 8-bromo-cAMP dramatically increased iIGF-I levels (6.3 +/- 0.72 times control). These data provide the first demonstration that gonadal iIGF-I secretion can be stimulated by the principal hormones involved in trophic regulation of the ovary. As with other gonadotropin-dependent functions of granulosa cells, this effect appears to be mediated by cAMP and enhanced by E2. This interface between circulating hormones and autocrine systems could provide an important mechanism to amplify the effects of gonadotropic hormones on a local level.
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