Insulin-like growth factor-I stimulates oxytocin and progesterone production by bovine granulosa cells in culture

The effect of insulin-like growth factor-I (IGF-I), epidermal growth factor (EGF), fibroblast growth factor (FGF) and nerve growth factor (NGF) on production of oxytocin and progesterone by cultured bovine granulosa and luteal cells was studied. Secretion of oxytocin was stimulated, in a dose-dependent manner, by IGF-I at 48 and 120 h of culture to levels much higher than those after stimulation with LH, FSH, EGF, FGF or NGF. A similar effect of IGF-I was observed for progesterone but, in contrast to oxytocin, secretion of progesterone was not increased by EGF, NGF or FGF. During primary culture, for 4 h, of dispersed bovine luteal cells obtained from corpora lutea between days 4 and 10 of the oestrous cycle, all the growth factors tested failed to stimulate secretion of oxytocin or progesterone. The data suggest the relevance of growth factors (especially IGF-I) for ovarian physiology and their possible importance for differentiation of follicles and luteinization.     

Regulation of insulin-like growth factor-I messenger ribonucleic acid expression in Leydig cells

In the present study, we evaluated insulin-like growth factor-I (IGF-I) messenger RNA expression in the rat testis. Crude interstitial cells were separated into three distinct bands on 15-60% Percoll density gradients. IGF-I mRNA was mainly localized in the Leydig cell-enriched fraction (band 3), while band 1 and band 2 cells did not contain significant amounts of IGF-I mRNA. Leydig cell IGF-I mRNA consisted of multiple species varying from 0.8 to 7.5 kb and was present in rat Leydig cells all ages examined, from 25 to 55 days old. To further document that IGF-I mRNAs are present in Leydig cells, the method of Klinefelter et al. (Biol. Reprod. (1987) 36, 769-783) was used to isolate highly purified (greater than 98% pure) Leydig cells. Most of the IGF-I mRNA was localized in these Leydig cells, while there was no detectable IGF-I mRNA in the whole testis or other interstitial cells. Furthermore, IGF-I mRNA in Leydig cells was increased more than 2-fold by growth hormone (GH) administration in vivo. This suggests that IGF-I mRNA in Leydig cells is also GH dependent. Interstitial IGF-I produced in Leydig cells may have both autocrine and paracrine effects in the testis.     

Thyroid hormone and growth hormone interact to regulate insulin-like growth factor-I messenger ribonucleic acid and circulating levels in the rat

Thyroid hormones influence growth in part by altering the secretion and effects of GH. GH, in turn, mediates its effects by regulating the synthesis and secretion of insulin-like growth factor-I (IGF-I). IGF-I is a pleiotropic growth factor that is synthesized by many tissues and acts on many tissues to regulate both cellular replication and differentiated function. We have studied the direct effects of thyroid hormones and the combined effects of thyroid hormones and GH on the regulation of IGF-I synthesis and secretion in hypophysectomized (hypox) rats in vivo. All rats, except normal littermates and a hypox control group, received 100 micrograms hydrocortisone/100 g BW for 10 days. Circulating IGF-I was measured by specific RIA (normal rats, 1 U/ml), and hepatic IGF-I mRNA was measured by Northern blot hybridization with an antisense cRNA probe. 1) Hypox rats treated with hGH (75 micrograms, ip, twice daily) for 10 days gained 17 g BW vs. 70 g for normal littermates. GH markedly increased hepatic IGF-I mRNA and circulating IGF-I (0.52 +/- 0.14 U/ml 12 h after the last GH injection vs. 0.03 +/- 0.02 for hypox controls). 2) T4 (1 micrograms/100 g BW, ip) for 10 days increased neither weight, hepatic IGF-I mRNA, nor circulating IGF-I. 3) Rats treated with T4 for 10 days followed by a single injection of 1 mg GH, ip, increased hepatic IGF-I mRNA and circulating IGF-I levels comparably as in rats receiving acute GH alone (IGF-I, 12 h, 0.31 +/- 0.09 vs. 0.36 +/- 0.06 U/ml). 4) Hypox rats treated with a single injection of T3 (1.5 micrograms/100 g BW, ip) had slightly increased hepatic IGF-I mRNA, but showed no significant change in circulating IGF-I levels. 5) A single injection of T3 plus GH to hypox rats increased IGF-I mRNA levels above those in rats injected with GH alone and increased serum IGF-I levels to 0.48 +/- 0.12 U/ml compared to 0.36 +/- 0.06 U/ml for GH alone. 6) After 10 days of GH treatment, a single injection of T3 lowered both hepatic IGF-I mRNA and circulating IGF-I (0.52 +/- 0.14 to 0.16 +/- 0.06 U/ml, 6 h after T3). These studies demonstrate that thyroid hormones have relatively little direct effect on IGF-I synthesis but can have major effects on GH-stimulated IGF-I synthesis and secretion. The pattern of these effects depends on the integrity of the pituitary gland, prior exposure of the liver to GH and/or thyroid hormones, and the temporal relationship between GH and thyroid hormone administration.     

Growth factors regulate immunoreactive insulin-like growth factor-I production by cultured porcine granulosa cells

The effects of various growth factors on the production of immunoreactive insulin-like growth factor I (iIGF-I) in short term (3-day) cultures of porcine granulosa cells was investigated. Epidermal growth factor (EGF) was shown to be a potent dose-dependent stimulator of iIGF-I production, achieving a 3.6-fold stimulation at a dose of 10 ng/ml. Transforming growth factor-alpha (10 ng EGF equivalents/ml) was also stimulatory. Platelet-derived growth factor (10 ng/ml) had no effect of its own, but enhanced EGF-stimulated iIGF-I production. The acidic and basic fibroblast growth factors (100 ng/ml) had no effect alone or in combination with EGF. Transforming growth factor-beta (10 ng/ml) had no effect of its own, but inhibited EGF-stimulated iIGF-I production. The interactive effects of EGF and FSH (200 ng/ml) on iIGF-I production were investigated in short term and longer term (7-day) cultures. In short term cultures under conditions optimized for EGF-dependent iIGF-I production, FSH had no effect of its own and inhibited EGF action. Conversely, in longer term cultures optimized for FSH-dependent iIGF-I production, EGF had no effect of its own and inhibited FSH action. Thus IGF production by cultured porcine granulosa cells is regulated in a complex manner and is highly dependent on the culture conditions. Our results suggest that IGF production in the ovary may also be regulated in a complex manner which is dependent on the developmental state of the follicle.     

Non-lactogenic effects of growth hormone on growth and insulin-like growth factor-I messenger ribonucleic acid of rat mammary gland

In contrast to established dogma that PRL is central in mammary development, and GH mimics PRL in affecting growth because of structural similarities, we found that both hGH, which is lactogenic, and rGH, which is non-lactogenic, were significantly more potent than hPRL and rPRL in stimulating mammary growth in rats. Additionally, hGH was more potent than hPRL in increasing mammary IGF-I mRNA content. These data indicate that GH has separate effects on parameters of mammary gland growth, suggesting an independent role for GH in mammary growth.     

Gonadotropins and estradiol stimulate immunoreactive insulin-like growth factor-I production by porcine granulosa cells in vitro

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.     

Regulation of insulin-like growth factor-binding protein messenger ribonucleic acid levels in sheep thyroid cells.

The insulin-like growth factors (IGFs) exist primarily bound to cell surface receptors or complexed to specific binding proteins (IGFBPs). The IGFBPs modulate the bioavailability of the IGFs and may enhance or inhibit IGF actions. Several distinct forms of IGFBPs have been described on the basis of size, immunological determinants, and distribution in biological fluids; the IGFBPs may differ as well in their biological function. Sheep thyroid cells produce IGFBPs under hormonal regulation. Cells grown in basal medium or with six-hormone (6H) medium supplements (transferrin, glycyl-histidyl-lysine, hydrocortisone, somatostatin, insulin, and TSH) release nonglycosylated BPs that migrate at 24, 27, 29, and 32 kDa on Western ligand blot. Cells cultured with the thyroid mitogens epidermal growth factor and phorbol ester release additional glycosylated IGFBPs of 40-44 kDa. Immunoprecipitation experiments indicate that 29- and 32-kDa IGFBPs are antigenically related to IGFBP-2, and the 40- to 44-kDa proteins are related to IGFBP-3. Using specific cDNA probes IGFBP-1, -2, and -3, we examined the regulation of IGFBP mRNA levels in sheep thyroid cultures. The rat IGFBP-2 cDNA probe hybridized to an approximately 1.6-kilobase mRNA species in cells under all culture conditions. However, IGFBP-3 mRNA was detectable only in epidermal growth factor- or phorbol ester-treated cells and appeared within 4 h, preceding the release of IGFBP-3 protein into the medium. The 6H additives, which stimulate differentiated function in thyroid cells, inhibited the mRNA levels of both IGFBP-2 and IGFBP-3. IGFBP-1 mRNA was not detectable. The distinct regulation of these IGFBPs suggest that they may play different biological roles in modulating thyroid physiology.     

Effects of gonadotropins, insulin and insulin-like growth factor I on ovarian oxytocin and progesterone production.

Oxytocin is produced in the granulosa-derived cells of the ruminant corpus luteum where its gene is dramatically up-regulated within days of ovulation. Regulation of these processes is poorly understood but oxytocin release can be increased by insulin, insulin-like growth factor I (IGF-I), and gonadotropins. Here we have assessed interactions between these regulatory systems. Follicle-stimulating hormone (FSH), luteinizing hormone (LH) and human chorionic gonadotropin (hCG) caused dose-dependent release of oxytocin from bovine granulosa cells cultured in medium containing 100 ng/ml insulin. The gonadotropins also increased oxytocin mRNA levels and their effects were mimicked by forskolin. The effects of these stimuli on oxytocin and progesterone release were synergistically increased by insulin or IGF-I. Binding studies revealed separate binding sites with characteristics of insulin and IGF-I receptors. Insulin potentiated the effects of hCG and forskolin on oxytocin mRNA levels and release of oxytocin and progesterone in cells from follicles containing greater than 50 ng/ml estradiol. In cells from follicles containing less than 5 ng/ml estradiol these stimuli had little effect on oxytocin release although progesterone release was synergistically increased by insulin and forskolin. The data suggest that gonadotropins regulate oxytocin synthesis and release and that these effects are amplified by insulin or IGF-I acting via their own receptors. Changes associated with maturation of the target cells in vitro appear prerequisite for oxytocin production in response to increased cAMP levels in the presence of insulin or IGF-I.     

Regulation of insulin-like growth factor I messenger ribonucleic acid levels by serum in cultured rat fibroblasts.

Fibroblasts represent one of the in vivo sites of extrahepatic insulin-like growth factor I (IGF-I) production. In this study, cultured fibroblasts prepared from the skin of neonatal rats were used as a model to assess the role of serum in regulating IGF-I messenger RNA (mRNA) levels. IGF-I mRNA, as demonstrated by Northern blot analysis, was present in the cultured fibroblasts, and serum free media which was conditioned by fibroblasts for 20 h contained 108 pg/ml of immunoreactive IGF-I. Fetal calf serum (FCS) decreased steady state IGF-I mRNA levels, as measured by solution hybridization/RNase protection assay, in fibroblasts in a time- and dose-dependent fashion. Incubation of fibroblasts for 18 h in the presence of 0.3%, 0.6%, or 1% FCS decreased IGF-I mRNA levels to 76%, 56%, and 46% of the levels present in control cells which were maintained in serum free media with 0.25% BSA. Maximal inhibition to approximately 20% of control levels was seen with 4-10% FCS. In contrast, basic fibroblast growth factor and beta-actin mRNA levels increased 2- and 4-fold, respectively, with increasing concentrations of FCS. Treatment of the cells with 10 micrograms/ml cycloheximide resulted in partial abrogation of the inhibitory effect of FCS while protein synthesis in the cells was decreased to 6% of control levels. The addition of 2 micrograms/ml of insulin or 15-100 ng/ml of IGF-I to the fibroblasts did not reproduce the inhibitory effect of FCS. Finally, the inhibitory factor(s) present in the FCS was partially removed/inactivated by charcoal stripping or heat inactivating the serum, but delipidation of the FCS by chloroform extraction had no effect on the inhibitory effect of FCS. In summary, FCS contains a factor(s) that decreases IGF-I mRNA levels in cultured fibroblasts in a time- and dose-dependent fashion. The partial abrogation of the inhibitory effect of FCS with cycloheximide treatment suggests that this effect is at least partially dependent upon new protein synthesis. Furthermore, the studies using delipidated, heat-inactivated, and charcoal-stripped serum suggest that the inhibitory factor(s) is a peptide.     

Estrus cycle-dependent co-variation of insulin-like growth factor-I (IGF-I) messenger ribonucleic acid and protein in the rat ovary

It has been suggested that insulin-like growth factor-I (IGF-I) exerts paracrine or autocrine actions in the ovary and may play a role in the regulation of ovarian function. We have examined ovarian levels of IGF-I mRNA and IGF-I protein throughout the estrus cycle. The lowest levels of IGF-I mRNA were found on proestrus (12.00 h). The mRNA levels on estrus (12.00 h) were significantly (P less than 0.05) higher than on proestrus. The correlation between the levels of IGF-I and IGF-I mRNA was linear and significant (r = 0.706; P less than 0.01). Our observations that the IGF-I gene expression and translation vary during the estrus cycle, with an increase between proestrus and estrus, suggest that the gonadotropin surge could be of importance for the regulation of IGF-I in vivo and that IGF-I may be involved in the cell proliferation and differentiation caused by these hormones.     

Oxytocin/neurophysin-I messenger ribonucleic acid in bovine granulosa cells increases after the luteinizing hormone (LH) surge and is stimulated by LH in vitro.

Bovine granulosa cells express the oxytocin/neurophysin-I (OT/NP-I) gene and secrete OT in vitro. We have shown previously that bovine granulosa cells isolated from the preovulatory follicle after the LH surge secrete 20 times more OT over 5 days in culture than granulosa cells obtained before the surge. LH or FSH stimulates OT secretion in vitro by granulosa cells isolated before the LH surge. We also observed that granulosa cells of preovulatory follicles isolated before the LH surge respond to OT with an increase in progesterone secretion, suggesting that OT may be involved in regulating the follicular/luteal phase shift, or ovulation, in an autocrine fashion. The objective of this study was to determine whether the increase in OT secretion from granulosa cells after the LH surge is regulated at the level of mRNA accumulation, peptide synthesis, and/or peptide secretion. Bovine preovulatory follicles were obtained during the early follicular phase (approximately 36 h before the LH surge), during the midfollicular phase (approximately 12 h before the LH surge), or during the late follicular phase (after the LH surge). Total RNA isolated from granulosa cells and theca interna at the time of cell isolation or after culture with or without LH was subjected to Northern analysis for OT/NP-I mRNA and quantified by densitometry. OT/NP-I mRNA was not detectable or was barely detectable in granulosa cells collected during the early or midfollicular phase (n = 6 and n = 4 follicles, respectively), but a strong hybridization signal was obtained from RNA isolated after the LH surge (n = 5 follicles; P < 0.01). In contrast, OT/NP-I mRNA was not detectable in theca interna before or after the LH surge. Although OT/NP-I mRNA was not detectable in granulosa cells isolated 24 h after prostaglandin F2 alpha injection, after 24 h in culture, a weak OT/NP-I mRNA hybridization signal was observed in RNA from granulosa cells in LH-containing cultures. After 72 h in culture, granulosa cells cultured in control, as well as in LH-containing medium, exhibited a strong signal for OT/NP-I mRNA, but granulosa cells treated with LH exhibited a stronger OT/NP-I hybridization signal than control cultures (P < 0.01). Theca interna did not yield any OT/NP-I hybridization signal initially, and none was induced in culture.(ABSTRACT TRUNCATED AT 400 WORDS)     

An insulin-like growth factor-binding protein in ovarian follicular fluid blocks follicle-stimulating hormone-stimulated steroid production by ovarian granulosa cells

An inhibitor of FSH action on granulosa cells has been purified from porcine follicular fluid using a combination of ammonium sulfate precipitation, dialysis in 30% (vol/vol) acetic acid, gel filtration chromatography under acidic conditions, and several steps of reverse phase HPLC. Activity was monitored by using an in vitro granulosa cell bioassay, measuring the effect of the inhibitor on FSH-stimulated estradiol production. The purified polypeptide dose-dependently inhibited production of both estradiol (EC50 = 0.7 nM) and progesterone (EC50 = 1.3 nM) by rat granulosa cells cultured in the presence of 20 ng/ml FSH. N-Terminal sequence analysis revealed a high degree of homology with the 53,000 mol wt human GH-dependent insulin-like growth factor-binding protein (IGF-BP). Coincubation of stoichiometric amounts of IGF-I or -II and the inhibitor (IGF-BP) resulted in complete neutralization of the inhibitory effect. Since both IGFs are produced locally in the ovary and exert stimulatory effects on granulosa cells, local production of IGF-BP may provide an important means of regulating ovarian follicle growth.     

2-hydroxyestradiol enhanced progesterone production by porcine granulosa cells: dependence on de novo cholesterol synthesis and stimulation of cholesterol side-chain cleavage activity and cytochrome P450scc messenger ribonucleic acid levels

2-Hydroxyestradiol (2-OH-E2) stimulates progestin secretion by granulosa cells, but the intracellular locus of the stimulatory effect has not been clarified. The objectives of the present studies were to 1) determine the role of de novo sterol synthesis in the effect of 2-OH-E2 on progestin biosynthesis, and 2) examine the effects of 2-OH-E2 on cholesterol side-chain cleavage (SCC) activity and the level of messenger RNA (mRNA) for P450scc. Inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A reductase with lovastatin (5 micrograms/ml) or mevinolin (5 micrograms/ml) reduced FSH- and 2-OH-E2-stimulated (but not E2-stimulated) progesterone production. Mevalonate (20 mM) enhanced basal progesterone production and reversed the inhibitory effect of lovastatin but did not affect progesterone biosynthesis in the presence of 2-OH-E2. As an index of the activity of cholesterol SCC enzyme, granulosa cells were exposed to 25-hydroxycholesterol (10 micrograms/ml) for 24 h and progesterone secretion monitored. Conversion of 25-hydroxycholesterol into progesterone was stimulated 2- to 3-fold by maximally effective concentrations of 2-OH-E2, E2, and FSH. 2-OH-E2 and/or E2 further enhanced 25-hydroxycholesterol conversion in the presence of FSH, LH, and epinephrine. Aminoglutethimide, an inhibitor of SCC, reduced 2-OH-E2- and 2-OH-E2 plus FSH-stimulated progesterone production by 97% and 95%, respectively. 2-OH-E2 also increased basal (by 2 to 3-fold) and FSH-stimulated (to 3.5-fold of FSH-treated controls) levels of mRNA for cytochrome P450scc. Collectively, our studies support the hypothesis that 2-OH-E2-enhanced progesterone biosynthesis by porcine granulosa cells is dependent on de novo cholesterol synthesis and is associated with increased levels of the mRNA encoding cytochrome P-450scc, which leads to increases in basal and gonadotropin-induced SCC activity.     

Dihydrotestosterone inhibits insulin-stimulated cyclin D2 messenger ribonucleic acid expression in rat ovarian granulosa cells by reducing the phosphorylation of insulin receptor substrate-1

The effect of 5alpha-dihydrotestosterone (DHT) on insulin-stimulated granulosa cell proliferation was examined using cyclin D2 mRNA as a marker. Granulosa cells from 3-d estradiol-treated immature rats showed a concentration-dependent increase in cyclin D2 mRNA expression in response to insulin. Exposure to DHT reduced the insulin-stimulated cyclin D2 mRNA expression. Inhibition of the two insulin-signaling pathways, ERK and phosphatidylinositol 3 kinase (PI3 kinase), by using specific inhibitors, also reduced this insulin-stimulated response. These results suggest that both ERK and PI3 kinase signaling are involved in insulin stimulated granulosa cell proliferation. DHT exposure resulted in reduced insulin-stimulated ERK phosphorylation. DHT treatment also reduced the insulin mediated insulin receptor substrate-1 and Raf-1 phosphorylation, the upstream molecules of ERK in insulin signaling pathway. Additionally, inhibition of insulin stimulated PI3 kinase activation reduced ERK phosphorylation. The present study therefore shows that the inhibitory effect of DHT on insulin-stimulated granulosa cell proliferation occurs early in the signaling pathway at the level of insulin receptor substrate-1 phosphorylation, leading to reduced ERK phosphorylation and subsequent inhibition of cyclin D2 mRNA expression.