Regulation of insulin-like growth factor I biosynthesis in porcine granulosa cells.

In the present studies we examined the regulation of insulin-like growth factor I (IGF-I) expression in porcine granulosa cells in vitro. Using Northern analysis and ribonuclease protection assays with exon-specific probes, we identified the IGF-I messenger RNA (mRNA) transcripts present in these cells under basal and hormone-stimulated conditions. We also assessed changes in secreted IGF-I using Western blots and correlated the change in protein secretion after hormone treatment with changes in mRNA levels. By analogy to the human IGF-I gene and its transcription, two major transcripts of approximately 1 and 7.5 kilobases, seen in freshly isolated granulosa cells and follicle wall and in single passaged granulosa (MDGp1) cells, most likely correspond to IGF-IA. Minor transcripts of 3-4 kilobases, which appeared after FSH or forskolin treatments or in control cells after long exposure of the autoradiographs, were attributed to incompletely processed RNA precursors. Ribonuclease protection assay analysis using probes to detect alternative use of exon 5 or exon 6 indicated that most, if not all, of the transcripts contained only exon 6 sequence (IGF-IA). Both class 1 and class 2 transcripts were identified using exon 1- and exon 2-specific probes, respectively. GH increased steady state levels of IGF-I mRNA 3-fold, FSH increased it approximately 10-fold, and forskolin maximally increased it 12- to 15-fold. Estradiol had no effect alone or in combination with the other treatments. All treatments that increased IGF-I mRNA coordinately increased both class 1 and class 2 transcripts, with the increase in class 1 greater than that in class 2. Multiple forms of IGF-I protein were seen under basal conditions and after hormone treatment. These were identified based on mRNA analysis and biochemical methods as both glycosylated and nonglycosylated IGF-IA prohormone, incompletely processed forms of prohormone, and the mature peptide. Changes in the levels of total protein were similar to the changes in mRNA (GH, 3-fold; FSH and forskolin, 10- to 20-fold). All forms of the protein changed coordinately, suggesting that these hormones had no major effect on the intracellular processing mechanism. IGF-binding protein-3 was able to bind to all IGF-I forms. These data conclusively demonstrate FSH and GH induction of ovarian IGF-I. The porcine granulosa cell culture system used in these studies should be an excellent system for studying the hormonal regulation of IGF-I expression.

Insulin-like growth factor binding protein-3 inhibits porcine granulosa cell function in vitro.

Recombinant human insulin-like growth factor binding protein-3 (rhIGFBP-3) effects on basal, insulin-like growth factor I (IGF-I)-, and follicle-stimulating hormone (FSH)-stimulated progesterone (P4) secretion and [3H]aminoisobutyric acid (AIB) uptake by primary porcine granulosa cells (MDGs) and MDGs that have been passaged once (MDGp1) were assessed. Cells were treated concurrently or were preincubated with rhIGFBP-3 followed by treatment. rhIGFBP-3 had no effect on MDG or MDGp1 cell numbers after 24 h. Cotreatment with rhIGFBP-3 inhibited P4 secretion after treatment with FSH, IGF-I, and FSH plus IGF-I. FSH did not stimulate [3H]AIB uptake. However, the IGF-I-stimulated increase in [3H]AIB uptake was completely prevented by concurrent treatment with IGFBP-3. Preincubation of MDGp1 cells with IGFBP-3 dose dependently inhibited FSH- and IGF-I-stimulated P4 secretion. This inhibition was associated with increased cell association of the binding protein and increased IGF-I binding to the cells. These results indicate that IGFBP-3 is inhibitory to a variety of crucial functions in porcine granulosa cells, supporting a role for it in the regulation of granulosa cell function.

Changes in the messenger ribonucleic acid for insulin-like growth factor-I and -II in the porcine testis during and between two waves of testicular development.

The boar testis was used as a model for examining the possible role of production of the insulin-like growth factors (IGF) in steroidogenesis and/or testicular growth as testicular development occurs in waves. Blood and testes were sampled from boars at different ages (100-102 days of gestation; 7, 19, and 30 days; and 10 and 25 wk), selected to occur during and between the last two waves of testicular development. Serum was analyzed for testosterone and RNA was extracted from the testes for Northern and dot-blot analysis of IGF mRNA. Testosterone concentrations declined (p = 0.01) from 7 days to 10 wk of age and rebounded at 25 wk, indicating completion of the second and third waves of testicular development. The quantity of testicular mRNA for IGF-I increased gradually with age, and that for IGF-II decreased. We therefore conclude that regulation of the expression of the mRNAs for IGF-I and -II in the pig testis is not a function of either the waves of testicular development or the level of steroidogenesis.

Effects of growth hormone and gonadotropin on the insulin-like growth factor system in the porcine ovary.

The effects of growth hormone (GH) +/- pregnant mare's serum gonadotropin (PMSG) on levels of insulin-like growth factor (IGF)-I and -II and IGF binding protein (BP)-2 and -3 in serum and follicular fluid (FFI) and on the expression of their mRNA in the ovaries of prepubertal gilts were determined. Steroids in FFI were also quantified. In the first experiment, GH, given for either 20 or 40 days, caused a distinct (threefold, p < 0.05) increase in IGF-I in both serum and FFI with no change in the FFI:serum ratio (0.65). Effects of GH on IGF-II were opposite, with a drop in circulating and FFI levels (p < 0.05). In contrast to data for IGF-I, FFI levels were higher than those in serum for IGF-II (1.42, FFI:serum); IGF-II levels and the ratio fell after GH treatment. GH for either 20 days or 40 days increased serum IGBP-3 to 140% and 250% of control values while decreasing serum IGFBP-2 by 46% and 31%, respectively (p < 0.001). FFI IGFBP-3 was increased to a similar extent by GH (p < 0.005), but IGFBP-2 was not affected. Neither progesterone (P4) nor estradiol (E2) was affected by treatment with GH. However, androstenedione (A4) was decreased by 20-day and 40-day GH treatment relative to the respective controls (p < 0.05). In the second experiment, PMSG resulted in a modest (28%) increase in intrafollicular IGF-I (p < 0.06).(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of the mRNAs for the insulin-like growth factors and their binding proteins during development of porcine ovarian follicles.

We examined the expression of the mRNAs for the insulin-like growth factors (IGFs) and two of their binding proteins (BPs), IGFBP-2 and IGFBP-3, in individual follicles of the porcine ovary. Follicular development was synchronized with a progestin (altrenogest). Individual follicles were isolated on days 1, 3, 5 and 7 after progestin withdrawal. No IGFBP-3 mRNA was detected. While IGF-II mRNA was easily detected, the levels of expression did not change. IGF-I and IGFBP-2 mRNAs increased and decreased, respectively, with follicle development until day 7 when IGF-I expression declined. Regression analysis of IGF-I and IGFBP-2 mRNA expression was performed to assess the relative strength of correlations with day, diameter and steroid concentrations as covariates. IGFBP-2 mRNA was correlated with both day and diameter (r = -.713 and -.705, respectively, n = 24) and neither estrogen (E2) nor progesterone (P4) contributed to the fit. While IGF-I mRNA expression was correlated to both day (r = .483) and diameter (r = .587), the strongest predictor was E2 concentration (r = .694, n = 27). In conclusion, the expression of IGF-I and IGFBP-2 mRNAs in the ovarian follicle are discordantly regulated during follicular growth and maturation. The observed changes in these parameters should result in increased bioavailable IGF-I. This supports a pivotal autocrine/paracrine role for these factors during follicle growth and development.

Divergent actions of prostaglandins-E2 and -F2 alpha on the regulation of insulin-like growth factor-binding protein-3 in luteinized granulosa cells.

Insulin-like growth factor (IGF)-I synergizes with gonadotropin to further stimulate ovarian steroidogenesis. In contrast, the IGF-binding proteins (IGFBPs) produced by granulosa cells have been shown to antagonize the stimulatory actions of the IGFs and gonadotropins. The purpose of the present study was to examine the effects on IGFBP-3 production of prostaglandin (PG)-E2, a compound known to stimulate luteal function and prevent/delay luteal regression (a luteotropic compound), and PGF2 alpha, a compound known to be luteolytic. PGF2 alpha significantly stimulated IGFBP-3 production to 2.6-fold of control (P < 0.05) while PGE2 attenuated its production to half of control (P < 0.05). In contrast to the effects of IGFBP-3, PGE2 stimulated progesterone production to 8-fold of control (P < 0.05) while PGF2 alpha had no effect. Possible mechanisms of action of PGE2 and PGF2 alpha were also examined. PGE2, but not PGF2 alpha, stimulated cAMP accumulation which has been previously shown to inhibit IGFBP-3 production. PGF2 alpha is suspected to act via activation of protein kinase-C. However, a phorbol ester did not mimic PGF2 alpha's action toward IGFBP-3. This study demonstrated that PGE2 and PGF2 alpha conversely modulate IGFBP-3 production. Since IGFBPs have been shown to antagonize gonadotropin and IGF actions, this action of the prostaglandins may impact on the synergism between IGFs and gonadotropin necessary for follicular growth and luteal function.

The role of insulin-like growth factors and epidermal growth factor-related peptides in intraovarian regulation in the pig ovary.

The autocrine and paracrine role of the insulin-like growth factors (IGFs) and epidermal growth factor (EGF)-related peptides in pig ovary are reviewed. For convenience, each of these regulatory systems is divided into several interactive components: regulated expression of the growth factors, growth factor reception at the cell surface and intracellular action of the growth factors. In addition, the concept of regulated bioavailability and targeting of growth factors in the extracellular space is developed as an important control locus and area for future study. With regard to the IGF system, these components include two ligands--IGF-I and -II, both expressed in the porcine ovary--and the possibility of three receptors. IGF-I and the type I IGF receptor appear to be the most important in stimulating ovarian function and amplifying hormone action. In addition, the 'set-point' of the ovarian IGF system may be determined by the activity of several IGF-binding proteins (IGFBPs). At least four of these proteins are expressed in the pig ovary. Studies of their regulation and action in ovarian cells indicate that they can function as antagonists to FSH and the IGFs. However, preliminary evidence suggests a more dynamic model in which these proteins may direct the site and timing of IGF effects. There are fewer data on the EGF system. At least four EGF-related peptides are expressed in pig ovaries, but insufficient information is available to predict their physiological regulation. These peptides are potent mitogens for ovarian cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of insulin-like growth factor-binding protein-2 and -3 messenger ribonucleic acid in the porcine ovary: localization and physiological changes.

Insulin-like growth factor-binding protein (IGFBP)-2 and -3 are the most prevalent IGFBPs in porcine follicular fluid, as determined on ligand blots, but little is known about the localization and regulation of their synthesis in vivo. This study was designed to investigate the localization and cyclic regulation of the mRNA for these two IGFBPs in the porcine ovary, RNA was extracted from whole ovaries morphologically classified as immature, preovulatory, and luteal. Northern hybridization analysis of this RNA showed no significant difference in the expression of IGFBP-2 mRNA in these ovaries (OD for preovulatory, luteal, and immature ovaries, 0.076 +/- 0.01, 0.071 +/- 0.01, and 0.10 +/- 0.008/micrograms RNA, respectively). IGFBP-3 mRNA was not different in immature and preovulatory ovaries, but was 10-fold greater (P less than 0.025) in luteal ovaries. Northern analysis of RNA extracted from ovaries also showed no significant change in IGFBP-2 mRNA on days (d) 11, 16, and 21 of the estrous cycle. IGFBP-3 mRNA tended to decrease between d11-16 with the onset of luteal regression and was significantly decreased in d21 preovulatory ovaries to 22% of the values in d11 ovaries. Granulosa, thecal, and luteal cells were also analyzed for IGFBP mRNA. IGFBP-2 mRNA was most abundant in granulosa cells, lower in thecal cells, and lowest in luteal cells. No IGFBP-3 mRNA could be detected in granulosa cells, and luteal cells expressed 15- to 63-fold greater levels than thecal cells. These results show that IGFBP-2 and -3 mRNAs are expressed in specific ovarian cell types and that their expression appears to be independently regulated during the reproductive cycle. This provides further evidence for the importance of these proteins as paracrine/autocrine regulators of ovarian function.

Gonadotropin and cAMP modulation of IGE binding protein production in ovarian granulosa cells.

Porcine granulosa cells (GC) produce insulin-like growth factor (IGF) binding protein (BP)-3 and IGFBP-2 in culture. A gonadotropin, follicle-stimulating hormone (FSH), dramatically inhibited GC production of these IGFBPs in control cultures and in cultures stimulated by insulin plus epidermal growth factor (EGF) or IGF-I plus EGF. Stimulators of adenylate cyclase (forskolin, cholera toxin) and a derivative of adenosine 3',5'-cyclic monophosphate (cAMP), 8-bromoadenosine 3',5'-cyclic monophosphate, inhibited IGFBP synthesis in a manner similar to FSH. In contrast, the antagonist of cAMP action, (R)-p-adenosine 3',5'-cyclic phosphorothioate [(R)-p-cAMPS], significantly stimulated production of IGFBP-3 and IGFBP-2 compared with controls. This stimulatory effect of (R)-p-cAMPS was counteracted by cotreatment with FSH in a dose-dependent manner. Finally, treatment of GC cultures with FSH plus 3-isobutyl-1-methylxanthine resulted in a significant reduction in cellular content of mRNA coding for IGFBP-3 with no change in IGFBP-2 mRNA. In summary, agents that elevate intracellular cAMP were found to mimic the effects of FSH on IGFBP production.

The ovarian insulin-like growth factors, a local amplification mechanism for steroidogenesis and hormone action.

The importance of the ovarian insulin-like growth factors (IGFs) has been suggested by data from numerous laboratories and several approaches in the last several years. In the aggregate, these data indicate that this system could function as an important local amplification mechanism for steroidogenesis and gonadotropin action. Studies supporting this hypothesis have described several interacting components of this autocrine/paracrine system. First, the several types of ovarian cells possess an IGF-response system, which includes receptors for IGFs and an effective intracellular transduction system. The IGFs can promote growth and/or differentiation of ovarian cells, and their predominant actions depend on the nature of the cells and the presence of additional modulating factors. The biochemical events leading to enhanced steroidogenesis are now understood in considerable detail and include induction of several steps in the cAMP-dependent steroidogenic cascade. The second component of the ovarian IGF system comprises hormone-responsive local production of IGFs. Both IGF-I and IGF-II may be secreted; gonadotropins, gonadal steroids and locally produced growth factors can regulate the IGF system at this level. Finally, ovarian cells secrete a heterogeneous and complex family of IGF-binding proteins (IGFBPs). These proteins can impact on multiple ovarian functions in a manner which is generally opposite to that of the IGFs themselves. As is the case for the IGFs, the secretion of these proteins by ovarian cells is regulated by gonadotropins and locally produced ovarian factors. Collectively, these several components provide an integrated, synergistically cooperative local network to promote gonadotropin-dependent growth and differentiation in the ovary.

The effect of severing the spinal cord of fetal lambs on length of gestation.

Lambs with spinal cords that had been severed near the head at 50 days of gestation were born after a shortened gestation of about 128 days. Lambs with severed spinal cords born as a twin with an intact lamb had a normal gestation of 148 days. The presence of an intact spinal cord or the signals that it might carry apparently influence the length of gestation in the ewe.