Follistatin inhibits activin-induced differentiation of rat follicular granulosa cells in vitro.

The effect of follistatin on activin-induced granulosa cell differentiation was investigated in freshly harvested granulosa cells from diethylstilbestrol (DES)-treated rats. Activin induced a remarkable change in granulosa cellular morphology from elongated fibroblast-like to round cells, which follistatin prevented. Follistatin itself had no influence on the cellular morphology. We studied the action of follistatin on activin-induced differentiation of granulosa cells cultured in a chemically defined medium. Addition of activin (30 ng/ml) to the culture increased the FSH binding site approximately 2-fold compared with the control (spontaneous expression) level, whereas follistatin reduced the activin-induced expression level to the control level in a concentration-dependent manner. Activin (30 ng/ml) markedly augmented FSH-induced hCG binding and progesterone production by approximately 20-fold, and these effects were suppressed by follistatin in a concentration-dependent manner. Similarly, addition of follistatin to the culture induced a concentration-dependent decrease of activin-enhanced inhibin-producing activity, but had no effect on FSH-induced inhibin production. These results suggest that follistatin/activin-binding protein binds to activin stoichiometrically to suppress the activin-induced differentiation of rat granulosa cell in vitro, but follistatin itself has no direct effect on activin-independent reactions.     

Activin-A, but not inhibin, regulates 17beta-hydroxysteroid dehydrogenase type 1 activity and expression in cultured rat granulosa cells

17beta-Hydroxysteroid dehydrogenase type 1 (17HSD type 1) catalyzes the reduction of estrone (E(1)) to biologically more active estradiol (E(2)). In the present study, the effect of activin, inhibin, and follistatin on 17HSD activity and 17HSD type 1 expression in cultured, unluteinized rat granulosa cells was examined. Furthermore, the effects of these hormones on 17HSD type 1 expression were compared with the expression of P450 aromatase (P450arom). Rat granulosa cells were pre-incubated in serum-free media for 3 days, followed by a 2-day treatment with activin, inhibin, follistatin and 8-Br-cAMP. Activin in increasing concentrations appeared to effect a dose-dependent increase in 17HSD activity. In addition, increasing concentrations of activin also increased 17HSD type 1 mRNA expression. Addition of 8-Br-cAMP at concentrations of 0.25 and 1.5 mmol/l together with activin significantly augmented the stimulatory effects of activin alone in the cultured cells. Neither inhibin, nor follistatin, either alone or in combination with 8-Br-cAMP, had any notable effects on 17HSD activity and 17HSD type 1 expression. Preincubation of activin with increasing concentrations of follistatin significantly diminished the stimulatory effect of activin. In the presence of follistatin, activin did not significantly increase the 8-Br-cAMP-induced 17HSD activity and 17HSD type 1 expression. The culturing of granulosa cells in the presence or the absence of inhibin or follistatin with or without 8-Br-cAMP did not alter the effect of these peptides on P450arom expression in rat granulosa cells as judged by Northern blot analysis of total RNA. However, cAMP-induced P450arom expression was enhanced by activin treatment, except when follistatin was present. This is in line with the suggested role of follistatin as an activin-binding protein, which limits the bioavailability of activin to its membrane receptors. Thus, the results support the notion of a paracrine/autocrine role of activin in follicular steroidogenesis of growing follicles.     

Activin-A up-regulates type I activin receptor mRNA levels in human immortalized extravillous trophoblast cells.

Activin is known to play an important regulatory role in reproduction, including pregnancy. To further examine the role and signaling mechanism of activin in regulating placental function, the steady-state level of activin type I receptor (ActRI) mRNA in immortalized extravillous trophoblasts (IEVT) cells was measured using competitive PCR (cPCR). An internal standard of ActRI cDNA for cPCR was constructed for the quantification of ActRI mRNA levels in IEVT cells. ActRI mRNA levels were increased in a dose-dependent manner by activin-A with the maximal effect observed at the dose of 10 ng/ml. Time course studies revealed that activin-A had maximal effects on ActRI mRNA levels at 6 hours after treatment. The effects of activin-A on ActRI mRNA levels was blocked by follistatin, an activin binding protein, in a dose-dependent manner. In addition, inhibin-A inhibited basal, as well as activin-A-induced ActRI mRNA levels. These findings provide evidence, for the first time, that activin-A modulates ActRI mRNA levels in human trophoblast cells.     

Activin-A, but not inhibin, regulates 17β-hydroxysteroid dehydrogenase type 1 activity and expression in cultured rat granulosa cells

17β-Hydroxysteroid dehydrogenase type 1 (17HSD type 1) catalyzes the reduction of estrone (E₁) to biologically more active estradiol (E₂). In the present study, the effect of activin, inhibin, and follistatin on 17HSD activity and 17HSD type 1 expression in cultured, unluteinized rat granulosa cells was examined. Furthermore, the effects of these hormones on 17HSD type 1 expression were compared with the expression of P450 aromatase (P450arom). Rat granulosa cells were pre-incubated in serum-free media for 3 days, followed by a 2-day treatment with activin, inhibin, follistatin and 8-Br-cAMP. Activin in increasing concentrations appeared to effect a dose-dependent increase in 17HSD activity. In addition, increasing concentrations of activin also increased 17HSD type 1 mRNA expression. Addition of 8-Br-cAMP at concentrations of 0.25 and 1.5 mmol/l together with activin significantly augmented the stimulatory effects of activin alone in the cultured cells. Neither inhibin, nor follistatin, either alone or in combination with 8-Br-cAMP, had any notable effects on 17HSD activity and 17HSD type 1 expression. Preincubation of activin with increasing concentrations of follistatin significantly diminished the stimulatory effect of activin. In the presence of follistatin, activin did not significantly increase the 8-Br-cAMP-induced 17HSD activity and 17HSD type 1 expression. The culturing of granulosa cells in the presence or the absence of inhibin or follistatin with or without 8-Br-cAMP did not alter the effect of these peptides on P450arom expression in rat granulosa cells as judged by Northern blot analysis of total RNA. However, cAMP-induced P450arom expression was enhanced by activin treatment, except when follistatin was present. This is in line with the suggested role of follistatin as an activin-binding protein, which limits the bioavailability of activin to its membrane receptors. Thus, the results support the notion of a paracrine/autocrine role of activin in follicular steroidogenesis of growing follicles.     

活化素和卵泡抑素对绍鸭卵泡颗粒细胞FSH受体mRNA表达作用的研究

分别用活化素（Activin）、卵泡抑素（FSP）及其组合（Activin FSP）来处理培养的鸭未成熟卵泡颗粒细胞,发现在FSH存在与不存在的情况下,Activin均能促进FSH受体mRNA的表达,且随着Activin浓度的增大,其刺激作用增强。FSP自身对FSH受体产生无显著作用,但能中和Activin对该受体产生的促进作用。这说明FSP和Activin对颗粒细胞具有自分泌作用,二者通过调节FSH受体mRNA的表达而在卵泡的生长发育过程中起着重要作用。     

Activin A and gonadotropin regulation of follicle-stimulating hormone and luteinizing hormone receptor messenger RNA in avian granulosa cells.

Activin A regulation of the expression of mRNA for the LH receptor, FSH receptor, and the inhibin alpha subunit as well as the effect of activin A on the secretion of progesterone were investigated in chicken granulosa cell cultures. Granulosa layers were isolated from the F(1) and F(3) + F(4) follicles from five hens, pooled according to size, dispersed, and cultured for 48 h. In experiment 1 (n = 3 replications), granulosa cells were cultured with or without highly purified ovine (o) FSH at 50 ng/ml and in the presence of 0, 10, or 50 ng/ml of recombinant chicken activin A. Experiment 2 (n = 4 replications) followed the same protocol as experiment 1, except that oFSH was replaced with oLH. Results from these experiments showed that addition of activin A to the granulosa cell cultures had no effect on the expression of mRNA for the inhibin alpha subunit or the FSH receptor, but it did affect the expression of mRNA for the LH receptor. Treatment of F(3) + F(4) granulosa cells with LH stimulated the expression of mRNA for the LH receptor; however, when LH was combined with either dose of activin A, this induction was prevented. The highest dose of activin A with or without LH resulted in decreased expression of the LH receptor compared to the untreated controls in the F(3) + F(4) cell cultures. Progesterone secretion by the granulosa cells from both follicle sizes was not altered by activin A. In experiment 3 (n = 3 replications), the effect of activin A on the growth of granulosa cells was examined with the following treatments: 0, 10, or 50 ng/ml of activin A; 50 ng/ml of either oLH or oFSH; and oLH or oFSH combined with 10 ng/ml of activin A. The highest dose of activin reduced the rate of granulosa cell proliferation in both follicle types. Growth of F(1) and F(3) + F(4) granulosa cells was stimulated by the addition of either gonadotropin, and the presence of 10 ng/ml of activin A with either gonadotropin did not alter this proliferation, except for the LH-treated F(3) + F(4) granulosa cells, in which the increase in proliferation was prevented. The results suggest that activin A could act as a local factor that regulates follicular maturation by preventing excessive or untimely LH receptor expression.     

Paracrine effect of folliculo-stellate cells on the growth factor-like action of activin A in anterior pituitary cultures.

Several studies have shown that pituitary folliculo-stellate (FS) cells exhibit local functions within the pituitary gland. On the other hand, we have shown previously that activin A increases the number of FSH-producing gonadotropes in cultured rat anterior pituitary cells. In this study, we investigated whether FS cells exert an influence on the action of activin A. FS cells were prepared by culturing the dispersed rat anterior pituitary cells in media containing 15% fetal calf serum and 6 mM glutamine for 15 days. Most cells had the morphological characteristics of FS cells and S-100 protein immunoreactivity, a specific marker of FS cells. The number of FSH cells, which was higher in activin A-treated than in control cultures, was reduced to the control level by incubation with activin A plus conditioned media from FS cell-enriched cultures (FSCM). This inhibitory effect of FSCM was neutralized by a follistatin antibody, but not by anti-S-100 protein or anti-basic fibroblast growth factor. Furthermore, follistatin suppressed activin A stimulated increases in the number of FSH cells in a similar inhibitory pattern to that of FSCM. Meanwhile, the number of FSH cells was not affected by FSCM or follistatin in the absence of activin A. These results suggest that FS cells are involved in the regulation of the function and/or the morphogenesis of the FSH cell-lineage by affecting the action of activin A, and that this paracrine effect of FS cells is mediated by follistatin.     

The role of the activin system in keloid pathogenesis

Keloid scars represent a pathological response to cutaneous injury under the regulation of many growth factors. Activin-A, a dimeric protein and a member of the transforming growth factor- superfamily, has been shown to regulate various aspects of cell growth and differentiation in the repair of the skin mesenchyme and the epidermis. Thus our aim was to study the role of activin and its antagonist, follistatin, in keloid pathogenesis. Increased mRNA expression for activin was observed in keloid scar tissue by performing RNase protection assay. Immunohistochemistry showed increased localization of both activin-A and follistatin in the basal layer of epidermis of keloid tissue compared with normal tissue. ELISA demonstrated a 29-fold increase in concentration of activin-A and an 5-fold increase in follistatin in conditioned media in keloid fibroblasts compared with normal fibroblasts. Although keloid keratinocytes produced 25% more follistatin than normal keratinocytes, the amounts of activin-A, in contrast, was 77% lower. Proliferation of fibroblasts was stimulated when treated with exogenous activin-A (46% increase in keloids fibroblasts) or following co-culture with hAHaCaT cells (66% increase). Activin-A upregulated key extracellular matrix components, namely collagen, fibronectin, and -smooth muscle actin, in normal and keloid fibroblasts. Co-treatment of follistatin with activin-A blocked the stimulatory effects of activin on extracellular matrix components. These findings emphasize the importance of the activin system in keloid biology and pathogenesis and suggest a possible therapeutic potential of follistatin in the prevention and treatment of keloids. collagen; fibroblasts; follistatin; keloid scar; keratinocytes; -smooth muscle actin; transforming growth factor-     

Follistatin, an activin-binding protein, associates with heparan sulfate chains of proteoglycans on follicular granulosa cells

Follistatin, an activin-binding protein secreted by cultured rat granulosa cells, was shown to associate with the cell surface by affinity labeling with 125I-activin. Addition of follistatin to the cultured cells demonstrated a typical ligand-binding saturation curve, suggesting that granulosa cells have a specific binding site for follistatin. This binding was markedly inhibited by heparin and heparan sulfate, but not by chondroitin sulfates A and C, keratan sulfate, and dermatan sulfate. When granulosa cells were treated with glycosaminoglycan-degrading enzymes before or after addition of follistatin to the cultures, heparinase and heparitinase treatments resulted in significant suppression of the binding, whereas treatment with chondroitinase ABC had no effect. A competition study of the binding using heparin derivatives demonstrated that follistatin seemed to recognize O-sulfate groups in the heparin molecule. Heparitinase-treated granulosa cells exhibited almost full responsiveness to activin, indicating that the enzyme treatment had no effect on activin and receptor interaction. These results suggest that follistatin/activin-binding protein binds to heparan sulfate side chains of proteoglycans on the granulosa cell surface to regulate the various actions of activin.     

Follicle-stimulating hormone regulation of inhibin alpha- and beta(B)-subunit and follistatin messenger ribonucleic acid in cultured avian granulosa cells

FSH regulation of inhibin alpha-, beta(B)-subunit and follistatin mRNA was investigated in cultured chicken granulosa cells, which were isolated and pooled according to size from the F(4) + F(5) follicles, small yellow follicles (SYF), and large white follicles (LWF). In experiment 1 (four replicate experiments), granulosa cells were cultured, and the effect of FSH (50 ng/ml) on the growth of cells from the different follicles was examined at 24 and 48 h of culture. Cell viability was >95% for all of the granulosa cell cultures at 24 and 48 h. At 24 h, the number of granulosa cells in both the FSH-treated and the untreated cultures for all follicle types was numerically greater than the number of cells originally plated. At 48 h, FSH-treated cultures for all follicle types had twice (P: < 0. 05) the number of cells as the untreated cultures. In experiment 2 (three replicate experiments), FSH increased expression of the mRNA for inhibin alpha-subunit in LWF granulosa cells at 4 and 24 h to detectable levels and increased inhibin alpha-subunit protein accumulation to detectable levels by 24 h in granulosa cells from the LWF. FSH also increased (P: < 0.05) mRNA levels for the inhibin alpha-subunit at 4 and 24 h in SYF granulosa cells and at 24 h in F(4) + F(5) granulosa cells. The effects of FSH on follistatin and ss(B)-subunit were variable with respect to follicle development and culture duration. These results suggest that FSH plays an important role in stimulating the production of mRNA and protein for the inhibin alpha-subunit in small prehierarchical follicles.     

Decidual activin: its role in the apoptotic process and its regulation by prolactin

Successful pregnancy requires profound differentiation and reorganization of the uterine tissues including, as pregnancy progresses, extensive apoptosis of decidual tissue to accommodate the developing conceptus. We have previously shown a positive correlation between expression of activin A and apoptosis in the decidua and have also shown that expression of activin A occurs at the time when prolactin (PRL) receptors disappear from decidual cells. The goals of this study were to examine whether activin A plays a role in decidual apoptosis and whether expression of activin A in the decidua is regulated by PRL and placental lactogens. Studies were carried out using primary rat decidual cells, a decidual cell line (GG-AD), and PRL null mice. Treatment of decidual cells with activin A significantly increased DNA degradation, caspase 3 activity, and caspase 3 mRNA expression. However, this effect was observed only in the absence of endogenous activin production by these cells. Addition of follistatin to decidual cells that were producing activin A decreased both caspase 3 activity and mRNA expression. Similarly, addition of activin-blocking antibodies to cultures of GG-AD cells, which also produce activin A, caused a reduction in both DNA degradation and caspase 3 activity. PRL and placental lactogens caused an inhibition of activin A mRNA expression in primary decidual cells. Even more convincingly, decidua of PRL null mice expressed abundant activin A at a time when no expression of this hormone is detected in wild-type mice and treatment of PRL null mice with PRL caused a profound inhibition of activin A mRNA expression. In summary, our investigations into the role and regulation of decidual activin have revealed that activin A can induce cell death in the decidua and that its expression is under tight regulation by PRL and placental lactogens.