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.     

Regulation of tissue-type plasminogen activator activity and messenger RNA levels by gonadotropin-releasing hormone in cultured rat granulosa cells and cumulus-oocyte complexes

Gonadotropin-releasing hormone (GnRH) acts directly on the ovary to induce ovulation in hypophysectomized proestrous rats. Because plasminogen activators (PAs) are implicated in gonadotropin-induced ovulation, we have studied the effect of GnRH on ovarian PA synthesis. GnRH induced tissue-type PA (tPA) secretion by cultured rat granulosa cells, but inhibited the secretion of urokinase-type PA. These effects were blocked by co-treatment with a GnRH antagonist, suggesting that stereospecific GnRH receptors are involved. Follicle-stimulating hormone (FSH) also induced tPA in granulosa cells but with a different time course than GnRH; the combined effect of FSH and GnRH was additive. The GnRH effect was mimicked by the calcium- and phospholipid-dependent protein kinase C activator, phorbol myristate acetate. In isolated cumulus-oocyte complexes and cumulus cells, GnRH treatment also increased tPA activity. In contrast, treatment of denuded oocytes with GnRH did not increase enzyme activity. After GnRH stimulation of the cumulus-oocyte complexes, tPA content in the denuded oocyte was elevated, suggesting that the cumulus cells mediate the action of GnRH to increase the oocyte enzyme levels. Hybridization experiments using a labeled rat tPA-specific DNA probe showed that both FSH and GnRH increased the level of tPA mRNA in cultured granulosa cells; the stimulatory effect of GnRH was blocked by the GnRH antagonist. Our results indicate that GnRH treatment increases tPA secretion by cultured granulosa cells and cumulus-oocyte complexes. The stimulation of enzyme activity in the granulosa cells is accompanied by increases in tPA mRNA levels.     

Corticosteroids suppress cyclooxygenase messenger RNA levels and prostanoid synthesis in cultured vascular cells

Prostacyclin synthesis by cultured vascular smooth muscle cells was inactivated by aspirin. Recovery required serum factors replaceable by EGF plus TGF-beta and was blocked by cycloheximide but not by actinomycin D. Recovery of cyclooxygenase activity was prevented by preincubation with dexamethasone (0.1 to 2 microM), which also suppressed basal enzyme activity by up to 70%. A full length 2.8 Kb cDNA hybridization probe for human cyclooxygenase identified a cyclooxygenase messenger RNA of approximately 2.8 Kb in these cells. Cyclooxygenase mRNA levels were enhanced by EGF/TGF-beta, but suppressed completely by corticosteroids. It is concluded that inhibition of prostanoid synthesis by corticosteroids is mediated by suppressing cyclooxygenase messenger RNA. These observations provide a new molecular mechanism for the anti-inflammatory activity of the corticosteroids.     

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.     

In vitro study on proopiomelanocortin messenger RNA levels in cultured rat anterior pituitary cells

The fundamental examination on the measurement of proopiomelanocortin (POMC) mRNA levels in cultured rat anterior pituitary (AP) cells was studied. In addition, the detailed study on time- and dose-related effects of corticotropin-releasing factor (CRF) and dexamethasone on the level of POMC mRNA in AP cells in vitro was examined. Basal levels of POMC mRNA in AP cells cultured with serum initially declined after 1-day culture, gradually increased and reached a peak after 3-day culture, and then slightly decreased after 4- and 5-day culture. These mRNA levels after 3-day culture did not change through subsequent 15-hr incubation without serum. CRF treatment caused a time- and dose-dependent increase in POMC mRNA levels. The minimum effective dose of CRF was 0.1 nM for 15-hr incubation. The significant increase in POMC mRNA levels was observed after 3 hrs of 1 nM CRF treatment with a 2-fold elevation seen after 15 hrs of exposure. Dexamethasone treatment caused a dose-dependent decrease in POMC mRNA levels in AP cells. The minimum effective dose was 0.1 microgram/ml and such mRNA levels did not decrease until 15 hrs of exposure.     

Regulation of follicle-stimulating hormone-receptor messenger RNA in hen granulosa cells relative to follicle selection

Both the viability of hen prehierarchal follicles and subsequent differentiation associated with the selection of a single follicle per day into the preovulatory hierarchy depend on circulating FSH and the expression of FSH receptor (FSH-R) in granulosa cells. The present study addresses mechanisms that mediate both basal expression plus selective up-regulation of FSH-R mRNA in granulosa cells from prehierarchal follicles. Results demonstrate that FSH-R mRNA is both expressed and functional in granulosa cells collected from growing prehierarchal follicles as small as those of 1-2 mm in diameter, as indicated by rapid induction of steroidogenic acute regulatory (StAR) protein expression by FSH in vitro. Real-time polymerase chain reaction determined that relative FSH-R expression within the granulosa layer from individual prehierarchal follicles of 6-8 mm in diameter was similar among the 8-13 follicles within this cohort, with the notable exception that the granulosa layer from a single follicle (presumably the selected follicle) showed elevated expression. Levels of FSH-R mRNA expression were enhanced by both recombinant human (rh) transforming growth factor (TGF) beta1 and, to a lesser extent, rh-activin A after 20 h of culture. This stimulatory effect was effectively blocked by mitogen-activated protein (MAP) kinase signaling induced by TGF alpha treatment. Finally, inhibition of MAP kinase signaling, using the selective inhibitor U0126, promoted FSH-R expression and further enhanced TGF beta1-induced FSH-R expression in vitro. Collectively, results suggest that premature granulosa cell differentiation normally is suppressed by tonic MAP kinase signaling. At the time of follicle selection, a release from inhibitory MAP kinase signaling is proposed to occur, which enables the full potentiation of FSH-R expression mediated by intrafollicular factors.     

Stimulation of progesterone secretion by cultured human granulosa cells with melatonin and catecholamines

Granulosa cells, aspirated from the follicles of patients undergoing treatment for in-vitro fertilization, were cultured in serum-supplemented medium. Adrenaline and noradrenaline stimulated a dose-related increase in progesterone secretion with a maximum stimulation at 10(-5) M, a response that was prevented by the beta-antagonist, propranolol. Adrenaline and hCG showed similar characteristics in their stimulation of progesterone secretion but there was no further increase in progesterone when the 2 compounds were added together. Melatonin stimulated progesterone secretion and, like adrenaline, this stimulation was prevented by propranolol. The ability of both adrenaline and melatonin to increase progesterone secretion was dependent on the degree of follicular development, as determined by peripheral oestradiol concentrations, on the day of laparoscopy. These results suggest that adrenaline and melatonin may have a physiological role in modulating luteal function and that melatonin may act by a beta-adrenergic-related mechanism.     

Thyrotropin stimulation of cultured thyroid cells increases steady state levels of the messenger ribonucleic acid for thyroid peroxidase

We have examined the effect of TSH on thyroid peroxidase (TPO) mRNA levels in dog thyroid cell primary cultures. Freshly dispersed dog thyroid cells were cultured for up to 5 days in the absence or presence of 5 mU/ml bovine TSH. At the outset of culture, and at daily intervals thereafter, total cytoplasmic RNA was extracted and applied to Nytran paper using a slot-blot apparatus. A nick-translated cDNA fragment of the porcine TPO gene was used to probe these filters. Autoradiographs were quantified by densitometry. Nonspecific binding was negligible as determined using a pUC18 probe. During the first 2 days of culture, TPO mRNA levels declined irrespective of whether or not TSH was present in the medium. TSH did not affect this decline. Between 3 and 5 days of culture, TPO mRNA levels in control (no TSH) cells increased to 3 times the initial level (expressed relative to cellular DNA). However, during the same period TSH stimulated TPO mRNA levels 8-fold above the initial level. To confirm that the signal with the cDNA probe was actually that of dog TPO mRNA, cellular RNA (day 4 of culture) was subjected to Northern blot analysis using the same cDNA probe. Specific bands of 2.9 kilobases were detected corresponding to the known size of TPO mRNA in pig thyroid tissue. The signal of this 2.9 kilobase species was enhanced by TSH. In conclusion, the data indicate that chronic TSH stimulation raises steady state levels of TPO mRNA and provide an explanation, at least in part, for the mechanism by which TSH enhances TPO bioactivity in thyroid tissue.     

Hormonal regulation of tissue plasminogen activator secretion and mRNA levels in rat granulosa cells

Granulosa cells from immature rats produce tissue plasminogen activator (tPA) in response to follicle stimulating hormone (FSH) or luteinizing hormone (LH) both in vitro and in vivo. We have used the in vitro system to investigate the level at which the hormonal induction of tPA is regulated. Within 12 h following FSH addition, a dramatic but transient increase in tPA secretion occurs for by 24 h secretion returns to basal levels. This pattern of enzyme induction is similar with LH, but the onset of the increase is delayed. When steady-state tPA mRNA levels are examined after hormone treatment, the results mirror those obtained if one measures enzyme activity; a large increase in tPA mRNA followed by a decrease to basal levels is observed with both hormones, and the lag in induction by LH is also apparent. These results demonstrate that the regulation of tPA activity by gonadotropins occurs at the level of the steady-state concentration of the mRNA. In the presence of cycloheximide, the induction of tPA mRNA by FSH or LH is not greatly affected, indicating that this phase of the response to gonadotropins does not require the synthesis of new protein. However, the decrease in tPA mRNA levels observed 24 h after FSH treatment is affected by cycloheximide, in that the drug delays the reduction in mRNA levels seen with hormone alone.     

Modulation of thyroglobulin messenger RNA level by thyrotropin in cultured thyroid cells.

To examine the influence of thyrotropin (TSH) on the thyroglobulin (Tgb) mRNA content, the latter was evaluated in the cytoplasm of hog thyroid cells cultured in the absence (control cells) or presence of TSH. The Tgb mRNA levels were determined by, (i) kinetics of hybridization to sheep Tgb cDNA, (ii) capacity of coding for peptides immunologically related to Tgb in reticulocyte lysate. In cells cultured for 4 days in the absence of TSH, the content of Tgb mRNA sequences decreased to 30% of its initial value and the messenger activity to 15%. Conversely, TSH maintained the initial Tgb mRNA level in cells cultured in its presence, and TSH concentrations 50 micronU/ml or 5 mU/ml gave identical results. At each period tested poly (A) content was the same in TSH-treated and control cells. When TSH was added to media after 4 or 8 days culture without TSH, the Tgb mRNA level was partially restored. These results suggest that TSH exerts a positive control on Tgb gene expression through modulation of Tgb mRNA content of thyroid cells.     

Activin-A inhibits proopiomelanocortin messenger RNA accumulation and adrenocorticotropin secretion of AtT20 cells.

The complexity of corticotropic cell regulation by multiple central and peripheral factors is well recognized. The present study provides evidence for the participation of an additional factor in the regulation of this cell type of the anterior pituitary. Using the clonal AtT20 cell line as a model for corticotropes, homodimeric activin-A was observed to suppress basal ACTH secretion and POMC mRNA accumulation by approximately 50%. These effects required prolonged treatment with activin-A and were concentration dependent; the half-maximum concentration was in the range of 30-50 pM. Consistently, AtT20 cells were found to express specific high affinity binding sites for [125I]activin-A. The simultaneous addition of inhibin-A along with increasing concentrations of activin-A did not alter the characteristics of the inhibition of ACTH secretion by activin-A alone. This is in contrast to observations with gonadotropes of the anterior pituitary as well as a number of other cell types in which inhibin-A can partially antagonize the biological actions of activin-A. The results may suggest the participation of a subclass of activin receptors that mediate effects on ACTH secretion and POMC mRNA accumulation. As previously shown, the incubation of AtT20 cells with a synthetic glucocorticoid, dexamethasone, attenuated basal ACTH secretion and POMC expression in a concentration-dependent manner. The inhibition of both of these parameters by activin-A, however, was independent of glucocorticoids, because the two agents were additive in their actions. In addition to effects on secretion and mRNA levels, treatment with activin-A also inhibited the rate of proliferation of AtT20 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

The methylation state of poly A-containing messenger RNA from cultured hamster cells.

The poly A-containing mRNA of cultured hamster (BHK-21) cells has been examined with regard to methylation status. Steady state-labeled mRNA was obtained by incubating cells for 20-22h in the presence of [methyl-3H]-methionine and 32Pi. The degree of methylation of this RNA was 1.8 methyl groups per 1000 nucleotides, or 4-5 methyl groups on the average per molecule. The nature of the methylated residues was determined by paper chromatography and electrophoresis of acid and alkaline hydrolysates, by DEAE cellulose chromatography of alkaline hydrolysates and of T2 RNase digests, and by examining the effect of subjecting samples to "beta-elimination." Approx. half of the methyl groups occurred in standard ("internal") linkage, 10% as m5Cp and 40% as m6Ap residues. The remainder occurred at least for the most part in "blocked" 5'-termini with the presumptive structure m7G(5')ppp(Nm)p.., where Nm was Gm, m6Am, Um, or Cm.     

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.