Regulation of luteinizing hormone receptor messenger ribonucleic acid levels by gonadotropins, growth factors, and gonadotropin-releasing hormone in cultured rat granulosa cells

The induction of LH receptors in granulosa cells is prerequisite for ovarian follicles to ovulate and form corpora lutea. Earlier studies have demonstrated the modulatory role of gonadotropins, growth factors, and GnRH on ovarian LH receptor content. We have now analyzed the influences of gonadotropins (FSH, LH, and PRL), several growth factors, and GnRH on LH receptor mRNA levels in cultured granulosa cells. Cells were obtained from immature estrogen-treated rats and cultured in medium containing FSH with or without growth factors or GnRH for 48 h. Some cells were also treated with FSH for 48 h, followed by treatment with FSH, LH, or PRL for another 2 days. Cellular total RNA was extracted, and blot hybridization with 32P-labeled LH receptor cRNA or 28S ribosomal RNA cDNA probes was performed. Treatment of granulosa cells with FSH increased the levels of five species of LH receptor mRNAs in a dose- and time-dependent manner. In FSH-primed cells, LH receptor mRNA levels were maintained by FSH, LH, and PRL. In contrast, treatment of cells with basic fibroblast growth factor or epidermal growth factor suppressed FSH induction of LH receptor mRNA in a dose-dependent manner, whereas treatment with insulin-like growth factor-I had no effect. In addition, GnRH suppressed FSH-stimulated LH receptor mRNA levels in a dose-dependent manner; the effects of GnRH could be counteracted by coincubation with a GnRH antagonist, suggesting mediation by specific GnRH-binding sites. These studies demonstrated that the observed stimulatory effects of gonadotropins (FSH, LH, and PRL) and the inhibitory effects of growth factors (epidermal growth factor and basic fibroblast growth factor) and GnRH on LH receptor content are correlated to their regulation of LH receptor mRNA levels. The granulosa cell culture system should provide a useful model for studying LH receptor gene regulation.     

Dynamic regulation of follicle-stimulating hormone-beta messenger ribonucleic acid levels by activin and gonadotropin-releasing hormone in perifused rat pituitary cells.

Maintenance of FSH biosynthesis requires ongoing exposure to pulsatile GnRH. Recent data demonstrate that activin also stimulates FSH biosynthesis. We used a perifused pituitary system to examine regulation of FSH beta mRNA levels by pulsatile GnRH and activin. Hourly pulses of 10 nM GnRH increased FSH beta mRNA levels by 3-fold. In the same experiment, continuous infusion of 50 ng/ml activin elicited a 50-fold increase in FSH beta mRNA. This magnitude of response to activin in perifusion was unexpected, as only a 2.7-fold increase in FSH beta mRNA was observed when activin was administered to pituitary cells that were cultured in dishes. Since perifusion columns, unlike culture dishes, are exposed to a continuous supply of fresh medium, we examined the possibility that endogenous factors produced by pituitary cells cultured in dishes were stimulating the cells in a paracrine fashion, thereby precluding the full response to exogenously added activin. The kinetics of FSH beta mRNA expression were examined immediately after pituitary dispersion and at different times after culturing the cells in plates. FSH beta mRNA levels fell rapidly after dispersion to 8% of initial levels and remained low over 8 h. Thereafter, FSH beta mRNA levels increased slowly and exceeded initial levels by the second day of culture. In a parallel set of experiments, when medium conditioned by exposure to plated cells was applied to the perifusion system, FSH beta mRNA levels were selectively stimulated (6-fold). These data suggest the removal during dispersion and subsequent accumulation in culture of pituitary-derived factors that are important for the maintenance of FSH beta mRNA levels. We conclude that activin plays a greater role in the regulation of FSH beta mRNA levels than was suggested by previous experiments employing static culture systems in which autocrine or paracrine stimulation may have obscured the effects of exogenously added activin.     

Differential regulation of thyroid hormone receptor messenger ribonucleic acid levels by thyrotropin-releasing hormone

In addition to its well known actions in stimulating TSH and PRL synthesis and secretion, TRH has been shown to decrease the concentration of thyroid hormone receptors (TRs) in GH4C1 cells as measured by nuclear thyroid hormone (T3) binding. In the present study we have investigated the effects of TRH on the levels of mRNA encoding the different forms of TR, TR beta-1, TR beta-2, and TR alpha-1 as well as that of the non-T3-binding variant, c-erbA alpha-2. GH3 cells were incubated with 100 nM TRH in the presence or absence of 1 nM T3 for 48 h, and mRNA levels were determined by Northern blot analysis. Results revealed that there is differential regulation of the individual TRs by TRH at the pretranslational level. The mRNA for the pituitary-specific form of TR, TR beta-2, was down-regulated by 60% by TRH in GH3 cells, while that of its alternative splice product, TR beta-1, was unchanged. A modest change was observed in TR alpha-1 mRNA levels, which were down-regulated by 20%; there was no change in c-erbA alpha-2 mRNA levels. Levels of nuclear T3 binding were assessed under the same conditions, and 100 nM TRH was found to decrease binding by 40% from 0.78 to 0.46 fmol/micrograms DNA. A similar change in nuclear T3 binding was seen after incubation with 1 nM T3. The effect of TRH on the GH mRNA response to T3 was investigated. In the absence of TRH there was a 4-fold induction of GH mRNA after incubation with 1 nM T3. In the presence of 100 nM TRH, no significant induction in GH mRNA by T3 was seen, indicating that T3 responsiveness as well as receptor concentration are diminished by TRH under these conditions.     

Beta-adrenergic agents stimulate tissue plasminogen activator activity and messenger ribonucleic acid levels in cultured rat granulosa cells

Expression of tissue plasminogen activator (tPA) gene is stimulated by gonadotropins in granulosa cells. Because adrenergic agents interact with specific granulosa cell receptors to increase progesterone biosynthesis, the effects of these pounds on tPA activity and mRNA levels were also investigated. Cells obtained from immature estrogen-treated rats were initially cultured with FSH or medium alone for 2 days. They were then reincubated with various adrenergic agents before measurement of medium tPA activity using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by a fibrin overlay technique. In addition, cellular RNA was extracted, and tPA mRNA levels were analyzed using a specific rat cRNA probe. Isoproterenol, a beta-adrenergic agonist, stimulated the secretion of tPA activity in a dose-dependent manner, with FSH-pretreated cells secreting higher levels of the enzyme than cells without FSH priming. Northern blot hybridization of total RNA showed the accumulation of a 22S species tPA message in cells treated with isoproterenol, suggesting increased expression of the tPA gene. Furthermore, slot blot hybridization of RNA from these cells indicated a time-dependent increase in tPA mRNA, with maximal induction between 1-3 h of incubation. A selective beta 2-adrenergic agonist, terbutaline, but not the beta 1-agonist dobutamine, stimulated tPA activity. Also, the stimulatory effect of isoproterenol was blocked by a beta 2-antagonist (ICI-118,551) but not by a beta 1-antagonist (practolol), suggesting the involvement of a beta 2-receptor. Like FSH and LH, isoproterenol increased extra- and intracellular cAMP levels. Cotreatment of a saturating dose of isoproterenol with FSH or LH did not further stimulate tPA activity. Similar to that in cells treated with FSH, inhibition of protein synthesis by cycloheximide resulted in the superinduction of tPA mRNA in isoproterenol-treated cells. Thus, activation of beta 2-adrenergic receptors in granulosa cells induces tPA mRNA and activity, presumably through the protein kinase-A pathway shared by gonadotropins. Adrenergic neurotransmitters may be potential intraovarian regulators of this important protease.     

Androgen inhibition of follicle-stimulating hormone-stimulated luteinizing hormone receptor formation in cultured rat granulosa cells

Since LH receptors are decreased in atretic follicles known to contain high androgen levels, we have studied the androgen modulation of LH receptor formation in vitro. Granulosa cells from hypophysectomized, diethylstilbestrol-treated rats were cultured for 3 days with FSH in the presence or absence of nonaromatizable androgens, dihydrotestosterone and 5 alpha-androstane-3 alpha, 17 beta-diol, or a synthetic androgen, R1881 (17 beta-hydroxy-17 alpha-methyl-4,9,11-estratrien-3-one). FSH increased LH receptor content in granulosa cells, while concomitant androgen treatment decreased LH receptor content in a dose- and time-dependent manner, without changing the equilibrium dissociation constant (Kd) for human CG. R1881 (10(-7) M), dihydrotestosterone (10(-6) M), and 5 alpha-androstane-3 alpha, 17 beta-diol (10(-6) M) inhibited LH receptor content by 68%, 65%, and 65%, respectively. Similar to earlier findings, these androgens enhanced FSH-stimulated progesterone biosynthesis and aromatase activity in the same cells. To study their LH responsiveness, androgen-treated cells were washed and reincubated for 2 more days with or without LH. Although basal progesterone production was elevated by R1881 pretreatment, the androgen-pretreated cells were less responsive to LH. Treatment with cyanoketone, an inhibitor of 3 beta-hydroxysteroid dehydrogenase, did not alter the inhibitory effects of R1881 on LH receptors, indicating that the androgen action is not mediated by endogenous progestins. Furthermore, R1881 inhibited the stimulation of LH receptor formation by forskolin, cholera toxin, and 8-bromo-cAMP, suggesting that androgens may inhibit LH receptor induction by affecting post-cAMP events. Estrogen treatment enhanced the FSH induction of LH receptor content, while concomitant addition of R1881 also suppressed the estrogen action. Thus, androgens inhibit FSH-induced functional LH receptors in cultured rat granulosa cells. The androgen effect is exerted, at least partially, at post-cAMP sites and is independent of changes in progestin biosynthesis.     

Follicle-stimulating hormone suppresses cytosolic 3,5,3'-triiodothyronine-binding protein messenger ribonucleic acid expression in rat granulosa cells

FSH plays crucial roles in differentiation of granulosa cells and development of follicles. Considering the broad scope of FSH effects, a large number of genes are likely responsive to the hormone. However, only a limited number of genes have been identified as FSH-regulated genes, particularly during the preantral stage. In an attempt to better define genes involved in follicular development, we examined primary granulosa cell cultures, an undifferentiated rat ovarian granulosa cell line and rat ovaries, using differential display, quantitative RT-PCR, Northern blot analysis, and in situ hybridization. We report, for the first time, that nicotinamide adenine dinucleotide phosphate-dependent cytosolic T(3)-binding protein mRNA is expressed in the ovary, particularly in the granulosa cell layer of preantral and early antral follicles, but not in large preovulatory follicles. Its expression markedly declines in response to FSH, which is dependent on the period of the exposure. This FSH-responsive down-regulation is dependent on granulosa cell differentiation and follicular development. FSH down-regulates the mRNA via the adenylyl cyclase/cAMP pathway, and the down-regulation requires de novo synthesis of a regulatory protein(s). The cytosolic T(3)-binding protein may play a significant role in the regulation of steroidogenesis and follicular development in the mammalian ovary.     

Regulation of steroidogenic acute regulatory protein and luteinizing hormone receptor messenger ribonucleic acid in hen granulosa cells.

The regulation of steroidogenic acute regulatory protein (StAR) in vitro by gonadotropins was investigated in granulosa cells from prehierarchal and preovulatory hen follicles. Basal levels of StAR messenger RNA (mRNA) in undifferentiated granulosa cells from prehierarchal (6- to 8-mm) follicles were consistently low, but detectable, and were significantly increased by treatment with 8-bromo-cAMP and FSH (but not LH) within 3-6 h of culture. After 20 h of culture, 8-bromo-cAMP, FSH, and LH each increased StAR mRNA levels above those in control cultured cells, and the delayed response to LH treatment was associated with increased levels of LH receptor (LH-R) mRNA. On the other hand, inhibition of mitogen-activated protein (MAP) kinase signaling, using the MAP kinase kinase inhibitors U0126 and PD98059, in the presence of FSH further increased StAR mRNA and protein levels, LH-R mRNA levels, and progesterone synthesis compared with those in cells cultured with FSH alone. The highest basal expression of StAR mRNA during follicle development was found in granulosa from the largest (F1) preovulatory follicle, with comparatively lower levels in granulosa from less mature (F2 plus F3) preovulatory follicles. Treatment with LH rapidly increased StAR mRNA and protein (but not LH-R mRNA) expression in cultures of F1 granulosa and in combined F2 plus F3 granulosa within 3 h, although the magnitude of stimulation was greater in F2 plus F3 granulosa. Compared with results from granulosa cells from prehierarchal follicles cultured for 20 h, inhibition of MAP kinase signaling in the presence of LH for 1 h failed to further enhance levels of StAR or LH-R expression or progesterone production in F2 plus F3 follicle granulosa compared with the effect of LH treatment alone. These results demonstrate that StAR expression in the hen ovary is up-regulated by gonadotropins at least in part via cAMP signaling. The ability of MAP kinase kinase inhibitors to potentiate gonadotropin-induced StAR and LH-R expression plus progesterone synthesis in prehierarchal follicle granulosa cells in vitro suggests that inhibition of paracrine or autocrine factor-mediated MAP kinase signaling in vivo may be a prerequisite for the full potentiation of granulosa cell steroidogenesis that occurs after recruitment into the preovulatory hierarchy. Finally, these results fail to support a role for MAP kinase signaling in acutely modulating LH-mediated StAR expression or progesterone production in hierarchal follicles, such as occurs during the preovulatory surge of progesterone.     

Growth hormone enhances follicle-stimulating hormone-induced differentiation of cultured rat granulosa cells

Suppression of serum GH levels in immature rats is associated with delayed onset of puberty and decreased ovarian steroidogenic responsiveness to FSH. To investigate possible direct effects of GH on the differentiation of ovarian cells, granulosa cells from hypophysectomized estrogen-treated rats were cultured with FSH in the presence or absence of GH for 3 days. FSH stimulated granulosa cell LH receptor formation and steroid production in a dose-dependent manner. Concomitant treatment with GH increased LH receptor content by enhancing the action of low doses of FSH without substantial increases in the maximal response. This increase was due to an elevation in the receptor number rather than changes in their affinity for hCG. At 3 ng/ml FSH, concomitant treatment with ovine or bovine GH increased LH/hCG binding in a dose-dependent manner, with 300 ng/ml GH increasing the FSH action by about 3-fold. LH receptors in the GH-treated cells were functional, as indicated by the enhanced cAMP production of these cells in response to LH treatment. The cellular protein content in the FSH-treated cultures was slightly increased by GH (18%), but cell number and viability were unaffected. The change in cell protein content could not account for the increases in the amount of LH receptors. In addition to its effects on LH/hCG receptor content, GH also augmented FSH-stimulated progesterone and 20 alpha-hydroxy-4-pregnen-3-one production in a dose-dependent manner, with 100 ng/ml GH causing significant increases in FSH-induced progesterone production. In contrast, GH treatment did not significantly affect FSH-stimulated estrogen production. The augmentating effects of GH on LH receptor formation and progestin biosynthesis were associated with an enhancement of FSH-stimulated cAMP production. In addition, GH increased forskolin- and 8-bromo-cAMP-induced LH receptor formation and progestin production. Thus, GH-augmented LH receptor induction and progestin biosynthesis may be due to both increased cAMP production and enhanced action of cAMP. The present data have demonstrated that GH augments gonadotropin-stimulated differentiation of ovarian granulosa cells, suggesting an important regulatory role of GH in follicular growth and pubertal development.     

The effect of thyroid hormone on fibronectin messenger ribonucleic acid levels in primary cultured rat hepatocytes.

Our previous in vivo studies demonstrated that thyroid hormone promotes the expression of the fibronectin (FN) gene in the rat liver, while it inhibits the synthesis in cultured human skin fibroblasts. These results can be interpreted as either different regulation of FN synthesis or gene expression among tissues, or divergent results of experiments performed in vivo or in vitro. Here we report on the action of thyroid hormone on FN gene expression in vitro using primary cultured hepatocytes compared to that in cultured skin fibroblasts. Hepatocytes were isolated from hypothyroid rats and were cultured in medium supplemented with thyroidectomized bovine serum (TxBS) or fetal bovine serum (FBS). T3 was added 2 or 24 h after plating, and cells were harvested after 2, 6, or 24 h. Total RNA was extracted, and mRNAs for rat FN and albumin were measured. The requirement of de novo protein synthesis for thyroid hormone-mediated induction of FN mRNA was examined by the addition of cycloheximide 15 min before T3 addition. The amount of FN mRNA significantly decreased in the hepatocytes cultured with TxBS compared with those cultured with FBS. The addition of T3 to TxBS resulted in the restoration of FN mRNA to the level in hepatocytes cultured in FBS. FN mRNA increased during the course of culture in the absence of T3; however, a further increase was observed 6 h after T3 addition. The abundance of albumin RNA decreased during the course of culture, but unlike FN mRNA, it was not changed by T3 addition. The increase in FN mRNA by T3 was not influenced by cycloheximide. These results indicate that thyroid hormone enhances FN gene expression in hepatocytes by its direct action without requiring de novo protein synthesis. In contrast, T3 decreased FN mRNA in cultured skin fibroblasts. Thus, the mode of thyroid hormone action on FN gene expression is different among tissues.     

Luteinizing hormone receptor appearance in cultured porcine granulosa cells requires continual presence of follicle-stimulating hormone.

During the differentiation of ovarian granulosa cells, follicle-stimulating hormone (follitropin; FSH) mediates the induction of cell surface receptors for luteinizing hormone (lutropin; LH). Using primary cultures of porcine granulosa cells, we demonstrate that both the induction and maintenance of LH receptors are critically dependent upon the continual presence of FSH. The termination of FSH-promoted LH receptor induction is paralleled by the termination of FSH-induced intracellular cAMP accumulation. Changing the medium is without effect on FSH-induced appearance of LH receptors. Furthermore, 1 mM aminoglutethimide, which completely blocks FSH-stimulated progesterone biosynthesis, does not decrease the induction of LH receptors by FSH. Thus, the induction of LH receptors by FSH does not appear to require the accumulation of a factor in the medium, nor does it appear to be mediated via FSH-stimulated progesterone synthesis. These data suggest that intracellular cAMP, produced while FSH remains bound to the cell surface, mediates the induction of LH receptors and that the continual presence of FSH is required for both the induction and maintenance of cell surface LH receptors.     

Rat granulosa cells express transforming growth factor-beta type 2 messenger ribonucleic acid which is regulatable by follicle-stimulating hormone in vitro

Freshly harvested granulosa cells (GC) from diethylstilbestrol (DES)-treated rats were examined for the presence of transforming growth factor-beta 1 (TGF-beta 1) and TGF-beta 2 mRNA by Northern analysis. TGF-beta 1 mRNA was not detectable, but hybridization of total RNA with a radiolabeled TGF-beta 2 cDNA probe revealed two mRNA species (5.1 and 3.6 kb) indicative of TGF-beta 2 mRNA. In response to FSH (50 ng/ml), relative TGF-beta 2 mRNA concentrations in cultured GC were 54% of control levels. Furthermore, the conditioned culture medium from FSH-treated GC contained significantly lower (p less than 0.01) TGF-beta-like activity relative to controls. These results demonstrate that rat GC express TGF-beta 2 mRNA which is regulatable by FSH in vitro.