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Endocrinology Vol. 140, No. 8 3444-3451
Copyright © 1999 by The Endocrine Society

ARTICLES

Effect of Prolactin on the Expression of Luteinizing Hormone Receptors during Cell Differentiation in Cultured Rat Granulosa Cells1

Takashi Hirakawa, Takashi Minegishi, Mari Tano2, Takashi Kameda, Hiroshi Kishi, Yoshito Ibuki, Tetsuya Mizutani and Kaoru Miyamoto

Department of Obstetrics and Gynecology School of Medicine, Biosignal Research Center Institute for Molecular and Cellular Regulation (T.M., K.M.), Gunma University, Maebashi, Gunma 371-8511, Japan

Address all correspondence and requests for reprints to: Dr. Takashi Minegishi, Department of Obstetrics and Gynecology, Gunma University School of Medicine, Maebashi, Gunma 371-8511, Japan. E-mail: tminegis{at}sb.gunma-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Chronic and transient hyperprolactinemia has been associated with luteal phase dysfunction. Recently, evidence has emerged to suggest that elevated PRL may exert its antigonadal effects through reducing available ovarian LH receptors. We have now examined the influences of PRL on LH receptor induction in cultured granulosa cells. Basal specific LH binding was negligible and remained unchanged in response to treatment with PRL by itself. Whereas treatment with FSH produced, as expected, a substantial increase in specific LH binding, concurrent treatment with PRL resulted in no significant change during the first 4 days of culture, followed by a significant decrease in LH binding on days 5 and 6 as well as an approximately 50% inhibition of FSH effect on day 6. Scatchard plot analysis showed that concurrent treatment with PRL resulted in inhibition of the granulosa cell LH binding capacity, whereas no difference could be detected in the binding affinity of LH to its receptor. Treatment with 8-bromo-cAMP produced a significant increase in specific LH binding; concurrent treatment with PRL (30 ng/ml) produced a significant attenuation of 8-bromo-cAMP action. In addition, treatment with FSH increased the intracellular accumulation of cAMP, and concurrent treatment with PRL did not result in inhibition of the FSH action, as assessed by the generation of intracellular cAMP. Taken together, these findings suggest that the ability of PRL to interfere with FSH action with regard to the induction of LH receptors is exerted at sites distal to those involved in cAMP generation. The effect of PRL on LH receptor messenger RNA (mRNA) levels was not significant during the increase in receptors, whereas after the maximal level of receptor expression was reached, the effect of PRL was apparent. Cotreatment with FSH (30 ng/ml) and increasing doses of PRL inhibited the levels of FSH-induced LH receptor mRNA in a dose-dependent manner, whereas PRL did not inhibit the effect of FSH on the FSH receptor mRNA. To investigate the hormonal regulation of the 5'-flanking region, we analyzed the effect of FSH on 1379 bp of LH receptor promoter in rat granulosa cells. Treatment with FSH (1–100 ng/ml) significantly enhanced the activity of 1379 bp of the LH receptor 5'-flanking region in dose-dependent manner. Treatment with 30 ng/ml PRL alone did not significantly influence the activity of the LH receptor promoter and did not affect the increased promoter activity induced by FSH. In addition, the rates of LH receptor mRNA gene transcription assessed by nuclear run-on transcription assay increased by the addition of FSH and were not affected by the addition of PRL in the presence of FSH. These data showed that PRL might not effect LH receptor gene transcription in the regulation of LH receptor mRNA. Next, an attempt was made to determine the effect of PRL on LH receptor mRNA stability by measuring the decay of LH receptor mRNA under conditions known to inhibit transcription. However, inhibitors of transcription were found to have a stabilizing effect on the LH receptor mRNA, thus potentially masking the effect of PRL. According to the expression of LH receptor mRNA, PRL might not affect the maximum level induced by FSH, but thereafter the maximum levels of LH receptor mRNA decreased faster than those of the control. Therefore, it may be possible that PRL acts to stimulate labile LH receptor mRNA-destabilizing factors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE INDUCTION of LH receptors in granulosa cells is an obligatory step for ovarian follicles to ovulate and develop into corpora lutea. Several studies have shown that the administration of FSH increases LH receptor numbers in rat granulosa cells through a protein synthesis-dependent event (1, 2, 3, 4).

Excessive secretion of PRL in physiological and pathological conditions, such as lactation and hyperprolactinemia, is a common cause of amenorrhea (5, 6). It has been well documented that in addition to the inhibitory effect of PRL on gonadotropic secretion at the hypothalamic-pituitary level, PRL exerts a direct inhibitory effect on gonadotropic action at the ovarian level (7, 8, 9, 10, 11). Excess PRL has been shown to inhibit gonadotropin-induced 17ß-estradiol secretion in a variety of preovulatory model systems (12, 13, 14, 15, 16, 17, 18, 19). This inhibition has been reported to be due to a reduction in aromatase activity (12, 14, 15, 19). However, the precise mechanism by which PRL alters the gonadotropin-induced steroidogenic capacity of granulosa cells remains undefined.

Recently, evidence has emerged to suggest that elevated PRL may exert its antigonadal effects through reducing available ovarian LH receptors. For example, the steroidogenic response of the gonads to gonadotropins is diminished in the face of PRL elevation in both sexes (20, 21); in women with hyperprolactinemia, higher doses of human menopausal gonadotropin and hCG are usually required for ovulation induction (22); infertile women undergoing ovarian hyperstimulation for in vitro fertilization frequently develop ovarian unresponsiveness, presumably due to a transient hyperprolactinemia (23, 24). Finally, Adashi et al. (25) showed that impaired ovarian function was associated with a significant reduction in available LH receptors. On the other hand, it has been reported that once induced, LH receptors are maintained by FSH (4, 10, 26), LH (4), or PRL (10, 26). These luteotropic actions of PRL in rats have been supported by data showing that PRL increased LH receptor messenger RNA (mRNA) levels in vitro (27). To ascertain whether PRL increases or decreases LH receptor mRNA induced by FSH in cultured granulosa cells, we analyzed FSH and LH receptor mRNA levels and their mechanisms of regulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Hormones and reagents
Rat FSH (I-8) and hCG (CR-119) were obtained from the National Hormone and Pituitary Distribution Program (Bethesda, MD). Diethylstilbestrol (DES), gentamicin sulfate, and 8-bromo-cAMP (8-Br-cAMP) were purchased from Sigma Chemical Co. (St. Louis, MO). DMEM, Ham’s F-12 medium, and fungizone were purchased from Life Technologies (Grand Island, NY). The RNA labeling kit and nucleic acid detection kit were purchased from Boehringer Mannheim (Mannheim, Germany).

Rat granulosa cell culture
Granulosa cells were obtained from immature female Wistar rats that received an injection of 2 mg DES in 0.1 ml sesame oil once daily for 4 days. The ovaries were then excised, and granulosa cells were released by puncturing follicles with a 25-gauge needle. At all times, the animals were treated as humanely as possible, following NIH guidelines. Granulosa cells were washed and collected by brief centrifugation, and cell viability was determined by trypan blue exclusion. The granulosa cells were then cultured in Ham’s F-12-DMEM (1:1, vol/vol) medium supplemented with 1.1 g/liter NaHCO3, 40 mg/liter gentamicin sulfate, 1 mg/liter fungizone, and 100 mg/liter BSA on collagen-coated plates in a humidified atmosphere containing 5% CO2 and 95% air at 37 C (28).

Preparation of complementary RNA (cRNA) probes
Rat FSH receptor complementary DNA (cDNA) was subcloned into the EcoRI site of the Bluescript KS+ vector and linearized with HindIII (28). Digoxigenin-labeled FSH receptor cRNA probes corresponding to bases 239-2368 were produced by in vitro transcription with T7 RNA polymerase and an RNA labeling kit (Boehringer Mannheim). Rat LH receptor cDNA was prepared as described previously and linearized with BglII (29). Digoxigenin-labeled LH receptor cRNA probes corresponding to bases 440-2560 were produced by in vitro transcription with T3 RNA polymerase and an RNA labeling kit (Boehringer Mannheim). A digoxigenin-labeled GAPDH probe was obtained by the same method.

RNA isolation and analysis
Granulosa cells were cultured in 60-mm dishes containing 5 x 106 viable cells in 5-ml of medium, and reagents were added to the medium after 24 h of cell culture. The granulosa cells were further incubated, and the cultures were stopped at the selected time as indicated for the guanidinium acid-thiocyanate-phenol-chloroform method (30). The final RNA pellet was dissolved in diethylpyrocarbonate-treated H2O. Total RNA was quantified by measuring the absorbance of samples at 260 nm. For Northern blot analysis, 15 µg total RNA from each dish were separated by electrophoresis on denaturing agarose gels and subsequently transferred to a nylon membrane (Biodyne, ICN Biomedicals, Inc., Glen Cove, NY). In accordance with the standard protocol for the nucleic acid detection kit (Boehringer Mannheim), Kodak X-Omat film (Eastman Kodak Co., Rochester, NY) was then exposed to the membranes. Luminescence detection was quantified with an LKB 2202 UnitroScan Laser Densitometer (LKB Produkter AB, Bromma, Sweden), normalized against a corresponding relative amount of GAPDH mRNA in each sample, and expressed as relative densitometric units.

cAMP assays
Granulosa cells (5 x 105 cells/culture dish) were washed with warm medium and then preincubated for 15 min at 37 C in 0.5 ml medium without serum in the presence of 0.5 mM 3-isobutyl-1-methylxanthine (Sigma Chemical Co.). Purified hormones were added to the dish, and the incubation was continued for 60 min at 37 C. After incubation, the medium was removed, and the cells were rinsed twice with PBS at 4 C and lysed with 0.5 ml 95% (vol/vol) ethanol. Aliquots of the resulting lysate were centrifuged at 4 C at 15,000 x g for 15 min. The supernatant was dried and resuspended in 0.3 M imidazole buffer, pH 6.5. Intracellular cAMP levels were determined by the double antibody RIA method. Triplicate plates were analyzed for each data point.

Receptor binding assay
Granulosa cells were cultured in Immulon-2 Removawell (Dynatech Corp., Chantilly, VA). Each well contained 1 x 105 viable cells in 0.1 ml medium. After 24-h incubation, hormone was added to the medium. At the times indicated, the cells were placed on ice and quickly washed three times with 0.2 ml cold medium. Then, the granulosa cells were incubated in a 1:1 (vol/vol) mixture of DMEM-Ham’s F-12 medium containing 0.1% BSA (pH 7.4) at 37 C with 5 x 104 cpm [125I]hCG (0.5 ng, 100,000 cpm/ng). hCG was iodinated according to the chloramine-T method. The incubation medium was removed after 2 h of incubation, and the cells were washed twice with 0.2 ml medium. Each well was then torn off from the Removawell strip, and the amount of radioactivity remaining in the well (cell-bound hormone) was quantified by {gamma}-spectrometry. Nonspecific binding was determined by adding excess unlabeled hCG (1.25 IU/well). Specific binding was analyzed by the method of Scatchard to determine the maximum binding capacity of the cells and the affinity of the binding sites for hCG.

Vector preparation and transfection
Plasmid pGL3-Basic is a luciferase vector lacking eukaryotic promotor and enhancer sequences (Promega Corp., Madison, WI). The pGL3-Control contains a simian virus 40 (SV40) promoter and a SV40 enhancer inserted into the structure of pGL3-Basic (Promega Corp.). The pRL-SV40 vector contains the SV40 early enhancer/promoter region, which provides strong, constitutive expression of Rluc in a variety of cell types. The pRL vector provides constitutive expression of Renilla luciferase. The rat LH receptor promotor from -1389 to -1 bp relative to the transcriptional initiation site was generated from genomic DNA via PCR using primers specific to the rat LH receptor sequence. For evaluating promotor activity, -1379 to -1 bp of the 5'-flanking sequence of the rat LH receptor promoter were ligated to a luciferase reporter vector (pGL3-Luc) and named LH receptor-Luc. Plasmid DNA was purified by alkaline lysis and centrifugation on two cesium chloride gradients as described previously (32). Using FuGENE 6 Transfection Reagent (Boehringer Mannheim, Mannheim, Germany), a total of 1 µg plasmid DNA was transfected, as described previously (33), into primary granulosa cell cultures plates (5 x 105 cells, 2 ml of that in a 35-mm dish). To assay regulatory elements, granulosa cells were cultured for 48 h in hormone-free conditions before transfection. Thirty hours after transfection, cells were treated with hormones for 6 h. After the incubation, cells were harvested, and luciferase activity was measured. In the luciferase assay, luciferin and Mg2+ ATP were added to cellular extracts, and the production of light was monitored conveniently by a luminometer. Luciferase activity was assayed as previously described (34).

Isolation of nuclei
Granulosa cells were cultured in 60-mm dishes containing 5 x 106 cells in 5 ml serum-free medium. After 24 h, granulosa cells were further incubated in the presence or absence of FSH (30 ng/ml) or FSH (30 ng/ml) plus PRL (30 ng/ml) for 24 h before isolating the nuclei. Cells were washed three times with ice-cold Dulbecco’s PBS without calcium and magnesium, collected by scraping in PBS without calcium and magnesium, and then centrifuged for 5 mm at 1000 rpm at 4 C. The cell pellet was resuspended in 500 µl Nonidet P-40 lysis buffer [10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, and 0.5% Nonidet P-40]. Lysed cells were incubated on ice for 10 min and centrifuged for 5 min at 3000 rpm.. The nuclear pellet was then resuspended in 500 µl Nonidet P-40 lysis buffer and centrifuged for 5 min. The final nuclear pellet was gently resuspended in 100 µl glycerol storage buffer [50 mM Tris-HCl (pH 8.3), 40% glycerol, 5 mM MgCl2, and 0.1 mM EDTA (pH 8.0)], frozen in liquid nitrogen, and stored at -80 C.

Run-on transcription assay
The nuclear run-on transcription assay was performed according to a previously described protocol (35). The relative amount of incorporation of label into specific RNAs was determined by DNA excess filter hybridization, as described previously (35), using cDNAs for rat LH receptor. Five micrograms each of LH receptor, Bluescript, and GAPDH cDNAs were included on the DNA filter during hybridization to correct for background and to serve as internal controls. Autoradiographic bands were quantified by a fluoroimage analyzer (BAS 2000, Fuji Photo Film Co., Ltd., Tokyo, Japan).

Data analysis
The relative abundance of a 2.4-kb signal for rat FSH receptor mRNA and that of a 5.4-kb signal for rat LH receptor mRNA in different preparations were quantified with a LKB 2202 UnitroScan Laser densitometer (LKB Produkter AB), normalized against levels of GAPDH mRNA in each sample, and expressed as a percentage of the control value (100%). The data are presented as the mean ± SE of measurements from triplicate cultures for one representative experiment. Comparisons between groups were performed by one-way ANOVA. The significance of differences between the mean values in the control group and each treated group was tested with Duncan’s multiple comparison test. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To examine the possible effect of PRL on the acquisition of LH receptors, granulosa cells were cultured in the presence of FSH (30 ng/ml) with or without PRL at 30 ng/ml for 168 h (Fig. 1Go). Basal specific LH binding was negligible and remained unchanged in response to treatment with PRL alone. Whereas treatment with FSH produced, as expected, a substantial increase in specific LH binding, concurrent treatment with PRL resulted in no significant change during the first 96 h of culture, followed by a significant decrease in LH binding on days 120 and 144 h as well as an approximately 60% inhibition of the FSH effect at 120 h.



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  		Figure 1. Time course of PRL’s effect on the FSH-induced LH receptor mRNA. Granulosa cells from DES-primed immature rats were cultured for 24 h, and then 30 ng/ml FSH, with or without 30 ng/ml PRL, were added. After various incubation times, the levels of LH receptor were determined by [125I]hCG binding assays. Values are the mean ± SE of quadruplicate determinations. This figure is representative of at least three different experiments. *, Difference from the control value at P < 0.05. A Scatchard plot (inset; B/F, bound to free ratio) is shown for rat granulosa cells cultured for 120 h in FSH (30 ng/ml) or FSH (30 ng/ml) plus PRL (30 ng/ml).

 
To determine whether the PRL-attenuated LH binding was due to decreased binding capacity, decreased binding affinity, or both, granulosa cells were cultured in the presence of FSH with or without PRL at the 30 ng/ml dose level for 5 days. Scatchard plot analysis showed that concurrent treatment with PRL resulted in 63% inhibition of the granulosa cell LH binding capacity, whereas no difference could be detected in the binding affinity of its receptors (mean ± SE Kd: FSH, 3.45 ± 0.34 x 10-11; FSH plus PRL, 3.62 ± 0.32 x 10-11). These findings suggest that the ability of PRL to attenuate the LH binding of FSH-stimulated granulosa cells is attributable to a decrease in binding capacity, but not to the affinity of the LH receptor. These observations also indicate that the effect of PRL represents a partial blockade of the inductive potential of FSH rather than an alteration of the binding characteristics of the LH receptors.

To further characterize the cellular mechanisms underlying the interaction between PRL and FSH, we next investigated the possibility that the PRL-attenuated FSH action may involve cAMP production. Treatment with 8-Br-cAMP for 72 h produced a significant increase in specific LH binding, whereas concurrent treatment with PRL (30 ng/ml) produced a significant (P < 0.05) attenuation of 8-Br-cAMP (2 mM) action (Fig. 2Go). Although treatment with FSH increased the intracellular accumulation of cAMP, concurrent treatment with PRL did not result in a significant inhibition of FSH action, as assessed by the generation of intracellular cAMP (Table 1Go). Moreover, concurrent treatment with PRL had no effect on the FSH receptor mRNA (Fig. 3Go). Because it has been reported that FSH increases the expression of its own receptors via cAMP signals, the lack of a detectable effect of PRL on FSH receptor mRNA agrees well with the result that PRL did not show any effect on FSH-induced cAMP accumulation. Taken together, these findings suggest that the ability of PRL to interfere with FSH action with regard to the induction of LH receptors is exerted at sites distal to those involved in cAMP generation.



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  		Figure 2. Effect of PRL on 8-Br-cAMP-induced LH receptor. Granulosa cells were stimulated by 8-Br-cAMP with or without 30 ng/ml PRL. After 96 h, the levels of LH receptor were determined by [125I]hCG binding assays. Values are the mean ± SE of quadruplicate determinations. This figure is representative of at least three different experiments. *, Difference from the control value at P < 0.05.

 

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  		Table 1. Effect of FSH and PRL on intracellular cAMP accumulation

 


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  		Figure 3. Dose-related effect of PRL on FSH-induced FSH receptor mRNA. Granulosa cells from DES-primed immature rats were cultured alone for 24 h (0, control, 0 h). These cells were then further incubated with 30 ng/ml FSH alone and with a combination of FSH (30 ng/ml) plus increasing concentrations of PRL. FSH-R mRNA levels were measured using Northern blot analysis as described in Materials and Methods. This figure is representative of at least three different experiments.

 
The number of LH receptors in granulosa cells from immature rats is increased by FSH in a time- and dose-dependent manner. Therefore, in the presence of FSH, the decrease in FSH receptor levels may result in LH receptor expression. We next examined the effect of PRL on FSH-induced FSH receptor mRNA. Although PRL did not inhibit the effect of FSH on FSH receptor mRNA, as shown in Fig. 3Go, Fig. 4Go shows that cotreatment with FSH (30 ng/ml) and increasing doses of PRL inhibited the levels of FSH-induced LH receptor mRNA in a dose-dependent manner. As shown in Fig. 5Go, PRL treatment reduced the amount of LH receptor mRNA significantly at 96 h. As can also be seen for this time course, the effect of PRL on mRNA levels was not significant during the increase in receptors, whereas after the maximal levels of expression of receptors, the effect of PRL was significant.



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  		Figure 4. Dose-related effect of PRL on FSH-induced LH receptor mRNA. A, Granulosa cells from DES-primed immature rats were cultured alone for 24 h (0, control, 0 h). These cells were then further incubated without FSH, with 30 ng/ml FSH alone, and with a combination of FSH (30 ng/ml) plus increasing concentrations of PRL. LH receptor mRNA levels were measured using Northern blot analysis as described in Materials and Methods. The Northern blot is representative of four experiments. B, Autoradiographs of LH receptor mRNA (5.4 kb) was quantified by densitometric scanning. The amount of LH receptor mRNA with FSH alone was taken as 100%. Data were normalized for GAPDH mRNA levels in each sample and expressed relative to the control value. The absorbance values obtained from this study as well as those from three other studies were standardized to the control and are represented (mean ± SE; n = 4) in the bar graphs. *, Difference from the control value at P < 0.05. **, Difference from the control value at P < 0.01.

 


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  		Figure 5. Time course of PRL’s effect on the FSH-induced LH receptor mRNA. A, Granulosa cells from DES-primed immature rats were cultured alone for 24 h (0, control, 0 h). These cells were then further incubated with 30 ng/ml FSH alone and with a combination of FSH (30 ng/ml) plus 30 ng/ml PRL. After various incubation times, total RNA was extracted, and LH receptor mRNA levels were measured using Northern blot analysis as described in Materials and Methods. The Northern blot is representative of three experiments. B, Autoradiographs of LH receptor mRNA (5.4 kb) were quantified by densitometric scanning. The amount of LH receptor mRNA with FSH alone was taken as 100%. Data were normalized for GAPDH mRNA levels in each sample and expressed relative to the control (FSH alone, 48 h) value. The absorbance values obtained from this study as well as those from three other studies were standardized to the 48 h control and are represented (mean ± SE; n = 3) in the graph. *, Difference from the control value at P < 0.05.

 
We next examined whether PRL regulation of LH receptor mRNA is dependent on gene transcription and/or receptor mRNA stability. The following set of experiments was designed to address the potential contributions made by changes in LH receptor gene transcription to the regulation of LH receptor mRNA. To determine whether the LH receptor 5'-flanking region plays a role in directing LH receptor mRNA expression, the proximal 1379 bp of the LH receptor 5'-flanking region was inserted into a transient expression vector, pGL3-Basic, which contains luciferase as the reporter gene, and the resulting plasmid (LH receptor-Luc) was transiently transfected into rat granulosa cells. Gene transfer studies were performed using FuGENE transfection, and luciferase enzyme activity was measured in light units as an indication of promoter activity. Cells were cotransfected with pRL as an internal control for transfection efficiency. To investigate the hormonal regulation of the 5'-flanking region, we analyzed the effect of FSH on 1379 bp of LH receptor promoter in rat granulosa cells. Treatment with FSH (1–100 ng/ml) significantly enhanced the activity of 1379 bp of the LH receptor 5'-flanking region in a dose-dependent manner (Fig. 6AGo). As shown in Fig. 6BGo, treatment with 30 ng/ml PRL alone did not significantly influence the activity of the LH receptor promoter and did not affect the increased promoter activity induced by FSH.



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  		Figure 6. Effect of PRL on FSH-induced expression of LH receptor-Luc in rat granulosa cells. A, Granulosa cells were cultured for 48 h in hormone-free conditions and cotransfected with LH receptor-Luc and pRL. After transfection 30 h later, cells were treated by FSH for 6 h and processed. Luciferase activity was corrected for the amount of Renilla luciferase activity detected in each lysate. Each bar represents the mean ± SE of three independent experiments. B, After transfection 30 h later, cells were treated with FSH and 30 and 100 ng/ml PRL for 6 h, then processed. Luciferase activity was corrected for the amount of Renilla luciferase activity detected in each lysate. Each bar represents mean ± SE of three independent experiments.

 
To test whether PRL has an effect on LH receptor gene transcription in the presence of FSH, we performed nuclear run-on assays on granulosa cells. As shown in Fig. 7Go, although FSH 30 ng/ml significantly enhanced the activity of LH receptor gene transcription, no difference in LH receptor gene transcription was observed in granulosa cells treated with FSH vs. those treated with FSH plus PRL. These data, therefore, suggest that the effects of PRL on LH receptor induction are not mediated by a decrease in LH receptor gene transcription.



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  		Figure 7. Effect of PRL on FSH-induced LH receptor gene transcription. A, Granulosa cells were cultured in 60-mm dishes containing 5 x 106 cells in 5 ml serum-free medium. After 24 h in culture, granulosa cells were further incubated with 30 ng/ml FSH alone or 30 ng/ml FSH and 30 ng/ml PRL for 24 h, and nuclear run-on assays were then performed as described inMaterials and Methods. B, Data acquired from the nuclear run-on experiments shown in A were quantitated by a fluoroimage analyzer (BAS 2000). Data were normalized for glyceraldehye-3-phosphate dehydrogenase levels in each sample and are expressed relative to the control (cont) value. Transcriptional activities are expressed relative to the activity in the control. The data shown are the mean ± SE of three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our present findings provide yet another example of the inhibitory nature of PRL as it acts in the stages of folliculogenesis (36). PRL was previously shown to inhibit progesterone biosynthesis by porcine (12), murine (13, 17, 37, 38, 39), and human (7, 40, 41) granulosa cells, an effect critically dependent on their degree of differentiation (12, 40). Our present observations thus complement previous findings regarding the inhibitory effect(s) of PRL at the level of the ovary, yet point, for the first time, to an additional facet of PRL action on LH receptor mRNA levels.

In this culture system, FSH treatment induced LH receptor mRNA in a dose- and time-dependent manner. Although treatment of cells with PRL alone had no effect, cotreatment of cells with FSH and PRL reduced FSH-induced LH receptor mRNA levels. Granulosa cells from preantral follicles have a negligible number of LH-binding sites, whereas priming with FSH induced functional LH and PRL receptors (1, 2, 3, 4). Once induced, LH receptors are maintained by FSH and LH (4, 10, 26). The maintenance of rat LH receptors by FSH and LH is consistent with the luteotropic action of LH in rats and indicates the important role played by LH receptors in corpora luteal development.

The present observations of stimulatory effects of FSH on granulosa LH receptor mRNA levels in vitro suggest that the previously observed increases in LH binding induced by these hormones are closely related to the regulation of LH receptor mRNA levels. As the actions of LH and FSH on LH receptor numbers are mediated through the protein kinase A pathway (10, 42), the present observation of induction and maintenance of LH receptor message levels by FSH presumably resulted from activation of the same second messenger system. In contrast to the LH receptor mRNA, PRL in combination with FSH had no effect on FSH receptor mRNA levels, which are also maintained by cAMP. Therefore, the result that PRL did not affect cAMP production is not in conflict with the data that FSH receptor mRNA did not decrease by PRL treatment. In addition, as PRL did not inhibit cAMP production, PRL can be considered to specifically affect the production of LH receptor mRNA. The present observations, like those of Dorrington and Gore-Langton (37), further indicate that the ability of PRL to attenuate FSH hormonal action also involves a post-cAMP site(s). The observed maintenance of message levels by these gonadotropins may be a result of increased LH receptor gene transcription and/or message stability. Our results showed that the suppression of LH-binding sites by PRL was correlated with the decreases in receptor mRNA levels. Additional trials were therefore required to further clarify the mechanisms of PRL action in LH receptor regulation. In these experiments, the promoter region of the LH receptor gene provided an interesting model for analyzing the mechanisms involved in the inhibitory actions of PRL. Previous studies have revealed that the 5'-flanking region of the rat LH receptor gene is characteristic of that in the so-called housekeeping genes, in that the region has no apparent TATA or CAAT boxes, is rich in G and C residues, and contains multiple potential transcriptional start sites (43). Interestingly, there are no clear cAMP or estrogen response elements within the 2.1 kb of the sequenced LH receptor gene 5'-flanking region (43). Both FSH and cAMP have also been shown to increase the transcription of other genes, including the aromatase, {alpha}-inhibin, in rat granulosa cells. Hormonal regulation of the aromatase mRNA and LH receptor mRNA occurs via a similar mechanism in rat ovaries (44). Recently, a hexameric motif, AGGTCA, that confers cAMP inducibility was identified within the rat aromatase promoter (45, 46). Because LH receptor-Luc includes this sequence, it is possible that LH receptor-Luc also responds to FSH stimulation in a dose-dependent manner. On the other hand, although FSH administration increased the transcription of the LH receptor gene in a dose-dependent manner, PRL did not have any effect on FSH-induced transcription. Data from the nuclear run-on assays demonstrated that the effect of PRL on the decrease of FSH-induced LH receptor mRNA is not brought about by transcriptional mechanisms.

Based on the observed expression of LH receptor mRNA, PRL might not affect the maximum level induced by FSH, but thereafter the maximum levels of LH receptor mRNA disappear faster than those in the control. Therefore, the half-life of FSH receptor and LH receptor mRNA were examined, and no significant effect of PRL on FSH receptor mRNA was detected; however, we were unable to estimate the half-life of LH receptor mRNA in the same experiment. In previous experiments, the cells were incubated in the absence or presence of inhibitors of transcription (47), and in the absence of transcription inhibitors, LH receptor mRNA decreased rapidly. These data further show that actinomycin D prevented the rapid decline in LH receptor mRNA. To ensure that this effect of actinomycin D was a consequence of inhibition of transcription rather than due to some unknown nonspecific effect of actinomycin D, {alpha}-amanitin, an agent that inhibits transcription via a distinct mechanism from actinomycin D (48), was also tested and showed the same effect as actinomycin D. These results suggest that there is normally a labile factor(s) that destabilizes the LH receptor mRNA and that inhibition of its synthesis results in an increased stability of LH receptor mRNA. It may be that PRL also acts to stimulate the labile LH receptor mRNA-destabilizing factors. However, standard methodologies for examining the half-life of the LH receptor mRNA might be unable to determine whether PRL affects LH receptor mRNA stability. Thus, examination of the rate of decay of LH receptor mRNA in the presence or absence of PRL when an inhibitor of transcription is present does not reveal a difference in the stability of the message, most likely due to the effect of the transcription inhibitor of LH receptor mRNA. Further studies will be required to determine what effect PRL has on LH receptor mRNA stability.

In summary, our studies present evidence for the contribution of increased LH receptor gene transcription to the induction of LH receptor and LH receptor mRNA by FSH in rat granulosa cell. Although PRL did not result in an inhibition of FSH action as assessed by the generation of FSH receptor mRNA, PRL decreased LH receptor mRNA at sites distal to those involved in cAMP generation. This study provides information regarding the mechanisms by which PRL inhibits the expression of LH receptor; however, many questions remain regarding this important phenomenon.


   Acknowledgments
 
We thank the National Hormone and Pituitary Agency, NIDDK, University of Maryland School of Medicine for the rat FSH.


   Footnotes
 
1 This work was supported by grants from the Ministry of Education, Science and Culture of Japan, Tokyo, Japan (no. 10044235, 09470353, and 10877253). Back

2 Supported by fellowships from the Japan Society for the Promotion of Science for Japanese Junior Scientists. Back

Received November 23, 1998.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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