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Biology of Reproduction 67, 610-615 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Effect of Transforming Growth Factor ß on the Expression of Luteinizing Hormone Receptor in Cultured Rat Granulosa Cells1

Kyoko Inouea, Kazuto Nakamuraa,c, Kazuko Abea, Takashi Hirakawaa, Megumi Tsuchiyaa, Hiroko Matsudaa,c, Kaoru Miyamotob,c, and Takashi Minegishi2,,a,c

a Department of Obstetrics and Gynecology, School of Medicine, Gunma University, Maebashi, Gunma 371-8511, Japan b Department of Biochemistry, Fukui Medical University, Matsuoka, Fukui 910-1193 and c CREST, JST (Japan Science and Technology Corporation), Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The present study was undertaken in order to identify the mechanism underlying the effect of transforming growth factor-ß (TGFß) on LH receptor (LH-R) expression in rat granulosa cells. Treatment with FSH produced a substantial increase in LH-R mRNA level, and concurrent treatment with increasing concentrations of TGFß brought about dose-dependent increases in FSH-induced LH receptor mRNA. TGFß, either alone or in combination with FSH, did not affect intracellular cAMP levels. We then investigated whether the effect of TGFß and FSH on LH-R mRNA levels results in increased transcription and/or altered mRNA stability. To determine whether the LH receptor 5'-flanking region plays a role in directing LH receptor mRNA expression, the proximal area of the LH receptor 5'-flanking regions were inserted into an expression vector, pGL-Basic, which contains luciferase as the receptor gene, and the resulting plasmids were transiently transfected into rat granulosa cells. FSH (30 ng/ml) significantly enhanced the activity of 1389 base pairs of the LH receptor 5'-flanking region, but treatment with TGFß did not significantly influence the activity induced by FSH. On the other hand, the decay curves for LH-R mRNA transcript in primary granulosa cells showed a significant increase in half-life after the addition of TGFß.

follicle-stimulating hormone, granulosa cells, growth factors, luteinizing hormone, ovary


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The pituitary gonadotropins are key hormones in the regulation of folliculogenesis. It is clear that the ability of gonadotropins to modulate ovarian function depends not only on the circulating levels of the gonadotropins but also on the expression of appropriate receptor proteins by potential target cells in the ovary. FSH and LH act through stimulatory G protein-coupled receptors expressed on target cells and transduce their signal at least in part by the activation of adenylyl cyclase and the production of second messenger cAMP. The expression of receptors for LH is one of the major markers of the FSH-induced differentiation of granulosa cells [1].

Primary cultures of rat granulosa cells obtained from immature female rats pretreated with estradiol are a frequently used model for studying cell differentiation. Using this defined model, the function of FSH to stimulate FSH and LH receptor induction has been shown to be mediated at least in part by cAMP, as exogenous cAMP or other agents that increase intracellular levels of cAMP mimic the action of FSH [2–6].

Transforming growth factor ß (TGFß) is recognized as a physiological mediator of the growth and differentiation of various cell types [7]. The negative effect of TGFß on steroidogenesis has been observed in testicular Leydig cells [8]. TGFß has also been proposed as an autocrine regulator of adrenocortical steroidogenesis, acting mainly by decreasing the expression of cytochrome P450c17 [9]. TGFß can decrease the steady-state level of steroidogenic acute regulatory gene (StAR) mRNA in a time- and concentration-dependent manner [10]. Using rat granulosa cell cultures, the present study also investigates the effect of TGFß on FSH-induced LH receptor mRNA expression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Hormones and Reagents

Rat FSH (I-8) was obtained from the National Hormone and Pituitary Distribution Program (Bethesda, MD). Diethylstilbestrol (DES) and gentamicin sulfate were purchased from Sigma Chemical Co. (St. Louis, MO). TGFß was purchased from Pepro Tech EC Ltd. (St. James' Square, London). Dulbecco modified Eagle medium (DMEM), Ham F-12 medium, and fungizone were purchased from Gibco Laboratories (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, which received an injection of 2 mg diethylstilbestrol in 0.2 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 National Institute of Health 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 F-12/DMEM (1:1 v/v) medium supplemented with 1.1 g/L NaHCO3, 40 mg/L gentamicin sulfate, 1 mg/L fungizone, and 100 mg/L BSA on collagen-coated plates in a humidified atmosphere containing 5% CO2, 95% air at 37°C [11].

Cyclic AMP 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-isobuty-1-methylxanthine (Sigma). 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% (v/v) ethanol. Aliquots of the resulting lysate were centrifuged 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 radioimmunoassay method [12]. Triplicate plates were analyzed for each data point.

Receptor Binding Assay

Granulosa cells were cultured in Immulon-2 Removawell plates (Dynatech Laboratories, Inc., Chantilly, VA). Each well contained 1 x 105 viable cells (determined by trypan blue exclusion) in 0.1 ml medium. After 24 h of 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 of cold medium. Then the granulosa cells were incubated in a 1:1 (v/v) mixture of DMEM/Ham 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). The hCG was iodinated according to the chloramine-T method [13]. 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 ß-spectrometry. Nonspecific binding was determined by adding excess unlabeled hCG (1.25 IU/well; Pregnyl, Organon Pharmaceuticals, Oss, The Netherlands).

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 in the guanidinium acid-thiocyanate-phenol-chloroform method [14]. The final RNA pellet was dissolved in diethyl pyrocabonate-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, Glen Cove, NY). Rat LH receptor cDNA was prepared as described previously and linearized with Bgl II [15]. 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 glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe was obtained by the same method. In accordance with the standard protocol for the nucleic acid detection kit (Boehringer Mannheim), Kodak X-Omat film (Eastman Kodak, 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.

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 fragment of the 5'-flanking region of -1389 to -1 base pairs (bp) relative to the translational initiation site was generated from genomic DNA via polymerase chain reaction (PCR) using primers specific to the rat LH receptor sequence. The cDNA was amplified 30 cycles by PCR containing Taq DNA polymerase and 1 µM each of the two primers at 94°C for 15 sec, 50°C for 30 sec, and 72°C for 3 min in each amplification cycle. Primers for the LH receptor were 5'-CAGAACCCGGGCCTAGTGAGCTAAG and 3'-TGTGAGTCCGACCGCCCGGTCTAGAGTGT. The isolated PCR-synthesized cDNA fragments were subcloned into T easy and characterized by nucleotide sequencing analysis. The sequence of the cDNA fragment was identical to the published sequence of rat LH receptor cDNA [16].

For evaluating promotor activity, these fragments were ligated to a luciferase reporter vector (pGL3-Basic) and named LH-R (1389)-Luc. The luciferase assay was performed by using the Dual-Luciferase Reporter System (Promega Corp.), in which the transfection efficiency was monitored by cotransfected pRL-CMV-Rluc, an expression vector of renilla luciferase.

Transient Transfection

Plasmid DNA was purified by alkaline lysis and centrifugation on two cesium chloride gradients as described previously [17, 18]. Using FuGENE, a total of 0.25 µg of plasmid DNA were transfected, as described previously [19], into primary granulosa cell cultures plates (2.5 x 105 cells, 0.5 ml of that in a 20-mm dish). To assay regulatory elements, granulosa cells were cultured 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. The cells were lysed in lysis buffer supplied by the manufacturer followed by measurement of the firefly and the renilla luciferase activities using a luminometer. The relative firefly luciferase activities were calculated by normalizing transfection efficiency according to the renilla luciferase activities. The experiments were performed in triplicate, and similar results were obtained from at least three independent experiments. In the luciferase assay, luciferin and Mg2+ ATP were added to cellular extracts, and the production of light was monitored conveniently using a luminometer. Luciferase activity was assayed as previously described [20].

Transcription Stability Analysis

Cells were preincubated with FSH alone or FSH and TGFß for 24 h before the addition of 5 µM actinomycin-D to arrest new RNA synthesis. Cells were harvested for RNA extraction and Northern blot analysis at 0, 3, 6, and 9 h after the addition of the inhibitor.

Data Analysis

The relative abundance of a 5.4-kilobase (kb) rat LH receptor mRNA in different preparations was quantified with a LKB 2202 UnitroScan Laser Densitometer (LKB Produkter AB, Bromma, Sweden), 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 ± SEM 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 were tested with Duncan multiple comparison test. A value of P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
To examine the possible effect of TGFß on the acquisition of LH receptors, granulosa cells were cultured in the presence of FSH (30 ng/ml) with or without TGFß at 10 ng/ml for 120 h (Fig. 1). Basal specific LH binding was negligible and remained unchanged in response to treatment with TGFß alone (data not shown). Treatment with FSH produced, as expected, a substantial increase in specific LH binding, and concurrent treatment with TGFß resulted in significant change compared with that of FSH alone except in the first 24 h of culture.



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  		FIG. 1. Time course of TGFß‘s effect on the FSH-induced LH-R. Granulosa cells were stimulated by FSH (30 ng/ml) with or without TGFß (10 ng/ml). After various incubation times, the level of LH-R was determined by 125I-hCG binding assays. Values are the mean ± SEM of quadruplicate determinations. This figure is representative of at least three different experiments. *Difference from the control (FSH alone) at each time, P < 0.01

We next examined the effect of TGFß on FSH-induced LH receptor mRNA. Granulosa cells were cultured for 24–96 h in the presence of FSH (30 ng/ml) with or without TGFß at a concentration of 10 ng/ml (Fig. 2). Basal LH receptor mRNA levels remained low throughout the 96-h incubation period and were not significantly affected by treatment with TGFß alone (data not shown). The concurrent treatment with TGFß resulted in significant augmentation of the FSH-induced LH receptor mRNA for all time points studied. In addition, granulosa cells were cultured in the absence or presence of FSH (30 ng/ml) with or without increasing concentrations of TGFß (0.1–10 ng/ml) (Fig. 3). Levels of basal LH receptor mRNA were negligible and remained unchanged in treatment with TGFß by itself. In contrast, treatment with FSH expectedly produced a substantial increase in LH receptor mRNA level, and concurrent treatment with increasing concentrations of TGFß brought about dose-dependent increases in FSH-induced LH receptor mRNA.



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  		FIG. 2. Time course of TGFß‘s effect on the FSH-induced LH-R mRNA. A) Granulosa cells from DES-primed immature rats were cultured alone for 24 h. These cells were then further incubated without FSH, with FSH (30 ng/ml), and with a combination of FSH (30 ng/ml) plus TGFß (10 ng/ml). After various incubation times, total RNA was extracted and LH-R mRNA levels were measured using Northern blot analysis as described in Materials and Methods. B) Luminescence detection of LH-R mRNA (5.4 kb) was quantified by densitometric scanning. The amount of LH-R mRNA with FSH alone (24 h) was taken as 100%. Data were normalized for GAPDH mRNA levels in each sample and expressed as a value relative to the control. The absorbance values obtained from this experiment as well as those from two other experiments were standardized in relation to the control and are represented (mean ± SEM; n = 3) in the bar graphs. The data shown are means ± SEM of the three independent experiments. *Difference from the control (FSH alone) at each time, P < 0.01



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  		FIG. 3. Dose-related effect of TGFß on FSH-induced LH-R mRNA. A) Granulosa cells from DES-primed immature rats were cultured for 24 h alone and were then cultured with 30 ng/ml FSH alone and then FSH (30 ng/ml) plus increasing concentrations of TGFß for 48 h. LH-R mRNA levels were measured using Northern blot analysis, as described in Materials and Methods. The Northern blot analysis is representative of the three experiments. B) Luminescence detection of LH-R (5.4 kb) mRNA was quantified by densitometric scanning. The amount of LH-R mRNA with FSH alone was taken as 100%. Data were normalized for GAPDH mRNA levels in each sample and expressed as a value relative to the control. The absorbance values obtained from this experiment as well as those from the two other experiments were standardized in relation to the control values and are represented (mean ± SEM; n = 3) in the bar graphs. The data shown are means ± SEM of three independent experiments. *Difference from the control (FSH alone) value, P < 0.05

It is known that the action of FSH is mediated by cAMP and that a considerable amount of cAMP is accumulated in granulosa cells. It has been shown that FSH increased intracellular cAMP levels; however, TGFß either alone or in combination with FSH did not have a significant effect on the intracellular cAMP levels (Table 1).


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  		TABLE 1. Effect of TGFß on FSH-induced intracellular cAMP accumulation in rat granulosa cells (means ± SD)

We next examined whether TGFß regulation of LH-R mRNA was dependent on gene transcription and/or receptor mRNA stability. In a previous experiment, a similar region of the rat LH receptor gene was studied and well analyzed; therefore, we selected the proximal 1389 bp of the LH-R 5'-flanking region for this experiment [16]. Rat granulosa cells transiently transfected with luciferase-reporting plasmids (LH-R[1389]-Luc) responded to FSH (30 ng/ml) with significantly enhanced activity of this reporting activity. However, treatment with 10 ng/ml TGFß alone did not significantly influence the activity of the LH-R promotor nor affect the increased promotor activity induced by FSH (Fig. 4).



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  		FIG. 4. Effect of FSH and TGFß on LH-R-Luc expression in rat granulose cells. Granulosa cells from DES-primed immature rats were cultured for 48 h in hormone-free conditions, then cotransfected with LH-R (1389)-Luc and pRL. Thirty hours after transfection, cells were treated with FSH (30 ng/ml) with or without 10 ng/ml of TGFß for 6 h and were then processed. Luciferase activity was corrected for the amount of renilla luciferase activity detected in each lysate. Each bar represents means ± SEM of three independent experiments

In order to assess the degradation rates of LH-R mRNA transcripts, granulosa cells were preincubated for 24 h with FSH alone or with FSH and TGFß. After this preincubation period, 5 µM actinomycin-D was added to arrest new RNA synthesis. Cells were harvested at 0, 3, 6, and 9 h after addition of the transcription inhibitor, and LH-R mRNA levels were quantified by Northern blot analysis. The amount of LH-R mRNA at Time 0 (the time of addition of actinomycin-D) in each treatment group was assigned a value of 100% and other values in each treatment group at different time points were expressed as a percent of the Time 0 value. As shown in Figure 5, the decay curves for the 5.4-kb LH-R mRNA transcript in primary granulosa cells were significantly altered in the presence of TGFß and increased the half-life of the LH-R mRNA transcript.



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  		FIG. 5. Effect of TGFß on LH-R mRNA transcripts. A) Granulosa cells were preincubated with FSH alone or FSH and TGFß for 24 h. After this preincubation period, 5 µM actinomycin-D was added to arrest new RNA synthesis. Cells were harvested at 0, 3, 6, and 9 h after the addition of the transcription inhibitor and LH-R mRNA levels were quantitated by Northern blot analysis. B) The mRNA levels at Time 0 were assigned a relative value of 100%, and mRNA levels at all other times are expressed as percentages of these values


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Other laboratories have demonstrated the presence of TGFß in adult ovaries of different species, and it seems to be involved closely in the process of oocyte development [21, 22]. Our results indicate that TGFß is capable of synergizing with FSH in the expression of LH-R mRNA in a dose- and time-dependent manner. Although the addition of TGFß did not increase levels of FSH-induced intracellular cAMP, the response to cAMP analogs is enhanced in granulosa cells in this experiment (data not shown), suggesting that TGFß potentiates the action of FSH at sites distal to cAMP generation in these rat cells. It has been shown that, when levels of LH-R mRNA are altered, all the transcripts (7.5, 5.4, 3.8, 2.3, and 1.2 kb) appear to be coordinately regulated. These results suggest that the turnover of the receptor transcripts could be occurring via a common mechanism. The observed maintenance of message levels by FSH and TGFß may be a result of increased LH-R gene transcription and/or message stability. The 5'-flanking region of the rat LH-R gene is very similar to the same region in the so-called housekeeping genes. This region has no apparent TATA or CAAT boxes, is rich in G and C residues, and contains multiple potential transcriptional start sites [16]. In this experiment, FSH significantly enhanced the activity of the 1389-bp LH-R 5'-flanking region, but the treatment with TGFß alone influenced neither the activity of the LH-R promoter nor the increased promoter activity induced by FSH. Thus, we may suggest that TGFß had no effect on the interaction between FSH and LH-R-Luc in this experiment, though it may be possible that TGFß does have an effect on the further 5' end of the LH-R gene. Further studies will be required to clarify this point.

The data presented suggest a possible role for changes in LH-R mRNA stability in the TGFß-induced regulation of LH-R in rat granulosa cells. Previous observations have suggested that a labile destabilizing factor may constitutively degrade LH-R mRNA [23]. Our data support the concept that TGFß may act to increase LH-R mRNA by preventing the synthesis or actions of an LH-R mRNA-destabilizing factor in the presence of FSH. Expression of specific, highly regulated mRNAs like c-fos, c-myc, and ß-adrenergic receptor are controlled, at least in part, at the level of mRNA degradation [24, 25]. Changes in stability of a particular mRNA often appear to result from changes in the binding of specific proteins to defined sequences and/or structures in the target mRNA. The RNA sequences recognized by regulatory proteins are often located within discrete regions of the mRNA. An LH-R mRNA binding protein, which is a candidate for a transacting factor involved in the hormonal regulation of LH-R mRNA stability in the rat ovary, has been reported [26, 27]. In the rat ovary, differences in LH-R number seen during follicular development, ovulation, and luteinization involve concomitant changes in receptor mRNA levels [28]. Since levels of LH-R mRNA closely parallel receptor number, it is likely that posttranscriptional regulation plays a very important role in mediating physiological changes in receptor expression during the ovarian cycle. TGFß enhances the expression of LH-R only in the presence of FSH, while TGFß alone does not induce LH-R mRNA. Therefore, it is tempting to speculate from these observations that TGFß has a primary effect on FSH receptor expression that secondarily potentiates FSH action and results in augmentation of LH-R mRNA. Because expression of LH-R is one of the major markers of the FSH-induced differentiation of granulosa cells and is essential for the response to LH increase for the induction of ovulation, the pathways might be interfered with at the level of LH-R expression. As we show in this article, TGFß has a direct effect on LH-R mRNA half-life in granulosa cells; therefore, it is possible that TGFß is essential for maintaining a critical level of LH-R expression in the ovary for the sake of ovulation.

Members of the TGFß superfamily bind to a distinctive combination of type I and type II serine/threonine kinase receptors and transduce signals through the phosphorylation of specific Smad proteins to alter transcription [29, 30]. The appearance of TGFß expression in granulosa cells at the time of antrum formation may be correlated with its ability to modulate gonadotropin receptor expression and enhance steroidogenic activity [31–33]. The expression of TGFß in granulosa cells bordering the basement membrane of large healthy follicles indicates that these are the follicles that will eventually luteinize. This is consistent with the expression of the luteal marker enzyme 3ß-hydroxysteroid dehydrogenase, which was colocalized in the same cells that expressed TGFß in the marmoset ovary [34]. It has also been reported that TGFß was localized in oocytes of all follicle stages and in the theca and granulosa cells of large antral follicles [35]. The conversion of cholesterol to pregnenolone is the rate-determining step in granulosa cell steroidogenesis. The rate of pregnenolone synthesis depends on the level and activity of the reaction-catalyzing enzyme, P450scc, and its access to its substrate cholesterol via stimulation of StAR [36]. It is well established that FSH- and LH-induced increases in intracellular cAMP, leading to the subsequent stimulation of P450scc and StAR mRNA synthesis and StAR protein phosphorylation, stimulate progesterone synthesis in vitro [37–39].

Taken together, these data indicate that TGFß plays an important role in the induction of LH-R expression in the presence of FSH. Moreover, the increase of LH-R expression might be correlated with the augmented effect of LH on steroidogenesis in granulosa cells. Members of the TGFß superfamily bind to a distinctive combination of type II and type I serine/threonine kinase receptors and transduce signals through phosphorylation of specific Smad proteins to alter transcription [29, 30]. Identification and characterization of the intracellular Smad signal transduction cascade and the downstream target genes for TGFß in granulosa cells remain to be further clarified.


   ACKNOWLEDGMENTS
 
We thank the National Hormone and Pituitary Agency, National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, University of Maryland School of Medicine, for the rat FSH.


   FOOTNOTES
 
First decision: 25 January 2002.

1 Supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (13470344), Tokyo, Japan, and by Fellowships of the Japan Society for the Promotion of Science for Japanese Junior Scientists (M.T.). Back

2 Correspondence. FAX: 81 27 220 8443; tminegis{at}showa.gunma-u.ac.jp Back

Accepted: March 18, 2002.

Received: December 27, 2001.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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