0013-7227/91/1284-2160$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 128, No. 4 Printed in U.S.A.
Epidermal Growth Factor Directly Stimulates Steroidogenesis in Primary Cultures of Porcine Leydig Cells: Actions and Sites of Action* C. SORDOILLETt, M. A. CHAUVIN, E. DE PERETTI, A. M. MORERA, AND M. BENAHMED INSERM CJF no. 90-08, Groupe de Recherches sur les Communications Cellulaires, Laboratoire de Biochimie, Hopital Sainte-Eugehie, Centre Hospitaller Lyon-Sud, 69310 Pierre-Benite, France
ABSTRACT. The actions and the mechanisms of action of epidermal growth factor (EGF) in testicular steroidogenesis were investigated using a model of primary culture of purified porcine Leydig cells from immature intact animals. EGF decreased (1.7-fold) human CG (hCG)-induced dehydroepiandrosterone (DHEA) accumulation in the medium whereas it enhanced (2.5fold) that of testosterone. The maximal and half-maximal effects on both DHEA and testosterone secretions were observed at similar concentrations which were, respectively, 3 (5 x 10"10 M) and 0.7 (11 X 10"11 M) ng/ml EGF, after 72-h treatment. EGF effect on DHEA and testosterone secretion was similarly observed whether the cells were acutely (3 h) stimulated with hCG (1 ng/ml) or with 8-bromo-cAMP (10~3 M). TO further localize the steroidogenic biochemical steps affected by EGF, the growth factor action on steroidogenic enzyme activities was investigated. EGF increased A5 steroid intermediate (i.e. pregnenolone and DHEA) formation [evaluated in the presence of 10~6 M of WIN 24540, an inhibitor of 3/3-hydroxysteroid dehydrogenase/ isomerase (3/J-HSDI) activity]. However, this stimulation was
T
HE POTENTIAL role of epidermal growth factor (EGF) in modulating certain aspects of the testis function by acting on different testicular cell types have been examined by several laboratories. It has been reported that spermatogenesis might be affected directly (1) and/or indirectly via testicular somatic cells, such as Sertoli and Leydig cells. For example, in Sertoli cells, EGF stimulates the production of lactate and inhibits aromatase activity (2). Although different in vitro models have been used to investigate the role of EGF on steroidogenesis, its role appears not well defined in Leydig cell physiology. Indeed, both inhibitory and stimulatory efReceived November 1, 1990. Address all correspondence and requests for reprints to: Dr. M. Benahmed, Groupe de Recherches sur les Communications Cellulaires, Laboratoire de Biochimie, Hopital Sainte-Eugenie, CH Lyon-Sud, 69310 Pierre-Benite, France. * This work was financially supported by Institut National de la Sante et de la Recherche Medicale and Ministere de la Recherche et de l'Enseignement Superieur (MRES). t Recipient of a grant from MRES.
observed in cells when acutely (3 h) stimulated with hCG (0.011 ng/ml) but not when incubated with 22R-hydroxycholesterol (0.01-10 Mg/ml). Such findings indicate that EGF did not affect cholesterol side chain cleavage cytochrome P450 activity but probably increased cholesterol substrate availability for this enzyme in the inner mitochondria. Moreover, EGF significantly (P < 0.001) increased A5 steroid intermediate (i.e. pregnenolone and DHEA) but not A4 steroid intermediate (i.e. progesterone and androstenedione) conversion into testosterone, indicating that EGF enhances 3/3-HSDI activity. Such effects of EGF are directly exerted on Leydig cells since EGF receptors (Kd = 16 x 10~u M) are present in primary cultures of purified porcine Leydig cells. Together, the present findings show that in Leydig cells from intact animals, EGF enhances the gonadotropin action on testosterone formation through an increase in the availability of cholesterol substrate in the mitochondria as well as an increase in the activity of 3/3-HSDI. {Endocrinology 128: 21602168,1991)
fects of the growth factor on testicular steroidogenesis have been reported. For example, EGF has been reported to inhibit human CG (hCG) -induced steroid production in a clonal line of cultured Leydig tumor cells (MA-10) (3). An inhibitory effect of EGF on testicular steroidogenesis has been also reported by Hsueh et al. (4) who used total interstitial testicular cells from hypophysectomized rats. More recently, by using Leydig cells from immature rats, Verhoeven and Cailleau (5) have reported an inhibitory effect of EGF in the absence of LH but a stimulatory effect of EGF in the presence of the gonadotropin on steroid hormone production. Although EGF receptors have been localized in the Leydig cell line MA10 (3) and in crude testicular interstitial cells (4), they have not yet been identified in purified Leydig cells from intact animals. In addition to the discrepancies between these results, the mechanisms of action of EGF on steroidogenesis in Leydig cells from normal intact animals have not been investigated further. Because of the potential role of EGF in the testis function, the present study
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MECHANISMS OF ACTION OF EGF ON LEYDIG CELLS was undertaken: 1) to determine the optimal conditions for EGF action on cultured purified porcine Leydig cells from intact animals, 2) to identify the biochemical mechanisms of action involved in EGF action on Leydig cell steroidogenesis, and 3) to determine whether EGF receptors are present on these cells, supporting a direct interaction between the growth factor and Leydig cells.
Materials and Methods EGF, 22R-hydroxycholesterol, 8-bromo-cAMP, insulin, transferrin, vitamin E, HEPES were purchased from Sigma Chemical Co. (St. Louis, MO). Transforming growth factor a (TGFa) was obtained from Biomedical Technologies Inc. (Stoughton, MA). hCG CR (121 13,450 IU/mg) was a gift of Dr. R. E. Canfield. Dulbecco's modified Eagle's (DME) medium and Ham's F12 medium were obtained from GIBCO (Grand Island Biological Co., Grand Island, NY) and collagenase/ dispase from Boehringer (Mannheim, FRG). Iodogen was purchased from Pierce Europe (Netherlands). WIN 24540 (4a, 5epoxy-17/3-hydroxy-5a-androstane) was kindly provided by Sterling-Wintrop (NY). Leydig cell preparation and culture Isolated Leydig cells were prepared from immature porcine testes (2- to 3-week old) by collagenase treatment as described by Mather and Phillips (6) and modified by Benahmed et al. (7). Briefly, decapsulated testes were minced and washed twice in DME/F12 (1:1) medium. After collagenase dissociation (0.5 mg/ml, 90-120 min at 32 C), cells were washed by centrifugation (200 x g for 10 min). The pellet was then resuspended and submitted to two successive sedimentations of 5 and 15 min. The crude interstitial cells were recovered from the supernatants and Leydig cells were prepared from this fraction by Percoll gradient centrifugation. The percentage of Leydig cells in the final preparation, as established by staining for 3/3hydroxysteroid dehydrogenase activity (8), ranged from 8090% (7). Leydig cells were plated in Falcon 24-multiwell plates (0.4 X 106 cells per dish) and cultured at 32 C in a humidified atmosphere of 5% CO2, 95% air in DME/F12 medium (1:1) containing sodium bicarbonate (1.2 mg/ml), 15 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES) and gentamicin (20 jig/ml). This medium was supplemented with insulin (2 Mg/ml), transferrin (5 Mg/ml), and vitamin E (10 ng/ ml). At the end of the experiments, the culture medium was collected and stored at -20 C until assayed for steroid hormone content. The cells were then detached from the culture dishes in trypsin-EDTA and counted in a Coulter counter (Coultronics, Margency, France). Leydig cell steroidogenic activity Porcine cultured Leydig cell steroidogenic activity was mainly evaluated through the secretion of dehydroepiandrosterone (DHEA) and testosterone, two major steroid hormones secreted in the medium (9). One of the major characteristics of this culture system is that the secretion of the steroid hormones in response to the gonadotropin LH/hCG stimulation remains stable for several days. Indeed, hCG-stimulated DHEA and
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testosterone secretion maximally increases at day 2 of culture, plateaues during the six following days of culture and then dramatically declines, probably reflecting a cell dedifferentiation process that may appear in cell cultures (6). Testosterone, DHEA, and pregnenolone were assayed in the culture medium by using previously reported specific RIAs (10-12). All the experimental data are represented as the mean ± SD of triplicate determinations of steroid production by three replicate cultures within each treatment group. All experiments reported here were repeated at least three times in different (independent) cell preparations. A representative experiment of each serie of experiments is presented. Statistical significance between groups was determined by Student's t test using the StatWorks software package on a Macintosh Plus computer. Differences are accepted as significant at P < 0.05. lode
125
I-labeling of EGF
Purified EGF (1 Mg) was labeled using 125I by the Iodogen procedure (13). Preparations of 125I-EGF with a specific activity of 200-300 nQi/iig was obtained by this method. Binding assays For measurements of 125I-EGF binding, immature Leydig cells were plated in Falcon 24-multiwell plates. Cells were washed once with DME/F12 medium and then incubated with 125 I-EGF and increasing amounts of unlabeled EGF for 14 h at 4 C. The cells were then washed three times with PBS BSA (1 mg/ml) (0-4 C) and solubilized in 0.5 N sodium hydroxide, 0.4% deoxycholate and the radioactivity was measured in a ycounter. Binding was analyzed by the method of Scatchard (14) and by using the EBDA and LIGAND computer program (15) which were converted to BIOSOFT by G. A. McPherson and distributed by Elsevier-Biosoft (Cambridge, UK).
Results Effects of EGF on Leydig cell steroidogenesis
In the following experiments, the optimal conditions for EGF action on testicular (Leydig cell) steroidogenesis were first investigated. The effect of EGF was tested on the secretion of testosterone and DHEA produced in porcine immature Leydig cells, between day 2 and day 6 of culture. Figure 1 shows dose-dependent effect of EGF on testosterone and DHEA secretion acutely stimulated with maximal effective hCG (1 ng/ml) concentration. EGF stimulates hCG-induced testosterone production with apparent maximal (2.5 fold) and half-maximal (ED50) effects, respectively, of 3 ng/ml (5 X 10~10 M) and 0.7 ng/ml (11 x 10~n M) EGF (Fig. 1A). In contrast to this effect on testosterone, EGF decreased in a dosedependent manner DHEA accumulation (1.7 fold) with apparent maximal and half-maximal (ID50) effects, respectively, observed with 3 ng/ml (5 X 10~10 M) and 0.7 ng/ml (11 x 10"11 M) EGF (Fig. IB). However, although these opposite effects of EGF were
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MECHANISMS OF ACTION OF EGF ON LEYDIG CELLS
2162
Endo«1991 Voll28.No4
14 - A
o
12
20
EGF
O
c
10 15
UJ
z o oc
UJ
10
-EGF
o UJ
UJ
-EGF
O.I
10
(0 O (A Ui
EGF ng/ml
B 0.01
30
0.1 hCG
ng/ml
25
-EGF
25 UJ
X
20
Q
EGF
! -EGF
10
0.1 EGF n|/rol
15
FIG. 1. Effect of EGF on hCG-stimulated steroid secretion: dose dependency. Leydig cells were cultured with increasing concentrations of EGF (0-10 ng/ml) for 72 h. The cells were then washed and stimulated with hCG (1 ng/ml, 3 h) before evaluating the testosterone (A) or the DHEA (B) content of the medium. The results represent the mean ± SD of three determinations in each of triplicate incubations.
also observed on the basal steroidogenesis, they appear to be relatively moderate, both on the accumulation of DHEA (-EGF: 0.58 ± 0.05 vs. +EGF: 0.47 ± 0.04 ng/ 106 cells/3 h, P < 0.05) and of testosterone (-EGF: 0.5 ± 0.01 vs. +EGF: 0.8 ± 0.04 ng/106 cells/3 h, P < 0.001). Figure 2 shows that EGF influences the secretion of the two steroid hormones stimulated with increasing concentrations of hCG (0.01-1 ng/ml, 3 h). In this context, the growth factor appears to increase and decrease, respectively, hCG-stimulated testosterone (Fig. 2A) and DHEA
5
L
0.1
0.01
hCG
fail
FIG. 2. Effect of hCG on EGF-treated Leydig cells. Leydig cells were cultured in the absence (•—•) or presence (O—O) of EGF (10 ng/ml) for 72 h. At the end of the treatment, the cells were washed and stimulated with increasing concentrations of hCG (0.01-1 ng/ml, 3 h) and testosterone (A) or DHEA (B) secretions were evaluated. The results represent the mean ± SD of three determinations in each of triplicate incubations.
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MECHANISMS OF ACTION OF EGF ON LEYDIG CELLS (Fig. 2B) secretions by affecting the maximal Leydig cell steroidogenic capacity but not by affecting hCG doses required for the maximal (0.1 ng/ml hCG) production of the two steroid hormones. Furthermore, the stimulatory effect of EGF on hCGstimulated testosterone production was evidenced after a long-term treatment. Indeed, no significant effect on hCG-stimulated testosterone was observed before 24-h treatment with EGF (10 ng/ml) (Table 1). A significant (P < 0.001) stimulatory effect was observed at 48 h and was maximal after 72 h of EGF treatment (Table 1). A similar stimulatory effect of EGF was observed on testosterone production whether Leydig cells were stimulated with hCG (-EGF: 7.75 ± 0.5 vs. +EGF: 13.1 ± 1.1 ng/106 cells/3 h, P < 0.001) or with 8-bromo-cAMP (-EGF: 8.4 ± 0.3 vs. +EGF: 13.7 ± 0.8 ng/106 cells/3 h, P < 0.001) indicating that the effects of EGF on steroidogenesis are subsequent to hCG-induced cAMP formation. These effects of EGF (10 ng/ml, 72 h) on steroidogenesis were not related to a change in Leydig cell number (-EGF: 38.8 ± 0.6 vs. +EGF: 38.7 ± 0.5 x 104 cells per dish). Taken together, the increase in testosterone secretion coupled with the decline in DHEA secretion in EGFtreated Leydig cells in response to acute hCG stimulation led us to hypothesize that EGF may modulate the formation and/or the catabolism of DHEA into A4 steroid hormones and particularly into testosterone. The following experiments were carried out to test these different possibilities. Effects of EGF on A5 steroid hormone formation In order to test whether EGF modulates the formation of A5 steroid hormones, namely DHEA, we used WIN 24540, a compound capable of rapidly suppressing the activity of 3/3-hydroxysteroid dehydrogenase/isomerase (3/3-HSDI), an enzyme responsible for the catabolism of TABLE 1. Time course of EGF effect on hCG (3 h) -stimulated testosterone secretion in Leydig cells Time (h) 0 6 24 48 72
Testosterone (ng/106 cells • 3 h) 13.1 ± 14.4 ± 14.8 ± 21.7 ± 26.6 ±
0.3 0.3 (NS) 1.0 (NS) 1.0° 2.0°
Leydig cells were cultured for the duration indicated (6-72 h) in the presence of EGF (10 ng/ml). At the end of the treatment, the medium was removed and the cells were washed before being stimulated with hCG (1 ng/ml, 3 h), and the testosterone content of the medium was measured. The results represent the mean ± SD of three determinations in each of triplicate incubations. NS, Not significant (P > 0.05). 0 P < 0.001, time of treatment us. to.
2163
A5 steroids into A4 steroids (16). Figure 3 shows the effects of EGF (10 ng/ml) on DHEA (Fig. 3A) and pregnenolone (Fig. 3B) secretions stimulated with increasing concentrations of hCG (0.01-1 ng/ml) in the presence of WIN 24540 at a concentration (10~5 M) which inhibits more than 90% of testosterone formation in these experimental conditions (-WIN 24540: 25.7 ± 1.0 vs. +WIN 24540: 2.1 ± 0.1 ng testosterone/106 cellules). EGF enhanced the hCG-induced accumulation of DHEA in the culture medium, indicating that the growth factor stimulates DHEA formation (Fig. 3A). Furthermore, the parallel and similar increase in pregnenolone formation after EGF treatment (Fig. 3B) indicates that the increase in DHEA formation induced by EGF is directly related to that of pregnenolone. Experiments were subsequently carried out to determine whether the increase in pregnenolone formation was related to a possible effect of EGF on cholesterol side chain cleavage enzyme (P450scc) activity. Such a possibility was tested by using hydroxylated cholesterol derivative, 22(R)-diol (22 R-hydroxy) cholesterol in excess as exogenous substrate for P45O8CC. Hydroxylated cholesterol derivatives pass readily between cell membranes and can be used to replace cholesterol as substrate for P450scc (17-19). As shown in Fig. 4, when Leydig cells were incubated with 22R-hydroxycholesterol (0.01-10 jig/ml, 3 h) in the presence of WIN 24540 (10~5 M), the stimulatory effect of EGF on DHEA formation was no longer observed. These observations indicate that the stimulatory effect of EGF on pregnenolone and DHEA formation was not related to an increase in P4508CC activity but more probably to an increase in cholesterol substrate availability in the inner mitochondria. Effects of EGF on the catabolism of A5 steroid hormones into testosterone The concomittant decrease in DHEA and increase in testosterone secretion in EGF-treated Leydig cells (Figs. 1-2) suggest that the growth factor may also enhance the catabolism of A5 steroid hormones into testosterone. The following experiments were thus carried out to clarify the role of steroidogenic enzymes metabolizing pregnenolone to testosterone as possible sites of EGF action. Testosterone production was measured after incubating Leydig cells for 1.5 h in the presence of different steroid substrates (500 ng/ml). As shown in Table 2, both A5 and A4 steroid substrates are converted into testosterone and as expected, the rate of this conversion was different for each steroid. That DHEA and androstenedione were the most converted into testosterone was not surprising since in porcine Leydig cells, testosterone biosynthesis from pregnenolone preferentially occurs via 17a-hydroxypregnenolone, DHEA and then androstenedione (9 and our unpublished data). Pretreatment of Leydig cells for
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MECHANISMS OF ACTION OF EGF ON LEYDIG CELLS
2164
Endo»1991 Voll28«No4
I
A
100
60 -
' v tEGF
•
-EGF
HI +EGF
80 40 T
-EGF
O)
60 20
40
0L 20
/
0.1
10
cholesterol 0
I
i
1
FIG. 4. Effect of EGF on A5 steroid hormone formation in Leydig cells incubated with 22R-hydroxycholesterol. Leydig cells were cultured in the absence or presence of EGF (10 ng/ml, 72 h). At the end of the treatment, the cells were washed and incubated with increasing concentrations of 22R-hydroxycholesterol (0.01-10 Mg/ml, 3 h) in the presence of WIN 24540 (10~5 M) and then DHEA secretion was evaluated. The results represent the mean ± SD of three determinations in each of triplicate incubations.
i
ai
0.01
pg/ml
hCG ng/ml B • EGF
TABLE 2. Effects of EGF on testosterone production in Leydig cells incubated with differents steroid substrates Testosterone (ng/106 cells • 1.5 h)
2.5
-EGF
-EGF
0)
Pregnenolone Dehydroepiandrosterone Progesterone Androstenedione
17.6 ± 33.7 ± 19.5 ± 207 ±
1.3 2.5 1.0 8.9
+EGF 29.6 ± 58.3 ± 21.6 ± 204 ±
0.9° 3.5° 0.9 (NS) 15.6 (NS)
Leydig cells were cultured for 72 h in the absence or presence of EGF (10 ng/ml). At the end of the treatment, the medium was removed, and the cells were incubated with different steroid substrates (namely pregnenolone, dehydroepiandrosterone, progesterone, and androstenedione, 500 ng/ml, 1.5 h), and the testosterone content of the medium was measured. The results represent the mean ± SD of three determinations in each of triplicate incubations. NS, Not significant (P > 0.05). a P < 0.001, +EGF vs. -EGF.
1.S
o o 0)
I 0.5 L i 9.01
at hCG
/ml
FIG. 3. Effect of EGF on A5 steroid hormone formation. Leydig cells were cultured in the absence (•—•) or presence (O—O) of EGF (10 ng/ml, 72 h). At the end of the treatment, the cells were washed and stimulated with increasing concentrations of hCG (0.01-1 ng/ml, 3 h) in the presence of WIN 24540 (10~5 M) and DHEA (A) or pregnenolone (B) secretions were evaluated. The results represent the mean ± SD of three determinations in each of triplicate incubations.
72 h with EGF (10 ng/ml) exhibited a differential effect on exogenous A5 steroid and on A4 steroid substrate conversion into testosterone. Indeed, the growth factor exerted a significant (P < 0.001) stimulatory effect on testosterone production in Leydig cells incubated with exogenous A5 steroid hormones (e.g. pregnenolone and DHEA) but not with exogenous A4 steroid hormones (e.g. progesterone and androstenedione). These observations clearly indicating that EGF enhances 3/3-HSDI activity were further confirmed by the findings reported
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MECHANISMS OF ACTION OF EGF ON LEYDIG CELLS in Fig. 5. Indeed, the stimulating effect of EGF on 3/3HSDI, reflected by the increase in DHEA conversion into testosterone was dependent upon EGF doses in the medium. The maximal (about 2-fold) and half-maximal increases were, respectively, observed with 1 ng/ml (1.7 X 1(T10 M) and 0.45 ng/ml (7.5 X KT11 M) EGF. Evidence for EGF receptors in cultured porcine Leydig cells
2165
30
* •o c
20
3
In order to determine whether the effects of EGF reported above are directly exerted on Leydig cells, the following experiments were performed to identify EGF receptors on primary cultures of porcine immature Leydig cells. Binding experiments were conducted using intact Leydig cells in near confluent monolayer cultures with various concentrations of EGF at 4 C for 14 h. As shown in Fig. 6, EGF binding was concentration dependent with saturation observed at 50 nM EGF. The Scatchard analysis of the binding data revealed the existence of two classes of binding sites (Fig. 6, inset). The binding parameters were calculated and yielded an apparent Kd value of 16 X 10~ u M with 1.2 x 103 sites per cell for the high affinity system and an apparent Kd value of 2.7 X 10"8 M with 27.6 X 103 sites per cell for the low affinity system.
Discussion The present findings demonstrate the stimulatory effect of EGF on testosterone production under acute term 85
65
45_r -EGF
0.1 EGF ng/ml
FiG. 5. Effect of EGF on 30-HSDI activity in Leydig cells. Leydig cells were cultured in the presence of increasing concentrations of EGF (010 ng/ml, 72 h). At the end of the treatment, the cells were washed and incubated with DHEA (500 ng/ml, 2 h) and then testosterone production was evaluated. The results represent the mean ± SD of three determinations in each of triplicate incubations.
o £
10
10
50 E6F
[nM]
125
FIG. 6. Binding of I-EGF to porcine immature Leydig cells. Cultured Leydig cells were incubated for 14 h at 4 C with 125I-EGF and various amounts of unlabeled EGF. The amount of specifically bound 125I-EGF was then assayed as described under Materials and Methods. The data were plotted as a saturation curve and treated by Scatchard analysis to calculate the binding parameters (inset).
LH/hCG stimulation in primary cultures of purified porcine Leydig cells. EGF enhances hCG-induced testosterone secretion to a maximal (2.5-fold) increase with ED50 of 11 x 10"11 M after a 72-h treatment. The increase in acutely hCG-stimulated testosterone production was not related to the well known cell growth stimulatory activity of EGF since no increase in the number of Leydig cells (cultured in serum-free defined medium) was observed after a 72-h treatment with EGF (10 ng/ml). In addition, this stimulatory effect of the growth factor is observed in porcine cultured Leydig cells in which the differentiated (steroidogenic) function is maximal and is not declining (i.e. between day 2 and day 6 of cultures). Such observations indicate that the enhancement of Leydig cell activity observed after EGF treatment is primarily not a reflection of the impact of the growth factor on the dedifferentiation process that appears in cell cultures. This effect of EGF is exerted through specific membrane receptors identified in the present study in cultured immature porcine Leydig cells. Leydig cells specifically bind EGF through a high affinity binding system with a Kd value of 16 X 10~ n M with 1.2 X 103 sites per cell. Although it has been reported that tumoral Leydig cells (3) and total interstitial testicular cells from hypophysectomized rats (4) exhibit EGF receptors, this is the first report indicating the presence of such receptors on purified Leydig cells from intact animals. Our observation is consistent with that of Suarez-Quian et al. (20) who localized immunocytochemically EGF receptors in rat interstitial tissues from intact animals. However the direct interaction between the growth factor and the
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MECHANISMS OF ACTION OF EGF ON LEYDIG CELLS
testicular steroidogenic cells will be definitely ascertained with the demonstration of EGF receptors on Leydig cells by using autoradiography, immunocytochemistry, and in situ hybridization technics. Although the concentration of EGF (or a related peptide) present in the testis is not determined, the ED50 of EGF was within the Kd range observed for EGF high affinity receptor identified on porcine Leydig cells. This observation suggests that the steroidogenic effect of EGF occurs within a concentration range that might be expected under physiological conditions. Moreover, the variation of high affinity EGF receptors during the male gonad development and during different physiological conditions as well as their regulation by the major factors controlling Leydig cell function will be an interesting possibility to investigate. With regard to the biochemical mechanisms involved in the growth factor effect, the increase in LH/hCG stimulated testosterone production in EGF-treated Leydig cells was probably not related to the potential changes in the structure and function of the Leydig cell membrane. Indeed, similar stimulatory effect of EGF was observed on testosterone production whether it was acutely stimulated with the gonadotropin or with 8bromo-cAMP, suggesting, in addition, that the biochemical steps involved in the growth factor action are located beyond cAMP formation. The growth factor appears to potentiate the gonadotropin LH/hCG action on testosterone production by affecting at least two different biochemical levels. The first step(s) that appears to be controlled by EGF in Leydig cells is related to an increase in pregnenolone formation. Such effect was not related to an increase in P450scc activity but probably to an increase in cholesterol substrate availability for this enzyme in the inner mitochondria. Indeed, our present findings indicate that the stimulatory effect of EGF on pregnenolone formation was only observed when Leydig cells were acutely stimulated with the gonadotropin but not when they were incubated in the presence of hydroxylated cholesterol derivatives that pass readily between cell membranes. These findings suggest that EGF action involves the cholesterol transport into the inner mitochondria and the cholesterol availability for P450scc. Since cholesterol mobilization from the cytoplasm into mitochondria involves microfilaments (21), sterol carrier proteins (22), labile regulatory protein(s) (probably the steroidogenesis activator polypeptide) (23), and the more recently identified GTP-regulatory protein(s) (24), it is tempting to speculate that these elements may be targets for the regulatory action of EGF in Leydig cells. The second biochemical step(s) involved in Leydig cell steroidogenesis that appears to be under EGF control is the 3/3-HSDI enzyme activity. The increase in this en-
Endo • 1991 Voll28«No4
zyme activity is dependent upon EGF concentration in the medium. Furthermore, it is of interest to note that this enzyme activity may well represent a very sensitive biochemical step(s) for EGF action in Leydig cells since the growth factor stimulatory effect is observed at very low concentrations (ED50 = 7 x 10"11 M EGF). This is the first report indicating that EGF enhances testicular steroidogenesis by increasing the activity of a key enzyme in Leydig cell maturation. However, it remains to be examined whether EGF is a positive effector controlling the enzyme expression at genetic or posttranscriptional levels. Moreover, it is known that EGF activates in different cellular systems a variety of intracellular signaling system(s). The binding of EGF induces the activation of EGF receptor tyrosine kinases which directly phosphorylate specific cellular substrates. The addition of the ligand is followed by several other early or delayed responses (25). Therefore, an important step toward an understanding of the stimulating action of EGF on Leydig cell steroidogenesis would be to establish which of the EGF-activated intracellular signaling system(s) is involved in the enhancement of cholesterol substrate availability in the mitochondria and of 3/3-HSDI activity reported here. Because of the negligible levels of EGF in the circulatory system (26), the effect of the growth factor on testicular function is likely related to a local production of an EGF-like substance(s). Initial observations have indicated that (receptor reactive) EGF-like substances may be produced by Sertoli cells (27). More recently, it has been reported that these substances are probably not related to EGF but to TGFa, a protein that has a structural homology with EGF and binds to the EGF receptor (28). Indeed, the gene encoding for TGFa, but not that for EGF, is transcribed in the testis (29). In addition, TGFa has been recently identified immunohistochemically in the rat testis (30). In this context, as expected, we have observed similar stimulatory effects on androgen production when porcine Leydig cells were treated with TGFa instead of EGF (unpublished data). Thus, the testicular ligand for Leydig cell EGF receptors might be related to TGFa and/or to a chemically undefined factor(s) such as Sertoli cell secreted growth factor, reported to be a receptor reactive EGF-like substance(s) but distinct from EGF and TGFa (27). Furthermore, although it is now well accepted that seminiferous tubules exert a control on Leydig function (31-37), the molecules involved in tubular-interstitial cell interactions remain poorly known. The tubular (Sertoli cell) origin of EGF-like substances (27, 29) coupled with the steroidogenic effects of EGF reported here and by others (3-5) suggest that the EGF-like substances might be involved in such interactions.
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MECHANISMS OF ACTION OF EGF ON LEYDIG CELLS
Finally, it should be borne in mind that the action of most growth factors is highly dependent on the presence of other growth factors (38). Thus, in vivo, the role of testicular EGF (or EGF-like substances) may lie in the interactions of this factor with other factors present in and released from testicular cells such as TGF/3, basic fibroblast growth factor, insulin growth factors, and /?nerve growth factor (39, 40). In this context, for example, the interactions between EGF and TGF0 on testicular steroidogenesis are of great interest. Indeed, we have already reported that TGF/3 also affects testicular (Leydig cell) steroidogenesis by controlling the same biochemical step(s) reported here to be affected by EGF but in a distinct manner (41, 42). TGF/3 effects differ from those of EGF in that they induce a decrease in cholesterol substrate availability for P450scc but are similar in that they elicit an increase in 30-HSDI enzyme activity (41). Consequently, because EGF (or a related factor) and TGF/3 are produced concomitantly in the testis, the results of their interactions is of major importance for testicular steroidogenesis. In summary, by using a model of primary cultures of immature porcine Leydig cells, we show that EGF directly (via specific membrane receptors) potentiates acute LH/hCG steroidogenic action by enhancing cholesterol substrate availability for P450scc and promotes Leydig cell differentiated function by increasing 3/3HSDI activity. This is the first report on the different biochemical mechanisms involved in EGF stimulating action on testicular steroidogenesis.
Acknowledgments We are grateful to Prof. E. Chambaz for his constant interest in our work and to Dr. M. G. Forest for testosterone antisera. We thank Mr. E. Villard and Mr. P. Bouteille for providing us with porcine testes.
References 1. Tsutsumi 0, Kurachi H, Oka T 1986 A physiological role of epidermal growth factor in male reproductive function. Science 233:975-977 2. Mallea LE, Machado AJ, Navaroli E, Rommerts FFG 1986 Epidermal growth factor stimulates lactate production and inhibits aromatization in cultured Sertoli cells from immature rats. Int J Andrology 9:201-208 3. Ascoli M 1981 Regulation of gonadotropin receptors and gonadotropin responses in a clonal strain tumor cells by epidermal growth factor. J Biol Chem 256:179-183 4. Hsueh AJW, Welsh TH, Jones PBC 1981 Inhibition of ovarian and testicular steroidogenesis by epidermal growth factor. Endocrinology 108:2002-2004 5. Verhoeven G, Cailleau J 1986 Stimulatory effects of epidermal growth factor on steroidogenesis in Leydig cells. Mol Cell Endocrinol 47:99-106 6. Mather JP, Phillips D 1984 Primary culture of testicular somatic cells. In: Barnes DW, Sirbasku DA, Sato GH (eds) Methods for Serum-free Culture of Cells of the Endocrine System. Alan R. Liss, New York, p 29-45 7. Benahmed M, Morera AM, Chauvin MA, de Peretti E 1987 Somatomedin C/ insulin-like growth factor I as a possible intrates-
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ticular regulator of Leydig cell activity. Mol Cell Endocrinol 50:6977 Mendelson C, Dufau M, Catt KJ 1975 Gonadotropin binding and stimulation of cyclic adenosine 3':5'-monophosphate and testosterone production in isolated Leydig cells. J Biol Chem 250:88188823 Orava M, Haour F, Leinonen P, Ruokonen A, Vihko R 1985 Relationships between unconjugated and sulphated steroids in porcine primary Leydig cell culture. J Steroid Biochem 22:507-512 Forest MG, Cathiard AM, Bertrand J, 1973 Total and unbound testosterone in the newborns and in hypogonadal children. Use of a sensitive radioimmunoassay for testosterone. J Clin Endocrinol Metab 36:1132-1142 De Peretti E, Forest MG 1976 Unconjugated dehydroepiandrosterone plasma levels in normal subjects from birth to adolescence in human: the use of a sensitive radioimmunoassay. J Clin Endocrinol Metab 43:982-991 De Peretti E, Mappus E 1983 Pattern of plasma pregnenolone sulfate levels in humans from birth to adulthood. J Clin Endocrinol Metab 57:550-556 Eppstein DA, Marsh YV, Schryver B, Bertics PJ 1989 Inhibition of epidermal growth factor/transforming growth factor a-stimulated cell growth by a synthetic peptide. J Cell Physiol 141:420430 Scatchard G 1949 The interaction of proteins for small molecules and ions. Ann NY Acad Sci 51:660-672 Munson PJ, Rodbard D 1980 LIGAND: a versatile computerized approach for characterization of ligand-binding systems. Anal Biochem 107:220-239 Neuman HC, Potts GO, Ryan WR, Stonner FW 1970 Steroidal heterocycles. XIII 4«, 5-epoxy-5c*-androst-2-eno (2,3-d) isoxazoles and related compounds. J Med Chem 13:948-951 Arthur JR, Blair HAF, Boyd GS, Mason JI, Suckling KE 1976 Oxidation of cholesterol and cholesterol analogues by mitochondrial preparations of steroid-hormone-producting tissue. Biochem J 158:47-51 Brinkmann AO, Leemborg FG, Rommerts FFG, van der Molen HJ 1984 Differences between the regulation of cholesterol side chain cleavage in Leydig cells from mice and rats. J Steroid Biochem 21:259-264
19. Mason JI, Robidoux WF 1978 Pregnenolone biosynthesis in isolated cells of Snell rat adrenocortical carcinoma 494. Mol Cell Endocrinol 12:299-308 20. Suarez-Quian CA, Dai M, Onada M, Kriss RM, Dym M 1989 Epidermal growth factor localization in the rat and monkey testes. Biol Reprod 41:921-932 21. Strott CA 1990 The search for the elusive adrenal steroidogenic "regulatory" protein. Trends Endocrinol Metab 1:312-314 22. Vahouny GV, Dennis P, Chanderbhan R, Fiskum G, Noland BJ, Scallen TJ 1984 Sterol carrier protein2 (SCP2)-mediated transfer of cholesterol to mitochondrial inner membrane. Biochem Biophys Res Commun 122:509-517 23. Pedersen RC, Brownie AC 1987 Steroidogenesis-activator polypeptide isolated from a rat Leydig cell tumor. Science 236:188-190 24. Xu X, Xu T, Robertson DG, Lambeth JD 1989 GTP stimulates pregnenolone generation in isolated rat adrenal mitochondria. J Biol Chem 264:17674-17680 25. Carpenter G, Cohen S 1990 Epidermal growth factor. J Biol Chem 265:7709-7712 26. Carpenter G, Zendegui J 1985 A biological assay for epidermal growth factor/Urogastrone and related peptides. Anal Biochem 153:279-282 27. Holmes SD, Spotts G, Smith RG 1986 Rat Sertoli cells secrete a growth factor that blocks epidermal growth factor (EGF) binding to its receptors. J Biol Chem 261:4076-4080 28. Marquardt H, Hunkapiller MW, Hood LE, Todaro G 1984 Rat transforming growth factor type I: structure and relation to epidermal growth factor. Science 233:1079-1082 29. Skinner MK, Takacs K, Coffey RJ 1989 Transforming growth factor a gene expression and action in the seminiferous tubule: peritubular cell-Sertoli cell interactions. Endocrinology 124:845854
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30. Teerds KJ, Rommerts FFG, Dorrington JH 1990 Immunohistochemical detection of transforming growth factor a in Leydig cells during the development of the rat testis. Mol Cell Endocrinol 69:R1-R6 31. Aoki A, Fawcett DW 1978 Is there a local feedback from seminiferous tubules affecting activity of the Leydig cells? Biol Reprod 19:144-158 32. Bergh A 1983 Paracrine regulation of Leydig cells by seminiferous tubules. Int J Androl 6:57-65 33. Sharpe RM, Cooper I 1984 Intratesticular secretion of a factor(s) with major stimulatory effects on Leydig cell testosterone secretion in uitro. Mol Cell Endocrinol 37:159-168 34. Benahmed M, Grenot C, Tabone E, Sanchez P, Morera AM 1985 FSH regulates cultured Leydig cell function via Sertoli cell proteins: an in vitro study. Biochem Biophys Res Commun 132:729734 35. Verhoeven G, Cailleau J 1985 A factor in spent media from Sertoli cell-enriched cultures that stimulates steroidogenesis in Leydig cells. Mol Cell Endocrinol 40:57-68 36. Payne A, Chase D, O'Shaugnessy PJ 1982 Regulation of steroidogenesis in Leydig cells. In: Conn M (ed) Cellular Regulation of Secretion and Release. Academic Press, New-York p 355-408 37. Benahmed M, Morera AM, Chauvin MA, de Peretti E, Revol A
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1987 FSH regulates testicular steroidogenesis through Sertoli cell factors: effects and preliminary characterization. In: Orgebin-Crist MC, Danzo BJ (eds) Cell Biology of the Testis and Epididymis. Ann NY Acad Sci 513:470-471 Sporn MB, Roberts A 1986 Transforming growth factor-beta: biological function and chemical structure. Science 233:532-534 Bellve AR, Zheng W 1989 Growth factors as autocrine and paracrine modulators of male gonade functions. J Reprod Fert 85:771793 Benahmed M, Sordoillet C, Chauvin MA, Morera AM 1990 Local regulators of testicular function. In: Bouchard P, Haour F, Franchimont P, Schatz B (eds) Recent Progress on GnRH and Gonadal Peptides. Elsevier, Paris, p 383-405 Benahmed M, Sordoillet C, Chauvin MA, de Peretti E, Morera AM 1989 On the mechanisms involved in the inhibitory and stimulating actions of TGF/3 on porcine testicular steroidogenesis: an in vitro study. Mol Cell Endocrinol 67:155-164 Benahmed M, Sordoillet C, Hartman DJ, Esposito G, Cochet C, Tabone E, de Peretti E, Chauvin MA, Morera AM 1990 Transforming growth factor beta and related peptides: a role in testicular intercellular communication. In: Findlay J, Haseltine F (eds) Growth Factors in Fertility Regulation. Cambridge University Press, Cambridge, pp 235-259
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Vol. 128, No. 4 Printed in U.S.A.
Epidermal Growth Factor Directly Stimulates Steroidogenesis in Primary Cultures of Porcine Leydig Cells: Actions and Sites of Action* C. SORDOILLETt, M. A. CHAUVIN, E. DE PERETTI, A. M. MORERA, AND M. BENAHMED INSERM CJF no. 90-08, Groupe de Recherches sur les Communications Cellulaires, Laboratoire de Biochimie, Hopital Sainte-Eugehie, Centre Hospitaller Lyon-Sud, 69310 Pierre-Benite, France
ABSTRACT. The actions and the mechanisms of action of epidermal growth factor (EGF) in testicular steroidogenesis were investigated using a model of primary culture of purified porcine Leydig cells from immature intact animals. EGF decreased (1.7-fold) human CG (hCG)-induced dehydroepiandrosterone (DHEA) accumulation in the medium whereas it enhanced (2.5fold) that of testosterone. The maximal and half-maximal effects on both DHEA and testosterone secretions were observed at similar concentrations which were, respectively, 3 (5 x 10"10 M) and 0.7 (11 X 10"11 M) ng/ml EGF, after 72-h treatment. EGF effect on DHEA and testosterone secretion was similarly observed whether the cells were acutely (3 h) stimulated with hCG (1 ng/ml) or with 8-bromo-cAMP (10~3 M). TO further localize the steroidogenic biochemical steps affected by EGF, the growth factor action on steroidogenic enzyme activities was investigated. EGF increased A5 steroid intermediate (i.e. pregnenolone and DHEA) formation [evaluated in the presence of 10~6 M of WIN 24540, an inhibitor of 3/3-hydroxysteroid dehydrogenase/ isomerase (3/J-HSDI) activity]. However, this stimulation was
T
HE POTENTIAL role of epidermal growth factor (EGF) in modulating certain aspects of the testis function by acting on different testicular cell types have been examined by several laboratories. It has been reported that spermatogenesis might be affected directly (1) and/or indirectly via testicular somatic cells, such as Sertoli and Leydig cells. For example, in Sertoli cells, EGF stimulates the production of lactate and inhibits aromatase activity (2). Although different in vitro models have been used to investigate the role of EGF on steroidogenesis, its role appears not well defined in Leydig cell physiology. Indeed, both inhibitory and stimulatory efReceived November 1, 1990. Address all correspondence and requests for reprints to: Dr. M. Benahmed, Groupe de Recherches sur les Communications Cellulaires, Laboratoire de Biochimie, Hopital Sainte-Eugenie, CH Lyon-Sud, 69310 Pierre-Benite, France. * This work was financially supported by Institut National de la Sante et de la Recherche Medicale and Ministere de la Recherche et de l'Enseignement Superieur (MRES). t Recipient of a grant from MRES.
observed in cells when acutely (3 h) stimulated with hCG (0.011 ng/ml) but not when incubated with 22R-hydroxycholesterol (0.01-10 Mg/ml). Such findings indicate that EGF did not affect cholesterol side chain cleavage cytochrome P450 activity but probably increased cholesterol substrate availability for this enzyme in the inner mitochondria. Moreover, EGF significantly (P < 0.001) increased A5 steroid intermediate (i.e. pregnenolone and DHEA) but not A4 steroid intermediate (i.e. progesterone and androstenedione) conversion into testosterone, indicating that EGF enhances 3/3-HSDI activity. Such effects of EGF are directly exerted on Leydig cells since EGF receptors (Kd = 16 x 10~u M) are present in primary cultures of purified porcine Leydig cells. Together, the present findings show that in Leydig cells from intact animals, EGF enhances the gonadotropin action on testosterone formation through an increase in the availability of cholesterol substrate in the mitochondria as well as an increase in the activity of 3/3-HSDI. {Endocrinology 128: 21602168,1991)
fects of the growth factor on testicular steroidogenesis have been reported. For example, EGF has been reported to inhibit human CG (hCG) -induced steroid production in a clonal line of cultured Leydig tumor cells (MA-10) (3). An inhibitory effect of EGF on testicular steroidogenesis has been also reported by Hsueh et al. (4) who used total interstitial testicular cells from hypophysectomized rats. More recently, by using Leydig cells from immature rats, Verhoeven and Cailleau (5) have reported an inhibitory effect of EGF in the absence of LH but a stimulatory effect of EGF in the presence of the gonadotropin on steroid hormone production. Although EGF receptors have been localized in the Leydig cell line MA10 (3) and in crude testicular interstitial cells (4), they have not yet been identified in purified Leydig cells from intact animals. In addition to the discrepancies between these results, the mechanisms of action of EGF on steroidogenesis in Leydig cells from normal intact animals have not been investigated further. Because of the potential role of EGF in the testis function, the present study
2160
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MECHANISMS OF ACTION OF EGF ON LEYDIG CELLS was undertaken: 1) to determine the optimal conditions for EGF action on cultured purified porcine Leydig cells from intact animals, 2) to identify the biochemical mechanisms of action involved in EGF action on Leydig cell steroidogenesis, and 3) to determine whether EGF receptors are present on these cells, supporting a direct interaction between the growth factor and Leydig cells.
Materials and Methods EGF, 22R-hydroxycholesterol, 8-bromo-cAMP, insulin, transferrin, vitamin E, HEPES were purchased from Sigma Chemical Co. (St. Louis, MO). Transforming growth factor a (TGFa) was obtained from Biomedical Technologies Inc. (Stoughton, MA). hCG CR (121 13,450 IU/mg) was a gift of Dr. R. E. Canfield. Dulbecco's modified Eagle's (DME) medium and Ham's F12 medium were obtained from GIBCO (Grand Island Biological Co., Grand Island, NY) and collagenase/ dispase from Boehringer (Mannheim, FRG). Iodogen was purchased from Pierce Europe (Netherlands). WIN 24540 (4a, 5epoxy-17/3-hydroxy-5a-androstane) was kindly provided by Sterling-Wintrop (NY). Leydig cell preparation and culture Isolated Leydig cells were prepared from immature porcine testes (2- to 3-week old) by collagenase treatment as described by Mather and Phillips (6) and modified by Benahmed et al. (7). Briefly, decapsulated testes were minced and washed twice in DME/F12 (1:1) medium. After collagenase dissociation (0.5 mg/ml, 90-120 min at 32 C), cells were washed by centrifugation (200 x g for 10 min). The pellet was then resuspended and submitted to two successive sedimentations of 5 and 15 min. The crude interstitial cells were recovered from the supernatants and Leydig cells were prepared from this fraction by Percoll gradient centrifugation. The percentage of Leydig cells in the final preparation, as established by staining for 3/3hydroxysteroid dehydrogenase activity (8), ranged from 8090% (7). Leydig cells were plated in Falcon 24-multiwell plates (0.4 X 106 cells per dish) and cultured at 32 C in a humidified atmosphere of 5% CO2, 95% air in DME/F12 medium (1:1) containing sodium bicarbonate (1.2 mg/ml), 15 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES) and gentamicin (20 jig/ml). This medium was supplemented with insulin (2 Mg/ml), transferrin (5 Mg/ml), and vitamin E (10 ng/ ml). At the end of the experiments, the culture medium was collected and stored at -20 C until assayed for steroid hormone content. The cells were then detached from the culture dishes in trypsin-EDTA and counted in a Coulter counter (Coultronics, Margency, France). Leydig cell steroidogenic activity Porcine cultured Leydig cell steroidogenic activity was mainly evaluated through the secretion of dehydroepiandrosterone (DHEA) and testosterone, two major steroid hormones secreted in the medium (9). One of the major characteristics of this culture system is that the secretion of the steroid hormones in response to the gonadotropin LH/hCG stimulation remains stable for several days. Indeed, hCG-stimulated DHEA and
2161
testosterone secretion maximally increases at day 2 of culture, plateaues during the six following days of culture and then dramatically declines, probably reflecting a cell dedifferentiation process that may appear in cell cultures (6). Testosterone, DHEA, and pregnenolone were assayed in the culture medium by using previously reported specific RIAs (10-12). All the experimental data are represented as the mean ± SD of triplicate determinations of steroid production by three replicate cultures within each treatment group. All experiments reported here were repeated at least three times in different (independent) cell preparations. A representative experiment of each serie of experiments is presented. Statistical significance between groups was determined by Student's t test using the StatWorks software package on a Macintosh Plus computer. Differences are accepted as significant at P < 0.05. lode
125
I-labeling of EGF
Purified EGF (1 Mg) was labeled using 125I by the Iodogen procedure (13). Preparations of 125I-EGF with a specific activity of 200-300 nQi/iig was obtained by this method. Binding assays For measurements of 125I-EGF binding, immature Leydig cells were plated in Falcon 24-multiwell plates. Cells were washed once with DME/F12 medium and then incubated with 125 I-EGF and increasing amounts of unlabeled EGF for 14 h at 4 C. The cells were then washed three times with PBS BSA (1 mg/ml) (0-4 C) and solubilized in 0.5 N sodium hydroxide, 0.4% deoxycholate and the radioactivity was measured in a ycounter. Binding was analyzed by the method of Scatchard (14) and by using the EBDA and LIGAND computer program (15) which were converted to BIOSOFT by G. A. McPherson and distributed by Elsevier-Biosoft (Cambridge, UK).
Results Effects of EGF on Leydig cell steroidogenesis
In the following experiments, the optimal conditions for EGF action on testicular (Leydig cell) steroidogenesis were first investigated. The effect of EGF was tested on the secretion of testosterone and DHEA produced in porcine immature Leydig cells, between day 2 and day 6 of culture. Figure 1 shows dose-dependent effect of EGF on testosterone and DHEA secretion acutely stimulated with maximal effective hCG (1 ng/ml) concentration. EGF stimulates hCG-induced testosterone production with apparent maximal (2.5 fold) and half-maximal (ED50) effects, respectively, of 3 ng/ml (5 X 10~10 M) and 0.7 ng/ml (11 x 10~n M) EGF (Fig. 1A). In contrast to this effect on testosterone, EGF decreased in a dosedependent manner DHEA accumulation (1.7 fold) with apparent maximal and half-maximal (ID50) effects, respectively, observed with 3 ng/ml (5 X 10~10 M) and 0.7 ng/ml (11 x 10"11 M) EGF (Fig. IB). However, although these opposite effects of EGF were
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MECHANISMS OF ACTION OF EGF ON LEYDIG CELLS
2162
Endo«1991 Voll28.No4
14 - A
o
12
20
EGF
O
c
10 15
UJ
z o oc
UJ
10
-EGF
o UJ
UJ
-EGF
O.I
10
(0 O (A Ui
EGF ng/ml
B 0.01
30
0.1 hCG
ng/ml
25
-EGF
25 UJ
X
20
Q
EGF
! -EGF
10
0.1 EGF n|/rol
15
FIG. 1. Effect of EGF on hCG-stimulated steroid secretion: dose dependency. Leydig cells were cultured with increasing concentrations of EGF (0-10 ng/ml) for 72 h. The cells were then washed and stimulated with hCG (1 ng/ml, 3 h) before evaluating the testosterone (A) or the DHEA (B) content of the medium. The results represent the mean ± SD of three determinations in each of triplicate incubations.
also observed on the basal steroidogenesis, they appear to be relatively moderate, both on the accumulation of DHEA (-EGF: 0.58 ± 0.05 vs. +EGF: 0.47 ± 0.04 ng/ 106 cells/3 h, P < 0.05) and of testosterone (-EGF: 0.5 ± 0.01 vs. +EGF: 0.8 ± 0.04 ng/106 cells/3 h, P < 0.001). Figure 2 shows that EGF influences the secretion of the two steroid hormones stimulated with increasing concentrations of hCG (0.01-1 ng/ml, 3 h). In this context, the growth factor appears to increase and decrease, respectively, hCG-stimulated testosterone (Fig. 2A) and DHEA
5
L
0.1
0.01
hCG
fail
FIG. 2. Effect of hCG on EGF-treated Leydig cells. Leydig cells were cultured in the absence (•—•) or presence (O—O) of EGF (10 ng/ml) for 72 h. At the end of the treatment, the cells were washed and stimulated with increasing concentrations of hCG (0.01-1 ng/ml, 3 h) and testosterone (A) or DHEA (B) secretions were evaluated. The results represent the mean ± SD of three determinations in each of triplicate incubations.
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MECHANISMS OF ACTION OF EGF ON LEYDIG CELLS (Fig. 2B) secretions by affecting the maximal Leydig cell steroidogenic capacity but not by affecting hCG doses required for the maximal (0.1 ng/ml hCG) production of the two steroid hormones. Furthermore, the stimulatory effect of EGF on hCGstimulated testosterone production was evidenced after a long-term treatment. Indeed, no significant effect on hCG-stimulated testosterone was observed before 24-h treatment with EGF (10 ng/ml) (Table 1). A significant (P < 0.001) stimulatory effect was observed at 48 h and was maximal after 72 h of EGF treatment (Table 1). A similar stimulatory effect of EGF was observed on testosterone production whether Leydig cells were stimulated with hCG (-EGF: 7.75 ± 0.5 vs. +EGF: 13.1 ± 1.1 ng/106 cells/3 h, P < 0.001) or with 8-bromo-cAMP (-EGF: 8.4 ± 0.3 vs. +EGF: 13.7 ± 0.8 ng/106 cells/3 h, P < 0.001) indicating that the effects of EGF on steroidogenesis are subsequent to hCG-induced cAMP formation. These effects of EGF (10 ng/ml, 72 h) on steroidogenesis were not related to a change in Leydig cell number (-EGF: 38.8 ± 0.6 vs. +EGF: 38.7 ± 0.5 x 104 cells per dish). Taken together, the increase in testosterone secretion coupled with the decline in DHEA secretion in EGFtreated Leydig cells in response to acute hCG stimulation led us to hypothesize that EGF may modulate the formation and/or the catabolism of DHEA into A4 steroid hormones and particularly into testosterone. The following experiments were carried out to test these different possibilities. Effects of EGF on A5 steroid hormone formation In order to test whether EGF modulates the formation of A5 steroid hormones, namely DHEA, we used WIN 24540, a compound capable of rapidly suppressing the activity of 3/3-hydroxysteroid dehydrogenase/isomerase (3/3-HSDI), an enzyme responsible for the catabolism of TABLE 1. Time course of EGF effect on hCG (3 h) -stimulated testosterone secretion in Leydig cells Time (h) 0 6 24 48 72
Testosterone (ng/106 cells • 3 h) 13.1 ± 14.4 ± 14.8 ± 21.7 ± 26.6 ±
0.3 0.3 (NS) 1.0 (NS) 1.0° 2.0°
Leydig cells were cultured for the duration indicated (6-72 h) in the presence of EGF (10 ng/ml). At the end of the treatment, the medium was removed and the cells were washed before being stimulated with hCG (1 ng/ml, 3 h), and the testosterone content of the medium was measured. The results represent the mean ± SD of three determinations in each of triplicate incubations. NS, Not significant (P > 0.05). 0 P < 0.001, time of treatment us. to.
2163
A5 steroids into A4 steroids (16). Figure 3 shows the effects of EGF (10 ng/ml) on DHEA (Fig. 3A) and pregnenolone (Fig. 3B) secretions stimulated with increasing concentrations of hCG (0.01-1 ng/ml) in the presence of WIN 24540 at a concentration (10~5 M) which inhibits more than 90% of testosterone formation in these experimental conditions (-WIN 24540: 25.7 ± 1.0 vs. +WIN 24540: 2.1 ± 0.1 ng testosterone/106 cellules). EGF enhanced the hCG-induced accumulation of DHEA in the culture medium, indicating that the growth factor stimulates DHEA formation (Fig. 3A). Furthermore, the parallel and similar increase in pregnenolone formation after EGF treatment (Fig. 3B) indicates that the increase in DHEA formation induced by EGF is directly related to that of pregnenolone. Experiments were subsequently carried out to determine whether the increase in pregnenolone formation was related to a possible effect of EGF on cholesterol side chain cleavage enzyme (P450scc) activity. Such a possibility was tested by using hydroxylated cholesterol derivative, 22(R)-diol (22 R-hydroxy) cholesterol in excess as exogenous substrate for P45O8CC. Hydroxylated cholesterol derivatives pass readily between cell membranes and can be used to replace cholesterol as substrate for P450scc (17-19). As shown in Fig. 4, when Leydig cells were incubated with 22R-hydroxycholesterol (0.01-10 jig/ml, 3 h) in the presence of WIN 24540 (10~5 M), the stimulatory effect of EGF on DHEA formation was no longer observed. These observations indicate that the stimulatory effect of EGF on pregnenolone and DHEA formation was not related to an increase in P4508CC activity but more probably to an increase in cholesterol substrate availability in the inner mitochondria. Effects of EGF on the catabolism of A5 steroid hormones into testosterone The concomittant decrease in DHEA and increase in testosterone secretion in EGF-treated Leydig cells (Figs. 1-2) suggest that the growth factor may also enhance the catabolism of A5 steroid hormones into testosterone. The following experiments were thus carried out to clarify the role of steroidogenic enzymes metabolizing pregnenolone to testosterone as possible sites of EGF action. Testosterone production was measured after incubating Leydig cells for 1.5 h in the presence of different steroid substrates (500 ng/ml). As shown in Table 2, both A5 and A4 steroid substrates are converted into testosterone and as expected, the rate of this conversion was different for each steroid. That DHEA and androstenedione were the most converted into testosterone was not surprising since in porcine Leydig cells, testosterone biosynthesis from pregnenolone preferentially occurs via 17a-hydroxypregnenolone, DHEA and then androstenedione (9 and our unpublished data). Pretreatment of Leydig cells for
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MECHANISMS OF ACTION OF EGF ON LEYDIG CELLS
2164
Endo»1991 Voll28«No4
I
A
100
60 -
' v tEGF
•
-EGF
HI +EGF
80 40 T
-EGF
O)
60 20
40
0L 20
/
0.1
10
cholesterol 0
I
i
1
FIG. 4. Effect of EGF on A5 steroid hormone formation in Leydig cells incubated with 22R-hydroxycholesterol. Leydig cells were cultured in the absence or presence of EGF (10 ng/ml, 72 h). At the end of the treatment, the cells were washed and incubated with increasing concentrations of 22R-hydroxycholesterol (0.01-10 Mg/ml, 3 h) in the presence of WIN 24540 (10~5 M) and then DHEA secretion was evaluated. The results represent the mean ± SD of three determinations in each of triplicate incubations.
i
ai
0.01
pg/ml
hCG ng/ml B • EGF
TABLE 2. Effects of EGF on testosterone production in Leydig cells incubated with differents steroid substrates Testosterone (ng/106 cells • 1.5 h)
2.5
-EGF
-EGF
0)
Pregnenolone Dehydroepiandrosterone Progesterone Androstenedione
17.6 ± 33.7 ± 19.5 ± 207 ±
1.3 2.5 1.0 8.9
+EGF 29.6 ± 58.3 ± 21.6 ± 204 ±
0.9° 3.5° 0.9 (NS) 15.6 (NS)
Leydig cells were cultured for 72 h in the absence or presence of EGF (10 ng/ml). At the end of the treatment, the medium was removed, and the cells were incubated with different steroid substrates (namely pregnenolone, dehydroepiandrosterone, progesterone, and androstenedione, 500 ng/ml, 1.5 h), and the testosterone content of the medium was measured. The results represent the mean ± SD of three determinations in each of triplicate incubations. NS, Not significant (P > 0.05). a P < 0.001, +EGF vs. -EGF.
1.S
o o 0)
I 0.5 L i 9.01
at hCG
/ml
FIG. 3. Effect of EGF on A5 steroid hormone formation. Leydig cells were cultured in the absence (•—•) or presence (O—O) of EGF (10 ng/ml, 72 h). At the end of the treatment, the cells were washed and stimulated with increasing concentrations of hCG (0.01-1 ng/ml, 3 h) in the presence of WIN 24540 (10~5 M) and DHEA (A) or pregnenolone (B) secretions were evaluated. The results represent the mean ± SD of three determinations in each of triplicate incubations.
72 h with EGF (10 ng/ml) exhibited a differential effect on exogenous A5 steroid and on A4 steroid substrate conversion into testosterone. Indeed, the growth factor exerted a significant (P < 0.001) stimulatory effect on testosterone production in Leydig cells incubated with exogenous A5 steroid hormones (e.g. pregnenolone and DHEA) but not with exogenous A4 steroid hormones (e.g. progesterone and androstenedione). These observations clearly indicating that EGF enhances 3/3-HSDI activity were further confirmed by the findings reported
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MECHANISMS OF ACTION OF EGF ON LEYDIG CELLS in Fig. 5. Indeed, the stimulating effect of EGF on 3/3HSDI, reflected by the increase in DHEA conversion into testosterone was dependent upon EGF doses in the medium. The maximal (about 2-fold) and half-maximal increases were, respectively, observed with 1 ng/ml (1.7 X 1(T10 M) and 0.45 ng/ml (7.5 X KT11 M) EGF. Evidence for EGF receptors in cultured porcine Leydig cells
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30
* •o c
20
3
In order to determine whether the effects of EGF reported above are directly exerted on Leydig cells, the following experiments were performed to identify EGF receptors on primary cultures of porcine immature Leydig cells. Binding experiments were conducted using intact Leydig cells in near confluent monolayer cultures with various concentrations of EGF at 4 C for 14 h. As shown in Fig. 6, EGF binding was concentration dependent with saturation observed at 50 nM EGF. The Scatchard analysis of the binding data revealed the existence of two classes of binding sites (Fig. 6, inset). The binding parameters were calculated and yielded an apparent Kd value of 16 X 10~ u M with 1.2 x 103 sites per cell for the high affinity system and an apparent Kd value of 2.7 X 10"8 M with 27.6 X 103 sites per cell for the low affinity system.
Discussion The present findings demonstrate the stimulatory effect of EGF on testosterone production under acute term 85
65
45_r -EGF
0.1 EGF ng/ml
FiG. 5. Effect of EGF on 30-HSDI activity in Leydig cells. Leydig cells were cultured in the presence of increasing concentrations of EGF (010 ng/ml, 72 h). At the end of the treatment, the cells were washed and incubated with DHEA (500 ng/ml, 2 h) and then testosterone production was evaluated. The results represent the mean ± SD of three determinations in each of triplicate incubations.
o £
10
10
50 E6F
[nM]
125
FIG. 6. Binding of I-EGF to porcine immature Leydig cells. Cultured Leydig cells were incubated for 14 h at 4 C with 125I-EGF and various amounts of unlabeled EGF. The amount of specifically bound 125I-EGF was then assayed as described under Materials and Methods. The data were plotted as a saturation curve and treated by Scatchard analysis to calculate the binding parameters (inset).
LH/hCG stimulation in primary cultures of purified porcine Leydig cells. EGF enhances hCG-induced testosterone secretion to a maximal (2.5-fold) increase with ED50 of 11 x 10"11 M after a 72-h treatment. The increase in acutely hCG-stimulated testosterone production was not related to the well known cell growth stimulatory activity of EGF since no increase in the number of Leydig cells (cultured in serum-free defined medium) was observed after a 72-h treatment with EGF (10 ng/ml). In addition, this stimulatory effect of the growth factor is observed in porcine cultured Leydig cells in which the differentiated (steroidogenic) function is maximal and is not declining (i.e. between day 2 and day 6 of cultures). Such observations indicate that the enhancement of Leydig cell activity observed after EGF treatment is primarily not a reflection of the impact of the growth factor on the dedifferentiation process that appears in cell cultures. This effect of EGF is exerted through specific membrane receptors identified in the present study in cultured immature porcine Leydig cells. Leydig cells specifically bind EGF through a high affinity binding system with a Kd value of 16 X 10~ n M with 1.2 X 103 sites per cell. Although it has been reported that tumoral Leydig cells (3) and total interstitial testicular cells from hypophysectomized rats (4) exhibit EGF receptors, this is the first report indicating the presence of such receptors on purified Leydig cells from intact animals. Our observation is consistent with that of Suarez-Quian et al. (20) who localized immunocytochemically EGF receptors in rat interstitial tissues from intact animals. However the direct interaction between the growth factor and the
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MECHANISMS OF ACTION OF EGF ON LEYDIG CELLS
testicular steroidogenic cells will be definitely ascertained with the demonstration of EGF receptors on Leydig cells by using autoradiography, immunocytochemistry, and in situ hybridization technics. Although the concentration of EGF (or a related peptide) present in the testis is not determined, the ED50 of EGF was within the Kd range observed for EGF high affinity receptor identified on porcine Leydig cells. This observation suggests that the steroidogenic effect of EGF occurs within a concentration range that might be expected under physiological conditions. Moreover, the variation of high affinity EGF receptors during the male gonad development and during different physiological conditions as well as their regulation by the major factors controlling Leydig cell function will be an interesting possibility to investigate. With regard to the biochemical mechanisms involved in the growth factor effect, the increase in LH/hCG stimulated testosterone production in EGF-treated Leydig cells was probably not related to the potential changes in the structure and function of the Leydig cell membrane. Indeed, similar stimulatory effect of EGF was observed on testosterone production whether it was acutely stimulated with the gonadotropin or with 8bromo-cAMP, suggesting, in addition, that the biochemical steps involved in the growth factor action are located beyond cAMP formation. The growth factor appears to potentiate the gonadotropin LH/hCG action on testosterone production by affecting at least two different biochemical levels. The first step(s) that appears to be controlled by EGF in Leydig cells is related to an increase in pregnenolone formation. Such effect was not related to an increase in P450scc activity but probably to an increase in cholesterol substrate availability for this enzyme in the inner mitochondria. Indeed, our present findings indicate that the stimulatory effect of EGF on pregnenolone formation was only observed when Leydig cells were acutely stimulated with the gonadotropin but not when they were incubated in the presence of hydroxylated cholesterol derivatives that pass readily between cell membranes. These findings suggest that EGF action involves the cholesterol transport into the inner mitochondria and the cholesterol availability for P450scc. Since cholesterol mobilization from the cytoplasm into mitochondria involves microfilaments (21), sterol carrier proteins (22), labile regulatory protein(s) (probably the steroidogenesis activator polypeptide) (23), and the more recently identified GTP-regulatory protein(s) (24), it is tempting to speculate that these elements may be targets for the regulatory action of EGF in Leydig cells. The second biochemical step(s) involved in Leydig cell steroidogenesis that appears to be under EGF control is the 3/3-HSDI enzyme activity. The increase in this en-
Endo • 1991 Voll28«No4
zyme activity is dependent upon EGF concentration in the medium. Furthermore, it is of interest to note that this enzyme activity may well represent a very sensitive biochemical step(s) for EGF action in Leydig cells since the growth factor stimulatory effect is observed at very low concentrations (ED50 = 7 x 10"11 M EGF). This is the first report indicating that EGF enhances testicular steroidogenesis by increasing the activity of a key enzyme in Leydig cell maturation. However, it remains to be examined whether EGF is a positive effector controlling the enzyme expression at genetic or posttranscriptional levels. Moreover, it is known that EGF activates in different cellular systems a variety of intracellular signaling system(s). The binding of EGF induces the activation of EGF receptor tyrosine kinases which directly phosphorylate specific cellular substrates. The addition of the ligand is followed by several other early or delayed responses (25). Therefore, an important step toward an understanding of the stimulating action of EGF on Leydig cell steroidogenesis would be to establish which of the EGF-activated intracellular signaling system(s) is involved in the enhancement of cholesterol substrate availability in the mitochondria and of 3/3-HSDI activity reported here. Because of the negligible levels of EGF in the circulatory system (26), the effect of the growth factor on testicular function is likely related to a local production of an EGF-like substance(s). Initial observations have indicated that (receptor reactive) EGF-like substances may be produced by Sertoli cells (27). More recently, it has been reported that these substances are probably not related to EGF but to TGFa, a protein that has a structural homology with EGF and binds to the EGF receptor (28). Indeed, the gene encoding for TGFa, but not that for EGF, is transcribed in the testis (29). In addition, TGFa has been recently identified immunohistochemically in the rat testis (30). In this context, as expected, we have observed similar stimulatory effects on androgen production when porcine Leydig cells were treated with TGFa instead of EGF (unpublished data). Thus, the testicular ligand for Leydig cell EGF receptors might be related to TGFa and/or to a chemically undefined factor(s) such as Sertoli cell secreted growth factor, reported to be a receptor reactive EGF-like substance(s) but distinct from EGF and TGFa (27). Furthermore, although it is now well accepted that seminiferous tubules exert a control on Leydig function (31-37), the molecules involved in tubular-interstitial cell interactions remain poorly known. The tubular (Sertoli cell) origin of EGF-like substances (27, 29) coupled with the steroidogenic effects of EGF reported here and by others (3-5) suggest that the EGF-like substances might be involved in such interactions.
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MECHANISMS OF ACTION OF EGF ON LEYDIG CELLS
Finally, it should be borne in mind that the action of most growth factors is highly dependent on the presence of other growth factors (38). Thus, in vivo, the role of testicular EGF (or EGF-like substances) may lie in the interactions of this factor with other factors present in and released from testicular cells such as TGF/3, basic fibroblast growth factor, insulin growth factors, and /?nerve growth factor (39, 40). In this context, for example, the interactions between EGF and TGF0 on testicular steroidogenesis are of great interest. Indeed, we have already reported that TGF/3 also affects testicular (Leydig cell) steroidogenesis by controlling the same biochemical step(s) reported here to be affected by EGF but in a distinct manner (41, 42). TGF/3 effects differ from those of EGF in that they induce a decrease in cholesterol substrate availability for P450scc but are similar in that they elicit an increase in 30-HSDI enzyme activity (41). Consequently, because EGF (or a related factor) and TGF/3 are produced concomitantly in the testis, the results of their interactions is of major importance for testicular steroidogenesis. In summary, by using a model of primary cultures of immature porcine Leydig cells, we show that EGF directly (via specific membrane receptors) potentiates acute LH/hCG steroidogenic action by enhancing cholesterol substrate availability for P450scc and promotes Leydig cell differentiated function by increasing 3/3HSDI activity. This is the first report on the different biochemical mechanisms involved in EGF stimulating action on testicular steroidogenesis.
Acknowledgments We are grateful to Prof. E. Chambaz for his constant interest in our work and to Dr. M. G. Forest for testosterone antisera. We thank Mr. E. Villard and Mr. P. Bouteille for providing us with porcine testes.
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