J. Endocrinol. Invest. 14: 93-97,1991
Epidermal growth factor stimulates testosterone production of human Leydig cells in vitro V. Syed , S.A. Khan, and E. Nieschlag Max Planck Clinical Research Unit for Reproductive Medicine and Institute of Reproductive Medicine of the University, D - 4400 MOnster, F. R. Germany ABSTRACT. Epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) have been shown to regulate Leydig cell steroidogenesis in several species. We have investigated the effects, if any, of EGF and IGF-I on in vitro testosterone production of human Leydig cells. Interstitial cells or Percoll purified Leydig cells were isolated from the testes obtained from patients (n = 9) undergoing orchidectomies for treatment of prostate cancer and were cultured for different time periods with hCG, dibutyryl cAMP, EGF and IGF-I. Testosterone in the culture media was measured by radioimmunoassay. While EGF had a stimulatory effect on basal testosterone production of isolated interstitial cells or pu-
rified Leydig cells, IGF-I was ineffective. When the interstitial cells were cultured in the presence of hCG or EGF for 3, 6 or 24 h, the stimulatory effects of EGF on testosterone production were only evident after 24 h. On the other hand, hCG stimulated testosterone production at all time points (i.e after 3, 6, 24 h of incubation). When added in the presence of maximal concentrations of hCG and dibutyryl cAMP, EGF did not further enhance steroidogenesis. On the other hand, IGF-I potentiated the effects of hCG on testosterone production. These studies suggest that EGF and IGF-I may playa regulatory role in steroidogenic function of the human testes.
INTRODUCTION
known to secrete IGF-I (8-11). Tres et al. (11) have shown that Sertoli cells secrete an IGF-Ilike peptide and these cells also contain receptors for IGF-I and are responsive to the mitogenic action of this peptide (12). Human testicular tissue also contains IGF-I receptors (13) and human semen contains IGF-I (14) which at least in part appears to be of gonadal origin. In pig Leydig cells IGF-I has been shown to stimulate hCG and cAMP-stimulated testosterone production, suggesting that Sertoli cells may exert regulatory influence on Leydig cells through the secretion of an IGF-Ilike factor (5,15). Although a role of EGF and IGF-I in the regulation of testicular function in rodents has been made likely by several studies, little is known about their possible physiological role in the primate testis. In the present study, we have explored the effects, if any, of EGF and IGF-I on human Leydig cell testosterone production in vitro.
Various growth factors such as insulin-like growth factor-1 (IGF-I), epidermal growth factor (EGF) and transforming growth factor-beta (TGFB) are known to influence Leydig cell function in rats, mice and pigs (1-5). Epidermal growth factor (EGF), a polypeptide (mol wt 6,100), originally identified in murine submaxillary glands, has been shown to both stimulate and inhibit Leydig cell steroidogenesis in vitro (1, 2). EGF enhanced basal and hCG stimulated production of C 19 and C21 steroids by immature and adult rat and mouse Leydig cells (2). On the other hand, Hsueh et al. (1) reported that EGF inhibited hCG stimulated testosterone production of rat Leydig cells obtained from hypophysectomized animals. The in vivo or in vitro effects of EGF on human Leydig cells are not known. Recent studies have also indicated a role of IGF - I or somatomedin C on expression of differentiated functions in selected endocrine tissues such as rat and porcine granulosa cells (6, 7). Rat testi$is
MATERIALS AND METHODS Chemicals and media Ham's F-12 nutrient mixture, Dulbecco's modified Eagles minimum essential medium (DMEM) and Lglutamine were purchased from Flow Laboratories, (Irvine, Scotland). Bovine serum albumin (BSA), collagenase (Type I) and dibutyryl adenosine 3,5
Key-words: Growth factors. IGF-I, EGF, Leydig cells, testis, steroidogenesis. Correspondence: Prof. Dr. E. Nieschlag, Institute of Reproductive Medicine, University of Munster, Steinfurter Str. 107, 0 - 4400 Munster, Federal Republic of Germany. Received December 1, 1989; accepted November 5, 1990.
93
V. Syed, S. A. Khan, and E. Nieschlag
cyclic monophosphate (dbcAMP) were obtained from Sigma Chemicals (MOnchen, F. R. Germany) PercollT M was purchased from Pharmacia Fine Chemicals (Uppsala, Sweden) . Epidermal growth factor and insulin-like growth factor-1 were obtained from Calibiochem GmbH (Frankfurt, F. R. Germany) and from Bachem Inc. (Torrance, CA , USA) , respectively. All cell isolations and incubations were carried out in Ham 's F12: DMEM (1: 1) containing 20 mmol/I Lglutamine and 0.2% BSA.
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Testes The testicular tissues were obtained from 9 patients undergoing orchidectomies for treatment of prostatic carcinoma. The mean age of the patients was 75.4 (range 63-81) years ; they were not suffering from any other endocrine disease. For each individual experiment, both testes from the same patient were transported to the laboratory in ice-cold medium and the interstitial cells were prepared immediately as described below. Every experiment was repeated at least three times using a different cell preparation obtained from testes of different patients.
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Epidermal growth factor stimulates testosterone production of human Leydig cells in vitro V. Syed , S.A. Khan, and E. Nieschlag Max Planck Clinical Research Unit for Reproductive Medicine and Institute of Reproductive Medicine of the University, D - 4400 MOnster, F. R. Germany ABSTRACT. Epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) have been shown to regulate Leydig cell steroidogenesis in several species. We have investigated the effects, if any, of EGF and IGF-I on in vitro testosterone production of human Leydig cells. Interstitial cells or Percoll purified Leydig cells were isolated from the testes obtained from patients (n = 9) undergoing orchidectomies for treatment of prostate cancer and were cultured for different time periods with hCG, dibutyryl cAMP, EGF and IGF-I. Testosterone in the culture media was measured by radioimmunoassay. While EGF had a stimulatory effect on basal testosterone production of isolated interstitial cells or pu-
rified Leydig cells, IGF-I was ineffective. When the interstitial cells were cultured in the presence of hCG or EGF for 3, 6 or 24 h, the stimulatory effects of EGF on testosterone production were only evident after 24 h. On the other hand, hCG stimulated testosterone production at all time points (i.e after 3, 6, 24 h of incubation). When added in the presence of maximal concentrations of hCG and dibutyryl cAMP, EGF did not further enhance steroidogenesis. On the other hand, IGF-I potentiated the effects of hCG on testosterone production. These studies suggest that EGF and IGF-I may playa regulatory role in steroidogenic function of the human testes.
INTRODUCTION
known to secrete IGF-I (8-11). Tres et al. (11) have shown that Sertoli cells secrete an IGF-Ilike peptide and these cells also contain receptors for IGF-I and are responsive to the mitogenic action of this peptide (12). Human testicular tissue also contains IGF-I receptors (13) and human semen contains IGF-I (14) which at least in part appears to be of gonadal origin. In pig Leydig cells IGF-I has been shown to stimulate hCG and cAMP-stimulated testosterone production, suggesting that Sertoli cells may exert regulatory influence on Leydig cells through the secretion of an IGF-Ilike factor (5,15). Although a role of EGF and IGF-I in the regulation of testicular function in rodents has been made likely by several studies, little is known about their possible physiological role in the primate testis. In the present study, we have explored the effects, if any, of EGF and IGF-I on human Leydig cell testosterone production in vitro.
Various growth factors such as insulin-like growth factor-1 (IGF-I), epidermal growth factor (EGF) and transforming growth factor-beta (TGFB) are known to influence Leydig cell function in rats, mice and pigs (1-5). Epidermal growth factor (EGF), a polypeptide (mol wt 6,100), originally identified in murine submaxillary glands, has been shown to both stimulate and inhibit Leydig cell steroidogenesis in vitro (1, 2). EGF enhanced basal and hCG stimulated production of C 19 and C21 steroids by immature and adult rat and mouse Leydig cells (2). On the other hand, Hsueh et al. (1) reported that EGF inhibited hCG stimulated testosterone production of rat Leydig cells obtained from hypophysectomized animals. The in vivo or in vitro effects of EGF on human Leydig cells are not known. Recent studies have also indicated a role of IGF - I or somatomedin C on expression of differentiated functions in selected endocrine tissues such as rat and porcine granulosa cells (6, 7). Rat testi$is
MATERIALS AND METHODS Chemicals and media Ham's F-12 nutrient mixture, Dulbecco's modified Eagles minimum essential medium (DMEM) and Lglutamine were purchased from Flow Laboratories, (Irvine, Scotland). Bovine serum albumin (BSA), collagenase (Type I) and dibutyryl adenosine 3,5
Key-words: Growth factors. IGF-I, EGF, Leydig cells, testis, steroidogenesis. Correspondence: Prof. Dr. E. Nieschlag, Institute of Reproductive Medicine, University of Munster, Steinfurter Str. 107, 0 - 4400 Munster, Federal Republic of Germany. Received December 1, 1989; accepted November 5, 1990.
93
V. Syed, S. A. Khan, and E. Nieschlag
cyclic monophosphate (dbcAMP) were obtained from Sigma Chemicals (MOnchen, F. R. Germany) PercollT M was purchased from Pharmacia Fine Chemicals (Uppsala, Sweden) . Epidermal growth factor and insulin-like growth factor-1 were obtained from Calibiochem GmbH (Frankfurt, F. R. Germany) and from Bachem Inc. (Torrance, CA , USA) , respectively. All cell isolations and incubations were carried out in Ham 's F12: DMEM (1: 1) containing 20 mmol/I Lglutamine and 0.2% BSA.
~
200
EGf
~
&.2",
0.02",
.,
100
100
800
200
:::l G>
0
""0.... ~
C'l
"'.....,"
2.5",
25",
bD
tl. 100 G>
d
Testes The testicular tissues were obtained from 9 patients undergoing orchidectomies for treatment of prostatic carcinoma. The mean age of the patients was 75.4 (range 63-81) years ; they were not suffering from any other endocrine disease. For each individual experiment, both testes from the same patient were transported to the laboratory in ice-cold medium and the interstitial cells were prepared immediately as described below. Every experiment was repeated at least three times using a different cell preparation obtained from testes of different patients.
...0 ...G> ...'"0 'G>" E-
