0013.7227/92/1302-0831$03.00/0 Endocrinology Copyright 0 1992 by The Endocrine
Vol. 130, No. 2 Printed in U.S.A.
Society
Transforming Growth Factor ,61 Inhibits Action in Cultured Porcine Sertoli Cells* A. M. MORERA, G. ESPOSITO, AND M. BENAHMED
C. GHIGLIERI,
M. A. CHAUVIN,
Gonadotropin
D. J. HARTMANN,
INSERM CJF 90-08, Groupe de Recherches SW les Communications Cellulaires, Laboratoire de Biochimie, H6pital Sainte Eugknie, Centre Hospitalier Lyon-Sud, 69310 Pierre-Bknite, and Centre de Radioanalyse, Znstitut Pasteur (D.J.H.), 69007 Lyon, France
ABSTRACT.
In the present study, we have tested the effects of transforming growth factor @l (TGFPl) on FSH action toward aromatase activity and lactate production in cultured Sertoli cells isolated from immature porcine testes. Whereas treatment of Sertoli cells with FSH resulted in a dose-dependent increase (about 7-fold) in aromatase activity (conversion of testosterone into estradiol) (ED,, = 80 rig/ml FSH), the addition of TGFfil reduced this gonadotropin -action. The inhibitory effect of TGFal on FSH aromatase activitv was dose denendent (ED,, = 0.1 r&ml, 4 pM TGF@l) with a maximal decrease (about 46%) observed after a long term (48-h) treatment. TGF@l exerted its inhibitory effect on FSH action at the level(s) of CAMP accumulation, exerting no apparent effect on the gonadotropin receptor or at a site(s) related to CAMP action. TGFfll (2 rig/ml) significantly (P < 0.002) reduced (52% decrease) FSH-stimulated CAMP levels in cultured porcine Sertoli cells. However, such an inhibitory effect of the growth factor was no longer observed when stimulation of CAMP accumulation with FSH occurred in the presence of methyl isobutyl xanthine (0.5 mM),
an inhibitor of CAMP-phosphodiesterase activity. This observation suggests that TGFPl decreased CAMP levels by increasing catabolism of the cyclic nucleotide through an enhancement of CAMP-phosphodiesterase activity. The inhibitory effect of TGFPl was not limited to the action of FSH on aromatase activity but also extended to the gonadotropin action (mediated by CAMP) on lactate production. As for the inhibitory effect of TGFPl on FSH-induced aromatase activity, the inhibitory effect of the growth factor on FSH-stimulated lactate production was dose and time dependent with a maximal decrease (about 30%) observed in the picomolar range (1 r&ml, 40 PM) after 48 h treatment with TGFPl. In conclusion,~the present study demonstrates that TGFal attenuates FSH action on Sertoli cell activity and that such inhibitory action is potentially exerted through a decrease in CAMP levels. Because of the local production of TGFbl, it is suggested that the effects of the growth factor reported here might be exerted in the context of the testicular paracrine mechanisms. (Endocrinology 130: 831-836, 1992)
A
GREAT body of evidence supports the concept that testicular function is not only under the control of systemic hormones, namely the gonadotropins LH and FSH but is also under the control of local factors among which several regulatory peptides and growth factors have recently been identified (for references and reviews, see l-3). Among these factors, transforming growth factor /?l (TGFPl), a 25,000 mol wt homodimeric protein, is a member of a family of structurally related, multifunctional polypeptide hormones with wide paracrine and autocrine biological activities including control of growth and differentiation (4-6). Recent studies have indicated that TGFP is secreted in the testis (7-9). Al-
though there is much evidence that, in the testis, Leydig cells are a target for TGFPl action (7, lo-12), little is known about the effects of this growth factor on the other testicular cell types and particularly on Sertoli cells. In the present study, we have investigated the possibility that TGFPl could affect Sertoli cell functions controlled by FSH. We report here that TGFPl antagonizes FSH stimulatory action on Sertoli cell functions particularly on aromatase activity and lactate production. Materials
and Methods
TGFPl purified to homogeneity from human platelets was a generousgift of Dr. J. P. Hendrick (Laboratoire de Radioimmunologie, Centre Hospitalier et Universitaire de Liege, Belgium) or obtained from British Bio-Technology Limited (BBL, Oxford, UK). Collagenase(catalog 103586) and lactoperoxidase were obtained from Boehringer (Mannheim, FRG). Dulbecco’s modified Eagle’smedium (DME) and Ham’sF12 mediumwere obtained from GIBCO (Grand Island, NY). Porcine FSH (NIHFSH p2) was kindly provided by the National Institute of
Received August 21,199l. Address all correspondence and requests for reprints to: Dr. M. Benahmed, Groupe de Recherche sur les Communications Cellulaires, Laboratoire de Biochimie, Hopital Sainte-Eugenic, Centre Hospitalier Lyon-Sud, 69310, Pierre-B&rite, France. * This work was supported by Institut National de la Sante et de la Recherche Medicale (INSERM, CJF 90-08), Minis&e de 1’Education Nationale, and in part by Fondation pour la Recherche en Hormonologie (FRH 69 9101) and Serono laboratories.
831
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832
TGFPl INHIBITS
FSH ACTION
Arthritis, Metabolic, and Digestive Diseases (Pituitary Hormone Distribution Program, Bethesda, MD). Human transferrin, insulin, a-tocopherol, HEPES, DNase type 1, lactate dehydrogenase (from rabbit muscle), and NAD were purchased from Sigma Chemical Co. (St. Louis, MO). [Y] and 2, 4, 6, 7[“Hlestradiol (94 Ci/ml) were purchased from Amersham International (Amersham, UK). Estradiol antiserum was obtained from BioM6rieux (Lyon, France). Isolation
and culture
of Sertoli
cells
Sertoli cells were isolated from immature porcine testes (23 weeks old) by collagenase treatment as described by Mather and Phillips (13). The testes were decapsulated, minced, and washed in DME/FlB (1:l) medium. After collagenase dissociation (0.5 mg/ml, 90-120 min at 32 C), cells were washed by mild centrifugation (200 x g for 10 min). The pellet was then resuspended and after a sedimentation of 5 min, the sedimented tubules were recovered and washed three times by unit gravity in DME/FlZ medium. These tubules were then incubated 10 min (room temperature) in 20 ml 1 M Glycine, 2 mM EDTA, and 20 IU/ml DNase 1 in Ca++, Mg++-free phosphate buffer solution, pH 7.2. This treatment led to the release of the contaminating interstitial (Leydig) cells. The glycine-treated tubules were then washed three times (in DME/FlB medium) by unit gravity and incubated in 100 ml DME/F12 medium containing collagenase (0.5 mg/ml), DNase (0.05 mg/ml) and soybean trypsin inhibitor (0.05 mg/ml) for 15 min (32 C). The supernatants containing the peritubular myoid cell fraction were removed, and the sedimented tubules were treated again as described above with collagenase (0.5 mg/ml, 20 min, 32 C) until small clumps resulted. Clumps were left to settle, and the supernatants were discarded. This procedure led to a purified Sertoli cell population not contaminated by Leydig cells or germ cells and containing between 2-5% peritubular myoid cells as evaluated by using fibronectin and desmin immunostainings (14; Tabone, E., M. Benahmed, D. J. Hartmann, and A. M. Morera, unpublished data). Sertoli cells were plated in Falcon (Oxnard, CA) 24-multiwell plates (0.25-0.4 X 10” cells per dish) cultured in DME/F12 medium (1:l) containing 1.2 mg/ml sodium bicarbonate, 15 mM HEPES, and 20 pg/ml gentamycin. This medium was supplemented with transferrin (5 pg/ml), insulin (2 pg/ml), and (Ytocopherol (10 kg/ml). Cells were cultured at 32 C in a humidified atmosphere of 5% C02, 95% air. Measurement
of
lactate production
The amounts of lactate present in Sertoli cell culture media were estimated with the enzymatic method described by Hohorst (15) by using a Kontron fluorimeter (Kontron Instruments, Zurich, Switzerland) at an excitation wavelength of 340 nm and an emission wavelength of 455 nm. The cell number was determined by using a Coulter Counter (Coultronics, Margency, France) once the cells were detached from the culture dishes in trypsin-EDTA. Aromatase
assay in cultured
Sertoli
cells
Sertoli cell aromatase activity was assessedby measuring the conversion of testosterone (1 mM, 8 h) into estradiol secreted
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Endo. 1992 Vol130. No 2
in the culture medium. The concentration of estradiol was determined through a RIA with a specific estradiol antibody and 2,4,6,7-[“Hlestradiol (94 Ci/mmol: cross-reactivity of the estradiol antiserum at 50% binding was estradiol lOO%,estrone 15%, dehydroepiandrosterone 0%, testosterone 0%, and androstenedione 0%). Similar amounts of RIA detectable estradiol were found before and after the ether extraction of the steroid from the culture medium and purification on a celite column. Therefore, unless specified otherwise in the described experiments, estradiol was directly measured in the culture medium. CAMP assay
Sertoli cells were stimulated with FSH for 30 min. At the end of this incubation, the culture media were collected into ethanol (80% vol/vol) at 4 C. After shaking, the supernatants were collected by centrifugation then evaporated to dryness under a stream of nitrogen. After reconstitution with 0.05 M phosphate buffer, pH 6.2, the samples were acetylated and assayed for CAMP using specific RIA (16) (CAMP lznI RIA, Pasteur kit, ERIA-Diagnostics Pasteur, Marnes, France). Binding
assays of FSH
Human FSH was labeled with “‘1 by the lactoperoxidase method (17). The specific activity was about 50 &!i/Fg. [12hI] FSH binding to cultured Sertoli cells was performed in 0.2 ml binding buffer (DME/F12, l:l, containing 1.2 mg/ml sodium bicarbonate, 15 mM HEPES, pH 7.5, and 0.5% BSA). Incubations were carried out for 2 h at 33 C. At the end of the incubation, the cells were quickly washed four times with icecold 150 mM sodium chloride containing 1% BSA. The cells were then dissolved in 0.5 M NaOH, 0.4% deoxycholate, and the radioactivity was measured in a y-counter. Data analysis
All the experimental data are presented as the mean + SD of triplicate determinations within each treatment group. All experiments reported here were repeated at least three times in different independent cell preparations. Statistical differences between two groups were determined using Student’s t test. Differences are accepted as significant at P < 0.05.
Results FSH-stimulated Sertoli cell.9
aromatase activity in immature porcine
Since all the data reported
aromatase
activity
so far on the regulation
of
in Sertoli cells have been obtained
using mainly cultured
cells from immature
rat testis, we
first tested whether cultured immature porcine Sertoli cells, an experimental model we use currently in our laboratory, is a suitable model for investigating the regulatory mechanisms involved in Sertoli cell aromatase activity. Figure 1 shows some of the characteristics of aromatase activity in response to FSH in porcine Sertoli cells. Immature cultured porcine Sertoli cells exhibited a very low basal aromatase activity but which is highly responsive to stimulation by the gonadotropin FSH in a
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TGFPl
INHIBITS
FSH
ACTION
A z =:
300 -
c fn 4 . bo
200 -
0
1
1
FSH
10
100
10000
1000
(nglml)
IN SERTOLI
CELLS
porcine Sertoli cells cultured in optimal conditions as indicated above. In porcine Sertoli cells, basal aromatase activity evaluated through the accumulation of estradiol in the medium is very low and appeared not to be significantly affected by TGFPl (2 rig/ml, 48 h) treatment (Fig. 2B). By contrast, TGFPl inhibited FSHinduced aromatase activity in cultured porcine Sertoli cells. Indeed, as shown in Fig. 2A, Sertoli cell treatment with TGFPl (0.01-10 rig/ml, 48 h) resulted in a concentration-dependent attenuation of FSH-induced aromatase activity to a maximal decrease of 41% observed with 1 rig/ml TGFPl and with an apparent IDS0 of 0.1 rig/ml (4 PM) TGFPl (Fig. 2A). Furthermore, the inhibitory effect of the growth factor was observed for all FSH (l3000 rig/ml) doses tested to stimulate Sertoli cell aromatase activity. TGFPl decreased the maximal response to the gonadotropin but without affecting the FSH concentrations required for the maximal and half-maximal activity of the enzyme (Fig. 2B). The inhibitory effect of TGFPl on FSH-induced aro-
24
Time
30
44
= c \o 2
72
Effect of TGF/31 on FSH-stimulated in cultured Sertoli cells
aromatase activity
We therefore next examined TGFPl action on basal and FSH-stimulated aromatase activity by using the
l-l“c,
;: loo II i
1. Effects of FSH on Sertoli cell aromatase activity. Sertoli cells were cultured for 48 h with or without increasing concentrations (O3000 rig/ml) of FSH (A) and for indicated times (O-72 h) with FSH (2000 rig/ml) (B), then testosterone substrate (200 rig/ml) was added for 8 h, and estradiol production was measured in the culture medium. The results represent the mean + SD of three separate determinations in three different dishes. FIG.
dose- and time-dependent manner. The maximal (about 7-fold increase) and half maximal effects were respectively observed with 1000 and 80 rig/ml FSH (Fig. 1A). The stimulating effect of FSH was time dependent since it was detected at 24 h FSH treatment and was maximal after 44 h treatment with the hormone (Fig. 1B). Furthermore, in control experiments, we have determined the optimal cell culture conditions for aromatase activity in response to FSH action. This stimulatory effect of FSH was: 1) maximal between day 2 and 12 in porcine Sertoli cell cultures; and 2) dependent upon cultured Sertoli cell densities (0.1-l x lo6 cells per dish). The response to FSH stimulation was maximal between 0.3-0.5 x lo6 cells per dish and then declined at a density higher than 0.5 X lo6 cells per dish (data not shown).
2ocI
‘M ,a
(hours)
-
I
A
300 -
a
0
a33
II
0’
I
’
’
I 0
“‘-.
FSH
‘s; L!p:
300
1
.l
““-1 10
I loo
FSH + TGFl31 (rig/ml)
-
-TGFDl
-
+TGFDl
rD z -
200
iit
0 0
.l
1
FSH
10
loo
loo0
II
00
(rig/ml)
2. Effects of TGFPl on Sertoli cell aromatase activity. Sertoli cells were cultured: for 48 h with a fixed dose of FSH (2000 rig/ml (A) and with or without increasing concentrations of TGFPl (O-10 rig/ml) (B), for 48 h with a fixed dose of TGFPl (2 rig/ml) and with or without increasing concentrations of FSH (l-3000 rig/ml); then testosterone substrate (200 rig/ml) was added for 8 h, and estradiol production was measured in the culture medium. The results represent the mean + SD of three separate determinations in three different dishes. FIG.
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TGFBl
834
INHIBITS
FSH
ACTION
matase activity was not related to a change in Sertoli cell number after the treatment with TGFPl (2 rig/ml, 48 h) (-TGFPl: 0.38 f 0.02 us. + TGFPl: 0.39 f 0.03 x lo6 cells per dish).
IN SERTOLI
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Endo. 1992 Vol130. No 2
80,
I
Sites of action of TGFpl To delineate the mechanisms involved in the inhibitory effect of TGFPl on FSH-induced aromatase activity, we have investigated the potential role of the growth factor on FSH binding to its receptors, on FSH-stimulated CAMP production, and on 8-bromo CAMP-stimulated aromatase activity. Our data indicated that after TGFPl treatment (2 rig/ml, 48 h), specific FSH binding to Sertoli cells was not affected (-TGF/31: 30.0 X 1 us. + TGFPl: 32.1 + 1.5 x lo3 cpm/106 cells). To examine whether TGFPl affected the accumulation of CAMP, Sertoli cells were pretreated with TGFPl (2 rig/ml, 48 h) before being stimulated with FSH (2000 rig/ml) in the absence or presence of methyl isobutylxanthine (MIX) (0.5 IrIM), used as an inhibitor of CAMP-phosphodiesterase (PDE) activity. Whereas TGFPl was without significant effect on the basal accumulation of extracellular CAMP, its addition significantly (P < 0.002) decreased 52% inhibition) the action of FSH. However, such an inhibitory effect of TGFPl on extracellular CAMP accumulation was no longer observed when Sertoli cells were stimulated with FSH but in the presence of MIX (Fig. 3). Finally, TGFPl appears to be without effect on 8bromo-CAMP-induced aromatase activity (Fig. 4), suggesting that TGFPl effect on FSH action probably involves site(s) proximal but not distal to CAMP accumulation. Effects of TGF/31 on FSH-stimulated
lactate production
To test the specificity of TGFPl action, we have studied the effect of the growth factor on another Sertoli cell
25 8Br
74 222 666 CAMP ( PM)
TGFI31 FSH + TGFBl
0 CAMP
500
(pmoles/
1000
lo6
FIG. 3. Effects of TGFPl on extracellular CAMP cells were cultured in the presence or absence of 48 h, then with or without MIX (0.5 mM) for 30 without FSH (2000 rig/ml) for 30 min as indicated. in the culture medium. The results represent the separate determinations in three different dishes. FSH + TGFPl.
1500
cells)
accumulation. Sertoli TGFPl (2 rig/ml) for min and then with or CAMP was measured mean k SD of three P < 0.002; +FSH us.
2000
FIG. 4. Effects of TGFpl on 8-bromo-CAMP-stimulated aromatase activity. Sertoli cells were cultured in the presence or absence of TGFPl (2 rig/ml, 48 h) and with or without increasing concentrations of 8bromo-CAMP (O-2 mM) for 48 h, then testosterone substrate (200 ng/ ml) was added for 8 h, and estradiol production was measured in the culture medium. The results represent the mean f SD of three separate determinations in three different dishes.
0 24 48 72 Time (hours)
2. al % z 2
0
FSH
1
1
10
100
FSH+TGFOl (rig/ml)
as :A -
0.3 1 FSH
Basal
FSH
0
10 100 hglml)
a . .. 1000 10000
FIG. 5. Effects of TGFPl on lactate production. Sertoli cells were cultured with TGFpl (2 rig/ml) for the different times indicated and then stimulated for 6 h with a maximal efficient dose of FSH (2000 rig/ml) (A); with increasing concentrations of TGFPl (0.01-10 rig/ml) for 48 h and then stimulated for 6 h with FSH (2000 rig/ml) (B); and with or without a fixed dose of TGFPl (2 rig/ml) for 48 h and then with increasing concentrations of FSH (l-2500 rig/ml) for 6 h (C). The culture media were collected for lactate determination. The results represent the mean + SD of three separate determinations in three different dishes.
parameter known to be highly regulated by FSH via the CAMP pathway, i.e. lactate production. Sertoli cells were pretreated with TGFPl before being acutely stimulated with the gonadotropin to increase lactate production. The data obtained indicate that TGF/31 inhibited in a time- (Fig. 5A) and dose (Fig. 5B)-dependent manner
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TGFBl
INHIBITS
FSH
ACTION
FSH stimulated lactate production with a maximal inhibition (31% decrease) observed with 1 rig/ml TGFPl after 48 h treatment. TGFPl (2 rig/ml, 48 hours) decreased lactate accumulation stimulated with all FSH doses tested (l-3000 rig/ml, 6 h). In these conditions, TGF@l significantly (P < 0.005) decreased the maximal response to FSH (observed at 1000 rig/ml) in terms of lactate accumulation, but it did not affect the FSH doses required for the maximal and half-maximal levels of lactate produced (respectively observed with 500 and 50 rig/ml FSH) (Fig. 5C). Discussion The purpose of the current study was to investigate the effect of TGFpl on the gonadotropin action toward Sertoli cell functions. The experimental model used was Sertoli cells obtained from immature (3-4 weeks old) porcine testes and cultured in defined medium. In regard to the regulation of aromatase activity (present study) and of lactate production (present study; 18), cultured porcine Sertoli cells appear comparable to other experimental models (particularly to the rat model). This study shows that TGF/31 antagonizes FSH action on aromatase activity and lactate production. Indeed, although TGF/31 by itself did not significantly affect basal aromatase activity and stimulated (in short term conditions) basal lactate production (18), however, it inhibited FSH-induced aromatase activity (about 40% decrease) and FSH-stimulated lactate production (about 30% decrease) with an IDso in the picomolar concentration range after a long term treatment. Although the concentration of TGF/31 present in the testis is not yet determined, the concentration of TGFPl required to inhibit FSH action on Sertoli cell aromatase activity was consistent with the dissociation constant range for TGFPl high affinity receptors we have previously identified in porcine Sertoli cells (18). Such observations suggest that the inhibitory effect of TGFPl action on gonadotropin action occurs within a concentration range that might be expected under physiological conditions. The mechanisms through which TGFPl antagonizes FSH action remain to be clarified. However, the present data indicate that the inhibitory effect of the growth factor is not related to a decrease in FSH binding to its receptors but more probably to a decrease in the levels of CAMP, the intracellular mediator of the gonadotropin action on Sertoli cell parameters studied here, i.e. aromatase activity (19), and lactate production (20; our unpublished data). Furthermore, that this decrease in the cyclic nucleotide levels induced by TGFPl was observed only in the absence but not in the presence of MIX would indicate that the growth factor reduced CAMP accumulation by increasing the Sertoli cell PDE
IN SERTOLI
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835
activity. Whether a modulation of the activity of this last enzyme may play a physiologically relevant role in the gonadotropin action remains to be studied. However, it is of interest to indicate that a cycling of the CAMPPDE activity has been suggested as a cause of the changes in FSH responsiveness of the Sertoli cell during the spermatogenic cycle of the seminiferous tubules (21). Therefore, in this context, the potential regulation of this Sertoli cell enzyme activity by TGFPl (or its related peptides) will be of interest to investigate. Further studies are required to examine whether TGF/31 is a positive effector controlling the Sertoli cell phosphodiesterase expression at genetic or posttranscriptional levels. In addition, an increased catabolism of CAMP through an enhancement of CAMP-PDE attenuating FSH action has been also reported in granulosa cells. Indeed, such a mechanism(s) is probably involved in the inhibitory effect of, at least, GnRH (22) and of epidermal growth factor (23) on gonadotropin action in cultured rat granulosa cells. Owing to the negligible levels of active TGFPl in the circulatory system (4-6), the effects of this factor on FSH action would depend on its local production. Indeed, we have shown previously that a biologically active and receptor reactive TGFP-like factor is secreted by immature cultured porcine Sertoli cells (8). Consistent with these findings, the TGF/31 gene is transcribed in the rat (Sertoli and peritubular myoid cell) testis (9). Furthermore, we also have reported that TGFP-like secretion in Sertoli cells is greatly inhibited after FSH treatment (8). Together with the data reported in the present study, one might speculate that: 1) the inhibitory effect of TGFPl on the gonadotropin action is probably not accomplished in an autocrine context; and 2) by dramatically reducing the secretion of TGFp in Sertoli cells, the gonadotropin may amplify its stimulatory action on aromatase activity. On the other hand, the possibility exists that the inhibitory effect of TGFPl on FSH action is exerted in the context of a paracrine control mainly originating from peritubular myoid cells and/or germ cells. Indeed, although the secretion of (active) TGF/31 from peritubular myoid cells and germ cells remains to be clearly demonstrated, TGF/31 is potentially present in these cells as shown by the presence of TGFPl messenger RNA in rat peritubular myoid cells (9) and by the identification of a positive TGFPl immunostaining in rat germ cells (Ghiglieri, C., E. Tabone, J. P. Hendrick, P. Franchimont, A. M. Morera, and M. Benahmed, in preparation). In this context, rat peritubular myoid cells (24) and germ cells (25) have been reported to secrete factors which inhibit FSH-induced Sertoli cell aromatase activity. Such factors remain as yet chemically undefined. In the light of our present findings, it will be of interest to determine whether these factors are distinct from TGFpl
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836
TGFPl
INHIBITS
FSH
ACTION
(or its related peptides). Finally, the data in the present study extend the concept related to TGF/31 as a potential testicular factor antagonizing LH action in Leydig cells (10-12) to FSH action in Sertoli cells. However, one must note that the biochemical mechanisms involved in the inhibitory effect of TGFPl on LH action are probably distinct from those reported here on FSH action. Indeed, although in Leydig cells, TGFPl decreased LH/hCG binding as well as LH/ hCG-stimulated CAMP formation (10, ll), its inhibitory effect is predominantly exerted at a point after CAMP formation, probably by decreasing the action of gonadotropin (via CAMP pathway) on cholesterol substrate availability in the mitochondria (12). Such differences in the mechanism(s) of action of TGFPl might be related to the different embryological, mesenchymal, and epithelial origins of, respectively, Leydig and Sertoli cells in which TGFPl may activate different intracellular signaling systems. In summary, by using a model of cultured immature porcine Sertoli cells we report, for the first time, that TGF@l is a potential local factor that antagonizes FSH action on Sertoli cell functions such as aromatase activity and lactate production. Such an inhibitory effect is probably related to a decrease in the gonadotropin-stimulated cyclic nucleotide accumulation. Acknowledgments We are grateful to Dr. J. P. Hendrick for TGFfll. We are indebted to the National Hormone and Pituitary Program of the NIH for supplying us with porcine FSH used in these studies. We are grateful to Mr E. Villar and P. Bouteille for providing us with porcine testes. We thank Dr. D. A. Lawrence for reading the manuscript.
References 1. Bellve AR, Zheng W 1989 Growth factors as autocrine and paracrine modulators of male gonadal functions. J Reprod Fertil 85:771-793 2. Benahmed M, Sordoillet C, Chauvin MA, Morera AM 1990 Local regulators of the testis function. In: Bouchard P, Haour F, Franchimont P, Schatz B (eds) Recent Progress on GnRH and Gonadal Peptides. Elsevier, Paris, pp 383-405 3. Skinner MK 1991 Cell-cell interactions in the testis. Endocr Rev 12~45-77 4. Lawrence DA 1985 Transforming -- growth factors: an overview. Biol Cell 53:93-98 5. Roberts AB, Flanders KC, Kondaiah P, Thompson NL, Van Obberghen-Shilling E, Wakefield L, Rossi P, De Crombrugghe B, Heine U, Sporn MB 1988 Transforming growth factor /3: biochemistry and roles in embryogenesis, tissue repair and remodeling, and carcinoeenesis. In: Clark JH fed) Recent Progress in Hormone Research. Academic Press, London, vol44:157-1197 J 1990 The transforming growth factor-p family. Annu 6. Massague Rev Cell Biol 6:597-641
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SERTOLI
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Endo. Voll30.
1992 No 2
7. Morera AM, Benahmed M, Cachet C, Chauvin MA, Chambaz E, Revol A 1987 A TGFP-like peptide is a possible intratesticular modulator of steroidogenesis. Ann NY Acad Sci 513:494-496 8. Benahmed M, Cachet C, Keramidas M, Chauvin MA, Morera AM 1988 Evidence for FSH dependent secretion of a receptor reactive transforming growth factor P-like material by immature Sertoli cells in primary culture. Biochem Biophys Res Commun 154:12221231 9. Skinner MK, Moses HL 1989 Transforming growth factor B gene expression and action in the seminiferous tubule: peritubular cellSertoli cell interactions. Mol Endocrinol 3:625-630 10. Lin T, Blaisdell J, Haskell JF 1987 Transforming growth factor-6 inhibits Leydig cell steroidogenesis in primary culture. Biochem Biophys Res Commun 146:387-394 11. Morera AM, Cachet C, Keramidas M, Chauvin MA, De Peretti E, Benahmed M 1988 Direct regulating effects of transforming growth factor 4 on the Levdig cell steroidoaenesis in urimarv culture. J Steroid Biochem 30:443-447 ” 12. Benahmed M. Sordoillet C. Chauvin MA. De Peretti E. Morera AM 1989 On the mechanisms involved’in the inhibitory and stimulating actions of transforming growth factor-p on porcine testicular steroidoeenesis: an in uitro studv. Mol Cell Endocrinol 67:155-164 13. Mather JP, Phillips DM 1984 Primary culture of testicular somatic cells. In: Barnes DW. Sirbasku DA, Sato GH teds) Methods for Serum-free Culture of the Endocrine System. Alan. R. Liss, New York, pp 29-45 14. Tung PS, Skinner MK, Fritz IB 1984 Fibronectin synthesis is a marker for peritubular cell contaminants in Sertoli cell-enriched cultures. Biol Reprod 30:199-201 15. Hohorst HJ 1970 L-(+)-Lactat, bestimmung mit lactat-dehydrogenase und NAD. In: Bermeyer HU (ed) Methoden der Enzymatischen Analysen, ed 2. Part II. Verlag Chemie GmgH, Weinheim, p 1425-1429 16. Harper JF, Booker G 1975 Femtomole sensitive radioimmunoassay for cyclic AMP and cyclic GMP after 2’-0-acetylation bv acetic anhydride in aqueous solution. J Cyclic Nucleotide Res 1:207-218 17. Ketelsleaers JM. Catt KJ 1974 Receutor bindine nronerties of lz51 FSH prepared by enzymatic iodination. J Clin Endocrinol Metab 391159-1162 18. Esposito G, Keramidas M, Mauduit C, Feige JJ, Morera AM, Benahmed M 1991 Direct regulating effect of transforming growth factor /31 on lactate production in cultured porcine Sertoli cells. Endocrinology 128:1441-1449 19. Amstrong DT, Dorrington HH 1977 Estrogen biosynthesis in the ovaries and testes. In: Thomas JA. and Sinehal RL feds) Advances in Sex Hormone Research: Regulatory Mechanisms Affecting Gonadal Hormone Action. University Park Press, Baltimore, vol, 3:217-258 20. Mita M, Price JM, Hall PF 1982 Stimulation by follicle stimulating hormone of synthesis of lactate by Sertoli cells from rat testes. Endocrinology 110:1535-1541 21. Parvinen M 1982 Regulation of the seminiferous epithelium. Endoer Rev 3:404-417 22. Knecht M, Katz MS, Catt KJ 1981 Gonadotropin releasing hormone inhibits cyclic nucleotide accumulation in cultured rat granulosa cells. J Biol Chem 25634-36 23. Knecht M, Catt KJ 1983 Modulation of CAMP-mediated differentiation in ovarian granulosa cells by epidermal growth factor and platelet-derived growth factor. J Biol Chem 25634-36 24. Verhoeven G, Cailleau J 1988 Testicular peritubular cells secrete a protein under androgen control that inhibits induction of aromatase activity in Sertoli cells. Endocrinology 123:2100-2110 25. Le Magueresse B, Jegou B 1986 Possible involvement of germ cells in the regulation of estradiol-170 and ABP secretion by immature rat Sertoli cells (in oitro studies). Biochem Biophys Res Commun 141:861-865
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Vol. 130, No. 2 Printed in U.S.A.
Society
Transforming Growth Factor ,61 Inhibits Action in Cultured Porcine Sertoli Cells* A. M. MORERA, G. ESPOSITO, AND M. BENAHMED
C. GHIGLIERI,
M. A. CHAUVIN,
Gonadotropin
D. J. HARTMANN,
INSERM CJF 90-08, Groupe de Recherches SW les Communications Cellulaires, Laboratoire de Biochimie, H6pital Sainte Eugknie, Centre Hospitalier Lyon-Sud, 69310 Pierre-Bknite, and Centre de Radioanalyse, Znstitut Pasteur (D.J.H.), 69007 Lyon, France
ABSTRACT.
In the present study, we have tested the effects of transforming growth factor @l (TGFPl) on FSH action toward aromatase activity and lactate production in cultured Sertoli cells isolated from immature porcine testes. Whereas treatment of Sertoli cells with FSH resulted in a dose-dependent increase (about 7-fold) in aromatase activity (conversion of testosterone into estradiol) (ED,, = 80 rig/ml FSH), the addition of TGFfil reduced this gonadotropin -action. The inhibitory effect of TGFal on FSH aromatase activitv was dose denendent (ED,, = 0.1 r&ml, 4 pM TGF@l) with a maximal decrease (about 46%) observed after a long term (48-h) treatment. TGF@l exerted its inhibitory effect on FSH action at the level(s) of CAMP accumulation, exerting no apparent effect on the gonadotropin receptor or at a site(s) related to CAMP action. TGFfll (2 rig/ml) significantly (P < 0.002) reduced (52% decrease) FSH-stimulated CAMP levels in cultured porcine Sertoli cells. However, such an inhibitory effect of the growth factor was no longer observed when stimulation of CAMP accumulation with FSH occurred in the presence of methyl isobutyl xanthine (0.5 mM),
an inhibitor of CAMP-phosphodiesterase activity. This observation suggests that TGFPl decreased CAMP levels by increasing catabolism of the cyclic nucleotide through an enhancement of CAMP-phosphodiesterase activity. The inhibitory effect of TGFPl was not limited to the action of FSH on aromatase activity but also extended to the gonadotropin action (mediated by CAMP) on lactate production. As for the inhibitory effect of TGFPl on FSH-induced aromatase activity, the inhibitory effect of the growth factor on FSH-stimulated lactate production was dose and time dependent with a maximal decrease (about 30%) observed in the picomolar range (1 r&ml, 40 PM) after 48 h treatment with TGFPl. In conclusion,~the present study demonstrates that TGFal attenuates FSH action on Sertoli cell activity and that such inhibitory action is potentially exerted through a decrease in CAMP levels. Because of the local production of TGFbl, it is suggested that the effects of the growth factor reported here might be exerted in the context of the testicular paracrine mechanisms. (Endocrinology 130: 831-836, 1992)
A
GREAT body of evidence supports the concept that testicular function is not only under the control of systemic hormones, namely the gonadotropins LH and FSH but is also under the control of local factors among which several regulatory peptides and growth factors have recently been identified (for references and reviews, see l-3). Among these factors, transforming growth factor /?l (TGFPl), a 25,000 mol wt homodimeric protein, is a member of a family of structurally related, multifunctional polypeptide hormones with wide paracrine and autocrine biological activities including control of growth and differentiation (4-6). Recent studies have indicated that TGFP is secreted in the testis (7-9). Al-
though there is much evidence that, in the testis, Leydig cells are a target for TGFPl action (7, lo-12), little is known about the effects of this growth factor on the other testicular cell types and particularly on Sertoli cells. In the present study, we have investigated the possibility that TGFPl could affect Sertoli cell functions controlled by FSH. We report here that TGFPl antagonizes FSH stimulatory action on Sertoli cell functions particularly on aromatase activity and lactate production. Materials
and Methods
TGFPl purified to homogeneity from human platelets was a generousgift of Dr. J. P. Hendrick (Laboratoire de Radioimmunologie, Centre Hospitalier et Universitaire de Liege, Belgium) or obtained from British Bio-Technology Limited (BBL, Oxford, UK). Collagenase(catalog 103586) and lactoperoxidase were obtained from Boehringer (Mannheim, FRG). Dulbecco’s modified Eagle’smedium (DME) and Ham’sF12 mediumwere obtained from GIBCO (Grand Island, NY). Porcine FSH (NIHFSH p2) was kindly provided by the National Institute of
Received August 21,199l. Address all correspondence and requests for reprints to: Dr. M. Benahmed, Groupe de Recherche sur les Communications Cellulaires, Laboratoire de Biochimie, Hopital Sainte-Eugenic, Centre Hospitalier Lyon-Sud, 69310, Pierre-B&rite, France. * This work was supported by Institut National de la Sante et de la Recherche Medicale (INSERM, CJF 90-08), Minis&e de 1’Education Nationale, and in part by Fondation pour la Recherche en Hormonologie (FRH 69 9101) and Serono laboratories.
831
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832
TGFPl INHIBITS
FSH ACTION
Arthritis, Metabolic, and Digestive Diseases (Pituitary Hormone Distribution Program, Bethesda, MD). Human transferrin, insulin, a-tocopherol, HEPES, DNase type 1, lactate dehydrogenase (from rabbit muscle), and NAD were purchased from Sigma Chemical Co. (St. Louis, MO). [Y] and 2, 4, 6, 7[“Hlestradiol (94 Ci/ml) were purchased from Amersham International (Amersham, UK). Estradiol antiserum was obtained from BioM6rieux (Lyon, France). Isolation
and culture
of Sertoli
cells
Sertoli cells were isolated from immature porcine testes (23 weeks old) by collagenase treatment as described by Mather and Phillips (13). The testes were decapsulated, minced, and washed in DME/FlB (1:l) medium. After collagenase dissociation (0.5 mg/ml, 90-120 min at 32 C), cells were washed by mild centrifugation (200 x g for 10 min). The pellet was then resuspended and after a sedimentation of 5 min, the sedimented tubules were recovered and washed three times by unit gravity in DME/FlZ medium. These tubules were then incubated 10 min (room temperature) in 20 ml 1 M Glycine, 2 mM EDTA, and 20 IU/ml DNase 1 in Ca++, Mg++-free phosphate buffer solution, pH 7.2. This treatment led to the release of the contaminating interstitial (Leydig) cells. The glycine-treated tubules were then washed three times (in DME/FlB medium) by unit gravity and incubated in 100 ml DME/F12 medium containing collagenase (0.5 mg/ml), DNase (0.05 mg/ml) and soybean trypsin inhibitor (0.05 mg/ml) for 15 min (32 C). The supernatants containing the peritubular myoid cell fraction were removed, and the sedimented tubules were treated again as described above with collagenase (0.5 mg/ml, 20 min, 32 C) until small clumps resulted. Clumps were left to settle, and the supernatants were discarded. This procedure led to a purified Sertoli cell population not contaminated by Leydig cells or germ cells and containing between 2-5% peritubular myoid cells as evaluated by using fibronectin and desmin immunostainings (14; Tabone, E., M. Benahmed, D. J. Hartmann, and A. M. Morera, unpublished data). Sertoli cells were plated in Falcon (Oxnard, CA) 24-multiwell plates (0.25-0.4 X 10” cells per dish) cultured in DME/F12 medium (1:l) containing 1.2 mg/ml sodium bicarbonate, 15 mM HEPES, and 20 pg/ml gentamycin. This medium was supplemented with transferrin (5 pg/ml), insulin (2 pg/ml), and (Ytocopherol (10 kg/ml). Cells were cultured at 32 C in a humidified atmosphere of 5% C02, 95% air. Measurement
of
lactate production
The amounts of lactate present in Sertoli cell culture media were estimated with the enzymatic method described by Hohorst (15) by using a Kontron fluorimeter (Kontron Instruments, Zurich, Switzerland) at an excitation wavelength of 340 nm and an emission wavelength of 455 nm. The cell number was determined by using a Coulter Counter (Coultronics, Margency, France) once the cells were detached from the culture dishes in trypsin-EDTA. Aromatase
assay in cultured
Sertoli
cells
Sertoli cell aromatase activity was assessedby measuring the conversion of testosterone (1 mM, 8 h) into estradiol secreted
IN SERTOLI
CELLS
Endo. 1992 Vol130. No 2
in the culture medium. The concentration of estradiol was determined through a RIA with a specific estradiol antibody and 2,4,6,7-[“Hlestradiol (94 Ci/mmol: cross-reactivity of the estradiol antiserum at 50% binding was estradiol lOO%,estrone 15%, dehydroepiandrosterone 0%, testosterone 0%, and androstenedione 0%). Similar amounts of RIA detectable estradiol were found before and after the ether extraction of the steroid from the culture medium and purification on a celite column. Therefore, unless specified otherwise in the described experiments, estradiol was directly measured in the culture medium. CAMP assay
Sertoli cells were stimulated with FSH for 30 min. At the end of this incubation, the culture media were collected into ethanol (80% vol/vol) at 4 C. After shaking, the supernatants were collected by centrifugation then evaporated to dryness under a stream of nitrogen. After reconstitution with 0.05 M phosphate buffer, pH 6.2, the samples were acetylated and assayed for CAMP using specific RIA (16) (CAMP lznI RIA, Pasteur kit, ERIA-Diagnostics Pasteur, Marnes, France). Binding
assays of FSH
Human FSH was labeled with “‘1 by the lactoperoxidase method (17). The specific activity was about 50 &!i/Fg. [12hI] FSH binding to cultured Sertoli cells was performed in 0.2 ml binding buffer (DME/F12, l:l, containing 1.2 mg/ml sodium bicarbonate, 15 mM HEPES, pH 7.5, and 0.5% BSA). Incubations were carried out for 2 h at 33 C. At the end of the incubation, the cells were quickly washed four times with icecold 150 mM sodium chloride containing 1% BSA. The cells were then dissolved in 0.5 M NaOH, 0.4% deoxycholate, and the radioactivity was measured in a y-counter. Data analysis
All the experimental data are presented as the mean + SD of triplicate determinations within each treatment group. All experiments reported here were repeated at least three times in different independent cell preparations. Statistical differences between two groups were determined using Student’s t test. Differences are accepted as significant at P < 0.05.
Results FSH-stimulated Sertoli cell.9
aromatase activity in immature porcine
Since all the data reported
aromatase
activity
so far on the regulation
of
in Sertoli cells have been obtained
using mainly cultured
cells from immature
rat testis, we
first tested whether cultured immature porcine Sertoli cells, an experimental model we use currently in our laboratory, is a suitable model for investigating the regulatory mechanisms involved in Sertoli cell aromatase activity. Figure 1 shows some of the characteristics of aromatase activity in response to FSH in porcine Sertoli cells. Immature cultured porcine Sertoli cells exhibited a very low basal aromatase activity but which is highly responsive to stimulation by the gonadotropin FSH in a
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TGFPl
INHIBITS
FSH
ACTION
A z =:
300 -
c fn 4 . bo
200 -
0
1
1
FSH
10
100
10000
1000
(nglml)
IN SERTOLI
CELLS
porcine Sertoli cells cultured in optimal conditions as indicated above. In porcine Sertoli cells, basal aromatase activity evaluated through the accumulation of estradiol in the medium is very low and appeared not to be significantly affected by TGFPl (2 rig/ml, 48 h) treatment (Fig. 2B). By contrast, TGFPl inhibited FSHinduced aromatase activity in cultured porcine Sertoli cells. Indeed, as shown in Fig. 2A, Sertoli cell treatment with TGFPl (0.01-10 rig/ml, 48 h) resulted in a concentration-dependent attenuation of FSH-induced aromatase activity to a maximal decrease of 41% observed with 1 rig/ml TGFPl and with an apparent IDS0 of 0.1 rig/ml (4 PM) TGFPl (Fig. 2A). Furthermore, the inhibitory effect of the growth factor was observed for all FSH (l3000 rig/ml) doses tested to stimulate Sertoli cell aromatase activity. TGFPl decreased the maximal response to the gonadotropin but without affecting the FSH concentrations required for the maximal and half-maximal activity of the enzyme (Fig. 2B). The inhibitory effect of TGFPl on FSH-induced aro-
24
Time
30
44
= c \o 2
72
Effect of TGF/31 on FSH-stimulated in cultured Sertoli cells
aromatase activity
We therefore next examined TGFPl action on basal and FSH-stimulated aromatase activity by using the
l-l“c,
;: loo II i
1. Effects of FSH on Sertoli cell aromatase activity. Sertoli cells were cultured for 48 h with or without increasing concentrations (O3000 rig/ml) of FSH (A) and for indicated times (O-72 h) with FSH (2000 rig/ml) (B), then testosterone substrate (200 rig/ml) was added for 8 h, and estradiol production was measured in the culture medium. The results represent the mean + SD of three separate determinations in three different dishes. FIG.
dose- and time-dependent manner. The maximal (about 7-fold increase) and half maximal effects were respectively observed with 1000 and 80 rig/ml FSH (Fig. 1A). The stimulating effect of FSH was time dependent since it was detected at 24 h FSH treatment and was maximal after 44 h treatment with the hormone (Fig. 1B). Furthermore, in control experiments, we have determined the optimal cell culture conditions for aromatase activity in response to FSH action. This stimulatory effect of FSH was: 1) maximal between day 2 and 12 in porcine Sertoli cell cultures; and 2) dependent upon cultured Sertoli cell densities (0.1-l x lo6 cells per dish). The response to FSH stimulation was maximal between 0.3-0.5 x lo6 cells per dish and then declined at a density higher than 0.5 X lo6 cells per dish (data not shown).
2ocI
‘M ,a
(hours)
-
I
A
300 -
a
0
a33
II
0’
I
’
’
I 0
“‘-.
FSH
‘s; L!p:
300
1
.l
““-1 10
I loo
FSH + TGFl31 (rig/ml)
-
-TGFDl
-
+TGFDl
rD z -
200
iit
0 0
.l
1
FSH
10
loo
loo0
II
00
(rig/ml)
2. Effects of TGFPl on Sertoli cell aromatase activity. Sertoli cells were cultured: for 48 h with a fixed dose of FSH (2000 rig/ml (A) and with or without increasing concentrations of TGFPl (O-10 rig/ml) (B), for 48 h with a fixed dose of TGFPl (2 rig/ml) and with or without increasing concentrations of FSH (l-3000 rig/ml); then testosterone substrate (200 rig/ml) was added for 8 h, and estradiol production was measured in the culture medium. The results represent the mean + SD of three separate determinations in three different dishes. FIG.
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TGFBl
834
INHIBITS
FSH
ACTION
matase activity was not related to a change in Sertoli cell number after the treatment with TGFPl (2 rig/ml, 48 h) (-TGFPl: 0.38 f 0.02 us. + TGFPl: 0.39 f 0.03 x lo6 cells per dish).
IN SERTOLI
CELLS
Endo. 1992 Vol130. No 2
80,
I
Sites of action of TGFpl To delineate the mechanisms involved in the inhibitory effect of TGFPl on FSH-induced aromatase activity, we have investigated the potential role of the growth factor on FSH binding to its receptors, on FSH-stimulated CAMP production, and on 8-bromo CAMP-stimulated aromatase activity. Our data indicated that after TGFPl treatment (2 rig/ml, 48 h), specific FSH binding to Sertoli cells was not affected (-TGF/31: 30.0 X 1 us. + TGFPl: 32.1 + 1.5 x lo3 cpm/106 cells). To examine whether TGFPl affected the accumulation of CAMP, Sertoli cells were pretreated with TGFPl (2 rig/ml, 48 h) before being stimulated with FSH (2000 rig/ml) in the absence or presence of methyl isobutylxanthine (MIX) (0.5 IrIM), used as an inhibitor of CAMP-phosphodiesterase (PDE) activity. Whereas TGFPl was without significant effect on the basal accumulation of extracellular CAMP, its addition significantly (P < 0.002) decreased 52% inhibition) the action of FSH. However, such an inhibitory effect of TGFPl on extracellular CAMP accumulation was no longer observed when Sertoli cells were stimulated with FSH but in the presence of MIX (Fig. 3). Finally, TGFPl appears to be without effect on 8bromo-CAMP-induced aromatase activity (Fig. 4), suggesting that TGFPl effect on FSH action probably involves site(s) proximal but not distal to CAMP accumulation. Effects of TGF/31 on FSH-stimulated
lactate production
To test the specificity of TGFPl action, we have studied the effect of the growth factor on another Sertoli cell
25 8Br
74 222 666 CAMP ( PM)
TGFI31 FSH + TGFBl
0 CAMP
500
(pmoles/
1000
lo6
FIG. 3. Effects of TGFPl on extracellular CAMP cells were cultured in the presence or absence of 48 h, then with or without MIX (0.5 mM) for 30 without FSH (2000 rig/ml) for 30 min as indicated. in the culture medium. The results represent the separate determinations in three different dishes. FSH + TGFPl.
1500
cells)
accumulation. Sertoli TGFPl (2 rig/ml) for min and then with or CAMP was measured mean k SD of three P < 0.002; +FSH us.
2000
FIG. 4. Effects of TGFpl on 8-bromo-CAMP-stimulated aromatase activity. Sertoli cells were cultured in the presence or absence of TGFPl (2 rig/ml, 48 h) and with or without increasing concentrations of 8bromo-CAMP (O-2 mM) for 48 h, then testosterone substrate (200 ng/ ml) was added for 8 h, and estradiol production was measured in the culture medium. The results represent the mean f SD of three separate determinations in three different dishes.
0 24 48 72 Time (hours)
2. al % z 2
0
FSH
1
1
10
100
FSH+TGFOl (rig/ml)
as :A -
0.3 1 FSH
Basal
FSH
0
10 100 hglml)
a . .. 1000 10000
FIG. 5. Effects of TGFPl on lactate production. Sertoli cells were cultured with TGFpl (2 rig/ml) for the different times indicated and then stimulated for 6 h with a maximal efficient dose of FSH (2000 rig/ml) (A); with increasing concentrations of TGFPl (0.01-10 rig/ml) for 48 h and then stimulated for 6 h with FSH (2000 rig/ml) (B); and with or without a fixed dose of TGFPl (2 rig/ml) for 48 h and then with increasing concentrations of FSH (l-2500 rig/ml) for 6 h (C). The culture media were collected for lactate determination. The results represent the mean + SD of three separate determinations in three different dishes.
parameter known to be highly regulated by FSH via the CAMP pathway, i.e. lactate production. Sertoli cells were pretreated with TGFPl before being acutely stimulated with the gonadotropin to increase lactate production. The data obtained indicate that TGF/31 inhibited in a time- (Fig. 5A) and dose (Fig. 5B)-dependent manner
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TGFBl
INHIBITS
FSH
ACTION
FSH stimulated lactate production with a maximal inhibition (31% decrease) observed with 1 rig/ml TGFPl after 48 h treatment. TGFPl (2 rig/ml, 48 hours) decreased lactate accumulation stimulated with all FSH doses tested (l-3000 rig/ml, 6 h). In these conditions, TGF@l significantly (P < 0.005) decreased the maximal response to FSH (observed at 1000 rig/ml) in terms of lactate accumulation, but it did not affect the FSH doses required for the maximal and half-maximal levels of lactate produced (respectively observed with 500 and 50 rig/ml FSH) (Fig. 5C). Discussion The purpose of the current study was to investigate the effect of TGFpl on the gonadotropin action toward Sertoli cell functions. The experimental model used was Sertoli cells obtained from immature (3-4 weeks old) porcine testes and cultured in defined medium. In regard to the regulation of aromatase activity (present study) and of lactate production (present study; 18), cultured porcine Sertoli cells appear comparable to other experimental models (particularly to the rat model). This study shows that TGF/31 antagonizes FSH action on aromatase activity and lactate production. Indeed, although TGF/31 by itself did not significantly affect basal aromatase activity and stimulated (in short term conditions) basal lactate production (18), however, it inhibited FSH-induced aromatase activity (about 40% decrease) and FSH-stimulated lactate production (about 30% decrease) with an IDso in the picomolar concentration range after a long term treatment. Although the concentration of TGF/31 present in the testis is not yet determined, the concentration of TGFPl required to inhibit FSH action on Sertoli cell aromatase activity was consistent with the dissociation constant range for TGFPl high affinity receptors we have previously identified in porcine Sertoli cells (18). Such observations suggest that the inhibitory effect of TGFPl action on gonadotropin action occurs within a concentration range that might be expected under physiological conditions. The mechanisms through which TGFPl antagonizes FSH action remain to be clarified. However, the present data indicate that the inhibitory effect of the growth factor is not related to a decrease in FSH binding to its receptors but more probably to a decrease in the levels of CAMP, the intracellular mediator of the gonadotropin action on Sertoli cell parameters studied here, i.e. aromatase activity (19), and lactate production (20; our unpublished data). Furthermore, that this decrease in the cyclic nucleotide levels induced by TGFPl was observed only in the absence but not in the presence of MIX would indicate that the growth factor reduced CAMP accumulation by increasing the Sertoli cell PDE
IN SERTOLI
CELLS
835
activity. Whether a modulation of the activity of this last enzyme may play a physiologically relevant role in the gonadotropin action remains to be studied. However, it is of interest to indicate that a cycling of the CAMPPDE activity has been suggested as a cause of the changes in FSH responsiveness of the Sertoli cell during the spermatogenic cycle of the seminiferous tubules (21). Therefore, in this context, the potential regulation of this Sertoli cell enzyme activity by TGFPl (or its related peptides) will be of interest to investigate. Further studies are required to examine whether TGF/31 is a positive effector controlling the Sertoli cell phosphodiesterase expression at genetic or posttranscriptional levels. In addition, an increased catabolism of CAMP through an enhancement of CAMP-PDE attenuating FSH action has been also reported in granulosa cells. Indeed, such a mechanism(s) is probably involved in the inhibitory effect of, at least, GnRH (22) and of epidermal growth factor (23) on gonadotropin action in cultured rat granulosa cells. Owing to the negligible levels of active TGFPl in the circulatory system (4-6), the effects of this factor on FSH action would depend on its local production. Indeed, we have shown previously that a biologically active and receptor reactive TGFP-like factor is secreted by immature cultured porcine Sertoli cells (8). Consistent with these findings, the TGF/31 gene is transcribed in the rat (Sertoli and peritubular myoid cell) testis (9). Furthermore, we also have reported that TGFP-like secretion in Sertoli cells is greatly inhibited after FSH treatment (8). Together with the data reported in the present study, one might speculate that: 1) the inhibitory effect of TGFPl on the gonadotropin action is probably not accomplished in an autocrine context; and 2) by dramatically reducing the secretion of TGFp in Sertoli cells, the gonadotropin may amplify its stimulatory action on aromatase activity. On the other hand, the possibility exists that the inhibitory effect of TGFPl on FSH action is exerted in the context of a paracrine control mainly originating from peritubular myoid cells and/or germ cells. Indeed, although the secretion of (active) TGF/31 from peritubular myoid cells and germ cells remains to be clearly demonstrated, TGF/31 is potentially present in these cells as shown by the presence of TGFPl messenger RNA in rat peritubular myoid cells (9) and by the identification of a positive TGFPl immunostaining in rat germ cells (Ghiglieri, C., E. Tabone, J. P. Hendrick, P. Franchimont, A. M. Morera, and M. Benahmed, in preparation). In this context, rat peritubular myoid cells (24) and germ cells (25) have been reported to secrete factors which inhibit FSH-induced Sertoli cell aromatase activity. Such factors remain as yet chemically undefined. In the light of our present findings, it will be of interest to determine whether these factors are distinct from TGFpl
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836
TGFPl
INHIBITS
FSH
ACTION
(or its related peptides). Finally, the data in the present study extend the concept related to TGF/31 as a potential testicular factor antagonizing LH action in Leydig cells (10-12) to FSH action in Sertoli cells. However, one must note that the biochemical mechanisms involved in the inhibitory effect of TGFPl on LH action are probably distinct from those reported here on FSH action. Indeed, although in Leydig cells, TGFPl decreased LH/hCG binding as well as LH/ hCG-stimulated CAMP formation (10, ll), its inhibitory effect is predominantly exerted at a point after CAMP formation, probably by decreasing the action of gonadotropin (via CAMP pathway) on cholesterol substrate availability in the mitochondria (12). Such differences in the mechanism(s) of action of TGFPl might be related to the different embryological, mesenchymal, and epithelial origins of, respectively, Leydig and Sertoli cells in which TGFPl may activate different intracellular signaling systems. In summary, by using a model of cultured immature porcine Sertoli cells we report, for the first time, that TGF@l is a potential local factor that antagonizes FSH action on Sertoli cell functions such as aromatase activity and lactate production. Such an inhibitory effect is probably related to a decrease in the gonadotropin-stimulated cyclic nucleotide accumulation. Acknowledgments We are grateful to Dr. J. P. Hendrick for TGFfll. We are indebted to the National Hormone and Pituitary Program of the NIH for supplying us with porcine FSH used in these studies. We are grateful to Mr E. Villar and P. Bouteille for providing us with porcine testes. We thank Dr. D. A. Lawrence for reading the manuscript.
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