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Desensitization of sertoli cell-enriched cultures by FSH, l -isoproterenol and glucagon. influence on subsequent stimulation of 17β-estradiol production
1. steroid Bioche~. Vol. 25, No. 1, pp. 45-51, Printed in Great Britain. All rights reserved
1986
0022473 I:86 $3.00 + 0.00
Copyright 0 1986Pergamon JournalsLtd
DESENSITIZATION OF SERTOLI CELL-ENRICHED CULTURES BY FSH, L-ISOPROTERENOL AND GLUCAGON. INFLUENCE ON SUBSEQUENT STIMULATION OF 17/?-ESTRADIOL PRODUCTION GUIDO VERHOEVEN andJEAN
CAILLEAU
Laborato~um voor Experimentele Geneeskunde en Endoc~nolo~e, advent of Developmental Biology, Onderwijs en Navorsing, Legendo, Gasthuisberg, B-3000 Leuven, Belgium (Received 11 September
1985)
Summary-Sertoli cell-enriched cultures derived from 19-day old rats were exposed to FSH, L-isoproterenol, glucagon and dbcAMP for 24 up to 96 h. The influence of primary stimulation with these agonists on the response of the cells to subsequent stim~ation with the homologous or heterologous agonists was investigated. Particular attention was paid to the response of the aromatase system defined as the ability of the cells to convert testosterone into 17/i-estradiol. The responsiveness of this system was compared with the responsiveness of two other systems: adenylate cyclase and phosphodiesterase. It could be demonstrated that preincubation with the mentioned agonists results in a decreased responsiveness (maximal response) and a decreased sensitivity (ED,) upon re-stimulation with the homologous agonists. Preincubation with FSH also provokes partial desensitization for glucagon and L-isoproterenol. This heterologous desensiti~tion can be mimicked by d&. The mentioned desensitization reactions are accompanied by a marked decrease in the a~um~ation of CAMP in the medium. Whereas desensitization of the adenylate cyclase occurs rapidly, desensitization of the aromatase response requires protracted stimulation for 34 days. In contrast with the adenylate cyclase and the aromatase system, the responsiveness of phosphodiesterase to FSH, L-isoproterenol, glucagon and dbcAMP is not affected by repeated stimulation for 96 h. It is concluded that hormonal desensitization affects both early (CAMP) and late (aromatase) responses of the Sertoli cell. However, some responses, such as phosphodiesterase activity seem to escape the-desensitization process.
gram and Dr A. F. Parlow. Steroids, L-isoproterenol, glucagon and dbcAMP were obtained from Sigma. 3-Isobutyl-1-methylxanthine (MIX) was purchased from Aldrich Chemical Company.
INTRODUCTION Rat Sertoli cells cultured in vitro respond to a number of hormonal agonists. From a physiological point of view, FSH and testosterone are the most important stimulators fl]. P-Agonists and glucagon also have marked effects on cultured cells, but their biological role remains unclear [2-4]. In previous studies, it has been demonstrated that stimulation with FSH or L-isoproterenol causes homologous desensitization of the CAMP response of Sertoli cells, and that stimulation with FSH results in heterologous desensitization to isoproterenol IS]. Homologous desensitization of the adenylate cyclase can also be achieved in Sertoli cell membranes and may be related to receptor phosphorylation [6]. The heterologous desensitization is at least in part due to the CAMPdependent induction of a specific CAMPp~osphodiesterase [‘?-IO]. In the present study we investigated whether desensitization at the level of the adenylate cylase affects more distal responses of Sertoli cells such as stimulation of aromatase activity (defined as the ability of the cells to convert testosterone into 17/J-estradiol) [I I] and induction of phosphodiesterase activity [7,8].
Media
Preincubations were performed in Medium A. This medium consists of RPM1 1640 (Flow Laboratories) supplemented with Hepes (Gibco, Biocult), 22.5 mM, L-glutamine (Flow Laboratories) 4.3 mM and phenol red (Gibco, Bio-cult) 56pM. Medium B consists of Medium A supplemented with streptomycine sulfate (100 pg/ml) and penicillin G (lOOU/ml). Medium C is used for the actual incubations and contains Medium A enriched with MIX, 0.1 mM and testosterone 0.5 PM. Phosphate buffered saline, Dulbecco’s formula (PBS) was obtained from Gibco, Bio Cult. Sertoli cell-enriched cultures
Testes were derived from 19-day old Wistar rats. Sertoli cells were prepared by a modification of the method of Welch and Wiebe[l2] as described previously [3]. Routinely, 2 x lo6 cells suspended in 2 ml medium B were seeded in multi~shes confining six 35 mm wells (Greiner). Cultures were maintained in a humidified incubator (32”C, 5% CO,). On days 3, 4, S or 6 the medium was aspirated, cells were washed twice with 2ml PBS and preincubation was started by the addition of 2ml medium A without
EXPERIMENTAL
Materials Ovine FSH (oFSH:NIH-FSH-S13) was a gift of the NIAMDD Pituitary Hormone Distribution Pro45
46
GUIDO VERHOEVEN and PREINCUBATION INCUBATION WITH FSH I*..l OR CONTROL IlEOlUtl lo,ol
PREINCUBATION
TlHf
WITH
PREINCUBATION
(~.ol
OR CONTROL
INCUBATION WITH L-ISOPROTERENOL OR CONTROL MEDIUM lo 01
PREINCUBATION
ltlOURS1
INCUBATION WITH FStl I*.ml OR CONTROL HEOUJU looI
FSH
JEANCAILLEAU
WITH
dbcAMP
l0.m)
MEDIUM
to.01
INCUBATION WITH GLUCAGON lo.1 OR CONTROL MfOlUH lo .I
TIWE (HOURS1
lm.ol OR CONTROL
INCUBATION WIT,, L-ISOPROTERENOL OR CONTROL MEDIUM lo.oi
I*..)
MEDIUM
lo.01
INCUBATION WITH GLUCAGON l*,mI OR CONTROL MEDIUM lo.01
1
21 b8 PREINCUBATION
72 96 TIME IHOURSI
2L 48 PREINCUBATION
12 96 TIME IHOURS
2r. I.8 PREINCUBATION
12 96 TIME (HOURS1
Fig. 1. FSH and dbcAMP induced desensitization as a function of time. Sertoli cell-enriched cultures were preincubated at 32°C for the indicated periods of time in Medium A (control) or in the same medium supplemented with oFSH (1 pg/ml) or dbcAMP (1 mM). After this preincubation (on day 7 of culture), the cells were rinsed twice with PBS and were incubated for another 24 h period at 32°C in Medium C (control: Medium A supplemented with testosterone, 0.5 p M and MIX, 0.1 mM) or in the same medium containing oFSH (1 pg/ml) L-isoproterenol (10 PM) or glucagon (10 pgg/ml). The aromatase activity (accumulation of estradiol in the medium) was evaluated during the incubation period. Each point represents the mean + SD of 4-6 incubations. Values between brackets indicate the percentage inhibition (preincubation with agonist vs preincubation with control medium). Significant inhibition (P < 0.01) is indicated by an asterisk.
(control) or with the additions specified in the Results section. The media were changed every 24 h. On day 7 the preincubation medium was discarded, the cells were washed with PBS and 2 ml Medium C without (control) or with the indicated agonists was added. After an incubation period of 24 h at 32°C the medium was collected and stored at -20°C for determination of 17B-estradiol. The cells were washed twice with PBS and were dissolved in 1 ml of 0.1 M NaOH for determination of protein content. Proteins were measured by the method of Lowry[ 131. Measurement of 178 -estradiol production The media were extracted with 4 vol of cyclohexane-ethylacetate (1: 1, v/v) and 17/I -estradiol was measured by a specific radioimmunoassay as described previously [2] W-90% of the radioimmunoassayable estradiol co-crystallized with authentic 17fi-estradiol on repeated crystallization (not shown). Measurement of phosphodiesterase activity To measure phosphodiesterase activity in Sertoli cells, culture conditions were slightly modified. 4 x lo6 Sertoli cells suspended in 3 ml medium were seeded in Petri dishes with a diameter of 6cm. The monolayers were preincubated and incubated as explained in the previous paragraphs. Cells were
washed, harvested and homogenized in 0.5 ml phosphodiesterase buffer as described elsewhere [7]. The homogenates were stored at -70°C and phosphodiesterase activity was measured in the unfractionated homogenates using the method of Thompson et af.[14]. RESULTS Injluence of preincubation with FSH, dbcAMP, L-isoproterenol and glucagon on the responsiveness of the aromatase to homologous and heterologous agonists
Preincubation with FSH (1 pg/ml) results in a decrease in the response of the aromatase on subsequent stimulation with the same agonist (Fig. 1). The decrease becomes statistically significant (P -C0.01) after 48 h of preincubation. After 96 h of preincubation a 82% reduction in the aromatase response is observed. Preincubation with FSH also decreases the response to L-isoproterenol and glucagon. The reduction in the responsiveness to these heterologous agonists, however, is less pronounced than the reduction in the responsiveness to FSH. Preincubation with dbcAMP (1 mM) results in desensitization for FSH, L-isoproterenol and glucagon (Fig. 1). The degree of desensitization is com-
Desensitization
of Sertoli cells
PREINCUBATION WITH L-ISOPROTERENOL (m,nl INCUBATION WITH FSH I..*1 OR CONTROL HEOIUM lo,nl
PREINCUBATION
TINE NUJURSI
PREINCUBATION WITH GLUCAGON INCUBATION WITH FSH IO.9 1 OR CONTROL ~OIUM lO.0)
CB PRE&BATKIN
72 9L TINE lHOURSI
OR CONTROL MEDIUM b.0)
INCUBATION WITH L-ISOPROTERENOL OR CfflTROL NEMUM b.4
24 b6 PREINCUBATION
l*.d
INCUBATION WITH GLUCAGON IV1 OR CONTROL MEOIUM lo.01
2l a PREINCUEATION
72 91 TINE iHOURS
12
PC
TINE IHOURSI
(m,olOR CONTROL MEDIUM (0.0)
lNCUBATl!JN WITH L-ISOPROTERENOL OR CONTROL KKHUN lo.nl
26 4B PRE,NCUBATION
47
72 96 TNIE RIOURSI
I*.Dl
INCUBATION WITH GLUCAGON f*.DI OR CONTROL NEDIUN lo,01
w
PREINCUBATION
TIME INOURS
Fig. 2. L-Isoproterenol and glucagon induced desensitization as a function of time. Cells were preincubated and incubated as explained in Fig. 1; the oFSH or the dbcAMP in the preincubation medium was replaced by L-isoproterenol (10 PM) or glucagon (lOpg/ml). The inhibition of aromatase activity is measured and expressed as explained in Fig. 1.
parable
for the three
hormones.
It may be noted,
however, that a more profound desensitization for FSH is obtained after 96 h of preincubation with FSH than after prein~ubation with dbcAMP. For the other two hormones the FSH induced reduction in responsiveness equals the dbcAMP induced desensitization. Preincubation with L-isoproterenol (10pM) or glucagon (10 pg/ml) results in nearly complete desensitization for the homologous agonists whereas the response to heterologous agonists is not affected (Fig. 2). Dose-response
relationships
The influence of preincubation with increasing concentratjons of FSH, dbcAMP, L-isoproterenoi and glucagon on the responsiveness to homologous and heterologous agonists is shown in Fig. 3. In this experiment, the cells were exposed to the indicated concentrations of the agonists on days 3, 4, 5 and 6 of culture and the induction of aromatase activity was studied on day 7. The data show that FSH and dbcAMP induce a dose-dependent desensitization for the homologous as well as for the heterologous agonists. At a concentration of 1 pg&/ml the FSHinduced homologous desensitization is nearly complete whereas the desensitization for the heterologous agonists barely reaches 50%. L-Isoproterenol and glucagon produce only homologous de~nsiti~tion. The ED,, values for the 4 homologous desensiti~tion reactions studied are: 0.05 pglrnl for FSH, 50pM
for dbcAMP, 5 PM for r_-isoproterenol and 0.5 pg/ml for glucagon. I@e~ce of preincubation with homologous agonists on the dose-respon~~e curves for FSH, dbcA~P, ~-~soproterenol and glucagon Preincubation during 96 h with 0.05 pg/ml FSH (a concentration producing 50% desensitization) results in marked changes in the dose-response curve for FSH. Besides the expected 50% reduction in the maximal response there is an obvious shift of the curve towards higher concentrations of FSH. In cells preincubated with control medium, half-maximal stimulation of aromatase activity is observed at a concentration of 10 ng/ml; in desensitized cells a 8.5fold higher concentration of FSH is required. A comparable decrease in responsiveness (maximal activity) and sensitivity (ED, value) is observed for the homologous desensitization reaction produced by L-isoproterenol and glucagon. The decrease in sensitivity is somewhat less pronounced for dbcAMP. Comparison of the eflects of preincubation with FSH, dbcAMP, L-isoproterenol and glucagon on various responses of Sertoli cells
Table 1 compares the influence of preincubation and incubation with various agonists on three Sertoli cell responses: accumulation of CAMP in the medium, stimulation of phosphodiesterase activity and stimulation of aromatase activity. The data on the stimuIation of aromatase activity largely confirm the findings discussed in the previous
48
Gur~o
VERHOEVENand JEAN CAILLEAU
Fig. 3. Dose-response relationship of the FSH, dbcAMP, L-isoproterenol and glucagon induced desensitization of aromatase activity. Sertoli cell-enriched cultures were preincubated for 96 h in the presence of the concentrations of oFSH bane1 A), dbcAMP (panel B), L-isoproterenol (panel C) and glucagon (panel D) indicated in the abcissa. After this preincubation (on day 7 of culture), the cells were washed twice with PBS and were incubated for 24 h in Medium C supplemented with oFSH (1 pg/ml; a-----•), dbcAMP (1 mM, O----O), glucagon (lOpg/ml, H-D) or L-isoproterenol (1OpM; n-0). Aromatase activity is expressed as a percentage of the activity observed in cells preincubated in control medium. 100% corresponds to 2.45 k 0.15 ng/mg protein for ceils incubated with FSH, 3.56 f 0.28 for cells incubated with dbcAMP, 1.81 f 0.17 for cells incubated with L-isoproterenol and I .03 f 0.05 for ceils incubated with glucagon (mean + SD; n = 16).
paragraphs. The response to FSH or dbcAMP is decreased after preincubation with FSH as well as after preincubation with dbcAMP. The response to L-isoproterenol and glucagon is markedly reduced after pretreatment with the homologous agonists but preincubation with FSH and dbcAMP also provokes a partial decrease in responsiveness. The agonist-induced accumulation of CAMP in the medium largely parallels the aromatase response, All the studied agonists provoke homologous desensitization, whereas FSH and dbcAMP also provoke partial desensiti~tion for the heterologous agonists. The phosphodiesterase response shows a completely different pattern. At the end of the preincubation period phosphodiesterase activity was 6.95 rf: 0.05 (mean f SD; n = 3) in cells preincubated during 96 h with control medium. In cells preincubated with FSH, L-isoproterenol, glucagon and dbcAMP, the respective values were: 35.39 zt 2.32, 15.77&0.53, 15.06+0.11 and 85.30&2.22. If these cells are incubated for a further 24 h in Medium C (Table 1) phosphodiesterase activity decreases (except for the cells preincubated with control medium where the MIX in Medium C may cause a slight increase in activity) but does not completely reach control values. If the incubation is performed in Medium C
supplemented with one of the studied agonists a further increase in phosphodiesterase activity is observed. Under none of the studied conditions is there any sign of desensitization. DISCUSSION
The results reported here demonstrate that FSH, L-isoproterenol and glucagon not only stimulate CAMP production, conversion of testosterone into estradiol (aromatase activity) and phosphodiesterase activity in Sertoli ceil-enriched cultures but also induce a state of refractoriness to subsequent stimulation. Desensitization with respect to CAMP production has been reported previously for FSH and L-isoproterenol [S, 7,9, 151. The major new findings from the present work are: (1) glucagon also induces a state of refractoriness; (2) the FSH, L-isoproterenol and glucagon-induced desensitization also affects later responses of these cells such as activation of the aromatase activity; (3) the phosphodiesterase remains responsive to all the mentioned agonists despite repeated stimulation for 96 h. The de~nsiti~tion of the aromatase response shows some remarkable analogy with the desensitization of the CAMP response as reported
of
Desensitization
Table
I.
Influence of preincubation
Sertoli
cells
with homologous or heterologous of Sertoli cell-enriched cultures
Accumulation of CAMP (nmolime. mot)
49
agonists
on various responses
Phosphodiesterase activity (U/me. mot)
Aromatase activity (nnime DrOt)
Preincubation
Incubation
Control FSH L-isoproterenol Glucagon dbcAMP
Control
0.06 0.07 0.07 0.09 0.07
+ + f f f
0.01’ 0.03” 0.02” 0.04a 0.02”
9. I I + 0.97’ 17.82 T2.Wb 12.67; l.OOh,C I I .43 + I .57b,’ 22.80 + 2.516
0.16 + 0.04” 0.18 ; 0.03d 0.15 f 0.03” 0.16F0.03” 0. I6 + 0.06”
Control FSH L-isoproterenol Glucagon dbcAMP
FSH
I .53 0.12 I .38 I .4l 0.50
i f f f +
0.08” o.oc 0.03” 0.03a 0.06b
62.03 13.13 51.77 58.07 81.43
f f f f *
2.64b 3.22” I .56b 4.35b 3.184
2.17 0.51 I .95 2.08
+ f * k 1.50f
0.03” 0.06’ 0.18” 0.03” 0.05b
Control FSH L-isoproterenol Glucagon dbcAMP
L-isoproterenol
0.76 0.40 0.1 I 0.67 0.37
f + * f f
0.04” 0.04b 0.05’ 0.05” 0.07b
28.83 42.11 37.16 35.04 49.06
f f + I F
l.51d 2.49’ I .57b,’ I .OO‘ I .OO”
I .60 I .08 0.32 I .45 0.95
0.05d 0.03’ 0.04d 0.02b 0.06’
Control FSH L-isooroterenol G&go” dbcAMP
Glucagon
0.63 0.29 0.55 0.09 0.33
f + f i f
0.04’ 0.02b 0.02” 0.02c 0.05b
30.77 41.07 36.51 34.09 52.42
f + + I f
2.47’ 2.lOb l.52b,C I .OO’ 2.06d
I .25 i 0.04” 0.81 + 0.02b 1.18iO.03” 0.29 f 0.02” 0.66 f 0.06’
Control FSH L-isooroterenol Gluc’agon dbcAMP
dbcAMP
96.76 i 1.53” 97.13 i 2.30a 96.81 + 2.07” 97. I I : 2.93” 106.13i4.24”
I f f F f
3.20 + 0.04” 2.33*0.11h 3.17 +0.03” 3.20 ; 0.05” I .08 + 0.04’
Sertoli cell-enriched cultures were preincubated for 96 h in Medium A (control) or in the same medium supplemented with oFSH (1 pg/ml), L-isoproterenol (I 0 PM), glucagon (I 0 fig/ml) or dbcAMP (I mM). After this preincubation, the cells were washed twice with PBS and were incubated for 24h in Medium C (control) or in the same medium supplemented with the mentioned concentrations of oFSH, L-isoprotenol, glucagon or dbcAMP. The accumulation of CAMP and estradiol was measured in the incubation medium. Phosphodiesterase activity was measured in the cells at the end of the incubation period. Values represent the mean + SD of 3 incubations. The effects of the preincubation conditions were analyzed by one-way analysis of variance. There are significant differences between groups indicated by different letters (P < 0.01).
previously [5]. Under the conditions used in both series of experiments (primary stimulation in the absence of a phosphodiesterase inhibitor and subsequent testing of the residual responsiveness in the presence of a phosphodiesterase inhibitor) there is homologous desensitization after stimulation with FSH, L-isoproterenol and glucagon. In addition, stimulation with FSH produces partial heterologous desensitization for all the other agonists. This partial heterologous desensitization can be mimicked by preincubation with dbcAMP. Several mechanisms have been invoked to explain the decrease in CAMP production after repeated stimulation of Sertoli cells: uncoupling of receptors from the catalytic moiety of the adenylate cyclase [6]; disappearance of specific receptors from the cell membrane [6,16] and CAMP dependent induction of a specific phosphodiesterase [7,8]. All these phenomena occur within a matter of hours, however, whereas desensitization of the aromatase response requires 4 days. The difference in timing cannot be explained simply on the base of a prolonged half-life of the aromatase system. In fact, no residual aromatase activity is measured in cells preincubated with FSH, L-isoproterenol or glucagon for 24 up to 96 h and
subsequently incubated with control medium containing testosterone as a substrate for the aromatase (Figs 1 and 2). At the present time we can only speculate on the mechanism(s) responsible for this slow decrease in the responsiveness of the aromatase system. It may be worthwhile to remember, however, that the timecourse of the CAMP response and the aromatase response of Sertoli cells after primary stimulation are completely different [3, 51. After primary stimulation the intracellular CAMP increases rapidly and transiently reaching maximal values within the first 2 h of stimulation. The CAMP in the medium increases for approx. 4 h but remains constant thereafter. The conversion of testosterone into estradiol (aromatase response), however, proceeds at a constant rate throughout the stimulation period for at least 48 h. Although this does not exclude that stimulation of Sertoli cells might provoke increased production of new aromatase enzyme, it rather suggests that stimulation might activate an inactive form of the enzyme already present under basal conditions. In such a model it is conceivable that the initial decrease in CAMP production (desensitization) would merely limit the “excessive” amount of CAMP pouring out
50
GUIDO VERHOEVENand JEAN CAILLEAU
10 ng ml.’
/
0
10-Z
10-l
t
10
tone. GLUCAGON l~g.ml~'l
Fig. 4. Influence of homologous desensitization on the dose-response curves for induction of aromatase activity by FSH, dbcAMP, L-isoproterenol and glucagon. Sertoli cell-enriched cultures were preincubated for 96 h either in control medium (e---•) or (0-O) in medium supplemented with oFSH (0.05 rg/ml; panel A), dbcAMP (50 PM; panel B), r.-isoproterenol (5 FM; panel C) or glucagon (0.05 pg/ml; panel D). After this preincubation period, the cells were washed twice with PBS and were incubated for 24 h in Medium C suppIemented with the concentrations of homologous agonists indicated in the abcissa. Estradiol was measured in the incubation medium. Values represent the mean & SD of 6 incubations. The ED, values of the various curves are indicated by an arrow.
of the cells during the first hours of stimulation. A more pronounced desensitization might be required to impede the intracellular accumulation of sufficient amounts of CAMP able to activate and maintain aromatase activity. The foregoing hy~thesis cannot explain al1 our observations. In fact, it may be noted that in cells pretreated with FSH or dbcAMP the aromatase response is reduced even after maximal stimulation with dbcAMP. (Fig. 4, Table 1). This indicates that some step beyond the production of CAMP has become rate limiting. It is tempting to speculate that the FSH or CAMP induced increase in the production of a protein kinase inhibitor described by Tash et a1./17] may be responsible for this decreasing responsiveness. Alternatively, it is conceivabie that prolonged stimulation of Sertoli cells wouid result in some form of “differentiation” of these cells accompanied by a decrease in aromatase activity. In fact, a similar decrease in aromatase activity is observed during pubertal development in vim [18, 191. It is of particular interest that the high affinity CAMP-phosphodiesterase remains responsive to FSH, L-isoproterenol and glucagon even after repeated stimulation with these agonists for 96 h. Although the mechanism of this “escape from desensitization” remains obscure, this observation further supports the contention [7-l@ 201 that this enzyme
may play a physiological role in the age-dependent decrease of FSH-induced accumulation of CAMP in the testis [21-231. In view of these observations it might be of interest to investigate whether other Sertoli cell responses such as the secretion of androgen binding protein (ABP) or transferrin (proteins that may be required to maintain the production of fertile spermatozoa) also escape the desensitization process. It can be concluded that all the studied agonists that stimulate Sertoli cells also desensitize these cells to subsequent stimulation. This desensitization not only results in decreased production of CAMP, but also provokes decreased responsiveness and sensitivity with respect to later responses such as the activation of the aromatase. However, the lack of desensitization with respect to phosphodiesterase activation, shows that some Sertoli cell functions may escape the desensitization process. Acknowledgements-This research was supported by a grant from the Nationaal Fonds voor Wetenschappelijk Onderzoek van Belgii (grant no. 3.0047.80), a “Krediet aan Navorsers” from the same institution and a grant from the Onderzoeksfonds K. U. Leuven. The authors appreciate the skilled technical assistance of MR Lodewijk Deboel and thank Dr W. Heyns for critical discussion of the manuscript. We gratefully acknowledge the National Pituitary Agency, NIH (Bethesda, Md, U.S.A.) and Dr Parlow for the gift of oFSH.
Desensitization REFERENCES
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of Sertoli
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12. Welch M. J. and Wiebe J. P.: Rat Sertoh cells: a rapid method for obtaining viable cells. Endocrinology 96 (1975) 618-624. 13. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J.: Protein measurement with Folin phenol reagent, J. biol. Chem. 193 (1951) 265-275. 14. Thompson W. J., Terasaki W. L., Epstein P. M. and Strada S. J.: Assay of cyclic nucleotide phosphodiesterase and resolution of multiple molecular forms of the enzyme. Adv. cyclic nucl. Res. 10 (1979) 69-92. 15. Jahnsen T., Verhoeven G., Purvis K., Cusan L. and Hansson V.: Desensitization of FSH-responsive adenylyl cyclase in cultured immature Sertoli cells by homologous hormone. Archs Androl. 8 (1982) 205-211. 16. Jahnsen T., Gordeladze J. O., Torjesen P. A. and Hansson V.: FSH-responsive adenylyl cyclase in rat testes: desensitization by homologous hormone. Archs Androl. 5 (1980) 169-177. 17. Tash J. S., Welsh M. J. and Means A. R.: Regulation of protein kinase inhibitor by follicle-stimulating hormone in Sertoli cells in vitro. Endocrinology 108 (1981) 427434. 18. Dorrington J. H., Fritz I. B. and Armstrong D. T.: Control of testicular estrogen synthesis. Biol. Reprod. 18 (1978) 55-64, 19. Rommerts F. F. G., de Jong F. H., Brinkmann A. 0. and van der Molen H. J.: Development and cellular localization of rat testicular aromatase activity. J. Reprod. Fer?. 65 (1982) 281-288. 20. Geremia R., Rossi P., Pezzotti R. and Conti M.: Cyclic nucleotide phosphodiesterase in developing rat testis. Identification of somatic and germ-cell forms. Molec. cell. Endocr. 28 (1982) 37-53. 21. Fakunding J. L., Tindall D. J., Dedman J. R., Mena C. R. and Means A. R.: Biochemical actions of folliclestimulating hormone in the Sertoli cells of the rat testis. Endocrinology 98 (I 976) 392402. 22. Steinberger A., Hintz M. and Heindel J. J.: Changes in cyclic AMP responses to FSH in isolated rat Sertoli cells during sexual maturation. Biol. Reprod. 19 (1978) 566572. 23. Van Sickle M., Oberwetter J. M., Birnbaumer L. and Means A. R.: Developmental changes in the hormonal regulation of rat testis Sertoli cell adenylyl cyclase. Endocrinology 109 (1981) 127&1280.
1986
0022473 I:86 $3.00 + 0.00
Copyright 0 1986Pergamon JournalsLtd
DESENSITIZATION OF SERTOLI CELL-ENRICHED CULTURES BY FSH, L-ISOPROTERENOL AND GLUCAGON. INFLUENCE ON SUBSEQUENT STIMULATION OF 17/?-ESTRADIOL PRODUCTION GUIDO VERHOEVEN andJEAN
CAILLEAU
Laborato~um voor Experimentele Geneeskunde en Endoc~nolo~e, advent of Developmental Biology, Onderwijs en Navorsing, Legendo, Gasthuisberg, B-3000 Leuven, Belgium (Received 11 September
1985)
Summary-Sertoli cell-enriched cultures derived from 19-day old rats were exposed to FSH, L-isoproterenol, glucagon and dbcAMP for 24 up to 96 h. The influence of primary stimulation with these agonists on the response of the cells to subsequent stim~ation with the homologous or heterologous agonists was investigated. Particular attention was paid to the response of the aromatase system defined as the ability of the cells to convert testosterone into 17/i-estradiol. The responsiveness of this system was compared with the responsiveness of two other systems: adenylate cyclase and phosphodiesterase. It could be demonstrated that preincubation with the mentioned agonists results in a decreased responsiveness (maximal response) and a decreased sensitivity (ED,) upon re-stimulation with the homologous agonists. Preincubation with FSH also provokes partial desensitization for glucagon and L-isoproterenol. This heterologous desensiti~tion can be mimicked by d&. The mentioned desensitization reactions are accompanied by a marked decrease in the a~um~ation of CAMP in the medium. Whereas desensitization of the adenylate cyclase occurs rapidly, desensitization of the aromatase response requires protracted stimulation for 34 days. In contrast with the adenylate cyclase and the aromatase system, the responsiveness of phosphodiesterase to FSH, L-isoproterenol, glucagon and dbcAMP is not affected by repeated stimulation for 96 h. It is concluded that hormonal desensitization affects both early (CAMP) and late (aromatase) responses of the Sertoli cell. However, some responses, such as phosphodiesterase activity seem to escape the-desensitization process.
gram and Dr A. F. Parlow. Steroids, L-isoproterenol, glucagon and dbcAMP were obtained from Sigma. 3-Isobutyl-1-methylxanthine (MIX) was purchased from Aldrich Chemical Company.
INTRODUCTION Rat Sertoli cells cultured in vitro respond to a number of hormonal agonists. From a physiological point of view, FSH and testosterone are the most important stimulators fl]. P-Agonists and glucagon also have marked effects on cultured cells, but their biological role remains unclear [2-4]. In previous studies, it has been demonstrated that stimulation with FSH or L-isoproterenol causes homologous desensitization of the CAMP response of Sertoli cells, and that stimulation with FSH results in heterologous desensitization to isoproterenol IS]. Homologous desensitization of the adenylate cyclase can also be achieved in Sertoli cell membranes and may be related to receptor phosphorylation [6]. The heterologous desensitization is at least in part due to the CAMPdependent induction of a specific CAMPp~osphodiesterase [‘?-IO]. In the present study we investigated whether desensitization at the level of the adenylate cylase affects more distal responses of Sertoli cells such as stimulation of aromatase activity (defined as the ability of the cells to convert testosterone into 17/J-estradiol) [I I] and induction of phosphodiesterase activity [7,8].
Media
Preincubations were performed in Medium A. This medium consists of RPM1 1640 (Flow Laboratories) supplemented with Hepes (Gibco, Biocult), 22.5 mM, L-glutamine (Flow Laboratories) 4.3 mM and phenol red (Gibco, Bio-cult) 56pM. Medium B consists of Medium A supplemented with streptomycine sulfate (100 pg/ml) and penicillin G (lOOU/ml). Medium C is used for the actual incubations and contains Medium A enriched with MIX, 0.1 mM and testosterone 0.5 PM. Phosphate buffered saline, Dulbecco’s formula (PBS) was obtained from Gibco, Bio Cult. Sertoli cell-enriched cultures
Testes were derived from 19-day old Wistar rats. Sertoli cells were prepared by a modification of the method of Welch and Wiebe[l2] as described previously [3]. Routinely, 2 x lo6 cells suspended in 2 ml medium B were seeded in multi~shes confining six 35 mm wells (Greiner). Cultures were maintained in a humidified incubator (32”C, 5% CO,). On days 3, 4, S or 6 the medium was aspirated, cells were washed twice with 2ml PBS and preincubation was started by the addition of 2ml medium A without
EXPERIMENTAL
Materials Ovine FSH (oFSH:NIH-FSH-S13) was a gift of the NIAMDD Pituitary Hormone Distribution Pro45
46
GUIDO VERHOEVEN and PREINCUBATION INCUBATION WITH FSH I*..l OR CONTROL IlEOlUtl lo,ol
PREINCUBATION
TlHf
WITH
PREINCUBATION
(~.ol
OR CONTROL
INCUBATION WITH L-ISOPROTERENOL OR CONTROL MEDIUM lo 01
PREINCUBATION
ltlOURS1
INCUBATION WITH FStl I*.ml OR CONTROL HEOUJU looI
FSH
JEANCAILLEAU
WITH
dbcAMP
l0.m)
MEDIUM
to.01
INCUBATION WITH GLUCAGON lo.1 OR CONTROL MfOlUH lo .I
TIWE (HOURS1
lm.ol OR CONTROL
INCUBATION WIT,, L-ISOPROTERENOL OR CONTROL MEDIUM lo.oi
I*..)
MEDIUM
lo.01
INCUBATION WITH GLUCAGON l*,mI OR CONTROL MEDIUM lo.01
1
21 b8 PREINCUBATION
72 96 TIME IHOURSI
2L 48 PREINCUBATION
12 96 TIME IHOURS
2r. I.8 PREINCUBATION
12 96 TIME (HOURS1
Fig. 1. FSH and dbcAMP induced desensitization as a function of time. Sertoli cell-enriched cultures were preincubated at 32°C for the indicated periods of time in Medium A (control) or in the same medium supplemented with oFSH (1 pg/ml) or dbcAMP (1 mM). After this preincubation (on day 7 of culture), the cells were rinsed twice with PBS and were incubated for another 24 h period at 32°C in Medium C (control: Medium A supplemented with testosterone, 0.5 p M and MIX, 0.1 mM) or in the same medium containing oFSH (1 pg/ml) L-isoproterenol (10 PM) or glucagon (10 pgg/ml). The aromatase activity (accumulation of estradiol in the medium) was evaluated during the incubation period. Each point represents the mean + SD of 4-6 incubations. Values between brackets indicate the percentage inhibition (preincubation with agonist vs preincubation with control medium). Significant inhibition (P < 0.01) is indicated by an asterisk.
(control) or with the additions specified in the Results section. The media were changed every 24 h. On day 7 the preincubation medium was discarded, the cells were washed with PBS and 2 ml Medium C without (control) or with the indicated agonists was added. After an incubation period of 24 h at 32°C the medium was collected and stored at -20°C for determination of 17B-estradiol. The cells were washed twice with PBS and were dissolved in 1 ml of 0.1 M NaOH for determination of protein content. Proteins were measured by the method of Lowry[ 131. Measurement of 178 -estradiol production The media were extracted with 4 vol of cyclohexane-ethylacetate (1: 1, v/v) and 17/I -estradiol was measured by a specific radioimmunoassay as described previously [2] W-90% of the radioimmunoassayable estradiol co-crystallized with authentic 17fi-estradiol on repeated crystallization (not shown). Measurement of phosphodiesterase activity To measure phosphodiesterase activity in Sertoli cells, culture conditions were slightly modified. 4 x lo6 Sertoli cells suspended in 3 ml medium were seeded in Petri dishes with a diameter of 6cm. The monolayers were preincubated and incubated as explained in the previous paragraphs. Cells were
washed, harvested and homogenized in 0.5 ml phosphodiesterase buffer as described elsewhere [7]. The homogenates were stored at -70°C and phosphodiesterase activity was measured in the unfractionated homogenates using the method of Thompson et af.[14]. RESULTS Injluence of preincubation with FSH, dbcAMP, L-isoproterenol and glucagon on the responsiveness of the aromatase to homologous and heterologous agonists
Preincubation with FSH (1 pg/ml) results in a decrease in the response of the aromatase on subsequent stimulation with the same agonist (Fig. 1). The decrease becomes statistically significant (P -C0.01) after 48 h of preincubation. After 96 h of preincubation a 82% reduction in the aromatase response is observed. Preincubation with FSH also decreases the response to L-isoproterenol and glucagon. The reduction in the responsiveness to these heterologous agonists, however, is less pronounced than the reduction in the responsiveness to FSH. Preincubation with dbcAMP (1 mM) results in desensitization for FSH, L-isoproterenol and glucagon (Fig. 1). The degree of desensitization is com-
Desensitization
of Sertoli cells
PREINCUBATION WITH L-ISOPROTERENOL (m,nl INCUBATION WITH FSH I..*1 OR CONTROL HEOIUM lo,nl
PREINCUBATION
TINE NUJURSI
PREINCUBATION WITH GLUCAGON INCUBATION WITH FSH IO.9 1 OR CONTROL ~OIUM lO.0)
CB PRE&BATKIN
72 9L TINE lHOURSI
OR CONTROL MEDIUM b.0)
INCUBATION WITH L-ISOPROTERENOL OR CfflTROL NEMUM b.4
24 b6 PREINCUBATION
l*.d
INCUBATION WITH GLUCAGON IV1 OR CONTROL MEOIUM lo.01
2l a PREINCUEATION
72 91 TINE iHOURS
12
PC
TINE IHOURSI
(m,olOR CONTROL MEDIUM (0.0)
lNCUBATl!JN WITH L-ISOPROTERENOL OR CONTROL KKHUN lo.nl
26 4B PRE,NCUBATION
47
72 96 TNIE RIOURSI
I*.Dl
INCUBATION WITH GLUCAGON f*.DI OR CONTROL NEDIUN lo,01
w
PREINCUBATION
TIME INOURS
Fig. 2. L-Isoproterenol and glucagon induced desensitization as a function of time. Cells were preincubated and incubated as explained in Fig. 1; the oFSH or the dbcAMP in the preincubation medium was replaced by L-isoproterenol (10 PM) or glucagon (lOpg/ml). The inhibition of aromatase activity is measured and expressed as explained in Fig. 1.
parable
for the three
hormones.
It may be noted,
however, that a more profound desensitization for FSH is obtained after 96 h of preincubation with FSH than after prein~ubation with dbcAMP. For the other two hormones the FSH induced reduction in responsiveness equals the dbcAMP induced desensitization. Preincubation with L-isoproterenol (10pM) or glucagon (10 pg/ml) results in nearly complete desensitization for the homologous agonists whereas the response to heterologous agonists is not affected (Fig. 2). Dose-response
relationships
The influence of preincubation with increasing concentratjons of FSH, dbcAMP, L-isoproterenoi and glucagon on the responsiveness to homologous and heterologous agonists is shown in Fig. 3. In this experiment, the cells were exposed to the indicated concentrations of the agonists on days 3, 4, 5 and 6 of culture and the induction of aromatase activity was studied on day 7. The data show that FSH and dbcAMP induce a dose-dependent desensitization for the homologous as well as for the heterologous agonists. At a concentration of 1 pg&/ml the FSHinduced homologous desensitization is nearly complete whereas the desensitization for the heterologous agonists barely reaches 50%. L-Isoproterenol and glucagon produce only homologous de~nsiti~tion. The ED,, values for the 4 homologous desensiti~tion reactions studied are: 0.05 pglrnl for FSH, 50pM
for dbcAMP, 5 PM for r_-isoproterenol and 0.5 pg/ml for glucagon. I@e~ce of preincubation with homologous agonists on the dose-respon~~e curves for FSH, dbcA~P, ~-~soproterenol and glucagon Preincubation during 96 h with 0.05 pg/ml FSH (a concentration producing 50% desensitization) results in marked changes in the dose-response curve for FSH. Besides the expected 50% reduction in the maximal response there is an obvious shift of the curve towards higher concentrations of FSH. In cells preincubated with control medium, half-maximal stimulation of aromatase activity is observed at a concentration of 10 ng/ml; in desensitized cells a 8.5fold higher concentration of FSH is required. A comparable decrease in responsiveness (maximal activity) and sensitivity (ED, value) is observed for the homologous desensitization reaction produced by L-isoproterenol and glucagon. The decrease in sensitivity is somewhat less pronounced for dbcAMP. Comparison of the eflects of preincubation with FSH, dbcAMP, L-isoproterenol and glucagon on various responses of Sertoli cells
Table 1 compares the influence of preincubation and incubation with various agonists on three Sertoli cell responses: accumulation of CAMP in the medium, stimulation of phosphodiesterase activity and stimulation of aromatase activity. The data on the stimuIation of aromatase activity largely confirm the findings discussed in the previous
48
Gur~o
VERHOEVENand JEAN CAILLEAU
Fig. 3. Dose-response relationship of the FSH, dbcAMP, L-isoproterenol and glucagon induced desensitization of aromatase activity. Sertoli cell-enriched cultures were preincubated for 96 h in the presence of the concentrations of oFSH bane1 A), dbcAMP (panel B), L-isoproterenol (panel C) and glucagon (panel D) indicated in the abcissa. After this preincubation (on day 7 of culture), the cells were washed twice with PBS and were incubated for 24 h in Medium C supplemented with oFSH (1 pg/ml; a-----•), dbcAMP (1 mM, O----O), glucagon (lOpg/ml, H-D) or L-isoproterenol (1OpM; n-0). Aromatase activity is expressed as a percentage of the activity observed in cells preincubated in control medium. 100% corresponds to 2.45 k 0.15 ng/mg protein for ceils incubated with FSH, 3.56 f 0.28 for cells incubated with dbcAMP, 1.81 f 0.17 for cells incubated with L-isoproterenol and I .03 f 0.05 for ceils incubated with glucagon (mean + SD; n = 16).
paragraphs. The response to FSH or dbcAMP is decreased after preincubation with FSH as well as after preincubation with dbcAMP. The response to L-isoproterenol and glucagon is markedly reduced after pretreatment with the homologous agonists but preincubation with FSH and dbcAMP also provokes a partial decrease in responsiveness. The agonist-induced accumulation of CAMP in the medium largely parallels the aromatase response, All the studied agonists provoke homologous desensitization, whereas FSH and dbcAMP also provoke partial desensiti~tion for the heterologous agonists. The phosphodiesterase response shows a completely different pattern. At the end of the preincubation period phosphodiesterase activity was 6.95 rf: 0.05 (mean f SD; n = 3) in cells preincubated during 96 h with control medium. In cells preincubated with FSH, L-isoproterenol, glucagon and dbcAMP, the respective values were: 35.39 zt 2.32, 15.77&0.53, 15.06+0.11 and 85.30&2.22. If these cells are incubated for a further 24 h in Medium C (Table 1) phosphodiesterase activity decreases (except for the cells preincubated with control medium where the MIX in Medium C may cause a slight increase in activity) but does not completely reach control values. If the incubation is performed in Medium C
supplemented with one of the studied agonists a further increase in phosphodiesterase activity is observed. Under none of the studied conditions is there any sign of desensitization. DISCUSSION
The results reported here demonstrate that FSH, L-isoproterenol and glucagon not only stimulate CAMP production, conversion of testosterone into estradiol (aromatase activity) and phosphodiesterase activity in Sertoli ceil-enriched cultures but also induce a state of refractoriness to subsequent stimulation. Desensitization with respect to CAMP production has been reported previously for FSH and L-isoproterenol [S, 7,9, 151. The major new findings from the present work are: (1) glucagon also induces a state of refractoriness; (2) the FSH, L-isoproterenol and glucagon-induced desensitization also affects later responses of these cells such as activation of the aromatase activity; (3) the phosphodiesterase remains responsive to all the mentioned agonists despite repeated stimulation for 96 h. The de~nsiti~tion of the aromatase response shows some remarkable analogy with the desensitization of the CAMP response as reported
of
Desensitization
Table
I.
Influence of preincubation
Sertoli
cells
with homologous or heterologous of Sertoli cell-enriched cultures
Accumulation of CAMP (nmolime. mot)
49
agonists
on various responses
Phosphodiesterase activity (U/me. mot)
Aromatase activity (nnime DrOt)
Preincubation
Incubation
Control FSH L-isoproterenol Glucagon dbcAMP
Control
0.06 0.07 0.07 0.09 0.07
+ + f f f
0.01’ 0.03” 0.02” 0.04a 0.02”
9. I I + 0.97’ 17.82 T2.Wb 12.67; l.OOh,C I I .43 + I .57b,’ 22.80 + 2.516
0.16 + 0.04” 0.18 ; 0.03d 0.15 f 0.03” 0.16F0.03” 0. I6 + 0.06”
Control FSH L-isoproterenol Glucagon dbcAMP
FSH
I .53 0.12 I .38 I .4l 0.50
i f f f +
0.08” o.oc 0.03” 0.03a 0.06b
62.03 13.13 51.77 58.07 81.43
f f f f *
2.64b 3.22” I .56b 4.35b 3.184
2.17 0.51 I .95 2.08
+ f * k 1.50f
0.03” 0.06’ 0.18” 0.03” 0.05b
Control FSH L-isoproterenol Glucagon dbcAMP
L-isoproterenol
0.76 0.40 0.1 I 0.67 0.37
f + * f f
0.04” 0.04b 0.05’ 0.05” 0.07b
28.83 42.11 37.16 35.04 49.06
f f + I F
l.51d 2.49’ I .57b,’ I .OO‘ I .OO”
I .60 I .08 0.32 I .45 0.95
0.05d 0.03’ 0.04d 0.02b 0.06’
Control FSH L-isooroterenol G&go” dbcAMP
Glucagon
0.63 0.29 0.55 0.09 0.33
f + f i f
0.04’ 0.02b 0.02” 0.02c 0.05b
30.77 41.07 36.51 34.09 52.42
f + + I f
2.47’ 2.lOb l.52b,C I .OO’ 2.06d
I .25 i 0.04” 0.81 + 0.02b 1.18iO.03” 0.29 f 0.02” 0.66 f 0.06’
Control FSH L-isooroterenol Gluc’agon dbcAMP
dbcAMP
96.76 i 1.53” 97.13 i 2.30a 96.81 + 2.07” 97. I I : 2.93” 106.13i4.24”
I f f F f
3.20 + 0.04” 2.33*0.11h 3.17 +0.03” 3.20 ; 0.05” I .08 + 0.04’
Sertoli cell-enriched cultures were preincubated for 96 h in Medium A (control) or in the same medium supplemented with oFSH (1 pg/ml), L-isoproterenol (I 0 PM), glucagon (I 0 fig/ml) or dbcAMP (I mM). After this preincubation, the cells were washed twice with PBS and were incubated for 24h in Medium C (control) or in the same medium supplemented with the mentioned concentrations of oFSH, L-isoprotenol, glucagon or dbcAMP. The accumulation of CAMP and estradiol was measured in the incubation medium. Phosphodiesterase activity was measured in the cells at the end of the incubation period. Values represent the mean + SD of 3 incubations. The effects of the preincubation conditions were analyzed by one-way analysis of variance. There are significant differences between groups indicated by different letters (P < 0.01).
previously [5]. Under the conditions used in both series of experiments (primary stimulation in the absence of a phosphodiesterase inhibitor and subsequent testing of the residual responsiveness in the presence of a phosphodiesterase inhibitor) there is homologous desensitization after stimulation with FSH, L-isoproterenol and glucagon. In addition, stimulation with FSH produces partial heterologous desensitization for all the other agonists. This partial heterologous desensitization can be mimicked by preincubation with dbcAMP. Several mechanisms have been invoked to explain the decrease in CAMP production after repeated stimulation of Sertoli cells: uncoupling of receptors from the catalytic moiety of the adenylate cyclase [6]; disappearance of specific receptors from the cell membrane [6,16] and CAMP dependent induction of a specific phosphodiesterase [7,8]. All these phenomena occur within a matter of hours, however, whereas desensitization of the aromatase response requires 4 days. The difference in timing cannot be explained simply on the base of a prolonged half-life of the aromatase system. In fact, no residual aromatase activity is measured in cells preincubated with FSH, L-isoproterenol or glucagon for 24 up to 96 h and
subsequently incubated with control medium containing testosterone as a substrate for the aromatase (Figs 1 and 2). At the present time we can only speculate on the mechanism(s) responsible for this slow decrease in the responsiveness of the aromatase system. It may be worthwhile to remember, however, that the timecourse of the CAMP response and the aromatase response of Sertoli cells after primary stimulation are completely different [3, 51. After primary stimulation the intracellular CAMP increases rapidly and transiently reaching maximal values within the first 2 h of stimulation. The CAMP in the medium increases for approx. 4 h but remains constant thereafter. The conversion of testosterone into estradiol (aromatase response), however, proceeds at a constant rate throughout the stimulation period for at least 48 h. Although this does not exclude that stimulation of Sertoli cells might provoke increased production of new aromatase enzyme, it rather suggests that stimulation might activate an inactive form of the enzyme already present under basal conditions. In such a model it is conceivable that the initial decrease in CAMP production (desensitization) would merely limit the “excessive” amount of CAMP pouring out
50
GUIDO VERHOEVENand JEAN CAILLEAU
10 ng ml.’
/
0
10-Z
10-l
t
10
tone. GLUCAGON l~g.ml~'l
Fig. 4. Influence of homologous desensitization on the dose-response curves for induction of aromatase activity by FSH, dbcAMP, L-isoproterenol and glucagon. Sertoli cell-enriched cultures were preincubated for 96 h either in control medium (e---•) or (0-O) in medium supplemented with oFSH (0.05 rg/ml; panel A), dbcAMP (50 PM; panel B), r.-isoproterenol (5 FM; panel C) or glucagon (0.05 pg/ml; panel D). After this preincubation period, the cells were washed twice with PBS and were incubated for 24 h in Medium C suppIemented with the concentrations of homologous agonists indicated in the abcissa. Estradiol was measured in the incubation medium. Values represent the mean & SD of 6 incubations. The ED, values of the various curves are indicated by an arrow.
of the cells during the first hours of stimulation. A more pronounced desensitization might be required to impede the intracellular accumulation of sufficient amounts of CAMP able to activate and maintain aromatase activity. The foregoing hy~thesis cannot explain al1 our observations. In fact, it may be noted that in cells pretreated with FSH or dbcAMP the aromatase response is reduced even after maximal stimulation with dbcAMP. (Fig. 4, Table 1). This indicates that some step beyond the production of CAMP has become rate limiting. It is tempting to speculate that the FSH or CAMP induced increase in the production of a protein kinase inhibitor described by Tash et a1./17] may be responsible for this decreasing responsiveness. Alternatively, it is conceivabie that prolonged stimulation of Sertoli cells wouid result in some form of “differentiation” of these cells accompanied by a decrease in aromatase activity. In fact, a similar decrease in aromatase activity is observed during pubertal development in vim [18, 191. It is of particular interest that the high affinity CAMP-phosphodiesterase remains responsive to FSH, L-isoproterenol and glucagon even after repeated stimulation with these agonists for 96 h. Although the mechanism of this “escape from desensitization” remains obscure, this observation further supports the contention [7-l@ 201 that this enzyme
may play a physiological role in the age-dependent decrease of FSH-induced accumulation of CAMP in the testis [21-231. In view of these observations it might be of interest to investigate whether other Sertoli cell responses such as the secretion of androgen binding protein (ABP) or transferrin (proteins that may be required to maintain the production of fertile spermatozoa) also escape the desensitization process. It can be concluded that all the studied agonists that stimulate Sertoli cells also desensitize these cells to subsequent stimulation. This desensitization not only results in decreased production of CAMP, but also provokes decreased responsiveness and sensitivity with respect to later responses such as the activation of the aromatase. However, the lack of desensitization with respect to phosphodiesterase activation, shows that some Sertoli cell functions may escape the desensitization process. Acknowledgements-This research was supported by a grant from the Nationaal Fonds voor Wetenschappelijk Onderzoek van Belgii (grant no. 3.0047.80), a “Krediet aan Navorsers” from the same institution and a grant from the Onderzoeksfonds K. U. Leuven. The authors appreciate the skilled technical assistance of MR Lodewijk Deboel and thank Dr W. Heyns for critical discussion of the manuscript. We gratefully acknowledge the National Pituitary Agency, NIH (Bethesda, Md, U.S.A.) and Dr Parlow for the gift of oFSH.
Desensitization REFERENCES
1. Fritz I. B.: Sites of action of androgens and Follicle stimulating hormone on cells of the seminiferous tubuli. In Biochemical Actions of Hormones (Edited by G. Litwack). Academic Press, New York, Vol. V (1978) pp. 249-28 1. 2. Verhoeven G., Dierickx P. and De Moor P.: Stimulation effect of neurotransmitters on the aromatization of testosterone by Sertoli cell-enriched cultures. Molec. cell. Endocr. 13 (1979) 241-253. and follicle 3. Verhoeven G.: Effects of neurotransmitters stimulating hormone on the aromatization of androgens and the production of adenosine 3’,5’-monophosphate by cultured testicular cells. J. steroid Biochem. 12 (1980) 315.-322. H., Jutte N. H. P. 4. Eikvar L., Levy F. O., Attramadal M., Froysa A., Tvermyr S. M. and Hansson V.: Glucagon-stimulated cyclic AMP production and formation of estradiol in Sertoli cell cultures from immature rats. Molec. cell. Endocr. 39 (1985) 107-113. 5 Verhoeven G., Cailleau J. and De Moor P.: Desensitization of cultured rat Sertoli cells by folliclestimulating hormone and by L-isoproterenol. Molec. cell. Endocr. 20 (1980) 113-126. H., Jahnsen T. and Hansson V.: Regulation 6 Attramadal of hormone-responsive Sertoli cell adenylyl cyclase in a cell-free system. Molec. cell. Endocr. 34 (1984) 221-228. I Verhoeven G., Cailleau J. and De Moor P.: Hormonal control of phosphodiesterase activity in cultured rat Sertoli cells. Molec. cell. Endocr. 24 (1981) 41-51. 8 Conti M., Toscano M. V., Petrelli L., Geremia R. and Stefanini M.: Regulation by follicle-stimulating hormone and dibutyryl adenosine 3’,5’-monophosphate of a phosphodiesterase isoenzyme of the Sertoli cell. Endocrinology 110 (1982) 1189-I 196. 9 Conti M., Toscano M. V., Petrelli L., Geremia R. and Stefanini M.: Involvement of phosphodiesterase in the refractoriness of the Sertoli cell. Endocrinology 113 (1983) 1845-1853. 10. Conti M., Toscano M. V., Geremia R. and Stefanini M.: Follicle-stimulating hormone regulates in vivo testicular phosphodiesterase. Molec. cell. Endocr. 29 (1983) 79-89. J. H.. Fritz I. B. and Armstrong D. T.: Site 11. Dorrington at which FSH regulates estradiol-17/I biosynthesis in Sertoli cell preparations in culture. Molec. cell. Endocr. 6 (1976) 117-122.
of Sertoli
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51
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