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Follicle-stimulating hormone regulates in vivo testicular phosphodiesterase
Molecular and Cellular Endocrinology, 29 (1983) 79-89 Elsevier Scientific Publishers Ireland, Ltd.
FOLLICLE-STIMULATING HORMONE TESTICULAR PHOSPHODIESTERASE Marco CONTI, Michele Mario STEFANINI
V. TOSCANO,
Instime of Histology and General Embryology, Rome (Italy) Received
29 April
1982; revision
received
REGULATES
Raffaele
University
9 September
IN VIVO
GEREMIA
and
of Rome, Via A. Scarpa 14, 00161
1982; accepted
14 September
1982
The effect of FSH on testicular phosphodiesterase was studied in immature rats in order to verify that the regulation of response to hormone in the gonad involves an increased cyclic AMP catabolism. Hydrolysis of cyclic AMP and cyclic GMP was measured in the homogenates of seminiferous tubules and interstitium of control animals and animals injected i.p. with 50 gg ovine FSH twice, 24 h and 12 h before necroscopy. After hormonal treatment, cyclic AMP phosphodiesterase activity in the seminiferous tubules was markedly increased whether results were expressed per testis or per mg protein, while cyclic GMP phosphodiesterase present in the same compartment was apparently unaffected. In the interstitium, cyclic nucleotide hydrolysis was usually decreased after FSH injection. The stimulation of phosphodiesterase, a slow process reaching maxima1 stimulation after 12 h, was dependent on the dose of FSH injected. In addition, DEAE-cellulose chromatography of cytosol prepared from control and treated seminiferous tubules confirmed that stimulation was restricted to a cyclic AMP hydrolysing enzyme while the activity of the cyclic GMP hydrolysing form was not modified. Thus it is demonstrated that testicular phosphodiesterase is under FSH control. It is proposed that this in viva regulation is a relevant phenomenon in the modulation of Sertoli cell function and contributes to the refractoriness that follows gonadotropin treatment. Keywords:
cyclic nucleotides;
desensitization;
phosphodiesterase;
gonadotropins.
Gonadotropic target cells share the ability of many hormone-dependent tissues to regulate the amplitude of their response, becoming insensitive to continued hormonal stimulation (Catt et al., 1979). This regulation is probably mediated by changes in the number of receptors (Hsueh et al., 1976; Conti et al., 1976) and by desensitization of adenylate cyclase (Hunzicker-Dunn and Birnbaumer, 1976a, b; Harwood’et al., 1978). A comparable modification of response occurs acutely in the Sertoli cell of the seminiferous tubules. Thus these cells, which are targets of FSH (Tung et al., 1975) and of /3-adrenergic agonists (Verhoeven et 0303-7207/83/0000-0000/$03.00
0 Elsevier Scientific
Publishers
Ireland,
Ltd.
M. Conti
80
et al.
al., 1979), become refractory to a second stimulation with gonadotropin, both in vivo (O’Shaughnessy, 1980) and in vitro (Verhoeven et al., 1980). Although several hypotheses have been proposed, the exact mechanism underlying this change in sensitivity is not fully understood. The present experiments explore the possibility that FSH regulates the responsiveness of the Sertoli cell by modulating the activity of phosphodiesterase. We present evidence that the injection of gonadotropin into immature rats produces an increase in the specific activity of a high-affinity CAMP phosphodiesterase. In addition, this regulation is elicited at physiological FSH concentrations and develops with a timecourse comparable to other FSH effects.
MATERIALS
AND
METHODS
Animal treatment Immature Wistar rats were used in all experiments. In order to alleviate variations within groups, pairs of control and treated animals were from the same litter. lo-day-old rats were injected with 50 pg oFSH (ovine FSH-S14 with 9 times the biological potency of NIH-FSH-Sl) dissolved in phosphate-buffered saline (PBS). Unless stated, animals received two injections 24 h and 12 h before necroscopy. Control animals received a saline vehicle only. At the end of hormonal treatment rats were sacrificed and testes removed and weighed. Materials [3H]cAMP and [ “H]cGMP (spec. act. 35-40 Ci/mmole) were obtained from New England Nuclear Corp. (Boston, MA). CAMP, cGMP, PMSF and Crotalus atrox snake venom were purchased from Sigma Chemical Co. All other reagents were of analytical grade from BDH (London, Great Britain) or Hoechst (Frankfurt, F.R.G.). Ovine FSH (oFSH-S14) was supplied by the NIAMDD Pituitary Hormone Distribution Program. Homogenate preparation Each pair of decapsulated testes was incubated in 0.1% collagenase (Worthington) solution in PBS at room temperature, with continuous shaking (50 cycles/min) for lo-15 min until the seminiferous tubule mass was dispersed. The resulting seminiferous tubules were separated from the remaining cell suspension by sedimentation at unit gravity. Supernatants of the first sedimentation were transferred to separate tubes, the cells were washed by sedimentation at 200 x g in a clinical
FSH regulation
81
ofphosphodiesterase
centrifuge and finally were resuspended in the homogenization buffer. This preparation will be referred to as the interstitium. Similarly, the seminiferous tubules were washed 3 times with PBS. After the last wash the two cell preparations were resuspended in 20 mM Tris-HCl, pH 8.0, 10 mM fi-mercaptoethanol, 2 mM phenylmethylsulphonylfluoride, homogenized with an all-glass Dounce tissue grinder and used directly in the phosphodiesterase assay. Phosphodiesterase
assay
Phosphodiesterase activity was measured according to the method of Thompson and Appleman (1971) with minor modifications (Conti et ai., 1981).The cyclic nucleotide concentration in the assay was 1 PM for CAMP and 1 PM for cGMP. Usually two or three homogenate concentrations were used in each assay. Recovery of adenosine, measured by adding a known amount of [ “C]adenosine (10000 dpm/tube) prior to the incubation with snake venom, varied between 40 and 50% in all assays performed. No correction for this recovery was introduced in the calculation of enzyme specific activity. Comparable specific activities were obtained when the products of the reaction were separated in the presence of methanol (Thompson et al., 1979). Cyclic nucleotide hydrolysis was linear up to 30 min and proportional to the crude enzyme preparation added to the assay. Protein content of homogenates was measured according to a modified Lowry procedure (Bensadoun and Weinstein, 1976) using bovine serum albumin as standard. DEAE-cellulose
chromatography
of phosphodiesterase
Cell homogenates were centrifuged at 20000 x g for 30 min at 4°C and at 100000 X g for 1 h at 4°C in a Beckman ultracentrifuge (SW40 rotor). The resulting supernatants from control and treated groups, containing most of the phosphodiesterase activity, were applied to DEAE-cellulose columns (Whatman DE52) preequilibrated with 70 mM acetate buffer, pH 6.5, containing 0.2 mM EGTA. After extensive washing with starting buffer, a 70-600 mM acetate gradient, pH 6.5, was applied to the columns. The gradient was formed in a single gradient former and split in two at the outlet of the chamber, in order to ensure uniformity of ionic strength applied to the two columns. A total of 140 fractions were collected, and every other fraction was assayed for phosphodiesterase activity using 1 PM CAMP and 1 PM cGMP as substrates, in the presence of 1 mM CaCl, and 100 ng purified brain calmodulin per tube (Jamieson and Vanaman, 1979).
82
M. Conti et al.
RESULTS Experiments were performed on lo-day-old Wistar rats, since at this age Sertoli cells are fully responsive to gonadotropin and represent the majority of the cells in seminiferous tubules. The stimulatory effect of FSH on the mitotic activity of testicular cells (Means, 1975) was taken into consideration by expressing the activity both on a per testis and a protein basis. The enzymatic activity of cGMP phosphodiesterase was also measured as an internal control. 24 h after the injection of 50 pg oFSH, the testicular weight of animals receiving hormone was consistently higher (19.84 + 4.66 mg/testis, mean _+ SD, N = 24) than saline-treated controls (14.44 * 2.55 mg/testis, N = 22). The increase in the testicular weight was accompanied by an increase in the protein recovered after hormonal treatment (seminiferous tubules: control 812 + 32 pg protein/testis, FSH-treated 1541 + 185 pg protein/testis; interstitium: control 54 + 10 pg protein/testis, FSHtreated 86 _t 11 pg protein/testis, mean& SE, N = 7). When phosphodiesterase was measured in the different compartments of the testis with 1 PM CAMP as substrate, a substantial increase in the activity of the seminiferous tubule followed hormone treatment (Table 1). This increase was in the order of 5-fold when the data were expressed per testis, and 2.5-fold after correction of the activity per mg of protein. On the other hand, phosphodiesterase activity measured with 1 PM cGMP was affected only on a per testis basis, and was not significantly different from the control when corrected per mg of protein. In addition, the stimulation of phosphodiesterase activity by FSH appeared to be restricted to the seminiferous tubules of the immature testis, since either no stimulation was observed in the interstitial compartment, or else a 20% decrease in CAMP phosphodiesterase activity was detected (Table 1). Stimulation of phosphodiesterase activity was evident 3 h after a single injection of FSH (Fig. l), and the activity continued to increase up to 12 h. A second injection given 12 h after the first dose further stimulated CAMP hydrolysis in the seminiferous tubules. Cyclic GMP phosphodiesterase activity was unchanged throughout the duration of the experiment, and the CAMP hydrolysis in the interstitium was decreased significantly only with multiple treatments (Fig. 1). The increase in phosphodiesterase activity was also dependent on the dose of FSH administered to the animals (Fig. 2). Stimulation of the activity was detected with doses ranging from 10 to 100 pg oFSH given in two separate injections, and near-maximal stimulation was obtained with 100 pg FSH, suggesting that the doses required to stimulate phosphodiesterase are comparable to those employed to stimulate androgen-
I
tubules
(control,
Seminiferous
Mean+SEM
on testicular
different
20.7 f 3.2 6.5f 1.0 from control;
24.2 i 3.0 30.6 rt 0.8
* P < 0.025 vs. control.
22.6k3.0 (NS) 12.1 rt I.0 *
Control
hCG
53.25 + 3.73
52.96 + 3.26 26.105 1.31
5 1.20 t 4.04 (NS)
40.79 i 2.08 ** 63.12k4.33 *
FSH
protein
26.6 + 1.0 (NS) 41.0+ 3.2 *
hCG
protein
* P < 0.001; ** P < 0.01.
Control
from control;
pmoles/min/mg
activity
different
70.51 rt 8.11 **
pmoles/min/testis
Phosphodiesterase
phosphodiesterase
N = 9; FSH, N = 7). NS, not significantly
36.09~4.12
I PM cGMP
3.53 If: 0.70 (NS) 93.73 rt 15.48 *
Control
Control
FSH
pmoles/min/mg
activity
pmoles/min/testis
Phosphodiesterase
2.89 f 0.42 20.08 f 2.13
activity
1 PM CAMP 1 FM CAMP
Assay condition
phosphodiesterase
Mean + SEM, N = 3. NS, not significantly
tubules
compartment
Seminiferous lnterstitium
Testicular
Effect of hCG treatment
Table 2
tubules
compartment
lnterstitium Seminiferous
Testicular
Effect of FSH on testicular
Table
p E i: 2
%-
$ 8 9 -8 :
M. Cami et
84
al.
80-
ii iii /.
0
6
12 18 Ttme 1hr.s)
24
Fig. 1. Time-course of FSH stimulation of testicular phosphodiesterase. Immature Wistar rats were injected i.p. with a single dose of 50 pg ovine FSH. Control and sham-treated rats received PBS only. At the indicated time intervals, rats were sacrificed and testes removed, weighed and decapsulated. Each pair of decapsulated testes was processed separately and seminiferous tubules were separated from the interstitium as described in Materials and Methods. Phosphodiesterase activity was measured with 1 PM CAMP (O0) and 1 PM cGMP (o- - - - - -0) in the homogenates of the seminiferous tubules (top panel) and with 1 PM CAMP in the interstitium homogenate (bottom panel). A group of rats received a second injection of 50 pg FSH 12 h after the first treatment (0). Each point represents the mean i SEM of 2-3 different animals assayed in triplicate at two protein concentrations.
binding protein accumulation in the epididymis (Hansson et al., 1975) and to those required to stimulate protein kinase inhibitor in the immature testis (Tash et al., 1979). Again, cGMP hydrolysis was not affected at any of the doses employed and inhibition of CAMP interstitial phosphodiesterase was evident. Furthermore, the stimulation by FSH was specific to the gonadotropin, since 200 IU hCG (Pregnyl, Organon) injected i.p. had no effect on the seminiferous tubule activity (Table 2). On the other hand, chorionic gonadotropin had a slight stimulatory effect on the phosphodiesterase activity of the interstitium (Table 2). The assay of CAMP and cGMP hydrolysis at low substrate concentrations represents only an indirect estimate of the different phos-
85
FSH regulation of phosphodiesterase
P “, 60. E ,
0
0.1
1.0 pg o FSH
10
100
- 514
Fig. 2. Dose dependence of FSH stimulation of testicular phosphodiesterase. Immature rats Enzyme were injected with increasing FSH doses twice, 24 and 12 h before necroscopy. digestion and homogenate preparation was as in Materials and Methods. Phosphodiesterase and 1nM cGMP(O------0) as activity was measured using 1 PM CAMP (0 -0) substrates in the seminiferous tubules (top panel) and with 1 pM CAMP in the interstitium (bottom panel). Each point represents the meanf SEM of 2 or 3 animals assayed in triplicate at two protein concentrations.
phodiesterase forms present in the crude preparation, since these enzymes, especially the cGMP hydrolysing form, use both nucleotides as substrate (Pichard and Cheung, 1976). In order to confirm that a specific phosphodiesterase is stimulated by FSH, we analysed the activity of soluble extracts of seminiferous tubules by ion-exchange chromatography. The first peak eluted in our chromatographic system hydrolyses cGMP more efficiently than CAMP (Fig. 3B). This form was not affected by the hormonal treatment. Conversely, the second form eluted was markedly stimulated after FSH injection (Fig. 3A). This form had a low K, for CAMP (approx. 2.0 PM) and its activity was not regulated by either Ca2+ or calmodulin (data not shown). The elution pattern, the affinity for the substrates and the Ca2+ insensitivity of this form closely resemble the characteristics of the Sertoli cell phosphodiesterase that is regulated in vitro by FSH and dibutyryl CAMP (Conti et al., 1982; Geremia et al., 1982).
M. Conti et al.
A
1 pMcAMP
fraction number
Fig. 3. DEAE-cellulose chromatography of phosphodiesterase activity in the seminiferous tubules of control and treated animals. Immature rats were divided into two groups. One group received 50 /.~g FSH twice, as previously described, while controls received saline only. Cell homogenization and chromatography were performed as in Materials and Methods. Every other fraction was assayed for phosphodiesterase activity using 1 PM CAMP (panel A) and 1 pM cGMP (panel B). Activity measured in each fraction was corrected for the cytosol protein applied to the column. l - - - - - -0, control cytosol; O0, FSH-treated cytosol.
DISCUSSION that phosphodiesterase activity is modThese studies demonstrate ulated by FSH in vivo and suggest that an additional regulatory mechanism that controls intracellular CAMP is present in the gonadotropic target cells. Thus, FSH not only stimulates adenylate cyclase to produce CAMP (Means et al., 1980) but also stimulates the activity of a phosphodiesterase form that exhibits high affinity for CAMP. This, in turn, is probably responsible for an accelerated CAMP turnover in the cells of the
FSH regulation of phosphodiesterase
87
seminiferous tubules and consequently for a decreased stimulation by subsequent gonadotropic treatment. It has been demonstrated that in vivo intratesticular injection of FSH produces refractoriness of the gonad (O’Shaughnessy, 1980) and that this is related to receptor down-regulation and refractoriness of adenylate cyclase. It is likely therefore that phosphodiesterase stimulation and other regulatory mechanisms such as receptor down-regulation and an adenylate cyclase refractory state are all operating in the same cell and contribute to a ‘fine tuning’ of the responsiveness of the target. It is not excluded that other regulatory feedback mechanisms are present in the FSH target cells beyond the generation of CAMP. It has been demonstrated, in fact, that FSH enhances the synthesis of a protein kinase inhibitor and that the intracellular concentration of this heat-stable molecule is sufficient to inhibit a large proportion of the protein kinase activity (Tash et al., 1979). Although no direct proof has been provided here, Sertoli cells are probably responsible for the increased phosphodiesterase activity, since these cells are the major site of FSH action in the seminiferous tubules. This is further supported by the recent observations that FSH in vitro stimulates phosphodiesterase activity of the Sertoli cell (Conti et al., 1981; Verhoeven et al., 1981). In addition, the enzyme stimulated in vivo is very similar, if not identical, to the form selectively stimulated in cultured cells (Conti et al., 1982). The data presented also show that a specific enzyme is stimulated after the in vivo gonadotropic treatment. This isoform is apparently the same form that naturally increases in the seminiferous tubule during puberty (Means et al., 1978; Geremia et al., 1982). It is therefore possible that phosphodiesterase regulation contributes to the refractoriness that ensues during testicular maturation. In agreement with the above hypothesis is the observation that an increase in plasma FSH precedes the onset of the refractory state during puberty (Keteslegers et al., 1978). This endogenous FSH might trigger a phenomenon similar to what we have described and therefore increase the activity of a high-affinity CAMP phosphodiesterase. This in turn might lead to refractoriness of the mature Sertoli cell. Our data are not in contrast with the observation (Fakunding et al., 1976) that FSH injection produces a decrease, rather than an increase, in phosphodiesterase activity: the two events occur at different times. The inhibition is fast and maximal in about 20 min while the stimulation of phosphodiesterase is a delayed effect of gonadotropin that develops to its maximum in about 12 h. Thus it is possible that the two events are triggered by FSH in the same cell but that one is subsequent to the other. Such a possibility is reinforced by our in vitro observations that stimula-
88
M. Conti et al.
tion rapidly follows a preliminary inhibition of the activity (Conti et al., 1982). We have also reported a slight but consistent decrease in interstitial cell phosphodiesterase activity following FSH injection. This is consistent with the repeated observations of trophic effects of FSH on immature interstitial tissue (Ode11 and Swerdloff, 1976) and could account, at least in part, for the observed increased sensitivity of the testis to LH-hCG after FSH priming of the gonad (Hsueh et al., 197X; Chen et al., 1977). It is possible that the stimulation of phosphodiesterase is a phenomenon common to all gonadotropic target cells of the testis and ovary, and represents a physiological event in the function of the gonad. This assumption is supported by our finding that hCG, which acts in a different compartment of the testis, also has a stimulatory effect on the phosphodiesterase activity of the interstitium. In addition, it has been reported that the activity of a high-affinity phosphodiesterase form increases in the ovarian follicle during the estrous cycle of the rat (Schmidtke et al., 1980). Our findings that FSH is capable of regulating a high-affinity CAMP phosphodiesterase strongly suggest that the increase in CAMP hydrolysis reported in the rat ovarian follicle is dependent on. the preceding FSH surge in proestrus.
ACKNOWLEDGEMENTS We are indebted to the NIAMDD Hormone Distribution Program and to Dr. J.F. Parlow for the generous gift of ovine FSH. This work was supported by CNR project ‘Biology of Reproduction’ grants 80.01166.85 and 80.01176.85 and by Ford Foundation Grant 790-0659.
REFERENCES Bensadoun, A. and Weinstein, D. (1976) Anal. Biochem. 70, 241-256. Catt, K.J., Harwood, J.P., Aguilera, G. and Dufau, M.L. (1979) Nature 109-I 16. Chen, Y.D.I., Shaw, M.J. and Payne, A.H. (1977) Mol. Cell. Endocrinok 8, Con& M., Harwood, J.P., Hsueh, A.J.W., Dufau. M.L. and Catt, K.J. (1976) 25 1, 7729-773 1. Conti, M., Geremia, R., Adamo, S. and Stefanini, M. (1981) Biochem. Commun. 98, 1044- 1050. Conti, M., Toscano, M.V., Petrelli, L., Geremia, R. and Stefanini, M. (1982) 110, 1189-1196. Fakunding, J.L.. Tindall, D.J., Dedman, J.R., Mena. C.R. and Means, A.R. crinology 98, 392-402.
(London)
280,
291-299. J. Biol. Chem. Biophys.
Res.
Endocrinology (1976)
Endo-
FSH regulation
ofphosphodresteruse
89
Geremia, R., Rossi, P., Pezzotti, R. and Conti, M. (1982) Mol. Cell. Endocrinol. 28, 37-53. Hansson. V., Weddington, S.C., Petrusz, P., Ritzen. E.M., Nayfeh, S.N. and French, F.S. (1975) Endocrinology 97, 469-473. Harwood, J.P., Conti, M., Conn, P.M., Dufau, M.L. and Catt, K.J. (1978) Mol. Cell. Endocrinol. 11, 121-135. Hsueh, A.J.W., Dufau, M.L. and Catt. K.J. (1976) Biochem. Biophys. Res. Commun. 72, 1145-1152. Hsueh, A.J.W., Dufau, M.L. and Catt, K.J. (1978) Endocrinology 103, 1096-l 102. Hunzicker-Dunn, M. and Birnbaumer, L. (1976a) Endocrinology 99, 185-197. Hunzicker-Dunn, M. and Birnbaumer, L. (1976b) Endocrinology 99, 211-222. Jamieson Jr., G.A. and Vanaman, T.C. (1979) Biochem. Biophys. Res. Commun. 90. 1048-1056. Ketelslegers, J.M., Hetzel, W.D., Sherins, R.J. and Catt, K.J. (1978) Endocrinology 103. 212-222. Means, A.R. (1975) In: Handbook of Physiology, Section 7. Vol. 5 (American Physiological Society, Washington, DC) pp. 203-217. Means, A.R.. Dedman, J.R., Tindall, D.J. and Welsh, M.J. (1978) Biol. Reprod. Suppl. 2, 403-421. Means, A.R., Dedman, J.R., Tash, J.S., Tindall. D.J., van Sickle, M. and Welsh, M.J. (1980) Annu. Rev. Physiol. 42, 59-70. Odell, W.D. and Swerdloff, R.S. (1976) Recent Progr. Hormone Res. 32, 245-280. O’Shaughnessy, P.J. (1980) Biol. Reprod. 23, 810-814. Pichard, A.L. and Cheung, W.Y. (1976) J. Biol. Chem. 251, 5726-5737. Schmidtke, J., Meyer, H. and Epplen. J.T. (1980) Acta Endocrinol. (Kbh.) 95, 404-411. Tash, J.S., Dedman, J.R. and Means, A.R. (1979) J. Biol. Chem. 254, 1241-1247. Thompson, W.J. and Appleman, M.M. (1971) Biochemistry 10, 311-320. Thompson, W.J., Terasaki, W.L.. Epstein, P.M. and Strada, S.J. (1979) Adv. Cyclic Nucleotide Res. 10, 69-92. Tung, P.S., Dorrington, J.H. and Fritz, I.B. (1975) Proc. Natl. Acad. Sci. (U.S.A.) 72, 1838-1842. Verhoeven, G., Dierickx, P. and de Moor, P. (1979) Mol. Cell. Endocrinol. 13. 241-253. Verhoeven, G., Cailleau, J. and de Moor, P. (1980) Mol. Cell. Endocrinol. 20, 113-126. Verhoeven, G., Cailleau, J. and de Moor, P. (1981) Mol. Cell. Endocrinol. 24, 41-51.
FOLLICLE-STIMULATING HORMONE TESTICULAR PHOSPHODIESTERASE Marco CONTI, Michele Mario STEFANINI
V. TOSCANO,
Instime of Histology and General Embryology, Rome (Italy) Received
29 April
1982; revision
received
REGULATES
Raffaele
University
9 September
IN VIVO
GEREMIA
and
of Rome, Via A. Scarpa 14, 00161
1982; accepted
14 September
1982
The effect of FSH on testicular phosphodiesterase was studied in immature rats in order to verify that the regulation of response to hormone in the gonad involves an increased cyclic AMP catabolism. Hydrolysis of cyclic AMP and cyclic GMP was measured in the homogenates of seminiferous tubules and interstitium of control animals and animals injected i.p. with 50 gg ovine FSH twice, 24 h and 12 h before necroscopy. After hormonal treatment, cyclic AMP phosphodiesterase activity in the seminiferous tubules was markedly increased whether results were expressed per testis or per mg protein, while cyclic GMP phosphodiesterase present in the same compartment was apparently unaffected. In the interstitium, cyclic nucleotide hydrolysis was usually decreased after FSH injection. The stimulation of phosphodiesterase, a slow process reaching maxima1 stimulation after 12 h, was dependent on the dose of FSH injected. In addition, DEAE-cellulose chromatography of cytosol prepared from control and treated seminiferous tubules confirmed that stimulation was restricted to a cyclic AMP hydrolysing enzyme while the activity of the cyclic GMP hydrolysing form was not modified. Thus it is demonstrated that testicular phosphodiesterase is under FSH control. It is proposed that this in viva regulation is a relevant phenomenon in the modulation of Sertoli cell function and contributes to the refractoriness that follows gonadotropin treatment. Keywords:
cyclic nucleotides;
desensitization;
phosphodiesterase;
gonadotropins.
Gonadotropic target cells share the ability of many hormone-dependent tissues to regulate the amplitude of their response, becoming insensitive to continued hormonal stimulation (Catt et al., 1979). This regulation is probably mediated by changes in the number of receptors (Hsueh et al., 1976; Conti et al., 1976) and by desensitization of adenylate cyclase (Hunzicker-Dunn and Birnbaumer, 1976a, b; Harwood’et al., 1978). A comparable modification of response occurs acutely in the Sertoli cell of the seminiferous tubules. Thus these cells, which are targets of FSH (Tung et al., 1975) and of /3-adrenergic agonists (Verhoeven et 0303-7207/83/0000-0000/$03.00
0 Elsevier Scientific
Publishers
Ireland,
Ltd.
M. Conti
80
et al.
al., 1979), become refractory to a second stimulation with gonadotropin, both in vivo (O’Shaughnessy, 1980) and in vitro (Verhoeven et al., 1980). Although several hypotheses have been proposed, the exact mechanism underlying this change in sensitivity is not fully understood. The present experiments explore the possibility that FSH regulates the responsiveness of the Sertoli cell by modulating the activity of phosphodiesterase. We present evidence that the injection of gonadotropin into immature rats produces an increase in the specific activity of a high-affinity CAMP phosphodiesterase. In addition, this regulation is elicited at physiological FSH concentrations and develops with a timecourse comparable to other FSH effects.
MATERIALS
AND
METHODS
Animal treatment Immature Wistar rats were used in all experiments. In order to alleviate variations within groups, pairs of control and treated animals were from the same litter. lo-day-old rats were injected with 50 pg oFSH (ovine FSH-S14 with 9 times the biological potency of NIH-FSH-Sl) dissolved in phosphate-buffered saline (PBS). Unless stated, animals received two injections 24 h and 12 h before necroscopy. Control animals received a saline vehicle only. At the end of hormonal treatment rats were sacrificed and testes removed and weighed. Materials [3H]cAMP and [ “H]cGMP (spec. act. 35-40 Ci/mmole) were obtained from New England Nuclear Corp. (Boston, MA). CAMP, cGMP, PMSF and Crotalus atrox snake venom were purchased from Sigma Chemical Co. All other reagents were of analytical grade from BDH (London, Great Britain) or Hoechst (Frankfurt, F.R.G.). Ovine FSH (oFSH-S14) was supplied by the NIAMDD Pituitary Hormone Distribution Program. Homogenate preparation Each pair of decapsulated testes was incubated in 0.1% collagenase (Worthington) solution in PBS at room temperature, with continuous shaking (50 cycles/min) for lo-15 min until the seminiferous tubule mass was dispersed. The resulting seminiferous tubules were separated from the remaining cell suspension by sedimentation at unit gravity. Supernatants of the first sedimentation were transferred to separate tubes, the cells were washed by sedimentation at 200 x g in a clinical
FSH regulation
81
ofphosphodiesterase
centrifuge and finally were resuspended in the homogenization buffer. This preparation will be referred to as the interstitium. Similarly, the seminiferous tubules were washed 3 times with PBS. After the last wash the two cell preparations were resuspended in 20 mM Tris-HCl, pH 8.0, 10 mM fi-mercaptoethanol, 2 mM phenylmethylsulphonylfluoride, homogenized with an all-glass Dounce tissue grinder and used directly in the phosphodiesterase assay. Phosphodiesterase
assay
Phosphodiesterase activity was measured according to the method of Thompson and Appleman (1971) with minor modifications (Conti et ai., 1981).The cyclic nucleotide concentration in the assay was 1 PM for CAMP and 1 PM for cGMP. Usually two or three homogenate concentrations were used in each assay. Recovery of adenosine, measured by adding a known amount of [ “C]adenosine (10000 dpm/tube) prior to the incubation with snake venom, varied between 40 and 50% in all assays performed. No correction for this recovery was introduced in the calculation of enzyme specific activity. Comparable specific activities were obtained when the products of the reaction were separated in the presence of methanol (Thompson et al., 1979). Cyclic nucleotide hydrolysis was linear up to 30 min and proportional to the crude enzyme preparation added to the assay. Protein content of homogenates was measured according to a modified Lowry procedure (Bensadoun and Weinstein, 1976) using bovine serum albumin as standard. DEAE-cellulose
chromatography
of phosphodiesterase
Cell homogenates were centrifuged at 20000 x g for 30 min at 4°C and at 100000 X g for 1 h at 4°C in a Beckman ultracentrifuge (SW40 rotor). The resulting supernatants from control and treated groups, containing most of the phosphodiesterase activity, were applied to DEAE-cellulose columns (Whatman DE52) preequilibrated with 70 mM acetate buffer, pH 6.5, containing 0.2 mM EGTA. After extensive washing with starting buffer, a 70-600 mM acetate gradient, pH 6.5, was applied to the columns. The gradient was formed in a single gradient former and split in two at the outlet of the chamber, in order to ensure uniformity of ionic strength applied to the two columns. A total of 140 fractions were collected, and every other fraction was assayed for phosphodiesterase activity using 1 PM CAMP and 1 PM cGMP as substrates, in the presence of 1 mM CaCl, and 100 ng purified brain calmodulin per tube (Jamieson and Vanaman, 1979).
82
M. Conti et al.
RESULTS Experiments were performed on lo-day-old Wistar rats, since at this age Sertoli cells are fully responsive to gonadotropin and represent the majority of the cells in seminiferous tubules. The stimulatory effect of FSH on the mitotic activity of testicular cells (Means, 1975) was taken into consideration by expressing the activity both on a per testis and a protein basis. The enzymatic activity of cGMP phosphodiesterase was also measured as an internal control. 24 h after the injection of 50 pg oFSH, the testicular weight of animals receiving hormone was consistently higher (19.84 + 4.66 mg/testis, mean _+ SD, N = 24) than saline-treated controls (14.44 * 2.55 mg/testis, N = 22). The increase in the testicular weight was accompanied by an increase in the protein recovered after hormonal treatment (seminiferous tubules: control 812 + 32 pg protein/testis, FSH-treated 1541 + 185 pg protein/testis; interstitium: control 54 + 10 pg protein/testis, FSHtreated 86 _t 11 pg protein/testis, mean& SE, N = 7). When phosphodiesterase was measured in the different compartments of the testis with 1 PM CAMP as substrate, a substantial increase in the activity of the seminiferous tubule followed hormone treatment (Table 1). This increase was in the order of 5-fold when the data were expressed per testis, and 2.5-fold after correction of the activity per mg of protein. On the other hand, phosphodiesterase activity measured with 1 PM cGMP was affected only on a per testis basis, and was not significantly different from the control when corrected per mg of protein. In addition, the stimulation of phosphodiesterase activity by FSH appeared to be restricted to the seminiferous tubules of the immature testis, since either no stimulation was observed in the interstitial compartment, or else a 20% decrease in CAMP phosphodiesterase activity was detected (Table 1). Stimulation of phosphodiesterase activity was evident 3 h after a single injection of FSH (Fig. l), and the activity continued to increase up to 12 h. A second injection given 12 h after the first dose further stimulated CAMP hydrolysis in the seminiferous tubules. Cyclic GMP phosphodiesterase activity was unchanged throughout the duration of the experiment, and the CAMP hydrolysis in the interstitium was decreased significantly only with multiple treatments (Fig. 1). The increase in phosphodiesterase activity was also dependent on the dose of FSH administered to the animals (Fig. 2). Stimulation of the activity was detected with doses ranging from 10 to 100 pg oFSH given in two separate injections, and near-maximal stimulation was obtained with 100 pg FSH, suggesting that the doses required to stimulate phosphodiesterase are comparable to those employed to stimulate androgen-
I
tubules
(control,
Seminiferous
Mean+SEM
on testicular
different
20.7 f 3.2 6.5f 1.0 from control;
24.2 i 3.0 30.6 rt 0.8
* P < 0.025 vs. control.
22.6k3.0 (NS) 12.1 rt I.0 *
Control
hCG
53.25 + 3.73
52.96 + 3.26 26.105 1.31
5 1.20 t 4.04 (NS)
40.79 i 2.08 ** 63.12k4.33 *
FSH
protein
26.6 + 1.0 (NS) 41.0+ 3.2 *
hCG
protein
* P < 0.001; ** P < 0.01.
Control
from control;
pmoles/min/mg
activity
different
70.51 rt 8.11 **
pmoles/min/testis
Phosphodiesterase
phosphodiesterase
N = 9; FSH, N = 7). NS, not significantly
36.09~4.12
I PM cGMP
3.53 If: 0.70 (NS) 93.73 rt 15.48 *
Control
Control
FSH
pmoles/min/mg
activity
pmoles/min/testis
Phosphodiesterase
2.89 f 0.42 20.08 f 2.13
activity
1 PM CAMP 1 FM CAMP
Assay condition
phosphodiesterase
Mean + SEM, N = 3. NS, not significantly
tubules
compartment
Seminiferous lnterstitium
Testicular
Effect of hCG treatment
Table 2
tubules
compartment
lnterstitium Seminiferous
Testicular
Effect of FSH on testicular
Table
p E i: 2
%-
$ 8 9 -8 :
M. Cami et
84
al.
80-
ii iii /.
0
6
12 18 Ttme 1hr.s)
24
Fig. 1. Time-course of FSH stimulation of testicular phosphodiesterase. Immature Wistar rats were injected i.p. with a single dose of 50 pg ovine FSH. Control and sham-treated rats received PBS only. At the indicated time intervals, rats were sacrificed and testes removed, weighed and decapsulated. Each pair of decapsulated testes was processed separately and seminiferous tubules were separated from the interstitium as described in Materials and Methods. Phosphodiesterase activity was measured with 1 PM CAMP (O0) and 1 PM cGMP (o- - - - - -0) in the homogenates of the seminiferous tubules (top panel) and with 1 PM CAMP in the interstitium homogenate (bottom panel). A group of rats received a second injection of 50 pg FSH 12 h after the first treatment (0). Each point represents the mean i SEM of 2-3 different animals assayed in triplicate at two protein concentrations.
binding protein accumulation in the epididymis (Hansson et al., 1975) and to those required to stimulate protein kinase inhibitor in the immature testis (Tash et al., 1979). Again, cGMP hydrolysis was not affected at any of the doses employed and inhibition of CAMP interstitial phosphodiesterase was evident. Furthermore, the stimulation by FSH was specific to the gonadotropin, since 200 IU hCG (Pregnyl, Organon) injected i.p. had no effect on the seminiferous tubule activity (Table 2). On the other hand, chorionic gonadotropin had a slight stimulatory effect on the phosphodiesterase activity of the interstitium (Table 2). The assay of CAMP and cGMP hydrolysis at low substrate concentrations represents only an indirect estimate of the different phos-
85
FSH regulation of phosphodiesterase
P “, 60. E ,
0
0.1
1.0 pg o FSH
10
100
- 514
Fig. 2. Dose dependence of FSH stimulation of testicular phosphodiesterase. Immature rats Enzyme were injected with increasing FSH doses twice, 24 and 12 h before necroscopy. digestion and homogenate preparation was as in Materials and Methods. Phosphodiesterase and 1nM cGMP(O------0) as activity was measured using 1 PM CAMP (0 -0) substrates in the seminiferous tubules (top panel) and with 1 pM CAMP in the interstitium (bottom panel). Each point represents the meanf SEM of 2 or 3 animals assayed in triplicate at two protein concentrations.
phodiesterase forms present in the crude preparation, since these enzymes, especially the cGMP hydrolysing form, use both nucleotides as substrate (Pichard and Cheung, 1976). In order to confirm that a specific phosphodiesterase is stimulated by FSH, we analysed the activity of soluble extracts of seminiferous tubules by ion-exchange chromatography. The first peak eluted in our chromatographic system hydrolyses cGMP more efficiently than CAMP (Fig. 3B). This form was not affected by the hormonal treatment. Conversely, the second form eluted was markedly stimulated after FSH injection (Fig. 3A). This form had a low K, for CAMP (approx. 2.0 PM) and its activity was not regulated by either Ca2+ or calmodulin (data not shown). The elution pattern, the affinity for the substrates and the Ca2+ insensitivity of this form closely resemble the characteristics of the Sertoli cell phosphodiesterase that is regulated in vitro by FSH and dibutyryl CAMP (Conti et al., 1982; Geremia et al., 1982).
M. Conti et al.
A
1 pMcAMP
fraction number
Fig. 3. DEAE-cellulose chromatography of phosphodiesterase activity in the seminiferous tubules of control and treated animals. Immature rats were divided into two groups. One group received 50 /.~g FSH twice, as previously described, while controls received saline only. Cell homogenization and chromatography were performed as in Materials and Methods. Every other fraction was assayed for phosphodiesterase activity using 1 PM CAMP (panel A) and 1 pM cGMP (panel B). Activity measured in each fraction was corrected for the cytosol protein applied to the column. l - - - - - -0, control cytosol; O0, FSH-treated cytosol.
DISCUSSION that phosphodiesterase activity is modThese studies demonstrate ulated by FSH in vivo and suggest that an additional regulatory mechanism that controls intracellular CAMP is present in the gonadotropic target cells. Thus, FSH not only stimulates adenylate cyclase to produce CAMP (Means et al., 1980) but also stimulates the activity of a phosphodiesterase form that exhibits high affinity for CAMP. This, in turn, is probably responsible for an accelerated CAMP turnover in the cells of the
FSH regulation of phosphodiesterase
87
seminiferous tubules and consequently for a decreased stimulation by subsequent gonadotropic treatment. It has been demonstrated that in vivo intratesticular injection of FSH produces refractoriness of the gonad (O’Shaughnessy, 1980) and that this is related to receptor down-regulation and refractoriness of adenylate cyclase. It is likely therefore that phosphodiesterase stimulation and other regulatory mechanisms such as receptor down-regulation and an adenylate cyclase refractory state are all operating in the same cell and contribute to a ‘fine tuning’ of the responsiveness of the target. It is not excluded that other regulatory feedback mechanisms are present in the FSH target cells beyond the generation of CAMP. It has been demonstrated, in fact, that FSH enhances the synthesis of a protein kinase inhibitor and that the intracellular concentration of this heat-stable molecule is sufficient to inhibit a large proportion of the protein kinase activity (Tash et al., 1979). Although no direct proof has been provided here, Sertoli cells are probably responsible for the increased phosphodiesterase activity, since these cells are the major site of FSH action in the seminiferous tubules. This is further supported by the recent observations that FSH in vitro stimulates phosphodiesterase activity of the Sertoli cell (Conti et al., 1981; Verhoeven et al., 1981). In addition, the enzyme stimulated in vivo is very similar, if not identical, to the form selectively stimulated in cultured cells (Conti et al., 1982). The data presented also show that a specific enzyme is stimulated after the in vivo gonadotropic treatment. This isoform is apparently the same form that naturally increases in the seminiferous tubule during puberty (Means et al., 1978; Geremia et al., 1982). It is therefore possible that phosphodiesterase regulation contributes to the refractoriness that ensues during testicular maturation. In agreement with the above hypothesis is the observation that an increase in plasma FSH precedes the onset of the refractory state during puberty (Keteslegers et al., 1978). This endogenous FSH might trigger a phenomenon similar to what we have described and therefore increase the activity of a high-affinity CAMP phosphodiesterase. This in turn might lead to refractoriness of the mature Sertoli cell. Our data are not in contrast with the observation (Fakunding et al., 1976) that FSH injection produces a decrease, rather than an increase, in phosphodiesterase activity: the two events occur at different times. The inhibition is fast and maximal in about 20 min while the stimulation of phosphodiesterase is a delayed effect of gonadotropin that develops to its maximum in about 12 h. Thus it is possible that the two events are triggered by FSH in the same cell but that one is subsequent to the other. Such a possibility is reinforced by our in vitro observations that stimula-
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M. Conti et al.
tion rapidly follows a preliminary inhibition of the activity (Conti et al., 1982). We have also reported a slight but consistent decrease in interstitial cell phosphodiesterase activity following FSH injection. This is consistent with the repeated observations of trophic effects of FSH on immature interstitial tissue (Ode11 and Swerdloff, 1976) and could account, at least in part, for the observed increased sensitivity of the testis to LH-hCG after FSH priming of the gonad (Hsueh et al., 197X; Chen et al., 1977). It is possible that the stimulation of phosphodiesterase is a phenomenon common to all gonadotropic target cells of the testis and ovary, and represents a physiological event in the function of the gonad. This assumption is supported by our finding that hCG, which acts in a different compartment of the testis, also has a stimulatory effect on the phosphodiesterase activity of the interstitium. In addition, it has been reported that the activity of a high-affinity phosphodiesterase form increases in the ovarian follicle during the estrous cycle of the rat (Schmidtke et al., 1980). Our findings that FSH is capable of regulating a high-affinity CAMP phosphodiesterase strongly suggest that the increase in CAMP hydrolysis reported in the rat ovarian follicle is dependent on. the preceding FSH surge in proestrus.
ACKNOWLEDGEMENTS We are indebted to the NIAMDD Hormone Distribution Program and to Dr. J.F. Parlow for the generous gift of ovine FSH. This work was supported by CNR project ‘Biology of Reproduction’ grants 80.01166.85 and 80.01176.85 and by Ford Foundation Grant 790-0659.
REFERENCES Bensadoun, A. and Weinstein, D. (1976) Anal. Biochem. 70, 241-256. Catt, K.J., Harwood, J.P., Aguilera, G. and Dufau, M.L. (1979) Nature 109-I 16. Chen, Y.D.I., Shaw, M.J. and Payne, A.H. (1977) Mol. Cell. Endocrinok 8, Con& M., Harwood, J.P., Hsueh, A.J.W., Dufau. M.L. and Catt, K.J. (1976) 25 1, 7729-773 1. Conti, M., Geremia, R., Adamo, S. and Stefanini, M. (1981) Biochem. Commun. 98, 1044- 1050. Conti, M., Toscano, M.V., Petrelli, L., Geremia, R. and Stefanini, M. (1982) 110, 1189-1196. Fakunding, J.L.. Tindall, D.J., Dedman, J.R., Mena. C.R. and Means, A.R. crinology 98, 392-402.
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Geremia, R., Rossi, P., Pezzotti, R. and Conti, M. (1982) Mol. Cell. Endocrinol. 28, 37-53. Hansson. V., Weddington, S.C., Petrusz, P., Ritzen. E.M., Nayfeh, S.N. and French, F.S. (1975) Endocrinology 97, 469-473. Harwood, J.P., Conti, M., Conn, P.M., Dufau, M.L. and Catt, K.J. (1978) Mol. Cell. Endocrinol. 11, 121-135. Hsueh, A.J.W., Dufau, M.L. and Catt. K.J. (1976) Biochem. Biophys. Res. Commun. 72, 1145-1152. Hsueh, A.J.W., Dufau, M.L. and Catt, K.J. (1978) Endocrinology 103, 1096-l 102. Hunzicker-Dunn, M. and Birnbaumer, L. (1976a) Endocrinology 99, 185-197. Hunzicker-Dunn, M. and Birnbaumer, L. (1976b) Endocrinology 99, 211-222. Jamieson Jr., G.A. and Vanaman, T.C. (1979) Biochem. Biophys. Res. Commun. 90. 1048-1056. Ketelslegers, J.M., Hetzel, W.D., Sherins, R.J. and Catt, K.J. (1978) Endocrinology 103. 212-222. Means, A.R. (1975) In: Handbook of Physiology, Section 7. Vol. 5 (American Physiological Society, Washington, DC) pp. 203-217. Means, A.R.. Dedman, J.R., Tindall, D.J. and Welsh, M.J. (1978) Biol. Reprod. Suppl. 2, 403-421. Means, A.R., Dedman, J.R., Tash, J.S., Tindall. D.J., van Sickle, M. and Welsh, M.J. (1980) Annu. Rev. Physiol. 42, 59-70. Odell, W.D. and Swerdloff, R.S. (1976) Recent Progr. Hormone Res. 32, 245-280. O’Shaughnessy, P.J. (1980) Biol. Reprod. 23, 810-814. Pichard, A.L. and Cheung, W.Y. (1976) J. Biol. Chem. 251, 5726-5737. Schmidtke, J., Meyer, H. and Epplen. J.T. (1980) Acta Endocrinol. (Kbh.) 95, 404-411. Tash, J.S., Dedman, J.R. and Means, A.R. (1979) J. Biol. Chem. 254, 1241-1247. Thompson, W.J. and Appleman, M.M. (1971) Biochemistry 10, 311-320. Thompson, W.J., Terasaki, W.L.. Epstein, P.M. and Strada, S.J. (1979) Adv. Cyclic Nucleotide Res. 10, 69-92. Tung, P.S., Dorrington, J.H. and Fritz, I.B. (1975) Proc. Natl. Acad. Sci. (U.S.A.) 72, 1838-1842. Verhoeven, G., Dierickx, P. and de Moor, P. (1979) Mol. Cell. Endocrinol. 13. 241-253. Verhoeven, G., Cailleau, J. and de Moor, P. (1980) Mol. Cell. Endocrinol. 20, 113-126. Verhoeven, G., Cailleau, J. and de Moor, P. (1981) Mol. Cell. Endocrinol. 24, 41-51.