FSH-Responsive Adenylyl Cyclase in Rat Testes: Desensitization by Homologous Hormone

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1 Archives of Andrology Journal of Reproductive Systems ISSN: (Print) (Online) Journal homepage: FSH-Responsive Adenylyl Cyclase in Rat Testes: Desensitization by Homologous Hormone T. Jahnsen, J. O. Gordeladze, P. A. Torjesen & V. Hansson To cite this article: T. Jahnsen, J. O. Gordeladze, P. A. Torjesen & V. Hansson (1980) FSH- Responsive Adenylyl Cyclase in Rat Testes: Desensitization by Homologous Hormone, Archives of Andrology, 5:2, , DOI: / To link to this article: Published online: 09 Jul Submit your article to this journal Article views: 5 View related articles Citing articles: 13 View citing articles Full Terms & Conditions of access and use can be found at

2 FSH-Responsive Adenylyl Cyclase in Rat Testes: Desensitization by Homologous Hormone T. JAHNSEN,' J. 0. GORDELADZE,' P. A. TORIESEN,~ and V. HANSSON' Injection of high doses of FSH causes a partial desensitization of the FSH-responsive adenylyl cyclase (AC) in adult rat testes. The loss of responsiveness to FSH is dependent on time. Two hours after injection of one dose of human FSH (256 pg hfsh-pt,) in vivo, no effect was seen on FSH-stimulated AC activity (K, and maximal activation). After 24 hr there was an approximately 40% decrease in FSH activated AC. Injection of two doses of FSH (256 pg hfsh-pt,) 24 and 48 hr before being killed, gave a loss in FSH-responsive AC activity of approximately 60%. The decreased FSH-stimulated AC activity after FSH injections in vivo was associated with a comparable decrease in specific FSH binding. Whether this is a reflection of receptor occupancy or down regulation of the number of FSH receptors is not known. Desensitization of the FSH-responsive AC caused by FSH was not associated with a change in its Kt,,. Key Words: Sertoli cell; FSH; Desensitization; Hormone receptor; Adenylyl cyclase. INTRODUCTION Several adenylyl cyclase (AC) systems show decreased responsiveness to hormone stimulation after exposure to high concentrations of homologous hormone. This phenomenon is called desensitization, and has been described for a number of hormones acting through activation of the AC [2, 5, 8, 11, 191. The process of hormone-induced desensitization of AC appears initially to be due to uncoupling of the AC from the hormone receptor, a process which precedes receptor down-regulation [ 191. Hormoneinduced desensitization of AC activity is receptor specific, and does not require protein synthesis [ The primary target cells for FSH within the testis are the Sertoli cell. Defective Sertoli cell function is often associated with elevated serum levels of FSH. To what extent such elevated FSH levels may cause desensitization of the Sertoli cell to FSH is not known as desensitization of the FSH responsive AC in seminiferous tubules has not been described. In a previous paper [lo], we have shown that FSH causes a three- to fivefold stimulation of AC activity in membrane particles of seminiferous tubules from adult rats. The purpose of the present study was to examine whether or not FSH-responsive AC in seminiferous tubules can be desensitized by injection of high doses of homologous hormone. Received December 20, 1979; revised March 5, 1980 From the 'Institute of Pathology, Rikshospitalet and,hormone Laboratory, Aker Hospital, Oslo, Norway. Address reprint requests to: Tore Jahnsen, Institute of Pathology, Rikshospitdet, Oslo, Norway. 0 Elsevier North Holland, Inc., ARCHIVES OF ANDROLOGY, 5, (1980) 52 Vanderbilt Ave., New York, New York i6/80/060 i69-09%02.25

3 170 T. Jahnsen et al. MATERIALS AND METHODS Animals Adult male Sprague-Dawley rats were injected S.C. with 256 pg of human FSH-PT2. Saline containing 0.1% BSA (bovine serum albumin) was injected into the control animals. The animals were anesthetized with ether and then killed by cervical dislocation. The testes were removed and immediately placed on ice. Chemicals and Enzymes Creatine phosphate and creatine kinase were obtained from Calbiochem and [a- '32P] ATP was obtained from The Radiochemical Center, Amersham, England. Myokinase, Tns, ATP, CAMP, EDTA and sodium dodecylsulfate (SDS) were from Sigma Chemical Company, and 5'-guanylylimidodiphosphate [GMP-P(NH)P] was obtained from Boehringer Mannheim. Hormones Two human FSH preparations were employed: hfsh-ft, (15,000 IU 2 IRP HMGimg by RIA, 2% hlh contamination) and hfsh-ftz (15,000 IU/mg by RIA, 0.4% hlh contamination) [21]. Ovine FSH (NIH-S12) was obtained from the National Institute of Healths Pituitary Hormone Program and treated with antiserum against ovine LH as described by Purvis et al. [16]. hcg " [ A-A o FSH-S12* anti LH *---*-o h F S H -? T1 hfsh-pt2 25 t C.- E 20' cn E 15 % 1 L 1 I FSH conc. (pg/ml 1 FIGURE I. Dose response curves indicating relative potencies between different FSH preparations. Seminiferous tubules were dissected from three 90-day-old normal rats as described in Materials and Methods. The seminiferous tubules were then pooled and tubular membrane particles were prepared. FSH-responsive AC activities (picomoles CAMP formed per milligram protein per minute) were determined in the presence of 1.9 mm ATP, 1.4 mm EDTA, 0.04 mm GMP-P(NH)P, 3.5 mm Mg2+ (0.2 mm in excess of ATP + EDTA), and an ATP-regenerating system at 35 C. hfsh-pt, and hfsh-pt, are human FSH preparations. Ovine FSH (NIH-Sl2) is an ovine FSH preparation absorbed with LH antiserum. Each value is the mean of duplicate determinations.

4 FSH Response in Rat Testes by Homologous Hormone 171 Basal Control 2h 24h Hormones - basal Control 2h 24 h FIGURE 2. The effect of hcg (1 pg/ml) and hfsh-pt, (2 pg/ml) on the activity of membranebound AC prepared from whole testes of 70-day-old control and FSH-treated rats. Animals in the two groups subjected to treatment received a single injection of 256 pg hfsh-pt2 2 and 24 hr, respectively, prior to being killed. AC activity was measured at 35 C in the presence of 1.0 mm ATP, 1.4 mm EDTA, 0.04 mm GMP-P(NH)P, 2.8 mm Mg2+ (0.4 mm in excess of ATP + EDTA), and an ATP-regenerating system. The top panel shows absolute AC activities (picomoles CAMP formed per milligram protein per minute). The middle panel shows relative hormonal responses (hormonestimulated AC activity divided by basal AC activity). The bottom panel shows hormone-stimulated AC activity minus basal AC activity. Each value is the mean 2 SD (standard deviation) of triplicate determinations. For further information see Materials and Methods. (Physex) was from LEO, Denmark. The hormone preparations were dissolved in distilled water containing 0.1% BSA to the desired final concentration. Preparation of Membrane Particles for Adenylyl Cyclase (AC) Assay and Gonadotropin Binding The testes were weighed, and after removal of the tunica albuginea homogenized in 20 volumes (with respect to the original tissue weight) of TE-buffer (10 mm Tris-HC1, 1 mm EDTA, ph 7.4) using an Ultra-Turrax homogenizer ( Janke-Kunkel AG, Germany) at rheostate setting 7 for 2 x 15 sec. The homogenates were then filtered through a nylon mesh and subjected to two centrifugations. The first centrifugation was carried out at 27,000 g for 30 min at 0"-4"C. The pellet was resuspended in 20 volumes (with respect to the initial tissue weight) of TE-buffer, rehomogenized using the Ultra-Turrax homogenizer for sec, and recentrifuged for 30 min at

5 172 T. Jahnsen et ai. the same speed and temperature. The final pellet was resuspended in 10 volumes of TE-buffer containing 0.1% BSA using the Ultra-Turrax for sec. Aliquots of 20 pl of the final membrane particle suspensions were used for the assay of AC activity and 50 yl for the determination of specific FSH and LH binding assay. AC Assay AC activity was determined in 20-4 aliquots of membrane particles (30-70 pg protein per assay tube) in afinal volume of 50 p1 containing 1.O or 1.9 mm ATP (including approximately 3 X lo6 cpm of [CU - "PIATP), mm MgCI2, 1 mm EDTA, 1 mm camp (approximately 10,000 cpm [3H]cAMP). 20 mm creatine phosphate, 0.2 mg/ml creatine kinase, 0.02 mg/ml myokinase, and 25 mm Tris-HCI buffer, ph 7.3 [lo]. Incubations were camed out at 35 C for 10 min. The reactions were stopped with 0.1 ml of a "stopping solution" containing 10 mm CAMP, 40 mm ATP, and 1% sodium dodecylsulfate (SDS) followed by mixing and immediate cooling to 0 C. The [32P]cAMP formed and ["]CAMP added to monitor recovery were then isolated according to the method of Salomon et al. [18] using Dowex [ 121 and alumina chromatography [ 17, 221 with the modification described by Birnbaumer et al. [ 11 and Bockaert et al. [2]. Overall recovery wasapproximately 70% and reaction blanks ranged from 3-6 cpm/106 cpm of labeled ATP added. The imidazol eluates from the alumina columns were collected in scintillation vials containing 5 ml of Hydro-Luma (Lumac Systems AG, Switzerland). Radioactivity was measured in a Nuclear Chicago, Mark I liquid scintillation counter. Assay precision was assessed by standard deviation (SD) of replicates and was never more than 5%. The concentrations of ATP and GMP-P(NH)P were calculated from UV-light Control *-a Km=36 ng/ml 5[ &--A 2h I 4. - o...c] 2G h I, I 0 'QOOl FSH (~g/rnl) FIGURE 3. Dose response curves showing the responsiveness of AC from 70-day-old-rat seminiferous tubular membrane particles from control and FSH-treated animals (see Fig. 2) to varying concentrations of hfsh- PT,. All responses are relative to basal activity (picomoles camp formed per milligram protein per minute): Basal control , basal 2 hr 7.1 & 0.1, and basal 24 hr 11.0 * 0.6. Each point on the curves represents the mean of duplicates. Cyclic AMP formation was measured at 35 C in the presence of 1.0 mm ATP, 1.4 mm EDTA, 0.04 mm GMP-P(NH)P, 2.8 mm Mg'* (0.4 mm in excess of ATP + EDTA), and an ATP-regenerating system (see Materials and Methods). 1 2

6 FSH Response in Rat Testes by Homologous Hormone 173 TABLE 1 Specific FSH and LH Binding to Testis Membrane Particles Varying Time Periods After One (Exp. 1) or Two (Exp. 2) Injections of 256 pg hfsh-pt,. Values Given are Mean 2 SD [1251]-~FSH BINDING ['251]-HLH BINDING [CPM/MG PROTEIN] PERCENT [CPM/MG PROTEIN] PERCENT x 103 OF CONTROL x 103 OF CONTROL Exp. 1 Control 1.8? hr hr Exp. 2 Control f hr t spectrophotometry at 260 nm. Protein was assayed according to the method of Lowry et al. [13], using BSA as the standard. Dissection of Seminiferous Tubules Seminiferous tubules were dissected in Eagles medium using the method of Christensen and Mason [3] at room temperature. The isolated seminiferous tubules were collected and weighed. Then the membrane particle suspensions were prepared as described above. Measurements of FSH and LH Binding Specific FSH and LH binding was estimated by equilibration of testicular membrane particles with [12sI]-hLH (specific activity 45 gcilpg) or [1251]-hFSH (specific activity 48 gci/pg) in a total volume of 200 pl. After incubation with approximately 25,000 cpm of labeled hormone at 22 C (LH) or 30 C (FSH), the tubes were centrifuged at 27,000 g for 30 min at 0"-4"C and washed twice with 2 ml of phosphate buffered saline containing 0.1% neomycin and 0.1% BSA. Nonspecific binding was assessed by incubating parallel tubes with an excess of unlabeled FSH (NIH-FSH-S12,2 pg) or hcg (10 IU). Details of the binding assays, iodination techniques, and the hormones used for iodination have been described previously [7]. RESULTS As seen in Fig. 1, FSH caused a fivefold stimulation of AC activity in membrane particles from seminiferous tubules. Human FSH-PTz was slightly more potent than the human FSH-PT,. An ovine FSH preparation absorbed with an excess of anti-lh antibody, although 20-fold less potent, caused the same maximal activation of the AC. hcg 10 IU/ml did not stimulate AC activity in seminiferous tubules (not shown). The effect of a single injection of human FSH (256 pg of human FSH-PT2) on FSH and hcg-responsive AC was studied in membrane particles from whole testes (Fig. 2) and also on the FSH-responsive AC in isolated seminiferous tubules (Fig. 3). The animals were killed 2 and 24 hr after the injection of FSH. As seen from Fig. 2, the injection of FSH caused a slight but gradual increase in basal activity, probably due to receptor occupancy. Two hours after the injection there was little change in the activation caused either by hcg or FSH. However, 24 hr after the injection of FSH, despite the fact that the basal activity was elevated, the FSH response was decreased both in relative and

7 174 T. Jahnsen et al f Basal fl hcg FSH 4-0 G4 31 ~ 2 :.:.:.... at z*,.... Control 48 h r : ~.... J i..:... ~ - E Control Hormones -basal... :.:.:. :.>>.:.:.:... FIGURE 4. Responsiteness of AC in membrane particles from whole testis homogenate to hcg (1 pgiml) and ofsh (NIH-Sl2) (25 pg/mlj absorbed with LH antiserum. Pretreated animals (80 days old) were given two doses of 256 pg hfsh-pt? 24 and 48 hr, respectivelj, before removal of organs. All responses are quoted as mean x SD of triplicates. Incubation of membrane particles was performed at 35 C in a medium containing 1.0 m ll 4TP, 1.4 mm EDTA, 0.04 mm GMP-P(NH)P, 2.8 mm Mg (0.4 rnm in exceqs of ATP + EDTA), and an ATP-regenerating system. The top panel shows absolute AC arthities (picomoles CAMP formed per milligram protein per minute). The middle panel \how$ relatite hormonal responses (hormone-stimulated AC activity divided by basal AC actitit)). The bottom panel shows hormone-stimulated AC activity minus basal AC activity. 48 h absolute terms. We also examined FSH-responsive AC in isolated seminiferous tubules from the Same animals. In this case the effect of multiple concentrations of FSH were tested. As seen from Fig. 3, there was no change in the FSH dose response curves 2 hr after injection of FSH. However, 24 hr after injection of FSH, the relative responses decreased from about 4.1 to 2.1. The K,,, for AC activation (the concentration of FSH giving 50% maximal response) was unchanged. FSH binding in membrane particles from total testes of the same animals showed little or no change in specific FSH binding after 2 hr. but a significant decrease 24 hr after the injection of FSH (Exp. 1 in Table I). Figures 4 and 5 show the results of another study in which adult male rats were given two injections of FSH (256 pg of human FSH-PT,) at 24 and 48 hr before removal of the organ\. The animals were killed 48 hr after the first injection and hcg and FSH-

8 FSH Response in Rat Testes by Homologous Hormone 175 responsive AC were determined in membrane particles from whole testes (Fig. 4) and from isolated seminiferous tubules (Fig. 5). In this case ovine FSH (NIH-Sl2) absorbed with an excess of anti-ovine LH antibody (kindly provided by M. Raj, North Carolina) was used to stimulate the FSH-responsive AC. As seen in Fig. 4 there was again a considerable decrease in FSH-responsive AC 48 hr after the first injection, and a significant but smaller increase in basal activity was recorded. In Fig. 5 it is shown that the relative FSH response in seminiferous tubules decreased from 4.5- to 2.0-fold. Once again there was no change in the K, of AC activation (K,,, 1.1 pg/ml, ovine FSH (NIH-S12) absorbed with anti-ovine LH antibody). Table 1 shows the effects of these treatments on specific FSH and LH binding in membrane particles from whole testes. As seen from Table 1 there was a gradual decrease in available FSH and LH binding sites with time. DISCUSSION The present study shows that injections of high doses of FSH may cause a reduced responsiveness of the FSH-stimulated AC in seminiferous tubules and whole testes. Concomitantly there is also a comparable decrease in specific FSH binding. Whether the decreased FSH binding capacity is due to occupancy or degradation of the FSH receptor was not investigated. A similar reduction in FSH binding capacity after hfsh injections has also recently been demonstrated by Gnanaprakasam et al. [6]. Desensitization of the FSH-responsive AC in seminiferous tubules is reminiscent of that in other systems, although in our studies more time is required to achieve a loss of 5- O-O Control I FSH (pg/ml I FIGURE 5. Dose response curves showing the responsiveness of AC from 80-day-old-rat seminiferous tubular membrane particles from controls and FSH-treated animals (see Fig. 4) to varying concentrations of ofsh (NIH-S12) absorbed with LH antiserum. All responses (mean of duplicates) are reiative to basal activity (picornotes CAMP formed per milligram protein per minute): Basal control 5.9 * 0.1, basal 48 hr 7.0? 0.1. The accumulation of CAMP was measured as described in legend to Fig. 4.

9 176 T. Jahnsen et al. response. Whereas the LH/hCG-responsive AC in corpus luteum can be desensitized within minutes [8], the desensitization of the FSH-responsive AC in seminiferous tubules of adult rat testes needs several hours. In other systems showing desensitization of the AC by homologous hormone, such as the isoproterenol-induced AC desensitization in human astrocytoma [ 191 and LH/hCG-induced AC desensitization in the rat ovary [5] or in the rat testes (Jahnsen et al., unpublished), the first step in the process of desensitization is a rapid loss of AC activity with little or no loss of specific membrane receptors. Subsequently this is followed by a decrease in receptor number. It may therefore be concluded from these studies that the initial desensitization of the AC is primarily due to the uncoupling of the membrane receptors from the AC, and that this uncoupling may be a prerequisite for subsequent receptor down-regulation. It is possible that such uncoupling of the AC from the hormone receptors may be mediated via membrane phosphorylation reactions [4, 91. In the present study, we found that a short time after a high dose of FSH there was a considerable increase in basal AC activity, in particular after 2 and 24 hr. Part of this increase is probably due to stimulation of Leydig cell AC (due to LH contamination) because the increased basal activity was less when investigating AC in isolated seminiferous tubules from the same animals. The fact that 24 and 48 hr after injection of FSH there is not only a decrease in the relative response of the AC but also a considerable decrease in absolute activities clearly reveals a desensitization of the FSH-responsive AC in the adult rat testis. Very similar results to these presented here have also been obtained by injection of high doses of FSH in 10-day-old rats (not shown). The physiological importance of the observed desensitization of the FSH-responsive AC in seminiferous tubules is unclear. Firstly, the loss of response is only pahial, and approximately 40% of the FSH-stimulated AC activity still remains 48 hr after the injection of FSH. Furthermore, the functional role of FSH in the postpubertal male is controversial. Many of the rapid responses to FSH, which are cyclic nucleotide mediated, become attenuated or are lost [15]. However, the fact that the adult rat testis has an FSH-responsive AC that can be desensitized by homologous hormones indicates that the regulation of FSH responses may occur at the membrane level. In addition, as shown by others, fluctuations in cyclic nucleotide metabolism (phosphodiesterase activities) and protein kinase activities 114, 15, 201 may be additional ways by which Sertoli cell response can be regulated. Acknowledgments: This study was supported by grants from Norwegian Research Council for Science and the Humanities (NAVF), World Health Organization (WHO), The Population Council, New York, and Landsforeningen mot Kreft (LMK). The authors gratefully acknowledge the technical assistance of Tone Varaas and Drude Andersen. We also wish to thank National Institute of Health s Pituitary Hormone Program for generously providing us with ovine FSH. REFERENCES I. Birnbaumer L, Yang P-C, Hunzicker-Dunn M, corpora lutea of rabbits, rats and pigs: Regulation Bockaert J, Duran JM (1976): Adenylyl cyclase by ATP, and some comparative properties. activities in ovarian tissues. I. Homogenization Endocrinology 99: and conditions of assay in Graafian follicles and 2. Bockaert J, Hunzicker-Dunn M, Birnbaumer L

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