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Biology of Reproduction 62, 1835-1843 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Regular Article

Somatostatin Inhibits Follicle-Stimulating Hormone-Induced Adenylyl Cyclase Activity and Proliferation in Immature Porcine Sertoli Cell via sst2 Receptor1

Slavica Krantic2,a, and Mohamed Benahmeda

a Institut National de la Santé et de la Recherche Médicale (INSERM) Unité 407, Faculté de Médecine Lyon Sud, F;ch69921 Oullins, France

ABSTRACT

The potential involvement of somatostatin (SRIF) in testicular function was studied by using as a model primary cultures of purified immature porcine Sertoli cells. In the present report we show that Sertoli cells express mRNA for sst2 SRIF receptor and display SRIF-sensitive adenylyl cyclase. Sensitivity of adenylyl cyclase to SRIF and its analogues is compatible with the pharmacological profile of this receptor type. Relevant cAMP production is similarly inhibited by SRIF in both basal and stimulated (by gonadotropin FSH or by forskolin) conditions. Moreover, the observed SRIF actions on Sertoli cells require functional coupling of specific membrane receptors to adenylyl cyclase via Gi proteins because pertussis toxin prevents SRIF-dependent inhibition of adenylyl cyclase in either basal or FSH-stimulated conditions. Given the potent antiproliferative actions of SRIF in other cell types, we further assessed the possible SRIF-dependent modulation of [3H]thymidine incorporation by Sertoli cells. Our data point to SRIF-mediated inhibition of both basal and FSH-stimulated [3H]thymidine uptake. This inhibition of Sertoli cell proliferation is, at least in basal conditions, also blocked by pertussis toxin pretreatment. Altogether, these data suggest that SRIF may play a role as an (local) inhibitor of FSH actions in testicular development.

INTRODUCTION

The endocrine control of spermatogenesis is provided by FSH and testosterone, two hormones targeting Sertoli cells. In addition, the central role of these cells in spermatogenesis is evidenced by germ cell dependency on Sertoli cells for structural and microenvironmental support as well as for essential regulatory (e.g., binding and transport proteins, growth factors and proteases) and nutritional (e.g., lactate) factor supply. The production of a great number of these key factors is regulated by FSH [1]. Additional argument for the critical importance of Sertoli cells in the regulation of spermatogenesis is given by the fact that the number of Sertoli cells in adult testis is critical in guaranteeing the production of mature germ cells in sufficient amount to insure fertility. Follicle-stimulating hormone is the major regulator of adult Sertoli cell number because it stimulates their proliferation in the course of fetal and perinatal development [2]. All these biological actions of FSH on immature and adult Sertoli cells are mediated by heptahelical transmembrane receptors [3] coupled to adenylyl cyclase activation via heterotrimeric guanine nucleotide binding Gs proteins [4].

In addition, such direct FSH actions on Sertoli cells can be either potentiated or inhibited indirectly by certain factors produced locally under the influence of FSH. For example, direct stimulatory actions of FSH on immature Sertoli cell proliferation are further potentiated by means of FSH-stimulated basic fibroblast growth factor (bFGF) expression in Sertoli cells [5]. However, many aspects of the local modulatin of FSH actions remain poorly understood especially in the context of putative inhibition of Sertoli cell proliferation during the prepubertal development.

Somatostatin (SRIF) is a regulatory peptide acting as hormone, neurohormone, and neurotransmitter. At the cellular level, one of the most remarkable SRIF actions is the inhibition of proliferation in both normal and tumoral cells [6]. Somatostatin is ubiquitously distributed in a wide variety of mammalian cells and tissues including those of endocrine, immune, and central nervous systems [7, 8]. In particular, SRIF has been identified in pig, rodent, and human testis [9–11]. The biological actions of SRIF involve five cloned (sst1–sst5) receptors belonging to the same superfamily of heptahelical transmembrane receptors as FSH receptors. All known SRIF receptors display multiple transduction pathway couplings, but they are all negatively coupled to (i.e., inhibit) adenylyl cyclase via heterotrimeric Gi proteins and trigger subsequent decrease in cAMP production [8, 12].

Concerning SRIF receptor expression in the male gonad, the only existing report has recently documented that, in rat testis, mRNAs corresponding to three (sst1–sst3) out of five SRIF receptors are expressed in Sertoli and germ cells [13]. However, testicular actions of SRIF remain poorly understood, and it is currently not clear whether it might act as a local regulatory factor. Given that FSH and SRIF impinge on the same (cAMP) transduction pathway in the opposite manners, we hypothesized that SRIF plays a role in control of FSH-dependent Sertoli cell functions and thereby in regulation of spermatogenesis. To test that hypothesis, we focused on possible SRIF actions on immature Sertoli cells and studied 1) the expression of SRIF receptors, 2) their coupling to adenylyl cyclase, and 3) the effects of SRIF on basal and FSH-induced Sertoli cell proliferation. To do that we used as a model purified primary cultures of immature porcine cells thus taking advantage of 1) the relatively long (15 days) time period over which these cells are responsive to FSH in terms of cAMP production in culture conditions; and 2) the great number of Sertoli cells per testis that can be obtained in this model.

MATERIALS AND METHODS

Chemicals

Dulbecco's minimum essential medium (DMEM)-Ham's F-12 medium, TRIzol reagent, and Moloney murine leukemia virus (MMLV) reverse transcriptase were obtained from Life Technologies (Eragny, France) and DNase-I from Pharmacia Biotech (Uppsala, Sweden). Taq polymerase was from Promega (Charbonière, France). [3H]Adenine (26 Ci/mmol), [{alpha}32P]ATP (30 Ci/mmol), and [3H]cAMP (40 Ci/mmol) were from Amersham (Les Ulis, France); [3H]CH3-thymidine (88.7 Ci/mmol) was from Dupont-NEN (Les Ulis, France). Solvable and Ultima Gold were purchased from Packard (Rungis, France). Complete, Mini-EDTA-free protease inhibitor cocktail, collagenase/dispase, creatine kinase, creatine phosphate, ATP, and GTP were from Boehringer (Mannheim, Germany). Somatostatin analogues SRIF14 and SRIF28 were obtained from Peninsula Laboratories (San Carlos, CA) while SMS 201995 was a kind gift from Novartis (Basel, Switzerland). Porcine FSH (USDA-pFSH-B-1) was generously provided by USDA Animal Hormone Program, Beltsville Agricultural Research Center (Beltsville, MD). All other chemicals were purchased from Sigma (L'Isle d'Abeau, France).

Cell Isolation and Culture

Sertoli cells were isolated from testes of 21-day-old pigs: at this age pigs are routinely castrated for the sake of body mass gain and meat taste improvement. The isolation of Sertoli cells was performed by collagenase dispersion as previously described [14]. After decapsulation, testes were chopped and extensively washed in DMEM-Ham's F-12 medium. They were then treated with collagenase–dispase (0.4 mg/ml, 90 min, 32°C). Collagenase was removed by centrifugation (200 x g for 10 min, 4°C). Resulting pellets were resuspended in the medium and allowed to sediment for 5 min. Sedimented tubules were recovered, washed, and decanted three times. The tubules were then incubated (room temperature, 20 min) in Ca2+/Mg2+-free PBS solution containing 1 M glycine, 2 mM EDTA, and DNase-I (0.1 mg/ml) in order to remove the remaining Leydig cells. After repeated washings by gravity sedimentation (three cycles), the seminiferous tubules were incubated in DMEM–Ham's F-12 medium supplemented with collagenase-dispase (0.4 mg/ml), DNase-I (0.1 mg/ml), and fetal calf serum (10%) for 30 min at 32°C. The supernatants containing peritubular myoid cells were discarded and the tubule pellets were treated with collagenase-dispase as described (0.4 mg/ml, 30 min, 32°C) until small clumps were obtained. Sedimented clumps contained highly enriched Sertoli cells, devoid of Leydig and germ cells, and containing 2–5% of myoid cells as evaluated by fibronectin, desmin, and alkaline phosphatase immunostaining [15].

Sertoli cells were cultured in DMEM–Ham's F-12 medium containing 1.2 mg/ml sodium bicarbonate, 15 mM Hepes, 20 µg/ml gentamicin, 5 µg/ml transferrin, 10 µg/ml {alpha}-tocopherol, 100 UI penicillin, 0.05 mg/ml streptomycin, and 2 µg/ml insulin at 32°C in a humidified atmosphere of 5% CO2 and 95% air. Cultures were run for at least 5 days before starting the experiments.

Reverse Transcription-Polymerase Chain Reaction Analysis of sst2 Receptor Expression

Total RNAs were extracted with TRIzol reagent according to the manufacturer's instructions from Sertoli cells either immediately after testis dispersion or 5 days after the beginning of culture.

An aliquot of each sample (10 µg/5 µl) was treated with 0.1 µl (0.75 U) of DNase-I for 10 min at 37°C in order to exclude any genomic DNA contamination. Complementary DNA was synthesized (1 h, 42°C) from 10 µg of cellular RNA by using MMLV reverse transcriptase (200 U) as previously described [16, 17]. To ascertain further that cDNA was not contaminated by genomic DNA, reverse transcription (RT) for each sample was also performed in the absence of MMLV reverse transcriptase.

One-tenth (2 µl) of the first strand of cDNA synthesis reaction was added to polymerase chain reaction (PCR) buffer (100 mM Tris) containing 0.2 mM dNTP, 1.25 U Taq DNA polymerase in a total volume of 50 µl. The concentration of MgCl2 in the reaction mixture was 2.5, 4, 1.5, and 1.5 mM for sst1–sst3 SRIF receptors and GAPDH, respectively. Primers for SRIF receptors were added in a 0.4 µM final concentration and have been previously described: for sst1 [18], for sst2 [19], and for sst3 [20]. Amplification of cDNA encoding human GAPDH was achieved by using the sense primer 5'-CACCACCAACTGCTTAGCAC-3' and the antisense primer 5'-CCACCCTGTTGCTGTAGCCAA-3'. These primers were defined on two different exons in order to allow additional monitoring of sample contamination by genomic DNA. Expected amplification products were the following lengths (bp): 233, 414, 222 (for sst1–sst3 receptors, respectively), and 520 for GAPDH. After initial denaturation (150 sec, 94°C), the samples were subjected to 40 cycles (for sst1–sst3 receptors) or 30 cycles (for GAPDH) of amplification including denaturation (60 sec, 94°C), hybridization (60 sec, 65°C for sst1 and sst3; 60°C for sst2 and GAPDH), and elongation (75 sec, 72°C). Final elongation was achieved at 72°C for 5 min. Products of amplification were visualized on 2% agarose gel with ethidium bromide staining. The absence of reactive contamination by genomic DNA or cDNA was checked by PCR amplifications systematically carried out in parallel by replacing cDNA by water.

Adenylyl Cyclase Assay

On semipurified membrane preparations Sertoli cells were scraped off from petri dishes (100 x 20 mm, density of 20 x 106 cells/dish) in 30 mM Tris-HCl buffer, pH 7.2, supplemented with 10 mM EGTA and 3% sucrose, washed, and then homogenized in the same buffer. Homogenates were centrifuged for 3 min at 1800 x g (4°C); pellets were discarded and supernatants were recentrifuged for 15 min at 28 000 x g (4°C). Resulting semipurified membrane pellets were resuspended in a small volume of Tris-HCl buffer (30 mM Tris-HCl, pH 7.2) containing 10 mM EGTA and 10% sucrose. The aliquots of these preparations were frozen and kept at -80° C until used.

Male Wistar rats (200–250 g) were sacrificed by decapitation in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC). Striatum was dissected on ice and processed as described for Sertoli cells to prepare the semipurified plasma membrane samples.

Adenylyl cyclase activity was measured by conversion of [{alpha}32P]ATP to [32P]cAMP. The final incubation medium (50 µl) contained 50 mM Tris-maleate buffer (pH 7.2), 1.5 mM MgSO4, 1 mM cAMP, 5 mM creatine phosphate, 0.1 mg/ml creatine kinase, 0.15 mM ATP, 0.01 mM GTP, 10 mM theophyline, 1 µCi [{alpha}32P]ATP, and 0.001 µCi of [3H]cAMP. The reaction was initiated by addition of 10 µl homogenate containing 10–20 µg of proteins. Incubation was performed under conditions of linear [32P]cAMP production (32°C for 30 min). [32P]cAMP was separated from [{alpha}32P]ATP by two-step elution as previously reported [21]. Recovery of [3H]cAMP on individual columns varied between 70 and 80%. Adenylyl cyclase activity was expressed in pmol of [32P]cAMP (corrected for elution efficiency of each individual column determined on the basis of [3H]cAMP recovery) formed per mg protein over 30 min. Protein concentration was determined according to the method of Lowry [22].

On whole cell preparations Sertoli cells cultured in 12-well plates at a density of 1 x 106 cells/well were incubated (24 h, 32°C) with [3H]adenine (2 µCi/106 cells) in order to label the intracellular pool of [3H]ATP. After extensive wash in cell culture medium, the cells were preincubated with an inhibitor of phosphodiesterase (isobutylmethylxantine, 1 mM) for 15 min at 32°C. The conversion of [3H]ATP to [3H]cAMP was subsequently measured over the 15-min incubation period (32°C) in the presence or in the absence of the drugs to be tested. An inhibitor of endopeptidases, bacitracin (0.5 g/L), was included in the medium to limit proteolytic degradation of SRIF. At the concentration used, bacitracin has no obvious toxic effect on Sertoli cells as evaluated by trypan blue exclusion test (data not shown).

The enzymatic reaction was stopped by discarding the supernatant, wash, and subsequent addition of 1 ml of ice-cold solution containing trichloroacetic acid (5%), an excess of nonradioactive cAMP (1 mM), and ATP (1 mM). The cells were scraped off in the latter solution and frozen at -20°C until chromatography.

[3H]cAMP enzymatically converted by adenylyl cyclase from [3H]ATP on the one hand, and the exceeding substrate [3H]ATP on the other hand were separated by two-step elution on Dowex and alumina columns as previously described [23]. The radioactivity of eluates was quantified by liquid scintillation spectrophotometry in Tri-carb 1900 TR (Packard) counter with 45% efficiency. The results were expressed as a ratio of [3H]cAMP recovered in alumina eluate over [3H]ATP recovered in Dowex eluate.

Cell Proliferation Assay

Five days after the culture beginning, the plated (24-well plates with 0.5 x 106 cells/well) Sertoli cells were washed twice in the medium devoid of insulin and cultures were treated by SRIF14 (10 nM), FSH (0.25 µg/ml), or both. All drugs were diluted in insulin-free medium because this hormone has potent mitogenic actions on immature porcine Sertoli cells [24]. Some cultures were left untreated to serve as a control for the basal rate of cell proliferation. All experiments were performed in the presence of the protease inhibitor cocktail. The cultures were carried on for 24 h and [3H]CH3-thymidine pulse (1 µCi/well) was performed during the last 5 h of culture. The criterion used to define the test period of 24 h was the time necessary for completion of one cell division cycle (16 h) by Sertoli cells [25]. At the end of the test period, plates were extensively washed and 0.5 ml of Solvable was added per well. The radioactivity was quantified by liquid scintillation beta counting (Tri-carb 1900 TR Packard counter with 45% efficiency) in the presence of 10 volumes of Ultima Gold liquid scintillation cocktail.

Pertussis Toxin Treatment

In some experiments, Sertoli cells were treated with pertussis toxin. Although toxin concentration (30 ng/ml) and cell time exposure (24 h) were rigorously identical in all experiments, the applied protocol of toxin treatment slightly differed between adenylyl cyclase and cell proliferation assays.

For adenylyl cyclase assays, the toxin was washed out prior to the beginning of the 30-min test period. The production of [3H]cAMP was therefore measured in the absence of the toxin and in the presence of either 1) SRIF14 alone, 2) FSH alone, or 3) a combination of both.

In proliferation assays, where the test period was of 24 h (see above), the toxin was added at the same time as either 1) SRIF14 alone, 2) FSH alone, or 3) a combination of both.

The effects of pertussis toxin alone on adenylyl cyclase activity and cell proliferation were determined in parallel for the two respective assays.

Data Analysis

If not otherwise specified, data are presented as the mean ± SEM of triplicate determinations performed in two to four independent experiments carried out with different cell and membrane preparations or cell cultures. Statistical significance of the differences observed between experimental groups was determined by one-way ANOVA using the InStat (Graph PAD Software) computer program. Posthoc comparisons between treatment group means were made with the Bonferroni test for multiple comparisons. Differences are accepted as significant if P < 0.05.

RESULTS

sst2 Receptor mRNA Expression in Porcine Sertoli Cells

In order to find out if immature porcine Sertoli cells are putative SRIF targets, we first studied whether they express mRNAs corresponding to the sole cloned porcine SRIF receptor gene, sst2 [26].

When cDNA corresponding to total mRNA isolated from porcine Sertoli cells at day 5 of culture was used as a template, a PCR product of expected size (414 bp) was observed with primers specific for sst2 (lane 5, Fig. 1). The identity of the amplified fragments was confirmed by sequencing (data not shown). As SRIF receptor genes are intronless [8], we ascertained that the size of the fragment obtained with genomic DNA and the one obtained with cDNA was identical (lanes 5 and 7, Fig. 1). The negative controls, i.e., reactions in which 1) mRNA was used as a template, and 2) template was replaced by water (corresponding respectively to lanes 6 and 8 of Fig. 1), allow to exclude any template or reactive contamination. The absence of contamination by genomic DNA was further confirmed by parallel PCR assays in which the same cDNA and genomic DNA samples were used as templates for amplification with primers specific for GAPDH defined in two different GAPDH gene exons. A single PCR product of the expected size (520 bp) was obtained when cDNA was employed as a template (lane 1, Fig. 1). A heavier molecular weight fragment that could be amplified from genomic DNA template (lane 3, Fig. 1) was absent in the reactions carried out with cDNA (lane 1, Fig. 1). The corresponding products of amplifications were sequenced to confirm the identity (data not shown).



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  		FIG. 1. Expression of sst2 SRIF receptor mRNA in porcine Sertoli cells. Lanes 1 and 5 correspond to the products of amplifications in which cDNA (+RT reactions) was used as a template, whereas lanes 2 and 6 correspond to the reactions performed with mRNA (-RT reactions). Lanes 3 and 7 show the fragments amplified from genomic DNA used as a positive control for the size of the amplified products. Negative controls consisted of replacing templates by water (lanes 4 and 8). M = DNA ladder indicating the size (in bp) of the amplified fragments. Lanes: 1–4 = GAPDH; lanes: 5–8 = sst2

Given that the recent in situ hybridization study reported the presence of sst1 and sst3 SRIF transcripts in adult rat Sertoli cells [13], we assessed their expression in immature porcine Sertoli cells. The results we could obtain under the same experimental conditions used to detect sst2 mRNAs indicated that sst1 and sst3 transcripts are not expressed. However, in positive controls in which genomic DNA was used as a template, the selected primers allowed the amplification of the fragments of the expected size (233 and 222 bp for sst1 and sst3, respectively; data not shown).

Results obtained on sst1–sst3 receptor mRNA expression by using cDNA corresponding to Sertoli cells that were used immediately after enzymatic dispersion (i.e., without culture) were identical (data not shown) to those presented above and obtained after 5 days of culture.

Sensitivity of Sertoli Cell Adenylyl Cyclase to SRIF14 and Its Analogues Is Compatible with sst2 Receptor Involvement

To determine whether the expression of sst2 receptor mRNA corresponds to the presence of functional SRIF receptors, we studied SRIF actions on adenylyl cyclase activity in cultured Sertoli cells. In the preliminary experiments, we optimized the experimental conditions because semipurified membranes from porcine Sertoli cells were never used before in adenylyl cyclase studies. In particular, we tested the effect of GTP concentration on adenylyl cyclase activity. Increasing GTP concentrations up to 1 mM was inefficient in triggering an additional increase of the basal adenylyl cyclase activity as compared with those measured in the presence of 0.1 mM GTP (data not shown). 0.1 mM GTP concentration (i.e., concentration routinely used in adenylyl cyclase assays) was therefore used in subsequent experiments. The effect of Mg2+ concentration on adenylyl cyclase activity in membranes from porcine Sertoli cells was also analyzed. Indeed, divalent cations are able to elicit the enzyme activity in the absence of Gs or other stimulators [27, 28]. Increasing Mg2+ concentration resulted in a sharp augmentation of the enzyme activity in the 0.1–1.5 mM concentration range but no additional stimulation was evidenced with increasing Mg2+ concentration from 1.5 up to 10 mM (data not shown). MgSO4 was therefore used at 1.5 mM concentration in order to keep the cation abundance at physiologically low levels.

The chosen assay conditions (especially in regard to GTP concentration and assay temperature) were also tested by parallel determination of basal adenylyl cyclase activity in membrane preparations obtained from porcine Sertoli cells on the one hand and rat brain striatum taken as the positive control, on the other hand. At 0.1 mM concentration of GTP, 1.5 mM of MgSO4 and 30 min incubation at 32°C, basal activity was almost 10-fold higher in rat brain than in Sertoli cell preparations (Table 1). In order to ensure further that such relatively low basal adenylyl cyclase activity in Sertoli cell membranes was not due to the inadequate experimental conditions or to the impairment of the enzyme integrity during the procedure of membrane preparation, we studied the enzyme capacity to be stimulated by forskolin. This alkaloid targets directly the adenylyl cyclase catalytic domain and is commonly used to estimate the maximal capacity of the enzyme to be activated [27]. In the same experimental conditions (0.1 mM GTP, 30 min 32°C), forskolin (10 µM) increased the basal adenylyl cyclase activity in Sertoli cell and striatum preparations up to 4- and 11-fold, respectively (Table 1).


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  		TABLE 1. Adenylyl cyclase activation in Sertoli cells and brain.a

We then assessed adenylyl cyclase sensitivity to SRIF14. Indeed, SRIF peptide family consists of two bioactive molecules: a tetradecapeptide (SRIF14) and its N-terminus extended form (SRIF28) [8] that have been both identified in mammalian testis [9–11]. Globally considered, the adenylyl cyclase activity was significantly lower in the presence of SRIF14 than in its absence (F = 5.14, P = 0.0032 for semipurified membrane preparations and F = 8.17, P = 0.0003 for whole cells). Somatostatin-14 inhibited basal adenylyl cyclase activity in a dose-dependent manner in either semipurified membrane preparations (Fig. 2A) or whole cells (Fig. 2C). In both experimental paradigms, the maximal inhibition was achieved in nanomolar concentration range and equaled 40–50% of the enzyme activity measured in the absence of the peptide. To confirm involvement of sst2 receptor in the observed SRIF14-dependent adenylyl cyclase inhibition, we measured the enzyme activity in the presence of the two SRIF14 analogues (SRIF28 and SMS 201995) known to be equally potent as SRIF14 on sst2 receptor [8]. At 10 nM concentration (this concentration was chosen because it corresponds to a 10-fold reported IC50 concentration for sst2-mediated SRIF14 inhibition of adenylyl cyclase [8, 29]; IC50 is pharmacologically defined as the analogue concentration necessary to inhibit 50% of maximal response) all three agonists inhibited cAMP production with identical efficiency in both membrane preparations (Fig. 2B) and whole cells (Fig. 2D). Given the equipotency of SRIF14, SRIF28, and SMS 201995 on sst2 receptor-mediated actions of the neuropeptide such as has been previously reported by others [8] and confirmed here, in further experiments we studied in detail only the SRIF14 actions.



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  		FIG. 2. Sertoli cells display SRIF-sensitive adenylyl cyclase. Somatostatin-14 and its agonists (SRIF28 and SMS 201995) inhibit basal adenylyl cyclase activity in an sst2-specific manner in both semipurified membranes (A, B) and whole cells (C, D). The depicted points represent the mean ± SEM of triplicate determination obtained in one representative experiment for either semipurified membrane preparations or whole cells. These experiments were repeated two to four times. a: P < 0.05 for adenylyl cyclase activity in the presence versus in the absence of SRIF. b: P < 0.01 for adenylyl cyclase activity in the presence versus in the absence of SRIF

Somatostatin-14 Inhibits Pharmacologically and Hormonally Stimulated Adenylyl Cyclase Activities

Somatostain-14 significantly inhibited forskolin-stimulated adenylyl cyclase activity in a similar manner in both semipurified membrane preparations (F = 5.81, P = 0.0018) and whole cells (F = 10.91, P = 0.0001). However, the maximal inhibition in membrane preparations equaled 20% of the enzyme activity measured in the absence of the neuropeptide (Fig. 3A), whereas in whole cells (Fig. 3C), inhibition reached -40% for the SRIF14 concentrations greater than 0.1 nM (compare Fig. 3, A and C). Such different degrees of adenylyl cyclase inhibition were consistently observed, although the forskolin concentration used was 10-fold greater in membrane preparation than in whole cell assays (10 µM versus 1 µM, respectively). These forskolin concentrations were chosen on the basis of preliminary experiments in which the EC50 concentration for its actions was determined (EC50 is pharmacologically defined as the analogue concentration sufficient to induce 50% of maximal response). At the EC50 concentration, the enzyme activity doubled in both membrane preparation and whole cell assays as compared to the basal values (data not shown).



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  		FIG. 3. Somatostain-14 inhibits forskolin- (A, C) and FSH- (B, D) stimulated adenylyl cyclase in semipurified membrane preparations (A, B) and whole Sertoli cells (C, D). The drug concentrations used to activate adenylyl cyclase were: 1 µM forskolin and 0.25 µg/ml FSH in whole cells and 10 µM forskolin and 1 µg/ml FSH in semipurified membrane preparations. Data are the mean ± SEM of the results obtained in (n) independent experiments all performed in triplicates. They are expressed as a % of adenylyl cyclase activity measured in the absence of SRIF in each particular experiment as this reference value differed from one culture to another. The range of reference values was in forskolin-stimulated conditions: 42–65 pmol cAMP mg-1 min-30 (n = 4) for A; 27–48 x 103 cAMP ATP-1 (n = 5) for C. For FSH-stimulated conditions, the reference values were: 38–52 pmol cAMP mg-1 min-30 (n = 3) for B; 33–67 x 103 cAMP ATP-1 (n = 5) for D. a: P < 0.05 for adenylyl cyclase activity in the presence versus in the absence of SRIF. b: P < 0.01 for adenylyl cyclase activity in the presence versus in the absence of SRIF

The FSH-stimulated adenylyl cyclase in Sertoli cell membrane preparations and whole cells was also significantly sensitive to inhibition by SRIF14 (F = 4.50, P = 0.0061 and F = 14.14, P = 0.0001 for semipurified membrane preparations and whole cells, respectively). The corresponding maximal inhibitions of FSH-stimulated enzyme activity were of the same order of magnitude (-20% and -40%) as those measured in the presence of forskolin in membrane preparations (Fig. 3B versus 3A) and whole cells (Fig. 3D versus 3C), respectively. In addition, as for stimulation with forskolin in the absence of SRIF14, more FSH was needed to elicit the half-maximal adenylyl cyclase activation in membrane preparations than in whole cells (1 µg/ml versus 0.25 µg/ml for membrane and cell assays, respectively). At these FSH concentrations, a similar (twofold) increase in adenylyl cyclase activity was observed in both experimental paradigms (data not shown).

Pertussis Toxin Prevents SRIF14-Mediated Inhibitionof Basal and FSH-Stimulated Adenylyl Cyclase

The involvement of plasma membrane SRIF-specific receptors in the described inhibitory effects of SRIF14 on adenylyl cyclase was further addressed functionally through assessment of their coupling to Gi proteins in Sertoli cells pretreated with pertussis toxin. The pretreatment conditions were determined in the preliminary experiments by measurement of cAMP production by Sertoli cells incubated, respectively, with either 10 ng/ml, 30 ng/ml, or 50 ng/ml of the toxin over a 16-h, 24-h, or 48-h time period. Cell viability was checked in parallel by trypan blue exclusion test. The presence of pertussis toxin altered neither basal cAMP production nor cell viability under any conditions tested (data not shown). In addition, the basal rate of cAMP accumulation was identical independent of whether the toxin was washed-out or not after the 24-h pretreatment period (data not shown). Experimental conditions (30 ng/ml, 24 h with toxin washout at the end of the preincubation period) were finally defined as such because they correspond to the generally used protocol for adenylyl cyclase assay with pertussis toxin pretreatment.

As expected for receptors coupled to adenylyl cyclase via Gi proteins, the toxin pretreatment completely abolished SRIF14-dependent inhibition of both basal (Fig. 4A) and FSH-stimulated (Fig. 4B) adenylyl cyclase activity. Toxin pretreatment did not modify basal adenylyl cyclase activity significantly (P = 0.2319) (Fig. 4A). However, the pretreatment of Sertoli cells with pertussis toxin was consistently associated with a slight decrease of FSH-stimulated cAMP production (Fig. 4B: compare empty and filled FSH points), although this decrease was not significant (P = 0.2046).



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  		FIG. 4. Pertussis toxin pretreatment (24 h, 30 ng/ml) abolishes the SRIF14 inhibition of both basal (A) and FSH (0.25 µg/ml)-stimulated (B) adenylyl cyclase in primary cultures of Sertoli cells. Data are the mean ± SEM obtained in one experiment representative out of three independent experiments, each performed in triplicate. Empty symbols correspond to the data obtained in pertussis toxin pretreated cultures while filled symbols correspond to the results obtained in control cultures (in the absence of toxin). a: P < 0.05 for adenylyl cyclase activity in the presence versus in the absence of SRIF. b: P < 0.01 for adenylyl cyclase activity in the presence versus in the absence of SRIF c: P < 0.001 for adenylyl cyclase activity in the presence versus in the absence of SRIF

Functional Relevance of SRIF Effects on FSH-Mediated Sertoli Cell Response

To correlate the observed SRIF actions on adenylyl cyclase activity with a biological response of Sertoli cells to FSH, we measured FSH-stimulated [3H]CH3-thymidine incorporation in Sertoli cell cultures incubated for 24 h in the absence or in the presence of SRIF14.

Follicle-stimulating hormone (0.25 µg/ml, 24 h), used in these experiments also as a positive control, increased Sertoli cell proliferation by about 40% over the basal level (Fig. 5A).



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  		FIG. 5. Somatostatin-14 inhibits both basal and FSH-stimulated Sertoli cell proliferation (A). Pertussis toxin blocks SRIF14 inhibition of basal Sertoli cell proliferation (B). Presented data are representative of two independent experiments in which identical results were obtained. Experimental values correspond to mean ± SEM of multiple (n = 5) determinations of [3H]CH3-thymidine incorporation. The scale was kept identical in A and B in order to allow the comparison of basal and FSH-stimulated proliferation in the absence (A) and in the presence (B) of the toxin. a: P < 0.05 compared to the reference indicated by lines above the respective columns. b: P < 0.01 compared to the reference indicated by lines above the respective columns. ns: nonsignificant

Considered globally, the presence of SRIF14 (10 nM, 24 h) significantly (F = 28.07, P = 0.0001) decreased basal [3H]CH3-thymidine incorporation by Sertoli cells (Fig. 5A). Similarly, the addition of SRIF14 significantly (P = 0.0018) decreased FSH-induced Sertoli cell proliferation (Fig. 5A).

We then assessed the possible alteration of SRIF14 actions on basal and FSH-induced Sertoli cell proliferation in the presence of pertussis toxin. The inhibitory effects of SRIF14 on Sertoli cell proliferation are completely prevented (i.e., became insignificant with F = 3.12 and P = 0.0883) when pertussis toxin (30 ng/ml, 24 h) was added to Sertoli cell cultures simultaneously with SRIF14. In the presence of the toxin, the basal rate of [3H]CH3-thymidine incorporation was not significantly (P = 0.0634) different than that observed in the absence of toxin (compare the first columns of Fig. 5, A and B).

By contrast, pertussis toxin significantly (P = 0.0061) lowered Sertoli cell proliferation in the presence of FSH (compare third columns of Fig. 5, A and B). Moreover, the toxin completely abolished the FSH-induced increase of [3H]CH3-thymidine incorporation over the basal rate (Fig. 5B). These findings rendered impossible the interpretation of eventual pertussis toxin effects on the SRIF14-mediated inhibition of FSH actions on Sertoli cell proliferation (Fig. 5B).

DISCUSSION

In the present work we report on SRIF-mediated negative regulation of FSH actions in immature Sertoli cells. Such a novel biological role for this regulatory peptide is reflected by its capacity to counteract the FSH-dependent increase of both cAMP production and [3H]CH3-thymidine incorporation. Data reported here thus suggest that SRIF might be a putative candidate for the negative regulator of immature Sertoli cell proliferation. Indeed, all studied hormones (FSH [2]; insulin [24]) and growth factors (epidermal growth factor, bFGF [24]) mediate an increase in immature Sertoli cell proliferation in either rat [2, 24] or pig [24].

The methods employed in our study were validated by several lines of control experiments because the immature porcine Sertoli cells have never been used before to functionally assess SRIF actions. First, the RT-PCR approach was validated by the fact that the length of fragments obtained for the amplification by using the primers specific for the sst2 receptor from either genomic or cDNA were identical in size as expected for intronless genes such as genes encoding SRIF receptors [8]. Moreover, the amplification of sst2-related fragments from the total RNA extracts obtained from Sertoli cells either immediately after enzymatic dispersion (i.e., without culture) or after 5 days of culture excluded any artifactual, culture-related alteration of sst2 receptor expression and allowed us to use the 5-day cultures in further experiments on SRIF actions in Sertoli cells. Second, the direct estimation of cAMP production in membrane preparations obtained from immature porcine Sertoli cells after 5 days of culture was validated by parallel measurement of the forskolin-dependent stimulation of adenylyl cyclase activity in membrane preparations obtained from rat brain striatum (commonly used as a reference in the literature). The extent of forskolin-mediated adenylyl cyclase activation reported here for immature porcine Sertoli cell membranes is in agreement with literature data for either rat brain or testis membrane preparations [30]. In addition, the present FSH-mediated increase of adenylyl cyclase activity in membrane preparations obtained from cultured porcine Sertoli cells is of the same order of magnitude as that measured in the former study by using plasma membranes of freshly isolated rat Sertoli cells [31]. Otherwise, FSH-stimulated cAMP production comparable to that seen here in whole porcine Sertoli cells after 5 days of culture has been previously reported in whole rat Sertoli cells [32, 33] therefore validating the chosen experimental conditions. Third, an increase in the proliferation rate of the same order of magnitude as that determined here for FSH-stimulated immature porcine Sertoli cells has already been reported in fetal and immature rat Sertoli cells treated with FSH [2, 34].

From the functional point of view, our findings suggest that immature porcine Sertoli cells can be directly targeted by SRIF. The presence of sst2 SRIF receptor transcrips in total RNA extracts of these cells corresponds to the expression of SRIF-specific receptor proteins with sst2-type selectivity. Indeed, SRIF14, SRIF28, and SMS 201995 inhibit adenylyl cyclase activity of Sertoli cells in the nanomolar concentration range and with equal efficiency as expected according to the pharmacological profile of the sst2 receptor [35].

The SRIF-sensitive adenylyl cyclase activity of Sertoli cells documented here is inhibited by SRIF14 in both basal and stimulated conditions. Stimulated enzyme activity is similarly inhibited after activation by either forskolin or FSH. Hence, SRIF14-mediated inhibition is independent of the mechanism of adenylyl cyclase activation (i.e., directly by forskolin or indirectly by FSH via Gs protein). However, the inhibitory actions of SRIF14 on activated adenylyl cyclase (normalized as a percentage of forskolin- or FSH-stimulated enzyme activity in the absence of the peptide) are globally more pronounced in whole cells than in semipurified membrane preparations. These differences might be a consequence of suboptimal experimental conditions (e.g., inadequate cofactor concentrations, incubation temperature) used in semipurified membrane assays. Given that altering incubation temperature, GTP, and Mg2+ concentrations cannot improve measurable adenylyl cyclase activity, this possibility seems less likely. Alternatively, the observed differences between SRIF-mediated inhibition of adenylyl cyclase in membrane preparations on the one hand and whole cells on the other hand might be related to the damage of SRIF receptors during purification of Sertoli cell plasma membranes. It is, however, reasonable to assume that this was not the case because SRIF14 inhibits basal adenylyl cyclase activity with equal efficiency (about -40%) in both membrane preparations and whole cells. Nevertheless, it is worth mentioning that an obviously disturbed integrity of signaling systems in membrane preparations might prevent the cross-talk between transduction pathways operating in whole cells and potentiate the agonist-induced adenylyl cyclase inhibition. For example, in whole cells (but not in membrane preparations), protein kinase C can be activated in response to SRIF [7] and FSH [4], and it might subsequently inhibit adenylyl cyclase by phosphorylation [28] in a fashion additive to direct SRIF-mediated adenylyl cyclase inhibition.

The blockage of SRIF signaling at the level of plasma membrane receptor coupling to adenylyl cyclase by pertussis toxin pretreatment further demonstrates that SRIF receptors expressed at the plasma membranes of Sertoli cells act as functional receptors. Their action involves {alpha}i/{alpha}o subunits of Gi/Go proteins (these proteins are inactivated by pertussis toxin-mediated ribosylation) [36]. The pertussis toxin-dependent suppression of SRIF14 inhibition of both basal and FSH-stimulated adenylyl cyclase activity is complete. Consequently, SRIF receptor coupling to adenylyl cyclase is direct as already suggested by our plasma membrane adenylyl cyclase assays (see above). Somatostatin-14 is therefore an extracellular signal able to interact with {alpha}i subunits of Sertoli cell Gi proteins. Relevantly, the presence of {alpha}i subunits of heterotrimeric Gi-proteins has already been reported in Sertoli cells: their expression is FSH regulated, but until our study, the endogenous ligands able to activate them have remained unknown [33]. However, pertussis toxin weakly but consistently inhibited FSH-dependent activation of adenylyl cyclase, although this inhibition was not significant. Noteworthy, the pertussis toxin sensitivity of FSH-induced cAMP accumulation has been reported previously in both immature hamster Sertoli cells [37] and in the Chinese hamster ovary cell line transfected with the human FSH receptor [38]. In addition, our findings extend to peptide messengers the previously reported results regarding actions of the neurotransmitter messenger acetylcholine on basal and FSH-stimulated adenylyl cyclase activity in immature hamster Sertoli cells [39].

Concerning the potential biological responses of Sertoli cells to SRIF, our data point to the correlation existing between the observed SRIF-dependent inhibition of adenylyl cyclase and its inhibitory actions on the proliferation of immature Sertoli cells. Indeed, in the nanomolar concentration range SRIF14 inhibits both FSH-stimulated and the basal rate of [3H]CH3-thymidine incorporation. Moreover, in the manner analogous to that observed for SRIF inhibition of basal adenylyl cyclase, pertussis toxin completely inhibited SRIF actions on Sertoli cell proliferation. Therefore, pertussis toxin-induced inactivation of Gi proteins and subsequent block of signal transduction at the level of receptor coupling to adenylyl cyclase are sufficient to prevent SRIF14-mediated inhibition of Sertoli cell proliferation.

By contrast, the implication of Gi proteins in SRIF14 actions on FSH-stimulated Sertoli cell proliferation cannot be assessed by using pertussis toxin inasmuch as in the absence of SRIF14, the toxin significantly inhibited FSH-dependent [3H]CH3-thymidine uptake. These observations imply that pertussis toxin inhibits the FSH-triggered pathway mediating the proliferative response. This pathway has been identified as being cAMP-dependent because the FSH-mediated increase in Sertoli cell proliferation can be mimicked by a stable cAMP analogue [2]. Given the insensitivity of Gs protein to pertusis toxin [36], altogether these findings suggest that the toxin affects other G-proteins that may be indirectly involved in adenylyl cyclase activation by FSH. The Go protein would be a good candidate for such a role because 1) its activation after the ligand–receptor interaction leads to the increase in intracellular Ca2+ concentration; and 2) Ca2+ has been involved in the positive regulation of adenylyl cyclase activity [28]. Concordant with such a presumption is the reported promiscuity of the FSH receptor interaction with G proteins (in addition to Gs, this receptor also binds Go [38]). Furthermore, inhibition of the FSH-triggered increase in cAMP production by pertussis toxin pretreatment (24 h with subsequent washout) has been demonstrated recently [38]. In agreement, we also found that pertussis toxin pretreatment always decreased the stimulation of adenylyl cyclase by FSH although the observed decrease was not significant. The unequivocal demonstration of Gi protein involvement in the SRIF-dependent inhibition of FSH-induced Sertoli cell proliferation must therefore await the development of Gi-selective antagonists (pertussis toxin inactivates both Gi and Go proteins and therefore does not allow these two proteins to be distinguished from each other [36]).

Finally, an important question raised by our study is the possible intratesticular source of SRIF. Our preliminary data indicate that spermatogonia of 21-day-old porcine testis are SRIF immunoreactivity positive (A. Gougeon, personal communication). In this light, it is worth mentioning that several years ago it has been proposed that germ cells might control FSH actions in Sertoli cells via production of unknown local regulatory factors [40]. Future studies are needed in order to answer whether or not SRIF might be one of these germ cell–born regulatory factors.

In conclusion, the reported anti-FSH actions of SRIF14 on cAMP production in Sertoli cells might turn out to be of particular interest not only in the course of development (through the regulation of the Sertoli cell number) but also in the adult testis through modulating FSH actions on the production of specific regulatory factors necessary for correct spermatogenesis. Further research is now required in order to assess the in vivo SRIF actions on fertility by using whole integrated systems. In this context, the recent development of stable nonpeptide SRIF receptor agonists [41] should help in conducting the relevant in vivo experiments.

ACKNOWLEDGMENTS

We are indebted to Drs. Stolz and Parlow for the generous gifts of SMS 201995 and FSH, respectively. We thank Miss Juliette Longin for her participation in the preliminary experiments.

FOOTNOTES

First decision: 7 January 2000.

1 Supported by INSERM (Unité 407), ARC grant 7244 (S.K.) and Ligue contre le Cancer, Comité de l'Ardèche (S.K.). Back

2 Correspondence: Slavica Krantic, Laboratoire de Communication cellulaire en Biologie de la reproduction, INSERM 407, Faculté de Médecine Lyon Sud, B.P. 12, F-69921 Oullins, Cedex, France. FAX: 33 4 78 86 31 16; krantic{at}lyon151.inserm.fr Back

Accepted: January 28, 2000.

Received: December 3, 1999.

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