Effects of follicle-stimulating hormone and vitamin A upon purinergic secretion by rat Sertoli cells

Molecular and Cellular Biochemistry 278: 185–194, 2005. c�Springer 2005

Effects of follicle-stimulating hormone andvitamin A upon purinergic secretion by ratSertoli cells

Daniel Pens Gelain, Emerson Andre Casali, Ramatis Birnfeld deOliveira, Luiz Fernando de Souza, Fabiano Barreto, Felipe Dal-Pizzoland Jose Claudio Fonseca MoreiraDepartamento de Bioquımica, Instituto de Ciencias Basicas da Saude, Universidade Federal do Rio Grande do Sul,Porto Alegre, RS, Brazil

Received 14 February 2005; accepted 18 May 2005

Abstract

Follicle-stimulating hormone (FSH) and vitamin A (retinol) are two of the main regulators of the male reproductive system.Recently, it has been described that extracellular purines can affect some important reproductive-related functions in Sertolicells and germinative cells, by activating specific purinergic receptors. In this work, we report that both FSH and retinol areable to induce changes in the levels of extracellular purines of cultured rat Sertoli cells. FSH induced an increase in adenosine,mainly caused by enhanced ecto-ATPase activity, while retinol increased xanthine and hypoxanthine levels, and decreased uricacid concentration by an unknown mechanism. These data indicate that purinergic signaling may be involved in the controland/or regulation of some of the reproductive-related actions of these hormones. (Mol Cell Biochem 278: 185–194, 2005)

Key words: Sertoli, FSH, retinol, extracellular purines, purinoceptors

Introduction

Purine nucleosides and nucleotides can act as important ex-tracellular messengers, besides their more established role incellular metabolism [1, 2]. These molecules mediate their ef-fects via triggering of distinct cell surface receptors, namedadenosine (or P1) and P2 purinergic receptors [3, 4]. WhileP1 receptors are activated by adenosine, P2 receptors are trig-gered mainly by ATP. Some works have shown that inosine isalso able to trigger A3 adenosine receptors [5]. P2 receptorscan be subclassified into P2Y (G-protein coupled receptors)or P2X (ligand-gated ion channels), and adenosine receptorsare subdivided into A1, A2A, A2B, and A3, according to their

Address for offprints: D. P. Gelain, Departamento de Bioquımica, UFRGS, Laboratorio 25. Rua Ramiro Barcelos 2600 anexo, CEP 90035-003, Porto Alegre,RS, Brazil (E-mail: [email protected])

effect on adenylyl cyclase activity, effect on IP3 turnover, andCa2+ mobilization.

Many authors have been showing that extracellularpurines, such as ATP and adenosine, can modulate cellularprocesses in the male reproductive system through triggeringof purinoceptors. Different subtypes of both adenosine andP2 receptors were detected on Sertoli and germinative cells,and their activation can induce important functional changesin these cells. Sertoli cells express A1 purinoceptors, recentlydemonstrated to be essential for acquisition of fertilizing ca-pacity [6]; these receptors couple to a G-inhibitory proteinwhen activated by adenosine, thus leading to inhibition ofadenylyl cyclase [7, 8] and participating in the modulation of

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inhibin secretion [9]. Indeed, these cells also express P2Y re-ceptors, which are coupled to phosphoinositide turnover andintracellular [Ca2+] mobilization [10]. The expression of dif-ferent subtypes of P2X receptors according to the stage of theseminiferous epithelium cycle was also observed [11], whichreinforced the suggestion that purinergic signaling may playa role in controlling the maturation of germ cell subsets ofdifferent developmental ages in seminiferous tubules. Also,ATP receptors activation in Sertoli cells can cause Na+ influx-dependent membrane depolarization, with consequent open-ing of voltage-gated Ca2+ channels [12].

The gonadotropin FSH is considered the pituitary key reg-ulator of gametogenesis. Its action is mainly mediated by aGs-protein-coupled receptor, which increases cAMP concen-tration in the cytosol, leading to activation of PKA and othereffects [13]. This hormone stimulates Sertoli cell prolifera-tion during fetal and peri-natal life of the rat [14] and, in sex-ual mature rats, activates PKC and calcium release [15]. Thereare several evidences indicating a link between FSH signal-ing and purinergic receptors in Sertoli cells. For instance, ithas been demonstrated that A1 receptor activation can inhibitsome responses of Sertoli cells to FSH, such as the eleva-tion in cAMP levels and androgen aromatization [8]. Also,P2Y receptor activation affects FSH-influenced PI turnoverand intracellular [Ca2+] mobilization, besides the hormonal-induced increase in cAMP [10]. The activation of P2 receptorsinduces other biological effects related to responsiveness toFSH, such as the increase of γ -glutamyl-transpeptidase activ-ity, estradiol, and transferrin secretion, and the hormonal in-duced decrease in aromatase activity [16]. Besides, our groupdemonstrated that Sertoli cells are able to secrete both ATPand adenosine [17], and that FSH influences the activity ofthe ecto-nucleotidases of Sertoli cells [18]; these enzymes, inturn, are known to regulate the concentration of extracellularpurinergic metabolites such as ATP and adenosine.

Besides exerting a fundamental role in many processessuch as vision, growth and differentiation of numerous typesof cell, vitamin A (retinol) and its biologically active deriva-tive, retinoic acid, are clearly involved in the functional reg-ulation of the male reproductive system [19, for review].Retinol and its derivatives were also demonstrated to inhibitthe stimulatory effect of FSH on the production of cAMP inneonatal and fetal rat Sertoli cells [20]. This effect was alsoobserved in mature Sertoli cells [21], besides the reduction ofthe expression of PKC and androgen receptors as well [22].However, the best documentated of retinol-regulated Sertolicell functions is secretion: it is reported that retinoids in-crease the secretion of transferrin, androgen-binding protein(ABP), insulin-like growth factor-binding protein 4 (IGFBP-4), inhibin-α and glycoproteins such as sulphated glycopro-tein (Sgp-2) [20, 23–26]. Up to now, however, there are noworks reporting the actions of retinol upon purinergic secre-tion of cells from the male reproductive system.

Our group has long been reporting the actions of retinolupon several oxidative parameters of Sertoli cells metabolism[27–30]. Our results indicate that many actions of retinol aremediated by an increase on reactive oxygen species (ROS)production in the cellular environment. Recently, we haveshown that extracellular purines of Sertoli cells can be mod-ulated by H2O2 [31], which lead us to investigate the effectof retinol upon purinergic secretion of Sertoli cells, verify-ing if the retinol-induced production of ROS is involved inthis process. Also, we have previously published that FSHregulates the activity of ecto-nucleotidases [17], which sug-gested that this gonadotropin could also affect the secretionof extracellular purinergic compounds. Thus, the goal of thisreport is to demonstrate the effect of two major regulatorsof Sertoli cell functions – retinol and FSH – on the levelsof extracellular purines of these cells. The results presentedhere can evoke new perspectives in the study of purinergicsignaling in Sertoli cells.

Materials and methods

Materials and animals

All drugs, kits and enzymes were purchased from SigmaChemical Co. (St. Louis, MO, USA) unless otherwise stated.Pregnant Wistar rats were housed individually in plexiglasscages. Litters were restricted to eight pups each. The animalswere maintained on a 12-h light/dark cycle at a constant tem-perature of 23 ◦C, with free access to commercial food andwater. Male immature rats (15–17 days old) were killed byether inhalation.

Isolation and culture of Sertoli cells

Sertoli cells were isolated as previously described [17, 18].Briefly, testes of immature rats were removed, decapsulatedand digested enzymatically with trypsin and deoxyribonucle-ase for 30 min at 37 ◦C, and centrifuged at 750 × g for 5 min.The pellet was mixed with soybean trypsin inhibitor, then cen-trifuged and incubated with collagenase and hyaluronidasefor 30 min at 37 ◦C. After incubation, this fraction was cen-trifuged (10 min at 40 × g). The pellet was taken to isolateSertoli cells and the supernatant was discarded. After count-ing, Sertoli cells were plated in 6 × dishes multiwell plates(3 × 105 cells/cm2) in Medium 199 or DMEM/F12 1:1, lowglucose, 1% FBS, supplemented with sodium bicarbonate,gentamicin, and fluconazol, and cultured for 24 h to attach.The medium was then changed to serum-free medium andcells were taken for assay after 48 h of culture. FSH or retinoltreatments were carried out together with culture medium inthe last 24 h of culture.

187

Assays

To evaluate the effects of retinol or FSH in the release ofpurinergic agonists, Sertoli cells were preincubated for 24 hwith retinol 7 µM in or ovine FSH 2 mU/ml, gently washedthree times to eliminate debris and dead/dying cells and in-cubated for various times in 5% CO2 at 34 ◦C with phenolred-free HBSS supplemented with HEPES 15 mM. This in-cubation medium was used for HPLC analysis of extracellu-lar purines. Kinetics of degradation of extracellular ATP andadenosine was assessed by adding ATP or adenosine 25 µMto the incubation medium, and the appearance/disappearanceof their metabolic products on the extracellular space wasevaluated by HPLC analysis. In order to block the forma-tion of extracellular adenosine from AMP, we used the mosteffective inhibitor of ecto-5′-nucleotidase, α,β-methyleneadenosine diphosphate (AOPCP) 25 µM. S-(4-Nitrobenzyl)-6-thioinosine (NBTI) 10 µM and dipyridamole (dip) 10 µMwere used to inhibit the transport of adenosine and/or inosine,and erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) 10 µMwas used to inhibit adenosine deaminase (ADA). Trolox100 µM, which is widely adopted as a standard antioxidant,was used to block retinol-induced ROS production. The con-centrations of retinol and FSH used were chosen according tocriteria followed by previous works from our group [27–30]and others [32], and correspond to physiologic levels com-monly reported for these molecules in the male reproductivesystem.

HPLC analysis of extracellular purines

Extracellular purines were measured as previously described[17, 31]. After incubation, the medium was removed and cen-trifuged to eliminate debris. Samples were treated with TFA7% to precipitate proteins, evaporated in a vacuum centrifuge(−61 ◦C) and resuspended at 1/10 of the original volume toallow the detection of the low concentrations of purinergiccompounds found in these cells. Purine content was deter-mined by a reverse-phase HPLC system equipped with aC-18 column (SupelcosilTM, Supelco R©, 25 cm×4.6 mm) andUV detector. Elution was carried out over a 25 min period, ata flow rate of 1.2 ml/min, using a linear gradient from 100%buffer A (KH2PO4 60 mM and tetrabutylammonium chloride5 mM, pH 6.0) to 100% buffer B (buffer A 70% plus methanol30%). Internal samples were used for identification.

ATP measurements

The cellular ATP content of Sertoli cells was determined aspreviously described [31] by the well-established luciferin–luciferase method. After assay, cells were washed and the

ATP content was extracted with 2% perchloric acid (PCA).Samples were neutralized with NaOH and diluted 200-foldin Tris 10 mM (pH 7.4). An aliquot of this cell extract(60 µl) was mixed with 300 µl of luciferin–luciferase solu-tion (2 mg/ml) and the bioluminescence produced by the re-action with ATP was counted in a luminometer. To assess ex-tracellular ATP release we collected the incubation mediumof Sertoli cells pretreated with FSH for 24 h after short pe-riods of incubation (5–60 min) with phenol red-free HBSS.This incubation medium (100 µl) was mixed with 300 µl ofluciferin–luciferase solution and ATP concentration quanti-fied as described above.

Culture purity and cellular integrity

Sertoli cell cultures were estimated to be 95–98% pure,as assessed by bright light and phase contrast microscopyand alkaline phosphatase assay. Cellular disruption and in-tegrity was controlled by comparing the lactate dehydroge-nase (LDH) activity of incubation medium with that of cellslysed with Triton X-100 1%, using a commercial kit fromSigma following the manufacturer instructions.

Protein quantification

Protein content was measured as described by Lowry et al.[33] and results were standardized against the protein content.

Statistical analysis

Extracellular purines were measured in at least three separatereplicates for each experiment and the mean and standard er-ror calculated. Statistical analysis was performed on the rawdata with the ANOVA, with Duncan’s post hoc test. Differ-ences were considered to be significant when p < 0.05.

Results

Selective HPLC analysis revealed that FSH appeared to in-duce little or no alterations in the extracellular purinergiccontent of Sertoli cells. Concentrations of ATP, ADP, AMP,and inosine accumulated during 60 min after a 24-h period ofincubation with FSH were not altered; however, the amountof adenosine was increased by 40% in FSH-treated cells(Fig. 1). Downstream metabolites hypoxanthine, xanthine,and uric acid were also not affected (not shown). Once wepreviously reported that Sertoli cells are able to convert ex-tracellular ATP into adenosine and inosine by the sequentialaction of ecto-nucleotidases and ecto-nucleosidases [15, 34],we used the ecto-5′-nucleotidase inhibitor AOPCP to blockthe production of adenosine from AMP degradation; a curve

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Fig. 1. Extracellular purines of Sertoli cells treated with FSH. Cultured Ser-toli cells were treated with FSH 2 mU/ml for 24 h; the medium was thenremoved and cells were incubated with phenol red-free HBSS for 60 min.After this period, incubation medium was removed and the purinergic con-tent quantified by HPLC as described in “Materials and methods” section.∗Different from control (ANOVA, Duncan’s post hoc, p < 0.05).

for inhibition of AMP degradation was performed with vary-ing concentrations (1, 25, 100, and 500 µM) of AOPCP, andwe utilized the concentration of 25 µM in further experi-ments. We found that AOPCP treatment completely reversedthe FSH-induced increase of adenosine (Table 1). Also, weobserved that this treatment caused an acute accumulationof ATP, ADP, and AMP in the incubation medium of FSH-treated cells, differently of cells with only AOPCP. Inhibitionof ecto-ADA by EHNA (10 µM) naturally causes an increasein adenosine accumulation in untreated cells, and this effectwas enhanced in FSH-treated cells (Table 1). A similar re-sult was also observed with the nucleoside uptake inhibitorsdipyridamole 10 µM and NBTI 10 µM (dip/NBTI), thus rein-forcing the role of ATP degradation cascade in FSH-inducedadenosine increase (Table 1).

Table 1. Effects of AOPCP, EHNA and dip/NBTI on purinergic secretion of FSH-treated Sertoli cells

Untreated AOPCP EHNA dip/NBTI

Control FSH Control FSH Control FSH Control FSH

ATP 9.26 ± 0.67 10.27 ± 2.45 12.24 ± 2.17 83.14 ± 12.44∗ 9.88 ± 0.94 12.44 ± 2.97 10.07 ± 2.44 9.54 ± 1.99

ADP 10.68 ± 1.25 11.42 ± 2.07 11.75 ± 1.54 130.54 ± 14.93∗ 9.41 ± 1.13 10.68 ± 2.15 9.82 ± 1.63 9.66 ± 2.14

AMP 31.21 ± 3.22 36.15 ± 3.18 98.58 ± 5.14 159.44 ± 17.47∗ 33.89 ± 2.5 31.08 ± 3.1 28.66 ± 3.21 33.24 ± 3.14

Adenosine 56.91 ± 0.96 87.54 ± 3.21∗ 34.28 ± 2.06 31.06 ± 4.14 112.27 ± 11.56 184.03 ± 6.7∗ 74.04 ± 2.66 110.28 ± 13.25∗

Inosine 306.44 ± 18.66 317.14 ± 6.89 214.63 ± 10.9 222.6 ± 14.62 194.35 ± 24.16 186.77 ± 16.33 367.12 ± 21.48 382.73 ± 12.24

Sertoli cell cultures were preincubated for 24 h with FSH 2 mU/ml then culture medium was discharged and cells were incubated with AOPCP 25 µM, EHNA10 µM or dipyridamole 10 µM/NBTI 10 µM (dip/NBTI) for 60 min in HBSS without phenol red. Extracellular purines accumulated during this period wereidentified and quantified by selective HPLC analysis, as described in “Materials and methods.” Data are expressed as picomoles of extracellular purines/mgcell protein (mean ± S.E.M.).∗Different from respective control (ANOVA, Duncan’s post hoc, p < 0.05). Data representative of three independent experiments, n = 3.

In an attempt to better understand the mechanism bywhich FSH increases extracellular adenosine levels in Ser-toli cells, we performed kinetic assays of ATP, AMP, andadenosine degradation, in order to establish the role of ecto-ATPase/apyrase, ecto-5′-nucleotidase, and ecto-ADA regu-lation in controlling the levels of adenosine. Figure 2 showsthat ATP 25 µM degradation is increased by FSH treatment,and that adenosine is the downstream metabolite which mostaccumulates from ATP degradation after 30 min. This isreversed by AOPCP treatment, which blocked the forma-tion of adenosine from ATP via ecto-5′-nucleotidase activity(Fig. 2c and d). AMP 25 µM degradation was not modifiedby FSH pretreatment (Fig. 3), thus suggesting that ecto-5′-nucleotidase is not regulated by FSH. Adenosine (25 µM)degradation was also observed not to be modified by FSHpretreatment (Fig. 4). Kinetics of ATP release was performedby the luciferin-luciferase luminescent detection in short pe-riods of time between 5 and 60 min of incubation after FSHtreatment. ATP levels were found to be increased right after5 min of FSH pretreatment; after 30 min, however, efflux ofextracellular ATP returned to basal levels (Fig. 5). After that,ATP levels do not differ in both treatments until 60 min ofincubation.

The 24-h preincubation of Sertoli cells with retinol causedno significant alterations in extracellular ATP, ADP, AMP,adenosine or inosine. However, significant changes wereobserved in the downstream products of adenosine/inosinedegradation: retinol treatment caused an increase in extra-cellular hypoxanthine and xanthine levels, whereas uric acidwas found to be slightly decreased compared to control cells(Fig. 6). These alterations were not found to be related to in-creased nucleotidase/nucleosidase activity: we did not foundalterations in exogenous ATP, AMP, and adenosine degra-dation in cells pretreated with retinol (not shown). AOPCPdid not exert any effect in these cells, as well as nucleosidere-uptake inhibition and EHNA treatment (Table 2). Since

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Fig. 2. Kinetics of extracellular ATP degradation in Sertoli cells preincubated with FSH in the presence of AOPCP. Cultured Sertoli cells were treatedwith FSH 2 mU/ml for 24 h, then the medium was removed and the degradation of ATP 25 µM was followed by HPLC analysis. (A) Control; (B)FSH pretreatment; (C) AOPCP 25 µM; (D) FSH pretreatment with AOPCP 25 µM. Values represent mean ± S.E.M. of a representative experiment,n = 3.

our group has reported that retinol 7 µM is able to increaseROS production and lead to cellular oxidative stress [27–30],we administered an antioxidant treatment in retinol-treatedSertoli cells. Thus, the co-treatment of retinol with the an-tioxidant Trolox 100 µM reversed these changes, indicatingthat ROS production is involved in this effect (Table 2). Also,we observed that retinol treatment decreased the intracellularlevel of ATP, a common effect observed in situations of cellu-lar oxidative stress (Fig. 7), and this effect was also reversedby Trolox, thus suggesting a correlation between intracellu-lar ATP consumption and extracellular levels of downstreamATP metabolites.

Discussion

Since the middle of the 1980 decade the influence of puriner-gic receptors on the regulation of the functions of the malereproductive system has been studied. Despite the fact thatworks on this subject have revealed that purinergic signal-ing is an important component of the intricate and complex

regulation system of the testicular function, affecting the re-sponses of Sertoli cells to hormones such as FSH [9, 35] andaltering functions like transferrin [13], inhibin α [36] andpyruvate secretion [37], little attention has been paid by re-searchers on the potential perspectives that the elucidation ofpurinergic and hormonal cross-talking can bring to the com-prehension of the male reproductive function. The actions ofboth retinol and FSH in this system have been studied for sev-eral years, and the role that these two molecules were foundto exert in the regulation and maintenance of male fertilitygave them the status of “classical” regulators of spermato-genesis, together with testosterone. Thus, the results of thiswork confirm previous data from other groups which indi-cated that purinergic signaling appears to be an importantcomponent of the testicular paracrine/autocrine system, oncethe effects of both the gonadotropin FSH and vitamin A uponpurinergic secretion suggest that extracellular purines may beinvolved in the metabolic response of Sertoli cells to thesetwo regulators.

Our results strongly suggest that the FSH-induced in-crease in extracellular adenosine concentration resulted from

190

Fig. 3. Kinetics of extracellular AMP degradation in Sertoli cells preincu-bated with FSH. Cultured Sertoli cells were treated with FSH 2 mU/ml for24 h, then the medium was removed and the degradation of AMP 25 µMwas followed by HPLC analysis. (A) Control; (B) FSH pretreatment. Valuesrepresent mean ± S.E.M. of a representative experiment, n = 3.

increased ecto-nucleotidase activity upon stimulated ATP se-cretion. These data agree with previous observations from ourgroup [18]. Besides, Lu et al. reported that FSH and cAMPpositively regulate the mRNA of ecto-ATPase in Sertol cells[38], which could also explain the increased degradation ofATP into adenosine in FSH-treated cells. It is generally ac-cepted that ecto-5′-nucleotidase is the rate-limiting step ofthe ecto-nucleotidase pathway converting ATP into adeno-sine; however, our data demonstrated that FSH induces nosuch alteration, once extracellular AMP degradation was notaffected by FSH-treatment. ATP degradation, on the otherhand, was found to be increased in cells pretreated with FSH,thus suggesting that degradation of extracellular ATP is themechanism by which FSH increases extracellular adenosinein Sertoli cells. Nevertheless, FSH seems to be also enhanc-ing ATP secretion, which is not detected by HPLC analysisprobably because the Km for the degradation of extracellu-lar ATP in these cells −131 ± 17.4 µM [34] is much greaterthan the physiologic concentrations of ATP found in their sur-

Fig. 4. Kinetics of extracellular adenosine degradation in Sertoli cells prein-cubated with FSH. Cultured Sertoli cells were treated with FSH 2 mU/mlfor 24 h, then the medium was removed and the degradation of adenosine25 µM was followed by HPLC analysis. (A) Control; (B) FSH pretreatment.Values represent mean ± S.E.M. of a representative experiment, n = 3.

face – about 70–100 nM [17]. Results obtained from AOPCPtreatment also reinforces this idea, once FSH-treated cells ac-cumulate not only extracellular AMP in the presence of theecto-5′-nucleotidase inhibitor, but also ATP and ADP (Table1), and the luciferin-luciferase assay for ATP release furtherdemonstrated the increased ATP release in early periods of in-cubation in FSH-treated cells. Thus, FSH-induced increasedadenosine seems to be a result of both increased ATP releaseand degradation via ecto-nucleotidase pathway. We did notfind, on the other hand, any evidence in our data pointingto a possible action of FSH directly upon adenosine releasevia regulation of nucleoside transporters: blockade of ecto-5′-nucleotidase abolished the effect of FSH upon extracellu-lar adenosine concentrations, and the results obtained withEHNA and dip/NBTI did not suggest a possible increase inadenosine release by FSH.

It is well-established that activation of A1 adenosine recep-tors in Sertoli cells decreases FSH-induced cAMP increase in

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Fig. 5. Kinetics of ATP release in Sertoli cells treated with FSH. CulturedSertoli cells were treated with FSH 2 mU/ml for 24 h, and then the mediumwas removed and ATP release was assessed in both control and treated cellsfor 60 min by bioluminescent quantification with luciferin-luciferase systemas described in “Materials and methods.” Values are mean ± S.E.M. of arepresentative experiment; ∗Different from control (ANOVA, Duncan’s posthoc, p < 0.05).

Fig. 6. Extracellular purines of Sertoli cells treated with retinol. CulturedSertoli cells were treated with retinol 7 µM for 24 h; the medium was thenremoved and cells were incubated with phenol red-free HBSS for 60 min.After this period, incubation medium was removed and the purinergic con-tent quantified by HPLC as described in “Materials and methods” section.∗Different from control (ANOVA, Duncan’s post hoc, p < 0.05).

Sertoli cells, which would affect some reproductive-relatedfunctions of these cells, such as androgen aromatization [8].Conti et al. suggested that activation of adenosine receptorsof Sertoli cells would result in increased metabolic supplyfor germinative cells [37]. It is possible that the ability ofFSH to evoke adenosine release by Sertoli cells is part of anautocrine mechanism of negative feedback for control of theFSH-dependent increase in cytosolic cAMP and secretion ofpyruvate and inhibin α, in which FSH-evoked extracellular

Fig. 7. Cytosolic ATP content of Sertoli cells treated with retinol. Sertolicells were treated with retinol 7 µM and cellular ATP content was evaluated60 min after the onset of incubation by the luciferin-luciferase assay, asdescribed in “Materials and methods.” ∗Different from control (ANOVA,Duncan’s post hoc, p < 0.05).

adenosine would activate Sertoli cell A1 receptors, thus de-creasing some effects of the gonadotrophin. Also, it is possi-ble that such increase would activate the different subtypes ofadenosine receptors of germinative cells, which are differen-tially expressed according to the stage of spermatic differen-tiation of these cells. Thus, FSH would exert different effectsin spermatogenesis regulation by evoking adenosine releasein Sertoli cells, which would activate A1 or A3 receptors ofthe associated germinative cells.

On the other hand, retinol-induced changes on the levelsof adenosine downstream metabolites hypoxanthine, xan-thine, and uric acid do not seem to be correlated to an ef-fect upon both ecto-nucleotidase/nucleosidase activity and/oradenosine release, once the use of AOPCP, EHNA, anddip/NBTI did not affect these changes. Retinol appears toaffect downstream steps in purinergic enzymatic degrada-tion pathway, such as purine-nucleoside-phosphorylase orxanthine-oxireductase/dehydrogenase activities; it is impor-tant to point, however, that neither of these enzymes was de-scribed to be expressed on extracellular surfaces of cell mem-branes. However, retinol demonstrated to affect intracellularlevels of ATP, an effect reversed by Trolox, thus suggestingthat this effect results from ROS production and consequentcellular oxidative stress. Thus, it is possible that increasedhypoxanthine/xanthine levels are a result of augmented in-tracellular production of these purines, which are describedto be highly affected by variations of cytosolic ATP levels[39].

We have demonstrated that retinol is able to causeoxidative-dependent changes in Sertoli cells, such as alter-ations in iron metabolism and cell cycle by enhancing ROSproduction [27–30], and we recently reported that oxidativestress is able to induce acute alterations on the levels of extra-cellular purines, namely inosine, hypoxanthine, and xanthine,

192

Tabl

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0.67

8.94

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289.

94±

2.6

9.15

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.26

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149.

54±

1.4

11.4

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2.4

8.69

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99

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.68

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.66

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4710

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699.

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2.04

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1.48

10.5

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9.66

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669.

58±

1.35

11.2

7±

2.54

10.3

7±

2.02

AM

P31

.21

±3.

2229

.24

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2286

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7492

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5528

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105.

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6.95

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59.3

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306.

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18.6

631

2.55

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5.33

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187.

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519

4.24

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387

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369.

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21.5

325.

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633

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193

which appeared to be involved on the response of Sertoli cellsto oxidative insults [31]. Despite the fact that the reversionof the modifications on the levels of extracellular puriner-gic compounds by the antioxidant Trolox (Table 2) indicatesthat these modifications are related to the previously reportedretinol-induced ROS production, it is impossible to say ifthey constitute a cellular response against oxidative stress.It is known, however, that the superoxide-producing confor-mation of the enzyme xanthine-oxireductase/dehydrogenase,namely xanthine oxidase, is able to catalyze the synthesis ofretinoic acid from retinaldehyde, a common cytosolic deriva-tive from retinol, in vitro [40]. The possibility that retinol af-fects the purinergic metabolism of Sertoli cells in such wayshould not be discharged, once we have previously reportedthat retinol induces superoxide production in Sertoli cells byan unknown mechanism [27] but more studies are necessaryto elucidate this issue and its consequences.

Concluding, this study shows that FSH and retinol, whichare important regulators of the male reproductive system, areable to induce different changes on the release of extracel-lular purines of Sertoli cells. While FSH-induced changesmay have consequences in purinoceptors signaling, the con-sequences of the modifications induced by retinol remains tobe clearly determined. The data presented here suggest thatpurinergic signaling must be involved in the regulation of im-portant reproductive-related functions of Sertoli cells, oncethese hormones control many of these functions in these cells.

Acknowledgments

This work was supported by the following Brazilian agen-cies for research grants: CNPq, CAPES, FAPERGS, andPROPESQ-UFRGS.

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Effects of follicle-stimulating hormone and vitamin A upon purinergic secretion by rat Sertoli cells

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