Regulation of steroidogenesis by atrial natriuretic peptide (ANP) in the rat testis: Differential involvement of GC-A and C receptors

peptides 29 (2008) 2024–2032

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Regulation of steroidogenesis by atrial natriuretic peptide (ANP) in the rat testis: Differential involvement of GC-A and C receptors Virgı´nia Mara Pereira a, Amilton P. Raposo Costa a,b, Alzira Amelia Martins Rosa-e-Silva c, Maria Aparecida Ribeiro Vieira a, Adelina Martha dos Reis a,* a

Department of Physiology and Biophysics, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil Federal University of Piaui, Teresina, PI, Brazil c Federal University of Brasilia, Brasilia, DF, Brazil b

article info

abstract

Article history:

Previous studies have established a stimulatory effect of natriuretic peptides (NP) on

Received 2 July 2008

testosterone production in mouse Leydig cells as intense as that of LH. Chronic adminis-

Received in revised form

tration of ANP in mice, on the other side, reduced testosterone levels. So, the understanding

5 August 2008

of the role of ANP on testicular steroidogenesis has been impaired by discrepant findings.

Accepted 5 August 2008

The aim of the present study was to clarify the physiological role of ANP in the rat testis

Published on line 20 August 2008

steroidogenesis using a model that preserves the interactions between testis cells and a medium devoid of any circulating factors that could interfere with testosterone production.

Keywords:

First, ANP was immunolocalized in the interstitial compartment of the rat testis, mainly in

Atrial natriuretic peptide

Leydig cells. We also determined the presence of ANP and both GC-A (guanylyl cyclase A)

Steroidogenesis

and C receptors by real-time PCR in testis. Perfusion in vitro of testis with ANP (1 and

Rat testis

3  107 M) stimulated testosterone production in a time- and dose-dependent manner. On

Testosterone

the other side, testosterone secretion induced by LH was blunted by ANP. Similar effect was

C receptor

obtained using the specific C receptor ligand, cANF, indicating the involvement of C receptor in such response. In conclusion, ANP stimulated testosterone production in the rat testis perfused in vitro but decreased testosterone production LH-induced, effect that seems to involve C receptor. To this extent, our results suggest the existence of a local and complex peptidergic system in the rat testis, involving ANP and its receptors that could importantly modulate the androgen biosynthesis. # 2008 Elsevier Inc. All rights reserved.

1.

Introduction

Natriuretic peptides (NP) constitute a family of hormones that actively participate in the maintenance of body fluid homeostasis and blood pressure control [8]. Three main biologically active forms of NP have been described to date, namely, atrial natriuretic peptide (ANP) [14], brain natriuretic peptide (BNP)

[50], C-type natriuretic peptide (CNP) [27]. NP act through different receptors: ANP and BNP act by coupling to guanylyl cyclase A (GC-A) receptors and CNP acts by coupling to guanylyl cyclase B (GC-B) [41,51,15]. Besides, all natriuretic peptides can bind to C receptor, initially described as a clearance receptor [31], but that can also be involved in the biological effects of NP [1,2,30]. With regard specifically to ANP,

* Corresponding author at: Dept Fisiologia e Biofı´sica, ICB, Universidade Federal de Minas Gerais, Av. Antonio Carlos 6627, CEP 31270-901, Belo Horizonte, MG, Brazil. Tel.: +55 31 3409 2931; fax: +55 31 3409 2924. E-mail address: [email protected] (A.M.d. Reis). 0196-9781/$ – see front matter # 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2008.08.005

peptides 29 (2008) 2024–2032

although its main source is the atrial cardiocyte, it is also found in a number of extra-atrial tissues and organs [17]. ANP is the most studied of the NP and its actions on kidneys, blood vessels, and adrenal glands result in diuresis, natriuresis and vasodilation [8]. In addition to these effects, ANP inhibits the release of aldosterone, renin, and vasopressin, while stimulating the release of androgens and progesterone through specific cell-surface receptors [22,47]. Natriuretic peptides and their receptors have been identified in male and female reproductive systems. In the female, ANP immunoreactivity was found in bovine corpus luteum [55], follicular fluid, and ovarian perfusate [26]. Expression of NP components as well as its regulation during estrous cycle was demonstrated in rat uterus [45,46] and ovaries [18,23]. With regard to the male reproductive systems, proANP [53] and CNP and its mRNA [34] were identified in the rat testis. Moreover, GC-A and GC-B receptors were detected in rat [44] and mouse Leydig cells [25]. Also, the expression of C receptors was reported in the rat testis [37], more conspicuously in the surroundings of the seminiferous tubules. Immunolocalization of ANP in the rat testis is still controversial in the literature. Immunohistochemical studies have shown the presence of ANP in the acrosomes of round and elongated spermatids [54,39] and in nuclei of spermatocytes and round spermatids [11]. Another study has shown ANP immunoreactivity in Leydig and Sertoli cells [4]. CNP has also been identified in rat testis [34], and endogenous production of CNP and expression of GC-B receptor were reported in Leydig cells [44]. Several studies conducted in mouse Leydig cell cultures have demonstrated the stimulatory effect of ANP on testicular steroidogenesis [35,42]. The effects of NP on testicular steroidogenesis were first demonstrated by Bex and Corbin [6], who observed an increase in testosterone production induced by hANP in homogenized mouse testes. Mukhopadhyay et al. [36] demonstrated a stimulatory effect of ANP on testosterone production, with the concomitant increase of cGMP levels. Khurana and Pandey [25] verified that ANP and BNP increased testosterone production in a dosedependent manner, whereas CNP was effective only in high concentrations. It is important to note that most studies, such as the ones cited above, which confirm the stimulatory effect of ANP on testicular steroidogenesis, were conducted in mouse Leydig cell cultures [35,42]. More recently Lee et al. [29] have shown that chronic administration of ANP in mice reduced testosterone levels in both plasma and testes. In humans, the acute injection of ANP did not elevate peripheral testosterone and LH levels, but it significantly increased testosterone concentrations in the spermatic vein, which suggests a direct positive influence of ANP on Leydig cells [16]. So, at this point, the effect of ANP on testicular steroidogenesis has been impaired by contradictory findings. In this context, the aim of the present study was to clarify the physiological role of ANP in the rat testis steroidogenesis, as well as to evaluate the participation of different types of ANP receptors in this response. To maintain the cellular interrelationship in the testis, which is crucial for physiological androgen production [33,48,38] and better mimics the in vivo situation, we used the in vitro perfusion model previously described [52].

2.

Materials and methods

2.1.

Animals

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Male Wistar rats were obtained from CEBIO (Centro de Bioterismo, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil) and were cared for according to the international guidelines for animal care. The experimental protocol was approved by the Ethics Committee in Animal Experimentation of the Federal University of Minas Gerais. The animals (5–6 per cage) were maintained under controlled light and temperature conditions (lights on from 05:00 to 19:00 h, 23  3 8C) and had free access to tap water and a standard rat chow (Nuvital Nutrientes Ltda, Colombo, PR, Brazil).

2.2.

Immunohistochemistry

Wistar adult rats (3 months old; n = 3) received an intraperitoneal injection of 5000 IU/kg heparin sulfate (Liquemine, Roche, Rio de Janeiro, Brazil) and were anesthetized with 30 mg/kg sodium pentobarbital. After laparotomy and thoracotomy, animals were perfused through the left ventricle with 0.05 M PBS containing protease inhibitors: 105 M phenylmethylsulfonylfluoride, 0.5  105 M Pepstatin A, 105 M EDTA, 105 M para-hydroxymercurybenzoate, and 9  104 M orthophenantroline, all of which were purchased from Sigma–Aldrich Corporation (St Louis, MO, USA). After perfusion with 4% paraformaldehyde, testes were removed, cleaned of fat, immersed in 4% paraformaldehyde and kept for 2 h in the refrigerator (6 8C). They were then fixed in Bouin’s solution for 4 h, at room temperature, and transferred to 70% alcohol until free from the yellowish coloration conferred by Bouin’s. Finally, the testes were embedded in paraffin, sectioned at 4 mm, and mounted on gelatinized slides. The testis sections were processed through the avidin– biotin–peroxidase method [21], with modifications, using the Vectastain ABC Kit (Vector Laboratories, Burlingame, CA, USA). Subsequently, they were deparaffinized in xylene and rehydrated in graded alcohols, followed by distilled water. The sections were then pre-incubated in methanol 1%/ hydrogen peroxide solution for 30 min, followed by normal goat serum for 30 min, in order to block endogenous peroxidase and nonspecific binding, respectively. After the completion of this procedure, slides were incubated overnight, at 4 8C, with primary antibodies diluted in PBS containing 1% bovine serum albumin (BSA): rabbit polyclonal against ANP (Peninsula Laboratories Inc., Belmont, CA, USA) 1:1000. Later, slides were rinsed with PBS and incubated for 30 min with biotinylated anti-rabbit IgG 1:200. They were finally washed with PBS and incubated with the avidin–biotin complex 1:200 for 1 h. The immunostaining was visualized with 3,30 -diaminobenzidine tetrahydrochloride (DAB, Sigma–Aldrich Corp.) and counterstained with hematoxylin. Negative controls were obtained by incubation of the slices with 1% PBS/BSA, instead of with the primary antibody. Additional negative controls for the ANP immunohistochemistry were performed by preabsorption of the primary antibody with ANP 103 M. Slides were analyzed using a qualitative method.

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2.3.

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Real-time polimerase chain reaction

To confirm the local synthesis of the natriuretic peptide family components, we evaluated the mRNAs expression in 25-day-old immature (n = 3) and 3 months old adult (n = 3) rat testis by Quantitative PCR (Q-PCR). Rats were anesthetized with Ketamine (90 mg/kg) plus Xylazine (5 mg/kg), testes were removed, cleaned of adherent connective tissue and immediately frozen in liquid nitrogen and stored at 80 8C. RNA was extracted using TRIZOL Reagent (Invitrogen, Carlsbad, CA, USA). Two micrograms of RNA was reverse transcribed (Superscript First Strand Synthesis – Invitrogen) and maintained at 20 8C. Quantitative PCR (Q-PCR) assay was conducted following the manufacturer’s procedures (Applied Biosystems) using the fluorescent dye SYBR Green Master Mix. All reactions were done in duplicate. Table 1 shows the specific primers manufactured by Invitrogen (Sa˜o Paulo, SP, Brazil). Each primer (2 pmol) was used in a final volume reaction of 25 ml. Q-PCR parameters were 1 cycle at 52 8C for 2 min, 1 cycle at 95 8C for 10 min, 40 cycles at 95 8C for 30 s and 50 8C for 1 min. No amplification of fragments occurred in control samples without reverse transcriptase. Quantity of mRNA was calculated using the DDCt method. For each Q-PCR, the threshold cycle (Ct) was determined. Expression levels were normalized to the GAPDH housekeeping gene according to the following formula: DCt = CtTarget GAPDH . Subsequently, the respective mRNA levels were  Ct calculated using the DDCt method, that is, DDCt = DCt adult  DCt immature (control) and the relative mRNA levels were calculated as 2DDCt based on the results of control experiments.

2.4.

Surgical procedure for testis perfusion

Rat testis perfusions were done in accordance with Costa et al. [12] adapted version of the surgical procedure described by Koos et al. [28] for rat ovary. Rats were anesthetized with sodium pentobarbital (30 mg/kg) and received, simultaneously, an intraperitoneal injection of 5000 IU/kg heparin sulfate (Liquemine, Roche, Rio de Janeiro, Brazil). After laparotomy, major aorta branches were doubly ligated and sectioned, kidneys were excised, and right testes separated from the epididymis and cleaned of adipose and connective tissues. The aorta was cannulated with a catheter, advanced until approximately 3–5 mm from the right testicular artery. Aorta and vena cava were severed just after the end of the cannula and the preparation was removed from the animal. The testes were perfused with cold saline solution to completely wash out the blood and immediately attached to the perfusion apparatus.

2.5.

Perfusion apparatus and medium

The perfusion system, consisting of a closed circuit system with recirculation of the medium, was similar to that used for ovary perfusion by Costa et al. [12] and was adapted from Bra¨nnstro¨m [7]. The medium perfusion consisted of 60 ml of medium 199 with Earle´s salt (Sigma–Aldrich Corp.) supplemented with 4% bovine serum albumin (Sigma–Aldrich Corp.), 18 mM HEPES buffer (Sigma–Aldrich Corp.), 0.2 IU/ml sodium heparin sulfate (Liquemine-Roche), 0.02 IU/ml insulin (Biohulin-Biobras, Montes Claros, Brazil), and 0.06 mg/ml gentamicin (Garamicina, Schering, Rio de Janeiro, Brazil). The system was prepared 1 h before placement of the testes and the temperature was maintained constant at 34 8C by a warming and circulation pump. The pH (7.4) was maintained constant by continuous oxygenation with 95% O2/5% CO2. The system pressure was maintained at 80 mmHg, supplying a constant flow of 2 ml/min to the testes. All tubes and glass components of the perfusion system were siliconized to avoid steroid adherence.

2.6.

Treatments

The hormones were added to the perfusion system immediately after collection of the first sample, which was carried out after a stabilization period of 1 h 30 min. The following hormones were used: LH (ovine-LH-26), from NIDDK (NIH, Bethesda, MD, USA); rANP1–28 and cANF4–23 (Peninsula Laboratories Inc., Belmont, CA, USA). LH was diluted in distilled water and the natriuretic peptides in acetic acid 0.01N and they were stored at 80 8C. Samples (2 ml) of the perfusion medium were collected immediately after the stabilization period and, thereafter, at 1-h intervals for up to 4 h. Each sample was replaced with 2 ml of fresh medium containing the peptides. The samples were stored at 20 8C until hormone measurements.

2.7.

Experimental groups

A total of 43 testes were used in this experiment, divided into 7 groups: (a) control group; (b) LH 3.3  109 M; (c) rANP 1  107 M; (d) rANP 3  107 M; (e) LH 3.3  109 M + ANP 3  107 M; (f) LH 3.3  109 M + cANF 6  107 M; (g) cANF 6  107 M.

2.8. Testosterone, progesterone and pregnenolone radioimmunoassay (RIA) Testosterone concentration in the perfusion medium was determined by direct RIA, using the double-antibody technique described by Be´langer et al. [5], with modifications. Testosterone was iodinated by Chloramine T method, using

Table 1 – The primers of natriuretic peptides family members for Q-PCR Genes

Forward primer

Reverse primer

ANP NPR-A NPR-C GAPDH

50 -GGATTTCAAGAACCTGCTAGA-30 50 -ATCACAGTCAATCACCAGGAGTTC-30 50 -CCTACAATTTCGACGAGACCAAA-30 50 -ATGTTCCAGTATGACTCCACTCACG-30

50 -CTTCATCGGTCTGCTCGCTCA-30 50 -AGATGTAGATAACTCTGCCCTTTC-30 50 -ACTCGCTCACTGCCCTGGATGTA-30 50 -GAAGACACCAGTAGACTCCACGACA-30

Length (bp) 95 97 73 102

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125 Iodine. The radioactivity was counted in a gamma counter (LKB-Wallac Minigamma 1275, Turku, Finland). The assay immunoreactivity was about 40%. The intra- and inter-assay coefficients of variation were 5.5% and 13%, respectively. Pregnenolone and progesterone concentrations were determined after extraction with ethyl ether (1:30). Hormone standards (purchased from Sigma–Aldrich Corp.) and [+H]progesterone and [+H]-pregnenolone (Amersham Pharmacia Biotech, Arlington Heights, IL) were used. Separation was performed by using a 200 ml suspension of 625 mg of activated charcoal (Merck & CO., Inc., Darmstadt, Germany) and 62.5 mg of dextran (D-1537, Sigma–Aldrich Corp.) in gelatin 0.1% phosphate buffer. The radioactivity was counted in a scintillation analyzer b-counter (TR-1600, Packard Instrument CO., Meridian, NJ) using a scintillation cocktail consisting of 5 g of 2,5-diphenyloxazole (Merck & CO., Inc.) and 625 mg of 1,4bis[5-phenyl-2-oxazolyl]benzene/2,2´-p-phenylene-bis[5-phenyloxazole] (Sigma–Aldrich Corp.) in toluene/methanol (980/ 20 ml; Merck & CO., Inc.).

2.9.

Statistical analysis

Hormone concentration values were subtracted from time 0 h values and the results were expressed in nanogram  ml of medium1  gram of testes1. Relative values of hormone

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concentrations were expressed as mean  SEM. The hormone levels were compared in the total time of perfusion, using twoway ANOVA, followed by Bonferroni test. One-way ANOVA followed by Student–Newman–Keuls were used for analysis of the areas under testosterone curves and Student t test for Q-PCR data. Comparisons with P < 0.05 were considered significant.

3.

Results

3.1.

Immunohistochemistry

Intense ANP immunoreactivity was observed in the interstitial compartment of the rat testis, particularly in Leydig cells (Fig. 1A–C). This pattern of labeling in Leydig cells surrounding seminiferous tubules was found in all testis slices, with homogeneous distribution, independently of the stage of the spermatic cycle. A weak immunoreactivity was observed in a few cells of the germinative epithelium (Fig. 1C).

3.2.

Real-time PCR

Adult rat testis presented higher mRNA expression for ANP (Fig. 2A) and C receptor (Fig. 2C) compared with immature rat testis. A tendency that did not reach significance of higher GC-

Fig. 1 – Immunohistochemical localization of ANP in paraffin-embedded tissue sections of rat testis. Note the stronger staining in the interstitial compartment (Leydig cells (LC)), compared to the seminiferous tubules (A–C). Weak staining was also observed in some germ cells (arrows) (C). No reaction was observed in negative controls (D). Original magnification: A, D: T80; B, C: T200.

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Fig. 2 – Quantitative PCR mRNA expression of natriuretic peptide system components in immature and adult rat testis. (A) QPCR of ANP mRNA; (B) Q-PCR of GC-A mRNA; (C) Q-PCR of C receptor mRNA. Relative amount of genes were normalized to the GAPDH housekeeping gene. Data were analyzed by Student t test. Each value represents the mean W SEM of three samples run in duplicate. (*) P < 0.05.

A mRNA expression was also observed in adult compared with immature rat testis (Fig. 2B).

3.3.

No alteration of testosterone production was observed in the cANF testis perfusion as compared to control group.

Steroidogenesis

4. Fig. 3A shows the effect of ANP on testosterone production in isolated rat testis perfused in vitro. The higher concentration of ANP (3  107 M) induced a significant enhancement of testosterone production as compared to control starting at the first hour of perfusion. The lower concentration of ANP (1  107 M) also increased testosterone secretion, but this effect was first observed in the second hour of perfusion. Fig. 3B shows the areas under testosterone curves, where the dose-dependent effect of ANP could be better observed. Neither of the ANP concentration was as efficient as LH in stimulating steroidogenesis (Fig. 3B). Data in Table 2 show that ANP also increased the production of testosterone precursors, i.e., pregnenolone and progesterone (P < 0.05 as compared to control), indicating that ANP may act at the very beginning of the steroidogenesis pathway. As observed with testosterone production, the increase in precursor production induced by ANP (3  107 M) was also less than 50% of the increase induced by LH. In order to determine the effect of ANP on LH-stimulated steroidogenesis, testes were perfused with LH (3.3  109 M) plus ANP (3.0  107 M). Surprisingly, rather than to enhance testosterone production induced by LH, ANP significantly reduced it (Fig. 4). The ring-deleted analog of ANP, cANF, which interacts specifically with C receptors, also inhibited the LHstimulated testosterone secretion as shown in Fig. 5 (P < 0.01).

Discussion

In the present study we investigated the physiological role of ANP in the rat testis steroidogenesis and evaluated the participation of different types of ANP receptors in this response. The mRNA expression for ANP and C receptors was higher in adult than in immature rat testis. An intense immunoreactivity for ANP was observed in the Leydig cells. ANP stimulated testosterone production in the rat testis perfused in vitro, but blunted the production induced by LH. Similarly, cANF also blunted testosterone production induced by LH, indicating the involvement of the C receptors in this response. ANP immunoreactivity was detected in Leydig cells, but not in germ cells, except for a few germinative cells that presented weak labeling. The location of ANP in the testis is still disputed in the literature. Earlier studies have detected ANP immunoreactivity in the rat testis, more specifically, in the acrosomes of round and elongated spermatids [54,39] or in the nuclei of spermatocytes and round spermatids [11]. However, a more recent study [4] detected the strongest and most conspicuous ANP staining in rat testes localized in Leydig cells, and such results are similar to ours. We cannot explain this discrepancy in the literature. However, it is important to note that the method we used for tissue preparation for IHC, which consisted of perfusion of the testis with protease inhibitors,

Table 2 – Effect of ANP on production of testosterone precursors in whole rat testis perfused in vitro Treatment Control LH ANP

Pregnenolone (ng/ml)

Progesterone (ng/ml)

1.4  0.1 27.6  2.6a 6.5  0.4a

3.9  0.7 20.2  4.1a 8.3  1.2b

Testosterone (ng/ml) 7.45  1.25 66.89  4.51a 24.44  5.50a

Testes were perfused with medium 199 in absence (control) or presence of LH (3.3  109 M) and ANP (3  107 M) for 4 h at 34 8C in 5% CO2/95% O2 atmosphere. Progesterone and pregnenolone production were determined by RIA after extraction with ethyl ether. Values represent the mean  SEM (n = 6) of the hormone concentrations at the fourth hour of perfusion. a P < 0.01 compared to control. b P < 0.05 compared to control.

peptides 29 (2008) 2024–2032

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of the NP components in adult than immature rat testis seems to indicate the involvement of this system in processes observed in adult animals such as steroidogenesis. Our results show that ANP stimulates testicular steroidogenesis in a dose- and time-dependent manner. However, differently from previous studies, the intensity of testosterone release induced by ANP 107 M is much lower than the intensity of testosterone release induced by LH 109 M, concentration usually used for maximal stimulation in most of the studies such as the perfusion of isolated rat and rabbit testes [9] or in vitro cell culture [42]. We have chosen the concentration of 107 M as the highest for ANP because 106 M is usually used for determination of non-specific binding. Several studies using mouse or rat isolated Leydig cells have shown that testosterone production stimulated by ANP reaches similar levels to that induced by LH [42,25,13]. In general, however, these studies used isolated Leydig cell cultures or mouse Leydig tumor cells (MA-10) – a cell line that contains, predominantly, a very high density of GC-A receptors [24]. The testosterone production induced by ANP in isolated rat Leydig cells was lower compared to mouse Leydig cells and this difference was attributed to a lower guanylyl cyclase activity in the rat cells [35]. However, the possibility of degradation of ANP receptor/guanylyl cyclase activity by the extensive enzymatic digestion necessary for the rat Leydig cells isolation was also raised. In the present study, using perfused rat testes, we could determine the effects of ANP in a more physiological situation, avoiding enzymatic digestion and maintaining the cellular interactions, which is important for steroidogenesis.

Fig. 3 – Effect of ANP on testosterone production in whole rat testes perfused in vitro. Testes were perfused with medium 199 in absence (control) or presence of ANP (1.0 or 3.0 T 10S7 M), for 4 h, at 34 8C, in an atmosphere of 5% CO2/ 95% O2. Testosterone production was estimated by direct RIA. (A) Time course of testosterone production. Each data point represents the mean W SEM of 6–8 testis perfusions. (*) P < 0.01 control vs. ANP 3 T 10S7 M; (**) P < 0.05 control vs. ANP 1 T 10S7 M; (#) P < 0.05, ANP 1 T 10S7 M vs. ANP 3 T 10S7 M. (B) Area under the testosterone curves. Values represent the mean W SEM (*) P < 0.05 and (**) P < 0.01 compared to control group. Basal values (0 h): control: 5.55 W 1.01; ANP 1.0 T 10S7 M: 5.44 W 0.71; ANP 3.0 T 10S7 M: 8.48 W 1.45; LH: 7.38 W 1.14 ng mlS1 gS1.

not only protects the local peptides from degradation, but also provides an organ free of blood and circulating peptides. The synthesis of ANP and its receptors by testis cells, determined by Q-PCR, strongly suggests the existence of a local natriuretic peptide system. Indeed, the higher expression

Fig. 4 – Effect of ANP on LH-stimulated testosterone production in whole rat testis perfused in vitro. Testes were perfused with medium 199 in absence (control) or presence of LH (3.3 T 10S9 M) and LH + ANP (3 T 10S7 M), for 4 h, at 34 8C, in an atmosphere of 5% CO2/95% O2. Testosterone production was estimated by direct RIA. Each data point represents the mean W SEM of 6–8 testis perfusions. (#) P < 0.01, control vs. LH; (*) P < 0.01, LH vs. LH + ANP. Basal values (0 h): control: 5.55 W 1.01; LH: 7.38 W 1.14; LH + ANP: 8.81 W 1.20 ng mlS1 gS1.

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Fig. 5 – Effect of cANF on LH-stimulated testosterone production by whole rat testis perfused in vitro. Testes were perfused with medium 199 in absence (control) or presence of cANF (6 T 10S7 M), LH (3.3 T 10S9 M) and LH + cANF for 4 h at 34 8C in 5% CO2/95% O2 atmosphere. Testosterone production was estimated by direct RIA. Each data point represents the mean W SEM of 5–8 testis perfusions. (*) P < 0.01, LH vs. LH + cANF. Basal values at 0 h: control: 5.55 W 1.01; LH: 7.38 W 1.14; LH + ANP: 8.81 W 1.20; cANF: 15.06 W 3.45; LH + cANF: 7.30 W 0.86 ng mlS1 gS1.

The stimulatory effect of ANP on the release of pregnenolone and progesterone, which are precursors for testosterone synthesis, was similar to its stimulatory effect on testosterone production, indicating that ANP acts on the beginning of the steroidogenic pathway. The exact intracellular mechanism by which the Leydig cells respond to the ANP stimulus is still not well characterized, but it is known that ANP binds to GC-A receptors in the cellular membrane and stimulates cGMP production [19,43]. The involvement of the GC-A receptor on testosterone secretion induced by ANP was well characterized by Pandey et al. [40] that demonstrated an higher concentration of serum testosterone in mice expressing four copies of Npr1, the gene encoding GC-A receptor. An increased testosterone production, accompanied by the increase of cGMP levels, has been observed in many studies using Leydig cells treated with ANP [42,36]. It has been suggested that ANP stimulates testosterone production in purified mouse Leydig cells through the activation of P450scc enzyme, since an ANP stimulated increase in testosterone production, beginning at the first step of steroidogenesis (i.e., on pregnenolone secretion), has been demonstrated [25]. Thus we believe that ANP could play a role in the process of steroidogenesis through the involvement of the steroidogenic acute regulatory protein (StAR), responsible for transport of cholesterol from the outer to the inner mitochondrial membrane in steroidogenic tissues [10,49]. StAR mRNA and StAR protein expression in response to hCG were identified in

mouse Leydig cells [32]. Besides, Khurana and Pandey [25] have shown that cycloheximide completely blocked the stimulatory effect of natriuretic peptides on testosterone production, by affecting the protein synthesis in the steroidogenic pathway. Previous studies in mouse Leydig cells have shown a synergistic effect of ANP on testosterone secretion induced by LH [20], suggesting an interaction between LH/cAMP and ANP/ cGMP systems. In our experiments, however, ANP did not enhance, as expected, but rather reduced testosterone production induced by LH. The same effect was observed when cANF, a specific C receptor ligand, was associated with LH, indicating that C receptor is involved in this response. cANF has been shown to inhibit adenylyl cyclase activity in a variety of tissues, such as rat aorta, anterior pituitary, and adrenal cortical membranes. Besides, cANF was found to inhibit cAMP levels and progesterone secretion stimulated by LH in MA-10 Leydig tumor cell line [2]. The inhibitory effect of cANF depended upon the presence of guanine nucleotides and was attenuated by pertussis toxin treatment, suggesting that C receptors are coupled to adenylyl cyclase through a Gi protein [3]. Our findings that cANF reduced the LH-stimulated production of testosterone together with Q-PCR results demonstrating the presence of C receptors in the rat testis, indicate a physiological role for C receptors in regulating testicular steroidogenesis. In brief, this study has shown that ANP regulates steroidogenesis in the whole rat testis. ANP stimulated testosterone production in the rat testis perfused in vitro, but blunted the production induced by LH. Similarly, cANF also blunted testosterone production induced by LH, indicating the involvement of the C receptors in this response. The presence of GC-A and C receptors and the distinct effects of ANP on testosterone production indicate that a dual mechanism participates in the steroidogenesis in rat testis. To this extent, our results suggest the existence of a local and more complex peptidergic system in the rat testis, involving ANP and its receptors, and which could importantly modulate the androgen biosynthesis.

Acknowledgments The authors thank Jacqueline Braga Pereira for her valuable technical assistance and Dr. Y.S. Bakhle for his helpful comments. This study was funded by FAPEMIG, CAPES, CNPq, and PRONEX.

references

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