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Testis; |
Departments of Anatomy3 and Physiology,4 the Centre of Heart, Brain, Hormone, and Healthy Aging,5 and the Centre of Reproduction, Development, and Growth,6 Faculty of Medicine, The University of Hong Kong, Hong Kong, China
ABSTRACT
Based on the finding of gene expression of adrenomedullin (Adm) and its receptor components in the rat testis, a paracrine effect of ADM on testicular steroidogenesis has been suggested by our group. The present study demonstrates the gene expression of Adm and the effect of ADM on testosterone production in the Leydig cell. The regulation of ADM by hCG and its interaction with endothelin 1 (EDN1) in the rat Leydig cells are also observed. Primary culture of Leydig cells produced Adm mRNA and secreted 275 ± 19 pg immunoreactive ADM per 106 cells in 24 h. In addition, the Leydig cell was shown to coexpress mRNAs encoding for the calcitonin receptor-like receptor (CALCRL) and receptor activity-modifying protein (RAMP1, RAMP2, and RAMP3). These may account for the specific binding of ADM to the Leydig cells. Administration of ADM to Leydig cells resulted in an inhibition of hCG- and EDN1-stimulated testosterone production. Correlated with this, ADM reduced EDN1 production, whereas its production was increased by EDN1. Furthermore, the production of ADM and the mRNA levels of Calcrl and Ramp2 were suppressed by hCG. Our results suggest that ADM has an autocrine effect on Leydig cell steroidogenesis, possibly by interacting with EDN1 and under the control of gonadotropin. We propose that there is an ADM/EDN1 local regulatory mechanism that may be important in modulating the control of testicular functions by gonadotropins.
adrenomedullin, endothelin 1, Leydig cells, testis
Adrenomedullin (ADM) is a peptide hormone first isolated from human pheochromocytoma in 1993. It elicits a potent and long lasting hypotensive effect [1]. ADM belongs to the calcitonin gene-related peptide (CALCA) family by virtue of its characteristic N-terminal ring with a disulphide bond and an amidated C-terminus [2]. Human ADM consists of 52 amino acids, and it shares about 25% structural homology with human CALCA [3]. The rat ADM has 50 amino acids. The biological actions of ADM and CALCA are mediated through the calcitonin receptor-like receptor (CALCRL) in association with one of the three receptor activity-modifying proteins (RAMPs); namely, RAMP1, RAMP2, and RAMP3 [4].
ADM has been found in various tissues, including reproductive organs such as the uterus [5, 6], the ovary [5, 7], the prostate [8, 9], the epididymis [10], and the testis [11, 12]. In the rat testis, ADM peptide level is found to be 50-fold higher than its level in the plasma, but it is relatively low compared with that in the adrenal gland [12]. The presence of Adm mRNA has been confirmed by solution hybridization-RNase protection assay and RT-PCR [12]. In addition, the presence of specific binding sites has been shown by Scatchard plot analysis and RT-PCR of mRNAs of Calcrl and all three types of Ramps, indicating the presence of both ADM and CALCA receptors in the rat testis [12]. In vitro study has shown that ADM inhibits hCG-stimulated testosterone release in a dose-dependent manner in the rat testis by acting on its specific receptors [12]. On the other hand, endothelin 1 (EDN1) is known to stimulate steroidogenesis [13, 14], and Santiemma et al. (2001) have demonstrated that ADM antagonizes the contraction of peritubular myoid cells (PMCs) induced by EDN1 [15]. Taken together, it is hypothesized ADM may act in an autocrine or/and paracrine manner to regulate testosterone production in the Leydig cell, possibly by interacting with EDN1. We therefore studied ADM secretion and the mRNA expression of Adm, Calcrl, and Ramps as well as its effects on testosterone production in the isolated Leydig cells. To understand the interaction between ADM and EDN1 in the Leydig cell, the effects of ADM and EDN1 on the production of each other were investigated. The regulation of the synthesis of ADM by hCG in the Leydig cell was also addressed.
Seven-wk-old male Sprague-Dawley rats were obtained from the Laboratory Animal Unit, Faculty of Medicine, University of Hong Kong. They were fed standard laboratory food and water and housed under 12L:12D cycles at 22°C–24°C. All animal experiments were conducted in compliance with the procedure approved by the Committee on the Use of Living Animals for Research and Teaching of the University of Hong Kong and were carried out in accordance with the guide for the Care and Use of Laboratory Animals (National Academy of Sciences).
Leydig cells were isolated using a method modified from Ko et al. [16]. Five rats were killed by an overdose of sodium pentobarbital (40 mg per 100 g of rat). The testes were excised rapidly and washed twice in 1x phosphate-buffered saline (PBS). The decapsulated testes were digested for 15 min at 34°C in Dulbecco modified Eagle medium/F12 Ham (1:1; GIBCO-BRL), which was supplemented with 1% phosphosaline and 15 mM HEPES (DMEM, pH 7.2; Sigma, St. Louis, MO) and contained 0.5 mg/ml collagenase type IA, 0.25 mg/ml soybean trypsin inhibitor, and 1 mg/ml bovine serum albumin (BSA; all from Sigma). The flask was incubated for 15 min with shaking at 80 cycles/min, and the cells and tubules were transferred to ice-cold medium to stop the digestion. The suspension was allowed to settle for 5 min, and the supernatant containing the interstitial cells was collected. The tubules were dispersed in another 50 ml medium, and the supernatant was pooled and centrifuged. The Leydig cells were separated by discontinuous Percoll (Amersham Biosciences, Uppsala, Sweden) gradients (with six density fractions ranging from 1.030 to 1.096 g/ml).
Leydig cells located at the boundary between fractions of 1.070 and 1.096 g/ml densities were collected and washed twice with ice-cold DMEM-BSA medium to remove any residual Percoll. The viability of the isolated Leydig cells was examined by the trypan blue exclusion test. A total of 1 x 106 cells were plated in a six-multiwell plate for all treatments, except for the studies of ADM effect on testosterone production, in which 2 x 105 cells were seeded in a NUNC 24-multiwell plate (Nunc, Roskilde, Denmark). The cells were cultured in DMEM-BSA at 34°C in a humidified atmosphere of 5% CO2/95% air to maintain the medium at pH 7.4. After 2 h, the Leydig cells were well attached to the surface of the plate. The purity of the isolated cells was determined by 3β-hydroxysteroid dehydrogenase (3β-HSD) cytochemistry by a modified method of Mendelson et al. [17]. Briefly, 105 freshly isolated Leydig cells were incubated with 2 ml staining mixture containing 0.25 mg/ml 4-nitro-blue tetrazolium chloride (Boehringer Mannheim Corp., Indianapolis, IN), 1.25 mg/ml β-nicotinamide adenine dinucleotide (Sigma), and 1 mg/ml BSA in 4 ml PBS with 0.2 mg pregnenolone (etiocholan-3β-ol-17-one; Sigma) in 0.2 ml dimethyl sulfoxide (Sigma) at 32°C in a humidified atmosphere of 5 % CO2/95% air for 90 min with occasional shaking.
Hormonal Treatments of Leydig Cells
All of the hormonal studies were carried out in DMEM-BSA at 34°C in a humidified atmosphere of 5% CO2/95% air. In the study of ADM secretion and the gene expression of Adm, Calcrl, and Ramps, the isolated cells were cultured in DMEM-BSA only for 24 h. To study the effects of ADM on basal, hCG-stimulated, and EDN1-stimulated testosterone production, the cells were incubated with 1–100 nM of ADM in the presence or absence of 0.05 mIU/ml hCG (Sigma) or 10 nM EDN1 in DMEM-BSA for 3 h. To study the effects of ADM on EDN1 and of EDN1 on ADM synthesis, the cells were incubated with 0.001–100 nM rat adrenomedullin (1–50) or endothelin 1 (Phoenix Pharmaceuticals Inc., Belmont, CA) for 4 or 12 h. To study the effect of hCG on ADM production and the gene expression of preproADM and the receptor component proteins of ADM, the cells were incubated with 0.05–50 nIU/ml hCG for 12 h. After incubation, the media were collected and centrifuged at 4000 x g at 4°C for 10 min. The supernatants were stored frozen at –70°C immediately until the amounts of ADM, EDN1, and testosterone secreted were measured. The cells of all of the experiments except for the studies of testosterone production were used for RNA extraction for the studies of gene expression.
Total RNA of Leydig cells in each well was extracted by TRIZOL reagent (Invitrogen Life Technologies, Carlsbad, CA) and subjected to RT-PCR as described previously [12]. Total RNA extract (2 µg) was used as a template for producing cDNA using MultiScribe Reverse Transcriptase (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. The primer sets used were summarized in Table 1. The PCR conditions were: (1) for Tbp, Adm, Ednra, and Edrnb, initial activation of 5 min at 95°C, followed by 28 cycles (Tbp), 29 cycles (Adm and Ednrb), or 30 cycles (Ednra) of 95°C for 45 sec (denaturation), 56°C for 30 sec (annealing), and 72°C for 45 sec (extension); (2) for Calcrl and Ramps, same activation, followed by 28 cycles (Calcrl and Ramp2) or 29 cycles (Ramp1 and Ramp3) of 95°C for 45 sec, 60°C for 30 sec, and 72°C for 1 min; (3) for Edn1, same activation, followed by 32 cycles of 95°C for 45 sec, 50°C for 30 sec, and 72°C for 90 seconds. The PCR products were fractionated on a 2% agarose gel with 0.25 µg/ml ethidium bromide (GIBCO-BRL) by electrophoresis, and the gel was visualized and photographed by UVP GelDoc-It Imaging system (Ultra-Violet Products Ltd., Cambridge, UK) and image acquisition and analysis software (LabWorks; Ultra-Violet Products) under ultraviolet illumination. Semiquantitative analysis was performed by comparing the optical densities (ODs) of the bands of target genes to the OD of a reference gene (Tbp) and expressed as fraction of the basal values. Linearity was established by measuring the ODs of different concentrations of the same cDNA samples. There was a linear relationship between OD and sample concentration. It should be noted that the percentage changes cited in Results represent the changes in the OD of the mRNA studied, which may not be the same as the changes in the absolute amounts of mRNA.
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ADM Measurement by Radioimmunoassay
The peptides in the culture medium were extracted, purified, and concentrated by reversed-phase extraction using Sep-Pak Vac 3cc C18 Cartridges (Waters Corp., Milford, MA). The eluate was dried in a speed-vacuum concentrator (Savant, CA). The dried extract was reconstituted in radioimmunoassay (RIA), buffer and the ADM levels were assayed as detailed previously [18, 19].
A total of 100 µl ADM standards (0–500 pg; rat ADM1–50 peptide; Phoenix Pharmaceuticals) or samples were incubated with 100 µl 125I-labeled ADM (
10 000 cpm/tube; Phoenix Pharmaceuticals) and 100 µl rabbit anti-ADM serum (Peninsula, San Carios, CA) at 4°C overnight. Then, the reaction mixtures were incubated with 100 µl normal rabbit gamma globulin (Antibodies Inc., Davies, CA) in 1:100 dilutions, 100 µl goat anti-rabbit immunoglobulin G (IgG) serum (Antibodies Inc.) in 1:10 dilution, and 100 µl of 10% polyethyleneglycol at 4°C for 3 h. The bound and free peptides were separated by centrifugation at 4000 rpm at 4°C for 1 h. The pellets of the bound 125I-labeled ADM were counted using a gamma counter (Packard Cobra II Auto-gamma counting system). The sensitivity of the assay was 1–5 pg per tube. The intraassay and interassay coefficients of variation for ADM were 7% and 10%, respectively.
The peptide was extracted and reconstituted as described above. EDN1 was measured with an endothelin 1 RIA kit purchased from Phoenix Pharmaceuticals. The sensitivity of the assay was 1–2 pg per tube. The intraassay and interassay coefficients of variation for EDN1 were <5% and <14%, respectively.
Testosterone Measurement by Enzyme Immunoassay
Testosterone levels in the culture media were measured directly with a testosterone immunoassay kit purchased from Diagnostic Systems Laboratories Inc. (Webster, TX). The sensitivity of the assay was 0.04 ng/ml. The intraassay and interassay coefficients of variation were 4.2% and 5.6%, respectively.
All of the results represent mean ± SEM, and statistical analysis of the data was performed by one-way ANOVA followed by Newman-Keuls multiple comparison test, with P values of <0.05 regarded as significant.
The viability of the isolated cells was 90%–95%. The yield of the cells was 1.2 to 1.5 x 106 per testis. The purity of the cells determined by 3β-HSD was about 85%.
ADM Immunoreactivity and mRNA Expression of Adm, Calcrl, and Ramps
The amount of ADM secreted by the rat Leydig cells was found to be 274.9 ± 18.9 pg/106 cells at 24 h. The mRNA of Adm, Calcrl, Ramp1, Ramp2, and Ramp3 was expressed in the rat Leydig cells, as indicated by the specific single band obtained by RT-PCR using primers specific to each gene (results not shown).
Effect of ADM on Testosterone Secretion
ADM treatment had a significant effect on basal testosterone production. As shown in Figure 1A, 1 and 10 nM ADM had no effect on testosterone production, but 100 nM ADM moderately increased testosterone production by 14% (P < 0.001). ADM inhibited hCG-stimulated testosterone production (P < 0.001; Fig. 1B). The minimal effective dose of ADM was 1 nM (P < 0.05), and the reduction was up to 21% at 100 nM (P < 0.01). Similarly, ADM had a negative effect on the testosterone production induced by EDN1 (P < 0.001; Fig. 1C). A total of 1 nM of ADM was able to exert an inhibitory effect (P < 0.01), and the reduction was up to 20% at 100 nM (P < 0.001).
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Effect of ADM on EDN1 Production and the Gene Expression of EDN Receptors
ADM induced significant decreases on End1 mRNA expression at both 4 h (P < 0.01) and 12 h (P < 0.01; Fig. 2A). The minimal effective dose of ADM was 0.001 nM at both time points (P < 0.01 at 4 h and P < 0.05 at 12 h). Up to 30% reduction of Edn1 mRNA level was observed at 12 h at 1 nM (P < 0.01), whereas the maximal reduction of Edn1 mRNA level at 4 h was 20% at ADM concentrations of 1 and 10 nM (P < 0.01). In contrast to the reduction in mRNA level, EDN1 secretion at 4 h was not reduced significantly by ADM (Fig. 2B; lower left panel). On the other hand, ADM inhibited EDN1 secretion at 12 h (P < 0.01; Fig. 2B; lower right panel). The reduction was significant at ADM concentrations of 100 and 1000 nM, and the maximal reduction was 42% at 1000 nM (P < 0.01).
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The effect of ADM on Ednra and Ednrb mRNA levels was studied at 12 h. ADM exerted an inhibitory effect on the mRNA expression of both Ednra (P < 0.0001; Fig. 3A) and Ednrb (P < 0.0001; Fig. 3B). The inhibitory effect of ADM on Ednra mRNA level was more pronounced than on Edrnb mRNA level. The minimal effective doses of ADM on Ednra and Ednrb were 0.001 nM (P < 0.05) and 100 nM (P < 0.01), respectively, and the maximal reductions on Ednra and Ednrb were 44% and 24%, respectively, at 1000 nM (P < 0.01).
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Effect of EDN1 on ADM Production and the Gene Expression of Adm and ADM Receptor Components
EDN1 markedly increased both the Adm mRNA level (P < 0.001; Fig. 4A) and ADM secretion at 4 h (P < 0.001; Fig. 4B). The maximal stimulations in Adm mRNA and peptide levels were 133% at 1 nM (P < 0.001) and 95% at 10 nM (P < 0.001). Although EDN1 enhanced the production of ADM, it exerted no significant effect on the mRNA expressions of Calcrl (P = 0.0826) and Ramp2 (P = 0.1118; data not shown).
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Effect of hCG on ADM Production and the Gene Expression of Adm and ADM Receptor Components
Human CG attenuated the expression of Adm mRNA between 0.05 and 50 mIU/ml at 12 h (P < 0.001; Fig. 5A), with a maximal reduction of 35% at 5 mIU/ml (P < 0.001). ADM secretion was inhibited by hCG in the same concentration range (P < 0.001; Fig. 5B), with a maximal reduction of 21% at 5 mIU/ml hCG (P < 0.001). Human CG also exerted an inhibitory effect on the mRNA expression of both Calcrl (P < 0.001; Fig. 6A) and Ramp2 (P < 0.001; Fig. 6B) in the Leydig cells between 0.05 and 50 mIU/ml. The maximal reductions for Calcrl and Ramp2 mRNA levels were 43% (P < 0.001) and 29% (P < 0.001), respectively, at 50 mIU/ml hCG.
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ADM and its receptors have been found to be expressed in the whole testis in a recent study by our laboratory [12]. The present studies show that ADM is expressed and secreted by the isolated Leydig cells, which is in line with our previous finding using immunocytochemistry of the presence of ADM peptide in both the rat Sertoli and Leydig cells [12]. The secretory rate of ADM was similar to that of macrophages, reported previously [20]. In addition to the production of ADM, the results of RT-PCR demonstrated the expression of Calcrl and Ramps in the Leydig cells. The result is consistent with previous studies showing that ADM receptor is predominantly expressed in the interstitium in the rat testis [21] and only the Leydig cell is weakly stained for CALCRL in the human testis [22]. CALCA and ADM receptors have been reported to consist of CALCRL in association with one of the three RAMPs [4]. CALCRL couples with RAMP1 to result in a CALCA receptor, whereas its association with RAMP2 or RAMP3 produces an ADM receptor. On this basis, the expression of Calcrl and all three types of Ramps in the Leydig cells indicates the presence of both ADM and CALCA receptors. On this basis, both ADM and CALCA receptors are present in the Leydig cells. Furthermore, RAMP3 may combine with CALCRL to give rise to a receptor that binds to both ADM and CALCA, but the expression of Ramp3 is very low in the Leydig cells and was not studied further. Previous study has also shown the mRNA expression of ADM receptor in PMCs [21], and the existence of specific ADM receptors in these cells has been confirmed by receptor-binding studies [23]. These findings suggest that both Leydig cells and PMCs are possible target sites for ADM. CALCA has been shown to be produced in the vasculature as well as the nerve endings in the rat testis [22, 24].
ADM has been shown to inhibit hCG-stimulated testosterone secretion in the whole testis by acting on its specific receptor [12]. Human CG has actions similar to LH and is used in place of LH because it is more readily available. The production of ADM and the expression of ADM receptors suggest that ADM may act in an autocrine mode in the regulation of testosterone production in the Leydig cells. Indeed, the present studies demonstrate a dual effect of rat ADM peptide on steroidogenesis. ADM stimulated basal testosterone production only at high doses, but it inhibited hCG- and EDN1-stimulated testosterone production. This suggests that some of the pathways activated by ADM may stimulate basal testosterone production, whereas the others may inhibit the stimulatory actions of hCG and EDN1, and that its inhibitory effects predominate in the presence of hCG and EDN1. At 100 nM, ADM stimulated testosterone secretion even when it inhibited EDN1 production. In the present study, ADM also significantly reduced mRNA levels of Edn1 and Ednra, which has been shown to mediate the stimulatory action of Edn1 on rat Leydig cells [2]. Therefore, ADM may inhibit EDN1-stimulated steroidogenesis by reducing both EDN1 production and the responsiveness of the Leydig cells to EDN1. Besides, EDN1 is known to enhance hCG-stimulated steroidogenesis by augmenting intracellular Ca2+ [14], and the intracellular EDN1 level is significantly increased by hCG treatment [25], so ADM may inhibit hCG-stimulated testosterone production partly by its suppressive effect on EDN1.
Although hCG-stimulated testosterone production is almost completely suppressed by ADM in the whole testis [12], the present study shows that the maximal reduction is only 21% in isolated Leydig cells. This discrepancy suggests that ADM may also regulate hCG-stimulated steroidogenesis indirectly by acting on other types of testicular cells. In fact, the production of ADM as well as the inhibitory effect of ADM on EDN1 production has been demonstrated in the Sertoli cells in a separate study by our group [26]. EDN1 is mainly secreted by the Sertoli cells and is known to stimulate Leydig cell steroidogenesis [13]. ADM may therefore act on the Sertoli cells in addition to the Leydig cells to inhibit steroidogenesis in the testis. Besides, nitric oxide generated by ADM may inhibit steroidogenesis in a paracrine fashion in addition to autocrine regulation in the Leydig cell. In the testis, nitric oxide is mainly produced by endothelial cells and macrophages, which are in intimate association with Leydig cells [27].
The finding of an inhibitory ADM effect on EDN1-stimulated testosterone production in the Leydig cell is in line with the result in the myoid cells, in which ADM inhibited EDN1-induced PMC contraction [15]. Both suggest that ADM may regulate testicular functions by antagonizing the action of EDN1. In the Leydig cell, although EDN1 production is reduced by ADM, ADM production is enhanced by EDN1. Thus, ADM and EDN1 are interrelated at both the production and functional levels, and these two peptides may form an important local regulatory mechanism to fine tune hCG-stimulated steroidogenesis. The testicular level of EDN1 is upregulated by hCG (LH) carried by the blood to the testis, and this EDN1 then enhances the hCG (LH)-stimulated testosterone production [14]. ADM production is stimulated by the elevated level of EDN1. ADM, in turn, suppresses hCG (LH)-stimulated and EDN1-stimulated testosterone production, partly by decreasing the response to EDN (Leydig cells) as well as by inhibiting EDN1 production (Leydig cells and Sertoli cells).
Although ADM inhibits hCG-stimulated testosterone production, ADM production and the expression of Calcrl and Ramp2 in the Leydig cells are reduced by hCG. Therefore, ADM production in the Leydig cells as well as the responsiveness of the cells to ADM and possibly CALCA are under hCG (LH) control. Conversely, EDN1 production is stimulated by hCG in the rat testis [25]. Together with the finding that EDN1 enhances hCG-stimulated testosterone production while ADM suppresses it, the opposite regulatory effects of LH on EDN1 and ADM would enhance its stimulatory effect on testosterone synthesis. A proposed local regulatory mechanism formed by ADM and EDN1 on the regulation of testosterone production stimulated by hCG (LH) in rat testis is shown in Figure 7.
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In summary, the effects of hCG/LH in simultaneously stimulating EDN production and inhibiting ADM production would augment its direct stimulatory effect on steroidogenesis. The increase in EDN would then increase ADM, which would inhibit LH-stimulated steroidogenesis directly and indirectly via a decrease in EDN secretion. The result is an augmented acute effect (i.e., a bigger increase in a short time) and a curtailed longer-term effect (i.e., return to normal sooner). The significance is that without an EDN or ADM effect, steroidgenesis would solely depend on the change of LH level. On top of this, an increase in testosterone also stimulates ADM gene expression (Tang, unpublished results) to increase the inhibitory effect of ADM on LH-stimulated steroidogenesis.
This is the first study to demonstrate the gene expression and secretion of ADM as well as the mRNA expression of Calcrl and Ramps in rat Leydig cells. This, together with the functional studies of the effects of ADM on Leydig cell testosterone, suggests that ADM may represent a novel local factor in the regulation of steroidogenesis, and possibly spermatogenesis. In addition, an autocrine/paracrine regulatory loop between ADM and EDN1 is established which may be important in fine-tuning the gonadotropin-mediated testicular functions in different part of the testis.
FOOTNOTES
1Supported by grant HKU 7451/04M from the Research Grants Council of Hong Kong and a merit award from the University of Hong Kong. 
Correspondence: 2Fai Tang, Department of Physiology, Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong, China. FAX: 852 28559730; e-mail: HUftang{at}hkucc.hku.hku
Received: 12 February 2007.
First decision: 28 March 2007.
Accepted: 20 November 2007.
REFERENCES
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  Y.-F. Chan, F. Tang, and W.-S. O Adrenomedullin in the Rat Testis. II: Its Production, Actions on Inhibin Secretion, Regulation by Follicle-Stimulating Hormone, and Its Interaction with Endothelin 1 in the Sertoli Cell Biol Reprod, April 1, 2008; 78(4): 780 - 785. [Abstract] [Full Text] [PDF]
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