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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
The present study demonstrates the expression of adrenomedullin (ADM) in the rat Sertoli cells and its effect on inhibin production. The regulation of ADM by FSH and its interaction with endothelin 1 (EDN1) in the rat Sertoli cells have also been established. Primary culture of Sertoli cells secreted 414 ± 27 pg immunoreactive ADM per 106 cells in 24 h and expressed Adm mRNA. In addition, the Sertoli cell was shown to co-express mRNAs encoding for the calcitonin receptor-like receptor (CALCRL) and receptor activity-modifying proteins (RAMPs) 1–3. These may account for the specific binding of ADM to the Sertoli cells. Administration of ADM to Sertoli cells resulted in an enhancement of basal and FSH-stimulated inhibin B production. On the other hand, the production of ADM and the mRNA levels of Calcrl and Ramp2 in the Sertoli cells were suppressed by FSH. The results suggest that ADM, via its control in the secretion of inhibin B, may play a role in regulating spermatogenesis as well as the hypothalamus-pituitary-gonad feedback system. In addition, like in the Leydig cell, ADM and EDN1 were found to regulate the production of each other in opposite directions in the Sertoli cells, suggesting the presence of yet another local regulatory mechanism in the rat testis that may be important in modulating testicular functions regulated by gonadotropins.
adrenomedullin, endothelin 1, Sertoli cells, testis
Adrenomedullin (ADM), a ubiquitous peptide hormone with multiple functions, its receptor, and its receptor activity-modifying proteins (RAMPs) have been shown in the rat testis, and a paracrine effect of ADM on testicular steroidogenesis is suggested by our group [1]. ADM has been found to inhibit testosterone production in isolated rat Leydig cells under stimulation by hCG and endothelin 1 (EDN1). In these cells, EDN1 production was reduced by ADM, whereas ADM production was increased by EDN1. Furthermore, administration of gonadotropin to the Leydig cells suppressed the production of ADM as well as the mRNA levels of calcitonin receptor-like receptor (CALCA) and RAMP2 [2].
We also have studied ADM secretion and the mRNA expression of Adm, Calcrl, and Ramps and their effects on inhibin production in isolated Sertoli cells. Inhibin is an important endocrine secretion, as it may stimulate testicular testosterone production [3] and inhibits pituitary FSH secretion [4]. It is a marker for both Sertoli cell function and spermatogenesis [5]. As EDN1 is produced by Sertoli cells [6], its interaction with ADM and the effects of ADM and EDN1 on the production of each other in these cells were investigated. The regulation of the synthesis of ADM by FSH in the Sertoli cell was also studied.
Three-week-old male Sprague-Dawley rats were obtained from the Laboratory Animal Unit, Faculty of Medicine, of the 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, the University of Hong Kong, and were carried out in accordance with the guidelines for the Care and Use of Laboratory Animals (National Academy of Sciences).
Sertoli cells were isolated from 3-wk-old male Sprague-Dawley rats using a method modified from Ko et al. [7]. For each study, three rats were killed by an overdose of sodium pentobarbital (40 mg per 100 g of rat; Dorminal 20%; Alfasan, Woerden, Holland). The testes were excised rapidly and washed twice in 1x phosphate-buffered saline (PBS) containing 100 U/ml penicillin G and 100 µg/ml streptomycin (1% PS) (GIBCO-BRL, Grand Island, NY). Decapsulated testes were cut and digested with 20 ml Dulbecco modified Eagle medium (DMEM)/F12 Ham (1:1; GIBCO-BRL) supplemented with 1% PS and 15 mM HEPES (Sigma, St. Louis, MO) (DMEM, pH 7.2) and containing 2.5 g/l trypsin, 1 g/l collagenase type IA, 1 g/l hyaluronidase, and 10 mg DNase I (all from Sigma) in a shaking water bath (Sheldon Manufacturing Inc., Cornelius, OR) at 80 cycles/min at 37°C for 10 min. The tubules were digested for no more than 10 min; at the end of incubation, 20 ml ice-cold DMEM medium was added to stop the digestion. The cell suspension was allowed to settle, and interstitial cells were removed by decanting the supernatant. The resulting cell pellet was resuspended in DMEM medium and filtered through 70-µm nylon mesh (BD Biosciences, Bedford, MA) to remove undigested fragments. The cells then were washed twice with the medium, followed by centrifuging at 900 rpm for 5 min. The washed cells were resuspended in DMEM medium containing 10 µg/ml bacitracin, 10 µg/ml insulin, and 5 µg/ml transferrin (all from Sigma). The viability of the isolated Sertoli cells was examined each time by trypan blue exclusion test and was not lower than 95%. Sertoli cells (1 x 106 cells per well) were plated in a six-multiwell plate (Iwaki; Asahi Techno Glass, Chiba, Japan) and cultured at 34°C. Two days after plating, the cells were treated with a hypotonic solution (20 mM Tris-HCl, pH 7.4) at room temperature for 2.5 min to lyse any contaminating germ cells. The Sertoli cells were allowed to recover for 24 h in DMEM supplemented with 0.1% (w/v) bovine serum albumin (DMEM-BSA; Sigma) before experimentation.
The purity of the cell was determined by vimentin immunofluorescence microscopy. Isolated Sertoli cells grown on a sterilized 24 x 24 mm cover glass in a six-multiwell plate were fixed with neutral-buffered formalin at room temperature. The fixed cells were rinsed twice with 1x PBS containing 0.5% BSA (w/v; PBS-BSA) at room temperature. Anti-mouse vimentin monoclonal antibody (Sigma) was diluted 1:40 in PBS-BSA and incubated with cells at room temperature for 1 h. Bound primary antibody was detected with an anti-mouse IgG (Fab specific) fluorescein isothiocyanate (FITC)-conjugated secondary antibody (Sigma) diluted 1:200 in PBS-BSA by incubation at 37°C for 30 min. Propidium iodide (PI) was employed as a nuclear counterstain. Images were digitized using a Zeiss Axiophot microscope equipped with Diagnostic Instruments SPOT RT digital camera systems. FITC and PI were detected by excitation filters at 450–490 nM and 546 nM, respectively. The purity of the Sertoli cells isolated was measured by determining the fraction of FITC/PI double-stained cells in the cell population (PI-stained nuclei) and was not lower than 95% for each isolation.
Hormonal Treatments in Sertoli 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 and FSH-stimulated inhibin production, the cells were incubated with 10 or 100 nM of ADM peptide in the presence or absence of 0.05 IU/ml recombinant FSH (Serono Laboratories Inc., Geneva, Switzerland) for 24 h. In the study of ADM effect on EDN1 synthesis, the cells were incubated with 0.001–100 nM of rat adrenomedullin (1–50) peptide (Phoenix Pharmaceuticals Inc., Belmont, CA) for 3 or 8 h while in the study of EDN1 effect on ADM synthesis the cells were incubated with 0.001-1000 nM of rat endothelin 1 peptide (Phoenix Pharmaceuticals) for 4 or 12 h. In the study of the effect of FSH on ADM production, the cells were incubated with 0.0005–2.5 IU/ml FSH for 24 h. After incubation, the supernatants were collected and stored frozen at –70°C immediately until ADM, EDN1, or inhibin secretion was measured. The cells of all the experiments except for the studies of inhibin production were used for RNA extraction for the studies of gene expression.
The total RNA of Sertoli cells in each well was extracted by TRIZOL, and the mRNA of Adm, Calcrl, Ramps, Edn1, and Edn receptors was determined by RT-PCR according to the method of Li et al. [1] with some modifications. The details have been previously described [2].
Measurement of ADM and EDN1 by Radioimmunoassay
ADM was measured by radioimmunoassay (RIA) using 125I-labeled ADM and antibody from Phoenix. EDN1 was measured by a commercial RIA kit available from Phoenix Pharmaceuticals. The details of the procedures have been previously described in Chan et al. [2].
Total Inhibin Measurement by Enzyme Immunoassay
Total inhibins (inhibins A and B) in the culture media were measured directly with specific two-site enzyme immunoassays (EIAs) to discriminate inhibin A and inhibin B. The EIA kits were purchased from Diagnostic Systems Laboratory (Webster, TX). The sensitivity of the assays was 1 pg/ml for inhibin A and 7 pg/ml for inhibin B. The intraassay and interassay coefficients of variation were <5% and <14%, respectively, for both assays.
All of the results represent mean ± SEM, and statistical analysis of the data was performed by one-way ANOVA (Figs. 1, 2, and 4) followed by Newman-Keuls multiple comparison test, with P < 0.05 regarded as significant.
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ADM Immunoreactivity and mRNA Expression of Adm, Calcrl, and Ramp
The amount of ADM secreted by the rat Sertoli cells was found to be 413.6 ± 26.5 pg/106 cells per 24 h. The mRNAs of Adm, Calcrl, and Ramp1 to Ramp3 (results not shown) were expressed in the rat Sertoli cells, as indicated by the specific single band obtained by RT-PCR using primers specific to each gene [2].
Effect of ADM on Total Inhibins Secretion
ADM stimulated basal inhibin A and inhibin B production in a dose-dependent manner at 10 and 100 nM (P < 0.001; Fig. 1). The stimulation was only 7% for inhibin A (P < 0.05), but it was greater than 100% for inhibin B (P < 0.001). FSH inhibited inhibin A secretion, whereas the stimulatory effect of ADM partially restored this level back toward the basal level. In contrast to inhibin A, ADM stimulated Sertoli cells to secrete 113% more inhibin B in the basal state. Under FSH stimulation, the Sertoli cells increased inhibin B secretion by 162% (P < 0.001), and the addition of ADM further enhanced the Sertoli cells to secrete inhibin B (at 100 nM by 46%; P < 0.001).
Effect of ADM on EDN1 Production and the Gene Expression of EDN Receptors
ADM inhibited Edn1 mRNA expression at 3 h between 1 nM and 1 µM (P < 0.001; Fig. 2A). The inhibitory effect continued up to 8 h at 1 pM to 1 µM ADM (P < 0.001; Fig. 2A). The maximal reductions at 3 h and 8 h were 39% (P < 0.001) and 30% (P < 0.001), respectively, at 1 µM ADM. In contrast to Edn1 mRNA expression, significant inhibition of EDN1 peptide secretion was observed only at 8 h (P < 0.001; Fig. 2B). The minimal effective dose of ADM was 1 nM (P < 0.05), whereas the maximal reduction of EDN1 peptide secretion was 52% at 1000 nM (P < 0.001; Fig. 2B).
ADM exerted an inhibitory effect on the mRNA expressions of both Ednra (P < 0.001; Fig. 3A) and Ednrb (P < 0.001; Fig. 3B) at 8 h. The maximal reductions on both receptor subtypes were similar, whereas the minimal effective doses of ADM on Ednra and Ednrb were 0.001 nM and 1 nM, respectively.
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Effect of EDN1 on ADM Production and the Gene Expression of Adm and Adm Receptor Components
EDN1 stimulated Adm mRNA expression at 4 h at all doses (P < 0.001; Fig. 4A, left panel). The maximal stimulation was 74% at 100 nM EDN1 (P < 0.001). The stimulatory effect of EDN1 persisted at 12 h at 1 and 100 nM (P < 0.001) but was much less than that at 4 h (Fig. 4A, right panel). The effects of EDN1 on ADM secretion were consistent with but more marked than its stimulatory effects on Adm mRNA expression. At 4 h, it stimulated ADM secretion between 1 and 1000 nM (P < 0.001; Fig. 4B, left panel), and there was a maximal stimulation of 125% at 1000 nM (P < 0.001). At 12 h, ADM secretion was enhanced at EDN1 concentrations of 1 to 1000 nM (P < 0.001), with a maximal stimulation of 47% at 10 nM (P < 0.001; Fig. 4B, right panel).
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The effects of EDN1 on the mRNA expressions of Calcrl and Ramp2 in the Sertoli cells were studied at 12 h. EDN1 concentrations at 1–1000 nM stimulated Calcrl mRNA expression significantly (P = 0.0006; Fig. 5A), with a maximal stimulation of 19% at 10 nM (P < 0.01). The mRNA expression of Ramp2 was upregulated at 10–1000 nM EDN1 (P < 0.001), with a maximal stimulation of 25% at 1000 nM (P < 0.001; Fig. 5B).
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Effect of FSH on ADM Production and the Gene Expression of Adm Receptor Components
The mRNA expression of Adm mRNA was inhibited by FSH between 0.0005 and 0.5 IU/ml at 24 h (P < 0.001), and the maximal reduction was 57% (P < 0.001) at 0.5 IU/ml FSH (Fig. 6A). The reduction of Adm mRNA expression was accompanied by a reduction of ADM secretion (P < 0.001), and the maximal reduction was 68% at 0.5 IU/ml FSH (P < 0.001; Fig. 6B).
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FSH also significantly reduced the mRNA expressions of Calcrl (P < 0.001; Fig. 7A) and Ramp2 (P < 0.001; Fig. 7B) in the Sertoli cells at 24 h. The reduction of Calcrl mRNA level was observed to have a maximal reduction of 55% at 0.5 IU/ml (P < 0.001). FSH decreased Ramp2 mRNA levels between 0.005 and 2.5 IU/ml, and the maximal reduction was 32% at 0.5 IU/ml (P < 0.01).
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The result that ADM is expressed and secreted by the isolated Sertoli cells is in line with our previous finding of the presence of ADM peptide in both the Sertoli and Leydig cells in the rat testis [1] and the expression of ADM in the Sertoli cell [8]. This is in contrast to the finding in the human testis, in which immunoreactive ADM is detected mainly in germ cells and in a small number of Leydig cells and peritubular myoid cells, but not in Sertoli cells [9]. In addition to the production of ADM, mRNA of Calcrl and all three Ramps were found in the isolated Sertoli cells. This indicates the presence of both ADM and CALCA receptors. CALCA has been shown to be produced in the vasculature as well as the nerve endings in the rat testis [10, 11].
The co-expression of ADM and ADM receptors suggests an autocrine regulation of Sertoli cell function. ADM was found to have a dual effect on testosterone production in the Leydig cells in that it stimulates basal but inhibits hCG-stimulated testosterone production [2]. Another dual effect of ADM has been found in aldosterone production in the zona glomerulosa [12–14]. However, a stimulatory effect of ADM on both basal and FSH-stimulated inhibin production in the Sertoli cells was reported here.
Using the specific two-site EIA kits, the production of inhibin A and inhibin B from the isolated Sertoli cells could be discriminated [15–17]. As Sertoli cells secrete very low levels of inhibin A compared with inhibin B (basal level seven times lower; FSH-stimulated level 20 times lower, Fig. 1), we only considered the changes in inhibin B secretion in our discussion. The ADM secreted by the Sertoli cells may have a paracrine inhibitory effect on steroidogenesis in the Leydig cells [1]. The stimulatory effect of ADM on inhibin production suggests that ADM secreted by these cells may also indirectly regulate testicular function via a modulation of inhibin secretion, although the stimulatory effect of inhibin on Leydig cell steroidogenesis is controversial [3, 18, 19].
ADM and EDN1 have opposite effects on the levels of each other in Sertoli cells, which suggests the presence of a local regulatory mechanism formed by ADM and EDN1 in the rat testis. ADM inhibited Edn1 mRNA expression, and there was a time lag between the reduction in Edn1 mRNA level and peptide secretion, suggesting that ADM may affect Edn1 gene transcription directly, with a subsequent reduction in EDN1 peptide secretion. ADM also may reduce the responsiveness of the Sertoli cells to EDN1 stimulation via an inhibition of Ednra and Ednrb gene expression. On the other hand, both ADM production and the gene expression of Calcrl and Ramp2 are enhanced by EDN1. In short, ADM and EDN1 have opposite effects on the levels of each other in Sertoli cells, which suggests the presence of a local regulatory mechanism formed by ADM and EDN1 in the rat testis. Similar regulatory loops between ADM and EDN1 have been demonstrated in vascular smooth muscle cells [20, 21], aortic endothelial cells [22], and Leydig cells [2]. Most of the EDN1 in the rat testis is produced by Sertoli cells, whereas EDN receptors are abundant in the Leydig cells and peritubular myoid cells [5]. On this basis, the main action of EDN1 may be paracrine.
Although the effect of EDN1 on inhibin production has not been studied, the findings that EDN1 is known to inhibit FSH-induced cAMP formation [23] and that cAMP increases inhibin B production [24] suggest that ADM may be inhibitory. ADM and EDN1 may therefore interact to regulate inhibin production in the Sertoli cells. However, the Sertoli cells are not like the Leydig cells, in which EDN1 stimulates while ADM inhibits steroidogenesis.
Although ADM was found to augment FSH-stimulated inhibin production, FSH markedly reduces ADM production in Sertoli cells. The inhibitory effects of FSH on Adm mRNA expression and ADM release were maintained over a 24-h incubation period. In addition, the mRNA levels of Calcrl and Ramp2 were reduced significantly by FSH. A similar suppressive effect of FSH on EDN1 has also been shown in the Sertoli cell [5]. Both FSH and ADM stimulated inhibin secretion in Sertoli cells. When FSH secretion increases, FSH inhibits ADM production and thereby increases EDN secretion, whereas its direct effect is to inhibit EDN production. A proposed local regulatory mechanism formed by ADM and EDN1 on the regulation of inhibin production stimulated by FSH in rat testis is shown in Figure 8.
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The Sertoli cell is similar to the Leydig cell [2] in the following aspects: (1) the effects of ADM and EDN on each other; (2) the effects of FSH or LH on ADM. These cells differ in the following ways: (1) In the Leydig cell, ADM increases basal testosterone secretion but decreases gonadotropin (LH)-stimulated testosterone production, whereas in the Sertoli cell, it stimulates both basal and gonadotropin (FSH)-stimulated inhibin production. (2) EDN may inhibit FSH-stimulated inhibin in the Sertoli cells, whereas it stimulates LH-stimulated testosterone secretion in the Leydig cell. (3) FSH inhibits both ADM and EDN secretion in the Sertoli cell, whereas LH inhibits ADM but stimulates EDN secretion in the Leydig cell.
This is the first study to demonstrate gene expression of Calcrl and Ramps as well as gene expression of Adm and secretion of ADM in rat Sertoli cells. This, together with the functional studies of the effects of ADM on inhibin production by the Sertoli cells, suggests that ADM may represent a novel local factor in the regulation of spermatogenesis. In addition, an autocrine regulatory loop between ADM and EDN1 is established, and this may be important in fine tuning the gonadotropin-mediated testicular functions in different parts of the testis. The regulation of ADM by gonadotropins and the stimulatory roles of ADM on inhibin secretions also suggest that it may play a role in regulating the hypothalamus-pituitary-gonad feedback system.
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: 2Wai-Sum O, Department of Anatomy, Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong, China. FAX: 852 2817 0857; e-mail: owaisum{at}hkucc.hku.hk
Received: 12 February 2007.
First decision: 28 March 2007.
Accepted: 6 December 2007.
REFERENCES
This article has been cited by other articles:
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  Y.-F. Chan, W.-S. O, and F. Tang Adrenomedullin in the Rat Testis. I: Its Production, Actions on Testosterone Secretion, Regulation by Human Chorionic Gonadotropin, and Its Interaction with Endothelin 1 in the Leydig Cell Biol Reprod, April 1, 2008; 78(4): 773 - 779. [Abstract] [Full Text] [PDF]
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