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Baker Heart Research Institute (G.C.D., D.K.L.L., M.A.Y., A.I.S., R.A.L.), Melbourne 8008; Monash University Institute of Reproduction and Development (G.C.D., M.K.O., M.P.H.), Clayton 3168; and the Australian Research Council Centre of Excellence in Biotechnology and Development (M.K.O.), Canberra 2601, Australia
Address all correspondence and requests for reprints to: A. Ian Smith, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia. E-mail: ian.smith{at}med.monash.edu.au.
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Introduction |
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The ACE gene encodes both a somatic isozyme, which is expressed in many tissues of the body, including testicular endothelial and Leydig cells, and a testis-specific isozyme (termed testicular or germinal ACE), which is found only in spermatids and spermatozoa (4). Testicular ACE has been shown to play an essential role in the control of the male reproductive system by the infertility of mice deficient in the testicular isoform of ACE but replete in somatic ACE (5, 6, 7).
A homolog of ACE, termed ACE2 or ACEH, which has recently been cloned, is most highly expressed in the heart, the kidney, and the testis, implicating the enzyme in both cardiovascular and reproductive function (8, 9). In vitro studies suggest that this enzyme is a carboxypeptidase with a limited substrate specificity and is insensitive to inhibitors of ACE (8, 9). Unlike the dipeptidase ACE, ACE2 removes only a single residue from the carboxyl terminus of certain peptides, including Ang II. Thus, ACE2 functions as an Ang II-degrading enzyme, forming the vasodilator peptide Ang (1, 2, 3, 4, 5, 6, 7) (10, 11). The importance of ACE2 in cardiovascular function is evident from the severe cardiac contractility defects observed in the ACE2 knockout mouse, a phenotype that was rescued by concomitant ACE ablation (12). These findings support the concept that ACE2 acts as a counter-regulator of ACE in vivo, limiting the level of Ang II, at least in the heart. The role of ACE2 in the testis, the tissue with the highest level of expression, however, remains largely unknown, particularly because ACE2 knockout male mice are fertile.
In this study, we report the cloning of ACE2 from a human testicular cDNA library and characterization of the expressed protein. Using a specific quenched fluorescent substrate (QFS), we demonstrate ACE2-like catalytic activity in membrane preparations of rat testis and enriched Leydig cells. Furthermore, we describe the cellular immunolocalization of ACE2 protein in rat and human testes and the developmental expression of ACE2 in the rat testis.
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Materials and Methods |
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Reagents
All reagents were obtained from Sigma Chemical Company (St. Louis, MO) unless otherwise stated. The ACE2 QFS, (7-methoxycoumarin-4-yl)-acetyl-Ala-Pro-Lys (2,4-dinitrophenyl) (10), was synthesized by Auspep (Parkville, Victoria, Australia). Z-Pro-prolinal (ZPP) was a generous gift from Dr. S. Wilk (Mount Sinai Hospital, New York, NY). N-[1(R, S)-carboxyl-3-phenylpropyl]-leucine (cFP-Leu) was synthesized by Karen Stewart (Department of Biochemistry and Molecular Biology, Monash University, Clayton, Australia). The specific ACE2 inhibitor, (S, S) 2-{1-carboxy-2-[3-(3,5-dichloro-benzyl)-3H-imidazol-4-yl]-ethylamino}-4-methyl-pentanoic acid (MLN-4760) (12), was a generous gift of Dr. Natalie Dales (Millenium Pharmaceuticals, Cambridge, MA). Ethane dimethane sulfonate (EDS) was manufactured by Dr. Michael Fuller (Department of Chemistry, Monash University) using the protocol of Edwards et al. (13).
Cloning of ACE2 from human testis cDNA library
Gene-specific primers were designed based on the published human ACE2 sequence (GenBank accession no. AF241254) (9). The forward primer was 5'-GGTACCATGTCAAGCTCTTCCTGGCTCC-3', and the reverse primer was 5'-GCGGCCGCCTAAAAGGAGGTCTGAACATCATCAGT-3'. These primers were used to amplify the cDNA from a human testis quick-clone cDNA library (catalog no. 7117–1; Clontech, Palo Alto, CA). This was accomplished by using a two-step PCR protocol using Advantage 2 DNA polymerase (Clontech). The PCR product was ligated into pcDNA3.1/V5-His-TOPO according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). The cDNA sequence was verified by DNA sequence analysis on a 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA).
Expression of recombinant ACE2 in P-selectin-transfected Chinese hamster ovary (CHO-P) cells
ACE2 was expressed in CHO-P cells using a modified diethylaminoethyl-dextran method described by al-Moslih et al. (14). Cells were harvested 72 h post transfection, and membranes were prepared as follows.
Preparation of an enriched population of rat Leydig cells
Adult rat Leydig cells were prepared by enzymatic dispersal followed by centrifugal elutriation essentially as described previously (15). The purity of the Leydig cell fraction, as determined by 3ß-hydroxysteroid dehydrogenase histochemistry, was approximately 90%.
Hormonal regulation of testicular ACE2
To investigate the possible hormonal regulation of ACE2 in the testis, adult rats (100 d old) received sc low-dose (3 cm long) and high-dose (3 x 8 cm long) testosterone implants and were maintained for a total of 10 d before assessment, as previously described (16, 17). The control group (no implants) and each treatment group consisted of three animals. At the end of the experimental period, testes were removed under ether anesthesia, weighed, and snap frozen in liquid nitrogen before homogenization and QFS assay.
Preparation of membrane fractions from testes, Leydig cells, and CHO-P cells
Testes were homogenized in ice-cold Tris-buffered saline (TBS; 100 mM Tris and 1 M NaCl, pH 6.5) using a small hand-held homogenizer. Testis homogenates, Leydig cells, and CHO-P cell preparations were subjected to a cycle of freeze/thaw (three times) and then sonicated briefly on ice. The homogenates were centrifuged at 4 C for 1 h at 100,000 x g, the supernatants were reserved (soluble fraction), and the membrane pellets were washed in ice-cold TBS and recentrifuged. After resuspension in TBS, the protein content of the membrane and soluble fractions was determined by the bicinchoninic acid method (Micro BCA Protein Assay Reagent Kit; Pierce, Rockford, IL), using BSA as standard.
QFS assay of ACE2 activity
Membranes from ACE2-transfected CHO-P cells, homogenized rat testis, and Leydig cell preparations were incubated with an ACE2-specific QFS, (7-methoxycoumarin-4-yl)-acetyl-Ala-Pro-Lys (2,4-dinitrophenyl), as previously published (10). Assays were performed in black 96-well microtiter plates with 50 µM QFS in a final volume of 100 µl/well TBS. Membranes were also assayed in the presence of MLN-4760, a specific inhibitor of ACE2, to confirm cleavage by this protease. The potential contribution of prolyl endopeptidase to QFS cleavage was determined by addition of ZPP (1 µM). Reactions proceeded at 37 C for 30–120 min within a temperature-controlled fluorescence microplate reader (fmax; Molecular Devices, Sunnyvale, CA) before reading the liberated fluorescence (
ex = 320 nm, em = 420 nm).
Cleavage of Ang II by testicular and expressed ACE2
The cleavage of synthetic Ang II (
10 µM) by membranes from ACE2-transfected CHO-P, mock-transfected CHO-P, or homogenized testis was investigated by HPLC analysis using a Zorbax Eclipse C18 column and an Agilent 1100 series LC with online mass spectrometric detector (Agilent Technologies, Palo Alto, CA) for identification of peptide fragments.
Generation of ACE2-specific antisera
Synthetic peptides corresponding to amino acids 107–116 (ACE2107) and 124–132 (ACE2124) of human ACE2 were synthesized by Auspep, with an additional C-terminal Cys residue for thiol coupling to keyhole limpet hemocyanin. The sequence of the 124–132 peptide (STIYSTGKV) was identical to the corresponding peptide in rat ACE2, whereas the 107–116 sequence (VLSEDKSKRL) differed from the rat ortholog at four residues. Both peptide sequences are unique to ACE2 and are not found in the homolog ACE. Coupling of synthetic peptides to the carrier protein, keyhole limpet hemocyanin, was carried out essentially as previously described (18). Antibodies were raised in New Zealand White rabbits and affinity purified from pooled sera using standard protocols (19, 20). Rabbit nonimmune IgG was also prepared from sera collected before immunization as previously described (21).
Immunohistochemistry of ACE2 in rat testis
The distribution of ACE2 protein was performed using the Dako Autostainer (Dako, Carpinteria, CA) as described previously (22). Testes were perfusion fixed with Bouin’s fixative and processed into paraffin (23). Endogenous peroxidase activity in dewaxed and rehydrated testis sections was blocked by a 5-min incubation in Peroxidase Block (Dako). Antibody access was improved using an antigen retrieval method as described previously (23). The sections were subsequently incubated with anti-ACE2124 (10 µg/ml) for 120 min and an Envision polymer-antirabbit-horseradish peroxidase (Dako) for 15 min and visualized using diaminobenzidine tetrahydrochloride (Dako) for 5 min. Sections were counterstained using Mayer’s hematoxylin and mounted under glass using p-xylene-bis(N-pyridinium bromide). Sections were analyzed and photographed using a BH2 microscope (Olympus, Tokyo, Japan).
Selective destruction of Leydig cells by EDS treatment
The cellular localization and developmental regulation of ACE2 in the rat testis was also investigated in testes depleted of Leydig cells using the specific Leydig cell cytotoxin EDS. Briefly, rats were injected (ip) with either EDS (7.5 mg/100 g body weight) dissolved in dimethylsulfoxide (DMSO) and water (1:3 vol/vol) or a single injection (ip) of DMSO and water vehicle only. Animals were killed at 7, 14, 21, 28, and 49 d after injection, and tissues were processed for immunohistochemistry as described earlier. After selective destruction of Leydig cells in mature rats treated with EDS, Leydig cells are completely absent from the rat testis for a period of 1–2 wk, but the Leydig cell population is progressively restored to normal within 49 d by recruitment of interstitial precursor cells (24).
Immunohistochemistry of ACE2 in human testis
Human testis tissue was obtained with consent from a healthy human donor with unexplained testicular pain requiring orchidectomy. A portion of testis was fixed with Bouin’s fixative and embedded in paraffin as described previously (23). The distribution of ACE2 protein within adult human testis was determined using anti-ACE2107 (10 µg/ml) in an avidin-biotin amplified immunohistochemical method (23). The specificity of the staining was established by replacing the primary ACE2 antiserum with either ACE2 antiserum preabsorbed overnight at 4 C with a 10-fold excess (wt/wt) of the immunizing ACE2 peptide before immunohistochemistry or the appropriate nonimmune rabbit sera.
Western blot analysis of testicular and expressed ACE2
Membranes from ACE2-transfected CHO-P cells, mock-transfected CHO-P cells, homogenized rat testis, or Leydig cell preparations were size fractionated on 6% SDS-PAGE gels by standard methods. Proteins from the gel were transferred onto polyvinylidene difluoride (ImmobilonTH-P; Millipore, Bedford, MA) membrane, blocked with 5% (wt/vol) skim milk powder in TST buffer [50 mM Tris-base, 500 mM NaCl, 0.05% Tween 20 (polyoxyethylenesorbitan monolaurate)] overnight at 4 C, and incubated with anti-ACE2124 (10 µg/ml) in TST containing 0.5% (wt/vol) skim milk for 1 h at room temperature. After washing, the membrane was incubated with an affinity-purified antirabbit IgG conjugated to horseradish peroxidase (Amrad, Richmond, Victoria, Australia) at a 1:1000 dilution for 1 h. Bound antibody was visualized on X-OMAT blue film (Eastman Kodak, Rochester, NY) using a chemiluminescence detection system as outlined by the manufacturer (Perkin-Elmer Life Sciences, Boston, MA).
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Results |
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). The expressed ACE2 was sensitive to the specific inhibitor designed by Millenium Pharmaceuticals, MLN-4760 (100 nM), as well as an inhibitor of our own design, cFP-Leu (Fig. 1B). Inhibition of ACE2 by cFP-Leu was dose dependent, with an IC50 of approximately 10 µM and the highest dose (100 µM) resulting in 87 ± 6% (mean ± SEM, n = 3) inhibition of activity. In contrast, the activity present in mock-transfected cells could not be substantially inhibited by either MLN-4760 (Fig. 1A) or by the prolyl endopeptidase inhibitor, ZPP (data not shown), suggesting the presence of other QFS-cleaving peptidases in CHO-P cell membranes. Membrane preparations of ACE2-transfected cells, but not mock-transfected cells, also showed significant catalytic activity against the putative physiological substrate, Ang II, to form Ang (1, 2, 3, 4, 5, 6, 7), as assessed by HPLC and mass spectrometry (data not shown).
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). The lack of QFS-cleaving activity in the soluble fraction of the testis when ZPP was present suggests that, unlike testicular ACE, ACE2 in the testis is membrane bound as it is in the heart and kidney. In support of this conclusion, QFS degradation by testis membranes increased in a time-dependent manner and was not inhibited by ZPP (Fig. 2, A and B). Substantial inhibition of testicular membrane ACE2 activity was obtained with cFP-Leu (100 µM), MLN-4760 (100 nM), or the metal ion chelator, EDTA (5 mM; Fig. 2B). ACE2-like activity was also detected in membrane preparations from enriched Leydig cells (Fig. 2A), which, like the activity in membranes from whole testis, was inhibited by MLN-4760 (Fig. 2B). Neither low- nor high-dose testosterone implants significantly altered the testicular levels of ACE2 catalytic activity (Fig. 3).
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, lanes A and B) (9). A band with comparable molecular mass was detected in the Leydig cell membrane fractions (Fig. 4, lane C), again suggesting that ACE2 in this tissue exists as the full-length, membrane-bound enzyme. The remaining bands were deemed nonspecific compared with identical blots probed with nonimmune serum (data not shown) or by their presence in mock-transfected CHO-P cells probed with anti-ACE2124 (Fig. 4, lane B).
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). No staining was observed within seminiferous tubules. There also appeared to be no staining of endothelial cells, macrophages, or peritubular cells. The staining of the Leydig cells was quite variable, with some cells showing little or no staining, whereas other cells displayed intense staining. The specificity of the ACE2 antiserum used for these immunohistochemical studies was confirmed by the lack of staining when the primary antibody was preabsorbed with a 10-fold excess of immunizing peptide before immunohistochemistry (Fig. 5B). Similarly, substitution of the primary antibody for nonimmune rabbit serum resulted in no staining (data not shown).
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), or 21 post treatment. By 28 d post treatment, scattered clusters of recovering Leydig cells were weakly positive, although many cells with distinct Leydig cell morphology remained unstained (Fig. 5D). By 49 d after EDS treatment, the staining pattern was indistinguishable from the normal adult pattern (data not shown). All DMSO-treated control testes also showed a normal adult staining pattern (data not shown).
Very few positive cells were observed in the interstitial tissue of testes from rats aged 1–5 d postpartum (Fig. 5E). There was a progressive increase in the number of positive cells between d 7 and 19 postpartum (Fig. 5F), particularly in interstitial areas adjacent to the testicular capsule. However, it was noted that many cells possessing the morphological characteristics of immature Leydig cells were not stained at these ages. The majority of Leydig cells were strongly positive for ACE2 from d 22 onwards (Fig. 5G).
Immunolocalization of ACE2 in human testes
ACE2 immunoreactivity was also observed in normal human testis, using an antiserum raised against residues 107–116 (Fig. 5H). There was no immunostaining of human testis sections probed with preabsorbed ACE2 antiserum (data not shown), indicating the specificity of the antisera. As in the rat, positive staining was observed in Leydig cells; however, ACE2 staining was also present in human Sertoli cells and was concentrated in the adluminal half of the cell, i.e. surrounding spermatocytes and spermatids (Fig. 5H). No other cell types, including germ cells, were positive for ACE2 in the human testis.
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Discussion |
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Membranes prepared from rat testicular homogenates could also efficiently cleave the ACE2 fluorescent substrate; this activity was significantly attenuated by the specific inhibitors MLN-4760 and cFP-Leu, indicating specific cleavage by ACE2. Interestingly, we observed significant apparent activity in the soluble fraction of rat testis; however, inhibition by ZPP indicates that this activity is due to prolyl endopeptidase, cleaving at the Pro-Lys bond. Thus caution must be exercised when using this ACE2 fluorescent substrate for assessing ACE2 activity in crude tissue homogenates because it is susceptible to cleavage by other peptidases. ACE2-like activity was also detected in membrane preparations of enriched Leydig cells, which is consistent with the immunolocalization of the protein in this cell type. Leydig cell membranes contained an immunopositive protein of similar size to the expressed ACE2 enzyme, as detected by Western immunoblot analysis, again confirming the presence of the full-length, membrane-bound enzyme in this cell type.
Immunohistochemistry also demonstrated that the peptidase ACE2 was localized primarily to Leydig cells within the rat testis and to both Leydig and Sertoli cells in the human. Significantly, ACE2 was not present in germ cells or endothelial cells, thereby showing a different testicular distribution to the homologous peptidase testicular ACE (25, 26) but overlapping with the distribution of the somatic form of ACE. The significance of the apparent species variation between ACE2 in the rat and human remains to be determined.
Interestingly, the expression of ACE2 in the testis is developmentally regulated. Although a few immunopositive cells were observed in the first 5 d after birth, there was a progressive increase in the number of positive cells between d 7 and 19 postpartum, which was coincident with the maturation of progenitor cells into steroidogenic Leydig cells. Ablation of Leydig cells with the toxin EDS also abolished ACE2 immunoreactivity, indicating that the Leydig cells are indeed the main testicular source of this protein, at least in the rat. During recovery of the Leydig cells after EDS treatment, ACE2-positive cells increased in parallel with the increase in Leydig cells. The fetal generation of Leydig cells during development and the early regenerating population of Leydig cells post EDS showed comparatively little or no staining. Taken together, these data suggest that ACE2 expression is correlated with the presence of adult-type Leydig cells.
It was noted that there was quite a degree of variability in the intensity of ACE2 staining among Leydig cells of the same testis in all groups investigated, which may indicate differences in the levels of expression between individual Leydig cells. The relationship between ACE2 staining and Leydig cell function remains to be determined, although clearly the data suggest some relationship with the adult Leydig cell phenotype. Interestingly, the fact that suppression of LH and local testosterone levels by the use of testosterone implants did not substantially alter the testicular levels of ACE2 catalytic activity suggests that the relationship is not simply with the level of steroidogenic activity of the cell. These data suggest that the enzyme is not hormonally regulated but is a constitutive product of the Leydig cell, which reaches its highest levels of expression at a comparatively late stage of Leydig cell development.
The expression of ACE2 by Leydig cells suggests a regulatory role for the enzyme in steroidogenesis or some other Leydig cell function. Indeed, evidence exists for the production of Ang II within the testicular interstitium, and the primary receptor, the AT1 receptor, is expressed on Leydig cell membranes (27, 28). Because Ang II has been shown to reduce both basal and LH-stimulated testosterone synthesis by these cells, one can speculate on a potential role of ACE2 in limiting Ang II levels in this tissue (28, 29).
Finally, the possibility that ACE2 is part of the local vascular regulatory system also must be considered. It is well established that the Leydig cells have direct and indirect effects on microvascular flow characteristics and permeability in the testis. Hyperstimulation of the Leydig cells with supraphysiological doses of human chorionic gonadotropin causes a dramatic change in testicular blood flow, leading to a transient decrease in interstitial fluid volume, followed by an increase in endothelial cell permeability and accumulation of fluid in the testis (30). Moreover, it is known that ablation of the Leydig cells causes a reduction in interstitial fluid and blood flow through the testis (31, 32). The increase in interstitial fluid volume after human chorionic gonadotropin treatment and the reduced fluid accumulation after Leydig cell ablation might be explained by changes in ACE2 expression within the testis, which would lead to changes in the conversion of Ang II to Ang (1, 2, 3, 4, 5, 6, 7) and subsequent alterations in the vasculature. However, because testosterone alone can prevent most of the vascular effects of Leydig cell ablation (32, 33) and we have shown that ACE2 is not hormonally regulated in the intact testis, it is apparent that much more study needs to be done on the control and effects of the entire renin-angiotensin pathway within the testis to explain these data.
In summary, the expression of ACE2 by Leydig cells suggests a regulatory role for the enzyme in steroidogenesis, possibly by limiting testosterone inhibition by Ang II. Moreover, one can also speculate a role for ACE2 in the local vascular regulatory system. Although the ACE2 knockout mice were reportedly fertile (12), the data in the present study suggest that a more detailed examination of testicular function in these animals may be warranted. Further study also is required to determine the exact role of ACE2, as well as the entire renin-angiotensin system, in testicular function.
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Acknowledgments |
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Footnotes |
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Abbreviations: ACE, Angiotensin-converting enzyme; Ang, angiotensin; cFP-Leu, N-[1(R, S)-carboxyl-3-phenylpropyl]-leucine; CHO-P, Chinese hamster ovary transfected with P-selectin; DMSO, dimethylsulfoxide; EDS, ethane dimethane sulfonate; MLN-4760, (S, S) 2-{1-carboxy-2-[3-(3,5-dichloro-benzyl)-3H-imidazol-4-yl]-ethylamino}-4-methyl-pentanoic acid; QFS, quenched fluorescent substrate; TBS, Tris-buffered saline; ZPP, Z-Pro-prolinal.
Received April 7, 2004.
Accepted for publication June 22, 2004.
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signaling protein to discrete amino acid sequences within the cytoplasmic domain of the platelet membrane glycoprotein Ib-IX-V complex. Biochemistry 37:638–647[CrossRef][Medline]
-subunit gene in prostate cancer cell lines. J Mol Endocrinol 32:55–67[Abstract]
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