Abstract
Mice lacking the gene (Npr1) encoding the natriuretic peptide receptor A (NPRA) have hypertension with elevated blood pressure and cardiac hypertrophy. In particular, Npr1 gene-deficient male mice exhibit lethal vascular events similar to those seen in untreated human hypertensive patients. Serum testosterone levels tend to be lower in hypertensive male humans than in normal males without hypertension, but the genetic basis for this tendency remains unknown. To determine whether Npr1 gene function affects the testosterone level, we measured serum testosterone in male hypertensive mice lacking a functional Npr1 gene, wild-type animals with two copies, and the gene-duplicated littermates expressing four copies of the gene. In the Npr1 gene-knockout (zero-copy) mice, the serum testosterone level was 62% lower than that in the two-copy control mice (80 ± 10 vs. 120 ± 14 ng/ml, respectively; P < 0.005). Serum testosterone in the four-copy mice was 144% (P < 0.005) of that in the two-copy wild-type control mice. To investigate the role of NPRA in testicular steroidogenesis, we analyzed atrial natriuretic peptide (ANP)-dependent guanylyl cyclase activation, accumulation of intracellular cGMP, and testosterone production in purified primary Leydig cells from animals with zero, two, or four copies of the Npr1 gene. Leydig cells lacking the Npr1 gene did not show ANP-stimulated guanylyl cyclase activation or cGMP accumulation and had no ANP-dependent testosterone production. ANP stimulation of Leydig cells from the four-copy males elicited a 2-fold greater production of cGMP compared to that in the two-copy wild-type counterparts (260 ± 12 vs. 126 ± 7 pmol/1 × 106 cells; P < 0.001). Similarly, ANP-dependent testosterone production in Leydig cells was nearly twice as high in four-copy mice as in two-copy wild-type controls (561 ± 18 vs. 325 ± 11 ng/1 × 106 cells; P < 0.001). ANP-dependent guanylyl cyclase activation and production of cGMP in Leydig cells increased progressively with the number of Npr1 gene copies. Our results establish the existence of an alternate mechanism for testicular steroidogenesis that is stimulated by NPRA-dependent cGMP signaling, in addition to that mediated by gonadotropins, via a cAMP pathway. These findings demonstrate the role of Npr1 gene function in the maintenance of serum testosterone levels and testicular steroidogenesis and provide a genetic link between hypertension associated with decreased NPRA and low testosterone levels.
ATRIAL NATRIURETIC peptide (ANP) is a hypotensive cardiac hormone involved in the regulation of renal and cardiovascular functions mainly directed toward reducing blood pressure and fluid volume (1–3). ANP is known to exert its effects on a variety of physiological responses, such as diuresis, natriuresis, vasorelaxation, steroidogenesis, and cell proliferation (4–6). The discovery of structurally related brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) suggests that the role of natriuretic peptides in the control of body fluid and blood pressure homeostasis is complex. Although three natriuretic peptides have highly homologous structures, they also have distinct sites of synthesis and probably elicit discrete biological functions (7, 8). The functional complexity of the natriuretic peptide system is increased by the existence of at least three types of natriuretic peptide receptors: NPRA, NPRB, and a clearance receptor, NPRC (9–11). Both NPRA and NPRB are members of the receptor guanylyl cyclase (GC) family and produce their own second messenger, cGMP, that mediates most of the biological effects of natriuretic peptides. The production of cGMP in response to natriuretic peptides results from their binding to the extracellular domain of NPRA and NPRB, which probably allosterically regulates an increased activity of the intracellular guanylyl cyclase catalytic domain. The extracellular domains of NPRA and NPRB are homologous to the extracellular domain of NPRC, which does not contain an intracellular GC catalytic domain and is thought to clear natriuretic peptides (12). ANP and BNP bind to NPRA, and CNP binds to NPRB; however, all three natriuretic peptides bind to NPRC.
Among the three natriuretic peptides, ANP has been studied in greatest detail and participates in multiple physiological responses. In addition to its principal effects on sodium balance and fluid volume regulation, ANP has also been shown to inhibit aldosterone secretion from adrenal gland, renin from kidney, and vasopressin from posterior pituitary and to stimulate androgen secretion from normal Leydig cells, progesterone from granulosa cells, and LH from anterior pituitary (8, 13). Natriuretic peptide receptors are ubiquitously distributed in diverse tissues and cell types; however, with respect to the reproductive tissues, functional NPRA has been shown to be present in both male (14–17) and female (18, 19) gonads. Although earlier reports have shown that ANP stimulates the synthesis of testosterone in isolated Leydig cells (15, 16, 20–22), the exact mechanisms of ANP-dependent Leydig cell steroidogenesis are not yet clearly understood. Previous studies have shown that ANP receptor density was drastically reduced in spontaneously hypertensive rats (SHR) compared with that in normotensive Wistar-Kyoto counterparts, and ANP weakly stimulated testosterone production in isolated Leydig cells of SHR (23). These results correlate well with the finding that male patients with hypertension show lower circulating testosterone levels and an increased risk of erectile dysfunction (24, 25). Men with a family history of hypertension have also been shown to have lower than normal serum testosterone levels (26), suggesting a possible genetic link between hypertension and low serum testosterone levels. However, the precise relationship between hypertension and low testosterone levels remains an enigma. Certain other peptide hormones known to be involved in the regulation of blood pressure have also been shown to influence fertility and steroidogenesis (27).
Our gene-targeting strategies, resulting in mice with either a gene knockout or a gene duplication of Npr1, have shown that the blood pressure-lowering effects of ANP are absent in Npr1 null mutant mice (28), whereas these ANP effects are increased linearly in Npr1 gene-duplicated mice (29). Thus, we found that animals lacking NPRA have elevated blood pressure (∼15 mm Hg over wild-type controls) and exhibit cardiac pathology similar to that described in human patients with hypertensive heart disease. To address whether Npr1 gene copy number affects testosterone production in vivo and to study the direct effect of NPRA on ANP-dependent steroidogenesis in hypertensive mouse models, we measured serum testosterone levels and Leydig cell steroidogenesis in males with zero, two, and four copies of the Npr1 gene.
Materials and Methods
Materials
ANP (rat-28), BNP (rat-32), and CNP (porcine-22) were purchased from Peninsula Laboratories, Inc. (Belmont, CA). Na125I (17 mCi/μg) was obtained from Amersham Pharmacia Biotech (Arlington Heights, IL), and the RIA kit used to measure testosterone was obtained from Pantex (Santa Monica, CA). Succinimido-4-azidobenzoate was purchased from Pierce Chemical Co. (Rockford, IL). Percoll was received from Pharmacia (Piscataway, NJ), and the cGMP assay kit was obtained from Biomedical Technology, Inc. (Stoughton, MA). The protein assay kit was purchased from Bio-Rad Laboratories, Inc. (Hercules, CA). Culture media and buffers were purchased from Life Technologies, Inc. (Grand Island, NY). All other chemicals were molecular biology reagent grade.
Production of mice lacking the Npr1 gene
Targeted disruption of the Npr1 gene to produce mice lacking functional NPRA has been reported previously (28). The targeting vector was made using a 6.5-kb fragment of strain 129 genomic DNA containing sequences upstream of the 5′-Npr1 coding region as the 5′-region of homology, and a PCR-derived 1.5-kb fragment, containing exon 2, intron 2, and part of exon 3 of the Npr1 gene as the 3′-homology arm. Animals were made from correctly targeted embryonic stem cells as previously described (30, 31). F1 heterozygous animals were determined by Southern blot analysis of DNA extracted from tail biopsies with BamHI and hybridizing with a PCR derived probe for exon 4 or a probe that hybridizes to the neomycin resistance gene. F1 heterozygotes were interbred to produce mutant mice homozygous for disruption of the Npr1 gene. Animals were handled under protocols approved by the institutional animal care and use committee.
Production of mice with a duplication of the Npr1 gene
Targeted gene duplication of Npr1 has been previously described (29). The duplication-targeting construct was made by inserting the same 6.5-kb fragment in the opposite orientation in the targeting vector and replacing the 1.5-kb fragment with a 1.3-kb HindIII fragment containing sequence 6.0-kb downstream of the last Npr1-encoding exon. Electroporation and cell culture were performed as previously described (29). Correctly targeted clones were identified by Southern blot, digesting the embryonic stem cell DNA with BamHI, and hybridizing the blot with an exon 1 probe. F1 heterozygotes were crossed to produce mice homozygous for duplication of the Npr1 gene. Homozygous animals were identified by the Massachusetts Institute of Technology (MIT) marker analysis, using primers D3MIT40 and D3MIT101 (32). Animals were handled under protocols approved by the institutional animal care and use committee.
Serum testosterone assay
Blood samples were collected from age-matched male mice with zero, two, and four copies of the Npr1 gene under CO2 anesthesia using noncoated glass pipette into ice-cold tubes and immediately centrifuged to isolate the plasma. Serum testosterone concentrations were determined using a direct RIA kit.
Leydig cell isolation and stimulation of testosterone
Mice were killed by cervical dislocation, and testis were removed, decapsulated, and placed in Waymouth medium containing 10 mm HEPES (pH 7.4) and 0.1% BSA. Testes from zero-, two-, and four-copy mice were dispersed mechanically using a 50-cc syringe, essentially as previously described (15, 22). Tubules were allowed to settle for 10 min, and supernatants were centrifuged at 100 × g for 10 min at 4 C to pellet the Leydig cells, which were then further purified on Percoll gradients. The viability of cells was determined by the trypan blue exclusion test. Leydig cells were treated with appropriate concentrations of ANP, BNP, CNP, or LH incubated at 37 C for 3 h in an atmosphere of 5% CO2 and 95% O2 in a shaking water bath. The reaction was stopped by the addition of 1 ml ice-cold Waymouth medium and centrifugation at 100 × g for 10 min. The release of testosterone in the medium and intracellular accumulation of cGMP were determined by the direct RIA kits.
Photoaffinity labeling of NPRA
The photoaffinity ligand azidobenzoyl-[125I]ANP was prepared as previously described (14). Isolated Leydig cells from Npr1 gene-knockout and wild-type mice were washed with assay medium and then incubated at 4 C in fresh medium containing azidobenzoyl-[125I]ANP for 10 min in the dark as described previously (33). After binding, cells were washed three times with cold assay medium and photolysed in fresh medium. Cells were rewashed four times with assay medium and lysed in a solution containing SDS (0.5%), Triton X-100 (1%), phenylmethylsulfonylfluoride (1 mm), N-ethylmalemide (2 mm), and leupeptin and aprotinin (10 μg/ml each). The aliquots of the cell lysates were boiled for 5 min with an equal volume of sample buffer and analyzed by SDS-PAGE using 7.5% gels under reduced conditions. Electrophoresis was carried out at a constant current of 25 mA until the bromophenol blue front reached the bottom of the gel. Proteins in the gel were stained with Coomassie brilliant blue R-250. After destaining, the gels were dried and autoradiographed at −70 C using Kodak X-Omat film (Eastman Kodak Co., Rochester, NY) and a Cronex Lightning Plus (DuPont, Wilmington, DE) intensifying screen. The proteins used for standard molecular mass calibration were as follows: myosin (Mr 205,000), β-galactosidase (Mr 116,000), phosphorylase b (Mr 97,000), BSA (Mr 67,000), ovalbumin (Mr 45,000), and carbonic anhydrase (Mr 29,000).
Guanylyl cyclase assay
The plasma membranes were prepared by suspending purified Leydig cells in 5 vol sodium phosphate buffer (10 mm; pH 7.4) containing sucrose (250 mm), NaCl (150 mm), EDTA (5 mm), leupeptin (10 μg/ml), and aprotinin (10μ g/ml). Cells were homogenized in a Dounce-type homogenizer and centrifuged at 800 × g for 5 min at 4 C. The supernatants were collected, and pellets resuspended in 5 vol buffer, homogenized, and centrifuged as described above. Both supernatants were pooled and centrifuged at 100,000 × g for 1 h at 4 C. The pellets were resuspended in 50 mm HEPES buffer (pH 7.4) containing 150 mm NaCl and protease inhibitors as described above and recentrifuged at 100,000 × g for 1 h at 4 C. The membrane pellets obtained were resuspended in the above buffer, and aliquots were frozen in liquid nitrogen and stored at− 75 C until used. The guanylyl cyclase activity was assayed as previously described (34, 35). Briefly, 10-μl aliquots of membranes were added to the 100-μl total reaction mixture containing Tris-HCl buffer (50 mm; pH 7.6), MnCl2 (4 mm), GTP (1 mm), BSA (1 mg/ml), creatine phosphate (7.5 mm), theophyline (10 mm), 3-isobutyl-1-methylxanthine (IBMX; 0.2 mm), and creatine phosphokinase (3 μm) in the presence or absence of ANP. Reaction mixtures were incubated at 37 C for 20 min and then stopped by the addition of 900 μl sodium acetate buffer (50 mm; pH 6.2) and by placing the samples in a boiling water bath for 3 min. The amount of cGMP generated was determined using a RIA kit. The protein contents were determined using the protein assay kit.
Determination of intracellular cGMP
Leydig cells from different genotypes of mice were treated with ANP at 37 C for 10 min in the presence of 0.2 mm IBMX. The reaction was stopped with 0.5 n HCl. Cells were disrupted by a rapid freezing and thawing five times, and the cell lysates were centrifuged at 100 × g for 10 min. Supernatants were lyophilized and reconstituted in water. cGMP was measured using RIA kit as previously described (35).
Statistics
All data are presented as the mean ± se. Statistical significance was established using Student’s t test or Duncan’s one-way ANOVA.
Results
The present studies were designed primarily to determine whether the hypertension observed in Npr1-deficient mice is accompanied by altered testosterone levels. We measured serum testosterone concentrations in male mice completely lacking the Npr1 gene (zero-copy) and in age-matched wild-type (two-copy) and gene-duplicated (four-copy) littermates. The serum testosterone concentration was 62% lower (P < 0.005) in Npr1 null mutant (zero-copy) hypertensive mice compared with the wild-type (two-copy) control mice (Fig. 1). To determine whether the testosterone level in vivo is quantitatively related to NPRA levels, we compared serum testosterone levels in animals with zero, two, or four copies of the Npr1 gene, which have been previously shown to have 0%, 100% (by definition), and 200% of normal NPRA levels. The results demonstrated that serum testosterone levels in four-copy animals are 144% of the levels in two-copy wild-type control mice (P < 0.005). These results indicate that NPRA plays an important role in androgen biosynthesis and the maintenance of circulating hormone in vivo.
Serum testosterone levels in Npr1gene-knockout (zero-copy), wild-type (two-copy), and gene-duplicated (four-copy) mice. Blood samples were collected under CO2 anesthesia from age-matched male mice with zero, two, and four copies of the Npr1 gene using a noncoated glass pipette into ice-cold tubes and immediately centrifuged to isolate the plasma. Serum testosterone concentrations from different samples were determined using a RIA kit. The bars represent the mean ± se of three separate determinations. P < 0.005, significantly different from two-copy wild-type control mice.
To further characterize the effect of Npr1 gene copy numbers on testosterone production, we measured the synthesis and release of testosterone in purified Leydig cells isolated from mice with zero, two, or four copies of the Npr1 gene. ANP treatments of purified Leydig cells showed a gene dose-dependent stimulation of testosterone biosynthesis (Fig. 2). Isolated Leydig cells from zero-copy null mutant mice showed no synthesis or release of testosterone in response to ANP treatment. However, similar treatment of Leydig cells from two- and four-copy animals stimulated testosterone production by almost 35- and 60-fold, respectively. LH induced testosterone production in Leydig cells from two-copy wild-type mice comparable to that induced by ANP (Table 1). Both ANP and BNP stimulated testosterone production in Leydig cells from two-copy animals by more than 25-fold, whereas CNP, a third member of the natriuretic peptide hormone family, was able to stimulate testosterone only 1.5- to 2-fold compared with the untreated control levels (Table 1). The stimulated levels of testosterone production in response to ANP, BNP, or LH were comparable in two-copy wild-type mice, and the LH-stimulated level of testosterone production was not significantly different among the Npr1 gene-knockout (zero-copy), wild-type (two-copy), or gene-duplicated (four-copy) mice. We performed LH dose-response curves for testosterone production in Leydig cells from zero-, two-, and four-copy mice, which showed that LH stimulated testosterone production in a concentration-dependent manner in all three genotypes with almost similar dose-response curves (Fig. 3).
ANP stimulation and dose-response curves of testosterone production in isolated Leydig cells from mice with zero, two, and four copies of the Npr1 gene. In each tube, purified Leydig cells (1 × 106 cells) were incubated with increasing concentrations of ANP in the presence of 0.2 mm IBMX for 3 h at 37 C in an atmosphere of 5% CO2 and 95% O2. At the end of incubation period, the reaction was stopped by placing the tubes on ice and centrifuging at 1500 rpm for 15 min. Testosterone in the medium was estimated using a direct RIA kit. Data indicate the mean ± se of three separate experiments. **, Significantly different from two-copy wild-type control mice.
![LH stimulation and dose-response curves of testosterone production in isolated Leydig cells in Npr1 gene-knockout (zero-copy), wild-type (two-copy), and gene-duplicated (four-copy) mice. Purified Leydig cells (1 [tiems] 10−6 cells) were incubated with increasing concentrations of LH in the presence of 0.2 mm IBMX for 3 h at 37 C in an atmosphere of 5% CO2 and 95% O2. At the end of incubation period, the reaction was stopped, and contents were centrifuged at 1500 rpm for 15 min as described in Materials and Methods. In the supernatants, testosterone was estimated using a direct RIA kit. Each point represents the mean ± se of three independent determinations.](https://oup.silverchair-cdn.com/oup/backfile/Content_public/Journal/endo/140/11/10.1210_endo.140.11.7121/1/m_ee1197121003.jpeg?Expires=1713422054&Signature=shEA1rD7M7tJ1GisJkDL8J4Smv9Y7lS58EcbkHZ5sdqo~JAFozfb~F1GDUNbVI7NcfzI2O64KXaWm0HvZ9vZDL4KsfiAtjQUqDBh4fsuFn-cXhdYBMfutOBfe-xfOdNiTRORECStd8MFhbWOmp-ZzBZ5gOauh~TjjanAhDPC7EN96RwWCNJeMSP0iuBhPgPB65j96jzIjResGY~KV560GCoHXGpbvDWlx4XzX7-qiVV~TjBaCDWf0IfFvIdKiZOGvD5FbKjihVlK6ULurJ-gLeJTauImIi5IyNJ9ZOleMm9pT64olTbdWz5hz4N952J7e6yD1Zw6jl3kserr6U7lWA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
LH stimulation and dose-response curves of testosterone production in isolated Leydig cells in Npr1 gene-knockout (zero-copy), wild-type (two-copy), and gene-duplicated (four-copy) mice. Purified Leydig cells (1 [tiems] 10−6 cells) were incubated with increasing concentrations of LH in the presence of 0.2 mm IBMX for 3 h at 37 C in an atmosphere of 5% CO2 and 95% O2. At the end of incubation period, the reaction was stopped, and contents were centrifuged at 1500 rpm for 15 min as described in Materials and Methods. In the supernatants, testosterone was estimated using a direct RIA kit. Each point represents the mean ± se of three independent determinations.
Differential effects of three natriuretic peptides on testosterone production in purified Leydig cells isolated from Npr1 gene knockout (zero-copy), wild-type (two-copy), and gene-duplicated (four-copy) mice
| Treatment | Testosterone produced (ng/1 × 106 cells·3 h) | ||||
|---|---|---|---|---|---|
| 0-copy mice | 2-copy mice | 4-copy mice | |||
| None | 8.5 ± 1.3 | 11.6 ± 1.1 | 10.4 ± 1.2 | ||
| ANP | 11.7 ± 1.2 | 324.11 ± 10.2a | 560.6 ± 18.5a | ||
| BNP | 10.5 ± 1.4 | 336.7 ± 8.6a | 534.4 ± 16.5a | ||
| CNP | 18.2 ± 1.6 | 20.4 ± 1.7 | 21.3 ± 1.2 | ||
| LH | 275.6 ± 8.2 | 322.8 ± 11.6 | 354.1 ± 12.7 | ||
| Treatment | Testosterone produced (ng/1 × 106 cells·3 h) | ||||
|---|---|---|---|---|---|
| 0-copy mice | 2-copy mice | 4-copy mice | |||
| None | 8.5 ± 1.3 | 11.6 ± 1.1 | 10.4 ± 1.2 | ||
| ANP | 11.7 ± 1.2 | 324.11 ± 10.2a | 560.6 ± 18.5a | ||
| BNP | 10.5 ± 1.4 | 336.7 ± 8.6a | 534.4 ± 16.5a | ||
| CNP | 18.2 ± 1.6 | 20.4 ± 1.7 | 21.3 ± 1.2 | ||
| LH | 275.6 ± 8.2 | 322.8 ± 11.6 | 354.1 ± 12.7 | ||
Purified Leydig cells (1 × 106) were suspended in 1 ml assay medium and then treated with ANP (1 × 10−7m), BNP (1 × 10−7m), CNP (1 × 10−7m), and LH (50 ng/ml) in the presence of 0.2 mm IBMX. The treated cells were incubated in an atmosphere of 5% CO2 and 95% O2 at 37 C for 3 h. The reaction was stopped by the addition of 2 ml ice-cold assay medium and centrifugation at 100× g for 10 min. Testosterone in supernatants was assayed using the direct RIA kit. The results represent the mean ± se of three separate experiments.
P < 0.001 compared with controls and between the genotypes.
Differential effects of three natriuretic peptides on testosterone production in purified Leydig cells isolated from Npr1 gene knockout (zero-copy), wild-type (two-copy), and gene-duplicated (four-copy) mice
| Treatment | Testosterone produced (ng/1 × 106 cells·3 h) | ||||
|---|---|---|---|---|---|
| 0-copy mice | 2-copy mice | 4-copy mice | |||
| None | 8.5 ± 1.3 | 11.6 ± 1.1 | 10.4 ± 1.2 | ||
| ANP | 11.7 ± 1.2 | 324.11 ± 10.2a | 560.6 ± 18.5a | ||
| BNP | 10.5 ± 1.4 | 336.7 ± 8.6a | 534.4 ± 16.5a | ||
| CNP | 18.2 ± 1.6 | 20.4 ± 1.7 | 21.3 ± 1.2 | ||
| LH | 275.6 ± 8.2 | 322.8 ± 11.6 | 354.1 ± 12.7 | ||
| Treatment | Testosterone produced (ng/1 × 106 cells·3 h) | ||||
|---|---|---|---|---|---|
| 0-copy mice | 2-copy mice | 4-copy mice | |||
| None | 8.5 ± 1.3 | 11.6 ± 1.1 | 10.4 ± 1.2 | ||
| ANP | 11.7 ± 1.2 | 324.11 ± 10.2a | 560.6 ± 18.5a | ||
| BNP | 10.5 ± 1.4 | 336.7 ± 8.6a | 534.4 ± 16.5a | ||
| CNP | 18.2 ± 1.6 | 20.4 ± 1.7 | 21.3 ± 1.2 | ||
| LH | 275.6 ± 8.2 | 322.8 ± 11.6 | 354.1 ± 12.7 | ||
Purified Leydig cells (1 × 106) were suspended in 1 ml assay medium and then treated with ANP (1 × 10−7m), BNP (1 × 10−7m), CNP (1 × 10−7m), and LH (50 ng/ml) in the presence of 0.2 mm IBMX. The treated cells were incubated in an atmosphere of 5% CO2 and 95% O2 at 37 C for 3 h. The reaction was stopped by the addition of 2 ml ice-cold assay medium and centrifugation at 100× g for 10 min. Testosterone in supernatants was assayed using the direct RIA kit. The results represent the mean ± se of three separate experiments.
P < 0.001 compared with controls and between the genotypes.
ANP stimulation of Leydig cells from wild-type (two-copy) and gene-duplicated (four-copy) mice elicited the accumulation of intracellular cGMP by 126 ± 7 and 260 ± 12 pmol/1 × 106 cells (P < 0.001), approximately 90- and 185-fold stimulations, respectively, compared with that in nontreated control cells (Fig. 4). However, ANP-dependent stimulation of cGMP in isolated Leydig cells from null mutant (zero-copy) mice was completely abolished (1.4 ± 0.2 pmol/1 × 106 cells, comparable to untreated control values). To assess the specificity of the ANP-dependent cGMP production and receptor activity, we tested the effect of the NPRA antagonist A-71915. The results showed that the NPRA antagonist effectively abolished the ANP-dependent accumulation of cGMP in Leydig cells from both wild-type (two-copy) and gene-duplicated (four-copy) mice. To confirm that the Npr1 gene copy numbers directly affect the density of cell surface receptors, we measured the ANP-dependent guanylyl cyclase activity in plasma membrane preparations of Leydig cells from animals with zero, two, or four copies of the Npr1 gene. The ANP-dependent guanylyl cyclase activation and cGMP production in Leydig cells were again found to be proportionate to the Npr1 gene copy number (Fig. 5). Plasma membrane preparations of Leydig cells from the Npr1 gene-deficient mice (zero-copy) do not yield any photoaffinity-labeled NPRA protein band (Fig. 6). Neither ANP nor BNP treatments of Leydig cells from the Npr1-deficient null mutant mice resulted in any increase in testosterone production, showing that both peptide hormones function by interacting with NPRA.
Effect of the NPRA antagonist A71975 on ANP-stimulated cGMP production in Leydig cells from wild-type and Npr1 gene-targeted mutant mice. Purified Leydig cells from each genotype of mice were preincubated with 0.2 mm IBMX and then treated with ANP (1 × 10−7m) in the presence or absence of the NPRA antagonist A71975 for 10 min at 37 C. The reaction was stopped with 100 μl HCLO4 (10N). Cells were disrupted by freezing and thawing four times, and cell lysates were centrifuged at 200 × g for 10 min. The supernatant was washed five times with water-saturated diethyl ether, and the pH of the solution was adjusted to 6.5. The amount of cGMP was estimated by RIA. Each data point represents the mean ± se of three or four separate experiments.
Stimulation of guanylyl cyclase activity in plasma membrane preparations of Leydig cells from wild-type (two-copy), Npr1 gene-knockout (zero-copy), and gene-duplicated (four-copy) mutant mice. Guanylyl cyclase activity was assayed by incubating 40 μg plasma membranes at 37 C for 30 min in the presence of ANP (1 × 10−7m) as described in Materials and Methods. Each reaction was terminated by the addition of 900 μl sodium acetate buffer (50 mm; pH 6.2) and subsequently placing the tubes in a boiling water bath for 3 min. The amount of cGMP formed was measured using a direct RIA kit. Each data point represents the mean ± se of three separate determinations.
![Autoradiogram showing the presence and absence of the photoaffinity-labeled 135-kDa NPRA protein band in Leydig cells isolated from two-copy wild-type and zero-copy Npr1 gene-knockout mutant mice. Freshly isolated Leydig cells from two-copy wild-type and zero-copy null mutant mice were labeled with azidobenzoyl-[125I]ANP at 4 C as described in Materials and Methods. After photolysis at 4 C for 10 min, cells were solubilized in a solution containing 0.5% SDS, 1% Triton X-100, and protease inhibitors. The proteins were separated by SDS-PAGE using 7.5% gel under reduced conditions, and the radiolabeled NPRA band was identified by autoradiography. The 135-kDa labeled NPRA band is indicated by an arrow. The positions of the standard molecular mass markers are indicated on the margin. Lane a indicates the presence of the labeled NPRA band in Leydig cells of two-copy wild-type mice, and lane b shows the absence of the NPRA protein band in Leydig cells of zero-copy null mutant mice. The autoradiogram is representative of two separate receptor-labeling experiments.](https://oup.silverchair-cdn.com/oup/backfile/Content_public/Journal/endo/140/11/10.1210_endo.140.11.7121/1/m_ee1197121006.jpeg?Expires=1713422054&Signature=fEvMucvPTugBmiGkxx77x5cC-Y1Tt-xbgWNBgiA8QITPiUoBGYOHykR6zcs8Vc2aSon3IQuFeD7IbYOkEQySLx8ilnj1ZZNtwQCWA8b5MEnxfBH6kmC1sMOfVPeTQIPy6sXQAt44~XPkJh5AGQWKetw8JR9niX1hZuDguoW3DPkm3B-5UOoVumQRuKH9WdFgmtW76Xdu3j~3XI195BM~R8jDsH5BULKqk-o4CDudtJz6-Objf2J6~4scf~dF4lkJLdQjFp0wV8e4R-VSXNXilxKFYFnDDiDwjlZUBYKnOREMIV6RwwsVWkcXQgjhdUOCB7xCfiT8mI7o6nn2azUTtQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Autoradiogram showing the presence and absence of the photoaffinity-labeled 135-kDa NPRA protein band in Leydig cells isolated from two-copy wild-type and zero-copy Npr1 gene-knockout mutant mice. Freshly isolated Leydig cells from two-copy wild-type and zero-copy null mutant mice were labeled with azidobenzoyl-[125I]ANP at 4 C as described in Materials and Methods. After photolysis at 4 C for 10 min, cells were solubilized in a solution containing 0.5% SDS, 1% Triton X-100, and protease inhibitors. The proteins were separated by SDS-PAGE using 7.5% gel under reduced conditions, and the radiolabeled NPRA band was identified by autoradiography. The 135-kDa labeled NPRA band is indicated by an arrow. The positions of the standard molecular mass markers are indicated on the margin. Lane a indicates the presence of the labeled NPRA band in Leydig cells of two-copy wild-type mice, and lane b shows the absence of the NPRA protein band in Leydig cells of zero-copy null mutant mice. The autoradiogram is representative of two separate receptor-labeling experiments.
Discussion
We have investigated the mechanisms of ANP/NPRA action using the Npr1 gene-deficient and gene-duplicated mutant mouse models and have studied the effect of Npr1 gene copy number on serum testosterone levels in vivo and testicular steroidogenesis in vitro. The serum testosterone concentration was decreased by 62% in Npr1 gene-deficient (zero-copy) mice and increased by 144% in gene-duplicated four-copy mice relative to that in the two-copy wild-type controls. Data presented herein demonstrate that ANP does not stimulate testosterone production in Leydig cells from Npr1 gene-knockout (zero-copy) mice, whereas it stimulated approximately 35- and 60-fold testosterone production in Leydig cells from wild-type (two-copy) and gene-duplicated (four-copy) mice, respectively. Previous studies have shown that ANP stimulated 4- to 5-fold testosterone production in Leydig cells from normotensive Wistar-Kyoto rats compared with that in the genetic strain of SHR (23). These results indicated that a defect at one or more loci in ANP and/or its receptor system in the testis might contribute to the development of hypertension in SHR. The findings that the hypertension is associated with lower than normal levels of circulating testosterone suggest that 1) serum testosterone affects blood pressure regulation; 2) high blood pressure can negatively regulate steroidogenesis; or 3) there are genes involved in the regulation of blood pressure that also affect steroidogenesis. The observations that certain young normotensive men with a family history of hypertension also exhibit low serum testosterone levels (26) are in agreement with our present findings and support the third possibility. Our present results show that NPRA deficiency in male mice is characterized by both high blood pressure and low circulating testosterone levels.
Male patients with hypertension are not only more likely to have low serum testosterone levels, but also have an increased risk for erectile dysfunction and infertility (25). Serum testosterone levels are thought to play an important role in male reproductive success; for example, a mouse model was recently described in which homozygous disruption of insulin-like growth factor I leads to an 82% reduction in serum testosterone levels accompanied by infertility (36). Npr1-deficient male mice are fertile and produce normal sized litters when crossed with both wild-type and Npr1-deficient females, indicating that in these mice serum testosterone levels may be reduced (to 38% of wild-type levels) without causing infertility. Indeed, LH is considered the main regulator of serum testosterone levels; however, the administration of ANP has been reported to alter steroidogenesis in young men without modifying gonadotropin (LH) secretion (37). In contrast, previous studies have indicated that ANP exerted a stimulatory effect on LH secretion (38); however, it negatively affected the secretion of ACTH as well as stress-induced GH release (39). Although in the present model of Npr1 gene-targeted mice, the serum level of LH was not determined, our results support the contention that unlike LH, Leydig cell steroidogenesis in response to ANP is mediated through the activation of NPRA involving cGMP as second messenger, in contrast to gonadotropin (LH), which involves the cAMP pathway. As in all three groups of the Npr1 gene-targeted animals regardless of Npr1 gene copy number, the responsiveness of Leydig cells to LH remained identical, the serum testosterone level in zero-copy mice is significantly lower than that in two-copy wild-type animals. The likely explanation for this effect would be that the ANP/NPRA signaling mechanism contributes to an additive effect in conjunction with gonadotropin (LH) in the biosynthesis and maintenance of circulating serum testosterone levels in vivo. Our previous studies have shown that the stimulation of Leydig cells by combined treatment with ANP and LH caused a marked increase in testosterone production compared to that with either ANP or LH alone (15). However, the effect of Npr1 gene copy number on LH secretion, which is the main regulator of serum testosterone levels, remains to be investigated. Our current work shows that ANP stimulation increases testosterone biosynthesis by acting directly through NPRA and that this response is affected by Npr1 gene copy number and hence by the level of NPRA expression.
BNP also activates NPRA and stimulates testosterone production in a manner comparable to that of ANP. On the contrary, CNP, which binds to NPRB, stimulates testosterone only minimally by 1.5- to 2.0-fold in gene-disrupted (zero-copy), wild-type (two-copy), or gene-duplicated (four-copy) mice, indicating that it does not require NPRA. Recent studies have also shown that ANP and BNP regulate blood pressures exclusively through NPRA, whereas CNP most likely regulates blood pressure through NPRB (40). Although CNP and NPRB have been shown to be expressed in Leydig cells (41), the exact role of CNP in testis is not immediately clear. It has also been recently suggested that the possible role of CNP in testis may not necessarily be confined to steroidogenesis alone, as it may influence yet undefined functions of testicular cells, including Leydig cells (42). We have previously reported the presence of ANP-like peptide molecules in mouse and rat testes, which indicated that ANP might serve as a physiological natriuretic peptide hormone involved in testicular steroidogenesis and spermatogenesis (43). Our results with the Npr1 gene-deficient (zero-copy) and gene-duplicated (four-copy) mice establish a direct involvement of NPRA in the maintenance of serum testosterone levels and testicular steroidogenesis in response to ANP. The present data demonstrate that ANP stimulates the production of cGMP and testosterone in isolated Leydig cells of wild-type (two-copy) mice, but fails to produce cGMP and testosterone in Leydig cells from Npr1 null mutant (zero-copy) mice even at micromolar concentrations. The NPRA antagonist A71915 blocked the production of cGMP and testosterone in Leydig cells from two-copy wild-type and four-copy gene-duplicated mice, supporting the functional role of NPRA in testicular steroidogenesis.
It is also important to note that ANP exerted only minimal effect or did not significantly stimulate testosterone production in rat Leydig cells compared with that in mouse Leydig cells (44). These researchers suggested that species differences between rat and mouse Leydig cells in the effect of ANP on testosterone production would probably result from a lower guanylyl cyclase activity in rat Leydig cells. In contrast, it has been shown that rat testis contained the highest ANP-dependent guanylyl cyclase activity of any tissue reported to date (45). It is interesting to note that species differences exist not only with regard to the ANP-dependent steroidogenesis of Leydig cells; however, both testosterone production and cAMP accumulation in response to gonadotropins were severalfold higher in mouse Leydig cells than in rat Leydig cells (46–48). Our unpublished data also support the contention that the isolated rat Leydig cells contain a much lower density of ANP receptor than mouse Leydig cells. As rat Leydig cells are isolated with extensive enzymatic digestion, this may have caused the degradation of ANP receptor/guanylyl cyclase activity, showing a low or even absent ANP response on testosterone stimulation in these cells. Bolus administration of ANP did not affect testosterone secretory activity of rat Leydig cells; however, chronically administered ANP was able to stimulate the steroidogenic capacity of rat Leydig cells (49). It has also been suggested that calcium ions may play a role in the mechanisms by which ANP exerts its stimulatory effects on rat Leydig cell steroidogenesis (50, 51). These researchers also observed that ANP at a lower concentration (1 × 10−11m) stimulated testosterone approximately 8-fold; however, at the higher concentration (1 × 10−7m) only 3-fold testosterone stimulation was observed compared with the basal levels. Indeed, the findings of those previous studies are controversial with regard to the capacity of ANP to stimulate the production of cGMP in various cells types. The common characteristics of the ANP effect across species is that ANP stimulates optimum levels of intracellular cGMP accumulation in a concentration range of 1 × 10−9 to 1 × 10−6m. It has also been postulated that the effect of cGMP in rat Leydig cells in possibly due to a specific activation of a cAMP-dependent protein kinase (44); however, valid experimental evidence supporting these postulates are still lacking. Nonetheless, the species differences between mouse and rat Leydig cell steroidogenesis exist in response to ANP as well as gonadotropins, but the exact cause of these differences among the species remains to be investigated.
In summary, the data obtained from our Npr1 gene-deficient and gene-duplicated mutant mouse models demonstrate an alternative mechanism for Leydig cell steroidogenesis in addition to the known cAMP-dependent steroidogenic pathways mediated by gonadotropins. Our results establish a role for Npr1 gene function in the maintenance of serum testosterone levels and testicular steroidogenesis in Leydig cells and indicate one possible genetic link between low testosterone levels and the molecular mechanisms governing the state of hypertension.
This work was supported by NIH Grants HL-62145, HL-49277, and GM-20069 and the American Heart Association (South East Affiliate).
Acknowledgments
We are indebted to Dr. Ming Li, Kamala Pandey and Huong Nguyen for their assistance during the course of this work. We thank Dr. Thomas W. von Geldern for the generous gift of A71915. We also thank the Pituitary Distribution Program of the NIDDK (Bethesda, MD) for the generous gift of bovine LH.
References
