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Department of Physiology, Tulane University School of Medicine (K.N.P.), New Orleans, Louisiana 70112; and the Department of Pathology, University of North Carolina (P.M.O., N.M., O.S.), Chapel Hill, North Carolina 27599
Address all correspondence and requests for reprints to: Kailash N. Pandey, Ph.D., Department of Physiology, SL-39, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, Louisiana 70112. E-mail: kpandey{at}mailhost.tcs.tulane.edu
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Introduction |
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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, 15, 16, 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, 21, 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.
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Materials and Methods |
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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 x
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 x 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), ß-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 (10
µg/ml). Cells were homogenized in a Dounce-type homogenizer and
centrifuged at 800 x 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 x 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 x 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 x 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.
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Results |
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). 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.
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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).
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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 x 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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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, 47, 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 x 10-11 M) stimulated testosterone approximately 8-fold; however, at the higher concentration (1 x 10-7 M) 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 x 10-9 to 1 x 10-6 M. 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.
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Acknowledgments |
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Footnotes |
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Received June 4, 1999.
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S in plasma membranes of Leydig cells. Arch
Biochem Biophys 316:392–398[CrossRef][Medline]
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