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Original Studies |
Institute of Anatomy (R.M., S.W., A.F.H., M.S.D.) and Institute for Hormone and Fertility Research (D.M.), University of Hamburg, 20246 Hamburg, Germany
Address all correspondence and requests for reprints to: Dr. Ralf Middendorff, Institute of Anatomy, University of Hamburg, Martinistraße 52, 20246 Hamburg, Germany.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 and
ß1), seems to be necessary for NO function (1). NO serves
as a neurotransmitter in the nervous system and as a mediator of
endothelium-dependent relaxation of blood vessels and mediates the
tumoricidal and bactericidal actions of macrophages (5, 6). Three
different subtypes of the enzyme NO synthase (NOS) [a neuronal (nNOS),
an endothelial (eNOS), and an inducible or macrophageal (iNOS) form]
are known and all of them are capable of producing NO (7). NOS activity was found to be present in male reproductive organs (8, 9). In Leydig cells of the human testis, nNOS (10) and a functionally active sGC (11) have been detected. Furthermore, an inhibitory effect of NO on testosterone secretion by rat Leydig cells has been described (12, 13).
In the human testis, Leydig cells are in close relationship with the peritubular lamina propria on the one hand (14) and with testicular blood vessels on the other hand (15). Both (blood vessels and the peritubular lamina propria) possess contractile elements; namely, vascular smooth-muscle cells (15, 16) and the myofibroblasts of the inner 3–5 layers of the peritubular lamina propria (14, 17, 18, 19).
Because NO was first described as a smooth muscle-relaxing factor (reviewed in Refs. 2–6), it is possible that NO, produced by Leydig cells, may counterbalance contractions of neighboring vascular myocytes and peritubular myofibroblasts.
The present report shows that NO-producing enzymes (NOS) and NO receptors are not only present in Leydig cells but also in peritubular lamina propria, Sertoli, and blood vessel cells, suggesting production and activity of NO in these structures.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Testes were obtained from 14 patients, 30–86 yr old, who were undergoing orchiectomy as the primary treatment of prostatic carcinoma.
Isolation of seminiferous tubules and testicular blood vessels
One to 2 h after surgery, pieces of chilled human testes were transferred to dishes containing Ham’s F12/DMEM culture medium (Gibco, Eggenstein, Germany) supplemented with 15 mmol/L NaHCO3, 20 mmol/L HEPES (pH 7.4), 100 IU/mL penicillin, 100 µg/mL streptomycin, 2.5 µg/mL amphotericin B, 10 µg/mL transferrin, 5 µg/mL hydrocortisone, and 2% FBS. Seminiferous tubules were then exposed as described (20). Blood vessels were pulled off from the connective tissue. Isolated tubules and vessels were either frozen in liquid nitrogen or transferred to 24-well microtiter plates (approximately 100 mg/well) containing the above mentioned culture medium.
Protein preparation
Frozen testicular blood vessels and seminiferous tubules were pulverized in a mortar, suspended in 9 mL homogenization buffer (0.05 mol/L Tris-HCl (pH 7.5) containing 10 mmol/L EDTA, 10 mmol/L dithiothreitol, and 0.1 mmol/L phenyl methyl sulfonyl fluoride) and homogenized by three strokes in a Potter-Elvehjem homogenizer. After centrifugation at 3,000 x g for 8 min to remove cell debris and nuclei, the supernatant fractions were centrifuged for 30 min at 100,000 x g. The resulting supernatant fractions, referred to as cytosolic extracts, were stored at -70 C, and the crude membrane pellets were washed once each in homogenization buffer plus 0.6 mol/L KCl, in homogenization buffer and were finally resuspended in 150 µL of 0.05 mol/L Tris-HCl buffer (pH 7.5). Protein concentration was determined with a Bio-Rad kit (Munich, Germany) using BSA (fraction V) as standard.
Immunoblotting
After separation by SDS-PAGE in 8% acrylamide gels, proteins
(individual preparations from six different testes) were transferred to
nitrocellulose membranes (Amersham, Braunschweig, Germany) at room
temperature for 18 h at 12 V in transfer buffer containing 100
mmol/L Tris base and 193 mmol/L glycine. The transferred proteins were
visualized by staining with Ponceau S (Sigma, Deisenhofen, Germany),
and the membranes were incubated for 2 h in a solution containing
1% blocking reagent (Boehringer, Mannheim, Germany) in 0.1 mol/L
maleic acid, 0.15 mol/L NaCl, and 0.005% thimerosal adjusted to pH
7.5. After washing for 5 min in tris-buffered saline plus Tween (20
mmol/L Tris (pH 7.6), 137 mmol/L NaCl, 0.05% Tween 20), the proteins
were incubated with monoclonal mouse (anti-nNOS, diluted 1:500;
anti-eNOS, 1:100; anti-iNOS, 1:2,500; all purchased from Transduction,
Lexington, KY) or polyclonal rabbit (directed against the
1-subunit of sGC, 1:200, kindly provided by D. Koesling,
Berlin, Germany) antibodies for 1 h at room temperature, rinsed
twice for 10 min in tris-buffered saline plus Tween, and then incubated
with antimouse IgG, linked to peroxidase (Pierce, Rockford, IL,
1:2,000) or antirabbit IgG-peroxidase (Sigma, 1:500). Enhanced
chemiluminescence fluorography, performed with enhanced
chemiluminescence fluorography-detection reagents from Amersham,
according to the manufacturer’s instructions, and Medical X-ray film
(Fuji) were used for the detection of bound secondary antibodies.
Immunohistochemistry
Tissue blocks derived from 14 human testes were used. Cryostat sections (10 µm), washed in 2% saponin in PBS and fixed with 4% paraformaldehyde in PBS, as well as paraffin sections (6 µm) prepared as described previously (10, 11), were mounted onto chrome-gelatin precoated slides. On paraffin sections, the following rabbit polyclonal antisera were employed: anti-nNOS (Biomol, Hamburg, Germany; working dilution 1:500), anti-cGMP (Biogenesis, Sandown, NH; 1:300), and anti-sGC (1:200; see above). Rabbit polyclonal anti-eNOS (Biomol, 1:500) and anti-iNOS (Biomol, 1:500) were used on cryostat sections. For visualization of immunoreactivity (IR), a combination of the peroxidase antiperoxidase with the avidin-biotin-peroxidase procedure was used (10, 11).
For negative controls, sections were used in which primary, secondary,
or tertiary antibodies were replaced by PBS and in which only the
development of the enzyme activity (peroxidase) was performed.
Furthermore, sections were incubated with normal rabbit or mouse serum
(Sigma), as well as with purified rabbit or mouse IgGs (Sigma), instead
of the primary antisera. In addition, anti-nNOS and anti-sGC antisera
were preadsorbed to the corresponding antigens (20 µg nNOS/mL
anti-nNOS, 1:500; 10 µg 1 sGC/mL anti-sGC, 1:200); and
anti-eNOS and anti-iNOS antisera were preadsorbed to lysates from
either endothelial cells or macrophages, known to contain eNOS and
iNOS, respectively, and delivered by one of the manufacturers of the
antibodies (Transduction) to serve as positive controls. For positive
controls, antisera were used on rat brain sections, where
immunohistochemical data for cGMP, sGC, eNOS, and nNOS are already
available (21, 22, 23). As further evidence for reaction specificity,
primary antibodies were replaced by differently generated ones [mouse
monoclonal anti-nNOS, 1:500; anti-eNOS, 1:100; and anti-iNOS, 1:500
(all purchased from Transduction) and rabbit polyclonal anti-cGMP,
1:500 (kindly provided by J. DeVente, Maastricht, The
Netherlands)].
Measurement of cGMP production by isolated seminiferous tubules and blood vessels
After 1 day of culture (see above) at 34 C (5% CO2/95% O2), the medium was removed; and tubule and vessel preparations (testes from five patients, four different tubule and vessel preparations from each testis) were washed twice in Locke’s salt solution (154 mmol/L NaCl, 5.6 mmol/L KCl, 2.2 mmol/L CaCl2, 1 mmol/L MgCl2, 6 mmol/L NaHCO3, 10 mmol/L glucose, 2 mmol/L HEPES, pH 7.4) supplemented with 20 µmol/L bacitracin. To measure the effects of sodium nitroprusside (SNP) and of L-nitro arginine methyl ester (L-NAME) on cGMP production, the vessels and tubules, respectively, were first incubated for 1 h at 34 C in 250 µL Locke’s solution containing additionally 0.25 mmol/L 3-isobutyl-1-methyl-xanthine (IBMX, purchased from Sigma) in the absence and then in the presence of either 1 mmol/L SNP (purchased from Sigma) or 1 mmol/L L-NAME (Sigma). Solutions were removed after each incubation, immediately frozen in liquid nitrogen, and stored at -70 C until use in the cGMP RIA.
cGMP was measured, as described (24), with RIA reagents kindly provided by IBL (Hamburg, Germany). The minimum detection limit was 10 fmol/tube and cross-reactivity with cAMP was less than 0.001%.
All measurements of cGMP were performed in triplicate. Treatment
effects, based on 20 different experiments [each in the absence or
presence of SNP (see Fig. 7a
) and L-NAME (see Fig. 7b), respectively]
were assessed statistically using t test as installed in the
GraphPad InStat Software (GraphPad Inc., Sorrento, CA), with
P 
0.05 as the criterion of significance. The results
(see Fig. 7) are mean ± SE (SEM) of
treatment effects of all experiments performed with tubule and vessel
preparations, respectively.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1-subunit of sGC, 82 kDa in size (1), could
be demonstrated in the cytosolic protein extracts of human testicular
vessels and tubules (Fig. 1a). The
presence of nNOS was analyzed in parallel immunoblots. A single protein
migrating at approximately 160 kDa (Fig. 1b), the molecular mass of
nNOS on denaturing SDS-polyacrylamide gels (7, 22), was found in
cytosolic protein fractions of blood vessels and seminiferous tubules
of the human testis and in extracts of a rat pituitary tumor cell line
delivered as positive control by the manufacturer of the anti-nNOS
antibody. eNOS-IR, migrating with an electrophoretic mobility
equivalent to 135 kDa (7, 22), was detected in crude membrane protein
fractions of testicular vessels and tubules (Fig. 1c). Aortic
endothelial cells, known to possess eNOS (4), served as positive
control.
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). IR for cGMP was found within
endothelial cells of small blood vessels (Fig. 2b), whereas in large
blood vessels, cGMP-IR was additionally detected in smooth-muscle cells
(Fig. 2c). Staining of testicular veins was not observed. A
subpopulation of peritubular myofibroblasts (Fig. 3a) showed a positive staining for sGC.
Some lamina propria cells of all layers showed cGMP-IR (Fig. 3b).
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). In contrast, only
weak-to-moderate nNOS-IR was found in the lamina propria (not shown).
Intensive staining for nNOS was present in endothelial cells of some
small vessels (Fig. 4a). IR for eNOS was
found in endothelial and smooth muscle cells of large blood vessels
(Fig. 4b and c). The endothelium of small vessels, however, showed only
weak eNOS-IR.
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), cGMP, eNOS, and nNOS
(not shown). Consistent with previous reports (8, 9), specific
immunostaining for sGC, cGMP, and nNOS was seen also in Leydig
cells.
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), but neither of the other testicular structures, showed any
iNOS-IR. In agreement (data not shown), Western blot analyses of
vessels and tubules did not reveal iNOS-specific signals.
To assure the validity of the immunochemical data obtained,
a variety of control experiments have been performed. Generally,
the immunohistochemical and Western blot data presented were
representative of independent analyses of at least six testes derived
from different patients. For each antigen examined, the specificity of
signals was confirmed by negative control reactions in which specific
antisera were replaced by either normal rabbit/mouse antisera or
purified rabbit/mouse IgGs. Staining specificity was also demonstrated
with antisera preincubated with an excess of the corresponding antigens
or with cell lysates known to contain the specific antigens (Fig. 6). Positive controls corroberated the
specificity of the antibody reactions, both by Western Blots in
different tissues (Fig. 1) and by immunohistochemistry on rat brain
sections, where comparable analyses were performed previously (21, 22, 23).
Moreover, immunohistochemical results were confirmed by staining
procedures on testicular sections with additional antibodies against
nNOS, eNOS, iNOS, and cGMP.
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).
SNP-induced increases of cGMP were measured in all experiments, and the
data revealed [on an average, 47-fold (vessels) and 14-fold (tubules),
respectively] stimulations of sGC activity. Note that the extent of
sGC stimulation in blood vessels varied considerably among individual
preparations, presumably because of differences in diameters of
vessels.
Additional studies served to prove the local production of NO in
vessels and tubules. For this, cGMP production during incubations, in
either the absence or presence of the NOS inhibitor L-NAME, was
determined. Addition of the NOS inhibitor decreased cGMP production in
both vessels and tubules by 51.6% and 42.6%, respectively (Fig. 7b).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NO is a major endogenous vasodilator (4, 5), and NO-induced increases in cGMP levels correlate with relaxation (reviewed in Ref.25). Therefore, the observed NO-mediated cGMP accumulation by testicular blood vessels and the immunohistochemical localization of sGC and cGMP in vascular smooth muscle cells are indicative of a vasorelaxant function (4). In addition, NO has been shown to influence permeability of vascular endothelial cells (26, 27). However, whether the occurrence of sGC and cGMP in testicular endothelial cells can be explained in this context, remains to be elucidated.
In seminiferous tubules, NO-induced cGMP production may mediate relaxation of myofibroblasts. Whereas endothelin, for example, has been shown to be involved in peritubular cell contraction in the rat (28), the agents responsible for relaxation have not yet been defined. Peritubular myofibroblasts express filaments characteristic of fibroblasts and smooth muscle cells (14, 19). An influence of NO on myofibroblasts may be postulated in context of the well-known NO effects on smooth muscle cells in other organs (6). In the peritubular lamina propria, NO may participate in the regulation of the peristaltic activity of the tubules, which in turn, is necessary for sperm transport (16). Furthermore, NO may influence the permeability of the lamina propria and, by this, the transport of nutrients into the tubular lumen (19).
Our findings that NOS is present and functionally active in testicular blood vessels and seminiferous tubules refers to a local production of NO in these structures. Thus, physiological effects on vessels and tubules may be elicited by endogenously-produced NO and by NO from neighboring Leydig cells previously shown to express nNOS (10, 11).
Based on our findings that some Sertoli cells show NOS-IR, the NO-induced increases in cGMP production observed in isolated tubules may, in part, be caused by NO produced by Sertoli cells. Thus, NO derived from these cells may contribute also to the presumed relaxation of lamina propria cells. However, the reported NO effects on sperm cells after spermiation (29, 30), cells which are devoid of NOS activity (31, 32), point to additional or alternative functions of Sertoli cell-produced NO.
The expression of the two NOS isoforms, nNOS and eNOS, in blood vessels and seminiferous tubules is a notable observation. Recent analyses of mutant mice with targeted disruptions of either the neuronal (23, 33) or the endothelial NOS gene (34) suggested that eNOS-produced NO may be involved in dilatation of blood vessels, whereas nNOS-derived NO might contribute to vasoconstriction in brain (33). It will be of particular interest to investigate whether there also is a functional heterogeneity of the two NOS subtypes in the testis.
IR for the third NOS isoform, iNOS, was not detected in blood vessels and lamina propria cells. However, a minority of Sertoli cells showed iNOS-IR. In this context, it should be noted that a measurable iNOS gene expression by isolated rat Sertoli cells was described to depend on induction by cytokines (35).
Taken together, our results demonstrate that testicular vasculature and
seminiferous tubules are sites of NO production and activity (Fig. 8). It is an attractive idea that NO acts
locally to regulate the distribution of oxygen, nutrients, and hormones
by testicular vessels, as well as the peristaltic activity of tubules
in context of sperm transport.
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Acknowledgments |
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Footnotes |
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Received October 28, 1996.
Revised February 25, 1997.
Revised July 9, 1997.
Accepted August 14, 1997.
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55 µm) blood vessels (b, longitudinal section; c,
cross-section) showed strong eNOS-IR in endothelial and smooth-muscle
cells (x630). The vascular lumen is marked by
asterisks, endothelial cells by arrows,
and smooth muscle cells by arrowheads.
