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From the * Endocrinology Division, Department of
Internal Medicine, the || Department of Economy,
School of Economy, and the Department of
Biochemistry, University of Ancona, Ancona, Italy; the
Department of Biomedical and Surgical
Sciences, University of Verona, Verona, Italy; and the
Endocrinology Division, Department of Medical
and Surgical Sciences, University of Padua, Padua, Italy.
| Correspondence to: Dr G. Balercia, Endocrinology Division, Department of Internal Medicine, Umberto I Hospital, Via Conca—Torrette, 60100 Ancona, Italy (e-mail: g.balercia{at}ao-umbertoprimo.marche.it). |
| Received for publication June 20, 2003; accepted for publication October 27, 2003. |
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Abstract |
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 Top Abstract Patients and MethodsResultsDiscussionReferences |
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Key words: Male infertility, spermatozoa motility, asthenozoospermia
NO is a free radical generated from the oxidation of L-arginine to L-citrulline by 3 isoforms of nicotinamide adenine dinucleotide phosphate (NADPH)-dependent NO synthases (NOS). Both neuronal NOS (nNOS), first described in neurones, and endothelial NOS (eNOS), first identified in endothelial cells, are constitutive Ca2+-dependent isoforms, whereas the macrophage NOS (iNOS) is an inducible Ca2+-independent isoform (Moncada et al, 1991; Fostermann et al, 1994). NOS protein and activity have been detected in both human and rat testis, epididymis, prostate, and seminal vescicles (Ehren et al, 1994; Burnett et al, 1995). Experiments performed with antibodies raised against NOS showed that this enzyme is associated with the acrosome and the tail of mouse spermatozoa (Herrero et al, 1996) and appears to be involved in the fertilization process, including sperm motility and acrosome reaction (Herrero et al, 1997). Furthermore, indirect immunofluorescence assays showed that human spermatozoa express constitutive NOS in the postacrosomal and equatorial segments (Lewis et al, 1996), and spin trapping experiments showed that NO is synthesized by the human male gamete (Herrero et al, 2000).
Low concentrations of NO cause a significant increase in capacitation (Zini et al, 1995) and zona pellucida binding (Sengoku et al, 1998), although the effects of NO on sperm motility and viability remain undefined (Hellstrom et al, 1994; Rosselli et al, 1995; Weinberg et al, 1995; Nobunaga et al, 1996). Together, these data suggest a relevant role of NO in the pathophysiology of sperm cells.
In the present study, we have correlated NO concentration in semen and kinetic features of sperm cells from normal and asthenozoospermic men in order to acquire a deeper insight about the role of NO in the pathophysiology of human spermatozoa.
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Patients and Methods |
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TopAbstractPatients and MethodsResultsDiscussionReferences |
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Semen Analysis
Semen samples were collected after 3 days of sexual abstinence. After
liquification at room temperature for 30 minutes, standard seminal parameters
were analyzed according to World Health Organization
(1999) guidelines.
Computer-assisted sperm analysis (CASA) for sperm motility assay was
additionally performed, as previously reported
(Balercia et al, 2003). One
semen aliquot (3 µL) was placed in a 20-µm-deep cell VU chamber
(Conception Technologies, La Jolla, Calif). Two chambers were loaded, 6
different fields per chamber were randomly examined, and at least 200
spermatozoa for each field of chambers were scored. Movement characteristics
were analyzed using an automated analyzer (CellTrack VP110, Motion Analysis
Corp, Palo Alto, Calif). Kinetic characteristics were evaluated only for
motile sperm and expressed as mean values considering: total sperm motility
(percent), curvilinear velocity (VCL, µm/s), straight progressive velocity
(VSL, µm/s) and lateral head displacement (ALH, µm).
Determination of NO Levels in Semen
For NO level evaluation, individual sperm samples were diluted to 5 x
106/mL with Dulbecco phosphate-buffered saline (PBS) (20 mM, pH
7.4), aliquoted to form 2 replicates and stored at –80°C in sterile
tubes until nitrite measurements were performed within 15 days. Each sample
was then suspended in a substrate buffer (HEPES 25 mM, NaCl 140 mM, KCl 5.4
mM, CaCl2 1 mM, and MgCl2 1 mM, pH 7.4) in the presence
of 1.44 mM of NADPH and 20 mU of nitrate reductase and incubated at room
temperature for 1 hour in order to convert all available nitrate to nitrite by
the enzyme. The reaction was stopped by freeze-thawing the samples, which were
then sonicated and centrifuged at 1500 x g for 15 minutes. NO
concentration, which is related to nitrite and nitrate levels, was determined
using a spectrophotometric assay based on the formation of a colored azo dye
product when a Griess reagent (1% sulfanilamide, 0.1% naphthylenediamine
dihydrochloride, and 2.5% H3PO4) was mixed in equal
volumes with the sample, as first described by Chen and Mehta
(1996) and then modified by
Camilletti et al (2001). The
chromophore absorption was then read at 543 nm, and nitrite concentrations,
expressed in nmol NO/106 cells, were determined from a standard
curve generated by using known concentrations of sodium nitrite.
Western Blot Analysis
Washed spermatozoa were lysed in RIPA buffer (1x PBS, 1% Igepal
CA-630, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 10 mg/mL
of PMSF, Aprotinin, 100 mM of sodium orthovanadate, and 4% protease inhibitor
cocktails) through a microcentrifugation at 10 000 x g for 10
minutes at 4°C. The supernatants were collected and treated with an equal
volume of sample application buffer (125 mmol/L Tris HCl, pH 6.8, 2% SDS, 5%
glycerol, 0.003% bromophenol blue, and 1% ß-mercaptoethanol). Forty
micrograms of protein was loaded and separated by 8% SDS-polyacrylamide gel
electrophoresis with a set of molecular-weight markers (Sigma-Broad range).
After electrophoresis, samples were transferred to polyvinylidine difluoride
membranes. The blots were blocked with 5% nonfat milk in 10 mmol/L Tris, pH
7.5, 100 mmol/L NaCl, and 0.1% Tween 20 and incubated with eNOS and iNOS
rabbit polyclonal antibodies (both from Sigma Chemical Co, St Louis, Mo)
diluted at 1:200 in 5% milk PBS/Tween for 1 hour at room temperature. Positive
controls were included in all experiments as provided by the manufacturer to
confirm antibody specificity. As an internal control, blots were reprobed with
an anti–ß-actin antibody (Santa Cruz Biotechnology Inc, Santa Cruz,
Calif). Blots were then washed with TTBS (10 mmol/L Tris, pH 7.5, 100 mmol/L
NaCl, and 0.1% Tween 20) and incubated with horseradish
peroxidase–conjugated anti-rabbit immunoglobulin G (IgG) (1:5000; Sigma)
for 1 hour at room temperature. Peroxidase activity was detected using
3,3'-diaminobenzidine (Sigma) as a substrate.
Statistical Analysis
Statistical analysis was performed using the SAS statistical package
(Statistical Analysis Systems Institute, Cary, NC). Results are reported as
mean value plus or minus standard deviation. Differences among the groups were
evaluated by t tests, and the Kolmogorov-Smirnov test was used to
determine whether the data were random samples from a normal distribution.
Finally, the linear dependence was measured using Pearson's correlation
coefficient.
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Results |
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TopAbstractPatients and MethodsResultsDiscussionReferences |
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A significant linear negative correlation was evident between NO
concentration and percentage of total sperm motility (
= –.50;
P = .0007) (Figure 2).
Furthermore, the CASA evaluation of sperm kinetic characteristics revealed a
significant linear negative correlation between NO concentration and VSL
( = –.49; P = .0006)
(Figure 3) and VCL ( =
–.56; P < .0001) (Figure
4). These data become more interesting from the statistical point
of view, in consideration of the reduced numerical size of the sample.
Finally, no correlation was evident between NO concentration and ALH (data not
shown).
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iNOS and eNOS expression was examined by Western blot in both normozoospermic and asthenozoospermic men to evaluate the NOS isoforms that are responsible of the observed NO levels. As shown in Figure 5, both iNOS and eNOS proteins were expressed in the 2 groups of considered patients.
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Discussion |
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TopAbstractPatients and MethodsResultsDiscussionReferences |
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Herrero et al (2000) have reported that NO is capable of regulating cyclic adenosine monophosphate (cAMP) concentration and, consequently, capacitation via stimulation of adenyl cyclase activity. This modulation could act directly by targeting the enzyme or by altering the action of a distinct regulatory protein. It has been speculated on the sensitivity of sulfhydryl groups of proteins to both nitrosative and oxidative events, which in turn may elicit distinct functional changes. However, this type of regulation remains to be elucidated in the male gamete.
Even though cAMP is the best-established messenger in human sperm capacitation, it is possible that NO has other targets for modulating this process. As demonstrated in other tissues, NO could modulate cyclic guanosine monophosphate levels (Murad, 1994). Alternatively, the mechanism by which NO leads to phosphotyrosine accumulation could conceivably involve the stimulation of tyrosine kinase activity, the inhibition of tyrosine phosphatase activity, or a combination of both of these effects.
In the present study, nitrite, the stable endproduct of the NOS/NO pathway, has been detected in the semen of a control group of normozoospermic fertile men and a group of idiopathic asthenozoospermic infertile patients. Our results provide evidence that NO concentration is significantly lower in the former than in the latter. Furthermore, a significant negative linear correlation between NO concentration and sperm motility is evident, as well as other kinetic characteristics of spermatozoa such as VCL and VSL. Since the leukocyte concentration was less than 1 x 106/mL in each sample, it is unlikely that a significant contribution to nitrite concentrations could have come from neutrophils, the only other contaminating cell type capable of NO production.
Our findings suggest that high concentrations of NO play a deleterious effect on spermatozoa kinetic characteristics. As a possible explanation, it has been reported that NO may react with superoxide or hydrogen peroxide, resulting in the formation of peroxinitrite, hydroxyl radical, NO2, or singlet oxygen, which cause oxidation of sperm membrane lipids and thiol proteins (Stamler et al, 1992). NO also may inhibit cellular respiration by nitrosylation of heme in mitochondrial enzymes, aconitase, and glyceraldehyde phosphate dehydrogenase, leading to a depletion of adenosine triphosphate and a consequent loss of motility by spermatozoa.
In conclusion, the main features of the present study are that the NO concentrations in the semen samples of infertile patients affected by asthenozoospermia are significantly higher than those in normozoospermic fertile subjects. The present data suggest that the overproduction of this free radical and the consequent excessive exposure to oxidative conditions have a potential pathogenetic role in the reduction of sperm motility. The role played by NO in spermatozoa capacitation (Aitken et al, 1995; Zini et al, 1995) leads us to speculate that such paradoxical involvement in both pathologic and physiologic processes depends on the alternative redox state and relative level of NO.
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References |
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