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From the * Department of Urology, St Marianna
University School of Medicine, Kawasaki, Japan; the
R&D Center, Biomedical Laboratories,
Kawagoe, Japan; and the Scientific Crime
Laboratory, Kanagawa Prefectural Police, Yokohama, Japan.
| Correspondence to: Dr Teruaki Iwamoto, Department of Urology, St Marianna University School of Medicine, 2-16-1, Sugao, Miyamae-ku, Kawasaki 216-8511, Japan (e-mail: t4iwa{at}marianna-u.ac.jp). |
| Received for publication April 14, 2003; accepted for publication July 21, 2003. |
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Abstract |
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Key words: Sperm motility inhibitor, seminal vesicles, asthenozoospermia
To evaluate the Sg quantity in liquefied seminal plasma, it is important that the stable antigen fragment is used to raise the antibody. Moreover, it is suggested that the criteria used when targeting the sperm motility region of the Sg antigen also focus on clarifying the role that it plays after the liquefaction of spermatozoa. Previously, we demonstrated that recombinant Sg I (rSg I) that lacks the SPMI region lost its inhibitory activity on demembranated spermatozoa (Miyano et al, 2003). Therefore, we produced an mAb that recognized the SPMI fragment of Sg and developed a sandwich ELISA system to quantify the original Sg concentration in seminal plasma. In the present study, the relationship between Sg levels in seminal plasma and semen parameters, which were obtained from semen analysis, was examined to elucidate the function of SPMI under physiological conditions.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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The tissue specimens of the seminal vesicle and prostate were obtained from patients (n = 2) who were undergoing a prostatectomy for prostate cancer. The epididymis and testis were from a patient who was undergoing an orchiectomy for a testicular tumor. These specimens were fixed with 10% formalin in phosphate-buffered saline (PBS) for 12 hours at 25°C and embedded in paraffin. The 5- to 7-µm-thick sections were cut with a microtome and mounted on glass slides (Mitsubishi Biochemical Laboratory, Tokyo, Japan). These sections were provided for immunohistochemistry after deparaffinization and dehydration.
Anti-Sg polyclonal antibody (pAb) was prepared by immunizing rabbits with human rSg II and purified by affinity-column chromatography with protein G-sepharose (HiTrap Protein G, Amersham Biosciences, Uppsala, Sweden). Mice were also immunized with human rSg II to produce an mAb. The mAb F11 was purified from mouse ascitic fluid by affinity-column chromatography with protein G-sepharose (HiTrap Protein G, Amersham).
The Ethical Committee/Institutional Review Board of St Marianna University approved the study. Informed consent was obtained from volunteers before the use of their tissue or semen for research.
Western Blotting
rSg proteins rSg I (aa 23-462), rSg I, which lacks the SPMI region (aa
108-159 were deleted from rSg I; rSg I [-SPMI]), and rSg I, which lacks the
repeat region (aa 320-379 were deleted from rSg I; rSg I [-repeat]) were
produced by the baculovirus system in Spodoptera frugiperda (Sf21)
cells. A recombinant SPMI fragment (aa 108-159; rSPMI) was produced by
Escherichia coli. Then, these recombinant proteins were purified with
the QIA express Ni-NTA Protein Purification System (QIAGEN, Hilden, Germany)
as previously reported (Murakami et al,
1998; Miyano et al,
2003). Seminal plasma and purified recombinant protein samples
were solubilized in sodium dodecyl sulfate (SDS) sample buffer (62.5 mM
Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 5% ß-mercaptoethanol, and 0.0125%
bromophenol blue), after the measurement of each protein concentration by the
bicinchoninic acid assay (Smith et al,
1985) using bovine serum albumin (BSA) as a standard.
Molecular-mass separations were carried out on 10%-20% (wt/vol) gradient
polyacrylamide SDS gels. These gels were electroblotted onto polyvinylidene
difluoride membranes, and the membranes were stained with Coomassie brilliant
blue R-250 or blocked for 1 hour at 37°C with a solution of 5% (wt/vol)
nonfat dried milk in Tris-buffered saline (20 mM, pH 7.8) supplemented with
Tween 20 (0.05%; TBS-T). The membranes were incubated either with anti-human
rSg mAb (F11) or with mouse normal immunoglobulin G (IgG) at 1 µg/mL in
TBS-T containing 5% (wt/vol) nonfat dried milk for 1 hour at 37°C. After 3
washes in TBS-T for 5 minutes, the membranes were incubated with goat
anti-mouse IgG conjugated with alkaline phosphatase (BioRad, Richmond, Calif)
at a dilution of 1:3000 in TBS-T for 30 minutes at 37°C. Following several
washes in TBS-T, detection was performed with
5-bromo-4-chloro-3-indolylphosphate/nitro blue tetrazolium (BCIP/NBT; Sigma
Chemical Co, St Louis, Mo).
Immunohistochemistry
The tissue sections were blocked for endogenous peroxidase activity with 3%
hydrogen peroxide in methanol for 15 minutes. Nonspecific antibody binding was
blocked in PBS containing 10% normal goat serum for 1 hour at room
temperature. The sections were incubated with the mAb F11 at 5 µg/mL or
with normal mouse IgG as a negative control at 5 µg/mL in PBS containing
10% normal goat serum for 1 hour at 37°C. Each section was treated with
peroxidase-conjugated anti-mouse IgG (Histofine Simple Stain MAX PO, Nichirei,
Tokyo) for 30 minutes at room temperature and developed with
3-amino-n-ethylcarbazole (Nichirei) for visualization. Sections were
counter-stained with Mayer hematoxylin for 10 seconds and mounted in aqueous
permanent mounting solution (Nichirei).
The Quartz-Crystal Microbalance
The fragment peptides of SPMI that were chemically synthesized and purified
by reverse-phase high-pressure liquid chromatography (HPLC) were purchased
from the manufacturer (QIAGEN K. K., Tokyo, Japan). Detection of peptides
binding to the mAb F11 was carried out using a quartz-crystal microbalance
(QCM) technique. A QCM is a very sensitive mass-measuring device. Its
resonance frequency decreases linearly upon the increase of the mass on the
QCM electrode at nanogram levels (Okahata
et al, 1999). Previously, this technique was applied for
protein-DNA interactions using a 27-MHz QCM
(Okahata et al, 1998);
however, there has been no study of protein-protein interactions. First, the
mAb F11 (0.8 µg) was immobilized on a gold electrode surface of a 27-MHz
QCM with hydrophobic interactions, and the frequency of the electrode was
detected and recorded with a frequency counter-equipped computer (Affinix Q
System, Initium Inc, Tokyo, Japan) in the mixing chamber containing PBS at
25°C. After stabilization, each peptide or rSPMI was added to the chamber
at 10 µg/mL, and the changes in frequency were recorded. We confirmed the
QCM result with Western blotting as described above. Purified Sg proteins were
separated using 10% SDS gel. For immunoblot, mAb F11 (0.3 µg/mL) and mAbF11
(0.3 µg/mL) preabsorbed with each peptide (5 µg/mL) were used.
Determination of SPMI/Sg in Seminal Plasma
Seminal plasma levels of Sg were measured using a sandwich ELISA system
that we developed for this study. This was an ELISA system with a quantitative
sandwich technique using microtiter plates. A human seminal plasma sample
diluted with a dilution buffer (0.1 M phosphate buffer, pH 7.5, containing
0.9% NaCl, 0.1% Tween 20, and 1% BSA) was placed into the wells of a
microtiter plate precoated with the mAb F11 at 400 ng/well. As the detecting
antibody, the anti-Sg pAb was added to the wells at 100 ng/well in the
dilution buffer. The secondary antibody, horseradish peroxidase-conjugated
goat anti-rabbit IgG (Zymed Laboratories Inc, San Francisco, Calif), was added
to the wells at 1:4000 diluted with the dilution buffer. The resulting
absorbance with o-phenylendiamine was proportional to the amount of
bound Sg, and was measured at 492 nm; concentrations were estimated using a
standard curve. HPLC-purified Sg II was prepared by a modified method
previously reported (Robert and Gagnon,
1996) and was used as the standard. Briefly, semen samples treated
with 8 M urea were reduced with dithiothreitol and treated with iodoacetamide;
then, the centrifuged supernatant was loaded onto an SP-Sepharose column
(Amersham Biosciences, Tokyo, Japan). Among the fractions of Sg that were
eluted with a linear gradient of NaCl (0-400 mM), the former fractions
containing Sg II were pooled and loaded onto an HPLC column (Vydac C4, The
Separation Group, Hesperia, Calif). The eluate containing purified Sg II was
lyophilized and stored at -80°C until use. Protein concentration was
measured by the bicinchoninic acid assay
(Smith et al, 1985) using BSA
as a standard. All measurements were performed in duplicate, and the mean was
taken. The minimum detectable concentration was 15 ng/mL, and intra- and
interassay coefficients of variation were 6.3% and less than 10%,
respectively. A typical standard curve for HPLC-purified native Sg II and the
mean ± standard deviation of the coefficient of variation (%) (n = 5)
are shown in Figure 1. We used
the linear segment of the standard curve that ranged from 31 to 250 ng/mL for
our measurements. The dilutions of seminal plasma samples were selected from 3
values [1) 1.6 x 105, 2) 3.2 x 105, or 3)
6.4 x 105] in order that the measured Sg level was within the
range.
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Statistical Analysis
Statistical analysis was conducted using the Mann-Whitney U test
for unpaired values and Spearman rank correlation coefficients for paired
values; a P-value <.05 was considered significant. Simple linear
regression analysis was evaluated between the log-transformed Sg
concentrations, sperm motility, and total protein concentrations. A log
transformation was used to obtain a normal distribution of Sg
concentrations.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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F =
9 Hz as a result of the binding of the peptide fragment EP3 onto the mAb F11
(Figure 3E and F). The addition
of rSPMI also showed the binding to the mAb F11
(Figure 3E). For the Western
blot analysis with preabsorbed mAb F11 with EP1-3, the incubations with mAb
F11 preabsorbed with excess EP3 peptides showed no immunological staining to
purified Sg I and II (Figure
3G) and the fragments of Sg in seminal plasma (data not
shown).
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Correlation of Sg Levels With Sperm Parameters
The average Sg concentration in liquefied seminal plasma was 19 ± 13
mg/mL (range, 4-68 mg/mL, n = 100). The SPMI/Sg concentrations were correlated
with the total protein concentrations but not with any other semen parameters
(Table 1). However, after log
transformation, the SPMI/Sg concentrations were correlated positively with the
total protein concentrations (r = 0.793, P < .0001) and
negatively with the percentages of sperm with forward progression (r
= -0.229, P = .0220) (Figure
4). No significant difference in the mean of SPMI/Sg concentration
was found between the oligozoospermic group (sperm concentration 
20
x 106/mL) and the group with the normal sperm concentration
(sperm concentration >20 x 106/mL) or between the
asthenozoospermic group (sperm motility 50%) and the group with normal
sperm progression (sperm motility >50%)
(Table 2).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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The mAb F11 was found suitable for the quantification of Sg in seminal plasma after liquefaction, because fragments of 14 and 18 kDa can be found that react with this antibody in seminal plasma after liquefaction. Sg is the physiological substrate of PSA, but the SPMI fragment region appears to be resistant to its protease activity (Robert et al, 1997). The detection antibody in this ELISA system, anti-Sg pAb, was demonstrated to be a suitable antibody for the detection of Sg fragments as a marker for human semen identification in forensic samples because of its high sensitivity and broad detection range of Sg fragments (Sato et al, 2001). Therefore, this ELISA system is suitable for the quantification of Sg as the precursor protein of SPMI in seminal plasma after liquefaction. The estimated Sg I concentration in seminal plasma immediately after ejaculation was 7-11 mg/mL (Robert and Gagnon, 1996). The present study showed that the mean Sg concentration, 19 ± 13 mg/mL, was the sum of Sg I and II. Analysis of the gene structure of Sg indicates that an equal number of molecules of both Sg I and II are expressed, so that the total Sg present was estimated to be twice that of Sg I, 14-22 mg/mL. This estimated value was within the range of the results from the present study. Another quantification of Sg in seminal plasma using an immunofluorometric assay showed that the concentration of Sg was 153 ± 61 µM (20-350 µM) (Koistinen et al, 2002). If the calculation was performed as the sum of Sg I and II, the concentration of Sg is estimated at 9.6 ± 3.8 mg/mL (1.3-2.2 mg/mL). The use of different methods and standards as well as dilutions of seminal plasma may cause such a difference.
In this study, we showed that the SPMI activity of Sg in liquefied human seminal plasma was estimated as the quantity of the SPMI fragment of Sg and that the log-transformed SPMI/Sg levels were negatively correlated with the sperm motility (r = -0.23, P = .0213). As previously reported, both Sg and fragmented Sg inhibit sperm motility; however, this effect on intact spermatozoa is less than on demembranated-reactivated spermatozoa (Iwamoto and Gagnon, 1988a). Previously, levels of SPMI activity in liquefied human seminal plasma measured by a biological assay using demembranated spermatozoa were not correlated with sperm motility (Iwamoto, 1999). The assay system was sensitive for detecting SPMI activity but not under physiological conditions. An immunofluorometric assay using an mAb, which was developed recently by another group, did not show a correlation between Sg levels and sperm motility (Koistinen et al, 2002). The antibody they used may be specific to the Sg region except for the SPMI fragment, but they did not show the epitope of the antibody. Moreover, these 2 previous studies were concerned with infertile patients. The use of healthy male volunteers was one reason we detected the correlation between SPMI/Sg levels and sperm motility.
There was no significant difference in the mean of the SPMI/Sg
concentration between the asthenozoospermic group (50%) and the group with
normal sperm progression (>50%) (Table
2). The results from this study suggest that the cause-and-effect
relationship between SPMI/Sg and asthenozoospermia depends on the condition of
the spermatozoa; therefore, the receptor and signaling mechanism of SPMI must
be elucidated before further discussion about the relationship between the
levels of SPMI/Sg and asthenozoospermia can occur.
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Acknowledgments |
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Footnotes |
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References |
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) and P-values for
correlations between Sg concentration and semen quality in healthy male
volunteers*
