Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita city, Osaka 565-0871, Japan
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Abstract |
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Key words: asthenozoospermia/fractalkine/seminal plasma
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Introduction |
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Human semen contains sperm and leukocytes, as well as various types of proteins such as enzymes, hormones and those with unknown functions. Large numbers of leukocytes have been detected in some semen samples associated with male genital tract infection and infertility (Caldamone et al., 1980; Berger et al., 1982
). Indeed, it has been reported that an elevated concentration of leukocytes impairs the fertilizing ability of sperm (Maruyama et al., 1985
; Thomas, et al., 1997
). There is also increasing evidence that chemokines are responsible for the elevation of lymphocytes in inflammatory regions in the reproductive tracts. Seminal plasma also contains various chemokines, such as interleukin (IL)-8, and monocyte chemotactic and activating factor (MCAF). The levels of cytokines produced in seminal plasma are markedly elevated in genital infections such as leukospermia (Shimoya et al., 1993
, 1995
; Comhaire et al., 1994
; Zalata et al., 1995
; Depuydt et al., 1996
; Eggert-Kruse et al., 2001
). These cytokines may accumulate and activate leukocytes in the male genital tract, where activated leukocytes produce large amounts of elastase (Jochum et al., 1986
; Wolff and Anderson, 1988
; Micic et al., 1989
). It has also been shown that polymorphonuclear (PMN) elastase is an inhibitor of sperm motion (Satoh et al., 1990
; Wolff et al., 1990
), and that secretory leukocyte protease inhibitor (a potent inhibitor of leukocyte elastase) in seminal plasma reduces the extent of motility inhibition caused by elastase (Moriyama et al., 1998
).
In the present study, fractalkine was quantified in ejaculates from normal donors and infertile patients, and the relationship between fractalkine and seminal parameters was examined. The existence of CX3CR-positive leukocytes in semen samples was also examined in order to determine their possible migration into semen.
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Materials and methods |
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Semen collection
Semen was obtained by masturbation after 5 days of abstinence. Samples were collected in a sterile container and examined within 1 h after ejaculation (World Health Organization, 1992, 1999
). Semen samples were obtained from 80 infertile and 10 proven fertile men. The fertile men had fathered at least one child and had no recent history of venereal infection. Informed consent to use seminal plasma was obtained from all patients in this study.
Separation of lymphocytes from semen
Semen was layered onto a four-step discontinuous Percoll®(Pharmacia, Uppsala, Sweden) gradient (the components of each density fraction are listed in Table I), and centrifuged at 800xg in a swing-out rotor for 25 min at room temperature. Enriched lymphocyte fractions were washed three times with phosphate-buffered saline (PBS).
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Preparation of seminal plasma
At 30 min after collection, liquefied semen samples were first centrifuged at 1000xg for 10 min, after which the supernatants were re-centrifuged at 10 000xg for 15 min to remove cellular elements and debris (Shimoya et al., 1993). After centrifugation, the clear seminal plasma was collected and stored at 80°C until determination of fractalkine and IL-8 concentrations (Shimoya et al., 1993
).
Western blot analysis
In order to determine fractalkine protein levels in the seminal plasma, Western blotting analysis was performed using an anti-human fractalkine polyclonal antibody. Seminal plasma (5 µl) was electrophoresed on a 7.5 % SDSpolyacrylamide gel and transferred onto nitrocellulose membranes (0.45 µm; Schleicher and Schuell, Dassel, Germany). The membrane was incubated with 5% dried milk protein followed by anti-human fractalkine polyclonal antibody. The primary antibody was used at a final concentration of 1.0 µg/l. Fractalkine immunoreactivity was visualized using an ECL Western blotting analysis system (Amersham, Aylesbury, UK).
Determination of IL-8 in seminal plasma
Seminal plasma levels of IL-8 were monitored using an enzyme-linked immunosorbent assay (ELISA) kit (R&D systems, Minneapolis, MN, USA). Dilution of the seminal plasma caused no adverse effect on IL-8 measurements using this kit. Seminal plasma levels of IL-8 detected were >31.2 pg/ml, and no cross-reactivity was identified with either cytokines (e.g. IL-1, IL-6), chemokines (e.g. MCAF) or growth factors (e.g. epidermal growth factor; EGF). The intra-assay variability of the IL-8 kit was 6.09.2%; inter-assay variability was 7.29.6%.
Fractalkine titre in seminal plasma
Fractalkine titres in seminal plasma were measured by densitometric analysis of the Western blots. The expression of fractalkine protein was quantified and analysed using a NIH image software program [developed and provided by the Research Services Branch (RSB) of the National Institute of Mental Health (NIMH)]. Intra-assay and inter-assay variabilities of the fractalkine titres were 6.09.8 and 6.99.7% respectively.
RNA extraction
RNA was extracted from lymphocytes in the semen using a guanidine thiocyanate-phenol-chloroform extraction technique (Chomczynski and Sacchi, 1987).
RT-PCR amplification
RT-PCR was performed using an RT-PCR high kit (Toyobo Co., Tokyo, Japan). The reaction was carried out in the presence of M-MLVRTase and 1 ml RNA sample in a 5x RTase buffer, random primers and dNTP mix for 40 min at 42°C. PCR amplification was performed using an RT mixture (10 µl), with sequence-specific primers against human CX3CR (5'-TTGAGTACGATGATTTGGCTGA-3'/5'-GGCTTTGGCTTTCTTGTGG-3') (Gene Bank accession number U28934). PCR was carried out for 35 cycles using a thermal cycler (Perkin-Elmer/Cetus, Norwalk, CT, USA). Each cycle consisted of denaturation at 94°C (40 s), annealing at 52°C (40 s) and extension at 72°C (40 s). The amplification yielded a 653-bp DNA product according to the published sequence of the fractalkine gene (Muehlhoefer et al., 2000). RT was performed with total RNA without reverse transcriptase (a mock RT sample). PCR of a mock RT sample was carried out to detect any possible contamination in the RNA samples by genomic DNA. An aliquot (20 µl) of a 50 µl PCR mixture was electrophoresed on 1% agarose gel and stained using ethidium bromide; the amplified products were visualized using ultraviolet illumination. Molecular sizes were estimated using a 100-bp DNA ladder. All primers were obtained from Becks (Tokyo, Japan).
Statistical analysis
All values were presented as mean ± SEM. Statistical analysis of fractalkine and IL-8 levels in seminal plasma was conducted using Welch's t-test, and a P-value < 0.05 was considered significant. The correlation was analysed by simple linear regression.
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Results |
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Discussion |
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IL-8 in seminal plasma has been associated with genital tract infection and is a good marker for leukospermia (Shimoya et al., 1993). It has also been reported (Depuydt et al., 1996
) that certain cytokines, including IL-8, IL-6, IL-1 receptor antagonist, and IL-1
were associated with genital tract infection, though no correlation was found between IL-8 and sperm motility in the present study. Several inflammatory mediators are known to enhance fractalkine mRNA. The up-regulation of fractalkine plays an important role not only in the binding of NK cells to endothelial cells, but also in NK cell-mediated endothelium damage (Yoneda et al., 2000
). However, in contrast to other chemokines such as IL-8, elevated fractalkine levels could not be detected in the seminal plasma of infertile patients with leukospermia. These results suggest that male genital tract infections, such as leukospermia, do not affect the production of fractalkine and that fractalkine is produced in the male genital tract by a different pathway to that used to produce IL-8. Taken together, the combination of IL-8 and fractalkine in seminal plasma might provide more useful information about male infertility.
A large number of leukocytes in the semen is associated with male genital tract infection and infertility (Caldamone et al., 1980; Berger et al., 1982
), and the value of flow cytometry in detecting leukocytes in human semen has been reported (Ricci et al., 2000
). In the present study, the presence of CX3CR mRNA in leukocytes in human semen was demonstrated. Seminal plasma constitutively contains a certain amount of fractalkine. Hence, these results show that fractalkine contained in the seminal plasma might induce the migration of CX3CR-positive cells into the male genital tract, where these lymphocytes might contribute to the immune defence system. However, further studies are necessary to investigate the mechanism by which fractalkine improves sperm motility.
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Acknowledgements |
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Notes |
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References |
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Submitted on September 10, 2001; resubmitted on November 26, 2001; accepted on February 22, 2002.