The expression of secretory leukocyte protease inhibitor (SLPI) in the Fallopian tube: SLPI protects the acrosome reaction of sperm from inhibitory effects of elastase

Yukinobu Ota1, Koichiro Shimoya1,4, Qing Zhang1, Akihiro Moriyama2, Rika Chin1, Kumiko Tenma1, Tadashi Kimura3, Masayasu Koyama1, Chihiro Azuma1 and Yuji Murata1

1 Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita City, Osaka565-0871, 2 Department of Obstetrics and Gynecology, Saiseikai Nakatsu Hospital, Osaka 543-8502 and 3 Department of Gynecology, Osaka Medical Center for Cancer and Cardiovascular Diseases, 1-3-3 Nakamichi, Higashinari-ku, Osaka 537-8511, Japan


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Secretory leukocyte protease inhibitor (SLPI) is a protein found in various fluids, including parotid secretions, cervical mucus, seminal plasma and ascites, and is a potent inhibitor of human leukocyte elastase activity. The objective of the study was 2-fold, to evaluate (i) the presence of SLPI in the Fallopian tube, and (ii) the effect of SLPI on the acrosome reaction of sperm. METHODS AND RESULTS: Western blot analysis revealed that SLPI protein was detected as a 12 kDa band in the isthmus, ampulla and infundibulum of the Fallopian tube. Immunohistochemistry using an anti-SLPI polyclonal antibody revealed positive staining of epithelial cells in the Fallopian tube. RT–PCR demonstrated that SLPI transcripts were expressed in the Fallopian tube. To determine the function of SLPI in the Fallopian tube, the effects of SLPI and elastase on the sperm acrosome reaction were examined. SLPI prevented the reduction of the acrosome reaction by elastase in a dose-dependent manner. CONCLUSION: The present findings suggest that SLPI in the Fallopian tube contributes to sperm–oocyte interaction.

Key words: acrosome reaction/Fallopian tube/secretory leukocyte protease inhibitor (SLPI)


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Secretory leukocyte protease inhibitor (SLPI), which has been found to be a potent inhibitor of human leukocyte elastase, cathepsin G and trypsin (Thompson and Ohlsson, 1986Go), is a 12 kDa protein that was originally isolated from human parotid gland secretions (Thompson and Ohlsson, 1986Go). SLPI has also been shown to inhibit mast cell chymase (Fink et al., 1986Go), a protease released during mast cell degranulation, and to inhibit histamine release from mast cells in vitro (Dietze et al., 1990Go). SLPI is found in various fluids, including parotid secretions (Thompson and Ohlsson, 1986Go), cervical mucus (Denison et al., 1999aGo; Moriyama et al., 1999Go), seminal plasma (Ohlsson et al., 1995Go; Moriyama et al., 1998Go; Denison et al., 1999bGo), ascites (Shimoya et al., 2000Go) and amniotic fluid (Denison, et al., 1999cGo; Zhang et al., 2001Go). The levels of SLPI in biological samples have been monitored to examine their correlation with pathological conditions (Kida et al., 1992Go; Kouchi et al., 1993Go; Sluis et al., 1994). Increased levels of SLPI in nasal secretions and in bronchoalveolar lavage fluids may be indicative of inflammatory lung conditions or allergic reactions (Fryksmark et al., 1989Go; Vogelmeier et al., 1991Go; Lee et al., 1993Go). We have also reported that SLPI modulates immunodefence function in the male and female genital tracts (Moriyama, et al., 1998Go; 1999Go; Shimoya et al., 2000Go). Ohlsson et al. suggested that SLPI serves a local protective function against proteolytic degradation of the male reproductive tract tissues (Ohlsson et al., 1995Go). King et al. reported that SLPI played an antibacterial protective role in the human endometrium (King et al., 2000Go). It has also been reported that the titres of SLPI in vaginal fluids are related to antiviral function and genital tract infection (Draper et al., 2000Go; Pillay et al., 2001Go)

Many of the early steps in human reproduction take place in the Fallopian tube. These include gamete transport, maturation, fertilization and early embryogenesis. It follows, therefore, that the environment in the Fallopian tube represents the optimal conditions for these and other important developmental processes. The Fallopian tube contains various factors, such as growth factors and cytokines (Buhi et al., 1999Go). However, little is known about defensive factors in the Fallopian tube. SLPI modulates immunodefence functions in various organs (Ohlsson et al., 1995Go; Moriyama et al., 1998Go, 1999Go). We have reported that SLPI has a role in the recovery of sperm motility reduced by elastase (Moriyama et al., 1998Go). The aim of this study was to investigate the expression of SLPI in the Fallopian tube and to clarify the functions of SLPI in the Fallopian tube during fertilization.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Reagents
Goat anti-SLPI polyclonal antibodies and recombinant (r)SLPI were purchased from R&D Systems (Minneapolis, MN, USA). Recombinant elastase was purchased from Sigma Chemical Co. (St Louis, MO, USA). Goat serum used for the control in histochemical analysis was purchased from Zymed Laboratories (San Francisco, CA, USA).

Samples
Nine samples of Fallopian tubes were obtained from gynaecological patients who underwent total hysterectomy and bilateral oophorectomy. The patients ranged in age from 35 to 47 years old. Patients with venereal infection complications were excluded from this study. This study was approved by the local ethics committee of the Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine. Informed consent was obtained from each patient. Semen samples were obtained from seven proven fertile men. The fertile men had fathered at least one child and had no recent history of venereal infection. Semen was obtained by masturbation after 5 days of abstinence. Samples were collected in a sterile container and examined within 1 h of ejaculation.

Tissue preparation for Western blot analysis
The homogenizing buffer for protein extraction from the Fallopian tubes consisted of 0.5 mol/l Tris–HCl (pH 6.8), 10% sodium dodecyl sulphate (SDS), 6% ß-mercaptoethanol and 1% bromophenol blue. The Fallopian tubes were homogenized in a 2 ml volume. Homogenates were centrifuged at 4°C for 30 min at 14 000 g to remove debris. Following protein determinations, the samples were aliquoted and subjected to polyacrylamide gel electrophoresis.

Western blot analysis of Fallopian tubes
To examine SLPI protein in the Fallopian tubes, we performed Western blotting analysis using an anti-human SLPI polyclonal antibody. A total of 10 µg of oviductal protein were electrophoresed on a 15% SDS–polyacrylamide gel and transferred onto a nitrocellulose membrane (0.45 µm; Schleicher and Schuell, Dassel, Germany). The membrane was incubated with 5% dried milk protein followed by anti-human SLPI polyclonal antibody. The primary antibody was used at a final concentration of 1.0 µg/ml. SLPI immunoreactivity was visualized using an enhanced chemiluminescence Western blotting analysis system (Amersham, Aylesbury, UK).

Protein assay
Protein levels were determined with BioRad (Hercules, CA, USA) Protein Determination Reagent, according to the method of Bradford (Bradford, 1976Go).

Determination of SLPI levels in the Fallopian tubes by densitometric analysis of Western blotting
To measure titres of SLPI levels in different parts of the Fallopian tubes, the expression of SLPI protein was quantified and analysed by an NIH image software program (developed and provided by the Research Services Branch of the National Institute of Mental Health). Intra- and inter-assay variabilities of the fractalkine titres were within 10%.

RNA extraction
RNA was extracted from Fallopian tube samples of 0.5 g wet weight by acid guanidine thiocyanate–phenol–chloroform extraction according to the method of Chomczynski and Sacchi (Chomczynski and Sacchi, 1987Go).

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 1 U/µl of M-MLV-RTase and 1 µl of RNA sample in a 1xRTase buffer, random primers (1.25 pmoles/µl) and dNTP mix (0.5 mmol/l) for 40 min at 42°C. PCR amplification was performed in a reaction volume of 10 µl with sequence-specific primers for human SLPI (5'-ACTCCTGCCTTCACCATGAA-3'/5'-CATTCGATCAACTGGCACTT-3') and against human neutrophil elastase (5'-GCTCAA-CGACATCGTGATTC-3'/5'-CTCACGAGAGTGCAGACGTT-3').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 570 bp DNA product that corresponded to the published sequence of the SLPI gene (Stetler et al., 1986Go) and a 231 bp DNA product that corresponded to the published sequence of the neutrophil elastase gene (Okano et al., 1990Go).

RT was performed with total RNA without reverse transcriptase (a mock RT sample) to detect possible contamination by genomic DNA in RNA samples. A total of 10 µl of a 20 µl PCR mixture was electrophoresed on 1.5% agarose gel and stained with ethidium bromide, and amplified products were visualized by UV illumination. PCR products were digested with BamHI to confirm that they were authentic SLPI transcripts. Molecular sizes were estimated using a 100 bp DNA ladder. All primers were obtained from Invitrogen life technologies (Tokyo, Japan).

Immunohistochemical staining of SLPI in the Fallopian tubes
To determine the localization of SLPI in the Fallopian tube, we performed immunohistochemical staining using an avidin–biotin peroxidase complex method kit (OminiTags Universal Streptavidin/ Biotin Affinity Immunostaining Systems, Lipshaw, Pittsburg, PA, USA). Paraffin sections of the Fallopian tube were incubated in 0.3% hydrogen peroxide to block endogenous peroxidase and covered with 2% goat IgG to minimize non-specific binding. The 1000-fold diluted goat polyclonal anti-SLPI antibody (R&D Systems) or control preimmune goat serum for the control was applied at RT and left for 1 h. After the sections were rinsed with phosphate-buffered saline solution, they were further incubated for 30 min with biotin-labelled goat anti-mouse IgG, and then with avidin–peroxidase complex at 4°C. Peroxidase activity in the sections was visualized with 0.1% 3,3-diaminobenzidinine-tetrahydrochloride containing 0.02% hydrogen peroxide in 0.1 mol/l Tris buffer (pH 7.2). The slides were counter-stained with Mayer’s haematoxylin. HE staining was performed on the same sections.

Preparation of motile sperm
Semen specimens were obtained after 5 days of abstinence. After liquefaction at room temperature, the semen was examined to determine the sperm count and motility using a Makler Counting Chamber (Sefi-Medical Instruments, Haifa, Israel). The absence of leukocytospermia (polymorphonuclear cells >1x106/ml) in the collected samples was verified. Motile sperm were obtained by the swim-up method (World Health Organization, 1992Go).

Incubation of motile sperm with SLPI and elastase
The motile sperm were adjusted to 4x106/ml and immediately incubated with various concentrations of elastase and SLPI. After incubation for 24 h, the acrosome reaction was determined using a kit specific for the acrosome reaction.

Determination of acrosome reaction using the acrobeads kit
The sperm stimulated with SLPI and elastase were washed twice in modified human tubal fluid (HTF) medium with 10% serum substitute supplement (SSS) and re-suspended in modified HTF medium with 3% SSS. To assess the acrosome reaction, we used an acrobeads kit (Fuso Pharmacy, Osaka, Japan) specific for the acrosome reaction. The reaction of sperm with MH61 beads (Ohashi et al., 1994Go) was carried out in four wells of a 60-well flat-bottomed human leukocyte antigen (HLA) multiplate (Sumitomo Bakellite, Tokyo, Japan).

In the first step, serial dilution of sperm suspension in the wells was performed as follows: (i) 10 µl of capacitation medium from the kit was added to the second, third and fourth wells; (ii) 10 µl of sperm suspension (4x106/ml) was added to the first and second wells; (iii) the mixture in the second well was mixed by pipetting, and then a volume of 10 µl was removed and placed into the third well; (iv) the same procedure was carried out between the third and fourth wells; (v) 10 µl of the mixture was removed from the fourth well.

In the next step, 10 µl of MH61-beads (2x105/ml) were added to each well, and the contents of each well were mixed gently with the tip of a pipette. The 60-well tissue culture plate was incubated at 37°C in 5% CO2 /95% air and agglutination of the bead–sperm complexes was observed at 100x magnification with an inverted phase-contrast microscope in five fields of each well after 6 h of incubation. After the acrosome reaction was completed, all of the MH61 beads were bonded to sperm. Positive agglutination was defined as the absence of MH61 beads free from bound sperm in the microscopic field. The grades of acrosome reaction were determined as grades 0–4, with a higher acrobeads’ score representing a higher rate of acrosome reaction (Ohashi et al., 1992Go, 1995Go). The use of acrobeads has been compared with conventional methods of acrosome reaction determination, such as Pisum sativum agglutinin staining (Kawamoto et al., 1999Go).

Statistical analysis
Values represent means ± SEM. Statistical analysis was conducted using the Wilcoxon test and P < 0.05 was considered significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
To detect SLPI protein in the Fallopian tubes, we performed Western blot analysis. As shown in Figure 1aGo, SLPI protein was detected as a 12 kDa band in all parts of the Fallopian tube. A second immunoreactive smaller band was also detected by Western blot analysis. This band was thought to represent a degradation product of SLPI. To determine SLPI levels in the parts of the Fallopian tubes, we analysed the densitometric intensity of the Western blot analysis using ‘NIH image’ software. Figure 1bGo shows the levels of SLPI protein in each part of the Fallopian tube. SLPI protein was present at similar levels in the isthmus, ampulla and infundibulum, and we concluded that SLPI was present in all parts of the Fallopian tube.



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Figure 1. (a) Western blot analysis of SLPI protein in the Fallopian tube. Approximately 10 µg of Fallopian tube protein were electrophoresed on a 15% SDS–polyacrylamide gel and transferred onto a nitrocellulose membrane. The SLPI signal was detected as described in the text. PC: positive control (200 ng rSLPI). Lane I: tissue lysate from the isthmus of the Fallopian tube of a woman with myoma uteri (case 1). Lane A: tissue lysate from the ampulla of the Fallopian tube of the case 1. Lane F: tissue lysate from the infundibulum of the Fallopian tube of the case 1. Lane NC: negative control. (b) The levels of SLPI protein in the parts of the Fallopian tubes by densitometric determination of Western blot analysis. Approximately 10 µg of Fallopian tube protein were electrophoresed on a 15% SDS–polyacrylamide gel and transferred onto a nitrocellulose membrane. The SLPI signal was detected as described in the text. Values represent means ± SEM (n = 4). I, A and F represent the isthmus, ampulla and infundibulum of the Fallopian tube respectively. There is no significant difference of the SLPI protein levels among the isthmus, ampulla and infundibulum.

 
RT–PCR was then performed to examine the expression of the SLPI gene in the Fallopian tube. Figure 2aGo shows that SLPI transcripts were present in the Fallopian tube. The PCR products were digested with BamHI to confirm that they were derived from authentic SLPI gene transcripts. The 570 bp DNA product was digested to 336 and 234 bp fragments, as expected (data not shown). To identify the origin of SLPI, we performed immunohistochemical staining of sections of the Fallopian tube using an anti-SLPI polyclonal antibody. The epithelial cells in the Fallopian tube were intensely stained (Figure 3Go). Both ciliated and non-ciliated epithelial cells were stained, but ciliated cells were slightly more strongly stained than non-ciliated cells. The expression of elastase mRNA in the Fallopian tubes was examined by RT–PCR. As shown in Figure 3Go, neutrophil elastase mRNA was detected in the Fallopian tube.



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Figure 2. (a) SLPI mRNA expression in the Fallopian tube. Agarose gel electrophoresis of RT–PCR-amplified SLPI cDNA. M: DNA size marker, 100 bp ladder. Second lane: cDNA from the Fallopian tubes of a woman with myoma uteri (case 1). Third lane: cDNA from a mock RT sample of the Fallopian tubes of case 1. Fourth lane: cDNA of the Fallopian tubes from a woman with endometriosis (case 2). Fifth lane: cDNA from mock RT sample of the Fallopian tubes of case 2. (b) Neutrophil elastase mRNA expression in the Fallopian tube. Agarose gel electrophoresis of RT–PCR-amplified elastase cDNA. M: DNA size marker, 100 bp ladder. Lane 1: cDNA from the Fallopian tubes of case 1. Lane 2: cDNA from the Fallopian tubes of case 2. Lane 3: cDNA from the Fallopian tubes of case 3. Lane 4: cDNA from the Fallopian tubes of case 4.

 


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Figure 3. Immunohistochemical staining of SLPI-producing cells in the Fallopian tube. The cells in the Fallopian tube were stained by the avidin–biotin complex method with a goat polyclonal anti-SLPI antibody (a,d) or control goat serum (b,e). HE staining was demonstrated in (c,f). The epithelial cells in the Fallopian tube were intensely stained. The scale bar represents 200 µm in (a,b,c) and 100 µm in (d,e,f).

 
To determine the function of SLPI in the Fallopian tube, motile sperm of proven fertile donors were cultured with various concentrations of SLPI and elastase. The acrosome reaction was assessed using a specific kit. Figure 4Go demonstrates that incubation of the sperm with elastase for 24 h reduced the acrosome reaction of sperm in a dose-dependent manner. This reduction was prevented by the addition of SLPI. Figure 4BGo shows that SLPI antagonized the reduction of the acrosome reaction by elastase in a dose-dependent fashion. These results demonstrate that SLPI in the oviduct might prevent elastase from interfering with the acrosome reaction of the sperm.



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Figure 4. (a) The effect of elastase on the acrosome reaction of sperm. Motile sperm (5x105 cells/ml) were cultured in the presence of various concentrations of recombinant (r)-elastase. The acrosome reaction was determined by the acrobeads test. The values represent the scoring of the acrobeads test in seven proven fertile donors. (b) Dose-dependent effect of SLPI on the reduction of the sperm acrosome reaction by elastase. Motile sperm were cultured for 24 h in the presence of various concentrations ofr-SLPI plus 100 mg/l r-elastase. The acrosome reaction was determined by the acrobeads test. The values represent the scoring of the acrobeads test in seven proven fertile donors.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In the present study, we demonstrated the expression of SLPI protein and mRNA in the Fallopian tube. The findings suggested that SLPI was constitutively present in all parts of the Fallopian tube. Proteases and protease inhibitors play essential interactive roles during the inflammatory process. SLPI, which is a potent inhibitor of human leukocyte elastase, cathepsin G and trypsin (Thompson and Ohlsson, 1986Go), participates in the body’s natural defence. This molecule might contribute to homeostasis of the immunodefence system in the oviduct in case of salpingitis, for example in chlamydia infection.

We previously reported the beneficial effect of SLPI in the cervical mucus during the menstruation cycle and suggested that SLPI might be a major defensive molecule of cervical tissues (Moriyama et al., 1999Go). King et al. demonstrated that SLPI in the human endometrium and decidua played an antibacterial protective role (King et al., 2000Go). It may play a protective role by preventing damage caused by various mechanisms such as macrophage digestion and the release of inhibitory factors. Further investigations will be necessary to examine the relationship between the SLPI level and genital tract infections such as salpingitis.

Our immunohistochemical analysis using anti-SLPI polyclonal antibody showed that the epithelial cells of the Fallopian tube were intensely stained, suggesting that these epithelial cells are the main source of SLPI in the Fallopian tube. The ciliated cells were slightly more strongly stained than the non-ciliated cells. The Fallopian tubes have bacteriostatic and bactericidal mechanisms that protect against infection through the Fallopian tubes into the peritoneal cavity. The up-regulation of SLPI plays a defensive role in the epithelial surface in inflammatory lung diseases (Abbinante et al., 1993Go). SLPI might protect the Fallopian tube epithelium from the leukocyte protease in the Fallopian tubes. Human semen also contains a certain amount of SLPI (Ohlsson et al., 1995Go; Moriyama et al., 1998Go; Denison et al., 1999bGo). It might be possible that SLPI on the sperm surface enters into the uterine cavity and the Fallopian tube. SLPI in semen also might protect the Fallopian tube epithelium from the leukocyte protease.

Elastase is a strong protease that is produced by leukocytes in the genital tract. Previously it was reported that proteases were present in the hamster oviduct (Diaz et al. 2000Go). In the present study, we demonstrated the expression of elastase mRNA in the Fallopian tube. We have reported that elastase is a strong inhibitor of sperm motility and that SLPI has a role in the recovery of the motility of sperm damaged by elastase (Moriyama et al., 1998Go). The Fallopian tubes have an important role in fertilization and oocyte maturation. SLPI in Fallopian tubes might play a defensive role in oocyte maturation in addition to a defensive role in promoting fertilization. Elastase reduced the acrosome reaction of sperm and this reduction was prevented by the addition of SLPI. Therefore, our previous and present results demonstrate that SLPI interferes with the reduction of sperm motility and the acrosome reaction by elastase. Human oviductal cells produce factors that are important for the maintenance of sperm motility in vitro (Yao et al., 2000Go). Boatman and Magnoni reported that an oviductal factor (oviductin) enhanced the penetration of follicular oocytes in hamsters (Boatman and Magnoni, 1995Go). An oviductal factor that was a potential in-vivo capacitating agent in cattle was reported (Parrish et al., 1989Go). SLPI expressed in the Fallopian tube is one of the important factors affecting sperm–oocyte interaction during fertilization.

Experiments using animal models have demonstrated that the vast majority of sperm that enter the oviduct remain in the lower segments of the isthmus without ascending to the ampulla, regardless of the type or time of insemination (Overstreet and Cooper, 1978Go; Hunter and Nichol, 1983Go; Hunter, 1984Go; Smith et al., 1987Go). These studies demonstrated that the caudal isthmus acts as a reservoir for sperm during the period from mating to ovulation. In the isthmus of the Fallopian tube, sperm bind to the oviductal epithelium. This interaction may represent part of the in-vivo capacitation process (Smith et al., 1987Go). Yanagimachi and Mahi observed that guinea pig sperm remained in the lower isthmus before ovulation and were transported to the ampulla at the time of ovulation (Yanagimachi and Mahi, 1976Go). The present study demonstrated that the levels of expression of SLPI protein in the isthmus are equal to those in the ampulla and the infundibulum. SLPI in the isthmus might play an important role in maintaining the motility of sperm and their ability to undergo the acrosome reaction while they are stored until ovulation.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This work was supported, in part, by Grants-in-Aid for Scientific research (Nos 13671712, 13671713, 13877273 and 12671596) from the Ministry of Education, Science, and Culture of Japan (Tokyo, Japan) and Akaeda medical research foundation.


    Notes
 
4 To whom correspondence should be addressed. E-mail: shimoya{at}gyne.med.osaka-u.ac.jp Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on December 14, 2001; resubmitted on May 10, 2002; accepted on June 13, 2002.