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
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Abstract |
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Key words: acrosome reaction/Fallopian tube/secretory leukocyte protease inhibitor (SLPI)
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Introduction |
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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., 1999). However, little is known about defensive factors in the Fallopian tube. SLPI modulates immunodefence functions in various organs (Ohlsson et al., 1995
; Moriyama et al., 1998
, 1999
). We have reported that SLPI has a role in the recovery of sperm motility reduced by elastase (Moriyama et al., 1998
). 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.
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Materials and methods |
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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 TrisHCl (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% SDSpolyacrylamide 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, 1976).
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 thiocyanatephenolchloroform extraction according to the method of Chomczynski and Sacchi (Chomczynski and Sacchi, 1987).
RTPCR amplification
RTPCR was performed using an RTPCR 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., 1986) and a 231 bp DNA product that corresponded to the published sequence of the neutrophil elastase gene (Okano et al., 1990
).
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 avidinbiotin 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 avidinperoxidase 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 Mayers 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, 1992).
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., 1994) 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 beadsperm 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 04, with a higher acrobeads score representing a higher rate of acrosome reaction (Ohashi et al., 1992, 1995
). The use of acrobeads has been compared with conventional methods of acrosome reaction determination, such as Pisum sativum agglutinin staining (Kawamoto et al., 1999
).
Statistical analysis
Values represent means ± SEM. Statistical analysis was conducted using the Wilcoxon test and P < 0.05 was considered significant.
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Results |
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Discussion |
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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., 1999). King et al. demonstrated that SLPI in the human endometrium and decidua played an antibacterial protective role (King et al., 2000
). 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., 1993). 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., 1995
; Moriyama et al., 1998
; Denison et al., 1999b
). 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. 2000). 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., 1998
). 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., 2000
). Boatman and Magnoni reported that an oviductal factor (oviductin) enhanced the penetration of follicular oocytes in hamsters (Boatman and Magnoni, 1995
). An oviductal factor that was a potential in-vivo capacitating agent in cattle was reported (Parrish et al., 1989
). SLPI expressed in the Fallopian tube is one of the important factors affecting spermoocyte 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, 1978; Hunter and Nichol, 1983
; Hunter, 1984
; Smith et al., 1987
). 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., 1987
). 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, 1976
). 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.
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Acknowledgements |
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Notes |
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References |
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Submitted on December 14, 2001; resubmitted on May 10, 2002; accepted on June 13, 2002.