1 Epithelial Cell Biology Research Center, Department of Physiology, Faculty of Medicine, The Chinese University of Hong Kong, 2 Department of Obstetrics & Gynecology, Faculty of Medicine, The University of Hong Kong, 3 Department of Anatomy, Faculty of Medicine, The Chinese University of Hong Kong, 4 Department of Pathology, Faculty of Medicine, The University of Hong Kong and 5 Department of Obstetrics & Gynecology, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, NT, Hong Kong
6 To whom correspondence should be addressed. e-mail: hsiaocchan{at}cuhk.edu.hk
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Abstract |
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Key words: acrosome reaction/conditioned medium/embryo development/hydrosalpinx/sperm motility
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Introduction |
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The role of epithelial cells in normal oviductal fluid formation and their importance in normal sperm functions and embryo development have been reported (Dickens et al., 1993, 1995, 1996; Dickens and Leese, 1994
; Downing et al., 1997
; Downing et al., 1999
). Epithelial cells from normal Fallopian tubes have been cultured (Bongso et al., 1989
; Ouhibi et al., 1989
; Henriksen et al., 1990
; Ménézo et al., 1990
; Takeuchi et al., 1991
). Co-culture of normal Fallopian tube epithelial cells with embryos has been shown to enhance in-vitro development of embryos to blastocyst stage (Bongso et al., 1989
; Ménézo et al., 1990
; Yeung et al., 1992
), improve pregnancy rates (Bongso et al., 1992
) and maintain sperm motility in vitro (Morales et al., 1996
; Murray et al., 1997
; Yao et al., 2000
). These results suggest that Fallopian tube fluid is important for a number of reproductive events. However, epithelial cells from hydrosalpinx have not been cultured and characterized, and thus the role of epithelial cells in the formation of hydrosalpinx and HF has not been investigated to any significant extent (Ajonuma et al., 2002
).
In an attempt to investigate the role of epithelial cells in the formation of hydrosalpinx and HF, epithelial cell cultures from human hydrosalpinx were established and characterized in the present study. The effects of hydrosalpinx epithelial cell culture conditioned medium (CM) on mouse embryo development, human sperm motility and acrosome reaction were also examined.
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Materials and methods |
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Epithelial cell culture and collection of conditioned medium
Normal Fallopian tubes (NFT) were obtained from healthy Chinese women within their reproductive ages and in different menstrual cycles who had bilateral salpingectomy for tubal sterilization. Patients undergoing IVF with hydrosalpinx observed during ultrasound monitoring were advised to undergo bilateral salpingectomy prior to the next treatment cycle. Normal and abnormal Fallopian tissues were collected into sterile tubes containing cold minimum essential medium (MEM; Life Technologies, USA) immediately after surgical removal, placed on ice and transported to the cell culture laboratory.
After fatty areas had been trimmed off, tissue pieces were rinsed with sterile Mg2+- and Ca2+-free phosphate-buffered saline (PBS) (Life Technologies) to remove blood and tissue fragments. The Fallopian tubes were cut at the ampulla and isthmus portion, incised longitudinally and the epithelium exposed. Tissue pieces were again rinsed thoroughly to remove any remaining trace of blood and transferred into a 15 ml centrifuge tube. Epithelial cells were enzymatically isolated. Tissue pieces were incubated overnight at 4°C in 10 mg collagenase (Sigma, St Louis, MO, USA) dissolved in 10 ml MEM that was previously filtered through 0.22 µm Millipore filter (Millipore, Bedford, MA, USA). After enzyme digestion, the contents of the tubes were poured into a Petri dish. Using a sterile scalpel blade, the epithelium was carefully and gently scraped to dislodge the remaining epithelial cells. The medium containing epithelial cells was aspirated into another sterile centrifuge tube and cell suspension spun down at 1000 g for 5 min. Epithelial cells were resuspended in 12 ml of pre-warmed Dulbeccos modified Eagle medium with nutrient mixture Hams F-12 (DMEM/F-12; Gibco-BRL, Invitrogen, Grand Island, NY, USA) supplemented with 10% FBS (Gibco-BRL, Invitrogen), 1% non-essential amino acids (Sigma) and 100 µg/ml of penicillin/streptomycin (Sigma); and washed twice by centrifugation for 5 min in the same amount of DMEM/F-12. Thereafter, cells were plated in Petri dishes and incubated in a humidified chamber containing 5% CO2 in air at 37°C for 810 h, to allow fibroblasts to settle down and attach. The medium containing mainly epithelial cells was the aspirated, washed, seeded in 10 ml of pre-warmed DMEM/F-12 in a culture dish and incubated in a humidified chamber containing 5% CO2 in air at 37°C. Some cells were cultured on collagen-coated coverslips. The collagen used in this study was C7521 type VI from human placenta (Sigma). Stock concentration (3 mg/ml): 3 mg of collagen in 1 ml of 10 mmol/l acetic acid and aliquotted at 250 µl in autoclaved microcentrifuge tubes and stored at 20°C. Coating concentration is at 1:4 dilution with ethanol. In brief, 250 µl of collagen stock solution was dissolved in 750 µl of 60% ethanol, mixed thoroughly and 50 µl applied to coat 1 cm3 area of the glass slide used.
Culture medium was changed every 2 days until cells had grown to confluence. CM was collected from normal and hydrosalpinx epithelial cell cultures after 36 h of culture when epithelial cells had grown to confluence, pooled, centrifuged (1000 g for 5 min), filtered using 0.22 µm Millipore filter and stored at 70°C until further use. Osmolality and pH were measured using Wescor digital vapour pressure osmometer (5500; Wescor Inc., Utah, USA) and Beckman pH meter (Beckman; Bora, CA, USA) respectively.
Upon confluence, culture medium DMEM/F-12 was removed and epithelial cells washed twice with sterile PBS. Mg2+- and Ca2+-free Hanks balanced salt solution (HBSS) (Life Technologies), supplemented with 0.025% trypsin and 0.01% of EDTA (Sigma), was added to the cell culture and incubated at 37°C for 510 min to detach the cells. After trypsinization, culture medium (v/v 1:1) was added to inactivate typsin and the cell suspension was washed twice by centrifugation and subjected to subculture.
Light microscopy
Fallopian tubes were cut into small pieces after removing fatty tissues and fixed in 4% paraformaldehyde overnight. Sections were cut at 5 µm using a ReichertJung, Biocut rotary microtome 1130, and dried onto Superfrost microscope slides (Fisher brand; Fisher Scientific, Pittsburgh, PA, USA). Epithelial cells cultured to confluence on slides were washed with PBS and fixed in 2% paraformaldehyde for 20 min. Cells were washed in PBS, non-specific binding sites blocked with 3% hydrogen peroxide and incubated in anti-human epithelial membrane antibody conjugated to horse-radish peroxidase (anti-EMA/HRP; Dako, Denmark) for 1 h at room temperature. Reaction product was revealed by incubation with 3,3-diaminobenzide hydrochloride substrate (DAB; Vector Laboratories, Inc., CA, USA) for another 15 min and sections were finally counterstained with methyl green.
Electron microscopy
Fallopian tube tissues and polarized epithelial cells cultured on collagen-coated glass slips were fixed in 2.5% glutaraldehyde (Electron Microscopy Science, Fort Washington, PA, USA) in 0.1 mol/l PBS (pH 7.4) and rinsed in Sorensens buffer and post-fixed in 2% osmium tetroxide (Electron Microscopy Science) for 1 h. Samples were rinsed again three times for 10 min each in Sorensens buffer, dehydrated in graded alcohol, critical point-dried in a LADD critical point dryer (Bulington, VT, USA), coated with palladium gold and examined with a scanning electron microscope (JSM 6301FE; JEOL, Tokyo, Japan). For transmission electron microscopy, fixed tissues were embedded in Epon 812 and ultrathin sections were cut with Leica Ultracut, double-stained with uranyl acetate and lead citrate. Sections were observed under an electron microscope (Hitachi H 7100, Tokyo, Japan).
Assessment of mouse embryo development
Mouse embryos were collected from superovulated adult female (810 weeks old) ICR mice. Mice were kept in the Laboratory Animal Service Center of the Chinese University of Hong Kong and were given laboratory chow and water ad libitum. They received intraperitoneal injections of 7.5 IU pregnant mares serum gonadotrophin (PMSG) (Sigma) followed up by additional injection of 7.5 IU hCG (Sigma) 48 h later. The mice were co-caged with sexually matured male ICR overnight. Female mice were examined for copulation plugs the following morning. Those that exhibited copulation plugs were killed 4042 h post hCG administration. Recovered 2-cell embryos were placed in pre-warmed modified human tubal fluid medium supplemented with 0.5% bovine serum albumin (BSA) (hTF; Irvine Scientific, Santa Ana, CA, USA). hTF is not very different from the culture medium DMEM/F-12 used for CM preparation in that both media contain all major essential nutrients in a simple culture medium. They are also supplemented with serum, HEPES and bicarbonate at similar concentrations. Both media support embryo development and can maintain sperm motility in vitro.
Eighty-four 2-cell mouse embryos were collected after superovulation and randomly allocated: 20 to hydrosalpinx CM in duplicate (40), 24 to NFT CM, and 20 to hTF. Both NFT CM and hTF served as controls. The embryos were cultured under embryo-grade mineral oil (Sigma). Embryo development was assessed after a further 3648 h of culture in vitro with an inverted microscope (Axiovert 25; Zeiss, Germany).
Measurement of sperm motility
Sperm collection and processing
The details of semen collection, sperm preparation, and sperm motility analysis had been previously described (Ng et al., 2000). In brief, semen samples were collected by masturbation from men attending the Assisted Reproduction Unit of The Prince of Wales Hospital, Chinese University of Hong Kong after abstinence of 34 days. Only samples with normal semen parameters according to World Health Organization (1999) criteria (>2 ml by volume, 20x106 sperm/ml, 50% forward progression, 30% normal morphology) were used. After complete liquefaction at room temperature, semen samples were processed with a two-step Gradient Isolate Sperm separation medium, i.e. 45 and 90% Isolate (Irvine Scientific). Semen was layered on top of 45 and 90% Isolate and centrifuged at 300 g for 20 min. The resulting sperm pellet was washed twice in hTF medium at 150 g for 10 min and finally resuspended in the same medium.
Sperm motility analysis
CM samples were thawed at room temperature and placed in a humidified chamber at 37°C under 5% CO2 in air. Sperm suspensions were adjusted with modified sperm washing medium to a concentration of 20x106/ml. Sperm from each sample were divided into three equal portions. The adjusted sperm suspension aliquots were centrifuged at 500 g for 5 min and the supernatants discarded as much as possible. The sperm pellets were resuspended in either 0.5 ml of NFT or hydrosalpinx CM and 0.5 ml of hTF (control).
Sperm motility and velocities were analysed at 1, 3, 5 and 24 h after incubation at 37°C under 5% CO2 using the TOXIVOS Sperm Analysis System (Hamilton Thorne Research, Beverly, MA, USA). TOXIVOS Sperm Analysis System version 10.8 set-up parameters were set at x4 objective in dark field, 30-frame shift rate at 60 Hz, minimum contrast of 80 and illumination intensity of 3200 at 0.82 magnifications. A 10 µl aliquot of the sperm suspension was transferred to a pre-warmed Hamilton Thorne Research 2X-CEL disposable semen analysis slide (Hamilton Thorne) with a chamber depth of 20 µm placed on a warmed microscope stage at 37°C. Five fields were randomly selected during evaluation and 200 sperm were analysed in each field.
The following CASA parameters were determined: percentage of motile sperm, mean curvilinear velocity (VCL, µm/s), mean straight line velocity (VSL, µm/s), average path velocity (VAP, µm/s), mean linearity (LIN: VSL/VCL), amplitude of lateral head displacement (ALH, µm), head beat cross frequency (BCF, Hz), and straightness (STR: VAP/VCLx100%).
Assessment of sperm acrosomal reaction (acrobead assay)
Paramagnetic beads (Dynalbeads M-450; Dynalas, Oslo) coated with monoclonal antibody MH61 (anti-CD46; Fuso Pharmaceuticals, Osaka, Japan), which binds to acrosome-reacted sperm were prepared for the assay by making an MH61 bead suspension. MH61 bead stock medium was vortexed for 30 s. Then, 20 µl was removed and added to 380 µl of BiggersWhittenWittingham (BWW) medium supplemented with BSA to make a concentration of 1.5x106/ml and stored at 4°C until used on the same day. A sterile 96-well culture plate was used for this assay. Four wells were used for one specimen and designated as wells 1, 2, 3 and 4 respectively. 100 µl of CM (test) and BWW/BSA (control) without sperm were placed in wells 2, 3 and 4 and 200 µl of sperm suspension for either test or control was placed in well 1. In order to make serial dilutions of x1, x2, x4 and x8 of sperm suspension, 100 µl aliquot was removed from well 1, added to well 2, mixed gently and thoroughly, and so on to well 4 and the final 100 µl discarded making final sperm concentrations of 4, 2, 1 and 0.5x106/ml. 10 µl of MH61 bead medium was then added to the wells and gently mixed in reverse order from wells 4 to 1. The tissue culture plate was incubated at 37°C in 5% CO2 and attachment of sperm to the beads was evaluated after 24 h.
Assessment of the degree of agglutination (attachment of sperm to the beads) after 24 h of incubation to ensure total attachment of all sperm that had undergone capacitation was done under an inverted phase-contrast microscope. According to the manufacturers protocol, each well was observed for agglutination in five different fields, top, bottom, left and right at random. If three or more of five fields in a well had positive agglutination, the well was judged as positive (Ohashi et al., 1995; Sharma et al., 1997
).
Statistical analysis
Data are presented as mean (SEM) for sperm motility analysis and median (range or %) for acrosome reaction. Analysis of variance was used for data assessment and difference between groups was compared using NewmanKeuls multiple comparison test. P < 0.05 was considered significant. Statistical analysis was carried out using GraphPad Prism (GraphPad Software Inc., San Diego, CA, USA).
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Results |
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Effects of CM on mouse embryo development
Osmolality and pH of both NFT and hydrosalpinx CM were within physiological ranges of 287300 mmol/kg and 7.27.5 respectively and comparable with control values. In all, 3/40 (8%,) mouse embryos cultured in the hydrosalpinx CM developed. Two of the three embryos developed up to 4-cell and one to 8-cell embryo stages. In NFT CM and hTF, 17/24 (71%) and 19/20 (95%) mouse embryos developed into morula and early blastocysts respectively (Figure 2). Embryo development in hydrosalpinx CM also showed poor embryo development with extensive blastomere fragmentation and degeneration compared with those of NFT CM and hTF in this study.
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Discussion |
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In the present study, CM of hydrosalpinx epithelial cell cultures demonstrated adverse effects on mouse embryo development, human sperm motility and acrosome reaction whereas CM from NTF had similar effects to that of hTF (control), suggesting that hydrosalpinx epithelial cells may be secreting substance(s) yet to be identified into the lumen. Those substances may be hostile to sperm affecting their motility, capacitation and acrosome reaction and early embryo development in post-infectious hydrosalpinx. Since hydrosalpinx yields different influences on IVF outcomes, some variations may have been possible and individual CM sample test would have provided more distinct results. However, sample size was a limiting factor in this study. Therefore, CM from all patients was pooled together prior to sperm motility and embryo development assay. Second, it would have been more clinically relevant to use human embryos, but for ethical reasons, human embryos were not used. Only two studies using human embryos in hydrosalpinx have been reported (Granot et al., 1998; Strandell et al., 1998
). Granot et al. used only four samples and abnormal (three pronuclear 3PN) embryos. Small sample size may be the reason for lack of toxicity seen in their study. On the other hand, Strandell et al. (1998
) used 12 samples and had 13.9% embryo (blastocyst) development rate (range 024%) in their 100% HF dilution, significantly different from 50% HF (33.3% range 1356%) and control (P = 0.0027).
We have suggested that abnormalities in the transepithelial ion transport across epithelia may be one of the most important factors in the pathophysiology of post-infectious hydrosalpinx formation (Ajonuma et al., 2001). Human Fallopian tube epithelial cells have been reconstituted in a polarized culture and chloride ion movement was found to be responsible for the generation of transepithelial potential difference across the cultured epithelial cells (Dickens et al., 1996
; Downing et al., 1997
). These chloride fluxes were sensitive to the inhibitors of some ion channels and co-transporters (Gott et al., 1988
). These transporters may include the sodium/hydrogen exchangers (NHE), anion Cl/HCO3 exchangers (AE), sodium bicarbonate co-transporter (NBC) and sodium epithelial channels (ENaC), aquaporin water channels (AQP) and cystic fibrosis transmembrane regulator (CFTR), a cAMP-activated chloride channel. Post-infectious hydrosalpinx pathology such as atrophy of mucosal folds, marked exfoliation and loss of epithelial cells may affect epithelial membrane ions channels and transporters located on the epithelial membrane necessary for electrolyte and fluid transport, leading to abnormal fluid secretion and reabsorption. CFTR, in particular, may also play an important role in the process of HF formation, considering its multifunctional properties, i.e. a channel, regulator of other epithelial channels, and more recently, a bacterial receptor (Pier et al., 1997
; Pier et al., 1998
; Zaidi et al., 1999
; Gerceker et al., 2000
; Goldberg and Pier, 2000
; Ajonuma et al., 2002
).
The loss of membrane polarity may lead to decreased expression of epithelial membrane transporters and ion channels. This could be responsible at least in part for the formation of HF following pelvic inflammatory disease and subsequent adverse effects (Ajonuma et al., 2002).
The electrolyte composition of CM was not analysed in this study but pH and osmolality values were within physiological ranges and comparable with the control. Therefore, reduction in the percentage of progressive motile sperm after 3 h of incubation, decreased acrosome reaction and poor embryo development in hydrosalpinx CM only were unlikely to be due to effects of pH or osmolality. Energy substrates in CM would not lead to adverse effects of hydrosalpinx CM as NFT CM prepared under the same condition exhibited CASA parameters, acrosome reaction and embryo development results comparable to hTF (control).
There are two possibilities which could account for the toxic effect of CM obtained from the epithelial cells of post-infectious hydrosalpinx. First, the hydrosalpinx epithelial cells may be producing less embryotrophic factors on which sperm motility, fertilization and early embryo development depend. Low protein values (Ng et al., 2000; Ajonuma et al., 2001
) have been suggested to contribute to HF adverse effects on sperm motility. The present finding that secretory vesicles in the NFT epithelial cell culture (Figure 1) are drastically reduced, and sometimes absent, in the hydrosalpinx epithelial cells indicated possible decreased production of oviduct-specific glycoproteins in hydrosalpinges. The alternative possibility is that infected epithelial cells in hydrosalpinx may produce toxic substance(s), not yet characterized, affecting sperm functions and embryo development. For instance, higher levels of 57 kDa heat shock proteins have been observed in HF and reported to be associated with less embryo development (Beatty et al., 1993
). It is also possible that hydrosalpinx epithelial cells may produce substance(s) that are hostile to sperm affecting their motility, capacitation and acrosome reaction. For example, the effect of reactive oxygen species (ROS) in HF on mouse embryo development has been reported (Bedaiwy et al., 2002
). It is well recognized that infection, such as that leading to hydrosalpinx and cytokine production, can induce the expression of nitrogen oxide synthase isoform II (NOS2) in a variety of cells including the female reproductive tract, generating high amounts of nitrogen oxide (NO) which is toxic to both gametes and developing embryos (Hunley et al., 1995
; Barroso et al., 1998
). A number of other factors including cytokines may also contribute to decreased sperm motility, acrosome reaction and or poor embryo development observed in the present study. David et al. (1969
) suggested that macrophages, plasmocytes and other cellular elements involved in late inflammatory reaction might release cytokines, prostaglandin, leukotrienes and other compounds that could be deleterious to intrauterine oocytes. Hydrosalpinges may secrete cytokines that adversely affect pregnancy outcome (Grifo et al., 1989
; Toth et al., 1992
) via haematogenous and lymphatic routes. Chen et al. (2002
) studied cytokines in HF and reported that their concentrations were not predictive of subsequent IVF outcomes. However, cytokine concentrations have been shown to be higher in severe pelvic adhesions (Cheong et al., 2002
). Inappropriate expression of growth factor genes and integrins can interfere with blastocyst formation and implantation. Meyer et al. (1997
) have demonstrated that integrins (
v
3) associated with the window of implantation are found to be decreased in women with hydrosalpinges compared with controls. These womens endometria were described as out of phase endometria due to the presence of gland/stromal dyssynchrony. However, 70% (14/20) of these women showed increased integrin expression after surgical correction of their hydrosalpinges. Bildirici et al. (2001
) reported that surgical removal of communicating hydrosalpinges increased the expression of integrin
v
3 and therefore may improve endometrial receptivity. Illera et al. (2000
) demonstrated that blockage of integrin
v
3 resulted in impaired implantation in the mouse. Collectively, these studies suggest that HF may contain yet unknown substance(s) that interfere with the expression of integrins. Although cytokines, NO and ROS measurements were not done in this study, further studies are required to identify the substance(s) secreted by hydrosalpinx epithelium involved in mediating the toxic effect of HF on sperm and embryo.
In summary, the present results suggest that hydrosalpinx epithelial cells may be producing a fluid milieu hostile to sperm affecting their motility, capacitation and acrosome reaction and early embryo development, thus providing evidence for salpingectomy for large hydrosalpinges prior to IVF. The reconstituted epithelial cell culture system provides a model to further investigate the mechanism of HF formation and possible role of epithelial secretions. In-depth characterization of hydrosalpinx epithelium and its secretions is necessary. Further analysis of hydrosalpinx epithelial cell culture CM may shed light on the mechanisms underlying the toxic effects of HF on embryo development and adverse IVF outcomes.
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Acknowledgement |
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Submitted on July 21, 2002; resubmitted on September 10, 2002; accepted on October 31, 2002.