Academic Unit of Reproductive and Developmental Medicine, Level 4, The Jessop Wing, Royal Hallamshire Hospital, Sheffield S10 2SF, UK
1 To whom correspondence should be addressed. e-mail: A.Pacey{at}Sheffield.ac.uk
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Abstract |
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Key words: endosalpinx/human/integrins/RGD/sperm
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Introduction |
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By contrast, our knowledge of spermendosalpingeal interactions in the human female reproductive tract remains limited. However, there is accumulating evidence to suggest that the uterine tube is not merely a passive conduit for gametes (Hunter, 1998), but provides a dynamic environment that may play a role in regulating and maintaining sperm function. It has been shown that human sperm bind to the endosalpinx (Pacey et al., 1995a
), and that more sperm bind to cells from the isthmic region of the uterine tube compared with the ampulla (Baillie et al., 1997
). In addition, a number of investigators have described the positive effects on human sperm function after co-incubation with endosalpingeal cells, including prolonged viability (Morales et al., 1996
; Ellington et al., 1998
), increased motility (Guerin et al., 1991
; Yeung et al., 1994
; Yao et al., 2000
), and a regulatory effect on sperm capacitation (Murray and Smith, 1997
; Petrunkina et al., 2001
).
Despite the increased awareness of the importance of spermendosalpingeal interaction to reproductive success, the specific molecular mechanisms mediating this interaction in the human are poorly understood. In non-human species, spermepithelial interaction has been shown to involve carbohydrate recognition (reviewed by Suarez, 2001), but in the human a number of workers have suggested that integrins, a family of cell adhesion molecules, may also be involved. This is because integrins are known to have a widespread role in many cellcell and cellmatrix interactions (Ruoslahti and Pierschbacher, 1986
; 1997) including fertilization and implantation (reviewed by Klentzeris, 1997
). Experimental evidence to support this hypothesis is, however, currently lacking but it has been demonstrated that the
1 integrins,
4,
5,
6 and
v chains (Glander and Schaller, 1993
; Trubner et al., 1997
) are expressed on the plasma membrane of human sperm. Also expressed are the proteins fibronectin and vitronectin (Fusi and Bronson, 1992
; Fusi et al., 1992
) which contain the amino acid sequence Arg-Gly-Asp (RGD) that is the recognition sequence for a number of integrins. Unfortunately, very little is known about the expression of integrins or RGD-containing matrix proteins on the apical surface of endosalpingeal cells, although
3 and
v integrin subunits are known to be expressed and may play a role in extrauterine implantation (Sulz et al., 1998
).
In their studies to investigate the role of the RGD adhesion sequence in sperm binding to the oolemma, Bronson and Fusi (1990a;b) used a novel technique in which oligopeptides containing the RGD sequence (or a scrambled peptide) were introduced into an established assay of spermoolemma interaction. Using this technique, they were able to demonstrate that the numbers of sperm binding to the oolemma were reduced in the presence of oligopeptides containing the RGD sequence but remained similar to control values when scrambled oligopeptides were present. In addition, it was shown that the RGD sequence is specifically recognized by integrins present on the oolemmal surface (Fusi et al., 1993
). Since this experimental system is very similar to an in-vitro assay we have used previously to quantify the numbers of sperm bound to explants of human endosalpinx (Baillie et al., 1997
), we have chosen to use this approach to study the role of integrins and the RGD sequence in mediating human sperm endosalpingeal interactions.
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Materials and methods |
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Patient recruitment and surgery
The specific details regarding the recruitment of patients, surgical procedures and the isolation of endosalpingeal explants for use in experiments have been fully described elsewhere (Pacey et al., 1995a). Briefly, tubal tissue was obtained from 19 pre-menopausal women undergoing routine abdominal hysterectomy for benign gynaecological conditions not affecting the uterine tubes, at the Royal Hallamshire Hospital, Sheffield, UK. The study was approved by the South Sheffield Research Ethics Committee, and all patients gave their written consent. Uterine tubes from patients at any stage of the menstrual cycle were obtained, since it has been shown previously that the level of sperm binding to the human endosalpinx is independent of reproductive hormones (Baillie et al., 1997
).
Material from the ampulla was taken from a region in the distal portion of the uterine tube, 12 cm from the cut fimbrial end and isthmic tissue from the region closest to the utero-tubal junction in the proximal portion of the tube. Epithelial mucosa was isolated from the underlying musculature by grasping the folds of mucosa with watchmakers forceps (Fisher Scientific, UK) and cutting them away with fine-pointed scissors (Fisher Scientific). Sections of mucosa were then carefully cut to produce explants of 1 mm3. The explants were placed into separate wells (three explants per well, four wells per tubal region) of an untreated, flat-bottomed tissue culture plate (Gibco BRL, UK) containing 500 µl of tissue culture medium composed of a 50/50 mixture of Hams F-12/Dulbeccos modified Eagles medium (DMEM) containing 1% L-glutamine (200 mmol/l), 5% of a 50/50 mixture of Nu-serum/fetal bovine serum, 1% penicillinstreptomycin solution (containing 5000 IU/ml penicillin and 5000 µg/ml streptomycin), 2% HEPES buffer (1 mol/l) and 3% sodium bicarbonate (7.5%) solution. Explants were then incubated overnight in a humid atmosphere of 5% CO2 in air at 37°C, before being used in experiments on the following day. Only explants obtained from a single patient were used in each experiment.
Preparation of sperm
Motile sperm were obtained from the semen of sperm donors attending the Andrology Laboratory in the Jessop Wing of the Royal Hallamshire Hospital. All donors adhered to a 2 day period of sexual abstinence prior to sample production and were all of proven fertility producing ejaculates containing ≥60x106 sperm/ml and 15% ideal morphological forms (World Health Organization, 1999). Sperm were isolated from seminal plasma using a direct swim-up technique (Mortimer, 1990
). Briefly, aliquots of 300 µl of semen were overlaid with 600 µl of EBSS containing 0.3% (w/v) human serum albumin (HSA) (Sigma Chemicals, UK) and incubated in a humid atmosphere of 5% CO2 in air at 37°C for 1 h. After 1 h the top 50% of medium was removed from each tube, the samples pooled and the concentration manually checked using a Neubauer haemocytometer. The sperm sample was adjusted to 5x106/ml with EBSS containing 0.3% (w/v) HSA, and the concentration manually checked as before using a Neubauer haemocytometer.
Experiment 1: The effect of RGD and non-RGD oligopeptides on sperm function
To assess the effect of the oligopeptides on sperm function prior to their use in spermendosalpingeal interaction assays, a number of preliminary experiments were performed. This was to preclude the possibility that any effect in the spermendosalpingeal interaction assay was a direct result of the peptide sequence blocking a receptor-integrin interaction and not as a result of a deleterious effect of the oligopeptides on sperm function. In addition, a spermhamster oocyte interaction assay was performed as a positive control to establish that the RGD oligopeptides were working as previously described (Bronson and Fusi, 1990a;b).
Measurement of sperm function
Sperm were isolated from the ejaculates of six donors and prepared as described above. To three aliquots, sperm were incubated with 250 µmol/l of each of the three oligopeptides, and a fourth incubated with medium alone served as a control. Both at the start of the incubation and after 3 h incubation in a humid atmosphere at 37°C, two 10 µl aliquots of the sperm suspension were removed from each incubation to determine the percentage motility and also the viability and acrosomal status of the sperm population.
To determine the percentage of motile sperm in each of the incubations, each 10 µl aliquot was loaded into a 20 µm depth Microcell chamber (Conception Technologies, USA) and analysed using a Hamilton Thorn Motility Analyser (Hamilton Thorn Research, USA). The machine used was running version 10.8 of the analysis software and was operated with the following settings: number of frames acquired, 60; minimum contrast, 80; minimum cell size, 2; and magnification factor, 4.78. At least 200 sperm were examined from each incubation condition from up to 16 fields of view.
To determine the viability and acrosomal status of sperm in the incubation, the second 10 µl aliquot was added to 100 µl of hypo-osmotic swelling (HOS) solution (World Health Organization, 1999) and incubated for 30 min at 37°C. The suspension was then spotted onto a poly-L-lysine-coated slide and allowed to air-dry before being fixed in absolute methanol for 2 min. The fixed slides were then stained to evaluate the acrosomal status using the technique described by Moore et al. (1987
) using a monoclonal antibody donated to this study by Professor Moore. Briefly, 20 µl of neat primary monoclonal antibody (mAb 18.6) was added to the slides and incubated for 1 h in a humid atmosphere at 37°C. The slides were then washed in 0.1 mol/l phosphate-buffered saline (PBS), and 20 µl of the secondary antibody (anti-mouse IgG FITC conjugate) was added at a 1:40 dilution. Slides were placed in a humid chamber as before and incubated for 30 min at 37°C before being washed twice in PBS and mounted with MOWIOL (Calbiochem, UK) and kept in the dark until analysis. Analysis was always performed within 2 days of the experiment and ≥200 sperm were counted per experimental condition. The numbers of live acrosome-intact, dead acrosome-intact, live acrosome-reacted and dead acrosome-reacted sperm were recorded. Live sperm with an intact cell membrane and an intact acrosome display coiled tails and fluorescence over the acrosome region, whereas non-viable or acrosome-reacted cells were those displaying straight tails and a lack of fluorescence or patchy fluorescence over the head region.
Inhibition of human spermhamster oocyte binding
Spermhamster oocyte binding assays were performed following the method developed by Yanagimachi et al. (1976), but with slight modifications as described below. Briefly, female golden hamsters (2530 days old) were induced to ovulate by an i.p. injection of 30 IU of pregnant mare serum gonadotrophin (PMSG; Folligon, UK) on the morning of post-estrous vaginal discharge. Approximately 72 h later, each animal received an i.p. injection with hCG (Chorulon; Intervet, UK). The animals were killed 2024 h later by an overdose of pentarbarbitone sodium (Euthatal; J.M.Loveridge plc, UK) and the oviducts removed. The oocytes were freed from the surrounding cumulus cells by a 15 min incubation with 0.1% (w/v) bovine testicular hyaluronidase type IV-S (1100 IU/mg; Sigma Chemicals) diluted in BiggersWhittenWhitingham medium (BWW) supplemented with 1% (w/v) bovine serum albumin (BSA). The oocytes were made zona pellucida (ZP)-free by incubation in 0.1% (w/v) bovine pancreatic trypsin type III for 1 min (Sigma Chemicals). The zona-free oocytes were rinsed thoroughly in BWW medium and incubated at 37°C in fresh BWW medium containing 1% (w/v) BSA.
Prior to each experiment, the zona-free oocytes were randomly allocated into four groups and then transferred into 100 µl droplets contained in a 35 mm diameter Corning plastic Petri dish (VWR Int. Ltd, UK), with 10 oocytes per drop. The drops were then overlain with 2 ml of mineral oil (BDH, UK). Into a separate Petri dish, 4x150 µl drops of a sperm suspension from a single donor (prepared as described above and containing 5x106 sperm per ml) were prepared and overlain with mineral oil. To three of the drops of sperm or zona-free oocytes, an aliquot of each oligopeptide was then added to give a final concentration of 250 µmol/l. To the fourth, the same volume of medium was added as a control. The drops of sperm and oocytes were then preincubated for 30 min at 37°C before removing 100 µl of sperm suspension and adding it to the corresponding incubation of oocytes, giving a total volume of 200 µl per droplet. This dish was incubated overnight for 1517 h at 37°C in a humid atmosphere of 5% CO2 in air.
The following morning, the oocytes were gently washed with fresh BWW to remove unbound sperm and were then mounted on a slide containing four wax spots, compressed with a coverslip and examined with a Nikon Diophot using a x40 objective. The number of bound sperm per oocyte was examined for each experimental group and control.
Experiment 2: The effect of RGD and non-RGD oligopeptides on spermepithelial binding
For each experiment, sperm and epithelial explants were preincubated with the oligopeptides separately for 30 min at 37°C, with a control group being incubated with medium alone under the same conditions. Three working concentrations of each oligopeptide were used in this experiment (250, 62.5 and 15.5 µmol/l) and these were prepared by serial dilution from a thawed aliquot of frozen stock solution on each day as required. After the 30 min preincubation period, the medium overlying the explants was removed and 500 µl of the corresponding sperm suspension (preincubated with the same concentration of oligopeptide) was added. As such, explants were exposed to 2.5x106 sperm. These were then incubated for 1 h and processed according to the methods of Baillie et al. (1997). Briefly, following the incubation, the suspension was removed from the wells and 500 µl of fresh (sperm free) warm EBSS was added and the well gently agitated for 1 min (to remove unbound sperm) and the procedure repeated once more before the addition of 500 µl of 3% (v/v) of 25% glutaraldehyde (Sigma Chemicals) in 0.1 mol/l PBS and left overnight at 4°C. The following day the tissue was rinsed several times with PBS, stained for 30 s with Gills haematoxylin and cleared with several changes of 50:50 PBS:glycerol over 4 h. Explants were then mounted in PBS:glycerol under a coverslip, sealed with silicone grease. These were then observed using a x100 oil immersion objective and the number of sperm bound to the epithelial surface in each field of view (equivalent to a surface area of 0.03 mm2) were recorded. A total of 25 microscope fields per experimental condition was examined in this way and the data used to determine a mean number of bound sperm per microscope field (0.03 mm2) per peptide concentration. The fields to be analysed were selected using a systematic random sampling method (Mayhew, 1991
) and the number of replicates (fields examined) to provide a result with a sampling error of less than ±5% determined by performing a progressive mean analysis (Williams, 1977
). Only those fields that contained a flat area of epithelium as identified by the presence of ciliated cells were included for analysis.
Experiment 3: Determining the location of the RGD adhesion sequence
To determine the location of the RGD adhesion sequence, the oligopeptides RGDV and GRGES were coupled to 6 µm diameter FluoresbriteTM YellowGreen (YG) carboxylate Polysciences Inc. (Warrington, USA) latex beads that were then employed in a variation of the binding assay used in experiment 2 and in a series of co-incubations with sperm. The beads were obtained as a 2.5% suspension, at a stock concentration of 1.05x108 and the oligopeptides were co-valently coupled to them following the manufacturers protocol and binding kit. As a control, an aliquot of beads was processed in the same manner but in the absence of any peptide. For each experiment, the beads were re-suspended in 500 µl of EBSS containing 0.3% HSA to concentrations of 2x106, 5x106 and 10x106 beads per ml.
For experiments with endosalpingeal explants, 500 µl of the bead suspension (oligopeptide-coupled or control) were added to each well of explants and placed in the incubator at 37°C for 1 h. During the 1 h incubation, the plate was randomly shaken to assist the beads in making contact with an area of epithelium. This step was deemed necessary since, unlike sperm, the beads are not motile. Following the incubation, the bead suspension was removed and the explants were washed twice in EBSS to remove unbound beads and subsequently processed as for the sperm binding assay described in experiment 2.
For incubations with sperm, highly motile suspensions of donor sperm were prepared and adjusted to 20x106 per ml. A 10 µl aliquot of each sperm suspension and a 10 µl aliquot of the bead suspension (containing 1.05x108 beads per ml) were placed onto a glass slide and mixed thoroughly using a pipette tip. A coverslip was applied and the slides were placed into a humid atmosphere at room temperature for 10 min prior to observing the preparation at x100 magnification using an Olympus BH2 microscope (Olympus, Japan). The number of beads that were bound to the surface of motile sperm was then determined when performing an immunobead test for sperm antibodies during semen analysis (Mortimer, 1994).
Experiment 4: Competitive inhibition experiments
To determine the specificity of the binding between the RGD sequence in the oligopeptides coupled to fluorescent beads and the RGD-binding receptors on sperm and/or epithelium (determined in experiment 3), the binding of RGDV-coupled beads in the presence of soluble RGDV or GRGES was assessed. Soluble RGDV and GRGES were added to the assay at the concentration of 250 µmol/l and RGDV-coupled beads were added to each incubation at a concentration of 5x106.
Statistical analysis
Regression analysis and analysis of variance were performed to compare the differences between the numbers of sperm or beads bound to each tubal region in the control groups and the various treatment groups. Students t-test was used to examine the differences between human spermhamster oocyte adhesion in the control and in the presence of oligopeptide. All statistical tests were performed using the Statistical Package for Social Scientists (USA).
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Results |
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Experiment 2
Figure 1 shows the mean number (± SEM) of sperm bound per 0.03 mm2 of epithelium obtained from the tubal isthmus and ampulla in the presence of three concentrations of each oligopeptide. As would be expected from our previous work (Baillie et al., 1997), in the absence of any of the oligopeptides significantly (P < 0.05) more sperm bound to isthmic tissue (3.34 ± 0.45) compared with tissue from the ampulla (1.81 ± 0.39). Moreover, in the presence of oligopeptides, the numbers of sperm bound to ampullary tissue was not significantly different at any of the concentrations or oligopeptides tested. However, with regard to tissue from the isthmus there were significantly fewer sperm bound in the presence of 62.5 µmol/l GRGDTP (1.18 ± 0.41, P < 0.05) as well as 250 µmol/l RGDV (1.17 ± 0.29, P < 0.01) when compared with the control (3.34 ± 0.45). Interestingly, in the presence of GRGES (which does not contain an RGD sequence), although the numbers of sperm bound at the three doses tested were numerically lower than that in the control incubation (no oligopeptide), they were not significantly different from it.
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During co-incubations with sperm from six ejaculates, <1% of sperm in each treatment were seen to have beads bound to them. This was despite sperm making numerous visible contacts with the beads. Incubations were observed at a variety of time-points between 5 and 60 min with the same behaviour being observed on each occasion. As such, it was concluded that the beads were unable to bind to sperm.
During co-incubation with epithelial cells the situation was rather different. Following co-incubations with explants from the tubal ampulla, less than one fluorescent bead was observed binding per 0.03 mm2 of epithelium and this was not significantly different between protein-coupled or control beads. Moreover, the number of beads binding to ampullary epithelium did not vary as a function of the bead concentration (Figure 2). However, when co-incubated with explants from the tubal isthmus, analysis of variance indicated that there was both a significant effect of bead type (P = 0.006) and concentration (P = 0.003) on the number bound to the epithelial surface (Figure 2). For example, at a bead concentration of 5x106 per ml, significantly more RGDV-coupled beads were bound per field of epithelium (1.47 ± 0.26) than GRGES-coupled (0.67 ± 0.24; P < 0.05) or uncoupled (0.30 ± 0.07; P < 0.01). Moreover, at a bead concentration of 10x106 per ml, there were significantly (P < 0.05) more RGDV-coupled beads bound per field of epithelium (2.38 ± 0.48) than uncoupled (0.77 ± 0.17). In addition, at a concentration of 10x106 RGDV-coupled beads, there were significantly more beads (P < 0.01) bound per field than at an RGDV-coupled bead concentration of 2x106 per ml (0.43 ± 0.06). This effect was not observed with GRGES-coupled or uncoupled (control) beads, although with increasing bead concentration there were numerically more beads bound to the epithelial surface.
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Experiment 4
Figure 3 shows the results of the experiments in which 5x106 RGDV-coupled beads per ml were incubated with explants from the tubal isthmus and ampulla in the presence or absence of 250 µmol/l of soluble RGDV and GRGES. With ampullary tissue, the presence of soluble RGDV or GRGES made no difference to the number of beads bound. However, the binding of RGDV-coupled beads to isthmic tissue was competitively inhibited in the presence of soluble RGDV (P < 0.05) but not GRGES.
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Discussion |
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In many non-human mammals, it has been proposed that an important mechanism mediating spermepithelial binding is the interaction between surface-associated sperm lectins and oligosaccharides present on the epithelial surface. This has been demonstrated by the ability of species-specific sugars to inhibit spermepithelial binding in both in-vivo (Demott et al., 1995) and in-vitro systems (Dobrinski et al., 1996
; Green et al., 2001
). Ignotz et al. (2001
) have recently proposed the identity of a carbohydrate-binding protein on bull sperm that may be involved in this process. However, it has been suggested that this carbohydrate-mediated interaction may only serve to first catch the sperm and to bring the sperm into close contact with the epithelial surface, and to prepare the underlying mechanisms that may still be involved in the communication between sperm and the endosalpinx (Wagner et al., 2002
).
This study has, therefore, examined the role in human spermendosalpingeal interactions of the widespread cell adhesion mechanism involving the recognition between the Arg-Gly-Asp (RGD) adhesion sequence and certain integrins (Ruoslahti and Pierschbacher, 1986). To do this, a methodological approach previously used to study spermoolemma binding (Bronson and Fusi, 1990a
;b) was modified for use in a simple in-vitro assay that can quantify the ability of the human endosalpinx to bind sperm (Baillie et al., 1997
). From the experiments described in this report, it is proposed that the binding of human sperm to the human endosalpinx in the isthmic region of the uterine tube could be mediated in part by a mechanism that involves the Arg-Gly-Asp (RGD) adhesion sequence. The evidence for this comes from the experiments shown in Figure 1, where the addition of RGD-containing oligopeptides, at some concentrations, can reduce the numbers of sperm bound to isthmic but not ampullary explants. However, a simple competitive inhibition was not observed for any of the peptides tested, suggesting that the mechanism could be more complicated than that observed for sperm oolemma binding. This is supported by the fact that in our experiments, it was not possible to reduce to zero the numbers of sperm bound to the epithelium in either isthmic or ampullary tissue. Moreover, the range of concentrations chosen was the same as that used by Bronson and Fusi (1990a
;b) and it may be possible that these are inappropriate to achieve the same level of inhibition seen by these authors. However, the limited supply of human tissue prevented a wider range of concentrations to be tested.
The fact that fluorescent beads, to which RGD oligopeptides have been coupled, preferentially bound to the isthmic but not ampullary epithelium, in a dose-dependent manner (Figure 2) suggest that the integrin is located on the epithelial surface. This could be inhibited in the isthmus (but not in the ampulla) by the addition of soluble RGD into the assay (Figure 3). However, the presence of the non-RGD-containing peptide GRGES could also inhibit binding to some extent (but not significantly). This was a somewhat surprising result but not entirely unexpected since Bronson and Fusi (1990b) described that high concentrations of the peptide GRGES could sterically interfere with RGD recognition sites, even if the recognition sequence was not occupied.
Surprisingly, very little is known about the integrin expression on the human endosalpinx. It has been shown by Sulz et al. (1998) that the integrin subunits
3 and
v are expressed, but any regional differences that may account for our findings in Figure 2 have yet to be observed. It is known that only five integrin molecules specifically recognize the RGD sequence (Ruoslahti and Pierschbachher, 1997
) and so these should be relatively easy to detect in the human endosalpinx using either immunohistochemistry and/or molecular techniques and to identify expression changes between the isthmus and the ampulla. Moreover, since the binding of integrins to their ligands (in this case presumably a sperm protein containing RGD) can trigger intracellular signalling pathways within the cell (Schwartz and Ginsberg, 2002
), it is interesting to speculate what events may be induced in the endosalpinx following sperm binding by this mechanism. For example, it has been shown previously in the horse (Ellington et al., 1993a
) that the intracellular calcium level is elevated following sperm binding to epithelial cells. Furthermore, in a separate study by the same authors (Ellington et al., 1993b
), it was shown that the proteins secreted by uterine tube epithelial cells of the bovine were altered following co-incubation with sperm. This implies the existence of a subtle mechanism of crosstalk between sperm and epithelial cells and this is an important concept in our understanding of these events in human reproduction. It is interesting that the development of unfertilized Xenopus eggs can be triggered by the addition of oligopeptides containing RGD (Iwao and Fujimura, 1996
) and therefore that intracellular signalling of the endosalpingeal cells can occur through this route should not be surprising.
In contrast to the identity of the integrin receptor on the endosalpinx, it is interesting to speculate on the nature of the corresponding ligand that is presumably present on sperm. There is a number of proteins known to be present on the outer leaflet of the sperm plasma membrane and which contain an RGD sequence and are therefore likely candidates for the sperm ligand in spermepithelial binding. These include p47 (Jansen et al., 2001) and fibronectin and vitronectin (Fusi and Bronson, 1992
; Fusi et al., 1992
). Whilst little is known about the expression of p47 on human sperm, it is known that both fibronectin and vitronectin are only expressed in capacitated (or acrosome-reacted) cells (Fusi and Bronson, 1992
). This is consistent with a role for them in binding to the oolemma, but is confusing in the context of sperm storage in the tubal isthmus. This is because it is generally considered that the mechanism by which sperm are ultimately released from binding sites on the epithelium is due to changes in the sperm plasma membrane associated with sperm capacitation, rather than by changes in the epithelial surface. There are two lines of evidence for this. The first is the fact that the number of binding sites on the epithelial surface appears to be independent of the changes in reproductive hormones, both in the human (Baillie et al., 1997
) and in other non-human mammals (Suarez et al., 1991
; Lefebvre et al., 1995
). The second is from experiments in non-human species that have shown that uncapacitated sperm are preferentially able to bind to the endosalpinx (Kervancioglu et al., 1994
; Lefebvre et al., 1996
; Fazeli et al., 1999
) and that the ability of sperm to bind sugars is reduced in capacitated sperm (Demott et al., 1995
; Revah et al., 2000
). As such, the fact that the only known RGD-containing proteins on the sperm plasma membrane are exposed following capacitation would seem inconsistent with the idea that uncapacitated human sperm are able to bind to the isthmic epithelium by this mechanism. However, it remains possible that there are other proteins on uncapacitated sperm that have yet to be identified and that contain an RGD sequence. Moreover, it is possible that capacitated human sperm still have the capacity to bind to the human endosalpinx. Since human sperm capacitation is difficult to study and it has not yet been possible to determine the capacitational status of bound sperm, it unclear whether capacitated human sperm are different from uncapacitated sperm in their epithelial binding characteristics. In addition, previous reports from our group (Pacey et al., 1995b
) have shown how hyperactivated motility is associated with the ability of human sperm to detach from their connection with the endosalpinx, yet hyperactivated sperm often go on to re-attach to other cells. Since hyperactivation is considered a visible marker of capacitation (Mortimer et al., 1997
), then this would imply that capacitated human sperm can bind to the endosalpinx at least in vitro. This therefore leads to the question as to the mechanism by which human sperm are released from their epithelial connections in order to take part in fertilization. Perhaps the answer lies in subtle changes in the endosalpinx rather than changes in sperm that have been previously suggested. In our experiments we have shown that fewer RGD-coupled beads bind to ampullary epithelium, implying that there are fewer sperm-binding integrins present in the ampulla. Perhaps, then, as human sperm slowly pass through the uterine tube it is the combined effect of the wider lumen and reduced sperm-binding ability of the epithelium that allows sperm to be available in the tubal lumen for fertilization.
Finally, although the data in this report would suggest an important role for the RGD sequence in human sperm epithelial interaction, it remains highly likely that other processes may also be involved. It is evident from our study that the addition of RGD oligopeptides cannot reduce to zero spermepithelial interaction in either isthmic or ampullary tissue and this in turn implies that other mechanisms may be involved in the process.
In conclusion, this work provides the first evidence for a molecular basis of how human sperm bind to the isthmic region of the uterine tube and how physiological changes in sperm and epithelial cells may be mediated. This is an important step forward in our understanding of the early events surrounding human conception, and, whilst there is much work to be done, it would be interesting to speculate whether dysfunction in the integrin expression in the endosalpinx of some women may be an as yet undefined contributor to their infertility.
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Acknowledgements |
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References |
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Submitted on December 24, 2002; accepted on April 2, 2003.