Hum. Reprod. Advance Access originally published online on January 29, 2004
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Human Reproduction, Vol. 19, No. 3, 639-644,
March 2004
© 2004 European Society of Human Reproduction and Embryology
Integrins are not involved in the process of human sperm–oolemmal fusion
Department of Obstetrics and Gynecology, Asahikawa Medical College, Midorigaoka-higashi 2-1, Asahikawa, 0788510, Japan
1 To whom correspondence should be addressed. e-mail: ksen{at}asahikawa-med.ac.jp
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Abstract |
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BACKGROUND: We investigated whether integrins are required for the human sperm–oocyte binding and fusion processes. METHODS: The expression of several integrin subunits at the human oocyte plasma membrane was investigated using immunofluorescence microscopy, and the functional role of integrins expressed at the human oocyte surface in sperm–oocyte interaction was studied using a zona-free human oocyte binding and fusion assay. A total of 144 unfertilized oocytes were stained with anti-integrin antibodies and 147 zona-free unfertilized oocytes were inseminated in the presence of various anti-integrin antibodies that were expressed in oocyte plasma membrane. RESULTS: The antibodies of six
integrin subunits (2, 3, 5, 6, V, M) and six 
integrin subunits (1, 2, 3, 4, 5, 6) were bound to the surface of fixed unfertilized oocytes. In contrast, the presence of 1 and 4 subunits could not be verified. The human sperm–oocyte binding was only partially inhibited by blocking antibodies of 2, 3, 5, 6, V, M, 1, 2 and 3 with a maximum of 55% inhibition, but antibodies of 4, 5 and 6 showed no effect on sperm–oolemmal binding. A similar reduction of the number of fused sperm was observed. However, the ratio of fused sperm to total sperm (bound and fused) was not impaired by all integrin antibodies, suggesting that integrins had no role in the sperm–oolemmal fusion process. CONCLUSIONS: These results suggest that one of the binding mechanisms can be inhibited by integrin antibodies but that this mechanism does not play an essential role in the human sperm–oolemmal binding and fusion processes. The other mechanisms, insensitive to integrins, may involve both binding and fusion processes in human oocytes.
Key words: human gamete fusion/integrin/oocyte plasma membrane/sperm
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Introduction |
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The molecular events of sperm–oocyte binding and fusion have been studied extensively, but identification of the molecules involved in sperm–oocyte interaction remains incomplete. Several candidate molecules for sperm–oocyte binding and fusion have been proposed. The best candidate ligand on sperm is ADAM (A Disintegrin and Metalloprotease) protein. In the mouse, antibodies and peptide of the disintegin domain of fertilin
and cyritestin have been reported to strongly inhibit sperm–oocyte binding and fusion (Primakoff et al., 1987
; Yuan et al., 1997; Evans et al., 1998; Bigler et al., 2000; Zhu et al., 2000; McLaughlin et. al., 2001). The evidence that disintegrin-like domains of fertilin and cyritestin might be responsible for sperm–oocyte interactions suggests that complementary binding molecules on the oocyte plasma membrane are integrins (Blobel et al., 1992; Myles et al., 1994; Bronson et al., 1999). Indeed, a number of integrin subunits have been detected in the oolemma of mammalian oocytes. It seems likely that one or more integrins are involved in the processes of sperm–oocyte binding and fusion. Since peptides with a sequence of fertilin disintegrin domains bound to 61 integrin and an anti-6 integrin function-blocking monoclonal antibody, GoH3, inhibits mouse sperm–oocyte binding and fusion, mouse oocyte integrin 61 could be a primary candidate for a sperm receptor (Almeida et al., 1995). This concept is further supported by the findings that integrin 61 has been reported to be associated with CD9 in various cells including mouse oocyte plasma membrane, because recent studies demonstrate that oocyte CD9 plays a key role in sperm–oocyte fusion (Miyado et al., 2000; Kaji et al., 2000; Le Naour et al., 2000). However, oocytes from mice null 6 integrin subunit were shown to have no reduction in sperm–oocyte binding and fusion, suggesting that 6 integrin is not critical for oocyte–sperm interactions (Miller et al., 2000). The involvement of other subfamilies of integrins, particulally 4 and 9 integrins, in the processes of sperm–oocyte binding and fusion has also been suggested (Zhu and Evans et al., 2002). This may reflect redundancy of 61 and other oocyte integrins, or it may mean that integrins have no role in binding and fusion.
In humans, several integrin subunits have been identified in the oolemma, but controversy still exists (de Nadai et al., 1996; Ji et al., 1998; Fusi et al., 1993; Campbell et al., 1995; Capmany et al., 1998). The involvement of the RGD-binding subfamily of integrins in the gamete interactions has also been demonstrated by the inhibition of interactions of human sperm with zona-free hamster and human oocytes by RGD peptides (Bronson and Fusi, 1990; Ji et al., 1998). However, limited information is available concerning the role of integrins in human sperm–oocyte interaction.
The aim of the present study was to investigate whether integrins are required for human sperm–oocyte binding and fusion process. The expression of several integrin subunits at the human oocyte plasma membrane was investigated using immunofluorescence microscopy, and the functional role of integrins expressed at the human oocyte surface in sperm–oocyte interaction was studied using a zona-free human oocyte binding and fusion assay.
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Materials and methods |
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Unfertilized oocytes in this study were obtained from patients undergoing IVF. Written informed consent was obtained from all patients and this study was approved by the local ethical committee. Follicular stimulation and IVF protocols were described previously (Sengoku et al., 1995
). Briefly, follicular stimulation was achieved with a desensitizing protocol using a combination of GnRH agonist (buserelin acetate, Spurecur; Hoechst, Japan) and hMG (Pergonal; Teikokuzouki, Japan). hCG 10 000 IU (mochida; Moshida Pharmaceutical, Japan) was administered when leading follicles were >16 mm in diameter. Oocyte recovery was performed 34–36 h after the hCG injection using transvaginal ultrasound-guided aspiration. The oocytes were examined for fertilization 16–18 h after insemination. Oocytes with no signs of fertilization and apparently normal morphology were included in this study.
Immunohistochemistry
One day old unfertilized oocytes were transferred to phosphate-buffered saline (PBS). The zona pellucida was removed by a brief exposure to acid Tyrode’s solution. After 3 h of incubation (recovery time) in human tubal fluid (HTF; Irvine Scientific, USA) supplemented with 10% synthetic serum substitute (SSS; Irvine Scientific), zona-free unfertilized oocytes were fixed in 2% paraformaldehyde in PBS for 30 min. After washing in PBS with 0.3% bovine serum albumin (BSA; Sigma Chemical Co., St Louis, MO) and 100 mmol/l glycine (blocking solution), the unfertilized oocytes were incubated for 1 h with various anti-human integrin antibodies diluted in PBS containing 3% fetal calf serum (to mask non-specific binding sites). Mouse monoclonal antibodies against human integrin subunits 1 (FB12), 2 (P1E6), 3 (P1B5), 4 (P4G9), 5 (P1D6), V (CLB706), M (ICRF44), 2 (P4H9), 3 (B3A), 4 (ASC3), 6 (CSb6) and rabbit polyclonal antibodies against 5 were supplied by Chemicon International (USA). Anti-human integrin subunit 6 (GoH3) monoclonal antibody was raised in the rat (Gibco BRL, Life Technologies, USA) and mouse monoclonal anti-human integrin subunit 1 (6S6) was purchased from Upstate Biotechnology Inc. (USA). They were all used at a 1:20 (v/v) dilution in PBS. The specimens were washed by several transfers to blocking solution and incubated with one of the following fluorescent conjugates for 45 min. The conjugated secondary antibodies (raised in mouse, goats or rabbit) were used at a dilution of 1:200 in PBS–BSA.
When staining was not detected, the detection was amplified by a biotinylated anti-mouse, anti-rat or anti-rabbit IgG and streptavidin–fluorescein isothiocynate (FITC). The unfertilized oocytes labelled by integrin antibodies were incubated for 45 min in a solution containing biotinylated goat anti-mouse, anti-rat or anti-rabbit IgG at a dilution of 1:200 in PBS–BSA (Sigma Chemical Co) and then reincubated for 30 min in PBS with streptavidin –FITC (at a dilution of 1:150, Sigma Chemical Co.; Ji et al., 1998).
Negative controls were obtained by substituting the incubation in primary antibody for an incubation in PBS containing 3% fetal calf serum. Moreover, negative controls were established during each staining procedure to confirm that the fluorescence observed was not attributable to non-specific binding of the secondary antibody.
Labelled specimens were mounted on slides in PBS supplemented with 25 mg/ml 1,4-diazabicyclo-(2.2.2)octane (DABCO; Sigma) and photographed on an Olympus BX60 fluorescent microscope (Olympus Optical Co., Tokyo, Japan). Overlays of captured images were processed with Adobe Photoshop 7.0.
The possibility of penetration of sperm into unfertilized oocytes, and the possible activation of oocytes, were confirmed by Hoechst 33342 (10 µg/ml) staining. Only metaphase II stage oocytes were included in this study.
Assessment of sperm–oocyte interaction
The dye transfer technique (Hinkley et al., 1986) was used to assess the sperm–oocyte interaction. Zona pellucida-free unfertilized oocytes were incubated in HTF medium containing 0.1 µg/ml Hoechst 33342 (Sigma) for 30 min, and then rinsed thoroughly in PBS over 15 min. Oocytes were preincubated with the 25 µg/ml anti-integrin antibodies for 1 h, washed free from unbound antibodies and then inseminated with 100 000/ml sperm in fresh HTF medium. The control group contained mouse IgG (Sigma). Two hours after insemination, the oocytes were washed using a narrow bore pipette to remove loosely adhering sperm. Oocytes were then fixed with 2.5% glutaraldehyde in PBS at pH 7.4 for 30 min, rinsed with PBS and mounted for observation under an Olympus BX60 fluorescent microscope. Sperm were considered fused when fluorescent-positive condensed or decondensed sperm heads were observed on the oocyte surface. Sperm attached to the oocyte surface which could be seen by light microscope, without fluorescence, were designated as binding sperm.
To confirm the validity of this dye transfer technique, we preloaded zona-free human oocytes with 0.1 µg/ml Hoechst dye and then inseminated with uncapacitated sperm (cultured in Ca2+-free HTF medium). When oocytes were inseminated with uncapacitated sperm, none of the attached sperm showed fluorescent-positive condensed sperm nuclei. Therefore, the present experiment in which 0.1 µg/ml Hoechst dye was preloaded made it possible to distinguish fused from unfused sperm.
Statistical analysis
Statistical significance of the data was determined by Student’s t-test and the 
2-test, as appropriate. Differences were considered significant at P < 0.05.
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A total of 144 unfertilized oocytes were stained with anti-integrin antibodies. The detection was amplified by a biotinylated anti-mouse, anti-rat or anti-rabbit IgG and streptavidin–FITC in six integrin subunits (
1, 2, 4, 5, M, 1 3, 6). Antibodies of six integrin subunits (2, 3, 5, 6, V, M) and six integrin subunits (1, 2, 3, 4, 5, 6) were bound to the surface of fixed unfertilized oocytes. In contrast, the presence of 1 and 4 subunits could not be verified. The heterogeneity of the intensity or distribution of fluorescence was found among oocytes. In some oocytes, surface staining was unevenly distributed, but the most typical pattern was intense and uniformly distributed at the oocyte surface. Furthermore, some integrin subunits were inconsistently expressed in unfertilized oocytes. Negative controls substituting the incubation in primary antibody for an incubation in PBS containing 3% fetal calf serum showed no immunofluorescence labellings. The representative patterns of staining are shown in Figure 1, and a summary of these patterns is shown in Table I and Table II.
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These immunocytochemical findings indicated a potential role for
and integrin subunits in the gamete binding and fusion process. We investigated whether integrins could be involved in binding and/or fusion of human gametes during fertilization. In all, 147 zona-free unfertilized oocytes were inseminated in the presence of various anti-integrin antibodies that were expressed in oocyte plasma membrane, and 135 zona-free oocytes served as the controls. All antibodies used in this study, except 4, 5 and 6, partially inhibited the binding process by 
55% as compared with controls. Similar reduction of the number of fused sperm was observed by anti-integrin subunits. No apparent effect on binding and fusion was observed in the presence of 4, 5 and 6 integrin subunits. However, the ratio of fused sperm to total sperm (bound and fused) was not impaired by all integrin antibodies, indicating no effect of integrins on the sperm–oolemmal fusion (Table III and Table IV). Dose-dependency of inhibition was not estimated due to limited number of samples and because higher concentrations of antibodies caused damage to the gametes. The above-mentioned inhibition was specific since mouse IgG control antibody had no effect on binding and fusion.
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Discussion |
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To investigate the expected role for integrins in human sperm–oocyte binding and fusion, we studied the expression of integrins on the human oocyte surface and whether antibodies against integrin subunits inhibit human sperm–oocyte membrane binding and fusion.
In this study, 2, 3, 5, 6, V, M, 1, 2, 3, 4, 5 and 6 subunits were detected in human oocyte plasma membrane; however, exposed surface localization of the 1 and 4 subunits could not be verified. These results extend previous observations of integrin expression on the human oocyte surface, although some controversy still exists. Integrin subunits 2, 3, 4, V, L, 1, 2, 3, 4, 5 and 7 have been demonstrated in human oocytes by Campbell et al. (1995). Using a rosetting technique, Fusi et al. (1992, 1993) demonstrated that 2, 5, 6, V, 1 but not 4 were detected on human oolemma. It has also been reported that 2, 5 and 6 were detected by immunofluorescense labelling, but 4 and 1 were not detected on the surface of human oocytes (de Nadai et al., 1996).
While 6 was not identified by Campbell at al. (1995), other studies including the present study have described its presence in human oocytes. These staining results were consistent with the report in which the expression of 5, 6 was detected in a serial analysis of gene expression (SAGE) in the human oocyte (Neilson et al., 2000).
The differences in labelling protocols and antibodies could explain the discrepancies between studies in the ability to detect specific integrin subunits. However, the consistent absence of labelling with 1 antibody in several studies, including our current observation, indicates that this subunit is not present or is present in extremely small numbers on the surface of human oocytes.
Recently, it has been suggested that 4/9 is involved in mouse sperm–oocyte membrane interaction from studies of fertilin binding assays to mouse zona-free oocytes using the peptides perturbing integrin-mediated interaction (Zhu and Evans, 2002). The 91 may be a major receptor for ADAM that lack RGD motifs, since 91 specifically binds to the disintegrin domain of fertilin synthesized in bacteria (Eto et al., 2000).
The 4 subunit was reported to be detected by Campbell et al. (1995), but Fusi et al. (1993) and de Nadai et al. (1996) failed to find the expression of 4 on human oocytes. Although a similar amplification step to Ji et al. (1998) was employed in our study to increase the sensitivity of the detection system, 4 subunit was not detected. Thus, it seems unlikely that 4 is involved in human sperm–oocyte membrane interactions. The 9 integrin has not yet been shown to be present on human oocytes, and we did not investigate the expression of 9, because antibody against 9 was not available in our current study.
These immunocytochemical findings might suggest a potential role for integrin subunits in the human gamete binding and fusion process. Zona-free unfertilized human oocytes were inseminated in the presence of various anti-integrin antibodies that were expressed in human oocyte plasma membrane.
Our observation demonstrated that human sperm–oocyte binding was only partially inhibited by blocking antibodies of 2, 3, 5, 6, V, M, 1, 2 and 3 with a maximum of 55% inhibition, but antibodies of 4, 5 and 6 showed no effect on sperm–oolemmal binding. In addition, the fusion of oocytes by sperm that had become bound to oolemma was not blocked.
It should be noted that the oocytes used in this study were 1 day old unfertilized oocytes and that alterations produced by the acid Tyrode’s treatment used for zona pellucida removal were possible. However, several experiments, including our previous study, show that ageing and acid Tyrode’s treatment in vitro do not affect the ability of oocytes to fuse with sperm (Tesarik, 1989; Sengoku et al., 1995; Ji et al., 1997, 1998).
It is also unlikely that these findings were due to use of a suboptimal concentration of the antibodies, since Ji et al. (1998) reported that the inhibition of sperm fusion with oocytes reached a plateau at 25 µg/ml of antibody against 1 integrin. Although we did not investigate the dose-dependency of inhibition due to a limited number of specimens and gamete toxicity of high concentrations of antibodies, concentrations of antibodies used in our study are similar to those used by Ji et al. (1998).
It has been demonstrated that blocking anti-human 1 integrin monoclonal antibody, RGD (Arg-Gly-Asp)-containing peptide, or both, did not result in full inhibition of human gamete fusion (Ji et al., 1998). These authors suggested that 1 integrins are involved in human gamete interaction, but that human gamete fusion can bypass the 1 requirement, or alternatively that a co-factor is required for the gamete fusion process. Both RGD-containing peptide and FEE (Phe-Glu-Glu)-containing peptide, a putative integrin recognition sequence in human fertilin , as is the QDE peptide in mice, has been reported to inhibit both adhesion and penetration of human sperm with zona free human oocyte, but it is not complete inhibition (Bronson et al., 1999). Bronson et al. (1999) proposed that integrins which recognize fertilin and RGD-containing sperm-associated proteins, such as vitronectin and fibronectin, each play a role in gamete interaction and that they may cooperate, although fertilin and cyritestin genes were determined to be non-functional pseudogenes in humans (Jury et al., 1997; Grzmil et al., 2001).
Similar results were reported in the heterologous system (human sperm–hamster oocytes). RGD-containing peptides can inhibit the adhesion and penetration of zona-free hamster oocytes by human sperm, suggesting that RGD-dependent integrins, such as 51 and V1, V3, are involved in the process of fertilization (Bronson and Fusi, 1990). Furthermore, fibronectin and vitronectin have been expressed on the surface of capacitated human sperm (Fusi and Bronson, 1992; Fusi et al., 1992). It has also been reported that antibody against 2 and 5 integrins inhibited both attachment and fusion of human sperm with hamster oocyte by 50% (de Nadai et al., 1996).
Echistatin, a disintegrin, inhibits the binding of vitronectin and fibronectin to integrins V3 and V1, and inhibits the binding of human sperm to the oolemma of zona-free hamster oocytes. Although oolemmal adhesion of sperm was reduced markedly by echistatin, it was not inhibited completely, and it had no apparent effect on oocyte penetration by sperm that did bind (Bronson et al., 1999). The authors suggested that oolemmal integrins facilitate sperm adherence to the oocyte surface but are not required for sperm penetration.
In mice, it has been reported that several lines of mice null for integrin subunits (6, 7, 3 and 5) are normally fertile (Mayer et al., 1997; Hodivala-Dilke et al., 1999; Huang et al., 2000; Miller et al., 2000). Recently, it has been demonstrated that 3 null oocytes and 1 integrin null oocytes function normally in sperm–oocyte binding and fusion, suggesting that none of the integrins known to be present on mouse oocytes are essential for sperm–oocyte binding and fusion (He et al., 2003).
Taken together including our findings of the partial inhibition binding and no apparent effect of fusion process by several antibodies against integrins, it seems likely that integrins are involved in human sperm–oolemmal interaction, but are redundant with each other or may play a merely marginal role, and that the membrane fusion is a separate event which is independent of integrin receptors.
CD9 has been reported to be essential for sperm–oocyte fusion in mouse (Kaji et al., 2000; Le Naour et al., 2000; Miyado et al., 2000), although the role of CD9 in gamete interaction in human has not been clearly determined.
It seems likely that an oocyte surface tetraspanin web involving 1 integrin and integrin- associated proteins may define or help maintain a site for sperm fusion (Takahashi et al., 2001) because 61 and CD9 are reported to be co-immunoprecipitated from mouse oocytes (Miyado et al., 2000). However, the combined evidence demonstrating that mouse oocyte lacking 61 fuse normally with sperm (Miller et al., 2000) and that the human sperm oolemmal fusion process was not impaired by antibodies against several integrin subunits in this study, would appear to indicate that a CD9 partner other than the integrins could function as sperm receptor to initiate the gameta fusion process.
In conclusion, the small but significant reduction of sperm–oocyte plasma membrane binding by antibodies against the several integrin subunits implies involvement of the integrins in human sperm–oocyte interaction. However, our data support the hypothesis that one of the binding mechanisms can be inhibited by integrin antibodies but that this mechanism does not play an essential role in the binding and fusion process. The other mechanisms, insensitive to integrins, might involve the binding and fusion processes in human oocytes.
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Acknowledgements |
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The authors acknowledge Dr W.Richard Dukelow (Former Endocrine Research Center, Michigan State University) for a critical reading of the manuscript. This work was supported by grant from the Japan Society for the Promotion of Science (Grant-in-Aid for Scientific Research 15591725).
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Submitted on August 7, 2003; resubmitted on October 15, 2003; accepted on October 30, 2003.
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