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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Key words: human gamete fusion/integrin/oocyte plasma membrane/sperm
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Introduction |
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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 spermoocyte interaction.
The aim of the present study was to investigate whether integrins are required for human spermoocyte 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 spermoocyte interaction was studied using a zona-free human oocyte binding and fusion assay.
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Materials and methods |
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Immunohistochemistry
One day old unfertilized oocytes were transferred to phosphate-buffered saline (PBS). The zona pellucida was removed by a brief exposure to acid Tyrodes 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 PBSBSA.
When staining was not detected, the detection was amplified by a biotinylated anti-mouse, anti-rat or anti-rabbit IgG and streptavidinfluorescein 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 PBSBSA (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 spermoocyte interaction
The dye transfer technique (Hinkley et al., 1986) was used to assess the spermoocyte 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 Students t-test and the 2-test, as appropriate. Differences were considered significant at P < 0.05.
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Results |
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Discussion |
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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 spermoocyte 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
9
1 may be a major receptor for ADAM that lack RGD motifs, since
9
1 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 spermoocyte 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 spermoocyte 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 spermoolemmal 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 Tyrodes treatment used for zona pellucida removal were possible. However, several experiments, including our previous study, show that ageing and acid Tyrodes 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 spermhamster 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 5
1 and
V
1,
V
3, 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 V
3 and
V
1, 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 spermoocyte binding and fusion, suggesting that none of the integrins known to be present on mouse oocytes are essential for spermoocyte 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 spermoolemmal 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 spermoocyte 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
6
1 and CD9 are reported to be co-immunoprecipitated from mouse oocytes (Miyado et al., 2000
). However, the combined evidence demonstrating that mouse oocyte lacking
6
1 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 spermoocyte plasma membrane binding by antibodies against the several integrin subunits implies involvement of the integrins in human spermoocyte 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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References |
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Submitted on August 7, 2003; resubmitted on October 15, 2003; accepted on October 30, 2003.