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Keywords:

  • fertilization;
  • integrin;
  • vitronectin;
  • sperm;
  • mouse

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Oocyte integrins have been described as essential for fertilization. But this concept has been challenged by deletion experiments. Recently, we have shown that sperm integrin α6β1 plays a determinant role in mouse gamete interaction. In this study, we demonstrate the presence of αvβ3 integrin by Western blot and immunofluorescence on the sperm membrane. Oocytes and/or sperm preincubations with anti-αv or anti-β3 antibodies were performed before in vitro fertilization on cumulus-intact and zona-free egg assays. We observed inhibitory effects on the fusion process mostly by means of sperm function. An antibody directed against vitronectin inhibited gametes fusion, whereas the presence of exogenous vitronectin increased its efficiency. We suggest that vitronectin (on multimeric forms) can play a first nonspecific link corresponding to loosely bound spermatozoa to oocyte and that this link could be mediated by means of oocyte proteoglycans or integrins, and sperm αvβ3 integrin. Developmental Dynamics 239:773–783, 2010. © 2010 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

The initial gamete interaction model that was conceived involved one integrin, α6β1, on the oocyte (Almeida et al., 1995), and its sperm ligand, fertilin β, also known as ADAM2 (A Disintegrin And Metalloprotease2; Chen and Sampson, 1999). In fact, sperm–egg interaction suggests a much more complex molecular process, involving several protein families such as ADAMs, CRISPs, SLLP1, SAMP14, SAMP32; immunoglobulins such as Izumo; integrins; GPI-APs; and tetraspanins. Among them, two molecules have been shown to be essential for fertilization: Izumo, a sperm protein belonging to the immunoglobulin superfamily (Inoue et al., 2005), and CD9 tetraspanin on the oocyte membrane (Kaji et al., 2000; Le Naour et al., 2000; Miyado et al., 2000). Deletions of these two genes result in a lack of fusion of spermatozoa with oocytes and a dramatic reduction of the knockout (KO) mice fertility. Moreover, many integrin subunits, that may play a role in gamete interaction, have been identified on the oocyte membrane: α2, α3, α4, α5, α6, α9, αM, αv, β1, β2, β3, and β5 (Campbell et al., 1995; Evans et al., 1995; Zuccotti et al., 1998; Burns et al., 2002; Sengoku et al., 2004; Vjugina et al., 2009). However, the essential role of such proteins upon fertilization was called into question in several publications. Indeed, α3 and α6 integrin subunit null eggs from KO mice and oocyte-specific β1 integrin conditional KO mice were designed and investigated. These animals were fertile and their oocytes were normally functional in in vitro fertilization (Miller et al., 2000; He et al., 2003). β3 integrin subunit KO mice are also fertile (Hodivala-Dilke et al., 1999). This led to the conclusion that “none of the integrins known to be present on the mouse egg or to be ADAM receptors are essential for sperm–egg binding and fusion” (He et al., 2003), but their involvement in this phenomenon was never excluded (Evans, 2009). In this context, recent study using wild-type (WT) and β1-null eggs and polymers of peptide from the fertilin β disintegrin domain, suggested that oocyte β1 integrin enhances the initial adhesion of sperm to the egg plasma membrane, subsequent attachment and fusion being mediated by additional egg and sperm proteins present in the β1 integrin complex (Baessler et al., 2009). Another recent study, using either knockdown by RNA interference directed against α9 integrin or anti-β1 integrin antibody revealed a reduction of sperm–egg binding and fusion (Vjugina et al., 2009). In addition, during total or conditional KO experiments, the deleted females were always mated with WT males and in vitro tests were also performed using WT sperm. But neither the presence of these integrins on mouse spermatozoa nor their involvements in gamete interaction were questioned. Yet, the expression of α6, αv and β3 integrins on human spermatozoa had been demonstrated for a long time (Fusi et al., 1996b). Recently, we have shown that α6β1 integrin is also expressed by mouse spermatozoa and involved in gamete adhesion and fusion (Barraud-Lange et al., 2007b). In addition, we have demonstrated that the cyclic FEE peptide, that mimics the disintegrin binding domain of fertilin β, ligand of integrin α6β1, potentiates the inhibitory effect of RGD peptide, which can bind to RGD sensitive integrins. RGD-binding integrin subfamily (including αvβ1, αvβ3, and αvβ5), expressed by the oocyte have been shown to be implicated in fertilization because the use of RGD peptide during IVF assays inhibits fertilization (Bronson and Fusi, 1990). This suggests that RGD insensitive and RGD sensitive integrins cooperate during fertilization (Ziyyat et al., 2005). Bronson et al. described a similar potentiation of the inhibitory effect of RGD peptide by FEE peptide (Bronson et al., 1999). They used a linear FEE-containing peptide and a cyclic RGD-containing peptide that exhibited a synergistic inhibition of both adhesion and fusion, suggesting cooperation between the two receptor ligands during fertilization in human. All these data suggests that at least two different oocyte integrins could be involved in human gamete interaction, one recognizing fertilin and the other recognizing RGD-containing sperm-associated proteins such as vitronectin or fibronectin, and that they may cooperate (Bronson et al., 1999). Indeed, αvβ3 integrin is RGD sensitive and is known to be the receptor of vitronectin (VN), a component of the extracellular matrix of acrosome reacted (AR) sperm (Fusi et al., 1996a; Bronson et al., 2000). Moreover, the colocalization of αvβ3 with α6β1 integrin on human zona pellucida (ZP) free oocytes supports this hypothesis (Ziyyat et al., 2005).

We postulate that such cooperation might also take place on sperm, which suggests that sperm would express not only the α6β1 integrin but also the αvβ3 integrin. In addition, after being released during the AR, VN has been described in human as the Velcro that binds gametes together through its adhesion to αvβ3 integrin (Fusi et al., 1996a; Bronson et al., 2000). This suggests that αvβ3 integrin is present on the two gametes, what is true in humans but not yet shown in mice. Thus, we decided to search whether αvβ3 is expressed by mouse sperm and involved in mouse gamete interaction or not. Western blot and immunofluorescence analysis enabled us to demonstrate that αvβ3 integrin is actually expressed on mouse spermatozoa membrane. Functional tests using antibodies directed against αv and β3 integrin subunits demonstrated its involvement in fertilization both on sperm and cumulus oocyte complex (COC) side. We also showed the role of vitronectin in mouse sperm–egg interaction. All these findings lead us to propose a model according to which macromolecules of VN, liberated during AR (Bronson et al., 2000), could bind to αvβ3 integrin both on sperm and oocyte, as a Velcro. It would constitute a first nonspecific adhesion process leading to what is called as loosely bound sperm. Afterward, other sperm –oocyte ligands and receptors could interact to make a tighter and more specific adhesion possible.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

αv and β3 Integrin Subunits and Vitronectin Are Expressed on Mouse Sperm Membrane

Western blot analysis.

Expression of αv and β3 integrin subunits and vitronectin on cauda epididymal sperm was evaluated by immunoblot analysis. As shown on Figure 1A, specific bands at approximately 125 and 135 kDa, were detected on sperm (10 μg proteins per 106 sperm) as in the F9 Whole Cell Lysate positive control (50 μg). It corresponds to the expected molecular weight of αv and β3 integrin subunits under reducing conditions. Under the same conditions, VN was detected using the anti-vitronectin antibody as a specific band at 65–75 kDa (Fig. 1B). Controls using a nonspecific rabbit and rat polyclonal antibodies were negative. These results demonstrated the presence of αv, β3 integrin subunits and VN on mouse sperm membrane.

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Figure 1. Expression and dynamic distribution of αvβ3 integrin on mouse sperm. A,B: Western Blot analysis of mouse sperm αvβ3 integrin and vitronectin expression. A: Alpha V and beta 3 integrin subunits and vitronectin expression was investigated by Western blot analysis. Proteins from epididymal sperm were extracted with deoxycholate/1% NP40, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions and blotted with anti-αv and anti-β3 integrin subunit and anti-VN antibodies. Specific bands (approximately 125 and 135 kDa and 65 kDa) were detected on sperm extract after rabbit polyclonal antibodies incubation against αv and β3 integrin subunits and vitronectin, respectively. A: These bands correspond to αv and β3 integrin subunits as confirmed by the use of the F9 Whole Cell Lysate as positive control (50 μg of proteins). No specific band appeared when a nonspecific rabbit IgG was used (Isotype lane). B: Band at 65 kDa corresponds to vitronectin. No specific band appeared when a nonspecific rabbit IgG was used. 10 μg proteins correspond to one million sperm. C: Dynamic distribution of alpha V beta 3 integrin on mouse sperm assessed by immunofluorescence. Distribution of αv and β3 integrin subunits was studied by immunofluorescence microscopy. Spermatozoa were exposed to Pisum sativum agglutinin conjugated to fluorescein (PSA-FITC; b,e,h) after anti-αv integrin subunit antibody (c,f,i) followed by anti-rat biotinylated secondary antibody and Alexa Fluor 594 conjugated Streptavidin. The following fluorescent patterns were recorded: negative noncapacitated sperm (c), 30% of capacitated acrosome intact spermatozoa (f) and 93% for capacitated acrosome reacted spermatozoa (i) were stained. Sperm nuclei were detected by DAPI (4′,6-diamidine-2-phenylidole-dihydrochloride) staining (a,d,g). These images correspond to the most common staining but other distributions have been observed. Nonimmune control did not give any signal. Approximately similar pattern staining was observed for β3 integrin except that β3 appeared on 40% of freshly recovered noncapacitated sperm. The same treatment was applied to sperm as for αv detection but using anti-β3 antibody (a′ to i′). % indicates the frequency of showed distribution. Scale bar = 1 μm.

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Immunofluorescence analysis.

Immunofluorescence was used to localize αv and β3 integrin subunits on the head of mouse sperm. In parallel, acrosomal status was assessed using Pisum sativum agglutinin (PSA) staining protocol. The detection of the αv integrin subunit varied according to the sperm state of maturity. Indeed, freshly recovered noncapacitated sperm did not show any distinct fluorescence (Fig. 1C.c). The staining detection appeared during capacitation, whereas only 30% of capacitated acrosome intact (AI) spermatozoa were stained (Fig. 1C.f), 93% of capacitated AR spermatozoa group (Fig. 1C.i) showed a specific labeling. Three different staining patterns could be observed: αv integrin subunit could be seen spreading over the acrosome, over the whole head or could be localized on the equatorial region. Whereas some spermatozoa showed a punctual staining others had a more uniform covering over the entire head. The most frequent pattern was the overall covering of the acrosomal region (75% of capacitated acrosome-reacted spermatozoa). Similar distribution, staining, and relative proportions were found for β3 integrin subunit, except that it appeared less dependent on the sperm maturity status. Forty percent of freshly recovered noncapacitated sperm were stained (Fig. 1C.c′). Nonimmune controls (Fig. 1C) and secondary antibodies gave no signal (data not shown).

αvβ3 Integrin Is Functional During Mouse Fertilization

Because the presence of αv and β3 integrin subunits as well as that of VN was demonstrated on the mouse sperm, we next investigated their involvement in fertilization process by studying the effects of their inhibition using their respective antibodies or the addition of VN on the fertilization rate. Fertilization rate (FR; the percentage of eggs fused with at least one sperm) and/or the fertilization index (FI; the mean number of fused sperm per egg) were assessed in cumulus-intact and zona-free eggs insemination, respectively. To discriminate the origin of the antibody effect, whether it was acting on integrin subunits from oocyte, sperm, or both, gametes were separately preincubated with the antibodies. We observed the involvement of cumulus oocyte complex (COC) αv integrin subunit in the fertilization process. Indeed, preincubation of COC with the anti-αv antibody at 50 μg/ml significantly inhibited the FR, as compared to the control group (32.9 ± 5.3% vs. 79.3 ± 3.9%, respectively; P < 0.0001, Fig. 2A). After sperm preincubation, the FR was also significantly decreased when compared with the control group (23.7 ± 3.9%; P < 0.0001). The inhibitory effect of the antibody was not further increased when both gametes were preincubated, suggesting that there is neither potentialization nor addition of inhibitory effects on both gametes (Fig. 2A).

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Figure 2. Effects of alpha V integrin subunit antibody (50 μg/ml) on mouse cumulus-intact and zona free in vitro fertilization (IVF) assays. A: Fertilization rate or mean (± SEM) percentage of fertilized eggs following cumulus-intact IVF assay at 106 spermatozoa per ml for 3 hr. B: Fertilization index or mean (± SEM) of sperm number fused by egg on zona-free IVF assay at 105 spermatozoa per ml for 3 hr. No antibodies (CTRL), in vitro fertilization in the presence of antibody but without preincubations (IVF), groups with cumulus oocyte complex (COC), oocyte (O), sperm (S), sperm and COC (S/COC) or sperm and oocyte (S/O) preincubations. (), number of oocytes. The studies were repeated at least three times.

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We confirmed on zona-free eggs insemination assays the effect of the anti-αv integrin subunit antibodies (50 μg/ml) on sperm when preincubated alone or with oocytes and when the antibody was present in the inseminating medium during the IVF (FI: 2.22 ± 0.19, 2.40 ± 0.17 and 2.77 ± 0.16, respectively, as compared to the control group at 4.01 ± 0.17; P < 0.0001; Fig. 2B). After sperm preincubation at 200 μg/ml, the inhibitory effect remained partial (Fig. 4). But, anti-αv mAb (at 50 μg/ml and 200 μg/ml) had no effect on zona-free oocytes (Figs. 2B, 4).

The same experiments were carried out using the anti-β3 antibody (10 μg/ml), which triggers a modest but significant decrease of the FR between the control group and the group in which IVF was performed in the presence of the antibody (54.5 ± 5.3%, 32.4 ± 5.7%; P = 0.039; Fig. 3A). No differences were observed when the other groups were compared with the controls (Fig. 3A). The modest inhibitory effect of the antibody (10 μg/ml) was also observed on FI in zona-free assays, only when sperm was preincubated alone or with oocytes, and in IVF group (FI: 2.68 ± 0.17; P < 0.0001; 3.01 ± 0.15; P = 0.01; and 2.85 ± 0.15; P = 0.002 compared with the control group, following analysis of variance (ANOVA) and post hoc tests at 3.63 ± 0.13; Fig. 3B). Incubation of oocytes alone did not induce any significant inhibition. Using the antibody at 50 μg/ml, these results remained unchanged except for IVF group in which no difference was observed when compared with the control group (FI: 3.04 ± 0.21 and 3.72 ± 0.19; P = 0.25; Fig. 3C). Similar tests were performed using either a nonspecific IgG or sodium azide, when the antibody solutions contained it, as negative controls. They did not impair FR or FI (data not shown). Altogether, these data demonstrate that the effect of anti-αv antibodies (and to a lesser extent those directed against anti-β3 integrin subunit), is specific, proving the involvement of sperm αvβ3 integrin in the fertilization process in mouse. This also shows that the anti-αv antibody is efficient on both the sperm and the cumulus-intact oocyte while anti-β3 antibody is effective only on sperm in a less efficient manner.

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Figure 3. Effects of beta 3 integrin subunit antibody on mouse cumulus-intact (10 μg/ml) and zona free in vitro fertilization (IVF) assays (10 and 50 μg/ml). A: Fertilization rate or mean (± SEM) percentage of fertilized eggs following cumulus-intact IVF assay at 106 spermatozoa per ml for 3 hr. B: Fertilization index or mean (± SEM) of sperm number fused by egg on zona-free IVF assay at 105 spermatozoa per ml for 3 hr. No antibodies (CTRL), in vitro fertilization in the presence of antibody but without preincubations (IVF), groups with cumulus oocyte complex (COC), oocyte (O), sperm (S), sperm and COC (S/COC) or sperm and oocyte (S/O) preincubations (antibody concentration:10 μg/ml). The studies were repeated at least three times. C: The same experiment that this described in B but at 50 μg/ml of antibody. (), number of oocytes. The study was repeated two times.

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Figure 4. Effects of alpha V and beta 3 integrin subunit antibodies on mouse zona free in vitro fertilization (IVF) assays at 200 μg/ml. Fertilization index or mean (± SEM) of sperm number fused by egg on zona-free IVF assay at 105 spermatozoa per ml for 3 hr. No antibodies (CTRL), after oocytes (O) preincubation followed by IVF in the presence of anti-alphav antibody (O/IVF) or sperm preincubation (S) with anti-alphav or anti-beta3 antibodies. (), number of oocytes.

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Vitronectin, the αvβ3 Integrin Ligand, Takes Part in Mouse Fertilization

It is known that mouse oocytes express αvβ3 integrin (Almeida et al., 1995; Evans et al., 1995). We showed that mouse spermatozoa also express both αvβ3 integrin and its ligand (VN), raising the possibility of an involvement of VN in gamete interaction. Indeed, VN is a macromolecule from the extracellular cell matrix, which is recognized by the αvβ3 integrin. Furthermore, VN is released by sperm during the AR, which occurs when the sperm crosses the ZP (Bronson et al., 2000). VN could, therefore, play a role of Velcro between gamete membranes, then creating a nonspecific first attachment between the gametes. Hence, we tested the effect of sperm preincubation with increasing concentrations of VN or of the anti-VN antibody on IVF assays. Sperm preincubation with VN at 0.1 μM significantly increased FI on zona-free assay from 3.16 ± 0.21 for the control group to 5.33 ± 0.21 for the VN group (P < 0.0001; Fig. 5A). Inversely, anti-VN antibody sperm preincubation induced a significant dose-dependent inhibition of the FR: 92 ± 5,5%, 29.9 ± 4,7%, 21.2 ± 3,9%, 5,3 ± 3% and 0.0% for 0 (control), 10, 20, 30, and 50 μg/ml antibody on COC assay, respectively (P < 0.0001; Fig. 5B). Of interest, no spermatozoa could be seen within the perivitelline space, suggesting that they could not cross the zona pellucida. A smaller inhibitory effect was also observed on zona-free assay with the anti-VN antibody from 3.38 ± 0.13 for the control group to 2.39 ± 0.13, 1.77 ± 0.07 and 1.53 ± 0.07 for the anti-VN groups at 25, 50, and 100 μg/ml, respectively (P < 0.0001; Fig. 5C). In this later test, we were unable to completely inhibit fertilization even using twice the concentration that abolished completely fertilization in COC assay. These data show that VN is actually involved in fertilization, probably by means of its recognition by αvβ3 integrin.

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Figure 5. Effect of vitronectin and anti-vitronectin on mouse IVF assays. A: Fertilization index or mean (± SEM) of sperm number fused by egg following exposure of sperm to the vitronectin (0.1 μM) pretreatment and insemination at 105 spermatozoa per ml for 3 hr on zona-free IVF assays. B: Fertilization rate or mean (± SEM) percentage of fertilized eggs following sperm preincubations at 0 (Ctrl), 10, 20, 30, and 50 μg/ml anti-vitronectin antibody on cumulus-intact IVF assays. C: Fertilization index or mean (± SEM) of sperm number fused by egg following exposure of sperm to 0, 25, 50, and 100 μg/ml anti-vitronectin antibody zona-free IVF assays. (), number of oocytes. The studies were repeated at least three times.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

In this study, using Western blot analysis and immunofluorescence microscopy, we show that mouse sperm express the αvβ3 integrin. Indeed, the αv integrin subunit appears in a small percentage of sperm after capacitation and at a larger extent after AR. Approximately 40% of freshly recovered noncapacitated sperm express β3 at their surface suggesting that only a proportion of β3 subunits are dimerized with αv. Indeed, β3 subunit can also form a heterodimer with αIIb subunit, another natural receptor for fibronectin and vitronectin, even if its presence on sperm surface has not been reported yet. Another explanation may be that αvβ3 heterodimerization could occur only after AR and the following sperm membrane reorganization.

The functional implication of the αvβ3 integrin in gamete interaction was demonstrated using antibodies directed against αv or β3 integrin subunits in cumulus-intact eggs assay by preincubating gametes separately. Indeed, the anti-αv antibody inhibited gametes fusion regardless of the gamete that was preincubated, while anti-β3 antibody had a weak sperm inhibition effect. This indicated that both COC and sperm αv and to a lesser extent sperm β3 are involved in the adhesion/fusion process. This difference of effect could be due to a greater efficiency of the anti-αv antibody or a partial combination of the two subunits as explained above. This is in agreement with the different kinetic of localization for the two subunits that we report. In turn, the αv integrin subunit might bind to other beta subunits such as β1, β5, β6, and β8. For example, we have already shown that the β1 subunit is present and functional on mouse sperm surface (Barraud-Lange et al., 2007b). Obviously, a combination with other subunits is not excluded, because all integrins have not been checked on the mouse sperm membrane.

Involvement of αvβ3 integrin in gamete interaction has already been indirectly suggested by other studies. αvβ3 integrin is RGD-sensitive, oligopeptides containing the RGD sequence inhibits sperm – oolemma adhesion and oocyte penetration in both heterologous (human–hamster) and homologous (hamster–hamster) gamete interactions (Fusi et al., 1993). We have also shown an inhibitory effect of RGD peptide in a homologous system (human–human; Ziyyat et al., 2005). But in all these studies as in others, the peptide was still present during the insemination and, therefore, could have had an action on sperm. Supporting this idea, a study performed in bovine with gametes separately preincubated has also shown that the RGD peptide, anti-αv and anti-α5 antibodies inhibit fusion whatever the gamete that was incubated (Goncalves et al., 2007, 2009). In contrast, experiments performed by He et al. did not show any inhibition using antibodies directed against αv or β3 integrin subunits (He et al., 2003). The major difference between the two studies lies in the fact that He et al. had not looked for the sperm effects of antibodies because the expression of αv and β3 integrin subunits by mouse sperm was not known. For the oocyte effect, results from the two studies are quite close because we found no effect of anti-αv and anti-β3 antibodies (even at 200 μg/ml; Fig. 4) on oocyte zona-free suggesting that these oocyte integrin subunits are probably not involved on gamete interaction. This finding is strengthened by the fact that He et al. used a different function-blocking anti-αv antibody at 200 μg/ml and saw no effects. Using immunofluorescence analysis, we failed to detect the oocyte αv and β3 integrin subunits (data not shown) suggesting that these proteins are at least weakly expressed on the oocyte surface and/or that used antibodies, which are nevertheless able to recognize the sperm proteins, fail to recognize oocyte proteins.

The inhibitory effects observed in the group where IVF occurs in the presence of antibodies but without gamete preincubation are probably due, in the case of zona-free assays, to the duration of insemination which was longer (3 hr vs. 40 min) and sperm concentration which was likely lower in our experiments. Indeed, the design of experimental assays for gamete function can be critical for detecting an antibody effect. Vjugina et al. observed reduced sperm–egg interactions as a result of oocyte α9 knockdown by RNA interference or β1 antibody treatment in inseminations with a sperm:egg ratio of 100:1, although not in inseminations with 500:1 (Vjugina et al., 2009). In fact, a sperm:egg ratio of 100:1 is closer to our experimental conditions. This ratio applied to 10 oocytes and 1 ml corresponds to our final sperm concentration (105/ml), while this concentration varies from 1 to 5 × 105/ml in the study of He and co-workers. Finally, despite a sperm concentration less than or equal to that used by He et al., we obtained a higher fertilization index. Our conditions seem more favorable to allow revealing a weak antibody inhibitory effect.

In experiments performed by He et al., only oocytes were preincubated and sperm were added to this medium for insemination (He et al., 2003). However, in the case of the zona-free assay, sperm preincubation may be crucial because the fusion occurs very quickly and the antibody might not have time to exert its inhibitory effect which is not immediate and requires a delay to be effective.

In the case of cumulus-intact assays, the effect observed in the group in which IVF is performed in the presence of antibodies is probably due, in addition to the possible effect on the cumulus cells that express at least αv integrin subunit on their surface (Tamba et al., 2008), to the delay required for sperm to cross the cumulus and the ZP before gametes fusion. It represents a necessary sperm preincubation step. In this regard, this effect is closer to that observed after sperm preincubation.

When only COC was preincubated, sperm effect could be due to residual antibodies remaining trapped by the viscous extracellular matrix. It is certainly difficult to completely get rid of antibodies with which it was incubated.

Sperm and cumulus cells inhibitory effect of anti-αv and anti-β3 integrin subunits antibodies remains partial, confirming that they participate but are not the only molecules involved in gamete interaction.

Deletions of αv integrin subunit gene led to the death of the majority of the fetuses at day 9.5, and those which survived until birth died from brain or intestine hemorrhage (Bader et al., 1998). These data highlight the necessity to explore the role of αv subunit in mouse fertilization. The fact that β3 integrin subunit null mice are viable and fertile (Hodivala-Dilke et al., 1999) does not exclude a possible redundancy for this integrin subunit. Conditional deletions of these integrin subunits have never been investigated, but even when such experiments were performed for other integrin subunits, focus was only on the oocyte, never on the spermatozoon or on the cumulus cells (Miller et al., 2000; He et al., 2003).

Our data reveal that the αvβ3 integrin plays a role in gamete interaction, and most probably by means of VN. Hence, we evaluated the role of this latter using exogenous VN or an anti-VN antibody. Anti-VN antibody inhibited the gamete fusion on cumulus-intact (completely at 50 μg/ml) and on ZP-free IVF assays in a dose-dependent manner. The fact that anti-VN antibody did not completely abolish fertilization in zona-free assays suggests that a substantial part of its effect possibly takes place through an action on the cumulus cells or on ZP sperm interaction. In addition, this result could be explained by membrane changes due to ZP removal. Actually, VN acts by means of its receptors, oocyte integrins, which organization is modified by ZP removal (Ziyyat et al., 2006; Barraud-Lange et al., 2007b). Addition of 0.1 μM VN to the medium during zona-free oocytes insemination promoted human gametes fusion, as already observed by Fusi et al. (Fusi et al., 1996a). They showed that this fusion promotion was probably due to the VN promotion of calcium dependent sperm – oolemma binding. They have also observed that repeated washings of the capacitated spermatozoa led to a reduced binding, which could be restored by the addition of VN to the medium. In our study, prolonged sperm incubation (3 hr) with the same VN concentration (0.1 μM) caused their aggregation (data not shown). This is probably due to the dynamic emergence of VN receptors (integrin αvβ3 and others) during capacitation and AR. The fact that αvβ3 integrin appears mostly at the moment of AR and that it is precisely at this time that VN is released (Bronson et al., 2000), leads us to propose the following scenario: VN is released during acrosome reaction when the sperm enters the perivitelline space. Gamete integrins capture some of the released VN, which after dimeric or multimeric formation could then play a role as Velcro between oocyte and sperm αvβ3 integrins. Such nonspecific adhesion would enable oocyte and spermatozoon to stick together, the time necessary for α6β1 integrin, fertilin β, cyritestin and probably other molecules to interact. As VN possesses only a single RGD sequence, we hypothesize that a VN molecule binds to another, through a non-RGD motif to form at least a dimer. The RGD sequence located at the carboxy-terminal boundary of the somatomedin B domain of VN (Suzuki et al., 1984) appears to mediate binding of VN to distinct members of integrins, in particular to the VN receptor, and is responsible for the cell attachment activity of VN and other adhesive proteins (Ruoslahti and Pierschbacher, 1986). Multimeric forms of VN have been shown to exist (Sane et al., 1988, 1990; Hess et al., 1995). In this scenario, within the multimeric molecule, the RGD site of each VN molecule remaining free can make a bridge between the two gametes through the recognition of these RGD sites by integrins. This scenario would also explain the phenomenon of calcium-dependent sperm agglutination in the presence of a large concentration of VN previously described (Fusi et al., 1996a; Fig. 6A). A second nonexclusive scenario could be that sperm capture some released VN by means of RGD site. VN is a conformationally labile molecule that possesses a cryptic heparin binding site which is not expressed in the soluble form, but which makes its appearance when VN is immobilized. Human and mouse eggs have been shown to synthesize proteoglycans (Tesarik and Kopecny, 1986; Zhuo and Kimata, 2001; Russell and Salustri, 2006; Romanato et al., 2008) which could provide a secondary binding site for VN (Fig. 6B). Because the used antibodies did not allow either to stain or inhibit oocyte integrins (at least on the zona-free oocytes), we favor this second scenario.

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Figure 6. Hypothetical models of participation of vitronectin (VN), integrins and related molecules on mouse sperm–egg adhesion and fusion. Important sperm molecules for interaction with oocyte oolemma are not detected on the surface of freshly recovered sperm. When sperm are capacitated, a part of some proteins such as integrins appear at the surface (1). It is the acrosome reaction that reveals all the molecules involved in the process of membrane–membrane interaction and in particular integrins and IZUMO (2). At this moment, a part of sperm released VN is captured by sperm αvβ3 integrin. As VN possesses only a single RGD sequence, two nonexclusive scenarios are possible: A: This VN would play a role as Velcro between oocyte and sperm αvβ3 integrins (3). In this case, VN acts as multimeric form (at least dimer) in which VN molecules bind to them by a site other than RGD to kept free the RGD site to link oocyte αvβ3 integrin. B: Alternatively and more likely, mouse eggs which have been shown to synthesize proteoglycans could provide a secondary binding site for VN. Indeed, VN is a conformationally labile molecule that possesses a cryptic heparin binding site which is not expressed in the soluble form, but which makes its appearance when VN adheres to solid phase surfaces. When spermatozoon is in the perivitelline space, due to repeated contact with oocyte membrane or Exosome-like vesicles present in the perivitelline space, it captures oocyte membrane fragments containing the tetraspanin CD9 and its partners. We postulate that this CD9 allows the sperm to organize molecular complexes that would deal with those existing on the oocyte side.

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We propose that these models account for the phenomena that occur in vitro. Two categories of spermatozoa are observed on the oolemma: the “loosely bound” that could correspond to spermatozoa linked to the oocyte membrane only through VN, and the “tightly bound” that could be doubly linked to the oolemma by both α6β1 and αvβ3 integrins and probably other molecules.

Homozygous null mice completely deficient in VN demonstrate normal survival, development, and fertility suggesting that its role in these processes may overlap with other adhesive matrix components (Zheng et al., 1995). Fibronectin, which is also expressed by sperm, could provide this redundancy. Because of the embryonic lethality of fibronectin null mutant mouse, the essential nature of its role in fertilization could not be assessed (George et al., 1993). However, its participation in fertilization has been widely documented in several species (Fusi et al., 1992, 1996a; Thys et al., 2009).

We and others demonstrated partial gamete membrane symmetry with α6β1 integrin, αvβ3 integrin and fertilinβ (personal observations) expressed by both gametes. This partial gamete membrane symmetry, often unknown or overlooked or not enough taken into account, could be significantly enhanced by the membrane fragment transfer that occurs in perivitelline space by trogocytosis and/or exosome-like vesicles (Barraud-Lange et al., 2007a; Miyado et al., 2008). According to this last publication, exosomes derived from WT CD9 oocytes can associate with sperm and make them able to fuse with either WT or CD9 null oocytes. But this assertion concerning functional role of these transferred exosomes has been questioned by other work (Gupta et al., 2009). However, CD9, which is not expressed by sperm (Chen et al., 1999), is actually transferred from the oocyte to the sperm present in the perivitelline space. We have shown that this transfer affects entire CD9-containing membrane fragments (Barraud-Lange et al., 2007a). Because integrins are associated with CD9 tetraspanin that controls their reorganization during fertilization (Ziyyat et al., 2006), it is likely that integrins are also transferred with CD9 tetraspanin. After membrane fragments transfer and adhesion step, it will then form the molecular complexes common to both membranes in which the absence of a molecule on one side could be offset by its presence on the other side. This is probably the reason why the absence of one of these “not essential but contributing” molecules does not prevent gamete interaction (Miller et al., 2000; He et al., 2003). In contrast, molecules whose absence leads to a drastic effect play a much more important role, as it is the case of CD9 to which the organizer role of molecular complex has been widely attributed.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Antibodies and Peptides

For Western blot analysis, rabbit polyclonal antibodies against mouse αv integrin subunit (Fitzgerald, USA), β3 integrin subunit (Biosource, USA), and vitronectin (Santa Cruz Biotechnology, USA) were used, the negative control being rabbit IgGH+L and the secondary antibody a biotinylated anti-rabbit (Abcys S.A., France). The bands were detected with streptavidin conjugated to HRP (horseradish peroxidase [HRP]) enzyme (Immunotech, France).

To perform functional blocking tests and immunofluorescence experiments, a rat monoclonal antibody against the mouse αv integrin subunit (RMV-7), its isotype, a rat IgG (R3-34), a hamster monoclonal antibody against the mouse β3 integrin subunit (2C9-G2) and its isotype, and an hamster IgG1 were purchased from BD Pharmingen (USA). Vitronectin and anti-vitronectin antibody (H-270) were purchased from Sigma and Santa Cruz, respectively. For immunolabeling purpose, an anti-rat biotinylated secondary antibody (BD Pharmingen) and an Alexa Fluor 594 conjugated streptavidin (Invitrogen, France) were used to detect the signal. Detection of the AR was performed using Pisum sativum agglutinin conjugated to fluorescein (PSA-FITC, Invitrogen).

Western Blot Analysis

Capacitated sperm were washed twice in phosphate buffered saline (PBS), the pellet snap-frozen in liquid N2 and stored at −80°C for further use. Sperm aliquots were lysed in 50 mM Tris (pH 8.0), 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.25% sodium deoxycholate, and 1% NP40, supplemented with Protease Inhibitor Cocktail (Sigma) for 1 hr on ice, gently sonicated, and then boiled for 5 min with an equal volume of 2× NuPAGE LDS sample buffer (Invitrogen) supplemented with 5% β-mercaptoethanol. F9 Whole Cell Lysate (Abcam, France) was used at 50 μg proteins as a positive control for the αv and β3 integrin subunits detection. Sample proteins were separated by a precast gel electrophoresis (SoftGel, USA) and electro-transferred to Immobilon-P membranes (Millipore, France). Membranes were blocked for 1 hr with 2% casein before incubation with appropriate primary antibodies (rabbit anti-αv integrin: 1:5,000; rabbit anti-β3 integrin: 0.5 μg/ml, and a nonspecific rabbit IgG: 0.5 μg/ml) for 90 min at 37°C, followed by biotinylated appropriate secondary antibodies (0.5 μg/ml) and HRP-Streptavidin (0.02 μg/ml), for 1 hr and 45 min at 37°C, respectively. Vitronectin expression was revealed by anti-vitronectin antibody at 1:200 dilution and by secondary HRP conjugated (0.5 μg/ml) antibody. HRP activity was revealed by ECL detection kit (GE Healthcare, France) and autoradiography (BioMax Light Film, Kodac). Film exposure was less than 3 min.

Sperm Immunolabeling and Fluorescence Microscopy

Freshly recovered, capacitated spermatozoa or calcium ionophore induced AR spermatozoa (10 μM Calcium ionophore A23187, for 30 min at 37°C, Sigma) were washed in PBS containing 1% bovine serum albumin (BSA), centrifuged at 300 g for 10 min. After washing, the unfixed spermatozoa were incubated in PBS containing 10 μg/ml of rat anti-αv or hamster anti-β3 integrin antibodies for 70 min at 37°C and then with 10 μg/ml of the anti-rat or anti-hamster biotinylated antibody (for αv and β3, respectively) for 45 min at room temperature (RT). To detect the staining, Alexa 594 coupled streptavidin (10 μg/ml) was applied during 30 min at RT. Negative controls were performed by omitting the first antibody. The cells were then submitted to the PSA-staining protocol for the sequential detection of integrin distribution and acrosomal status. The spermatozoa were stained with fluorescein isothiocyanate (FITC) -conjugated lectin PSA (25 μg/ml in PBS) for 10 min. After repeated washing with double distilled water, a drop of sperm suspension was smeared on slide, air-dried, mounted with Vectashield/DAPI (4′,6-diamidine-2-phenylidole-dihydro chloride) and covered with a coverslip for analysis. Detection was performed using a Zeiss Axiophot epifluorescence microscope and images were digitally acquired with a camera (Coolpix 4500, Nikon).

Gamete Preparation and In Vitro Fertilization

Oocytes.

B6CBA F1 female mice (5–8 weeks old), purchased from Charles River Laboratories (France), were superovulated with 5 IU PMSG and 5 IU hCG (Intervet, France) 48 hr apart. At 12 to 14 hr after hCG injection, the animals were killed by cervical dislocation. Cumulus oophorus were collected by tearing the ampulla wall of the oviduct, placed in Whittingham's medium (Whittingham, 1971) supplemented with 3% BSA (Sigma), and maintained at 37°C under 5% CO2 in air under mineral oil (Sigma). When experiments were run with zona-free oocytes, cumulus cells were removed by a brief exposure to hyaluronidase (0.01%, Sigma). The ZP was then dissolved with acidic Tyrode's (AT) solution (pH 2.5) (Sigma) under visual monitoring. The zona-free eggs were rapidly washed five times and kept at 37°C under 5% CO2 in air for 2 to 3 hr to recover their fertilization ability.

Sperm preparation.

Mouse spermatozoa were obtained from the caudae epididymis of B6CBA F1 male mice (8–13 weeks old) and capacitated at 37°C under 5% CO2 for 90 min in a 500-μl drop of Whittingham's medium supplemented with 30 mg/ml BSA, under mineral oil.

In vitro fertilization.

Cumulus-intact and zona-free eggs were inseminated with capacitated spermatozoa for 3 hr in a 50-μl drop of medium at a final concentration of 106/ml or 105/ml, respectively. Then, they were washed and directly mounted in Vectashield/DAPI for observation under UV light (Zeiss Axioskop 20 microscope). The oocytes were considered fertilized when they showed at least one fluorescent decondensed sperm head within their cytoplasm.

To test the effect of antibodies on the fertilization rates (FR) or fertilization index (FI), before in vitro fertilization (IVF), either one or both gametes were separately preincubated for 30 min in medium supplemented with anti-αv or anti-β3 antibodies at 50 or 10 and 50 μg/ml, respectively. Oocytes were then washed, and sperm were diluted 1:100 into the final droplet containing the oocytes. In parallel, insemination was performed in medium containing antibodies without any gamete preincubations.

Effects of VN or anti-VN antibody were assessed by sperm preincubation in a medium containing either VN at 0.1 μM or the anti-VN antibody at 10, 20, 30, or 50 μg/ml for 30 min.

Statistical Analysis

Statistical analysis was performed using standard ANOVA. Differences were considered significant at P < 0.05.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

We thank K. Senni for constructive discussion about the role of the vitronectin.

REFERENCES

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  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
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