Membrane transfer from oocyte to sperm occurs in two CD9-independent ways that do not supply the fertilising ability of Cd9-deleted oocytes

in Reproduction
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  • 1 Service d'Histologie Embryologie, Université Paris Descartes, Inserm U1016, Biologie de la Reproduction, Hôpital Cochin, Assistance Publique-Hôpitaux de Paris, Université Paris Descartes, 123, Boulevard Port Royal, 75013 Paris, France

Spermatozoa undergo regulation of their functions along their lifespan through exchanges via vesicles or interactions with epithelial cells, in the epididymis, in the seminal fluid and in the female genital tract. Two different ways of oocyte membrane transfer to spermatozoa have been described: trogocytosis and exosomes. We here report an analysis of in vitro exchanges between the membranes of unfertilised oocytes and capacitated spermatozoa. We showed that optimum conditions are fulfilled when unfertilised oocytes interact with acrosome-reacted spermatozoa, a scenario mimicking the events occurring when the fertilising spermatozoon is inside the perivitelline space. Although CD9 tetraspanin is an essential molecule for fertilisation, exosome and trogocytosis transfer persists in Cd9-null oocytes in spite of their dramatic fusion failure. These exchanges are CD9 tetraspanin independent. We also confirm that mice sperm express CD9 tetraspanin and that when Cd9-null oocytes were inseminated with sperm covered with oocyte membrane materials, including CD9 tetraspanin, no rescue of the oocytes' fertilisability could be obtained. Thus, the existence of two ways of exchange between gametes during fertilisation suggests that these events could be of a physiological importance in this process.

Abstract

Spermatozoa undergo regulation of their functions along their lifespan through exchanges via vesicles or interactions with epithelial cells, in the epididymis, in the seminal fluid and in the female genital tract. Two different ways of oocyte membrane transfer to spermatozoa have been described: trogocytosis and exosomes. We here report an analysis of in vitro exchanges between the membranes of unfertilised oocytes and capacitated spermatozoa. We showed that optimum conditions are fulfilled when unfertilised oocytes interact with acrosome-reacted spermatozoa, a scenario mimicking the events occurring when the fertilising spermatozoon is inside the perivitelline space. Although CD9 tetraspanin is an essential molecule for fertilisation, exosome and trogocytosis transfer persists in Cd9-null oocytes in spite of their dramatic fusion failure. These exchanges are CD9 tetraspanin independent. We also confirm that mice sperm express CD9 tetraspanin and that when Cd9-null oocytes were inseminated with sperm covered with oocyte membrane materials, including CD9 tetraspanin, no rescue of the oocytes' fertilisability could be obtained. Thus, the existence of two ways of exchange between gametes during fertilisation suggests that these events could be of a physiological importance in this process.

Introduction

Mammalian sperm collected from the testis are immotile and unable to fertilise the oocyte. They acquire these properties while going through the epididymal duct. One of the most important changes that occur is the transfer to the sperm membrane of hydrophobic proteins found in the epididymal compartment that normally bear glycosylphosphatidylinositol tails. As these proteins normally exist in membrane microdomains and are not free in the fluid, a transport system such as vesicles should occur in the epididymis (Caballero et al. 2011). Evidences have been provided that such vesicles exist in ram and humans, and in vitro experiments have shown that such vesicles can transfer proteins to the sperm in zinc- and pH-dependent manners (Gatti et al. 2005). Interestingly, the transfer of membrane molecules from the oocyte to the fertilising sperm when it is into the perivitelline space (PVS) also exists (Barraud-Lange et al. 2007a). Indeed, membrane fragments containing CD9 tetraspanin are transferred from the oocyte to the fertilising spermatozoon. This transfer has been related to trogocytosis, a process first described in the immune system where activated lymphocytes can capture plasma membrane fragments from antigen-presenting cells with which they establish a tight contact (Hudrisier et al. 2001). This acquisition by the spermatozoon of membrane fragments containing proteins could supply sperm membrane with proteins it does not express or not sufficiently. This could influence its membrane molecular reorganisation and then substantially modify its avidity for the oocyte membrane at the time of fertilisation. Supporting this hypothesis, Miyado et al. (2008) have confirmed the transfer of CD9 tetraspanin from the oocyte to the spermatozoon located into the PVS, but they have suggested that this transfer is supported by ‘exosome-like’ vesicles released from the oolemma into the PVS. Exosomes are small membrane vesicles of endocytic origin secreted by many different cells (Thery et al. 2002). They are implicated in intercellular communication particularly in the immune system. Miyado et al. (2008) have proposed that the release of CD9-containing vesicles from eggs before fertilisation bestows the sperm-fusing ability and renders the sperm competent to fuse even with Cd9−/− eggs. This would suggest that the essential role of oocyte CD9 tetraspanin is played once the molecule is transferred to the sperm. However, this result has been challenged by a correspondence from Myles and Primakoff's group (Gupta et al. 2009) who failed to reproduce the Cd9−/− egg rescue experiments.

In this work, we studied these new ways of gamete exchanges and communication either by trogocytosis or by exosome release, as two ways of membrane transfer from oocyte to the fertilising spermatozoon. We observed that fertilisability of Cd9−/− eggs could not be rescued by sperm covered by CD9 oocyte membrane fragments and showed that these gamete exchanges are CD9 tetraspanin independent.

Results

Spermatozoa interact with material released from the egg plasma membrane

We have described the existence of a transfer of oocyte membrane fragments to the spermatozoon via a trogocytosis process (Rubinstein et al. 2006, Barraud-Lange et al. 2007a). Miyado et al. (2008) have confirmed the existence of a membrane transfer but via an ‘exosome-like’ mechanism. Unlike gamete trogocytosis, exosome transfer of material does not require a direct contact between gamete membranes as sperm can capture exosomes released into the culture medium by zona pellucida (ZP)-free oocytes. Therefore, we explored the ability of eggs to perform material transfer via exosomes and trogocytosis.

As described in the experimental design (Fig. 1), sperm were incubated with ZP-free eggs or in a medium in which oocytes have been incubated and then withdrawn (no contact between gametes). Free-swimming sperm recovered from either trogocytosis or exosome models were examined by fluorescent microscopy.

Figure 1
Figure 1

Experimental design of trogocytosis and exosome models with WT oocytes. ZPs of WT oocytes were first removed by acidic Tyrode's solution. ZP-free eggs were then labelled with the PKH67 (Green Fluorescent Cell Linker for General Cell Membrane Labelling). After 2 h of recovery time, oocytes were retrieved from the drop of medium, so-called ‘vesicles-enriched medium’. Those oocytes were then inseminated in a 60 μl new drop of medium with 1.5×105 sperm/ml for 1 h (trogocytosis set-up). In parallel, the same sperm concentration was incubated in the vesicle-enriched medium for 1 h (exosome set-up). Spermatozoa from both media were analysed for green fluorescence by confocal microscopy and by flow cytometry analysis. CD9 tetraspanin has also been checked by western blot analysis in the vesicle-enriched medium.

Citation: REPRODUCTION 144, 1; 10.1530/REP-12-0040

We confirmed the presence of Paul Karl Horan (PKH)-labelled spermatozoa in the trogocytosis (Fig. 2a, b and c) and exosome (Fig. 2d, e and f) models. The egg membrane material transferred to the sperm head was then quantified by flow cytometry (FCM) analysis and compared in the two models. On a total of three independent experiments, an average of 8.8±3.7% stained sperm were obtained after trogocytosis protocol vs an average of 2.3±0.8% after exosome protocol. Figure 2 (g and h) with 6.4% labelled sperm after trogocytosis protocol vs 2.2% sperm after exosome is an illustrative example of this series of experiments. The difference in exchange rates between gametes was amplified when the oocytes remained only 5 min in the medium after ZP removal and PKH staining (4.7% after trogocytosis protocol vs 0.3% after exosome protocol; data not shown). In all sperm FCM analysis, the sperm autofluorescence was used as a negative control (Fig. 2i). These results lead us to conclude that both ways (trogocytosis and exosome) of membrane transfer from oocyte to sperm coexist.

Figure 2
Figure 2

Identification of membrane fragment transfer from PKH67-labelled oocytes to spermatozoa by both trogocytosis and exosome. Confocal microscopic analysis of spermatozoa recovered 1 h after insemination with PKH67-labelled oocytes (a, b and c) or after incubation in vesicle-enriched medium provided by PKH67-labelled oocytes (d, e and f). In both cases, oocyte membrane materials were detected on sperm head, which presents PKH67 dots associated with a slightly diffuse staining (b and e). Merged images of blue (DNA DAPI staining (a and d) and green signals are represented in c and f). Scale bar=2 μm. Flow cytometric analysis of sperm membrane capture in the trogocytosis (g) and exosome (h) set-up. Dot plots are representative of an independent experiment and show 6.4% (g, square C4) and 2.2% (h, square C4) of stained sperm for trogocytosis and exosome membrane transfer models respectively. Sperm autofluorescence was used as a negative control (i).

Citation: REPRODUCTION 144, 1; 10.1530/REP-12-0040

The egg membrane transfer preferentially occurs with acrosome-reacted sperm

During gamete interaction, the fertilising spermatozoon that reaches the PVS is acrosome reacted and interacts with the oolemma through trogocytosis and/or exosome pathways (Barraud-Lange et al. 2007a, Miyado et al. 2008). In our in vitro experimental model using ZP-free oocytes, the sperm population capacitated in vitro was a mixture of acrosome-reacted and acrosome intact sperm (Okabe et al. 1987). To clarify whether this exchange is favoured by the conditions just preceding fertilisation, we checked the sperm ability to interact with egg membrane material according to its acrosomal status. In this experiment, we analysed the acrosome status of sperm that have captured membrane fragments following interaction with ZP-free oocytes. The Izumo 1 protein present on equatorial segment of acrosome-reacted sperm was used as a marker of acrosome reaction (Fig. 3A; Inoue et al. 2005). FCM analysis of free sperm recovered 1 h post-insemination with ZP-free PKH-labelled oocytes revealed that the percentage of acrosome-reacted sperm was significantly higher in the group of sperm binding PKH-membrane fragments compared with the group of PKH-free sperm (56.7±7.6 vs 18.4±8.5%, respectively, P<0.001; Fig. 3B). This result indicates that although the egg membrane capture is not strictly specific to the acrosome-reacted sperm, it preferentially occurs following the acrosome reaction.

Figure 3
Figure 3

Acrosomal status of sperm bound to PKH-stained egg membrane fragments. Free-swimming sperm recovered after insemination with PKH26-stained oocytes (trogocytosis set-up) were stained with a polyclonal anti-Izumo 1 antibody (10 μg/ml) and revealed with a FITC anti-rabbit antibody to assess the sperm acrosomal status. Epifluorescence and flow cytometric analysis were performed. (A) Epifluorescence images of a PKH26-positive sperm, which present an Izumo 1 staining specific of an acrosome-reacted status. (B) Column graph presenting the percentage of acrosome intact (AR−) and acrosome-reacted (AR+) sperm within the sperm populations that have bound (membrane fragment +) or not (membrane fragment −) PKH26-egg membrane fragments. The measures performed by flow cytometric analysis revealed that on average 56.7±7.6% of PKH-positive sperm realised their acrosome reaction vs 18.4±8.5% in the population of PKH-free sperm (P<0.001). Number of experiments=4.

Citation: REPRODUCTION 144, 1; 10.1530/REP-12-0040

Exosomes transferred from oocyte to spermatozoa contain CD9 tetraspanin

As trogocytosis leads to the capture of oocyte tetraspanin CD9 by sperm head (Barraud-Lange et al. 2007a), we wondered whether, in our system, exosome membrane transfer could also provide sperm with egg CD9 tetraspanin.

First, we analysed by electron microscopy (EM) the content of oocyte PVS by collecting it during ZP removal. This medium was subjected to a series of centrifugations, and EM analysis of the final pellet revealed the presence of vesicles ranging in size from 50 to 150 nm in diameter (Fig. 4A) as described for exosomes in somatic cells (Thery et al. 2001, Couzin 2005).

Figure 4
Figure 4

Presence of CD9 in membrane material released by oocyte. (A) The electron microscopy analysis of the pellet obtained after centrifugation at 100 000 g of the PVS content collected during ZP removal revealed that it contained vesicles ranging in size from about 50 to 150 nm in diameter. (B) WB analysis of the vesicle-enriched medium (provided by 50 WT oocytes) was performed with anti-CD9 antibody (WT Ex). Granulosa cells (1.5×105 per lane) were used as positive control. Vesicle-enriched medium provided by 50 Cd9−/− oocytes (Cd9−/− Ex) and Cd9−/− granulosa cells were used as negative controls. GC, granulosa cells; Ex, exosomes.

Citation: REPRODUCTION 144, 1; 10.1530/REP-12-0040

Secondly, the western blot (WB) analysis of the ‘vesicle-enriched medium’ pellet showed the presence of CD9 tetraspanin with a specific band at the expected molecular weight of 24 kDa (Fig. 4B). Altogether, the EM and WB analysis demonstrated the presence of CD9 in the vesicles released by eggs indicating that, as trogocytosis does, the membrane material transferred by exosomes from oocyte to sperm can supply it with CD9 molecules.

CD9 tetraspanin is expressed by sperm and unmasked after acrosome reaction

WB analysis of mouse sperm

Contrary to what has been initially described (Chen et al. 1999, Li et al. 2004), spermatozoa do also express CD9 tetraspanin, as recently reported (Kimura et al. 2009, Ito et al. 2010) and as confirmed here. Indeed, we observed the presence of CD9 tetraspanin on cauda epididymal mature sperm. A characteristic band of 24 kDa was obtained by WB analysis under non-reducing conditions, corresponding to the CD9 tetraspanin expected molecular weight. Wild-type (WT) oocytes (n=20) were used as positive control, and sperm from Cd9-null male mice were used as negative control (Fig. 5A).

Figure 5
Figure 5

Expression and distribution of CD9 tetraspanin on mouse sperm. (A) WB analysis of cauda epididymal sperm from WT (lane 1) and Cd9−/− (lane 2) males. Oocytes from WT female (lane 3) were used as positive control. The molecular weight of CD9 in WT sperm (lane 1) as in WT oocytes (lane 3) was ∼24 kDa. CD9 was absent in the sperm from Cd9−/− males (lane 2). (B) CD9 immunodetection in mature (cauda epididymal) sperm. Acrosome intact (a, b and c), spontaneously acrosome-reacted (d, e and f) and ionophore-induced acrosome-reacted sperm (g, h and i) were observed. Sperm were exposed to rhodamine–Pisum sativum agglutinin (PSA) (b, e and h) after anti-CD9 tetraspanin antibody (KMC8) staining (c, f and i) revealed by goat anti-rat IgG Alexa Fluor 488 (green) secondary antibody. The following CD9 staining fluorescent patterns were observed: only 10% of acrosome intact sperm were stained (c), 60% of spontaneously acrosome-reacted sperm (f) and 75% of ionophore-induced acrosome-reacted sperm were stained (i). Sperm nuclei were detected with DAPI staining (a, d and g).

Citation: REPRODUCTION 144, 1; 10.1530/REP-12-0040

Immunofluorescence analysis of mouse sperm

To precisely localise CD9 tetraspanin on sperm head and assess its dynamic appearance according to the sperm status (acrosome intact or acrosome reacted), immunofluorescence analysis of mouse sperm was carried out using anti-CD9 antibody on freshly recovered, capacitated and acrosome-reacted spermatozoa. Acrosomal status was assessed by Pisum sativum agglutinin (PSA) staining protocol. Only 10% of freshly recovered or capacitated non-acrosome-reacted spermatozoa presented a fluorescent signal that appeared mainly as a thin line in the acrosomal region (Fig. 5B a, b and c). On 60–75% of acrosome-reacted sperm, bright fluorescent dots were observed (Fig. 5B f and i). Secondary antibody alone and sperm from Cd9−/− male served as negative controls (data not shown).

Egg membrane transfer to sperm is CD9 independent

Few years ago, the indispensability of the oocyte CD9 tetraspanin in gamete fusion has been demonstrated by the severely impaired fertilising ability of Cd9−/− oocytes (Kaji et al. 2000, Le Naour et al. 2000, Miyado et al. 2000). We were interested in studying whether the CD9 tetraspanin could be involved in the functional aspect concerning these exchanges. To explore the involvement of CD9 tetraspanin in the transfer of oocyte membrane material to sperm, we measured trogocytosis and exosome membrane material release using Cd9-null oocytes. As expected, no fertilisation was observed but trogocytosis and exosome release occurred. Indeed, sperm incubated either in the medium containing vesicles from PKH67-labelled Cd9-null oocytes (3.3%) or directly with PKH67-labelled Cd9 null oocytes (5.5%) presented a PKH67 staining (data not shown). These results indicated that Cd9-deleted oocytes are able to release membrane materials that can be captured by sperm and, then, that the presence of CD9 tetraspanin on the oocytes is not necessary for this phenomenon to take place.

The loss of CD9 tetraspanin alters morphology of microvilli but not exosome synthesis and secretion

To further confirm the independence of exosome secretion with regard to the expression of the CD9 tetraspanin, we analysed Cd9-null oocytes by EM. No difference was observed between WT and Cd9-null oocytes regarding vesicular material present into the PVS (Fig. 6A, B, C and D and A′, B′, C′ and D′ respectively). We noticed numerous vesicles in the PVS of both WT and Cd9-null oocytes, often gathered and with variable diameters ranging from 50 to 150 nm. Some of these WT oocyte vesicles were labelled with anti-CD9 antibody (Fig. 6A, B, C and D). Cd9-null oocytes were used as negative staining control (Fig. 6A′, B′, C′ and D′). The appearance of these vesicles was also variable, with or without dense content inside and some vesicles presented a cup-shaped morphology characteristic of exosomes (Fig. 6B and B′). These results suggest that among the vesicles found into the PVS, there are exosomes and that the synthesis and secretion of these vesicles in PVS are CD9 independent. Nevertheless, we observed morphological differences that accompanied the loss of CD9 concerning the microvilli structure with an altered length, thickness and density on Cd9-null oocytes (Fig. 6D′ vs D for WT oocytes) confirming the previous reports (Runge et al. 2007).

Figure 6
Figure 6

Electron microscopic analysis of WT and Cd9−/− oocytes. ZP intact WT (A, B, C and D) and Cd9−/− (A′, B′, C′ and D′) eggs were observed after staining using an anti-CD9 mAb revealed by 10 nm gold bead-conjugated anti-rat IgG. Cd9−/− oocytes were considered as negative controls (A′, B′, C′ and D′). Vesicles with various sizes (50–150 nm) and appearances were observed in the PVS of both WT (A, B, C and D) and Cd9-deleted oocytes (A′, B′, C′ and D′). Small vesicles displaying a cup-shaped morphology characteristic of exosomes were observed in the PVS (B and B′). Some vesicles in the PVS of WT eggs presented a CD9 labelling (A, B, C and D). The WT oocytes showed a normal structure of the microvilli (enlargement in D) while Cd9−/− oocytes were observed with altered microvillar morphology (enlargement in D′).

Citation: REPRODUCTION 144, 1; 10.1530/REP-12-0040

Sperm retrieval from oocyte CD9-containing material medium is not sufficient to rescue the fertilisation defect of Cd9-deleted mouse oocytes

Miyado et al. (2008) have assigned to exosome transfer, a specific and essential role in gamete fusion as they claimed that it allows sperm to fertilise Cd9-null oocytes. In order to further investigate the role of CD9 tetraspanin in gamete fusion, we assessed whether trogocytosis and exosome oocyte membrane transfer could compensate for the loss of fusing ability of Cd9-null oocytes.

As already described, no passive transfer of membrane material exists between oocytes (Barraud-Lange et al. 2007a, Miyado et al. 2008). Moreover, when PKH67-stained and non-stained WT oocytes were co-inseminated, sperm that have captured PKH67-stained membrane fragments were observed bound to stained (Fig. 7a, b and c) and non-stained oocytes (Fig. 7d, e and f). This observation implies that the capture of membrane fragment does not alter sperm viability and that a spermatozoon is able to capture membrane fragments from one egg and to bind to another one.

Figure 7
Figure 7

Acquisition of oocyte membrane fragments by sperm does not impair their ability to bind to other oocytes. Mixed PKH67-stained and non-stained oocytes were inseminated with sperm for 1 h. PKH67-stained spermatozoa were detected attached on both stained (a, b and c) and non-stained oocytes (d, e and f). Transmission images (a and d); superimposition of 15 consecutive PKH67 staining confocal sections at 0.4 μm intervals (b and e); superimposition of transmission and fluorescent signal (c and f). Scale bar=20 μm.

Citation: REPRODUCTION 144, 1; 10.1530/REP-12-0040

Co-insemination experiments of ZP-free WT and Cd9-null oocytes were performed (Fig. 8A). After co-incubation, mixed WT and Cd9-null oocytes were analysed by fluorescence microscopy i) to detect the presence of CD9 tetraspanin at the egg membrane in order to differentiate WT from Cd9-null oocytes and ii) to observe decondensed sperm nuclei in their cytoplasm to assess their fertilisation status (Fig. 9A and B respectively). WT oocytes (n=169) showed a fertilisation rate and fertilisation index of 100% and 2.5±0.3 sperm heads per oocyte, respectively, while none of the Cd9-null oocytes (n=68) were fertilised (Table 1). Then stained Cd9-deleted oocytes with PKH26 were co-inseminated with WT oocytes labelled with PKH67. The fluorescent microscopy analysis revealed the adhesion of PKH67-covered sperm on unfertilised Cd9-null oocytes (Fig. 9C and D). Despite this transfer, we confirmed the absence of fertilised Cd9-null oocytes. These results show that transfer of egg membrane fragment to sperm, via trogocytosis and/or exosomes, cannot rescue the Cd9-null oocytes' fusion defect despite the fact that sperm have captured CD9 tetraspanin from WT oocytes.

Figure 8
Figure 8

Experimental design of Cd9-null oocyte insemination with CD9 recovered sperm. (A) Fresh ZP-free WT and Cd9-null oocytes were mixed together in a 60 μl drop of medium for 2 h of recovering time. They were next inseminated with 1.5×105 sperm/ml for 1 h. After washing, all oocytes were incubated with anti-CD9 antibody (KMC8) for 1 h and then with Alexa Fluor 488 to distinguish WT from Cd9-null oocytes. Oocytes were then mounted in Vectashield/DAPI for observation. Oocytes were considered fertilised when fluorescent decondensed sperm head visible under u.v. light were present in their cytoplasm. To assess the fertilisation index, the number of decondensed sperm heads was recorded. Epifluorescence microscope analysis was conducted in parallel to check the presence of CD9 at the egg surface. (B) ZP-free WT oocytes were incubated for 2 h in a 60 μl drop of medium. WT oocytes were then retrieved from the medium. Zona-free Cd9-null oocytes were inseminated in this drop with 1.5×105 sperm/ml for 3 h. In parallel, ZP-free WT oocytes were inseminated with 1.5×105 sperm/ml for 3 h. Cd9-null and WT oocytes were mounted and observed as described previously.

Citation: REPRODUCTION 144, 1; 10.1530/REP-12-0040

Figure 9
Figure 9

The interaction of sperm covered by oocyte CD9-containing material with Cd9-deleted oocytes is not sufficient to rescue their fertilisation defects. (A) Co-inseminated WT and Cd9−/− oocytes were analysed under epifluorescence microscope after anti-CD9 antibody labelling. WT oocytes appeared intensely labelled while Cd9−/− oocytes were not. (B) Fertilisation status and index were assessed by the number of decondensed sperm heads in the cytoplasm of all oocytes examined under u.v. light. As presented, WT oocytes had at least one decondensed sperm head in their cytoplasm while Cd9−/− oocytes had none. (C) In another experiment, PKH67-stained WT oocytes (green) and PKH26-stained Cd9−/− oocytes (red) were co-inseminated. Some PKH67-covered sperm (green) were present at the surface of PKH26-Cd9-null egg. Picture represents the superimposition of DAPI and fluorescence detection after confocal microscopic analysis. (D) Enlargement of PKH67-stained sperm in contact with Cd9-null egg surface obtained by zooming on boxed area in image C.

Citation: REPRODUCTION 144, 1; 10.1530/REP-12-0040

Table 1

Fertilisation of Cd9 null oocytes with sperm expressing the oocyte CD9 tetraspanin.

Wild-typeCd9 null
Co-insemination of WT and Cd9-null oocytes
 Number of oocytes16968
 Fertilisation rate (%)1000
 Fertilisation index (mean±s.e.m.)2.5±0.30
Fertilisation of Cd9-null oocytes according to exosome protocol
 Number of oocytes16173
 Fertilisation rate (%)1000
 Fertilisation index (mean±s.e.m.)2.2±0.30

Fertilisation rate: the percentage of eggs fused with at least one sperm. Fertilisation index: the total number of fused sperm/total number of eggs. The results represent the mean±s.e.m. from three different experiments.

In another experiment series, the drop of culture medium where the ZP-free WT oocytes (n=161) have been incubated for 2 h served as a fertilisation medium to Cd9-null oocytes (n=73) (Fig. 8B). For a total of three independent experiments, none of the Cd9-null oocytes were fertilised (Table 1) despite the foregoing evidence that this medium was enriched in CD9-containing material that interacts with sperm (see Fig. 4). The discrepancy between our results and those of Miyado et al. cannot be explained by the different medium used, as we also performed the experiment using their conditions, TYH medium and B6C3 F1 mouse male line, without success (data not shown).

Sperm are able to achieve membrane exchanges with other cell types than oocytes

Along their journey from the testis to the oocytes, sperm have multiple interactions essential for their maturation process in the epididymis (Yanagimachi et al. 1985, Fornes et al. 1995, Frenette & Sullivan 2001), with seminal fluid (Ronquist et al. 1978, Breitbart & Rubinstein 1982) and while transiting in the female genital tract (Suarez 1987, Baillie et al. 1997). As sperm can exchange with different cell types, we wondered whether there were membrane exchanges between spermatozoa and cumulus cells with which they closely interact at fertilisation.

In order to study this possible membrane exchange, we conducted experiments in which sperm were incubated with previously labelled PKH cumulus cells. One hour after co-incubation, spermatozoa and cumulus cells were collected, washed with acidic Tyrode's solution to remove any possible bound cellular debris and analysed by immunofluorescence. PKH-stained sperm heads were observed showing that membrane fragment transfer occurred between cumulus cells and sperm (Fig. 10b). Interaction with cumulus cell has yet to be explored but it is interesting to note that there is a very lower fertilisation rate of eggs when cumulus cells have been removed before insemination, suggesting an essential function of the cumulus cells during fertilisation process (Tanghe et al. 2002, Jin et al. 2011).

Figure 10
Figure 10

Identification of membrane fragment transfer from PKH26-labelled somatic cells to spermatozoa by ‘trogocytosis-like’ pathway. Confocal microscopic analysis of spermatozoa recovered 1 h after incubation with PKH26-labelled granulosa cells (a, b and c) or with PKH26-labelled fibroblasts (a′, b′ and c′). In both cases, membrane materials were detected on sperm head, which presented PKH26 staining (b and b′). Dots (up arrow) and diffusion (down arrow) of PKH26 dye can coexist on some sperm head (b). Phase transmission (a and a′), fluorescent (b and b′), and merge file (c and c′) images.

Citation: REPRODUCTION 144, 1; 10.1530/REP-12-0040

We next wondered whether sperm were able to achieve membrane exchanges with cell types they do not naturally meet. We incubated sperm with previously labelled PKH fibroblast cells. Observation by fluorescent microscopy after incubation revealed that sperm heads were also stained with PKH, indicating that this phenomenon is not sperm–oocyte specific (Fig. 10b′).

Discussion

The essential role of the oocyte CD9 tetraspanin in the adhesion/fusion process has been demonstrated for long. However, the eventual role played by the CD9 tetraspanin after its transfer from oocyte to sperm on the gamete fusion is still controversial (Miyado et al. 2008, Gupta et al. 2009). In order to go further in investigating the role of CD9 tetraspanin in gamete fusion, we studied the transfer ways of the CD9 tetraspanin from the oocyte to the spermatozoa.

In this study, we showed that both phenomena, namely the trogocytosis and the exosome, coexist. It should be noted that when we quantified the trogocytosis exchange, which requires a contact between sperm and oocytes, a possible exchange via exosomes could also have taken place at the same time. The trogocytosis quantification can therefore include the two phenomena. As the percentage of stained spermatozoa was always more important in the trogocytosis protocol than in the exosome one, we suggest that the two ways coexist. When we analysed sperm after exosome protocol, gametes having never been in contact with each other and the trogocytosis phenomenon being absolutely excluded from this analysis, the rate of exchange was weak, confirming again that more efficient exchanges exist because of the contact between both gametes. Moreover, the fact that exosome transfer was very weak 5 min after ZP removal clearly confirmed that a delay is necessary for vesicle synthesis and/or excretion as described previously (Miyado et al. 2008). The existence of two mechanisms of exchange reinforces its potential involvement in the fertilisation process.

We demonstrated that membrane exchanges preferentially occur with acrosome-reacted spermatozoa. These data indicate that the acrosome-reacted status, that of fertilising sperm in the PVS, promotes oocyte membrane capture. We propose two nonexclusive hypotheses for this result: i) inner acrosomal membrane, exposed after the acrosome reaction, is more favourable to the oocyte membrane fragment capture due to its physicochemical properties and ii) capture of membrane fragments could involve a ‘ligand–receptor’ system, which is usefully unmasked by the acrosome reaction. This second hypothesis is supported by the sperm dynamic expression of some proteins. Indeed, some sperm proteins, like α6β1, αvβ3 integrins and Izumo 1, have been shown to appear gradually with capacitation and acrosome reaction (Inoue et al. 2005, Barraud-Lange et al. 2007b, Boissonnas et al. 2010). The exchanges between the oocyte and sperm membranes are more important when the egg is not fertilised (Barraud-Lange et al. 2007a) and the sperm acrosome reacted (this study), i.e. under the conditions that just precede fertilisation when the sperm is into the PVS. Thus, trogocytosis is favoured by the conditions just preceding natural fertilisation suggesting this process being an important step in fertilisation.

Using EM, we confirmed the presence of exosomes in the oocyte PVS and we demonstrated their CD9 tetraspanin expression by WB analysis of the exosome-enriched medium. However, the goal of the exchange between oocytes and sperm is not to supply spermatozoa in CD9 tetraspanin. In fact, as described previously (Kimura et al. 2009, Ito et al. 2010) and confirmed here, the CD9 tetraspanin is also expressed on mouse sperm at a high level and is localised on the equatorial segment after acrosome reaction. This result shows that CD9 is mostly detected at the surface of the fertilising sperm at the time of membrane interaction and fusion. Therefore, if CD9 passes from the oocyte to the sperm, it cannot constitute the key element of this transfer unless if it carries some essential partners. Moreover, spermatozoa are able to capture the membrane material by trogocytosis and exosomes from Cd9-deleted and WT oocytes. Membrane material exchange between both gametes is thus totally independent of the presence of oocyte CD9 tetraspanin. Furthermore, EM analysis of WT and Cd9−/− oocytes showed the presence of vesicular material in their PVS confirming that the synthesis and secretion of vesicles are CD9 independent.

In the immune system, trogocytosis modulates the immune response to the antigen but also enables the transfer of new molecules that the lymphocyte does not express (Joly & Hudrisier 2003, Hudrisier et al. 2005). Similarly, Miyado et al. attributed to sperm covered with CD9-containing vesicles the capacity to rescue the fertilising ability of Cd9-deleted oocytes. However, this result was challenged by other authors who were unable to replicate this result (Gupta et al. 2009). Similarly, we showed that none of these two ways of membrane transfer rescue the fusing ability of Cd9-null oocytes. Indeed, incubating Cd9-null oocytes in vesicle-enriched medium or co-incubating them with WT oocytes did not allow the fusion of oocyte membrane-enriched sperm with Cd9-deleted oocytes. As also reported by Gupta et al. (2009), experiments were repeated several times without allowing us to reproduce the fusion rescue of Cd9−/− eggs.

Moreover, we observed that gamete membrane exchange is not sperm–oocyte specific, as sperm are able to achieve it with other cell types along its travel from the testis to the oocyte. Indeed, sperm acquire their fertilising ability during epididymal transit. Particularly, they acquire proteins from membranous vesicles called epididymosomes released in the epididymal intraluminal compartment (Yanagimachi et al. 1985, Fornes et al. 1995, Frenette & Sullivan 2001). Epididymosomes could be considered as ‘exosome-like’ vesicles insuring a way of communication between the epididymis epithelium and the maturing sperm. Sperm are then in contact with seminal secretions and encounter prostasomes produced by the prostate (Ronquist et al. 1978, Breitbart & Rubinstein 1982). Interactions with prostasomes increase sperm motility (Stegmayr & Ronquist 1982, Carlsson et al. 1997), have a role in the sperm capacitation process (Cross & Mahasreshti 1997) and in the stabilisation of their plasma membrane (Saez et al. 2003). Once in the female genital tract, spermatozoa transiently bind to the epithelial cells of the caudal isthmus leading to the concept of ‘sperm reservoir’ (Suarez 1987, Baillie et al. 1997). These interactions preserve sperm fertility particularly by preventing precocious capacitation. Indeed, viability of sperm can be extended by incubating them with vesicles prepared from the apical membranes of the endosalpinx (Dobrinski et al. 1997, Murray & Smith 1997, Gwathmey et al. 2006). Similarly, human and equine sperm incubated with endosalpingeal membrane vesicles capacitate more slowly than sperm incubated in capacitating medium alone (Dobrinski et al. 1997, Murray & Smith 1997).

In this context, it is important to note that other data indicate that sperm are able to not only bind to (Scofield et al. 1992, Almeida et al. 1995) but also fuse with somatic cells including granulosa cells (Mattioli et al. 2009). Mouse sperm may also penetrate into fibroblast cells (Bendich et al. 1974) or lymphocytes (Ashida & Scofield 1987). It is then obvious that sperm can make membrane fragment exchanges with many different cell types even with cells they would never encounter in natural conditions. However, as the sperm are the only cells that are able to cross the ZP to reach the PVS, exchange between oocyte and the fertilising spermatozoon is a very specific event. Moreover, whenever sperm interact with vesicles from epididymis, prostate and oviduct epithelium, they acquire functions such as motility, recognition of the ZP and delay of capacitation. One can thus hypothesise that interaction with the oocyte membrane fragments also provides new features, such as activation of molecular complexes involved in binding to and/or fusion with the oocyte membrane, for sperm.

Conclusion

The sperm maturation is modulated by numerous exchanges in the epididymis, in the seminal fluid and in the female genital tract. We here report exchanges between sperm, cumulus cells and oocyte itself. We observed that membrane fragment exchanges between gametes are insured by two CD9-independent ways indicating that it is probably an important step during the fertilisation process. The fact that this phenomenon happens just before fertilisation suggests a substantial role for intercellular communications, taking place during these exchanges. Finally, we showed that these exchanges did not allow the rescue of the fertility of Cd9-deleted oocytes. Furthermore, the mouse spermatozoon expresses itself CD9 tetraspanin that is particularly unmasked after acrosome reaction. To clarify the importance of this transfer, inhibition experiments of these membrane exchange phenomena have to be conducted. The aim would be to determine whether they have an essential or only a facilitating role during fertilisation by activating the adhesion molecules present on sperm membrane. Recently, it has been shown that knockdown of Rab27A and Rab27B prevented the exosome-release process in somatic cells (Ostrowski et al. 2010). It is therefore towards the extinction of genes involved in the oocyte synthesis or secretion of exosomes that we need to pursue. Applied to the oocyte during in vitro maturation, this approach would allow blocking the synthesis or secretion of exosomes in the PVS and evaluate their importance in the gamete fusion process.

Materials and Methods

Ethics statement

Animals were maintained in an animal facility at room temperature (RT; 21–23 °C) and 14 h light:10 h darkness photoperiods with free access to water and food. Procedures for handling and experimentation followed ethical guidelines established by the Federation of European Laboratory Animal Science Associations. The experiments were approved by the departmental veterinary services of Paris (approval number: A75 14-02).

Gamete preparation

Preparation of oocytes

Cd9−/− female mice were generously given by C Boucheix (Le Naour et al. 2000). Cd9−/− and C57BL/6 WT female mice (6- to 10-week old) were superovulated with 5 UI pregnant mares serum gonadotrophin and 5 UI hCG (Intervet, Beaucouze, France) 48 h apart. Fifteen to 16 h after hCG injection, animals were killed by cervical dislocation. Cumulus–oocyte complexes were collected by tearing the oviductal ampulla and then disseminated in a drop of Ferticult IVF medium (FertiPro, Beernem, Belgium) supplemented with 3% BSA (Sigma–Aldrich) or in TYH medium (Kamya Biomedical Company, Seattle, WA, USA; Miyado et al. 2008). Cumulus cells were removed by a brief exposure to hyaluronidase (0.01%; Sigma–Aldrich). The ZP was removed from eggs with acidic Tyrode's solution (pH 2.5; Sigma–Aldrich) under visual monitoring.

Sperm preparation

Mouse spermatozoa were obtained from the cauda epididymis of B6CBA F1 mice (8- to 13-week old) and B6C3 F1 mice (8- to 13-week old) (Miyado et al. 2008). Sperm were capacitated at 37 °C for 90 min in a 500 μl drop of Ferticult medium supplemented with 3% BSA or in TYH under mineral oil.

Immunolabelling and fluorescence microscopy

Oocyte immunolabelling

When Cd9−/− and WT oocytes were co-incubated, a CD9 labelling was performed to differentiate between deleted and WT oocytes. Sperm were washed away from oocytes and the oocytes fixed in PBS-paraformaldehyde (PFA) 4% for 1 h at 4 °C. After washing, oocytes were incubated in 1% PBS–BSA with rat anti-CD9 antibody (KMC8, 20 μg/ml; BD Pharmingen, San Diego, CA, USA) for 1 h and then with 10 μg/ml goat anti-rat IgG Alexa Fluor 488 (Molecular Probes, Invitrogen, Carlsbad, CA, USA) for 1 h. Oocytes were washed and mounted in Vectashield/4′,6′-diamidine-2-phenylidole-dihydrochloride (DAPI; Vector Laboratories, Burlingame, USA) and covered with a coverslip for analysis.

Sperm immunolabelling

Freshly recovered or capacitated spermatozoa were washed in PBS–BSA 1%, centrifuged at 600 g for 5 min and fixed in PBS–PFA 4% for 15 min at RT. Some of the capacitated spermatozoa were incubated with calcium ionophore A23187 (15 μM; Sigma–Aldrich) for 15 min at 37 °C, then washed and fixed in the same way. After washing, the fixed spermatozoa were incubated in PBS–BSA 1% with 20 μg/ml anti-CD9 antibody for 1 h and then with 10 μg/ml goat anti-rat Alexa Fluor 488 for 1 h at RT. Control immunofluorescence study was performed with secondary antibody alone and with Cd9−/− sperm. For the detection of CD9 tetraspanin distribution according to the acrosomal status, labelled sperm were then submitted to PSA staining protocol. They were stained with rhodamine-conjugated lectine PSA (25 μg/ml; Vector Laboratories) for 10 min and washed in PBS. A drop of sperm suspension was then mounted with Vectashield/DAPI and covered with a coverslip for analysis. In another experiment series, the Izumo 1 protein present on equatorial segment of acrosome-reacted sperm was used as a marker of acrosome reaction. Free-swimming sperm recovered after insemination with PKH26-stained oocytes (trogocytosis protocol) were stained with a polyclonal anti-Izumo 1 antibody (10 μg/ml) and revealed with a FITC anti-rabbit antibody (anti-Izumo 1 antibody was generously given by M Okabe, Osaka University, Osaka, Japan). Detection was performed using a Nikon Eclipse E600 epifluorescence microscope and images were digitally acquired with a camera (Nikon DXM 1200). Statistical analysis was performed using χ2 test. Differences were considered significant at P<0.01.

Cell membrane labelling with the PKH fluorescent cell linker

ZP-free oocytes were stained with PKH26 (red) or 67 (green) fluorescent cell linker for general cell membrane labelling (Sigma–Aldrich) by a short incubation in a drop of 5 μM labelling solution at 37 °C and washed in several drops of culture medium (Ferticult, BSA 3%). Cumulus cells were recovered after treatment of cumulus–oocyte complexes with hyaluronidase solution and washed twice in Ferticult BSA 3%. NIH3T3 fibroblastic cells were harvested after Accutase treatment (Thermo Scientific, Villebon sur Yvette, France) and washed twice in Ferticult 3% BSA. Cumulus or fibroblastic cells were incubated for 5 min at RT in a 5 μM PKH labelling solution and washed three times in Ferticult 3% BSA. Cell pellets were next re-suspended in 100 μl Ferticult 3% BSA and deposited in a drop under mineral oil for sperm insemination.

Preparation of vesicle-enriched medium

CD9-containing vesicle medium

The medium containing vesicles was collected from ZP-free WT eggs cultured in a 60 μl drop of medium. After 2 h recovery, WT denuded oocytes were retrieved from the medium, permitting to obtain an enriched vesicles medium. Oocytes were counted before and after the recovery time. When necessary, oocyte membrane was labelled with PKH (5 μM final concentration; Sigma–Aldrich) or anti-CD9 (KMC8) just after ZP removal as described earlier.

Incubation sperm procedure in trogocytosis and exosome models

Oocytes were inseminated in a 60 μl new drop of medium with 1.5×105 sperm/ml for 1 h (trogocytosis model). In parallel, the same sperm concentration was inseminated in the vesicle-enriched medium for 1 h (exosome model) (Fig. 1). Cumulus cells or fibroblastic cells (1×105) were incubated with 1.5×105 sperm/ml for 1 h. Spermatozoa from those mediums were analysed for green or red fluorescence by microscopy and/or FCM.

WB analysis

Immediately after ZP removal, ZP-free eggs were rapidly washed five times and kept in Ferticult medium drops at 37 °C under 5% CO2 in air for 2 h to recover their fertilisability. Oocytes were retrieved for later studies and remnant Ferticult medium was collected and ultracentrifuged for 1 h at 100 000 g. Pellet was washed in PBS supplemented with Protease Inhibitor Cocktail (Sigma) and then solubilised in sample buffer (NuPAGE LDS sample buffer, Invitrogen).

Capacitated sperm, isolated cumulus cells or oocytes were washed twice in PBS supplemented with Protease Inhibitor Cocktail and the pellet was stored at −80 °C for further use. The cumulus cells or oocytes were lysed in sample buffer (NuPAGE LDS sample buffer).

Sperm aliquots were lysed in 50 mM Tris (pH 8), 150 mM NaCl, 1 mM EDTA, 0.25% sodium deoxycholate and 1% NP40 for 1 h on ice and then centrifuged at 15 000 g for 15 min at 4 °C. An equal volume of 2× NuPAGE LDS sample buffer was then added to the supernatant.

Protein samples analysed by WB corresponding to 1×106 spermatozoa, 1×105 granulosa cells or the medium of 50 oocyte remnants were loaded per well in Novex Tris–glycine precast gel (Invitrogen) under non-reducing conditions and electrotransferred to Immobilon-P membranes (GE Healthcare, Aulnay sous bois, France). After 1 h saturation in WBR reagent (Roche), membranes were incubated for 2 h at RT with anti-CD9 mAb (KMC8) at 1:250 dilution in Tris buffer saline (TBS) supplemented with 0.1% Tween 20 and 0.5% WBR reagent. After washing, the membrane was incubated with rabbit anti-rat HRP-conjugated antibody (0.2 μg/ml; Stressgen, Brussels, Belgium) for 1 h at RT. HRP activity was revealed by ECL detection kit (GE Healthcare).

Detection of sperm interacting with membrane material

Flow cytometric analysis of sperm after incubation according to trogocytosis and exosome protocols

Sperm capture of vesicles and membrane fragments labelled with PKH67 was evaluated by FCM using a Cytomics TM FC500 flow cytometer (Beckman Coulter, Villepinte, France) equipped with argon laser (wavelength: 488 nm). Flow cytometer was driven with the CXP cytometer software (Beckman Coulter). For each sample, 5000 cells were analysed at a flow rate of 200–300 cells per second using FSC/FL1 (cell size/relative fluorescence) parameters. Autofluorescence was assessed with the same concentration of sperm incubated in a drop of vesicle-free medium.

Microscopic analysis of sperm after trogocytosis and exosome protocols with eggs

Sperm incubated for 1 h, either in the vesicle-enriched medium or with PHK67-labelled oocytes, were washed with acidic Tyrode's solution to remove any possible bound cellular debris and fixed in PBS–BSA 1%, mounted in Vectashield/DAPI medium and analysed for fluorescence staining.

Microscopic analysis of sperm after trogocytosis and exosome procedures with somatic cells

Sperm incubated for 1 h with PHK26-labelled cells were washed with acidic Tyrode's solution to remove any possible bound cellular debris, fixed in PBS–PFA 1%, mounted between slide and cover slide in Vectashield/DAPI medium and finally analysed for fluorescence. Images were captured using an Eclipse E600 epifluorescence microscope and were digitally acquired using a camera (Nikon DXM 1200). Image analysis was performed using ImageJ free software.

IVF

ZP-free oocytes (Cd9−/− or WT) were inseminated for 3 h with capacitated sperm in a 100 μl drop of medium. They were then washed and directly mounted in Vectashield/DAPI for observation under u.v. light. We considered as fertilised the oocytes showing fluorescent decondensed sperm heads within their cytoplasm. To assess the fertilisation index (FI), the number of decondensed sperm heads was recorded.

Transmission EM

Oocyte preparation

For transmission EM, oocytes were collected from WT or Cd9−/− mice. Their cumulus cells were removed by a brief exposure to hyaluronidase and they were washed and fixed in a 100 μl drop of 0.25% glutaraldehyde in PBS 1% BSA for 30 min and then washed in PBS 1% BSA. For pre-embedding CD9 labelling, WT and Cd9−/− oocytes were incubated with 10 μg/ml rat anti-CD9 antibody for 1 h at RT, three times washed in PBS 1% BSA and then incubated with 10 nm gold bead-conjugated goat anti-rat IgG at 1:50 dilution (BBInternational, Cardiff, UK). After three washes, the oocytes were fixed in 2.5% glutaraldehyde in Sorensen buffer supplemented with 1% BSA for 30 min at RT and 1 h at 4 °C. After three washes in Sorensen buffer with 1% BSA, the oocytes were post-fixed with 1% osmium tetroxide in 0.1 M phosphate buffer and then dehydrated in 70, 90 and 100% ethanol. After 10 min in a 1:2 mixture of epoxy propane and epoxy resin, the oocytes were embedded in gelatin capsules with freshly prepared epoxy resin and polymerised at 60 °C for 24 h. Samples were then mounted into epon blocks and 70 nm thin sections were cut with an ultramicrotome (Reichert ultracut S), stained with uranyl acetate and Reynold's lead citrate, and observed under a transmission electron microscope (Philips CM10).

Exosome preparation

The acidic Tyrode's medium in which oocytes were incubated to remove the ZP (about 100 μl medium for 100 oocytes) was recovered and rapidly neutralised with Tris 1 M, pH 8.8, and next treated according to the modified protocol of Thery et al. (2001) to obtain exosome fractions. Briefly, neutralised medium was subjected to two successive centrifugations at 1500 g for 10 min and 15 000 g for 30 min to remove cellular debris. Then the supernatant was collected and centrifuged at 100 000 g for 1 h to pellet exosomes. Pellet in 10 μl PBS was deposited on formwar/carbon-coated EM grids and allowed to settle for 10–15 min. Then grids were negatively stained with 4% uranyl acetate before to be observed under a transmission electron microscope (Philips CM10).

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This work was supported by the ANR-09-PIRI-0011 grant.

Acknowledgements

The authors thank C Boucheix (Inserm, Villejuif) for providing Cd9 knockout mice. They also thank C Lesaffre for technical assistance, P. Bourdoncle for confocal microscopy analysis assistance (Plate-Forme Imagerie, Institut Cochin) and B Chanaud and L Stouvenel for flow cytometry analysis assistance (Plate-Forme de Cytométrie en flux, Institut Cochin).

References

  • Almeida EA, Huovila AP, Sutherland AE, Stephens LE, Calarco PG, Shaw LM, Mercurio AM, Sonnenberg A, Primakoff P & Myles DG et al. 1995 Mouse egg integrin α 6 β 1 functions as a sperm receptor. Cell 81 10951104. doi:10.1016/S0092-8674(05)80014-5.

    • Search Google Scholar
    • Export Citation
  • Ashida ER & Scofield VL 1987 Lymphocyte major histocompatibility complex-encoded class II structures may act as sperm receptors. PNAS 84 33953399. doi:10.1073/pnas.84.10.3395.

    • Search Google Scholar
    • Export Citation
  • Baillie HS, Pacey AA, Warren MA, Scudamore IW & Barratt CL 1997 Greater numbers of human spermatozoa associate with endosalpingeal cells derived from the isthmus compared with those from the ampulla. Human Reproduction 12 19851992. doi:10.1093/humrep/12.9.1985.

    • Search Google Scholar
    • Export Citation
  • Barraud-Lange V, Naud-Barriant N, Bomsel M, Wolf JP & Ziyyat A 2007a Transfer of oocyte membrane fragments to fertilizing spermatozoa. FASEB Journal 21 34463449. doi:10.1096/fj.06-8035hyp.

    • Search Google Scholar
    • Export Citation
  • Barraud-Lange V, Naud-Barriant N, Saffar L, Gattegno L, Ducot B, Drillet AS, Bomsel M, Wolf JP & Ziyyat A 2007b α6β1 integrin expressed by sperm is determinant in mouse fertilization. BMC Developmental Biology 7 102 doi:10.1186/1471-213X-7-102.

    • Search Google Scholar
    • Export Citation
  • Bendich A, Borenfreund E & Sternberg SS 1974 Penetration of somatic mammalian cells by sperm. Science 183 857859. doi:10.1126/science.183.4127.857.

    • Search Google Scholar
    • Export Citation
  • Boissonnas CC, Montjean D, Lesaffre C, Auer J, Vaiman D, Wolf JP & Ziyyat A 2010 Role of sperm αvβ3 integrin in mouse fertilization. Developmental Dynamics 239 773783. doi:10.1002/dvdy.22206.

    • Search Google Scholar
    • Export Citation
  • Breitbart H & Rubinstein S 1982 Characterization of Mg2+- and Ca2+-ATPase activity in membrane vesicles from ejaculated ram seminal plasma. Archives of Andrology 9 147157. doi:10.3109/01485018208990233.

    • Search Google Scholar
    • Export Citation
  • Caballero J, Frenette G & Sullivan R 2011 Post testicular sperm maturational changes in the bull: important role of the epididymosomes and prostasomes. Veterinary Medicine International 2011 757194 doi:10.4061/2011/757194.

    • Search Google Scholar
    • Export Citation
  • Carlsson L, Ronquist G, Stridsberg M & Johansson L 1997 Motility stimulant effects of prostasome inclusion in swim-up medium on cryopreserved human spermatozoa. Archives of Andrology 38 215221. doi:10.3109/01485019708994880.

    • Search Google Scholar
    • Export Citation
  • Chen MS, Tung KS, Coonrod SA, Takahashi Y, Bigler D, Chang A, Yamashita Y, Kincade PW, Herr JC & White JM 1999 Role of the integrin-associated protein CD9 in binding between sperm ADAM 2 and the egg integrin α6β1: implications for murine fertilization. PNAS 96 1183011835. doi:10.1073/pnas.96.21.11830.

    • Search Google Scholar
    • Export Citation
  • Couzin J 2005 Cell biology: the ins and outs of exosomes. Science 308 18621863. doi:10.1126/science.308.5730.1862.

  • Cross NL & Mahasreshti P 1997 Prostasome fraction of human seminal plasma prevents sperm from becoming acrosomally responsive to the agonist progesterone. Archives of Andrology 39 3944. doi:10.3109/01485019708987900.

    • Search Google Scholar
    • Export Citation
  • Dobrinski I, Smith TT, Suarez SS & Ball BA 1997 Membrane contact with oviductal epithelium modulates the intracellular calcium concentration of equine spermatozoa in vitro. Biology of Reproduction 56 861869. doi:10.1095/biolreprod56.4.861.

    • Search Google Scholar
    • Export Citation
  • Fornes WM, Sosa MA, Bertini F & Burgos MH 1995 Vesicles in rat epididymal fluid. Existence of two populations differing in ultrastructure and enzymatic composition. Andrologia 27 233237. doi:10.1111/j.1439-0272.1995.tb01099.x.

    • Search Google Scholar
    • Export Citation
  • Frenette G & Sullivan R 2001 Prostasome-like particles are involved in the transfer of P25b from the bovine epididymal fluid to the sperm surface. Molecular Reproduction and Development 59 115121. doi:10.1002/mrd.1013.

    • Search Google Scholar
    • Export Citation
  • Gatti JL, Metayer S, Belghazi M, Dacheux F & Dacheux JL 2005 Identification, proteomic profiling, and origin of ram epididymal fluid exosome-like vesicles. Biology of Reproduction 72 14521465. doi:10.1095/biolreprod.104.036426.

    • Search Google Scholar
    • Export Citation
  • Gupta S, Primakoff P & Myles DG 2009 Can the presence of wild-type oocytes during insemination rescue the fusion defect of CD9 null oocytes? Molecular Reproduction and Development 76 602 doi:10.1002/mrd.21040.

    • Search Google Scholar
    • Export Citation
  • Gwathmey TM, Ignotz GG, Mueller JL, Manjunath P & Suarez SS 2006 Bovine seminal plasma proteins PDC-109, BSP-A3, and BSP-30-kDa share functional roles in storing sperm in the oviduct. Biology of Reproduction 75 501507. doi:10.1095/biolreprod.106.053306.

    • Search Google Scholar
    • Export Citation
  • Hudrisier D, Riond J, Mazarguil H, Gairin JE & Joly E 2001 Cutting edge: CTLs rapidly capture membrane fragments from target cells in a TCR signaling-dependent manner. Journal of Immunology 166 36453649.

    • Search Google Scholar
    • Export Citation
  • Hudrisier D, Riond J, Garidou L, Duthoit C & Joly E 2005 T cell activation correlates with an increasedproportion of antigen among the materials acquiredfrom target cells. European Journal of Immunology 35 22842294. doi:10.1002/eji.200526266.

    • Search Google Scholar
    • Export Citation
  • Inoue N, Ikawa M, Isotani A & Okabe M 2005 The immunoglobulin superfamily protein Izumo is required for sperm to fuse with eggs. Nature 434 234238. doi:10.1038/nature03362.

    • Search Google Scholar
    • Export Citation
  • Ito C, Yamatoya K, Yoshida K, Maekawa M, Miyado K & Toshimori K 2010 Tetraspanin family protein CD9 in the mouse sperm: unique localization, appearance, behavior and fate during fertilization. Cell and Tissue Research 340 583594. doi:10.1007/s00441-010-0967-7.

    • Search Google Scholar
    • Export Citation
  • Jin M, Fujiwara E, Kakiuchi Y, Okabe M, Satouh Y, Baba SA, Chiba K & Hirohashi N 2011 Most fertilizing mouse spermatozoa begin their acrosome reaction before contact with the zona pellucida during in vitro fertilization. PNAS 108 48924896. doi:10.1073/pnas.1018202108.

    • Search Google Scholar
    • Export Citation
  • Joly E & Hudrisier D 2003 What is trogocytosis and what is its purpose? Nature Immunology 4 815 doi:10.1038/ni0903-815.

  • Kaji K, Oda S, Shikano T, Ohnuki T, Uematsu Y, Sakagami J, Tada N, Miyazaki S & Kudo A 2000 The gamete fusion process is defective in eggs of Cd9-deficient mice. Nature Genetics 24 279282. doi:10.1038/73502.

    • Search Google Scholar
    • Export Citation
  • Kimura M, Kim E, Kang W, Yamashita M, Saigo M, Yamazaki T, Nakanishi T, Kashiwabara S & Baba T 2009 Functional roles of mouse sperm hyaluronidases, HYAL5 and SPAM1, in fertilization. Biology of Reproduction 81 939947. doi:10.1095/biolreprod.109.078816.

    • Search Google Scholar
    • Export Citation
  • Le Naour F, Rubinstein E, Jasmin C, Prenant M & Boucheix C 2000 Severely reduced female fertility in CD9-deficient mice. Science 287 319321. doi:10.1126/science.287.5451.319.

    • Search Google Scholar
    • Export Citation
  • Li YH, Hou Y, Ma W, Yuan JX, Zhang D, Sun QY & Wang WH 2004 Localization of CD9 in pig oocytes and its effects on sperm–egg interaction. Reproduction 127 151157. doi:10.1530/rep.1.00006.

    • Search Google Scholar
    • Export Citation
  • Mattioli M, Gloria A, Mauro A, Gioia L & Barboni B 2009 Fusion as the result of sperm–somatic cell interaction. Reproduction 138 679687. doi:10.1530/REP-08-0316.

    • Search Google Scholar
    • Export Citation
  • Miyado K, Yamada G, Yamada S, Hasuwa H, Nakamura Y, Ryu F, Suzuki K, Kosai K, Inoue K & Ogura A et al. 2000 Requirement of CD9 on the egg plasma membrane for fertilization. Science 287 321324. doi:10.1126/science.287.5451.321.

    • Search Google Scholar
    • Export Citation
  • Miyado K, Yoshida K, Yamagata K, Sakakibara K, Okabe M, Wang X, Miyamoto K, Akutsu H, Kondo T & Takahashi Y et al. 2008 The fusing ability of sperm is bestowed by CD9-containing vesicles released from eggs in mice. PNAS 105 1292112926. doi:10.1073/pnas.0710608105.

    • Search Google Scholar
    • Export Citation
  • Murray SC & Smith TT 1997 Sperm interaction with fallopian tube apical membrane enhances sperm motility and delays capacitation. Fertility and Sterility 68 351357. doi:10.1016/S0015-0282(97)81528-2.

    • Search Google Scholar
    • Export Citation
  • Okabe M, Adachi T, Takada K, Oda H, Yagasaki M, Kohama Y & Mimura T 1987 Capacitation-related changes in antigen distribution on mouse sperm heads and its relation to fertilization rate in vitro. Journal of Reproductive Immunology 11 91100. doi:10.1016/0165-0378(87)90014-3.

    • Search Google Scholar
    • Export Citation
  • Ostrowski M, Carmo NB, Krumeich S, Fanget I, Raposo G, Savina A, Moita CF, Schauer K, Hume AN & Freitas RP et al. 2010 Rab27a and Rab27b control different steps of the exosome secretion pathway. Nature Cell Biology 12 1930.(sup pp 11–13) doi:10.1038/ncb2000.

    • Search Google Scholar
    • Export Citation
  • Ronquist G, Brody I, Gottfries A & Stegmayr B 1978 An Mg2+ and Ca2+-stimulated adenosine triphosphatase in human prostatic fluid – part II. Andrologia 10 427433. doi:10.1111/j.1439-0272.1978.tb03064.x.

    • Search Google Scholar
    • Export Citation
  • Rubinstein E, Ziyyat A, Prenant M, Wrobel E, Wolf JP, Levy S, Le Naour F & Boucheix C 2006 Reduced fertility of female mice lacking CD81. Developmental Biology 290 351358. doi:10.1016/j.ydbio.2005.11.031.

    • Search Google Scholar
    • Export Citation
  • Runge KE, Evans JE, He ZY, Gupta S, McDonald KL, Stahlberg H, Primakoff P & Myles DG 2007 Oocyte CD9 is enriched on the microvillar membrane and required for normal microvillar shape and distribution. Developmental Biology 304 317325. doi:10.1016/j.ydbio.2006.12.041.

    • Search Google Scholar
    • Export Citation
  • Saez F, Frenette G & Sullivan R 2003 Epididymosomes and prostasomes: their roles in posttesticular maturation of the sperm cells. Journal of Andrology 24 149154.

    • Search Google Scholar
    • Export Citation
  • Scofield VL, Clisham R, Bandyopadhyay L, Gladstone P, Zamboni L & Raghupathy R 1992 Binding of sperm to somatic cells via HLA-DR. Modulation by sulfated carbohydrates. Journal of Immunology 148 17181724.

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  • Stegmayr B & Ronquist G 1982 Promotive effect on human sperm progressive motility by prostasomes. Urological Research 10 253257. doi:10.1007/BF00255932.

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    • Export Citation
  • Suarez SS 1987 Sperm transport and motility in the mouse oviduct: observations in situ. Biology of Reproduction 36 203210. doi:10.1095/biolreprod36.1.203.

    • Search Google Scholar
    • Export Citation
  • Tanghe S, Van Soom A, Nauwynck H, Coryn M & de Kruif A 2002 Minireview: functions of the cumulus oophorus during oocyte maturation, ovulation, and fertilization. Molecular Reproduction and Development 61 414424. doi:10.1002/mrd.10102.

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  • Thery C, Boussac M, Veron P, Ricciardi-Castagnoli P, Raposo G, Garin J & Amigorena S 2001 Proteomic analysis of dendritic cell-derived exosomes: a secreted subcellular compartment distinct from apoptotic vesicles. Journal of Immunology 166 73097318.

    • Search Google Scholar
    • Export Citation
  • Thery C, Zitvogel L & Amigorena S 2002 Exosomes: composition, biogenesis and function. Nature Reviews. Immunology 2 569579.

  • Yanagimachi R, Kamiguchi Y, Mikamo K, Suzuki F & Yanagimachi H 1985 Maturation of spermatozoa in the epididymis of the Chinese hamster. American Journal of Anatomy 172 317330. doi:10.1002/aja.1001720406.

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V Barraud-Lange and C Chalas Boissonnas contributed equally to this work

 

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    Experimental design of trogocytosis and exosome models with WT oocytes. ZPs of WT oocytes were first removed by acidic Tyrode's solution. ZP-free eggs were then labelled with the PKH67 (Green Fluorescent Cell Linker for General Cell Membrane Labelling). After 2 h of recovery time, oocytes were retrieved from the drop of medium, so-called ‘vesicles-enriched medium’. Those oocytes were then inseminated in a 60 μl new drop of medium with 1.5×105 sperm/ml for 1 h (trogocytosis set-up). In parallel, the same sperm concentration was incubated in the vesicle-enriched medium for 1 h (exosome set-up). Spermatozoa from both media were analysed for green fluorescence by confocal microscopy and by flow cytometry analysis. CD9 tetraspanin has also been checked by western blot analysis in the vesicle-enriched medium.

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    Identification of membrane fragment transfer from PKH67-labelled oocytes to spermatozoa by both trogocytosis and exosome. Confocal microscopic analysis of spermatozoa recovered 1 h after insemination with PKH67-labelled oocytes (a, b and c) or after incubation in vesicle-enriched medium provided by PKH67-labelled oocytes (d, e and f). In both cases, oocyte membrane materials were detected on sperm head, which presents PKH67 dots associated with a slightly diffuse staining (b and e). Merged images of blue (DNA DAPI staining (a and d) and green signals are represented in c and f). Scale bar=2 μm. Flow cytometric analysis of sperm membrane capture in the trogocytosis (g) and exosome (h) set-up. Dot plots are representative of an independent experiment and show 6.4% (g, square C4) and 2.2% (h, square C4) of stained sperm for trogocytosis and exosome membrane transfer models respectively. Sperm autofluorescence was used as a negative control (i).

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    Acrosomal status of sperm bound to PKH-stained egg membrane fragments. Free-swimming sperm recovered after insemination with PKH26-stained oocytes (trogocytosis set-up) were stained with a polyclonal anti-Izumo 1 antibody (10 μg/ml) and revealed with a FITC anti-rabbit antibody to assess the sperm acrosomal status. Epifluorescence and flow cytometric analysis were performed. (A) Epifluorescence images of a PKH26-positive sperm, which present an Izumo 1 staining specific of an acrosome-reacted status. (B) Column graph presenting the percentage of acrosome intact (AR−) and acrosome-reacted (AR+) sperm within the sperm populations that have bound (membrane fragment +) or not (membrane fragment −) PKH26-egg membrane fragments. The measures performed by flow cytometric analysis revealed that on average 56.7±7.6% of PKH-positive sperm realised their acrosome reaction vs 18.4±8.5% in the population of PKH-free sperm (P<0.001). Number of experiments=4.

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    Presence of CD9 in membrane material released by oocyte. (A) The electron microscopy analysis of the pellet obtained after centrifugation at 100 000 g of the PVS content collected during ZP removal revealed that it contained vesicles ranging in size from about 50 to 150 nm in diameter. (B) WB analysis of the vesicle-enriched medium (provided by 50 WT oocytes) was performed with anti-CD9 antibody (WT Ex). Granulosa cells (1.5×105 per lane) were used as positive control. Vesicle-enriched medium provided by 50 Cd9−/− oocytes (Cd9−/− Ex) and Cd9−/− granulosa cells were used as negative controls. GC, granulosa cells; Ex, exosomes.

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    Expression and distribution of CD9 tetraspanin on mouse sperm. (A) WB analysis of cauda epididymal sperm from WT (lane 1) and Cd9−/− (lane 2) males. Oocytes from WT female (lane 3) were used as positive control. The molecular weight of CD9 in WT sperm (lane 1) as in WT oocytes (lane 3) was ∼24 kDa. CD9 was absent in the sperm from Cd9−/− males (lane 2). (B) CD9 immunodetection in mature (cauda epididymal) sperm. Acrosome intact (a, b and c), spontaneously acrosome-reacted (d, e and f) and ionophore-induced acrosome-reacted sperm (g, h and i) were observed. Sperm were exposed to rhodamine–Pisum sativum agglutinin (PSA) (b, e and h) after anti-CD9 tetraspanin antibody (KMC8) staining (c, f and i) revealed by goat anti-rat IgG Alexa Fluor 488 (green) secondary antibody. The following CD9 staining fluorescent patterns were observed: only 10% of acrosome intact sperm were stained (c), 60% of spontaneously acrosome-reacted sperm (f) and 75% of ionophore-induced acrosome-reacted sperm were stained (i). Sperm nuclei were detected with DAPI staining (a, d and g).

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    Electron microscopic analysis of WT and Cd9−/− oocytes. ZP intact WT (A, B, C and D) and Cd9−/− (A′, B′, C′ and D′) eggs were observed after staining using an anti-CD9 mAb revealed by 10 nm gold bead-conjugated anti-rat IgG. Cd9−/− oocytes were considered as negative controls (A′, B′, C′ and D′). Vesicles with various sizes (50–150 nm) and appearances were observed in the PVS of both WT (A, B, C and D) and Cd9-deleted oocytes (A′, B′, C′ and D′). Small vesicles displaying a cup-shaped morphology characteristic of exosomes were observed in the PVS (B and B′). Some vesicles in the PVS of WT eggs presented a CD9 labelling (A, B, C and D). The WT oocytes showed a normal structure of the microvilli (enlargement in D) while Cd9−/− oocytes were observed with altered microvillar morphology (enlargement in D′).

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    Acquisition of oocyte membrane fragments by sperm does not impair their ability to bind to other oocytes. Mixed PKH67-stained and non-stained oocytes were inseminated with sperm for 1 h. PKH67-stained spermatozoa were detected attached on both stained (a, b and c) and non-stained oocytes (d, e and f). Transmission images (a and d); superimposition of 15 consecutive PKH67 staining confocal sections at 0.4 μm intervals (b and e); superimposition of transmission and fluorescent signal (c and f). Scale bar=20 μm.

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    Experimental design of Cd9-null oocyte insemination with CD9 recovered sperm. (A) Fresh ZP-free WT and Cd9-null oocytes were mixed together in a 60 μl drop of medium for 2 h of recovering time. They were next inseminated with 1.5×105 sperm/ml for 1 h. After washing, all oocytes were incubated with anti-CD9 antibody (KMC8) for 1 h and then with Alexa Fluor 488 to distinguish WT from Cd9-null oocytes. Oocytes were then mounted in Vectashield/DAPI for observation. Oocytes were considered fertilised when fluorescent decondensed sperm head visible under u.v. light were present in their cytoplasm. To assess the fertilisation index, the number of decondensed sperm heads was recorded. Epifluorescence microscope analysis was conducted in parallel to check the presence of CD9 at the egg surface. (B) ZP-free WT oocytes were incubated for 2 h in a 60 μl drop of medium. WT oocytes were then retrieved from the medium. Zona-free Cd9-null oocytes were inseminated in this drop with 1.5×105 sperm/ml for 3 h. In parallel, ZP-free WT oocytes were inseminated with 1.5×105 sperm/ml for 3 h. Cd9-null and WT oocytes were mounted and observed as described previously.

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    The interaction of sperm covered by oocyte CD9-containing material with Cd9-deleted oocytes is not sufficient to rescue their fertilisation defects. (A) Co-inseminated WT and Cd9−/− oocytes were analysed under epifluorescence microscope after anti-CD9 antibody labelling. WT oocytes appeared intensely labelled while Cd9−/− oocytes were not. (B) Fertilisation status and index were assessed by the number of decondensed sperm heads in the cytoplasm of all oocytes examined under u.v. light. As presented, WT oocytes had at least one decondensed sperm head in their cytoplasm while Cd9−/− oocytes had none. (C) In another experiment, PKH67-stained WT oocytes (green) and PKH26-stained Cd9−/− oocytes (red) were co-inseminated. Some PKH67-covered sperm (green) were present at the surface of PKH26-Cd9-null egg. Picture represents the superimposition of DAPI and fluorescence detection after confocal microscopic analysis. (D) Enlargement of PKH67-stained sperm in contact with Cd9-null egg surface obtained by zooming on boxed area in image C.

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    Identification of membrane fragment transfer from PKH26-labelled somatic cells to spermatozoa by ‘trogocytosis-like’ pathway. Confocal microscopic analysis of spermatozoa recovered 1 h after incubation with PKH26-labelled granulosa cells (a, b and c) or with PKH26-labelled fibroblasts (a′, b′ and c′). In both cases, membrane materials were detected on sperm head, which presented PKH26 staining (b and b′). Dots (up arrow) and diffusion (down arrow) of PKH26 dye can coexist on some sperm head (b). Phase transmission (a and a′), fluorescent (b and b′), and merge file (c and c′) images.

  • Almeida EA, Huovila AP, Sutherland AE, Stephens LE, Calarco PG, Shaw LM, Mercurio AM, Sonnenberg A, Primakoff P & Myles DG et al. 1995 Mouse egg integrin α 6 β 1 functions as a sperm receptor. Cell 81 10951104. doi:10.1016/S0092-8674(05)80014-5.

    • Search Google Scholar
    • Export Citation
  • Ashida ER & Scofield VL 1987 Lymphocyte major histocompatibility complex-encoded class II structures may act as sperm receptors. PNAS 84 33953399. doi:10.1073/pnas.84.10.3395.

    • Search Google Scholar
    • Export Citation
  • Baillie HS, Pacey AA, Warren MA, Scudamore IW & Barratt CL 1997 Greater numbers of human spermatozoa associate with endosalpingeal cells derived from the isthmus compared with those from the ampulla. Human Reproduction 12 19851992. doi:10.1093/humrep/12.9.1985.

    • Search Google Scholar
    • Export Citation
  • Barraud-Lange V, Naud-Barriant N, Bomsel M, Wolf JP & Ziyyat A 2007a Transfer of oocyte membrane fragments to fertilizing spermatozoa. FASEB Journal 21 34463449. doi:10.1096/fj.06-8035hyp.

    • Search Google Scholar
    • Export Citation
  • Barraud-Lange V, Naud-Barriant N, Saffar L, Gattegno L, Ducot B, Drillet AS, Bomsel M, Wolf JP & Ziyyat A 2007b α6β1 integrin expressed by sperm is determinant in mouse fertilization. BMC Developmental Biology 7 102 doi:10.1186/1471-213X-7-102.

    • Search Google Scholar
    • Export Citation
  • Bendich A, Borenfreund E & Sternberg SS 1974 Penetration of somatic mammalian cells by sperm. Science 183 857859. doi:10.1126/science.183.4127.857.

    • Search Google Scholar
    • Export Citation
  • Boissonnas CC, Montjean D, Lesaffre C, Auer J, Vaiman D, Wolf JP & Ziyyat A 2010 Role of sperm αvβ3 integrin in mouse fertilization. Developmental Dynamics 239 773783. doi:10.1002/dvdy.22206.

    • Search Google Scholar
    • Export Citation
  • Breitbart H & Rubinstein S 1982 Characterization of Mg2+- and Ca2+-ATPase activity in membrane vesicles from ejaculated ram seminal plasma. Archives of Andrology 9 147157. doi:10.3109/01485018208990233.

    • Search Google Scholar
    • Export Citation
  • Caballero J, Frenette G & Sullivan R 2011 Post testicular sperm maturational changes in the bull: important role of the epididymosomes and prostasomes. Veterinary Medicine International 2011 757194 doi:10.4061/2011/757194.

    • Search Google Scholar
    • Export Citation
  • Carlsson L, Ronquist G, Stridsberg M & Johansson L 1997 Motility stimulant effects of prostasome inclusion in swim-up medium on cryopreserved human spermatozoa. Archives of Andrology 38 215221. doi:10.3109/01485019708994880.

    • Search Google Scholar
    • Export Citation
  • Chen MS, Tung KS, Coonrod SA, Takahashi Y, Bigler D, Chang A, Yamashita Y, Kincade PW, Herr JC & White JM 1999 Role of the integrin-associated protein CD9 in binding between sperm ADAM 2 and the egg integrin α6β1: implications for murine fertilization. PNAS 96 1183011835. doi:10.1073/pnas.96.21.11830.

    • Search Google Scholar
    • Export Citation
  • Couzin J 2005 Cell biology: the ins and outs of exosomes. Science 308 18621863. doi:10.1126/science.308.5730.1862.

  • Cross NL & Mahasreshti P 1997 Prostasome fraction of human seminal plasma prevents sperm from becoming acrosomally responsive to the agonist progesterone. Archives of Andrology 39 3944. doi:10.3109/01485019708987900.

    • Search Google Scholar
    • Export Citation
  • Dobrinski I, Smith TT, Suarez SS & Ball BA 1997 Membrane contact with oviductal epithelium modulates the intracellular calcium concentration of equine spermatozoa in vitro. Biology of Reproduction 56 861869. doi:10.1095/biolreprod56.4.861.

    • Search Google Scholar
    • Export Citation
  • Fornes WM, Sosa MA, Bertini F & Burgos MH 1995 Vesicles in rat epididymal fluid. Existence of two populations differing in ultrastructure and enzymatic composition. Andrologia 27 233237. doi:10.1111/j.1439-0272.1995.tb01099.x.

    • Search Google Scholar
    • Export Citation
  • Frenette G & Sullivan R 2001 Prostasome-like particles are involved in the transfer of P25b from the bovine epididymal fluid to the sperm surface. Molecular Reproduction and Development 59 115121. doi:10.1002/mrd.1013.

    • Search Google Scholar
    • Export Citation
  • Gatti JL, Metayer S, Belghazi M, Dacheux F & Dacheux JL 2005 Identification, proteomic profiling, and origin of ram epididymal fluid exosome-like vesicles. Biology of Reproduction 72 14521465. doi:10.1095/biolreprod.104.036426.

    • Search Google Scholar
    • Export Citation
  • Gupta S, Primakoff P & Myles DG 2009 Can the presence of wild-type oocytes during insemination rescue the fusion defect of CD9 null oocytes? Molecular Reproduction and Development 76 602 doi:10.1002/mrd.21040.

    • Search Google Scholar
    • Export Citation
  • Gwathmey TM, Ignotz GG, Mueller JL, Manjunath P & Suarez SS 2006 Bovine seminal plasma proteins PDC-109, BSP-A3, and BSP-30-kDa share functional roles in storing sperm in the oviduct. Biology of Reproduction 75 501507. doi:10.1095/biolreprod.106.053306.

    • Search Google Scholar
    • Export Citation
  • Hudrisier D, Riond J, Mazarguil H, Gairin JE & Joly E 2001 Cutting edge: CTLs rapidly capture membrane fragments from target cells in a TCR signaling-dependent manner. Journal of Immunology 166 36453649.

    • Search Google Scholar
    • Export Citation
  • Hudrisier D, Riond J, Garidou L, Duthoit C & Joly E 2005 T cell activation correlates with an increasedproportion of antigen among the materials acquiredfrom target cells. European Journal of Immunology 35 22842294. doi:10.1002/eji.200526266.

    • Search Google Scholar
    • Export Citation
  • Inoue N, Ikawa M, Isotani A & Okabe M 2005 The immunoglobulin superfamily protein Izumo is required for sperm to fuse with eggs. Nature 434 234238. doi:10.1038/nature03362.

    • Search Google Scholar
    • Export Citation
  • Ito C, Yamatoya K, Yoshida K, Maekawa M, Miyado K & Toshimori K 2010 Tetraspanin family protein CD9 in the mouse sperm: unique localization, appearance, behavior and fate during fertilization. Cell and Tissue Research 340 583594. doi:10.1007/s00441-010-0967-7.

    • Search Google Scholar
    • Export Citation
  • Jin M, Fujiwara E, Kakiuchi Y, Okabe M, Satouh Y, Baba SA, Chiba K & Hirohashi N 2011 Most fertilizing mouse spermatozoa begin their acrosome reaction before contact with the zona pellucida during in vitro fertilization. PNAS 108 48924896. doi:10.1073/pnas.1018202108.

    • Search Google Scholar
    • Export Citation
  • Joly E & Hudrisier D 2003 What is trogocytosis and what is its purpose? Nature Immunology 4 815 doi:10.1038/ni0903-815.

  • Kaji K, Oda S, Shikano T, Ohnuki T, Uematsu Y, Sakagami J, Tada N, Miyazaki S & Kudo A 2000 The gamete fusion process is defective in eggs of Cd9-deficient mice. Nature Genetics 24 279282. doi:10.1038/73502.

    • Search Google Scholar
    • Export Citation
  • Kimura M, Kim E, Kang W, Yamashita M, Saigo M, Yamazaki T, Nakanishi T, Kashiwabara S & Baba T 2009 Functional roles of mouse sperm hyaluronidases, HYAL5 and SPAM1, in fertilization. Biology of Reproduction 81 939947. doi:10.1095/biolreprod.109.078816.

    • Search Google Scholar
    • Export Citation
  • Le Naour F, Rubinstein E, Jasmin C, Prenant M & Boucheix C 2000 Severely reduced female fertility in CD9-deficient mice. Science 287 319321. doi:10.1126/science.287.5451.319.

    • Search Google Scholar
    • Export Citation
  • Li YH, Hou Y, Ma W, Yuan JX, Zhang D, Sun QY & Wang WH 2004 Localization of CD9 in pig oocytes and its effects on sperm–egg interaction. Reproduction 127 151157. doi:10.1530/rep.1.00006.

    • Search Google Scholar
    • Export Citation
  • Mattioli M, Gloria A, Mauro A, Gioia L & Barboni B 2009 Fusion as the result of sperm–somatic cell interaction. Reproduction 138 679687. doi:10.1530/REP-08-0316.

    • Search Google Scholar
    • Export Citation
  • Miyado K, Yamada G, Yamada S, Hasuwa H, Nakamura Y, Ryu F, Suzuki K, Kosai K, Inoue K & Ogura A et al. 2000 Requirement of CD9 on the egg plasma membrane for fertilization. Science 287 321324. doi:10.1126/science.287.5451.321.

    • Search Google Scholar
    • Export Citation
  • Miyado K, Yoshida K, Yamagata K, Sakakibara K, Okabe M, Wang X, Miyamoto K, Akutsu H, Kondo T & Takahashi Y et al. 2008 The fusing ability of sperm is bestowed by CD9-containing vesicles released from eggs in mice. PNAS 105 1292112926. doi:10.1073/pnas.0710608105.

    • Search Google Scholar
    • Export Citation
  • Murray SC & Smith TT 1997 Sperm interaction with fallopian tube apical membrane enhances sperm motility and delays capacitation. Fertility and Sterility 68 351357. doi:10.1016/S0015-0282(97)81528-2.

    • Search Google Scholar
    • Export Citation
  • Okabe M, Adachi T, Takada K, Oda H, Yagasaki M, Kohama Y & Mimura T 1987 Capacitation-related changes in antigen distribution on mouse sperm heads and its relation to fertilization rate in vitro. Journal of Reproductive Immunology 11 91100. doi:10.1016/0165-0378(87)90014-3.

    • Search Google Scholar
    • Export Citation
  • Ostrowski M, Carmo NB, Krumeich S, Fanget I, Raposo G, Savina A, Moita CF, Schauer K, Hume AN & Freitas RP et al. 2010 Rab27a and Rab27b control different steps of the exosome secretion pathway. Nature Cell Biology 12 1930.(sup pp 11–13) doi:10.1038/ncb2000.

    • Search Google Scholar
    • Export Citation
  • Ronquist G, Brody I, Gottfries A & Stegmayr B 1978 An Mg2+ and Ca2+-stimulated adenosine triphosphatase in human prostatic fluid – part II. Andrologia 10 427433. doi:10.1111/j.1439-0272.1978.tb03064.x.

    • Search Google Scholar
    • Export Citation
  • Rubinstein E, Ziyyat A, Prenant M, Wrobel E, Wolf JP, Levy S, Le Naour F & Boucheix C 2006 Reduced fertility of female mice lacking CD81. Developmental Biology 290 351358. doi:10.1016/j.ydbio.2005.11.031.

    • Search Google Scholar
    • Export Citation
  • Runge KE, Evans JE, He ZY, Gupta S, McDonald KL, Stahlberg H, Primakoff P & Myles DG 2007 Oocyte CD9 is enriched on the microvillar membrane and required for normal microvillar shape and distribution. Developmental Biology 304 317325. doi:10.1016/j.ydbio.2006.12.041.

    • Search Google Scholar
    • Export Citation
  • Saez F, Frenette G & Sullivan R 2003 Epididymosomes and prostasomes: their roles in posttesticular maturation of the sperm cells. Journal of Andrology 24 149154.

    • Search Google Scholar
    • Export Citation
  • Scofield VL, Clisham R, Bandyopadhyay L, Gladstone P, Zamboni L & Raghupathy R 1992 Binding of sperm to somatic cells via HLA-DR. Modulation by sulfated carbohydrates. Journal of Immunology 148 17181724.

    • Search Google Scholar
    • Export Citation
  • Stegmayr B & Ronquist G 1982 Promotive effect on human sperm progressive motility by prostasomes. Urological Research 10 253257. doi:10.1007/BF00255932.

    • Search Google Scholar
    • Export Citation
  • Suarez SS 1987 Sperm transport and motility in the mouse oviduct: observations in situ. Biology of Reproduction 36 203210. doi:10.1095/biolreprod36.1.203.

    • Search Google Scholar
    • Export Citation
  • Tanghe S, Van Soom A, Nauwynck H, Coryn M & de Kruif A 2002 Minireview: functions of the cumulus oophorus during oocyte maturation, ovulation, and fertilization. Molecular Reproduction and Development 61 414424. doi:10.1002/mrd.10102.

    • Search Google Scholar
    • Export Citation
  • Thery C, Boussac M, Veron P, Ricciardi-Castagnoli P, Raposo G, Garin J & Amigorena S 2001 Proteomic analysis of dendritic cell-derived exosomes: a secreted subcellular compartment distinct from apoptotic vesicles. Journal of Immunology 166 73097318.

    • Search Google Scholar
    • Export Citation
  • Thery C, Zitvogel L & Amigorena S 2002 Exosomes: composition, biogenesis and function. Nature Reviews. Immunology 2 569579.

  • Yanagimachi R, Kamiguchi Y, Mikamo K, Suzuki F & Yanagimachi H 1985 Maturation of spermatozoa in the epididymis of the Chinese hamster. American Journal of Anatomy 172 317330. doi:10.1002/aja.1001720406.

    • Search Google Scholar
    • Export Citation