Reagents and Antibodies

All the chemicals were obtained from Sigma Chemical Co. (St. Louis, MO) unless otherwise stated. Affinity-purified mouse monoclonal antibody against syntaxin 1 (STX1), rabbit polyclonal antibodies against syntaxin 2 (STX2), SNAP23, VAMP1, VAMP2, and VAMP3 (specific for each isoform and with no cross-reactivity with the other two) as well as complexin 1/2 (binds to both isoforms) were purchased from Synaptic System (Göttingen, Germany). Rabbit polyclonal antibody against SNAP25 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse IgG1 monoclonal antibodies against raft marker protein flotillin 1 and against a marker protein for caveolae (caveolin 1) were obtained from BD Biosciences (San Jose, CA). Mouse IgG antibody against clathrin heavy chain (X22) was a kind gift from J.J. Hölzenspies (Utrecht University, Utrecht, the Netherlands). Control SNAP23, VAMP1, and complexin 1/2 peptides were purchased from Synaptic System, and control flotillin-1 peptide was purchased from Abcam (Cambridge, U.K.).

In Vitro Maturation and Fertilization

Sow (Sus scrofa) ovaries were obtained from adult fertile sows at a special slaughterhouse (Groenlo, the Netherlands). This abattoir is special as it is unique in facilitating a continuous slaughter line exclusively for adult fertile sows. Routinely, prepubertal gilts are slaughtered for meat production. Prepubertal gilts are not yet sexually active and thus contain irrelevant ovaria for our studies. Cumulus-oocyte complexes (COCs) were collected by aspiration of 3–6 mm follicles and subsequently selected using well-established morphological criteria [16]. The yield per daily collection session was usually above 100 oocytes. In vitro maturation (IVM) was performed as previously described [16]. COCs were collected in HEPES buffered M199 (Gibco Laboratories Inc., Grand Island, NY) and washed in preequilibrated M199 supplemented with 2.2 mg/ml NaHCO₃, 0.1% (w/v) polyvinylpyrrolidone (PVP), 100 μM cysteamine, 75 μg/ml potassium penicillin G, and 50 μg/ml streptomycin sulfate (oocyte maturation medium, OMM). Selected COCs were cultured at 38°C with 5% CO₂ for 22 h in OMM supplemented with 0.05 international units/ml recombinant human follicle-stimulating hormone (rhFSH, a kind gift from Organon, Oss, the Netherlands). After 22 h IVM, COCs were transferred into OMM without rhFSH for another 22 h to reach the metaphase II stage. The CR was induced by in vitro fertilization (IVF) by coincubating M-II oocytes with freshly ejaculated porcine sperm cells from fertile boars (Cooperative Boar Artificial Insemination Center, Bunnik, the Netherlands). In short, the freshly ejaculated sperm cells were washed with IVF medium (113.1 mM NaCl, 3.0 mM KCl, 20 mM Tris, 11.0 mM glucose, 0.1% bovine serum albumin (BSA), 1.0 mM caffeine, 7.5 mM CaCl₂, 0.05 mM sodium pyruvate, 75 μg/ml potassium penicillin G, and 50 μg/ml streptomycin sulfate; pH 7.4) for 5 min and then centrifuged at 700 × g at room temperature (RT). Washed sperm cells were resuspended in the IVF medium and adjusted to a concentration of 1 × 10⁶ spermatozoa/ml. This sperm suspension was added to the oocytes in a ratio of 1000 sperm cells/oocyte and coincubated at 38°C with 5% CO₂ in air for 8 h. The maturation and fertilization competence of the oocytes were determined based on their overall morphology and DNA-staining pattern, which were assessed as described previously [17] by confocal laser scanning microscopy (Leica TCS SP2, Leica Microsystems GmbH, Wetzlar, Germany). Oocytes could be divided into three categories: 1) germinal vesicle (0 h, GV), as indicated by the presence of the germinal vesicle structure, 2) meiosis-II (44 h after IVM, M-II), as indicated by the presence of the metaphase plate and the first polar body, and 3) fertilized (52 h, IVF), as indicated by the presence of two pronuclei. Three independent experiments were conducted, and for each experiment, at least 20 oocytes per group were evaluated. Oocytes that could not be categorized according to the criteria described above were excluded from this study.

Immunoblotting

Protein concentrations from all the samples were standardized according to the Lowry method [18]. For both sperm and control brain samples, 15 μg of total protein extract was mixed with an appropriate amount of lithium dodecyl sulfate loading buffer (Invitrogen, Carlsbad, CA) in the presence of 0.1 M dithiothreitol. Cumulus-free oocytes were obtained as previously described [17], and 100 oocytes were used per sample, which corresponds to 10–12 μg of total protein. All the samples were heated for 10 min at 100°C prior to immunoblotting. Proteins were separated in a 4% stacking and 12% running SDS-PAGE gel and wet-blotted onto polyvinylidene difluoride membranes (GE Healthcare, Buckinghamshire, U.K.). After blocking for 1 h with ReliaBLOT (Bethyl Laboratories, Inc., Montgomery, TX) at RT, the blots were incubated overnight at 4°C with primary antibodies diluted in ReliaBLOT (a 1:250 dilution from a 1 mg/ml stock was used for all experiments except for syntaxin2 where a 1:1000 dilution was used). After washing the blots in TBST (50 mM Tris, pH 7.4, 250 mM sucrose, and 0.2% v/v Tween-20), secondary antibodies (dilutions of 1:500 from a 2 mg/ml stock) were added for 30 min. After rinsing with TBST, protein was visualized using chemiluminescence (ECL-detection kit; Supersignal West Pico, Pierce, Rockford, IL).

Labeling of CGs

Labeling of the CGs was performed as previously described [1, 19] with minor modifications. For efficient lectin binding, partially denuded oocytes were used. To remove the surrounding cumulus cells, oocytes of different maturation stages were gently pipetted in HEPES-buffered M199; partially denuded oocytes were separately collected and washed two times in HEPES-buffered M199. After washing, oocytes were fixed in freshly prepared 4% (v/v) para-formaldehyde (Electron Microscopy Sciences, Hatfield, PA) in phosphate-buffered saline (PBS; 137 mM NaCl, 8.1 mM Na₂HPO₄, 2.7 mM KCl, 1.5 mM KH₂PO₄, pH 7.4) for 1 h at RT. After fixation, oocytes were washed twice in PBS-PVP (0.1 M PBS, pH 7.4, containing 0.3% (w/v) PVP) and permeabilized in PBSS (0.1 M PBS, pH 7.4, containing 0.1% saponin) for 10 min. Nonspecific-binding sites were blocked using blocking buffer glycine (PBSS with 1% (w/v) BSA and 2% (v/v) normal goat serum (Vector Lab, Burlingame, CA) supplemented with 100 mM glycine) for 2 h at RT. After rinsing in PBS, oocytes were incubated in fluorescein isothiocyanate-conjugated peanut agglutinin (PNA-FITC; EY Laboratory, San Mateo, CA) diluted in PBSSc (PBSS containing 0.1g/L CaCl₂) for 1 h at RT in the dark. As a negative control, PNA-FITC was preincubated in PBSSc containing 100 mM lactose for 30 min at RT before use.

Immunofluorescent Staining for SNARE Proteins, Complexin, and Clathrin

Immunolabeling was performed as previously described [17]. Both primary and secondary antibodies were diluted in blocking buffer and centrifuged at 100 000 × g for 1 h before use to prevent aggregation of the antibody. Oocytes were incubated with primary antibodies overnight at 4°C. For negative controls, purified mouse or rabbit IgG (BD Biosciences) that matched the host species of the primary antibodies (a 1:250 dilution from a 1 mg/ml stock was used for all experiments except for syntaxin2 where a 1:1000 dilution was used) were used with the dilutions of the negative controls being identical to the dilution of the primary antibodies used in the same experiment. Oocytes were rinsed three times in PBS after primary antibodies incubation. SNARE-labeled oocytes were subsequently incubated with (dilutions of 1:500 from a 2 mg/ml stock) Alexa-568 conjugated goat anti-mouse IgG or goat anti-rabbit IgG (Molecular Probes, Eugene, OR) for 1 h at RT. DNA was labeled with 10 μM ToPro-3 iodide (Molecular Probes, Leiden, the Netherlands) in PBSS for 20 min. After three rinses in PBSS, oocytes were mounted in a 0.12 mm, 8-well Secure-Seal Spacer (Molecular Probes) on a cover slip, covered in a drop of Vectashield (Vector Lab), and sealed with a microscope slide (Superfrost Plus; Menzel, Braunschweig, Germany). In control experiments, 1 μg/ml primary antibodies were blocked with 5 μg/ml synthetic peptides epitopes of the corresponding proteins the antibodies were raised against. The synthetic blocking peptides for anti-SNAP23 antibody (sequence: DRIDIANARAKKLIDS), anti-VAMP1 (sequence: SAPAQPPAEGTEG), anti-complexin 1/2 (sequence: KYLPGPLQDMFKK), and anti-flotillin-1 (sequence: SISQVNHKPLRTA) were coincubated with the corresponding antibodies for 2 h prior to their use in the immunofluorescent experiments. These conditions were recommended by the company that delivered the blocking peptides and antibodies and were effective in preventing antibody binding to oocyte structures (see Supplemental Fig. S1; all the supplemental data are available online at  www.biolreprod.org).

Confocal Laser Scanning Microscopy and Image Acquisition

Images were obtained through a 40× oil immersion objective using a Leica TCS SP2 confocal system (Leica Microsystems GmbH), equipped with 488, 568, and 633 nm lasers. Dual and triple channel images were obtained by sequential scanning. ImageJ (National Institutes of Health,  http://rsb.info.nih.gov/ij/) software was used for the analysis of the images. Laser power and acquisition settings were adjusted to produce submaximal pixel values in the oocyte, and the settings used to image control IgG stainings were matched to the highest settings used to image primary antibody staining in the same experiment. Images made at the equatorial region of the oocytes were used to discriminate on representative DNA structures for germinal vesicles for the GV stage or for metaphase plate/first polar body for the M-II stage. Background subtraction and contrast/brightness enhancement (up to ∼20% enhancement using the maximum slider in Image J) were performed identically for all the images in the same experiment.

Distribution of SNARE Proteins in Porcine Oocytes

To determine how SNARE proteins are organized just prior to CR, we used M-II arrested oocytes (the final maturation stage of mammalian oocytes that leaves them ready to become fertilized by sperm). With indirect immunofluorescence and immunoblotting techniques, we demonstrated that the complementary SNARE proteins syntaxin 2, SNAP23, VAMP1, and VAMP2 were present in porcine M-II oocytes, whereas syntaxin 1, SNAP25, and VAMP3 were below the detection level (Fig. 1A and Supplemental Fig. S2). Therefore, all the SNARE members required to form a SNARE complex that enables intracellular membrane fusion are present in porcine oocytes.

FIG. 1.

Cortex orientation of SNARE proteins in M-II porcine oocytes. A) Immunofluorescent labeling of Q-SNARE proteins (red) syntaxin 2 and SNAP23 and of R-SNARE proteins (red) VAMP1 and VAMP2 in M-II porcine oocytes (O); absence of labeling for syntaxin 1, SNAP25, and VAMP3. Western blot analysis of SNARE proteins present in the M-II oocytes shows comparable molecular weights to that of control brain tissues (B) and of boar sperm cells (S). B) PNA-FITC staining (green) indicates a defined cortex arrangement of cortical granules adjacent to the oolemma at M-II arrested oocyte. PB, first polar body; MP, metaphase plate. The dashed circles indicate the area of the zona pellucida. Chromatin was stained with Topro-3 (blue). Bars = 20 μm.

In M-II oocytes, SNARE proteins were predominately observed in the cortical region of the oocyte and showed punctuate staining patterns that are characteristic for SNARE proteins (Fig. 1A). Note that at the light resolution limits of confocal microscopy the immunofluorescent signals at the cortex can originate either from membrane proteins that reside at the oolemma or from membrane proteins that reside in secretory vesicles that are associated with the oolemma. The fact that SNARE-staining patterns coincided with that of the PNA-FITC (Fig. 1B) could imply that the SNARE proteins are associated with the CGs. To our surprise, from the immunoblotting studies, SNAP25 was not detected in the porcine oocytes and only the low molecular weight form of VAMP1, which is similar to that found in porcine sperm, was seen (Fig. 1A immunoblot and Supplemental Fig. S2); this is in contrast to our observations in control pig brain tissues and the report on mouse oocytes [13]. Moreover, VAMP3 was below the detection level in our oocyte samples preparation, and a lower molecular weight form of SNAP23 was observed (Fig. 1A immunoblot and Supplemental Fig. S2).

Peripheral Migration of Secretory Granules and VAMP During Oocyte Maturation Indicate SNARE-Mediated Docking of CGs to the Oolemma

Because we found that SNARE proteins are enriched in the cortex of M-II arrested oocytes, we further analyzed whether this localization pattern is caused by a redistribution of SNARE proteins during oocyte maturation. To this end, immature GV-stage oocytes were harvested from follicles and were stained for the SNARE proteins. Simultaneously, CGs were identified by labeling with secretory granule-specific binding lectin (i.e., PNA-FITC). During maturation, the CGs moved to a sharply defined region under the oolemma as was shown by changes in PNA labeling (Fig. 2, A1 and A2). A similar relocalization was observed for VAMP1-labeled structures (Fig. 2, B1 and B2). SNAP23-labeled structures, however, showed a similar peripheral redistribution but were not present under the oolemma (Fig. 2, C1 and C2). In contrast to the maturation-dependent redistribution of secretory granules and the above mentioned SNARE protein relocalization of VAMP1 and SNAP23, we observed that syntaxin 2 labeled structures did not show relocation upon meiotic maturation, being already associated with the oolemma at the GV stage and remaining there until the end stages of oocyte maturation (Fig. 2, D1 and D2).

FIG. 2.

Secretory granules and SNARE proteins migrate to the oocyte surface upon meiotic maturation. PNA-FITC was used to label secretory granules (green) and to demonstrate the migration of these granules upon oocyte maturation. Antibodies against different SNARE proteins and SNARE regulator (complexin) were used to indicate the localization of these proteins (red) at different maturation stages. DNA of the maturating oocytes was labeled with ToPro-3 (blue). Oocytes of two maturation stages (0 h: GV, and 44 h IVM: M-II) were used. PNA-positive secretory granules and SNARE-positive membrane vesicles showed dispersed punctuate pattern throughout the entire ooplasm at the GV stage (A1–C1), and syntaxin 2 was observed already in association with oolemma in GV oocyte (D2). Both PNA-positive secretory granules and SNARE-positive membrane showed either a pronounced oocyte surface-enriched (or oolemma-associated) staining pattern (A2–B2, D2) or they were concentrated to the cortex of the oocyte in M-II arrested oocytes (C2). Complexin in both GV and M-II stages showed a sharply plasma membrane-associated pattern (E1–E2). GV, germinal vesicle; PB, first polar body; MP, metaphase plate; V1, VAMP1; SN23, SNAP23; Sy2, syntaxin 2. Dashed circles indicate the relative position for the zona pellucida. Representative images of three independent experiments (n = 20 per group per experiment) are presented; the percent of oocytes with this labeling ± SD are indicated, while the other oocytes either had aberrant or very dim labeling. Bars = 20 μm.

The detected concentration of VAMP1- and PNA-positive structures just under the surface of the oolemma in M-II stage oocytes suggests that the CGs become docked at the oolemma by the use of SNARE proteins. SNARE-mediated membrane priming and docking requires additional components such as complexin to stabilize the formed SNARE complex [15]. Therefore, we subsequently analyzed maturing oocytes for the presence of complexin. Our results showed that the presence of complexin was confined to a sharply defined region underneath the oolemma in M-II oocytes (Fig. 2E2). However, in contrast to the redistribution of PNA- and VAMP1-positive structures, no redistribution of complexin was observed because it was already present near the oolemma in the GV-stage oocytes (Fig. 2E1) and remained at this location. This suggests that docking of secretory granules to the oolemma is mediated by SNARE proteins, and the subsequent involvement of complexin present in close proximity to the oolemma could contribute to the stabilization of this interaction and thus prevent exocytosis. To examine this possibility, we performed a series of colocalization studies in both immature (GV) and matured (M-II) unfertilized oocytes. We found in the majority of the immature oocytes that PNA-containing cortical granules and syntaxin 2 as well as VAMP1 to some extent colocalized at the oolemma but not at other areas in the oocyte (Fig. 3 arrows and Table 1). Moreover, in the matured M-II oocytes, the amount of colocalization between PNA-positive structures (i.e., CGs) and SNARE proteins was substantially increased at the oocyte surface (see Fig. 4, where the colocalization is indicated with arrows as well as in the enlarged panels, and Table 1), and this colocalization also matched the topology of complexin.

FIG. 3.

Minimal colocalization of cortical granules and SNARE proteins in GV oocyte. Oocytes of germinal vesicle stage were used to demonstrate the distribution of cortical granules (labeled with PNA-FITC, green) and SNARE proteins (indirect immunolabeling, red). Chromatin was stained with ToPro-3 (blue). The majority of the secretory granules did not associate with SNARE proteins in GV-arrested oocytes; a small amount of colocalization between cortical granules and SNARE proteins can be observed just underneath the oolemma (arrows). Rabbit IgG (RIgG) was used as a negative control for SNARE proteins, demonstrating the absence of signal. PNA (L): PNA-binding sites were inhibited with 100 mM lactose, which was used as a negative control for PNA labeling. Dashed circles indicate the relative position for the zona pellucida. Representative images of three independent experiments (n = 20 per group per experiment) are presented. Bars = 20 μm.

TABLE 1.

In vitro maturation coincides with colocalization of cortical granules and the SNARE proteins at the entire surface of the oocyte.^a

FIG. 4.

Cortical granules are docked to the oolemma and colocalized with SNARE proteins in M-II-arrested oocytes. Oocytes of meiosis-II stage were used to demonstrate the colocalization of cortical granules (labeled with PNA-FITC, green) and SNARE proteins (indirect immunolabeling, red). Chromatin was stained with ToPro-3 (blue). Cortical granules are positioned in close proximity to the oolemma and show colocalization (indicated with arrows) with SNARE proteins over the entire oocyte surface. Areas indicated by the arrows are shown in the enlarged panels and clearly indicate colocalization. PNA (L): PNA-binding sites were inhibited with 100 mM lactose, which was used as a negative control for PNA labeling. Dashed circles indicate the relative position for the zona pellucida. Representative images of three independent experiments (n = 20 per group per experiment) are presented. Bars = 20 μm.

Functional Implications of CG Docking to the Oolemma for Endocytosis in Maturing Oocytes

Cortical reaction is a calcium-dependent exocytotic process, and in most of the cell types, exocytosis and endocytosis are indirectly coupled processes. Therefore, we extended our investigations with the immunolocalization of endocytotic proteins during oocyte maturation. Immunolocalization of clathrin (for clathrin-dependent endocytosis) showed that clathrin was not only present at the oolemma but also in intracellular vesicular structures in GV oocytes (Fig. 5A1). Clathrin migrated to the same sharply defined area just under the oolemma during oocyte maturation (Fig. 5A2). This indicated that clathrin was targeted to the oolemma (and/or oolemma-docked structures) in M-II oocytes and was not sequestered further by endocytosis or released deeper in the oocyte. Another protein-mediated endocytotic pathway is initiated by the formation of membrane caveolae enriched with flotillin and caveolin. Both membrane raft marker proteins were present in oocytes, but they did not show obvious redistribution upon oocyte maturation (Fig. 5, B1–B2 and C1–C2).

FIG. 5.

Clathrin-dependent endocytosis is arrested in meiosis-II oocyte. Indirect immunolabeling of endocytosis-related proteins (red) and chromatin (blue) in two maturation stages (GV and M-II) oocytes. A) Clathrin migrated from the cytoplasm (A1) to the surface of the oocyte where it becomes arrested adjacent to the oolemma at the M-II stage (A2). B1–B2) Flotillin-1 concentrates to the cortex region of the oocyte but does not associate with or reside underneath the oolemma. C1–C2) Caveolin 1 does not show meiotic maturation-dependent redistribution. D1–D2, E1–E2) Purified mouse IgG (MIgG) and rabbit IgG (RIgG) were used as negative controls, demonstrating the absence of signal. GV, germinal vesicle; PB, first polar body; MP, metaphase plate. Dashed circles indicate the relative position for the zona pellucida. Representative images of three independent experiments (n = 20 per group per experiment) are presented; the percent of oocytes with this labeling ± SD are indicated, while the other oocytes either had aberrant or very dim labeling. Bars = 20 μm.

Cortical Reaction Leads to Clathrin and Complexin Dissociation from the Oolemma

The fusion of a sperm cell with the oocyte leads to the initiation of the CR. During the CR, CGs fuse with the oolemma. Because this results in an enlargement of the oocyte surface, a compensatory reduction of this surface must be accomplished by an immediate induction of endocytosis. We tested whether fertilization induces the release of complexin and clathrin and as such reestablishes membrane traffic (exocytosis and endocytosis at the oolemma) in the fertilized oocyte. For this reason, we have analyzed the fate of SNARE proteins, complexin, and clathrin in fertilized oocytes until 8 h after IVF. At this stage, fertilized oocytes contain both a male and female pronucleus (Fig. 6, A–E). As expected, the intracellular PNA staining decreased below the detection level when compared to M-II oocytes as the CGs fused with the oolemma and the granule contents were released and diluted (Fig. 6A). In the fertilized oocytes, the CR coincided with the release of complexin from the oocyte's surface into the cytosol and surrounding lipid droplets (Fig. 6B), most likely a result of the dissociation of complexin from the SNARE protein complex. When SNARE distributions of fertilized and unfertilized mature M-II oocytes were compared, VAMP1 was found to be present at intracellular structures (Fig. 6C), indicating that VAMP1 was endocytosed or at least the VAMP1-containing membrane structure was transported away from the oolemma (where they were originally located, see Fig. 1A) after fertilization. The noted release of clathrin into the cytosol may be of importance for reestablishing endocytosis at the oolemma after the CR (Fig. 6D).

FIG. 6.

Cortical reaction releases enzymatic content and initiates the endocytosis-driven recycling of VAMP1 and the disassociation of complexin into the cytosol. Indirect immunolabeling of endocytosis-related proteins (red), chromatin (blue), and cortical granules (green, stained with PNA-FITC) in oocytes 8 h after IVF. Both monospermic-fertilized (A–E) and polyspermic-fertilized (F–J) porcine oocytes are shown. A, F) The cortical reaction has taken place as evidence from the absence of PNA labeling of cortical granules in the oocytes. B, G) Complexin 1/2 are released from the SNARE complex after sperm-induced fertilization and therefore have lost their oolemma-specific orientation. A portion of complexin appears to be liberated in the cytosol while another part is detected around lipid droplets. However, this redistribution of clathrin is less apparent in the polyspermic-fertilized oocytes. C, H) VAMP1 is endocytosed into an interior area of the monospermic-fertilized oocyte as the defined oolemma-specific pattern is lost and VAMP1 is detected at intracellular structures inside the ooplasm. More pronounced plasma membrane-orientated staining was observed for VAMP1 in polyspermic-fertilized oocyte. D, I) Clathrin is released from the oocytes surface after fertilization and diffuses into the oocyte's cytosol when compared to the oolemma-specific pattern in M-II oocytes. E, J) Syntaxin 2 showed consistent plasma membrane-specific staining in both types of fertilized oocytes. PN, pronuclei; S, sperm cell (indicative of IVF). Dashed circles indicate the relative position for the zona pellucida. Representative images of three independent experiments (n = 20 per group per experiment) were presented. In all cases, >85% of the monospermic- and polyspermic-fertilized oocytes showed the type of labeling presented here. Bars = 20 μm. The relative percent of unfertilized, monospermic-fertilized, and polyspermic-fertilized oocytes is given in the lowest panel.

While the majority of the metaphase II-arrested oocytes were fertilized by one sperm (55% of all oocytes, Fig. 6), a relative low number of oocytes remained unfertilized (18%) and also some oocytes showed polyspermic fertilization (27% of all oocytes, Fig. 6, F–J), which is normal after porcine IVF. Both monospermic-fertilized and polyspermic-fertilized oocytes (Fig. 6, A–E) showed similar normal cortical secretion of PNA-containing vesicles (Fig. 6, A and F) and redistribution of complexin1/2 (Fig. 6, B and G). In contrast, VAMP1 and clathrin did not demonstrate redistribution into intracellular membrane structures in polyspermic-fertilized oocytes (Fig. 6, H and I) but did demonstrate redistribution in monospermic-fertilized oocytes (Fig. 6, C and D). Syntaxin 2 did not change its distribution after both types of fertilization (Fig. 6, E and J).