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1 Graduate School of Science and Technology,
2 Research Center for Environmental Genomics, Kobe University, Nada-ku, Kobe 657-8501, Japan
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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Experimental approaches focusing on egg cytoplasmic proteins have also inferred an importance of the egg plasma membranes in the sperm-induced egg activation. In earlier reports, protein-tyrosine kinase (PTK)-specific inhibitors (e.g. genistein, lavendustin A, peptide A7, PP2) were shown to block sperm-induced egg activation in Xenopus (Sato et al. 1998, 1999, 2000b; Glahn et al. 1999). Later, a 57-kDa Src family PTK, xSrc (formerly called as Xyk), was identified as a target protein of the PTK inhibitors (Sato et al. 1996; Iwasaki et al. 2006). It has been demonstrated that xSrc becomes activated within minutes of insemination and that the activated xSrc interacts, phosphorylates and activates phospholipase C
(PLC), by which inositol 1,4,5-trisphosphate, a second messenger for the transient Ca2+ release, is produced through breakdown of phosphatidylinositol 4,5-bisphosphate (Sato et al. 2000b, 2001). Importantly, xSrc localizes exclusively to the egg plasma membranes in unfertilized Xenopus eggs, suggesting that the egg plasma membranes serve as a platform of sperm–egg interaction and subsequent xSrc activation signaling. A similar cascade of events involving egg Src-like proteins and PLC has been demonstrated in invertebrates such as sea urchin (Abassi et al. 2000), starfish (Giusti et al. 1999) and ascidians (Runft et al. 2000) (for review see Jaffe et al. 2001; Runft et al. 2002; Townley et al. 2006) and fish (Wu & Kinsey 2000; Sharma & Kinsey 2006); however, a molecular detail of the signal transduction from the sperm–egg membrane interaction to Src activation has not yet been fully documented.
Under these circumstances, we have been interested in structure and function of microenvironment in the plasma membranes, in other words, membrane microdomains or rafts. Signaling molecules that contain extracellular (e.g. cell surface receptors) and/or intracellular portions (e.g. Src family PTKs) often localize to the membrane microdomains that are enriched in cholesterol and glycosphingolipids (Brown & London 1998; Simons & Toomre 2000; Hemler, 2003, 2005; Pike 2005). Several normal cellular (e.g. lymphocyte activation) as well as pathological processes (e.g. infection with microbes) have been shown to require proper organization of the membrane microdomains. Therefore, it is attractive to suggest that the egg plasma membrane microdomains play an important role in the sperm–egg membrane interaction and signaling. In fact, the egg membrane microdomains could be isolated from Xenopus unfertilized eggs as non-ionic detergent-insoluble low density fractions that were enriched in cholesterol, ganglioside GM1 and xSrc (Sato et al. 2002). Further studies have demonstrated that sperm-dependent xSrc activation is taking place in the egg membrane microdomains in vivo and in vitro and that disruption of the microdomains leads to a failure of successful fertilization (Sato et al. 2002, 2003).
More recently, we have identified an egg membrane microdomain-associated protein uroplakin III (xUPIII) that is tyrosine phosphorylated upon fertilization (Sakakibara et al. 2005). xUPIII is a Xenopus homologue of mammalian UPIIIa that has been originally identified as a member of UP family proteins that constitute an asymmetrical membrane unit in the apical surface of the urothelial tissues (Sun et al. 1999; Garcia-Espana et al. 2006). xUPIII, as like mammalian UPIIIa, consists of an amino-terminal, N-glycosylated extracellular domain, a transmembrane sequence and a carboxyl-terminal cytoplasmic sequence that contains a tyrosine phosphorylation site (Tyr-249) (Sakakibara et al. 2005). It has been shown that a specific antibody against the extracellular domain of xUPIII (anti-xUPIII ED) inhibits fertilization in a dose-dependent manner, suggesting that xUPIII is involved in the sperm–egg membrane interaction. This idea has been supported by the facts that xUPIII is identified as a target of sperm-derived protease, which is essential for sperm-induced egg activation and that the anti-xUPIII ED antibody blocks the protease action (Mahbub Hasan et al. 2005).
In the present study, we attempted to characterize further the organization and function of the egg membrane microdomain containing xUPIII. As in the case of the mammalian UPIII, xUPIII constitutes a hetero-complex with uroplakin Ib (xUPIb), a tetraspanin member of UP proteins (Mahbub Hasan et al. 2005). In the mouse, two other tetraspanin molecules, CD9 and CD81, have been shown to be involved in sperm–egg fusion (Kaji et al. 2000, 2002; Le Naour et al. 2000; Miller et al. 2000; Miyado et al. 2000; Rubinstein et al. 2005, 2006; Ziyyat et al. 2006). Therefore, experiments were designed to examine whether Xenopus egg CD9 (xCD9) is also in association with xUPIII in the egg membrane microdomains. We also analyzed interaction of GM1, a highly enriched ganglioside in the microdomains, with xUPIII and its functional importance. Finally, we tried to reconstitute the xUPIII-xUPIb complex in the membrane microdomains using HEK293 cell expression system. Results obtained suggest that GM1, but not xCD9, is in association with UPIII and plays an important role in the sperm–egg membrane interaction, and that xUPIII-xUPIb complex formation is required for their proper cellular localization and contributes to negative regulation of the tyrosine kinase xSrc.
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Results |
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To evaluate whether xUPIII is in association with tetraspanins xUPIb and xCD9, we prepared low density, detergent-insoluble membrane microdomains of Xenopus unfertilized eggs. We employed two types of non-ionic detergent: Brij 98 and Triton X-100, the former has a lesser hydrophobicity. In both conditions, sucrose density ultra centrifugation yielded detergent-insoluble membrane microdomain or raft fractions (Fig. 1, fractions 4–6) that contained only a small fraction of egg proteins (Fig. 1A–C) but high levels of GM1 (Fig. 1A,B, AB: CTB) and cholesterol (Fig. 1D). A recovery of raft-associated proteins was relatively higher in the preparation using Brij 98 than that using Triton X-100 (Fig. 1A–C). In accordance with this, immunoblotting analysis demonstrated that the recovery of xCD9 in the raft fractions was significantly higher in the Brij 98 preparation than in the Triton X-100 preparation, while other molecules such as xSrc, xUPIb and xUPIII showed a similar pattern of raft-enriched distribution in these two conditions (Fig. 1E, tubulin served as a non-raft marker). These results show that depending on the species of detergents used, components of Xenopus egg membrane microdomains can vary, and in particular, that xCD9 has a different detergent-solubility from xUPIII, xUPIb and xSrc, whose localization to the microdomains are not affected by the use of Brij 98 and Triton X-100.
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We examined the interaction of xUPIII, xUPIb and xCD9 more directly by co-immunoprecipitation experiments. Whole cell lysates were prepared from Xenopus eggs that had been treated with or without a chemical cross-linker DSP, which would stabilize molecular interactions, if any, on the egg surface. The lysates were then analyzed by immunoprecipitation and immunoblotting with antibodies against xCD9, xUPIb, or xUPIII. xUPIb and xUPIII were found to co-immunoprecipitate with each other irrespective of DSP treatment (Fig. 2A). Co-immunoprecipitation of xCD9 with xUPIb and xUPIII was hardly detectable even in the presence of DSP (Fig. 2A). Therefore, we concluded that xCD9 is not in association with xUPIII-containing membrane microdomains in Xenopus eggs.
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To evaluate functional importance of GM1 in the sperm-dependent signal transduction, we performed in vitro kinase assay using the Brij 98-rafts as an enzyme source. As in the case of the rafts prepared by the Triton X-100 method (Sato et al. 2002, 2003), the Brij 98 rafts contained an enriched amount of xSrc (Fig. 1E), whose kinase activity, as judged by autophosphorylation, could be elevated by the addition of sperm (265 ± 47% against egg rafts alone control, n = 3) and canceled in the presence of PP2, a Src-specific inhibitor (55 ± 27%, n = 3) (Fig. 2E). Under this system, exogenously added GM1 inhibited the sperm-induced phosphorylation of xSrc (95 ± 33%, n = 3) (Fig. 2E), suggesting that GM1 is involved in the sperm–egg raft interaction and/or xSrc activation by the sperm. In vitro fertilization experiments demonstrated that GM1, but not asialo-GM1, had an inhibitory effect on cortical contraction (not shown) as well as first cell cleavage of inseminated eggs (Table 1). Indirect fluorescent studies using CTB demonstrated that GM1 consistently localized to the apical surface of the egg plasma membranes as the extracellular domain of xUPIII did (Fig. 3A–F). It is notable that GM1 and UPIII co-localize on distinct, patch-like structures with 1–2 µm in a diameter (Fig. 3G–I). Taken together, we suggest that GM1 serves an important role in the sperm–egg membrane interaction and subsequent egg activation signaling.
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It has been reported that, in mammalian cells, co-expression of UPIb is required for UPIII to localize to the plasma membranes (Deng et al. 2002; Tu et al. 2002). Thus, we examined whether this is the case in xUPIII. To this end, we prepared HEK293 cells transiently expressing xUPIII and/or xUPIb and analyzed their expression, interaction and cellular localization. Immunoblotting analysis of Triton X-100-solubilized whole cell lysates demonstrated that expression level of xUPIII augmented when it was co-expressed with xUPIb (Fig. 4A), suggesting that co-expression leads to an increased stability of xUPIII. Reciprocal co-immunoprecipitation experiments confirmed that xUPIb and xUPIII interacted with each other only in the co-expressing cells (Fig. 4B,C). Surface biotinylation experiments showed that xUPIII, but not xUPIb, could be effectively biotinylated when co-expression was done (data not shown). Immunoprecipitation experiments with anti-biotin antibody demonstrated that both xUPIII and xUPIb could be effectively recovered in the immunoprecipitates when they were co-expressed (Fig. 4D), suggesting that these proteins require co-expression to be exposed properly on the cell surface. Consistently with this idea, indirect immunofluorescent studies demonstrated that xUPIII localized to the plasma membranes only when it was co-expressed with xUPIb (Fig. 4E). On the contrary, xUPIb showed a peripheral localization even in the absence of xUPIII (Fig. 4E). This is consistent with the previous report on the mammalian UPIb (Tu et al. 2002).
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We next examined the raft localization of xUPIb and xUPIII expressed in HEK293 cells. Sucrose density ultracentrifugation fractions were prepared from singly (xUPIb or xUPIII) or doubly transfected HEK293 cells and subjected to immunoblotting analysis. When expressed alone, a small population of xUPIb, but not xUPIII, localized to the detergent-insoluble membrane microdomain fractions, where endogenous GM1 was enriched (Fig. 5A, fractions 4–6). When co-expressed, however, both UPIb and UPIII effectively localized to the membrane microdomain fractions (Fig. 5B). Pretreatment of doubly transfected HEK293 cells with MßCD led to a dramatic decrease of xUPIb, xUPIII and GM1 in the membrane microdomain fractions (Fig. 5C), suggesting that localization of these components depends upon cholesterol. We also examined whether the raft localization of xUPIII could be supported by xCD9 in HEK293 cells. When expressed alone, xCD9 localized to the membrane microdomain fractions to some extent as xUPIb did (Fig. 6A). Such raft localization of xCD9 was also diminished by the pretreatment of the transfected cells with MßCD (Fig. 6B). When xCD9 and xUPIII were co-expressed in HEK293 cells, however, no increase of the amount of xCD9 or xUPIII in the membrane microdomain fractions was observed (Fig. 6C). Thus, the raft localization of xUPIII seems to be supported preferentially by xUPIb, and such molecular interaction cannot be reconstituted by xCD9.
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In Xenopus unfertilized eggs, membrane microdomains should be pre-organized with xUPIb, xUPIII and xSrc, in which xSrc is maintained to be inactive. Therefore, we performed transient expression of all of these components in HEK293 cells. As shown in Fig. 7A, kinase activity of the wild-type xSrc (xSrc-WT), as judged by autophosphorylation of Tyr-415, was suppressed when it was co-expressed with xUPIb and xUPIII. Such an effect was not seen when xSrc-WT was expressed alone or with either xUPIb or xUPIII (Fig. 7A, IB: pY415). A similar inhibitory effect on xSrc-WT was also seen when membrane microdomain-associated xSrc-WT was analyzed; that is, when co-expressed with xUPIb and xUPIII, phosphorylation of Tyr-415 in xSrc-WT was not detectable, whereas single-expressed xSrc-WT was found to be phosphorylated on Tyr-415 (data not shown). Expression level, phosphorylation at Tyr-526, a negative regulatory phosphorylation site, and localization to the membrane microdomains (not shown) of xSrc-WT were similar with each other (Fig. 7A). On the other hand, when the kinase-active version of xSrc (xSrc-KA), in which Tyr-526 residue was replaced by phenylalanine, was co-expressed with xUPIb and xUPIII, no suppression of the phosphorylation of Tyr-415 was observed (Fig. 7B). The results suggest that suppression of xSrc activity by the xUPIb and xUPIII complex requires the presence of Tyr-526 residue in xSrc (see Discussion). The suppressive effect of the xUPIb and xUPIII complex on xSrc was reversible, because the addition of H2O2, a strong trigger of xSrc activation (Sato et al. 2001), promoted a rapid activation of xSrc-WT in triply transfected HEK293 cells (Fig. 7C). Upon H2O2 treatment, both xUPIII and xSrc-WT became tyrosine-phosphorylated (Fig. 7D), as shown in the case of xUPIII and xSrc-KA (Sakakibara et al. 2005).
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Discussion |
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From the use of two different non-ionic detergents (Brij 98 and Triton X-100), we have learned that there may be more than two different types of membrane microdomains in Xenopus unfertilized eggs. A selective and augmented enrichment of xCD9 in the Brij 98-preparation suggested that it resides in a microdomain that is distinct from those containing other components examined (e.g. xUPIII and xSrc). Chemical cross-linking and co-immunoprecipitation experiments also supported this idea. These results may reflect that canonical tetraspanin-enriched microdomains, called as TEMs, are thought to be different from so-called membrane microdomains, which can be characterized by several properties, most typically detergent-resistance. Nevertheless, xUPIb, a tetraspanin binding-partner of xUPIII, has been shown to reside in the membrane microdomain containing xUPIII, suggesting that compartmentalization of transmembrane proteins into distinct membrane microdomains rely not only on their primary structures but on their partnership with other molecules as well.
We still don't know whether xCD9 is important in Xenopus egg fertilization as in the mouse system. Results presented in this paper do not support the idea that xCD9 is involved in signaling events associated with fertilization. However, in Xenopus, it has been shown that integrin-interacting peptides containing Arg-Gly-Asp or Lys-Thr-Cys sequence can promote sperm-independent (parthenogenetic) egg activation and that Xenopus sperm express xMDC16, a potential egg integrin ligand containing the or Lys-Thr-Cys sequence (Iwao & Fujimura 1996; Shilling et al. 1998). Therefore, it still seems that CD9 participates in egg activation signaling through its interaction with egg integrins. To assess this, it will be interesting to examine the effect of microinjection of anti-CD9 carboxyl-terminal sequence antibody, which might interfere with integrin-dependent signal transduction, on fertilization or peptide-induced egg activation. We should note that a monoclonal antibody against the extracellular domain of mouse CD9, which is reportedly inhibitory toward mouse fertilization (Miyado et al. 2000), is not cross-reactive to Xenopus egg CD9.
One question arises as to the presence of other UP family proteins, that is, UPIa and UPII, in Xenopus eggs and their function in fertilization. As far as we have examined by using commercially available specific antibodies against mammalian proteins, UPIa and UPII were not detectable in Xenopus eggs. However, this could be due to inability of those antibodies to react with Xenopus UPIa and UPII sequence. So, we are in the process of preparing antibodies against UPIa and UPII of Xenopus origin. It has recently been demonstrated that all UP members (except for UPIIIb, a UPIII subspecies that has been identified in addition to UPIIIa, which is equivalent to Xenopus egg UPIII in this paper) are expressed at mRNA level (Garcia-Espana et al. 2006). In the light of the fact that UPIb and UPIII are present at protein level in eggs, we prefer the possibility that UPIa and UPII are also expressed at protein level, and if so, that they should be responsible for structural basis of egg membrane microdomains through formation of UP complexes as seen in mammalian cells.
We have demonstrated that GM1, a ganglioside that is enriched in both Brij 98- and Triton X-100-preparation of the egg membrane microdomains, associates with xUPIII. Several lines of evidence have shown that GM1 and other GMs (e.g. GM3) localize to the membrane microdomains of both sperm and eggs and that they would play an important role in the gametogenesis, fertilization and early embryogenesis (Belton et al. 2001; Cross 2004; Ng et al. 2005; Buttke et al. 2006; Selvaraj et al. 2006). In particular, carbohydrate-to-carbohydrate interaction mediated by GMs has been implicated in the sperm–egg membrane interaction in some species including rainbow trout (Yu et al. 2002), sea urchin (Ohta et al. 2000; Maehashi et al. 2003) and pig (Bou Khalil et al. 2006). Results obtained with in vitro kinase assay and in vitro fertilization suggested that Xenopus egg GM1 is involved in sperm–egg membrane interaction and egg activation signaling including xSrc activation. Further study will be necessary to analyze whether the GM1-xUPIII interaction is important for the gamete interaction and what kind of molecular events are operated by GM1 and other GMs in eggs and sperm at fertilization.
Transfection studies using HEK293 cells have allowed us to reconstitute the xUPIII-xUPIb complex in the membrane microdomains. Previously, a similar reconstitution study has been done to analyze the mammalian UPIII-UPIb complex (Tu et al. 2002). In that study, UPIb was shown to localize to the plasma membrane without co-expression of UPIII, whereas UPIII required the co-expression of UPIb to exit from the endoplasmic reticulum. In this study, however, xUPIII and xUPIb were shown to be effectively surface-biotinylated only when they were co-expressed. Subcellular fractionation studies also showed that xUPIII and xUPIb localize effectively to the detergent-insoluble membrane microdomains when they were co-expressed. These results suggest that the formation of heterogeneous complex is prerequisite for both xUPIII and xUPIb to manage their proper subcellular (i.e. membrane microdomain-associated) localization in HEK293 cells and maybe in Xenopus eggs. Under the same reconstitution systems, xCD9 was without effect on the association of itself and UPIII to the detergent-insoluble membrane microdomains, although xCD9 could localize to the microdomain to some extent by itself, arguing again that xCD9 is not in close relationship to the xUPIII-containing membrane microdomains.
Another important finding with use of the HEK293 cell expression system is that kinase activity of xSrc is suppressed when it was co-expressed with xUPIII and xUPIb. Although the molecular detail of the negative regulation of xSrc by the UPIII/UPIb complex is currently unknown, the fact that the kinase-active form of xSrc (xSrc-KA) is not suppressed by the xUPIII/xUPIb complex suggests that carboxyl-terminal Src kinase or Csk (Okada et al. 1991), whose target phosphorylation site is missing in the xSrc-KA protein, is involved in this phenomenon. At present, however, this idea is not supported because the phosphorylation level at Tyr-526 in the xSrc-WT protein was not augmented in the xUPIII/xUPIb/xSrc-triple expressing cells (Fig. 7A). Restoration of the kinase activity of xSrc-WT by H2O2 in the triple-expressing cells (Fig. 7C) suggests that the xUPIII/xUPIb complex contributes to the inactivation of xSrc-WT as a kind of kinase regulator, but not as a sole inhibitor for xSrc. Our previous study has demonstrated that xUPIII is a target of sperm-derived protease that is essential for egg activation (Mahbub Hasan et al. 2005). Our current model of sperm-induced egg activation involves proteolytic cleavage of the extracellular domain of xUPIII that would act as a trigger of xSrc activation by unknown mechanism. Thus, it will be attractive to test what kind of intracellular signal transducers are in association with the xUPIII/xUPIb complex in Xenopus eggs to modulate xSrc activity before and after fertilization, candidates of which include Csk and heterotrimeric GTP-binding proteins, the latter of which have been shown to act as molecular switches in some protease-activated receptor system (O’Brien et al. 2001). Further systematic identification of protein components and functional analysis in the egg membrane microdomains will serve as effective strategy for the understanding of the signaling network at Xenopus fertilization.
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Experimental procedures |
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Anti-phosphotyrosine mouse monoclonal antibody (PY99) was purchased from Santa Cruz (Santa Cruz, CA, USA). Antibodies against the recombinant extracellular domain (xUPIII ED, residues 1–191 of xUPIII) or carboxyl-terminal peptide (xUPIII CT, residues 244–265 of xUPIII) of xUPIII, a non-catalytic region of xSrc (residues 22–36 of xSrc1/2), a carboxyl-terminal sequence of mouse CD9 (residues 217–226 of mouse CD9) and the recombinant extracellular domain of xUPIb (residue 109–230 of xUPIb) were prepared according to the described methods (Harlow & Lane 1988; Sakakibara et al. 2005; Iwasaki et al. 2006). Monoclonal mouse antibodies against tubulin-ß and the FLAG epitope were obtained from Amersham Biosciences (Uppsala, Sweden) and Sigma-Aldrich (St. Louis, MO, USA), respectively. Rabbit polyclonal antibodies against the phosphorylated forms of Src (phospho-Tyr418 or phospho-Tyr529 in the human Src protein) were purchased from Oncogene Science (Cambridge, MA, USA). Protein A-Sepharose was from Amersham Biosciences. A Src-specific tyrosine kinase inhibitor PP2 (4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) was obtained from Calbiochem (San Diego, CA, USA), dissolved in dimethylsulfoxide and kept at –80 °C until use. Biotinylated cholera toxin B (CTB), horseradish peroxidase (HRPase)- or avidin-conjugated or unconjugated B subunit of cholera toxin, ganglioside GM1 and asialoganglioside GM1 were purchased from Sigma-Aldrich. Avidin-conjugated agarose beads and a chemical cross-linker dithiobis(succinimidyl propionate) (DSP) were purchased from Pierce (Rockford, IL, USA). Hydrogen peroxide (H2O2) was from Santoku Chemical Industries (Tokyo, Japan). Other chemicals were analytical grade and purchased from Sigma, Wako Pure Chemicals (Osaka, Japan), Peptide Institute (Osaka, Japan), or Nacalai Tesque (Kyoto, Japan).
Expression plasmids for xUPIb, xUPIII and xSrc
First strand DNA for the full-length xUPIb gene (777 bp) was isolated and amplified from the Xenopus ovarian mRNAs. Oligonucleotides used for amplification were: the sense primer, 5'-AGG ACA GGT GTT TCC CAT CTC TCA GGC AGG-3'; the antisense primer, 5'-GCC CAA AAG GCA TGT CCT TCA TGC TGG CCA-3', each corresponds to a part of the 5'- or the 3'-untranslated region of the xUPIb gene deposited in the nucleotide sequence database (accession number: BC041516). The amplified cDNA (909 bp) was purified and cloned into pCR2.1-TOPO vector (Invitrogen, Tokyo, Japan), and analyzed by DNA sequencer (3100 Genetic Analyzer, Applied Biosystems, Tokyo, Japan). Mammalian expression plasmid for xUPIb was prepared by polymerase chain reaction of the xUPIb DNA using pCR2.1-TOPO/xUPIb as a template, followed by subcloning of the amplified DNA into multi-cloning site of pCMVtag5A vector (Stratagene, CA, USA). Primers used were: the sense primer, 5'-CTG AAT TCA TGA AGG ACG ATT CTG GAG TTC-3': the antisense primer, 5'-GAA CTC TCG AGA TAC TCA ATG CGG GTG TAG-3'. The sense and antisense primers contained restriction site for EcoRI and XhoI, respectively, as indicated by underlines. The resulting plasmid pCMVtag5A/xUPIb expresses xUPIb tagged with 11 additional amino acids that contain the myc epitope, in the carboxyl-terminus (total amino acid residue of 270). Pfu DNA polymerase (Stratagene) was used for amplification of cDNAs. To express xUPIb as a FLAG-tagged protein, pCMVtag5A/xUPIb was digested by XhoI. The 3'-cohesive end of the resulting fragment was filled up with Klenow fragment (Takara, Kyoto, Japan), and further digested with EcoRI. The digest containing the xUPIb gene with the 5'-EcoRI-cleaved site and the 3'-blunt end was inserted into p3xFLAG-CMV-14 vector (Sigma) that was cleaved with EcoRI and EcoRV. The resulting construct, p3xFLAG-CMV-14/xUPIb, was confirmed by DNA sequencing. A plasmid CMV containing DNA encoding the full-length xUPIII was prepared as described previously (Sakakibara et al. 2005). p3xFLAG-CMV-14 plasmids containing wild type xSrc2 gene (xSrc-WT) or kinase-active mutant of xSrc2 (xSrc-KA) were prepared as described (Sato et al. 2003; Sakakibara et al. 2005).
Animals, gametes and embryos
Adult Xenopus laevis females and males were purchased from local dealers. Maintenance of frogs, collection of eggs and sperm, removal of jelly water from eggs and jelly water treated sperm were carried out as describe previously (Sato et al. 2000b). Briefly, ovulation was induced in females by administration of human chronic gonadotropin (500 IU/animal, Teikokuzoki, Tokyo, Japan). Ovulated eggs were immediately washed with 1x DeBoer's buffer (DB, 110 mM NaCl, 1.3 mM KCl, 0.44 mM Ca2Cl, 5.7 mM Tris–HCl, pH 7.2). The eggs were kept at ambient temperature (18–22 °C), and used within 3 h of spawning. When required, the jelly coat was removed from eggs by incubation with an excess volume of 1x DB containing 2% cysteine and 0.06 N NaOH for 3 min at ambient temperature. The resulting jelly coat-free eggs were washed 5 times with 1x DB and subjected to further manipulations (see below). To obtain sperm, a pair of testes were surgically removed from males, immediately washed 3 times with 1x DB and macerated. The macerated testes were dispersed in fivefold volume of 1x DB and centrifuged at 100 g for 2 min at 4 °C. The supernatant was taken and further centrifuged at 1000 g for 5 min at 4 °C. The resulting pellet was resuspended with 1x DB and used as "sperm suspension" for further manipulations within 1 day of the preparation. When required, the sperm suspension at the specified sperm concentrations was mixed with equal volume of egg jelly water (Sato et al. 2000b). The mixture was brought to be pH 7.8 by the addition of a final concentration of 10 mM of HEPES–NaOH buffer, and was incubated at 21 °C for 5 min and gently rocked for 15 min at ambient temperature. After the incubation, the suspension was centrifuged at 1000 g for 3 min at 4 °C. The sperm pellet was washed once with 1x DB and centrifuged again. The resulting sperm was suspended in 1 mL 0.33x DB and designated as jelly water-treated sperm.
In vitro fertilization and extraction
In vitro fertilization was done by incubating the jelly coat-free eggs (monolayer) with the jelly water-treated sperm (0.4–2.0 x 105/mL) in plastic dishes, the bottom of which was coated with 2-mm layer of 1% agarose in 1x DB. Successful activation of eggs was scored by monitoring the occurrence of cortical contraction and first cell cleavage that occurred 15 and 90 min after insemination, respectively. For extraction, 1-mL packed volume of dejellied eggs (ca. 800 eggs) was homogenized in 2.5-mL extraction buffer A (20 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate, 10 mM 2-mercaptoethanol, 10 µg/mL leupeptin, 20 µM p-amidinophenyl)methane-sulphonyl fluoride hydrochloride (APMSF), pH 7.5) containing 1% Triton X-100 or 1% Brij 98 with a 7-mL Dounce tissue grinder (Wheaton, NJ, USA). The homogenates were incubated on ice for 10 min and centrifuged at 500 g for 10 min to remove debris and yolk platelets. The resulting supernatant was collected as "Triton X-100-solubilized egg extract." In some experiments, the effect of exogenous GM1 on fertilization was examined: various amounts (0–10 µg/mL) of GM1 or asialo-GM1, a structurally related negative control for GM1, were included in the insemination mixture. Alternatively, sperm alone or unfertilized eggs alone were preincubated with 10 µg/mL GM1. After the preincubation, the preincubated sperm and eggs were washed several times with 1x DB to remove excess GM1, and subjected to in vitro fertilization.
Cells, transfection, cell stimulation and extraction
Human embryonic kidney cell line 293 (HEK293) was kindly provided from Dr Kazuyoshi Yonezawa and maintained in Dulbecco's Modified Eagle's medium (DMEM) (Sigma) supplemented with 10% fetal calf serum at 37 °C in a humidified 5% CO2 atmosphere. Cells of 40%–50% confluence in 100-mm dishes were transfected with 2-µg plasmid DNA per dish for each expression construct using EffecteneTM reagent (Qiagen, Hilden, Germany) according to the manufacturer's protocol. After the transfection treatment (24 h), cells were washed twice with ice-cold phosphate-buffered saline (PBS) and added with 0.2 mL/dish of buffer A containing 1% Triton X-100 (see above). The cells/buffer mixture was collected and homogenized with a Dounce tissue grinder by 10–15 strokes, followed by incubation on ice for 3 min. The homogenates were then centrifuged at 500 g for 10 min to remove debris, and the supernatants were collected and further centrifuged at 150 000 g for 20 min. The resulting supernatants were collected as Triton X-100-solubilized whole cell lysate (WCL). In some experiments, transfected cells were serum-starved for 24 h and treated without or with H2O2 for the specified times. When detergent-insoluble membrane microdomains were prepared, the debris-free cell extracts were used without the 150 000-g centrifugation (see below).
Discontinuous sucrose-density gradient ultracentrifugation
Triton X-100-solubilized egg extract was subjected to preparation of detergent-insoluble membrane microdomains as described previously with a little modifications (Sato et al. 2002, 2006). The supernatant was collected and brought to 42.5% (w/v) sucrose by mixing with equal volume of ice-cold buffer A containing 85% sucrose. The resulting mixture (about 5 mL) was layered with 19-mL 35% (first) and 12-mL 5% sucrose (second) in buffer A. The samples were centrifuged at 144 000 g for 20 h in an SW28 rotator (Beckman, Fullerton, CA, USA). After the centrifugation, 3-mL aliquots of 12 fractions were collected from the top to the bottom of the tube. A similar method was employed when detergent-insoluble membrane microdomains were prepared from HEK293 cells. In this case, Triton X-100-solubilized cell extract (see above, about 400 µL), which had been prepared from two cell dishes of 100-mm diameter, was brought to 42.5% (w/v) sucrose by mixing with equal volume of ice cold buffer A containing 85% sucrose. The resulting mixture (about 0.8 mL) was layered with 2.6-mL 35% (first) and 1.6-mL 5% sucrose (second) in buffer A. The samples were centrifuged at 200 000 g for 20 h in an SW55 rotator (Beckman). After the centrifugation, 0.425-mL aliquots of 12 fractions were collected from the top to the bottom of the tube. In some experiments, detergent-insoluble membrane microdomain fractions (fractions 3–6) were combined and dialyzed against the buffer containing 20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate, 10 mM 2-mercaptoethanol with a dialysis membrane (MW 3500, Spectrum Medical Industries, Houston, TX, USA). The dialysis buffer was changed every 2 h for 3 times. The dialyzed fractions were centrifuged at 300 000 g for 1 h. The pellet was resuspended in 1.5 mL of buffer A and ultrasonicated for 1 min with ultrasonic disrupter (UD-201, TOMY, Tokyo, Japan). The suspension solution was centrifuged with 1800 g for 5 min to remove the debris. The supernatant was collected as "concentrated membrane microdomains" and used for further manipulations (see below).
Immunoprecipitation, SDS-PAGE, immunoblotting and silver stain
Pre-cleared protein samples (50–1000 µg, 1 mg/mL) were immunoprecipitated with an appropriate amount of antibodies as specified in the text for 3–6 h at 4 °C. After the incubation, samples were centrifuged at 10 000 g for 10 min at 4 °C and the supernatants were collected. The immune complexes in the supernatants were adsorbed onto 10 µL of protein A-Sepharose beads by gentle rotation for 30 min at 4 °C. The beads were washed 3 times with 500 µL of washing buffer containing 1% Triton X-100, 1% sodium deoxycholate, 0.15 M NaCl, 1 mM sodium orthovanadate, 10 µg/mL leupeptin, 20 µM APMSF and 50 mM Tris–HCl, pH 7.5. The washed beads were then treated with Laemmli's SDS sample buffer (Laemmli 1970) for 5 min at 98 °C. The SDS-denatured proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on 8%–12.5% gels and analyzed by immunoblotting or silver stain (Sato et al. 2002). In immunoblotting analysis, detection of the immune complex between proteins and the primary antibodies used was made by enzyme-linked color development with alkaline phosphatase (APase) (Cappel, Belgium) or horseradish peroxidase (HRPase) (Wako, Osaka, Japan) that was conjugated to the secondary antibodies. Silver stain of proteins in SDS gels was done by using the Bio-Rad Silver Stain Plus kit (Bio-Rad).
Cholesterol, GM1 and protein assays
All manipulations described here were performed at room temperature. Spectrophotometric measurement of cholesterol and protein was made at 592.5 and 595 nm, respectively, according to the described methods (Sato et al. 2002) using a Wako assay kit and a Bio-Rad assay kit, respectively. GM1 was detected by the dot-blotting method (Karacsonyi et al. 2004). Briefly, 3–5 µL of the samples was spotted on nitrocellulose filter (Bio-Rad, Japan, Tokyo, Japan) and air-dried for 2 h. The membrane was blocked with 0.5% bovine serum albumin (BSA) (Calbiochem) in buffer containing 20 mM Tris–HCl (pH 7.5), 110 mM NaCl and 0.05% Tween 20 (T-TBS) for 2 h, and then incubated with 140 ng/mL of HRPase-cholera toxin B (HRPase-CTB) for 2 h. After the membrane was washed by T-TBS, GM1 on the membranes was visualized by enzyme-linked chemiluminescence with use of an Amersham enzyme reaction kit (Amersham Biosciences, London, UK) and a LAS1000 fluoroimager (Fujifilm, Tokyo, Japan).
Analysis of GM1-xUPIII interaction
To evaluate physical interaction between GM1 and xUPIII, 100 µL of concentrated membrane microdomains was incubated in the presence of 1% Triton X-100 for 30 min at room temperature and then centrifuged at 7600 g for 10 min. The supernatant was collected and incubated with 10 µg/mL biotinylated CTB (biotin-CTB) for 2 h. After the incubation, 30 µL of avidin-conjugated beads (Pierce) was added into the mixture and incubated for 1 h. The beads were then washed 3 times with buffer A containing 1% Triton X-100. The washed gel was treated with Laemmli's SDS sample buffer for 5 min at 98 °C. The SDS-denatured proteins were separated by SDS-PAGE and analyzed by immunoblotting. Reconstitution of the GM1-xUPIII complex was done as follows. Anti-UPIII immunoprecipitates with an antibody against either xUPIII-ED or xUPIII-CT were prepared from Triton X-100-solubilized unfertilized egg extracts (100 µg, 1 mg/mL) as described above (see immunoprecipitation), and the immune complexes on protein A-Sepharose beads were incubated with 1 µg/mL GM1 for 20 h at 4 °C. After the treatments, the beads were washed 4 times with buffer A containing 1% Triton X-100, and were extracted 2 times by 400 µL of chloroform:methanol (ratio of 1 : 2, v/v). The extracted solution was collected and centrifuged at 7600 g for 5 min. The supernatant was lyophylized and dissolved in 30 µL of chloroform:methanol (ratio of 1 : 2, v/v). The GM1 in extract solution was detected by dot blotting as described above.
Surface biotinylation, chemical cross-linking and cholesterol depletion
Surface biotinylation of HEK293 cells expressing xUPIb and/or xUPIII was performed according to the described method (Mery et al. 2002) with little modifications. Briefly, untransfected or transfected HEK293 cells (see above) at full confluency were washed twice with PBS supplemented with 1 mM MgCl2 and 0.5 mM CaCl2 (PBS-MC). Cells were then incubated for 30–45 min at 4 °C with 0.5 mg/mL sulfo-NHS-biotin (EZ-LinkTM, Pierce). Biotinylation was terminated by rinsing the cells twice with PBS-MC containing 0.1% BSA and twice with PBS. The cell samples were extracted, separated by SDS-PAGE, and analyzed for the presence of biotinylated proteins with use of HRPase-conjugated avidin (ABC kit, Vector Laboratories, Burlingame, CA, USA). When the effect of cholesterol depletion was examined in transfected HEK293 cells, cells were washed twice with PBS and then treated with DMEM supplemented with 10 mM methyl-ß-cyclodextrin (MßCD) (Wako) for 30–45 min at 37 °C. After the treatments, cells were collected and washed twice with PBS and subjected to further manipulations. We also performed chemical cross-linking of Xenopus egg surface proteins. Briefly, monolayer of jelly coat-free unfertilized eggs (see above) were washed 3 times with PBS and gently rocked in the absence or the presence of 0.5 mM DSP for 20 min at room temperature. After the treatments, the eggs were washed 3 times with 50 mM 2-ethanolamine (pH 8.0) in PBS and subjected to further analysis.
In vitro protein kinase assay
The protein kinase assay was carried out as described previously (Sato et al. 2002). Briefly, 20 µL of concentrated detergent-insoluble membrane microdomains were incubated with or without jelly water-treated sperm (107 sperm/mL) for 2 min in the presence of 5 mM MgCl2, 1 mM ATP, 50 mM Tris–HCl (pH 7.5), 1 mM DTT for 2 min at 21 °C (total volume of 200 µL) and then incubated at 30 °C for 5 min for phosphorylation of proteins. The protein kinase assay was stopped by putting the assay mixtures on ice for 10 min and sodium orthovanadate was added to final concentrations as 1 mM. The assay mixtures were then centrifuged at 1000 g for 3 min to remove most sperm and the supernatant was centrifuged at 300 000 g for 1 h. The pellet was resuspended in Laemmli's SDS sample buffer and proteins was denatured at 98 °C for 5 min. Phosphorylated proteins were analyzed by SDS-PAGE and immunoblotting with anti-tyrosine phosphorylation antibody PY99. The effect of PP2 or CTB on sperm-dependent tyrosine phosphorylation was also examined as specified in the text.
Indirect immunofluorescent study
Immunostaining of Xenopus eggs and transfected HEK293 cells was done essentially as described previously (Sakakibara et al. 2005). Cells for immunostaining were cultured in glass-bottomed dishes of 35-mm diameter (MatTek, Ashland, MA, USA) and transfected with expression plasmids as described above. After 24 h of transfection, cells were washed 3 times with PBS, fixed with PBS supplemented with 4% paraformaldehyde for 10 min, permeabilized with PBS supplemented with 0.2% Triton X-100 for 2–5 min, and blocked with PBS supplemented with 30 mg/mL BSA for 1 h. Cells were then treated with primary antibodies of interest, diluted in the blocking solution, for 2 h. After the treatments, unbound primary antibodies were removed by washing with PBS (5 min x 3 times). Cells were then treated with secondary antibodies conjugated with either fluorescein isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (rhodamine), diluted in the blocking solution, for 1 h. After washing with PBS (5 min x 3 times), cell specimens were mounted in PBS containing 50 mg/mL 1,4-diazabucyclo[2,2,2]octane and 90% glycerol, and subjected to confocal laser scanning microscopic analysis by use of FV300 system (Olympus, Tokyo, Japan). The FITC was acquired at 488-nm argon laser excitation using 510-nm long-pass and 530-nm short-pass filters. The rhodamine signal was acquired at 543-nm helium-neon laser excitation using a 560–600-nm band-pass filter. Phase contrast images of cells were also taken under the same views. When egg surface GM1 was analyzed, FITC-labeled CTB (Sigma) was used.
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Acknowledgements |
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Footnotes |
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aContributed equally to this work.
bPresent address: Department of Applied Chemistry, School of Science, Northwestern Polytechnical University, Xi’an 710072, People's Republic of China
cPresent address: Department of Reproductive Biology and Pathology, National Research Institute for Child Health and Development, Setagaya-ku, Tokyo 157-8535, Japan
dPresent address: Division of Diabetes, Digestive, and Kidney Disease, Kobe University School of Medicine, Chuo-ku, Kobe 650-0017, Japan
* Correspondence: E-mail: kksato{at}kobe-u.ac.jp |
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Received: 8 September 2006
Accepted: 13 November 2006
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