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Genes to Cells (2005) 10, 345-356. doi:10.1111/j.1365-2443.2005.00838.x
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Generation of an antibody specific to Xenopus fertilized eggs by subtractive immunization

Keiichi Sakakibara1, Ken-ichi Sato2,*, Tetsushi Iwasaki2, Kazuyuki Kitamura3 and Yasuo Fukami1,2

1 Graduate School of Science and Technology, and 2 Research Center for Environmental Genomics, Kobe University, Kobe 657-8501, Japan
3 Yanaihara Institute, Fujinomiya 418-0011, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Here we report the generation and characterization of a monoclonal antibody, mAb 5H7-G1, which recognizes egg antigens in the animal cortex of fertilized, but not unfertilized, Xenopus eggs. The mAb 5H7-G1 was generated by subtractive immunization of mice: primary immunization with unfertilized egg extract followed by immunosuppression treatment with cyclophosphamide and repeated immunization with fertilized egg extract. In immunoblotting analysis, mAb 5H7-G1 recognizes multiple protein bands of fertilized (but not unfertilized or the ionophore-activated) Xenopus eggs. N-linked polysaccharide is most likely the target of mAb 5H7-G1 because immunoreactivity of mAb 5H7-G1 is effectively diminished when protein samples are treated with N-glycosidase F. Moreover, mAb 5H7-G1 recognizes some, but not all, tyrosine-phosphorylated proteins in eggs treated with H2O2, an artificial activator of the egg tyrosine kinase Src, suggesting that these proteins also contain N-linked sugars. When microinjected into fertilized Xenopus embryos, mAb 5H7-G1 causes a retardation or complete inhibition of first cell cleavage, suggesting that the mAb 5H7-G1-reactive antigens play an important role in this event. These results demonstrate that mAb 5H7-G1 is useful to analyze differential proteome display during fertilization and early development. More generally, subtractive immunization may work as a strategy to uncover cellular events that operate during different cellular conditions of interest.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Fertilization promotes a dynamic rearrangement of the structure and function of maternally stored components in unfertilized egg. It includes de novo synthesis or degradation, post-translational modifications and relocalization of cellular molecules such as proteins and mRNAs (Hardy 2002; Sato et al. 2004a). Enzymatic modulation, molecular interactions of egg membrane-associated molecules, or both are known to be important for sperm-induced egg activation events including transient Ca2+ release, which is necessary for the initiation of development (Runft et al. 2002; Stricker 1999). Subsequent Ca2+-dependent modulation of enzyme activity and degradation and synthesis of proteins play a key role in the transition of cell cycle from that of the gamete (metaphase II in most mammals and amphibians) to that of the somatic cell (Tunquist & Maller 2003). Recruitment of maternally stored mRNAs and its translational control have been implicated in acquisition for patterning and polarity of the early embryo (Groisman et al. 2002). On the other hand, fertilization and early embryogenesis involves only a limited or no genomic activity at the level of gene transcription. Gene transcription is not required for early embryonic development until the appropriate timing of the development. In the mouse, gene transcription from the zygotic genome is detectable after 10–12 h of fertilization. In Xenopus, zygotic gene transcription is taking place at the mid-blastula transition, which occurs about 4–5 h after fertilization. These facts tell us that cellular molecules that are pre-existing at the level of postgene transcription (i.e., proteins, mRNAs, etc.) organize and regulate functional cellular network for fertilization and early embryogenesis (Wickens et al. 2000).

While several kinds of cellular function at fertilization have been extensively analyzed, there may still be unidentified, unknown events, or both that serve as regulatory mechanism of fertilization. In this sense, global analysis of cellular function is a trend to investigate the cell system of interest as a whole. It includes genomics, transcriptome analysis and proteome analysis (Mann & Jensen 2003; Sato et al. 2002). However, such global and systematic analysis of the cell system often leads us to miss an important although minor event or molecule, which will be masked by those in a higher abundance. Under these circumstances, we have taken a unique approach to identify egg antigens associated with fertilization. We have generated a pool of monoclonal antibodies by immunizing mice, first with unfertilized egg extracts, and second with fertilized egg extracts. The first and second immunizations were intervened by treatment with the immunosuppressant drug, cyclophosphamide. This immunization scheme, called subtractive immunization (Matthew & Sandrock 1987; Ou et al. 1991; Williams et al. 1992; Zijlstra et al. 2003), has been expected to generate hybridoma clones that produce antibodies against fertilized egg antigens, which appear only after fertilization. In fact, we have obtained several clones that react specifically to fertilized egg proteins, but not to unfertilized egg proteins. Here we present generation and characterization of one monoclonal antibody, named mAb 5H7-G1. The mAb 5H7-G1 recognizes multiple egg antigens that are localized to the animal cortex of fertilized egg. Therefore, further experiments were designed to analyze developmental stage-dependent appearance, structural determinant or epitope, and biological function of the egg antigens recognized by mAb 5H7-G1.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
To obtain monoclonal antibody from Xenopus egg antigens that appear only after fertilization, mice were immunized first with extracts from unfertilized eggs. A subset of the immunized mice was then treated with the immunosuppressant drug cyclophosphamide (CP). We repeated this scheme four times in the same mice. Another subset of mice was immunized four times with extracts from unfertilized eggs without CP treatment. We examined whether immunosuppression is successful in CP-treated mice. In ELISA, all sera from normally immunized mice show a concentration-dependent response to unfertilized egg proteins (Fig. 1A, closed symbols). On the other hand, all sera from CP-treated mice do not show response to unfertilized egg proteins at any serum concentration (Fig. 1A, opened symbols), indicating that immunosuppression is successful. After clearing treatment of the drug, immunosuppressed mice were immunized several times with extracts from fertilized eggs (60 min post insemination). ELISA data demonstrate that all sera tested exhibit a concentration-dependent reactivity to fertilized egg proteins (Fig. 1B, closed symbols), but not to an equivalent amount of bovine serum albumin (opened symbols).



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  		Figure 1  Characterization of subtractive antibodies by ELISA. (A) Sera were prepared from four mice that had received four rounds of immunosubtraction with unfertilized egg extract and cyclophosphamide (CP) treatment (opened symbols) and from four others that had received the same scheme of immunization without CP treatment (filled symbols), and were assayed for reactivity with Triton X-100-solubilized Xenopus unfertilized egg extracts using ELISA at the indicated serial dilutions (1 : 500–1 : 500 000). (B) Sera were prepared from four mice that had received second immunization after CP treatment and its clearing, and subjected to ELISA at the indicated dilution points for either fertilized egg extracts (60 min post insemination) (filled symbols) or bovine serum albumin alone (opened symbols). (C-E) Purified IgG were obtained from three different hybridoma clones; 5H7-G1 (C), 7B4-E9 (D), and 8C8-B1 (E), and subjected to ELISA at the indicated IgG concentrations for unfertilized egg extracts (opened circles), fertilized egg extracts (closed circles), or bovine serum albumin alone (filled triangles). All data points are the means absorbance at 492 nm.

 
The results shown in Fig. 1A and 1B indicate that mice can produce antibodies against fertilized egg proteins after immunosuppression treatment. We performed hybridoma fusion using spleen cells from mice that had been immunosuppressed and immunized with fertilized egg proteins. We then screened hybridoma clones that produce antibodies. Twenty-four clones that produce antibodies out of more than 600 were selected and further characterized. Shown in Fig. 1C–1E are ELISA data of three different antibodies prepared from three independent hybridoma clones, named 5H7-G1, 7B4-E9 and 8C8-B1. All antibodies tested show a distinct pattern of dose-dependent response to fertilized egg antigens (opened circles), and do not react to bovine serum albumin (triangles). Unexpectedly, however, the same antibodies also recognize unfertilized egg antigens in a very similar dose-response to fertilized egg antigens (closed circles). Antibodies from 21 other clones show the same character (data not shown). At this point, it is not clear whether subtractive antibody to fertilized egg antigens is generated. There are two possibilities to explain this issue: one is that immunosuppression is somehow cancelled during the secondary immunization with fertilized egg extracts and the other is that ELISA analysis using egg extracts is not an appropriate method to identify subtractive antibody. Further characterization of one antibody with different methods concurred with the second possibility.

Figure 2 shows whole-mount, indirect immunofluorescent analysis of Xenopus eggs with antibody prepared from a hybridoma clone 5H7-G1 (hereafter, mAb 5H7-G1). The results demonstrate that the mAb 5H7-G1 signals, as visualized by FITC, are evident in fertilized egg (60 min post insemination, Fig. 2A), but not in unfertilized egg (Fig. 2B). Images obtained with serial optical sections demonstrate that the FITC signals in fertilized egg are a result of the binding of mAb 5H7-G1 through the entire cortical area in the animal hemisphere (Fig. 2Ab–2Ag, and Fig. 2Ca-2Cf). A similar result was also obtained from other hybridoma clone tested (mAb 8C8-B1, data not shown). Therefore, we proceeded to characterize further immunochemical property and biological function of mAb 5H7-G1.



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  		Figure 2  Spatial distribution of the mAb 5H7-G1 antigens in Xenopus egg before and after fertilization. Whole-mount immunocytochemistry was performed for (A) fertilized (60 min post insemination) and (B) unfertilized egg with mAb 5H7-G1 (1 µg/mL) as described in Experimental procedures. In each condition, a series of optical section (obtained with 50-µm intervals) from the bottom to the top of the dissected egg sample was visualized by fluorescent signal of FITC (b–g). Nomarski's images of the entire egg samples are shown in a. Bars are 250 µm. (C) Magnified images for a part of the animal hemisphere (a and b), the marginal zone (c and d), and the vegetal hemisphere (e and f) of fertilized egg specimens, as visualized by fluorescent signal of mAb 5H7-G1 and FITC (a, c, e) and Nomarski's interference (b, d, f). Bars are 62.5 µm.

 
We examined next whether the fertilized egg-specific mAb 5H7-G1 recognizes protein antigens. To this end, Triton X-100-solubilized whole egg extracts were prepared from unfertilized eggs, fertilized eggs (60 min after insemination), and H2O2-treated eggs (10 mM, 10 min). Normalized amounts of proteins were separated by SDS-PAGE (as revealed by protein staining, Fig. 3D) and analyzed by immunoblotting with several antibodies, including mAb 5H7-G1. As shown in Fig. 3, each of the mAbs 5H7-G1 (panel A), 7B4-E9 (panel B) and 8C8-B1 (panel C) recognize multiple proteins of ≥ 50 kDa in fertilized eggs. Although the three antibodies exhibited a different efficiency of binding, they shared a similar immunochemical property: (1) all mAbs can recognize some proteins in unfertilized eggs, but they bind more efficiently to fertilized egg proteins; and (2) all mAbs do not recognize proteins in H2O2-treated artificially activated eggs, where tyrosine phosphorylation of egg proteins is strongly stimulated (see Fig. 3E). These results demonstrate that mAbs 5H7-G1, 8C8-B1, and 7B4-E9 are actually subtractive antibodies against egg protein antigens, whose appearance is strongly augmented after fertilization, but not after H2O2-induced egg activation.



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  		Figure 3  Fertilization-specific Xenopus egg proteome display as revealed by immunoblotting analysis with subtractive antibodies. Triton X-100-solubilized whole egg extracts (30 µg protein/lane) were prepared from unfertilized (Uf), fertilized (F, 60 min post insemination), or H2O2-treated eggs (H, 10 min at 10 mM) and separated by SDS-PAGE on 8% polyacrylamide gels. Proteins were analyzed by immunoblotting with (A) mAb 5H7-G1 (1 µg/mL) and (B) mAb 8C8-B1 (1 µg/mL) (C) mAb 7B4-E9 (1 µg/mL), (D) staining with Coomassie Brilliant Blue, (E) anti-phosphotyrosine antibody (PY99, 1 µg/mL), or (F) normal mouse IgG (10 µg/mL), as described in Experimental procedures. Prestained molecular size markers are maltose binding protein (MBP)-fusion ß-galactosidase (175 kDa), MBP-fusion paramyosin (80 kDa), glutamic dehydrogenase (62 kDa), aldolase (47.5 kDa) and triosephosphate isomerase (32.5 kDa).

 
Results shown in Figs 2 and 3 led us to ask if the appearance of the mAb antigens is regulated in the course of egg fertilization. So, we performed immunoblotting analysis of Triton X-100-solubilized egg extracts prepared at different time points of insemination. As shown in Fig. 4A, multiple protein bands recognized by mAb 5H7-G1 become evident only after 40 min of insemination, while they are not so evident in unfertilized eggs and 20 min-inseminated eggs. We also determined whether mAb 5H7-G1 recognizes proteins of the Ca2+ ionophore-activated eggs. As shown in Fig. 4A, mAb 5H7-G1 do not show an efficient binding to Triton X-100-solubilized egg proteins at any time points of the Ca2+ ionophore treatment (0.5 µM A23187 [GenBank] ). The results suggest that antigens recognized by mAb 5H7-G1 appear in a sperm-dependent manner. One possibility is that sperm-derived molecules are a target of mAb 5H7-G1. However, mAb 5H7-G1 did not recognize any protein from sperm even at 1000 sperm per lane in immunoblotting (data not shown), arguing against this possibility (see Discussion).



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  		Figure 4  Time-dependent and fertilization-specific appearance of the mAb 5H7-G1 antigens. (A) Triton X-100-solubilized whole extracts (30 µg protein/lane) were prepared from Xenopus eggs at different times, either post insemination (0–90 min) or post A23187 [GenBank] treatment (20–60 min), and subjected to SDS-PAGE and immunoblotting with mAb 5H7-G1 (1 µg/mL). (B) Triton X-100-solubilized whole extracts from unfertilized (Uf), fertilized (F, 60 min post insemination), and A23187 [GenBank] -treated eggs (A, 60 min at 0.5 µM) were analyzed by immunoblotting with mAb 5H7-G1 before (WCL, 30 µg protein/lane) or after immunoprecipitation with the same antibody (IP, 300 µg protein/lane). Arrowheads indicate the positions of IgG heavy and light chains used for immunoprecipitation.

 
In Fig. 4B, we performed immunoprecipitation and immunoblotting of egg proteins with mAb 5H7-G1. Although multiple proteins of 50-175 kDa in fertilized eggs are efficiently recognized in direct immunoblotting of the whole cell lysates (WCL) (Fig. 4B), only a limited fraction of the mAb 5H7-G1-reactive antigens is recovered by immunoprecipitation (Fig. 4B, IP). At the same conditions, proteins from unfertilized eggs or the Ca2+ ionophore-activated eggs were not efficiently recognized by either immunoblotting or immunoprecipitation with mAb 5H7-G1 (Fig. 4B).

To determine whether egg antigens for mAb 5H7-G1 are functionally important in fertilization and early embryogenesis, we performed microinjection of the antibody. Results obtained are summarized in Table 1. When unfertilized eggs were injected with mAb 5H7-G1 at the egg cytoplasmic concentration of 40 µg/mL IgG, all injected eggs showed a normal rate of sperm-induced egg activation, judging from the occurrence of cortical contraction within 20 min of insemination. However, subsequent first embryonic cleavage, which should occur within 90 min of insemination, was 5-20 min delayed in some eggs (nine of 30 eggs). Moreover, when fertilized and activated eggs (25–40 min after insemination) were injected with the same antibody, more than 80% of eggs showed a delayed (five of 30 eggs) or a complete lack (19 of 30 eggs) of embryonic cleavage. These effects of mAb 5H7-G1 are specific and dose-dependent, as the 10 times lower amount of mAb 5H7-G1 IgG (4 µg/mL) or control IgG at 40 µg/mL is not inhibitory (Table 1, data not shown). These results suggest that the egg cytoplasmic molecule(s) containing the mAb 5H7-G1 epitope are important in first embryonic cleavage rather than in earlier events of sperm-induced egg activation. Similarly, mAb 8C8-B1, but not mAb 7B4-E9, showed an inhibitory effect on first embryonic cleavage when microinjected into fertilized embryos (data not shown). We think that the inability of mAb 7B4-E9 is a result of its relatively low immunoreactivity against fertilized egg antigens (see Fig. 3). It is possible that functional molecules containing the mAb 5H7-G1 epitope are exposed on the egg surface. Therefore, we examined the effect of adding mAb 5H7-G1 to the egg culture medium on sperm-induced egg activation and first cell cleavage. However, we did not observe any effect of mAb 5H7-G1 on the developmental processes at 40 µg/mL IgG (Table 1) or more (100 µg/mL, data not shown).


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  		Table 1  Summary of activation and first cell division of eggs bath-incubated or microinjected with mAb 5H7-G1 or normal mouse IgG
 
Recognition of multiple protein bands by mAb 5H7-G1 in immunoblotting indicates that post-translational modification rather than a certain amino acid sequence of egg proteins is likely to be the target of this antibody. However, it is also possible that the target is a certain amino acid sequence exposed as a result of multiple protein degradation. Among several post-translational modifications tested (phosphorylation, glycosylation, ubiquitination, nitration: data not shown), a certain type of glycosylation has been found to be a candidate epitope for mAb 5H7-G1. Shown in Fig. 5A is immunoblotting data of fertilized egg proteins with mAb 5H7-G1 before or after glycosidase treatments. The results demonstrate that binding of mAb 5H7-G1 to fertilized egg proteins is abolished when protein samples are treated with N-glycosidase F. This effect is not observed when protein samples are treated with endoglycosidase H or heat-inactivated N-glycosidase F (Fig. 5A). We also confirmed that after pretreatment of fertilized egg proteins with N-glycosidase F, mAb 5H7-G1 could no longer immunoprecipitate any antigenic proteins from the samples (data not shown). These results suggest that N-linked polysaccharides, not the high molecular weight mannose moiety, serve as an epitope of mAb 5H7-G1.



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  		Figure 5  Inhibition of immunoreactivity of mAb 5H7-G1 to fertilized egg antigens using N-glycosidase treatment. (A) Triton X-100-solubilized whole extracts from unfertilized (Uf) or fertilized (F, 60 min post insemination) eggs were treated in the absence or the presence of N-glycosidase or endoglycosidase H as described in Experimental procedures. Effect of heat-inactivated N-glycosidase, as indicated by +*, was also examined. Reaction products (30 µg protein/lane) were analyzed by immunoblotting with mAb 5H7-G1. (B) Triton X-100-solubilized whole cell lysates were prepared from unfertilized (Uf), fertilized (F, 60 min post insemination), or H2O2-treated eggs (H, 10 min at 10 mM), and immunoprecipitated (IP) with mAb 5H7-G1 (5 µg/mL) or anti-phosphotyrosine antibody (PY99, 10 µg/mL). The immunoprecipitates (300 µg protein/lane) as well as the whole cell lysates (WCL, 30 µg protein/lane) were separated by SDS-PAGE on 10% polyacrylamide gels and analyzed by immunoblotting with anti-phosphotyrosine antibody (PY99, 1 µg/mL). The position of a 42-kDa MAP kinase (MAPK) is indicated. Asterisks indicate the positions of protein bands that are immunoprecipitated with mAb 5H7-G1 and recognized by PY99 in H2O2-treated eggs.

 
Our previous study has shown that tyrosine phosphorylation of egg proteins via Src tyrosine kinase is important in sperm-induced egg activation and embryonic development in Xenopus eggs. So, we aimed to determine whether subtractive antibody mAb 5H7-G1 recognizes tyrosine-phosphorylated proteins in fertilized eggs. In Fig. 5B, total egg extracts from unfertilized, fertilized, or H2O2-activated eggs were analyzed by immunoprecipitation with mAb 5H7-G1 or anti-phosphotyrosine antibody PY99, followed by immunoblotting with PY99. The mAb 5H7-G1 immunoprecipitates of fertilized egg samples do not contain any detectable tyrosine-phosphorylated protein; however, those of H2O2-activated eggs contain at least two tyrosine-phosphorylated proteins at 90 kDa and 25-30 kDa. Specificity of the immunoprecipitation was verified by the fact that normal mouse IgG did not immunoprecipitate any tyrosine-phosphorylated protein from H2O2-activated egg samples (data not shown). Also, this binding is not a result of the direct binding of IgG to phosphotyrosine because a 42-kDa tyrosine-phosphorylated protein (MAPK) and some other tyrosine-phosphorylated proteins are not immunoprecipitated by mAb 5H7-G1 (Fig. 5B). Rather, mAb 5H7-G1 may immunoprecipitate N-glycosylated proteins that are tyrosine-phosphorylated, associate with tyrosine-phosphorylated proteins, or both.


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
Egg fertilization accompanies a number of biochemical and cell biological events that culminate in the creation of zygotic nucleus and initiation of embryonic development. Here we describe the generation and characterization of the monoclonal antibody, mAb 5H7-G1, which recognizes egg antigens that appear after fertilization of Xenopus eggs.

A unique immunization scheme, termed as subtractive immunization, has been employed in the present study to obtain the mAb 5H7-G1. As the means to detect potentially rare, localized, and unique antigens against a background of abundant, unlocalized antigens, subtractive immunization has been used successfully in several laboratories (for review see Zijlstra et al. 2003). It has been applied to several different levels of cell system: the whole cell level (Hooper et al. 2003; Sleeman et al. 1999; Wulf et al. 1996), wherein cell-surface antigens expressed specifically in tumor cells are detected; the subcellular localization level, wherein Xenopus egg proteins localized to the vegetal hemisphere but not in animal hemisphere or neuronal cell-specific antigens are detected (Denegre et al. 1997; Riggott & Matthew 1996); and the single protein molecule level, wherein two proteins with more than 90% identity of the amino acid sequence are discriminated (Sleister & Rao 2001, 2002). In all cases, donor mice are immunized first with the control antigens, immediately followed by treatment with the immunosuppressant drug, cyclophosphamide. After these treatments, the mice would have no immune response to the first antigens that act as tolerogens. Then the same mice are immunized with the second antigens that have very similar but distinct antigenic properties. According to this scheme, we have utilized unfertilized and fertilized egg extracts as the first and second antigens, respectively. To our knowledge, the present study represents the first application of subtractive immunization to analyze the whole cell system in a different time course of early development.

Twenty-four hybridoma clones out of more than 600 have been selected for the ability to produce a significant amount of antibody. In the ELISA analysis, all of them showed a similar immunoreactivity to the unfertilized and the fertilized egg extracts (Fig. 1). At this point, there is a concern that all these clones may fail to produce subtractive antibody specific to the fertilized egg antigens. However, further analysis by immunoblotting or immunocytochemical methods has demonstrated that eight out of 24 clones really produce specific antibody to fertilized egg antigens. This discrepancy may be because of one or more reasons indicated as follows: (1) unfertilized egg extracts contain a significant amount of target antigens, whose abundance becomes more evident in fertilized egg extracts and (2) both unfertilized and fertilized egg extracts share background materials, whose immunoreactivity is considerably high in the ELISA system but not in other methods. Whichever, we can conclude that the ELISA system may be too sensitive or inappropriate to evaluate the production of subtractive antibody to fertilized egg antigens.

We have identified some hybridoma clones whose mAbs recognize the cortical membrane area in the animal hemisphere of fertilized, but not unfertilized eggs. Immunoblotting of egg extracts with antibody mAb 5H7-G1 has demonstrated that the target antigens are recognized as multiple protein bands by immunoblotting, indicating that the target molecules of mAb 5H7-G1 are proteinous substances. Treatment of egg extracts with N-glycosidase F resulted in the loss of immunoreactivity of mAb 5H7-G1 to the egg proteins. This suggests that N-linked oligosaccharides are the target epitope for this antibody. It is also possible that mAb 5H7-G1 recognizes other epitope structure on the antigenic molecules containing N-linked oligosaccharides. However, we think this is unlikely because after the N-glycosidase F treatment, mAb 5H7-G1 does not recognize any protein bands in the lower molecular range, where at least a part of deglycosylated molecules should be recognized. Because of the difficulty in purifying the target molecules for mAb 5H7-G1, we do not know now the molecular structure of the N-linked oligosaccharides, which should be clarified by further investigation. Interestingly, all the other subtractive antibodies from different hybridoma clones also show very similar properties to the mAb 5H7-G1 as previously discussed. So, we note that the mAb 5H7-G1 antigens, which may involve N-linked oligosaccharides, seem to be highly immunogenic in fertilized eggs.

Immunoblotting analysis has also demonstrated that the mAb 5H7-G1 antigens are present in eggs for more than 40–90 min of insemination, but not in eggs of less than 20 min of insemination. This is consistent with our scheme of subtractive immunization, in which fertilized egg extracts of 60-min insemination have been used as the immunogens after immunosuppression. In Xenopus eggs, 40 min after insemination is the time period for inactivation of cytostatic factor, which consists of multiple protein complexes including a 42-kDa mitogen-activated protein kinase, and for the formation of zygotic nucleus via pronuclear fusion after DNA synthesis (for review see Sato et al. 2004b). Both events can be reproduced in unfertilized eggs by parthenogenetic activation with the calcium ionophore A23187 [GenBank] , which can cause artificial increase of intracellular Ca2+ and subsequent early developmental processes. Therefore, we have expected that the mAb 5H7-G1 antigens also appear in the eggs activated by A23187 [GenBank] . It is interesting to note that proteins prepared from the A23187 [GenBank] -activated eggs are not recognized by the mAb 5H7-G1. The results suggest that an insemination-specific, in other words, a sperm-dependent event is the target of this antibody.

This idea is potentially important because it is believed that sperm-induced egg activation and early developmental events can be essentially reproduced by artificial elevation of intracellular Ca2+ within unfertilized egg. On the other hand, only a limited series of early events of fertilization may be dependent on sperm function (e.g., sperm-egg contact or fusion and sperm incorporation). Further experiments will be necessary to determine whether the mAb 5H7-G1 antigens are related to fertilization-induced and Ca2+-independent cellular function of fertilized egg (see also below). Sperm-derived molecules by itself could also be a candidate for the target of the mAb 5H7-G1. We believe this is unlikely because immunoblotting analysis of extracts prepared from up to 1000 sperm did not show any visible band (data not shown). However, we cannot exclude the possibility that sperm-derived molecules are modified to be the target of the mAb 5H7-G1 during the fertilization processes (i.e., acrosome reaction and sperm-egg contact or fusion, both of which are difficult to analyze in vitro). There is a need to overcome a technical problem to analyze sperm components that will contribute to a differential display of egg or embryo components during fertilization.

The localization of the mAb 5H7-G1 antigens to the cortical membrane area in the animal hemisphere of fertilized eggs allows us to suggest its possible biological function. In unfertilized eggs of Xenopus, a marginal line between the animal and the vegetal hemispheres serves as an axis that defines a polarized nature of the eggs. It is well known that the vegetal hemisphere is enriched with several maternal components including mRNAs and proteins that contribute to the formation of germ lines as well as to the organization of somatic cell and tissue structures. On the other hand, the animal hemisphere provides a sperm entry point that defines a secondary, dorsal-ventral axis of the embryo. Although the appearance of the mAb 5H7-G1 antigens is rather slow (approximately 40 min post insemination), it could be important to sperm-egg interaction and subsequent embryonic polarization events. Supporting evidence of this idea has been obtained from microinjection experiments. The mAb 5H7-G1 caused a delay or complete block of first cell division in fertilized embryos. The results suggest that the mAb 5H7-G1 antigens are necessary components of this event. The inhibitory effect of the antibody is seen when microinjection is done after fertilization, but not before fertilization. This may be because the structure and function of mAb 5H7-G1 IgG injected into the egg cytoplasm cannot be maintained for a long time. A question as to which step of the first cell cleavage is impaired by the antibody remains to be answered.

It is noteworthy that the mAb 5H7-G1 could immunoprecipitate tyrosine-phosphorylated proteins from extracts of H2O2-treated eggs. In Xenopus eggs, sperm-dependent activation of tyrosine phosphorylation involving the tyrosine kinase Src is a critical step to initiate intracellular Ca2+ release and egg activation events (Sato et al. 2004b). Importantly, fertilization-induced tyrosine phosphorylation is a localized event to the cortical membrane area in the animal hemisphere, wherein sperm entry is taking place. Although sperm-induced tyrosine phosphorylation of egg proteins is very minor and hard to detect, we can use an H2O2 treatment of eggs as a parthenogenetic activation scheme of egg, in which egg activation is triggered from the point of tyrosine kinase activation (Sato et al. 2001). In fact, H2O2 treatment of eggs accompanies tyrosine phosphorylation of egg proteins as observed in fertilized eggs and almost replicates a series of events of egg activation. The mAb 5H7-G1 may recognize tyrosine-phosphorylated proteins via N-linked oligosaccharide moiety in the same molecule, the protein complex containing N-glycosylated proteins, or both and tyrosine-phosphorylated proteins. So, further study should determine whether the 5H7-G1 antigens in fertilized eggs also involves tyrosine-phosphorylated proteins.

In summary, we have demonstrated that a monoclonal antibody obtained with the subtractive immunization scheme can be used to analyze a fertilization-induced differential display of Xenopus egg proteins or egg proteome. Our present study has shed light on the implementation of subtractive immunization that can be a powerful means to generate antibodies unobtainable by standard immunization. We believe that the analysis of differential display of the Xenopus egg system, as shown in this paper, is an important issue for exploring functional proteomics of egg fertilization, which we term ‘fertilizome project’ (Sato et al. 2004a).


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Materials

African clawed frogs, Xenopus laevis, were purchased from Hamamatsu Seibutsu Kyozai (Hamamatsu, Japan). Frogs were maintained in polypropylene tanks filled with dechlorinated tap water or deionized water (1–1.5 L per animal) at 18–21 °C and fed twice a week. A23187 [GenBank] was obtained from Sigma (USA) and dissolved in dimethyl sulfoxide (DMSO). H2O2 was purchased from Santoku Chemical Industries (Tokyo, Japan). Protease inhibitors, leupeptin and (p-amidinophenyl)methanesulfonyl fluoride hydrochloride (APMSF), purchased from Peptide Institute (Osaka, Japan) and Wako Pure Chemicals (Osaka, Japan), respectively, were dissolved in water at 100 mg/mL and 100 mM, respectively, and kept at –30 °C until use. Mouse monoclonal anti-phosphotyrosine antibody PY99 was purchased from Santa Cruz (Santa Cruz, USA) and Sigma. Protein A-Sepharose CL-4B was from Amersham Pharmacia (UK). N-glycosidase F (Cat. no. 1365169) and endoglycosidase H (Cat. no. 1088726) were obtained from Roche (Mannheim, Germany). Unless otherwise indicated, other chemicals used were from Sigma, Wako, or Nacalai (Kyoto, Japan).

Eggs and embryos

Unless otherwise stated, all procedures were carried out at ambient temperature. To obtain eggs, Xenopus adult females were injected subcutaneously with 40 units of pregnant mare serum gonadotropin. After 3–6 days of the injection, the same animals were induced to ovulate with an injection of 500 units of human chorionic gonadotropin (Teikokuzoki, Tokyo, Japan). Ovulation began 6–8 h after the second injection. Eggs were washed three times with DeBoer's buffer (DB: 110 mM NaCl, 1.3 mM KCl, 0.44 mM CaCl2, pH 7.2–7.4, adjusted by NaHCO3) and then gently incubated with more than a 2-fold volume of DB supplemented with 2% cysteine and 0.06 N NaOH, pH 7.8 for 3–5 min. The resulting dejellied eggs were used within 2 h. Eggs were activated by insemination, H2O2, or A23187 [GenBank] , as previously described (Sato et al. 2000, 2001). After 5–90 min of the activation treatment, eggs were washed with DB, snap frozen in liquid nitrogen, and kept at –80 °C. Unfertilized eggs in the same batch of the preparation were also frozen, and kept at –80 °C as control.

Immunosubtraction and immunization

Antigen was prepared by extracting frozen eggs or embryos. All manipulations were conducted on ice or at 4 °C. Groups of eggs or embryos were mixed with 5-fold volume of ice-cold extraction buffer (1% Triton X-100, 20 mM Tris-HCl, 1 mM EDTA, 1 mM EGTA, 10 mMß-mercaptoethanol, 1 mM sodium orthovanadate, 10 µg/mL leupeptin, 20 µM APMSF, pH 7.5) supplemented with 150 mM NaCl and 250 mM sucrose and homogenized with a 7-mL Dounce tissue grinder (Wheaton, Millville, NJ, USA). The homogenates were centrifuged at 500 x g for 10 min, and the supernatants were collected and centrifuged at 150 000 x g for 20 min. The supernatants were collected and held at –80 °C until use, then thawed at room temperature. The protein concentration was determined by the Bradford dye-binding assay (Bio-Rad, USA). Antigens were diluted to a protein concentration of 4 mg/mL in extraction buffer and suspended in equal volume of phosphate-buffered saline and 3x volume of the Freund complete adjuvant (Difco), just prior to immunization. Seven-week old Balb/c mice (Yanaihara Institute, Fujinomiya, Japan) were injected intraperitoneally with 200 µL of the unfertilized egg antigen/emulsion (200 µg protein/animal), and then immediately injected with 200 µL of 12.5 mg/mL cyclophosphamide (Sigma, 2.5 mg/animal, 100 mg/kg body wt). Cyclophosphamide injection was repeated 24 and 48 h after immunization, and this drug was cleared for 2 weeks after the final administration. The successful immunosuppression by cyclophosphamide was verified by monitoring the antibody titre in mice 10 days post immunization, with enzyme-linked immunosorbent assay (see succeeding text). Mice were then immunized with the Freund complete adjuvant containing fertilized egg extract (60 min post insemination, 300 µg protein/animal) by intraperitoneal injection. Four or five booster immunizations followed in 2-week intervals, with a final boost the next 2 weeks, using 150 µg/animal/boost of fertilized egg extract. Spleens were harvested from mice 3 days after the final boost. We performed the first fusion and selection of hybridomas using the myeloma cell line P3U1. Isolated spleen cells and the P3U1 cells were washed two times in RPMI medium (Sigma) and mixed together at a 10 : 1 ratio of cell number. The mixture was centrifuged at 800 r.p.m. and the cell pellets were added with excess volume of 50% (w/v) polyethylene glycol (MW: 1450, Sigma). After 3-min incubation at 37 °C, the mixture was centrifuged at 800 r.p.m. for 5 min, and the cell pellets were suspended in HAT medium (Sigma) at 1-1.5 x 106 spleen cells/mL. After 12 days of incubation in HAT medium at 37 °C, culture media were collected and subjected to enzyme-linked immunosorbent assay for selection of the antibody-productive clones.

Enzyme-linked immunosorbent assay (ELISA)

Hybridoma clones with the antibody-producing ability were selected using ELISA, which was done essentially as described earlier (Harlow & Lane 1988). Specifically, extracts from either unfertilized or fertilized eggs were diluted in 0.1 M NaHCO3 to give a final concentration of 1 µg/mL, and were applied to a 96-well plate (Sepaplate 8FH or ELISA Plate H, Sumilon) for one night at 4 °C. After the overnight incubation, wells were blocked with BlockAce (Dainihon-seiyaku, Japan) for another night at 4 °C. Wells were washed three times in phosphate-buffered saline containing 0.05% Tween 20 (PBS-T). Media from hybridoma cell culture were directly applied to the washed wells at 100 µL/well, while sera from the positive mice, as positive controls, were applied at a dilution series of 1 : 500–500 000 in PBS containing 0.1% BSA. The incubation proceeded for 2–3 h at room temperature and terminated by the four-time wash with PBS-T. Horseradish peroxidase-conjugated anti-mouse IgG (Cappel, Germany) was then applied to wells at a dilution of 1 : 5000 in PBS containing 0.1% bovine serum albumin (BSA) 100 µL/well. The incubation proceeded for 2–3 h at room temperature and terminated by the four-time wash with PBS-T. The antibody-binding was visualized by adding a development solution containing 66 mM NaHPO4, 33 mM citric acid, 0.015% H2O2, and 1 mg/mL OPD compound (Sigma) (100 mL/well) for 10–30 min at room temperature, followed by the addition of 2 N H2SO4, a stop reagent. Colorimetric determination was done using a spectrophotometer set at 492 nm.

Immunoprecipitation

Protein samples (Triton X-100-solubilized egg extract, 100–500 µg protein) were incubated with the specified amount of antibody for 3–5 h at 4 °C. Immune complexes were collected by adsorption on to protein A-Sepharose. To remove nonspecifically bound materials, the Sepharose beads were washed three times with RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM Na3VO4, 10 µg/mL leupeptin, and 20 µM APMSF). The washed beads were then used for SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting (see succeeding text).

SDS-PAGE and immunoblotting

Protein samples (Triton X-100-solubilized egg extracts with or without immunoprecipitation) were mixed with a concentrated SDS sample buffer (Laemmli 1970) and boiled for 3 min. The SDS-treated proteins were separated by SDS-PAGE using 8% polyacrylamide gels and transferred to polyvinylidene difluoride membranes using a semidry blotting apparatus (Bio-Rad). Membranes were blocked with T-TBS buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl and 0.05% Tween 20 supplemented with 3 mg/mL bovine serum albumin for 1 h and then incubated for 2–4 h with culture media obtained from the hybridoma clones (diluted 1 : 1 in T-TBS) or from a mouse monoclonal or rabbit polyclonal antibody (diluted to 1 µg/mL IgG in T-TBS) as specified in the text. After the primary antibody treatment, the membranes were washed with T-TBS and then incubated for 1 h with alkaline phosphatase-conjugated goat polyclonal anti-rabbit IgG antibody (Cappel) at a dilution of 1 : 1000. When culture media or mouse antibody is used as the primary antibody, the membranes are treated for 1 h with rabbit polyclonal anti-mouse IgG antiserum (Cappel) at a 500-fold dilution before the treatment with the enzyme-conjugated antibody. Immune complexes were visualized by incubating the membranes with buffer containing 0.1 M Tris-HCl, pH 9.5, 5 mM MgCl2, 0.1 M NaCl, 50 µg/mL 5-blomo-4-chloro-3-indolyl phosphate p-toluidine salt and 150 µg/mL nitro blue tetrazolium. The color development proceeded for 15 min and was terminated by washing of the membranes with deionized water.

Whole-mount immunocytochemistry

Dejellied unfertilized eggs and fertilized embryos were fixed in methanol at –20 °C overnight. Fertilized embryos were collected at 60 min post insemination, and the successful fertilization of the same batch of eggs was verified by observing the first embryonic cleavage at 90 min post insemination. The fixed samples were rehydrated in a series of excess volume of methanol : PBS-T (9 : 1, 8 : 2, 7 : 3, 5 : 5, 2.5 : 7.5, 1 : 9 and 0 : 10) for 20 min at room temperature in each rehydration series. The rehydrated samples were further washed two times with PBS-T for 20 min, and then incubated in primary antibody solution containing the specified amount of monoclonal antibody, PBS-T, and 1% foetal calf serum, overnight at 4 °C. After wash treatment with PBS-T (60 min x 2), samples were incubated in secondary antibody solution containing fluorescein isothiocyanate (FITC)-conjugated goat polyclonal anti-mouse IgG antibody (Cappel) at a dilution of 1 : 1000, PBS-T and 1% foetal calf serum, overnight at 4 °C. After wash treatment with PBS-T (60 min x 2), samples were incubated in methanol (40 min x 4) and were then cleared for optical sectioning by two cycles of 10-min incubation in Murray's clear solution containing 1 : 2 volume of benzyl alcohol and benzyl benzoate. Whole-mount specimens were mounted in Murray's clear solution and kept under dark conditions. Fluorescence was observed with a confocal laser-scanning microscope (Model LSM410 Invert, Carl Zeiss, Germany) at 488-nm argon excitation using a 515-nm long-pass barrier filter. The same sample was also observed using a microscope equipped with Nomarski interference.

Other methods

Purification of IgG was carried out according to the method of Harlow & Lane (1988). Deglycosylation by using N-glycosidase F or endoglycosidase H was carried out according to the manufacturer's instruction. Microinjection of eggs or embryos with IgG was performed according to the method previously described (Sato et al. 2000). Sperm-induced activation and first cell cleavage of egg or embryo were scored under a dissecting microscope as described (Sato et al. 1998).


   Acknowledgements
 
We thank Misato Teraguchi for making our works smooth. This study was partly supported by the ‘Priority-C Area’ grants (12202027, 13202038) from the Ministry of Education, Culture, Sports, Science and Technology of Japan to K.S.


   Footnotes
 
Communicated by: Yoshimi Takai

* Correspondence: E-mail: kksato{at}kobe-u.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
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Received: 23 September 2004
Accepted: 21 December 2004




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