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Proteins of the sea urchin egg vitelline layer
DEVELOPMENTAL
BIOLOGY
102, 390-401 (1984)
Proteins of the Sea Urchin Egg Vitelline Layer H. L. NIMAN,* B. R. HOUGH-EVANS,?V. D. VACQUIER,* R. J. BRITTEN,~ R. A. LERNER,* AND E. H. DAVIDSON-~ *Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, Cal@rnia 9.X%37, ~Division of Biology, California of Technology, Pasadena, California 91125,and *Marine Biology Research Division, Scripps Institution of Oceanography, University of CalQbrnia, San Diego, La Jolla, Cal$brnia 92093 Received June SO,1983;accepted in revised form November
Institute
18, 1983
The vitelline layers (VL) of unfertilized sea urchin eggs were isolated, and the diversity of their polypeptide constitutents estimated by two-dimensional polyacrylamide gel electrophoresis. At least 25 components are reproducibly observed. While VL polypeptides are almost certainly synthesized in the growing oocyte, they are not among the more prevalent newly synthesized proteins detected in oocytes that were isolated and labeled in vitro for 4 hr. A set of monoclonal antibodies was raised against VL components and partially characterized. The 31 monoclonals analyzed fell into 11 classes with respect to their avidity for VL proteins solubilized under mild and under strongly denaturing conditions, and to their reactions with surface components of the VLs of living eggs. Fluorescence microscopy showed diverse patterns of surface reactivity when different monoclonal antibodies were compared. Two of the monoclonal antibodies reacted with specific sets of three proteins each on VL protein blots. It is concluded that the VL is a complex structure containing a large number of different polypeptide components, the genes for several of which should now be experimentally accessible.
INTRODUCTION
The vitelline layer (VL) of the sea urchin egg is an extracellular glycoprotein layer that coats the outer surface of the plasma membrane. During their growth phase in the ovary, sea urchin oocytes lack follicle cells, and it is likely that the components of the VL are synthesized and secreted by the oocytes themselves. In this respect the sea urchin egg VL differs from the extracellular coats that protect the oocytes and eggs of many other animals, e.g., the chorion of the silk moth egg (Kafatos et al, 197’7;Mazur et al, 1980), or the Drosophila egg (Spradling et al., 1980), which are formed in place from secretions of contiguous follicle cells. However, growing oocytes have been shown to synthesize major components of their own vitelline layers in several species. For example, the oocytes of the ascidian Ciona synthesize several fucose binding proteins that are incorporated into their VLs (Rosati et aL, 1982), and, similarly, growing mouse oocytes synthesize the major glycoprotein of the analogous structure, the zona pellucida (Greve et al, 1982). The oocyte of the starfish Astropecten also appears to secrete its VL (Santella et aL, 1983). Both ultrastructural and biochemical data indicate the considerable complexity of the sea urchin egg VL. Viewed by scanning electron microscopy (Tegner and Epel, 1973, 1976; Veron et al, 1977), the VL displays a regular, closely packed array of small (0.3 pm) projections, which are casts of the underlying microvilli of the egg surface. In section the VL appears to consist of an outer sheet about 100-200 A thick, connected to the
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plasma membrane by closely spaced processes called “vitelline posts” (Kidd, 1978). In quick freeze-deep etch preparations the VL is seen as a dense, irregular network of relatively coarse fibers (Chandler and Heuser, 1980). Immediately following fertilization the vitelline posts disappear and the VL is elevated away from the egg surface. The coarse fibers are replaced by a thicker layer of fine fibers, to which is applied on both inside and outside surfaces a close, regular array of macromolecules deriving from the paracrystalline protein released from the cortical granules (Chandler and Heuser, 1980; Villacorta-Moeller and Carroll, 1982; Carroll and Endress, 1982; Bryan, 1970; Kay et al, 1982). The VL can be disaggregated in 1 M urea, and is hydrolyzed by trypsin (Showman and Foerder, 1979). Glabe and Vacquier (1977) found that VLs isolated from unfertilized eggs are composed of 90% protein, 3.5% carbohydrate, and 0.43% sulfate and that glutamic and aspartic acids together account for 25% of the VL amino acids. Their analysis of solubilized VL proteins using SDS-polyacrylamide gel electrophoresis reveals numerous protein bands, ranging in molecular weight from about 15,000 to >200,000 ds. The functions of the VL are also complex. It provides a continuously expanding, protective, external surface for the egg membrane throughout oogenesis, as it is found even in small (30 pm) oocytes (our observations). Its properties are such as to permit transport of micro and macromolecules into the growing oocyte. In fertilization the initial role of the VL is to provide binding sites for sperm attachment, after the acrosome reaction
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40,5% 2-mercaptoethanol, to which 1% SDS was added to prevent protein degradation. Discrete, reproducible patterns of VL proteins could be obtained only when this amount of SDS was included. Protein was estimated using the procedure of Bradford (1976). In later preparations, 10 mM benzamidine, 3% aprotinin, and 1 mM PMSF were added to all media to reduce protein degradation. Protein gel electrophwesis. Two-dimensional electrophoresis of VL proteins was carried out by the method of O’Farrell(l975). The range of the isoelectric focusing gels used for the first dimension was pH 4-7. The gradient was measured as follows. The gel was sectioned into l-mm slices which were eluted with water. The pH of the eluate was measured with a pH meter. SDS-polyacrylamide gels (12.5%) were used for the second dimension. Labeling oocyte proteins. Ovaries containing developing oocytes were washed in MFSW, cut in small pieces, and stirred in an ice bath in a small volume of MFSW. Aliquots of the mixture were placed in cold MFSW (in an ice bath) under the dissecting microscope, and oocytes sorted by hand using a hair loop. Oocytes (50-150) of the same approximate diameter (30,40,50,60, or 70 pm) were placed in 50 ~1 MFSW containing approximately 300 &i/ml [%S]methionine (specific activity 800 Ci/ mmol) and incubated 4 hr at 15°C [the VL proteins contain about 3% methionine (Glabe and Vacquier, MATERIALS AND METHODS 1977)]. The mixture was dissolved in O’Farrell’s (1975) lysis buffer. Preparaticm of vitelline layers. Vitelline layers were prepared from unfertilized eggs, essentially by the Preparation of monoclonal antibodies; immunization method of Glabe and Vacquier (1977). Eggs were ob- and fusion. The exact immunization protocol used for tained by injection of l-2 ml 0.5 M KC1 into gravid the strain 129 mice, and for fusion of their spleen cells female sea urchins (Strong¢rotus purpuratus). The has been described (Niman and Elder, 1980,1982a). The eggs were washed twice by settling in Millipore-filtered mice were immunized intraperitoneally with 50 pg of seawater (MFSW, pH 8), resuspended in MFSW pH 5 soluble VL protein in complete Freund’s adjuvant preto remove egg jelly, and washed again 3X in MFSW, pared as follows. VLs were suspended in 3 volumes of pH 8. One milliliter of washed eggs (2.9 X lo6 eggs) was 0.5% NP-40 in phosphate-buffered saline (PBS) and sonresuspended in 25 ml isolation medium (30 mM sodium icated. Solubilized VL protein, at a concentration of apacetate (pH 6), 20 mM EDTA, 0.4% Triton X-100, 0.1 proximately 1 mg/ml, was mixed with an equal volume mg/ml soybean trypsin inhibitor (SBTI), 1 mM phen- of complete Freund’s adjuvant. Two weeks later an adylmethylsulfonyl fluoride (PMSF)); the eggs were ho- ditional 50 pg of VL protein in 5 mg/ml alum was admogenized in a Thomas homogenizer (glass vessel, Teflon ministered intraperitoneally. This protocol produced pestle), 5-10 strokes until all were disrupted. The VLs readily detected antibody in the sera of these mice 1 were sedimented in a tabletop centrifuge at 1400g for week after the second injection. One to two months after 3 min, and washed by centrifugation 3X in isolation the second injection, 50 pg of VL was administered inmedium. After the third wash, the VLs were sedimented travenously. Five days later the spleen was removed. and after removal of the supernatant, the fluffy VL layer The red blood cells in the spleen cell suspension were was pipetted off, leaving a dark pellet of contaminating lysed in 0.1 M NH&l in 10 mM Tris pH 7.2 at 4°C for nuclei and pigment granules. Isolated VLs were stored 10 min. The nucleated cells were mixed with 2 X 10’ SPin “VL saline” (0.15 M NaCl, 0.2% Na azide, 0.1 mg/ml 2/O myeloma cells which had been washed with miniSBTI, 10 mM Tris, pH 7.4) or dissolved immediately in mum essential medium (MEM) supplemented with 10% lysis buffer A of O’Farrell (1975), 9.5 M urea, 2% NP- fetal bovine serum (FBS), followed by a wash with MEM
has been triggered by the egg jelly (Shapiro et aL, 1980; Monroy and Rosati, 1983). A high-molecular-weight glycoprotein fraction with sperm binding activity has been identified on the sea urchin egg surface (Tsuzuki et aL, 197’7; Schmell et aL, 19’7’7;Glabe and Vacquier, 1978; Rossignol et CAL,1981; Glabe and Lennarz, 1981; Kinsey and Lennarz, 1981), but as yet the specific glycopeptides involved in sperm binding have not been characterized. During the first 3 min after sperm-egg fusion the VL serves as a substrate for various cortical granule enzymes, including vitelline delaminase, which is responsible for elevation (Carroll and Epel, 1975), and a peroxidase (Foerder and Shapiro, 1977) that results in rapid hardening. The VL also provides an array of binding sites for the paracrystalline protein. Throughout early development the fertilization envelope functions as a freely permeable, but extremely tough, elastic container that protects the spherical embryo from compression or other physical insult. At the blastula stage it dissolves in a specific proteolytic reaction mediated by a soluble hatching enzyme (Schuel, 1978). We are interested in the VL as a structurally and developmentally discrete complex of proteins produced during oogenesis. In this paper we present additional evidence regarding the diversity of this set of proteins, and we describe monoclonal antibodies that should be useful for isolation of the genes for the VL proteins.
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without FBS (the myeloma cells are routinely grown in the presence of 10Y4M 8-azaguanine). The nucleated spleen cells were mixed with the washed myeloma cells and pelleted. These cells were spread out and 200 ~1 of 30% polyethylene glycol (PEG) 1500 (Baker) in MEM was added. After a 2 min exposure to PEG, the cells were pelleted for 4 min. Two minutes later the PEG was removed and the cells were gently resuspended in MEM supplemented with 10% FBS. After overnight incubation at 37”C, the cells were pelleted and resuspended in 200 ml of HMT (hypoxanthine, 1.0 X 10T4il!I; methotrexate, 1.0 X 10e6M; thymidine 1.6 X low5 M) in MEM supplemented with 10% FBS. Cells were plated out into 40 microtiter plates (96 well, Falcon). Cells were fed weekly with one drop of HT (HMT without methotrexate). Macroscopic colonies were transferred to 24-well plates (Lindbro) and assayed for binding activity 4-8 days later. Antibody binding assay. Antibody to VL was detected using an ELISA assay as described (Niman and Elder, 1982b), with 25 pg of soluble VL protein, dried onto 96well microtiter plates. The protein was fixed with methanol (50 ~1, 5 min, 25°C). The plates were blocked for nonspecific adsorption by incubation at 37°C for 4-6 hr with 3% bovine serum albumin (BSA) in phosphatebuffered saline. The albumin was removed and each well was incubated overnight at 25°C with 25 ~1 of tissue culture supernatant. The plates were then washed 10 times with distilled Hz0 and then incubated with 25 ~1 rabbit anti-mouse K and X (Litton or Miles) diluted l/ 500 with 1% BSA in PBS. For isotype analysis, the wells were also incubated with various rabbit anti-mouse heavy chain sera. After 2 hr incubation at 37”C, the plates were again washed 10 times with distilled Hz0 and incubated with 25 ~1 glucose oxidase conjugated immunoaffinity purified goat anti-rabbit Ig diluted l/ 500 with 1% BSA. After 1.5 hr incubation at 37”C, the plates were washed 10 times with distilled Hz0 and incubated at 25°C with 50 ~1 of developing reagent [O.l M phosphate, pH 6.0, 1.2% glucose (mutorated), 10 pg/ ml horseradish peroxidase, and 100 pg/ml ABTS (Boehringer Mannheim) dye]. The optical density at 414 was read with a Titertech microscanner. Subcloning. Cultures which were positive on the ELISA assay (3- to lo-fold higher than the media control of the VL or BSA plate) were subcloned by limiting dilution. Cells were grown in a 1:l mixture of fresh HT and the culture fluid conditioned by the hybridoma. The cells were plated out in 96-well microtiter plates, transferred to 24-well plates and assayed as described above. Immunc&orescence method.s.Fresh, living eggs were dejellied by a 2 min exposure to seawater (pH 5.0) and washed into either seawater or a low ionic strength isosmotic medium consisting of 2 parts l’ M dextrose and 1 part seawater. The medium also contained 5 mM
sodium azide and 0.2 mg/ml soybean trypsin inhibitor. Two hundred microliters of a 4% v/v egg suspension was incubated 60 min at 23°C with 200 ~1 hybridoma culture supernatant in borosilicate glass tubes (12 X 75 mm). The eggs were then washed three times by settling through 4 ml portions of either seawater or fresh dextrose-seawater and the pellet resuspended in 200 ~1 of a 1:200 dilution of rhodamine-conjugated goat antimouse IgG (Cappel Laboratories). (The labeled antibody had been previously adsorbed for 30 min at a 1:lO dilution with a great excess of living dejellied eggs in dextroseseawater.) After 30 min the eggs were washed three times in 4 ml portions of fresh seawater or dextroseseawater and the final pellet resuspended in seawater or dextrose-seawater containing 5% formalin. Electrophoretic transfer and immunologic detection of VL proteins. Electrophoretic transfer and immunologic detection of VL proteins were carried out as described (Towbin et al, 1979). Approximately 20 pg of soluble VL protein per lane was used for polyacrylamide electrophoresis as described (Niman and Elder, 1980). The protein was transferred electrophoretically to nitrocellulose (Schleicher and Schuell) and soaked in 3% bovine serum albumin in phosphate-buffered saline for 1 hr at 25°C. The blots were incubated for 16 hr at 25°C in 5 ml. hybridoma tissue culture supernatant which had been diluted l/50 with 3% BSA, 0.1% Triton X-100. The blots were washed with 0.1% Triton X-100 in PBS for 20 min with 3 changes (100 ml each). The nitrocellulose was then incubated with 3% BSA for 5 min at 25°C followed by a 15 hr incubation at 25°C with peroxidase conjugated goat anti-mouse IgG (Cappel) diluted l/1000 in 3% BSA, 0.1% Triton X-100. The nitrocellulose was washed with 0.1% Triton in PBS for 20 min with 3 changes (100 ml each). The bound antibody was visualized by incubation in 10 mM Tris pH 7.4, 0.009% HzOz, 0.0025% 3,3’-dimethoxybenzidine dihydrochloride (Kodak). RESULTS
Protein Components of the Sea Urchin Egg Vitelline Layer Isolated vitelline layers were shown to contain a complex population of proteins when analyzed by polyacrylamide gel electrophoresis. Figure 1A shows the SDS single dimension separation, and Fig. 1B the twodimensional separation in the range of pH 4 to pH 7. The molecular weight and p1 of the most prominent spots detected by Coomassie blue are listed in Table 1. The gels clearly show the presence of at least 25 protein components in the VL. This observation has been repeated a number of times on different VL preparations, and the protein species listed in Table 1 are reproducibly observed.
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Sea Urchin Egg VL
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-31
PH 4
PH 7
FIG. 1. Electrophoretic display of VL components, stained with Coomassie blue. (A) SDS gel separation. Molecular weight markers indicated at the left (X10’) were phosphorylase B, BSA, and DNase. (B) Two-dimensional electropherogram carried out as described under Materials and Methods, with 150 pg VL protein. The protein spots are very similar to the circled areas of Fig. 2. TABLE 1 VITELLINE LAYER PROTEINS DETE(;TED BY TWO-DIMENSIONAL POLYACRYLAMIDE GEL ELE(;TROPHORESIS
Number 1 2 3 5O 6” 7 8” 9” 10A” 10B 11” 12” 13 14” 15” 16” 17” 18” 19” 20” 210 22 23 24 25
Approximate PI 4 5.5 4.6 5.5 4.5 5 4.2 5 4.2 6.3 5.5 5.8 4.8 4.9 5.1 5.9 5.2 5.5 6.1 5.5 5.3 5.9 6.0 6.1 5.5
a More abundant proteins.
Approximate molecular weight (XIOs) 240 160 150 140 120 130 110 110 90 90 89 80 75 75 75 74 72 72 62 60 55 55 54 53 53
Vitelline Layer Compments Are Not Among the Prevalent Newly Synthesized Proteins of the Oocyte The VL comprises about 1.2% of the total protein of the unfertilized S. purpuratus egg [calculated from results of Glabe and Vacquier (197’7)].VLs are present on small immature oocytes as well, and it is likely that formation of the VL during oogenesis requires accretion of newly synthesized VL proteins. Were the VL to consist of only a small number of protein species their synthesis in the oocyte should be easily detectable. Though a considerable amount of radioactivity can be incorporated by oocytes within a few hours of incubation in seawater containing r5S]methionine, we could not demonstrate that during labeling periods of this duration any newly synthesized VL proteins were assembled into the external VL structures. That is, when isolated, the VLs of living oocytes labeled in vitro failed to display incorporation of r5S]methionine into discrete proteins. An experiment in which the total [%]methionine-labeled proteins synthesized by 70-pm-diameter late vitellogenic oocytes were extracted from homogenates and displayed on two-dimensional gels together with authentic carrier VL proteins is shown in Fig. 2. Over 400 newly synthesized proteins are visualized in the autoradiogram, but few if any comigrate with the stained VL proteins. Results similar to those in Fig. 2 were obtained with 30, 40,50, and go-pm-diameter oocytes, and with unfertilized eggs. Nor could newly synthesized VL proteins be ob-
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FIG. 2. Prevalent newly synthesized proteins of the 70 pm oocyte. Living oocytes were isolated and labeled with [%]methionine for 4 hr; 62 oocytes were used in this experiment. Total incorporation into protein was 1.4 X lo5 cpm, of which 1.25 X lo5 cpm were loaded onto the gel together with 150 gg of unlabeled VL protein prepared separately as a carrier; 5 X lo4 cpm were recovered from the gel. (Most of the remaining input cpm failed to enter the second dimension). The gel was stained with Coomassie blue, dried, and autoradiographed. The black spots in the figure are the newly synthesized proteins resolved in the autoradiogram, and the circled areas represent the stained VL proteins (cf. Fig. 1). The arrow indicates a prominent newly synthesized protein, in which over 3700 cpm were incorporated. Note that the VL protein preparation, as indicated by the circled areas, includes none of this prevalent cytoplasmic protein.
served when small sections of ovary, rather than isolated oocytes, were incubated with [35S]methionine and tested in the same way. The multiplicity of VL proteins indicated in Fig. 1 suggests that on the average 4.8 X 10e4 of the oocyte proteins synthesized at any one time would be assigned to an average VL protein species (i.e., 0.012/25). The overall radioactivity recovered from the two-dimensional gel in the experiment of Fig. 2 was 5 X lo4 cpm. Expectation per spot for newly synthesized VL proteins, on the above assumption, is only about 24 cpm, compared to 100-3000 cpm for most of the proteins visualized autoradiographically and to general interspot radioactivity of 40 cpm per 2 mm2 over background. The result observed could be due in part to failure of modification reactions in the isolated oocytes, but the most likely explanation is simply that an insufficient number of cpm was incorporated per VL protein species.
Anti-VL Monoclmal Antibodies As synthesis of VL protein is evidently not easily detected, and none of the individual VL proteins has yet been characterized at the sequence level, we prepared a series of monoclonal antibodies at least some of which recognize VL components. These reagents should provide an alternative approach to the isolation of the VL protein genes. We now describe briefly the isolation and preliminary characterization of these antibodies, which were raised against VLs that had been sonicated and at least partially dissolved in NP-40 (see Materials and Methods). Hybridomas were obtained from three fusions. In the first fusion 73 cell lines were assayed for binding activity. Seven of these lines initially displayed activity, but only one was stable. The second fusion produced 105 cell lines, of which 16 were positive and seven were stable. The third fusion resulted in 224 testable
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Sea Urchin
cell lines of which 59 were positive and 33 were stable. Of the 41 stable cell lines, the 31 that displayed the highest titer and binding activity were further characterized. The isotype of the antibody produced by each hybridoma was determined, with the results listed in Table 2. The antibodies are heterogeneous in regard to isotype. Light chain frequencies were 84% K and 16% h, and heavy chain frequencies were 26% p, 3% y3, 39% yl, and 32% Yzb. An initial problem to be considered is whether the monoclonal antibodies recognize intrinsic VL determinants, as opposed to cytoplasmic contaminants. Convincing evidence that at least a majority of the 31 antibodies included in Table 2 indeed react with VL components was obtained in immunofluorescence experiments that indicated for each antibody the cytological locus of the reactive structures. Some of these reactions are illustrated in Fig. 5, and additional data have been presented elsewhere by Gache et aL (1983). Twenty-one of the 31 monoclonal antibodies display surface immunofluorescence as shown in Table 2. If the VL is first digested with pronase or trypsin as described by Gache et al. (1983), followed by extensive washing, 11 of these 21 antibodies now fail completely to react and four show very severely diminished reaction. A critical observation is that for all 21 of the antibodies that react with the surface structures of unfertilized eggs, the fluorescence elevates completely from the egg surface as the VL transforms into the fertilization envelope following activation with the ionophore A23187 or normal fertilization. Proteins of the outer egg surface that are incorporated in the fertilization envelope are by definition VL proteins. A preliminary classification of the 31 monoclonal antibodies was obtained by determining their ability to bind VL proteins that had been solubilized in two different ways. VLs that had been sonicated and then dissolved in NP-40 were used for immunization and initial screening of the hybridomas. An alternative and more strongly denaturing method was to dissolve the VLs in 8 M urea and then dialyze out the urea against PBS. The monoclonal antibodies were incubated with the two different preparations in an ELISA binding assay, as illustrated in Fig. 3. Figure 3A shows the dilution curve for the activity of monoclonal antibody X28G07, which bound equally well to the two different VL preparations. In contrast, as shown in Fig. 3B, antibody X27D02 binds significantly less well to the urea VL preparation. A more drastic effect is shown in Fig. 3C, where antibody X27CO9 can be seen to react well with the NP-40 solubilized VL, but not at all with VL dissolved in urea. We do not know whether the avidity of X27CO9 and X28G07 for the urea-solubilized VL determinants was
Egg
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VL
TABLE 2 SUMMARY OF CHARACTERISTICS OF ANTI-VI,
Isotype*
Binding pattern’
MONOCLONAL ANTIBODIES
Fluorescence low saltd
Fluorescence high salt”
Class
Name”
I
X25F09 X12G05
A A
+++ +++
II
W38F06 X27D08 X18FO8 X18ElO
A A A A
+++ +++ +++ +++
III
X2%06 X27B02 X28D05
A A A
IV
X28G07
A
+ + + -
-
v
XO5ClO X27D02 X23CO7 X19D07
B B B B
+++ +++ +++ +++
+++ +++ +++ +++
VI
X17FlO X23Cll WlOEll
B B B
+++ +++ +++
-
VII
X13G06 X08D06
B B
+ +
VIII
X24F05 X23Fll X15Gll
B B B
IX
x27co9 W37FlO
C C
+++ +++
X
X23F06
C
XI
X19E03 X26B02 X14F06 X25B09 X27B08 X29Dll
C C C C C C
+ -
+++ +++ -
-
+++ +++
-
-
a Hybridoma names beginning with W originated from the second fusion described in Results, while those beginning with X originated from the third fusion. b Isotype analysis was carried out as described under Materials and Methods. Abbreviations are K, kappa light chain; X, lambda light chain; p, IgM heavy chain; ~3, IgGa heavy chain; yl, IgGi heavy chain; y&,, IgGsb heavy chain. ‘See Fig. 3 for illustrations of the binding patterns denoted A, B, and C. d Surface immunofluorescence in low salt as shown in Fig. 5. BSurface immunofluorescence when incubated and washed in seawater.
decreased as a direct consequence of antigen denaturation, or for other reasons. It is, evident, however, that the three antibodies included in Fig. 3 recognize different determinants. Reactivity patterns determined similarly
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01 ANTIBODY
01334567
234567 DILUTION
1112”
I
FIG. 3. Differential binding of monoclonal antibodies to VL preparations solubilized by two different methods. Antibody binding was determined by the ELISA method, as described under Materials and Methods. Each monoclonal antibody preparation (A, X23G07; B, X27DO2; C, X27CO9) was assayed for binding to VL that had been sonicated and dissolved in NP-40. (+), VL solubilized with 8 M urea (a), or BSA (0).
are listed for all 31 hybridomas in Table 2. Ten of the 70 kDa. Antibody X19E03 (lane B) also reacted with cell lines produced antibodies that exhibited little dif- three different proteins of apparent mass 200, 95, and ference when reacted with VLs solubilized by the two 85 kDa. The multiple reactions observed in these blots could have been due to shared determinants, or possibly methods (pattern (A) of Fig. 3); 12 of the antibodies bound to urea solubilized VL proteins only about half to the presence of specific degradation fragments. The reactions observed for these antibodies can be tentaas well at maximum as to VL solubilized by sonication tively assigned, on the basis of relative mobility, to pro(pattern (B) of Fig. 3); and nine lost almost all binding teins 2,12, and 19 of Table 1 for antibody XOSDOG,and activity when reacted with the urea extract (pattern to proteins 1,lO (A or B), and 11 for antibody X19E03. (C) of Fig. 3). Clear identification of specific VL antigens by the nitrocellulose transfer procedure was obtained for two of L?$wential Reactions of Monoclinal Antibodies with the monoclonal antibodies. Initial attempts to immuSurface Components of Living Eggs noprecipitate specific labeled VL proteins failed because Hybridoma culture supernatants were incubated with these proteins display a strong tendency to form complexes that include most of the labeled components when unfertilized sea urchin eggs, and the reactions were viincubated with antibody. Two of the antibodies reacted sualized by fluorescence microscopy. Fifteen of the 31 with determinants that were stable to SDS denaturation antibodies exhibited bright surface fluorescence when and boiling in 2-mercaptoethanol, and so the antigens assayed under low salt conditions (+++ Table 2). Six which they recognized could be identified by nitrocelgave weak positive reactions (+). Figure 5 shows some lulose transfer. VL proteins were separated by gel elec- of the specific staining patterns produced by the various trophoresis, transferred to nitrocellulose, and reacted antibodies. The same patterns were observed with each with the hybridoma tissue culture supernatant as de- antibody in formaldehyde fixed as in living eggs (see scribed under Materials and Methods. In lane A of Fig. Gache et d, 1983). However, some variability is observed 4, antibody X08D06 is shown to react with three different from egg to egg even with a given antibody, as illustrated proteins, the apparent masses of which are 16580, and in panels G and H of Fig. 5. Although the antibodies
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antibodies. Seven of the 15 monoclonal antibodies that produced very strong surface fluorescence when reacted in low salt medium lost their activity when assayed under high salt conditions, i.e., seawater, while the reactivity of the remaining eight was unaffected. Gache et al. (1983) showed that all of these eight monoclonal antibodies block fertilization if reacted with the eggs prior to sperm addition, and that this effect is species specific. The antibodies interfere specifically with the sperm binding reaction. These eight monoclonals thus recognize VL components that are involved in the sperm binding reaction, such as the binding receptor protein, or contiguous structural elements. The effect of salt concentration on the binding of the monoclonal antibodies to the surface of the egg can also be used to classify the antibody activities. Taking into account both the in vitro assays of binding to solubilized VL proteins and their surface reactions with living eggs, the 31 monoclonal antibodies thus fall into 11 separate classes (Table 2). DISCUSSION
FIG. 4. Immunologic detection of VL proteins. Soluble VL protein was electrophoresed and transferred to nitrocellulose as described under Materials and Methods. Immunoreactive components were detected with peroxidase conjugates to the mouse antibody. Hybridoma supernatants were (A) XOSDOG,(B) X19E03, (C) X23CO’i’.Antibody X23CO7is included as an example of a null reaction. Molecular weight standards were myosin, 200,000,@-galactosidase,116,000,phosphorylase B, 94,000, bovine serum albumin, 68,000, and ovalbumin, 43,000.
used in panels C (X27D08), F (X12G05), and G or H (XlSFO8) gave similar binding curves when tested against soluble VL protein, the structures with which they react are obviously distinct. Furthermore, the hybridomas of panels D (X23CO7) and E (X19D07) which are not distinguishable by the binding assay, produce clearly different surface fluorescence patterns. The ionic strength of the medium was important in determining the surface reactivity of the monoclonal
Several estimates of the number of different S. purpuratus VL polypeptides have been offered. Carroll and Endress (1982) isolated that portion of the fertilization envelope that consists of the elevated VL by preventing assembly of the paracrystalline protein fraction released at exocytosis of the cortical granules. When dissociated, the elevated VL is found to be composed of eight prominent polypeptides. Since the procedure of Carroll and Endress relies on the normal process of proteolytic deamination of the VL, their preparations can be regarded as biologically “purified,” with respect to possible cytoplasmic contaminants. On the other hand, proteins such as those that constitute the “vitelline posts,” which connect the VL surface with the plasmalemma, might well be hydrolyzed or otherwise removed during the proteolytic deamination process. When isolated directly from unfertilized eggs by the method of Glabe and Vacquier (1977), essentially the method used here, the VL displays significantly more than eight components. Glabe and Vacquier reported 40 bands in one-dimensional SDS gels, and as shown in Table 1 of this paper a minimum of 25 reproducible polypeptides can be observed in two-dimensional analyses of solubilized VLs. We believe this is if anything a minimum estimate of the number of polypeptides for the following reasons: (a) Glabe and Vacquier showed that such preparations contain no more than about 2% contamination with other egg proteins; (b) addition of the protease inhibitors benzamidine, aprotinin, and PMSF, as well as soybean trypsin inhibitor did not reduce the number of spots observed
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FIG. 5. Patterns of monoclonal antibody binding to the vitelline layer of living eggs. (A) phase contrast micrograph of an egg treated with tissue culture supernatant from a hybridoma (Rz06B08) against Rauscher murine leukemia virus gp’70 (Niman and Elder, 1980). (B) Rhodamine fluorescence micrograph of A. (C)-(H) Fluorescence micrographs of eggs treated in low salt medium with hybridoma supernatants directed against the VL. (C) X27D08; (D) X23CO7; (E) X19D0’7; (F) X12G05; (G and H) X18F08. For photography of the egg surface, the fixed eggs were compressed by allowing the medium to evaporate; the surface against the overlayering coverslip was then photographed. Magnification was approximately X1060.
on two-dimensional gels, or affect their distribution; (c) when these gels were stained by the more sensitive silver procedure (Switzer et al, 1979) an even greater number of spots was observed; (d) the intensity and size of Coomassie blue stained spots (or bands, in SDS gels) were consistent and reproducible from preparation to preparation, and within a several fold factor of uncertainty, suggests that the number of molecules of the 15 more prominent proteins in the VL (i.e., those starred in Table 1) is roughly equivalent; (e) actin, a known cytoplasmic protein that is prevalent in eggs, and particularly in the egg cortex (reviewed by Vacquier, 1981) is completely undetectable in the VL protein preparation. We conclude that there are some 25 major VL compo-
nents, if not more, of which at least 8 are retained in the elevated VL fraction incorporated in the fertilization envelope. The complexity of the VL is implied in a completely different manner by the diversity of the set of monoclonal antibodies that we describe. Figure 5 illustrates six different patterns of surface reaction with the VLs of living eggs, and as shown in Table 2, many antibodies that react with dissolved VLs in ELISA fail to bind to the exposed surface of the living VL. A probable interpretation is that their determinants occupy internal positions within the VL. The effect of low salt is most likely due to the greater affinity of most antibodies in media of low ionic strength. Furthermore, Fig. 4 displays
NIMAN ET AL.
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Egg
VL
399
FIG. 5-Continued
reactions of two monoclonal antibodies with specific sets development is utilized for construction of subcellof three polypeptides. The remaining antibodies that ular, intracellular, and extracellular three-dimensional did not give clear blot reactions may include many that structures. It now seems likely that the VL of the sea urchin egg recognize carbohydrate rather than polypeptide deterwill provide another example of a complex structural minants. Three-dimensional extracellular structures may re- product, specific to a particular stage of the life cycle, oogenesis. The VL of the Xenopus egg is in some ways quire a relatively large amount of genomic information for their construction. This is suggested by the few ex- similar. It too is elevated following fertilization, to form amples of such structures that have been carefully an- a protective envelope, and Wolf et al (1976) have shown alyzed. The most germane example is the chorion of the that this VL contains at least 11 polypeptides, of which 10 are retained after elevation. Lewis et al. (1982) have moth egg. Kafatos and his colleagues have demonstrated that at least 180 distinct polypeptides are present in suggested that the Xenopus and sea urchin VLs are this structure (Regier et al, 1980). Similarly, the ubiq- functionally complex, because they undergo elevation uitous flagellum contains at least 120 diverse polypep- after fertilization. External egg coats that do not untides (Piperno et aL, 1977). Fifty polypeptides are in- dergo elevation often but not always contain fewer comvolved in the formation of the T4 phage coat (Wood and ponents. For example, the VL of Ciono contains three Revel, 1976). It may be that a significant fraction of the prominent components (Rosati et d, 1982) as does the protein-coding information being translated in early zona pellucida of the mouse egg (Bleil and Wassarman,
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1978,1980), and the VL of the abalone egg contains only four or five major components (Lewis et aL, 1982), while the zona pellucida of the pig egg contains three or four major and several minor proteins (Dunbar et aL, 1980). We assume that biosynthesis of VL polypeptides occurs in the growing oocyte, though as shown in Fig. 2 they are not sufficiently prevalent to be easily detected among newly synthesized proteins. As noted above, about 1.2% of the mature egg protein is included in the VL, or 0.47 ng of VL protein per egg. Were this quantity divided equally among 25 VL polypeptide components, there would be only about 19 pg of each component. Given about 6 weeks to complete oocyte growth and vitellogenesis (Gonor, 1973; Leahy et aL, 1978, 1981), during which most of the mass of the VL is formed only a small number of steady-state mRNAs would be required for the synthesis of each of the VL protein species. We conclude that a prominent structural component of the egg, the VL, is probably synthesized on a set of many different very low abundance messages. The fact that oogenesis is a slow process, requiring some weeks to complete, makes it possible for the oocyte to accommodate an enormous variety of such low prevalence messages, and yet to accumulate significant quantities of their products. We thank D. Schloeder for help with the isolation and maintenance of the hybridomas, and C. O’Connor for instruction in two-dimensional gel electrophoresis. The assistance of Patrick Leahy in the preparation of the VLs used for this study is gratefully acknowledged. E. J. Carroll, Jr. and E. Rothenberg provided helpful criticisms of the manuscript. This research was supported by NIH Grants BRSG-RR-07003, HD05753 (to E.H.D.), CA-25803 (to R.A.L.), and HD-12986 (to V.D.V). This is manuscript number 3105 IMM from the Research Institute of Scripps Clinic. REFERENCES BLEIL, J. D., and WASSARMAN, P. M. (1978). Identification and characterization of the proteins of the zona pellucida. J. CeU Bid 79, 173a. RLEIL, J. D., and WASSARMAN, P. M. (1980). Synthesis of zona pellucida proteins by denuded and follicle-enclosed mouse oocytes during culture in vitro. Pnx: Nat. Acud Sci USA 77.1029-1033. BRADFORD, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. And B&&em 72,248-254. BRYAN, J. (1970). The isolation of a major structural element of the sea urchin fertilization membrane. J. Cell Bid 44, 867-875. CARROLL, E. J., JR., and ENDRESS, A. G. (1982). Sea urchin fertilization envelope: uncoupling of cortical granule exocytosis from envelope assembly and isolation of an envelope intermediate from Shmgglocentrotus purpuratus embryos. Develop. Biol 94, 252-258. CARROLL, E. J., JR., and EPEL, D. (1975). Isolation and biological activity of the proteases released by sea urchin eggs following fertilization. Develop. Bid 44,22-32. CHANDLER, D. E., and HEUSER, J. (1980). The vitelline layer of the sea urchin egg and its modification during fertilization. A freeze-fracture
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study using quick-freezing and deep-etching. J. CeU BioL 84, 618632. DUNBAR, B. S., WARDRIP, N. J., and HEDRICK, J. L. (1980). Isolation, physicochemical properties, and macromolecular composition of zona pellucida from porcine oocytes. Biochemistry 19, 356-365. FOERDER, C. A., and SHAPIRO, B. M. (1977). Release of ovoperoxidase from sea urchin eggs hardens the fertilization membrane with tyrosine crosslinks. Proc. Nat. Auul L&i. USA 74,4214-4218. GACHE, C., NIMAN, H. L., and VACQUIER, V. D. (1983). Monoclonal antibodies to the sea urchin egg vitelline layer inhibit fertilization by blocking sperm adhesion. Exp. Cell Res. 147, 63-74. GLABE, C. G., and LENNARZ, W. J. (1981). Isolation of a high molecular weight glycoconjugate derived from surface of Shmg&cm~mtus purpuratus eggs that is implicated in sperm adhesion. J. Supramol Struct. Cell Biochern 15, 387-394. GLABE, C. G., and VACQUIER, V. D. (1977). Isolation and characterization of the vitelline layer of sea urchin eggs. J. CeU Biol 75,410-421. GLABE, C. G., and VACQUIER, V. D. (1978). Egg surface glycoprotein receptor for sea urchin sperm binding. Proc. Nat. Ad Sci USA 75, 881-885. GONOR, J. J. (1973). Reproductive cycles in Oregon populations of the echinoid Strolzgylocatrotus purpuratus (Stimpson). I. Annual gonad growth and ovarian gametogenic cycles. J. e~p. mur, Bid Ed 12, 45-64. GREVE, J. M., SALZMANN, G. S., ROLLER, R. J., and WASSARMAN, P. M. (1982). Biosynthesis of the major zona pellucida glycoprotein secreted by oocytes during mammalian oogenesis. Cell 31, 749-759. KAFATOS, F. C., REGIER, J. C., MAZUR, G. D., NADEL, M. R., BLAU, H. M., PETRI, W. H., WYMAN, A. R., GELINAS, R. E., MOORE, P. B., PAUL, M., EFSTRATIADES, A., VOURNAKES, J. N., GOLDSMITH, M. R., HUNSLEY, J. R., BAKER, B., NARDI, J., and KOEHLER, M. (1977). The egg-shell of insects: Differentiation-specific proteins and the control of their synthesis and accumulation during development. In “Results and Problems in Cell Differentiation” (W. Beermann, ed.), Vol. 8, pp. 45-145. Springer-Verlag, Berlin. KAY, E., EDDY, E. M., and SHAPIRO, B. M. (1982). Assembly of the fertilization membrane of the sea urchin. Isolation of a divalent cation-dependent intermediate and its crosslinking in vitro. CeU 29, 867-875. KIDD, P. (1978). The jelly and vitelline coats of the sea urchin egg: new ultrastructural features. J. Ultrastructure Res. 64,204-215. KINSEY, W. H., and LENNARZ, W. J. (1981). Isolation of a glycopeptide fraction from the surface of the sea urchin egg that inhibits spermegg binding and fertilization. J. CeU Biol 91, 325-331. LEAHY, P. S., TUTSCHULTE, T. C., BRIT~EN, R. J., and DAVIDSON, E. H. (1978). A large-scale maintenance system for gravid purple sea urchins (Strongylocentrotus purpuratus). J. Exp. ZooL 204,369-380. LEAHY, P. S., HOUGH-EVANS, B. R., BRITTEN, R. J., and DAVIDSON, E. H. (1981). Synchrony of oogenesis in laboratory-maintained and wild populations of the purple sea urchin (Strongylocentrotus purpuratus). J. Exp. Zoo1 215,7-22. LEWIS, C. A., TALBOT, C. F., and VACQUIER, V. D. (1982). A protein from abalone sperm dissolves the egg vitelline layer by a nonenzymatic mechanism. DeueZop. Bid 92,227-239. MAZUR, G. D., REGIER, J. C., and KAFATOS, F. C. (1980). The silkmoth chorion: Morphogenesis of surface structures and its relation to synthesis of specific proteins. Develop Bid 76, 305-321. MONROY, A., and ROSATI, F. (1983). Review article: a comparative analysis of sperm-egg interaction. Gamete Res. 7.85-102. NIMAN, H. L., and ELDER, J. H. (1980). Molecular dissection of Rauscher virus gp70 by using monoclonal antibodies: Localization of acquired sequences of related envelope gene recombinants. Proc Nat Acud Sci USA 77,4524-4528.
NIMAN, H. L., and ELDER, J. H. (1982a). Structural analysis of Rauscher virus gp’70 using monoclonal antibodies: Sites of antigenicity and P15(E) linkage. Virobgy 123,187-205. NIMAN, H. L., and ELDER, J. H. (1982b). mAbs as probes of protein structure: Molecular diversity among the envelope glycoproteins (gp7Os) of the murine retroviruses. In “Monoclonal Antibodies and T Cell Products” (David H. Katz, ed.) pp. 23-51. CRC Press, Boca Raton, Florida.
O’FARRELL, P. H. (1975). High resolution two-dimensional phoresis of proteins.
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PIPERNO,G., HUANG, B., and LUCK, D. J. L. (1977). Two-dimensional analysis of flagellar proteins from wild-type and paralyzed mutants of Chlamydomonos 1604.
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SCHMELL,E., EARLES,B. J., BREAUX, C., and LENNARZ,W. J. (1977). Identification of a sperm receptor on the surface of eggs of the sea urchin A. punctulata. J. Cell Biol 72,35-46,
SCHUEL,H. (1978). Secretory functions of cortical granules in fertilization and development: A critical review. Gamete Res 1,299-382. SHAPIRO,B. M., SCHACKMANN,R. W., GABEL, C. A., FOERDER,C. A., FARANCE,M. L., EDDY,E. M., and KLEBANOFF,S. F. (1986). Molecular alterations in gamete surfaces during fertilization and early development.
SHOWMAN, R. M., and FOERDER,C. A. (1979).Removal of the fertilization membrane of sea urchin embryos employing aminotriazole. Exp. Cell Res. 120,253-255.
SPRADLING,A. C., DIGAN, M. E., MAHOWALD,A. P., SCOTT,M., and CRAIG,E. A. (1980). Two clusters of genes for major chorion proteins of Drosophila
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SWITZER,R. C., III, MERRIL, C. R., and SHIFRIN, S. (1979). A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels. Anal Biochem 98, 231-237. TEGNER,M. J., and EPEL, D. (1973). Sea urchin sperm-egg interactions studied with the electron microscope. Science 179, 685-688. TEGNER, M. J., and EPEL, D. (1976). Scanning electron microscope studies of sea urchin fertilization. 1. Eggs with vitelline layers. J. Exp. Zool. 197,31-58.
REGIER,J. C., MAZUR,G. D., and KAFATOS,F. C. (1980). The silkmoth chorion: Morphological and biochemical characterization of four surface regions. Lkvelop. Biol. 76, 286-304. ROSATI,F., COTELLI, F., DE SANTIS, R., MONROY, A., and PINTO, M. R. (1982). Synthesis of fucosyl-containing glycoproteins of the vitelline coat in oocytes of Ciono intestinalis (Ascidia). Proc Nat Acud. Sci
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TOWBIN,H., STAEHELIN, T., and GORDON,J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Nat. Acad Sci USA 76, 4356-4354. TSUZUKI,H., YOSHIDA,M., ONITAKE, K., and AKETA, K. (1977). Purification of the sperm-binding factor from the egg of the sea urchin H. pulcherimua Biochem. Biophys. Res. Commun 75.502-511. VACQUIER,V. D. (1981). Dynamic changes of the egg cortex. Develop. Biol. 84, l-26.
VERON, M., FOERDER,C., EDDY, E. M., and SHAPIRO,B. M. (1977). Sequential biochemical and morphological events during assembly of the fertilization
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VILLACORTA-MOELLER, M. N., and CARROLL,E. J., JR. (1982). Sea urchin embryo fertilization envelope: Immunological evidence that soluble envelope proteins are derived from cortical granule secretions. L&u. Biol
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WOLF,D. P., NISHIHARA, T., WEST,D. M., SYRICK,R. E., and HEDRICK, J. L. (1976). Isolation, physicochemical molecular composition of the vitelline from Xenopus Levis eggs. Biochemists
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WOOD,W. B., and REVEL, H. R. (1976). The genome of bacteriophage T4. Bacterial Rev, 40, 847-868.
BIOLOGY
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Proteins of the Sea Urchin Egg Vitelline Layer H. L. NIMAN,* B. R. HOUGH-EVANS,?V. D. VACQUIER,* R. J. BRITTEN,~ R. A. LERNER,* AND E. H. DAVIDSON-~ *Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, Cal@rnia 9.X%37, ~Division of Biology, California of Technology, Pasadena, California 91125,and *Marine Biology Research Division, Scripps Institution of Oceanography, University of CalQbrnia, San Diego, La Jolla, Cal$brnia 92093 Received June SO,1983;accepted in revised form November
Institute
18, 1983
The vitelline layers (VL) of unfertilized sea urchin eggs were isolated, and the diversity of their polypeptide constitutents estimated by two-dimensional polyacrylamide gel electrophoresis. At least 25 components are reproducibly observed. While VL polypeptides are almost certainly synthesized in the growing oocyte, they are not among the more prevalent newly synthesized proteins detected in oocytes that were isolated and labeled in vitro for 4 hr. A set of monoclonal antibodies was raised against VL components and partially characterized. The 31 monoclonals analyzed fell into 11 classes with respect to their avidity for VL proteins solubilized under mild and under strongly denaturing conditions, and to their reactions with surface components of the VLs of living eggs. Fluorescence microscopy showed diverse patterns of surface reactivity when different monoclonal antibodies were compared. Two of the monoclonal antibodies reacted with specific sets of three proteins each on VL protein blots. It is concluded that the VL is a complex structure containing a large number of different polypeptide components, the genes for several of which should now be experimentally accessible.
INTRODUCTION
The vitelline layer (VL) of the sea urchin egg is an extracellular glycoprotein layer that coats the outer surface of the plasma membrane. During their growth phase in the ovary, sea urchin oocytes lack follicle cells, and it is likely that the components of the VL are synthesized and secreted by the oocytes themselves. In this respect the sea urchin egg VL differs from the extracellular coats that protect the oocytes and eggs of many other animals, e.g., the chorion of the silk moth egg (Kafatos et al, 197’7;Mazur et al, 1980), or the Drosophila egg (Spradling et al., 1980), which are formed in place from secretions of contiguous follicle cells. However, growing oocytes have been shown to synthesize major components of their own vitelline layers in several species. For example, the oocytes of the ascidian Ciona synthesize several fucose binding proteins that are incorporated into their VLs (Rosati et aL, 1982), and, similarly, growing mouse oocytes synthesize the major glycoprotein of the analogous structure, the zona pellucida (Greve et al, 1982). The oocyte of the starfish Astropecten also appears to secrete its VL (Santella et aL, 1983). Both ultrastructural and biochemical data indicate the considerable complexity of the sea urchin egg VL. Viewed by scanning electron microscopy (Tegner and Epel, 1973, 1976; Veron et al, 1977), the VL displays a regular, closely packed array of small (0.3 pm) projections, which are casts of the underlying microvilli of the egg surface. In section the VL appears to consist of an outer sheet about 100-200 A thick, connected to the
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8 1984 by Academic Press, Inc. of reproduction in any form reserved.
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plasma membrane by closely spaced processes called “vitelline posts” (Kidd, 1978). In quick freeze-deep etch preparations the VL is seen as a dense, irregular network of relatively coarse fibers (Chandler and Heuser, 1980). Immediately following fertilization the vitelline posts disappear and the VL is elevated away from the egg surface. The coarse fibers are replaced by a thicker layer of fine fibers, to which is applied on both inside and outside surfaces a close, regular array of macromolecules deriving from the paracrystalline protein released from the cortical granules (Chandler and Heuser, 1980; Villacorta-Moeller and Carroll, 1982; Carroll and Endress, 1982; Bryan, 1970; Kay et al, 1982). The VL can be disaggregated in 1 M urea, and is hydrolyzed by trypsin (Showman and Foerder, 1979). Glabe and Vacquier (1977) found that VLs isolated from unfertilized eggs are composed of 90% protein, 3.5% carbohydrate, and 0.43% sulfate and that glutamic and aspartic acids together account for 25% of the VL amino acids. Their analysis of solubilized VL proteins using SDS-polyacrylamide gel electrophoresis reveals numerous protein bands, ranging in molecular weight from about 15,000 to >200,000 ds. The functions of the VL are also complex. It provides a continuously expanding, protective, external surface for the egg membrane throughout oogenesis, as it is found even in small (30 pm) oocytes (our observations). Its properties are such as to permit transport of micro and macromolecules into the growing oocyte. In fertilization the initial role of the VL is to provide binding sites for sperm attachment, after the acrosome reaction
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40,5% 2-mercaptoethanol, to which 1% SDS was added to prevent protein degradation. Discrete, reproducible patterns of VL proteins could be obtained only when this amount of SDS was included. Protein was estimated using the procedure of Bradford (1976). In later preparations, 10 mM benzamidine, 3% aprotinin, and 1 mM PMSF were added to all media to reduce protein degradation. Protein gel electrophwesis. Two-dimensional electrophoresis of VL proteins was carried out by the method of O’Farrell(l975). The range of the isoelectric focusing gels used for the first dimension was pH 4-7. The gradient was measured as follows. The gel was sectioned into l-mm slices which were eluted with water. The pH of the eluate was measured with a pH meter. SDS-polyacrylamide gels (12.5%) were used for the second dimension. Labeling oocyte proteins. Ovaries containing developing oocytes were washed in MFSW, cut in small pieces, and stirred in an ice bath in a small volume of MFSW. Aliquots of the mixture were placed in cold MFSW (in an ice bath) under the dissecting microscope, and oocytes sorted by hand using a hair loop. Oocytes (50-150) of the same approximate diameter (30,40,50,60, or 70 pm) were placed in 50 ~1 MFSW containing approximately 300 &i/ml [%S]methionine (specific activity 800 Ci/ mmol) and incubated 4 hr at 15°C [the VL proteins contain about 3% methionine (Glabe and Vacquier, MATERIALS AND METHODS 1977)]. The mixture was dissolved in O’Farrell’s (1975) lysis buffer. Preparaticm of vitelline layers. Vitelline layers were prepared from unfertilized eggs, essentially by the Preparation of monoclonal antibodies; immunization method of Glabe and Vacquier (1977). Eggs were ob- and fusion. The exact immunization protocol used for tained by injection of l-2 ml 0.5 M KC1 into gravid the strain 129 mice, and for fusion of their spleen cells female sea urchins (Strong¢rotus purpuratus). The has been described (Niman and Elder, 1980,1982a). The eggs were washed twice by settling in Millipore-filtered mice were immunized intraperitoneally with 50 pg of seawater (MFSW, pH 8), resuspended in MFSW pH 5 soluble VL protein in complete Freund’s adjuvant preto remove egg jelly, and washed again 3X in MFSW, pared as follows. VLs were suspended in 3 volumes of pH 8. One milliliter of washed eggs (2.9 X lo6 eggs) was 0.5% NP-40 in phosphate-buffered saline (PBS) and sonresuspended in 25 ml isolation medium (30 mM sodium icated. Solubilized VL protein, at a concentration of apacetate (pH 6), 20 mM EDTA, 0.4% Triton X-100, 0.1 proximately 1 mg/ml, was mixed with an equal volume mg/ml soybean trypsin inhibitor (SBTI), 1 mM phen- of complete Freund’s adjuvant. Two weeks later an adylmethylsulfonyl fluoride (PMSF)); the eggs were ho- ditional 50 pg of VL protein in 5 mg/ml alum was admogenized in a Thomas homogenizer (glass vessel, Teflon ministered intraperitoneally. This protocol produced pestle), 5-10 strokes until all were disrupted. The VLs readily detected antibody in the sera of these mice 1 were sedimented in a tabletop centrifuge at 1400g for week after the second injection. One to two months after 3 min, and washed by centrifugation 3X in isolation the second injection, 50 pg of VL was administered inmedium. After the third wash, the VLs were sedimented travenously. Five days later the spleen was removed. and after removal of the supernatant, the fluffy VL layer The red blood cells in the spleen cell suspension were was pipetted off, leaving a dark pellet of contaminating lysed in 0.1 M NH&l in 10 mM Tris pH 7.2 at 4°C for nuclei and pigment granules. Isolated VLs were stored 10 min. The nucleated cells were mixed with 2 X 10’ SPin “VL saline” (0.15 M NaCl, 0.2% Na azide, 0.1 mg/ml 2/O myeloma cells which had been washed with miniSBTI, 10 mM Tris, pH 7.4) or dissolved immediately in mum essential medium (MEM) supplemented with 10% lysis buffer A of O’Farrell (1975), 9.5 M urea, 2% NP- fetal bovine serum (FBS), followed by a wash with MEM
has been triggered by the egg jelly (Shapiro et aL, 1980; Monroy and Rosati, 1983). A high-molecular-weight glycoprotein fraction with sperm binding activity has been identified on the sea urchin egg surface (Tsuzuki et aL, 197’7; Schmell et aL, 19’7’7;Glabe and Vacquier, 1978; Rossignol et CAL,1981; Glabe and Lennarz, 1981; Kinsey and Lennarz, 1981), but as yet the specific glycopeptides involved in sperm binding have not been characterized. During the first 3 min after sperm-egg fusion the VL serves as a substrate for various cortical granule enzymes, including vitelline delaminase, which is responsible for elevation (Carroll and Epel, 1975), and a peroxidase (Foerder and Shapiro, 1977) that results in rapid hardening. The VL also provides an array of binding sites for the paracrystalline protein. Throughout early development the fertilization envelope functions as a freely permeable, but extremely tough, elastic container that protects the spherical embryo from compression or other physical insult. At the blastula stage it dissolves in a specific proteolytic reaction mediated by a soluble hatching enzyme (Schuel, 1978). We are interested in the VL as a structurally and developmentally discrete complex of proteins produced during oogenesis. In this paper we present additional evidence regarding the diversity of this set of proteins, and we describe monoclonal antibodies that should be useful for isolation of the genes for the VL proteins.
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without FBS (the myeloma cells are routinely grown in the presence of 10Y4M 8-azaguanine). The nucleated spleen cells were mixed with the washed myeloma cells and pelleted. These cells were spread out and 200 ~1 of 30% polyethylene glycol (PEG) 1500 (Baker) in MEM was added. After a 2 min exposure to PEG, the cells were pelleted for 4 min. Two minutes later the PEG was removed and the cells were gently resuspended in MEM supplemented with 10% FBS. After overnight incubation at 37”C, the cells were pelleted and resuspended in 200 ml of HMT (hypoxanthine, 1.0 X 10T4il!I; methotrexate, 1.0 X 10e6M; thymidine 1.6 X low5 M) in MEM supplemented with 10% FBS. Cells were plated out into 40 microtiter plates (96 well, Falcon). Cells were fed weekly with one drop of HT (HMT without methotrexate). Macroscopic colonies were transferred to 24-well plates (Lindbro) and assayed for binding activity 4-8 days later. Antibody binding assay. Antibody to VL was detected using an ELISA assay as described (Niman and Elder, 1982b), with 25 pg of soluble VL protein, dried onto 96well microtiter plates. The protein was fixed with methanol (50 ~1, 5 min, 25°C). The plates were blocked for nonspecific adsorption by incubation at 37°C for 4-6 hr with 3% bovine serum albumin (BSA) in phosphatebuffered saline. The albumin was removed and each well was incubated overnight at 25°C with 25 ~1 of tissue culture supernatant. The plates were then washed 10 times with distilled Hz0 and then incubated with 25 ~1 rabbit anti-mouse K and X (Litton or Miles) diluted l/ 500 with 1% BSA in PBS. For isotype analysis, the wells were also incubated with various rabbit anti-mouse heavy chain sera. After 2 hr incubation at 37”C, the plates were again washed 10 times with distilled Hz0 and incubated with 25 ~1 glucose oxidase conjugated immunoaffinity purified goat anti-rabbit Ig diluted l/ 500 with 1% BSA. After 1.5 hr incubation at 37”C, the plates were washed 10 times with distilled Hz0 and incubated at 25°C with 50 ~1 of developing reagent [O.l M phosphate, pH 6.0, 1.2% glucose (mutorated), 10 pg/ ml horseradish peroxidase, and 100 pg/ml ABTS (Boehringer Mannheim) dye]. The optical density at 414 was read with a Titertech microscanner. Subcloning. Cultures which were positive on the ELISA assay (3- to lo-fold higher than the media control of the VL or BSA plate) were subcloned by limiting dilution. Cells were grown in a 1:l mixture of fresh HT and the culture fluid conditioned by the hybridoma. The cells were plated out in 96-well microtiter plates, transferred to 24-well plates and assayed as described above. Immunc&orescence method.s.Fresh, living eggs were dejellied by a 2 min exposure to seawater (pH 5.0) and washed into either seawater or a low ionic strength isosmotic medium consisting of 2 parts l’ M dextrose and 1 part seawater. The medium also contained 5 mM
sodium azide and 0.2 mg/ml soybean trypsin inhibitor. Two hundred microliters of a 4% v/v egg suspension was incubated 60 min at 23°C with 200 ~1 hybridoma culture supernatant in borosilicate glass tubes (12 X 75 mm). The eggs were then washed three times by settling through 4 ml portions of either seawater or fresh dextrose-seawater and the pellet resuspended in 200 ~1 of a 1:200 dilution of rhodamine-conjugated goat antimouse IgG (Cappel Laboratories). (The labeled antibody had been previously adsorbed for 30 min at a 1:lO dilution with a great excess of living dejellied eggs in dextroseseawater.) After 30 min the eggs were washed three times in 4 ml portions of fresh seawater or dextroseseawater and the final pellet resuspended in seawater or dextrose-seawater containing 5% formalin. Electrophoretic transfer and immunologic detection of VL proteins. Electrophoretic transfer and immunologic detection of VL proteins were carried out as described (Towbin et al, 1979). Approximately 20 pg of soluble VL protein per lane was used for polyacrylamide electrophoresis as described (Niman and Elder, 1980). The protein was transferred electrophoretically to nitrocellulose (Schleicher and Schuell) and soaked in 3% bovine serum albumin in phosphate-buffered saline for 1 hr at 25°C. The blots were incubated for 16 hr at 25°C in 5 ml. hybridoma tissue culture supernatant which had been diluted l/50 with 3% BSA, 0.1% Triton X-100. The blots were washed with 0.1% Triton X-100 in PBS for 20 min with 3 changes (100 ml each). The nitrocellulose was then incubated with 3% BSA for 5 min at 25°C followed by a 15 hr incubation at 25°C with peroxidase conjugated goat anti-mouse IgG (Cappel) diluted l/1000 in 3% BSA, 0.1% Triton X-100. The nitrocellulose was washed with 0.1% Triton in PBS for 20 min with 3 changes (100 ml each). The bound antibody was visualized by incubation in 10 mM Tris pH 7.4, 0.009% HzOz, 0.0025% 3,3’-dimethoxybenzidine dihydrochloride (Kodak). RESULTS
Protein Components of the Sea Urchin Egg Vitelline Layer Isolated vitelline layers were shown to contain a complex population of proteins when analyzed by polyacrylamide gel electrophoresis. Figure 1A shows the SDS single dimension separation, and Fig. 1B the twodimensional separation in the range of pH 4 to pH 7. The molecular weight and p1 of the most prominent spots detected by Coomassie blue are listed in Table 1. The gels clearly show the presence of at least 25 protein components in the VL. This observation has been repeated a number of times on different VL preparations, and the protein species listed in Table 1 are reproducibly observed.
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-31
PH 4
PH 7
FIG. 1. Electrophoretic display of VL components, stained with Coomassie blue. (A) SDS gel separation. Molecular weight markers indicated at the left (X10’) were phosphorylase B, BSA, and DNase. (B) Two-dimensional electropherogram carried out as described under Materials and Methods, with 150 pg VL protein. The protein spots are very similar to the circled areas of Fig. 2. TABLE 1 VITELLINE LAYER PROTEINS DETE(;TED BY TWO-DIMENSIONAL POLYACRYLAMIDE GEL ELE(;TROPHORESIS
Number 1 2 3 5O 6” 7 8” 9” 10A” 10B 11” 12” 13 14” 15” 16” 17” 18” 19” 20” 210 22 23 24 25
Approximate PI 4 5.5 4.6 5.5 4.5 5 4.2 5 4.2 6.3 5.5 5.8 4.8 4.9 5.1 5.9 5.2 5.5 6.1 5.5 5.3 5.9 6.0 6.1 5.5
a More abundant proteins.
Approximate molecular weight (XIOs) 240 160 150 140 120 130 110 110 90 90 89 80 75 75 75 74 72 72 62 60 55 55 54 53 53
Vitelline Layer Compments Are Not Among the Prevalent Newly Synthesized Proteins of the Oocyte The VL comprises about 1.2% of the total protein of the unfertilized S. purpuratus egg [calculated from results of Glabe and Vacquier (197’7)].VLs are present on small immature oocytes as well, and it is likely that formation of the VL during oogenesis requires accretion of newly synthesized VL proteins. Were the VL to consist of only a small number of protein species their synthesis in the oocyte should be easily detectable. Though a considerable amount of radioactivity can be incorporated by oocytes within a few hours of incubation in seawater containing r5S]methionine, we could not demonstrate that during labeling periods of this duration any newly synthesized VL proteins were assembled into the external VL structures. That is, when isolated, the VLs of living oocytes labeled in vitro failed to display incorporation of r5S]methionine into discrete proteins. An experiment in which the total [%]methionine-labeled proteins synthesized by 70-pm-diameter late vitellogenic oocytes were extracted from homogenates and displayed on two-dimensional gels together with authentic carrier VL proteins is shown in Fig. 2. Over 400 newly synthesized proteins are visualized in the autoradiogram, but few if any comigrate with the stained VL proteins. Results similar to those in Fig. 2 were obtained with 30, 40,50, and go-pm-diameter oocytes, and with unfertilized eggs. Nor could newly synthesized VL proteins be ob-
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FIG. 2. Prevalent newly synthesized proteins of the 70 pm oocyte. Living oocytes were isolated and labeled with [%]methionine for 4 hr; 62 oocytes were used in this experiment. Total incorporation into protein was 1.4 X lo5 cpm, of which 1.25 X lo5 cpm were loaded onto the gel together with 150 gg of unlabeled VL protein prepared separately as a carrier; 5 X lo4 cpm were recovered from the gel. (Most of the remaining input cpm failed to enter the second dimension). The gel was stained with Coomassie blue, dried, and autoradiographed. The black spots in the figure are the newly synthesized proteins resolved in the autoradiogram, and the circled areas represent the stained VL proteins (cf. Fig. 1). The arrow indicates a prominent newly synthesized protein, in which over 3700 cpm were incorporated. Note that the VL protein preparation, as indicated by the circled areas, includes none of this prevalent cytoplasmic protein.
served when small sections of ovary, rather than isolated oocytes, were incubated with [35S]methionine and tested in the same way. The multiplicity of VL proteins indicated in Fig. 1 suggests that on the average 4.8 X 10e4 of the oocyte proteins synthesized at any one time would be assigned to an average VL protein species (i.e., 0.012/25). The overall radioactivity recovered from the two-dimensional gel in the experiment of Fig. 2 was 5 X lo4 cpm. Expectation per spot for newly synthesized VL proteins, on the above assumption, is only about 24 cpm, compared to 100-3000 cpm for most of the proteins visualized autoradiographically and to general interspot radioactivity of 40 cpm per 2 mm2 over background. The result observed could be due in part to failure of modification reactions in the isolated oocytes, but the most likely explanation is simply that an insufficient number of cpm was incorporated per VL protein species.
Anti-VL Monoclmal Antibodies As synthesis of VL protein is evidently not easily detected, and none of the individual VL proteins has yet been characterized at the sequence level, we prepared a series of monoclonal antibodies at least some of which recognize VL components. These reagents should provide an alternative approach to the isolation of the VL protein genes. We now describe briefly the isolation and preliminary characterization of these antibodies, which were raised against VLs that had been sonicated and at least partially dissolved in NP-40 (see Materials and Methods). Hybridomas were obtained from three fusions. In the first fusion 73 cell lines were assayed for binding activity. Seven of these lines initially displayed activity, but only one was stable. The second fusion produced 105 cell lines, of which 16 were positive and seven were stable. The third fusion resulted in 224 testable
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cell lines of which 59 were positive and 33 were stable. Of the 41 stable cell lines, the 31 that displayed the highest titer and binding activity were further characterized. The isotype of the antibody produced by each hybridoma was determined, with the results listed in Table 2. The antibodies are heterogeneous in regard to isotype. Light chain frequencies were 84% K and 16% h, and heavy chain frequencies were 26% p, 3% y3, 39% yl, and 32% Yzb. An initial problem to be considered is whether the monoclonal antibodies recognize intrinsic VL determinants, as opposed to cytoplasmic contaminants. Convincing evidence that at least a majority of the 31 antibodies included in Table 2 indeed react with VL components was obtained in immunofluorescence experiments that indicated for each antibody the cytological locus of the reactive structures. Some of these reactions are illustrated in Fig. 5, and additional data have been presented elsewhere by Gache et aL (1983). Twenty-one of the 31 monoclonal antibodies display surface immunofluorescence as shown in Table 2. If the VL is first digested with pronase or trypsin as described by Gache et al. (1983), followed by extensive washing, 11 of these 21 antibodies now fail completely to react and four show very severely diminished reaction. A critical observation is that for all 21 of the antibodies that react with the surface structures of unfertilized eggs, the fluorescence elevates completely from the egg surface as the VL transforms into the fertilization envelope following activation with the ionophore A23187 or normal fertilization. Proteins of the outer egg surface that are incorporated in the fertilization envelope are by definition VL proteins. A preliminary classification of the 31 monoclonal antibodies was obtained by determining their ability to bind VL proteins that had been solubilized in two different ways. VLs that had been sonicated and then dissolved in NP-40 were used for immunization and initial screening of the hybridomas. An alternative and more strongly denaturing method was to dissolve the VLs in 8 M urea and then dialyze out the urea against PBS. The monoclonal antibodies were incubated with the two different preparations in an ELISA binding assay, as illustrated in Fig. 3. Figure 3A shows the dilution curve for the activity of monoclonal antibody X28G07, which bound equally well to the two different VL preparations. In contrast, as shown in Fig. 3B, antibody X27D02 binds significantly less well to the urea VL preparation. A more drastic effect is shown in Fig. 3C, where antibody X27CO9 can be seen to react well with the NP-40 solubilized VL, but not at all with VL dissolved in urea. We do not know whether the avidity of X27CO9 and X28G07 for the urea-solubilized VL determinants was
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TABLE 2 SUMMARY OF CHARACTERISTICS OF ANTI-VI,
Isotype*
Binding pattern’
MONOCLONAL ANTIBODIES
Fluorescence low saltd
Fluorescence high salt”
Class
Name”
I
X25F09 X12G05
A A
+++ +++
II
W38F06 X27D08 X18FO8 X18ElO
A A A A
+++ +++ +++ +++
III
X2%06 X27B02 X28D05
A A A
IV
X28G07
A
+ + + -
-
v
XO5ClO X27D02 X23CO7 X19D07
B B B B
+++ +++ +++ +++
+++ +++ +++ +++
VI
X17FlO X23Cll WlOEll
B B B
+++ +++ +++
-
VII
X13G06 X08D06
B B
+ +
VIII
X24F05 X23Fll X15Gll
B B B
IX
x27co9 W37FlO
C C
+++ +++
X
X23F06
C
XI
X19E03 X26B02 X14F06 X25B09 X27B08 X29Dll
C C C C C C
+ -
+++ +++ -
-
+++ +++
-
-
a Hybridoma names beginning with W originated from the second fusion described in Results, while those beginning with X originated from the third fusion. b Isotype analysis was carried out as described under Materials and Methods. Abbreviations are K, kappa light chain; X, lambda light chain; p, IgM heavy chain; ~3, IgGa heavy chain; yl, IgGi heavy chain; y&,, IgGsb heavy chain. ‘See Fig. 3 for illustrations of the binding patterns denoted A, B, and C. d Surface immunofluorescence in low salt as shown in Fig. 5. BSurface immunofluorescence when incubated and washed in seawater.
decreased as a direct consequence of antigen denaturation, or for other reasons. It is, evident, however, that the three antibodies included in Fig. 3 recognize different determinants. Reactivity patterns determined similarly
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I
FIG. 3. Differential binding of monoclonal antibodies to VL preparations solubilized by two different methods. Antibody binding was determined by the ELISA method, as described under Materials and Methods. Each monoclonal antibody preparation (A, X23G07; B, X27DO2; C, X27CO9) was assayed for binding to VL that had been sonicated and dissolved in NP-40. (+), VL solubilized with 8 M urea (a), or BSA (0).
are listed for all 31 hybridomas in Table 2. Ten of the 70 kDa. Antibody X19E03 (lane B) also reacted with cell lines produced antibodies that exhibited little dif- three different proteins of apparent mass 200, 95, and ference when reacted with VLs solubilized by the two 85 kDa. The multiple reactions observed in these blots could have been due to shared determinants, or possibly methods (pattern (A) of Fig. 3); 12 of the antibodies bound to urea solubilized VL proteins only about half to the presence of specific degradation fragments. The reactions observed for these antibodies can be tentaas well at maximum as to VL solubilized by sonication tively assigned, on the basis of relative mobility, to pro(pattern (B) of Fig. 3); and nine lost almost all binding teins 2,12, and 19 of Table 1 for antibody XOSDOG,and activity when reacted with the urea extract (pattern to proteins 1,lO (A or B), and 11 for antibody X19E03. (C) of Fig. 3). Clear identification of specific VL antigens by the nitrocellulose transfer procedure was obtained for two of L?$wential Reactions of Monoclinal Antibodies with the monoclonal antibodies. Initial attempts to immuSurface Components of Living Eggs noprecipitate specific labeled VL proteins failed because Hybridoma culture supernatants were incubated with these proteins display a strong tendency to form complexes that include most of the labeled components when unfertilized sea urchin eggs, and the reactions were viincubated with antibody. Two of the antibodies reacted sualized by fluorescence microscopy. Fifteen of the 31 with determinants that were stable to SDS denaturation antibodies exhibited bright surface fluorescence when and boiling in 2-mercaptoethanol, and so the antigens assayed under low salt conditions (+++ Table 2). Six which they recognized could be identified by nitrocelgave weak positive reactions (+). Figure 5 shows some lulose transfer. VL proteins were separated by gel elec- of the specific staining patterns produced by the various trophoresis, transferred to nitrocellulose, and reacted antibodies. The same patterns were observed with each with the hybridoma tissue culture supernatant as de- antibody in formaldehyde fixed as in living eggs (see scribed under Materials and Methods. In lane A of Fig. Gache et d, 1983). However, some variability is observed 4, antibody X08D06 is shown to react with three different from egg to egg even with a given antibody, as illustrated proteins, the apparent masses of which are 16580, and in panels G and H of Fig. 5. Although the antibodies
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200116940 6843-
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Sea Urchin Egg VL
antibodies. Seven of the 15 monoclonal antibodies that produced very strong surface fluorescence when reacted in low salt medium lost their activity when assayed under high salt conditions, i.e., seawater, while the reactivity of the remaining eight was unaffected. Gache et al. (1983) showed that all of these eight monoclonal antibodies block fertilization if reacted with the eggs prior to sperm addition, and that this effect is species specific. The antibodies interfere specifically with the sperm binding reaction. These eight monoclonals thus recognize VL components that are involved in the sperm binding reaction, such as the binding receptor protein, or contiguous structural elements. The effect of salt concentration on the binding of the monoclonal antibodies to the surface of the egg can also be used to classify the antibody activities. Taking into account both the in vitro assays of binding to solubilized VL proteins and their surface reactions with living eggs, the 31 monoclonal antibodies thus fall into 11 separate classes (Table 2). DISCUSSION
FIG. 4. Immunologic detection of VL proteins. Soluble VL protein was electrophoresed and transferred to nitrocellulose as described under Materials and Methods. Immunoreactive components were detected with peroxidase conjugates to the mouse antibody. Hybridoma supernatants were (A) XOSDOG,(B) X19E03, (C) X23CO’i’.Antibody X23CO7is included as an example of a null reaction. Molecular weight standards were myosin, 200,000,@-galactosidase,116,000,phosphorylase B, 94,000, bovine serum albumin, 68,000, and ovalbumin, 43,000.
used in panels C (X27D08), F (X12G05), and G or H (XlSFO8) gave similar binding curves when tested against soluble VL protein, the structures with which they react are obviously distinct. Furthermore, the hybridomas of panels D (X23CO7) and E (X19D07) which are not distinguishable by the binding assay, produce clearly different surface fluorescence patterns. The ionic strength of the medium was important in determining the surface reactivity of the monoclonal
Several estimates of the number of different S. purpuratus VL polypeptides have been offered. Carroll and Endress (1982) isolated that portion of the fertilization envelope that consists of the elevated VL by preventing assembly of the paracrystalline protein fraction released at exocytosis of the cortical granules. When dissociated, the elevated VL is found to be composed of eight prominent polypeptides. Since the procedure of Carroll and Endress relies on the normal process of proteolytic deamination of the VL, their preparations can be regarded as biologically “purified,” with respect to possible cytoplasmic contaminants. On the other hand, proteins such as those that constitute the “vitelline posts,” which connect the VL surface with the plasmalemma, might well be hydrolyzed or otherwise removed during the proteolytic deamination process. When isolated directly from unfertilized eggs by the method of Glabe and Vacquier (1977), essentially the method used here, the VL displays significantly more than eight components. Glabe and Vacquier reported 40 bands in one-dimensional SDS gels, and as shown in Table 1 of this paper a minimum of 25 reproducible polypeptides can be observed in two-dimensional analyses of solubilized VLs. We believe this is if anything a minimum estimate of the number of polypeptides for the following reasons: (a) Glabe and Vacquier showed that such preparations contain no more than about 2% contamination with other egg proteins; (b) addition of the protease inhibitors benzamidine, aprotinin, and PMSF, as well as soybean trypsin inhibitor did not reduce the number of spots observed
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FIG. 5. Patterns of monoclonal antibody binding to the vitelline layer of living eggs. (A) phase contrast micrograph of an egg treated with tissue culture supernatant from a hybridoma (Rz06B08) against Rauscher murine leukemia virus gp’70 (Niman and Elder, 1980). (B) Rhodamine fluorescence micrograph of A. (C)-(H) Fluorescence micrographs of eggs treated in low salt medium with hybridoma supernatants directed against the VL. (C) X27D08; (D) X23CO7; (E) X19D0’7; (F) X12G05; (G and H) X18F08. For photography of the egg surface, the fixed eggs were compressed by allowing the medium to evaporate; the surface against the overlayering coverslip was then photographed. Magnification was approximately X1060.
on two-dimensional gels, or affect their distribution; (c) when these gels were stained by the more sensitive silver procedure (Switzer et al, 1979) an even greater number of spots was observed; (d) the intensity and size of Coomassie blue stained spots (or bands, in SDS gels) were consistent and reproducible from preparation to preparation, and within a several fold factor of uncertainty, suggests that the number of molecules of the 15 more prominent proteins in the VL (i.e., those starred in Table 1) is roughly equivalent; (e) actin, a known cytoplasmic protein that is prevalent in eggs, and particularly in the egg cortex (reviewed by Vacquier, 1981) is completely undetectable in the VL protein preparation. We conclude that there are some 25 major VL compo-
nents, if not more, of which at least 8 are retained in the elevated VL fraction incorporated in the fertilization envelope. The complexity of the VL is implied in a completely different manner by the diversity of the set of monoclonal antibodies that we describe. Figure 5 illustrates six different patterns of surface reaction with the VLs of living eggs, and as shown in Table 2, many antibodies that react with dissolved VLs in ELISA fail to bind to the exposed surface of the living VL. A probable interpretation is that their determinants occupy internal positions within the VL. The effect of low salt is most likely due to the greater affinity of most antibodies in media of low ionic strength. Furthermore, Fig. 4 displays
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FIG. 5-Continued
reactions of two monoclonal antibodies with specific sets development is utilized for construction of subcellof three polypeptides. The remaining antibodies that ular, intracellular, and extracellular three-dimensional did not give clear blot reactions may include many that structures. It now seems likely that the VL of the sea urchin egg recognize carbohydrate rather than polypeptide deterwill provide another example of a complex structural minants. Three-dimensional extracellular structures may re- product, specific to a particular stage of the life cycle, oogenesis. The VL of the Xenopus egg is in some ways quire a relatively large amount of genomic information for their construction. This is suggested by the few ex- similar. It too is elevated following fertilization, to form amples of such structures that have been carefully an- a protective envelope, and Wolf et al (1976) have shown alyzed. The most germane example is the chorion of the that this VL contains at least 11 polypeptides, of which 10 are retained after elevation. Lewis et al. (1982) have moth egg. Kafatos and his colleagues have demonstrated that at least 180 distinct polypeptides are present in suggested that the Xenopus and sea urchin VLs are this structure (Regier et al, 1980). Similarly, the ubiq- functionally complex, because they undergo elevation uitous flagellum contains at least 120 diverse polypep- after fertilization. External egg coats that do not untides (Piperno et aL, 1977). Fifty polypeptides are in- dergo elevation often but not always contain fewer comvolved in the formation of the T4 phage coat (Wood and ponents. For example, the VL of Ciono contains three Revel, 1976). It may be that a significant fraction of the prominent components (Rosati et d, 1982) as does the protein-coding information being translated in early zona pellucida of the mouse egg (Bleil and Wassarman,
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DEVELOPMENTAL BIOLOGY
1978,1980), and the VL of the abalone egg contains only four or five major components (Lewis et aL, 1982), while the zona pellucida of the pig egg contains three or four major and several minor proteins (Dunbar et aL, 1980). We assume that biosynthesis of VL polypeptides occurs in the growing oocyte, though as shown in Fig. 2 they are not sufficiently prevalent to be easily detected among newly synthesized proteins. As noted above, about 1.2% of the mature egg protein is included in the VL, or 0.47 ng of VL protein per egg. Were this quantity divided equally among 25 VL polypeptide components, there would be only about 19 pg of each component. Given about 6 weeks to complete oocyte growth and vitellogenesis (Gonor, 1973; Leahy et aL, 1978, 1981), during which most of the mass of the VL is formed only a small number of steady-state mRNAs would be required for the synthesis of each of the VL protein species. We conclude that a prominent structural component of the egg, the VL, is probably synthesized on a set of many different very low abundance messages. The fact that oogenesis is a slow process, requiring some weeks to complete, makes it possible for the oocyte to accommodate an enormous variety of such low prevalence messages, and yet to accumulate significant quantities of their products. We thank D. Schloeder for help with the isolation and maintenance of the hybridomas, and C. O’Connor for instruction in two-dimensional gel electrophoresis. The assistance of Patrick Leahy in the preparation of the VLs used for this study is gratefully acknowledged. E. J. Carroll, Jr. and E. Rothenberg provided helpful criticisms of the manuscript. This research was supported by NIH Grants BRSG-RR-07003, HD05753 (to E.H.D.), CA-25803 (to R.A.L.), and HD-12986 (to V.D.V). This is manuscript number 3105 IMM from the Research Institute of Scripps Clinic. REFERENCES BLEIL, J. D., and WASSARMAN, P. M. (1978). Identification and characterization of the proteins of the zona pellucida. J. CeU Bid 79, 173a. RLEIL, J. D., and WASSARMAN, P. M. (1980). Synthesis of zona pellucida proteins by denuded and follicle-enclosed mouse oocytes during culture in vitro. Pnx: Nat. Acud Sci USA 77.1029-1033. BRADFORD, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. And B&&em 72,248-254. BRYAN, J. (1970). The isolation of a major structural element of the sea urchin fertilization membrane. J. Cell Bid 44, 867-875. CARROLL, E. J., JR., and ENDRESS, A. G. (1982). Sea urchin fertilization envelope: uncoupling of cortical granule exocytosis from envelope assembly and isolation of an envelope intermediate from Shmgglocentrotus purpuratus embryos. Develop. Biol 94, 252-258. CARROLL, E. J., JR., and EPEL, D. (1975). Isolation and biological activity of the proteases released by sea urchin eggs following fertilization. Develop. Bid 44,22-32. CHANDLER, D. E., and HEUSER, J. (1980). The vitelline layer of the sea urchin egg and its modification during fertilization. A freeze-fracture
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