Intact sea urchin spermatozoa were successfully biotinylated with NHS-LC-Biotin and the biotinylated spermatozoa retained the viability. Analysis of the membrane prepared from the biotinylated spermatozoa of the sea urchin Hemicentrotus pulcherrimus by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that several proteins such as wheat-germ agglutinin (WGA)-binding protein (220 kDa), guanylyl cyclase (131 kDa), sperm-activating peptide-l (SAP-l)-crosslinking protein (71 kDa), GPI-anchored protein (63 kDa) and functionally unknown proteins (50 kDa and 30 kDa) were specifically biotinylated. Experiments using spermatozoa of sea urchins, Anthocidaris crassispina and Clypeasterjaponicus showed that several proteins similar to those of H. pulcherrimus spermatozoa were also labeled with NHS-LC-Biotin.
Fucose sulfate glycoconjugate (FSG) isolated from the jelly coat of H. pulcherrimus was mixed with solubilized biotinylated sperm membrane proteins of H. pulcherrimus, A. crassispina or C. japonicus and then subjected to gel filtration chromatography on a Sepharose 2B column, indicating that only two biotinylated H. pulcherrimus sperm proteins were coeluted with H. pulcherrimus FSG.
For successful fertilization, spermatozoa must locate the egg, penetrate the egg's extracellular matrix and bind to and fuse with the egg plasma membrane. When exposed to the extracellular matrix (jelly coat) of the unfertilized sea urchin eggs, sea urchin spermatozoa undergo the acrosome reaction in which the acrosomal vesicle is exocytosed and an acrosomal process of filamentous actin is extended from the tip of the sperm head (Dan, 1967). The jelly coat is mainly composed of two large acidic glycoconjugates and oligopeptides (Garbers et al., 1982; Garbers et al., 1983; Hotta et al., 1970; Isaka et al., 1970; Ishihara et al., 1973; SeGall and Lennarz, 1979; Suzuki et al., 1981; Suzuki, 1989) and has been shown to activate sperm metabolism (Christen et al., 1983; Epel, 1978; Ohtake, 1976) and induce the acrosome reaction (Collins and Epel, 1977; Dan, 1952,1954,1956; Decker et al., 1976; Kinsey et al., 1979; Kinsey et al., 1980; Lopo and Vacquier, 1980; SeGall and Lennarz, 1979; Summers and Hylander, 1975; Summers et al., 1975; Tilney et al., 1973). The acrosome reaction of sea urchin spermatozoa is accompanied by ionic changes which cause an increase in the intracellular pH and trigger plasma membrane depolarization. In the last 15 years, substances responsible for activation of sperm metabolism were intensively studied and found to be oligopeptides. These oligopeptides are called sperm-activating peptides. Sperm-activating peptide-l (SAP-I, GFDLNGGGVG) is the first oligopeptide isolated from the egg jelly coats of Hemicentrotus pulcherrimus and Strongylocentrotus purpuratus (Garbers et al., 1982; Suzuki et al., 1981). SAP-I induces a number of physiological and biochemical events in sea urchin spermatozoa such as 1) a transient elevation of intracellular levels of cAMP and cGMP (Garbers et al., 1982) and Ca2+ (Hoshino et al., 1992; Schackmann and Chock, 1986), and 2) a transient activation of the membrane form of guanylyl cyclase (Bentley et al., 1986). It also induces a proton efflux across the sperm plasma membrane, resulting in an increase in the intracellular pH (Hoshino et al., 1992; Repaske and Garbers, 1983; Schackmann and Chock, 1986). In addition to the above, the peptide has been shown to promote the acrosome reaction in H. pulcherrimus spermatozoa as a specific co-factor of a major acrosome reaction-inducing substance, fucose sulfate glycoconjugate (FSG) (Yamaguchi et al., 1989). Prior to the induction of these physiological and biochemical events, the peptide seems to bind to specific receptors on the sperm plasma membrane (Smith and Gabers, 1983). SAP-I is known to crosslink specifically to a 77 kDa protein in S. purpuratus spermatozoa (Dangott et al., 1989) or to a 71 kDa protein in H. pulcherrimus spermatozoa (Shimizu et al., 1994).
FSG, a large acidic glycoconjugate in the jelly coat, has been shown to be the natural inducer of the acrosome reaction (SeGall and Lennarz, 1979; Garbers et al., 1983; Shimizu et al., 1990; Keller and Vacquier, 1994). It increases cAMP concentration, activates a cAMP-dependent protein kinase and stimulates Ca2+-accumulation by spermatozoa. However, information about the sperm surface molecules which may interact with FSG is limited although several sperm surface glycoproteins have been reported to be involved in the induction of the acrosome reaction (Podell and Vacquier, 1985; Trimmer et al., 1987). In S. purpuratus spermatozoa, several glycoproteins (320 kDa, 210 kDa, 140 kDa, 80 kDa and 60 kDa) were accessible to radioiodination of intact spermatozoa a s well a s isolated sperm membrane vesicles (Lopo and Vacquier, 1980; Podell et al., 1984; Trimmer et al., 1987). In H. pulcherrimus spermatozoa, several proteins such as WGA-binding protein (220 kDa), guanylyl cyclase (131 kDa for phosphorylated form and 128 kDa for dephosphorylated form) and sperm-activating peptide l-crosslinking protein (71 kDa) have been suggested to locate on the sperm surface (Sendai and Aketa, 1991; Harumi et al., 1991, 1992; Shimizu et al., 1994).
To obtain a deeper understanding of the sperm surface proteins which are involved in sperm-egg interaction, we attempted to label and characterize the sperm surface proteins (Hardy and Garbers, 1994). Here, we report that several new proteins in addition to proteins mentioned above were biotinylated and two proteins (63 kDa and 50 kDa) showed specific interaction with FSG.
MATERIALS AND METHODS
Sea urchins, H. pulcherrimus, Anthocidaris crassispina and Clypeaster japonicus were collected near Noto Marine Laboratory of Kanazawa University.
Sulfosuccinimidyl-6-(biotinamido)hexanoate (NHS-LC-Biotin) and disuccinimidyl suberate were products of Pierce (Rockford, IL, USA). ECL Western blotting detection system, Hybond-C-super membrane, streptavidin biotinylated horseradish peroxidase complex, anti-mouse lg (biotinylated species-specific whole antibody from sheep) and anti-rabbit lg (biotinylated species-specific whole antibody from donkey) were purchased from Amersham-Japan (Tokyo, Japan). Biotinylated standard proteins (low and high ranges) for SDS-PAGE were obtained from Bio-Rad Laboratories (Richmond, CA, USA). Prestained SDS molecular weight markers and raw wheat germ were purchased from Sigma Chemical Co. (St. Louis, MO, USA). GGGY-SAP-I (GGGYGFDLNGGGVG) was synthesized for us at National Institute for Basic Biology, Okazaki, Japan. Na125l(3.7GBq/ml NaOH solution, pH 10) was obtained from Du Pont/New England Nuclear (Boston, MA, USA). 3-[(3-cholamidopropyl)-dimethylammnio]-1-propanesulfonate (CHAPS) was a product of Dojindo Chemical Institute Co. (Kumamoto, Japan).
Rabbit anti-serum against a synthetic peptide (KPPPQKLTQEAIEVAANRVIPDDV) corresponding to the C-terminal amino acid sequence of S. purpuratus sperm guanylyl cyclase was generously provided by Dr. T. Quill in Professor David L. Garbers' laboratory (University of Texas Southwestern Medical Center, Dallas, Texas, USA). A monoclonal antibody against a 63 kDa sperm protein (GPI-anchored protein) of S. purpuratus was a kind gift of Professor Victor D. Vacquier (Scripps Institution of Oceanography, University of California, San Diego, CA, USA) (Mendoza et al., 1993). Rabbit anti-serum against H. pulcherrimus sperm creatine kinase was made in our laboratory (Harumi et al., 1992).
Purification of H. pulcherrimus FSG
H. pulcherrimus eggs were collected in filtered sea water after intracoelomic injection of 0.5 M KCl. The egg suspension was adjusted to pH 5.0 with 0.1 N HCl to dejelly and the dejellyed eggs were allowed to sink. The supernatant was centrifuged at 10,000 xg for 20 min at 4°C, and the resulting supernatant (jelly solution) was stored at −20°C until use.
FSG was purified from the jelly solution by sequential chromatography on a Sepharose 2B column as reported previously (Shimizu et al., 1990). A frozen and thawed jelly solution was centrifuged at 15,000 xg for 30 min at 4°C. The precipitate was dissolved in deionized and distilled water (DDW), followed by the addition of an equal volume of 0.2 M NaCl. The mixture was applied to a Sepharose 2B column (5 × 86 cm) equilibrated with 0.1 M NaCl and eluted with 0.1 MNaClat a flow rate of 50 ml/hr at 4°C. Fractions containing fucose were pooled, concentrated using an ultrafiltration apparatus with a YM-10 Diaflo membrane (Amicon Division, W. R. Grace of Co. MA, USA) and centrifuged at 10,000 xg 30 min at 4°C. The resulting supernatant was stored at −20°C until use.
Biotinylation of sea urchin spermatozoa
Spermatozoa obtained by intracoelomic injection of 0.5M KCl were collected as “dry sperm” at room temperature and stored on ice until use. Usually the dry sperm were used for experiments within 24 hr- Dry sperm (4 ml) were suspended in 40ml of Buffer A (0.5 M NaCl, 1 mM EDTA and 50 mM HEPES, pH 7.5) and centrifuged at 130 xg for 2 min to remove cell debris. The spermatozoa in the supernatant were pelleted by centrifugation at 4,000 xg for 30 min at 4°C and then suspended in 40 ml of Buffer A. The suspension was mixed with 0.5 ml of freshly prepared 25 mM NHS-LC-Biotin and incubated at 20°C for 1 hr with gentle shaking. The suspension, after being mixed with 0.5 ml of freshly prepared 25 mM NHS-LC-Biotin, was incubated at 20°C for 1 hr with gentle shaking and then 0.25 ml of 1 M Tris-HCl (pH 8.0) was added to terminate the reaction. After a 10 min-incubation at 20°C, the sperm suspension was centrifuged at 4,000 xg for 30 min at 4°C. The resulting sperm pellet was suspended in 40 ml of Buffer A, centrifuged at 4,000 xg for 30 min at 4°C, resuspended in 10 ml of Buffer A and centrifuged at 12,000 xg for 20 min at 4°C. The pellet (biotinylated spermatozoa) was used for immediate experiments or otherwise stored at −70°C until use.
Fertilization of H. pulcherrimus eggs with biotinylated spermatozoa
H. pulcherrimus eggs were collected in filtrated sea water after intracoelomic injection of 0.5 M KCl. A 5% washed egg suspension was fertilized in artificial sea water (ASW; 454 mM NaCl, 9.7 mM KCl, 24.9 mM MgCl2, 9.6 mM CaCl2H20, 27.1 mM MgS04H20, 4.4 mM NaHC03) using spermatozoa which were incubated with or without NHS-LC-Biotin for varying time periods. At 2 min after sperm addition, 100 eggs were randomly taken and numbers of eggs with or without the fertilization envelope were counted under a microscope. Fertilization rates (%) were calculated from the results of three independent experiments using the same batch of eggs and spermatozoa.
Preparation of sperm membranes
Unfrozen biotinylated or non-biotinylated spermatozoa were suspeneded in 20 volumes of ASW containing 2 mM benzamidine HCI, 0.01% (w/v) streptomycin, 0.01% (w/v) penicillin G and 20 mM Tris-HCl (pH 9.0) (Suzuki et al. 1987). The suspension was incubated with gentle agitation for 12 hr at 4°C and then centrifuged at 8,000 xg for 30 min at 4°C. The supernatant was saved and re-centrifuged under the same conditions. The resulting supernatant was then centrifuged at 100,000 xg for 60 min to pellet the sperm membranes. The pellet was resuspended in an appropriate volume of ASW and stored at −70°C until use.
Isolation of WGA-binding protein
The 220 kDa WGA-binding protein was isolated from the membranes of non-biotinylated spermatozoa by the method reported previously (Shimizu et al., 1994).
Gel filtration of biotinylated sperm proteins with FSG
Biotinylated sperm membranes prepared as described above were suspended in solubilization buffer (1% CHAPS and 20 mM TrisHCI pH 8.2 in 2-fold concentrated ASW) and subjected to sonication for 30 sec on ice. The homogenate was centrifuged at 4°C for 1 hr at 100,000 xg and the supernatant (biotinylated sperm protein solution) was stored on ice until use.
Five ml of H. pulcherrimus FSG solution (227μgprotein/ml) was mixed with an equal volume of biotinylated sperm protein solution (170μgprotein/ml) and allowed to stand for 30 min on ice. The mixture was then applied to a Sepharose 2B column (2.5 × 85 cm) equilibrated with a buffer (0.1% CHAPS and 10 mM Tris-HCl, pH 8.2 in ASW). Proteins were eluted with the equilibration buffer at a flow rate of 20 ml/hr and fractions of 5 ml were collected. After determination of the protein concentration of each fraction, an aliquot (1μl)of each fraction was spotted on a Hybond-C-super membrane and biotinylated proteins were detected by ECL. An equal volume of each fraction was also analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). One of the two equivalent gels was silver-stained to detect proteins and the other one was analyzed by ECL Western blotting. For control purposes, the sample containing only H. pulcherrimus FSG or only biotinylated sperm proteins was subjected to gel filtration on the same Sepharose 2B column.
SDS-PAGE and Western blotting
SDS-PAGE was carried out essentially as described by Laemmli (1970) using a 5-15% linear gradient gel for analysis of biotinylated sperm proteins and a 6% gel for analysis of FSG. Proteins in the gel were visualized by silver-staining (Morrissey, 1981). For ECL Western blotting, proteins in the gel were electrophoretically transferred onto a nitrocellulose membrane (Amersham Hybond C-super) using a Multiphor II Electrophoresis System (Pharmacia LKB Biotechmology) and a transfer buffer [39 mM glycine, 48 mM Tris, 0.0375%(w/v) SDS and 20% (v/v) methanol ] at 4°C for 60-120 min at constant current (0.8 mA/cm2). The nitrocellulose membrane was blocked with TBS-T [137 mM NaCl, 0.1%(v/v) Tween 20 and 20 mM Tris-HCl, pH7.6] containing 5% skim milk at 4°C for overnight, washed twice with TBS-T, and was shaken in 15 ml TBS-T once for 15 min and twice for 5 min. The membrane was then incubated with streptavidin biotinylated horseradish peroxidase complex solution diluted 1:5000 with TBS-T for 1 hr at room temperature and washed with TBS-T once for 15 min and twice for 5 min. Finally, the membrane was incubated with streptavidin biotinylated horseradish peroxidase complex solution diluted 1:5000 with TBS-T for 1 hr at room temperature and washed with TBS-T once for 15 min and four times for 5 min. The proteins that reacted with antibody were located by ECL Western blotting according to the manufacturer's instructions. After transferring of proteins, the SDS-gel was silver-stained to confirm whether the transferring was completed.
Crosslinking of GGGY(125I)-SAP-I to H. pulcherrimus spermatozoa
GGGY-SAP-I was iodinated by the chloramine-T method using radioactive sodium iodide and purified as described previously (Yoshino and Suzuki, 1992). Sperm membranes prepared from 16.7 mg wet weight of spermatozoa were incubated with GGGY(125I)-SAPI (10 pmol) in 1 ml of ASW buffered with 10 mM HEPES (pH 8.2) for 10 min at 20°C. The reaction was stopped by the addition of ice-cold ASW (0.9 ml, 10 mM HEPES, pH 8.2), and the mixture was centrifuged at 15,000 xg for 5 min at 4°C. The resulting pellet was resuspended in ASW (90 μl, 10 mM HEPES, pH 8.2) and incubated with a crosslinking reagent, disuccinimidyl suberate (1 mM) in dimethylsulfoxide (10 μl) for 30 min at 20°C. The incubation was terminated by the addition of ASW (0.9 ml, 10 mM Tris, pH 8.2) and then 0.5 ml of 30% (w/v) trichloroacetic acid was added.
Determination of protein concentration
The concentration of protein was determined by the Lowry method (1951) modified by Schacterle and Pollack (1973) using bovine serum albumin as a standard.
Purification ofH. pulcherrimus FSG
The precipitate obtained by centrifugation of frozen-thawed egg jelly solution contained almost entire FSG presented in the original solution. Approximately 15 mg of FSG proteins were obtained from one time chromatography of the resolubilized precipitate fraction which contained approximately 20 mg proteins. As reported previously, when the purified FSG was analyzed by SDS-PAGE in the presence of 2-mercaptoethanol two major protein bands (258 kDa and 237 kDa) and one minor red-stained protein band (120 kDa) were detected. However, these three components could not be detected in SDS-PAGE without 2-mercaptoethanol.
Biotinylation of sea urchin sperm proteins
Spermatozoa of three different species of sea urchins (H. pulcherrimus, A. crassispina and C. japonicus) were biotinylated as described in Methods, and the sperm membranes were prepared by the nitrogen cavitation. The sperm membrane proteins were analyzed by SDS-PAGE and subsequent ECL Western blotting. As shown in Fig. 1, several biotinylated proteins were detected in samples from spermatozoa of these species.
Viability of biotinylated spermatozoa
As shown in Table 1, H. pulcherrimus spermatozoa treated with NHS-LC-Biotin for 120 min retained the fertilizing ability although the motility of the spermatozoa began to decline at 60 min after the treatment. The eggs fertilized with the biotinylated spermatozoa developed normally at least to the blastula stage. When the spermatozoa were incubated with NHS-LC-Biotin for 4 hr the fertilizing ability was decreased to 80% of zero time control spermatozoa.
The viability of biotinylated spermatozoa
Identification of H. pulcherrimus biotinylated sperm membrane proteins
When biotinylated H. pulcherrimus sperm membrane proteins were analyzed by SDS-PAGE, one biotinylated protein with 220 kDa was electrophoresed to the same position of the WGA lectin-binding protein purified from H. pulcherrimus spermatozoa (Fig. 2). Similarly, an other biotinylated protein was electrophoresed to a position which corresponds to the molecular mass (71 kDa) of a 125l-GGGY-SAP-l-crosslinking protein (Fig. 3). Subsequently other biotinylated H. pulcherrimus sperm membrane proteins were separated by SDS-PAGE using a 5-15% linear gradient gel and then analyzed by ECL Western blotting. Three biotinylated proteins (137 kDa, 131 kDa and 63 kDa) reacted with antibodies specific for H. pulcherrimus sperm tail creatine kinase, S. purpuratus sperm guanylyl cyclase or S. purpuratus GPI-anchored protein (Fig. 3). Major biotinylated proteins in H. pulcherrimus spermatozoa are summarized in Table 2.
Major biotinylated proteins in H. pulcherrimus spermatozoa
Gel filtration of biotinylated sperm proteins with H. pulcherrimus FSG
When proteins solubilized from membranes prepared by hypotonic treatment of H. pulcherrimus biotinylated spermatozoa were combined with H. pulcherrimus FSG and subjected to chromatography on a Sepharose 2B column, only 63 kDa and 50 kDa biotinylated proteins were coeluted with FSG (Fig. 4). On the other hand, when H. pulcherrimus biotinylated sperm membrane proteins alone were applied to the column, almost all biotinylated proteins including 63 kDa and 50 kDa biotinylated proteins were eluted after the expected peak fraction of FSG. Co-elution of proteins solubilized from biotinylated sperm membranes of C. japonicus or A. crassispina with H. pulcherrimus FSG was not detected except weak signals by dot blot analysis.
Prior to the induction of the acrosome reaction, a major acrosome reaction-inducing substance in sea urchin egg jelly, FSG, must interact specifically with sperm surface substance(s). In this sense, it is important to have knowledge of sperm surface proteins. In the present study, we demonstrated that in H. pulcherrimus, several sperm proteins such as WGA-binding protein, membrane form of guanylyl cyclase, SAP-l-crosslinking protein and GPI-anchored protein, all of which have been demonstrated to be plasma membrane proteins, can be labeled by incubation with NHS-LC-Biotin, and the biotinylated spermatozoa retained the viability for at least 120 min. This suggests that biotinylation using NHS-LCBiotin is a useful technique for labeling sperm surface proteins. Biotinylation has several advantages over radioiodination to labele sperm surface proteins: 1) the reaction time is much shorter than radiolabeling and the resulting biotinylated proteins can be easily detected by ECL Western blotting with high sensitivity and 2) biotinylated spermatozoa or sperm membranes prepared from them can be stored in a freezer at −40°C for a long time (at least 1 year) without any problems in further characterization and analysis of the biotinylated proteins.
Of the biotinylated H. pulcherrimus, A. crassispina and C. japonicus sperm proteins, only two proteins (63 kDa and 50 kDa) from H. pulcherrimus spermatozoa were coeluted with H. pulcherrimus FSG in gel filtration chromatography. This sugests that these proteins have specific interaction with H. pulcherrimus FSG. To characterize these proteins further, we are currently isolating these proteins from the biotinylated H. pulcherrimus spermatozoa by affinity chromatography using anti-biotin agarose column.
The authors thank staffs of Noto Marine Laboratory, Kanazawa University for their kind help and supply of the sea urchins. This work was supproted by Grants-in-Aid for Scientific Research (Nos. 02404006 and 06454025) from the Ministry of Education, Science, Sports and Culture of Japan.