Micromere Differentiation in the Sea Urchin Embryo: Two-Dimensional Gel Electrophoretic Analysis of Newly Synthesized Proteins

A method for large-scale culture of isolated blastomeres of sea urchin embryos in spinner flasks was developed. Micromeres and meso-, macromeres isolated from sea urchin embryos at the 16-cell stage were cultured by this method and the patterns of protein synthesis by their descendants were examined by two-dimensional gel electrophoresis of [³⁵S] methionine-labeled proteins. Six distinct proteins with molecular weights of 140–kDa, 105–kDa, 43–kDa, 32–kDa, and 28–kDa (two components) were specifically synthesized by differentiating micromeres. Quantitative analysis of the two-dimensional gel patterns demonstrated that all these proteins, except the 32–kDa protein, appeared at the time of ingression of primary mesenchyme cells (PMC's) in vivo , several hours earlier than the onset of spicule formation. The synthesis of 32–kDa protein was paralleled to active spicule formation and the uptake of Ca²⁺. Cell-free translation products directed by poly (A)⁺ RNAs isolated from descendant cells of micromeres and meso-, macromeres were compared by two-dimensional gel electrophoresis. Several spots specific to the micromere lineage were detected. However, none of them comigrated with the proteins synthesized specifically by the cultured micromeres. The results suggest that the expression of these proteins specific to differentiating micromeres may involve post-translational modification.     

Spiculogenesis in the sea urchin embryo: Studies on the SM30 spicule matrix protein

When proteins isolated from spicules of Strongylocentrotus purpuratus embryos were examined by western blot analysis, a major protein of approximately 43 kDa was observed to react with the monoclonal antibody, mAb 1223. Previous studies have established that this antibody recognizes an asparagine-linked, anionic carbohydrate epitope on the cell surface glycoprotein, msp130. This protein has been shown to be specifically associated with the primary mesenchyme cells involved in assembly of the spicule. Moreover, several lines of evidence have implicated the carbohydrate epitope in Ca²⁺ deposition into the growing spicule. The 43 kDa, spicule matrix protein detected with mAb 1223 also reacted with a polyclonal antibody to a known spicule matrix protein, SM30. Further characterization experiments, including deglycosylation using PNGaseF, two-dimensional electrophoresis, and immunoprecipitation, verified that the 43 kDa spicule matrix protein had a pl of approximately 4.0, contained the carbohydrate epitope recognized by monoclonal antibody mAb 1223 and reacted with anti-SM30. Electron microscopy confirmed the presence of proteins within the demineralized spicule that reacted with mAb 1223 and anti-SM30. We conclude that the spicule matrix protein, SM30, is a glycoprotein containing carbohydrate chains similar or identical to those on the primary mesenchyme cell membrane glycoprotein, msp130.     

Micromere Differentiation in the Sea Urchin Embryo: Immunochemical Characterization of Primary Mesenchyme Cell-Specific Antigen and Its Biological Roles

Primary mesenchyme cell (PMC)-specific antigens in developing sea urchin embryos of five different species have been studied by using two different monoclonal antibodies, P4 and B2C2. Like B2C2 in Strongylocentrotus purpuratus (Anstrom et al. , 1987) P4 reacted with the N-linked carbohydrate in Strongylocentrotus intermedius embryo. Although both antibodies recognize the same group of glycoproteins in S. intermedius , P4 epitopes appeared earlier than B2C2 epitopes in Clypeaster japonicus embryo. PMCs of Anthocidaris crassispina blastulae raised in sulfate-deficient sea water were immuno-reactive with P4 but not with B2C2, although the embryos raised in normal sea water reacted with both antibodies at similar intensity. These results suggest that the epitopes of P4 and B2C2 are formed by glycosylation and sulfation, respectively. PMCs may display differential modification in their surface glycoprotein synthesis during differentiation. Furthermore, P4 inhibited cultured micromere descendant cells of Hemicentrotus pulcherrimus from attaching to the plastic dishes and forming spicules in vitro without detectable cytotoxic effect. P4-reactive glycoproteins may play important roles in cell-substrate interaction and spicule formation.     

Quantitative Changes in Periplasmic Proteins of the Macronucleus-Specific Bacterium Holospora obtusa in the Infection Process of the Ciliate Paramecium caudatum

ABSTRACT. The Gram-negative bacterium Holospora obrusa is a macronucleus-specific symbiont of the ciliate Paramecium caudatum. The infectious form of this bacterium infects the host macronucleus through digestive vacuoles and differentiates into the reproductive form two days after the infection in the nucleus. The monoclonal antibodies IF-3–1 and IF-3–2 reacted with 39 and 1S kDa periplasmic proteins, respectively, that were specific for the infectious form of H. obrusa. Because the antigens were not detected in the reproductive form of the bacterium, it appears that expression of the proteins decreases during or soon after the infection. Using these antibodies, quantitative changes in the antigens in the early infection process were examined by immunoblotting and immunogold electron microscopy. Immunoblotting showed that the amounts of both antigens were reduced within 1 h after the bacteria were engulfed into the digestive vacuoles of the paramecia, but that the amounts of IF-3–2 antigens declined earlier than the IF-3–1 antigen. Immunogold labeling showed that the level of IF-3–2 antigens became very low in the bacteria in the host digestive vacuoles, whereas there was no similar decrease in amount of IF-3–1 antigens. Possible functions of the antigens are discussed. The IF-3–1 antigens decrease in concentration in parallel with the decrease in the periplasmic region.     

SPICULE FORMATION IN VITRO BY THE DESCENDANTS OF PRECOCIOUS MICROMERE FORMED AT THE 8-CELL STAGE OF SEA URCHIN EMBRYO*

The micromeres at the 16-cell stage of sea urchin embryo have already been endowed with a faculty to self-differentiate into spicule-forming cells (11). The present experiment was designed to test whether the factor(s) necessary for such self-differentiation had already been localized at the 8-cell stage in an area corresponding to the presumptive micromere region in Hemicentrotus pulcherrimus. Since the blastomeres at the 8-cell stage are all equal in size in normal embryo, unequal 3rd cleavage, by which small blastomeres are pinched off toward the vegetal pole (precocious micromeres), was experimentally induced either by treatment with 4NQO (4-nitroquinoline-1-oxide) at the 2-cell stage or by continuous culture in Ca-free sea water. The precocious micromeres were cultured in vitro in natural sea water containing horse serum. Descendants of the precocious micromeres formed spicules. In comparison their spicule formation with that by the descendants of the micromere of normal embryo, no differences were found regarding 1) time of initiation of spicule formation, 2) rate of growth of spicule, 3) size and shape of resultant spicule and 4) percentage of clones which formed spicule. The fact indicates that factor(s) indispensable for self-differentiation into spicule-forming cells have already been localized near the vegetal pole as early as the 8-cell stage.     

Binding of [3H]Nitrendipine to Proteins in the Plasma Membrane of Sea Urchin Sperm

The plasma membrane fractions of the sperm of four species of sea urchin, obtained by the method by Podell et al. (24), gave similar electrophoretic profiles of proteins. Several proteins in the membrane fraction from Hemicentrotus pulcherrimus bound [³H]nitrendipine, a specific antagonist of voltage-dependent Ca²⁺channels, added at concentration of about 10⁴times those reported to be effective in muscle and nerve cells. Nifedipine, a close analogue of nitrendipine, decreased the bindings of [³H]nitrendipine to 210, 140, 130 and 110 kDa and increased its bindings to several other proteins. Diltiazem, another type of Ca²⁺channel blocker, enhanced the bindings of [³H]nitrendipine to proteins of 210, 140, 130 and 110 kDa, and decreased its bindings to the other proteins. This effect of diltiazem on the binding of [³H]nitrendipine to proteins in the membrane fraction was similar to its effect on the mammalian excitable membrane fraction. The proteins whose binding to [³H]nitrendipine was blocked by nifedipine and enhanced by diltiazem are Ca²⁺channels.     

Enhancement of spicule formation and calcium uptake by monoclonal antibodies to fibronectin-binding acid polysaccharide in cultured sea urchin embryonic cells

The effect of monoclonal antibodies to fibronectin-binding acid polysaccharide (anti-FAPS) on differentiation of primary mesenchyme cells and spicule formation was examined in cultured embryonic cells isolated from the sea urchin, Hemicentrotus pulcherrimus. Spicule formation of micromere-derived cells was enhanced by anti-FAPS. The increase of spicule formation correlated with the increase of calcium uptake into micromere-derived cells and spicules. Furthermore, both spicule formation and calcium uptake were inhibited by calcium-channel blockers (verapamil, diltiazem and nicardipine) and divalent ions (manganese and cobalt). These results suggest that FAPS, a component of the blastocoelic extracellular matrix surrounding the primary mesenchyme cells, may regulate the level of calcium uptake and spicule formation.     

The isolation and purification of surface specific proteins of somatic and reproductive protoplasts of lily and rapeseed

The plasma membrane surface proteins of intact somatic (leaf) and reproductive (pollen, generative cell or sperm cell) protoplasts of lily ( Lilium longiflorum ) and rapeseed ( Brassica napus cv. Midas) were compared after probing with N-hydroxysuccinimido- (NHS) or sulfo-NHS-biotin. The plasma membranes of intact protoplasts are impermeable to these biotin probes, which bind covalently to the free amino groups of surface proteins. Enzyme-labelled streptavidin was used to detect membrane proteins after separation by SDS-PAGE and western blotting. In lily, six proteins specific to the surface membrane of leaf protoplasts were identified varying from 25–64 kDa, three proteins to pollen protoplasts in the range 35–64 kDa and two proteins to generative cell protoplasts, 63 and 67 kDa. In rapeseed leaf protoplasts, seven proteins in the range 22–69 kDa were detected, while in the sperm enriched fraction five proteins were present in the same kDa range. The proteins identified as membrane specific for generative cell protoplasts of lily have been isolated and were used as antigens for monoclonal antibody production. Preliminary results indicate the successful production of antibodies to surface antigens. These antibodies will be used to localise surface specific epitopes which are likely to be involved in cell-cell recognition at fertilization.     

Disappearance of an epithelial cell surface-specific glycoprotein (Epith-1) associated with epithelial-mesenchymal conversion in sea urchin embryogenesis

Cell surface modification during mesenchyme ingression was examined using a monoclonal antibody (mAb), anti-Epith-1 mAb, raised against a protein (Epith-1) that was confined to the lateral surface of the epithelial cells in embryo of the sea urchin, Temnopleurus hardwicki. The mAb epitope was N-glycosylated oligosaccharides of 160 kDa monomeric Epith-1 protein. The glycoprotein was negatively charged, and its isoelectric point (IP) was 4.98. The mAb, however, is not immunologically cross-reactive with other sea urchin embryos including Hemicentrotus pulcherrimus, Strongylocentrotus nudus, and Scaphechinus mirabilis. Epith-1 is present initially in the cytoplasm of unfertilized eggs. Cytoplasmic Epith-1 shifted to the cell surface to be integrated in plasma membrane during the first cleavage, and remained there during early embryogenesis by retaining the same relative molecular mass (Mr). During primary and secondary mesenchyme ingression periods, however, Epith-1 disappears from the presumptive mesenchyme cell surface that was associated with internalization of the protein. In plutei, an additional anti-Epith-1 mAb-positive protein appears at the 142 kDa region, which was not associated with any visible alteration of the histologic localization of the protein in larvae. Anti-Epith-1 mAb IgG did not inhibit the reaggregation of epithelial cells in vitro, which suggests that either the protein is not involved in cell-cell adhesion or that the mAb is not recognizing the active site of the protein.     

The isotopic effects of D2O in developing sea urchin eggs

When developing sea urchin eggs were treated with sea water containing 40% D2O (D2O-SW) at the 8-cell stage, the micromere formation was delayed and micromeres were larger than those seen in the control. But eggs returned to normal artificial sea water (NASW) at the 16- to 32-cell stage did not form abnormal spicules in larvae of Pseudocentrotus depressus. Little effect on the spicule formation of Hemicentrotus pulcherrimus was also noted. If the culture period in D2O-SW was extended until the hatching stage, the number of plutei with abnormal spicules increased. Primary mesenchyme cells of Pseudocentrotus depressus larvae failed to make two aggregated spicule rudiments on the ventral side of the larva and developed a ring-like spicule. This ring-like spicule, however, only occasionally occurred in the larvae of Hemicentrotus pulcherrimus. The cell cycle was longer in the presence of D2O. However, blastomeres managed to divide throughout the development. Larvae reared in 20% D2O-SW after the hatching stage developed into quasi-normal plutei but smaller than control. We found no exogastrulation in these larvae. Exogastrulation was found only in larvae continuously cultured in 40% D2O-SW from the early development. These results are inconsistent with previous reports made by other authors.     

Monoclonal antibody inhibition of Neospora caninum tachyzoite invasion into host cells

Monoclonal antibodies were produced against Neospora caninum tachyzoites to identify antigens which may play a role during invasion of host cells. Confocal laser microscopy showed that most antigens recognised by the mAb were located on the surface, but one mAb, 1A5, reacted to the apical end of the parasite. Some mAbs, which recognised 70, 42 and 36kDa parasite proteins, significantly inhibited the invasion of the parasite in vitro. The mAbs which recognised 42 and 36kDa parasite protein, reacted with Nc-p43 and Nc-p36 expressed by vaccinia virus and Escherichia coli, respectively. These results suggest that a 70kDa protein, Nc-p43 and Nc-p36 are involved in the invasion of the parasite into host cells.     

The 5-HT receptor cell is a new member of secondary mesenchyme cell descendants and forms a major blastocoelar network in sea urchin larvae

A gene encoding the serotonin (5-hydroxytryptamine, 5-HT) receptor (5-HT-hpr) was identified from the sea urchin, Hemicentrotus pulcherrimus. Partial amino acid sequence deduced from the cDNA showed strong similarity to Aplysia californica 5-HT2 receptor. Immunoblotting analysis of this 5-HT-hpr protein (5-HT-hpr) with an antibody raised against a deduced peptide showed two bands. Their relative molecular masses were 69 and 53 kDa, respectively. The larger band alone disappeared after N-glycopeptidase F digestion, indicating the protein was N-glycosylated. Immunolocalization analysis showed that cells expressing the 5-HT-hpr (SRC) first appeared near the tip of the archenteron in 33-h post-fertilization (33 hpf) prism larvae. Their cell number doubled in 2 h, and 5-HT-hpr protein expression increased further without cell proliferation. SRC spread ventrally on the basal surface of the oral ectoderm in 36 hpf prism larvae, and then clockwise on the ventral ectoderm to the posterior region to complete formation of the SRC network in 48 hpf early plutei. The SRC network was comprised of 7 main tracts: 4 spicule system-associated tracts and 3 spicule system-independent tracts. The network extended short fibers to the larval body surface through the ectoderm, implicating a signal transmission system that receives exogenous signal. Double-stain immunohistochemistry with antibodies to primary mesenchyme cells showed that SRC were not stained by the antibody. In embryos deprived of secondary mesenchyme cell (SMC) by microsurgery, the number of SRC decreased considerably. These two data indicate that SRC are SMC descendants, adding a new member to the SMC lineage.     

A switch in the cellular basis of skeletogenesis in late-stage sea urchin larvae

Primary mesenchyme cells (PMCs) are solely responsible for the skeletogenesis during early larval development of the sea urchin, but the cells responsible for late larval and adult skeletal formation are not clear. To investigate the origin of larval and adult skeletogenic cells, I first performed transplantation experiments in Pseudocentrotus depressus and Hemicentrotus pulcherrimus, which have different skeletal phenotypes. When P. depressus PMCs were transplanted into H. pulcherrimus embryos, the donor phenotype was observed only in the early larval stage, whereas when secondary mesenchyme cells (SMCs) were transplanted, the donor phenotype was observed in late and metamorphic larvae. Second, a reporter construct driven by the spicule matrix protein 50 (SM50) promoter was introduced into fertilized eggs and their PMCs/SMCs were transplanted. In the resultant 6-armed pluteus, green fluorescent protein (GFP) expression was observed in both PMC and SMC transplantations, suggesting SMC participation in late skeletogenesis. Third, transplanted PMCs or SMCs tagged with GFP were analyzed by PCR in the transgenic chimeras. As a result, SMCs were detected in both larval and adult stages, but GFP from PMCs was undetectable after metamorphosis. Thus, it appears that SMCs participate in skeletogenesis in late development and that PMCs disappear in the adult sea urchin, suggesting that the skeletogenesis may pass from PMCs to SMCs during the late larval stage.     

Determination of antigenic domain in GST fused major surface protein (Nc-p43) of Neospora caninum

The antigenic domain of the major surface protein (Nc-p43) of Neospora caninum was examined by polymerase chain reaction of its gene fragments and recombinant expression as GST fusion proteins. The fragments of Nc-p43 were as follow: a total open reading frame (OFR), T; OFR without signal sequence and C-terminal hydrophobic sequence, S; N-terminal 2/3 parts of S, A; C-terminal 2/3 parts, P; N-terminal 1/3 part, X; middle 1/3 part, Y; and C-terminal 1/3 part, Z, respectively. The DNA fragments were cloned into pGEX-4T vector. Recombinant plasmids transformed into Escherichia coli of BL21 pLysS (DE3) strain were induced to express GST or GST fused fragments of Nc-p43 such as 69 kDa protein for T, 66 kDa for S, 52 kDa for A, 53 kDa for P, and 40 kDa proteins for X, Y, and Z, respectively in SDS-PAGE. The Nc-p43 fragments of T, S, and P reacted with a bovine serum of neosporosis while those of A, X, Y, and Z together with GST did not in the western blot. These findings suggest that the antigenic domain of Nc-p43 of N. caninum may be localized in the C-terminal 2/3 parts. Together with A19 clone in SAG1 of Toxoplasma gondii (Nam et al., 1996), the P fragment of Nc-p43 could be used as efficient antigens to diagnose and differentiate those infections with both species.     

Purification and Characterization of the Egg Jelly Macromolecules, Sialoglycoprotein and Fucose Sulfate Glycoconjugate, of the Sea Urchin Hemicentrotus Pulcherrimus

A sialoglycoprotein and a fucose sulfate glycoconjugate (FSG) were purified from egg jelly of the sea urchin Hemicentrotus pulcherrimus . Sialoglycoprotein which consisted of sialic acid (90%, w/w) and protein (10%, w/w) did not cause induction of the acrosome reaction and sperm isoagglutination. FSG which contained one mol sulfate/mol fucose possessed 2.0 times protein to fucose by weight. The proteins in intact FSG were separated to two major (258 kDa and 237 kDa) and one minor (120 kDa) proteins by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of 2-mercaptoethanol (2-ME) while the proteins could not be separated by HPLC in the presence of 0.1% SDS or SDS-PAGE without 2-ME. However, after carboxymethylation of FSG, two major (260 kDa and 240 kDa) proteins and two minor (140 kDa and 135 kDa) proteins were separated from the fucose sulfate moiety by HPLC in the presence of 0.1% SDS or SDS-PAGE without 2-ME. When FSG was first carboxymethylated with non-radioactive iodoacetic acid and then reduced with 2-ME and finally carboxymethylated with ¹⁴C-iodoacetic acid, the most of radioactivity was detected in 140 kDa and 135 kDa proteins. Carboxymethylted-FSG was less potent than intact FSG in induction of the acrosome reaction. Fucoidan, a fucose sulfate polymer, did not induce the acrosome reaction.     

Surface proteins localized in ruffling membranes and filopodia of human glioma cells detected using Mab S-11E10

We prepared mouse monoclonal antibody (Mab S-11E10) for the surface antigen specifically distributed in ruffling membranes and filopodia of cultured human glioma cells. The antibody reacted to the specified structures of other glioma cells (U251MG, KNS 60) as well as those of KNS 42 cells, which were the immunizing source. The antigens, identified by Mab S-11E10, had molecular weights of 65 and 66 kDa. Immunohistochemically, the antibody reacted only to astrocytes in the human brain, but the cross-reactivity was noted in extraneural tissues such as lymphocytes and epithelium of the bowel ducts. In 5 out of 6 low grade astrocytomas, most of the tumor cells were strongly stained, while tumor cells of highly cellular areas of anaplastic astrocytomas and glioblastomas were either not stained or only weakly stained. The specific localization of 65-66 kDa doublet proteins may suggest relation to spreading and locomotion of cells, and may correlate to astrocytic differentiation and/or function.     

Flagellasialin: a novel sulfated alpha2,9-linked polysialic acid glycoprotein of sea urchin sperm flagella

A novel alpha2,9-linked polysialic acid (polySia)-containing glycoprotein of sea urchin sperm flagella was identified and named "flagellasialin." Flagellasialin from Hemicentrotus pulcherrimus shows a diverse relative molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of 40-80 kDa. Flagellasialin is a 96-amino acid, threonine-rich, heavily O-glycosylated (80-90% by weight) glycoprotein with a single transmembrane segment at its C-terminus and no apparent cytosolic domain. Of 12 extracellular Thr residues, eight are O-glycosylated and three are nonglycosylated. Flagellasialin is highly expressed in the testis but cannot be detected in the ovary. The amino acid sequences of flagellasialin from three sea urchin species (H. pulcherrimus, Strongylocentrotus purpuratus, and Strongylocentrotus franciscanus) are identical, but some species differences exist in the three core glycan structures to which the sulfated alpha2,9-linked polyNeu5Ac chain is linked. Finally, the treatment of sperm with a specific antibody against the alpha2,9-linked polyNeu5Ac structure results in the elevation of intracellular Ca(2+) and inhibition of sperm motility and fertilization, implicating flagellasialin as a regulator of these critical processes.     

Exogastrulation induced by heavy water in sea urchin larvae.

Replacement of H2O with D2O in seawater causes exogastrulation in larvae of the sea urchins, Hemicentrotus pulcherrimus, Strongylocentrotus intermedius and S. nudus. When larvae at any stages before mesenchymal blastula stage are transferred to 40% D2O-seawater all of them develop gradually to exogastrulae and finally up to plutei with evaginated archenterons. Effects of D2O are partly reversible at limited steps of the way to exogastrulation. Fertilisation and cleavage are not affected appreciably by D2O (50% or less) except for the delay of cleavage.     

Trichinella spiralis: murine strain variation in response to monoclonally defined, protective, nonstage-specific antigens

Nine hybridoma cell lines secreting monoclonal antibodies (mAbs) against Trichinella spiralis muscle larvae (ML) excretory/secretory antigens (ESA) were developed. Two mAbs, 6-D8-E3 (6D8) and 6-B1-G10 (6B1), were studied in detail. Western blot analysis using ML ESA showed that 6D8 recognized 35- and 40-kDa constituents whereas 6B1 identified a doublet of 33 kDa. However, Western blots of SDS-PAGE of crude ML homogenate showed that 6D8 identified proteins of approximately 35 and 43-60 kDa, whereas 6B1 recognized bands of 42-50 kDa. These results indicated substantial apparent MW differences between secreted and nonsecreted proteins recognized by both mAbs. Neither 6D8 nor 6B1 reacted with adult worm ESA, but both recognized antigens in aqueous extracts of homogenates of whole adult worms. Competitive inhibition experiments using ML ESA as a target demonstrated that the antigen epitopes recognized by monoclonals 6D8, 6B1, a rat mAb, 9D4, and a 37-kDa antigen previously defined were noncross-reactive. MAbs 6D8, 6B1, and 9D4 were used to isolate proteins possessing target determinants by affinity chromatography from crude ML homogenates. Each mAb isolated distinct protein species as determined by SDS-PAGE (6B1, approximately 42 kDa; 6D8, approximately 28, 37, and 61 kDa; 9D4, approximately 29, 33, 38-57, 80, and 86 kDa). NFS mice responded in a dose-dependent manner to affinity-purified antigens and were 25-fold more effective (by weight of antigen) than either C3Heb/Fe(C3H) or B10.BR mice. Immunization of mice with 6D8, 6B1, or 9D4 antigens induced strong protection against a subsequent challenge infection in NFS mice as indicated by accelerated intestinal adult worm expulsion, reduced fecundity of the female worms, and reduction of ML burden. Affinity-isolated antigens stimulated in vitro proliferation of spleen and MLN cells from immune mice; however, the mitogenic response to these antigens barely varied among NFS, C3H, and B10.BR strains.     

Biochemical characterization of two ram cauda epididymal maturation-dependent sperm glycoproteins

Rabbit polyclonal antibodies were raised against ram cauda epididymal sperm proteins solubilized by N-octyl-beta-D-glucopy-ranoside (anti-CESP) and against proteins of the fluid obtained from the cauda epididymidis (anti-CEF). The anti-CESP polyclonal antibody reacted with several bands from 17 to 111 kDa with different regionalization throughout the epididymis. The strongest epitopes at 17 kDa and 23 kDa were restricted to the cauda epididymidis. The anti-CEF polyclonal antibody reacted mainly with a 17-kDa and a 23-kDa compound in the cauda sperm extract. These cauda epididymal 17- and 23-kDa proteins disappeared after orchidectomy, but they reappeared in the same regions after testosterone supplementation, indicating that they were secreted by the epithelium. The fluid and membrane 17- and 23-kDa antigens had a low isoelectric point and were glycosylated. The fluid 17- and 23-kDa proteins had hydrophobic properties: they were highly enriched in the Triton X-114 detergent phase and could be extracted from the cauda epididymal fluid by a chloroform-methanol mixture. These proteins were further purified, and their N-terminal sequences did not match any protein in current databases. A polyclonal antibody against the fluid 17-kDa protein recognized the protein in the cauda epididymal sperm extract and immunolocalized it on the sperm flagellum membrane and at the luminal border of all cells in the cauda epididymal epithelium. These results indicated that secreted glycoproteins with hydrophobic properties could be directly integrated in a specific domain of the sperm plasma membrane.