Involvement of protein serine and threonine phosphorylation in human sperm capacitation.

The involvement of serine and threonine phosphorylation in human sperm capacitation was investigated. Anti-phosphoserine monoclonal antibody (mAb) recognized six protein bands in the 43-55-kDa, 94 +/- 2-kDa, 110-kDa, and 190-kDa molecular regions, in addition to a faint band each in the 18-kDa and 35-kDa regions. Anti-phosphothreonine mAb recognized protein bands in six similar regions, except that the 18-kDa, 35-kDa, and 94 +/- 2-kDa protein bands were sharper and thicker, and an additional band was observed in the 110-kDa molecular region. In the 43-55-kDa molecular region, there was a well-characterized glycoprotein, designated fertilization antigen, that showed a further increase in serine/threonine phosphorylation after exposure to solubilized human zona pellucida. In a cell-free in vitro kinase assay carried out on beads or in solution, four to eight proteins belonging to similar molecular regions, namely 20 +/- 2 kDa, 43-55 kDa, 94 +/- 2 kDa, and 110 +/- 10 kDa, as well as in 80 +/- 4 and 210 +/- 10 kDa regions, were phosphorylated at dual residues (serine/tyrosine and threonine/tyrosine). Capacitation increased the intensity of serine/threonine phosphorylation per sperm cell, increased the number of sperm cells that were phosphorylated, and induced a subcellular shift in the serine/threonine-specific fluorescence. These findings indicate that protein serine/threonine phosphorylation is involved and may have a physiological role in sperm capacitation.     

Effects of experimentally generated bull antisperm antibodies on in vitro fertilization.

To test the hypothesis that bull antisperm antibodies have the capacity to interfere with fertilization, antisperm antibodies were generated in three 13-mo-old Holstein bulls by auto-immunizing each bull with sperm three times. All bulls produced serum antisperm IgG1 and IgG2 antibodies. No serum antisperm IgA nor seminal plasma antisperm antibodies of any isotype could be detected by ELISA. Western blots were performed with immunopurified IgG1 and IgG2 from pre- and post-immunization sera from one test bull. Both post-immunization IgG1 and IgG2 recognized a 45-kDa sperm antigen. Serum samples from a normal bull stud population tested by ELISA had significantly higher levels of antisperm antibodies than did heifers. The bull stud serum samples giving the highest ELISA values differed from those of the immunized bulls in that their antisperm antibodies were of the IgM isotype only. Bull sperm were incubated with serum from the immunized and control bulls, then added to bovine oocytes in vitro. Incubation of sperm with post-immunization serum reduced in vitro fertilization rates (p < 0.01). This study demonstrated that antisperm IgG1 and IgG2 generated by sperm auto-immunizations reduced fertility in vitro, and therefore naturally occurring antisperm antibodies may affect fertility in bulls.     

SOB3, a human sperm protein involved in zona pellucida binding: Physiological and biochemical analysis,purification

LB5 antibody was selected from a monoclonal antibody (mAb) library directed against human sperm proteins. LB5 mAb detected the corresponding protein SOB3 in the neck region and the flagellum of most live ejaculated sperm while it labelled, in addition, the acrosome of about 10–20% of spermatozoa. The percentage of LB5 acrosome-stained sperm was significantly correlated with the percentages of either spontaneous or A23187-induced acrosome-reacted sperm. While SOB3 could not be detected in the testis, it appeared in spermatozoa from the corpus epididymis segment. LB5 mAb impaired neither sperm motion parameters, acrosomal reaction triggering, nor sperm binding to zona-free hamster oocytes. By contrast, LB5 Fab fragments (200 μg/ml) inhibited sperm binding to human zonae pellicidae by 35.7%. If sperm were induced to acrosome react with A23187 prior to LB5 treatment, the inhibitory effect shifted to 59.9%, while no significant effect was observed following A23187 incubation alone. Western blotting of human sperm and cauda epididymis extracts revealed two bands of 18 and 19 kDa. While no cross-reaction was observed with other tested organs, a similar 18-kDa band was revealed in erythocytes and one of 19 kDa in B-lymphocytes. No cross-reactivity could be evidenced in any animal sperm analyzed. SOB3 was first separated in a 17- to 20-kDa preparative electrophoresis fraction and finally purified by isoelectrofocusing according to its pI of 9.8. These results suggest that SOB3 is localized under the outer acrosomal membrane, that it participates in secondary sperm binding to the zona pellucida, and that it shares homologies with the immune system. Mol. Reprod. Dev. 49:286–297, 1998. © 1998 Wiley-Liss, Inc.     

Follicle size-dependent effects of sow follicular fluid on in vitro cumulus expansion, nuclear maturation and blastocyst formation of sow cumulus oocytes complexes

Follicular fluid from 2 to 4 and 5 to 8 mm diameter non-atretic follicles (SFF and LFF, respectively) of sows was added during IVM of cumulus oocytes complexes (COCs) to study its effects on cumulus expansion, nuclear maturation, and subsequent fertilization and embryo development in presence or absence of recombinant human FSH. COCs aspirated from 2 to 5 mm follicles of sow ovaries, were cultured for the first 22 h in TCM-199 and 100 microM cysteamine, with or without 10% pFF and/or 0.05 IU/ml recombinant hFSH. For the next 22 h, the COCs were cultured in the same medium, but without pFF and FSH. After culture, cumulus cells were removed and the oocytes were either fixed and stained to evaluate nuclear stages or co-incubated with fresh sperm. Twenty-four hours after fertilization, presumptive zygotes were fixed to examine fertilization or cultured for 6 days to allow blastocyst formation. Subsequently, embryos were evaluated and the blastocysts were fixed and stained to determine cell numbers. When LFF was added to maturation medium, cumulus expansion and percentage of nuclear maturation (277 +/- 61 microm and 72%, respectively) of COCs were significantly higher (P < 0.05) than those in SFF (238 +/- 33 microm and 55%, respectively). However, in the presence of FSH both FF stimulated cumulus expansion and nuclear maturation to a similar degree. No differences were observed with regards to sperm penetration, male pronucleus formation, and to polyspermia between fertilized oocytes matured either in SFF or LFF. Fertilized oocytes matured in the presence of LFF without or with FSH showed a higher cleavage (45 +/- 7% and 51 +/- 7%, respectively) and blastocyst (14 +/- 4% and 22 +/- 6%, respectively) formation rate compared to SFF (cleavage, 35 +/- 8% and 41 +/- 4%, blastocyst: 8 +/- 3 and 13 +/-3, respectively; P < 0.05). The mean number of cells per blastocyst did not differ significantly between treatments. These findings indicate that factor(s) within follicles at later stages of development play an important role during oocyte maturation and thereby enhance developmental competence to occur.     

Antisperm antibodies associated with infertility: properties and encoding genes of target antigens

Infertility among couples of reproductive age is a perplexing condition when the cause is indeterminate. These cases are classified as unexplained infertility. In a subset of subjects, antisperm antibodies with sperm agglutinating and/or immobilizing activities have been detected in the blood or fluids of the reproductive tract. These cases are designated as immunologic infertility although a cause and effect relationship of the antibodies to infertility has not been established. In this review, seven target sperm antigens to antibodies associated with infertility and their encoding genes are described. The antisperm antibodies (ASAs) examined were obtained from infertile women or were monoclonal antibodies (mAb) raised against human sperm proteins. All the ASAs studied possessed potent sperm agglutinating and/or immobilizing activities. The target antigens were isolated from human and other mammalian sperm, and the encoding genes identified. The seven antigens are YWK-II, BE-20, rSMP-B, BS-63 (nucleoporin-related), BS-17 (calpastatin), HED-2 (zyxin), and 75- kDa. Each antigen is a distinct and separate entity and is produced by different cells of the reproductive tract, (e.g., germ cells, epididymal epithelial cells, and Sertoli cells). No single predominant target component has been found to interact with the ASAs. It is proposed that immunologic infertility is the consequence of the combined actions of multiple ASAs in immobilizing and/or agglutinating spermatozoa, blocking spermegg interaction, preventing implantation, and/or arresting embryo development.     

Hybridoma antibody characterization of stage-specific and stage-cross-reactive antigens of Eimeria tenella

Hybridoma antibodies (HAb) have been raised against the sporozoite stage of 3 species of avian coccidia. These HAb were utilized in Western blot analysis, resulting in the immunoenzymatic detection of sporozoite and merozoite antigens of 1 species, Eimeria tenella. The 5 HAb specific for the sporozoite stage showed either single bands at 22 and 28 kDa or a large diffuse band in the 7-10-kDa range. The 4 HAb that cross-reacted with both asexual stages recognized either a single sporozoite or merozoite antigen of 90 kDa, or multiple antigens (47-69 kDa) for both stages. The 9 HAb demonstrated 5 different immunofluorescent antibody (IFA) patterns, and the 4 cross-reactive HAb showed similar IFA patterns with both asexual stages of E. tenella. The sporozoite-specific HAb which identified the 22, 7-10, and 7-8 kDa antigens showed surface, surface-internal, or internal IFA patterns. The other sporozoite-specific HAb, which labeled the 28-kDa antigen, stained the refractile body. The IFA of the 4 stage-cross-reactive HAb, which recognized the 45-60-kDa and the 90- or 47-69-kDa antigens, localized these antigens to the surface and tip, respectively. Rabbit anti-sporozoite serum appeared to recognize all of the sporozoite and merozoite antigens identified by the HAb as well as a variety of additional stage-cross-reactive antigens.     

Pre-treatment of cattle sperm and/or oocyte with antibody to lipocalin type prostaglandin D synthase inhibits in vitro fertilization and increases sperm-oocyte binding

The present study was conducted to determine the affect of pre-treating of oocytes and/or sperm with a rabbit polyclonal antibody against recombinant cattle lipocalin type prostaglandin D synthase (alpha L-PGDS) on in vitro sperm-oocyte binding and fertilization. In vitro matured cattle oocytes were incubated (39 degrees C, 5% CO(2) in air) for 1h in the following treatments either 500 microL of fertilization medium (FM) or FM with alpha L-PGDS (1:2000). Frozen-thawed spermatozoa were washed by a 45/90% layered Percoll gradient centrifugation and incubated for 1h either FM or FM with alpha L-PGDS. This study utilized five different treatments: (1) no antibody (control); (2) a rabbit IgG against a non-bovine antigen, bacterial histidase (alpha-hist); (3) alpha L-PGDS at fertilization time (with fertilization medium); (4) alpha L-PGDS-treated oocytes; or (5) alpha L-PGDS-treated sperm. Pre-treated oocytes were incubated with 10 x 10(4) washed spermatozoa per 25 oocytes. Oocytes used to assess sperm binding were stained with Hoescht 33342, and the number of sperm bound per zonae pellucidae counted. The remaining oocytes were fixed in acid alcohol, stained with 1% acetate-orcein and observed to determine the presence of pronuclei. More sperm bound to the zonae pellucidae when oocytes and/or sperm were pre-treated with alpha L-PGDS: (1) 26.4+/-3.0; (2) 25.6+/-3.0; (3) 59.7+/-3.0; (4) 56.4+/-3.0; and (5) 57.1+/-3.0. Addition of alpha L-PGDS with sperm, oocytes, or both, decreased fertilization (P<0.05) compared with the control: (1) 89.2+/-2.0%; (2) 87.5+/-2.0%; (3) 19.4+/-2.0%; (4) 27.2+/-3.1%; and (5) 14.1+/-3.4%. The alpha L-PGDS reacts with both oocytes and spermatozoa, resulting in increases of in vitro sperm-oocyte binding and inhibition of fertilization. These observations suggest that L-PGDS may have a role in cattle fertilization.     

Antibodies to sperm surface antigens and the c-myc proto-oncogene product inhibit early embryonic development in mice.

The effects of antibodies against sperm antigens and the c-myc proto-oncogene product on early embryonic development were investigated in mice. Affinity-purified Fab' antibodies against lithium diiodosalicylate (LIS)-solubilized murine sperm extract and fertilization antigen (FA-1) reduced (p less than 0.01 to p less than 0.001) blastulation rates of in vitro cultured 2-cell murine embryos primarily because of an arrest of development at the morula stage. Similarly, the c-myc monoclonal antibody (mAb) affected early embryonic development in a dose-dependent manner. These effects were specific, since immunoabsorption, with its respective peptide, completely blocked the inhibitory effect of the c-myc mAb. Anti-LIS sperm Fab' identified four protein bands (approx. 36, 29, 24.6, and 17.6 kDa) on Western blots of extracts from unfertilized and fertilized ova, one band (approx. 68 kDa) each on 4-8-cell embryo and morula extracts, and one band (approx. 53 kDa) on blastocyst extracts. Anti-FA-1 Fab' did not react with unfertilized or fertilized ova, but specifically identified two protein bands (approx. 53 and 25.7 kDa) on blots of 2-cell-embryo extract, one band (approx. 25.7 kDa) on morula extract, and one band (approx. 53 kDa) on blastocyst extract. The c-myc mAb did not react with any band corresponding to the c-myc protein on blots of extracts from unfertilized or fertilized ova, 2-cell embryos, 4-8-cell embryos, morulae, or blastocysts. These results suggest that some of the cross-reacting sperm antigens that are expressed during early cleavages, and the product of the c-myc proto-oncogene may have a role in normal early embryonic development.     

Characterization of a cleavage product in the human choriogonadotropin beta-subunit

The various molecular forms of human chorionic gonadotropin present in a crude preparation of urine from pregnant women were analyzed by two-dimensional gel electrophoresis and immunoblotting with monoclonal antibodies directed to synthetic peptides corresponding to the carboxyl-terminal part of either the alpha or beta-subunit. Under reducing conditions, immunoblotting with antibodies directed to the beta-subunit revealed the presence of a low-molecular-weight material of 22 kDa. This molecular form had large heterogeneity, as analyzed by isoelectrofocusing; it was immunoreactive with antibodies directed to the 111-145 region. Using microsequencing techniques, we found that the fragment had a NH2 terminal portion corresponding to the sequence of the beta-subunit appearing from residue 48. Thus, the 22-kDa fragment comprises the 48-145 portion of the beta-subunit and is probably a cleavage product of the native protein with intrachain nicking.     

Terminal beta-D-galactofuranosyl epitopes recognized by antibodies that inhibit Trypanosoma cruzi internalization into mammalian cells

Polyclonal antisera to 80 - 90-kDa and to 50 - 60-kDa polypeptides of tissue-culture trypomastigotes which inhibit the interiorization of trypomastigotes of the Y strain (up to 70%) and of metacyclic trypomastigotes of the CL-14 clone (up to 50%) in cultured mammalian cells, were obtained. Both sera immunoprecipitate surface polypeptides of 90 kDa, 80 kDa, 72 kDa and 58 kDa in trypomastigotes and of 80 kDa, 77 kDa and 74 kDa in metacyclic trypomastigotes. These antigens are glycoproteins with affinity for concanavalin A. The antibodies (IgG class) of the inhibitory sera are mainly directed against carbohydrate epitopes, which were identified as being beta-D-galactofuranosyl units by radioimmunoinhibition assays. The direct involvement of the beta-D-galactofuranosyl unit in the process of parasite infection was verified using the synthetic disaccharide beta-D-galactofuranose (1----3)-alpha-D-mannopyranose which promoted, at 1 mg/ml concentration, 50% inhibition of internalization.     

Identification of human sperm peptide sequence involved in egg binding for immunocontraception

Development of a vaccine based on sperm antigens represents a promising approach to contraception. The sperm-zona pellucida (ZP) interaction constitutes the most important event in the fertilization process, and the molecular sequences involved at this site may provide the most attractive candidates for immunocontraception. In the present study, using the phase peptide display technique, a novel dodecamer sequence, designated as YLP(12), was identified that is involved in sperm-ZP recognition/binding. The synthetic 12-mer peptide based on this sequence and its monovalent Fab' antibodies specifically and significantly (P < 0.05) inhibited human sperm-ZP binding. In Western blot and immunoprecipitation procedures, the YLP(12) peptide recognized the ZP3 component of solubilized human ZP proteins. In the Western blot procedure involving 10 different human tissue extracts, the anti-YLP(12) Fab' antibodies recognized a protein band of approximately 72 +/- 2 kDa only in the testis lane. The peptide sequence was localized on the acrosomal region of the human sperm cell. These findings indicate that the novel testis-specific 12-mer YLP(12) that is present in the acrosomal region and is involved in human sperm-ZP interaction may find applications in contraceptive vaccine development, as well as in diagnosis and treatment of male infertility mediated through sperm dysfunction.     

Monoclonal antibodies against unfertilized zona-free mouse oocytes: Characterization and effects on fertilization

A panel of anti-oocyte antibodies was raised against unfertilized zona-free mouse oocytes by intrasplenic immunization and checked for their effects on in vitro fertilization. Four antibodies decreased the fertilization rate from about 90% in controls to 8% (B5-2 F7), 12% (A2-2 A7), 13% (4-G1), and 25% (A2-2 F2), when the sperm cell concentration was 1 × 10⁵ to 1 × 10⁶. Antigen localization: All the antibodies labelled components in the cell membrane of zona-free oocytes as demonstrated by indirect immunofluorescence and/or by complement-mediated oocyte lysis. In various patterns, the ooplasm and zona pellucida were also labelled with different intensities. Western blotting: A2-2 A7 and A2-2 F2 recognized a protein with a molecular weight of approximately 65 kDa, while antibody B5-2 F7 bound a 97 kDa protein. Complement activation and complement-mediated oocyte lysis: Systemically injected antibodies, C3 and C4 were detected on zona-free oocytes recovered from the mouse oviduct indicating the activation of C3 and C4 by antigen-antibody complexes. The recovered oocytes were not damaged, suggesting a presence of complement-regulating factors. In vitro, however, a large number of zona-free oocytes preincubated with antibodies were lysed or protruded ooplasma vesicles in complement-active serum. Stage, tissue, and species specificity: None of the antibodies, except A2-2 A7, showed a positive immunolabelling to the pronuclear stage. Antibodies 4-G1 and A2-2 F2 cross-reacted with the ovarian oocytes. No antibodies bound to any of the tissues tested, indicating that the corresponding antigen epitopes are not commonly expressed. A2-2 A7, A2-2 F2, and B5-2 F7 cross-reacted with hamster and human unfertilized oocytes, suggesting the presence of developmentally conserved molecules and the possibility to apply these antibodies in hamster and human in vitro fertilization. It is concluded that the approach used could be a useful strategy in searching for anti-fertilization antibodies for human contraception. © 1996 Wiley-Liss, Inc.     

The use of in vitro fertilization techniques to investigate the fertilizing ability of bovine sperm with proximal cytoplasmic droplets

The objective of this experiment was to determine the effect of proximal droplets on sperm-oocyte binding, zona penetration, fertilization, and the developmental competence of oocytes fertilized by sperm with proximal droplets (PD) in an in vitro fertilization (IVF) and culture system. Frozen semen from three bulls (PD1, PD2 and PD3) with varying proportions of normal appearing sperm with proximal droplets and semen from a normal control bull (C) were used in this experiment. The mean number of sperm bound to the zona pellucida (26.8 +/- 2.0, n=100; 15.2 +/- 1.1, n=100; 16.2 +/- 1.0, n=100) for bulls PD1, PD2, and PD3, respectively, were significantly lower (P<0.05) than that of the control bull C (47.4+/- 1.9; n=114). No spermatozoa with PD were found bound to the zona pellucida and this finding was consistent among the three bulls. The percentage penetration of zonae for the bulls PD1, PD2 and PD3 (74%, 74/100; 71%, 71/100 and 69%, 69/100, respectively) were not different than that of bull C (72%, 179/245). Similarly, the mean number of sperm penetrating the zona pellucida (1.43+/- 1.2, 1.24 +/- 1.1 and 1.20 +/- 1.1, for bulls PD1, PD2 and PD3, respectively) were not different than that of bull C (1.45 +/- 1.1). However, fertilization rates (8.8%, 8/90; 16.8%, 16/95; and 10.6%, 11/103, for bulls PD1, PD2 and PD3, respectively) were lower (P<0.001) than that of bull C (68.7%; 77/112). Similarly, cleavage rates (5%, 10/200; 8%, 8/100 and 14%, 15/111) for the bulls PD1, PD2 and PD3, respectively, were lower than that of the control bull, C (60.7%; 79/130). Cleaved zygotes resulting from the fertilization of oocytes by apparently normal sperm from bulls with PD did not develop beyond cleavage, whereas, 43.8% (57/130) morulae and 20% (26/130) blastocysts were produced by oocytes fertilized by sperm from bull C. In summary, normal appearing sperm with PD did not bind to the zona pellucida. Apparently normal sperm with out proximal droplets co-existing in the semen along with sperm containing PD were also functionally deficient, resulting in reduced zonae binding and zygotes resulting from insemination with semen with a high percentage of PD did not develop beyond cleavage.     

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.     

Cross-antigenicity between the major surface proteins (ospA and ospB) and other proteins of Borrelia burgdorferi

Two of the major surface Ag of Borrelia burgdorferi, the 31-kDa OspA and 34-kDa OspB proteins, are encoded by a 49-kb plasmid. In this study, mAb and monospecific polyclonal antibodies were used to define cross-antigenicity of the OspA and OspB protein to each other and to other lower molecular mass proteins by Western blot analysis. Two mAb studied, 105.5 and 184.1, were directed predominantly against the 31-kDa OspA protein. However, each also reacted with other minor bands, though with different specificities. Using V8 protease digestion and cleavage by cyanogen bromide, we demonstrated that each mAb reacted to the 31-kDa protein differently. Monospecific polyclonal rabbit and human antibodies directed against the 34-, 31-, 22-, and 20-kDa proteins were eluted from blots and used to further corroborate the cross-reactivity among these Ag. Rabbit antibodies to the 31- and 22-kDa Ag gave remarkably similar peptide maps after V8 protease digestion of the 31-kDa OspA protein, as did mAb 184.1, suggesting that this mAb recognized an immunodominant epitope common to the 22- and 31-kDa proteins. It seems likely therefore that the humoral immune response to Borrelia surface Ag may be due to a limited number of cross-reactive epitopes on distinct, but related, gene products.     

Toxoplasma gondii: characterization and localization of antigens secreted from tachyzoites

Since we had previously demonstrated the protective role played by Toxoplasma excreted-secreted antigens, the aim of the present work was to produce monoclonal antibodies directed against these antigens in order to determine if their localization in the parasite is compatible with a mechanism of excretion or secretion. Western immunoblotting analysis revealed three monoclonal antibodies (TG17-179, TG17-43, and TG17-113) raised against excreted-secreted antigens of 28.5, 27, and 21 kDa, respectively. The TG17-179 which reacts with antigens isolated by Concanavalin A affinity chromatography is directed against a glycosylated 28.5-kDa component. Colloidal immunogold labeling showed the ultrastructural localization of the 21-, 27-, and 28.5-kDa antigens in the matrix of the dense granules of tachyzoites and associated with the microvilli network of the parasitophorous vacuole, after host cell invasion. These observations suggest the following mechanism of Toxoplasma secretion: secreted antigens are first stored in tachyzoite-dense granules and are then released inside the parasitophorous vacuole. Among the secretory molecules characterized here, the native 27-kDa antigen recognized by TG17-43 is a calcium-binding protein found to be intermixed with the 21- and 28.5-kDa antigens inside the dense granules and hence could play a role in the packaging of secretory products. In addition, the 21- and 28.5-kDa antigens were also located beneath the parasite plasma-lemma. This particular location could reflect a transient step characteristic of T. gondii secretion.     

Characterization of two murine monoclonal antibodies (P10, P12) directed against different determinants on human blood platelet thrombospondin

Thrombospondin, a 450-kDa glycoprotein composed of three disulphide linked chains, is located in human blood platelet alpha-granules and is released from platelets upon stimulation. This glycoprotein is thought to play a major role in platelet aggregation. The aim of this study was to characterize two monoclonal antibodies (P10 and P12) directed against human blood platelet thrombospondin. When the released material obtained after stimulation of platelets with thrombin in the presence of 2 mM calcium was immediately treated with EDTA, labelled with 125I and incubated with monoclonal antibodies P10 and P12, both immunoprecipitated a major labelled protein band with a molecular mass of 160 kDa and a weaker band at 146 kDa, as analysed on reduced dodecyl sulphate/polyacrylamide gels. The major band corresponds in molecular mass to the thrombospondin subunits. If, however, the released material was left in the presence of Ca2+ for 48 h, then the main band was at 130 kDa and in addition one minor protein band (75 kDa) was immunoprecipitated by P10 whereas P12 recognized two minor protein bands (75 and 60 kDa). When P10 and P12 were incubated with 125I-labelled platelet releasates treated for 48 h at 4 degrees C with 10mM EDTA, three major protein bands (160, 146 and 130 kDa) were immunoprecipitated in addition to the minor bands mentioned above. These results indicate that thrombospondin is probably degraded by the endogenous platelet calcium-dependent protease. Investigation of tryptic peptide fragments of thrombospondin isolated by fast protein liquid chromatography showed that 125I-labelled antibody P10 bound to 400-kDa and 120-kDa fragments whereas 125I-labelled P12 only recognized a 400-kDa fragment. Competition studies involving solid-phase antibody binding and double antibody sandwich assays showed that P10 and P12 were directed against different determinants of thrombospondin. Purified thrombospondin, isolated in the presence of calcium, either directly or after treatment with EDTA, haemagglutinated trypsinized, formaldehyde-fixed sheep erythrocytes identically. The haemagglutination activity of EDTA-treated thrombospondin was inhibited by P10 and enhanced by P12. On the other hand, P10 and P12, despite their binding to calcium-treated thrombospondin, had no effect on its haemagglutination activity. Monoclonal antibodies P10 and P12 could be useful tools to investigate the role of thrombospondin in platelet aggregation.     

Characteristics of monoclonal antibodies reactive with human sperm and seminal plasma

The characteristics of monoclonal antibodies developed against human spermatozoa are described. Out of 10 monoclonal antibodies 9 did not react in ELISA with human RBC, WBC, platelets, Raji cells nor mouse sperm. Four monoclonal antibodies reacted with monkey sperm and all 10 reacted with human seminal plasma. Monoclonal antibodies showed differential reactivity with pre- and post-capacitated sperm. Four monoclonal antibodies were able to agglutinate sperm whereas none of these were positive in sperm-immobilization assay. Interestingly, two monoclonal antibodies (MA-46 and MA-50) were able to block the attachment of pre-capacitated sperm to zona denuded hamster oocytes. MA-46 and MA-50 recognized in immunoblot spermatozoa antigens having apparent molecular weights of 14 and 20 K Da and greater than 200 K Da respectively. The monoclonal antibodies reported in this study will be useful in further delineating the spermatozoa antigens involved in regulation of fertility.     

Association of egg zona pellucida glycoprotein mZP3 with sperm protein sp56 during fertilization in mice.

Purified mouse sperm receptor, a zona pellucida glycoprotein called mZP3, binds to plasma membrane overlying acrosome-intact sperm heads (P.M. Wassarman, 1999, Cell 96, 175-183). Some evidence suggests that mZP3 binds to sp56, a protein reported to be associated peripherally with the plasma membrane of acrosome-intact sperm heads (J.D. Bleil and P.M. Wassarman, 1990, Proc. Natl. Acad. Sci., USA 87, 7215-7219; A. Cheng et al., 1994, J. Cell Biol. 125, 867-878). Here, we report that membrane vesicles prepared from acrosome-intact sperm contain sp56. When these vesicles are incubated with eggs they inhibit binding of sperm to eggs in vitro (ID50 approximately 50-100 microg protein/ml). On the other hand, a monoclonal antibody directed against sp56 relieves the inhibition of binding of sperm to eggs by membrane vesicles. As expected, incubation of intact sperm with the antibody directed against sp56 inhibits binding of the sperm to eggs. Results of immunoprecipitation of sperm extracts incubated with mZP3, by either a polyclonal antibody directed against mZP3 or a monoclonal antibody directed against sp56, suggest that mZP3 is specifically associated with sp56. Results of laser scanning confocal microscopy of fixed sperm probed with antibodies directed against either sp56 or a approximately 155 kDa acrosomal protein, suggest that the two proteins are present in the acrosome, but with different distributions. Furthermore, confocal images of sperm, fixed after exposure to purified mZP3 and probed with antibodies against mZP3 and sp56, reveal overlap between mZP3 and sp56 at the surface of the sperm head. The possible implications of these results are discussed in the context of mammalian fertilization.     

Monoclonal antibodies as probes for processing of the mosquito yolk protein; a high-resolution immunolocalization of secretory and accumulative pathways

A library of monoclonal antibodies (mAB) directed against yolk polypeptides of the mosquito Aedes aegypti was utilized to visualize the secretory pathway of these polypeptides in the fat body and their accumulative pathway in developing oocytes. Single and double immunolabelling using mABs and colloidal gold of different sizes confirmed biochemical observation that 200 +/- 5 and 65 +/- 3 kDa polypeptides represent subunits of the yolk protein. This immunocytochemical analysis showed that, in trophocytes of the fat body, both the subunits of the yolk protein were routed simultaneously through the Golgi complex into secretory granules and were subsequently secreted. The yolk protein subunits were also directed together through all the steps of the accumulative pathway in the oocyte. Double immunogold labelling revealed that the subunits were present together during their binding to the oocyte membrane, transportation into and accumulation in the transitional yolk body, and, finally, crystallization in the mature yolk body. Electron microscopical immunocytochemistry also confirmed immunofluorescent data and showed that mABs directed against different steps in the biosynthetic processing of the yolk protein in the fat body, as well as in its accumulative pathway in oocytes.