Protein Of Bull Seminal Plasma Research Articles

Two-dimensional polyacrylamide gel electrophoresis of Bali bull (Bos javanicus) seminal plasma proteins and their relationship with semen quality

The present study evaluated the relationship between Bali bull (Bos javanicus) seminal plasma proteins and different semen quality parameters. Semen samples from 10 mature Bali bulls were evaluated for conventional semen parameters (general motility, viability, and normal morphology), sperm functionality (acrosome reaction, sperm penetration rate, sperm penetration index), sperm kinetics (computer-assisted semen analysis parameters such as sperm velocity), and sperm morphology (acrosome and membrane integrity). Frozen–thawed semen with higher sperm motility, viability, acrosome integrity, and membrane integrity (P < 0.05) are consistently higher in acrosome reaction and sperm penetration assay. Three bulls showed the highest, four bulls displayed the medium, and the remaining three bulls showed the lowest for all sperm parameters and SPA. The proteome maps of seminal plasma from high-quality and low-quality Bali bulls were also established. Seminal plasma of both high-quality and low-quality Bali bulls was subjected to two-dimensional SDS-PAGE with isoelectric point ranged from 3 to 10 and molecular weight from 10 to 250 kDa. Approximately 116 spots were detected with Blue Silver stain, and of these spots, 29 were selected and identified by MALDI-TOF/TOF-MS/MS. A majority of the proteins visualized in the seminal plasma two-dimensional maps was successfully identified. An essential group of the identified spots represented binder of sperm 1 (BSP1), clusterin, spermadhesin, tissue inhibitor of metalloproteinases 2 (TIMP-2), and phospholipase A2 (PLA2). Other proteins found in high abundance included seminal ribonuclease, serum albumin, cationic trypsin, and peptide similar to β2 microglobulin. Thus, a reference map of Bali bull seminal plasma proteins has been generated for the very first time and can be used to relate protein pattern changes to physiopathologic events that may influence Bali bull reproductive performance.

Lipid Selectivity in Lipid Efflux Induced by Proteins and Peptides

Some proteins and peptides have the ability to extract lipids from membranes. In some systems, such as some toxins, it leads to cell death. In other systems, this process is vital. For example, bovine seminal plasma contains phosphocholine-binding proteins that associate with sperm membranes upon ejaculation and cause lipid efflux; this effect modifies the membrane lipid composition, an essential step in the maturation process of fertile sperm. In general, the affinity of these peptides/proteins for membranes and their impact on membrane permeability are well characterized. However the induced lipid efflux and particularly its lipid specificity are much less investigated. Using model membranes with different compositions, we identified the lipid selectivity of the membrane lipid efflux of two systems. First, we characterized the lipid efflux induced by BSP1, the most abundant Binder-of-Sperm protein in bull seminal plasma. The protein binding displays a distinct affinity for phosphatidylcholine. However, even though the protein anchoring on membranes is PC-specific, we demonstrated that the lipid extraction is practically without any lipid specificity; BSP1 molecules solubilise a lipid patch to form small complexes with a stoichiometry of 10-15 lipids per protein. Second, we characterized the lipid specificity of lipid efflux induced by melittin, a helical peptide with a secondary amphipathic character. Phosphatidylethanolamine, and cholesterol, two molecular species ordering bilayers, reduce the membrane affinity of melittin. We determined that, in parallel, the lipid fraction extracted by melittin is depleted of these lipids. It is proposed that the peptide accumulates in regions of bilayers where the bilayers are less ordered and, when its concentration is sufficient, detaches this patch from the membrane. These findings illustrate the need to investigate globally lipid efflux processes, from the peptide/protein binding to the mixed lipid-peptide/protein self-assembly formation.

Chemical and physical requirements for lipid extraction by bovine binder of sperm BSP1

The bovine seminal plasma contains phosphocholine-binding proteins, which associate to sperm membranes upon ejaculation. These binder-of-sperm (BSP) proteins then induce a phospholipid and cholesterol efflux from these membranes. In this work, we determined physical and chemical parameters controlling this efflux by characterizing the lipid extraction induced by BSP1, the most abundant of BSP protein in bull seminal plasma, from model membranes with different composition. The model membranes were formed from binary mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) with 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (Lyso-PC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) or cholesterol. The modulation of BSP1-induced lipid extraction from membranes by their chemical composition and their physical properties brings us to propose a 3-step extraction mechanism. First, the protein associates with membranes via specific binding to phosphocholine groups. Second, BSP1 penetrates in the membrane, essentially in the external lipid leaflet. Third, BSP1 molecules solubilize a lipid patch coming essentially from the outer lipid leaflet, without any lipid specificity, to ultimately form small lipid/protein auto-assemblies. The stoichiometry of these complexes corresponds to 10–15 lipids per protein. It is also shown that fluid-phase membranes are more prone to BSP1-induced lipid extraction than gel-phase ones. The inhibition of the lipid extraction in this case appears to be related to the inhibition of the protein penetration in the membrane (step 2) and not to the protein association with PC head groups (step 1). These findings contribute to our understanding of the mechanism by which BSP1 modify the lipid composition of sperm membranes, a key event in sperm capacitation.

Aggregated Forms of Bull Seminal Plasma Proteins and Their Heparin-Binding Activity

Heparin-binding activity of bull seminal plasma proteins was shown to be dependent on their aggregation state. The protein fraction interacting with immobilized heparin was characterized by large polydispersity in the region of molecular weight of 60 000-10 000, while that not retained on the affinity carrier was present as aggregates with molecular weight &gt;100 000. Components of heparin-binding and non-heparin-binding fractions were separated by RP HPLC (reversed-phase HPLC) and analyzed by SDS (sodium dodecyl sulfate) electrophoresis and N-terminal sequencing. Size exclusion chromatography of whole seminal plasma and heparin-binding proteins in the presence of D-fructose (as a component of seminal plasma) showed that the region of molecular weights of protein-associated forms was shifted to lower values. An increase of heparin-binding activity of bull proteins, as determined by ELBA (Enzyme-Linked Binding Assay), correlates with a decrease of their aggregation state. The modulation of the aggregation state of bull proteins by seminal plasma components and, in this way, also of their heparin-binding properties suggests possible mechanisms for capacitation mediated by these proteins.

Novel purification method for mammalian seminal plasma phospholipid-binding proteins reveals the presence of a novel member of this family of protein in stallion seminal fluid.

A family of bull seminal plasma (BSP) phospholipid-binding proteins (BSP proteins), potentiate heparin- and HDL-induced capacitation. The homologous proteins have been purified from stallion and boar seminal plasma, and detected in low concentrations in other mammalian seminal plasma. In this study, we developed a new isolation method for mammalian seminal plasma choline phospholipid-binding proteins wherein they are present in low concentrations. The method is based on the interaction of this family of proteins with egg yolk low-density lipoprotein fraction (LDF). In order to demonstrate the feasibility of the method, we incubated LDF with alcohol precipitates of bull, boar, and stallion seminal plasma. LDF were re-isolated by ultracentrifugation along with bound proteins. LDF with associated proteins were dialyzed, lyophilized, and delipidated. BSP homologous proteins were finally purified by p-aminophenyl phosphorylcholine (PPC)-agarose and/or gelatin-agarose chromatographies, and analyzed by SDS-PAGE. With this new protocol, phospholipid-binding proteins of bull, boar, and stallion seminal plasma were recovered almost 100%. A new 12 kDa stallion seminal plasma protein of the same family was also isolated and partially sequenced. The radio-immunoassay (RIA) data showed that 10 mg of LDF can bind all BSP proteins present in 120 mg of alcohol precipitated BSP proteins. These results confirm the efficiency of the method and that the LDF step could be used for the isolation of all BSP proteins homologs from different mammalian species.

Affinity chromatography of bull seminal proteins on mannan–Sepharose

The interaction of bull seminal plasma proteins and sperm with mannan was investigated using an enzyme-linked binding assay (ELBA). A high mannan-binding activity was found in the protein fraction interacting with heparin. Mannan binding to seminal plasma proteins was inhibited by d-mannose and d-fructose, but not by d-mannose-6-phosphate, d-glucose-6-phosphate, ovalbumin and ovomucoid. Mannan inhibited the binding of bovine zona pellucida glycoproteins both to bull sperm and seminal plasma proteins. Yeast mannan immobilized to divinyl sulfone-activated Sepharose was used for the isolation of mannan-binding proteins. The protein components of this fraction were identified on the basis of relative molecular mass determination and N-terminal amino acid sequencing: RNAase dimer, PDC-109 and a protein homologous to BSP-30K (relative molecular mass 14 500). The isolated proteins were characterized by a high zona pellucida binding activity.

Radioimmunoassays for bull seminal plasma proteins (BSP-A1/-A2, BSP-A3, and BSP-30-Kilodaltons), and their quantification in seminal plasma and sperm.

Three proteins, BSP-A1/-A2, BSP-A3, and BSP-30 kilodaltons (collectively called BSP proteins), represent the major proteins of bovine seminal plasma (BSP). At ejaculation, these proteins bind to the sperm surface and induce molecular changes in the plasma membrane that are deemed to be essential for sperm capacitation. The present study was carried out to develop specific radioimmunoassays (RIAs) for the quantification of each of the BSP proteins in BSP and sperm. RIAs were developed using polyclonal antibodies raised in rabbits against each BSP protein. The purified and iodinated BSP proteins were used as standard and tracer, respectively. The RIAs that were developed were shown to be specific for each protein and the cross-reactivity toward various antigens was negligible (<2%). The average sensitivity limit was 5 ng/ml of sample for BSP-A1/-A2 and BSP-A3, and 40 ng/ml of sample for BSP-30-kDa. The concentration of BSP proteins was determined by analyzing the RIA data with spline function. BSP proteins represented 40% to 57% of seminal plasma total protein (25% to 47% of BSP-A1/-A2, 3% to 5% of BSP-A3, and 3% to 7% of BSP-30 kDa) and 4% to 6% of sperm total protein (2.5% to 4% of BSP-A1/-A2, 0.4% to 0.9% of BSP-A3, and 0.5% to 1% of BSP-30-kDa). We also determined the concentration of BSP proteins that were sperm-bound after semen cryopreservation in Tris-egg yolk-glycerol extender. A significant decrease (70%-80%) in sperm-bound BSP proteins was noted after cryopreservation. The availability of reliable RIA procedures should aid in the further understanding of the role of BSP proteins in sperm function as well as their effect on sperm cryopreservation.

Immunocytochemical localization of lipocalin-type prostaglandin D synthase in the bull testis and epididymis and on ejaculated sperm.

Previously, we identified a 26-kDa fertility-associated protein in bull seminal plasma as lipocalin-type prostaglandin D synthase. The objective of the present study was to immunohistochemically localize this enzyme to the various cell types within the bull testis and seven subsegments of the epididymis, and on ejaculated sperm in order to gain further insight into its potential function in male reproduction. In the testis, immunoperoxidase staining was localized within the elongating spermatids and Sertoli cells of the seminiferous tubules, varying with the stage of the spermatogenic cycle. The highest level of staining occurred during stages III-VII. The cuboidal epithelial cells of the rete testis and efferent ducts were also immunoreactive. Expression of lipocalin-type prostaglandin D synthase was not uniform in the seven epididymal subsegments, suggesting a possible role in sperm maturation. In all epididymal regions, expression was limited to the epithelial principal cells; no immunoreactivity was apparent in other cell types. Lipocalin-type prostaglandin D synthase was strikingly localized in the caput epididymidis, while moderate to weak staining was observed in the remainder of the epididymis. Droplets of reaction product observed within the lumen increased progressively from the caput to cauda. Using fluorescence microscopy, we also localized lipocalin-type prostaglandin D synthase to the apical ridge of the acrosome on ejaculated sperm.

Biophysical Study of the Perturbation of Model Membrane Structure Caused by Seminal Plasma Protein PDC-109

PDC-109, the major heparin-binding protein of bull seminal plasma, binds specifically to sperm choline lipids at ejaculation and mediates capacitation by stimulating cholesterol and phospholipid efflux. We carried out a biophysical study to investigate the membrane perturbation effect caused by PDC-109. Binding of PDC-109 to phosphatidylcholine model membranes was maximal at a 12:1 phosphatidylcholine to protein molar ratio. The process was independent of the membrane structure and involved a slight conformational change of the protein, compatible with an increased exposure to the solvent. PDC-109 binding to dimyristoylphosphatidylcholine prevented lipid molecules from participating in the gel-to-liquid phase transition, due to enhancement of both acyl chain disorder and interfacial hydration. Visualization of the lipid–protein complexes by electron microscopy showed surface irregularities and the presence of 10-nm particles. Permeability assays confirmed the PDC-109-induced disruption of the vesicles. This effect was not modified by heparin. However, presence of cholesterol inhibited the process in a concentration-dependent manner.

Characterisation of the conformational and quaternary structure-dependent heparin-binding region of bovine seminal plasma protein PDC-109

PDC-109, the major heparin-binding protein of bull seminal plasma, binds to sperm choline lipids at ejaculation and modulates capacitation mediated by heparin. Affinity chromatography on heparin-Sepharose showed that polydisperse, but not monomeric, PDC-109 displayed heparin-binding capability. We sought to characterise the surface topology of the quaternary structure-dependent heparin-binding region of PDC-109 by comparing the arginine- and lysine-selective chemical modification patterns of the free and the heparin-bound protein. A combination of reversed-phase peptide mapping of endoproteinase Lys-C-digested PDC-109 derivatives and mass spectrometry was employed to identify modified and heparin-protected residues. PDC-109 contains two tandemly arranged fibronectin type II domains (a, Cys 24-Cys 61; b, Cys 69-Cys 109). The results show that six basic residues (Lys 34, Arg 57, Lys 59, Arg 64, Lys 68, and Arg 104) were shielded from reaction with acetic anhydride and 1,2-cyclohexanedione in heparin-bound PDC-109 oligomers. In the 1H-NMR solution structures of single fibronectin type II domains, residues topologically equivalent to PDC-109 Arg 57 (Arg 104) and Lys 59 lay around β-strand D on the same face of the domain. In full-length PDC-109, Arg 64 and Lys 68 are both located in the intervening polypeptide between domains a and b. Our data suggest possible quaternary structure arrangements of PDC-109 molecules to form a heparin-binding oligomer.

Two-dimensional polyacrylamide gel electrophoresis map of bull seminal plasma proteins.

A two-dimensional polyacrylamide gel electrophoresis map of bull seminal plasma proteins has been established. About 250 spots were detected after silver staining and polypeptides from 24 spots have been N-terminally sequenced. Major proteins already described in bull seminal plasma, like PDC-109 and aSFP, have been located on the map; proteins not yet reported in male reproductive tracts have been evidenced; for some polypeptides showing a previously unknown N-terminal sequence, structural similarities with proteins described in other organisms have been found. A reference map of seminal plasma proteins could be useful in relating protein pattern changes to physiopathological events influencing the reproductive sphere.

Conformational features and thermal stability of bovine seminal plasma protein PDC-109 oligomers and phosphorylcholine-bound complexes.

At ejaculation, PDC-109, the major heparin-binding protein of bull seminal plasma, binds to the phosphorylcholine group of sperm lipids and modulates capacitation promoted by glycosaminoglycans during sperm residence in the female genital tract. Combination of size-exclusion chromatography, analytical ultracentrifugation, circular dichroism, Fourier-transform infrared spectroscopy, and differential scanning calorimetry has allowed us to biophysically characterize PDC-109 and its interaction with phosphorylcholine. PDC-109 can be regarded as a polydisperse molecule whose aggregation state can be modulated by the solute composition of its solution environment. Dissociation of PDC-109 oligomers occurs upon increasing the concentration of either NaCl, EDTA, CaCl2, or phosphorylcholine, suggesting that both ionic and hydrophobic interactions are responsible for the aggregation tendency of PDC-109 monomers. Dissociation processes are accompanied by exposure of peptide bonds to the solvent, changes in the environment of tyrosine and tryptophan residues, and a slight increase in the turn content at the expense of non-regular structure. Analysis of the heat-induced denaturation of PDC-109 oligomers revealed two melting transitions at about 36 degrees C (irreversible) and 55 degrees C (partially reversible) characterized by calorimetric enthalpy changes of 42 kJ/mol and 217 kJ/mol, respectively. These transitions could be assigned to the dissociation of oligomers and to the cooperative unfolding of PDC-109 monomers, respectively. The modulation of the aggregation state of PDC-109 by its molecular environment and by phosphorylcholine binding suggests possible mechanisms for capacitation mediated by the seminal plasma protein.

Crystallization and preliminary X-ray diffraction analysis of bovine seminal plasma PDC-109, a protein composed of two fibronectin type II domains.

PDC-109 is a 13 kDa glycoprotein and the major phosphorylcholine- and heparin-binding protein of bull seminal plasma. It is built by an acidic 23-residue N-terminal sequence followed by a tandem of fibronectin type II domains. Full-length PDC-109 was crystallized in complex with o-phosphorylcholine by vapor diffusion in sitting drops. Crystals grew to maximal size of 0.5 x 0.3 x 0.2 mm3, diffract x-rays beyond 2.6 A resolution, and belong to space group P321 with unit cell dimensions a = b = 93.6 A, c = 52.7 A.

Immunolocalization and quantitation of acidic seminal fluid protein (aSFP) in ejaculated, swim-up, and capacitated bull spermatozoa.

Acidic seminal fluid protein (aSFP, 12.9 kDa), a major protein of bull seminal plasma, belongs to the spermadhesin protein family. Boar spermadhesins become bound to the sperm head's surface at ejaculation and are thought to play a role as capacitation factors and/or in gamete recognition and binding. Here, we have investigated the topographical distribution and fate of bovine spermadhesin aSFP during sperm capacitation in order to assess whether aSFP could be involved in similar aspects of the fertilization process as its boar homologous proteins. 5.7 +/- 2.1 x 10(6) molecules/spermatozoa were quantitated on the surface of fresh ejaculated and washed sperm. The binding site of aSFP was restricted to a thin coat at the apical part of the acrosomal cap. The amount of aSFP in swim-up sperm was 1.8 +/- 1.0 x 10(6) molecules/spermatozoa, but decreased dramatically to 22 +/- 10 x 10(3) and to undetectable levels after incubation of sperm for 1.5 h and 18 h, respectively, in capacitation medium. This indicates that the bull spermatozoa surface may be completely depleted of spermadhesin aSFP before spermatozoa reach the surroundings of the investing egg. Therefore, our results suggest that aSFP may act as a decapacitation factor on bull spermatozoa rather than as a zona pellucida binding molecule.

Immunosuppressive and leucolytic effect of phospholipid binding protein in bull seminal plasma

Phospholipid binding protein (PBP) isolated from bull seminal vesicle fluid has a haemolytic effect in addition to immunosuppressive and leucolytic activities. Leucolysis of PBP is not rigidly dependent on haemolysis. Erythrocytes are more sensitive to PBP than leucocytes.

Seminalplasmin, the major basic protein of bull seminal plasma, is a secretory protein of the seminal vesicles

From the experimental results of three independent methods: (1) indirect immunofluorescence employing monospecific anti-seminalplasmin-IgGs, (2) cell-free translation of poly(A) + RNA from seminal vesicle and testicular tissue, as well as (3) Northern analysis of poly(A) + RNA of the latter tissues with a synthetic seminalplasmin-specific antisense DNA probe, it is concluded that the biosynthesis of seminalplasmin occurs in seminal vesicles but not in testis.

The major protein of bull seminal plasma: Biosynthesis and biological function

We isolated the major protein of apparent Mr of 15,000-16,000 from seminal plasma as well as from seminal vesicle secretion of bull and proved by amino acid analysis and tryptic peptide mapping that the two proteins were identical. An antiserum against this major protein was employed to quantitate and identify the major protein in seminal plasma as well as seminal vesicle secretion. The antiserum did not cross-react with proteins from bovine or human plasma or follicular fluid respectively. Cell-free translation of poly(A)RNA from seminal vesicle tissue and immunoprecipitation yielded one major species with apparent Mr of 18,000. Using the anti-major protein antiserum, this major species was specifically immuno absorbed. Cloning and sequencing of a major protein-specific cDNA led to the identification of clone pMP17, encoding a precursor of the major protein of 128 amino acid residues. We proved that the major protein is identical to protein PDC 109 (Esch et al., Biochem. Biophys. Res. Comm. 113:861-867, 1983). The seminal vesicles synthesize major protein in an androgen-dependent fashion. In addition to intraluminal secretion of the vas deferens, ampullary spermatozoa revealed an intense immunoreaction which was restricted to the neck region of the sperm head and the middle piece, while the principal piece of the tail as well as the sperm head were devoid of immunoreactive material. Epididymal epithelium (as well as calf seminal vesicle epithelium) showed no immunoreactivity with major protein antiserum. Immunoelectron microscopy demonstrated that only spermatozoa devoid of a plasma membrane around the middle piece were able to bind the antiserum against major protein. After removal of the plasma membrane from epididymal spermatozoa, binding of major protein to subplasmalemmal binding sites was visualised using gold-labeled MP. Transblotting with gold-labeled MP demonstrated a protein of about 66 kDa which appears to represent the major protein-receptor. Binding of major protein to the receptor (after loss of the plasma membrane in the mid-piece region of the spermatozoa after contact with secretions from seminal vesicles) is interpreted as a physiological process presumably related to the onset of sperm motility.

CDNA cloning of the mRNA for the major protein of bull seminal plasma

Journal Article cDNA cloning of the mRNA for the major protein of bull seminal plasma Get access M Kemme, M Kemme Max-Planck-Institut für Biophysikalische Chemie, Göttingen Search for other works by this author on: Oxford Academic Google Scholar K H Scheit K H Scheit Max-Planck-Institut für Biophysikalische Chemie, Göttingen Search for other works by this author on: Oxford Academic Google Scholar Acta Endocrinologica (Norway), Volume 117, Issue 4_Supplement, Apr 1988, Page S155, https://doi.org/10.1530/acta.0.117S155 Published: 01 April 1988

Molecular cloning and expression of a synthetic DNA coding for the antimicrobial protein of bull seminal plasma.

A DNA carrying the coding sequence for the antimicrobial protein from bull seminal plasma (SAP) was obtained by enzymic ligation of six synthetic oligonucleotides. The 162 bp synthetic DNA fragment was cloned into the C-terminal part of the lacZ-gene employing the vector pUR289. Expression in E. coli in the presence of the inducer isopropylthiogalactoside (IPTG) led to the formation of a fusion protein, which was shown by immuno-blotting to contain immuno-reactive antimicrobial protein. Approximately 90 min after induction, the cells stopped growing and the culture was found to contain no viable cells 3 h after induction. We conclude from this observation that the beta-galactosidase-antimicrobial protein fusion product was toxic for the E. coli cell and that the SAP-residue attached to beta-galactosidase was responsible for the cytotoxicity.

The major protein of bull seminal plasma is a secretory product of seminal vesicle

We isolated the major protein with apparent molecular weight, M r, 15000–16000 from seminal plasma as well as from seminal vesicle secretion of bull and proved by amino acid analysis and tryptic peptide mapping that the two proteins were identical. An antiserum against this major protein was employed to quantitate and identify the major protein in seminal plasma as well as in seminal vesicle secretion. The antiserum did not cross-react with proteins from bovine or human plasma or follicular fluid, respectively. Cell-free translation of poly(A +)RNA isolated from seminal vesicle tissue resulted in formation of one major species with apparent M r 18 000. Using the anti-major protein antiserum, this major species was specifically immuno absorbed. We thus provided evidence that the major protein component of bull seminal plasma is a secretory protein of seminal vesicle. Furthermore, it appeared that the isolated major protein may be closely related the protein PDC109, purified from bulll seminal plasma and sequenced by Esch et al. (Biochem. Biophys. Res. Commun. 113, 861–867 (1983)).