BSP A1/A2-like proteins in ram seminal plasma

The objective of this study was to assess the protein profile of ovine seminal plasma using 2D-PAGE and verify if BSP A1/A2 are present in ovine seminal plasma. Seminal plasma was collected from three mature rams and pooled to eliminate individual differences. Seminal plasma samples were submitted to 2D-PAGE using 12% acrylamide gels. The image analysis software identified 21 protein spots on the air-dried gel, with molecular weight ranging from 15 to 115 kDa and pI 3.2 to 8.7. The most prominent spots were those <30 kDa. The most intensely stained spots were: 3 (18-19 kDa, pI 4.8-5.0), 5 (17-18 kDa, pI 5.0-5.2), 7 (15-16 kDa, pI 6.2-6.4), and 23 (105-108 kDa, pI 6.8-7.0). Three of these spots (spots 3, 5 and 7, respectively) accounted for 41.1% of the relative intensity of the spots of the gels, based on the intensity of the Comassie blue staining. Western blot analysis indicated that spots 3 and 5 were similar to BSP A1/A2 (16.5, pI 4.7-5.0 and 16 kDa, pI 4.9-5.2) identified in Manjunath's studies [Manjunath P, Sairam MR. Purification and biochimical characterization of three major acid proteins (BSP A1, BSP A2 and BSP A3) from bovine seminal plasma. Biochem J 7 (1987) 685-92.], based on the specific reaction of the polyclonal antibody to those spots.     

Identification of PDC-109-like protein(s) in buffalo seminal plasma

The FN-2 family of seminal plasma proteins represents the major protein fraction of bovine seminal plasma. These proteins also constitute the major seminal plasma proteins fraction in horse, goat and bison seminal plasma and are present in pig, rat, mouse, hamster and human seminal plasma. BSP-A1 and BSP-A2, the predominant proteins of the FN-2 family, are collectively termed as PDC-109. Fn-2 proteins play an important role in fertilization, including sperm capacitation and formation of oviductal sperm reservoirs. Significantly, BSP proteins were also shown to have negative effects in the context of sperm storage. No conclusive evidence for the presence of buffalo seminal plasma protein(s) similar to PDC-109 exists. Studies with buffalo seminal plasma indicated that isolation and identification of PDC-109-like protein(s) from buffalo seminal plasma by conventional methods might be difficult. Thus, antibodies raised against PDC-109 isolated, and purified from cattle seminal plasma, were used for investigating the presence of PDC-109-like protein(s) in buffalo seminal plasma. Buffalo seminal plasma proteins were resolved on SDS-PAGE, blotted to nitro cellulose membranes and probed for the presence of PDC-109-like protein(s) using the PDC-109 antisera raised in rabbits. A distinct immunoreactive band well below the 20-kDa regions indicated the presence of PDC-109-like protein(s) in buffalo seminal plasma.     

Isolation and characterization of gelatin-binding bison seminal vesicle secretory proteins

Bovine seminal plasma (BSP) contains a family of major proteins designated BSP-A1/A2, BSP-A3, and BSP-30kDa (collectively called BSP proteins) that bind to sperm at ejaculation and potentiate sperm capacitation. Homologous proteins have been identified in stallion, boar, goat, and ram seminal plasma. We report here the isolation and characterization of homologous proteins from bison seminal vesicle secretions. Seminal vesicle secretory proteins were precipitated by adding cold ethanol and recovered by centrifugation. The precipitates were resuspended in ammonium bicarbonate, dialyzed, and lyophilized. Lyophilized proteins were dissolved in 0.05 M phosphate buffer (PB) and loaded onto a gelatin-agarose column. The unadsorbed proteins and adsorbed proteins were eluted with PB and 5 M urea in PB, respectively. The gelatin-adsorbed fraction was analyzed by SDS-PAGE and revealed the presence of four major proteins designated BiSV-16kDa, BiSV-17kDa, BiSV-18kDa, and BiSV-28kDa (BiSV: bison seminal vesicle proteins). Heparin-Sepharose chromatography allowed the separation of BiSV-16kDa, which did not bind heparin from other BiSV proteins, which bound heparin. Immunoblotting revealed that BiSV-16kDa cross-reacted with BSP-A3 antibodies, BiSV-17kDa and BiSV-18kDa cross-reacted with BSP-A1/-A2 antibodies, and BiSV-28kDa cross-reacted with BSP-30kDa antibodies. Radioimmunoassays indicated that approximately 25% of bison seminal vesicle total proteins are related to BSP proteins. The amino-terminal sequencing indicated that BiSV proteins share almost 100% sequence identity with BSP proteins. In addition, BiSV proteins bind to low-density lipoproteins isolated from hen's egg yolk. These results confirm that BSP protein homologs are present in mammalian seminal plasma and they may share the same biological role.     

Isolation and characterization of gelatin-binding proteins from goat seminal plasma

A family of proteins designated BSP-A1, BSP-A2, BSP-A3 and BSP-30 kDa (collectively called BSP proteins for Bovine Seminal Plasma proteins) constitute the major protein fraction in the bull seminal plasma. These proteins interact with choline phospholipids on the sperm surface and play a role in the membrane stabilization (decapacitation) and destabilization (capacitation) process. Homologous proteins have been isolated from boar and stallion seminal plasma. In the current study we report the isolation and preliminary characterization of homologous proteins from goat seminal plasma. Frozen semen (-80°C) was thawed and centrifuged to remove sperm. The proteins in the supernatant were precipitated by the addition of cold ethanol. The precipitates were dissolved in ammonium bicarbonate and lyophilised. The lyophilised proteins were dissolved in phosphate buffer and loaded onto a gelatin-agarose column, which was previously equilibrated with the same buffer. The column was successively washed with phosphate buffer, with phosphate buffer saline and with 0.5 M urea in phosphate buffer saline to remove unadsorbed proteins, and the adsorbed proteins were eluted with 5 M urea in phosphate buffer saline. Analysis of pooled, dialysed and lyophilised gelatin-agarose adsorbed protein fraction by SDS-PAGE indicated the presence of four protein bands that were designated GSP-14 kDa, GSP-15 kDa, GSP-20 kDa and GSP-22 kDa (GSP, Goat Seminal Plasma proteins). Heparin-affinity chromatography was then used for the separation of GSP-20 and -22 kDa from GSP-14 and -15 kDa. Finally, HPLC separation permitted further isolation of each one from the other. Amino acid sequence analysis of these proteins indicated that they are homologous to BSP proteins. In addition, these BSP homologs bind to hen's egg-yolk low-density lipoproteins. These results together with our previous data indicate that BSP family proteins are ubiquitous in mammalian seminal plasma, exist in several forms in each species and possibly play a common biological role.     

Seasonal variations in antioxidant enzyme activity in ram seminal plasma

Seminal oxidative stress status is emerging as a significant prognostic tool in assisted reproductive technology. A dynamic interplay between pro- and anti-antioxidant substances in the ejaculate is essential. In this study, we determined seasonal changes in the activity of the antioxidant enzyme defense system comprising superoxide dismutase (SOD), glutathione reductase (GR), glutathione peroxidase (GPx) and catalase (CAT) in seminal plasma (SP) of mature Rasa Aragonesa rams. This breed corresponds to a local Spanish genotype with a short seasonal anoestrus between May and July. In addition, the activity of these enzymes was measured in protein fractions isolated from ram SP by exclusion chromatography. Total protein content in ram SP was higher during the breeding season (October-February) with a significantly higher value in first ejaculates. Antioxidant enzyme activities were higher during the non-breeding season (March-September). Comparing first and second ejaculates, SOD and CAT activities were higher in the first of all months. However, GR and GPx activities changed throughout the year. Thus, GR activity was higher in July and August in first ejaculates, this difference being significant in July (4.53 versus 2.37 nmol substrate/minmg protein, P<0.05). Conversely, GPx activity was significantly higher in September and November in second ejaculates (21.1 versus 6.81 and 10.91 versus 5.33, respectively, P<0.05). After SP fractionation by exclusion chromatography, GR activity was located in fractions 1 and 2 being irrelevant in the following peaks, and CAT activity was not detected all along the chromatographic profile. GPx and SOD activities were spread out along all fractions with a main peak in fractions 6 and 7. Given that these two fractions showed the greatest capacity to recover and prevent cold-shock membrane injury [Barrios B, Pérez-Pé R, Gallego M, Tato A, Osada J, Muino-Blanco T, Cebrián-Pérez JA. Seminal plasma proteins revert the cold-shock damage on ram sperm membrane. Biol Reprod 2000;63:1531-7, Barrios B, Fernández-Juan M, Muino-Blanco T, Cebrián-Pérez J. Immunocytochemical localization and biochemical characterization of two seminal plasma proteins that protect ram spermatozoa against cold shock. J Androl 2005;26:539-49], we could suggest that the protective effect might be, at least partially, due to the antioxidant enzyme activity.     

Isolation and characterization of the major proteins of ram seminal plasma

Mammalian seminal plasma contains among others, two major families of proteins, namely spermadhesins and those proteins that contain fibronectin type II domains. Spermadhesins are the major proteins of boar and stallion seminal plasma and homologous proteins have been identified in the bull. These proteins appear to be involved in capacitation and sperm-egg interaction. In bovine seminal plasma, proteins containing fibronectin type II domains are the major proteins and are designated BSP proteins. These proteins play a role in sperm capacitation. In this study, we present the isolation and characterization of the major proteins of ram seminal plasma. Precipitated proteins from Suffolk ram seminal plasma were loaded onto a gelatin-Agarose column. The unadsorbed (fraction A) and retarded proteins (fraction B) were removed by washing the column with phosphate buffered-saline and the adsorbed proteins (fraction C) were eluted with 5 M urea. SDS-PAGE of fraction B indicated the presence of a 15.5 kDa protein, which is the major protein of ram seminal plasma (approximately 45% of total protein by weight) and was identified as a spermadhesin by N-terminal sequencing. SDS-PAGE analysis of fraction C revealed the presence of four proteins, which represented approximately 20% of total ram seminal plasma proteins by weight, and were identified as proteins of the BSP family and named RSP proteins. These RSP proteins were designated RSP-15 kDa, RSP-16 kDa, RSP-22 kDa, and RSP-24 kDa. Only RSP-15 kDa and -16 kDa proteins cross-reacted with antibodies against BSP proteins. Ram spermadhesin and RSP proteins interact with heparin but only RSP proteins bind to hen's egg yolk low-density lipoprotein. In conclusion, spermadhesin is the major protein of ram seminal plasma and other major proteins belong to the BSP protein family.     

Identification of heparin-binding proteins in bovine seminal plasma

A group of four similar proteins, BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa, represent the major acidic proteins found in bovine seminal plasma (BSP). These proteins are secretory products of the seminal vesicles; they bind to spermatozoa upon ejaculation and could represent decapacitation factors. It has been shown that the glycosaminoglycans present in the female reproductive tract are involved in the capacitation of spermatozoa. Therefore, it was of interest to investigate whether BSP-A1, -A2, -A3, and -30-kDa proteins of bovine seminal fluid interact with heparin. Chromatography of alcohol precipitates of bovine seminal fluid on a heparin-Sepharose column resolved these proteins into three peaks. Peaks 1 and 2 (retarded proteins) were eluted upon extensive washing of the column with 0.05 M phosphate buffer, pH 7.4 (equilibrating buffer), and accounted for approximately 25% of the applied proteins. Proteins in peak 3 represented adsorbed proteins and were eluted with phosphate buffer containing 1 M NaCl. Proteins in each peak were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. Peak 1 contained proteins with molecular weights ranging from 8 to 350 kDa, peak 2 contained a single protein with a molecular weight of 14 kDa, and peak 3 contained proteins with molecular weights of 15.5, 16, 25, and 30 kDa. The proteins in peak 3 were further resolved into unadsorbed (peak 4) and adsorbed (peak 5) proteins on a gelatin-Agarose column. Separation of the proteins of peak 3 and peak 5 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and reducing agents followed by transfer to nitrocellulose and probing with antibodies against the previously well-characterized BSP proteins indicated the presence of BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a fucose-binding protein from bull sperm and seminal plasma that may be responsible for formation of the oviductal sperm reservoir

Oviductal sperm reservoirs have been found in cattle, mice, hamsters, pigs, and horses. In cattle (Bos taurus), the reservoir is evidently formed when sperm bind to fucosylated ligands resembling Le(a) trisaccharide on the surface of oviductal epithelium. The aim of this study was to characterize the fucose-binding protein on bull sperm. Fresh ejaculated sperm were extracted with 0.5 M KCl in Hepes-balanced salts. Extracts were subjected to affinity chromatography using immobilized Le(a) trisaccharide (alpha-L-Fuc[1,4]-beta-D-Gal[1,3]-D-GlcNAc). Two-dimensional PAGE of the affinity chromatography eluates revealed a prominent protein of approximately 16.5 kDa and a pI of 5.8. This protein inhibited binding of sperm to oviductal explants. A similar analysis of proteins extracted from capacitated sperm (which do not bind to oviductal epithelium) showed a reduction in the amount of the 16.5-kDa protein. When examined by epifluorescence microscopy, live uncapacitated sperm labeled over the acrosome with a fucose-BSA-fluorescein isothiocyanate (FITC) conjugate, while capacitated sperm did not. When capacitated sperm were treated with 16.5-kDa protein, labeling with fucose-BSA-FITC was partially restored. The comparative ease with which the protein was removed from sperm and its apparent reassociation with sperm suggested that it could be a peripheral protein derived from epididymal or accessory gland fluids. Blots of SDS-PAGE gels of seminal plasma proteins revealed the presence of a Le(a)-binding protein with an apparent mass of 16.5 kDA: Amino acid sequencing of two tryptic fragments of the protein purified from sperm extracts identified it as PDC-109 (BSP-A1/A2), a product of the seminal vesicles.     

Phosphorylcholine-binding proteins from the seminal fluids of different species share antigenic determinants with the major proteins of bovine seminal plasma

The major proteins of bovine seminal plasma, BSP-A1, BSP-A2, BSP-A3, and BSP-30kDa (collectively named BSP proteins) bind to phospholipids containing the phosphorylcholine moiety. An affinity purification method using a p-aminophenyl phosphorylcholine-Agarose (PPC-Agarose) affinity matrix was developed for their purification. In this study, we investigated the distribution of BSP-like analogues in seminal fluid of the human, porcine, hamster, mouse, and rat using this affinity matrix. Alcohol precipitates of the seminal plasma/seminal vesicle secretions (SP/SVS) were further delipidated using isopropyl ether:n-butanol (60:40). The protein preparations obtained were solubilized in a minimal volume of buffer A (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.02% NaN₃), dialyzed against the same buffer, and applied to a PPC-Agarose column connected to a FPLC system. The unbound material was washed out and the adsorbed proteins eluted with buffer A containing 10 mM phosphorylcholine (PrC) and 10 M urea. The fractions were separated by SDS-PAGE, stained or transferred onto a nitrocellulose membrane, and probed with rabbit polyclonal anti-BSP antibodies. Anti-BSP cross-reacting proteins were detected in the seminal fluids of all the species investigated. Moreover, many of these proteins bound to the affinity matrix. The BSP proteins and their immunoreacting analogues appear to be ubiquitous in mammals and may possibly be involved in a common function such as in the modification of the lipid content of the sperm plasma membrane. © 1993 Wiley-Liss, Inc.     

Characterization of bovine seminal plasma by proteomics

Previous investigations of bovine seminal plasma (BSP) have revealed the identities of the three major proteins, BSP-PDC109, BSP-A3 and BSP-30 kDa, which together constitute about half of the total protein, as well as about 30 of the minor proteins. Analyses of BSP by 2-DE have revealed about 250 protein spots, suggesting that much of the BSP proteome remains undescribed. In this study, BSP has been analyzed by 2-D LC-based and SDS-PAGE-based proteomic methods. Ninety-nine proteins were identified, including 49 minor proteins that have not previously been described in seminal plasma of any species.     

Characterization of the cDNA and in vitro expression of the ram seminal plasma protein RSVP14

In previous studies we have shown that seminal plasma (SP) proteins can prevent and repair cold-shock membrane damage to ram spermatozoa. Three proteins of approximately 14, 20 and 22 kDa, mainly responsible for this protective ability, were identified in ram SP. They are exclusively synthesized in the seminal vesicles and, consequently, named RSVP14, RSVP20 and RSVP22. The aim of this study is to characterize and express the RSVP14 gene to provide new insights into the mechanisms through which SP proteins are able to protect spermatozoa. Additionally, a first approach has been made to the recombinant protein production. The cDNA sequence obtained encodes a 129 amino acid chain and presents a 25-amino acid signal peptide, one potential O-linked glycosylation site and seven phosphorylation sites on tyrosine, serine and threonine residues. The sequence contains two FN-2 domains, the signature characteristic of the bovine seminal plasma (BSP) protein family and related proteins of different species. More interestingly, it was shown that RSVP14 contains four disulphide bonds and a cholesterol recognition/interaction amino acid consensus (CRAC) domain, also found in BSP and similar proteins. Analysis of the relationships between RSVP14 and other mammalian SP proteins revealed a 76–85% identity, particularly with the BSP protein family. The recombinant protein was obtained in insect cell extracts and in Escherichia coli in which RSVP14 was detected in both the pellet and the supernatant. The results obtained corroborate the role of RSVP14 in capacitation and might explain its protective effect against cold-shock injury to the membranes of ram spermatozoa. Furthermore, the biochemical and functional similarities between RSVP14 and BSP proteins suggest that it might play a similar role in sperm functionality.     

Cloning of complementary DNA encoding the pB1 component of the 54-kilodalton glycoprotein of boar seminal plasma

Complementary DNA (cDNA) encoding a protein component pB1 (also pAIF-1 and DQH) of the 54-kilodalton glycoprotein of boar seminal plasma was cloned and its nucleotide sequence was determined (Gene Bank accession no. AF047026). The pB1 precursor protein is a 130-amino-acid-long polypeptide containing a 25-amino-acid-long signal peptide. The amino acid sequence of the pB1 is homologous to that of SFP1_BOVIN (named also BSP-A1/A2, PDC-109/ major protein and SVSp109), SFP3_BOVIN (BSP-A3), SFP4 BOVIN (BSP-30 KD), and SP1_HORSE (HSP-1) seminal plasma proteins. The homology extends also for the signal peptide of SFP1_BOVIN protein. All these seminal plasma proteins contain two fibronectin type-II domains that differ from those found in other proteins such as colagenases, fibronectins, and mannose receptors. The first domain located in the N-terminal region of pB1 is four amino acids shorter than those present in other proteins. High homology is also observed between 3' noncoding regions of the nucleotide sequences of cDNAs of pB1_PIG and SFP1_BOVIN (Gene Bank accession nos. AF047026 and P02784, respectively).     

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.     

Induction of epididymal boar sperm capacitation by pB1 and BSP-A1/-A2 proteins, members of the BSP protein family

A family of proteins designated BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa, collectively called BSP (bovine seminal plasma) proteins, constitute the major protein fraction of bull seminal plasma. BSP proteins can stimulate sperm capacitation by inducing cholesterol and phospholipid efflux from sperm. Boar seminal plasma contains one homologous protein of the BSP family, named pB1; however, its physiological role is still unknown. In the current study, we report a novel method to purify pB1 from boar seminal plasma by chondroitin sulfate B-affinity chromatography and reverse-phase-high performance liquid chromatography. We also studied the effect of pB1, BSP-A1/-A2, and whole boar seminal plasma on boar sperm capacitation. Boar epididymal sperm were washed, preincubated in noncapacitating medium containing pB1 (0, 2.5, 5, 10 or 20 microg/ml), BSP-A1/-A2 (0 or 20 microg/ml) proteins, or whole seminal plasma (0, 250, 500, or 1000 microg/ml), then washed and incubated in capacitating medium. Acrosomal integrity was assessed by chlortetracycline staining. The status of sperm capacitation was evaluated by the capacity of sperm to undergo the acrosome reaction initiated by the addition of the calcium ionophore, A23187. The pB1 and BSP-A1/-A2 proteins increased epididymal sperm capacitation as compared with control (sperm preincubated without proteins). This effect reached a maximum level at 10 microg/ml pB1 and at 20 microg/ml BSP-A1/-A2 (2.3- and 2.2-fold higher than control, respectively). Whole boar seminal plasma did not induce sperm capacitation. In addition, pB1 bound to boar epididymal sperm and was lost during capacitation. These results indicate that BSP proteins and their homologs in other species induce sperm capacitation in a similar way.     

Characterization of the major proteins of bovine seminal fluid by two-dimensional polyacrylamide gel electrophoresis

Recently, we demonstrated that the major proteins from bovine seminal plasma BSP-A1, -A2, -A3 and -30-kDa (collectively called BSP proteins) specifically interact with choline phospholipids. These proteins coat the surface of the spermatozoa after ejaculation and are believed to play an important role in membrane modifications occurring during capacitation. In this study we determined the isoelectric point (pl) and analysed the molecular heterogeneity of BSP proteins. Total protein from bovine seminal plasma (CBSP) and purified BSP proteins were iodinated using chloramine T. Samples were reduced, denatured, separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and visualized by autoradiography. Analysis of CBSP proteins demonstrated the presence of polypeptides migrating in the pH range of 3.5–7.8 and at molecular weights (Mr) between 6 and 100 kDa. Many isoforms of each BSP protein were found when purified iodinated proteins were analysed by 2D-PAGE. BSP-A1 was found at a Mr of 16.5 kDa and in the range of pl of 4.7–5.0; BSP-A2 at 16 kDa and at a pl of 4.9–5.2; BSP-A3 at 15 kDa and at a pl of 4.8–5.2, and BSP-30-kDa at 28 kDa and at a pl of 3.9–4.6. Similar results were obtained with immunolocalization of BSP proteins after Western blot using specific antibodies. The treatment of purified iodinated BSP proteins with neuraminidase increased the pl of BSP-30-kDa to 4.8–5.0 and decreased its Mr to 25 kDa, but no change was observed for BSP-A1, -A2 and -A3. The treatment of BSP proteins with sulfatase or acid phosphatase modified neither their Mr nor their pl. Furthermore, when CBSP proteins were separated in 2D-PAGE and the gels stained for glycoproteins with dansyl hydrazine, BSP proteins were among the major glycoproteins found in the bovine seminal plasma. In conclusion, BSP proteins are acidic and have several isoforms. Furthermore, the heterogeneity of BSP-30-kDa is mainly due to its sialic acid content. © 1994 Wiley-Liss, Inc.     

Seasonal variations in the composition of ram seminal plasma and its effect on frozen-thawed ram sperm

It has been proposed that seminal plasma (SP) in the extender or in post-thaw media can prevent and revert cold-shock damage in cryopreserved ram sperm; however, this was dependent on season. We evaluated sperm parameters from Frisian ram semen incubated for various intervals with SP from all seasons and stored at -18 or -196 degrees C. At both temperatures, SP from autumn or winter increased (P<0.05) sperm motility, whereas no SP, or SP from spring or summer, had no effect. However, neither viability nor membrane or acrosomal status were modified by SP. Thirteen SP proteins were bound to the sperm surface (16.1, 16.7, 17.4, 23.3, 25.2, 27.5, 35.0, 40.0, 49.0, 53.5, 55.5, 61.0, and 86.0kDa). The SP proteins that bound to sperm were affected by season, but not by conservation temperature. Sperm incubated with SP from autumn had increased concentrations of five proteins; two were identified (with specific antibodies) as RSVP14 and RSVP20. In conclusion, SP from autumn and winter improved sperm motility of frozen-thawed ram sperm, and storage of ram SP at -18 or -196 degrees C did not affect protein composition. The SP proteins that bound to the sperm surface may be responsible for sperm membrane stabilization and should be further investigated.     

Two-dimensional polyacrylamide gel electrophoresis of bovine seminal plasma proteins and their relation with semen freezability

The objective of this study was to evaluate the low weight (10-30 kDa) protein profile of bovine seminal plasma using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and to determine if any of these proteins was associated with semen freezability. Seminal plasma was collected from 16 bulls of high or low semen freezability. Twelve protein spots were identified from the 2D gel (15%); six of these were present in all samples. Of the 12 proteins found, three spots, present in all samples, 3 (15-16 kDa), 5 (16-17 kDa), and 7 (10-12 kDa) had nonsignificant variation among bulls, regardless of their freezability classification. Four proteins were more abundant (P<0.05) in seminal plasma samples collected from bulls with high semen freezability than in samples of bulls with low semen freezability: the spots 3 (15-16 kDa, pI 4.7-5.2), 7 (11-12 kDa, pI 4.8-4.9), 11 (13-14 kDa, pI 4.0-4.5), and 23 (20-22 kDa, pI 4.8-5.2). On the other hand, spot 25 (25-26 kDa, pI 6.0-6.5) was more abundant (P<0.05) on seminal plasma samples from bulls with low semen freezability. The N-terminus sequence of protein 7 was identical to the acidic seminal fluid protein (aSFP). Protein 23 (after trypsin digestion) had structural similarity to bovine clusterin. We concluded that there were differences in the seminal plasma protein profile from bulls with low and high semen freezability; aSFP, clusterin, proteins 3 and 11 may be used as semen freezability markers; and protein 25 was related to low semen freezability.     

Two-dimensional polyacrylamide gel electrophoresis of bovine semen after cryopreservation in half-milliliter straws

One of the problems encountered with two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is the streaking of proteins so that individual spot identification is compromised. This study was conducted to determine whether a low loading dose (50 microg) of protein would permit resolution of more discrete protein spots using megapixel camera technology, and if so, to present a nomenclature for future comparisons of the identified proteins. If the major proteins could be identified in a 50-microg sample we aimed to determine whether they could be identified in the supernatant (seminal plasma plus extender) of cryopreserved semen. Two ejaculates were obtained from each of 6 bulls and bovine seminal plasma (BSP) protein concentration was standardized to 50 microg/10 microl. Isoelectric points (pI) and molecular weights (MWt) of BSP proteins were determined by measuring spot mobility on 2-D PAGE (15% polyacrylamide). Three distinct protein spot constellations (a,b,c) could be readily seen by the naked eye and a faintly stained constellation "d" was identified by the megapixel camera. The image analysis software located 6 protein spots in both constellation "a" (MWt 26 kDa; pI 4.2 to 4.8) and "b" ( MWt 27 kDa; pI 6.6 to 8.0). Constellation "c" contained 13 protein spots distributed in a right-angled triangle with its base towards the acidic end of the gel (MWt 14.7 to 18.8 kDa; pI 5.3 to 7.4). Only spots c(2), c(3), c(5), c(8), and c(13) were present in all 12 samples. Streaking can be eliminated by using 50 microg protein for 2-D PAGE, and the major protein spots are readily identified by megapixel camera technology. Protein spots c(3), c(5), c(13) and constellation "a" appear to correspond with Manjunath's proteins (BSP-A(1), -A(2); -A(3); -30 kDa). Killian's 2 low fertility proteins may lie in the "c" constellation, and 1 of the high fertility proteins may lie in the "b" constellation. The 3 major BSP proteins can be visualized in the supernatant of cryopreserved semen. We believe that the technique may prove useful for retrospective analysis of processed semen batches that achieve less than satisfactory results in the field.     

The inhibitory effect of BSP-A1/-A2 on protein kinase C and tyrosine protein kinase

Bovine seminal plasma contains a group of similar proteins, namely BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa (collectively called BSP proteins), and they are secreted by the seminal vesicles. In our study, we purified the BSP-A1/-A2 through affinity chromatography and found for the first time that BSP-A1/-A2 can inhibit the activity of protein kinase C (PKC) and tyrosine protein kinase (TPK). The inhibition was dose dependent. When the PKC and TPK activities are expressed as the logarithm of percentage activity taking the activity in the absence of the BSP-A1/-A2 as 100%, there is a linear relationship between the their activities and the dose of BSP-A1/-A2.     

Conserved ram seminal plasma proteins bind to the sperm membrane and repair cryopreservation damage

Whole seminal plasma (SP) enhances the function and fertility of frozen/thawed ram sperm. The objective of the current study was to investigate whether SP proteins capable of binding to molecules from the sperm plasma membrane were conserved among ram breeds, and whether these proteins were sufficient to overcome cryopreservation-induced reductions in sperm quality. Whole ram SP, obtained from rams of various breeds, improved progressive motility of frozen/thawed sperm at all times evaluated (P < 0.05); however, it did not improve total motility (15 min, P = 0.480; 30 min, P = 0.764; and 45 min, P = 0.795). To identify SP proteins responsible for this effect, a new method was developed to retain SP proteins that bound specifically to the sperm membrane by immobilization of sperm membrane proteins. These proteins specifically bound to the sperm surface, especially the acrosomal region. Lactotransferrin, epididymal secretory protein E1, Synaptosomal-associated protein 29, and RSVP-20 were identified (mass spectrometry) in this fraction. The retained SP proteins fraction repaired ultrastructural damage of frozen/thawed sperm and, with the addition of fructose, significantly improved motility of frozen/thawed sperm. We concluded that SP proteins that bound to the sperm membrane were conserved among ram breeds, and that when added to frozen/thawed semen (along with an energy source), they repaired ram sperm damage and enhanced sperm motility.