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.     

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.     

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

The objective was to evaluate protein profiles of equine seminal plasma using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and to determine whether any of these proteins were related to semen freezability. Seminal plasma was collected from 10 stallions, of high and low semen freezability, housed at the State Stud of Lower Saxony, and routinely used in AI programs. Twenty-five protein spots were identified from the two-dimensional gel (12%), seven of which were present in all samples (all proteins were identified by MALDI-MS). Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been used to generate ion images of samples in one or more mass-to-charge (m/z) values, providing the capability of mapping specific molecules to two-dimensional coordinates of the original sample. Of the 25 proteins identified, two spots had greater relative content (P < 0.05) in seminal plasma samples collected from stallions with high semen freezability: spot 5 (80-85 kDa, isoelectric point [pI] 7.54), identified as CRISP-3; and spot 45 (18.2 kDa, pI 5.0-5.2), identified as HSP-2. Conversely, protein content was greater (P < 0.05) in seminal plasma samples from stallions with low semen freezability: spot 7 (75.4 kDa, pI 6.9-7.4), identified as lactoferrin; spot 15 (26.7 kDa, pI 5.51), identified as kallikrein; spot 25 (25 kDa, pI 7.54), identified as CRISP-3; and spot 35 (13.9 kDa, pI 3.8-4.2), identified as HSP-1. In conclusion, there were differences in the seminal plasma protein profile from stallions with high and low semen freezability. Furthermore, CRISP-3 and HSP-2 were potential seminal plasma markers of high semen freezability.     

Monthly variations in ovine seminal plasma proteins analyzed by two-dimensional polyacrylamide gel electrophoresis

This study was conducted to evaluate monthly changes in the ram seminal plasma protein profile using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) with a polyacrylamide linear gradient gel. Likewise, comparative analyses of the protein composition of ovine seminal plasma (SP) from ejaculates obtained along the year, and its relationship with sperm motility, viability and concentration of ejaculate were carried out. Western-blot analysis was performed to specifically detect P14, a ram SP protein postulated to be involved in sperm capacitation and gamete interaction [Barrios B, Fernández-Juan M, Mui?o-Blanco T, Cebrián-Pérez JA. Immunocytochemical localization and biochemical characterization of two seminal plasma proteins which protect ram spermatozoa against cold-shock. J Androl 2005;26:539-49], and its variations along the year have also been established. The experiment was carried out from May 2003 to April 2004, with nine Rasa Aragonesa rams. Ejaculates obtained every 2 days were pooled and used for each assay, to avoid individual differences, and three two-dimensional SDS-PAGE gels were run for each month. The high resolution of the gradient gel allowed the image analysis software to detect around 252 protein spots, with pIs ranging from 4.2 to 7.6, and molecular weight (M(r)) from 12.5 to 83.9 kDa. Four protein spots (1, 2, 3 and 4) of low M(r) (15.1, 15.7, 15.9 and 21.0 kDa) and acidic pI (5.9, 5.3, 5.7 and 6.6), respectively, had the highest relative intensity in the SP map (11.2, 9.3, 4.7 and 7.7%, respectively). Spot 3 was more abundant (P<0.05) from May to December, and negatively correlated (P<0.05, r=-0.34) with sperm viability and concentration (P<0.05, r=0.36). Another 12 protein spots also had significant quantitative differences (P<0.05) along the year, and 17 protein spots, which correlated with some seminal quality parameter, did not show quantitative monthly changes. Western-blot analysis indicated that spots 1 and 2 reacted with the anti-P14 antibody, raised against the P14 band (approximate M(r) 14 kDa) of ram SP. This indicates that spots 1 and 2 are similar to RSP15 [Bergeron A, Villemure M, Lazure C, Manjunath P. Isolation and characterization of the major proteins of ram seminal plasma. Mol Reprod Dev 2005;71:461-70], bovine PDC-109 [Esch FS, Ling NC, Bohlen P, Ying S, Guillemin R. Primary structure of PDC-109, a major protein constituent of bovine seminal plasma. Biochem Biophys Res Commun 1983;113:861-7] (also called BSP A1/A2 [Manjunath P, Sairam MR. Purification and biochemical characterization of three major acidic proteins (BSP-A1, BSP-A2 and BSP-A3) from bovine seminal plasma. Biochem J 1987;241:685-92]) and goat GSP-14/15 kDa [Villemure M, Lazure C, Manjunath P. Isolation and characterization of gelatine-binding proteins from goat seminal plasma. Reprod Biol Endocrinol 2003;1:39], based on our previous results on the P14 amino acid sequence [Barrios B, Fernández-Juan M, Mui?o-Blanco T, Cebrián-Pérez JA. Immunocytochemical localization and biochemical characterization of two seminal plasma proteins which protect ram spermatozoa against cold-shock. J Androl 2005;26:539-49].     

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.     

Proteomic analysis of the reproductive tract fluids from tropically-adapted Santa Ines rams

The present study is focused on the proteome of reproductive tract fluids from tropically-adapted Santa Ines rams. Seminal plasma, cauda epididymal (CEF) and vesicular gland fluid (VGF) proteins were analyzed by 2-D electrophoresis and mass spectrometry. Seminal plasma maps contained 302 ± 16 spots, within the 4-7 pH range. From these maps, 73 spots were identified, corresponding to 41 proteins. Ram Seminal Vesicle Proteins (RSVP) 14 and 22kDa and bodhesins 1 and 2 represented the most abundant seminal components. Other seminal proteins included clusterin, angiotensin-converting enzyme, matrix metalloproteinase-2, tissue-inhibitor of metalloproteinase-2, plasma glutamate carboxypeptidase, albumin, lactoferrin, alpha enolase, peroxiredoxin, leucine aminopeptidase, β-galactosidase, among others. Later, seminal plasma gels were run within narrow pH intervals (3.9-5.1; 4.7-5.9; 5.5-6.7), allowing the additional identification of 21 proteins not detected in 4-7 pH maps. Major proteins of CEF and VGF were albumin and transferrin, and RSVPs, respectively. Western blots confirmed that RSVPs were mainly present in VGF while bodhesins, in VGF and CEF. Based on RT-PCR, RSVP and bodhesin genes were primarily expressed in the vesicular glands. In summary, the reproductive tract fluids of Brazilian hairy rams contain several categories of proteins, with potential roles in sperm protection, capacitation, acrosome reaction and sperm-oocyte interaction.     

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.     

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.     

A comprehensive proteomic analysis of the accessory sex gland fluid from mature Holstein bulls

The expression of proteins in accessory sex gland fluid (AGF) of proven, high use mature Holstein bulls was evaluated. Thirty-seven bulls with documented fertility based on their non-return rates were studied. AGF was obtained by artificial vagina after bulls were surgically equipped with cannulae in the vasa deferentia. Samples of AGF were evaluated by two-dimensional SDS-PAGE, gels stained with Coomassie blue and polypeptide maps analyzed by PDQuest software. A master gel generated by the software representing the best pattern of spots in the AGF polypeptide maps was used as a reference for protein identification. Proteins were identified by Western blots and capillary liquid chromatography-nanoelectrospray ionization tandem-mass spectrometry (CapLC-MS/MS). The product ion spectra were processed using Protein Lynx Global Server 2.1 prior to database search with both PLGS and MASCOT (Matrix Science) software. The entire NCBI database was considered for mass fingerprint matching. An average of 52+/-5 spots was detected in the AGF 2D gels, which corresponded to proteins potentially involved in capacitation (bovine seminal plasma protein-BSP-A1/A2 and A3, BSP 30 kDa, albumin); sperm membrane protection, prevention of oxidative stress, complement-mediated sperm destruction and anti-microbial activity (albumin, clusterin, acidic seminal fluid protein--aSFP, 5'-nucleotidase--5'-NT, phospholipase A2--PLA2); acrosome reaction and sperm-oocyte interaction (PLA2, osteopontin); interaction with the extracellular matrix (tissue inhibitor of metalloproteinase 2, clusterin) and sperm motility (aSFP, spermadhesin Z13, 5'-NT). The 20 spots distinguished in all gels were matched to proteins associated with these functions. Proteins identified by tandem mass spectrometry as ecto-ADP-ribosyltransferase 5 and nucleobindin, never described before in the accessory sex gland secretions, were also detected. In summary, we identified a diverse range of components in the accessory sex gland fluid of a select group of Holstein bulls with documented fertility. Known characteristics of these proteins suggest that they play important roles in sperm physiology after ejaculation.     

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.     

Two-dimensional polyacrylamide gel electrophoresis of equine seminal plasma proteins and their correlation with fertility

The objectives of this study were to 1) identify proteins found in stallion seminal plasma utilizing two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) in conjunction with Western blot analysis; and 2) to determine if any of these individual proteins were correlated with stallion fertility utilizing regression analysis. Fertility was quantified by assigning a breeding score for each stallion. Each score was calculated by dividing the number of conceptions by the number of breedings for each stallion for four successive breeding seasons (1992-1995). Ejaculates from stallions of known fertility (n = 6) were collected with a Missouri-style artificial vagina. Immediately after collection, the semen sample was filtered and the gel fraction removed. The resultant sperm-rich fraction was centrifuged in a Beckman Microfuge E at 10,000 x g and the seminal plasma aspirated from the pelleted sperm cells. Two-dimensional PAGE of the seminal plasma was performed under denaturing conditions which revealed that 14 proteins were common in all stallions in the research population. Four of these proteins (SP-1, SP-2, SP-3, and SP-4) were found to be significantly (P < 0.05) correlated with the breeding score assigned for each stallion. Regression analysis of protein optical densities with breeding score indicated that SP-1 (72 kDa, pI 5.6) was positively correlated with fertility (P < 0.05, r2 = 0.706), while SP-2 (75 kDa, pI 6.0), SP-3 (18 kDa, pI 4.3), and SP-4 (16 kDa, pI 6.5) were found to be negatively correlated (P < 0.05, r2 = 0.762, 0.730, 0.775 respectively) with fertility. Western blot analysis of SP-1 indicated there was an antigenic homology with a bovine 55 kDa fertility-associated seminal plasma protein identified in a study by Killian et al. (19). This suggests that the two proteins may have a similar physiological role and therefore common biological properties. These results indicate that analysis of stallion seminal plasma proteins can be used as an indicator of fertilizing capacity. Identification of such proteins in stallion seminal plasma could lead to better insight into the nature of subfertility or infertility in the horse, as well as to indicate better cryopreservation strategies.     

Acidic cysteine proteinase inhibitor in seminal plasma

A cysteine proteinase inhibitor with acidic isoelectric point (pI = 4.7-5.0) was found in human seminal plasma. Its apparent molecular mass is 16 kDa. It inhibits cysteine proteinases like ficin, cathepsin H, cathepsin B and papain. The inhibitory activity of seminal plasma against ficin is almost the same as that of human serum.     

Phospholipases A2 in the reproductive system of the bull

1. Phospholipase A2 activities were studied in the reproductive organs, seminal plasma and spermatozoa of adult bulls. 2. Phosphatidylethanolamine and phosphatidylcholine with 14C-labelled linoleic (lino-PE, lino-PC) or arachidonic acid (ara-PE, ara-PC) at sn-2 position as well as a fluorescent derivative (4-pyrenylbutyric acid) of phosphatidylcholine (PPC) were used as substrates. 3. The radioactive substrates were hydrolysed most strongly by homogenates of the prostate and Cowper's gland, but also seminal vesicle and its secretory fluid, seminal plasma and ejaculated spermatozoa contained hydrolytic activity. The fluorescence substrate was most strongly hydrolysed by homogenates of ampulla and seminal vesicle as well as its secretory fluid, seminal plasma and ejaculated spermatozoa. 4. Seminal plasma and seminal vesicle fluid contained a Ca2(+)-independent enzyme (enzyme I), which hydrolysed only PPC, while another Ca2(+)-dependent enzyme (enzyme II) hydrolysed only the radioactive substrates. 5. Both enzymes were purified from the seminal vesicle fluid and their biochemical properties were analysed. In SDS-PAGE enzyme I preparation resulted in two major bands with molecular weights of 16,000 and 60,000 in equal quantities and minor band at 15,000. The binding of the enzyme I to Con A-Sepharose indicated that it is a glycoprotein and it had multiple pI-values from 3.75 to 5.0. Enzyme II gave in SDS-PAGE two closely located bands with molecular weights of about 15,000 and 16,000 (major band). Isoelectric focusing showed one band at pI 4.7. Both enzymes appear to bind to spermatozoa at ejaculation but their function remains to be shown.     

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.     

Proteome analysis of Nelore bull (Bos taurus indicus) seminal plasma

The Nelore bull (Bos taurus indicus) seminal plasma proteome was analyzed by MALDI-TOF MS and two-dimensional gel electrophoresis. A total of 260 spots were visualized in the 2-DE gel (pI range 3-10) and 13 spots could be identified by peptide mass fingerprinting corresponding to 11 different polypeptides. The results allowed the creation of the first proteomic map of Bos taurus indicus seminal plasma. The roles of the identified proteins in the bull seminal plasma are discussed.     

Lipocalin-type prostaglandin D synthase in milk: a new biomarker for bovine mastitis.

The whey protein pattern of milk from animals affected by mastitic inflammation was resolved by two-dimensional gel electrophoresis (2D-PAGE) and compared to milk from unaffected cows. Inflammation caused the appearance of four spots aligned at a molecular weight level of 26 kDa and over a pH-region of 5.0 to 6.4. The spots excised from 2D gels were treated with chymotrypsin and the resulting peptides analyzed by MALDI-TOF mass spectrometry and RP-HPLC. All four spots yielded highly similar chymotryptic peptide mass fingerprints as well as chromatographic peak patterns. A database search could identify the four spots as isoforms of the bovine prostaglandin D synthase (PGD-S). In one of the isoforms a defined cysteine residue was shown to be oxidized to a sulfonic acid.     

Binder of Sperm Proteins protect ram spermatozoa from freeze-thaw damage

Cryopreservation causes sub-lethal damage which limits the fertility of frozen thawed spermatozoa. Seminal plasma has been investigated as a cryoprotectant, but has yielded inconsistent results due to considerable variation in its constituents. Individual seminal plasma proteins offer an ideal alternative to whole seminal plasma, and several have been correlated with freezing success. Binder of Sperm Proteins (BSPs) are abundant ram seminal plasma proteins which have been suggested to have significant protective effects on ram spermatozoa during cold shock. This is in direct opposition to bull spermatozoa, where BSPs cause sperm deterioration during in vitro handling. We investigated the potential of BSP1 and BSP5 to prevent freezing associated damage to important functional parameters of ram spermatozoa. BSPs purified by size exclusion chromatography improved post thaw motility and penetration through artificial mucus. Highly purified BSP1 and BSP5, isolated by gelatin affinity and RP-HPLC, improved motility and membrane integrity, and reduced post thaw protein tyrosine phosphorylation. Exposure to BSP5 before freezing increased the amount of phosphatidylethanolamine on the sperm surface after thawing. Neither BSP1 nor BSP5 prevented freezing associated changes in membrane lipid disorder. These results suggest that BSPs may significantly improve freezing outcomes of ram spermatozoa.     

New Insights into the Phylogeny and Gene Context Analysis of Binder of Sperm Proteins (BSPs)

Seminal plasma (SP) proteins support the survival of spermatozoa acting not only at the plasma membrane but also by inhibition of capacitation, resulting in higher fertilizing ability. Among SP proteins, BSP (binder of sperm) proteins are the most studied, since they may be useful for the improvement of semen diluents, storage and subsequent fertilization results. However, an updated and detailed phylogenetic analysis of the BSP protein superfamily has not been carried out with all the sequences described in the main databases. The update view shows for the first time an equally distributed number of sequences between the three families: BSP, and their homologs 1 (BSPH1) and 2 (BSPH2). The BSP family is divided in four subfamilies, BSP1 subfamily being the predominant, followed by subfamilies BSP3, BSP5 and BSP2. BSPH proteins were found among placental mammals (Eutheria) belonging to the orders Proboscidea, Primates, Lagomorpha, Rodentia, Chiroptera, Perissodactyla and Cetartiodactyla. However, BSPH2 proteins were also found in the Scandentia order and Metatheria clade. This phylogenetic analysis, when combined with a gene context analysis, showed a completely new evolutionary scenario for the BSP superfamily of proteins with three defined different gene patterns, one for BSPs, one for BSPH1/BSPH2/ELSPBP1 and another one for BSPH1/BSPH2 without ELSPBP1. In addition, the study has permitted to define concise conserved blocks for each family (BSP, BSPH1 and BSPH2), which could be used for a more reliable assignment for the incoming sequences, for data curation of current databases, and for cloning new BSPs, as the one described in this paper, ram seminal vesicle 20 kDa protein (RSVP20, Ovis aries BSP5b).     

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.     

Soluble and membrane-associated heat shock proteins in soybean root

Summary Localization of heat shock proteins (Hsp) in endomembranes and determination of whether they are integral or peripheral membrane proteins will aid in understanding the physiological function of the heat shock response. Radiolabeled endomembranes (endoplasmic reticulum, Golgi, and plasma membrane), obtained by sucrose gradient centrifugation of heat-shocked soybean (Glycine max L.) root tissue were solubilized and the polypeptides separated by two-dimensional IEF-SDS-PAGE. Autoradiography revealed three groups of Hsp. A diverse group fo 25 low mol wt Hsp (18 to 24 kDa) with isoelectric point (pI) between 5 and 7; an intermediate mol wt group (30 to 47 kDa) with pI of 5.5 to 6.0; and a group of two high mol wt Hsp (75 to 80 kDa) with pI 4.8 to 5.2. The plasma membrane fraction lacked the Hsp pair of 47 kDa detected in the endoplasmic reticulum and Golgi fractions but possessed a unique Hsp of 30 kDa, pI 5.5.Comparison of soluble and microsome fractions revealed a difference in the pattern of the low mol wt Hsp class. The soluble fraction contained Hsp of 16–20 kDa with pI between 5 and 7.8 while the microsome fraction was characterized by Hsp of 18–24 kDa with pI between 5.8 and 6.5.The microsomal Hsp were not released by 1 M KCl. Treatment of the microsome fraction with Triton X-100 selectively released several Hsp, and Na₂CO₃ treatment removed additional Hsp from the membrane fraction.Abbreviations Hsp heat-shock protein(s) - GA Golgi apparatus - PM plasma membrane - 2 D two-dimensional