Background
Egg yolk (EY), heated skim milk (SM) and whole milk are components commonly used in extenders for sperm preservation (reviewed in [
1,
2]). Being products of animal origin, their compositions are not constant, and moreover they present potential risks of microbial contamination of semen. Because of these drawbacks, there is a keen interest to find substitutes. The development of novel extenders free of products of animal origin is difficult considering that the mechanisms by which EY and milk protect sperm are poorly understood.
Bovine seminal plasma contains a family of proteins designated as Binder of SPerm (BSP) proteins, which have been extensively characterized [
3‐
6]. These proteins positively modulate the induction of sperm capacitation, a process that is deemed to be essential for fertilization [
7,
8]. However, in the context of sperm storage, BSP proteins are detrimental to sperm as they extract cholesterol and phospholipids from sperm membranes (reviewed in [
2,
9]). We previously demonstrated that the low-density lipoproteins (LDL) of EY interact with BSP proteins and that this interaction prevents cholesterol and phospholipid extraction from the sperm membrane, thereby protecting sperm during preservation (reviewed in [
2]).
Whole milk and SM used in extenders are also known to protect sperm during storage. While whole milk contains lipoproteins, which could bind BSP proteins and protect sperm, SM does not, and yet is as efficient as whole milk in protecting sperm [
10‐
12]. Based on those observations, we postulated that the milk proteins could be involved in sperm protection. We have shown that casein micelles isolated from milk could interact with BSP proteins, the detrimental factors to sperm membranes [
13]. The association of casein micelles with BSP proteins was shown to preclude cholesterol and phospholipid extraction from membranes induced by BSP proteins, while maintaining sperm viability and motility during sperm storage [
13]. Further studies showed that bovine BSP proteins bind to several milk proteins, namely casein micelles, α-lactalbumin and β-lactoglobulin [
14]. These studies led us to propose that the interaction between milk proteins and bovine BSP proteins is the basis for sperm protection during storage using milk-based extenders.
Bovine species express three BSP members: BSP1, BSP3 and BSP5 [
3,
4]. Previous results showed that BSP genes and proteins are in fact a superfamily [
5,
6]. Homologs of BSP proteins have been isolated and characterized from the seminal plasma (SP) or seminal vesicle secretions of many mammals, including bison [
15], goat [
16], stallion [
17,
18], boar [
8,
19] and ram [
20]. In addition, a BSP1-like proteins has recently been detected in buffalo, camel and alpaca [
21,
22].
Interestingly, milk extender is used for conservation of semen from stallion (reviewed in [
23]), goat (reviewed in [
24]), ram (reviewed in [
12]) and buffalo (reviewed in [
25]). Phosphocaseinates, a milk component, has also been used to preserve stallion semen [
26‐
28]. More recently, an extender containing whey proteins has been used to preserve boar semen [
29]. BSP homologs have been identified in the semen of all these species. Therefore, we postulated that the mechanism underlying sperm protection by milk in bovine species could include similar features for all those mammals. It should be noted that many differences exist between semen from different mammalian species including seminal plasma composition, and protein concentration. These factors could have an impact on semen conservation and therefore it is essential to determine the general features as well as the putative specific characteristics of BSP proteins—milk fractions interactions for each species in order to develop a detailed view of the mechanism of sperm preservation.
The goal of the current study was to determine whether BSP homologous proteins found in boar, ram and stallion seminal plasma have an affinity for milk proteins similar to the BSP proteins in bovine species. Thus, we investigated by gel filtration and immunoblot the affinity of BSP homologous proteins found in the SP of these species for the milk proteins and compared the results with those obtained with BSPs from bovine species. Commercial milk-based extenders generally include glycerin and antibiotics [
1], but we focused our work on BSP protein interactions with milk proteins to highlight their specific contribution in sperm preservation. Based on these results, we believe that the mechanism of sperm protection by milk proteins previously described for BSP from bull could be broadened to include BSP proteins from more species of farm animals.
Methods
Materials
Tris(hydroxymethyl)aminomethane (Tris) was purchased from Sigma (St-Louis, MO) and Sepharose CL-4B and Sephadex G-25 medium were from Pharmacia Biotech Inc (Baie d’Urfé, QC, Canada). Acrylamide and bisacrylamide were purchased from MP Biomedical (Irvine, CA). Sodium-dodecyl sulfate (SDS) and other electrophoresis products were from Bio-Rad (Mississauga, ON, Canada). Low molecular weight (LMW) calibration kit was from GE Healthcare (Baie d’Urfé, QC, Canada). Immobilon-P polyvinylidene fluoride (PVDF) membranes were purchased from Millipore (Nepean, ON, Canada). Western Lightning Chemiluminescence Reagent kit was from Perkin-Elmer Life Sciences (Boston, MA). All other chemicals used were of analytical grade and obtained from commercial suppliers.
Preparation of skimmed milk and milk fractions
Skimmed milk was purchased at a local food store. It generally contains less than 0.1 % fat (mostly triglycerides). Before use, the skimmed milk was heated in a water bath maintained at 92–95 °C for 10 min. The heated skimmed milk was then cooled at room temperature and filtered through metal mesh to remove the coagulum [
30]. The skimmed milk fraction 1 (milk F1) and fraction 2 (milk F2) were prepared by gel filtration on Sepharose CL-4B column as described previously [
14].
Preparation of seminal plasma (SP) proteins
Semen used in this study was collected from fertile animals that were handled by qualified technicians, according to the Guide for the Care and Use of Agricultural Animals established by the Quebec Ministry of Agriculture and Fisheries. Stallion semen (pool of 3 ejaculates) was obtained from the Veterinary Medical School (St-Hyacinthe, Qc, Canada). Ram semen (a pool of over 100 ejaculates) was provided by the Centre d’Insémination Ovine du Québec (LaPocatière, Qc, Canada). Alcohol precipitates of SP proteins from stallion and ram semen were prepared as described previously [
3,
18,
20]. Boar semen (pool of 3 ejaculates) was obtained from F. Ménard Inc. (St-Pie-de-Bagotte, Qc, Canada). Boar SP proteins were prepared as previously described [
8] and concentrated using an YM-3 membrane (molecular weight cut-off, 3000 Dalton) in Amicon stirring ultrafiltration cell.
Isolation of BSP proteins from boar, stallion and ram SP proteins
Gelatin-adsorbed ram and stallion BSP proteins were isolated as described previously [
20]. Briefly, gelatin purified from calf skin was coupled to Affi-gel 15 (Bio-Rad) as previously described [
31]. Lyophilysed seminal plasma proteins (~130 mg) were then dissolved in 0.05 M phosphate-buffered saline (PBS) and loaded on a gelatin–agarose column (1.5
× 28 cm) at a flow rate of 30 ml/h. The adsorbed proteins were eluted with PBS containing 7 M urea, pooled, concentrated to ~3 ml and desalted at room temperature on a Sephadex G-25 column (1.5 × 25 cm) equilibrated with a 50 mM ammonium bicarbonate solution. The eluted proteins were then freeze-dried and stored at 4 °C. Purified boar BSP1 was prepared by a combination of chondroitin sulfate B-affinity chromatography and reverse-phase-high performance liquid chromatography as previously described [
8].
Generation of polyclonal antibodies directed against stallion and ram BSP proteins
Polyclonal antibodies were raised in rabbits by the Biotechnology Research Institute (Montréal, QC, Canada) using gelatin-adsorbed ram or stallion proteins dissolved in saline (1 mg/ml). Polyclonal antibodies were purified from anti-sera by affinity chromatography on a protein A-Sepharose column. Their specificity was assessed by immunoblot analysis as described previously [
32].
Gel filtration chromatography
SP proteins and gelatin-bound ram proteins were dissolved in Tris-buffered saline (20 mM Tris–HCl, 150 mM NaCl, 0.02 % sodium azide, pH 7.4; TBS). Skim milk, milk F1 or milk F2 prepared as described above were filtered through a 5-μm filter. SP proteins (boar, ram and stallion), purified boar BSP1 or gelatin-bound ram BSP proteins were filtered through a 0.45-μm filter. Mixtures of SP (BSP proteins) with milk, milk F1 or milk F2 were incubated 1 h at room temperature before loading on a Sepharose CL-4B column.
Gel filtration chromatography was carried out on a Sepharose CL-4B column (78 × 2.5 cm) equilibrated with TBS at room temperature at a flow rate of 70 ml/h. Fractions of 3.2 ml were collected and their absorbance was measured at 280 nm. The presence of BSP proteins in the various fractions was analyzed by immunoblotting using respective polyclonal antibodies.
The concentration of BSP protein homologs in the SP varies from species to species [
2]. In bovine species, the BSP proteins constitute the major portion of the total SP proteins (~50 %). Approximately 20–30 % of the total proteins in stallion and ram SP correspond to the BSP proteins whereas in boar, the amount of BSP1 is ~1 % of the total SP proteins. Bovine semen is generally diluted with milk extender at a dilution ratio of 1:10 to as high as 1:50 depending on the sperm count. In our previous studies with bovine species, we used 1:20 dilution ratio (corresponds to 1.75 mg SP proteins and 35 mg milk proteins) to study the interaction of BSP proteins and milk proteins [
8]. With this dilution factor, an adequate amount of milk proteins was available to sequester all the BSP proteins present in the bovine, ram or stallion seminal plasma. For comparison purposes, we used the same ratio (1:20) to study the interaction of BSP proteins from ram and stallion with milk proteins. At this ratio, the amount of BSP distributed in various fractions after gel filtration chromatography was sufficient to detect by immunoblot. Since the concentration of BSP1 in boar SP is relatively low (~1 %), we used 1:1 ratio (35 mg boar SP proteins and 35 mg milk proteins) to study the interaction of BSP1 with milk proteins in this species. This dilution ratio permitted detection of BSP proteins in elution fractions following gel filtration chromatography. The protein content of the samples was determined by the modified Lowry procedure [
33]. All gel filtration experiments using total seminal plasma proteins were performed in triplicate. Experiments performed using purified BSP proteins were done in duplicate.
SDS-page and immunoblotting
Proteins were precipitated with trichloroacetic acid (TCA, 15 % (w/v) final concentration), reduced, denatured, and separated by electrophoresis on 15 % polyacrylamide gels. Analyses were performed by immunoblot or by Coomassie Blue R-250 staining. For immunoblots, following the transfer of proteins to PVDF membrane, membranes were blocked with PBS containing 1 % bovine serum albumin (BSA) and 0.02 % Tween-20 and then probed with affinity-purified polyclonal antibodies directed against a C-terminal 15-mer of the boar BSP1 (as described in [
8]), stallion BSP proteins (antibodies isolated from the third boost at 1:1000 dilution) or ram BSP proteins (antibodies isolated from the third boost at 1:3000 dilution). All washings and antibody dilutions were done in PBS containing 0.1 % BSA and 0.02 % Tween-20. For identification, some proteins were also extracted from polyacrylamide gel and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as described by Havlis
et al. [
34].
LC-MS/MS data analysis
Peaks were generated using Mascot Daemon v.2.1.6 while protein identification was performed with the Mascot software package v.2.1.03 (Matrix Science, London, UK) [
35]. MASCOT was set up to search the nr_20090402 database (selected for Mammalia—720673 entries as on January 14
th, 2010). The search criteria were as follow: Tryptic digestion; Variable modifications include carbamidomethylation (Cys) and oxidation (Met) with a peptide mass tolerance of ± 15 ppm and a fragment mass tolerance of ± 0.6 Da. The maximum missed cleavage number was set at 2.
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Competing interests
The author(s) declare that they have no competing interests.
Authors’ contributions
MFL purified BSP proteins from seminal plasma, produced polyclonal antibodies, performed the binding studies by gel filtration and western blots. GP did western blots. Both MFL and GP planned part of the experiments and analyzed the results. All authors participated in the redaction of the manuscript.