Boar Sperm Protein Research Articles

Profiling of differentially expressed proteins between fresh and frozen-thawed Duroc boar semen using ProteinChip CM10.

Many studies have been conducted to improve technology for semen cryopreservation in pigs. However, computer-assisted analysis of sperm motility and morphology is insufficient to predict the molecular function of frozen-thawed semen. More accurate expression patterns of boar sperm proteins may be derived using the isobaric tags for relative and absolute quantification (iTRAQ) technique. In this study, the iTRAQ-labeling system was coupled with liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis to identify differentially expressed CM10-fractionated proteins between fresh and frozen-thawed boar semen. A total of 76 protein types were identified to be differentially expressed, among which 9 and 67 proteins showed higher and lower expression in frozen-thawed than in fresh sperm samples, respectively. The classified functions of these proteins included oxidative phosphorylation, mitochondrial inner membrane and matrix, and pyruvate metabolic processes, which are involved in adenosine triphosphate (ATP) synthesis; and sperm flagellum and motile cilium, which are involved in sperm tail structure. These results suggest a possible network of biomarkers associated with survival after the cryopreservation of Duroc boar semen.

Effect of Boar Sperm Proteins and Quality Changes on Field Fertility.

Simple SummaryArtificial insemination with extended liquid boar semen is widely used in the swine industry. The identification of the relationship between boar sperm characteristics and fertility could be of substantial importance to reproduction management. This study evaluated the relationship between boar sperm characteristics and sperm/seminal plasma proteins with main parameters of field fertility. Immotile spermatozoa and spermatozoa with biochemically active plasma membranes affected the number of live-born piglets and litter size of ≥12 piglets. The proteins osteopontin 70 and glutathione peroxidase 5, both separately and in combination, affected the farrowing rate. The combination of immotile sperm and protein osteopontin 70 explained the variation regarding litter size with ≥12 piglets. In conclusion, the evaluation of semen quality variables combined with the evaluation of specific sperm or seminal plasma proteins could provide useful information on in vivo fertilizing capacity of semen doses.This study aimed to evaluate boar sperm characteristics and proteins, in relation to their importance regarding in vivo fertility. Sixty-five ejaculates were used and 468 sows (parity ≥ 2) were inseminated. Sperm CASA kinetics, morphology, viability, DNA fragmentation, mitochondrial membrane potential, sperm membrane biochemical activity (HOST) and sperm proteins (Heat Shock Protein 90-HSP90, glutathione peroxidase-5-GPX5, Osteopontin 70-OPN70) were assessed and related to field fertility (number of live-born piglets—NLBP, litter size ≥ 12 piglets—LS, farrowing rate—FR). Statistical analysis was conducted with simple and multiple regression models. Simple regression analysis showed that immotile sperm (IM) significantly affected the NLBP and LS, explaining 6.7% and 6.5% of their variation, respectively. The HOST positive spermatozoa significantly affected the NLBP and LS, explaining 24.5% and 7.8% of their variation, respectively. Similarly, sperm with activated mitochondria significantly affected the NLBP, explaining 13.5% of its variation. Moreover, the OPN70 affected LS and FR, explaining 7.5% and 10.8% of their variation, respectively. Sperm GPX5 protein affected FR, explaining 6.7% of its variation. Multiple regression analysis showed that the combination of IM and/OPN70 explains 13.0% of the variation regarding LS, and the combination of GPX5 and OPN70 explains 13.6% of the variation regarding FR. In conclusion, the estimation of parameters IM, membrane biochemical activity, mitochondrial membrane potential, OPN and GPX5 can provide useful information regarding semen doses for field fertility.

Transwell isolation and difference analysis of capacitated boar sperm proteins based on the iTRAQ technique

During capacitation, proteins in boar sperm are released to maintain the stability of their own state and membrane structure. No studies have analyzed the differences between retained proteins and released proteins during sperm capacitation. In the present study, a Transwell chamber and polycarbonate membrane were used to separate the proteins of boar sperm and their released proteins. Isotopically labeled relative and absolute quantification (iTRAQ) was used to analyze each compartment protein. A total of 108 differential proteins were identified in the upper and lower chambers of the Transwell, among which 27 were significantly upregulated (p-value≤0.05 and |log2 (fold change)|≥1) and 81 were significantly downregulated (p-value≤0.05 and |log2 (fold change)|≤1). These differential proteins were mainly involved in biological processes (e.g., the regulation of cysteine peptidase activity, transmembrane transportation, ion transportation and ATP synthesis) and major signaling pathways (e.g., glutathione/galactose metabolism, cellular adhesion and PI3K-Akt), and most of them interacted with each other to some extent. In conclusion, retained proteins and released proteins of capacitated sperm were effectively separated using a Transwell chamber, and differential proteins were successfully identified from among the proteins. Bioinformatics analysis suggested that these differential proteins affect sperm capacitation mainly by adjusting sperm energy metabolism, motion characteristics and acrosome membrane status.

Effect of seasonal infertility period on boar sperm proteins and quality characteristics

Swine seasonal infertility reduces the productivity and profitability of a pig farm. The main causes of this condition are elevated environmental temperatures and long photoperiod during the summer season. The aim of this study was to investigate which sperm proteins and parameters are affected during the period of seasonal infertility. Depending on the environmental temperatures, the period from October to June was considered as cold and the period from July to September as warm season. A total of 65 ejaculates from 18 boars were collected over a year. Each semen sample was evaluated for kinetics (Computer Assisted Semen Analyzer), morphology (Sperm Blue stain), viability (Propidium Iodide - Calcein AM stain), mitochondrial membrane potential (Rhodamine 123 – Propidium Iodide stain), membrane integrity and functionality (Hypo-osmotic swelling test) and sperm DNA integrity (Acridine Orange Test). Moreover, selected proteins (HSP90, GPX5, OPN) were detected and quantified. The kinetic parameters VSL, LIN and the midpiece abnormalities were significantly higher in the warm compared to the cold season (p<0.05), while a strong tendency towards higher values for HSP90 and GPX5 was observed in warm compared to cold season (p=0.07and p=0.06, respectively). In conclusion, among the boar sperm characteristics tested in our study, seasonal infertility period negatively affected VSL and LIN kinetics, while GPX5 seminal plasma enzyme and HSP90 sperm surface protein increased their sperm protective effects.

Boar sperm protein tyrosine phosphorylation in the presence of egg yolk soluble and low density lipoprotein fractions during cooling

Increased protein tyrosine phosphorylation and the appearance of a phosphorylated protein of 32 kD (p32) are reported among the capacitation-like changes in cryopreserved boar sperm. Egg yolk freezing extenders are composed by two fractions: insoluble granules and soluble plasma, which contains the low density lipoproteins (LDL) proposed as responsible for the egg yolk cryoprotective action. The aim of this work was to analyze the effects of complete egg yolk and its insoluble, soluble and LDL fractions on boar sperm quality and protein tyrosine phosphorylation after the first stage of a standard cryopreservation protocol. Semen samples in Androstar® Plus diluent were centrifuged and resuspended in the different egg yolk extenders. Temperature was decreased from 17 °C to 5 °C and sperm quality, protein tyrosine phosphorylation and protein pattern were analyzed. Results showed that complete egg yolk as well as soluble and LDL egg yolk fractions maintained sperm quality after temperature decrease. Cooling without any lipid component or in the presence of the insoluble fraction, significantly reduced sperm motility. About sperm protein tyrosine phosphorylation analysis, the p32 band appeared before treatments or after cooling in Androstar® Plus diluent. Complete egg yolk and its insoluble fraction interfered with sperm tyrosine phosphorylation even after cells were extensively washed. Analysis of extenders revealed a high amount of tyrosine phosphorylated proteins in the insoluble fraction, which may have co-precipitate with sperm in experiments. Samples submitted to temperature decrease from 17 °C to 5 °C in the presence of soluble and LDL egg yolk fractions in Androstar® Plus diluent did not show any change in the p32 band associated with sperm capacitation. However, a tyrosine-phosphorylated protein of 33 kD present in clarified egg yolk was also observed in sperm treated with this extender. Protein transference from plasma and LDL egg yolk extenders was also observed in sperm protein profile. Results suggested that soluble and LDL fractions might have a protective action preventing sperm protein tyrosine phosphorylation during cooling from 17 °C to 5 °C. Further studies are needed to expand the knowledge of the LDL protection mechanism as well as to determine the possible benefits of clarified egg yolk in freezing protocols.

Electrophoretic and zymographic characterization of proteins isolated by various extraction methods from ejaculated and capacitated boar sperms

The presented work focuses on electrophoretic and zymographic characterization of boar sperm proteins isolated by various extraction methods and on comparison of the protein profiles obtained from ejaculated and in vitro capacitated spermatozoa. Sperm proteins of ejaculated and in vitro capacitated boar sperms were isolated with the following agents: 1% v/v Triton X-100, 1% v/v Triton X-114, 2% v/v acetic acid, 1% m/v sodium dodecyl sulphate (SDS), 30 mM N-octyl-β-D-glucopyranoside (OBG), rehydration buffer (RHB) for isoelectric focusing and finally by the freezing-thawing approach. The extracts were characterized in terms of 1-DE, 2-DE protein profiles, 1-DE glycoprotein staining and proteinase and hyaluronidase substrate zymographic profiles. The results have shown quantitative and qualitative differences in 1-DE protein and glycoprotein profiles with respect to the employed isolation approach. These differences were seen even more clearly in 2-DE protein profiles, where it was possible to distinguish the presence/absence, changes in relative abundance and pI/M(r) shifts of various protein spots. Proteinase and hyaluronidase zymograms supported the prediction that various isolation protocols result in various profiles of enzymatically active molecules.

Rapid PKA-catalysed phosphorylation of boar sperm proteins induced by the capacitating agent bicarbonate.

In boar spermatozoa, the capacitating agent bicarbonate has been shown to induce rapid changes both in plasma membrane lipid architecture and in motility; in each case, a PKA-dependent pathway is involved. Early bicarbonate-induced changes in protein phosphorylation were probed using a commercial antibody against the phosphorylated form of the consensus substrate site for cyclic AMP-dependent protein kinase. The antibody detected relatively few bands in sperm extracts, of which only a small number showed incubation-dependent changes. While the quantitative response varied between boar ejaculates, in general terms bicarbonate induced phosphorylation increases in bands of 96, 64, and 59 kDa within 80 sec. The changes reached a maximum after about 160 sec, declined somewhat thereafter, and then increased again slowly as incubation progressed further (up to 21 min). The bicarbonate-induced increases were strongly dependent on the presence of BSA in the incubation medium. They were inhibited by H89 (PKA inhibitor) but not by GF (PKC inhibitor), and were enhanced by papaverine (phosphodiesterase inhibitor) and by calyculin (protein phosphatase inhibitor). The cyclic AMP analogue cBIMPS was able to mimic bicarbonate action though its effect was less dramatic. Stearated Ht31, a permeable inhibitor of PKA's binding to A-kinase anchoring protein, did not affect either the intensity or the specificity of the bicarbonate-induced phosphorylation changes, though it blocked motility entirely. Immunocytochemical studies revealed marked bicarbonate-dependent phosphorylation changes in the post-acrosomal region of the head and in the neck, midpiece, and anterior regions of the tail. Fractionation of stimulated spermatozoa showed that all bands detectable with the antibody were bound to heads and to midpieces and associated large tail fragments; no bands were detected in either small tail or membrane fragments or in the cytoplasmic fraction. Differential extraction of the midpiece/large tail fraction revealed two protein bands with closely similar electrophoretic mobilities to the 96- and 59-kDa phosphorylated bands; MALDI-TOF analyses of these bands revealed both to be members of the Odf2 family.

Amino Acid Sequences of Porcine Sp38 and Proacrosin Required for Binding to the Zona Pellucida

We previously purified a boar sperm protein, sp38, and demonstrated that this protein bound to the 90-kDa family of zona pellucida (ZP) glycoprotein in a calcium-dependent manner. Sp38 competed with proacrosin for the binding to the zona pellucida. Herein we have isolated cDNA clones encoding sp38 from a boar testis cDNA library in λgt11. The amino acid sequence deduced from the cDNA sequence indicated that sp38 is initially synthesized as a 350-residue precursor protein. The N-terminal 51-residue sequence preceded the N-terminus of the mature sp38. Thus, the sp38 precursor is post-translationally modified to produce the mature protein of 299 residues. Immunostaining of sperm cells using an antibody prepared against a fusion protein of sp38 with T7 gene10 protein suggested that sp38 is localized at the intraacrosomal region and is released after the acrosome reaction. The 11-residue sequence, KRLSKAKNLIE, in sp38 shared a significant degree of similarity with the 8-residue sequence, KRLQQLIE, in the C-terminal region of porcine proacrosin. Both synthetic oligopeptides corresponding to these two sequences inhibited the binding of 125 I-labeled sp38 to zona pellucida glycoprotein.

Purification and characterization of a boar sperm protein interacting with immunoglobulins from an infertile woman.

Immunoglobulin G (IgG) fraction was prepared from a serum obtained from an infertile woman (IS) that induced sperm agglutination of human and boar sperm. The antisperm antibodies interacted with a 50-kD boar sperm protein, determined by immunoblot. The 50-kD protein was extracted with deoxycholate (DOC) and purified by affinity chromatography on concanavalin A column, ion exchange chromatography on CM52 column, affinity chromatography on IS IgG-protein G-agarose, and by preparative electrophoresis with electroelution. The purified 50-kD protein migrated as a single homogeneous band when analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by Western blot. The production of antibodies against specific sperm proteins may be a cause of immunological infertility.

Identification and expression of boar sperm proteins in the reproductive tract that interact with antibodies in serum from an infertile woman

Serum designated as IS obtained from a young healthy infertile woman induced a head-to-head agglutination of ejaculated boar sperm. The immunoglobulin G (IgG) prepared from IS localized to the acrosomal region of the sperm head obtained from the corpus and cauda epididymis as determined by an indirect immunofluorescent method. The IgG interacted with a boar sperm protein with an estimated molecular weight of 45-kDa, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoretic (SDS-PAGE) immunoblotting technique. However, the IgG did not interact with proteins extracted from sperm obtained from the testis and caput epididymis or from non-gonadal tissues including liver, kidney, spleen, muscle and serum. The IgG interacted with additional proteins of about 75- and 38-kDa present in the corpus and cauda epididymal fluids but not those in the caput epididymal fluid. The staining intensity of the 75-kDa band was reduced and that of the 38-kDa was nullified with ejaculated seminal plasma proteins. The interacting proteins were adsorbed when chromatographed on Concanavalin A Sepharose column, suggesting that they are glycoproteins.

Isolation, characterization, and localization of the zona pellucida binding proteins of boar sperm

In order to isolate the putative zona pellucida-binding proteins (ZPBPs) from boar spermatozoa, a new, simple method has been developed. The new isolation strategy made the most of the highly specific interactions between the components of the gametes. Detergent-extracted boar sperm proteins were submitted to affinity chromatography on a ZP-Sepharose column. SDS-PAGE analyses of the retained fraction under reducing conditions revealed that in addition to a component of Mr 38,000, the predominant ZPBPs contained at least three low molecular weight proteins (Mr less than 20 kDa). The isolated ZPBPs were effective in blocking sperm-oocyte binding in vitro. Using immunofluorescence microscopy, the ZPBPs were shown to be localized primarily in the sperm head, especially in the acrosomal cap region.

Proacrosin conversion inhibitor. Purification and initial characterization of a boar sperm protein which prevents the conversion of proacrosin into acrosin.

A proacrosin conversion inhibitor present in boar spermatozoa has been purified and initially characterized. Purification methods included sequential acid extractions of washed spermatozoa at pH 4.0, pH 3.5, and pH 2.5 followed by successive gel filtrations of the pH 2.5 sperm extract supernatant over Sephadex G-75 and G-50. The resulting 8.8-fold purified materials were judged to be homogeneous by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis, had an estimated molecular weight of 12,800, and a constant specific activity of 65 units/mg. Treatment with the proteinases acrosin, trypsin, or chymotrypsin destroyed the highly purified proacrosin conversion inhibitor, indicating that it is a protein. Additional properties of the inhibitor included stability to long periods of storage at pH 3.0 and 4 degrees C, stability to boiling and lyophilization, and an absolute requirement for divalent cations to maintain activity. The highly purified proacrosin conversion inhibitor does not inhibit acrosin. Therefore, it apparently acts to prevent proacrosin conversion by selectively inhibiting the zymogen's self-catalyzed conversion mechanism.