Porcine spermadhesin AQN-3 binds to negatively charged phospholipids.

The spermadhesin AQN-3 is a major component of porcine seminal plasma. While various studies suggest that this protein binds to boar sperm cells, its attachment to the cells is poorly understood. Therefore, the capacity of AQN-3 to interact with lipids was investigated. For that purpose, AQN-3 was recombinantly expressed in E. coli and purified via the included His-tag. Characterizing the quaternary structure by size exclusion chromatography revealed that recombinant AQN-3 (recAQN-3) is largely present as multimer and/or aggregate. To determine the lipid specificity of recAQN-3, a lipid stripe method and a multilamellar vesicle (MLV)-based binding assay were used. Both assays show that recAQN-3 selectively interacts with negatively charged lipids, like phosphatidic acid, phosphatidylinositol phosphates, and cardiolipin. No interaction was observed with phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, or cholesterol. The affinity to negatively charged lipids can be explained by electrostatic interactions because binding is partly reversed under high-salt condition. However, more factors have to be assumed like hydrogen bonds and/or hydrophobic forces because the majority of bound molecules was not released by high salt. To confirm the observed binding behavior for the native protein, porcine seminal plasma was incubated with MLVs comprising phosphatidic acid or phosphatidyl-4,5-bisphosphate. Attached proteins were isolated, digested, and analyzed by mass spectrometry. Native AQN-3 was detected in all samples analyzed and was - besides AWN - the most abundant protein. It remains to be investigated whether AQN-3, together with other sperm associated seminal plasma proteins, acts as decapacitation factor by targeting negative lipids with signaling or other functional roles in fertilization.

Boar Sperm Cryopreservation Improvement Using Semen Extender Modification by Dextran and Pentaisomaltose.

The long-term storage of boar sperm presents an ongoing challenge, and the modification of the cryoprotective compounds in semen extenders is crucial for improving cryopreservation's success rate. The aim of our study was to reduce the percentage of glycerol in the extender by elimination or substitution with biocompatible, non-toxic polysaccharides. For boar semen extender improvement, we tested a novel modification with the polysaccharides dextran and pentaisomaltose in combination with unique in silico predictive modeling. We targeted the analysis of in vitro qualitative sperm parameters such as motility, viability, mitochondrial activity, acrosome integrity, and DNA integrity. Non-penetrating polysaccharide-based cryoprotective agents interact with sperm surface proteins such as spermadhesins, which are recognized as fertility markers of boar sperm quality. The in silico docking study showed a moderate binding affinity of dextran and pentaisomaltose toward one specific spermadhesin known as AWN, which is located in the sperm plasma membrane. Pentaisomaltose formed a hydrophobic pocket for the AWN protein, and the higher energy of this protein-ligand complex compared with dextran was calculated. In addition, the root mean square deviation (RMSD) analysis for the molecular dynamics (MD) of both polysaccharides and AWN simulation suggests their interaction was highly stable. The in silico results were supported by in vitro experiments. In the experimental groups where glycerol was partially or entirely substituted, the use of pentaisomaltose resulted in improved sperm mitochondrial activity and DNA integrity after thawing when compared with dextran. In this paper, we demonstrate that pentaisomaltose, previously used for cryopreservation in hematopoietic stem cells, represents a promising compound for the elimination or reduction of glycerol in extenders for boar semen cryopreservation. This novel approach, using in silico computer prediction and in vitro testing, represents a promising technique to help identify new cryoprotectants for use in animal breeding or genetic resource programs.

Boar spermadhesin AWN: novel insights in its binding behavior and localization on sperm.

As a major spermadhesin first found in the seminal plasma (SP) of boars, AWN is described to fulfil a variety of reproduction related tasks. Although being the best investigated boar spermadhesin, information about its interaction with membranes is inconsistent. In this regard, previous reports locate AWN either inside or on the surface of sperm cells and at different regions, depending on the method and antibody used. Here, we localize native AWN (natAWN) in/on epididymal, ejaculated, capacitated, and acrosome-reacted boar sperm using epifluorescence and electron microscopy as well as an analysis of potential lipid-binding partners of natAWN and recombinant AWN (recAWN). By applying a custom-made anti-AWN antibody, localization of AWN in the equatorial segment (EQS) of ejaculated, capacitated, and acrosome-reacted boar sperm was discovered. Electron microscopy showed that AWN is localized both on the sperm surface and on the cytoplasmic side of the plasma membrane and in close vicinity to the nuclear and both acrosomal membranes of sperm. Analysis of epididymal sperm indicated migration of AWN from the retral postacrosomal part to the EQS during the epididymal passage. In contrast to hypotheses claiming a specific association of AWN to phosphatidylethanolamine (PE) and in line with our previous study describing an interaction with phosphatidic acid (PA), the current results show a rather electrostatically driven binding mechanism of AWN to negative lipids. In conclusion, this work provides new insights into the arrangement of AWN in the EQS, which suggest a possible role in sperm-oocyte fusion.

Structural and biophysical characterization of the major proteins from the seminal plasma of Dorper rams.

Pregnancy rates using frozen semen from rams are higher than for horses. One of the factors that positively influences this effect is the composition of low-molecular-weight proteins from seminal plasma, since the amounts of these proteins are much lower in horses. The aim of this work was to purify the major protein components from ram seminal plasma for structural and biophysical characterization. First, the ram semen was collected and the plasma separated by centrifugation. The protein fractions were isolated by gel filtration chromatography, analyzed by circular dichroism spectroscopy and the amino acid sequence identified by mass spectrometry (LC-MSE), the results of which were used to model the protein structure by bioinformatics techniques. This protein was identified by LC-MSE as a spermadhesin, being an unglycosylated monomer with Tm = 69.3 °C and ΔHm= 371 kJ mol-1 at pH 7.0. This work describes for the first time the structural characterization of a spermadhesin from seminal plasma of Dorper rams.

Purification, structural and biophysical characterisation of the major seminal plasma protein from Texel rams.

Spermadhesins are a group of low molecular weight proteins present in seminal plasma. In Texel rams, they represent more than 70% of the seminal plasma proteins. Although their functions have not yet been fully clarified, there is much discussion about the role of these proteins in maintaining sperm viability during and after the semen freezing process. This work sought to isolate the major component of the seminal plasma from rams of the Texel breed (O. aries SPD2) and to evaluate its structural and biophysical characteristics in order to better understand its role in spermatic viability. The protein was isolated by centrifugation and ion exchange chromatography and its biophysical properties were evaluated by circular dichroism spectrometry. Molecular dynamics simulations of the modelled protein compared to the homologous bovine protein were also carried out. The results showed that O. aries SPD2 has a transition temperature (Tm) of 65 °C and a ΔHm of 322.5 kJ mol-1, similar to the results for other spermadhesins described in the literature. The estimated composition of the secondary structure elements for the native protein is in agreement with that observed for the theoretical model. Its structural characteristics were preserved in simulations at temperatures of 27 °C and 40 °C, as was the case for bull spermadhesin. Taken together, these results suggest that the major component of the spermadhesins of Texel rams (O. aries SPD2) may play an important role in maintaining the viability of spermatozoa in fresh semen as well as after thawing.

Glycosidases in porcine follicular fluid and their effect on zona pellucida-AWN 1 spermadhesin interaction.

Oligosaccharide moieties on the surface of the oocyte belong to the key molecules that direct the course of fertilization and are subjected to changes during oocyte maturation in the follicle. In our study, we focused on the activities of five glycosidases in the fluids from porcine secondary and preovulatory follicles (α-l-fucosidase, α-d-galactosidase, ß-d-galactosidase, ß-D-N-acetylhexosaminidase, and α-d-mannosidase). All of them were detected active at neutral and acidic pH. However, changes in their activities associated with follicle development were observed only in the case of α-d-mannosidase, which was increased (P < 0.001), and ß-d-galactosidase, which was decreased (P < 0.001) at neutral pH, and of α-d-galactosidase and ß-N-acetylhexosaminidase, which were decreased (P < 0.0001) at the acidic pH. The comparison of glycosidases from follicular fluid and from blood plasma using red native electrophoresis revealed that most of the glycosidases are present in more than one isoenzyme form; some of them were detected mainly in the follicular fluid. Finally, we tested the effect of glycosidases on the interaction between zona pellucida and AWN 1 spermadhesin (putative sperm receptor of zona pellucida) and demonstrated that the effect of both ß-d-galactosidase and to a lesser degree α-d-mannosidase led to a decrease in this interaction. We can hypothesize that these two glycosidases modulate the amount of zona pellucida oligosaccharide moieties and/or their structures for an optimal sperm binding in pigs.

Ion-exchange chromatography used to isolate a spermadhesin-related protein from domestic goat (Capra hircus) seminal plasma

Mammalian seminal plasma contains among others, proteins called spermadhesins, which are the major proteins of boar and stallion seminal plasma. These proteins appear to be involved in capacitation and sperm-egg interaction. Previously, we reported the presence of a protein related to spermadhesins in goat seminal plasma. In the present study, we have further characterized this protein, and we propose ion-exchange chromatography to isolate this seminal protein. Semen was obtained from four adult Saanen bucks. Seminal plasma was pooled, dialyzed against distilled water and freeze-dried. Lyophilized proteins were loaded onto an ion-exchange chromatography column. Dialyzed-lyophilized proteins from the main peak of DEAE-Sephacel were applied to a C2/C18 column coupled to an RP-HPLC system, and the eluted proteins were lyophilized for electrophoresis. The N-terminal was sequenced and amino acid sequence similarity was determined using CLUSTAL W. Additionally, proteins from DEAE-Sephacel chromatography step were dialyzed and submitted to a heparin-Sepharose high-performance liquid chromatography. Goat seminal plasma after ion-exchange chromatography yielded 6.47 +/- 0.63 mg (mean +/- SEM) of the major retained fraction. The protein was designated BSFP (buck seminal fluid protein). BSFP exhibited N-terminal sequence homology to boar, stallion and bull spermadhesins. BSFP showed no heparin-binding capabilities. These results together with our previous data indicate that goat seminal plasma contains a protein that is structurally related to proteins of the spermadhesin family. Finally, this protein can be efficiently isolated by ion-exchange and reverse-phase chromatography.