Porcine model for the study of sperm capacitation, fertilization and male fertility.

Mammalian fertilization remains a poorly understood event with the vast majority of studies done in the mouse model. The purpose of this review is to revise the current knowledge about semen deposition, sperm transport, sperm capacitation, gamete interactions and early embryonic development with a focus on the porcine model as a relevant, alternative model organism to humans. The review provides a thorough overview of post-ejaculation events inside the sow's reproductive tract including comparisons with humans and implications for human fertilization and assisted reproductive therapy (ART). Porcine methodology for sperm handling, preservation, in vitro capacitation, oocyte in vitro maturation, in vitro fertilization and intra-cytoplasmic sperm injection that are routinely used in pig research laboratories can be successfully translated into ART to treat human infertility. Last, but not least, new knowledge about mitochondrial inheritance in the pig can provide an insight into human mitochondrial diseases and new knowledge on polyspermy defense mechanisms could contribute to the development of new male contraceptives.

Compartmentalization of the proteasome-interacting proteins during sperm capacitation.

Ubiquitination is a stable, reversible posttranslational modification of target proteins by covalent ligation of the small chaperone protein ubiquitin. Most commonly ubiquitination targets proteins for degradation/recycling by the 26S proteasome in a well-characterized enzymatic cascade. Studies using human and non-human mammalian spermatozoa revealed the role of the ubiquitin-proteasome system (UPS) in the regulation of fertilization, including sperm-zona pellucida (ZP) interactions as well as the early events of sperm capacitation, the remodeling of the sperm plasma membrane and acrosome, and for the acquisition of sperm fertilizing ability. The present study investigated the activity of UPS during in vitro capacitation of fresh boar spermatozoa in relation to changes in sperm proteome. Parallel and sequential treatments of ejaculated and capacitated spermatozoa under proteasome permissive/inhibiting conditions were used to isolate putative sperm proteasome-associated sperm proteins in a compartment-specific manner. A differential proteomic approach employing 1D PAGE revealed differences in accumulated proteins at the molecular weights of 60, 58, 49, and 35 kDa, and MS analysis revealed the accumulation of proteins previously reported as proteasome co-purifying proteins, as well as some novel proteins. Among others, P47/lactadherin, ACRBP, ADAM5, and SPINK2 (alias SAAI) were processed by the proteasome in a capacitation dependent manner. Furthermore, the capacitation-induced reorganization of the outer acrosomal membrane was slowed down in the presence of proteasomal inhibitors. These novel results support the proposed role of UPS in sperm capacitation and open several new lines of inquiry into sperm capacitation mechanism.

Ubiquitin-proteasome system participates in the de-aggregation of spermadhesins and DQH protein during boar sperm capacitation.

We studied the participation of the ubiquitin-proteasome system (UPS) in spermadhesin release during in vitro capacitation (IVC) of domestic boar spermatozoa. At ejaculation, boar spermatozoa acquire low molecular weight (8-16 kDa) seminal plasma proteins, predominantly spermadhesins, aggregated on the sperm surface. Due to their arrangement, such aggregates are relatively inaccessible to antibody labeling. As a result of de-aggregation and release of the outer layers of spermadhesins from the sperm surface during IVC, antibody labeling becomes feasible in the capacitated spermatozoa. In vivo, the capacitation-induced shedding of spermadhesins from the sperm surface is associated with the release of spermatozoa from the oviductal sperm reservoir. We took advantage of this property to perform image-based flow cytometry to study de-aggregation and shedding of boar spermadhesins (AQN, AWN, PSP protein families) and boar DQH (BSP1) sperm surface protein which induces higher fluorescent intensity in capacitated vs ejaculated spermatozoa. Addition of a proteasomal inhibitor (100 µM MG132) during IVC significantly reduced fluorescence intensity of all studied proteins (P < 0.05) compared to vehicle control IVC. Western blot detection of spermadhesins did not support their retention during IVC with proteasomal inhibition (P > 0.99) but showed the accumulation of DQH (P = 0.03) during IVC, compared to vehicle control IVC. Our results thus demonstrate that UPS participates in the de-aggregation of spermadhesins and DQH protein from the sperm surface during capacitation, with a possible involvement in sperm detachment from the oviductal sperm reservoir and/or sperm-zona pellucida interactions. The activity of sperm UPS modulates de-aggregation of boar spermadhesins and DQH sperm surface protein/binder of sperm1 (BSP1) during the sperm capacitation.

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.

Acrosin inhibitor detection along the boar epididymis.

Epididymal sperm maturation represents a key step in the reproduction process. Spermatozoa are exposed to epididymal fluid components representing the natural environment essential for their post-testicular maturation. Changes in sperm membrane proteins are influenced by proteolytic, glycosylation and deglycosylation enzymes present in the epididymal fluid. Accordingly, the occurrence of inhibitors of these enzymes in the epididymis is very important for the regulation of sperm membrane protein processing. In the present study, we monitored acrosin inhibitor distribution in boar epididymal fluid and in spermatozoa from different segments of the organ. Using specific polyclonal antibody we registered increasing signal of the acrosin inhibitor (AI) from caput to cauda epididymis. Mass spectroscopy examination of the immunoprecipitated acrosin inhibitor (12 kDa) unequivocally identified sperm-associated acrosin inhibitor (SAAI) in the epididymal tissue. Lectin staining showed N-glycosylation in AI from boar epididymis. Protein detection of AI was supported by the results of semi-quantitative RT-PCR showing the presence of mRNA specifically coding for SAAI and similarly increasing throughout the epididymal duct, from its proximal to distal part. Additionally, the immunofluorescence technique showed the AI localization in the secretory tissue of caput, corpus and cauda epididymis, and in the acrosome region and midpiece of the sperm.

Panel of monoclonal antibodies to sperm surface proteins as a tool for monitoring localization and identification of sperm-zona pellucida receptors.

Primary binding of the sperm to the zona pellucida (ZP) is one of the many steps necessary for successful fertilization. Sperm bind ZP by means of membrane receptors which recognize carbohydrate moieties on ZP glycoproteins according to a well-defined sequential process. Primary binding receptors, many of which have been disclosed in various mammals, are localized throughout the acrosomal region of the sperm surface. A panel of monoclonal antibodies against proteins from the sperm surface was prepared. Antibodies were screened by immunofluorescence for protein localization and Western blotting. Proteins localized on the sperm head and simultaneously detected by Western blotting were further studied in terms of immunolocalization in reproductive tissues and fluids, binding to ZP, immunoprecipitation and sequencing. Of 17 prepared antibodies, 8 recognized proteins localized on the sperm head and also detected proteins of interest by Western blotting. Only three other antibodies recognized proteins that also coincided in binding to ZP. These three antibodies were used for immunoprecipitation, and further protein sequencing of immunoprecipitates revealed that these antibodies distinguished acrosin precursor, RAB-2A protein, and lactadherin P47. This is not the first time we have detected acrosin on the surface of ejaculated and capacitated sperm. However, to our knowledge, this is the first time RAB-2A has been detected on the sperm surface. Lactadherin P47 has already been characterized and its physiological function in reproduction has been proposed.

Characterization of sperm surface protein patterns of ejaculated and capacitated boar sperm, with the detection of ZP binding candidates.

Complementary molecules on the surface of both gametes are responsible for the interaction of sperm protein receptors with zona pellucida (ZP) saccharide structures, and many primary sperm receptors for ZP glycoproteins have been disclosed in various mammals. For our study, proteins were obtained from the surface of ejaculated and in vitro capacitated boar sperm. The isolated proteins were characterized by 1D- and 2D-electrophoretic protein profiles, and by glycoprotein staining. Our results show quantitative and qualitative differences in protein and glycoprotein patterns between ejaculated and capacitated sperm. Far-western blotting with ZP glycoproteins identified 17 interactions in the subproteome of the ejaculated sperm and 14 interactions in the subproteome of the capacitated sperm. High-molecular-mass proteins, coincident with binding to ZP, were sequence-identified. Angiotensin-converting enzyme (ACE), polycystic kidney disease receptor and egg jelly receptor (PKDREJ), and acrosin precursor were successfully identified. This is the first time PKDREJ has been identified on the surface of boar spermatozoa.

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-ß-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.

Reproductive tissue expression and sperm localization of porcine beta-microseminoprotein.

Beta-microseminoprotein (MSP) is a predominant protein of human seminal plasma and originates from prostate secretions. MSP from boar seminal plasma has been sequenced and shows only 50%-52% homology with that of human. Porcine MSP is synthesized by the post-natal prostate gland and is identical with the sperm motility inhibitor. Although MSP is a protein characteristic of the prostate gland, we have established the presence of its mRNA transcript not only in boar prostate but also in other reproductive organ tissues. In extracts of all these organs, specific polyclonal antiMSP antibody recognizes a 12-kDa protein band identified by mass spectrometry as MSP. Immunofluorescence (IMF) has revealed the occurrence of MSP in the epithelial tissue of the prostate, epididymis, seminal vesicles and Cowper's glands. MSP has been localized on epididymal spermatozoa in the acrosomal region and on the flagellum of ejaculated spermatozoa. The absence of MSP on the surface of capacitated spermatozoa together with the antibody detection of MSP in the sperm acidic extract after in vitro capacitation indicates its acrosomal origin. Additionally, MSP has been localized by IMF in the sperm acrosome in capacitated spermatozoa with a permeabilized plasma membrane and by electron microscopy in ejaculated spermatozoa. The function of MSP in seminal plasma and spermatozoa is not fully understood. Nevertheless, the secretion of porcine MSP by various reproductive organs indicates its multiple roles in the reproductive process. For the first time in mammalian species, MSP has been localized in various physiological stages of sperm.

Biochemical and binding characteristics of boar epididymal fluid proteins.

During the passage through the epididymis, testicular spermatozoa are directly exposed to epididymal fluid and undergo maturation. Proteins and glycoproteins of epididymal fluid may be adsorbed on the sperm surface and participate in the sperm maturation process, potentially in sperm capacitation, gamete recognition, binding and fusion. In present study, we separated proteins from boar epididymal fluid and tested their binding abilities. Boar epididymal fluid proteins were separated by size exclusion chromatography and by high-performance liquid chromatography with reverse phase (RP HPLC). The protein fractions were characterized by SDS-electrophoresis and the electrophoretic separated proteins after transfer to nitrocellulose membranes were tested for the interaction with biotin-labeled ligands: glycoproteins of zona pellucida (ZP), hyaluronic acid and heparin. Simultaneously, changes in the interaction of epididymal spermatozoa with biotin-labeled ligands after pre-incubation with epididymal fluid fractions were studied on microtiter plates by the ELBA (enzyme-linked binding assay) test. The affinity of some low-molecular-mass epididymal proteins (12-17 kDa and 23 kDa) to heparin and hyaluronic acid suggests their binding ability to oviductal proteoglycans of the porcine oviduct and a possible role during sperm capacitation. Epididymal proteins of 12-18 kDa interacted with ZP glycoproteins. One of them was identified as Crisp3-like protein. The method using microtiter plates showed the ability of epididymal fluid fractions to change the interaction of the epididymal sperm surface with biotin-labeled ligands (ZP glycoproteins, hyaluronic acid and heparin). These findings indicate that some epididymal fluid proteins are bound to the sperm surface during epididymal maturation and might play a role in the sperm capacitation or the sperm-zona pellucida binding.

Interference with the 19S proteasomal regulatory complex subunit PSMD4 on the sperm surface inhibits sperm-zona pellucida penetration during porcine fertilization.

Proteolysis of ubiquitinated sperm and oocyte proteins by the 26S proteasome is necessary for the success of mammalian fertilization, including but not limited to acrosomal exocytosis and sperm-zona pellucida (ZP) penetration. The present study examined the role of PSMD4, an essential non-ATPase subunit of the proteasomal 19S regulatory complex responsible for proteasome-substrate recognition, in sperm-ZP penetration during porcine fertilization in vitro (IVF). Porcine sperm-ZP penetration, but not sperm-ZP binding, was blocked in the presence of a monoclonal anti-PSMD4 antibody during IVF. Inclusion in the fertilization medium of mutant ubiquitins (Ub+1 and Ub5+1), which are refractory to processing by the 19S regulatory complex and associated with Alzheimer's disease, also inhibited fertilization. This observation suggested that subunit PSMD4 is exposed on the sperm acrosomal surface, a notion that was further supported by the binding of non-cell permeant, biotinylated proteasomal inhibitor ZL3VS to the sperm acrosome. Immunofluorescence localized PSMD4 in the sperm acrosome. Immunoprecipitation and proteomic analysis revealed that PSMD4 co-precipitated with porcine sperm-associated acrosin inhibitor (AI). Ubiquitinated species of AI were isolated from boar sperm extracts by affinity purification of ubiquitinated proteins using the recombinant UBA domain of p62 protein. Some proteasomes appeared to be anchored to the sperm head inner acrosomal membrane, as documented by co-fractionation studies. In conclusion, the 19S regulatory complex subunit PSMD4 is involved in the sperm-ZP penetration during fertilization. The recognition of substrates on the ZP by the 19S proteasomal regulatory complex is essential for the success of porcine/mammalian fertilization in vitro.

Inhibition of 19S proteasomal regulatory complex subunit PSMD8 increases polyspermy during porcine fertilization in vitro.

The 26S proteoasome is a multi-subunit protease specific to ubiquitinated substrate proteins. It is composed of a 20S proteasomal core with substrate degradation activity, and a 19S regulatory complex that acts in substrate recognition, deubiquitination, priming and transport to the 20S core. Inhibition of proteolytic activities associated with the sperm acrosome-borne 20S core prevents fertilization in mammals, ascidians and echinoderms. Less is known about the function of the proteasomal 19S complex during fertilization. The present study examined the role of PSMD8, an essential non-ATPase subunit of the 19S complex, in sperm-ZP penetration during porcine fertilization in vitro (IVF). Immunofluorescence localized PSMD8 to the outer acrosomal membrane, acrosomal matrix and the inner acrosomal membrane. Colloidal gold transmission electron microscopy detected PSMD8 on the surface of vesicles in the acrosomal shroud, formed as a result of zona pellucida-induced acrosomal exocytosis. Contrary to the inhibition of fertilization by blocking of the 20S core activities, fertilization and polyspermy rates were increased by adding anti-PSMD8 antibody to fertilization medium. This observation is consistent with a possible role of PSMD8 in substrate deubiquitination, a process which when blocked, may actually accelerate substrate proteolysis by the 26S proteasome. Subunit PSMD8 co-immunoprecipitated with acrosomal surface-associated spermadhesin AQN1. This association indicates that the sperm acrosome-borne proteasomes become exposed onto the sperm surface following the acrosomal exocytosis. Since immunological blocking of subunit PSMD8 increases the rate of polyspermy during porcine fertilization, the activity of the 19S complex may be a rate-limiting factor contributing to anti-polyspermy defense during porcine fertilization.

Expression and localization of acrosin inhibitor in boar reproductive tract.

Proteinases and proteinase inhibitors play key roles in almost all physiological processes. Proteinase inhibitors are present in all tissues and body fluids. They interfere with the activity of proteinases and thus maintain homeostasis. The main role of proteinase inhibitors in the reproductive tract is the inactivation of prematurely released hydrolytic enzymes from damaged spermatozoa and the protection of reproductive tracts and spermatozoa against proteolytic degradation. In the boar reproductive system, acrosin inhibitors are found in seminal plasma and on spermatozoa. The amino acid sequence of seminal plasma and sperm-associated acrosin inhibitors is 90% identical, and their biochemical properties have been completely resolved. However, their origin and localization have not been fully elucidated. Using rabbit polyclonal antibody, we have studied the expression and localization of the seminal plasma acrosin inhibitor in the boar reproductive tract. The antibody recognizes a 12-kDa band in extracts from the cauda epididymidis, seminal vesicles, prostate, and Cowper's glands, and immunofluorescence has revealed the acrosin inhibitor in the epithelium and lumen of these organs. Gene expression of the acrosin inhibitor has been studied by reverse transcription together with the polymerase chain reaction. Acrosin inhibitor mRNA transcript is detectable in the epididymis, seminal vesicles, prostate, and Cowper's glands. The antibody has localized the acrosin inhibitor on the surface of epididymal and ejaculated spermatozoa in the acrosomal region. In extracts from epididymal and ejaculated spermatozoa, the specific antibody recognizes acrosin inhibitor at 8 kDa and 12 kDa. The presence of acrosin inhibitor on the sperm surface as a protective molecule for receptors mediating the sperm-zona pellucida binding suggests that it is crucial for the reproductive process.

Ubiquitin C-terminal hydrolase-activity is involved in sperm acrosomal function and anti-polyspermy defense during porcine fertilization.

The 26S proteasome, which is a multi-subunit protease with specificity for substrate proteins that are postranslationally modified by ubiquitination, has been implicated in acrosomal function and sperm-zona pellucida (ZP) penetration during mammalian fertilization. Ubiquitin C-terminal hydrolases (UCHs) are responsible for the removal of polyubiquitin chains during substrate priming for proteasomal proteolysis. The inhibition of deubiquitination increases the rate of proteasomal proteolysis. Consequently, we have hypothesized that inhibition of sperm acrosome-borne UCHs increases the rate of sperm-ZP penetration and polyspermy during porcine in vitro fertilization (IVF). Ubiquitin aldehyde (UA), which is a specific nonpermeating UCH inhibitor, significantly (P < 0.05) increased polyspermy during porcine IVF and reduced (P < 0.05) UCH enzymatic activity measured in motile boar spermatozoa using a specific fluorometric UCH substrate, ubiquitin-AMC. Antibodies against two closely related UCHs, UCHL1 and UCHL3, detected these UCHs in the oocyte cortex and on the sperm acrosome, respectively, and increased the rate of polyspermy during IVF, consistent with the UA-induced polyspermy surge. In the oocyte, UCHL3 was primarily associated with the meiotic spindle. Sperm-borne UCHL3 was localized to the acrosomal surface and coimmunoprecipitated with a peripheral acrosomal membrane protein, spermadhesin AQN1. Recombinant UCHs, UCHL3, and isopeptidase T reduced polyspermy when added to the fertilization medium. UCHL1 was detected in the oocyte cortex but not on the sperm surface, and was partially degraded 6-8 h after fertilization. Enucleated oocyte-somatic cell electrofusion caused polarized redistribution of cortical UCHL1. We conclude that sperm-acrosomal UCHs are involved in sperm-ZP interactions and antipolyspermy defense. Modulation of UCH activity could facilitate the management of polyspermy during IVF and provide insights into male infertility.

Saccharide-mediated interactions of boar sperm surface proteins with components of the porcine oviduct.

The role of boar seminal plasma proteins attached to the sperm plasma membrane during ejaculation has been studied in saccharide-mediated events in the female reproductive tract. Heparin-binding (Hep(+)) proteins (DQH sperm surface protein, and AQN and AWN spermadhesins) and their aggregated forms (fractions II and III) interacted more strongly with both oviductal epithelium cells and fluid than non-heparin-binding (Hep(-)) proteins (PSP I and PSP II spermadhesins) and their heterodimer (fraction IV), and interactions correlate with affinity of these proteins to yeast mannan. Indirect immunofluorescence (IMF) showed that the AQN 1 spermadhesin and fraction II bind to the apical glycocalyx of the ampulla, as well as the isthmic and uterine tubal junction regions of the oviductal sections. IMF demonstrated the recognition of AQN 1 and fraction II and mannosyl components of oviductal epithelium. We suggest that Hep(+) proteins (especially AQN 1, fraction II) on sperm could enable sperm binding to oviductal epithelium and thus participate in formation of the sperm oviductal reservoir. Interactions of Hep(+) proteins to oviductal epithelium were inhibited by mannan, hyaluronic acid and sialylated O-glycoproteins. No or slight inhibition was observed with sulphated polysaccharides (heparin, chondroitin sulphate) and simple monosaccharides. Besides that, attachment of boar seminal plasma proteins to oviductal epithelium cells was affected by oviductal fluid, the natural environment in the oviduct. Moreover, the ability of hyaluronic acid to inhibit the interaction of sperm surface proteins to the oviduct might play a role in sperm release from the oviductal reservoir and in the capacitation process.

Characterization of human seminal plasma proteins homologous to boar AQN spermadhesins.

Spermadhesins, proteins secreted by the boar sexual accessory glands, are believed to play an important role in sperm capacitation and primary contact of sperm and egg. We have previously found human seminal plasma proteins immunobiochemically related to boar AQN and AWN spermadhesins. In this study, we characterized further the AQN spermadhesin-related proteins, here designated as hSA (human spermadhesin-like) proteins. On Western blot, we immunodetected 14, 16 and 18 kDa forms of hSA proteins (hSA-14, hSA-16 and hSA-18, respectively) cross-reacting with rabbit antibody against AQN spermadhesins. Each relative molecular-mass form of hSA comprised three isoelectric isoforms (6.0, 6.8 and 8.4) as shown by 2D-PAGE. Glycoprotein analysis revealed that all hSA-16 and hSA-18 isoforms were N-glycosylated, and those of hSA-14 were non-glycosylated. Two isoforms of hSA-14 (pI 6.0 and 8.4) had affinity to heparin. Size-exclusion chromatography of human seminal plasma indicated that hSA proteins formed high molecular-mass complexes either with other hSA proteins or with seminal plasma lactoferrin and/or its fragments. Similarity of biochemical properties (relative molecular masses, isoelectric points and existence of non- and N-glycosylated forms) of hSA proteins and those of boar AQN spermadhesins, together with a previously described N-terminal amino acid sequence of one hSA protein identical to AQN spermadhesins, imply that hSA proteins are structurally related to boar AQN spermadhesins. However, localization of hSA proteins on the sperm tail and neck suggests that their biological role differs from that of boar AQN spermadhesins located on the sperm head.

Mannan-binding proteins from boar seminal plasma.

The interaction of boar seminal plasma proteins and sperm with yeast mannan was investigated by the enzyme-linked binding assay (ELBA) and specific detection of proteins after SDS electrophoresis and blotting using biotinylated derivative of the polysaccharide. Heparin-binding proteins (especially AQN 1 and DQH proteins) and their aggregated forms showed affinity to yeast mannan. Besides that, these proteins were shown to bind to oviductal epithelium. The mannan-binding activity of boar proteins and sperm was inhibited most efficiently by ovomucoid, ovalbumin and N-glycans released from ovalbumin, but not with d-glucose, d-mannose and their phosphates. On the other hand, yeast mannan inhibited both the interaction of boar seminal plasma and sperm with heparin and the binding of these proteins to porcine oviductal epithelium. Yeast mannan immobilized to divinyl sulfone-activated Sepharose was used for the isolation of mannan-binding proteins. Proteins adsorbed to the immobilized polysaccharide were analyzed by RP-HPLC, SDS electrophoresis and N-terminal amino acid sequencing. AQN and AWN spermadhesins and DQH protein (names are derived from the N-terminal amino acid sequence) were identified as components of the isolated fraction. The results suggest an involvement of mannan-binding proteins in the formation of the sperm oviductal reservoir in pig. The ability of these proteins to interact both the complex d-mannose-containing saccharide structures and the heparin may also play an important role in sperm release from the oviductal reservoir or the capacitation process.

Proteinase inhibitors in aggregated forms of boar seminal plasma proteins.

Boar seminal plasma proteins were separated by gel chromatography on Sephadex G-75 into five fractions (I-V). Serine proteinase inhibitors were found mainly in the protein fraction with relative molecular weight 5-25kDa. Small amounts of these inhibitors were also found in the high molecular weight protein fraction (M(r)>100kDa). The protein fraction containing most of the proteinase inhibitory activity was further separated by RP HPLC. Isolated proteins were characterized by SDS electrophoresis and immunoblotting, N-terminal amino acid sequencing and by determination of the proteinase inhibitory activity. In the fraction containing proteinase inhibitors, also beta-microseminoprotein (beta-MSP), AQN 1 and lactoferrin were identified. The possible existence of complexes of protein components in the fraction with relative molecular weight 5-25kDa was studied in detail using gel chromatographic separation on Sephadex G-50. A part of proteinase inhibitors with M(r) 8kDa was eluted together with AQN 1 spermadhesin. An interaction of isolated spermadhesin AQN 1 and proteinase inhibitor was shown.

Mutual interactions of boar seminal plasma proteins studied by immunological and chromatographic methods.

PROBLEM: Boar seminal plasma contains various types of proteins. Most of them belong to spermadhesins and some of them contain fibronectin type II domain. Almost all of these proteins are present under physiological conditions in aggregated forms differing in their relative molecular mass, composition, and binding properties. The study of mutual specific interactions between proteins of boar seminal plasma that could be involved in the formation of aggregated forms and most probably of sperm coating layers is the subject of our present communication. METHODS OF STUDY: Aggregated forms of boar seminal plasma proteins separated by size exclusion chromatography were analysed by Reversed-phase High-performance Liquid Chromatography (RP HPLC), SDS-PAGE and immunochemical methods (ELISA and immunoblotting). Mutual interactions between proteins were investigated by size exclusion chromatography of a mixture of separated monomer proteins and affinity chromatography of boar seminal plasma on immobilized spermadhesins. RESULTS: Composition of proteins that are adsorbed from boar seminal plasma to immobilized spermadhesins corresponds to that of aggregated forms found in seminal plasma. Mutual interactions between monomer forms observed by size exclusion chromatography are in good agreement with results of affinity chromatography. An existence of interactions between spermadhesins of the AQN and AWN families and the DQH sperm surface protein, as well as between proteins involved in the formation of heterodimer porcine seminal plasma I (PSP I)/PSP II was proved. CONCLUSION: Mutual specific interactions between protein components of boar seminal plasma were shown. These interactions participate in the formation of aggregated forms of proteins in seminal plasma and probably also in the arrangement and remodelling of protein coating layers of sperm. Aggregation of seminal plasma proteins is probably an important phenomenon in the fertilization process.

Isolation of non-heparin-binding and heparin-binding proteins of boar prostate.

Proteins of boar prostate secretion were separated by affinity chromatography on heparin-polyacrylamide to non-heparin-binding (H) and heparin-binding (H+) protein fractions. H- and H+ fractions were then subjected to RP HPLC. Elution profiles of H-and H+ fractions of prostate secretion were compared with those of seminal plasma and the amounts of corresponding proteins were compared. Besides, the isolated proteins were characterized by SDS-PAGE. In the H- fraction of prostate secretion, PSP I and PSP II spermadhesins and in the H+ fraction AQN 2 and AWN 1 spermadhesins were found in substantially lower amounts than in seminal plasma. On the contrary, beta-microseminoprotein was identified in abundant amounts both in H- and H+ fractions of boar prostate secretion. AQN 2 and AWN 1 spermadhesins were proved by their antibodies. Some seminal plasma proteins originating mainly in seminal vesicles could also be secreted by the prostatic gland. beta-Microseminoprotein was found to be produced mainly by the prostate.