Binder Of Sperm Proteins Research Articles

Implication of the human Binder of SPerm Homolog 1 (BSPH1) protein in capacitation

Binder of SPerm (BSP) proteins are a family of proteins expressed exclusively in the male reproductive tract (seminal vesicles or epididymis) of several mammalian species. They are known to promote capacitation, a sperm maturation step essential for fertilization. Our recent studies have shown that in human, the Binder of SPerm Homolog 1 (BSPH1) is expressed solely in epididymal tissues. The goal of the current study was to characterize BSPH1 and evaluate its effect on different sperm functions. A human recombinant BSPH1 (rec-BSPH1) was produced, purified and refolded. Rec-BSPH1 was found to share many characteristics with other members of the BSP superfamily, as it was able to bind gelatin and heparin as well as capacitate sperm. Rec-BSPH1 had no effect on sperm acrosome reaction or any sperm motility parameters. Native BSPH1 was localized on the equatorial segment, post-acrosomal segment and neck of ejaculated human sperm. Rec-BSPH1, following incubation with washed ejaculated human sperm, exhibited binding patterns similar to the native protein. These results show that the human epididymal BSPH1 shares many biochemical and functional characteristics with BSP proteins secreted by seminal vesicles of ungulates, and behaves similarly to its murine epididymal orthologue BSPH1. This study of human BSPH1 brings us one step closer to understanding the importance of this protein in male fertility.

An Overview on Binder of Sperm Proteins of Cattle

Bovine seminal plasma is a protein rich fluid, which plays major role in the fertility of sire. Proteins are derived from blood as well as accessory sex glands. Binder of sperm proteins are major proteins in seminal plasma characterized by their binding ability to heparin like glycosaminoglycans. These proteins destabilize the membrane of spermatozoa by interacting with glycosaminoglycans of female reproductive tract. Relatively much less proteomic has been done in seminal fluid as compare to other protein rich fluids. The functional and expression pattern studies of bulls with different fertility abilities can provide insights into the selection of sires.

Proteomic characterization and cross species comparison of mammalian seminal plasma

Seminal plasma contains a large protein component which has been implicated in the function, transit and survival of spermatozoa within the female reproductive tract. However, the identity of the majority of these proteins remains unknown and a direct comparison between the major domestic mammalian species has yet to be made. As such, the present study characterized and compared the seminal plasma proteomes of cattle, horse, sheep, pig, goat, camel and alpaca. GeLC-MS/MS and shotgun proteomic analysis by 2D-LC-MS/MS identified a total of 302 proteins in the seminal plasma of the chosen mammalian species. Nucleobindin 1 and RSVP14, a member of the BSP (binder of sperm protein) family, were identified in all species. Beta nerve growth factor (bNGF), previously identified as an ovulation inducing factor in alpacas and llamas, was identified in this study in alpaca and camel (induced ovulators), cattle, sheep and horse (spontaneous ovulators) seminal plasma. These findings indicate that while the mammalian species studied have common ancestry as ungulates, their seminal plasma is divergent in protein composition, which may explain variation in reproductive capacity and function. The identification of major specific proteins within seminal plasma facilitates future investigation of the role of each protein in mammalian reproduction. This proteomic study is the first study to compare the protein composition of seminal plasma from seven mammalian species including two camelid species. Beta nerve growth factor, previously described as the ovulation inducing factor in camelids is shown to be the major protein in alpaca and camel seminal plasma and also present in small amounts in bull, ram, and horse seminal plasma.

Cryopreservation increases coating of bull sperm by seminal plasma binder of sperm proteins BSP1, BSP3, and BSP5

Artificial insemination with frozen semen allows affordable, worldwide dissemination of gametes with superior genetics. Nevertheless, sperm are damaged by the cryopreservation process. Elucidating the molecular effects of cryopreservation on sperm could suggest methods for improving fertility of frozen/thawed semen. This study was undertaken to examine the effect of cryopreservation on the coating of sperm by binder of sperm (BSP) proteins in seminal plasma. BSP proteins are secreted by the seminal vesicles and coat the surface of sperm by partially intercalating into the outer leaflet of the sperm plasma membrane. The BSP proteins are known to play roles in the formation of the oviductal sperm storage reservoir and in sperm capacitation. We investigated the effects of cryopreservation on the sperm BSP protein coat using Bovipure to separate live sperm from extended semen and then assaying the amounts of BSP proteins on sperm using quantitative western blotting with custom-made antibodies against unique sequences of each BSP protein. Greater amounts of all three BSP proteins (BSP1, BSP3, and BSP5) were detected on frozen/thawed sperm than on fresh sperm. Furthermore, the reduction of BSP3 from 15 to 13 kDa in mass, which occurs during incubation of sperm under mild capacitating conditions, was enhanced by cryopreservation. We concluded that freezing alters the BSP protein coating on sperm, which could account in part for reduced fertility of cryopreserved semen samples.

Membrane-associated proteins of ejaculated sperm from Morada Nova rams

The objective was to describe the profile of membrane proteins from sperm of tropically adapted Morada Nova rams (N = 5). Samples from protein-enriched fractions of ejaculated sperm (containing 400 μg of protein) were separated by two-dimensional electrophoresis and respective maps analyzed using PDQuest software (version 7.3.0; Bio-Rad). Proteins were identified using tandem mass spectrometry. Also, membrane proteins were incubated with antibodies against binder of sperm protein (BSP) 1 and bodhesin 2 (Bdh-2), components of vesicular gland secretion. For membrane proteins of ejaculated sperm, an average of 133 ± 4.6 spots were detected per gel, of which, 107 spots were consistently present on all gels. Sixty-eight spots and 37 proteins were identified using mass spectrometry, corresponding to 71.6% of the intensity of all spots detected. Three major spots identified as ram seminal vesicle protein (RSVP) 14 represented approximately 30% of the intensity of all spots. Two of the most intense spots in the gel reacted against anti-BSP1, at 14 kDa. In addition, four low molecular weight spots reacted with anti-Bdh-2 antibodies. Proteins RSVP and Bdh-2 belong to the BSP and spermadhesin families, respectively, and were previously reported as major components of ram seminal proteins. Additional proteins identified in the sperm membrane two-dimensional maps included alpha-2-heparan sulfate-glycoprotein, plasma glutamate carboxypeptidase, arylsulfatase A, cathelicidin, heat shock protein 70 kDa, angiotensin-converting enzyme, leucine aminopeptidase, and clusterin. Some proteins were present as multiple isoforms, such as tubulin (12), alpha-2-heparan sulfate-glycoprotein (5), ATP synthase (5), Bdh-2 (4) and RSVP14 (3). Based on gene ontology analysis, the most common biological processes associated with the membrane proteins were cellular processes (34%), response to stimulus (14%), and metabolic processes (11%). Binding (37%) and catalytic activity (32%) corresponded to the most frequent molecular functions for those proteins. In conclusion, we identified a diverse cohort of components of membrane proteins in ram sperm. Major proteins previously reported in seminal plasma, such as RSVP14 and Bdh-2, were also extracted from sperm membranes. Knowledge of sperm proteins is crucial for elucidating mechanisms underlying their association with sperm function.

Major heparin-binding proteins of the seminal plasma from Morada Nova rams

The present study was conducted to identify the major heparin-binding proteins (HBPs) in the seminal plasma from tropically adapted Morada Nova rams, raised in the Northeast of Brazil. Seminal plasma samples from six adult, reproductively sound rams were collected and subjected to heparin affinity chromatography. HBPs were separated by one-dimensional SDS-PAGE and the main 13 bands were excised from the gel, digested with trypsin and identified by tandem mass spectrometry. Based on the integration of the area under the chromatographic peaks, the vast majority of proteins in the ram seminal plasma (83.5%) matched to those without affinity for heparin. However, only 16.5% of these proteins effectively bind to heparin. HBPs identified in this study include clusterin, Bodhesin 2, matrix metalloproteinase 2, beta mannosidase and dentin matrix acidic phosphoprotein 1, among others. Given the putative biochemical characteristics of HBPs, we suggest that heparin-binding proteins of the ram seminal plasma modulate several aspects of the reproductive events, such as sperm capacitation, formation of the oviduct reservoir, sperm–egg binding, and protection against microorganisms and oxidative stress. In contrast to what has been reported for other species, binder of sperm proteins (BSP) expressed in ram seminal plasma were not detected among HBPs. These findings were confirmed by both mass spectrometry and Western blot. These findings are novel and raise questions about BSPs ability to capacitate ram sperm, as they function in other mammals, such as the bovine. The approach used in the present study also allowed the identification of proteins never reported before in the ram seminal plasma, such as dentin matrix acidic phosphoprotein, cathelicidin and lactoperoxidase. Considering the importance of heparin as a binding partner in reproductive events, our findings give new insights on the roles of seminal plasma proteins in the ram.

Alterations to the Bull Sperm Surface Proteins That Bind Sperm to Oviductal Epithelium1

Three Binder of SPerm proteins (BSP1, BSP3, BSP5) are secreted by bovine seminal vesicles into seminal plasma and adsorbed onto sperm. When sperm inseminated into the female reach the oviduct, the BSP proteins bind them to its epithelial lining, forming a sperm storage reservoir. Previously, we reported that binding of capacitated sperm to oviductal epithelium in vitro is lower than that of uncapacitated sperm and we proposed that reduced binding was due to loss of BSP proteins during capacitation. Because of differences in amino acid sequences, we predicted that each BSP would respond differently to capacitating conditions. To test whether all three BSP proteins were lost from sperm during capacitation and whether the kinetics of loss differed among the three BSP proteins, ejaculated bull sperm were incubated under various capacitating conditions, and then the amounts of BSP proteins remaining on the sperm were assayed by Western blotting. Capacitation was assayed by analysis of protein tyrosine phosphorylation. While loss of BSP1 was not detected, most of the BSP5 was lost from sperm during incubation in TALP medium, even without addition of the capacitation enhancers heparin and dbcAMP-IBMX. Surprisingly, a smaller molecular mass was detected by anti-BSP3 antibodies in extracts of incubated sperm. Its identity was confirmed as BSP3 by mass spectrometry, indicating that BSP3 undergoes modification on the sperm surface. These changes in the composition of BSP proteins on sperm could play a role in releasing sperm from the storage reservoir by modifying sperm interactions with the oviductal epithelium.

Chemical and physical requirements for lipid extraction by bovine binder of sperm BSP1

The bovine seminal plasma contains phosphocholine-binding proteins, which associate to sperm membranes upon ejaculation. These binder-of-sperm (BSP) proteins then induce a phospholipid and cholesterol efflux from these membranes. In this work, we determined physical and chemical parameters controlling this efflux by characterizing the lipid extraction induced by BSP1, the most abundant of BSP protein in bull seminal plasma, from model membranes with different composition. The model membranes were formed from binary mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) with 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (Lyso-PC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) or cholesterol. The modulation of BSP1-induced lipid extraction from membranes by their chemical composition and their physical properties brings us to propose a 3-step extraction mechanism. First, the protein associates with membranes via specific binding to phosphocholine groups. Second, BSP1 penetrates in the membrane, essentially in the external lipid leaflet. Third, BSP1 molecules solubilize a lipid patch coming essentially from the outer lipid leaflet, without any lipid specificity, to ultimately form small lipid/protein auto-assemblies. The stoichiometry of these complexes corresponds to 10–15 lipids per protein. It is also shown that fluid-phase membranes are more prone to BSP1-induced lipid extraction than gel-phase ones. The inhibition of the lipid extraction in this case appears to be related to the inhibition of the protein penetration in the membrane (step 2) and not to the protein association with PC head groups (step 1). These findings contribute to our understanding of the mechanism by which BSP1 modify the lipid composition of sperm membranes, a key event in sperm capacitation.

Cryopreservation Increases Coating of Bull Sperm by Binder of SPerm Proteins BSP3 and BSP5.

Cryopreserved semen is used widely in the dairy industry to inseminate cows. This practice has allowed the affordable dissemination of semen from superior bulls throughout the world, but the fertility of cows has decreased since the 1950s, more so when artificial insemination with cryopreserved semen has been used. In order to address this problem, it is necessary to learn more about how sperm processing affects sperm quality and function, particularly at a molecular level. This study focused on the Binder of SPerm (BSP) proteins, which are secreted by the seminal vesicles and coat bull sperm. There is evidence that BSPs serve to bind sperm to the oviductal epithelium in order to hold the sperm in a storage reservoir in the oviduct, and that BSPs play a role in the capacitation of sperm. The sperm plasma membrane is modified by extension and cryopreservation of semen. Some of these modifications resemble those caused by capacitation. During incubation under capacitating conditions, we found that BSP5 is rapidly shed from sperm and BSP3 on sperm is quickly reduced in apparent molecular mass from 15 to 13 kDa. To determine if cryopreservation modifies the sperm BSP protein coat, we compared the amounts of 15 and 13 kDa BSP3 and BSP5 on frozen and fresh sperm. We also tested whether dilution of fresh semen in a commercially used milk extender modifies the BSP coat, by comparing the BSPs on fresh sperm diluted in milk extender with those diluted in TALP medium. Fresh semen (n = 6 bulls for BSP3, n = 7 bulls for BSP5) was diluted 1:5 in milk extender or TALP and incubated at 38.5 °C for 1 h. Then, seminal plasma, extender, and dead sperm were removed from live sperm by density gradient centrifugation through BoviPure (Nidacon). Frozen/thawed sperm from 3 straws from each of 4 bulls were isolated in the same manner and then pooled for use in all experiments. Proteins were extracted from 5 million sperm per sample, resolved by 18% SDS-PAGE and electroblotted to nitrocellulose. Seminal plasma was used as a positive control. Blots were labeled with custom-made antibodies against peptides in the N terminal domains of BSP3 and BSP5 (GenScript) and a commercially available β-tubulin antibody (abcam) to calibrate the loading of each lane. HRP-conjugated secondary antibodies and chemiluminescence were used to visualize the protein bands, which were recorded using the Bio-Rad ChemiDoc XRS+ imaging system. The intensity of each BSP band was normalized to the intensity of the β-tubulin band within the same lane. The amounts of 15 and 13 kDa BSP3 and of BSP5 detected in frozen sperm samples were greater (P < 0.03) than in fresh sperm diluted in either milk extender or TALP, but no significant differences were detected between the two dilution media. We concluded that the cryopreservation process enhances the binding of BSP5 and both forms of BSP3 to sperm. It is plausible that abnormally high binding of both BSP3 forms and BSP5 to cryopreserved sperm hinder the release of sperm from the oviductal epithelium, which is required for sperm to move out of the storage reservoir, and/or that excess binding of BSPs modifies the timing of capacitation, thereby reducing the chance that fertilization will occur at the optimal time after ovulation. This may explain the finding that the amount of BSP5 in semen samples is negatively correlated with bull fertility. Semen provided by Genex Cooperative, Inc; research supported by USDA NIFA NRICGP 2008-35203-19031.

Cleavage of Binder of Sperm Protein 3 (BSP3), Which Binds Bull Sperm to Oviductal Epithelium, by Sperm Surface Serine Protease.

Cleavage of Binder of Sperm Protein 3 (BSP3), Which Binds Bull Sperm to Oviductal Epithelium, by Sperm Surface Serine Protease.

Characterization of Recombinant Murine Binder of Sperm Protein Homolog 1 and Its Role in Capacitation1

Sperm capacitation is a maturation step that is deemed to be essential for sperm to fertilize an oocyte. A family of proteins, the binder of sperm (BSP), are known to bind choline phospholipids on sperm membranes and promote capacitation in bulls and boars. Recently, BSP-homologous genes have been identified in the epididymal tissues of human (BSPH1) and mouse (Bsph1, Bsph2). The aim of this study was to determine the binding characteristics of the murine binder of sperm protein homolog 1 (BSPH1) and evaluate its effects on sperm capacitation. Since it is not possible to purify the native BSP proteins from human and mouse in sufficient quantity, a murine recombinant BSPH1 (rec-BSPH1) was produced and used for the functional studies. Similarly to BSP proteins from other species, rec-BSPH1 bound to gelatin, heparin, phosphatidylcholine liposomes, and sperm. Both native BSPH1 and rec-BSPH1 were detected on the head and the midpiece region of sperm, although a stronger signal was detected on the midpiece region when sperm were incubated in a capacitating media containing bovine serum albumin. More importantly, murine rec-BSPH1 was able to capacitate sperm, but was unable to induce the acrosome reaction. These results show that murine epididymal BSPH1 shares many biochemical and functional characteristics with BSP proteins secreted by seminal vesicles of ungulates, and suggest that it might play a similar role in sperm functions.

Binder of Sperm Protein 3 (BSP3), Which Enhances Bull Sperm Binding to Oviductal Epithelium, Is Modified on the Sperm Surface During Capacitation.

Binder of Sperm Protein 3 (BSP3), Which Enhances Bull Sperm Binding to Oviductal Epithelium, Is Modified on the Sperm Surface During Capacitation.

Major proteins from the seminal plasma of adult Santa Ines rams

Santa Ines is a native Brazilian breed of hair sheep, representing a genotype developed in and adapted to tropical regions. Seminal plasma of the rams was obtained, subjected to two-dimensional electrophoresis and the major spots identified by tandem mass spectrometry. There were 143 spots consistently present on seminal plasma maps. The most abundant spots represented two families, binder of sperm proteins (BSP) and spermadhesins. The proteins included ram seminal vesicles proteins (RSVP) 14 kDa and 22 kDa, and bodhesin 1 and 2. This is the first report of identification of seminal plasma proteins of tropically-adapted sheep. These proteins are involved in several aspects of reproductive function, including sperm capacitation, sperm survival in the oviduct and gamete interaction in mammals.

Thermodynamics of the interaction between bovine binder of sperm BSP1 and low-density lipoprotein from hen's egg yolk

Egg yolk is used in extender to protect sperm from cold shock and freezing during preservation. It is the low-density lipoprotein (LDL) fraction of egg yolk that protects sperm. Even though essential for sperm capacitation, the major proteins from bull seminal plasma, the Binder of SPerm (BSP) proteins, are detrimental for sperm preservation because they induce a continual phospholipids and cholesterol efflux from sperm membranes. The BSP proteins were proposed to bind to egg yolk LDL, preventing the sperm membrane damage. We characterized the binding between the BSP proteins and the LDL by isothermal titration calorimetry, providing the thermodynamics and quantitative description of this putative association. The association between BSP1 (major BSP proteins) and LDL is characterized by an affinity constant ( K a) of 3.4 ± 0.4 μM −1. A protein/LDL ratio of 104 ± 5 was determined indicating that 104 molecules of BSP1 would bind to one LDL particle. This stoichiometry leads to proposing that the association involves 1.6 ± 0.1 phosphatidylcholines (PC) per BSP protein. This finding is satisfactorily consistent with the fact that each BSP1 protein has 2 binding sites for choline group. In conclusion, the formation of a high affinity complex between BSP1 and LDL is proposed to be important for the protection of sperm by egg yolk extender.

Bovine binder-of-sperm protein BSP1 promotes protrusion and nanotube formation from liposomes

Binder-of-sperm (BSP) proteins interact with sperm membranes and are proposed to extract selectively phosphatidylcholine and cholesterol from these. This change in lipid composition is a key step in sperm capacitation. The present work demonstrates that the interactions between the protein BSP1 and model membranes composed with phosphatidylcholine lead to drastic changes in the morphology of the lipidic self-assemblies. Using cryo-electron microscopy and fluorescence microscopy, we show that, in the presence of the protein, the lipid vesicles elongate, and form bead necklace-like structures that evolve toward small vesicles or thread-like structures. In the presence of multilamellar vesicles, where a large reservoir of lipid is available, the presence of BSP proteins lead to the formation of long nanotubes. Long spiral-like threads, associated with lipid/protein complexes, are also observed. The local curvature of lipid membranes induced by the BSP proteins may be involved in lipid domain formation and the extraction of some lipids during the sperm maturation process.

The N-terminal part of Binder of SPerm 5 (BSP5), which promotes sperm capacitation in bovine species is intrinsically disordered

Bovine BSP5 belongs to the Binder of SPerm (BSP) family. BSP5 plays a role in the bovine sperm capacitation by promoting cholesterol and phospholipid efflux. The variable N-terminal part in the BSP proteins is the uncharacterized region with no known function. Full-length, N-terminal part, and individual fibronectin type II domains of bovine BSP5 were cloned, expressed and purified from Escherichia coli. His-S tagged N-terminal part showed large variation in migration on SDS–PAGE in comparison to other constructs. Using mass spectrometry it was demonstrated that the His-S–N-terminal part has the expected molecular mass (13 kDa). The recombinant N-terminal part was sensitive to E. coli endogenous proteases during purification. Denaturing purification involving boiling lysis of cells was carried out, as the protein was thermostable. The His-S–N-terminal part lacked structure as determined by CD analysis. Bioinformatics analyses confirmed that the N-terminal part of bovine BSP5 is intrinsically disordered. In addition, bioinformatics analysis indicated that rabbit BSP and multiple forms of BSP proteins of bovine and equine species possess partially or completely disordered N-terminus. The conservation of disorder at the N-terminus in BSP members belonging to different species suggests a role in biological process such as sperm capacitation and/or sperm–egg interactions.

Recombinant expression and affinity purification of a novel epididymal human sperm-binding protein, BSPH1

Mammalian sperm undergo a series of maturation steps before acquiring fertilization competence. Our previous work demonstrated the importance of binder of sperm (BSP) proteins in bovine sperm capacitation. Recent studies identified a BSP-homologous DNA sequence in the human genome (BSPH1) and mRNA expression in the epididymis. The aim of this study was to develop an efficient method to express and purify recombinant human BSPH1. BSPH1 accumulates in inclusion bodies when expressed with an N-terminal hexahistidine tag in BL21 (DE3) Escherichia coli cells. Similar to other BSP proteins, BSPH1 contains two fibronectin type-II (Fn2) domains, each consisting of two disulfide bonds. Therefore, when expressed in Origami B (DE3)pLysS cells, a strain favouring disulfide bond formation, an improvement in soluble protein yield was observed. However, protein was aggregated, which complicated subsequent purification steps. Expression of glutathione-S-transferase-tagged BSPH1 in both cell types also led to accumulation in inclusion bodies. Finally, successful production of soluble and active protein was achieved when BSPH1 was expressed as a His(6)-thioredoxin-tagged protein. Recombinant protein bound phosphatidylcholine liposomes, low-density lipoproteins and human sperm, therefore displayed binding activities common to all BSP-family proteins, which may indicate similar biological function(s). This approach was also successful in producing the murine orthologue of BSPH1 in the soluble and active form. Thus, fusion to thioredoxin and expression in Origami B (DE3)pLysS cells may constitute a strategy applicable to all BSP-family proteins, and possibly to other proteins containing Fn2 domains. This work is important to elucidate the role of BSPH1 in human sperm functions and fertility.