Preparation of Biotinylated and FITC-Labelled Phosphorylcholine Poly(acrylamide) Derivatives and Their Application for Protein Ligand-Binding Studies

Biotin- and FITC-labelled water-soluble poly(acrylamide) derivatives of phosphorylcholine were prepared by coupling either maleinylated (a) or periodate-oxidized (b) L-glyceryl phosphorylcholine to poly-(acrylamide-allylamine) copolymer. Biotinylated phosphorylcholine poly(acrylamide) derivatives of both types were tested with Limulus polyphemus C-reactive protein and were used for the study of the phosphorylcholine-binding properties of boar seminal plasma proteins. Binding sites for phosphorylcholine on the surface of bull sperms were visualized using a FITC-labelled derivative of the ligand

The ubiquitin–proteasome system is involved in the regulation of activity of spermadhesin aqn1 and acrosin inhibitor, the two sperm surface proteins, during porcine fertilization

The spermadhesin AQN1and acrosin inhibitor (AI/SPINK2) proteins bind to the sperm plasma membrane at ejaculation. The AQN1 has been implicated in sperm binding to zona pellucida (ZP) of the oocyte as well as in sperm interactions with the epithelium of the oviductal sperm reservoir. The SPINK2 protects spermatozoa from proteolytic degradation during their trip up the female genital tract toward the oocyte. This study examined the role of two components of the 19S proteasome regulatory complex, the ubiquitin C-terminal hydrolase UCHL3 and PSMD8 in the AQN1-mediated boar sperm binding to zona pellucida. Interaction of PSMD4 subunit with the acrosomal surface-associated acrosin inhibitor AI/SPINK2 provided another line of evidence for the presence of 26S proteasomes on the sperm surface. Detection of the ubiquitinated forms of SPINK2 supports the hypothesis that SPINK2 activity is controlled by ubiquitin-proteasome system (UPS). The activity of the porcine AQN1, and thus the efficiency of sperm-oocyte recognition/binding, may be controlled by elements of the sperm surface-bound UPS, in particular by UCHL3, and by proteasomal regulatory complex subunit PSMD8. Ubiquitinated isoforms of AQN1 were also detected in boar sperm extracts. The UCHL inhibitor ubiquitin aldehyde and the antibodies against UCHL3 or PSMD8 increased the rate of sperm-ZP penetration and polyspermy during porcine in vitro fertilization (IVF). In contrast, the addition of recombinant UCHL3 to fertilization medium significantly reduced polyspermy rates, while maintaining satisfactory rate of monospermic fertilization (~50%). These results are significant for production agriculture. The high level of polyspermy that hinders porcine IVF for commercial embryo transfer could be mitigated by the modulation of the UCHL3 and/or PSMD8 activity

Vazebné vlastnosti proteinů kančí semenné plazmy v reprodukčním procesu

Boar seminal plasma proteins (spermadhesins and DQH protein)bind the sperm surface at ejaculation. binding of sperm to oviductal epithelial cells and to the glycoproteins of zona pellucida are mediated by the protein-saccharide (lectin-like)interactions.These intractions play role in the formation of sperm oviductal reservoir, in the release of capacitated sperm from the surface of oviduct and in sperm-zona pellucida interaction

Role of sperm surface proteins in gamete binding

Mammalian fertilization includes highly regulated biochemical interactions between complementary molecules located on the surface of both gametes. Sperm surface proteins play various roles in the fertilization process

Interaction of sperm surface proteins in the reproduction process

Mammalian fertilization includes highly regulated biochemical interactions between complementary molecules located on the surfaces of both gametes as well as those present in the natural environment of the gametes. Boar seminal plasma proteins AQN, AWN spermadhesins and DQH protein that are bound to the sperm at ejaculation play important role in the reproduction process. Binding of sperm to epithelial cells, recognition of gametes and binding of sperm to glycoprotein of zona pellucida are mediated by protein-saccharide interaction, lektin-type interaction. Another type of interaction (protein-protein) participates in the formation of aggregated forms, which seem to be the natural functional forms of seminal plasma proteins. Protein aggregates form the sperm coating layers and their re-modeling in the female reproductive tract is imporatnt for the sperm capacitation and for the primary binding of spermto the ovum

Exprese a lokalizace inhibitoru akrosinu v kančím reprodukčním traktu

Proteinase inhibitors present in seminal plasma inactivate prematurely released sperm acrosin and protect spermatozoa and reproductive epithelium of the male and female reproductive tracts against proteolytic degradation. Acrosin inhibitor mRNA was detectable in epididymis, seminal vesicles, prostate and Cowperś glands. By indirect immunofluorescence, acrosin inhibitors were detected in the secretory tissues of cauda epididymis, prostate and SV, and on epididymal and ejaculated spermatozoa in the acrosomal region. The presence of inhibitors of the proteolytic enzymes in epididymis is very important for the regulation of the sperm membrane protein processing

Role of sperm surface proteins in reproduction

Seminal plasma proteins bind to the sperm surface at ejaculation and may modulate sperm properties during reproduction. DQH sperm surface protein shows affinity to oviductal epithelium and zona pellucida (ZP) glycoproteins. The mRNA transcript of DQH protein was found in seminal vesicles (SV). DQH was immunodetected by monoclonal antibodies (MAbs) in SV extract and fluid, on SV tissue sections and on the membrane-associated acrosome part of ejaculated spermatozoa. These results confirmed the ability of DQH protein to bind to the sperm surface at ejaculation and to participate in the formation of the sperm reservoir in the porcine oviduct. Moreover, monoclonal antibodies reduced binding of sperm to oocytes and proved the role of DQH protein in the sperm-ZP primary binding

Sperm surface proteins of spermatozoa and proteins of seminal plasma in fertilization

Mammalian fertilization involves highly regulated biochemical events. We aimed at study of interaction of seminal plasma proteins with sperm and at mutual interactions of proteins from seminal plasma

Povrchové proteiny spermie: původ, biochemické vlastnosti a úloha v reprodukci

Seminal plasma proteins bind to the sperm surface at ejaculation and may modulate sperm properties during reproduction. Porcine spermadhesins (AQN, AWN, PSP) are secreted mainly by the seminal vesicles (SV), but their mRNAs have been found also in the cauda epididymis and prostate. Using specific polyclonal antibodies, PSP-I and PSP-II proteins were immunodetected in tissue extracts from cauda epididymis, prostate, SV and Cowper´s glands on the blots, and in secretory tissues of cauda epididymis, prostate and SV by indirect immunofluorescence. We localized PSP spermadhesins on epididymal and ejaculated spermatozoa. PSP proteins are produced not only by SV and prostate, but also by epididymis. Characterization of seminal plasma proteins expressed in the individual reproductive organs might help to understand their role in the reproduction process