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Isolation and study of proteins with zona pellucida binding activity
Zigo, Michal ; Postlerová, Pavla (advisor) ; Ryšlavá, Helena (referee) ; Antalíková, Jana (referee)
Binding of sperm to the oocyte is mediated by complementary molecules on the surface of both gametes and involves the interaction of sperm protein receptors with the zona pellucida (ZP) saccharide structures. It has been shown that many proteins receptors are involved in the sperm-ZP interaction, and potential primary sperm receptors for ZP glycoproteins have been investigated in various mammals. The majority of proteins with identified sperm-ZP binding activity belong to the plasma membrane proteins. However, the exact methods for isolation of sperm membrane proteins are still to be standardized. This study is focused on investigating how employment of various isolation protocols leads to acquisition of various protein mixtures. Further in the work, two possible approaches towards identification of potential ZP-binding partners are implemented, in order to disclose novel primary ZP-binding receptor candidates. Sperm proteins of ejaculated and in vitro capacitated boar sperms were isolated by: Triton X-100, Triton X-114, acetic acid, sodium dodecyl sulphate (SDS), N-octyl-β-D- glucopyranoside (OBG), rehydration buffer (RHB), and finally by freezing-thawing extraction and they were characterized by 1-D, 2-D protein profiles, glycoprotein staining and substrate zymographic methods. The results have...
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Secreted proteins by male reproductive tract
Cozlová, Nina ; Postlerová, Pavla (advisor) ; Železná, Blanka (referee) ; Antalíková, Jana (referee)
1 AbstractAbstractAbstractAbstract Proteins secreted in the male reproductive tract play a key role in post-testicular development of sperm and in further steps needed for fertilization. Sperm maturation represents a key step in the reproduction process. Sperm, during the passage through the epididymis undergoes significant changes due to proteolytic and glycolytic activities in the epididymal fluid. Inhibitor of acrosin protects spermatozoa and reproductive epithelium against proteolytic degradation and also protects binding sites for ZP on sperm plasma membrane. In boar reproductive system acrosin inhibitor (AI) was found in seminal plasma and on sperm plasma membrane. Polyclonal antibody recognized AI in extracts of the cauda epididymidis, seminal vesicles, prostate, and Cowper's glands. Using immunofluorescence method has revealed the AI in the epithelium and lumen of these organs but also on the surface of epididymal and ejaculated spermatozoa. We registered the increasing signal of AI from caput to cauda epididymis. Gene expression of AI mRNA was detected in the epididymis, seminal vesicles, prostate, and Cowper's glands and increased gradually throughout the epididymal duct. In present study, we also monitored AI in boar epididymal fluid and spermatozoa along the organ. In the epididymis, AI may...
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Isolation and study of proteins with zona pellucida binding activity
Zigo, Michal ; Postlerová, Pavla (advisor) ; Ryšlavá, Helena (referee) ; Antalíková, Jana (referee)
Binding of sperm to the oocyte is mediated by complementary molecules on the surface of both gametes and involves the interaction of sperm protein receptors with the zona pellucida (ZP) saccharide structures. It has been shown that many proteins receptors are involved in the sperm-ZP interaction, and potential primary sperm receptors for ZP glycoproteins have been investigated in various mammals. The majority of proteins with identified sperm-ZP binding activity belong to the plasma membrane proteins. However, the exact methods for isolation of sperm membrane proteins are still to be standardized. This study is focused on investigating how employment of various isolation protocols leads to acquisition of various protein mixtures. Further in the work, two possible approaches towards identification of potential ZP-binding partners are implemented, in order to disclose novel primary ZP-binding receptor candidates. Sperm proteins of ejaculated and in vitro capacitated boar sperms were isolated by: Triton X-100, Triton X-114, acetic acid, sodium dodecyl sulphate (SDS), N-octyl-β-D- glucopyranoside (OBG), rehydration buffer (RHB), and finally by freezing-thawing extraction and they were characterized by 1-D, 2-D protein profiles, glycoprotein staining and substrate zymographic methods. The results have...
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Secreted proteins by male reproductive tract
Cozlová, Nina ; Postlerová, Pavla (advisor) ; Železná, Blanka (referee) ; Antalíková, Jana (referee)
1 AbstractAbstractAbstractAbstract Proteins secreted in the male reproductive tract play a key role in post-testicular development of sperm and in further steps needed for fertilization. Sperm maturation represents a key step in the reproduction process. Sperm, during the passage through the epididymis undergoes significant changes due to proteolytic and glycolytic activities in the epididymal fluid. Inhibitor of acrosin protects spermatozoa and reproductive epithelium against proteolytic degradation and also protects binding sites for ZP on sperm plasma membrane. In boar reproductive system acrosin inhibitor (AI) was found in seminal plasma and on sperm plasma membrane. Polyclonal antibody recognized AI in extracts of the cauda epididymidis, seminal vesicles, prostate, and Cowper's glands. Using immunofluorescence method has revealed the AI in the epithelium and lumen of these organs but also on the surface of epididymal and ejaculated spermatozoa. We registered the increasing signal of AI from caput to cauda epididymis. Gene expression of AI mRNA was detected in the epididymis, seminal vesicles, prostate, and Cowper's glands and increased gradually throughout the epididymal duct. In present study, we also monitored AI in boar epididymal fluid and spermatozoa along the organ. In the epididymis, AI may...
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