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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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Isolation and study of proteins with zona pellucida binding activity
Zigo, Michal
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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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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Isolation and study of proteins with zona pellucida binding activity
Zigo, Michal
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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Biochemical methods as tool for study of reproductive proteins
Postlerová, Pavla ; Zigo, Michal ; Pohlová, Alžběta ; Jonáková, Věra
Study of molecular mechanisms in reproduction is essential for the understanding of this outstanding process. Our lab studies proteins secreted by reproductive organs and sperm using various biochemical methods for a long time. We have expertise in protein extraction from spermatic cells using different approaches, and by kits for proteins from the sperm surface and distinct subcellular compartments. The proteins of reproductive organ fluids are separated by chromatographic methods, such as size exclusion chromatography, high-performance liquid chromatography with reverse phase (RP-HPLC) and affinity chromatography on matrices with various ligands. Proteins are subjected to SDS- or 2D-electrophoresis for their characterization and comparison of various extraction methods, different mammalian species, and sperm in different functional development. Electrophoretically separated proteins may be transferred onto nitrocellulose membrane (Western blot) for antibody detection or binding studies with lectin-labelled ligands (lectins, polysaccharides, zona pellucida glycoproteins). We use immunoprecipitation method with specific antibody for protein determination followed by the MALDI identification. Proteins are localized by immunofluorescent techniques on/in spermatic cells and tissue sections of reproductive organs. Isolation of proteins from reproductive tissues and fluids, and the antibody detection is crucial for the studying of reproductive protein origin.
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Expression of selected proteins in sperm in men with normal and pathological spermiograms using monoclonal antibodies
Pěknicová, Jana ; Čapková, Jana ; Dorosh, Andriy ; Margaryan, Hasmik ; Kubátová, Alena ; Děd, Lukáš
Recent studies show that infertility in human populations it affects an estimated 15% of couples of reproductive age. Male infertility is the primary cause for 60% of these cases. For these reasons, we analyzed the acrosomal and sperm surface proteins in men with normal and pathological spermiograms. We found that intra-acrosome proteins: TERA (Transitional endoplasmic reticulum ATPase), GAPDHS (Sperm Glyceraldehyde-3-phosphate dehydrogenase), and PRKAR2A (C-AMP-dependent protein kinase II, PRKAR2A), which can be identified using our monoclonal antibodies, are different express in healthy men and men with astenozoospermia (with reduced sperm motility), and with a significantly reduced expression in the astenozoospermia. These proteins are involved in energy metabolism and apoptosis of the cells, and some of them in the sperm-egg interaction; therefore, they have an important role in reproduction. On the other hand, there were no statistically significant differences in the expression of surface proteins (Appolipoprotein J (Clusterin) and Semenogelin). Our findings show that astenozoospermia as a complex disorder of the semen is often combined with other pathological conditions that are not diagnosed by the semen analysis. Therefore monoclonal antibodies are so suitable instrument for the detection of proteins associated with the pathology of the sperm in the semen with low sperm motility. In general, monoclonal antibodies against the sperm proteins are an appropriate tool to detect sperm quality in reproductive medicine.
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Interaction of sperm surface proteins in the reproduction process
Jonáková, Věra ; Kraus, Marek ; Maňásková, Pavla ; Liberda, J. ; Tichá, M.
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.
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