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Proteomic architecture of sperm-egg interactions
Otčenášková, Tereza ; Stopka, Pavel (advisor) ; Petr, Jaroslav (referee) ; Vrbacký, Marek (referee)
Recent advances in proteomic methods provide new insights for biological research including the field of reproductive biology. Determination of the proteomic basis of spermatozoa is pivotal for understanding the complex process of gamete interactions during fertilization such as acrosome reaction. Great differences imposed by postcopulatory sexual selection and phylogeny can be observed regarding the size, shape, and molecular composition of sperm across animal taxa. The first objective of this doctoral thesis is to characterize the protein contents of the acrosome to ascertain its further functional significance in sperm-egg interaction. Also, we aim to investigate the potential relationships between sperm protein composition and sperm morphology diversification, risk of sperm competition, and species phylogenetic background. Wild-caught males from natural populations of species of Mus musculus musculus, Apodemus flavicollis, Microtus arvalis (order Rodentia), Acrocephalus palustris, Chloris chloris, Phylloscopus collybita, Cinclus cinclus, Hirundo rustica, and Taeniopygia guttata from a captive population (order Passeriformes) were subject to the analyses. Nano-liquid chromatography with tandem mass spectrometry was applied as the main methodological approach in this thesis. Our data implicate...
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Sperm acrosomal reactien in selected species of mammals
Frolíková, Michaela ; Stopka, Pavel (advisor) ; Jonáková, Věra (referee) ; Petr, Jaroslav (referee)
Mammalian sperm must undergo the process of capacitation - series of physiological and biochemical modifications prior fertilization. In last stage of capacitation sperm undergoes acrosome reaction (AR). During AR the cell membrane of the sperm fuses with the outer acrosomal membrane and the contents of acrosomal vesicle are released into extracellular space. Sperm which did not undergo AR or sperm missing acrosome at all are unable to fertilize. AR results into dramatical changes in the sperm head. Most of the proteins present in plasmatic and outer acrosomal membrane are reorganized or lost. There are also significant changes in cytoskeletal and intraacrosomal proteins are released to extracellular space uncovering new surface domains. Some sperms undergo AR even without presence of inductor of AR during capacitation in vitro. This event is called spontaneous (accelerated) AR. The latest research indicates that spontaneous AR is natural part of the process of fertilization. Field mice (Apodemus) show high level of promiscuity leading to significant risk of sperm competition. Unique reproduction strategy where the sperms form so-called sperm trains was evolved in field mice. Spontaneous AR is probably enabling the dissociation of sperms from the sperm train. The spontaneous AR rate is dependent on...
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Roles of acrosomal proteins in fertilization
Macíčková, Eliška ; Stopka, Pavel (advisor) ; Čermák, Lukáš (referee)
The acrosome is a crucial organelle for sperm cells. Defects in the biogenesis of the acrosome often lead to the impaired development of sperm cells and consequently, to the inability to fertilize the oocyte. Although it is evident that the acrosome is an essential structure, its main function has not been solved yet. The initial hypothesis was that the acrosome helps sperm to pass through the extracellular layers of the egg. This statement is based on evidence that acrosome is full of proteolytic enzymes, and inhibition of these enzymes reduces the ability to fertilize the oocyte. However, this hypothesis began to be questioned when it was found that (i) acrosome-reacted sperm cells are still able to pass through extracellular layers and fertilize the oocyte (ii) most of the sperm cells undergo premature acrosomal reaction before they reach the oocyte. Therefore, we aimed to characterize proteomes of the acrosome and acrosome- reacted sperm cells, identify important proteins using advanced bioinformatics approaches and discuss the function of the acrosome as a vesicle that serves to store and process metabolites after the previous maturation process. In this work, we revealed new findings that contribute to the hypothesis that acrosome is a lysosome-related organelle. Key words: sperm, acrosome,...
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Rab-2A protein: its localization and function in human spermatozoa and fertilization
Sadílková, Lucie ; Postlerová, Pavla (advisor) ; Lánská, Eva (referee)
The Rab-2A protein belongs to the Rab family of monomeric G-proteins involved in the regulation of intracellular vesicular transport. Although several studies describing the role of Rab-2A protein in mammalian sperm have been published, the exact localization and function of this protein in male gametes have not been fully understood yet. In the diploma thesis, we were the first to describe the presence of Rab-2A in human sperm using 5C5, produced by the Laboratory of Reproductive Biology. Using the 5C5 antibody, we were able to identify several Rab-2A isoforms with molecular weights of 26, 24, 22, and 18 kDa in the human sperm lysate, confirmed by mass spectrometry analysis. Using the indirect immunofluorescence method, we localized Rab-2A in the acrosome area of both ejaculated and capacitated sperm. We noticed that Rab-2A leaves the sperm during the acrosomal exocytosis. We also found a reduced Rab-2A protein level in sperm of patients with various pathologies of ejaculate. Based on studies describing the role of Rab-2A in acrosome biogenesis and the presence of Rab-2A in bovine sperm perinuclear theca, we tried to detect Rab-2A in this protein layer in human sperm. However, the chosen method is apparently not applicable to human sperm and isolation has failed. Additionally, we demonstrated the...
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Inhibins in reproduction
Babčová, Katarína ; Ulčová-Gallová, Zdeňka (advisor) ; Kužel, David (referee) ; Crha, Igor (referee)
Inhibin A and B participate to regulation of gametogenesis. We investigated their applicability as a marker of gametogenesis of men fertility disorders. We monitored the levels of inhibins during the treatment. We interested in their paracrine activity, relationship in sera, follicular fluid and seminal plasma depending on cause of fertility failure. We studied the levels of inhibin B in serum and seminal plasma from 355 men treated for fertility failure, in the context of their andrological and immunological findings (quality of spermiogrammes and acrosome area). We monitored concentration of levels of inhibin A and B in serum and follicular fluids depending on cause of fertility failure, on course and treatment outcome. We took blood samples in the time of the oocytes collection, of the embryotransfer and early pregnancy. The follicular fluids were obtained during the oocytes collection. The levels of both of inhibins were measured by ELISA in all medium (serum, follicular fluid, seminal plasma). We confirm, that inhibin B is useful marker of spermatogenesis in men, but is necessary to examine patient in complex with determination of immunology profile or quality of acrosome. Seminal plasma is, in some indicated cases, more suitable diagnostics material. Similarly inhibin B in women seems to be...
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The acrosome reaction in mammalian sperm
Picková, Jana ; Frolíková, Michaela (advisor) ; Kuntová, Barbora (referee)
The acrosome reaction (AR) of the sperm is a prerequisite for egg fertilization, which takes place in the female reproductive tract. The AR allows sperm to penetrate extracellular egg coats and fuse with the egg. At first, the sperm must undergo the process called capacitation, then AR is initiated and acrosomal content is released. While it is not clear, what initiates the AR, it is probably the egg's extracellular coats - the zona pellucida and cumulus cells, secreting progesterone and some other substances, which can initiate the AR. Lately, it was demonstrated, that in the mouse the most sperms undergo the AR in the upper isthmus of the oviduct. Only a few sperms reach the ampulla, but all of them can fertilize eggs. During the AR, the acrosomal content is released into the extracellular space. It was discovered, that the release of acrosomal proteins is not synchronous, soluble components are released faster from the acrosome than are acrosomal matrix proteins. Before the acrosomal release, G-coupled receptors and tyrosine kinase receptors activate phospholipases. Protein kinases are also activated, which results in the opening of Ca2+ channels in the acrosome and sperm plasma membrane and the release of Ca2+ . The increase of Ca2+ leads to actin depolymerization, membrane fusion, and finally,...
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Sperm acrosomal reactien in selected species of mammals
Frolíková, Michaela
Mammalian sperm must undergo the process of capacitation - series of physiological and biochemical modifications prior fertilization. In last stage of capacitation sperm undergoes acrosome reaction (AR). During AR the cell membrane of the sperm fuses with the outer acrosomal membrane and the contents of acrosomal vesicle are released into extracellular space. Sperm which did not undergo AR or sperm missing acrosome at all are unable to fertilize. AR results into dramatical changes in the sperm head. Most of the proteins present in plasmatic and outer acrosomal membrane are reorganized or lost. There are also significant changes in cytoskeletal and intraacrosomal proteins are released to extracellular space uncovering new surface domains. Some sperms undergo AR even without presence of inductor of AR during capacitation in vitro. This event is called spontaneous (accelerated) AR. The latest research indicates that spontaneous AR is natural part of the process of fertilization. Field mice (Apodemus) show high level of promiscuity leading to significant risk of sperm competition. Unique reproduction strategy where the sperms form so-called sperm trains was evolved in field mice. Spontaneous AR is probably enabling the dissociation of sperms from the sperm train. The spontaneous AR rate is dependent on...
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Sperm acrosomal reactien in selected species of mammals
Frolíková, Michaela
Mammalian sperm must undergo the process of capacitation - series of physiological and biochemical modifications prior fertilization. In last stage of capacitation sperm undergoes acrosome reaction (AR). During AR the cell membrane of the sperm fuses with the outer acrosomal membrane and the contents of acrosomal vesicle are released into extracellular space. Sperm which did not undergo AR or sperm missing acrosome at all are unable to fertilize. AR results into dramatical changes in the sperm head. Most of the proteins present in plasmatic and outer acrosomal membrane are reorganized or lost. There are also significant changes in cytoskeletal and intraacrosomal proteins are released to extracellular space uncovering new surface domains. Some sperms undergo AR even without presence of inductor of AR during capacitation in vitro. This event is called spontaneous (accelerated) AR. The latest research indicates that spontaneous AR is natural part of the process of fertilization. Field mice (Apodemus) show high level of promiscuity leading to significant risk of sperm competition. Unique reproduction strategy where the sperms form so-called sperm trains was evolved in field mice. Spontaneous AR is probably enabling the dissociation of sperms from the sperm train. The spontaneous AR rate is dependent on...
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Inhibins in reproduction
Babčová, Katarína ; Ulčová-Gallová, Zdeňka (advisor) ; Kužel, David (referee) ; Crha, Igor (referee)
Inhibin A and B participate to regulation of gametogenesis. We investigated their applicability as a marker of gametogenesis of men fertility disorders. We monitored the levels of inhibins during the treatment. We interested in their paracrine activity, relationship in sera, follicular fluid and seminal plasma depending on cause of fertility failure. We studied the levels of inhibin B in serum and seminal plasma from 355 men treated for fertility failure, in the context of their andrological and immunological findings (quality of spermiogrammes and acrosome area). We monitored concentration of levels of inhibin A and B in serum and follicular fluids depending on cause of fertility failure, on course and treatment outcome. We took blood samples in the time of the oocytes collection, of the embryotransfer and early pregnancy. The follicular fluids were obtained during the oocytes collection. The levels of both of inhibins were measured by ELISA in all medium (serum, follicular fluid, seminal plasma). We confirm, that inhibin B is useful marker of spermatogenesis in men, but is necessary to examine patient in complex with determination of immunology profile or quality of acrosome. Seminal plasma is, in some indicated cases, more suitable diagnostics material. Similarly inhibin B in women seems to be...
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Sperm acrosomal reactien in selected species of mammals
Frolíková, Michaela ; Stopka, Pavel (advisor) ; Jonáková, Věra (referee) ; Petr, Jaroslav (referee)
Mammalian sperm must undergo the process of capacitation - series of physiological and biochemical modifications prior fertilization. In last stage of capacitation sperm undergoes acrosome reaction (AR). During AR the cell membrane of the sperm fuses with the outer acrosomal membrane and the contents of acrosomal vesicle are released into extracellular space. Sperm which did not undergo AR or sperm missing acrosome at all are unable to fertilize. AR results into dramatical changes in the sperm head. Most of the proteins present in plasmatic and outer acrosomal membrane are reorganized or lost. There are also significant changes in cytoskeletal and intraacrosomal proteins are released to extracellular space uncovering new surface domains. Some sperms undergo AR even without presence of inductor of AR during capacitation in vitro. This event is called spontaneous (accelerated) AR. The latest research indicates that spontaneous AR is natural part of the process of fertilization. Field mice (Apodemus) show high level of promiscuity leading to significant risk of sperm competition. Unique reproduction strategy where the sperms form so-called sperm trains was evolved in field mice. Spontaneous AR is probably enabling the dissociation of sperms from the sperm train. The spontaneous AR rate is dependent on...
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