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Dýchání spermií ryb: druhová specifičnost a vliv teploty prostředí
RAHI, Deepali
The presented studies were designed to investigate the role of mitochondrial respiration, glycolysis, and fatty acid oxidation in the energy supply of spermatozoa in freshwater, externally fertilizing fish species possessing differences in sperm motility duration and spawning temperature. The first study conducted on Siberian sturgeon (Acipenser baerii, Acipenseriformes) revealed that mitochondrial respiration is essential for the energy supply of spermatozoa, while glycolysis and fatty acid oxidation have minor importance at both states - before and after attaining motility. Spermatozoon oxygen consumption rate (OCR) was higher than one detected for coldwater teleosts, such as trout, and lower than in marine species, for example, turbot. The presented study also revealed that sturgeons belong to the group of fishes whose spermatozoa possess the capacity to enhance oxidative metabolism after motility activation and uncoupling of oxidative phosphorylation (OXPHOS). In the second study conducted on spermatozoa of coldwater species burbot (Lota lota, Gadiformes), the functioning of the most important bioenergetic pathways (mitochondrial respiration, glycolysis, and fatty acid oxidation) was investigated at spawning temperature (4 °C) and maximum critical temperature (CTmax, 15 °C). At spawning temperature, similar to the results revealed for Siberian sturgeon spermatozoa, the role of glycolysis and fatty acid oxidation in energy supply was insignificant. Mitochondrial respiration was detectable at both quiescent and motile states, but no inhibition was observed after exposure of sperm to the respiratory inhibitor. Also, the OCR range was the lowest compared to the available data for OCR in teleost spermatozoa at motile or immotile state. Additionally, the OCR was not enhanced after motility activation or after treatment with an uncoupling agent. Contrastingly, at CTmax, the activity of OXPHOS became predominant. The OCR was enhanced, and that could be inhibited by using a respiratory inhibitor. Furthermore, to explore the long-lasting motility of sturgeon spermatozoa, succeeding steps were taken by studying the phosphocreatine-creatine (PCr-Cr) shuttle system apart from the previously studied pathways - OXPHOS, glycolysis, and fatty acid oxidation. The results obtained for sterlet (A. ruthenus, Acipenseriformes) revealed that PCr-Cr shuttle, known for maintaining a homogenous concentration of ATP molecules throughout the flagellar length, plays an active role while spermatozoa are at a quiescent state. No significant contribution of this shuttle in energy supply after sterlet sperm motility activation was detected. In addition to this, presented results also revealed that sturgeon spermatozoa, similar to carp and trout sperm, can be reactivated after the termination of the first motility period, and these secondly activated spermatozoa can successfully fertilize eggs. Moreover, for the first time, it was revealed that OXPHOS and PCr-Cr shuttle systems are the most crucial bioenergetic pathways during the sperm reactivation process. Overall, it was concluded that in the studied fish species, despite taxonomical differences or varying spawning temperature , sperm energy production and utilization strategy remained the same: most of the spermatozoon energy was derived from stored ATP that had been synthesized via OXPHOS during a quiescent but bioenergetically active state. Also, sturgeons are the group of fishes that possess an ability of sperm reactivation, with mitochondrial respiration and PCr-Cr shuttle system being the major contributing pathways for energy supply during reactivation process.
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LINC complex: The link between chromatin integrity and sperm motility
Šanovec, Ondřej ; Komrsková, Kateřina (advisor) ; Lánská, Eva (referee)
The LINC complex (Linker of the Nucleoskeleton and Cytoskeleton) is a protein structure located in the nuclear membrane that connects the cytoskeleton with the nucleoskeleton. This complex can be found in every mammalian cell including the gametes. However, here the LINC complex is more diverse and less studied than in the somatic cells. In this thesis, the LINC complex and its role in spermiogenesis have been studied in wild-type and Protamine 2 knockout (Prm2-/- ) mice. Protamines are small proteins that replace histones during spermiogenesis. The mouse model generated by the group of prof. Hubert Schorle has a deletion in Prm2 in exon 1 and its sperm possess a surprising phenotype including complete loss of motility. Therefore, it was hypothesized that the LINC complex might be responsible for miscommunication between the sperm head and tail which leads to the loss of sperm motility. Results from this study suggest that the LINC complex is not influenced by Prm2 deletion, however, actin dynamics, cytoskeletal motor proteins and tubulin acetylase/ histone deacetylase activity might be impaired. Prm2-/- sperm have a significantly higher abundance of β-actin compared to the wild type. Next, Prm2-/- sperm also show a different pattern of acetylation of α-tubulin but no change in the abundance of...
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Evolution of sperm morphology in birds
Zemanová, Aneta ; Stopka, Pavel (advisor) ; Frolíková, Michaela (referee)
The sperm is one of the most variable cells that carry various morphological differences across species and is subject to selective pressures that may drive changes in sperm morphology. Different morphological adaptations that emerged may enhance successful fertilization. This thesis focuses on the analysis of sperm morphology in different species of songbirds, the representation of proteins in both sperm and seminal fluid, and their classification into different categories of gene ontology. The main aim of my thesis was to determine whether differences in proteomes follow the level of sperm competition or rather their phylogeny. Six species of songbirds were selected for this study: Marsh Warbler (Acrocephalus palustris), White-throated dipper (Cinclus cinclus), European greenfinch (Chloris chloris), Barn swallow (Hirundo rustica), Common chiffchaff (Phylloscopus collybita) and Zebra finch (Taeniopygia guttata). Sperm morphometric measurements were made in all the species, with the greatest variability recorded in the central part of the sperm flagellum. Using mass spectrometry, were analysed sperm and seminal fluid proteins separately and discriminant analysis was performed to determine the proteins that most differentiated the selected songbird species from each other. Obtained data provide...
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Evaluation of antisperm antibodies (ASA) in diagnostics of idiopathic infertility
Pastyříková, Soňa ; Postlerová, Pavla (advisor) ; Novák, Jan (referee)
Infertility is a problem affecting almost 15% of couples. It can be caused by genetic, hormonal, anatomical or infectious problems, and it can also be caused by an immunological factor. One of the immunological causes of infertility is the presence of anti-sperm antibodies (ASA), which bind to sperm and adversely affect their function. The occurrence of ASA is manifested, for example, by reduced sperm motility, agglutination, and impaired sperm capacitation. Alternatively, ASA reduces the sperm ability to pass through the female urogenital tract, negatively affecting the sperm-oocyte interaction and the development of the embryo. The aim of this bachelor thesis was to compare existing methods for ASA determination. The main parameters were the complexity of the method and the assessment of results according to clinical studies focused on the detection and evaluation of ASA. The exact comparison of ASA detection methods based on the performed studies proved to be complicated, mainly due to the development of individual methods over time, different approaches to the evaluation of results, and low standardization of the methods. However, the most commonly recommended methods are MAR, ELISA and IBT tests, which are standardized and, due to commercially available kits, are considered to be easy to...
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Chemical communication of gametes
Otčenášková, Tereza ; Komrsková, Kateřina (advisor) ; Petr, Jaroslav (referee)
Fertilization is a multiple step process leading to fusion of female and male gametes resulting in a formation of a zygote. Besides direct gamete interaction via binding receptors localized on both oocyte and sperm surface, fertilization also involves communication based on chemical molecules triggering various signalling pathways. This work is aimed to characterize chemical communication of gametes of a model organism Mus musculus. For this purpose, modern proteomic and visualisation methods like nano-liquid chromatography tandem mass spectrometry (nLC-MS/MS), selected reaction monitoring (SRM) and immunofluorescent microscopy were used. Lipocalins were identified as candidate proteins involved in communication including those from major urinary proteins (MUPs), LCN lipocalins and fatty acid binding proteins (FABPs). For the first time, we report their presence in the sperm acrosome. Based on lipocalins capacity to bind and transport other molecules, we propose that these proteins have a protective and/or signalling role for gametes. Furthermore, chemical communication between sperm and oocyte is based on chemotaxis which enables their interaction before their fusion. In this work, we detected that spermatozoa show chemotactic responses in the presence of L-glutamate. This amino acid naturally...
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Cytoskeleton-membrane protein interaction network in sperm
Adamová, Zuzana ; Komrsková, Kateřina (advisor) ; Tolde, Ondřej (referee)
In order to fertilize the egg, sperm cell undergoes several subsequent maturation processes. The final one called acrosome reaction is an exocytosis of acrosome vesicle, which is filled with lytic enzymes. Acrosome reaction is crucial for penetration of the sperm cell through the egg surroundings, especially zona pellucida, as well as for reorganization of a membrane protein composition on its surface. This rearrangement leads to the exposure of proteins essential for fertilization, mainly for gamete recognition, binding and fusion in specific compartments of the sperm head. One of such protein is CD46, which is located in the acrosomal membrane of an intact sperm and after acosomal exocytosis it relocates to the equatorial segment of a sperm head, which is known to be the initial site of interaction of sperm with the egg plasma membrane. The relocation of CD46 is disrupted by inhibition of actin, which reorganization within sperm head is known to play a role in onset of acrosome reaction, however, the precise mechanism of CD46 interaction with actin in sperm is unknown. In this thesis, ezrin - a crosslinker of membrane proteins and actin - has been studied in context of CD46 and its relocation across the sperm head. Analysis of the immunofluorescent detection of ezrin revealed its mutual...
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The role of integrins in gametes prior to fertilization and during their interaction
Foldynová, Veronika ; Komrsková, Kateřina (advisor) ; Tlapáková, Tereza (referee)
Integrins are heterodimeric transmembrane glycoproteins that represent a large group of cell adhesion receptors involved in cell-cell, cell-extracellular matrix and cell-pathogen interacti- ons. Up to now, 24 different integrin heterodimers have been detected in mammals. They are involved in a wide range of processes such as immune response, lymphocyte homing, platelet aggregation, also in wound healing, cell differentiation, migration, proliferation and even in cell survival. Integrins have also been detected on germ cells and are now known to play an important role in reproductive processes such as fertilization, embryo implan- tation, and embryonic development. The main aim of this thesis is to introduce integrins from the perspective of reproduction, integrin physiology, occurrence and localization of individual subunits in male and female germ cells. A significant part of the work is devoted to a discussion of the role of integrins in gametes, both during maturation processes (egg maturation in the ovary, capacitation and the acrosomal reaction of sperm), in sperm mi- gration, oviductal reservoir formation and their direct and indirect involvement in adhesion and fusion of the gametes during fertilization. Keywords: integrins, sperm, egg, fusion, fertilization
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Analysis of chromosomal aberrations in sperm by fluorescence in situ hybridization
Bendová, Petra ; Diblík, Jan (advisor) ; Novotná, Drahuše (referee)
The presented bachelor work is focused on the determination of frequency chromosomally abnormal sperm in the semen of healthy men (donors) with normal karyotype (46, XY). The important process, which plays an irreplaceable role in the development of numerical aberrations of chromosomes or structural abnormalities in the segregation of the gametes, is meiosis. Therefore, I devote much attention on meiosis in the theoretical part. The theoretical part is focused on the process of pre mature sperm (spermatogenesis), and the consequences of fertilize the oocyte by aneuploid sperm. In my work I present an overview of numerical abnormalities in autosomes and gonosomes and their frequency and distribution of gametes in healthy men. I also focused on the distribution and a brief description of structural aberrations affecting chromosomes and not least I paid attention on method of multicolor interphase fluorescence in situ hybridization, which in combination with sperm chromatin dekondenzation become irreplaceable and valuable research tool for rapid analysis of chromosomal abnormalities in large sperm samples. The experimental part of bachelor work deals with monitoring the frequency of selected numerical abnormalities in sperm samples of five donors aged 23 to 30 years with the use of I-FISH (fluorescence in situ...
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The effect of diabetes mellitus on reproductive parameters and expression of selected testicular genes in diabetic mice
Valášková, Eliška ; Pěknicová, Jana (advisor) ; Kaňka, Jiří (referee)
According to the World Health Organization (WHO), 15% of couples in reproductive age suffer from infertility problems, and up to 60% of cases are caused by male factor. Causes of this condition could be genetic background, environmental factors and various diseases, including diabetes mellitus (DM). The aim of this study was to investigate the effects of DM on reproductive parameters and expression of selected testicular genes using mouse model (FVB inbred mouse strain). DM (type 1) was artificially induced by chemical substance streptozotocin, which causes destruction of pancreatic β cells. These mice were exposed to diabetic condition for 6 weeks and then subjected to analysis. Our results have shown that diabetic condition had an impact on body weight, weight of reproductive organs as well as kidneys and livers. We also observed decreased concentration and viability of diabetic sperm compared to control. Moreover, we noticed increased staining with apoptotic marker annexin V. Further, we evaluated changes of sperm nuclear proteins - protamines. In diabetic animals, we observed higher number of sperm with insufficient protamination. Nevertheless, protamine 1 to protamine 2 ratio (P1/P2), a marker of male fertility, was not altered in sperm of diabetic animals compared to control. Regarding the...
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