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Effect of cryopreservation on mouse sperm.
Veselá, Kateřina ; Hortová, Kateřina (advisor) ; Pěknicová, Jana (referee)
Cryopreservation or freezing of sperm in the reproductive biology is still actual topic. Today is the only method used for sperm storage, whether for the purposes of assisted reproduction, or for scientific purposes. However, this method has a negative impact on such stored cells and is therefore still a subject of many studies. Among the main causes of sperm damage in rodents there are inappropriately selected cryoprotective agents, poor or no elimination of oxidative stress generated during cryopreservation, as well as a poorly chosen speed and temperature of freezing. Correctly chosen procedures and the composition of the media in which sperm are stored, can significantly affect the quality of sperm. This bachelor theses focuses on the influence of cryopreservation on mouse spermatozoa. The main impact of this method includes the effect of freezing on sperm DNA, plasma membrane, acrosome, and sperm motility.
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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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Use of interspecific ICSI method for assessing the quality of the retained biological material from genetic resources
Vacková, Kristýna ; Benc, Michal (advisor) ; Barnetová, Irena (referee)
Intracytoplasmic sperm injection, or ICSI, is a commonly used method in assisted reproduction centers and is also an effective tool for a fertilization research. The interspecific ICSI is the suitable method for production of zygotes, which are formed by association of gametes of different, distantly unrelated species. It can bypass natural interspecies barriers and create so-called interspecific zygotes. This can be used to evaluation properties of genetic material. In this work, this method was used for evaluation of the quality of stored sperm originating from male goats of the white shorthair goat genetic resource. The aim of this work was to evaluate the effect of cryopreservation on the quality of goat sperm, which was frozen in three different ways at two different breeding stations. Freshly collected semen served as a control group. The heads of xenogeneic goat sperm were injected into mature mouse oocytes. The injected oocytes were allowed to develop to the zygote stage, in which both female (mouse) and male (goat) pronuclei was formed. In male pronuclei, DNA damage was assessed by immunofluorescence staining with anti-γH2AX antibody, a marker of DNA breaks. The results obtained by measuring the level of γH2AX fluorescence intensity in the paternal pronuclei show that the values differ...
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Expression and localization of alpha V and alpha 3 integrins in male germ cells of selected mammalian species
Bajerová, Kateřina ; Frolíková, Michaela (advisor) ; Krejčová, Tereza (referee)
Integrins are transmembrane glycoproteins mediating cell-cell adhesion and ensuring communication between extracellular matrix and cell cytoskeleton. The presence of integrin subunits has already been described on somatic cells, as well as on oocytes and sperm cells. In oocyte plasma membrane, integrins together with tetraspanins are involved in the formation of vast molecular webs. Analogical structures have been recently discovered in sperm plasma membrane as well. While the role of integrins on oocyte has been researched quite extensively, there is much less known about its presence and function on sperm. This Master's thesis focuses on expression and localization of integrins alpha V and alpha 3 on acrosome-intact sperm and its comparison between mouse, pig and human sperm cells. Furthermore, this thesis also describes relocation of the alpha V integrin after the acrosome reaction, which is an essential step of sperm maturation. Using confocal microscopy, the alpha V integrin localization was detected in acrosomal cap area of acrosome-intact sperm of all selected species. After acrosome reaction, the alpha V integrin relocated and was depicted in postacrosomal region and equatorial segment of acrosome-reacted sperm. Immunodetection revealed a decreasing expression of the alpha V integrin in...
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Effect of sperm ubiquitination in early embryonic development of porcine embryos
Petelák, Aleš
The PhD thesis is focused on the effect of porcine sperm cell extracellular ubiquitination on early embryonic development up to the blastocyst stage after ICSI. In addition, it also presents a potential improvement of the technique of in vitro fertilization using oocyte incubation with ion channels regulators. To address these aims, we established an entirely novel methodology for sperm cell sorting using flow cytometry and subsequent cryopreservation. We determined the conditions for successful sperm cell sorting based on extracellular ubiquitination rate providing highly specific selection as well as sufficient numbers of viable sperms for fertilization using the ICSI method. Concerning the following cryopreservation, established methods were optimized to enable freezing of a minimal sperm cell suspension volume with low cell numbers. The performed experiments showed a direct relationship between the rate of extracellular ubiquitination and the capability of sperms to give rise to a properly developing embryo. Highly ubiquitinated sperm cells were less successful regarding the embryonic development to the blastocyst stage if compared with the lowly ubiquitinated group (6,2 % vs. 16,7 %, P<0,001). Interestingly, the rate of extracellular ubiquitination showed no effect on the pronuclear formation...
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Gametogenesis and Fertilization in Humans
Crhová, Michaela ; Pavlasová, Lenka (advisor) ; Ehler, Edvard (referee)
This bachelor thesis summarises the topic of gametogenesis and fertilization in humans. A search in available literature was performed. The thesis concisely describes the cell cycle and cell division. The main focus is on meiosis, a reductional division that takes the lead part in sexual reproduction. The process of this division is described in detail, and also the mechanism that creates variability of protected cells is described. The product of meiosis is gametes, also known as germ cells. Production of these cells is a key moment in sexual reproduction. This thesis approaches this topic in the context of evolutionary biology and explains its importance in the survival of species on Earth. The main topic of the thesis is the production and maturing of human germ cells. This process is called gametogenesis. This thesis provides a cohesive description of the reproduction system of both sexes and describes the gradual maturing of the germ cells in gonads. The structure of both gametes, egg, and sperm is described with emphasis on the parts of these cells that take part in the process of fertilization. Female reproductive cycles are depicted, and their connection is explained. In contrast to this periodicity, the male continual maturing is also explained. This thesis compares the main differences in...
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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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Effect of sperm ubiquitination in early embryonic development of porcine embryos
Petelák, Aleš ; Krylov, Vladimír (advisor) ; Šušor, Andrej (referee) ; Procházka, Radek (referee)
The PhD thesis is focused on the effect of porcine sperm cell extracellular ubiquitination on early embryonic development up to the blastocyst stage after ICSI. In addition, it also presents a potential improvement of the technique of in vitro fertilization using oocyte incubation with ion channels regulators. To address these aims, we established an entirely novel methodology for sperm cell sorting using flow cytometry and subsequent cryopreservation. We determined the conditions for successful sperm cell sorting based on extracellular ubiquitination rate providing highly specific selection as well as sufficient numbers of viable sperms for fertilization using the ICSI method. Concerning the following cryopreservation, established methods were optimized to enable freezing of a minimal sperm cell suspension volume with low cell numbers. The performed experiments showed a direct relationship between the rate of extracellular ubiquitination and the capability of sperms to give rise to a properly developing embryo. Highly ubiquitinated sperm cells were less successful regarding the embryonic development to the blastocyst stage if compared with the lowly ubiquitinated group (6,2 % vs. 16,7 %, P<0,001). Interestingly, the rate of extracellular ubiquitination showed no effect on the pronuclear formation...
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Sperm motility and postmating prezygotic isolation in two nightingale species
Baránková, Lucie ; Reifová, Radka (advisor) ; Frolíková, Michaela (referee)
The motility of male gametes (sperm) is one of the important factors influencing the reproductive success of males. Because sperms are often subjected to strong postmating sexual selection and even closely related species often differ in sperm morphology, sperm motility could also differ between species, which may contribute to reproductive isolation between species. As part of my diploma thesis, I studied sperm motility in two closely related species of songbirds, the common nightingale (Luscinia megarhynchos) and the thrush nightingale (Luscinia luscinia). These two species of nightingales are an ideal model system because the areas of these two species overlap in the secondary contact zone across Central and Eastern Europe, where they occasionally hybridize and thus allow the study of speciation mechanisms in the natural environment. Both species also differ greatly in total sperm length. As part of my diploma thesis, I studied the possible influence of different sperm morphology on their motility. I further tested whether the motility of nightingale sperm differs in the fluid from the cloaca of a female of the same species and a different species, which would demonstrate the presence of postmating prezygotic reproductive isolation between species. The results of my work showed that despite the...
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