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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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Role of importins in reproduction
Mertová, Irem ; Frolíková, Michaela (advisor) ; Bubeníčková, Filipa (referee)
Importins are proteins from a group of karyopherins, which provide transport of proteins into the nucleus both in somatic cells and gametes. Transport of specific transcription factors plays an important role in gametes and affects key events of reproduction. In mammals, reproduction is mediated by the process of sexual reproduction. In this event, a haploid gamete is formed in each parent, which in later stages of fertilization combine and make a diploid zygote. Gene expression in gametes differs dramatically from somatic cells. In gametes, translation is suspended and specific proteins are expressed only at certain stages of cell development. Proteins determined for transport into the nucleus contain various nuclear localization signals, which are detected by different types of importins. Based on studying differences in expression of individual types of importins in various phases of gametogenesis, we can deduce their roles in these processes. Role of importins was proven in gametogenesis, differentiation of stem cells and maturation of germ cells. On top of that, importins appear to play a role in defense of male germ cells against oxidative stress.
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Kryoprezervace a transplantace spermatogonií kapra obecného
FUČÍKOVÁ, Michaela
Cryopreservation and transplantation of germ cells in fish provides a suitable tool for preserving genetic information. By method of surrogate reproduction, the offspring with characters of the chosen donor can be obtained. In this case of our commercially important species common carp. However, for the successful cryopreservation of the germ cells, a suitable protocol for each species must be established. Several cryoprotectants were tested. The best of them, Me2SO, regarding the viability of spermatogonia, was tested for its different concentrations depending also on the rate of freezing. Further testing, related to the effect of tissue size, incubation time and added sugar, was performed. The result of the assay identified best cryomedium composed of 2.5M dimethylsulfoxide, added sugar of 0.3M glucose, 1.5% BSA and 25nM Hepes dissolved in PBS. The most suitable size of tissue was 100 mg, incubation time was 30 min and coolig rate was -1 ° C/min. This protocol ensures the highest viability rate of cryopreserved spermatogonia of common carp. The second part of the work was to verify the success of the transplantation of cryopreserved and fresh spermatogonia into a suitably chosen recipient, the goldfish, which shares similar reproductive characteristics with carp, but also offers reduction of space requirements or resistance to koi-herpes virus. The transplanted germ cells colonized the germ line and started gametogenesis in 42.5% (cryopreserved spermatogonia) and 52.5% (fresh spermatogonia) goldfish recipients, which demonstrated that the transplantation of cryopreserved spermatogonia of common carp can be successfully achieved.
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