|
|
Ezrin-Radixin-Moesin (ERM) proteins: The link between tetraspanin web and cytoskeleton in gametes
Picková, Jana ; Frolíková, Michaela (advisor) ; Lánská, Eva (referee)
Ezrin, which belongs to the ERM protein family along with radixin and moesin, plays an important role in linking membrane proteins to the actin cytoskeleton. In sperm, ezrin was described so far in bovine and humans, where it probably contributes in actin polymerization during capacitation. During the acrosome reaction, actin plays a significant role in relocation of protein from the inner acrosomal membrane to the equatorial segment, which is an essential process for successful fertilization. One of the proteins that relocates to the equatorial segment, the site of initiation of gamete fusion, is protein CD9. This protein probably stabilizes protein interactions between sperm and oocyte during gamete fusion. In this diploma thesis, we focused on the role of ezrin in linking the CD9 protein to the actin cytoskeleton in mouse sperm. Using indirect immunofluorescence, we detected ezrin, CD9 and actin in acrosome intact mouse sperm. The co-localization of these proteins in the apical acrosomal region suggests their interactions. However, this was not confirmed by our co-immunoprecipitation experiments. Nevertheless, in the apical acrosomal region we also detected EWI-2 protein, which could act as linker between CD9 and ezrin. In this thesis, we also investigated changes in levels of active...
|
|
|
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...
|
|
|
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...
|
guest ::
