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Sperm morphology and molecular mechanism associated with sperm fertilizing ability in mice.
Šebková, Nataša ; Hortová, Kateřina (advisor) ; Jonáková, Věra (referee) ; Linhart, Otomar (referee)
The process of sperm capacitation and acrosome reaction (AR) are highly dynamic processes essential for the fertilization, including cytoskeleton proteins in the sperm head. The study of the distribution of actin, spectrin and α-tubulin in the head of representative mammalian sperm indicates that before and after the AR there were changes in the distribution of the cytoskeleton structures in the sperm head of all representatives, mainly in the apical part of the acrosome, but also in the equatorial and postacrosome part of the sperm head. The particularly well-studied actin cytoskeleton plays an important role during the capacitation and before and after the AR. Environmental estrogens can interfere with the function of endogenous hormones in very low concentrations. They can interact with specific receptors affecting several signalling pathways leading to sperm capacitation and AR. The effect of 17β-estradiol, estrone, estriol and synthetic 17α-ethynylestradiol was evaluated by the ratio of tyrosine phosphorylation and the state of the acrosome during in vitro capacitation in the sperm head. This study has provided the evidence that estrogens significantly stimulate capacitation progress in a concentration-dependent manner. Estrogens decrease number of sperm after the induced AR too. The raising...
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Gene mutations affecting the development of human testes
Macháčová, Simona ; Šebková, Nataša (advisor) ; Ševčíková Jonáková, Jana (referee)
Development of mammalian sex organs is remarkable process. Both ovaries and testes rise from the same precursor but then differentiate into morphologically and functionally different structure. The determining factors that decide the fate of undifferentiated structures are not just the genes themselves but also the timing of transcription regulatory genes and specific amount of their products. Development of male and female gonads manages a large set of regulatory genes that interact with each other. Together it forms the gene regulatory network. Crucial role in male sex differentiation plays an SRY gene which regulates the time and quantity of the other factors expression. Even slight errors (mutations) in genes sequences of regulatory genes could change their expression. This leads to disruption of the development of sex organs or even death of the whole organism. Naturally occurring mutations and their phenotype in humans are suitable for studies of gene sex regulatory factors. Thanks to these studies we are able to detect the cause of many birth defects what is a first step toward their eventual treatment.
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The involvement of mitotic kinases AURKA and PLK1 in the oocyte meiotic maturation
Brzáková, Adéla ; Šolc, Petr (advisor) ; Šebková, Nataša (referee)
Aurora A and Plk1 belongs to the extended family of serine/threonine kinases controlling the cell cycle. Both are involved in the control of mitosis and contribute also to the regulation of meiotic division. Although the role of Aurora A in the resumption of meiosis after the first meiotic block seems to be, according to present studies, a bit questionable matter, it is virtually certain that AURKA plays an irreplaceable role in the construction of the spindle. In the oocytes with inhibited (by RNAi or small molecular inhibitors) or overexpressed Aurora A, abnormal spindles with aberrant number of poles or otherwise unusually shaped spindles are created. Disruption of Plk1 function in oocytes also leads to the appearance of damaged spindles. In addition, Plk1, almost certainly, plays a role in the timing of the nuclear membrane breakdown - at GVBD.
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Influence of estrogens on mammalian sperm.
Šidlová, Adéla ; Šebková, Nataša (referee) ; Hortová, Kateřina (advisor)
For many years, estrogens have been considered typically female sex hormones. It is now certain that they are also very important in the regulation of male reproduction, whether their effect is positive or negative. Endogenous estrogens in mammalian males are an important part of the endocrine system. Estrogens play an important role in the growth and maintenance of bone mass, as well as in the development of germ cells and in the marches of spermatogenesis. Healthy sperm are essential for successful reproduction. Effect of endogenous estrogens is partly involved in the regulation of proper development of sperm. Besides the positive effect of endogenous estrogens, there is also a negative effect of estrogens from the external environment, so-called environmental estrogens. Between environmental estrogens are man-made chemicals, xenoestrogens, which increasingly contaminate the environment. Hazards of these estrogenic substances in nature is hidden in their ability to act as endocrine disruptors, which cause defects in many aspects of mammalian reproduction. With regards to the fact that there are more and more substances in current environment that can interfere with hormonal path of mammalian reproduction, it is important to look at this issue. Reproductive disorders that may be caused by...
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Distribution of CD46 and β1 integrin molecules with respect to different membrane structures of the sperm head
Šebková, Nataša ; Frolíková, Michaela ; Děd, Lukáš ; Dvořáková-Hortová, Kateřina
CD46 protein plays an important role during fertilization and its role is associated with acrosome stability. CD46 is probably involved in signalling pathways triggering the acrosome reaction (AR). It also associates through membrane integrins with specific MAP kinases involved in the AR. Our aim was to monitor the dynamics of relocation of CD46 and β1 integrin during sperm maturation and its preparation for the fertilization. The dependence of this localization changes on the dynamic of actin cytoskeleton was studied. Our results show the changes in the localization of these proteins associated with the AR and their co-localization was observed using proximity ligation assay. After the AR CD46 and β1 integrin spreads across the sperm head, entering the post-acrosomal compartment, and permeates the borders of different domains. It was shown previously that actin dynamics is necessary for acrosome reaction-associated translocation of Izumo1 protein that is required for sperm-egg fusion. Therefore Latrunculin A was used during sperm incubation. The co-incubation of capacitated sperm with Latrunculin A leads to a decrease of the percentages of sperm, which express relocation pattern after induced AR. 3D models and visualizations of potential membrane processes responsible for the relocation of proteins from the acrosomal area to the other compartments of the sperm head were prepared. Our results deliver new information that proteins CD46 and β1 integrin undergo dynamic relocation towards the sites of sperm-egg fusion during the AR in vitro. The inhibitor of actin dynamics abrogates significantly the AR-associated changes in proteins localization. We speculate that this relocation is of importance for the successful sperm-egg interaction, adhesion and subsequent gamete fusion.
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CD46 and β1integrin interaction in mouse sperm head
Šebková, Nataša ; Frolíková, Michaela ; Dvořáková-Hortová, Kateřina
CD46 protein plays an important role during fertilization and its role is associated with acrosome stability. CD46 is probably involved in signalling pathways triggering the acrosome reaction. Integrins interact with many cytoskeletal proteins such as actin, therefore changes in the actin cytoskeleton before and after AR may lead to changes in the association and localization of CD46 and β1integrin. Our aim was to monitor mutual CD46 and β1integrin interaction detected by the proximity ligation assay. It generates a localized signal in a form of spots revealing the exact position of the recognition event. Proteins interaction was study in freshly released sperm and sperm during the acrosome reaction, during which there is a gradual relocation of these proteins towards the equatorial segment and the whole sperm head. Proteins α and β tubulin were used as a positive control, α tubulin and β1 integrin as a negative control. In situ PLA showed a distinct spotted signal indicating the mutual interaction of CD46 and β1integrin. A positive response was demonstrated not only in freshly released sperm but also in sperm during the acrosome reaction. Freshly released sperm were distinctively labelled in the acrosome region and the neck, similarly to the positive control. Sperm during the acrosome reaction showed the signal across the whole sperm head region. No signal or sporadic nonspecific staining was detected in the case of the negative control. In summary, our results deliver new information that proteins CD46 and β1 integrin interact with each other. These results suppose the theory that β1 integrin can mediate a connection between CD46 and sperm cytoskeleton thereby molecules of signalling pathways leading to activation of the acrosome reaction.
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Dynamics of mouse sperm capacitation and acrosome reaction
Dvořáková-Hortová, Kateřina ; Frolíková, Michaela ; Děd, Lukáš ; Šebková, Nataša
Capacitation followed by the acrosome reaction (AR), is a very complex event of molecular changes, including acrosome matrix rearrangement and actin polymerization, which mammalian sperm must undergo in the female reproductive tract in order to obtain the ability to penetrate and fertilize the egg. CD46 and β1-integrin belong to specific proteins, which are predicted to interact during molecular reorganization of capacitating sperm. The IZUMO1 as the primary fusion protein of the mammalian sperm is also involved in this dynamic network. We investigated the relationship between the Izumo, CD46 and β1 integrin relocation in the sperm head during the capacitation and AR in vitro. We have already successfully monitored by immunofluorescent labelling the dynamics of proteins CD46 and β1-integrin. The changes in the localization of these proteins associated with the AR and their mutual co-localization was observed. The original β1-integrin location in the freshly released epididymal sperm is in the acrosome and it relocates during the AR further through the sperm head compartments into the equatorial segment and over the whole sperm head. Its density over the equatorial segment is decreasing with the extended time of the AR. Also its presence in the perforatorium of the mouse sperm head is very prominent. The pattern for protein CD46 is extremely similar if not identical in both aspects such as compartment localization and time progress during capacitation and AR in vitro. The molecular interaction of CD46 and β1-integrin was investigated using the Proximity Ligation Assay and Super resolution microscopy STED. The data were statistically analysed. The newly obtained results from CD46 and β1-integrin relocation are in correlation with IZUMO1 dynamics and giving a substantial knowledge on the studied protein network rearrangement during capacitation and AR in mouse spermatozoa.
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