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The localization of maternal RNAs during early development
Kováčiková, Petra ; Šindelka, Radek (advisor) ; Děd, Lukáš (referee)
The maternal determination of a developmental plan has been described in many animal species. The most well-known examples are found in the embryogenesis of C. elegans and D. melanogaster. In vertebrates, these studies are often limited by the size of oocytes to just a small subset of model organisms. The most comprehensive research can be found dealing with the early development in the frog, Xenopus laevis. Its oocytes, displaying differently pigmented animal and vegetal hemispheres, are patterned by an asymmetric distribution of maternal factors. These proteins and RNAs of maternal origin are stored in the oocyte and provide the only source of cell differentiation until the zygotic genome activation. Here, we address the role of RNA localization in this process. We assessed the localization patterns of both, maternal mRNAs and lncRNAs and found that, in addition to protein coding transcripts, lncRNAs also form concentration gradients along the animal-vegetal axis. Furthermore, using RT-qPCR tomography, we have now been able to demonstrate a difference between actively localized extremely animal group and spontaneously localized animal group of transcripts, a distinction emerging from the transcriptome characterization performed in our laboratory. Additionally, we found that the extremely animal...
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Post-copulatory sexual selection and sperm phenotypes
Bílková, Karolína ; Albrecht, Tomáš (advisor) ; Děd, Lukáš (referee)
Postcopulatory sexual selection has two basic components, sperm competition and cryptic female choice. In this thesis I summarize available data of the influence of postcopulatory sexual selection on sperm morhology and phenotype, focusing on both vertebrates and selected invertebrate taxa. In the first part of my thesis, I provide an overview of sperm phenotype adaptations that are influenced by the sperm competition and may closely determine male fertilization success. These are the length of sperm, sperm swimming speed and motility, longevity of sperm and the amount of ejaculate produced. Sperm phenotypes that evolve under strong postocopulatory selection may also involve morphological adaptations alowing sperm to mutually cooperate in the female reproductive tract. The cryptic female choise, in which female selectively use sperm after the sperm enters her reproductive tract, and the coevolution of sperm morphology and female reproductive tract is presented in the second part of this thesis. Powered by TCPDF (www.tcpdf.org)
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Effect of estrogens and endocrine distruptors on mammalian sperm in vitro and male reproductive parameters in vivo
Děd, Lukáš ; Pěknicová, Jana (advisor) ; Jonáková, Věra (referee) ; Linhart, Otomar (referee)
The endocrine system is one of the most important regulatory systems in human and animal bodies and almost all physiological processes are regulated by it. Human and animal reproduction are largely regulated by different hormones and their proper and well regulated functions are essential for successful production of the gametes, fertilization and related processes, development of the embryo and fetus, initiation of puberty and production of the offspring. Estrogens have generally been considered as primarily female hormones. However, there is increasing evidence of their important impact on male reproductive functions. This was well documented by production of estrogen receptor knock-out male mice, which are infertile. Estrogens and estrogen receptors are also very frequent targets of endocrine disruption. Endocrine disruptors are natural or artificial compounds that, at certain doses, can interfere with the endocrine functions in human and wildlife. This interference can include specific binding to the hormonal receptor, influencing the production, elimination and transport of the natural hormones and also not well documented mechanisms including epigenetic regulations. In the presented work, our aim was to evaluate the effects of natural estrogens and endocrine disruptors on male reproductive...
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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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Expression of selected proteins in sperm in men with normal and pathological spermiograms using monoclonal antibodies
Pěknicová, Jana ; Čapková, Jana ; Dorosh, Andriy ; Margaryan, Hasmik ; Kubátová, Alena ; Děd, Lukáš
Recent studies show that infertility in human populations it affects an estimated 15% of couples of reproductive age. Male infertility is the primary cause for 60% of these cases. For these reasons, we analyzed the acrosomal and sperm surface proteins in men with normal and pathological spermiograms. We found that intra-acrosome proteins: TERA (Transitional endoplasmic reticulum ATPase), GAPDHS (Sperm Glyceraldehyde-3-phosphate dehydrogenase), and PRKAR2A (C-AMP-dependent protein kinase II, PRKAR2A), which can be identified using our monoclonal antibodies, are different express in healthy men and men with astenozoospermia (with reduced sperm motility), and with a significantly reduced expression in the astenozoospermia. These proteins are involved in energy metabolism and apoptosis of the cells, and some of them in the sperm-egg interaction; therefore, they have an important role in reproduction. On the other hand, there were no statistically significant differences in the expression of surface proteins (Appolipoprotein J (Clusterin) and Semenogelin). Our findings show that astenozoospermia as a complex disorder of the semen is often combined with other pathological conditions that are not diagnosed by the semen analysis. Therefore monoclonal antibodies are so suitable instrument for the detection of proteins associated with the pathology of the sperm in the semen with low sperm motility. In general, monoclonal antibodies against the sperm proteins are an appropriate tool to detect sperm quality in reproductive medicine.
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Estrogen receptor beta (ERβ) in testicular cells and sperm
Dostálová, Pavla ; Žatecká, Eva ; Děd, Lukáš ; Dorosh, Andriy ; Postlerová, Pavla ; Jonáková, Věra ; Dvořáková-Hortová, Kateřina ; Pěknicová, Jana
Estrogen is a steroid hormone that plays an important role during sperm development in the male and female reproductive tract. Estrogen signalling is a complex process that depends on cell milieu and presence of receptors. Thanks to the steroid nature of estrogens, they can pass through the plasmatic membrane and bind to the intracellular estrogen receptors (ERs). Within the cell, there are several pools of ERs. One of them is localized to the cell nucleus and their activation leads to direct or indirect binding to DNA and ultimately to alternation in gene expression (genomic pathway). Other pools of ERs are associated with plasma membrane or are located in cytosol. Activation of membrane associated ERs leads to rapid non-genomic responses. Nowadays, two classical estrogen receptors are known – ERα and ERβ. Since ERβ is a predominant variant in testes, we focused our study on expression of ERβ variants in murine testes and sperm. We detected two variants of ERβ at mRNA level in both, testes and sperm. These variants differ in 54 nucleotids within the ligand binding domain and this variability results in different affinity to estrogen. We analyzed individual testicular cell types (spermatogonia, spermatocytes, spermatids, Sertoli cells) by RT-qPCR. Our results suggest that both ERβ variants are coexpressed in the same cell type and may therefore interact together. This may have consequences in mediating of estrogen signalling. Moreover, ERβ is expressed more in the later stages of spermatogenesis suggesting the role of ERβ in these stages or alternatively in spermatozoa alone. At the protein level, we detected ERβ in nuclear, membrane and cytosolic fraction prepared from testicular tissue suggesting the involvement of both, genomic and non-genomic, pathways of estrogen signaling in testes. In sperm, anti-ERβ antibodies localized ERβ in acrosome region and tail which is in accordance with the known role of estrogen on capacitation, acrosome reaction and motility.
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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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