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Fluorescent analysis of the differential protein expression in normozoospermic and asthenozoospermic sperm samples
Děd, Lukáš ; Čapková, Jana ; Kubátová, Alena ; Teplá, O. ; Pěknicová, Jana
Asthenozoospermia is one of the main seminal pathologies underlying male infertility. Previous proteomic studies have demonstrated the significant differences in the protein profiles between normozoospermic and asthenozoospermic sperm samples. Since these studies were primarily focused on the identification of differentially expressed proteins by mass spectrometry, we aimed to evaluate the ability of our diagnostic antibodies to detect the differential expression of selected protein markers by fluorescent microscopy and flow cytometry techniques. Therefore, we analyzed sperm samples from 30 men with normal and 30 men with astheno spermiograms, average by the panel of our diagnostic anti-human sperm (Hs) antibodies. Fluorescent microscopy and flow cytometry analysis revealed quantitative differences in the protein abundances between normo and astheno sperm samples, namely, in GAPDHs, evaluated with Hs-8 MoAb, VCP, evaluated with Hs-14 MoAb, and ATP synthase, evaluated with MoAb Hs-36. From the methodological point of view, we observed very high correlation between the data obtained by fluorescent microscopy and flow cytometry techniques and therefore both methods are useful for evaluation of protein differences associated with asthenozoospermia. From the clinical point of view, we observed the strong association of the low sperm motility in the sample with the expression of proteins, playing an important role in sperm energy metabolism (expected), but also with the expression of all tested intra-acrosomal proteins. These findings further demonstrate asthenozoospermia as a complex semen disorder frequently associated with other semen pathologies, which are not diagnosed by basic semen analysis, and the possibility to use monoclonal antibodies as a tool for diagnosis of protein associated sperm pathologies in the semen with the low sperm motility.
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Distribution of Invasive plants in PLA Kokořínsko
Váňová, Petra ; Pěknicová, Jana (advisor) ; Martin, Martin (referee)
The thesis is focused on the occurence of invasive plants in the Protected Landscape Area (PLA) of Kokořínsko-Máchův kraj. Three invasive species - Canadian goldenrod (Solidago canadensis), Bohemian knotweed (Reynoutria x bohemica) and Himalayan Balsam (Impatiens glandulifera) were mapped in the southern part of the PLA. The aim of the research was to identify the distribution of these invasive plants and to create maps of their occurrence in the monitored area using ESRI ArcMap software. Part of the work was to determine the incidence of invasive species in Natura 2000 habitats and ecosystems, according to the data set of the Nature Conservation Agency of the Czech Republic (Layer of Natura 2000 habitats; Consolidated layer of ecosystems of the Czech Republic, Czech: KVES). Most invaded Natura 2000 habitats and KVES ecosystems were identified. From all three of the studied plants growing within the study area, Solidago canadensis occurred the most. From the resulting field research and ongoing expertise, show that most areas of invaded habitats by Solidago canadensis are athropogenically influenced - grassland, introduced shrub vegetation and urbanized areas. During mapping of the invaded habitats only a limited, eradicative management by SCI employees was found, although part of the surveyed area is located in the SCI Kokořínsko.
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Influence of environmental variables for invasive plants spread in PLA Kokořínsko
Heřmanová, Lucie ; Pěknicová, Jana (advisor) ; Markéta, Markéta (referee)
Bachelor's dissertation is orientated towards issue of presence of invasive plants. In chosen part of PLA Kokořínsko proceeded mapping of current extension of chosen invasive kinds of goldenrod (Solidago) and knotweed (Reynoutria). The goal of this bachelor's dissertation was mapping of affected invasive plants and consecutive evaluation of results, when there was investigated, which biotope affected kinds colonize. As complementary analysis there was investigated preference of type of soil of individual kinds. Most often there was affected S. canadensis, which also covered the biggest area. From final survey resulted which biotopes and ecosystems the invasive plants mostly invade. From the data is also possible to see that invasive weeds colonize most frequently man-made affected habitats and brown soil is preferred kind of soil.
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Invasive plants in selected SCI of Karlovy Vary region
Bernasová, Nikola ; Pěknicová, Jana (advisor) ; Markéta, Markéta (referee)
In the area of interest, Protected Landscape Area (PLA) Slavkov Forest two locations were chosen where invasive plants populations were mapped. The field work focused on mapping the occurrence of giant hogweed, all species of knotweed, Canadian goldenrod, tall goldenrod and Himalayan balsam. The aim was to determine the invaded sites in the area of PLA Slavkov Forest. It was found that the mapped areas are most prone to giant hogweed. A considerable number of invaded sites have been chemically treated. In the studied area, Canadian goldenrod and tall goldenrod were present only in a small extent and Himalayan balsam was almost absent. No knotweed invaded locations were found. The invaded sites were most often observed in the vicinity of mesophilic meadows, urbanized area and arable land. This issue is dealt with in the Management plan for the protected landscape area Slavkov Forest, and in various projects aimed at mapping and disposal of invasive species.
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Habitat sutability for invasive plants spread in PLA Kokořínsko
Kofránková, Petra ; Pěknicová, Jana (advisor) ; Martin, Martin (referee)
The aim of this thesis is to analyze the occurrence of main invasive species in PLA Kokořínsko_Máchův kraj using field data acquisition method
Three invasive plant species have been identified in the area of interest. The geographical information on these tree species were collected via on-site terrain mapping using a GPS device and the aerial imagery as supporting mapping tool.
Invasive species are found at 12 of 16 cadastral areas. Most affected are the human-influenced habitats, soil type cambisol.
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Endocrine disruptors induce transgenerational alterations of the male reproductive parameters and mirna expression profiles in mouse primordial germ cells
Děd, Lukáš ; Brieno-Enríquez, M.A. ; García-López, J. ; Cárdenas, D.B. ; Guibert, S. ; Hourcade, J.de D. ; Pěknicová, Jana ; Weber, M. ; del Mazo, J.
Primordial germ cells (PGCs) are the embryonic precursors of the germ cell linage, which are restricted to form only sperm and oocytes following their specification from pluripotent cells. PGC precursors are specified in the epiblast around 6.25 days post coitum (dpc), and around 7.25 dpc become identifiable in a 40 cell-cluster. In the present study, we used a mouse model to evaluate the trans-generational (F1-F3) effects of vinclozolin (VCZ) administrated in two doses on male reproductive parameters. We observed decreased fertility rate, higher apoptotic rate and histopathologic alterations in adult testis, PGC number reduction, increments of PGCs apoptosis and changes in PGCs gene expression among all three generations. In the attempt to clarify the trans-generational transmition of the altered phenotypes, we performed the microRNA expression and DNA methylation analysis. We observed the significant alteration in the expression of multiple microRNA and microRNA-regulated genes which are important for PGCs specification, including LIN28, let-7 and BLIMP1. Trans-generational deregulation in the expression of factors involved in the Lin28-let-7-Blimp1 pathway can lead to specific VCZ-induced phenotype observed in our study.
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The ubiquitin–proteasome system is involved in the regulation of activity of spermadhesin aqn1 and acrosin inhibitor, the two sperm surface proteins, during porcine fertilization
Jonáková, Věra ; Yi, Y.J. ; Postlerová, Pavla ; Pěknicová, Jana
The spermadhesin AQN1and acrosin inhibitor (AI/SPINK2) proteins bind to the sperm plasma membrane at ejaculation. The AQN1 has been implicated in sperm binding to zona pellucida (ZP) of the oocyte as well as in sperm interactions with the epithelium of the oviductal sperm reservoir. The SPINK2 protects spermatozoa from proteolytic degradation during their trip up the female genital tract toward the oocyte. This study examined the role of two components of the 19S proteasome regulatory complex, the ubiquitin C-terminal hydrolase UCHL3 and PSMD8 in the AQN1-mediated boar sperm binding to zona pellucida. Interaction of PSMD4 subunit with the acrosomal surface-associated acrosin inhibitor AI/SPINK2 provided another line of evidence for the presence of 26S proteasomes on the sperm surface. Detection of the ubiquitinated forms of SPINK2 supports the hypothesis that SPINK2 activity is controlled by ubiquitin-proteasome system (UPS). The activity of the porcine AQN1, and thus the efficiency of sperm-oocyte recognition/binding, may be controlled by elements of the sperm surface-bound UPS, in particular by UCHL3, and by proteasomal regulatory complex subunit PSMD8. Ubiquitinated isoforms of AQN1 were also detected in boar sperm extracts. The UCHL inhibitor ubiquitin aldehyde and the antibodies against UCHL3 or PSMD8 increased the rate of sperm-ZP penetration and polyspermy during porcine in vitro fertilization (IVF). In contrast, the addition of recombinant UCHL3 to fertilization medium significantly reduced polyspermy rates, while maintaining satisfactory rate of monospermic fertilization (~50%). These results are significant for production agriculture. The high level of polyspermy that hinders porcine IVF for commercial embryo transfer could be mitigated by the modulation of the UCHL3 and/or PSMD8 activity.
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Changes in the expression of selected testicular genes in mice
Valášková, Eliška ; Margaryan, Hasmik ; Žatecká, Eva ; Pěknicová, Jana
The decrease in population fertility has become a major concern in many developed countries. Recent studies show that infertility is affecting an estimated 15% of all couples (World Health Organization, WHO, 2010). Male infertility is the primary or contributing cause in 60% of cases. Male infertility is caused by a number of factors, such as genetic background, various environmental factors and disease. Diabetes mellitus (DM), a serious health problem on its own, is also suspected to be a contributing factor to male infertility. The aim of this project was to analyze the cellular, molecular and genetic effects of diabetic environment on spermatogenesis and sperm quality and to determine the impact of DM on the in vivo reproduction, using the mouse model (Mus musculus) inbred FVB. Diabetes was induced using streptozotocin. We used our knowledge and tools (unique monoclonal antibodies developed by our group) to determine the status of reproductive organs, anogenital distance, and the quality of sperms. Genetic analysis was performed by a quantitative Reverse Transcription Polymerase Chain Reaction (qPCR). We tested selected genes which are expressed in testicular tissue and thus can influence process of spermatogenesis and consequently the sperm quality. Our preliminary data strongly suggest that DM impairs male fertility. We have found significant changes in the body and reproductive organ weight of mice with DM. We have identified qualitative and quantitative changes in the expression of proteins in epididymal fluid and sperms. We have also detected an increased number of apoptotic cells in sperm of diabetic mice compared to the control group. To our knowledge, there is no study assessing the correlation between DM and “unexplained infertility”. In view of this, it is essential to analyze the effects of DM on male fertility, sperm quality, and reproduction parameters.
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