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Účinky xenobiotik na oxidační stres, metabolizmus lipidů, integritu DNA a životaschopnost lidských buněk a rybích spermií in vitro
LINHARTOVÁ, Pavla
Pollution of the aquatic environment by inorganic and organic chemicals is a major factor posing a serious threat to the survival of aquatic organisms including fish. In addition balancing risks and benefits of fish consumption is nowadays an intensively discussed public health topic. Spermatozoa of almost all fish species are released into water environment where they can be directly exposed to various compounds, such as xenobiotics including toxic metals, prior to fertilization. In addition, exposure of parental adults to various xenobiotics may affect gamete quality, which may subsequently reduce fertilization success. On the other hand the advantages of eating fish are well-known, not only in the point that fish is a healthy source of protein and other nutrients, but eating contaminated fish may also confer various health benefits. Research over the past few decades has shown that the nutrients and particularly the n-3 fatty acids (FA) found in fish and seafood, are for examples protective against cardiac diseases and have a positive impact on brain development. The thesis provides a focus on two different cell model types. Firstly, human hepatocellular cells (Hep G2, ATCC) were used as in vitro tool for studying the effect of the intake of cadmium (Cd2+) contaminated fish on cytotoxicity, oxidative stress and fatty acid and phospholipid class compositions. Secondly, spermatozoa of one threatened species of fish, sterlet (Acipenser ruthenus) were used as in vitro model for studying effect of potentially hazardous xenobiotic compounds' occurring in open waters. Sperm from sterlet were exposed for 2h to environmentally relevant concentrations of DQ (0-150
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Iniciace pohybu byčíku, signalizace a regulace pohyblivosti spermií ryb: fyzikální a biochemické řízení
PROKOPCHUK, Galina
The current study attempted to shed light on the regulatory processes and response arrangements of fish spermatozoa during the course of maturation and motility initiation. The first intent of this study was to improve the understanding of the mechanism underlying the acquisition of potential for sperm motility in sturgeon. Up to present work, the physiological process underlying sperm maturation in this species has not been described at all. Our results showed that sperm maturation in sturgeon occurs outside the testes because of dilution of sperm by urine and involves the participation of high molecular weight substances as well as calcium ions present in seminal fluid and/or urine. The second aim of the present study was to investigate the coping mechanisms in fish spermatozoa with osmotic and ionic activating mode, as well as in spermatozoa of euryhaline fishes, to various environmental conditions. We showed that alteration of environmental osmolality might affect the fish sperm in different ways, depending on fish species and modes of spermatozoa motility activation either osmotic or ionic mode. In response to osmotic stress caused by hypotonicity, carp spermatozoa regulated the flow of water across their cell membrane and increased their cytoplasmic volume during their short motility period. In contrast, no indications of sperm volume changes were observed neither in sterlet nor in brook trout spermatozoa, both of which having an ionic mode of motility activation. We also examined the mechanism by which sperm motility triggering in euryhaline fishes can adapt to a broad range of environmental salinity. Our results demonstrated that spermatozoa of euryhaline tilapia, Sarotherodon melanotheron heudelotii, reared in fresh-, sea- or hypersaline water can be activated in hypotonic, isotonic or hypertonic conditions of swimming milieu, provided Ca2+ ions are present at various levels. It was established that the higher the fish rearing salinity or the more hypertonic ambient media at spermatozoa activation, the higher extracellular concentration of Ca2+ ions is required. The results obtained in the present study allow suggesting that osmolality is not the main factor inhibiting sperm motility inside the testis in the S. melanotheron heudelotii. A third aim of this study was investigation of the regulation of motility initiation process and description of flagellar beating initiation in chondrostean spermatozoa. We detected that K+ inhibition of sperm motility in sturgeon can be by-passed due to the pre-exposure of sperm cells to a high osmolality shock prior to its transfer to K+-rich swimming media. Thus, we hypothesized that sturgeon spermatozoa may be activated by use of an unexpected signaling pathway, independent from regular ionic stimulation. The successive activation steps in sturgeon spermatozoa were investigated by high-speed video microscopy, using specific experimental situation, where sperm motility initiation was delayed in time up to several seconds. At motility initiation, the first couple of bends formed at the basal region begins to propagate towards the flagellar tip, but gradually fades when reaching the mid-flagellum. This behavior repeats several times until a stage where the amplitudes of bends gradually reach similar value, what eventually leads to sperm progressive displacement. The total period needed for the flagellum to switch from immobility with rigid shape to full activity with regular propagating bends ranges from 0.4 to 1.2 seconds. In conclusion, the results of the current study bring valuable pieces of information into the general understanding of the processes of maturation of fish spermatozoa, their adaptability to different physical and biochemical circumstances, the extra- and intra-cellular signaling as well as the regulatory mechanisms of motility activation in fish spermatozoa.
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