|
|
Role reaktivních forem kyslíku a proteinové fosforylace na funkci spermií ryb
GAZO, Ievgeniia
Spermatozoa of externally fertilizing fish species after releasing into aqueous environment are particularly vulnerable to damage mainly due to alterations in composition of media surrounding sperm. Among factors affecting spermatozoa movement in external medium are water pollutants, temperature, pH and osmotic conditions. The goal of this thesis was to investigate the possible effects of oxidative stress on sperm performance and intracellular signaling, particularly the effect of pollutants occurring in water environment. In addition the molecular mechanisms of stress response and motility activation for spermatozoa of several freshwater fish species were analyzed. Our results show that xenobiotics, such as vinclozolin, induce a dose-dependent reduction in sterlet (Acipenser ruthenus) spermatozoa motility and velocity at environmentally relevant concentrations. Increased levels of lipid oxidation (LO) and protein carbonylation (CP), as well as changes in antioxidant activity of superoxide dismutase (SOD) indicate the development of an oxidative stress in spermatozoa exposed to xenobiotic. Moreover, increased DNA fragmentation as well as a reduction of the level of ATP was observed in spermatozoa incubated with xenobiotic in vitro. These results demonstrated that sterlet spermatozoa are highly susceptible to the presence of pollutants, which induce excessive ROS production even at low concentrations. Further studies were performed in order to evaluate the role of ROS production in fish sperm and protective properties of seminal plasma. The ROS were generated in carp (Cyprinus carpio L.) spermatozoa by in vitro incubation with xanthine - xanthine oxidase system (X-XO). A time- and dose-dependent reduction in spermatozoa motility and velocity was observed as well as increased LO, CP and DNA fragmentation. Moreover, it was shown that O-linked N-acetylglucosamine transferase and septin-8-A changed their phosphorylation state on tyrosine residue, and acid phosphatase activity decreased in response to oxidative stress. On the other hand, catalase (CAT), superoxide dismutase (SOD), and glutathione (GTH) in combination with seminal plasma can reduce oxidative stress in carp spermatozoa and improve sperm quality. Our next study was applied to determine how the protein phosphorylation pattern changed after motility activation in carp and sterlet spermatozoa, where phosphorylated proteins are located in spermatozoon and to identify proteins involved in sperm motility. It was shown that the pattern of protein phosphorylation and their localization differs significantly between two species. Phosphorylation on serine and tyrosine residues, as well as protein kinase A (PKA) and protein kinase C (PKC) substrates play an important role in spermatozoa motility activation and regulation in both species. Numerous signaling proteins involved in carp and sterlet spermatozoa movement were identified in this study, giving a better understanding of molecular mechanisms underlying sperm motility. As a conclusion, the results of this study provide new data on the effect of xenobiotics and oxidative stress on fish spermatozoa motility, DNA integrity, lipid and protein oxidation, antioxidant defense system and intracellular signaling. These data proved the toxicity of water pollutants and ROS for fish spermatozoa and proposed the use of CAT, SOD, or GTH in combination with seminal plasma to reduce oxidative stress in these cells. Moreover, we identified many spermatozoa proteins involved in stress response and motility. In practice, the data presented in this thesis could be useful for elaboration of suitable medium for cryopreservation and artificial propagation of freshwater fish species.
|
guest ::
RSS feed.