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Úloha regulačních proteinů pro pohyblivost rybích spermií
DZYUBA, Viktoriya
The investigation of the energetic aspects of spermatozoon motility implementation (Chapter 2) was carried out using demembranated spermatozoa of taxonomically distant fish species (common carp and sterlet). Special attention was given to the functioning of ATP regeneration systems: adenylate kinase (AK), and creatine kinase (CK). It was shown for the first time that the phosphocreatine/CK system is present in sterlet spermatozoa and plays an essential role in ATP regeneration. Spermatozoa of carp and sterlet were shown to have similar systems for ATP regeneration, while the efficacy of the studied systems differs in these species. The low baseline activity of CK in carp and AK in sterlet suggest these to be the source of the most pronounced effects of their inhibition on energy supply for flagella movement in the respective species. The presence of a maturational process during the post-testicular transit of sperm in sturgeon was recently ascertained in our laboratory (Chapter 3). This discovery prompted investigation of the factors that regulate this process including the involvement of proteolysis regulators and prooxidant-antioxidant system. As a result of this study (Chapter 3.3), we found that there was no significant difference between proteolytic profiles of seminal fluids (SF) of testicular sperm (TS) and Wolffian duct sperm (WS). It suggests that the majority of proteases present in SF of mature sperm originate in the testis. Measure of amidase and anti-proteolytic activities in the SF of sterlet sperm showed significant decrease in activities as the sperm passed through the kidneys and Wolffian ducts. Considering our observation that trypsin inhibition during in vitro TS maturation blocked the maturation process (Chapter 3.1), and based on zymography, amidase and anti-proteolytic activity determination, we think that the decrease in anti-proteolytic activity of spermatozoa surroundings during their post-testicular transit could be needed to prepare them for maturation. The present study showed that maturation of sturgeon spermatozoa and different times of storage in Wolffian ducts (in vivo storage), are accompanied by significant alterations in motility parameters as well as in SF redox balance (Chapter 4.1). A high level of TBA-reactive substances (TBARS) and a high activity of antioxidant enzymes were found in immature TS compared to those in WS. The high activity of the enzymatic antioxidant system (AOS) allows TS to cope with the deleterious effects of excessive reactive oxygen species production and to retain the ability to become motile after post-testicular transit, or after in vitro maturation. The increase in TBARS content during in vivo storage was not accompanied by a corresponding increase in activity of AOS. We suggest that extended time in the Wolffian ducts resulted in sperm oxidative damage resulting from inadequate AOS efficacy and, finally, in decreases in motility parameters. Short-term hypothermic in vitro storage of sterlet sperm resulted in a significant decrease in motility and velocity without changes of AOS activity (Chapter 4.2). It means that AOS of sterlet sperm is effective enough to prevent the development of oxidative stress during short-term storage. Short period of tench sperm exposure to hypotonic conditions was shown to induce oxidative stress and, as a result, sperm quality decline (Chapter 4.3). The combined results of the study concerning the regulation of sperm prooxidant-antioxidant status (Chapter 4) during spermatozoa maturation, motility activation and sperm in vivo and in vitro storage may confirm a dual role of reactive oxygen species (regulatory or damaging depending from the levels of their formation and elimination) in fish sperm physiology.
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