In sturgeon, the acquisition of the potential for motility activation called spermatozoon maturation takes place outside testes. This process can be accomplished in vitro by pre-incubation of immature testicular spermatozoa in seminal fluid collected from fully mature Wolffian duct sperm. Addition of trypsin inhibitor to the pre-incubation medium disrupts spermatozoon maturation. There are no available data for the role of proteolysis regulators in fish spermatozoon maturation, while their role is recognized in mammalian sperm maturation. The present study evaluated the involvement of seminal fluid proteases and anti-proteolytic activity in the sterlet spermatozoon maturation process. Casein and gelatin zymography and quantification of amidase and anti-proteolytic activity were conducted in sturgeon seminal fluid from Wolffian duct sperm and seminal fluid from testicular sperm, along with spermatozoon extracts from Wolffian duct spermatozoa, testicular spermatozoa, and testicular spermatozoa after in vitro maturation. We did not find significant differences in proteolytic profiles of seminal fluids from Wolffian duct sperm and ones from testicular sperm. Zymography revealed differences in spermatozoon extracts: Wolffian duct spermatozoon extracts were characterized by the presence of a broad proteolytic band ranging from 48 to 41 kDa, while testicular spermatozoon extracts did not show such activity until after in vitro maturation. The differences in amidase activity coincided with these results. It may not be the levels of proteolytic and anti-proteolytic activity per se, but the alterations in their interactions triggering a cascade of signaling events, that is crucial to the maturation process.

Department of Gamete and Embryo Biology Institute of Animal Reproduction and Food Research Polish Academy of Sciences in Olsztyn Tuwima 10 10 748 Olsztyn Poland

Institute of Chemistry and Biochemistry Faculty of Science University of South Bohemia in Ceske Budejovice Branisovska 1760 37005 Ceske Budejovice Czech Republic

Institute of Parasitology Biology Centre of ASCR Branisovska 31 37005 Ceske Budejovice Czech Republic

South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses Faculty of Fisheries and Protection of Waters University of South Bohemia in Ceske Budejovice Zatisi 728 2 389 25 Vodnany Czech Republic

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