Dzyuba Viktoriya, Słowińska Mariola, Cosson Jacky, Ciereszko Andrzej, Boryshpolets Sergii, Štĕrba Ján, Rodina Marek, Linhart Otomar, Dzyuba Borys
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/II, 389 25, Vodnany, Czech Republic.
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.
Fish Physiol Biochem. 2016 Dec;42(6):1755-1766. doi: 10.1007/s10695-016-0255-x. Epub 2016 Jul 12.
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.
在鲟鱼中，精子获得运动激活潜能即精子成熟的过程在睾丸外发生。这个过程可以通过将未成熟的睾丸精子在从完全成熟的沃尔夫管精子收集的精液中预孵育来在体外完成。在预孵育培养基中添加胰蛋白酶抑制剂会破坏精子成熟。目前尚无关于蛋白水解调节因子在鱼类精子成熟中作用的数据，而其在哺乳动物精子成熟中的作用已得到认可。本研究评估了精液蛋白酶和抗蛋白水解活性在小体鲟精子成熟过程中的作用。对来自沃尔夫管精子的鲟鱼精液、睾丸精子的精液以及来自沃尔夫管精子、睾丸精子和体外成熟后的睾丸精子的精子提取物进行了酪蛋白和明胶酶谱分析以及酰胺酶和抗蛋白水解活性的定量分析。我们没有发现来自沃尔夫管精子的精液和来自睾丸精子的精液在蛋白水解谱上有显著差异。酶谱分析揭示了精子提取物中的差异：沃尔夫管精子提取物的特征是存在一条从48 kDa到41 kDa的宽蛋白水解带，而睾丸精子提取物在体外成熟之前没有显示出这种活性。酰胺酶活性的差异与这些结果一致。可能不是蛋白水解和抗蛋白水解活性本身的水平，而是它们相互作用的改变引发了一系列信号事件，这对成熟过程至关重要。
