Determination of acrosin activity in sperm by two new flow cytometric methods

Authors: Alexandrina RUGINA¹, Ana-Maria GHEORGHE¹, Catalin IORDACHEL¹, Maria CALOIANU², Daniela BRATOSIN^1,3*, Jean MONTREUIL⁴

Affiliation: ¹ National Institute for Biological Science Research and Development, Bucharest, Romania; ² University of Bucharest, Faculty of Biology, Bucharest, Romania; ³ “Vasile Goldis” Western University of Arad, Faculty of Natural Sciences, Arad, Romania; ⁴ Université des Sciences et Technologie de Lille 1, Laboratoire de Chimie Biologique, UMR-CNRS/USTL n°8576, Villeneuve d’Ascq Cedex, France

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ABSTRACT. During fertilization, sperm utilizes a specific serine-dependent protease, known as acrosin, an enzyme with trypsin-like activity. In the present paper, we describe two rapid and sensitive flow cytometric assays we have developed for the determination of the activation level of proacrosin into acrosin in sperm using two original fluorescent staining. In the first method, the proportion of sperm with active acrosin was determined using the Alexa Fluor® 488 trypsin inhibitor conjugate (SBTI) and the presence of bound inhibitor in sperm was  determined by flow cytometric analysis. In the second method we used (CBZ-Ala-Arg)2-R110 trypsin, a specific proteinase substrate for trypsin activity determination. Our results demonstrate that valuable results concerning the sperm quality can be obtained using the two new flow cytometric techniques.

Keywords: goat spermatozoa, acrosin, flow cytometry, Allium extracts