Characterization by conventional flow cytometric parameters and apoptotic markers of goat sperm under the action of acrosinic natural inhibitors

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Article Title:	Characterization by conventional flow cytometric parameters and apoptotic markers of goat sperm under the action of acrosinic natural inhibitors
Authors:	Alexandrina Rugina, Luminita Takacs, Catalin Iordachel, Stela Zamfirescu, Maria Caloianu, Daniela Bratosin
Affiliation:	1 National Institute of Biological Science Research & Development (INSB), Bucharest, Romania 2 University of Bucharest, Faculty of Biology, Romania 3 University “ Ovidius” of Constanta, Faculty of Biology, Romania
Abstract:	During fertilization, sperm utilizes a specific serine-dependent proteolytic enzyme, known as acrosin, to aid in penetrating the zona pellucida and which is also involved in the dispersal of the acrosomal matrix. Recently, flow cytometry has been extensively used to evaluate several different characteristics of sperm. The objectives of this study were to evaluate the action of acrosinic natural inhibitors on the sperm viability and on a possible cell death, correlated with the determination of acrosomal reaction. The sperm cells exposed to diferents concentrations of acrosinic natural inhibitors were evaluated by flow cytometry after 30 min. incubation endpoints. We investigated cellular viability (calcein-AM assay) and apoptosis (light scattering properties of sperma, study of cell death using annexin-V-FITC and propidium iodide double – labelling, mitochondrial function assessed using DIOC6 and caspase-3 activity determination. Our results indicate that the percentage of viable cells and MFI of calcein fluorescence, on the one hand, and the percentage of apoptotic or necrotic cells, on the other hand, provide a rapid and ideally adapted methodology for screening and evaluation of acrosinic natural inhibitors action in sperm cells preservation and for assessing the functional status of cells.
Keywords:	goat spermatozoa, Allium extracts, apoptosis, cell viability, flow cytometry
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*Correspondence:	Daniela BRATOSIN, National Institute for Biological Science Research and Development, Spl. Independentei nº 296, Bucharest, Romania, Tel/Fax: 40.21.2200881, E-mail: bratosind@yahoo.com