Fluorescent Analysis of Boar Sperm Capacitation Process In Vitro.

Abstract

Capacitation involves physiological changes that spermatozoa must undergo in the female reproductive tract or in vitro to obtain the ability to bind, penetrate and fertilize the egg. Up to date, several methods have been developed to characterize this complex biological process. The goal of the presented study is to mutually compare several fluorescent techniques and determine their ability to detect changes in molecular processes during the capacitation progress and their ability to determine the real, physiological status of capacitated sperm. The capacitation process was analyzed using four fluorescent techniques (chlortetracycline (CTC) staining, anti-acrosine antibody (ACR.2) assay, anti-phosphotyrosine (pY) antibody assay and fluorescein isothiocyanate-conjugated phalloidine (FITC-phall) assay). All these methods were used in fluorescent microscopy and flow cytometry experiments. According to our results, all selected methods are capable to detect the capacitation progress of boar sperm in vitro, but there are significant differences in this ability in fluorescent microscopy and flow cytometry experimental arrangements as well as in the ability to determine the physiological status of capacitated sperm among the selected methods. All advantages and drawbacks of the selected methods are extensively discussed. Our study thus further contributes to better characterization and understanding of an important step in mammalian reproduction - capacitation. The work was supported by the Grants of the Ministry of Education of the Czech Republic Nos. VC 1M06011 and VZ 0021620828, the Grant Agency of the Czech Republic Nos. 523/08/H064 and 523/09/1793, and by the Institutional Research Support AVOZ 50520701. (poster)