Kinetische Untersuchungen zur in vitro Kapazitation und Zona pellucida-induzierte Akrosomreaktion am Eberspermatozoon

The aim of this study was to characterize quantitatively the main physiological processes related to the capacitation of boar spermatozoa and order them sequentially. Furthermore, it should be tested whether the modification of zona pellucida during in vitro maturation of oocytes affects the capability of zona pellucida to induce acrosome reaction. In order to perform the kinetic characterization of the capacitation process, the functional changes in diluted and 24-h stored spermatozoa have been observed for 180 minutes during incubation under capacitating conditions. The dynamic changes in the cytosolic calcium ion content, in the overall  phosphorylation of the tyrosine residues of sperm proteins und in the spontaneous acrosome reaction were examined in live sperm suspensions by means of flow cytometry. For investigation of effect of maturation state of oocytes on the induced acrosome reaction, zonae pellucidae were collected from ovaries of gilts slaughtered at the local abattoir. Then zona pellucida was either isolated directly from the collected oocytes (GV) or after in vitro maturation of oocytes (48 h). The experiments were organized in two sections: First, the effect of zona pellucida glycoproteins on porcine spermatozooa under capacitating conditions was examined using a flow cytometer. As marker were used the cytosolic concentration of calcium ions, the phosphorylation of tyrosine residues of sperm proteins and the induced acrosome reaction. Subsequently, the effects of intact zona pellucida from oocytes at different state of maturation were examined on the time course of acrosome reaction and on the changes in the cytosolic calcium ion content in frozen-thawed spermatozoa. The investigation of acrosome reaction process on the intact zona pellucida was performed by means of confocal laser scanning microscope. Following results were obtained: 1.        The dynamic flow cytometric analysis supports the notion that the sequence of processes related to the in vitro capacitation of boar spermatozoa is specifically ordered. The markedly increase in cytosolic calcium ion content is followed by the massive overall rise in protein phosphorylation of tyrosine residues in live sperm suspensions. These both events precede the initiation of spontaneous acrosome reaction and appear therefore to be related to the capacitation process.  2.        The effect of zona pellucida on the processes occurring during capacitation in sperm populations depends on the maturation state of the oocytes. Zonae pellucidae collected from in vitro matured oocytes induced an acceleration of the examined processes. 3.        Within used experimental system, intact zonae pellucidae had a stronger effect on the examined processes in cryopreserved spermatozoa than solubilized zona pellucida glycoproteins in liquid-preserved spermatozoa. These results let suggest that the biochemical and structural modifications of the zona pellucida occurring during in vitro maturation correlate with the functional properties of the zona pellucida. Due to the apparent amplified effects of the intact zona pellucida on the capacitation-related processes in comparison with the effects of zona pellucida glycoproteins it appears that the three-dimensional structure of the ligands of the zona pellucida is another key factor besides the maturation-conditioned modification. The role of in vivo maturation of the oocyte on the functionality of zona pellucida has to be clarified in further studies on zona pellucida and on zona pellucida-glycoproteins.