Organisation und Dynamik der Phospholipide in der Zell- und Akrosommembran von Eberspermien während der Kapazitation und Akrosomreaktion

One of the essential qualities of cell membranes in Eucaryotae, including boar and trout sperm, is a stable transverse phospholipid asymmetry. Cytoplasmatic accumulation of the aminophospholipids, phosphatidylethanolamine and phosphatidylserine characterises this asymmetry and is a major prerequisite for cell homeostasis. It is regulated and maintained by ATP-dependent action of an aminophospholipid translocase. The plasma membranes of several mammalian cells show moreover lateral lipid domains, which are imputed to play a significant role in signal transduction. The transverse and lateral distribution of membrane lipids is of functional importance for numerous intraand intercellular interactions. Mammalian spermatozoa are highly specialised cells and their membranes undergo significant changes during genesis. The localisation and dynamics of phosphatidylserine in the cell as well as acrosome membranes of boar sperm cells was studied during capacitation and acrosome reaction to assess the relevance of lipid asymmetry for sperm function. The localisation of endogenous phosphatidylserine in morphologically differentiated cells was followed using the selective calcium depending binding of annexinV. Information on the transverse dynamics of phospholipids in the plasma membrane was obtained by labelling the cells with a NBD-phospholipid analogues. The morphological status of the cells was assessed by flow cytometry, fluorescence and electron microscopy. Propidium iodide was used to discriminate between living and dead cells. PNA, a lectin which binds selectively to the outer acrosome membrane, was applied to distinguish the membranes in the acrosome region of the sperm cell. The results of this study indicate both a transversal and lateral inhomogenous distribution of lipids in the cell membrane as well as in the outer acrosome membrane during sperm genesis. The plasma membrane of freshly ejaculated, liquid-preserved, boar sperm shows a stable transversal lipid asymmetry characterised among other things by an accumulation of phosphatidylserine in the cytoplasmic monolayer. Moreover a cytoplasmic localisation of phosphatidylserine on the outer acrosome membrane could be detected for the first time. Therefore the two facing cytoplasmic leaflets of the outer acrosome and cell membrane contain phosphatidylserine. Neither during capacitation nor after induction of acrosome reaction with calcium ionophore do living spermatozoa expose phosphatidylserine on their surface. The translocation of NBD-phosphatidylserine in living capacitated cells is certainly slowed down, but its transverse asymmetry in the cell membrane is maintained. Applying fluorescence and electron microscopy substantiated the hypothesis that there are close interactions between the cell membrane and the outer layer of the acrosome membrane because of capacitation. The cytoplasmic accumulation of phosphatidylserine in lateral lipid domains is probably essential for the strong association of plasma and outer acrosome membrane finally leading to local fusions of both membranes. In the postacrosomal region of the boar sperm cell the plasma membrane shows an excellent structural stability. Especially in this region an interaction between cytoplasmically accumulated phosphatidylserine and proteins and/or cytoskeletal elements may be attributed to its stabilisation. An indication for the functional meaning of lateral lipid domains in membranes of sperm cells was deduced from the mainly methodical works on preparations of so called “raft domains” in membranes of trout sperm cells. The isolation of a membrane fraction with increased cholesterol to phospholipid ratio and characteristic protein pattern served as a starting point for futher investigations on mammalian sperm cells. The involvement of lateral lipid domains in capacitation and acrosome reaction with regard to the occuring fusion processes and the recognition of the oocyte is of particular interest. The results of the present work substantiate the importance of the transverse phospholipid asymmetry in sperm cell function, during capacitation and acrosome reaction as well. The cytoplasmic localisation of phosphatidylserine could mediate the fusion between cell membrane and outer acrosome membrane during acrosome reaction.