Studien zur Aufklärung des Funktionsmechanismus der Akrosomenreaktion in Spermien: Das Multi-PDZ-Domänen Protein MUPP1 als molekularer Organisator beteiligter Signaltranduktionsmoleküle

The acrosome reaction in sperm is a special type of the calcium regulated exocytosis in somatic cells. It is induced by the binding of the sperm to the zona pellucida of the oocyte. During acrosome reaction the acrosomal vesicle which is filled with hydrolysing enzyms fuses with the overlying plasma membrane by many fusion pores leading to the release of the acrosomal content. This process can be subdivided in two sequential reactions. First the binding of the sperm to the zona pellucida induces a sustained increase in the cytosolic calcium which leads to the early processes of fusion like the tethering, docking and priming of the acrosomal vesicle. Afterwards a efflux of Calcium form the acrosome mediates the final processes leading to the fusion pore building. Recently it was shown that the acrosomal exocytosis is comparable to the vesicle fusion at the synapse also mediated by the SNARE fusionmachinery. However one distinction has to be drawn concerning the fact that during acrosome reaction many fusion pores are formed simultaneously. The molecular mechanisms mediating this are mostly unkown. Within my PhD thesis I was able to show that the multi-PDZ-domain protein 1 (MUPP1) is localized to the acrosomal region of mammalian sperm. Therefore and because of its 13 PDZ domains it is a good candidate to organize signaling molecules participating in acrosome reaction. To check this hypothetis I analysed wether MUPP1 is functional involved in acrosome reactio by incubating permeablized mouse sperm with MUPP1 antibodies. The results show that the antibodies significantly inhibit the induced acrosome reaction. To identify potential new interaction partners of MUPP1 I performed a yeast two hybrid screen with a mouse testis cDNA library. Thereby I was able to identify the glutamte receptor interacting protein (GRIP1) as a good candidate. It was already shown that it forms a complex with the PLC 4 which is a key enzym of acrosomal exocytosis. Besides the Calcium/Calmodulin kinase II (CaMKII) was identified as a interacting partner of MUPP1. It is comparable to MUPP1 localized to the acrosomal region of sperm as well as to lipid rafts. Functional studies further show that a inhibition of the kinase leads to a potentiation of the spontaneous acrosome reaction of the sperm. This indicates that the kinase functioned like a clamp inhibiting spontaneous acrosome reaction in unstimulated sperm.