The initial interaction between gametes takes place at the level of the sperm surface and the zona pellucida (ZP), the extracellular matrix of the egg in mammals. Successful fertilization requires the proper molecular recognition of the ZP by the sperm. Recently, human ZP was demonstrated to be composed of four proteins: ZP1, ZP2, ZP3, and ZP4. The goals of this study were to determine the effects of recombinant human ZP2, ZP3, and ZP4 on human sperm acrosomal exocytosis and sperm motility. Exposure of sperm to ZP proteins, alone or in combination, promoted acrosomal exocytosis in a time-dependent manner. This effect occurred in parallel with a considerable decrease in progressive motility, coincident with an increase in nonprogressive sperm motility. An analysis of kinetic parameters of ZP-treated sperm demonstrated that a characteristic motility pattern could be defined by values of curvilinear velocity > 63.9 mum/s and linearity

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BackgroundThe mammalian oocyte extracellular matrix known as the zona pellucida (ZP) acts as a barrier to accomplish sperm fusion with the female gamete. Although penetration of the ZP is a limiting event to achieve fertilization, this is one of the least comprehended stages of gamete interaction. Even though previous studies suggest that proteases of sperm origin contribute to facilitate the passage of sperm through the ZP, in human this process is not yet fully understood. The aim of this study was to determine the ability of human sperm to degrade recombinant human ZP (rhZPs) proteins and to characterize the proteases involved in this process.MethodsPurified rhZP2, rhZP3 and rhZP4 proteins were incubated with capacitated sperm and the proteolytic activity was determined by Western blot analysis. To further characterize the proteases involved, parallel incubations were performed in the presence of the protease inhibitors o-phenanthroline, benzamidine and MG-132 meant to block the activity of metalloproteases, serine proteases and the proteasome, respectively. Additionally, protease inhibitors effect on sperm-ZP binding was evaluated by hemizona assay.ResultsThe results showed that rhZPs were hydrolyzed in the presence of capacitated sperm. O-phenanthroline inhibited the degradation of rhZP3, MG-132 inhibited the degradation of rhZP4 and benzamidine inhibited the degradation of the three proteins under investigation. Moreover, hemizona assays demonstrated that sperm proteasome inhibition impairs sperm interaction with human native ZP.ConclusionsThis study suggests that sperm proteasomes could participate in the degradation of ZP, particularly of the ZP4 protein. Besides, metalloproteases may be involved in specific degradation of ZP3 while serine proteases may contribute to unspecific degradation of the ZP. These findings suggest that localized degradation of ZP proteins by sperm is probably involved in ZP penetration and may be of help in understanding the mechanisms of fertilization in humans.

Background: Expression and activity of the potassium channel ether-à-go-go-1 (EAG1) are strongly related to carcinogenesis and tumor progression, which can be exploited for therapeutic purposes. EAG1 activity may be reduced by preventing its phosphorylation with epidermal growth factor receptor (EGFR) kinase inhibitors and by astemizole, which blocks the channel pore and downregulates its gene expression. Objective: We aimed to study the potential cooperative antiproliferative effect of the EGFR inhibitor gefitinib and the EAG1-blocker astemizole, in breast cancer cells. Materials and Methods: The cells were characterized by immunocytochemistry. Inhibitory concentrations were determined by non-linear regression analysis using doseresponse curves. The nature of the pharmacological effect was evaluated by the combination index equation while cell cycle analysis was studied by flow cy tometry. Results: Astemizole and gefitinib inhibited cell proliferation in a concentration-dependent manner, with inhibitory concentrations (IC 50) values of 1.72 µM and 0.51 µM, respectively. All combinations resulted in a synergistic antiproliferative effect. The combination of astemizole and gefitinib diminished the percentage of cells in G2/M and S phases, while increased accumulation in G0/G1 of the cell cycle. Conclusions: Astemizole and gefitinib synergistically inhibited proliferation in breast cancer cells expressing both EGFR and EAG1. Our results suggest that the combined treatment increased cell death by targeting the oncogenic activity of EAG1.