A PERMEABLE TETANUS TOXIN IS CAPABLE OF TRANSLOCATING INTO THE OOCYTES AND ALTER THE CORTICAL GRANULES EXOCYTOSIS

KLINSKY LAHOZ O.G.; WETTEN P.A.; ZANNI RUIZ E.; PAVAROTTI M.; BERBERIAN M.V.; MICHAUT M.A.

Congreso; LVII Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research (SAIB) and XVI Annual Meeting of the Argentinean Society for General Microbiology (SAMIGE); 2021

The cortical reaction in oocytes is a fundamental process that occurs during gamete fusion at fertilization. It entails the exocytosis of cortical granules and is involved in blocking polyspermy. There are multiple proteins that intervene in the fusion of cortical granules and plasma membrane, among which proteins of the VAMP family can be mentioned. It has been proven in our laboratory that by microinjecting tetanus toxin (TxTe), VAMP proteins are cleaved, and cortical granules exocytosis (CGE) can be avoided. On the other hand, cell penetration components are molecules that can cross plasma membranes due to characteristics of their chemical nature. They have received attention as biotechnological tools because of their ability to transport useful substances to cells. However, the ability of these components to enter the cytoplasm of oocytes is unknown. To determine if permeable components can be used as biotechnological tools to deliver molecules into the oocytes, the aim of this work was 1) to study if a permeable tetanus toxin (p-TxTe) was able to cross the zona pellucida and the membrane of mature oocytes (MII), and 2) to analyze if this toxin might inhibit the cortical granule exocytosis (CGE). Permeable TxTe bound to a cell-penetrating peptide with multiple arginine residues was purified from transformed bacteria Escherichia coli. MII oocytes were collected from hormonally stimulated female CF-1 mice, and incubated in p-TxTe at different times, at 37 ° C. Then, a group of oocytes was fixed and treated with primary and secondary antibodies to perform an indirect immunofluorescence assay (IFI). From the analysis of cells by confocal microscopy, it was determined that p-TxTe was able to translocate into the oocytes, through the zona pellucida and the plasma membrane. In turn, to analyze if CGE had been affected by incubation in TxTe, oocytes were parthenogenetically activated by strontium chloride (SrCl2). Compared with control oocytes, oocytes treated with p-TxTe were not able to respond to the activator. These results show that p-TxTe inhibited cortical granules exocytosis and that permeable components can be used as biotechnological tools to deliver useful molecules into the oocytes.