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

KLINSKY LAHOZ OG; WETTEN PAULA; ZANNI RUIZ EMILIA.; PAVAROTTI MARTÍN A; BERBERIAN MV; MICHAUT MA

Congreso; LVII Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research (SAIB); 2021

SAIB - SAMIGE

The cortical reaction in oocytes is a fundamental process that occurs during gamete fusion at fertilization. It entails theexocytosis of cortical granules and is involved in blocking polyspermy. There are multiple proteins that intervene in the fusionof cortical granules and plasma membrane, among which proteins of the VAMP family can be mentioned. It has been provenin 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 dueto characteristics of their chemical nature. They have received attention as biotechnological tools because of their ability totransport 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 ofthis work was 1) to study if a permeable tetanus toxin (p-TxTe) was able to cross the zona pellucida and the membrane ofmature oocytes (MII), and 2) to analyze if this toxin might inhibit the cortical granule exocytosis (CGE). Permeable TxTebound 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, at37°C. Then, a group of oocytes was fixed and treated with primary and secondary antibodies to perform an indirectimmunofluorescence assay (IFI). From the analysis of cells by confocal microscopy, it was determined that p-TxTe was ableto translocate into the oocytes, through the zona pellucida and the plasma membrane. In turn, to analyze if CGE had beenaffected by incubation in TxTe, oocytes were parthenogenetically activated by strontium chloride (SrCl2). Compared withcontrol oocytes, oocytes treated with p-TxTe were not able to respond to the activator. These results show that p-TxTe inhibitedcortical granules exocytosis and that permeable components can be used as biotechnological tools to deliver useful moleculesinto the oocytes.