IS IT POSSIBLE TO USE A CELL-PENETRATING PEPTIDE AS A VECTOR TO THE INTRACELLULAR DELIVERY OF MOLECULES INTO THE OOCYTE?

KLINSKY LAHOZ, OG; MICHAUT, MA; BERBERIAN, V; BERBERIAN, V; WETTEN, PA; WETTEN, PA; KLINSKY LAHOZ, OG; MICHAUT, MA

Salta

Congreso; Joint LV Annual SAIB Meeting and XIV PABMB Congress; 2019

The oocyte possesses a cell membrane named oolema that, in addition to the zona pellucida, protects the cytosol from the extracellular space and is essential in the sperm-egg interaction during fertilization. Nevertheless, it can be bypassed by intracytoplasmic microinjection, a physical technique used to introduce sperm or a number of different molecules into the oocyte cytosol. However, it is an invasive process because it implies the penetration of the plasma membrane and the zona pellucida. Cell penetrating peptides (CPPs) have been introduced as novel biocarriers, since they are able to translocate the cellular membranes without damaging it. These CPPs are small molecules composed of positive charged amino acids that can be attached to fluorophores, proteins or nanoparticles. CPPs are used as carriers or vectors to introduce these molecules into the cell. The mechanisms by which these permeable peptides manage to enter the cell depend on the concentration of CPP and the incubated cellular type. However, the capacity of the oocyte to allow or deny the entrance of CPPs into the cytoplasm remains unknown. Therefore, the aim of this work was to study if a CPP is capable of penetrating the oocyte oolema, in order to determine if it constitutes an alternative of intracytoplasmic microinjection for the intracellular delivery of different molecules. Thus, CF-1 mouse oocytes of two maturation stages, immature (Germinal Vesicle, GV) and mature (Metaphase II; MII) oocytes were incubated in medium with increasing concentrations of an arginine-rich CPP, attached to a fluorophore. The incubation was carried out at different times and different temperatures (4 °C and 37 °C). The cells were analyzed with confocal microscopy and the fluorescence intensity was used to graph concentration and time curves. Apparently, the MII oocytes incorporated CPP in a concentration and time-independent manner, at 4 °C and 37 °C. On the other hand, the penetration of peptide into GV oocytes was concentration and time-dependent, only at 37 °C. The comparison of GV and MII oocyte showed that the first one incorporated more CPP than the second one, in all concentrations used during the incubation. These results show that CPP penetrates the oolema in both maturation stages and that the penetration was higher in GV oocytes.