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

KLINSKY LAHOZ, OMAR; WETTEN, PAULA A; BERBERIAN, VICTORIA ; MICHAUT, MARCELA A.

Salta

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

Theoocyte possesses a cell membrane named oolema that, in addition to the zonapellucida, protects the cytosol from the extracellular space and is essentialin the sperm-egg interaction during fertilization. Nevertheless, it can bebypassed by intracytoplasmic microinjection, a physical technique used to introducesperm or a number of different molecules into the oocyte cytosol. However, itis an invasive process because it implies the penetration of the plasmamembrane and the zona pellucida. Cell penetrating peptides (CPPs) have beenintroduced as novel biocarriers, since they are able to translocate the cellularmembranes without damaging it. These CPPs are small molecules composed ofpositive charged amino acids that can be attached to fluorophores, proteins ornanoparticles. CPPs are used as carriers or vectors to introduce these moleculesinto the cell. The mechanisms by which these permeable peptides manage to enterthe 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 thecytoplasm remains unknown. Therefore, the aim of this work was to study if a CPPis capable of penetrating the oocyte oolema, in order to determine if itconstitutes an alternative of intracytoplasmic microinjection for theintracellular delivery of different molecules. Thus, CF-1 mouse oocytes of two maturationstages, immature (Germinal Vesicle, GV) and mature (Metaphase II; MII) oocytes wereincubated in medium with increasing concentrations of an arginine-rich CPP,attached to a fluorophore. The incubation was carried out at different timesand different temperatures (4 °C and 37 °C). The cells were analyzed withconfocal microscopy and the fluorescence intensity was used to graph concentrationand time curves. Apparently, the MII oocytes incorporated CPP in aconcentration 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 oneincorporated more CPP than the second one, in all concentrations used during theincubation. These results show that CPP penetrates the oolema in bothmaturation stages and that the penetration was higher in GV oocytes.