CONICET | Buscador de Institutos y Recursos Humanos

Exocytosis of cortical granules occurs in the egg a few minutes after the entry of the fertilizing sperm as part of ?egg activation? and it is involved in the block to polyspermy. In this regard, we have recently described for the first time the occurrence of compensatory endocytosis (CE) after massive cortical granule exocytosis, probably as a plasma membrane homeostasis mechanism. The aim of this work was to get further insights into the endocytic pathways involved in this internalization. CE was evaluated through pulse-chase experiments in SrCl2-activated eggs employing rhodamine-coupled Lens Culinaris Agglutinin (LCA), which binds to cortical granule exudate, in the presence of inhibitors of different endocytic pathways. We first observed that after treatment with SrCl2, cortical exudate in the egg membrane decreased significantly over time, consistent with an operating endocytic mechanism. By contrast, Ca2+ ionophore-activated eggs, in which CE is impaired, did not exhibit any reduction in cortical exudate after several hours, highlighting once again the mechanistic differences between activation methods. We next evaluated the role of actin filament dynamics in CE through the use of cytoskeleton disruptors (Cytochalasin D (10 μM), Latrunculin A (10 μM)) or a microfilament stabilizer (Jasplakinolide (0.5 μM) after SrCl2 activation. All of them produced a remarkable decrease in the relative amount of internalized LCA. Neither 5 μM filipin, which inhibits caveolae-mediated endocytosis, nor PitStop 2 (15 μM), a clathrin N-terminal domain blocker, produced a reduction in the internalized LCA-labeling compared to control. Finally, the addition of 80 μM dynasore, a dynamin inhibitor, produced a significant decrease in LCA internalization. These results indicate that CE in activated mouse eggs is not dependent on clathrin and caveolae, but on actin dynamics and dynamin activity.