Compensatory endocytosis occurs after cortical reaction during mouse egg activation

COHEN DJ; GOMEZ ELIAS M; CUASNICU PS

Congreso; XVIII Jornadas Multidisciplinarias de la Sociedad Argentina de Biología; 2016

COMPENSATORYENDOCYTOSIS OCCURS AFTER CORTICAL REACTION DURING MOUSE EGG ACTIVATIONGómez Elías MD, Cuasnicú PS, Cohen DJ; IBYME-CONICET. (mati.gomezelias@gmail.com)The fertilizingsperm triggers in the egg a series of events collectively referred to as ?eggactivation?. Among them, massive exocytosis of cortical granules occurs a fewminutes after fertilization and is involved in the block to polyspermy. It hasbeen described that after exocytotic activity cells maintain their surface areaby triggering compensatory endocytosis (CE) mechanisms, with the retrieval ofexcess surface membrane. In particular, CE occurring in sea urchin eggs isessential to maintain embryonic cell surface homeostasis during earlydevelopment. Therefore, the aim of this work was to evaluate the occurrence ofCE as a consequence of the cortical exocytosis in mouse eggs. For this purpose,we developed pulse-chase experiments employing rhodamine-coupled Lens CulinarisAgglutinin (LCA), which binds to cortical granule exudate. First, zona-freemouse eggs were activated by different agents, and incubated at 4°C for 30? in the presence of LCA(pulse). Then, cells were incubated for 30? at 4°C or 37°C to allow the internalization of membrane-attachedLCA (chase) and analyzed by confocal microscopy. Results showed a massive labelinginternalization in Sr2+-activated eggs when chasing at 37ºC. When the assay wasperformed in the presence of different CE inhibitors (25nM staurosporine or 1uMcyclosporin A) a significant decrease in the internalized labeling was observed.Interestingly, activation with A23187 ionophore did not induce labelinginternalization after exocytosis, highlighting once again the mechanisticdifferences between activation methods. Finally, fluorescence internalizationwas also observed in in vitrofertilized eggs. Altogether, these results demonstrate for the first time theoccurrence of a CE mechanism after massive cortical granule exocytosis, as aconsequence of egg activation.