PHOSPHATIDYLSERINE EXPOSURE DURING MOUSE EGG ACTIVATION: CALCIUM REQUIREMENTS AND CORRELATION WITH COMPENSATORY ENDOCYTOSIS

CUASNICU PS; GOMEZ ELIAS M; COHEN DJ

Congreso; Gordon Research Conferences on Fertilization and Activation of Development; 2017

The fertilizing sperm triggers inthe egg a series of events collectively referred to as ?egg activation?. Amongthese events, we have recently described the occurrence of a transient exposureof phosphatidylserine (PS) in the plasma membrane of fertilized or Sr2+-activatedmouse eggs, which is not associated with apoptosis. This event has shown to bea consequence of intracellular Ca2+ increase, so we next evaluatedthe source of calcium involved in PSexposure. Only those compounds which evoked Ca2+ influx from theextracellular medium, as Sr2+, ethanol or 2-APB, induced PStranslocation, whereas this effect was not observed after incubation with Ca2+ionophore A23187 or thimerosal in a Ca2+-free medium. In order toevaluate whether the cytoskeleton modifications that take place in the eggduring activation could influence PS exposure, treatment with Sr2+was performed after incubation with Cytochalasin D (CytD), which disrupts actinpolymerization, or Jasplakinolide (Jas), which stabilizes actin microfilaments.While PS exposure occurred normally in CytD-pretreated eggs, pretreatment withJas prevented the mobilization of this phospholipid, indicating that this is anactin-dependent process. Finally, we attempted to elucidate the functional roleof this event for the activation of development. In this regard, it has beenreported that after extensive exocytotic activity in neuroendocrine cells thereis a PS exposure in their plasma membrane that would be responsible forcompensatory endocytosis (CE) mechanisms essential to maintain cell surface homeostasis.Based on this, we first evaluated if CE occurred in mouse eggs after massiveexocytosis of cortical granules. For this purpose, we developed pulse-chaseexperiments employing rhodamine-coupled Lensculinaris agglutinin (LCA), which binds to cortical granule exudate. Wenoticed that fertilization or a short-time activation with Sr2+,enough for cortical exocytosis to occur, triggered in the egg theinternalization of LCA, which was partially impaired by known CE inhibitors(Staurosporine and Cyclosporin A), thus demonstrating for the first time theexistence of this mechanism in the mouse egg. Interestingly, CE was not detectedwhen activating eggs with Ca2+ ionophore A23187, suggesting a possibleconnection between PS exposure and CE. Altogether, we have described two novelevents occurring during mouse activation that could be interconnected and importantfor embryonic cell surface homeostasis.