Phosphatidylserine Exposure During Mouse Egg Activation: Calcium Requirements And Correlation With Compensatory Endocytosis

CUASNICÚ, PATRICIA S.; COHEN, DÉBORA J.; GÓMEZ ELÍAS, MATÍAS D.

Holderness NH

Congreso; Gordon Research Conference on Fertilization & Activation of Development (GRC); 2017

The fertilizing sperm triggers in the egg a series of events collectively referred to as ?egg activation?. Among these events, we have recently described the occurrence of a transient exposure of phosphatidylserine (PS) in the plasma membrane of fertilized or Sr2+-activated mouse eggs, which is not associated with apoptosis. This event has shown to be a consequence of intracellular Ca2+ increase, so we next evaluated the source of calcium involved in PS exposure. Only those compounds which evoked Ca2+ influx from the extracellular medium, as Sr2+, ethanol or 2-APB, induced PS translocation, whereas this effect was not observed after incubation with Ca2+ ionophore A23187 or thimerosal in a Ca2+-free medium. In order to evaluate whether the cytoskeleton modifications that take place in the egg during activation could influence PS exposure, treatment with Sr2+ was performed after incubation with Cytochalasin D (CytD), which disrupts actin polymerization, or Jasplakinolide (Jas), which stabilizes actin microfilaments. While PS exposure occurred normally in CytD-pretreated eggs, pretreatment with Jas prevented the mobilization of this phospholipid, indicating that this is an actin-dependent process. Finally, we attempted to elucidate the functional role of this event for the activation of development. In this regard, it has been reported that after extensive exocytotic activity in neuroendocrine cells there is a PS exposure in their plasma membrane that would be responsible for compensatory endocytosis (CE) mechanisms essential to maintain cell surface homeostasis (Ory et al., 2013). Based on this, we first evaluated if CE occurred in mouse eggs after massive exocytosis of cortical granules. For this purpose, we developed pulse-chase experiments employing rhodamine-coupled Lens culinaris agglutinin (LCA), which binds to cortical granule exudate. We noticed that fertilization or a short-time activation with Sr2+, enough for cortical exocytosis to occur, triggered in the egg the internalization of LCA, which was partially impaired by known CE inhibitors (Staurosporine and Cyclosporin A), thus demonstrating for the first time the existence of this mechanism in the mouse egg. Interestingly, CE was not detected when activating eggs with Ca2+ ionophore A23187, suggesting a possible connection between PS exposure and CE. Altogether, we have described two novel events occurring during mouse activation that could be interconnected and important for embryonic cell surface homeostasis.