TRPV3 channels mediate Ca²⁺ influx induced by 2-APB in mouse eggs

Abstract

Fertilization in mammals is initiated when a sperm fuses with a mature MII oocyte, also known as egg, and triggers a plethora of finely controlled processes identified as egg activation. The completion of all events of egg activation is driven by and depends on a series of repetitive calcium (Ca(2+)) increases (Ca(2+) oscillations), which rely on Ca(2+) influx from the extracellular media. Ca(2+) channels on the egg plasma membrane (PM) are thought to mediate this influx. The TRP Ca(2+) channel TRPV3 is differentially expressed during oocyte maturation, being most active at the MII stage. Specific stimulation of TRPV3 channels promotes Ca(2+) influx sufficient to induce egg activation and parthenogenesis. Here, we explore the function and distribution dynamics of the TRPV3 channel protein during maturation. Using dsRNA, TrpV3 overexpression, and inhibitors of protein synthesis, we modified the expression levels of the channel and showed that the TRPV3 protein is synthesized and translocated to the PM during maturation. We demonstrated that 2-APB at the concentrations used here to promote Ca(2+) influx in eggs, specifically and reversibly targets TRPV3 channels without blocking IP3R1. Finally, we found that the activity of TRPV3 channels is dependent upon an intact actin cytoskeleton, suggesting an actin-based regulation of its expression and/or function on the PM. Collectively, our results show TRPV3 is a target of 2-APB in eggs, a condition that can be used to induce parthenogenesis. The need of an intact actin cytoskeleton for the function of TRPV3 channels in oocytes is a novel finding and suggests the rearrangements of actin that occur during maturation could regulate both the presence on the PM and/or the function of TRPV3 and of other Ca(2+) channels involved in oocyte maturation and fertilization.

Keywords: 2-APB; Actin cytoskeleton; Ca(2+) influx; IP(3)R1 receptor; TRPV3 channel.