Two-pore channels function in calcium regulation in sea star oocytes and embryos

Egg activation at fertilization is an excellent process for studying calcium regulation. Nicotinic acid adenine dinucleotide-phosphate (NAADP), a potent calcium messenger, is able to trigger calcium release, likely through two-pore channels (TPCs). Concomitantly, a family of ectocellular enzymes, the ADP-ribosyl cyclases (ARCs), has emerged as being able to change their enzymatic mode from one of nucleotide cyclization in formation of cADPR to a base-exchange reaction in the generation of NAADP. Using sea star oocytes we gain insights into the functions of endogenously expressed TPCs and ARCs in the context of the global calcium signals at fertilization. Three TPCs and one ARC were found in the sea star (Patiria miniata) that were localized in the cortex of the oocytes and eggs. PmTPCs were localized in specialized secretory organelles called cortical granules, and PmARCs accumulated in a different, unknown, set of vesicles, closely apposed to the cortical granules in the egg cortex. Using morpholino knockdown of PmTPCs and PmARC in the oocytes, we found that both calcium regulators are essential for early embryo development, and that knockdown of PmTPCs leads to aberrant construction of the fertilization envelope at fertilization and changes in cortical granule pH. The calcium signals at fertilization are not significantly altered when individual PmTPCs are silenced, but the timing and shape of the cortical flash and calcium wave are slightly changed when the expression of all three PmTPCs is perturbed concomitantly, suggesting a cooperative activity among TPC isoforms in eliciting calcium signals that may influence localized physiological activities.