Protease-activated receptor 2 links protease activity with calcium waves during egg activation and blastomere cleavage

Successful initiation of animal development requires activation of the egg immediately prior to fusion of gamete pronucleii. In all taxa, this is initiated by waves of calcium transients which transverse across the egg. Calcium waves also occur at cleavage furrows during later blastula cytokinesis. Calcium is released from the endoplasmic reticulum through activation of 1,4,5-trisphosphate (IP3) receptors. Only a subset of the mechanisms employed to generate IP3 during vertebrate egg activation are defined, with strong evidence that other critical mechanisms exist. Serine proteases have been long implicated in egg activation and fertilisation. Here we report that treatment of zebrafish eggs with serine protease inhibitors leads to defective calcium wave propagation and failed egg activation. We further show that mutation of zebrafish Protease-activated receptor 2a (Par2a) also results in severe disruption of egg activation, leading to failed chorion elevation and ooplasmic segregation. Milder par2a mutants progress further, but then show abnormal blastomere cleavage. We observed that par2a mutants show decreased amplitude and duration of calcium transients. Restoring Ca++ or direct injection of IP3 ligand rescues egg activation aborted by either serine protease inhibitor treatment or by mutation of Par2a. We thus show that serine protease activity is a critical regulator of IP3 and subsequent calcium wave amplification during zebrafish egg activation, and link this to intracellular calcium release via the protease receptor, Par2a. This constitutes a novel signalling pathway critical for successful fertilisation. Significance StatementBoth sperm and egg must undergo a series of important steps to become competent for successful fertilisation. Defining these steps is central to our understanding of reproductive biology and our ability to improve fertility treatments. As the process of gamete maturation and fertilisation has highly conserved principles across the animal kingdom, there are also important implications for aqua- and agriculture. One of the first signalling events of your life leads to the release of bursts of calcium in the egg. We know the importance of this for fertilisation but have only a partial picture of how this occurs. Our work here, using fish genetics, identifies a new signalling pathway regulating these first important flashes of calcium in the egg.