Calcium pathway machinery at fertilization in echinoderms

Abstract

Calcium signaling in cells directs diverse physiological processes. The calcium waves triggered by fertilization is a highly conserved calcium signaling event essential for egg activation, and has been documented in every egg tested. This activity is one of the few highly conserved events of egg activation through the course of evolution. Echinoderm eggs, as well as many other cell types, have three main intracellular Ca(2+) mobilizing messengers - IP3, cADPR and NAADP. Both cADPR and NAADP were identified as Ca(2+) mobilizing messengers using the sea urchin egg homogenate, and this experimental system, along with the intact urchin and starfish oocyte/egg, continues to be a vital tool for investigating the mechanism of action of calcium signals. While many of the major regulatory steps of the IP3 pathway are well resolved, both cADPR and NAADP remain understudied in terms of our understanding of the fundamental process of egg activation at fertilization. Recently, NAADP has been shown to trigger Ca(2+) release from acidic vesicles, separately from the ER, and a new class of calcium channels, the two-pore channels (TPCs), was identified as the likely targets for this messenger. Moreover, it was found that both cADPR and NAADP can be synthesized by the same family of enzymes, the ADP-rybosyl cyclases (ARCs). In this context of increasing amount of information, the potential coupling and functional roles of different messengers, intracellular stores and channels in the formation of the fertilization calcium wave in echinoderms will be critically evaluated.

Figure 1. Calcium-release mechanisms in the endoplasmic reticulum (ER)

Ligand-binding: Specific ligands, IP3 and cADPR, bind to their channels (IP3 and RyR receptors, respectively), triggering Ca2+-release. Calcium-induced calcium release (CICR): IP3 and RyR receptors in the ER are biphasically regulated by Ca2+ concentrations - low calcium concentrations activate the channels, whereas higher concentrations inhibit the channels. Thus, calcium released from one receptor can diffuse to neighboring receptors, triggering further calcium release, forming a trail along a cluster of channels, and amplifying the original Ca2+ signal. Two-pool mechanism: Ca2+ released from one compartment may be taken up by a second compartment, which would overload and spontaneously release Ca2+.

Figure 2. ARC topological paradox

NAADP and cADPR are cytosolic messengers targeting channels in the ER (RyR receptors) or in acidic vesicles (TPCs). Paradoxically, ARCs in general, have a “targeted for secretion” signal peptide and are found in the plasma membrane or in the lumen of secretory vesicles (Type II transmembrane protein orientation). Putative nucleoside transporters would be responsible for transporting the messengers to the cytoplasm. In human lymphocytes, ARCs were also found in an alternative orientation (Type III), with the C-terminal facing the cytoplasm.

Figure 3. Calcium signaling events at fertilization in echinoderms - coupling of activity of multiple Ca2+ stores, channels and messengers

Sperm contact with the egg jelly triggers production of NAADP (1), which has been implicated in vesicle membrane fusion for the acrosomal reaction (2) and/or suggested to be delivered as the activating molecule to the eggs. Sperm-egg contact (3) has been shown to trigger a NAADP-dependent Ca2+ current that activate voltage-operated Ca2+ channels (VOC) in the membrane allowing Ca2+ influx for the cortical Ca2+ flash and the membrane depolarization responsible for the fast block to polyspermy (4). Coordinately, sperm egg interaction also triggers PLC activation and IP3 production, leading to IP3 receptors activation and amplification of the Ca2+ signal to form the wave by coupling with RyR receptors via CICR (5). The Ca2+ wave triggers the exocytosis of the cortical granules leading to the elevation of the fertilization envelope (FE) (6), which works as a shield for additional sperm, the slow block to polyspermy mechanism. Additionally, TPCs in acidic vesicles are found in the egg periphery and may play a role in local Ca2+ release events triggered by NAADP. ARCs are located in secretory vesicles, cortical granules and/or the plasma membrane, and may be responsible for differential production of either cADPR or NAADP.