The signaling pathway leading to IICR at fertilization has been a central subject since the mid-80s. IP3 is produced by hydrolysis of membrane phosphatidylinositol 4,5-bisphosphate (PIP2) by the aid of phospholipase C (PLC), referred to as the PI pathway. The receptor/G protein /PI pathway or receptor/protein tyrosine kinase (PTK)/PI pathway is well known in a wide variety of somatic cells. Although there are surface proteins that mediate sperm-egg binding (Fig. 1), no evidence for their link to intracellular signaling has been obtained (12). In 1990, Swann (13) showed that injection of hamster sperm extract into an egg is capable of inducing Ca2+ oscillations similar to those seen at fertilization. In mouse eggs, Ca2+ spikes induced by hamster sperm extract have higher frequency than those at fertilization (Fig. 2B). Although there was some difference in the Ca2+ spike frequency, the finding lead to the sperm factor hypothesis that predicts a cytosolic sperm factor, Ca2+ oscillation-inducing protein (COIP), is introduced into the egg cytoplasm upon sperm-egg fusion (Fig. 1). The predicted COIP is very important because it implies the existence of the sperm-derived egg-activation factor. The idea was reinforced by the following results (11, 12): Ca2+ oscillations are induced in human eggs upon intracytoplasmic (single) sperm injection (14), ICSI, which is a current powerful therapy of sterility. Namely, fertilization is possible without any sperm-egg surface interaction, if an assisted reproduction technology is used. Upon in vitro fertilization by usual insemination, Ca2+ oscillations in mouse eggs begin a few minutes ‘after’ sperm-egg fusion takes place. Ca2+ oscillations are completely prevented in CD9-knockout mouse eggs in which sperm-egg fusion is defective while sperm-egg binding still occurs (15). Thus, cytoplasmic sperm-egg continuity is prerequisite for inducing the Ca2+ response at fertilization (Fig. 1).
Ca2+ oscillation-inducing sperm protein
Studies have focused on the identification of COIP since 1990 by isolation and purification from sperm extract, associated with bioassay of Ca2+ oscillationinducing activity by injection of a sperm protein fraction into eggs. Several groups including us have been engaged in this work for 10 years, but no decisive candidate of COIP has been identified by this approach (2). Another approach is based upon the supposition that any messenger system related to the PI pathway is likely involved. PLCs are prime candidates for COIP. Actually, PLCβ1, γ1, γ2, δ1, and δ4 are known to be expressed in the mammalian sperm (16). However, recombinant PLCβ1, γ1, γ2, and δ1 all failed to cause Ca2+ release in the egg cytoplasm or caused Ca2+ release only at extremely high doses. In 2002, Saunders et al. identified a novel isozyme of PLC, PLC-zeta, that is specifically expressed in the mouse sperm (17). They also presented that injection of cRNA encoding PLCζ into mouse eggs can produce fertilization-like Ca2+ oscillations and subsequent early embryonic development up to the balatocyst and that the expressed level of PLCζ for initiation of Ca2+ oscillations was comparable to the amount estimated to be contained in a single mouse sperm. Furthermore, the Ca2+ oscillationinducing activity of sperm extract is lost when PLCζ is immunodepleted from the sperm extract. Thus, PLCζ has received much attention as the putative COIP (16, 17). Ca2+ oscillation-inducing activity of PLCζ Recently, characteristics of PLCζ has been extensively examined in relation to a candidate of the sperm factor. PLCζ is the smallest PLC isozyme identified to date; it is composed of four EF-hand domains in the N-terminus, X and Y catalytic domains, and C2 domain in the C-terminus (Fig. 3A) common to other PLC isozymes, but lacks a N-terminal pleckstrin homology (PH) domain (17). We expressed PLCζ in mouse eggs by injection of cRNA encoding PLCζ fused with a fluorescent protein ‘Venus’ which enabled us to monitor the expression level and distribution of PLCζ in the egg (18). Fertilization-like Ca2+ oscillations appear at 30 – 40 min after RNA injection (Fig. 3B), when expressed PLCζ reached 10 – 40 × 10−15 g in the egg, comparable to the estimated content in a single sperm (18). The frequency of Ca2+ spikes is progressively increased (Fig. 3B) because PLCζ is continuously expressed in this experiment. The egg is activated by PLCζ-mediated Ca2+ oscillations and forms the (female) pronucleus (PN).
Kouchi et al. (19) succeeded in synthesizing functional PLCζ using baculovirus/Sf9-cell expression system. Microinjection of recombinant PLCζ protein into mouse eggs induced serial Ca2+ spikes (Fig. 2C) quite similar to those produced by injection of sperm extract (Fig. 2B). PLCζ is similar to PLCδ1 (38% identity and 49% similarity in 647 amino acid residues of PLCζ), although the PH domain is present in PLCδ1 but absent in PLCζ. Recombinant PLCδ1 induced Ca2+ oscillations as well, but 20-fold higher concentration was required, compared with PLCζ (19). Since PLCζ as well as PLCδ lacks a regulatory domain such as the G protein-binding site of PLCβ or the SH domain of PLCγ for phosphorylation by PTK, the activation mechanism of PLCζ and PLCδ is unknown. It is necessary to access how PLCζ undergoes the active state for production of IP3. It is generally known that the enzymatic activity of PLC is enhanced by an increase in [Ca2+]. In vitro assay of PIP2-hydrolyzing activity at various [Ca2+] (plotting the specific activity in terms of percentage to the maximal activity) revealed that recombinant PLCζ has a significant activity at [Ca2+] as low as 10 nM and 70% maximal activity at 100 nM [Ca2+] (19) that is usually the basal [Ca2+]i level of cells. PLCζ activity is maximal at 1 μM [Ca2+], which is the peak [Ca2+]i level of each Ca2+ spike during Ca2+ oscillations in fertilized mouse eggs. EC50 was 52 nM for PLCζ, whereas it was 5.7 μM for PLCδ1; PLCζ has approximately 100-fold higher Ca2+-sensitivity. Such high Ca2+-sensitivity of PIP2-hydrolyzing activity of PLCζ is an appropriate characteristic as the sperm factor that is driven into the egg at fertilization, first triggers Ca2+ release without any preceding [Ca2+]i rise, and maintains long-lasting Ca2+ oscillations after fertilization.
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