Recombinant Phospholipase Cζ Has High Ca2+ Sensitivity and Induces Ca2+ Oscillations in Mouse Eggs

Abstract

] i rises are a pivotal signal for egg activation and are responsible for early embryogenesis (2, 3). Accumulated evidence suggests that Ca 2ϩ oscillations are induced by cytosolic sperm factor introduced into the ooplasm upon sperm-egg fusion (2, 4). Therefore, the identification of the Ca 2ϩ oscillation-inducing sperm factor, which is the egg-activating sperm factor, is currently being studied as a central subject to elucidate the mechanisms of fertilization. Recently, Saunders et al.(5) have reported a novel type of PLC (the enzyme that produces InsP 3 and diacylglycerol from membrane PtdInsP 2 ), PLC, which is specifically expressed in mammalian sperm. The injection of RNA-encoding PLC into mouse eggs causes Ca 2ϩ oscillations and subsequent early embryonic development by expressed PLC at an estimated level comparable with the content in a single sperm (5). The Ca 2ϩ oscillation-inducing activity of sperm extract (4, 6) is lost when pretreated with an antibody against PLC (5). Thus, PLC is considered a strong candidate of the sperm factor. To assure this possibility, it is primarily necessary to examine whether purified PLC protein induces Ca 2ϩ oscillations in the egg. It has been shown that PLC␤1 (7), -␥1 (8, 9), -␥2 (8), -␦1 (10), and -␦4 (7) are expressed in mammalian sperm and that recombinant PLC␤1, -␥1, -␥2, and -␦1 failed to cause Ca 2ϩ release in the ooplasm (11). PLC is the smallest PLC identified to date, lacking the N-terminal PH domain (Fig. 1A) (5) that is found in all isoforms of PLC␤, -␥, and -␦ and is the site for interaction with membrane phospholipids (12). Because PLC as well as PLC␦ lacks a regulatory domain such as the G protein-binding site of PLC␤ or the SH domain of PLC␥ for phosphorylation by tyrosine kinase, the activation mechanism of PLC and PLC␦ is unknown. Therefore, it is also necessary to access how PLC undergoes the active state for production of InsP 3 . Here we first show that recombinant PLC protein induces Ca 2ϩ oscillations in mouse eggs and that PLC possesses an extremely high Ca 2ϩ sensitivity in the PtdInsP 2 hydrolyzing activity to be active even at the resting state of cells. EXPERIMENTAL PROCEDURESCloning of PLC and PLC␦1-cDNA encoding full-length PLC (GenBank TM accession number AF435950) was cloned from a cDNA library originated from mouse testis mRNAs. PLC cDNA was amplified by PCR using Pfu polymerase and the following primers: forward, 5Ј-GAC AAGCGGCCCAGATCATG-3Ј; internal forward primer involving an EcoRI site, 5Ј-GGAATTCATATGGAAAGCCAACTTCATGAG-3Ј; re-* This work was supported by a grant-in-aid for general scientific research (Category B) (to S. M.) from the Japan Ministry of Education, Science, Sports, and Culture. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.The nucleotide sequence (s)

The Role of EF-hand Domains and C2 Domain in Regulation of Enzymatic Activity of Phospholipase Cζ

“…We have recently shown (7) that the PI(4,5)P 2 -hydrolyzing activity of recombinant PLC is ϳ100-fold more sensitive to Ca 2ϩ than that of PLC␦1 and is 70% maximal at 100 nM [Ca 2ϩ ], which is usually the basal [Ca 2ϩ ] i of cells. Recombinant PLC has ϳ20-fold higher Ca 2ϩ oscillation-inducing activity than PLC␦1 (7). Thus, PLC possesses remarkable properties that are favorable for the sperm factor to initiate (7) and maintain (3) Ca 2ϩ oscillations via continuously produced IP 3 .…”

“…Ca 2ϩ oscillations similar to those at fertilization are produced by PLC expressed in a mouse egg at an estimated level comparable with the content in a single mouse sperm (1,2). Injection of recombinant PLC into mouse eggs induces Ca 2ϩ oscillations as well (7). The Ca 2ϩ oscillation-inducing activity of sperm extract (4,8) is lost when the extract is pretreated with an antibody against PLC (1).…”

Regulation of Phospholipase C-δ1 through Direct Interactions with the Small GTPase Ral and Calmodulin

“…The sperm-specific PLC-isoform was found to trigger fertilizationlike Ca 2ϩ oscillations in eggs (4). In vitro measurements demonstrated that, although enzymatic activity is lower, PLC-has a 100-fold greater sensitivity to Ca 2ϩ indicating that this isoform is active at Ca 2ϩ concentrations equivalent to those in cells at the resting state (12). Recently, two groups independently cloned novel PLC-isoforms that are activated in a calcium dependent fashion (4,13).…”

Ca2+-independent Binding of Anionic Phospholipids by Phospholipase C δ1 EF-hand Domain

“…A splicing variant of PLC lacking 110 residues from the N terminus corresponding to the EF-hands 1-3 but identical to PLC in EF4 and the succeeding region failed to elicit a Ca 2ϩ spike (36). This indicates that the EF-hand domain is important for PLC to induce Ca 2ϩ oscillations (which presumably requires correct membrane binding).…”