The Role of X/Y Linker Region and N-terminal EF-hand Domain in Nuclear Translocation and Ca2+ Oscillation-inducing Activities of Phospholipase Cζ, a Mammalian Egg-activating Factor

Abstract

Sperm-specific phospholipase C-zeta (PLC) causes intracellular Ca2؉ oscillations and thereby egg activation and is accumulated into the formed pronucleus (PN) when expressed in mouse eggs by injection of cRNA encoding PLC, which consists of four EF-hand domains (EF1-EF4) in the N terminus, X and Y catalytic domains, and C-terminal C2 domain. Those in the N terminus of EF1. Ca 2؉ oscillation-inducing activity was lost by the former mutation and was remarkably inhibited by the latter. A short sequence 374 -383 fused with Venus showed active translocation into the nucleus of COS-7 cells, but 296 -309 or 1-19 did not. Despite the presence of these special regions, both activities were deprived by deletion of not only EF1 but also EF2-4 or C2 domain. Thus, PLC is driven into the nucleus primarily by the aid of NLS and putative regulatory sites, but coordinated three-dimensional structure, possibly formed by a folding in the X/Y linker and close EF/C2 contact as in PLC␦1, seems to be required not only for enzymatic activity but also for nuclear translocation ability. PLC2 is a novel isozyme of PLC (the enzyme that hydrolyzes membrane PIP 2 into IP 3 and diacylglycerol) and a strong candidate of the mammalian sperm-derived egg-activating factor (1). PLC is specifically expressed in the sperm (2) and induces repetitive increase in [Ca 2ϩ ] i called Ca 2ϩ oscillations and subsequent early embryonic development when expressed in mouse eggs by injection of RNA encoding PLC (2, 3). In mammalian fertilization, accumulated evidence indicates that a cytosolic sperm factor is driven into the ooplasm upon sperm egg fusion and induces Ca 2ϩ oscillations (4, 5), which are caused by Ca 2ϩ release from the endoplasmic reticulum mainly through type 1 IP 3 receptor (6) and are a pivotal signal for egg activation characterized by resumption of the second meiosis and formation of PN (5). PLC is a strong candidate of the sperm factor, because 1) fertilization-like Ca 2ϩ oscillations are produced by PLC expressed in a mouse egg at an estimated level comparable to the content in single mouse sperm (2, 3). 2) Injection of recombinant PLC into mouse eggs induces Ca 2ϩ oscillations as well (7). 3) Ca 2ϩ oscillation-inducing ability of sperm extract injected into eggs (4, 8) is lost when pretreated with an antibody against PLC (2). 4) PLC content in the mouse sperm and the number of Ca 2ϩ spikes at fertilization are reduced by transgenic RNA interference of PLC (9). 5) PLC has such a high Ca 2ϩ sensitivity of PIP 2 -hydrolyzing activity that the enzyme can be active in the resting cells at ϳ100 nM Ca 2ϩ (7, 10), suitable for the sperm factor as the first stimulus in the egg cytoplasm at fertilization.Another important property of PLC is nuclear translocation ability. PLC expressed by RNA injection is accumulated into the formed PN (3,11,12). This is consistent with earlier observation that sperm-derived Ca 2ϩ oscillation-inducing activity is concentrated into PN formed several hours after fertilization, as examined by transfer of the oopla...

Birth of Normal Offspring from Mouse Eggs Activated by a Phospholipase C.ZETA. Protein Lacking Three EF-hand Domains

“…It has been reported that an RNA concentration of at least 0.5 mg/ml is required for s-PLCζ to induce Ca 2+ oscillations, whereas an RNA concentration of 0.001-0.002 mg/ml is enough for PLCζ to induce Ca 2+ oscillations [16,26]. In the case of s-PLCζ, small Ca 2+ oscillations begin later than when using PLCζ and they are followed by discrete small Ca 2+ spikes [24,26]. Coinciding with these reports, we found that resumption of meiosis as well as pronuclear formation was delayed in s-PLCζ RNA (1 mg/ml)-injected eggs compared with eggs injected with PLCζ RNA (0.002 mg/ml; Fig.…”

“…The Ca 2+ oscillation-inducing activity is lost by immunodepletion of PLCζ in sperm extracts [16], and transgenic RNA interference of PLCζ results in a low rate of egg activation [22]. Moreover, PLCζ containing the consensus nuclear localization signal between the X and Y domains accumulates in the pronuclei [23][24][25][26], which is consistent with the Ca 2+ oscillation-inducing activity derived from sperm being localized in the pronuclei of fertilized eggs [7,27,28]. It is thus likely that PLCζ acts as the sperm factor that triggers the Ca 2+ oscillations required for egg activation.…”

Molecular approaches related to the European eel (Anguilla anguilla) reproductive process

“…Furthermore, EF-hand domains seem also to play a role in nuclear translocation (Kouchi et al, , 2005Yoda et al 2004). According to Kuroda et al (2006), Trp13, Phe14, and Val18, which may be necessary for appropriate conformation for nuclear translocation, may also be necessary to keep normal Ca 2+ oscillationinducing activity as well. Nevertheless, despite the lack of a part of the N-terminal of all teleosts studied so far, at least some of these Plcζs still can trigger Ca 2+ oscillations.…”

“…In teleost species, studies showed that a deletion of a part of the EF-hand domain reduces the Ca 2+ oscillatory activity of Plcζ (Kouchi et al, 2005;Kuroda et al, 2006). It remains possible that eel Plcζ1 does not trigger Ca 2+ oscillation, however medaka Plcζ, which is similarly truncated at its N-terminus, can induce Ca 2+ oscillation in mouse oocytes but at a lower activity than for fulllength mammalian PLCζ (Coward et al, 2011;Kuroda et al, 2006;Ito et al, 2008). These results suggest that these domains are involved in the Ca 2+ signal but are not obligatory to induce Ca 2+ oscillation.…”

“…Furthermore, due to its loss, eel PLCζ1 misses these two nuclear targeting regions localised in the lost part, which may affect the nuclear translocation of the protein. According to Kuroda et al (2006), in the mouse, nuclear targeting was absent for point mutation of Lys299 and/or Lys301 in the C terminus of X domain, and nuclear translocation was lost when the residues from the NLS were replaced by glutamate. Nevertheless, these substitutions did not affect PLCζ ability to induce the Ca 2+ oscillation.…”