Sperm-specific phospholipase C zeta (PLCζ) is widely considered to be the physiological stimulus that evokes intracellular calcium (Ca2+) oscillations that are essential for the initiation of egg activation during mammalian fertilisation. A recent genetic study reported a male infertility case that was directly associated with a point mutation in the PLCζ C2 domain, where an isoleucine residue had been substituted with a phenylalanine (I489F). Here, we have analysed the effect of this mutation on the in vivo Ca2+ oscillation-inducing activity and the in vitro biochemical properties of human PLCζ. Microinjection of cRNA or recombinant protein corresponding to PLCζI489F mutant at physiological concentrations completely failed to cause Ca2+ oscillations and trigger development. However, this infertile phenotype could be effectively rescued by microinjection of relatively high (non-physiological) amounts of recombinant mutant PLCζI489F protein, leading to Ca2+ oscillations and egg activation. Our in vitro biochemical analysis suggested that the PLCζI489F mutant displayed similar enzymatic properties, but dramatically reduced binding to PI(3)P and PI(5)P-containing liposomes compared with wild-type PLCζ. Our findings highlight the importance of PLCζ at fertilisation and the vital role of the C2 domain in PLCζ function, possibly due to its novel binding characteristics.

Introduction

In mammalian oocytes (eggs), the fertilising sperm evokes a series of preprogrammed biochemical and morphological events collectively known as ‘egg activation’. It is now well established in all mammalian species studied that the earliest step of egg activation involves marked increases in the levels of the egg cytosolic calcium concentration [Ca2+], which are both necessary and sufficient for activation and early embryonic development [13]. Despite recent controversies, a gamete-specific phospholipase C (PLC) isoform, PLCζ is widely considered as the physiological stimulus that induces the characteristic series of large cytoplasmic Ca2+ transients, known as Ca2+ oscillations, within the fertilising oocyte [38]. Sperm PLCζ is delivered from the fertilising sperm into the egg cytoplasm and catalyses the hydrolysis of its membrane-bound phospholipid substrate, phosphatidylinositol 4,5-bisphosphate (PIP2), triggering the cytoplasmic Ca2+ oscillations through the inositol 1,4,5-trisphosphate (InsP3) signalling pathway [3,4,9]. PLCζ appears to be the smallest currently known mammalian PLC isozyme, with the most basic domain organisation among all PLC isoforms. PLCζ consists of four EF-hand domains at the N-terminus, the characteristic X and Y catalytic domains in the centre, followed by a C-terminal C2 domain [4,9]. Thus, PLCζ possesses a similar domain organisation to PLCδ1 with the remarkable exception that it lacks an N-terminal pleckstrin homology (PH) domain [9]. The notable lack of a PH domain makes it unclear how this sperm-specific PLC isoform directly interacts and targets biological membranes. A recent report suggested that the N-terminal lobe of the EF-hand domain of PLCζ together with its positively charged XY linker has an essential role to provide a tether that facilitates proper PIP2 substrate access and binding in the PLCζ active site [10]. However, the exact mechanism by which PLCζ targets the PIP2-containing membrane is still unknown.

Evidence for the clinical importance of PLCζ has been provided by studies that have directly linked reduced expression levels and abnormal forms of PLCζ with male infertility [5,1115]. A very recent genetic study, using whole-exome sequencing analysis, identified a homozygous missense mutation in the PLCζ gene of two infertile brothers from Tunisia, presenting oocyte activation failure [8]. This mutation was located in the C2 domain of PLCζ, where an isoleucine at position I489 had been replaced with a phenylalanine (I489F) (Figure 1A) [8]. Interestingly, this is the first male infertility-linked PLCζ point mutation to be reported within the C2 domain of PLCζ, a domain, although well known to be essential for PLCζ function, whose exact role still remains unclear. It was shown that I489F mutation results in some loss of Ca2+ oscillation-inducing activity of PLCζ in eggs, but the degree of loss of activity was not quantified and the poor ability of the PLCζ mutant to trigger Ca2+ oscillations was not extensively characterised [8].

Effect of I489F mutation on Ca2+ oscillation-inducing activity of human PLCζ in mouse eggs.

Figure 1.
Effect of I489F mutation on Ca2+ oscillation-inducing activity of human PLCζ in mouse eggs.

(A) Schematic representation of human PLCζ domain organisation identifying the location of I489F mutation within the C-terminal C2 domain. (B and C) Fluorescence and luminescence recordings reporting the Ca2+ changes [fluorescence (black traces) and luciferase expression (red traces; luminescence) in c.p.s., respectively] in unfertilised mouse eggs following microinjection of cRNA encoding luciferase-tagged hPLCζWT and hPLCζI489F mutant (see also Table 1).

Figure 1.
Effect of I489F mutation on Ca2+ oscillation-inducing activity of human PLCζ in mouse eggs.

(A) Schematic representation of human PLCζ domain organisation identifying the location of I489F mutation within the C-terminal C2 domain. (B and C) Fluorescence and luminescence recordings reporting the Ca2+ changes [fluorescence (black traces) and luciferase expression (red traces; luminescence) in c.p.s., respectively] in unfertilised mouse eggs following microinjection of cRNA encoding luciferase-tagged hPLCζWT and hPLCζI489F mutant (see also Table 1).

Table 1
Properties of luciferase-tagged PLCζWT and PLCζI489F mutant expressed in unfertilised mouse eggs

Ca2+ oscillation-inducing activity and luciferase luminescence levels (c.p.s.) in the first hour of spiking are summarised for mouse eggs microinjected with luciferase-tagged PLCζWT and PLCζI489F mutant. Results are expressed as means ± SEM.

Construct Pipette cRNA concentration (µg/µl) Number of eggs injected Number of eggs oscillating Mean number of spikes in the first hour of spiking Average expression in the first hour of spiking (c.p.s.) 
PLCζWT-LUC 0.015 20 19/20 4 ± 0.290 0.06 ± 0.007 
PLCζI489F-LUC 0.015 19 1/19 0.15 ± 0.016 0.06 ± 0.004 
PLCζI489F-LUC 0.1 16 15/16 2.3 ± 0.250 0.74 ± 0.038 
Construct Pipette cRNA concentration (µg/µl) Number of eggs injected Number of eggs oscillating Mean number of spikes in the first hour of spiking Average expression in the first hour of spiking (c.p.s.) 
PLCζWT-LUC 0.015 20 19/20 4 ± 0.290 0.06 ± 0.007 
PLCζI489F-LUC 0.015 19 1/19 0.15 ± 0.016 0.06 ± 0.004 
PLCζI489F-LUC 0.1 16 15/16 2.3 ± 0.250 0.74 ± 0.038 

In the present study, we introduced the infertility-linked PLCζ I489F mutation into human PLCζ sequence and we analysed the effect of this mutation on both the in vivo Ca2+ oscillation-inducing activity and the in vitro biochemical/enzymatic properties of human PLCζ. For comparison, cRNA encoding luciferase-tagged versions of wild-type (WT) and PLCζI489F mutant or bacterially expressed recombinant proteins were microinjected into unfertilised mouse and bovine eggs. Circular dichroism (CD) spectroscopy was used to investigate whether the I489F mutation interferes with the proper folding of the C2 domain. The enzymatic and biochemical properties of PLCζWT and PLCζI489F mutant were analysed using an in vitro [3H]PIP2 hydrolysis and liposome-binding assays.

Materials and methods

Plasmid construction

Human phospholipase C zeta (PLCζ)-luciferase in pCR3 vector [5] was subjected to site-directed mutagenesis (QuikChange II; Stratagene) to generate the PLCζI489F mutant. PLCζWT and PLCζI489F mutant were amplified by polymerase chain reaction (PCR) from the corresponding pCR3 plasmid using Phusion polymerase (Finnzymes) and the appropriate primers to incorporate a 5′-SalI site and a 3′-NotI site and were cloned into a modified pET expression vector (pETMM41). The primers used for the amplification of PLCζWT and PLCζI489F mutants were: 5′-CCTAGTCGACATGGAAATGAGATGGTTTTTGTC-3′ (forward) and 5′-CTAAGCGGCCGCTCATCTGACGTACCAAACATAAA-3′ (reverse). Similarly with regard to the full-length PLCζ constructs, the C2 domains (480–608 aa) of PLCζWT and PLCζI489F mutants were amplified by PCR from the aforementioned corresponding pCR3 plasmids using Phusion polymerase (Finnzymes) and the appropriate primers to incorporate a 5′-SalI site and a 3′-NotI site, and were cloned into the pETMM41 vector. The primers used for the amplification of C2ζWT and C2ζI489F mutants were: 5′-CACCGTCGACATGCCAATTACACTTACAATAAGG-3′ (forward) and 5′-CTAAGCGGCCGCTCATCTGACGTACCAAACATAAA-3′ (reverse). Successful mutagenesis and cloning of the above constructs were confirmed by dideoxynucleotide sequencing (Applied Biosystems BigDye Version 3.1 chemistry and model 3730 automated capillary DNA sequencer by DNA Sequencing & Services™).

cRNA synthesis

Luciferase-tagged PLCζWT and PLCζI489F constructs were linearised by restriction digests and then cRNA was synthesised using the mMessage Machine T7 kit (Ambion) and a poly(A)tailing kit (Ambion), as per the manufacturer's instructions.

Protein expression and purification

Escherichia coli [BL21-CodonPlus(DE3)-RILP; Stratagene] cells were transformed with the appropriate pETMM41 construct and cultured at 37°C until the A600 reached 0.6. Then, protein expression was induced for 18 h at 16°C with 0.1 mM isopropyl β-d-thiogalactopyranoside (IPTG; ForMedium). Induced cells were then harvested by centrifugation at 6000 g for 10 min at 4°C and resuspended in ice-cold amylose column buffer [10 mM Tris–HC1 (pH 7.4), 200 mM NaCl, l mM EDTA and protein inhibitor mixture (Roche)]. Then, the resuspended cells were sonicated four times for 15 s on ice. After 20 min of centrifugation at 20 000 g at 4°C to remove the insoluble proteins, the soluble MBP-tagged fusion proteins were purified by affinity chromatography using an amylose resin column following standard procedures (New England Biolabs). Eluted proteins were then dialysed and concentrated using centrifugal concentrators (Sartorius; 10 000 molecular mass cut-off).

SDS–PAGE and Western blot

Recombinant MBP-fusion proteins were separated by SDS–PAGE and immunoblot analysis was performed as described recently [10]. Proteins were probed with a monoclonal penta-His antibody (1:5000 dilution).

Preparation and handling of mouse oocytes

Mature MII oocytes (eggs) were collected from female MF1 mice (Envigo Ltd) 6–8 weeks old, 15 h after injection with 10 IU human chorionic gonadotrophin (hCG). Approximately 48 h before hCG injection, mice were injected with 10 IU pregnant mare's serum gonadotrophin. Following collection, cumulus cells were removed using hyaluronidase treatment and eggs were maintained in M2 media (Sigma–Aldrich) under mineral oil at 37°C until use. Injected eggs were transferred for development in KSOM media (Embryomax by Millipore). All animal work was conducted according to Home Office Licensing procedures and approved by the Animals Ethics Committee at Cardiff University.

Preparation and handling of bovine oocytes

Ovaries were collected after slaughter from the local abattoir and transported to the laboratory at 24°C in PBS (Sigma P4417) within 1 h. Cumulus–oocyte complexes (COCs) were collected by slashing the surface of the ovary with a scalpel in Medium 199 — HEPES-buffered, and the solution was passed through a 100 µm mesh filter in order to retain the COCs. COCs were placed in maturation media for 22 h [16]. After completion of maturation, COCs were vortexed for 3–4 min in hyaluronidase in order to isolate the mature eggs, which were immediately washed and transferred in M2 media until use.

Protein microinjection and measurements of intracellular Ca2+

Eggs were incubated in M2 media containing Cal-520 AM (5 µM) for 30 min at 37°C before the experiment. Eggs were held in M2 for microinjection [17]. Recombinant PLCζWT and PLCζI489F mutant fusion proteins were diluted in injection buffer [120 mM KCl and 20 mM HEPES (pH 7.4)] prior to introduction into oocytes using a high pressure injection method. Alexa Fluor 594 (1 mM) was used as a loading control to ensure equal protein volume injection. Eggs were imaged in HKSOM media using a Nikon TE2000 inverted epifluorescence microscope connected to a cooled intensified CCD camera (Photek, U.K.), and fluorescence was recorded by photon-counting software (Photek, U.K.). In cases where luciferase expression was measured, the fluorescence (to quantify Ca2+ changes) was recorded alternately using a 10 s switching cycle [18]. The fluorescence signal was normalised to relative fluorescence by plotting the absolute fluorescence divided by the basal level fluorescence (F/F0). All egg experiments were conducted within a 3-week period.

cRNA microinjection and measurements of intracellular Ca2+ and luciferase expression

Eggs were held in M2 for microinjection. cRNA was diluted in injection buffer [120 mM KCl and 20 mM HEPES (pH 7.4)] and in the case of cRNA also mixed with 1 mM Oregon Green BAPTA dextran OGBD (Life Technologies) prior to introduction into oocytes using a high pressure injection method [17]. Bolus injection calculated the amount of injection solution microinjected that was ∼3–5% of the oocyte volume. Eggs were imaged in HKSOM media containing 100 µM luciferin using a Nikon TE2000 inverted epifluorescence microscope connected to a cooled intensified CCD camera (Photek, U.K.), and both luminescence and fluorescence were recorded by photon-counting software (Photek, U.K.). Luminescence (quantifying luciferase expression) and fluorescence (quantifying Ca2+ changes) were recorded alternately using a 10 s switching cycle [18], with these 2 signals being plotted individually for each oocyte over the same timescale. The fluorescence signal was normalised to relative fluorescence by plotting the absolute fluorescence divided by the basal level fluorescence (F/F0), and the luminescence was plotted as a running average over 5 min. All egg experiments were conducted within a 3-month period.

CD spectroscopy

CD spectra of MBP, MBP-C2ζWT and MBP-C2ζI489F were recorded on an Aviv model 215 instrument (Aviv Biomedical, Inc., Lakewood, NJ) using a 0.1 cm quartz cell at 4°C. Proteins were dissolved in 100 mM NaF and 20 mM KH2PO4/NaOH (pH 7.0), at a concentration of ∼0.15 mg/ml. Concentrations were determined based on the absorbance at 280 nm, assuming extinction coefficients derived from the amino acid composition [19]. Secondary structure content was analysed using the CDsstr algorithm [20] as implemented on DichroWeb [21] using the SMP180 reference spectra [22].

Thermal stability was monitored at 221 nm in 0.5°C intervals from 4 to 70°C (MBP) or up to a maximum temperature when protein aggregation was observed as indicated by a sharp increase in the dynode voltage with settings resulting in an average heating rate of ∼30°C/h. Apparent melting temperatures Tm and van't Hoff's enthalpies ΔHvH were estimated from non-linear curve fitting assuming a two-state folded-to-unfolded transition as described previously [23], with the ellipticity of the unfolded state set as that observed for MBP.

Molecular modelling

Structural models of MBP, MBP-C2ζWT and MBP-C2ζI489F were generated using the SWISS-MODEL, with the corresponding parts of the PDB co-ordinates 3mq9 (residues 5–366) and 1djg (rat PLCδ1, residues 496–624) as templates [24].

PIP2 hydrolysis assays

The PIP2 hydrolytic activity of recombinant MBP-tagged PLCζ proteins was determined as described previously [5,10,25]. The final PIP2 concentration in the reaction mixture was 220 µM, containing 0.05 µCi of [3H]PIP2. For the assays examining the Ca2+-dependence of PLC enzymatic activity, the Ca2+ buffers were prepared by EGTA/CaCl2 admixture, while in assays to determine the dependence on substrate PIP2 concentration, 0.05 µCi of [3H]PIP2 was mixed with cold PIP2 to give the appropriate final concentration [10,26]. Km and EC50 values of Ca2+-dependence for PIP2 hydrolysis for the MBP-tagged PLCζ recombinant proteins were determined by non-linear regression analysis (GraphPad Prism 5).

Liposome preparation and binding assays

Unilamellar liposomes were prepared as previously described [10,26]. For the protein–liposome-binding experiments, liposomes (100 µg) were incubated with 1 µg of each MBP-tagged recombinant protein for 30 min at room temperature and centrifuged for 5 h at 4°C. Supernatants and pellets were analysed either by SDS–PAGE and Coomassie Brilliant Blue staining or by the [3H]PIP2 hydrolysis assay, as recently described [10].

Results

Microinjection of cRNA encoding PLCζI489F mutant completely failed to trigger Ca2+ oscillations when expressed at physiological concentrations in mouse eggs

To investigate the impact of I489F mutation on the in vivo Ca2+ oscillation-inducing activity of human PLCζ, we used site-directed mutagenesis to generate the PLCζI489F mutant. To enable direct comparative analysis with PLCζWT and to verify that this construct was faithfully expressed as protein in cRNA-microinjected unfertilised mouse eggs, we produced this mutant as a luciferase-fusion construct, as previously described [10,26,27]. Microinjection of cRNA encoding luciferase-tagged PLCζWT (PLCζWT-LUC) caused prominent Ca2+ oscillations in all injected eggs (∼4.0 spikes in the first hour of oscillating), similar to those observed during fertilisation, following successful protein expression to a level indicated by a luminescence reading of 0.06 c.p.s. (Figure 1B; left panel and Table 1). In contrast, microinjection of cRNA corresponding to the PLCζI489F-LUC mutant failed to cause any Ca2+ oscillations at equivalent protein expression levels (0.06 c.p.s.; Figure 1B; right panel and Table 1). Interestingly, expression of the PLCζI489F-LUC mutant at significantly higher levels (0.74 c.p.s.) led to low-frequency Ca2+ oscillations (∼2.3 spikes in the first hour of oscillating; Figure 1C and Table 1), suggesting that overexpression of PLCζI489F-LUC, which leads to overload of this mutant within the egg cytoplasm, can induce Ca2+ release from intracellular stores.

Microinjection of MBP-PLCζI489F protein is less effective at triggering Ca2+ oscillations in mouse eggs

PLCζWT and PLCζI489F mutants were then subcloned into the pETMM41 expression vector to allow prokaryotic expression of these constructs, as 6xHis-MBP-fusion recombinant proteins. We have previously demonstrated that NusA is a powerful fusion partner for PLCζ, significantly enhancing the bacterial expression, protein solubility and the purified recombinant protein stability over time [5,10]. In this study, we demonstrate for the first time that in addition to NusA, MBP tag is an extremely effective protein tag for PLCζ. Our comparative experiments with NusA tag showed that MBP is an effective protein fusion partner for PLCζ, preserving its stability over time (data not shown).

Optimal protein production for these MBP-fusion PLCζ constructs required induction of protein expression with 0.1 mM IPTG for 18 h at 16°C. Following bacterial expression in E. coli and isolation by amylose resin affinity chromatography, the purified recombinant proteins were analysed by SDS–PAGE and immunoblot analysis using an anti-His (penta-His) monoclonal antibody (Figure 2A). The dominant protein band with mobility corresponding to the predicted molecular mass for MBP-PLCζWT and MBP-PLCζI489F mutants was observed for both recombinant fusion proteins (∼118 kDa). These major bands were also confirmed by immunoblot analysis by the penta-His antibody (Figure 2A; right panel). Some additional, fainter, low molecular mass bands could be observed, which were also detected by the penta-His antibody and were probably the result of protease degradation occurring during the various stages of protein expression and purification. Microinjection of MBP-PLCζWT into mouse eggs at a concentration of 0.0375 mg/ml revealed that it possesses a potent ability to trigger cytoplasmic Ca2+ oscillations (Figure 2B; upper panel), matching that observed after microinjection of native sperm extracts [28]. Microinjection of the MBP protein alone was unable to induce Ca2+ release. Interestingly, microinjection of MBP-PLCζI489F mutant protein at equivalent levels that MBP-PLCζWT triggered physiological Ca2+ oscillations (0.0375 mg/ml) was either unable to induce any Ca2+ oscillations (13/23 eggs) or could only trigger very low-frequency Ca2+ oscillations (10/23 eggs; Figure 2B; lower panel).

MBP-hPLCζI489F recombinant protein fails to induce physiological pattern of Ca2+ oscillations in mouse eggs, when microinjected in equivalent concentration levels to MBP-hPLCζWT.

Figure 2.
MBP-hPLCζI489F recombinant protein fails to induce physiological pattern of Ca2+ oscillations in mouse eggs, when microinjected in equivalent concentration levels to MBP-hPLCζWT.

(A) SDS–PAGE of affinity-purified recombinant MBP-tagged hPLCζWT and hPLCζI489F proteins (2 µg) analysed by 8% SDS–PAGE and Coomassie Brilliant Blue staining (left panel) or immunoblot analysis using the penta-His antibody (1:5000 dilution; right panel). (B) Patterns of Ca2+ oscillations in unfertilised mouse eggs following microinjection of MBP-hPLCζWT and MBP-hPLCζI489F recombinant proteins. F/F0 represents a fluorescence intensity of Cal-520 relative to baseline.

Figure 2.
MBP-hPLCζI489F recombinant protein fails to induce physiological pattern of Ca2+ oscillations in mouse eggs, when microinjected in equivalent concentration levels to MBP-hPLCζWT.

(A) SDS–PAGE of affinity-purified recombinant MBP-tagged hPLCζWT and hPLCζI489F proteins (2 µg) analysed by 8% SDS–PAGE and Coomassie Brilliant Blue staining (left panel) or immunoblot analysis using the penta-His antibody (1:5000 dilution; right panel). (B) Patterns of Ca2+ oscillations in unfertilised mouse eggs following microinjection of MBP-hPLCζWT and MBP-hPLCζI489F recombinant proteins. F/F0 represents a fluorescence intensity of Cal-520 relative to baseline.

To investigate whether it was possible to rescue the low-frequency Ca2+ oscillations, MBP-PLCζI489F recombinant protein was microinjected at higher levels. We found that a two-fold increase in the amount of the MBP-PLCζI489F mutant (0.075 mg/ml) microinjected into mouse eggs was able to rescue the defective Ca2+ oscillation-inducing phenotype and trigger egg activation, as indicated by cleavage of the two-cell stage (Figure 3). These findings agree with our previous observations suggesting that overload of the PLCζI489F mutant within the mouse egg cytoplasm can induce Ca2+ oscillations.

Microinjection of higher (non-physiological) levels of MBP-hPLCζI489F recombinant protein in mouse eggs induces physiological pattern of Ca2+ oscillations and triggers egg activation.

Figure 3.
Microinjection of higher (non-physiological) levels of MBP-hPLCζI489F recombinant protein in mouse eggs induces physiological pattern of Ca2+ oscillations and triggers egg activation.

F/F0 represents the fluorescence intensity relative to baseline. The right panel shows two-cell stage mouse embryos 22–24 h after injection of MBP-hPLCζI489F recombinant protein.

Figure 3.
Microinjection of higher (non-physiological) levels of MBP-hPLCζI489F recombinant protein in mouse eggs induces physiological pattern of Ca2+ oscillations and triggers egg activation.

F/F0 represents the fluorescence intensity relative to baseline. The right panel shows two-cell stage mouse embryos 22–24 h after injection of MBP-hPLCζI489F recombinant protein.

MBP-PLCζWT protein is more potent in triggering Ca2+ oscillations in bovine eggs than MBP-PLCζI489F

To examine whether our previous observations regarding the ability of the PLCζI489F mutant to trigger Ca2+ oscillations in mouse eggs are consistent with eggs of a different species, we compared the abilities of MBP-PLCζWT and MBP-PLCζI489F recombinant proteins to induce Ca2+ oscillations in bovine eggs. The optimal concentration for MBP-PLCζWT to induce a physiological pattern of Ca2+ oscillations in bovine eggs was 0.15 mg/ml. In contrast, microinjection of MBP-PLCζI489F mutant protein at this concentration was able to trigger very low-frequency Ca2+ oscillations in all microinjected eggs (Figure 4).

MBP-hPLCζWT recombinant protein has greater potency in triggering Ca2+ oscillations in bovine eggs compared with MBP-hPLCζI489F.

Figure 4.
MBP-hPLCζWT recombinant protein has greater potency in triggering Ca2+ oscillations in bovine eggs compared with MBP-hPLCζI489F.

Bovine eggs were injected with MBP-hPLCζWT recombinant protein (top trace) or MBP-hPLCζI489F protein (bottom trace) and Ca2+ oscillations recorded. F/F0 represents the fluorescence intensity of Ca2+ dye relative to baseline.

Figure 4.
MBP-hPLCζWT recombinant protein has greater potency in triggering Ca2+ oscillations in bovine eggs compared with MBP-hPLCζI489F.

Bovine eggs were injected with MBP-hPLCζWT recombinant protein (top trace) or MBP-hPLCζI489F protein (bottom trace) and Ca2+ oscillations recorded. F/F0 represents the fluorescence intensity of Ca2+ dye relative to baseline.

I489F does not alter the folding and the thermal stability of the PLCζ C2 domain

To investigate whether I489F mutation within the C2 domain of PLCζ interferes with the proper folding of this domain, we analysed the MBP-tagged WT and mutant C2 domains (C2ζWT and C2ζI489F) by CD spectroscopy. Attempts to produce an untagged or 6xHis-tag version of the C2 domain of PLCζWT using the bacterial expression system proved unsuccessful, as the protein appeared to be completely insoluble, accumulating into inclusion bodies. Thus, C2ζWT and C2ζI489F (Figure 5A) were cloned into the pETMM41 expression vector to allow bacterial expression of these domains as 6xHis-MBP-fusion recombinant proteins. The presence of the MBP tag significantly enhanced the expression of soluble C2 domains and the affinity-purified MBP-tagged C2 domains, after SDS–PAGE and immunoblot analysis using the penta-His antibody, displayed the predicted molecular mass (∼59 kDa; Figure 5B). It is worth noting that, prior to the CD experiments, removal of the MBP moiety from the C2 domains was attempted, but this resulted in rapid degradation of the proteins. Thus, the intact MBP-C2 domains were used for our CD studies, while the MBP moiety alone served as a control for our experiments. Spectra recorded at 4°C were indistinguishable from each other, but different from that of MBP alone (Figure 6A). Deconvolution of the spectra resulted in 39% α-helix/18% β-strand and 35% α-helix/24% β-strand for MBP by itself and the two fusion proteins, respectively. These values are in very good agreement with those of homology-based models of MBP (41% α-helix and 17% β-strand) and a PLCζ C2 domain combined with MBP (35% α-helix and 27% β-strand). The thermal stability was measured by monitoring the CD signal at 221 nm (Figure 6B). All samples showed a steep decrease in ellipticity. In contrast with MBP, the fusion proteins precipitated upon unfolding as indicated by an increase in light scattering. Assuming a two-state folded-to-unfolded transition and extrapolating to the ellipticity observed for unfolded MBP, fitting of the data resulted in melting temperature Tm = 60.5 ± 0.5°C and van't Hoff's enthalpies of ∼560 and ∼250 kJ/mol for MBP and the two fusion proteins, respectively. For MBP, these values agree with those previously reported [29].

Expression of WT and I489F mutant PLCζ C2 domains as MBP-tagged recombinant proteins.

Figure 5.
Expression of WT and I489F mutant PLCζ C2 domains as MBP-tagged recombinant proteins.

(A) Schematic representation of the MBP-fusion protein PLCζ C2 domains with numbers denoting their amino acid co-ordinates. (B) SDS–PAGE of affinity-purified recombinant MBP-tagged PLCζ C2 domains (1 µg) analysed by 10% SDS–PAGE and Coomassie Brilliant Blue staining (left panel) or immunoblot analysis using the penta-His antibody (1:5000 dilution; right panel).

Figure 5.
Expression of WT and I489F mutant PLCζ C2 domains as MBP-tagged recombinant proteins.

(A) Schematic representation of the MBP-fusion protein PLCζ C2 domains with numbers denoting their amino acid co-ordinates. (B) SDS–PAGE of affinity-purified recombinant MBP-tagged PLCζ C2 domains (1 µg) analysed by 10% SDS–PAGE and Coomassie Brilliant Blue staining (left panel) or immunoblot analysis using the penta-His antibody (1:5000 dilution; right panel).

CD analysis of MBP and MBP-tagged PLCζ C2 fusion proteins.

Figure 6.
CD analysis of MBP and MBP-tagged PLCζ C2 fusion proteins.

(A) CD spectra were recorded at 4°C for MBP (black), MBP-C2ζWT (blue) and MBP-C2ζI489F (red). (B) Thermal stability was monitored at 221 nm upon heating. In contrast with MBP, the fusion proteins were precipitated at ∼63°C. Dashed lines represent best fits assuming a two-state unfolding mechanism and an ellipticity of the unfolded state common with MBP.

Figure 6.
CD analysis of MBP and MBP-tagged PLCζ C2 fusion proteins.

(A) CD spectra were recorded at 4°C for MBP (black), MBP-C2ζWT (blue) and MBP-C2ζI489F (red). (B) Thermal stability was monitored at 221 nm upon heating. In contrast with MBP, the fusion proteins were precipitated at ∼63°C. Dashed lines represent best fits assuming a two-state unfolding mechanism and an ellipticity of the unfolded state common with MBP.

I489F does not alter the in vitro enzymatic properties of human PLCζ

We then examined the impact of I489F mutation on the in vitro enzymatic properties of PLCζ. To determine the specific PIP2 hydrolytic enzyme activities for PLCζWT and PLCζI489F, a micellar [3H]PIP2 hydrolysis assay was used as previously described [10,27,30]. The histograms of Figure 7A and Table 2 reveal that the enzymatic activities of PLCζWT and PLCζI489F were almost identical (960 ± 43 vs. 953 ± 49 nmol/min/mg), suggesting that I489F mutation has no effect on the ability of PLCζ to hydrolyse in vitro PIP2. To investigate the effect of I489F mutation on Ca2+ sensitivity of PLCζ enzyme activity, we assessed the ability of PLCζWT and PLCζI489F to hydrolyse [3H]PIP2 at different Ca2+ concentrations ranging from 0.1 to 0.1 mM. The resulting EC50 values for PLCζWT (66 nM) and PLCζI489F (60 nM) were very similar (Figure 7B and Table 2). In addition, calculation of the Michaelis–Menten constant Km, for PLCζWT and PLCζI489F, also yielded comparable values (81 vs. 93 µM; Table 2), indicating that I489F mutation has no effect on the Ca2+ sensitivity or on the in vitro enzymatic affinity of PLCζ for its substrate, PIP2.

I489F mutation does not affect the in vitro enzyme specific activity and the Ca2+ sensitivity of PLCζ.

Figure 7.
I489F mutation does not affect the in vitro enzyme specific activity and the Ca2+ sensitivity of PLCζ.

(A) PIP2 hydrolysis enzyme activity of MBP-PLCζWT and MBP-PLCζI489F obtained with the standard [3H]PIP2 hydrolysis assay. Values are means ± SEM (n = 4), using two different preparations of recombinant protein, and each experiment was performed in duplicate. (B) Effect of various [Ca2+] on the normalised activity of MBP-PLCζWT and MBP-PLCζI489F recombinant proteins. For these assays, values are ±SEM (n = 4), using two different batches of recombinant proteins and with each experiment performed in duplicate (see Table 2).

Figure 7.
I489F mutation does not affect the in vitro enzyme specific activity and the Ca2+ sensitivity of PLCζ.

(A) PIP2 hydrolysis enzyme activity of MBP-PLCζWT and MBP-PLCζI489F obtained with the standard [3H]PIP2 hydrolysis assay. Values are means ± SEM (n = 4), using two different preparations of recombinant protein, and each experiment was performed in duplicate. (B) Effect of various [Ca2+] on the normalised activity of MBP-PLCζWT and MBP-PLCζI489F recombinant proteins. For these assays, values are ±SEM (n = 4), using two different batches of recombinant proteins and with each experiment performed in duplicate (see Table 2).

Table 2
In vitro enzymatic properties of MBP-tagged PLCζWT and PLCζI489F mutants

Summary of specific enzyme activity, Km and EC50 values of Ca2+-dependence for PIP2 hydrolysis, determined by non-linear regression analysis (GraphPad Prism 5; see Figure 7).

PLCζ protein PIP2 hydrolysis enzyme activity (nmol/min/mg) Ca2+-dependence EC50 (nM) Km (μM) 
PLCζWT 960 ± 43 66 81 
PLCζI489F 953 ± 49 60 93 
PLCζ protein PIP2 hydrolysis enzyme activity (nmol/min/mg) Ca2+-dependence EC50 (nM) Km (μM) 
PLCζWT 960 ± 43 66 81 
PLCζI489F 953 ± 49 60 93 

I489F dramatically reduces the binding of PLCζ to PI(3)P and PI(5)P

The only specific binding partners for the PLCζ C2 domain that have been in vitro identified up to date are PI(3)P and PI(5)P [31,32]. To examine the binding properties of PLCζI489F to PIP2, PI(3)P and PI(5)P, we employed two different approaches: a liposome-binding (pull-down) and a liposome-binding/enzyme assay, as previously described [10,26]. For these experiments, we prepared unilamellar liposomes, which were composed of PC:CHOL:PE (4:2:1) with incorporation of 1% PIP2, 5% PI(3)P or 5% PI(5)P. For diminishing the non-specific protein binding to highly charged lipids, the liposome-binding experiments were performed in the presence of a near-physiological concentration of MgCl2 (0.5 mM) [26]. The MBP moiety alone served as the negative control for our experiments. As shown in Figure 8, MBP did not exhibit any specific liposome binding in the absence or presence of PIP2, PI(3)P and PI(5)P, whereas our positive control MBP-PLCζWT displayed robust binding to liposomes containing 1% PIP2, 5% PI(3)P or 5% PI(5)P. On the other hand, MBP-C2ζWT showed significant binding only to liposomes containing either 5% PI(3)P or 5% PI(5)P. In contrast, although MBP-PLCζI489F was able to bind strongly to liposomes containing 1% PIP2, its binding to liposomes containing either 5% PI(3)P or 5% PI(5)P significantly diminished (Figure 8). Similarly, MBP-C2ζI489F was unable to bind liposomes containing 5% PI(3)P or 5% PI(5)P. These findings clearly suggest that I489F mutation affects the binding of the PLCζ C2 domain to PI(3)P and PI(5)P. For more quantitative analysis, a liposome-binding/enzyme assay was employed to analyse the binding of MBP-PLCζWT and MBP-PLCζI489F to PIP2, PI(3)P and PI(5)P. Thus, 1 µg of recombinant protein corresponding to MBP-PLCζWT and MBP-PLCζI489F was incubated with liposomes containing 1% PIP2, 5% PI(3)P or 5% PI(5)P. After centrifugation, the supernatants were separated for the precipitated liposomes and the PIP2 hydrolytic activity was determined using the standard [3H]PIP2 hydrolysis assay. Based on the % of the PIP2 hydrolytic activity pre- and post-liposome binding, we estimated the relative binding of each PLCζ protein to the different phosphoinositide specific-containing liposomes [10]. As shown in the histograms in Figure 9, the binding of PLCζWT and PLCζI489F to PIP2-containing liposomes was almost identical, whereas the binding of PLCζI489F to PI(3)P- and PI(5)P-containing liposomes had been reduced by ∼50% compared with PLCζWT, suggesting that I489F mutation indeed dramatically reduces the binding of PLCζ to PI(3)P and PI(5)P.

I489F mutation reduces the binding of PLCζ to PI(3)P- and PI(5)P-containing liposomes.

Figure 8.
I489F mutation reduces the binding of PLCζ to PI(3)P- and PI(5)P-containing liposomes.

Liposome ‘pull-down assays’ of MBP-tagged PLCζWT and PLCζI489F proteins. Unilamellar liposomes containing PIP2 (1%), PI(3)P (5%) or PI(5)P (5%) were incubated with PLCζ recombinant proteins. Following liposome centrifugation, both the supernatant (s) and liposome pellet (p) were subjected to SDS–PAGE and Coomassie Brilliant Blue staining.

Figure 8.
I489F mutation reduces the binding of PLCζ to PI(3)P- and PI(5)P-containing liposomes.

Liposome ‘pull-down assays’ of MBP-tagged PLCζWT and PLCζI489F proteins. Unilamellar liposomes containing PIP2 (1%), PI(3)P (5%) or PI(5)P (5%) were incubated with PLCζ recombinant proteins. Following liposome centrifugation, both the supernatant (s) and liposome pellet (p) were subjected to SDS–PAGE and Coomassie Brilliant Blue staining.

Quantitative analysis suggests that I489F mutation reduces by ∼50% the binding of PLCζ to PI(3)P- and PI(5)P-containing liposomes.

Figure 9.
Quantitative analysis suggests that I489F mutation reduces by ∼50% the binding of PLCζ to PI(3)P- and PI(5)P-containing liposomes.

Normalised binding of MBP-PLCζWT and MBP-PLCζI489F recombinant proteins to unilamellar liposomes containing (A) 1% PIP2, (B) 5% PI(3)P and (C) 5% PI(5)P. Following centrifugation, the supernatants were assayed for their ability to hydrolyse PIP2in vitro, using the standard [3H]PIP2 hydrolysis assay (n = 4 ± SEM, using two different preparations of recombinant protein). Based on the percentage of the PIP2 hydrolytic activity pre- and post-liposome binding, the relative binding of each PLCζ protein to the liposomes was determined. Significant statistical differences (asterisks) were calculated by an unpaired Student's t-test; ***P < 0.0005 (GraphPad, Prism 5).

Figure 9.
Quantitative analysis suggests that I489F mutation reduces by ∼50% the binding of PLCζ to PI(3)P- and PI(5)P-containing liposomes.

Normalised binding of MBP-PLCζWT and MBP-PLCζI489F recombinant proteins to unilamellar liposomes containing (A) 1% PIP2, (B) 5% PI(3)P and (C) 5% PI(5)P. Following centrifugation, the supernatants were assayed for their ability to hydrolyse PIP2in vitro, using the standard [3H]PIP2 hydrolysis assay (n = 4 ± SEM, using two different preparations of recombinant protein). Based on the percentage of the PIP2 hydrolytic activity pre- and post-liposome binding, the relative binding of each PLCζ protein to the liposomes was determined. Significant statistical differences (asterisks) were calculated by an unpaired Student's t-test; ***P < 0.0005 (GraphPad, Prism 5).

Discussion

Mounting experimental and clinical evidence strongly supports the notion that sperm-specific PLCζ is the sole physiological stimulus of egg activation during mammalian fertilisation [4,8,1114,3336]. Sperm-delivered PLCζ triggers the repetitive Ca2+ oscillations within the fertilised egg, by catalysing the hydrolysis of PIP2 stimulating the InsP3 signalling pathway [3,9]. Although PLCζ is the smallest PLC isoform with the most basic domain structure organisation, its discrete biochemical properties contribute to its supreme effectiveness in triggering the Ca2+ signalling phenomenon within the fertilised mammalian eggs [9].

In the past few years, clinical reports have directly linked defects in human PLCζ with documented cases of male infertility. First, Yoon et al. [11] reported many infertile patients presenting oocyte activation failure, providing evidence that it was due to the absence or reduced levels of PLCζ within their sperm [11]. Then, the first direct link between male infertility and a mutation on the PLCζ gene came from a study that reported a mutation on the catalytic domain of PLCζ (H398P) of a patient who failed fertilisation after ICSI [12]. Interestingly, another study reported a second PLCζ mutation on the same heterozygous infertile patient, also in the catalytic domain (H233L) [14]. Recently, Escoffier et al. [8] reported a homozygous missense mutation in the PLCζ gene of two infertile brothers from Tunisia, presenting egg activation failure [8]. This mutation was located in the C2 domain of PLCζ where an Ile had been replaced with a Phe residue (I489F) (Figure 1A), making it the first PLCζ mutation located in a domain different from the catalytic XY domain.

Here, we introduced the infertility-linked I489F PLCζ mutation in human PLCζ sequence and we assessed the effects of this mutation upon the in vivo Ca2+ oscillation-inducing activity and the in vitro biochemical and enzymatic properties of human PLCζ. Our study provides evidence and extends the recent work of Escoffier et al. [8] by revealing that (i) PLCζ I489F mutation dramatically reduces the Ca2+ oscillation-inducing activity of PLCζ in mouse and bovine eggs (Figures 1B, 2B and 4). Although microinjection of physiological levels of the PLCζ mutant are either unable to trigger or triggered very low-frequency Ca2+ oscillations in mouse and bovine eggs, a two-fold increase in the amount of PLCζ microinjected in mouse eggs was capable of rescuing the defective Ca2+ oscillation-inducing phenotype, triggering egg activation of these PLCζI489F-microinjected eggs (Figure 3). This suggests that overloading the egg with significantly higher amounts of this PLCζ mutant can lead to successful oocyte activation, explaining the infertility of the two heterozygous brothers carrying this mutation. (ii) CD spectroscopy showed that the I489F C2 mutation has no effect on the proper folding and the thermal stability of this domain (Figure 6). This was consistent with our observations regarding the enzymatic properties of the PLCζI489F mutant, which were almost identical with the enzymatic properties of PLCζWT (Figure 7 and Table 2), suggesting that I489F mutation has no effect on the ability of PLCζ to hydrolyse PIP2. (iii) More importantly, our liposome-binding experiments revealed that I489F mutation dramatically reduces (∼50%) the binding of PLCζ to PI(3)P and PI(5)P (Figures 8 and 9), two phosphoinositides, which have been previously reported to interact with PLCζ in vitro [31,32]. To the best of our knowledge, the PLCζ C2 domain is the first C2 domain among the C2 domains of all PLC isoforms that have been shown to directly interact with PI(3)P and PI(5)P, in vitro. Although it is difficult to predict which amino acid residues play a role on these interactions, we have shown that I489 is a key residue for efficient binding of PLCζ to both PI(3)P and PI(5)P. High-resolution three-dimensional structure analysis of the PLCζ C2 domain by X-ray crystallography could help to reveal the critical binding sites for these interactions. It is now also necessary to understand the physiological role of the binding of PLCζ to PI(3)P and PI(5)P and also to extensively search for any other unidentified membrane egg proteins, which might interact with the C2 domain of PLCζ, assisting with the proper localisation and targeting of this enzyme within the egg cytoplasm.

It has been proposed, at least for mouse PLCζ, that its nuclear translocation ability regulates the cell-cycle dependent Ca2+ oscillations [3]. It is worth noting that there is no scientific evidence for the C2 domain playing a role in the nuclear sequestration of PLCζ, as it has been clearly demonstrated that the nuclear localisation signal of mouse PLCζ is located within the XY linker region, close to the start of the Y domain [3].

Understanding the complex mechanism of action of PLCζ requires further investigation. However, based on our previous and recent findings, we propose that after sperm–egg fusion and the delivery of PLCζ from the sperm into the egg cytoplasm, the high Ca2+ sensitivity of this prototypic PLC is conferred by its EF-hand domains, allowing it to be active at resting egg Ca2+ levels [27,31,37]. PLCζ may then associate with a specific intracellular membrane by interaction of the C2 domain with PI(3)P, PI(5)P or another unidentified egg membrane protein. Finally, the positively charged XY linker together with the first EF-hand domain provides a tether to facilitate proper PIP2 substrate access for the catalytic XY domain to proceed with the catalysis of the hydrolysis of PIP2 to produce InsP3 (Figure 10) [10].

Schematic illustration of a proposed intracellular targeting mechanism of PLCζ.

Figure 10.
Schematic illustration of a proposed intracellular targeting mechanism of PLCζ.

Our study suggests that association of PLCζ with a specific vesicular membrane may be mediated by interaction of the C2 domain with PI(3)P, PI(5)P or an as-yet-unidentified membrane protein. Then, association of PLCζ with the negatively charged PIP2 involves electrostatic interactions with the positively charged first EF-hand domain and the XY-linker region. The catalytic XY domain subsequently proceeds with the enzymatic cleavage of PIP2. The high Ca2+ sensitivity of the enzyme is conferred by the EF-hand domain enabling PLCζ to be active at resting nanomolar Ca2+ levels (figure modified from ref. [10]).

Figure 10.
Schematic illustration of a proposed intracellular targeting mechanism of PLCζ.

Our study suggests that association of PLCζ with a specific vesicular membrane may be mediated by interaction of the C2 domain with PI(3)P, PI(5)P or an as-yet-unidentified membrane protein. Then, association of PLCζ with the negatively charged PIP2 involves electrostatic interactions with the positively charged first EF-hand domain and the XY-linker region. The catalytic XY domain subsequently proceeds with the enzymatic cleavage of PIP2. The high Ca2+ sensitivity of the enzyme is conferred by the EF-hand domain enabling PLCζ to be active at resting nanomolar Ca2+ levels (figure modified from ref. [10]).

In conclusion, the identification of the first male infertility-linked PLCζ point mutation located in the C2 domain of PLCζ provides the first clinical support for the vital role of this domain in PLCζ function. As our study proposes, this is purely due to the novel binding properties of this domain to PI(3)P and PI(5)P or other unidentified “egg factors”. In addition, the identification of another male infertility-linked PLCζ mutation necessitates the use of recombinant PLCζ protein in a clinical setting with the aim to rescue such cases of oocyte activation failure.

Abbreviations

     
  • Ca2+

    calcium

  •  
  • CD

    circular dichroism

  •  
  • COCs

    cumulus–oocyte complexes

  •  
  • hCG

    human chorionic gonadotrophin

  •  
  • ICSI

    intracytoplasmic sperm injection

  •  
  • IP3

    inositol 1,4,5-trisphosphate

  •  
  • IPTG

    isopropyl β-d-thiogalactopyranoside

  •  
  • MBP

    maltose-binding protein

  •  
  • PH

    pleckstrin homology

  •  
  • PI(3)P

    phosphatidylinositol 3-phosphate

  •  
  • PI(5)P

    phosphatidylinositol 5-phosphate

  •  
  • PIP2

    phosphatidylinositol 4,5-bisphosphate

  •  
  • PLCζ

    phospholipase C zeta

  •  
  • PCR

    polymerase chain reaction

  •  
  • WT

    wild type.

Author Contribution

M.N., K.S. and F.A.L. devised the project strategy. M.N., K.B., K.S. and F.A.L. designed the experiments, which were performed by M.N., P.S., J.S., K.B., B.L.C., L.B., M.L. and Z.S. M.N. prepared the first manuscript draft, which was revised and approved by all authors.

Funding

This work was supported by an EU-FP7 Marie-Curie Intra-European Fellowship 628634 (to M.N.), an Institute for Molecular and Experimental Medicine Research Scholarship (to J.R.S.) and partially by a research grant from Cook Medical Technologies (to K.S. and F.A.L.). F.A.L and K.S. hold patents on PLCζ with Cardiff University.

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

References

References
1
Stricker
,
S.A.
(
1999
)
Comparative biology of calcium signaling during fertilization and egg activation in animals
.
Dev. Biol.
211
,
157
176
doi:
2
Runft
,
L.L.
,
Jaffe
,
L.A.
and
Mehlmann
,
L.M.
(
2002
)
Egg activation at fertilization: where it all begins
.
Dev. Biol.
245
,
237
254
doi:
3
Nomikos
,
M.
,
Kashir
,
J.
,
Swann
,
K.
and
Lai
,
F.A.
(
2013
)
Sperm PLCζ: from structure to Ca2+ oscillations, egg activation and therapeutic potential
.
FEBS Lett.
587
,
3609
3616
doi:
4
Saunders
,
C.M.
,
Larman
,
M.G.
,
Parrington
,
J.
,
Cox
,
L.J.
,
Royse
,
J.
,
Blayney
,
L.M.
et al. 
(
2002
)
PLCζ: a sperm-specific trigger of Ca(2+) oscillations in eggs and embryo development
.
Development
129
,
3533
3544
PMID:
[PubMed]
5
Nomikos
,
M.
,
Yu
,
Y.
,
Elgmati
,
K.
,
Theodoridou
,
M.
,
Campbell
,
K.
,
Vassilakopoulou
,
V.
et al. 
(
2013
)
Phospholipase Cζ rescues failed oocyte activation in a prototype of male factor infertility
.
Fertil. Steril.
99
,
76
85
doi:
6
Nomikos
,
M.
,
Sanders
,
J.R.
,
Kashir
,
J.
,
Sanusi
,
R.
,
Buntwal
,
L.
,
Love
,
D.
et al. 
(
2015
)
Functional disparity between human PAWP and PLCζ in the generation of Ca2+ oscillations for oocyte activation
.
Mol. Hum. Reprod.
21
,
702
710
doi:
7
Satouh
,
Y.
,
Nozawa
,
K.
and
Ikawa
,
M.
(
2015
)
Sperm postacrosomal WW domain-binding protein is not required for mouse egg activation
.
Biol. Reprod.
93
,
94
doi:
8
Escoffier
,
J.
,
Lee
,
H.C.
,
Yassine
,
S.
,
Zouari
,
R.
,
Martinez
,
G.
,
Karaouzene
,
T.
et al. 
(
2016
)
Homozygous mutation of PLCZ1 leads to defective human oocyte activation and infertility that is not rescued by the WW-binding protein PAWP
.
Hum. Mol. Genet.
25
,
878
891
doi:
9
Nomikos
,
M.
(
2015
)
Novel signalling mechanism and clinical applications of sperm-specific PLCζ
.
Biochem. Soc. Trans.
43
,
371
376
doi:
10
Nomikos
,
M.
,
Sanders
,
J.R.
,
Parthimos
,
D.
,
Buntwal
,
L.
,
Calver
,
B.L.
,
Stamatiadis
,
P.
et al. 
(
2015
)
Essential role of the EF-hand domain in targeting sperm phospholipase Cζ to membrane phosphatidylinositol 4,5-bisphosphate (PIP2)
.
J. Biol. Chem.
290
,
29519
29530
doi:
11
Yoon
,
S.-Y.
,
Jellerette
,
T.
,
Salicioni
,
A.M.
,
Lee
,
H.C.
,
Yoo
,
M.-s.
,
Coward
,
K.
et al. 
(
2008
)
Human sperm devoid of PLC, zeta 1 fail to induce Ca(2+) release and are unable to initiate the first step of embryo development
.
J. Clin. Invest.
118
,
3671
3681
doi:
12
Heytens
,
E.
,
Parrington
,
J.
,
Coward
,
K.
,
Young
,
C.
,
Lambrecht
,
S.
,
Yoon
,
S.-Y.
et al. 
(
2009
)
Reduced amounts and abnormal forms of phospholipase C zeta (PLCζ) in spermatozoa from infertile men
.
Hum. Reprod.
24
,
2417
2428
doi:
13
Kashir
,
J.
,
Heindryckx
,
B.
,
Jones
,
C.
,
De Sutter
,
P.
,
Parrington
,
J.
and
Coward
,
K.
(
2010
)
Oocyte activation, phospholipase C zeta and human infertility
.
Hum. Reprod. Update
16
,
690
703
doi:
14
Kashir
,
J.
,
Konstantinidis
,
M.
,
Jones
,
C.
,
Lemmon
,
B.
,
Lee
,
H.C.
,
Hamer
,
R.
et al. 
(
2012
)
A maternally inherited autosomal point mutation in human phospholipase C zeta (PLCζ) leads to male infertility
.
Hum. Reprod.
27
,
222
231
doi:
15
Nomikos
,
M.
,
Elgmati
,
K.
,
Theodoridou
,
M.
,
Calver
,
B.L.
,
Cumbes
,
B.
,
Nounesis
,
G.
et al. 
(
2011
)
Male infertility-linked point mutation disrupts the Ca2+ oscillation-inducing and PIP2 hydrolysis activity of sperm PLCζ
.
Biochem. J.
434
,
211
217
doi:
16
Chung
,
J.T.
,
Keefer
,
C.L.
and
Downey
,
B.R.
(
2000
)
Activation of bovine oocytes following intracytoplasmic sperm injection (ICSI)
.
Theriogenology
53
,
1273
1284
doi:
17
Swann
,
K.
,
Campbell
,
K.
,
Yu
,
Y.
,
Saunders
,
C.
and
Lai
,
F.A.
(
2009
)
Use of luciferase chimaera to monitor PLCζ expression in mouse eggs
.
Methods Mol. Biol.
518
,
17
29
doi:
18
Campbell
,
K.
and
Swann
,
K.
(
2006
)
Ca2+ oscillations stimulate an ATP increase during fertilization of mouse eggs
.
Dev. Biol.
298
,
225
233
doi:
19
Pace
,
C.N.
,
Vajdos
,
F.
,
Fee
,
L.
,
Grimsley
,
G.
and
Gray
,
T.
(
1995
)
How to measure and predict the molar absorption coefficient of a protein
.
Protein Sci.
4
,
2411
2423
doi:
20
Johnson
,
W.C.
(
1999
)
Analyzing protein circular dichroism spectra for accurate secondary structures
.
Proteins Struct. Funct. Genet.
35
,
307
312
doi:
21
Whitmore
,
L.
and
Wallace
,
B.A.
(
2008
)
Protein secondary structure analyses from circular dichroism spectroscopy: methods and reference databases
.
Biopolymers
89
,
392
400
doi:
22
Abdul-Gader
,
A.
,
Miles
,
A.J.
and
Wallace
,
B.A.
(
2011
)
A reference dataset for the analyses of membrane protein secondary structures and transmembrane residues using circular dichroism spectroscopy
.
Bioinformatics
27
,
1630
1636
doi:
23
Greenfield
,
N.J.
(
2004
)
Analysis of circular dichroism data
.
Methods Enzymol.
383
,
282
317
doi:
24
Biasini
,
M.
,
Bienert
,
S.
,
Waterhouse
,
A.
,
Arnold
,
K.
,
Studer
,
G.
,
Schmidt
,
T.
et al. 
(
2014
)
SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information
.
Nucleic Acids Res.
42
,
W252
W258
doi:
25
Nomikos
,
M.
,
Elgmati
,
K.
,
Theodoridou
,
M.
,
Georgilis
,
A.
,
Gonzalez-Garcia
,
J.R.
,
Nounesis
,
G.
et al. 
(
2011
)
Novel regulation of PLCζ activity via its XY-linker
.
Biochem. J.
438
,
427
432
doi:
26
Theodoridou
,
M.
,
Nomikos
,
M.
,
Parthimos
,
D.
,
Gonzalez-Garcia
,
J.R.
,
Elgmati
,
K.
,
Calver
,
B.L.
et al. 
(
2013
)
Chimeras of sperm PLCζ reveal disparate protein domain functions in the generation of intracellular Ca2+ oscillations in mammalian eggs at fertilization
.
Mol. Hum. Reprod.
19
,
852
864
doi:
27
Nomikos
,
M.
,
Blayney
,
L.M.
,
Larman
,
M.G.
,
Campbell
,
K.
,
Rossbach
,
A.
,
Saunders
,
C.M.
et al. 
(
2005
)
Role of phospholipase C-ζ domains in Ca2+-dependent phosphatidylinositol 4,5-bisphosphate hydrolysis and cytoplasmic Ca2+ oscillations
.
J. Biol. Chem.
280
,
31011
31018
doi:
28
Swann
,
K.
and
Yu
,
Y.
(
2008
)
The dynamics of calcium oscillations that activate mammalian eggs
.
Int. J. Dev. Biol.
52
,
585
594
doi:
29
Beena
,
K.
,
Udgaonkar
,
J.B.
and
Varadarajan
,
R.
(
2004
)
Effect of signal peptide on the stability and folding kinetics of maltose binding protein
.
Biochemistry
43
,
3608
3619
doi:
30
Nomikos
,
M.
,
Theodoridou
,
M.
,
Elgmati
,
K.
,
Parthimos
,
D.
,
Calver
,
B.L.
,
Buntwal
,
L.
et al. 
(
2014
)
Human PLCζ exhibits superior fertilization potency over mouse PLCζ in triggering the Ca2+ oscillations required for mammalian oocyte activation
.
Mol. Hum. Reprod.
20
,
489
498
doi:
31
Kouchi
,
Z.
,
Shikano
,
T.
,
Nakamura
,
Y.
,
Shirakawa
,
H.
,
Fukami
,
K.
and
Miyazaki
,
S.
(
2005
)
The role of EF-hand domains and C2 domain in regulation of enzymatic activity of phospholipase Cζ
.
J. Biol. Chem.
280
,
21015
21021
doi:
32
Nomikos
,
M.
,
Elgmati
,
K.
,
Theodoridou
,
M.
,
Calver
,
B.L.
,
Nounesis
,
G.
,
Swann
,
K.
et al. 
(
2011
)
Phospholipase Cζ binding to PtdIns(4,5)P2 requires the XY-linker region
.
J. Cell Sci.
124
,
2582
2590
doi:
33
Cox
,
L.J.
,
Larman
,
M.G.
,
Saunders
,
C.M.
,
Hashimoto
,
K.
,
Swann
,
K.
and
Lai
,
F.A.
(
2002
)
Sperm phospholipase C zeta from humans and cynomolgus monkeys triggers Ca2+ oscillations, activation and development of mouse oocytes
.
Reproduction
124
,
611
623
doi:
34
Knott
,
J.G.
,
Kurokawa
,
M.
,
Fissore
,
R.A.
,
Schultz
,
R.M.
and
Williams
,
C.J.
(
2005
)
Transgenic RNA interference reveals role for mouse sperm phospholipase Cζ in triggering Ca2+ oscillations during fertilization
.
Biol. Reprod.
72
,
992
996
doi:
35
Kashir
,
J.
,
Nomikos
,
M.
,
Lai
,
F.A.
and
Swann
,
K.
(
2014
)
Sperm-induced Ca2+ release during egg activation in mammals
.
Biochem. Biophys. Res. Commun.
450
,
1204
1211
doi:
36
Amdani
,
S.N.
,
Yeste
,
M.
,
Jones
,
C.
and
Coward
,
K.
(
2016
)
Phospholipase C zeta (PLCζ) and male infertility: clinical update and topical developments
.
Adv. Biol. Regul.
61
,
58
67
doi:
37
Kouchi
,
Z.
,
Fukami
,
K.
,
Shikano
,
T.
,
Oda
,
S.
,
Nakamura
,
Y.
,
Takenawa
,
T.
et al. 
(
2004
)
Recombinant phospholipase Cζ has high Ca2+ sensitivity and induces Ca2+ oscillations in mouse eggs
.
J. Biol. Chem.
279
,
10408
10412
doi:

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