Phospholipase Cζ rescues failed oocyte activation in a prototype of male factor infertility

Objective To determine the effect of infertility-linked sperm phospholipase Cζ (PLCζ) mutations on their ability to trigger oocyte Ca2+ oscillations and development, and also to evaluate the potential therapeutic utility of wild-type, recombinant PLCζ protein for rescuing failed oocyte activation and embryo development. Design Test of a novel therapeutic approach to male factor infertility. Setting University medical school research laboratory. Patient(s) Donated unfertilized human oocytes from follicle reduction. Intervention(s) Microinjection of oocytes with recombinant human PLCζ protein or PLCζ cRNA and a Ca2+-sensitive fluorescent dye. Main Outcome Measure(s) Measurement of the efficacy of mutant and wild-type PLCζ-mediated enzyme activity, oocyte Ca2+ oscillations, activation, and early embryo development. Result(s) In contrast to the wild-type protein, mutant forms of human sperm PLCζ display aberrant enzyme activity and a total failure to activate unfertilized oocytes. Subsequent microinjection of recombinant human PLCζ protein reliably triggers the characteristic pattern of cytoplasmic Ca2+ oscillations at fertilization, which are required for normal oocyte activation and successful embryo development to the blastocyst stage. Conclusion(s) Dysfunctional sperm PLCζ cannot trigger oocyte activation and results in male factor infertility, so a potential therapeutic approach is oocyte microinjection of active, wild-type PLCζ protein. We have demonstrated that recombinant human PLCζ can phenotypically rescue failed activation in oocytes that express dysfunctional PLCζ, and that this intervention culminates in efficient blastocyst formation.

O ocyte (or egg) activation, the earliest step of mammalian embryonic development after fertilization, is triggered by a characteristic series of large cytoplasmic Ca 2þ transients known as Ca 2þ oscillations (1,2). This striking Ca 2þ signaling phenomenon is both necessary and sufficient for the completion of all the events of egg activation such as cortical granule exocytosis, which acts to prevent polyspermy, the resumption and completion of meiosis, and pronuclei formation (3). Over the last decade, there has been growing evidence indicating that the physiologic agent responsible for generating Ca 2þ oscillations and the subsequent egg activation is a testisspecific isoform of phospholipase C, PLCz (4)(5)(6)(7). These studies culminate in the proposal that PLCz is delivered by the fertilizing sperm into the ooplasm, whereupon it hydrolyzes the membrane phospholipid substrate, phosphatidylinositol 4,5 bisphosphate (PIP 2 ), to trigger cytoplasmic Ca 2þ oscillations via the inositol 1,4,5trisphosphate (InsP 3 ) intracellular Ca 2þ signaling pathway (3)(4)(5). The smallest known mammalian PLC isozyme of $70 kd, PLCz consists of four EF hands, the catalytic X and Y domains, and a C2 domain (4,5). Each of the individual PLCz domains appears to have an essential role in conferring the distinct biochemical characteristics and the unique mode of regulation of this gamete-specific PLC isozyme (8)(9)(10)(11)(12)(13)(14).
The fundamental role of PLCz in mammalian fertilization has been further highlighted by recent clinical studies that have directly linked abnormal PLCz protein expression profiles with documented cases of male infertility (15)(16)(17)(18). Sperm from patients that displayed either reduced PLCz protein abundance or that expressed mutated forms of PLCz were correlated specifically with failed fertilization after intracytoplasmic sperm injection (ICSI) treatment, which was due empirically to the inability of such sperm to initiate the vital Ca 2þ oscillations required for egg activation (15)(16)(17)(18). The observation of aberrant sperm PLCz protein expression in infertile males suggests that the wild-type PLCz protein could be used as a potential therapy to overcome such cases of infertility. However, it is not known whether the wild-type human PLCz protein is able to physiologically activate eggs in the presence of mutant PLCz and if this would successfully lead to normal embryo development.
We now show that purified recombinant human PLCz protein is capable of hydrolyzing PIP 2 with a similar Ca 2þ dependence to mouse PLCz, and that it can also induce cytoplasmic Ca 2þ oscillations after microinjection into both mouse and human eggs, leading to successful egg activation and early embryo development. We also demonstrate the deleterious effect of male-infertility-linked PLCz mutations on both Ca 2þ oscillations and PIP 2 hydrolysis activity. Notably, mouse eggs expressing the mutant human PLCz were unable to activate normally and failed to commence embryo development. However, this infertile phenotype could be effectively rescued by microinjection of the wild-type human PLCz protein, leading to Ca 2þ oscillations and successful early embryogenesis up to the blastocyst stage. Our findings demonstrate the potential utility of PLCz protein in in vitro fertilization (IVF) treatment, thus providing a novel therapeutic agent that may help to overcome those cases of egg activation failure caused by deficient or defective forms of PLCz in human sperm.

Expression Plasmid Construction and cRNA Synthesis
A pCR3 plasmid construct encoding human PLCz-luciferase (19) was subjected to site-directed mutagenesis (QuikChange II; Stratagene) to generate the PLCz H233L and PLCz H398P mutants. Wild-type human PLCz (GenBank #AF532185) and the H233L and H398P mutants were amplified by polymerase chain reaction (PCR) from the corresponding pCR3 plasmid by use of Phusion polymerase (Finnzymes) to incorporate a 5 0 SalI site and a 3 0 NotI site and were cloned into a modified pET expression vector (pETMM60). The primers used for amplification of wild-type and mutant PLCz were: 5 0 -CCTAGTCGACA TGGAAATGAGATGGTTTTTGTC-3 0 (forward) and 5 0 -CTAA GCGGCCGCTCATCTGACGTACCAAACATAAAC-3 0 (reverse).

Protein Expression and Purification
For the NusA-PLC fusion protein expression studies, Escherichia coli (Rosetta [DE3]; Novagen) transformed with the appropriate pETMM60 plasmid was cultured at 37 C until A 600 reached 0.6, and NusA-fusion protein expression was induced for 18 hours at 16 C with 0.1 mM isopropyl-b-D-thiogalactopyranoside (IPTG; Promega). Cells were harvested (6,000 Â g for 10 minutes), resuspended in phosphate-buffered saline (PBS) containing a protease inhibitor mixture (EDTA-free; Roche), and sonicated 4 Â 15 seconds on ice. Soluble NusAfusion proteins were then purified by affinity chromatography on Ni-NTA resin after standard procedures (Qiagen) and elution with 275 mM imidazole. Eluted proteins were dialyzed overnight (10,000 MWCO; Pierce) at 4 C against 4 L of PBS, and concentrated with centrifugal concentrators (Sartorius; 10,000 MWCO).

Assay of PLC Activity
The PIP 2 hydrolytic enzyme activity of recombinant PLC proteins was assayed as previously described elsewhere (8,11,13). The final concentration of PIP 2 in the reaction mixture was 220 mM, containing 0.05 mCi of [ 3 H]PIP 2 . The hydrolysis assay conditions were optimized for linearity of enzyme kinetic activity, requiring a 10-minute incubation of 20 pmol of PLCz protein sample at 25 C. In assays to determine dependence on PIP 2 concentration, 0.05 mCi of [ 3 H]PIP 2 was mixed with cold PIP 2 to give an admixture of the appropriate final PIP 2 concentration. In assays examining PLC Ca 2þ sensitivity, Ca 2þ buffers were prepared by EGTA/CaCl 2 admixture, as previously described elsewhere (8,13).

Preparation of Gametes and Analysis of Embryos
Experiments were carried out with mouse eggs in HEPESbuffered potassium simplex optimized medium (H-KSOM) as previously described elsewhere (8,12,14). Eggs obtained from superovulated mice were microinjected 14.5 to 15.5 hours after human chorionic gonadotropin (hCG) administration (14,19). All procedures were in accordance with the UK Home Office Animals Procedures Act and were approved by the Cardiff University Animals Ethics Committee.
For the egg activation and embryo development studies, recombinant human PLCz protein-injected mouse eggs were kept in KSOM containing 5 mg/mL cytochalasin B for 6 hours. After pronuclei formation was observed, the activated eggs were cultured in KSOM at 37 C in 5% CO 2 , and the different stages of the early embryo development process were observed and counted at 6, 24, 48, 72, and 96 hours.

Microinjection and Measurement of Intracellular Ca 2D and Luciferase Expression
Mouse eggs were washed in M2 and microinjected either with complementary RNA (cRNA) or recombinant protein diluted in injection buffer (120 mM KCl, 20 mM HEPES, pH 7.4). All injections were 3% to 5% of the egg volume (10,12). The cRNA or recombinant protein was mixed with an equal volume of 1 mM Oregon Green BAPTA dextran (Molecular Probes). Eggs were maintained in H-KSOM containing 100 mM luciferin and were imaged on a Nikon TE2000 or Zeiss Axiovert 100 microscope equipped with a cooled intensified CCD camera (Photek Ltd.). Cytoplasmic Ca 2þ changes were monitored in these eggs for 4 hours after injection by measuring the Oregon Green BAPTA-dextran fluorescence with low-level excitation light from a halogen lamp (11,14).
At the end of Ca 2þ measurements, the same set of eggs was then monitored for luminescence (i.e., indicating recombinant protein concentration) by integrating light emission (in the absence of fluorescence excitation) for 20 minutes using the same intensified CCD camera (18,19). Notably, the fluorescence signals were typically 10 to 100 times greater than the luminescence signals. The Ca 2þ measurements for an egg were further analyzed only if the same egg was also luminescent. The luminescence reading from eggs was converted into an amount of luciferase by use of a standard curve that was generated by placing eggs in a luminometer that had been previously calibrated by microinjection with known amounts of luciferase protein (Sigma) (8,21).

Immunofluorescence of Sperm PLCz
The anti-PLCz, V-37, polyclonal antibody was raised in rabbits against a 16-mer-peptide sequence ( 8 SKIQDDFRGGKIN-LEK 23 ) of human PLCz protein and was affinity-purified as per the manufacturer's instructions (Invitrogen). Anti-NusA and anti-b-actin mouse monoclonal antibodies were purchased from Santa Cruz Biotechnology.
Human sperm samples washed with PBS (pH 7.4) were fixed with 4% ethanol-free formaldehyde (Polysciences Inc.) for 30 minutes at 4 C. Fixed samples were resuspended in PBS and spotted onto 0.1% poly L-lysine-coated (Sigma-Aldrich) coverslips and dried for 2 hours at 37 C before permeabilization with 1% Triton X-100 for 1 hour at 23 C. After blocking with 5% normal goat serum (Invitrogen) for 30 minutes, the samples were incubated with V-37 antibody (rabbit IgG in PBS containing 5% normal goat serum) overnight at 4 C, washed with PBS, then incubated with Alexa-488conjugated goat anti-rabbit antibody (Invitrogen) for 45 minutes. The samples were mounted on slides with antifading reagent (Invitrogen) and observed using a SP5 confocal microscope (Leica) under Â100 oil immersion objective; the collected images were edited with ImageJ (http://rsbweb.nih. gov/ij).

SDS-PAGE and Immunoblot Analysis
Fresh human sperm samples washed with PBS (pH 7.4) were mixed with 5Â SDS sample buffer, vortexed briefly, and sonicated for 5 seconds on ice. Sperm samples and recombinant proteins were separated by SDS-PAGE, as previously described elsewhere (8,18). Separated proteins were transferred onto polyvinylidene difluoride membrane and incubated overnight at 4 C with the appropriate primary antibody. Detection of horseradish peroxidase-coupled secondary antibody was achieved by use of Super Signal West Dura (Pierce) and a Bio-Rad ChemiDoc gel documentation system for image capture (11,20).
The human sperm and oocytes used in this study were donated by patients attending the IVF Wales clinic at the University Hospital of Wales, Cardiff, UK. The current project and all associated procedures were approved by the local South East Wales Research Ethics Committee and also by the UK Human Fertilisation and Embryology Authority (R0161).

Native and Recombinant Human PLCz Analysis
The expression and distribution of native PLCz in fertile human sperm was examined by immunoblot and immunofluorescence analysis on ejaculated sperm from a man whose partner had achieved successful pregnancy via ICSI. An affinity-purified, anti-PLCz polyclonal antibody positively detected a single, immunoreactive 70 kd protein corresponding to human PLCz (5), with the control anti-b-actin antibody identifying a 42 kd human b-actin band (Fig. 1A). Immunofluorescence analysis revealed native PLCz localization primarily in the equatorial region of the sperm head with some additional acrosomal staining (see Fig. 1B). Equatorial localization of PLCz is congruent with fusion of this sperm region with the oocyte plasma membrane at fertilization, thus facilitating early entry of PLCz into the ooplasm (1-3). The acrosomal staining suggests either an additional role of PLCz in earlier steps of fertilization that remains undefined, or is due to nonspecific immunoreactivity, although the immunoblot detection of only a single 70 kd protein (see Fig. 1A) would be consistent with the former suggestion.
Recombinant human PLCz was expressed as a NusAhexahistidine fusion protein in E. coli and purified by Ni-NTA affinity chromatography. Our earlier use of plasmid vectors comprising only the hexahistidine tag (i.e., without a fusion protein, such as NusA) provided reliable recombinant PLCz protein expression, but it did not effectively yield soluble, functional PLCz (unpublished data). In contrast, significant expression of soluble NusA-PLCz was observed, and the affinity-purified human PLCz fusion protein, after SDS-PAGE and immunoblot analysis, displayed the predicted $130 kd molecular mass (NusA $60 kd þ 70 kd hPLCz) ( Fig. 2A). Enzymatic determination of [ 3 H]PIP 2 hydrolysis activity for the purified human PLCz, mouse PLCz, and rat PLCd1 fusion proteins (8,11,13,18) (see Fig. 2B) reveals the human PLCz to have 42% higher specific activity than mouse PLCz (655 AE 36 vs. 460 AE 24 nmol/min/mg), but both of these PLCs had much lower specific activity (27% to 38%) relative to PLCd1 (1,703 AE 52 nmol/min/mg) ( Table 1). The relative Ca 2þ sensitivity of [ 3 H]PIP 2 hydrolysis was determined between 0.1 nM to 0.1 mM Ca 2þ (see Fig. 2C), yielding an EC 50 value for human PLCz that was near identical with mouse PLCz (70 vs. 64 nM Ca 2þ ), but this was in sharp contrast with the $80-fold higher PLCd1 EC 50 value of 5,327 nM (see Table 1). The marked EC 50 disparity is consistent with previous studies of PLC isoform Ca 2þ sensitivity that indicated that only PLCz would be near-optimally activated to hydrolyze its PIP 2 substrate at the $100 nM resting Ca 2þ levels in mammalian eggs (6,8).
Microinjection of recombinant wild-type human PLCz into mouse and human eggs revealed that it possesses a potent ability to induce cytoplasmic Ca 2þ oscillations (Fig. 3A, top and bottom traces, respectively), matching that observed after microinjection of native sperm extracts (2, 3). The NusA protein microinjection alone did not cause any Ca 2þ  changes (see Fig. 3A, middle trace). The minimal PLCz concentration required for a physiologic pattern of Ca 2þ oscillations was 0.0167 mg/mL, indicating that the amount of human PLCz in mouse eggs able to induce Ca 2þ oscillations and early embryogenesis was $80 fg/egg. This is in the same range as the estimated PLCz content within a single sperm (4). Moreover, we observed that highly efficient early embryo development, from pronuclei formation up to the multicellular blastocyst stage, was also specifically initiated by the human PLCz protein microinjection (see Fig. 3B). The successful early development to the blastocyst embryo stage observed with wild-type PLCz-injected eggs was >50% (see Fig. 3B), a value that is very similar to that previously obtained after microinjection of cRNA encoding luciferasetagged human PLCz (19).

In Vivo and In Vitro Analysis of Infertility-Linked PLCz Mutations
The first direct link between male infertility and a defective PLCz gene was made after identification of a PLCz point mutation in an infertile man with failed fertilization after ICSI treatment (16). This PLCz catalytic domain mutation of a conserved histidine residue to a proline (H398P) (Fig. 4A) disrupts both enzymatic PIP 2 hydrolysis and Ca 2þ release activity in mouse eggs (18). A second PLCz mutation, also in the Note: Summary of the specific hydrolytic enzyme activity and the EC50 values of Ca 2þ -dependent enzyme activity for [ 3 H]PIP2 hydrolysis that was determined as described in Materials and Methods; the data were analyzed by nonlinear regression analysis (GraphPad Prism 5) for the affinity-purified NusA-hexahistidine fusion proteins for hPLCz, mPLCz, and rPLCd1 (see Fig. 2B and C).  catalytic domain (H233L) (see Fig. 4A), has recently been identified (17), although this particular histidine residue is not conserved.
To enable the comparison of relative recombinant protein expression by luminescence measurement (8,21), we prepared luciferase-fusion constructs of each of these human PLCz mutants as well as wild-type PLCz for microinjection into mouse eggs. Prominent Ca 2þ oscillations ($9 spikes/2 hours) were observed in wild-type PLCz cRNA-injected mouse eggs, with the first Ca 2þ spike occurring after $25 minutes at a luminescence reading of 0.07 counts per second (Table 2), corresponding to expression of $29 fg PLCz/egg (see Fig. 4B, top trace; see Table 2). Microinjection of mutant PLCz H398P cRNA totally failed to cause any Ca 2þ oscillations in mouse eggs (see Fig. 4B, middle trace) (16), consistent with our recent findings for the equivalent mouse mutant (PLCz H435P ) (18). It is interesting that, with the other catalytic domain mutation, the PLCz H233L cRNA produced a dramatic reduction in Ca 2þ oscillation frequency compared with that of wild type (see Fig. 4B, bottom trace), with only $2.8 spikes/2 hours observed (see Table 2). Moreover, there was also a significant delay in initiation of cytoplasmic Ca 2þ

FIGURE 4
Effect of H233L and H398P mutations on Ca 2þ oscillation-inducing activity of human phospholipase Cz (PLCz) in mouse eggs. (A) Schematic representation of human PLCz domain structure identifying the location of H233L and H398P mutations within the X and Y catalytic domains, respectively. (B) Fluorescence and luminescence recordings reporting the cytosolic Ca 2þ changes (black traces; Ca 2þ ) and luciferase-PLCz expression level (in counts per second, cps), respectively, in unfertilized mouse eggs after the microinjection of cRNA encoding luciferasetagged, wild-type human PLCz, and the PLCz H233L and PLCz H398P mutants. Panels on the right display the integrated luminescence image of individual mouse eggs after cRNA microinjection of either wild-type or mutant PLCz. The relatively low luminescence values achieved, corresponding to femtogram levels of PLCz protein expressed in each cRNA-microinjected egg, are intended to mimic the approximate amount of PLCz that is delivered by entry of a single sperm. oscillations in the egg, with the first Ca 2þ spike appearing after $190 minutes at a luminescence value of 0.34 counts per second. Hence, whereas PLCz H398P completely abrogates, the PLCz H233L mutation substantially reduces the frequency of Ca 2þ oscillations in mouse eggs, with both resulting in a failure to activate embryo development.
The in vitro PIP 2 hydrolysis activity of the wild-type human PLCz, and the PLCz H233L and PLCz H398P mutant proteins was compared after their expression in E. coli, purification by Ni-NTA affinity chromatography, and gel/immunoblot analysis (Fig. 5A). Enzyme specific activity values obtained for each protein reveal that the PLCz H233L mutant retains only 24% of the activity of wild-type PLCz (157 AE 48 vs. 655 AE 36 nmol/min/mg), and the PLCz H398P mutant almost completely fails to hydrolyze [ 3 H]PIP 2 . These enzymatic data indicate that both of these histidine mutations when introduced into human PLCz dramatically diminish their PIP 2 hydrolytic activity, thus directly explaining why the cRNA microinjection of these PLCz mutants into unfertilized mouse eggs fails to induce normal egg activation.

Rescue of Egg Activation Failure by Microinjection of Human PLCz Protein
We further investigated whether the purified, recombinant wild-type human PLCz protein would be able to rescue the egg activation failure observed after expression of the infertility-linked human PLCz H398P and PLCz H233L mutants in mouse eggs (see Fig. 4B, middle and bottom traces, respectively). For this experiment, two different sets of mouse eggs were microinjected with cRNA encoding either the human PLCz H398P or PLCz H233L mutant. During the 3-hour time period after the injection of the mutant cRNAs, which enabled both of the mutant PLCz proteins to be expressed (>0.30 counts per second) at the physiologic level required for fertilization (i.e., the amount of PLCzeta normally present in a single sperm), there were no detectable Ca 2þ changes observed in either set of mouse eggs (Fig. 6, short arrow-traces on left). At this 3-hour post-cRNA time point, the same eggs were again microinjected, but this time with $80 fg of the purified recombinant, human wild-type PLCz protein.
This intervention with microinjected protein immediately resulted in the highly effective induction of a normal pattern of Ca 2þ oscillations (see Fig. 6, long arrow-traces in middle), leading to efficient physiologic egg activation and successful early embryo development up to the multicellular blastocyst stage (see Fig. 6, micrographs on right). The efficiency of development to the blastocyst stage for the wild-type PLCz protein-injected eggs was close to 60%. The observation of efficacious phenotypic rescue of mutant PLCz-mediated egg -0.39 AE 0.020 --Note: Values are mean AE standard error of the mean. The Ca 2þ oscillation-inducing activity (Ca 2þ spike number in 2 hours; time to first spike) and the simultaneously-measured PLCz-luciferase fusion protein luminescence levels (peak luminescence; luminescence at first spike) are summarized for mouse eggs that had been microinjected, as described in Materials and Methods, with cRNA encoding one of the following human PLCz-luciferase constructs: wild-type PLCz, the PLCz H233L or PLCz H398P mutant (see Fig. 4B). cps ¼ counts per second.
Nomikos. PLCz rescue of failed egg activation. Fertil Steril 2013. activation failure suggests that the direct microinjection of active, wild-type human PLCz protein could potentially be used as a therapy in specific cases of failed ICSI due to defective PLCz in human sperm.

DISCUSSION
Since the discovery of PLCz a decade ago (4), mounting evidence has strongly supported the notion that sperm-derived PLCz is the sole physiologic trigger of egg activation during mammalian fertilization (3,22,23). Upon sperm-egg fusion, it is believed that PLCz is introduced into the ooplasm and catalyses PIP 2 hydrolysis to generate InsP3. The intracellular Ca 2þ release triggered by InsP3 produces the characteristic cytoplasmic Ca 2þ oscillations that result in egg activation, and this initiates the embryo development process. Since then, PLCz has been identified in many different mammalian species, suggesting that it could play a pivotal role at fertilization in all mammals. Furthermore, recent clinical reports have linked reduced protein expression levels and abnormal forms of PLCz with human male infertility (15)(16)(17)(18)24). Although ICSI is a powerful technique that is extensively used by IVF clinics to overcome many conditions of male infertility, clinical studies have identified men whose sperm repeatedly fail to fertilize after ICSI due to egg activation failure. The sperm that fail at ICSI cannot induce the Ca 2þ oscillations required for activation, and recent evidence indicates that this infertile phenotype is associated with defective sperm PLCz protein in these patients, caused either by a low level of sperm PLCz protein expression or by genetic mutations resulting in a dysfunctional PLCz in sperm (15)(16)(17)(18)24).
Despite the major role of PLCz in mammalian fertilization, thus far only purified recombinant mouse PLCz has been successfully used to study in vitro biochemical properties and the regulatory mechanisms underlying PLCz function (6,8,9,11,13). In this study, we prepared recombinant human PLCz protein fused to NusA, a fusion protein known to greatly enhance the solubility and stability of recombinant proteins (25). Human PLCz is present as a 70 kd protein at the equatorial region in sperm (see Fig. 1). The purified human PLCz protein exhibited higher in vitro PIP 2 hydrolysis activity than recombinant mouse PLCz, whereas the EC 50 for Ca 2þ sensitivity was very similar for both recombinant proteins (see Fig. 2; see Table 1). Microinjection of recombinant, wild-type human PLCz protein induced Ca 2þ oscillations in both mouse and human eggs (see Fig. 3) and successfully activated mouse early embryo development up to the blastocyst stage.
The estimated amount of human PLCz protein in mouse eggs that was required to efficiently induce Ca 2þ oscillations and embryogenesis was $80 fg/egg (3-5 pL of 0.0167 mg/ mL), which is entirely consistent with the PLCz levels previously shown to be able to trigger egg activation and efficient development of mouse eggs (4,19). Recombinant mouse PLCz synthesized by baculovirus expression was less efficient at inducing Ca 2þ oscillations in mouse eggs compared with recombinant human PLCz, requiring an estimated 300 fg/ egg (6). In our preliminary studies using recombinant Egg activation failure with mutant forms of human phospholipase Cz (PLCz) rescued by microinjection of recombinant, wild-type human PLCz protein. The traces on the left report the Ca 2þ concentration changes observed in unfertilized mouse eggs after microinjection with the cRNA for the mutants PLCz H398P (short arrow, upper panel) and PLCz H233L (short arrow, lower panel). After a period of 3 hours to enable femtogram expression of the mutant PLCz proteins, a second microinjection of $80 fg of the affinity-purified, wild-type hPLCz recombinant protein was performed as described in Figure 3 (long arrows, upper and lower panels), approximating the amount of native hPLCz in a single sperm. The two panels on the right display representative micrographs illustrating the mouse embryos at the blastocyst developmental stage that were observed 96 hours after microinjection of the human PLCz recombinant protein into each mouse egg. human PLCz expressed alone, without the accompanying presence of a fusion protein to assist in stabilizing enzyme activity, we observed very poor ability to generate Ca 2þ oscillations. These observations are entirely consistent with the very recent report using human PLCz expressed without a fusion protein partner that required injection of 5,000 to 10,000 fg/egg to cause Ca 2þ oscillations and did not result in embryo development to the blastocyst stage (29). Thus, our strategic use of NusA as an efficient fusion protein partner appears to be important for the recovery of significant levels of soluble human PLCz. Importantly, this enzymatically active PLCz is capable of effecting successful embryo development when injected into mammalian eggs (see Fig. 3B), via generation of the characteristic Ca 2þ oscillations that mimic the physiologic egg activation phenomenon observed at fertilization (see Fig. 3A).
To investigate whether injection of recombinant human PLCz protein would be able to rescue the failed egg activation caused by infertility-linked PLCz mutants, we assessed the effect of two novel point mutations identified in the PLCz gene that have previously been specifically linked to male infertility (16,17,24). Both of these point mutations, H233L and H398P, are located on the X and Y catalytic domains of human PLCz, respectively (see Fig. 4A), and they have been found to dramatically reduce in vitro PIP 2 hydrolysis activity (see Fig. 5B), fully consistent with their inability to produce the normal pattern of Ca 2þ release in mouse eggs, resulting in egg activation failure (see Fig. 4B). However, microinjection of wild-type human PLCz protein into mouse eggs that were expressing these infertility-linked PLCz mutants effectively rescued the failure of egg activation by inducing a normal pattern of Ca 2þ oscillations, leading to successful early embryo development up to the blastocyst stage (see Fig. 6).
These findings promote the potential application of PLCz protein into IVF clinics as an effective therapeutic option for egg activation failure due to male factor deficiencies related to PLCz dysfunction. It has previously been demonstrated that egg activation failure due to defective PLCz can be approached by using a Ca 2þ ionophore treatment during ICSI (26), even though this procedure does not specifically induce the characteristic Ca 2þ oscillations observed at fertilization (27). However, it currently remains to be determined whether such ionophore treatment represents the safest or most effective method for overcoming egg activation failure, as it is known that the precise pattern of Ca 2þ oscillations after fertilization in mouse eggs can exert potentially deleterious downstream, longer-term effects on both gene expression and embryo development (28).
The co-microinjection of PLCz cRNA during ICSI could, in principle, be used to rescue egg activation failure of PLCz-deficient sperm. This method would, however, present difficulties in practice because the rate of synthesis and total amount of PLCz protein expressed in the egg cannot be readily controlled using a bolus of microinjected cRNA. Previous studies have shown that successful embryo development requires PLCz to be present within the egg at a relatively precise concentration range to closely match the specific amount of PLCz that would be provided physiologically by the entry of a single mature sperm at fertilization (19). Thus, the availability of purified, active recombinant human PLCz protein appears to represent both a highly practical and the most physiologic therapeutic agent for overcoming failed ICSI cases resulting from aberrant sperm PLCz. Recombinant human PLCz protein could potentially also be used in regenerative medicine approaches via generation of parthenogenetic embryos and blastocysts that may facilitate stem cell derivation and differentiation.