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

Abstract

Objective: To determine the effect of infertility-linked sperm phospholipase Cζ (PLCζ) mutations on their ability to trigger oocyte Ca(2+) 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 Ca(2+)-sensitive fluorescent dye.

Main outcome measure(s): Measurement of the efficacy of mutant and wild-type PLCζ-mediated enzyme activity, oocyte Ca(2+) 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 Ca(2+) 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.

Figure 1

Expression and distribution of phospholipase Cζ (PLCζ) protein in human sperm. (A) Immunoblot analysis of PLCζ protein in human sperm. Sperm cells (25,000 per lane) were analyzed by 9% SDS-PAGE, proteins were electrophoretically transferred, and the blot membrane was incubated either with affinity-purified, anti-PLCζ polyclonal antibody (V-37; 1:7,500 dilution; left panel) or with anti-β-actin monoclonal antibody (1:2,500 dilution; right panel). (B) Representative confocal microscope images of PLCζ immunofluorescence in human sperm after fixing and immunostaining with anti-PLCζ polyclonal antibody (V-37; 1:1,000 dilution) showing that the native PLCζ in this IVF patient localizes to the equatorial and acrosomal region of the sperm head.

Figure 2

Expression and enzymatic characterization of recombinant wild-type human phospholipase Cζ (PLCζ) protein. (A) One μg of bacterially-expressed, affinity-purified NusA-hPLCζ fusion protein analyzed by 7% SDS-PAGE (left panel) or by immunoblot analysis with either anti-PLCζ polyclonal (V-37; 1:10,000 dilution; middle panel) or anti-NusA monoclonal antibody (1:20,000 dilution; right panel). (B) The PIP₂ hydrolysis enzyme activities of recombinant hPLCζ, mPLCζ, and rPLCδ1 purified by nickel affinity chromatography as NusA-fusion proteins (20 pmol) determined with the [³H]PIP₂ cleavage assay, n = 3 ± standard error of the mean (SEM), using two different preparations of recombinant protein and with each experiment performed in duplicate. In control experiments with NusA alone, no specific PIP₂ hydrolysis activity was observed (data not shown). (C) Effect of varying [Ca²⁺] on the normalized PIP₂ hydrolysis enzyme activity of purified, recombinant hPLCζ, mPLCζ, and rPLCδ1 NusA-fusion proteins. For these assays, n = 2 ± SEM using two different batches of recombinant proteins and with each experiment performed in duplicate.

Figure 3

Recombinant human phospholipase Cζ (PLCζ) protein induces Ca²⁺ oscillations in mouse and human eggs and initiates early embryo development. (A) Representative fluorescence (au: arbitrary units) recordings reporting the Ca²⁺ concentration changes in a mouse and human egg after microinjection of human PLCζ recombinant protein (top and bottom trace, respectively). Microinjection of NusA alone does not induce Ca²⁺ release in mouse eggs (middle trace). (B) Micrographs illustrating mouse embryos at the various early developmental stages (pronuclear formation [PN], two-cell and eight-cell stages, and blastocyst stage) achieved after egg microinjection with ∼80 fg of purified, wild-type human PLCζ recombinant protein (0.0167 mg/mL). The optimal efficiency of blastocyst formation achieved by microinjection of hPLCζ into mouse eggs was 50% to 60%.

Figure 4

Effect of H233L and H398P mutations on Ca²⁺ oscillation-inducing activity of human phospholipase Cζ (PLCζ) in mouse eggs. (A) Schematic representation of human PLCζ 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 Ca²⁺ changes (black traces; Ca²⁺) and luciferase-PLCζ expression level (in counts per second, cps), respectively, in unfertilized mouse eggs after the microinjection of cRNA encoding luciferase-tagged, wild-type human PLCζ, and the PLCζ^H233L and PLCζ^H398P mutants. Panels on the right display the integrated luminescence image of individual mouse eggs after cRNA microinjection of either wild-type or mutant PLCζ. The relatively low luminescence values achieved, corresponding to femtogram levels of PLCζ protein expressed in each cRNA-microinjected egg, are intended to mimic the approximate amount of PLCζ that is delivered by entry of a single sperm.

Figure 5

Expression, purification, and enzyme activity of the PLCζ^H233L and PLCζ^H398P mutant proteins. (A) The affinity-purified NusA-fusion proteins for PLCζ^H233L and PLCζ^H398P (1 μg) analyzed by 7% SDS-PAGE (left panel) or by immunoblot analysis using anti-PLCζ polyclonal (V-37; 1:10,000 dilution; middle panel) or anti-NusA monoclonal antibody (1:20,000 dilution; right panel). (B) The [³H]PIP₂ hydrolysis activity of the purified PLCζ^H233L and PLCζ^H398P proteins, n = 3 ± standard error of the mean, determined using two different preparations of recombinant protein and with each experiment performed in duplicate.

Figure 6

Egg activation failure with mutant forms of human phospholipase Cζ (PLCζ) rescued by microinjection of recombinant, wild-type human PLCζ protein. The traces on the left report the Ca²⁺ concentration changes observed in unfertilized mouse eggs after microinjection with the cRNA for the mutants PLCζ^H398P (short arrow, upper panel) and PLCζ^H233L (short arrow, lower panel). After a period of 3 hours to enable femtogram expression of the mutant PLCζ proteins, a second microinjection of ∼80 fg of the affinity-purified, wild-type hPLCζ recombinant protein was performed as described in Figure 3 (long arrows, upper and lower panels), approximating the amount of native hPLCζ 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 PLCζ recombinant protein into each mouse egg.