Chimeras of sperm PLCζ reveal disparate protein domain functions in the generation of intracellular Ca2+ oscillations in mammalian eggs at fertilization

Abstract

Phospholipase C-zeta (PLCζ) is a sperm-specific protein believed to cause Ca(2+) oscillations and egg activation during mammalian fertilization. PLCζ is very similar to the somatic PLCδ1 isoform but is far more potent in mobilizing Ca(2+) in eggs. To investigate how discrete protein domains contribute to Ca(2+) release, we assessed the function of a series of PLCζ/PLCδ1 chimeras. We examined their ability to cause Ca(2+) oscillations in mouse eggs, enzymatic properties using in vitro phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and their binding to PIP2 and PI(3)P with a liposome interaction assay. Most chimeras hydrolyzed PIP2 with no major differences in Ca(2+) sensitivity and enzyme kinetics. Insertion of a PH domain or replacement of the PLCζ EF hands domain had no deleterious effect on Ca(2+) oscillations. In contrast, replacement of either XY-linker or C2 domain of PLCζ completely abolished Ca(2+) releasing activity. Notably, chimeras containing the PLCζ XY-linker bound to PIP2-containing liposomes, while chimeras containing the PLCζ C2 domain exhibited PI(3)P binding. Our data suggest that the EF hands are not solely responsible for the nanomolar Ca(2+) sensitivity of PLCζ and that membrane PIP2 binding involves the C2 domain and XY-linker of PLCζ. To investigate the relationship between PLC enzymatic properties and Ca(2+) oscillations in eggs, we have developed a mathematical model that incorporates Ca(2+)-dependent InsP3 generation by the PLC chimeras and their levels of intracellular expression. These numerical simulations can for the first time predict the empirical variability in onset and frequency of Ca(2+) oscillatory activity associated with specific PLC variants.

Keywords: sperm, PLC-zeta, calcium oscillations, egg activation, fertilization.

Figure 1

Construction of various PLCζ/PLCδ1 chimeras. Schematic linear representation of the domain structure of PLCζ (red), PLCδ1 (blue), ΔPH/PLCδ1 and their corresponding PLCζ/PLCδ1 chimeras; PHδ1/PLCζ, EFδ1/PLCζ, PLCζ/XYlinkδ1, PLCζ/XYδ1 and PLCζ/C2δ1. The various amino acid lengths and respective coordinates are indicated for each construct.

Figure 2

Expression of PLCζ/PLCδ1 chimeras in unfertilized mouse eggs. Fluorescence and luminescence recordings reporting the Ca²⁺ changes (black traces; Ca²⁺) and luciferase expression [red traces; Lum, in counts per second (cps)], respectively, in unfertilized mouse eggs following microinjection of cRNA encoding luciferase-tagged PLCζ, PHδ1/PLCζ, EFδ1/PLCζ, PLCζ/XYlinkδ1, PLCζ/XYδ1, PLCζ/C2δ1, PLCδ1 and ΔPH/PLCδ1 proteins. Panels on the right display representative integrated luminescence image of individual mouse eggs following cRNA microinjection of each PLC construct (see Table I).

Figure 3

Expression of recombinant NusA-tagged PLCζ/PLCδ1 chimeric proteins. Affinity-purified, NusA-6His-PLC fusion proteins (1 μg) were analyzed by 7% SDS–PAGE followed by either Coomassie Brilliant Blue staining (left-hand side panels) or immunoblot analysis using the anti-NusA monoclonal antibody at 1:25 000 dilution (right-hand side panels).

Figure 4

Enzyme activity of PLCζ/PLCδ1 chimeras. PIP₂ hydrolysis enzyme activities of the purified NusA-6His-PLC fusion proteins (20 pmol) were determined in vitro with the standard [³H]PIP₂ cleavage assay, n = 3 ± SEM, using three different preparations of recombinant protein and with each experiment performed in duplicate. In control experiments with NusA protein alone, there was no specific PIP₂ hydrolysis activity observed (data not shown).

Figure 5

Ca²⁺-dependent enzyme activity of PLCζ/PLCδ1 chimeras. Effect of varying [Ca²⁺] on the normalized activity of NusA-6His-tagged, wild-type PLCζ, PLCδ1, ΔPH/PLCδ1 and PLCζ/PLCδ1 chimeric fusion proteins; PHδ1/PLCζ, EFδ1/PLCζ, PLCζ/XYlinkδ1, PLCζ/XYδ1 and PLCζ/C2δ1. For these assays n = 2 ± SEM, using three different batches of recombinant proteins and with each experiment performed in duplicate. Color used for each construct matches the corresponding construct in Fig. 4.

Figure 6

In vitro binding of wild-type PLCζ, PLCδ1, ΔPH/PLCδ1 and PLCζ/PLCδ1 chimeras to PIP₂ and PI(3)P. Liposome ‘pull-down’ assay of various PLC constructs. Unilamellar liposomes comprising PtdCho:CHOL:PtdEtn (4:2:1) with or without 1% PIP₂ or 5% PI(3)P were incubated in the presence of 0.5 mM Mg²⁺ with the various purified PLC recombinant proteins. Following centrifugation, both the supernatant (s) and liposome pellet (p) were subjected to SDS–PAGE and Coomassie Brilliant Blue staining.

Figure 7

Mechanisms of PLCζ, InsP₃ and Ca²⁺ regulation in the egg cytosol during mammalian fertilization and associated numerical simulations of intracellular Ca²⁺ oscillations. (a) Simplified schematic to illustrate the interactions occurring between the PLCζ, InsP₃ and Ca²⁺ pathways within the egg cytosol. Following sperm–egg fusion, PLCζ diffuses from the sperm head into the egg cytosol and targets a distinct vesicular PIP₂-containing endomembrane. PLCζ-mediated hydrolysis of PIP₂ produces InsP₃ which stimulates Ca²⁺ release through binding to endoplasmic reticulum InsP₃ receptors. This mode of InsP₃-stimulated Ca²⁺ release forms the basis of the intracellular Ca²⁺ oscillations at fertilization, which ultimately lead to egg activation and embryo development. The model allows for Ca²⁺ influx and Ca²⁺ extrusion across the plasma membrane. (b) Numerical simulations of Ca²⁺ oscillations in the egg cytosol closely match experimental traces presented in Fig. 2 for corresponding parametric conditions. Individual simulations of oscillatory activity associated with PLCζ, PHδ1/PLCζ, EFδ1/PLCζ and PLCδ1 are generated with the following pairs of PLC and EC₅₀ (x_p) values: 0.88 μM s⁻¹/0.068 μM, 0.35 μM s⁻¹/0.084 μM, 0.83 μM s⁻¹/0.69 μM and 33.8 μM s⁻¹/4.2 μM, in agreement with experimental values presented in Tables I and II. (c) Contour plot mapping the oscillatory frequency of Ca²⁺ oscillations throughout the physiological range of PLC sensitivity to Ca²⁺ (EC₅₀) and PLC protein expression levels. The operating points of PLCζ, PHδ1/PLCζ, EFδ1/PLCζ and PLCδ1 proteins are indicated by circles/arrows. The nanomolar Ca²⁺ sensitivity of PLCζ enables the enzyme to be active at resting Ca²⁺ levels (nM). In contrast, the relatively low, micromolar Ca²⁺ sensitivity of PLCδ1 leads to low-frequency oscillations even at much higher protein expression levels.