Male infertility-linked point mutation disrupts the Ca2+ oscillation-inducing and PIP(2) hydrolysis activity of sperm PLCζ

Abstract

A male infertility-linked human PLCζ (phospholipase Cζ) mutation introduced into mouse PLCζ completely abolishes both in vitro PIP(2) (phosphatidylinositol 4,5-bisphosphate) hydrolysis activity and the ability to trigger in vivo Ca2+ oscillations in mouse eggs. Wild-type PLCζ initiated a normal pattern of Ca2+ oscillations in eggs in the presence of 10-fold higher mutant PLCζ, suggesting that infertility is not mediated by a dominant-negative mechanism.

Figure 1. Ca²⁺ oscillation-inducing activity of PLCζ-luciferase and mutants in mouse eggs

(A) Schematic representation of mouse PLCζ domain structure identifying the location of the H435P mutation within the catalytic Y domain, as well as the D210R control mutation in the X domain. (B) The left-hand panels show representative fluorescence (a.u.; arbitrary units) and luminescence (c.p.s.) recordings reporting the Ca²⁺ concentration changes (black traces; Ca²⁺) and luciferase expression (red traces; Lum) respectively in a mouse egg following microinjection of the indicated PLCζ-luciferase cRNA (encoding either PLCζ^WT, PLCζ^H435P, PLCζ^H435A or PLCζ^D210R). Right-hand panels show integrated images of luciferase luminescence from eggs microinjected with the corresponding PLCζ-luciferase cRNA. The peak luminescence (Lum) recorded is shown in c.p.s.

Figure 2. Expression, purification and enzyme activity of GST-fusion proteins for PLCζ, PLCδ1 and mutants

(A) Affinity-purified GST-fusion proteins for PLCζ^WT, PLCζ^H435P and PLCζ^D210R (2 μg) analysed by SDS/PAGE (8% gels) (left-hand panel) or immunoblot analysis using a polyclonal anti-GST antibody (right-hand panel). (B) Affinity-purified GST-fusion proteins for PLCδ1^WT and PLCδ1^H542P (2 μg) analysed by SDS/PAGE (8% gels) (left-hand panel) or immunoblot analysis using a polyclonal anti-GST antibody (right-hand panel). (C) [³H]PIP₂ hydrolysis activity of the purified GST–PLC fusion proteins. Values are means±S.E.M., n=3.

Figure 3. Co-expression of PLCζ^H435P and PLCζ^WT in mouse eggs

Left-hand panels show representative fluorescence and luminescence recordings reporting Ca²⁺ concentration changes (black traces; Ca²⁺) and luciferase expression (red traces; Lum) respectively in a mouse egg. The egg was co-microinjected with equal amounts of PLCζ-luciferase cRNA encoding PLCζ^WT and PLCζ^H435P (top panel), or was initially microinjected with cRNA for PLCζ^H435P followed, after a period of 1 h (middle panel) or 2 h (bottom panel), by the microinjection of cRNA for PLCζ^WT. Right-hand panels show the integrated image of luciferase luminescence from eggs microinjected with PLCζ^H435P and PLCζ^WT cRNA. The peak luminescence (Lum) recorded is shown in c.p.s.

Figure 4. Effect of PLCζ^H435P on sperm-induced Ca²⁺ oscillations

Mouse eggs were either untreated (IVF control) or injected with PLCζ^H435P cRNA (IVF+PLCζ^H435P) 3 h prior to the start of recording. PLCζ^H435P expression produced luminescence of 41.7±1.8 c.p.s. (value is mean±S.E.M., n=11). Fluorescence recordings [arbitrary units (a.u.)] reporting Ca²⁺ concentration changes were monitored after the addition of capacitated mouse sperm. Following IVF, both control and PLCζ^H435P cRNA-injected eggs exhibited robust Ca²⁺ oscillations and formed pronuclei. The number of Ca²⁺ spikes in control eggs was 33.4±3.8 (mean±S.E.M., n=7), and the number of Ca²⁺ spikes in PLCζ^H435P cRNA-injected eggs was 32.9±4.5 (mean±S.E.M., n=11).