Human PLCζ exhibits superior fertilization potency over mouse PLCζ in triggering the Ca(2+) oscillations required for mammalian oocyte activation

Abstract

A sperm-specific phospholipase C-zeta (PLCζ) is believed to play an essential role in oocyte activation during mammalian fertilization. Sperm PLCζ has been shown to trigger a prolonged series of repetitive Ca(2+) transients or oscillations in oocytes that precede activation. This remarkable intracellular Ca(2+) signalling phenomenon is a distinctive characteristic observed during in vitro fertilization by sperm. Previous studies have notably observed an apparent differential ability of PLCζ from disparate mammalian species to trigger Ca(2+) oscillations in mouse oocytes. However, the molecular basis and confirmation of the apparent PLCζ species difference in activity remains to be provided. In the present study, we provide direct evidence for the superior effectiveness of human PLCζ relative to mouse PLCζ in generating Ca(2+) oscillations in mouse oocytes. In addition, we have designed and constructed a series of human/mouse PLCζ chimeras to enable study of the potential role of discrete PLCζ domains in conferring the enhanced Ca(2+) signalling potency of human PLCζ. Functional analysis of these human/mouse PLCζ domain chimeras suggests a novel role of the EF-hand domain in the species-specific differences in PLCζ activity. Our empirical observations are compatible with a basic mathematical model for the Ca(2+) dependence of generating cytoplasmic Ca(2+) oscillations in mammalian oocytes by sperm PLCζ.

Keywords: PLCzeta; fertilization; male infertility; oocyte activation; phospholipase C; sperm.

Figure 1

Schematic representation of the domain structure of human PLCζ, mouse PLCζ and human/mouse PLCζ chimeras; hPLCζ, mPLCζ, hPLCζ/mEF, hPLCζ/mC2 and hPLCζ/mXYlink. The various amino acid lengths and respective coordinates are indicated for each construct.

Figure 2

Expression of human PLCζ, mouse PLCζ and human/mouse PLCζ chimeras in unfertilized mouse oocytes. 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 oocytes following microinjection of cRNA encoding luciferase-tagged hPLCζ, mPLCζ, hPLCζ/mEF, hPLCζ/mC2 and hPLCζ/mXYlink.

Figure 3

Expression and purification of recombinant NusA-6xHis-tagged human, mouse and human/mouse PLCζ chimeric proteins. Affinity-purified, NusA-6His-PLC fusion proteins (1 µg) were analysed by 7.5% SDS-PAGE followed by either Coomassie Brilliant Blue staining (upper panel) or immunoblot analysis using the Penta-His monoclonal antibody (1:5000 dilution) (lower panel).

Figure 4

In vitro enzymatic properties of human/mouse PLCζ chimeras. (A) [³H]PIP₂ hydrolysis activities of the purified NusA-6His-PLC fusion proteins (20 pmol), n = 3 ± SEM, determined using two different preparations of recombinant protein and with each experiment performed in duplicate. In control experiments with NusA, there was no specific PIP₂ hydrolysis activity observed (data not shown). (B) Effect of varying [Ca²⁺] on the normalized activity of NusA-6His-tagged, human and mouse PLCζ and human/mouse PLCζ chimeric fusion proteins. For these assays n = 3 ± SEM, determined using two different preparations of recombinant protein and with each experiment performed in duplicate.

Figure 5

(A) Ca²⁺ oscillations in a mouse oocyte were simulated using a mathematical model (Equations 1–3, Appendix) based upon the enzymatic properties of human PLCζ (top panel) and mouse PLCζ (middle panel). Simulations were performed under a sigmoidal increase of PLCζ expression (bottom panel). (B) Clustal alignment of human and mouse sperm PLCζ XY-linkers. Identical amino acids are shown in shaded yellow boxes and conservative substitutions in grey.