Homozygous mutation of PLCZ1 leads to defective human oocyte activation and infertility that is not rescued by the WW-binding protein PAWP

Abstract

In mammals, sperm-oocyte fusion initiates Ca(2+) oscillations leading to a series of events called oocyte activation, which is the first stage of embryo development. Ca(2+) signaling is elicited by the delivery of an oocyte-activating factor by the sperm. A sperm-specific phospholipase C (PLCZ1) has emerged as the likely candidate to induce oocyte activation. Recently, PAWP, a sperm-born tryptophan domain-binding protein coded by WBP2NL, was proposed to serve the same purpose. Here, we studied two infertile brothers exhibiting normal sperm morphology but complete fertilization failure after intracytoplasmic sperm injection. Whole exomic sequencing evidenced a missense homozygous mutation in PLCZ1, c.1465A>T; p.Ile489Phe, converting Ile 489 into Phe. We showed the mutation is deleterious, leading to the absence of the protein in sperm, mislocalization of the protein when injected in mouse GV and MII oocytes, highly abnormal Ca(2+) transients and early embryonic arrest. Altogether these alterations are consistent with our patients' sperm inability to induce oocyte activation and initiate embryo development. In contrast, no deleterious variants were identified in WBP2NL and PAWP presented normal expression and localization. Overall we demonstrate in humans, the absence of PLCZ1 alone is sufficient to prevent oocyte activation irrespective of the presence of PAWP. Additionally, it is the first mutation located in the C2 domain of PLCZ1, a domain involved in targeting proteins to cell membranes. This opens the door to structure-function studies to identify the conserved amino acids of the C2 domain that regulate the targeting of PLCZ1 and its selectivity for its lipid substrate(s).

PLCζ sequence, protein levels, and distribution in human sperm do not correlate with semen characteristics and fertilization rates after ICSI

“…Finally, our results suggest that PLCζ functions as an absolutely necessary sperm-borne activation factor [40] but that other yet unknown components in the complex activation pathway, especially in the oocytes, make PLCζ expression not sufficient to guarantee correct oocyte activation. …”

The eggstraordinary story of how life begins

“…The human I489F mutation is located at the EF‐hand–C2 domain interface. Adapted from Escoffier et al () [Color figure can be viewed at wileyonlinelibrary.com]…”

“…Eggs injected with recombinant mRNAs coding for H233L and H398P mutants display similar reduced Ca 2+ signaling. Adapted from Escoffier et al (), Heytens et al () and Kashir et al () [Color figure can be viewed at wileyonlinelibrary.com]…”

“…In vitro biochemical analysis of the mutant I489F PLCζ protein by Nomikos et al () showed that it had similar enzymatic properties to WT PLCζ, but dramatically reduced binding to PI 3 P and PI 5 P‐containing liposomes, confirming previous structure–function studies indicating the important role for the C2 domain binding to such lipids, which is required to trigger Ca 2+ oscillations in the egg. This is consistent with modeling predictions that suggested that the I489F mutation creates an unusually large hydrophobic area in the C2 domain that might compromise the targeting and/or binding of PLCζ to a specific substrate(s) (Escoffier et al, ). These studies of the effect of naturally occurring mutations in human PLCζ demonstrate their importance, both for identifying the molecular basis of certain types of male infertility, and also because they revealed important clues about the structure–function relationships of the PLCζ protein.…”

“…Escoffier et al () also examined the expression of the protein PAWP in the sperm of these two patients. PAWP, which is coded by the WBP2NL gene, has been proposed as an alternative candidate for the physiological agent of egg activation (A. T. H. Wu et al, ).…”