A catalytically competent sperm PLCzeta variant causes fertilization failure through protein insufficiency

The PLCζ p.Arg385Gln (R385Q) variant was recently identified in infertile patients with recurrent fertilization failure and markedly reduced sperm PLCζ levels. However, whether this variant causes infertility through loss of catalytic function or protein insufficiency remains unknown.

The structural and functional consequences of the R385Q variant were investigated using structural modelling, protein stability prediction, recombinant PLCζ enzymatic assays, and mouse oocyte microinjection followed by Ca²⁺ oscillation analysis.

Structural modelling predicted that the R385Q substitution destabilizes the catalytic domain without disrupting the active site. Consistent with this prediction, recombinant PLCζ^R385Q exhibited phosphatidylinositol 4,5-bisphosphate (PIP₂) hydrolytic activity comparable to wild-type PLCζ. Likewise, microinjection of PLCζ^R385Q cRNA into mouse oocytes induced Ca²⁺ oscillations with frequencies and patterns indistinguishable from those elicited by wild-type PLCζ, demonstrating preserved catalytic and oocyte activation activity.

These findings indicate that the R385Q variant is catalytically competent and does not cause infertility through loss of enzymatic function. Instead, together with our previous demonstration of markedly reduced PLCζ protein levels in patient sperm, the results support a protein insufficiency mechanism in which fertilization failure arises when PLCζ levels fall below the threshold required for physiological oocyte activation. This study expands the spectrum of pathogenic mechanisms underlying PLCζ-associated male infertility.