Sperm phospholipase C signaling via PLCζ1 and PLCδ4 is essential for Ca2+ oscillations and fertility in mice

Fertilization-induced calcium (Ca 2+ ) oscillations are essential for egg activation and embryonic development in mammals. The sperm-specific phospholipase C isoform, PLCζ1, is the primary trigger of these oscillations. Functional and clinical studies have linked mutations or reduced expression of PLCζ1 to male infertility. However, whether PLCζ1 alone accounts for Ca 2+ release at fertilization in all mammals remains uncertain. Male mice lacking Plcζ1 exhibit subfertility and reduced litter sizes; their sperm induce delayed, low-frequency Ca 2+ oscillations, indicating a compensatory mechanism. Notably, mouse sperm express high levels of PLCδ4, essential for completing the acrosome reaction (AR). In Plcζ1 -null sperm, PLCδ4 is upregulated and retained post-AR. These observations suggest a secondary functional role for PLCδ4. Using CRISPR/Cas9 genome editing, we generated Plcδ4 knockout (KO) mice. Plcδ4 -null sperm trigger attenuated, delayed Ca 2+ oscillations and significantly reduced fertilization rates, indicating that PLCδ4 contributes to but does not independently drive Ca 2+ signaling. We therefore generated a Double Knockout (DKO) mouse line lacking both sperm-enriched PLCs. DKO males were sterile, with sperm failing to induce Ca 2+ oscillations, and the isolated residual Ca 2+ rise was insufficient to activate eggs. These findings reveal that PLCζ1, the primary oscillator, and PLCδ4, a compensatory factor, work cooperatively within a complex, multi-component PLC system that ensures robust fertilization. This work reveals molecular specialization among mammals in initiating the fertilization Ca 2+ signals. It has implications for understanding male infertility, improving assisted reproductive technologies, and developing contraceptive strategies.