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

<p>by Neha Gupta, Lei Zhang, Hiroki Akizawa, Maria G. Gervasi, Darya A. Tourzani, Pablo E. Visconti, Ana Romarowski, Mariano Buffone, Wei Cui, Rafael A. Fissore</p> Fertilization-induced calcium (Ca²⁺) 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 Ca²⁺ release at fertilization in all mammals remains uncertain. Male mice lacking <i>Plcζ1</i> exhibit subfertility and reduced litter sizes; their sperm induce delayed, low-frequency Ca²⁺ oscillations, indicating a compensatory mechanism. Notably, mouse sperm express high levels of PLCδ4, essential for completing the acrosome reaction (AR). In <i>Plcζ1</i>-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 <i>Plcδ4</i> knockout (KO) mice. <i>Plcδ4</i>-null sperm trigger attenuated, delayed Ca²⁺ oscillations and significantly reduced fertilization rates, indicating that PLCδ4 contributes to but does not independently drive Ca²⁺ signaling. We therefore generated a Double Knockout (DKO) mouse line lacking both sperm-enriched PLCs. DKO males were sterile, with sperm failing to induce Ca²⁺ oscillations, and the isolated residual Ca²⁺ 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 Ca²⁺ signals. It has implications for understanding male infertility, improving assisted reproductive technologies, and developing contraceptive strategies.