Requirement of Lysosomal Two-Pore Channels for Normal Fertilization and Artificial Oocyte Activation in Mice

Lysosomes serve not only in the degradation of cellular components but also as calcium (Ca²⁺) stores. In this study, we investigated the effects of trans-Ned19, an inhibitor of lysosomal calcium channels known to block two-pore channels (TPCs), on fertilization and oocyte activation in mice.

Pronuclear formation was assessed via Hoechst 33342 staining, cortical granule release was evaluated using Lens culinaris agglutinin-fluorescein isothiocyanate (LCA-FITC) staining, intracellular Ca²⁺ levels were monitored with Cal-520 AM, and sperm motility was analyzed using a sperm motility analysis system (SMAS).

In strontium (Sr²⁺)-induced oocyte activation, trans-Ned19 significantly reduced pronuclear formation at 8 h post-activation. Cortical granule release and Ca²⁺ oscillations were also markedly suppressed. In contrast, during in vitro fertilization (IVF), trans-Ned19 treatment significantly decreased the fertilization rate; however, pronuclear formation and cortical granule release remained comparable to controls in fertilized embryos. Notably, when IVF was performed using zona pellucida-free oocytes, the fertilization rate in the trans-Ned19 group was similar to that of the controls. However, a significant increase in polyspermy was observed. Furthermore, trans-Ned19 significantly impaired sperm motility parameters, including straight-line velocity, curvilinear velocity, and average path velocity.

These findings suggest that lysosomal TPCs are essential for both normal fertilization and artificial oocyte activation in mice.