In mammals, mature oocytes are transformed into zygotes through fertilization with sperm. This process, known as oocyte activation, begins with a series of intracellular calcium (Ca²⁺) oscillations, followed by events such as cortical granule release and pronuclear formation, ultimately leading to embryonic development [1, 2]. During physiological oocyte activation, sperm-derived phospholipase C zeta (PLC$\zeta$) is known to trigger Ca²⁺ oscillations via activation of the inositol 1,4,5-trisphosphate (IP₃) receptor pathway [1]. Oocyte activation can also be artificially induced using methods such as electrical stimulation, ionophores, ethanol, and strontium (Sr²⁺) [3]. However, the precise mechanisms underlying these activation methods remain unclear. Sr²⁺ has been reported to induce Ca²⁺ oscillations similar to those observed during fertilization, although several studies suggest mechanistic differences between Sr²⁺-induced and sperm-induced activation [4, 5]. In our previous study, we found that chloroquine (CQ), an antimalarial drug, does not fundamentally inhibit sperm-induced oocyte activation but does impair Sr²⁺- and ethanol-induced activation by disrupting Ca²⁺ oscillations [6]. This result supports the notion that Sr²⁺ activates oocytes via a mechanism distinct from that of sperm. Given that CQ affects lysosomal function [7, 8], we hypothesized that its effects on oocyte activation might be mediated through lysosomes.

Lysosomes not only function as degradative organelles for intra- and extracellular components but also serve as intracellular Ca²⁺ stores [9, 10]. They interact with other calcium-regulating organelles, such as the endoplasmic reticulum and mitochondria, thereby modulating cytoplasmic Ca²⁺ signaling [11, 12]. Lysosomal membranes harbor calcium -permeable two-pore channels (TPCs), which play a key role in regulating intracellular Ca²⁺ dynamics [13]. The animal TPC family comprises three subtypes: TPC1, TPC2, and TPC3 [14]. In humans and mice, only TPC1 and TPC2 are expressed, with distinct subcellular localizations-TPC1 is distributed across various endolysosomal compartments, whereas TPC2 is predominantly localized to late endosomes and lysosomes [15]. TPCs are activated by nicotinic acid adenine dinucleotide phosphate (NAADP) [16]. And the NAADP-TPC pathway has been implicated in fertilization. For example, TPC knockdown in sea star oocytes disrupts embryonic development due to impaired Ca²⁺ signaling [17]. In mice, NAADP-mediated Ca²⁺ signaling via TPC1 regulates the acrosome reaction in sperm, and the presence of NAADP synthase has also been reported in human sperm [18, 19, 20]. Nonetheless, the role of TPCs in mammalian fertilization and oocyte activation remains largely unexplored.

Trans-Ned19 is a selective antagonist of NAADP and effectively inhibits NAADP-induced Ca²⁺ release [21]. Its inhibitory activity has been characterized across a broad concentration range, from 1 µM to 300 µM [21, 22].

In this study, we investigated the potential involvement of TPC-mediated Ca²⁺ signaling in fertilization and oocyte activation using the NAADP antagonist trans-Ned19.

For oocyte activation, calcium is released from intracellular Ca²⁺ stores, primarily the endoplasmic reticulum (ER), through the activation of IP₃-gated Ca²⁺ release channels (IP₃ receptors) [1]. In animal cells, NAADP, another Ca²⁺-mobilizing messenger, initiates Ca²⁺ release from the endolysosomal system via activation of TPCs [13]. However, the involvement of TPCs in oocyte activation remains unknown in mammals. In this study, we demonstrated that inhibition of TPCs by trans-Ned19 had little effect on sperm-induced oocyte activation, whereas it significantly suppressed Sr²⁺-induced oocyte activation by inhibiting Ca²⁺ oscillations. These results suggest that the cascade of sperm-induced oocyte activation is distinct from that of Sr²⁺-induced activation, consistent with our previous findings [6].

Although both TPC1 and TPC2 are present in mice [15], at least TPC1, which is localized to lysosomes and endosome, is expressed in mouse oocytes [23]. In sperm, TPC1 has been reported to localize to the acrosome and nuclear redundant vesicles [18]. Trans-Ned19, a selective and cell-permeant NAADP antagonist, is widely used to study the functional roles of TPCs [24]. In smooth muscle cells, 100 µM trans-NED 19 reduces TPC1-mediated Ca²⁺ elevation by nearly 60% [25]. In the present study, 300 µM trans-NED19 significantly suppressed Sr²⁺-induced Ca²⁺ oscillations, leading to a failure of normal pronuclear formation in approximately 50% of embryos. In mice, Sr²⁺ influx—which induces Ca²⁺ oscillations and triggers oocyte activation—occurs via transient receptor potential vanilloid 3 (TRPV3) channels [26]. Therefore, it is reasonable to propose that increased intracellular Sr²⁺ may induce oocyte activation via Ca²⁺ release from lysosomes, likely through TPCs, either directly or indirectly. However, even under trans-Ned19 treatment, expanded pronuclei were observed in approximately 50% of embryos, suggesting that pronuclear formation during Sr²⁺-induced activation is not solely dependent on TPC function. Consistently, it has been suggested that Sr²⁺ can sensitize IP₃ receptors and promote Ca²⁺ oscillations [5]. Furthermore, when trans-Ned19 was added 2 h after the start of Sr²⁺-induced activation, it did not affect pronuclear formation. In mice, pronuclei are typically formed approximately 3 h after fertilization, suggesting that TPCs are not involved in the later stages of pronuclear formation [27].

In contrast, in IVF embryos, expanded pronuclei were observed in nearly all fertilized embryos, even under trans-Ned19 treatment. This suggests that lysosomal TPCs are not essential for sperm-induced oocyte activation. These findings support the notion that TPC1-mediated Ca²⁺ release contributes to Sr²⁺-induced oocyte activation, whereas sperm-induced oocyte activation likely proceeds through a distinct Ca²⁺ signaling pathway independent of TPC1. Together, our results highlight mechanistic differences between artificial and physiological modes of oocyte activation and underscore the selective involvement of TPCs in non-physiological activation pathways.

Although trans-Ned19 treatment significantly reduced the fertilization rate in IVF, the rate became comparable to that in the control group after removal of the zona pellucida. Furthermore, trans-Ned19 treatment significantly decreased sperm motility, suggesting that TPCs in sperm may be required for maintaining normal motility. While the role of TPCs in sperm motility remains to be fully elucidated, Ca²⁺ signaling is known to regulate sperm motility, and inhibition of TPC-derived Ca²⁺ signaling by trans-Ned19 may contribute to the observed reduction [28]. In addition, TPCs have been reported to be involved in the regulating the acrosome reaction. Inhibition of TPCs by trans-Ned19 has been reported to impair the acrosome reaction in sperm, and a recent study suggests that this may occur via TPC-mediated Ca²⁺ signaling, potentially contributing to reduced fertilization rates [18, 19].

Although trans-Ned19 has been widely used as a selective TPC inhibitor, its effects may not be entirely specific and can vary depending on the experimental context. While trans-Ned19 has been employed in numerous studies investigating NAADP-sensitive Ca²⁺ signaling, its specificity and potential off-target effects may differ depending on cell type, dosage, and exposure conditions. In the present study, we focused on identifying the potential involvement of TPCs in fertilization and oocyte activation using trans-Ned19 as a pharmacological tool. However, we acknowledge the limitation of relying on a single inhibitor at a high concentration and recognize the need for further validation. Future studies incorporating additional pharmacological agents—such as SG-094, cis-Ned19, and TPC agonists (e.g., TPC2-A1-N and TPC2-A1-P)—will be essential to strengthen causal interpretation and expand upon the current findings.

On the other hand, the significantly increased incidence of polyspermy observed in zona-free IVF under trans-Ned19 treatment suggests that TPCs are involved in the mechanisms that prevent polyspermy in oocytes.

Rejection of polyspermy is known to occur at both the zona pellucida and the oocyte membrane, referred to as the “zona block” and “membrane block”, respectively. In IVF using zona-free oocytes, the membrane block is considered the primary barrier to polyspermy [29]. Although this block is thought to be associated with the loss of the oocyte membrane protein IZUMO1 receptor, also known as folate receptor 4 (JUNO) following fertilization [30], the underlying mechanisms remain largely unclear. Since cortical granule release was observed even under trans-Ned19 treatment, it is suggested that TPC involvement in polyspermy prevention occurs mainly at the level of the oocyte membrane. Interestingly, an increase in cytosolic Ca²⁺ alone is not sufficient to establish the membrane block [18]. In normal fertilization, TPC-mediated activity may be required for the establishment of this membrane block. Future studies should aim to elucidate how TPCs contribute to this process.

Cortical granules undergo Ca²⁺-dependent exocytosis, releasing their contents into the perivitelline space and modifying the zona pellucida to prevent polyspermy—a process known as the cortical reaction. This secretion mediated by the soluble NSF-attachment protein receptor (SNARE) protein pathway [31]. Trans-Ned19 treatment did not suppress cortical granule release in fertilized embryos, suggesting that TPC activity is not required for the cortical reaction during sperm-induced oocyte activation, consistent with its dispensability in pronuclear formation. In contrast, during Sr²⁺-induced oocyte activation, trans-Ned19 treatment significantly decreased the number of embryos exhibiting normal granule signals and significantly increased those with small granule signals. This suggests that TPC function is required for normal cortical reactions in Sr²⁺-induced activation. Even under trans-Ned19 treatment, 53% of embryos exhibited cortical granule release patterns similar to the control group, and the pronuclear formation rate was 47%, suggesting that some embryos successfully completed cortical granule release but failed to form expanded pronuclei. Conversely, embryos with small granule signals may have failed to form pronuclei, indicating a correlation between the granule signal pattern and pronuclear formation. These observations suggest that incomplete activation signals under trans-Ned19 treatment result in abnormal cortical reactions and defective pronuclear formation.

Inhibition of TPCs by trans-Ned19 had minimal impact on sperm-induced oocyte activation during IVF, whereas Sr²⁺-induced oocyte activation was inhibited from the initiation stage—specifically, at the onset of Ca²⁺ oscillations—leading to significant reductions in cortical granule release and pronuclear formation. Additionally, trans-Ned19 treatment was associated with a trend toward decreased sperm motility and fertilization rates, which were restored upon removal of the zona pellucida. These effects were similar to those observed with CQ treatment [6], suggesting that lysosome-derived Ca²⁺ signaling, including TPC-mediated pathways, may be a common target of both agents. More importantly, TPCs may contribute to the prevention of polyspermy, highlighting their role in post-fertilization events. Together, these findings provide new insights into the mechanisms of oocyte activation and hold potential implications for the advancement of assisted reproductive technologies.

CQ, chloroquine; TPC, two-pore channel; NAADP, nicotinic acid adenine dinucleotide phosphate; IVF, in vitro fertilization.

All raw data for this study are available from the corresponding author.

TY and SK designed the research study. TY and MWB performed the experiments. SK provided guidance and support throughout the research. TY analyzed the data. TY and SK wrote the manuscript. All authors contributed to revisions of the manuscript, reviewed and approved the final version, and agreed to be accountable for all aspects of the work.

All the animal experiments were approved by the Animal Experimentation Committee at the University of Yamanashi, Japan, and conducted in accordance with the ethical guidelines (protocol number A4-10). Animal handling and experimentation adhered to the 3Rs principle and followed the Guidelines for Proper Conduct of Animal Experiments issued by the Science Council of Japan.

We gratefully acknowledge discussions and technical support with Dr. H. Harayama in Kobe University, Drs. T. Wakayama, S. Wakayama, D. Ito, and Ms. Y. Kanda at the Advanced Biotechnology Center, and also Ms. S. Furusato at the Center for advanced Assisted Reproductive Technologies, University of Yamanashi as well as all the lab members.

This work was supported by JSPS KAKENHI Grant (Numbers 20K06443 and 24K01937 to SK).

The authors declare no conflict of interest.

Supplementary material associated with this article can be found, in the online version, at https://doi.org/10.31083/FBL42710.