Precise mimicry of physiological Ca ²⁺ oscillations for mammalian oocyte activation by nanosecond pulsed electric field

Oocyte activation deficiency is a primary cause of fertilization failure following intracytoplasmic sperm injection, a problem that can potentially be overcome through artificial oocyte activation (AOA). However, concerns persist regarding the safety and efficacy of AOA in clinical practice. We demonstrated that single-pulse nanosecond pulsed electric field (nsPEF) stimulation induced Ca²⁺ signaling patterns that depend on intensity in both mouse and human oocytes, facilitating parthenogenetic activation and blastocyst formation. The sperm-initiated physiological Ca²⁺ oscillations were effectively replicated by a series of Ca²⁺ signals triggered by repeated nsPEF at low or medium intensities, resulting in a significantly higher developmental potential for activated oocytes compared to those treated with A23187 (78.13% vs. 26.70%). The nsPEF stimulation achieved precise manipulation of calcium signaling through two distinct mechanisms: low-intensity nsPEF pulses mediated repetitive extracellular Ca²⁺ influx in an electro-permeable manner, while medium-intensity nsPEF stimulation triggered periodic Ca²⁺ release from the endoplasmic reticulum via the PIP₂-IP₃-IP₃R pathway, generating intracellular Ca²⁺ oscillations that resemble physiological patterns. The non-invasive nsPEF procedure ensured the safety of oocyte activation by maintaining cellular integrity and minimizing stress responses. The efficacy of nsPEF exposure in precisely manipulating Ca²⁺ signaling patterns is also demonstrated in human mature oocytes. This study establishes a quantitative, non-invasive nsPEF protocol for AOA that mimics the activation signaling delivered by sperm. This innovative approach overcomes the limitations of conventional chemical activators by enhancing biosafety and clinical efficacy, particularly for patients experiencing total fertilization failure due to severe male infertility. Its ability to accurately regulate Ca²⁺ signaling presents significant potential for advancing research in various fields, including embryonic development and germ cell differentiation.