# Precise mimicry of physiological Ca2+ oscillations for mammalian oocyte activation by nanosecond pulsed electric field

## TL;DR

A new non-invasive method using electric pulses mimics natural calcium signals in oocytes, improving activation and development into embryos.

## Contribution

A non-invasive nsPEF protocol is introduced that mimics sperm-initiated calcium oscillations in oocytes, enhancing activation and developmental potential.

## Key findings

- nsPEF stimulation at low or medium intensities mimics physiological Ca2+ oscillations, leading to higher developmental potential in activated oocytes.
- nsPEF induces Ca2+ influx via nanopores at low intensity and ER Ca2+ release via the PIP2–IP3–IP3R pathway at medium intensity.
- nsPEF outperforms A23187 in activating oocytes, with 78.13% developmental potential versus 26.70%.

## Abstract

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.

The figure shows the formation of cytoplasmic Ca2+ oscillations induced by nanosecond pulsed electric field (nsPEF). First, nsPEFs stimulation induces extracellular Ca2+ and/or Ca2+ in endoplasmic reticulum (ER) flow into the cytoplasm through nanopores, then the elevated [Ca2+]i is rapidly restored by replenishment of organelles. Under the medium‐intensity stimulation, phosphatidylinositol 4,5‐bisphosphate hydrolysis and inositol 1,4,5 triphosphate generation trigger Ca2+ release from ER to form a spontaneous secondary [Ca2+]i increase, and its restoration is in dependence on Ca2+‐permeable channels and refilling of calcium reservoirs.