IVF media shape PLCζ-dependent Ca²⁺ oscillatory excitability and developmental potential at fertilization

The aim of this study was to analyse how fertilization-associated [Ca²⁺]ᵢ dynamics and metabolic responses evolve across IVF media, to identify relationships with developmental potential. Cytosolic Ca²⁺ oscillations initiate egg activation and couple Ca²⁺ signalling to mitochondrial metabolism, yet how IVF media shape this Ca²⁺-metabolic response remains poorly understood. In the standard media tested here, full-term development remained close to 20%, highlighting the need to understand how fertilization media constrain developmental competence. Simultaneous microfluidic recordings of [Ca²⁺]ᵢ using Fura-2 and FAD autofluorescence were obtained from 319 oocytes across 19 conditions, including standard mouse media, commercial IVF media, and experimental formulations differing in energy substrates and extracellular [Mg²⁺]ₒ/[Ca²⁺]ₒ ratio. These recordings were analysed with dedicated algorithms to extract dynamic descriptors of Ca²⁺ excitation, Ca²⁺ release, inter-spike timing, and FAD redox state. The analysis revealed that IVF media modulate two interdependent functional layers of egg activation. The [Mg²⁺]ₒ/[Ca²⁺]ₒ ratio controlled PLCζ/InsP₃-dependent Ca²⁺ excitability, shaping oscillation number, mean frequency, and train duration. Energetic substrates provided a second level of control: glucose stabilized redox dynamics, whereas pyruvate or lactate were associated with redox imbalance, prolonged Ca²⁺ release, extended refractory periods, and lower oscillation frequency. These functional signatures provided a rationale for designing M16_CORRECTED, combining adjustment of the [Mg²⁺]ₒ/[Ca²⁺]ₒ ratio with reduced pyruvate and lactate. This formulation stabilized FAD redox dynamics, prolonged the oscillatory regime, and increased full-term survival to 52%. These findings indicate that IVF media can be rationally tuned by balancing Ca²⁺ excitation with metabolic compensation to improve developmental competence.