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

Area of Science:

• Reproductive Biology
• Cellular Metabolism
• Biophysics

Background:

• Cytosolic calcium ([Ca²⁺]ᵢ) oscillations are crucial for egg activation and link signaling to mitochondrial metabolism.
• The influence of in vitro fertilization (IVF) media on this Ca²⁺-metabolic coupling and its impact on developmental potential is not well understood.
• Standard IVF media yield suboptimal developmental competence, with full-term development rates around 20%.

Purpose of the Study:

• To analyze how fertilization-associated [Ca²⁺]ᵢ dynamics and metabolic responses vary across different IVF media.
• To identify relationships between these dynamics and the developmental potential of oocytes.
• To determine how IVF media composition constrains developmental competence.

Main Methods:

• Simultaneous microfluidic recordings of intracellular calcium ([Ca²⁺]ᵢ) using Fura-2 and flavin adenine dinucleotide (FAD) autofluorescence in 319 oocytes across 19 conditions.
• Analysis using dedicated algorithms to extract dynamic descriptors of Ca²⁺ excitation, Ca²⁺ release, inter-spike timing, and FAD redox state.
• Testing standard mouse media, commercial IVF media, and experimental formulations with varied energy substrates and extracellular Mg²⁺/Ca²⁺ ratios.

Main Results:

• IVF media modulate two interdependent functional layers of egg activation: Ca²⁺ excitability and metabolic response.
• The extracellular Mg²⁺/Ca²⁺ ratio controlled PLCζ/InsP₃-dependent Ca²⁺ excitability, affecting oscillation parameters.
• Energy substrates influenced redox dynamics; glucose stabilized FAD redox, while pyruvate/lactate induced imbalance, prolonged Ca²⁺ release, and reduced oscillation frequency.

Conclusions:

• A novel IVF medium formulation (M16_CORRECTED) was designed by adjusting the Mg²⁺/Ca²⁺ ratio and reducing pyruvate/lactate.
• This optimized medium stabilized FAD redox dynamics, prolonged the oscillatory Ca²⁺ regime, and increased full-term survival to 52%.
• Rational tuning of IVF media by balancing Ca²⁺ excitation and metabolic compensation can significantly improve developmental competence.