Supplementary Table S9. Proteomic analysis of bisected oocytes.

MII oocytes were incubated in Hoechst 33342 (1 µg/mL) dissolved in α-MEM medium containing 0.2 % (w/v) BSA and 50 µg/mL gentamicin, for 15 min at 37 °C under 6 % CO2. This way the spindle could be later visualized under UV light, allowing for consistent orientation of the oocytes relative to the spindle position. Immediately prior to bisection, oocytes were deprived of the zona pellucida by treatment with acidic Tyrode solution. Bisections took place in a drop of micromanipulation medium on a glass-bottomed dish on the stage of the micromanipulator (same as that used for ICSI). The micromanipulation medium consisted of a mixture of 10% α-MEM and 90% PVP-HCZB (v/v), supplemented as described, added with 5 µM Latrunculin B (CAT No. AG-CN2-0031-M001, AdipoGen Life Sciences). The room temperature was 27°C to facilitate the depolymerization of the actin cytoskeleton by Latrunculin B. Each MII oocyte was oriented so as to have the spindle at the 12 o’clock position in case of equatorial bisection conducted to separate A and V hemispheres, or 3 o’clock position in case of meridional bisection conducted to separate ‘left’ and ‘right’ hemispheres. Bisection was operated using a borosilicate capillary with O.D. 15 µm at the tip, connected to a piezo device, but the capillary was not filled with mercury. The capillary was pressed downward to divide the oocyte into two parts. Usually the two oocyte-halves remain connected by a thin cytoplasmic bridge, which was severed with the help of piezo pulses. Oocytes were processed in batches of 10 at a time in approximately 20 min, keeping the two halves of the same oocyte next to each other but far enough from those of other oocytes. The two halves were collected individually keeping track of the source oocyte and of which half was A, V, left or right relative to the spindle. The oocyte halves were processed for proteomics largely following the method of Zhang et al. with modifications.Zhang, H., et al., Deep Profiling of Oocyte Aging Enabled by Simple One-Step Vial-Based Pretreatment and Single-Cell Proteomics.JACS Au, 2025. 5(5): p. 2321-2333.