Mmo 4

Each spermatozoon was collected from an MHM-C drop into microinjection pipette (30′Bend, LISR, Thebarton, Australia) installed on micromanipulator (MN-4, NARISHIGE, Tokyo, Japan) with matching controlling system and injector (MMO-4 and IM-11-2, respectively, NARISHIGE) and captured upon movement stabilization. Sperm images were acquired individually using a camera (Olympus U-TVO-35XC-2) installed on an Olympus CKX53 microscope with an X60/1.42oil lens (ꝏ/0.17/OFN26.5 Olympus, Tokyo, Japan).

Glass capillaries (Harvard Apparatus (Holliston, MA, USA) Glass capillaries with 780-μm inner diameter) are pulled using a needle puller and micro forged to forge a holding pipette and an injection needle. The resulting injection needles are filled with mRNA solution diluted to 1 μg/μL in injection buffer (5 mM Tris-HCl pH = 7.4, 0.1 mM EDTA). The filled needle is positioned on a micromanipulator (Narishige MMO-4) and connected to a positive pressure pump (Eppendorf FemtoJet 4i). Embryos are placed in FHM drops covered with mineral oil under Leica (Wetzlar, Germany) TL Led microscope. Two-cell stage embryos were injected while holding with holding pipette connected to a Micropump CellTram Oil.

Five microliter of cell suspension, at a density of 4 × 10⁷ cells mL⁻¹, was deposited on 2% water agar with or without 1 mM EGTA and incubated at 21 °C for 12–15 h. Following slug formation, a piece of agar containing slugs was excised and placed upside down on a spacer attached to a 35 mm glass bottom dish (12 mm diameter glass, Iwaki). The spacer was filled with liquid paraffin to prevent desiccation during observation and to avoid light scattering. Slugs covered with agar were pushed with a 5 mm diameter plastic rod using a micromanipulator system (MM-94 and MMO-4, Narishige, Japan) (Supplementary Fig. 4a). In the micropipette assay, a piece of agar with slugs was excised and placed directly on a 35 mm glass bottom dish (12 mm diameter glass, Iwaki). A wet paper was placed in the dish and the agar piece was covered with liquid paraffin. A Femtotip microcapillary (1 µm tip diameter, Eppendorf, Germany) was mounted onto a Femtojet pump and micromanipulator (Eppendorf). The slug was pricked with the pipette using manual operation with the manipulator (Supplementary Fig. 4b). During mechanical stimulation, images of slugs expressing GCaMP6s were acquired at 5 s intervals using epifluorescence microscopy.