Sperm deposition at specifically predefined sites of the mouse ooplasm relative to the position of the MII spindle. (A–C) MII mouse oocytes (B6C3F1) at 14–15 h post-hCG were held in position by gentle suction of the zona pellucida with a holding pipette at the 3 o’clock position and subcortically injected with a single sperm head (CD1) using a piezo-driven needle approaching from the 9 o’clock position. (A′–C′) A single sperm head (encircled) was deposited in the cortical ooplasm opposite the spindle, i.e. contralateral (vegetal pole), half-way between poles (equator), or next to the spindle, i.e. ipsilateral (animal pole) of MII oocytes preloaded with Hoechst 33342. (A″–C″) Two pronuclei (♂♀) had formed 5 h after ICSI, attesting that the activation of the oocytes had been successful. (A″′–C″′) Pronuclei had enlarged and moved further toward the centre of the oocyte 10 h after ICSI. Blue colour in photographs is from DNA staining with Hoechst 33342 (1 µg/ml). MII, metaphase II.