Impaired calcium oscillations and PLCζ dysfunction as key contributors to the low efficiency of ICSI and artificial activation in sheep.

Intracytoplasmic sperm injection (ICSI) is less effective in ovine oocytes than in vitro fertilization (IVF), but the reasons remain unclear. Calcium oscillations, driven by phospholipase C zeta 1 (PLCζ1), are critical for fertilization success. This study evaluated Ca2+ wave dynamics in ovine oocytes matured in vitro and fertilized via IVF or ICSI. Metaphase II oocytes were fertilized by IVF (n = 140) or ICSI (n = 105) using frozen-thawed ram spermatozoa. Calcium oscillations were monitored by time-lapse microscopy with Fluo-4 pentapotassium salt, and PLCζ1 expression/localization was analysed via immunofluorescence. IVF-fertilized oocytes exhibited prolonged Ca2+ oscillations lasting up to 16 h, with peaks persisting during pronuclear formation. ICSI-fertilized oocytes showed restricted Ca2+ responses, with peaks limited to the first 4 h. In frozen-thawed sperm, PLCζ1 was mainly in the acrosomal region but relocated to the subequatorial region after ionomycin-induced acrosome reaction, mimicking IVF conditions. In contrast, in PVP-treated sperm used for ICSI, PLCζ1 mislocalized or was depleted, likely due to premature acrosome loss. These findings indicate that abnormal Ca2+ responses underlie ICSI's poor performance in ovine oocytes. Our preliminary results suggest that PLCζ1 mislocalization in sperm during ICSI could impair Ca2+ signalling. This study, the first to describe Ca2+ patterns in sheep oocytes fertilized via IVF, highlights key fertilization failure mechanisms in ICSI and underscores the need to optimize reproductive biotechnologies in farm animals.