The reduced embryonic development of sheep oocytes fertilized by ICSI depends on an abnormal calcium pattern

In mammals, Ca2+ is the universal activator of development at fertilization, playing a central role in early events associated with egg activation and the egg-to-embryo transition. In details, oocyte activation depends on a rise in intracellular free calcium ions (Ca2+) that is triggered by the sperm - specific phospholipase C zeta (PLCζ) and consists in a series of characteristic oscillations [1]. Intracytoplasmic sperm injection (ICSI), which bypasses fusion of the gametes, has been widely used in several species, with variable efficiency. While a great progress has been achieved in humans, the success of this technique remains limited in ruminants [2], particularly in sheep where the first cleavage is very low (35%) due to failure of pronuclear (PN) apposition and fusion, despite a high rate (80%) of activation and PN formation [3]. Given that PN migration and fus ion are Ca-dependent processes, we have recently hypothesized that an abnormal Ca2+ response could be the limiting factor in sheep ICSI oocytes. So, we compared the oscillation patterns of in vitro matured (IVM) oocytes fertilized by ICSI or in vitro fertilization (IVF), microinjected with the Ca-sensitive dye Fluo-4, and analysed by time lapse microscopy. Our results clearly demonstrated that an oscillatory response occurs in IVF oocytes. In particular, all responding oocytes (44%, n=140) showed repetitive Ca2+ oscillations from the time of gamete fusion until 2PN stage (frequency: 1 spike every 14±6 min). Interestingly, all ICSI oocytes (n=120) did not evoke the normal calcium response, showing (29%) a single spike or only a few (2-4) abnormal Ca2+ increments with low frequency (1 spike every 50-65 min). Hoechst staining at the end of the Ca2+ analysis revealed that all IVF oocytes responding with Ca2+ oscillations reached the 2PN stage, while all ICSI oocytes that showed a Ca2+ response formed only a single PN (78%) or 2PN without apposition (22%). In conclusion, our results confirm our hypothesis demonstrating for the first time that in sheep the failure of cleavage of ICSI oocytes depends on an abnormal Ca2+ response after sperm microinjection, due to lacking or prematurely ending Ca2+ oscillations. This pattern only sustains the early events of activation, such as the end of meiosis and formation of female PN, whereas other Ca-dependent events that could be more strictly related the sperm contribution appear deficient and fail, so compromising the development to blastocyst stage. [1] Saunders et al. PLCζ: a sperm-specific trigger of Ca2+ oscillations in eggs and embryo development, DEVELOPMENT, 129:3533- 3544, 2002. [2] Shirazi et al. Male pronuclear formation and embryo development following intracytoplasmic injection of ovine pretreated sperm. AVICENNA J MED BIOTECHNOL (AJMB), 10:41-48, 2018. [3] Ressaissi et al. The impaired development of sheep ICSI derived embryos is not related to centriole dysfunction, THERIOGENOLOGY, 159:7-12, 2021.