CALCIUM RESPONSES IN SHEEP OOCYTES FERTILIZED BY IVF OR ICSI

CALCIUM RESPONSES IN SHEEP OOCYTES FERTILIZED BY IVF OR ICSI Luisa Gioia (1), Luca Palazzese (2), Marta Czernik (2), Pasqualino Loi (2) (1) Università degli Studi di Teramo, Facoltà di Bioscienze e Tecnologie Agro-Alimentari e Ambientali. (2) Università degli Studi di Teramo, Facoltà di Medicina Veterinaria. Corresponding author: L. Gioia (lgioia@unite.it) In mammalian oocytes, at fertilization a rise in the concentration of intracellular free calcium ions (Ca2+) occurs. In particular, repetitive Ca2+ transients initiate shortly after sperm-oocyte fusion and stop around the time of the first mitotic cell cycle. Although the mechanism by which the sperm initiates this calcium response has not been fully clarified, it is widely accepted that this series of Ca2+ oscillations is sustained by the oocyte’s calcium store sensitized by the soluble PLCζ, released from the fertilizing sperm [1], and is necessary for supporting the events of oocyte activation. ICSI, which bypasses fusion of the gametes, has been widely used in several species, with variable efficiency [2]. A great progress has been achieved in humans, making of ICSI the golden standard for embryo production. In this species, it has been also demonstrated that after the injection of spermatozoon, Ca2+ oscillations are evoked with a pattern similar to that of IVF embryos that is able to activate the oocytes. In contrast, despite the efforts of several working groups around the world, the success of this technique has been limited in farm animals, especially in ruminants [3]. Particularly in sheep, ICSI outcomes are very limited and despite the resort to chemical activation the development to blastocyst as well as the number of newborns remain critically low compared to IVF [4]. Our previous reports revealed a discrepancy between the activation rate in sheep ICSI, assessed by pronuclear formation (80%), and the first cleavage (35%). Hoechst staining revealed two pronuclei in presumptive zygotes, indicating in the absence of pronuclear apposition and fusion the limiting factor [5]. Given that pronuclear migration and fusion are calcium-dependent processes, we hypothesized that current activation protocol affects the first calcium spikes, but does not induce the later, crucial ones. To date, there are no reports that compare oscillations patterns of ICSI vs IVF in sheep oocytes; therefore, the aim of this study is to analyse the response of Ca2+ in IVM oocytes fertilized by ICSI or IVF and microinjected with the Ca-sensitive dye Fluo-4. We have optimized the conditions to analyse the fertilized oocytes over the time to cover the time of pronuclei formation until the first cleavage by using time lapse microscopy and maintaining the oocyte in IVC medium at 38.5°C in 5% CO2. We demonstrated for the first time that in sheep oocytes fertilized by ICSI Ca2+ response is different from that observed after IVF. In fact, the majority of oocytes are unable to produce Ca2+ oscillations and, although in some oocytes the initiation of Ca2+ response takes place like in IVF oocytes, altogether a defective Ca2+ pattern is observed. Once identified the specific defective Ca2+ signal occurring after ICSI procedure, it could be interesting to develop ICSI/activation protocols in which the missing Ca2+ rises are artificially provided for as long as necessary, in order to better mimicking the temporal Ca2+ pattern occurring after IVF and improve the impact on embryo development. [1] Saunders et al. PLCζ: a sperm-specific trigger of Ca2+ o s c i l l ati o ns i n eggs and em bry o dev el o pm ent , Development, 129:3533-3544, 2002. [2] Salamone et al. Intracytoplasmic sperm injection in domestic and wild mammals, Reproduction, 154:F111 - F124, 2017. [3] Briski & Salamone. Past, present and future of ICSI in livestock species. Anim Reprod Sci, 19;106925. 2022. [4] 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. [5] Ressaissi et al. The impaired development of sheep ICSI derived embryos is not related to centriole dysfunction, Theriogenology, 159:7-12, 2021.