Development of novel oocyte activation approaches using Zn²⁺ chelators in pigs

Artificial oocyte activation is an essential step in somatic cell nuclear transfer (SCNT) and can enhance viability of embryos as a form of assisted reproductive technology (ART) in clinics. Most artificial activation methods have been developed to increase cytosolic calcium (Ca²⁺) level in oocytes. Interestingly, recent studies have demonstrated that mammalian oocytes can be activated using N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), a Zn²⁺ chelator. Although effective, TPEN is also known to induce apoptosis and shows poor selectivity between free Zn²⁺ and protein-bound Zn²⁺. The aim of this study was to identify different Zn²⁺ chelators that can activate pig oocytes. Among five Zn²⁺ chelators examined, 1,10-phenanthroline (Phen), and tris(2-pyridylmethyl)amine (TPA) successfully activated pig oocytes. The level of available Zn²⁺ was reduced without any increase in Ca²⁺ in oocytes incubated with Phen or TPA, indicating that the oocyte activation occurred independently of Ca²⁺ signal. When various concentrations (100-500 μM) and incubation durations (10-120 min) of Phen and TPA were used to activate pig oocytes, 500 μM for 60 min and 100 μM for 60 min of Phen and TPA treatments, respectively, were found to be most effective in supporting embryo development. The frequency of blastocyst formation after the treatments was higher than 40% at day 7. When oocytes were incubated with TPEN, Phen, or TPA under their optimal treatment conditions, there was no significant difference in the frequencies of day 7 blastocyst formation among the three treatments. However, day 5 blastocyst formation was observed from the Phen- and TPA-treated oocytes, whereas no blastocyst was formed at day 5 in the TPEN-treated oocytes. The average total cell number in day 7 blastocysts was higher in the Phen treatment group than in the TPEN treatment (P < 0.05). These results suggest that Phen and TPA can be used as powerful agents to artificially activate oocytes and to increase the developmental potential of SCNT embryos or embryos going through clinical ART procedures.