Pig oocyte activation using a Zn²⁺ chelator, TPEN.

Artificial oocyte activation is a critical step during SCNT. Most current activation protocols focus on inducing an increase in the intracellular free Ca(2+) concentration of the oocyte. Here, we have used a zinc chelator, TPEN, to enhance the efficiency of oocyte activation during SCNT. TPEN treatment of matured pig oocytes resulted in the reduction of available Zn(2+) in pig oocytes; however, the cytosolic Ca(2+) concentration in the oocytes was not affected by the TPEN treatment. When various concentrations (100-250 μM) and incubation durations (45 minutes-2.5 hours) of TPEN were used to activate oocytes, the efficiency of oocyte activation was not different from conventional activation methods. When oocytes that were activated by conventional activation methods were incubated with a lower concentration of TPEN (5-10 μM), a significant increase in embryos developing to the blastocyst stage was observed. In addition, when oocytes receiving a small Ca(2+) stimulus were further activated by higher concentration of TPEN (100-200 μM), a significant increase in the frequency of blastocyst formation was observed, compared to a conventional activation method. This result indicated that TPEN can be a main reagent in oocyte activation. No increase in the cytosolic Ca(2+) level was detected when oocytes were exposed to various concentrations of TPEN, indicating the ability of TPEN to induce oocyte activation is independent of an intracellular Ca(2+) increase. We were able to produce clones through SCNT by using the TPEN-assisted activation procedure, and the piglets produced through the process did not show any signs of abnormality. In this study, we have developed an efficient way to use TPEN to increase the developmental potential of cloned embryos.