Pig oocyte activation using a Zn2+ chelator, TPEN

Abstract

Artificial oocyte activation is a critical step during somatic cell nuclear transfer (SCNT). Most of current activation protocols focus on inducing an increase in the intracellular free Ca2+ concentration of the oocyte. Here, we have utilized 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 Zn2+ in pig oocytes; however, the cytosolic Ca2+ concentration in the oocytes was not affected by the TPEN treatment. When various concentrations (100 – 250 μM) and incubation durations (45 min – 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 Ca2+ 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 Ca2+ 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 Ca2+ increase. We were able to produce clones through SCNT 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 utilize TPEN to increase the developmental potential of cloned embryos.

The zinc spark is an inorganic signature of human egg activation

“…Because of this narrow window, reducing intracellular zinc availability may be a more targeted and specific approach for driving egg activation, and may thereby increase the efficacy of AOA. Importantly, TPEN-mediated AOA has resulted in healthy live offspring in murine and porcine models 14 17 .…”

“…Recently, we and others have demonstrated in rodent, porcine, and nonhuman primate species that zinc is another essential element involved in meiosis and egg activation 8 9 10 11 12 13 14 15 16 17 18 . In mouse, meiotic maturation – or the progression of the gamete from a cell arrested in prophase of meiosis I into a mature egg arrested at metaphase of meiosis II - is accompanied by a substantial (50%) increase in total zinc content that is required for proper meiotic progression 11 .…”

Single Ca2+ transients vs oscillatory Ca2+ signaling for assisted oocyte activation: limitations and benefits

“…CAMKII cRNA allowed oocyte activation in mouse (Knott et al 2006 (Suzuki et al 2010). Artificial Zn 2+ sequestration, bypassing the Ca 2+ mobilization and mediated by the specific Zn 2+ chelator N,N,N′,Ntetrakis (2-pyridylmethyl)ethane-1,2-diamine (TPEN), allows oocyte activation in several mammalian species, including human (Suzuki et al 2010, Lee et al 2015, Duncan et al 2016. In addition, full-term embryonic development was achieved in mice and pigs by using TPEN as the only agent for oocyte activation.…”

The fertilization-induced zinc spark is a novel biomarker of mouse embryo quality and early development

“…Recently, zinc emerged as an essential element required for the completion of meiosis and egg activation 1 2 3 4 5 6 7 8 9 10 11 . In mouse, meiotic maturation is accompanied by a substantial (50%) increase in total zinc content 4 , which is required to transition through anaphase.…”