151 A NOVEL METHOD TO INCREASE THE DEVELOPMENTAL POTENTIAL OF ACTIVATED OOCYTES BY USING THE Zn²⁺ CHELATOR TPEN [N,N,N',N'-TETRAKIS(2-PYRIDYLMETHYL)ETHYLENEDIAMINE]

Abstract

An increase in intracellular Ca²⁺ concentration is essential for oocyte activation. Thus most artificial oocyte-activation methods focus on increasing the Ca²⁺ concentration in the oocytes. Recently, full-term development was reported in mice when oocytes were activated with no increase in intracellular Ca²⁺ using the Zn²⁺ chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). During oocyte maturation, Zn²⁺ is responsible for Cdc25c recognition. Once attached, the Cdc25c-zinc complex dephosphorylates maturation-promoting factor (MPF)/cyclin-dependent kinase 1 (cdk1), thus activating the Cdc25c-MPF positive feedback loop and keeping the oocyte suspended in metaphase II. The TPEN can inhibit the Cdc25c-MPF positive feedback loop, indirectly giving TPEN the power to degrade MPF, allowing the oocyte to exit metaphase II. First, we tested if incubation of porcine oocytes with TPEN could induce oocyte activation. Second, we examined whether the combination of TPEN with conventional activation methods could increase the developmental potential of activated oocytes. Last, based on the results, somatic cell nuclear transfer (SCNT) embryos were further activated with the optimum condition of TPEN to produce clones to confirm developmental competence. Frequencies of blastocyst formation were recorded and analysed by using ANOVA following arcsin transformation. Total cell numbers in blastocysts were counted and compared by using the Student's t-test. Differences at P < 0.05 were considered significant. When oocytes were incubated with a high concentration of TPEN (100–250 μM) for 10 to 120 min, blastocyst formation was comparable with conventional activation methods; however, the total cell number in the blastocysts was significantly lower (31.3 ± 3.1 v. 24.8 ± 1.9). When oocytes were activated with conventional methods and then incubated with the high concentration of TPEN, embryo development was drastically decreased; no blastocyst development was achieved from TPEN-treated oocytes. Interestingly, when activated oocytes were incubated with a low concentration of TPEN (5–10 μM), surprisingly, the TPEN-treated group showed higher developmental potential compared with the control group. Specifically, the average percent blastocyst formation of TPEN-treated oocytes (5 μM for 30 min) was 27.2 ± 1.7%, but only 10.6 ± 2.5% developed to blastocyst in the control group. Moreover, the average cell number in blastocysts was significantly higher in TPEN-treated oocytes compared with the control group (33.1 ± 2.6 v. 28.2 ± 2.1, respectively). When 290 chemically activated SCNT embryos were treated with 5 μM TPEN for 30 min and transferred into a surrogate, 2 healthy piglets were born. The results indicate that incubation of oocytes with TPEN alone can activate porcine oocytes. Also, when activated oocytes are incubated with the right concentration of TPEN, it can increase embryo quality in vitro. Embryo transfer results also show that TPEN-incubated SCNT embryos are developmentally competent. Additional studies would guide us to develop more efficient way to use TPEN in the activation of SCNT embryos.