Objective: Somatic cell nuclear transfer (SCNT) protocols remain technically challenging and inefficient. Although demecolcine or nocodazole (Noc) has been used for chemically assisted enucleation to improve cloning efficiency, the process remains labor-intensive. This study aimed to use lysophosphatidic acid (LPA) to enhance Noc-mediated chemical enucleation (CE), thereby simplifying the SCNT procedure. Methods: Porcine MII oocytes underwent CE by incubation with Noc and various concentrations of LPA for 1 h. Actin signals were evaluated to determine the optimal LPA concentration. Following treatment with Noc and 25 µM LPA for 1 h and subsequent washout, spindle reformation was assayed. Noc/LPA CE-treated oocytes were used for SCNT via direct microinjection of donor cells into the ooplasm. Reconstructed embryos were recovered for 2 h in PZM-5 prior to activation with ionomycin and N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine. Activated reconstructed embryos were cultured in 6-DMAP for 4 h, then in chaetocin for an additional 20-24 h to promote demethylation and acetylation. Cleavage and blastocyst rates were evaluated on Day 2 and Day 6, respectively. Results: When matured oocytes were cultured in Noc/LPA for 1 h, the percentage of oocytes exhibiting actin signals increased significantly from 12.5% to 87.6% with the addition of 25 µM LPA. Although spindles reformed in 59.0% of oocytes 1 h after Noc/LPA washout, 83.9% of oocytes retained spindles enclosed by actin rings; this facilitated the direct microinjection of donor cells into the ooplasm within 30 min. Currently, blastocyst development rates range from 9.5% to 16.6% after 6 days of culture in PZM-5. Conclusion: Although the blastocyst rate requires further improvement, we demonstrated that enucleation of MII pig oocytes can be successfully achieved via Noc/LPA treatment. This protocol simplifies the SCNT procedure while maintaining consistent blastocyst development in pig IVM oocytes.
Wong et al. (Mon,) studied this question.