Effect of RGFP966 application in key steps of sheep somatic cell nuclear transfer on cloned embryo development

This study explored the effect and mechanism of the histone deacetylase 3 (HDAC3)-selective inhibitor (E)-N-(2-amino-4-fluorophenyl)-3-(1-cinnamyl-1H-pyrazol-4-yl)acrylamide (RGFP966) in optimizing key steps of sheep somatic cell nuclear transfer (SCNT). Using an "18 h + 4 h" two-step oocyte maturation protocol, 16 μM RGFP966 significantly increased the first polar body (PB1) extrusion rate to 63.0% ± 4.6% (vs. 51.7% ± 4.5% in the control group, P < 0.05). Given HDAC3's high expression in embryos arrested at the 8-cell and 16-cell stages (P < 0.05), RGFP966 was applied combinatorially in SCNT key steps: 16 μM for recipient oocyte maturation, 10 μM for donor cell pretreatment, and 16 μM for 1-cell reconstructed embryo culture. Results showed this combined treatment significantly improved cloned embryo cleavage rate (95.6% ± 0.7% vs. 85.7% ± 0.4%, P < 0.05), enhanced blastocyst epigenetic reprogramming (increased H3K27ac, decreased H3K9me3, P < 0.05), and promoted trophectoderm development (increased CDX2, P < 0.05). However, it had no significant effect on morula or blastocyst rates, while morula apoptotic levels in the treatment group were significantly elevated (P < 0.01). In conclusion, RGFP966 combined treatment optimizes early development of sheep cloned embryos by promoting oocyte maturation, improving reprogramming, and enhancing trophectoderm function, but RGFP966-induced morula apoptosis limits blastocyst rate improvement. This study provides a theoretical basis for optimizing SCNT via targeted HDAC3 regulation.