Cycloleucine enhances the development of porcine cloned embryos by inhibiting m6A methylation in fibroblasts.

The efficiency of somatic cell nuclear transfer (SCNT) is closely related to the reprogramming capacity of donor cells. The role of N6-methyladenosine (m6A) RNA methylation in regulating chromatin accessibility during porcine SCNT remains unclear. This study investigated the effects of the RNA methylation inhibitor cycloleucine (CL) on chromatin state in porcine fetal fibroblasts (PFFs) and the development of cloned embryos. The results showed that treatment of PFFs with 10 mmol/L CL for 48 h significantly decreased the expression of METTL3, WTAP, YTHDC1, and YTHDF2, increased the expression of the demethylase FTO, and reduced m6A levels. These results suggest that CL treatment reduces m6A modification and is associated with decreased expression of YTHDC1 and SETDB1, accompanied by reduced H3K9me3 levels and increased chromatin accessibility. Using CL-treated PFFs as donor cells, the blastocyst rate of cloned embryos was significantly increased. The expression of ZGA-related genes (eIF3A, TFIIA) and pluripotency-related genes (Nanog, Oct-4) was significantly upregulated. Immunofluorescence analysis revealed lower m6A and H3K9me3 levels in the CL-treated group than in the control group during the 2-8-cell stages. Collectively, these findings suggest that CL treatment of PFFs is associated with reduced m6A and H3K9me3 levels and increased chromatin accessibility, which may contribute to improved in vitro development of porcine cloned embryos. These findings provide a potential molecular target for improving the efficiency of porcine SCNT.