Bobcat339, a specific TET family inhibitor, impaired oocyte maturation and early embryogenesis in pig

Abstract

Abstract Background: Ten-eleven translocation (TET) enzyme family, which includes TET1/2/3, participates in active DNA demethylation in the eukaryotic genome; however, TET1/2/3 are functionally redundant. The effect of TET1/2/3 triple-gene knockdown or knockout on the porcine oocytes and embryos is unclear. In this study, using Bobcat339, a specific small-molecule inhibitor of the TET family, we explored the combined effects of TET enzymes on oocyte maturation and early embryogenesis in pigs. Method: First, porcine cumulus oocyte complexes were cultured in the IVM medium with various concentrations of Bobcat339, and the effects on the expansion of cumulus cells and oocyte maturation were investigated. Further, the apoptotic level of oocytes was assessed using RT-PCR and Annexin-V staining. The spindle architecture and chromosomal alignment were investigated using immunofluorescence staining. Furthermore, the fluorescent signals of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) were quantified. Subsequently, the effect of Bobcat339 on porcine parthenogenetic embryos was assessed. We recorded the blastocyst formation and developmental rates. Furthermore, the mRNA levels of zygotic gene activation (ZGA)- and pluripotency-related and imprinted genes were assessed using RT-PCR. Finally, RNA sequencing analysis was performed in the embryos at the 4-cell stage to identify differentially expressed genes. Result: Our results revealed that Bobcat339 treatment blocked porcine oocyte maturation and triggered early apoptosis. Furthermore, in the Bobcat339-treated oocytes, spindle architecture and chromosome alignment were disrupted, probably due to huge loss of 5hmC and concurrent increase in 5mC. Furthermore, after Bobcat339 treatment, early parthenogenetic embryos exhibited abnormal 5mC and 5hmC levels, which resulted in compromised cleavage and blastocyst rate. The mRNA levels of EIF1A and DPPA2 (ZGA marker genes) were significantly decreased, which may explain why the embryos were arrested at the 4-cell stage after Bobcat339 treatment. In addition, the mRNA levels of pluripotency-related genes OCT4 and NANOG were declined after Bobcat339 treatment. RNA sequencing analysis revealed differentially expressed genes in Bobcat339-treated embryos at the 4-cell stage, which were significantly enriched in cell proliferation, cell component related to mitochondrion, and cell adhesion molecule binding. Conclusion: Our results indicated that TET proteins are essential for porcine oocyte maturation and early embryogenesis, and they act by mediating 5mC/5hmC levels and gene transcription.