In most Hymenoptera, the caste differentiation is influenced by differential environmental cues (e.g., food), which affect larvae development. However, Melipona stingless bees represent an exception to this rule. Workers and queens can emerge from brood cells of the same size, with the same quantity and quality of food. The different phenotypes of female bees depend on the interaction of genetic and environmental components. This mechanism is driven by the juvenile hormone, a key physiological factor synthetized by the corpora allata glands. In addition to that, epigenetic mechanisms were recently associated with caste development in Melipona scutellaris . In this study, using images captured by confocal microscopy and analyzed by 3D computational reconstruction, we show the similarities and differences in the pattern and volume of heterochromatin in the cell nuclei of corpora allata glands of M. scutellaris . Our results imply that heterochromatin territorial dispersion may act as a major player on the female phenotypic plasticity of M. scutellaris stingless bees.

Cloning using somatic cell nuclear transfer (SCNT) has many potential applications such as in transgenic and genomic-edited animal production. Abnormal epigenetic reprogramming of somatic cell nuclei is probably the major cause of the low efficiency associated with SCNT. Strategies to alter DNA reprogramming in donor cell nuclei may help improve the cloning efficiency. In the present study, we aimed to characterize the effects of procaine and S-adenosyl-l-homocysteine (SAH) as demethylating agents during the cell culture of bovine skin fibroblasts. We characterized the effects of procaine and SAH on the expression of genes related to the epigenetic machinery, including the DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3 alpha (DNMT3A), DNA methyltransferase 3 beta (DNMT3B), TET1, TET2, TET3, and OCT4 genes, and on DNA methylation levels of bovine skin fibroblasts. We found that DNA methylation levels of satellite I were reduced by SAH ( p = 0.0495) and by the combination of SAH and procaine ( p = 0.0479) compared with that in the control group. Global DNA methylation levels were lower in cells that were cultivated with both compounds than in control cells (procaine [p = 0.0116], SAH [p = 0.0408], and both [p = 0.0163]). Regarding gene expression, there was a decrease in the DNMT1 transcript levels in cells cultivated with SAH ( p = 0.0151) and SAH/procaine (0.0001); a decrease in the DNMT3A transcript levels in cells cultivated with SAH/procaine ( p = 0.016); and finally, a decrease in the DNMT3B transcript levels in cells cultivated with procaine ( p = 0.0007), SAH ( p = 0.0060), and SAH/procaine ( p = 0.0021) was found. Higher levels of TET3 transcripts in cells cultivated with procaine ( p = 0.0291), SAH ( p = 0.0373), and procaine/SAH ( p = 0.0013) compared with the control were also found. Regarding the OCT4 gene, no differences were found. Our results showed that the use of procaine and SAH during bovine cell culture was able to alter the epigenetic profile of the cells. This approach may be a useful alternative strategy to improve the efficiency of reprogramming the somatic nuclei after fusion, which in turn will improve the SCNT efficiency.

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