The study of DNA methylation profiles of bull germ cells and somatic cells : lineage difference and effect of in vitro cell culture

Abstract

The DNA methylation profiles of various tissues collected from three Holstein bulls aged between 2-3 years old were evaluated by the arbitrarily-primed PCR technique originally designed for plant DNA research called Amplified methylation polymorphisms (AMPs)-PCR in combination with a methylation-sensitive restriction enzyme. Experiment 1 was aimed to study the difference of DNA methylation patterns between germ cells and somatic cells in bulls. DNA was extracted from sperm, white blood cells and fibroblast cell culture passage number 1, and was digested with HpaII enzyme. The genomic and digested DNA samples were subjected for AMPs-PCR. The PCR products were separated on polyacrylamide gel and stained with silver nitrate. The result evaluation based on the presence-absence of three types of marker: digestion resistant-, digestion sensitive- and digestion dependent marker. From twenty-seven sets of primer, approximately 1,000 markers could be scored in each bull. The samples from all bulls showed a similar but not identical pattern. Most of the markers were digestion-resistant markers signifying that both germ cells and somatic cells are generally methylated at the HpaII sites. Leukocytes had the highest percentage of digestion resistant markers (p < 0.05), whereas sperm cells showed a highest percentage of digestion sensitive markers (p < 0.05). Fibroblast cells yielded the highest percentage of digestion dependent markers (p < 0.05). The results showed that germ cells have less methylation than somatic cells. There are different methylation patterns among somatic cell types: leukocyte DNA is more methylated than fibroblast cells and partial differentiated somatic cells such as fibroblasts may have a different chromatin structure. Experiment 2 was aimed to study the effect of long-term cell culture on the DNA methylation profile of cultured fibroblast cells. The ear fibroblast cells were cultured in a standard culture protocol using basic culture medium supplemented by fetal calf serum and broad spectrum antibiotics until passage number 30. The cells from odd number passages were collected for DNA extraction. The digestion of DNA was carried out with HpaII enzyme. The samples were categorized into 3 groups: early-, medium- and late passages. The AMPs-PCR revealed that there was no alteration at the approximately 1,500 DNA methylation locations among groups. This can be concluded that the culture condition applied in this experiment did not affect the DNA methylation content in ear fibroblast cells culture maintained continuously for 5 months under the conventional cell culture conditions. Overall conclusions of this study are 1) the AMPs-PCR technique could be applied in the study of mammalian DNA methylation; 2) DNA methylation profiles of germ cells and somatic cells are different. At the HpaII sites investigated, somatic cells have more methylation content than germ cells and partial differentiated somatic cell lineage tends to have different genomic structure and 3) in vitro cell culture condition used in this study did not affect the DNA methylation profiles of fibroblast cells cultured up to passage number 30.