277 PRELIMINARY STUDY OF THE DIFFERENCE IN THE GENOME-WIDE METHYLATION PROFILES BETWEEN GERM CELLS AND SOMATIC CELLS OF BULLS

Abstract

DNA methylation is tissue-specific and is thought to be one of the factors that regulates gene expression. This recent study was carried out in order to examine the difference of the genome-wide methylation profiles at the HpaII sites in germ cells and somatic cells of bulls. Ejaculated spermatozoa, leukocytes, and ear fibroblasts were collected from 3 Holstein bulls of ages 1 to 3 years. The genomic DNA was extracted and treated overnight with a methylation-sensitive restriction endonuclease (HpaII) to digest unmethylated sites throughout the genome. Both undigested and digested DNA samples were used as templates in the PCR-based technique developed by researchers at the University of Queensland, which allows the amplification of the methylation sites by short oligonucleotide primers of arbitrary sequence containing the HpaII recognition site (CCGG). The amplicons were separated in 4% polyacrylamide gel by electrophoresis and the gel was stained with silver nitrate. The results were evaluated on the basis of the presence–absence of the band(s) in the digested template compared with the undigested counterpart, and the difference between types of marker was analyzed using the chi-square test. From 10 sets of primer, approximately 400 markers in the genomic samples could be scored. The samples from the 3 bulls showed similar but not identical patterns. Statistical analysis showed that the difference between marker types was dependent on the individual. Generally, most of the markers were digestion-resistant markers signifying that most of the HpaII sites in the genome of both germ cells and somatic cells are methylated. Leukocytes had a significantly higher methylation content compared to fibroblasts (94.1 vs. 90.1%; P = 0.0004), but did not differ from those in sperm (92.3%; P = 0.09). Sperm cells showed a slightly higher percentage of unmethylated sites than did somatic cells (3.5 vs. 2.6% in leukocytes and 3.3% in fibroblasts), and yet the difference was non-significant. Moreover, fibroblastic cells had a higher portion of the digestion-dependent markers than did other cell types, and this difference was statistically significant (6.6 vs. 4.2% in sperm, P = 0.009, and 3.3% in leukocytes, P = 0.001). In conclusion, the DNA of the germ cells and somatic cells is highly methylated at the HpaII sites, with some variation in methylation pattern between the 2 cell lineages. The markers found only in the digested template of the ear fibroblasts suggest the difference in genome structure between the versatile cell types and the fully differentiated ones. Further investigation is required to elucidate any possible relationship between the variation of the methylation pattern found in sperm and fibroblasts and the failure of the reprogramming process in cloned animals derived from somatic cell nuclear transfer.

This study was supported by The Faculty of Veterinary Science, Chulalongkorn University, and The Royal Golden Jubilee PhD program of Thailand Research Fund.