Analysis of H19/Igf2 Methylation Status in the Sperm of Cloned Goats and Their Offspring.

The H19/insulin-like growth factor 2 (Igf2) gene plays vital roles during development, but their expression and methylation status have not been elucidated in male cloned animals. In this study, we investigated the methylation status of imprinting control region (ICR) and differentially methylated region (DMR) of the H19/Igf2 locus in the sperm and testes of naturally reproduced Saanen dairy goats (NG, control), transgenic cloned dairy goats (CG), and the first generation of mating the CGs with the female NGs (FG). Bisulfite sequencing polymerase chain reaction revealed that the H19 ICR was highly methylated in the sperm of all testing groups (97.07% ± 0.47%, 93.73% ± 0.85%, and 96.67% ± 0.54%). Notably, the methylation level of H19 ICR was decreased in the testes of CG goats compared with that of NG goats (73.83% ± 3.28% vs. 97.27% ± 0.71%, p < 0.05), while the Igf2 DMR2 was highly methylated in the sperm of these three groups (89.57% ± 1.74%, 87.23% ± 1.11%, and 83.2% ± 1.27%). However, the Igf2 DMR2 was abnormally hypomethylated in the testes of CG goats (27.33% ± 1.005%, p < 0.001), and moderately methylated in FG goats (53.1% ± 1.47%, p < 0.01) compared with that in NG goats (82.67% ± 0.70%). Furthermore, we found significantly decreased expression of H19 (p < 0.01) and increased expression of Igf2 (p < 0.01) in CG goats. Thus, we infer that somatic cell nuclear transfer has clear effect on genomic imprinting in the testes, but not sperm of cloned goats.