Establishment of the paternal methylation imprint of the human H19 and MEST/PEG1 genes during spermatogenesis

Abstract

Parental-specific epigenetic modifications are imprinted on a subset of genes in the mammalian genome during germ cell maturation. However, the precise timing of their establishment remains to be determined. Methylation of CpG dinucleotides has been shown to be a part of the parental imprint. We have examined how the methylation pattern characteristic of the paternal allele in germ cells are established during human spermatogenesis. Two representative imprinted genes, H19 and MEST/PEG1, were studied. The experiments were performed using the bisulphite sequencing method on microdissected individual cells at different stages of male germ cell differentiation. We show that both genes are unmethylated in fetal spermatogonia, suggesting that all pre-existing methylation imprints are already erased by this stage. The MEST/PEG1 gene remains unmethylated at all subsequent post-pubertal stages of spermatogenesis, including mature spermatozoa. The methylation of H19 typical of the paternal allele first appears in a subset of adult spermatogonia and then is maintained in spermatocytes, spermatids and mature spermatozoa. Our results suggest that the methylation imprint inherited from the parents is first erased in the male germ line at an early fetal stage. The paternal-specific imprint is re-established only later, during spermatogonial differentiation in the adult testis.

IGF2/H19 hypomethylation in Silver–Russell syndrome and isolated hemihypoplasia

“…This result is consistent with a normal sperm-specific methylation pattern as published by Kerjean et al 23 The remaining H19 MLPA probe sets did not show consistent methylation patterns in six sperm DNA samples from normal controls (data not shown). Methylation sensitive MLPA probe sets within the KvDMR (7171-L6780, 7172-L6781, 7173-L6782 and 6276-L5782) and IGF2 regions (probe set 6269-L5775) revealed an unaltered methylation pattern in all patients when compared with blood DNA samples from normal controls but showed complete loss of methylation in all sperm DNAs (data not shown).…”

“…We found both sites to be fully methylated, consistent with a normal sperm-specific methylation pattern. 23,26,27 These results suggest that the epimutation is reversed in the male germ line of patient Esr3. However, it is possible that the primordial germ cells of patient Esr3, who is mosaic for the epimutation, are not affected by ICR1 hypomethylation.…”

Methylation analysis of the intergenic differentially methylated region of DLK1-GTL2 in human

“…Studies at the imprint control region of H19/Igf2 in mice as well as in the human showed around 5 and 6% intermediate methylation patterns (40 -50%) respectively. 28,29 These percentages are lower than the relaxation of allele-specific methylation observed at the IG-DMR.…”

Seeking the origin of female germline stem cells in the mammalian ovary

“…It might be that the quiescent state of putative mouse FGSCs is maintained through modulation of chromatin structure, particularly by histone deacetylation, and through R126 M De Felici and F Barrios inhibition of CDK2 (Johnson et al 2005b, Lee et al 2007). In the male germ line, spermatogonial stem cells (SSCs) originating from PGCs are intrinsically pluripotent and posses both cell cycle quiescence and self-renewal capability; in SSCs, however, imprinting is already partly re-established (Davis et al 2000, Kerjean et al 2000. Two other types of pluripotent stem cells that originate from PGCs either in vitro or in vivo are the embryonic germ (EG) cells and the embryonic carcinoma (EC) cells respectively.…”