Reproduction Abstracts

Introduction: Genome-wide demethylation reprograms the genome after fertilization and re-establishes totipotency: 5-methylcytosine (5 mC) in the paternal pronucleus is rapidly converted to 5-hydroxymethylcytosine (5 hmC) by the dioxygenase 10–11 translocation (TET) 3, while 5 mC in the maternal pronucleus is protected by the binding of developmental pluripotency-associated (DPPA) 3 to histone H3 dimethyl Lys9 (H3K9me2). This process is essential for normal development, because deletion of TET3 or DPPA3 in the maternal germline alters 5 mC and 5 hmC levels in parental pronuclei, resulting in lethality. This study examined the developmental significance of demethylation events by evaluating 5 hmC and H3K9me2 levels in oocytes and preimplantation embryos.

Materials and methods: Oocytes and embryos were fixed, pretreated with 2N HCl, and labeled with primary antibodies against 5 mC, 5 hmC, and H3K9me2, and then washed and treated with fluorophore-conjugated secondary antibodies and visualized by confocal microscopy.

Results and discussion: The signal of 5 hmC was detected at heterochromatin regions from growing stage oocytes, and the signal was strengthened up to fully grown oocytes, spreading into whole chromatin. Interestingly, 5 hmC was mainly localized to retrotransposon regions such as IAP and MLV during oogenesis, but was restricted to the male pronucleus in zygotes, and was not detected after the 4-cell stage. In contrast, the H3K9me2 signal was constant during early development, and was associated with a copy number increase via retrotransposon reactivation. These results suggest that demethylation process of female genome begin from oocyte growth stage, and the conversion of 5 mC to 5 hmC contributes to retrotransposon activation after fertilization.