To gain insights into the potential contribution of DNA methylation to the regulation of puberty, we inhibited DNA methylation by treatment with 5′-Azacytidine (Aza), a well-established DNA methyl transferase (DNMT) inhibitor 22, 23. The treatment (2 mg/Kg BW/day, i.p) was initiated on postnatal day (PND) 22, which in the rat corresponds to the initiation of the early juvenile (EJ) phase of pubertal development 2. We first evaluated the effect of Aza on the timing of puberty and estrous cyclicity, by continuing the treatment until PND44, i.e., nearly two weeks after all control animals had reached puberty. In all subsequent studies, the animals were treated only for the duration of the juvenile period, i.e., from PND22 to PND28. Rats subjected to long-term Aza treatment had delayed vaginal opening (Fig. 1a), (mean age at vaginal opening: C= 31.33 ± 0.21, n=6 vs Aza= 36.67 ± 0.67 days; t=−7.628, p<0.001, Student t Test), failed to reach puberty as assessed by the lack of ovulation, and showed no estrous cyclicity, as determined by daily vaginal lavages after vaginal opening (Fig. 1b). These alterations did
