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 not appear to result from a general, non-specific effect of Aza, because the animals treated with the inhibitor weighed significantly more (20 g) than control animals at the time of vaginal opening, and had not achieved puberty at the time of euthanasia even though they weighed 35 g more than the weight reached by controls at the time of first ovulation (Supplementary Fig. 1). Morphological examination of the ovaries either at PND 28, which marks the transition between late juvenile (LJ) development and the initiation of puberty 2 or on PND 44 showed that the ovaries of Aza-treated rats had no overt abnormalities, but were developmentally delayed (Fig. 1c). By PND 28, these ovaries had only small antral follicles and were about half the size of a control ovary. At PND 44, the ovaries of Aza treated rats had antral follicles, but no corpora lutea, indicating that they had not ovulated, and consequently, puberty had failed to occur.
