Defining functional puberty in female C57BL/6J mice using endocrine, cytological and morphological markers.

Puberty marks the transition to reproductive competence and is driven by activation of the hypothalamo/pituitary/gonadal axis, culminating in first ovulation in females. In mice, puberty onset is commonly inferred from vaginal opening and estrous cyclicity, but the precise timing of first ovulation remains unclear. Here, we aimed to define reliable parameters of functional puberty in female C57BL/6J mice by integrating external, endocrine, and morphological measures. Vaginal opening and daily vaginal cytology were combined with daily serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH) measurements and ovarian histology using the Pubertal Ovarian Maturation Score (Pub-Score), which estimates ovulation based on follicular development and corpus luteum morphology. Notably, we observed substantial inter-individual variability in puberty-related events, and no link between estrous cytology and first ovulation. Pub-Score analysis indicated that first ovulation occurred between 36 and 54 days postnatal, with a mean around 44 days, with no correlation with vaginal cytology. LH surges were detected in some of the females that had ovulated and showed limited temporal correspondence with vaginal cytology or Pub-Score estimates. Together, these findings demonstrate that puberty in female mice is a gradual, asynchronous process and that external or cytological markers alone are insufficient to define functional reproductive maturity.