Abstract Disclosure: M. Devillers: None. C.M. François: None. M. Chester: None. R. Corre: None. V. Cluzet: None. F. Giton: None. J. Cohen-Tannoudji: None. C.J. Guigon: None. Introduction: In females, androgens contribute to ovarian diseases such as polycystic ovary syndrome (PCOS), but their action is also crucial for ovarian physiology, i.e., follicular growth and estradiol (E2) synthesis during reproductive life, in interaction with the gonadotropins LH and FSH. However, it is unclear whether androgens already play a role in the ovary at mini-puberty, a phase of postnatal development with active follicular growth and high E2 levels. Hypothesis/question: Given their role in the adult ovary, we hypothesized that androgens may regulate early ovarian function at mini-puberty. Therefore, we analyzed their potential actions on the ovary and their possible interaction with gonadotropins during this period. Methods: We used the mouse as an experimental model to measure the intra-ovarian content and serum levels of testosterone by gas chromatography-mass spectrometry (GC-MS) at different prepubertal ages, including mini-puberty. We performed molecular-based studies (immunohistochemistry, RT-qPCR), morphometric studies and pharmacological approaches in vivo and in cultured ovaries. Main results: We found that mini-pubertal ovaries produce significant amounts of testosterone and display androgen receptor (AR) expression in growing follicles, both under the control of LH. By blocking AR signaling with flutamide either in vivo or in ovarian cultures, we found that this pathway may participate in the regulation of prepubertal E2 synthesis and follicular growth, possibly by regulating the expression of a number of key intra-ovarian regulators, including FSH receptor (Fshr), the aromatase enzyme converting androgens into estrogens (Cyp19a1) and the cell cycle inhibitor p27KIP1 (Cdkn1b). We further showed that AR may stimulate FSH-mediated regulation of Cyp19a1 through its action on Fshr mRNA abundance. Conclusion: This work supports the idea that AR signaling is already activated in mini-pubertal ovaries to regulate E2 synthesis and follicular growth, in interaction with LH and FSH signaling. This early physiological action of androgens may contribute to the implementation of early ovarian function with possible impacts on reproductive function. Presentation: Friday, June 16, 2023