Reproductive function in female mammals is largely orchestrated by the hypothalamic-pituitary-gonadal axis, which generates rhythmic hormonal fluctuations underlying the estrous cycle. Part of this cycle, the preovulatory LH surge, is tightly gated by the circadian system. The suprachiasmatic nucleus (SCN)-the central circadian clock-plays a critical role in this temporal regulation and among SCN-derived signals, neuropeptides such as arginine vasopressin (AVP) and vasoactive intestinal peptide (VIP) have been proposed to mediate this process. Notably, most SCN neurons are GABAergic; however, the contribution of SCN-derived GABAergic transmission in the female reproductive system remains unclear. To investigate the role of GABAergic output from the SCN, we first performed AAV-mediated SCN ablation in Vgat-IRES-Cre mice (Vgat; encoding the vesicular GABA transporter), resulting in disrupted estrous cycles. To assess GABAergic transmission from specific SCN populations, we next examined Avp-Vgat-/- and Vip-Vgat-/- mice, in which the Vgat gene is selectively deleted in AVP or VIP neurons. Vip-Vgat-/- females showed regular cycles. However, Avp-Vgat-/- females exhibited marked disruptions, and AAV-mediated Vgat rescue in AVP neurons in the SCN (SCN-AVP) restored normal estrous cycles. Anterograde tracing revealed dense SCN-AVP terminals in the anteroventral periventricular nucleus (AVPV), which contains kisspeptin neurons, but few projections to other major reproductive neuroendocrine populations. These findings suggest GABAergic output from SCN-AVP neurons stabilizes the estrous cycle, potentially via kisspeptin neurons in the AVPV, thereby highlighting that GABAergic signaling also contributes to female reproductive regulation alongside AVP and VIP.Significance Statement The circadian system must precisely coordinate the timing of ovulation, and is essential for maintaining a stable estrous cycle. Although most neurons in the master clock are GABAergic, their role in reproductive control remains unknown. Here, our study identifies GABAergic signaling from arginine vasopressin neurons in the master clock as a key regulator of the estrous cycle. Loss of this signaling disrupts estrous cyclicity, and its restoration rescues regular cycling. This finding highlights GABA release from arginine vasopressin neurons as an additional component of reproductive control, alongside established peptidergic regulators such as arginine vasopressin and vasoactive intestinal peptide. This work advances our understanding of how the brain's circadian system organizes complex reproductive physiology through multiple neural output pathways.
