Simple SummaryOxytocin (OT) is a hypothalamic hormone that controls key aspects of behavior and female and male reproductive systems as well as playing an important role during the estrous cycle, pregnancy and lactation. Depending on the physiological state, the activity of OT neurons is associated with sex steroid interactions and/or local synthesis of neurosteroids in the brain. This study aimed to determine the central effects of neurosteroids and stress on OT synthesis and release in the hypothalamic-posterior pituitary system in non-pregnant and pregnant sheep. The results showed that allopregnanolone (AL) alone differentially modulated OT synthesis at the hypothalamic and posterior pituitary levels, and markedly inhibited OT secretion induced by stress in non-pregnant sheep. In pregnant animals, an inhibitory effect of neurosteroids on OT secretion was demonstrated after blocking the enzymatic pathway of neurosteroid synthesis. In conclusion, the present study demonstrated the involvement of neurosteroids in the regulation of oxytocin secretion in sheep. The central inhibitory effect of neurosteroids in pregnant sheep is consistent with the concept of protecting the fetus from premature labor.AbstractOxytocin (OT) is a neuropeptide synthesized in the hypothalamic nuclei that modulates both behavioral and reproductive functions, associated with the increased neurosteroid synthesis in the brain. Therefore, the present study tested the hypothesis that manipulation of central neurosteroid levels could affect oxytocin synthesis and release in non-pregnant and pregnant sheep under both basal and stressful conditions. In Experiment 1, luteal-phase sheep were subjected to a series of intracerebroventricular (icv.) infusions of allopregnanolone (AL, 4 × 15 μg/60 μL/30 min) for 3 days. In Experiment 2, pregnant animals (4th month) received a series of infusions of the neurosteroid synthesis blocker, finasteride (4 × 25 μg/60 μL/30 min), conducted for 3 days. In non-pregnant sheep AL alone was shown to differentially modulate OT synthesis in basal conditions, and strongly inhibit OT response to stress (p< 0.001). In contrast, in pregnant animals, basal and stress-induced OT secretion was significantly (p< 0.001) increased during finasteride infusion compared to controls. In conclusion, we showed that neurosteroids were involved in the control of OT secretion in sheep, particularly under stress and pregnancy conditions and are part of an adaptive mechanism which is responsible for protecting and maintaining pregnancy in harmful situations.