Gut microbiota shapes how male and female mice react to acute stress

• Gut microbiota influences neuroendocrine stress responses in a sex-dependent manner. • Germ-free females exhibit amplified stress-induced corticosteroid production. • Microbiota suppresses stress-induced adrenal steroidogenesis in females. • Microbiota modulates gonadal steroidogenic gene expression. The gut microbiota plays an important role in regulating the hypothalamic-pituitary-adrenal (HPA) axis and modulating stress responses. However, its interaction with host sex in this context remains poorly understood. We investigated how sex and microbial status interact to shape the neuroendocrine response to stress by subjecting male and female germ-free (GF) and specific pathogen-free (SPF) mice to acute restraint stress. We analyzed plasma steroid profiles and the expression of genes involved in HPA and hypothalamic-pituitary-gonadal axis function across multiple brain regions, the pituitary, adrenal glands, and gonads. GF and SPF mice displayed distinct patterns of steroid release and metabolism following stress, with marked sex-specific differences. In males, stress elicited a robust increase in progesterone, particularly under SPF conditions, whereas in females, the absence of microbiota amplified the rise in deoxycorticosterone, corticosterone, and their metabolites. Stress-induced expression of adrenal steroidogenic enzymes and ACTH receptor was strongly microbiota-sensitive in females but only modestly affected in males. In the paraventricular nucleus, microbiota absence blunted the induction of gene encoding corticotropin-releasing hormone (CRH) in stressed males but not females, while in the pituitary, microbiota decreased the expression of CRH receptor ( Crhr1 ) and proopiomelanocortin ( Pomc ) in both sexes. Furthermore, microbiota influenced the expression of key steroidogenic genes in the gonads, with potential consequences for testosterone and estrogen synthesis. Together, these findings indicate that microbial signals shape the neuroendocrine stress response in a sex-dependent manner, leading to divergent hormonal and gene expression patterns.