Paternal Caffeine Exposure Programs Offspring Stress Vulnerability via Sperm Dlk1‐Dio3 Imprinting‐Directed Remodeling of a Novel Neural Circuit

ABSTRACT Paternal environmental exposures program offspring neurodevelopment via sperm epigenetics, yet mechanisms for intergenerational hypothalamic–pituitary–adrenal (HPA) axis dysregulation, a core hub for stress disorders, remain elusive. Using a paternal preconception caffeine exposure (PPCE) rat model with in vitro fertilization to exclude maternal confounders, we uncover a novel pathway linking sperm epigenetics to offspring HPA axis hyperresponsivity. By elevating paternal corticosterone, PPCE induces hypomethylation at the intergenic differentially methylated region (IG‐DMR) within sperm Dlk1‐Dio3 domain. This epigenetic alteration evades postfertilization reprogramming, persists in offspring hippocampus, and derepresses the maternally expressed miRNA cluster, causing posttranscriptional downregulation of glutaminase (GLS). Hippocampal GLS deficiency impairs glutamatergic neurotransmission in a novel circuit: ventral hippocampal CA1 glutamatergic neurons (vCA1 ^Glu ) → piriform cortex γ‐aminobutyric acid‐ergic neurons (Pir ^GABA ) → paraventricular nucleus corticotropin‐releasing hormone neurons (PVN ^CRH ). Chemogenetic activation of this circuit rescues HPA axis hyperresponsivity and affective phenotypes. Clinically, sperm IG‐DMR hypomethylation correlates with elevated plasma cortisol in prospective fathers. Importantly, paternal folic acid supplementation prevents these epigenetic alterations and restores offspring stress homeostasis. Our study delineates an intergenerational mechanism and identifies a potentially translatable prenatal intervention strategy.