AgRP neurons shape the sperm small RNA payload

Dietary habits and obesity in fathers imprint upon progeny through epigenetic signals carried by sperm RNA. By activating hypothalamic AgRP neurons in mice to mimic obesity's effects, we observed significant alterations in sperm tsRNAs, miRNAs, and rsRNAs. These changes, particularly in tsRNAs, mirror those seen with short-term high-fat diets, suggesting a shared upstream regulatory mechanism involving AgRP neurons that influence metabolic epigenetic inheritance. We aimed to replicate the AgRP neuronal state that will occur in obesity. To achieve this, we employed Designer Receptors Exclusively Activated by Designer Drugs (DREADD) .We bilaterally injected an activator DREADD adeno-associated viral (AAV-hM3Dq-mCherry) vector into the arcuate nucleus of the hypothalamus (ARH) in both AgrpCre/+ and Agrp+/+. The DREADD ligand, Clozapine-N-Oxide (CNO), was administered to AgrpCre/+ (activated) and Agrp+/+ (control) mice in the absence of food. Four hours post-administration, mature sperm was harvested from the caudae epididymides, and RNAs were extracted from the sperm. These RNAs were then used to prepare a small non-coding RNA (sncRNA) library, which was subsequently sequenced.