Abstract
This study investigated 17α-estradiol's effects on aged hypothalamic physiological activity via long-term administration. Single-nucleus transcriptomic sequencing (snRNA-seq) was performed on pooled hypothalami from each group: aged male Norway brown rats treated with 17α-estradiol (O.T), aged controls (O), and young controls (Y). Supervised clustering of neurons (based on neuropeptides/receptors) evaluated subtype responses to aging and 17α-estradiol. Aging-induced elevation of neuronal cellular metabolism, stress, and reduced synapse formation-related pathways were significantly attenuated by 17α-estradiol. Neuron population analysis showed that subtypes regulating food intake, reproduction, blood pressure, stress response, and electrolyte balance were sensitive to 17α-estradiol. 17α-estradiol increased serum oxytocin (Oxt) and hypothalamic-pituitary-gonadal (HPG) axis activity (elevated plasma Gnrh, total testosterone; reduced estradiol). Gnrh1 upregulation mediated its effects on energy homeostasis, neural synapse, and stress response. Notably, Crh neurons in O.T showed prominent stress phenotypes, distinct from Agrp/Ghrl neurons. Thus, HPG axis and energy metabolism may be key 17α-estradiol targets in male hypothalamus. Additionally, our results demonstrate that supervised clustering (based on neuropeptides/receptors) effectively assesses the responses of hypothalamic neuron subtypes to aging and 17α-estradiol treatment.