Sex differences in microglia morphology and function across the lifespan are mediated by the early hormone environment

Microglia, the resident immune cell of the central nervous system (CNS), contribute to a range of physiological processes across the lifespan. Microglia exhibit notable sex differences in morphology, reactivity, and transcriptomic profiles. Steroid hormones in early life are believed to elicit sex differences in many cells, including microglia, in the CNS. However, few studies have examined how neonatal hormone environment impacts microglial morphology and function across the lifespan. Therefore, here we used steroid hormones to manipulate the early hormone environment to assess the appearance and persistence of sex differences in a rat model of healthy aging. Rat pups were dosed with steroid hormones on postnatal day (P)0 and 1: females received testosterone to "masculinize" them and males received flutamide, an androgen antagonist, to "feminize" them. Brain tissue was then collected at three distinct developmental timepoints: adolescence (P30), adulthood (P150), and aging (P700) for immunohistochemistry and ex vivo microglial stimulation. Transcriptomic changes in hippocampal tissue of aged animals were also assessed using 3'UTR biased transcriptome sequencing (Tag-seq). We report that testosterone treatment in females leads to lifelong alterations in body size and vaginal morphology and results in microglia that display a more "masculinized" phenotype compared to controls. Flutamide had more moderate effects on microglia morphology in males, contributing to a more "feminized" phenotype in the hippocampus in adult and aged males. Testosterone treatment also resulted in greater transcriptomic changes in the aged hippocampus compared to flutamide treatment, especially in genes related to mitochondrial function and inflammation. These results indicate that (1) early hormone environment is critical for the induction of sex differences in microglial morphology and (2) sex differences in microglial morphology reverse during aging, and this reversal is also recapitulated with early hormone treatment.