Brain Sex Steroid-Mediated Intergenerational Sex-Specific Neurocognitive Effects of Paternal Sevoflurane Exposure in Rats.

Abstract

The biological basis for the male-biased prevalence of many neurodevelopmental disorders remains poorly understood. We found that preconception exposure of male rats to sevoflurane induces persistent dysregulation of stress and inflammatory signaling, while their offspring, almost exclusively males, develop neurobehavioral abnormalities. These effects in both generations are prevented by paternal pretreatment with either the Na+/K+/2Cl- cotransporter inhibitor bumetanide or the glucocorticoid receptor antagonist RU486. Using offspring from the same treatment groups, we tested the hypothesis that dysregulation of male-specific, sex hormone-dependent brain masculinization processes during the perinatal critical period contributes to the observed male-biased abnormalities. Sprague-Dawley F0 males were exposed to 2.1% sevoflurane for 3 hours on postnatal days (P) 56, 58, and 60. Subsets received bumetanide or RU486 30 minutes before each exposure. On P90, F0 males were mated to generate F1 offspring. On P0-P1, F1 males of sevoflurane-exposed sires received the androgen receptor (AR) antagonist flutamide, the estradiol (E2) synthesis inhibitor formestane, the estrogen receptor α (ERα) antagonist 1,3-Bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride (MPP), or the estrogen receptor β (ERβ) antagonist 4-[2-Phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP) (n = 18/group). Sex steroid concentrations and hippocampal mRNA expression of enzymes and receptors involved in brain masculinization were measured in P0 F1 males from control sires (F1M_C), sevoflurane-exposed sires (F1M_S), or sires pretreated with bumetanide (F1M_BS) or RU486 (F1M_RS) (n = 6-8/group). Behavioral and stress-responsivity outcomes were assessed in adulthood. Compared with F1M_C, P0 F1M_S exhibited increased hippocampal testosterone (mean ± standard error of the mean [SEM]: 1.979 ± 0.527 vs 0.524 ± 0.122 ng/mg; P = .008) and E2 levels (10.997 ± 3.756 vs 2.793 ± 0.719 ng/mg; P = .027). Hippocampal mRNA expression of the testosterone-synthesizing enzyme 17β-hydroxysteroid dehydrogenase (1.629 ± 0.135 vs 1.000 ± 0.066; P = .001) and the E2-synthesizing enzyme aromatase (1.617 ± 0.093 vs 1.000 ± 0.070; P = .001) was also increased. In contrast, serum testosterone levels and hippocampal Ar, Erα, and Erβ mRNA expression were unchanged. F1M_BS and F1M_RS did not differ from F1M_C in any of these measures. Perinatal flutamide treatment reduced stress and neuroinflammatory markers and improved sociability, anxiety-like behavior, sensorimotor gating, and spatial memory in adulthood. Formestane normalized inflammatory and behavioral abnormalities; MPP improved sociability and spatial memory; and PHTPP improved spatial memory only. These findings suggest that male-specific neurodevelopmental abnormalities following paternal preconception sevoflurane exposure arise from stress- and inflammation-programmed increases in brain-derived testosterone during the critical brain masculinization period, involving both AR and E2 signaling.