Estradiol-dependent axogenesis and Ngn3 expression are determined by XY sex chromosome complement in hypothalamic neurons

Abstract

Hypothalamic neurons show sex differences in neuritogenesis, female neurons have longer axons and higher levels of the neuritogenic factor neurogenin 3 (Ngn3) than male neurons in vitro. Moreover, the effect of 17-β-estradiol (E2) on axonal growth and Ngn3 expression is only found in malederived neurons. To investigate whether sex chromosomes regulate these early sex differences in neuritogenesis by regulating the E2 effect on Ngn3, we evaluated the growth and differentiation of hypothalamic neurons derived from the "four core genotypes" mouse model, in which the factors of "gonadal sex" and "sex chromosome complement" are dissociated. We showed that sex differences in neurite outgrowth are determined by sex chromosome complement (XX > XY). Moreover, E2 increased the mRNA expression of Ngn3 and axonal length only in XY neurons. ERα/β expressions are regulated by sex chromosome complement; however, E2-effect on Ngn3 expression in XY neurons was only fully reproduced by PPT, a specific ligand of ERα, and prevented by MPP, a specific antagonist of ERα. Together our data indicate that sex chromosomes regulate early development of hypothalamic neurons by orchestrating not only sex differences in neuritogenesis, but also regulating the effect of E2 on Ngn3 expression through activation of ERα in hypothalamic neurons.

X-Linked Histone H3K27 Demethylase Kdm6a Regulates Sexually Dimorphic Differentiation of Hypothalamic Neurons

“…Finally, neuronal cultures derived from FCG transgenic mice were treated with the GSK-J4 inhibitor and processed for Ngn3 relative expression analysis. In agreement with our previous results [34, 35], sexually dimorphic expression of Ngn3 was proven to be determined by sex chromosome complement in control conditions, with XX neurons expressing significantly higher levels than XY neurons regardless of gonadal sex. Remarkably, the inhibition of Kdm6 H3K27 demethylase activity in FCG transgenic cultures showed sex chromosomes-specific effects in line with the results observed in wild-type cultures: a decrease in Ngn3 mRNA levels in XX neurons and an increase in XY neurons due to GSK-J4 treatment and irrespective of gonadal type (Fig.…”

“…Accordingly, calculation of the BI estimated a higher complexity in the neuritic arborization of female (BI = 14.91 ± 0.85) than male (BI = 11.26 ± 1.25) neurons, without treatment effect (ANOVA: F(1, 12) = 6.067, p = 0.03). Regarding axonal length analysis, in agreement with results previously reported by our laboratory [34, 35], a clear sexual dimorphism was observed in control conditions, showing female neurons significantly longer axons on average than male neurons. Remarkably, GSK-J4 treatment nullified this difference, decreasing axonal length only in female derived neurons (Fig.…”

“…Similar conditions with female neurons showing greater neuritic development compared to male neurons were observed from 1 to 5 DIV, but no longer at 6 and 7 DIV [34]. Interestingly, these sexual differences disappear under E2 stimulation, as only XY neurons respond to the estrogen by accelerating their development, elongating their axons in a clear axogenic effect, and matching XX neurons, suggesting that a similar effect could occur in vivo with the arrival of early gonadal secretions and their organizing actions on brain during perinatal male development [34, 35, 78, 79]. Reisert et al .…”

“…On the other hand, XY neurons exhibit lower expression of neuritogenic-related escapee genes. However, these cells are influenced by E2 produced intraneuronally from testosterone secreted by testes during the critical period of perinatal development, presenting, in addition, a higher expression and enzymatic activity of aromatase [86, 87] and a higher sensitivity to E2 than XX neurons by expressing more estrogen receptors Esr1 and Esr2 [35, 88]. E2 organizational actions during the critical period in XY neurons would ensure the increase in Ngn3 expression necessary for the promotion of neuritogenesis.…”

“…Our previous results using FCG mice showed that sex chromosome complement (XY/XX) determines a sexually dimorphic expression of the proneural basic Helix-Loop-Helix transcription factor neurogenin 3 (Ngn3), with XX hypothalamic neurons presenting higher levels of Ngn3 mRNA than XY neurons, independently of gonadal sex [34]. In turn, XX neurons showed faster growth and maturation in vitro than XY neurons, a characteristic that was dependent on the higher Ngn3 expression in XX cultures [34,35]. These and other results [36][37][38][39][40][41] point to the importance of sex chromosome complement in the sexual differentiation of the brain, regulating the expression of autosomal genes involved in neurodevelopment and modulating neuronal differentiation in a sex-specific way independently of sex hormones.…”