Sex-Specific Aromatization of Testosterone in Mouse Hypothalamic Neurons

Abstract

Conversion of androgens to oestrogens by neural aromatase during brain development appears to be a prerequisite for sexual differentiation of the mammalian central nervous system. In order to investigate the pre- and perinatal patterns of testosterone (T) aromatization in the male and female mouse brain, aromatase activity (AA) was measured in hypothalamic and cerebral homogenates of embryonic day (ED) 17 fetuses and neonates using an in vitro ³H₂O product formation microassay. In addition, AA was examined in gender-specific neuronal cell cultures prepared from ED 15 mouse cerebral hemisphere and hypthalamus at 3 and 6 days in vitro (DIV), and this was compared with enzyme activities in homogenates. The aromatase has also been evaluated in glial-enriched cultures from ED 20 mouse hypothalamus and cortex as well as in ED 15 cultures treated with the neurotoxin kainic acid in order to localize AA to neurons and/or glial cells. Significant sex differencees in AA were observed in hypothalamic tissue homogenates as early as ED 17, becoming even more distinct in neonates, AA being always higher in males compared to females. Similar AA was also found in cells from both sexes from cultured ED 15 hypothalamus after 3 DIV. However, significantly higher AA was observed after 6 DIV in ED 15 male hypothalamic cultures compared to female. ED 20 glial-enriched hypothalamic culutures (purity > 95%) from both brain regions exhibited very low AA after 6 DIV, and no sex differences were found. After treatment of ED 15 hypothalamic cultures with kainic acid, a 70–80% decrease in AA was observed in both sexes compared to non-treated cultures and sex differences were no longer present. Kinetic studies performed on aromatase in male and female hypothalamic cells from ED 15 cultures after 6 DIV, from ED 17 fetuses, and from neonatal homogenates with T as substrate indicate similar T binding affinities in cultures as in embryonic and neonatal homogenates from both genders, with an apparent K_m of ∼ 40 nM. In cortex, from either ED 15 cultures, ED 20 cultures, ED 17 fetuses or neonates, AA was low and sex differences were not observed. Taken together, our results demonstrate: (1) sex differences in hypothalamic AA during pre- and perinatal development in the mouse with a higher capacity for oestrogen formation in male than in female; (2) no sex differences in AA from cortical tissues; (3) no sexual or developmental differences in hypothalamic aromatase T binding affinity; (4) aromatase is neuronal rather than astroglial, and (5) sex differences in AA are consequentially restricted to neurons.

Developmental Sex Differences in Estrogen Receptor-β mRNA Expression in the Mouse Hypothalamus/Preoptic Region

“…Specifically, estrogens are implicated in the development of structural and functional sex differences in the Hyp/POA. It is important to note that estrogens are actively synthesized in the brain by the enzyme aromatase which is perinatally expressed in hypothalamic neurons, and that higher aromatase expression and estrogen synthesis rates are found in the male Hyp/POA [3, 17, 18, 19]. This led to the concept of estrogenic masculinization of the mammalian brain [1, 2].…”

“…In the CNS, the classical ER-α is found in different brain regions including the Hyp/POA in adults and during development [6, 7, 20, 21]. Although estrogen synthesis is several times higher in the male hypothalamus compared to the female around birth [3, 17], inverse sex differences in the expression of the ER-α have been reported in the Hyp/POA with higher levels in females [7]. Moreover, it was suggested that estrogens downregulate the expression of its ER-α in the male Hyp/POA [7].…”

“…Moreover, this is the first description of sex differences in ER-β mRNA expression in the rodent Hyp/POA during the critical period of brain ontogeny when the structural and functional sexual dimorphic organization of the brain is brought about by estrogens [1, 2]. Thus, higher perinatal ER-β expression levels coincide with higher estrogen synthesis rates [3]and aromatase expression [17]in the male Hyp/POA. Sexual dimorphic expression of ER-β as shown in this study was limited to the late prenatal period until 2 weeks postnatally but was not found in adults, thereby confirming a previous report about the absence of ER-β sex differences in the adult Hyp/POA [10].…”

“…In particular, estrogens synthesized perinatally by the neuronal enzyme aromatase are required for the sexual differentiation of brain structures and functions and the permanent masculinization of the male central nervous system [2]. This is supported by observations which show higher estrogen-forming rates in the male hypothalamus/preoptic region (Hyp/POA) around birth [3]. Despite data which suggest the existence of membrane binding sites for estrogens [4]and rapid membrane-mediated estrogen effects on neurons [5], estrogen action in the brain and other tissues is primarily transmitted through ligand-dependent nuclear estrogen receptors (ER).…”

The ram as a model for behavioral neuroendocrinology

“…Further support for the idea that male-oriented behavior in rams may result, in part, from variations in brain differentiation was the observation by Resko et al (Resko et al, 1996) that aromatase activity is significantly lower in the medial preoptic area of maleoriented than of female-oriented rams. Previous studies in rats demonstrated that the capacity for aromatization in the MPOA is a sexually differentiated characteristic of the brain that is organized by perinatal exposure to gonadal testosterone (Beyer et al, 1993;.…”