Research reportEstrogen receptor β (ERβ) protein levels in neurons depend on estrogen receptor α (ERα) gene expression and on its ligand in a brain region-specific manner
Introduction
It has been well established that testosterone plays a major role in the expression of male typical sexual and aggressive behaviors. Testosterone exerts its function in the central nervous system (CNS) not only by acting through androgen receptors (AR), in its original form or as the 5α-reduced metabolite, dihydrotestosterone, but also by acting through estrogen receptors (ER), after being aromatized to estradiol (E2). Two subtypes of ERs, ERα and ERβ, which bind to E2 with a similar affinity, are identified in the CNS [13], [14], [37], [38]. ERα and β are very similar estrogen-binding proteins, perhaps gene duplication products, which can act as ligand-dependent transcription factors. Several lines of evidence have demonstrated that ER-dependent mechanisms as well as AR-dependent mechanisms may be involved in the regulation of reproductive and aggressive behaviors in male mice [23], [28], [34], [35]. It has not been studied until recently, however, how two types of ERs are involved in these behavioral regulations.
Behavioral studies using single (αERKO and βERKO) or double knockout (αβERKO) mice of ER genes suggest that both types of ERs are indeed involved in the regulation of a number of behaviors in male mice [17], [18], [19], [20], [21], [22]. These studies also showed that the lack of ERα and/or ERβ had differential effects on various behaviors. For instance, male sexual behavior was reduced in αERKO mice [18], [19], but not in βERKO mice [20], while it was completely abolished in αβERKO mice [21]. Male aggression, on the other hand, was greatly reduced in both αERKO and αβERKO mice [18], [19], [21], but increased in βERKO mice [17], [20]. Furthermore, facilitation of running wheel activity by estrogen was completely abolished in αERKO, but not in βERKO mice [22]. These findings support the notion that the two subtypes of ERs may be either synergistically or antagonistically involved in the regulation of brain function and behavior. It is not completely understood, however, how these two receptor systems affect each other’s message and protein expression in different parts of the brain.
Gonadal steroids regulate ERα mRNA and protein in a tissue-specific manner. In general, they are up-regulated by removal of gonadal steroids (gonadectomy) and down-regulated by estrogen replacement in the brain regions such as the medial preoptic area (MPOA), the bed nucleus of the stria terminalis (BNST), the ventromedial nucleus of hypothalamus (VMH), and arcuate nucleus [6], [10], [12], [16], [24], [29], [33], [39], although a different sensitivity of ERα regulation was observed depending on the sex, species, and steroid treatment regimens (e.g. short-term vs. long-term). In the medial amygdala (MeAMY), on the other hand, gonadectomy (GDX) and estrogen treatment do not affect ERα mRNA and protein [16], [24].
Very little is known about how estrogen regulates ERβ protein in the CNS and whether ERα plays a role in the expression of ERβ protein and its regulation by estrogen. Using RNase protection assay, it was shown that there were no differences in ERβ mRNA in hypothalamic tissues between αERKO and WT mice [4]. As this assay was carried out on RNA extracted from whole hypothalamic tissues, we cannot rule out the possibility that the expression of ERβ mRNA and protein in specific brain regions is indeed affected by the lack of ERα gene. Furthermore, it is not known whether the lack of ERα in αERKO mouse brain may affect the regulation of ERβ protein levels by estrogen.
In the present study, therefore, we compared the effects of GDX and short-term or long-term treatment of estrogen on ERβ immunoreactivity (ir) in αERKO and WT male mouse brains. We examined ERβ ir in six different brain regions where previous studies have shown that ERβ mRNA and immunoreactive cells are widely distributed [1], [3], [15], [29], [30], [31], [32]. Those included the MPOA, the BNST, the paraventricular nucleus (PVN), the VMH, the MeAMY and the dorsal raphe nucleus (DRN). It should be noted that distributions of ERα and ERβ are known to overlap in the MPOA and the BNST [30] whereas those in the DRN are quite distinctive [2], [3].
Section snippets
Mice
Adult (9–14 weeks old) male αERKO mice and their WT littermates were used. They were obtained from the αERKO breeding colonies maintained at the Rockefeller University by mating heterozygous male and female mice. Original breeding pairs (mixed background of C57BL/6J and 129) were obtained from the National Institute of Environmental Health Sciences. All procedures were approved by IACUC. Mice were group-housed (4–5 mice/cage) in plastic cages (30×20×12 cm) at constant temperature (22 °C) on a
Results
In both WT and αERKO mice, ERβ ir was localized in the nucleus of the neurons in all six brain regions examined. No apparent ERβ ir was observed in the cytoplasm or processes of the neurons.
In the MPOA, there was a significant genotype difference in the number of ERβ ir expressing cells in intact mice (P<0.01; Fig. 1, Fig. 4 and Table 1). αERKO intact mice showed a significantly lower number of ERβ ir expressing cells in the MPOA, compared to WT mice. In the MPOA of WT mice, GDX increased the
Site-specific effects of gonadectomy and estrogen treatment on the levels of ERβ immunoreactivity in WT mouse brains
We found in the present study that gonadectomy and estrogen treatment, site-specifically, affected the number of ERβ ir expressing cells in WT mouse brains. In the brain regions such as the MPOA, BNST and VMH, the numbers of ERβ ir cells were increased by gonadectomy in comparison to the gonadally intact mouse levels, and decreased by estrogen treatment (Table 1). We also found that the duration of exposure to ligand made a difference in the regulation of ERβ protein in WT mice. For instance,
Acknowledgements
Support for this work was provided by NIMH 62147 to SO. MN was a recipient of postdoctoral fellowships for research abroad from the Japan Society for Promotion of Science.
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