Changes in estrogen receptor-alpha mRNA in the mouse cortex during development

Abstract

Estrogen plays a critical role in brain development and is responsible for generating sex differences in cognition and emotion. Studies in rodent models have shown high levels of estrogen binding in non-reproductive areas of the brain during development, including the cortex and hippocampus, yet binding is diminished in the same areas of the adult brain. These binding studies demonstrated that estrogen receptors decline in the cortex during development but did not identify which of the two estrogen receptors was present. In the current study, we examined the expression of estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) in the mouse cortex during the first month of life. Messenger RNA was isolated from cortical tissue taken from C57BL/6 mice on postnatal day (PND) 1, 4, 10, 18 and 25 and expression levels were determined by real-time PCR. ERalpha mRNA expression in the mouse cortex at PND 25 was significantly reduced as compared to PND 1 (p<0.01). ERbeta mRNA expression at PND 25 was significantly increased as compared to PND 1 (p<0.05). Although the increase in ERbeta mRNA was statistically significant, the ERbeta levels were extremely low in the isocortex compared to ERalpha mRNA levels, suggesting that ERalpha may play a more critical role in the developmental decrease of estradiol binding than ERbeta. Additionally, we measured ERalpha mRNA expression in organotypic explant cultures of cortex taken from PND 3 mice. Explants were maintained in vitro for 3 weeks. mRNA was isolated at several time points and ERalpha and ERbeta mRNA was measured by real-time RT-PCR. ERalpha and ERbeta mRNA levels reflected a similar pattern in vitro and in vivo, suggesting that signals outside the cortex are not needed for this developmental change. This study lays the groundwork for an understanding of the mechanisms of the developmental regulation of ERalpha mRNA.

Figure 1

ERα mRNA expression in mouse cortex decreases during development in both male and female mice. Quantitative real-time PCR was performed on RNA isolated from PND1, PND4, PND10, PND18 and PND25 mouse cortex. Data was normalized to the housekeeping gene Histone 3.1 and expressed relative to PND1. Bars represent the mean +/- SEM, n=4. * = significantly different from PND1.

Figure 2

ERβ mRNA expression in the mouse cortex increases during postnatal development in both male and female mice. Quantitative real-time PCR was performed on RNA isolated from PND1, PND4, PND10, PND18 and PND25 mouse cortex. Data was normalized to the housekeeping gene Histone 3.1 and expressed relative to PND1. Bars represent the mean +/- SEM, n=4. * = significantly different from PND1.

Figure 3

ERα and ERβ mRNA expression in vitro follows in vivo trends across developmental time points. Quantitative Real-time PCR utilizing ERα specific primers was performed on RNA isolated from explants from PND3 males and females kept in culture for varying lengths of time (A). Data was normalized to the housekeeping gene Histone 3.1 and expressed relative to PND1. (B) Quantitative Real-time PCR utilizing ERβ specific primers was performed on RNA isolated from explants from PND3 males and females kept in culture for varying lengths of time. Data was normalized to the housekeeping gene Histone 3.1 and expressed relative to PND1. Three explants were pooled at each time point. Bars represent the mean +/- SEM, n=3. * = significantly different from PND4.

Figure 3

ERα and ERβ mRNA expression in vitro follows in vivo trends across developmental time points. Quantitative Real-time PCR utilizing ERα specific primers was performed on RNA isolated from explants from PND3 males and females kept in culture for varying lengths of time (A). Data was normalized to the housekeeping gene Histone 3.1 and expressed relative to PND1. (B) Quantitative Real-time PCR utilizing ERβ specific primers was performed on RNA isolated from explants from PND3 males and females kept in culture for varying lengths of time. Data was normalized to the housekeeping gene Histone 3.1 and expressed relative to PND1. Three explants were pooled at each time point. Bars represent the mean +/- SEM, n=3. * = significantly different from PND4.

Figure 4

Control gene expression in explant cultures. (A) GFAP mRNA expression remains consistent across age of organotypic explant cultures. Quantitave real-time PCR was performed on the RNA isolated from the organotypic explant cultures of both males and females using mouse GFAP specific primers. Bars represent the mean +/- SEM, n=3. (B) Neuronal content during the experiment was assessed by quantitative real-time PCR of MtAP-2 mRNA levels. Data was normalized to the housekeeping gene Histone 3.1 Bars represent the mean +/- SEM, n=3. (C) Cell death in the cultures was monitored by propidium iodide uptake, which is indicative of cell death. The number of dead cells per six random fields (200X) were counted and averaged. Bars represent the mean +/- SEM, n=3.

Figure 4

Control gene expression in explant cultures. (A) GFAP mRNA expression remains consistent across age of organotypic explant cultures. Quantitave real-time PCR was performed on the RNA isolated from the organotypic explant cultures of both males and females using mouse GFAP specific primers. Bars represent the mean +/- SEM, n=3. (B) Neuronal content during the experiment was assessed by quantitative real-time PCR of MtAP-2 mRNA levels. Data was normalized to the housekeeping gene Histone 3.1 Bars represent the mean +/- SEM, n=3. (C) Cell death in the cultures was monitored by propidium iodide uptake, which is indicative of cell death. The number of dead cells per six random fields (200X) were counted and averaged. Bars represent the mean +/- SEM, n=3.

Figure 4

Control gene expression in explant cultures. (A) GFAP mRNA expression remains consistent across age of organotypic explant cultures. Quantitave real-time PCR was performed on the RNA isolated from the organotypic explant cultures of both males and females using mouse GFAP specific primers. Bars represent the mean +/- SEM, n=3. (B) Neuronal content during the experiment was assessed by quantitative real-time PCR of MtAP-2 mRNA levels. Data was normalized to the housekeeping gene Histone 3.1 Bars represent the mean +/- SEM, n=3. (C) Cell death in the cultures was monitored by propidium iodide uptake, which is indicative of cell death. The number of dead cells per six random fields (200X) were counted and averaged. Bars represent the mean +/- SEM, n=3.

Figure 5

ERα protein expression in the cortex decreases during development. ERα specific immunohistochemistry was performed on slices from PND7 (A,C) and Adult (B,D) animals. A representative micrograph of cortex (A, B) and medial preoptic area (C, D) is shown (magnification = 100X). Dark spots represent immunopositive nuclei.