Regulation of oestrogen receptor gene expression: new insights and novel mechanisms.

17Beta-oestradiol is a pleiotropic hormone with organisational, activational and protective actions in both male and female mammals. It is responsible for numerous aspects of brain development, including sexual differentiation of the brain. The organisational effects of early oestrogen exposure are essential for long-lasting cognitive and behavioural functions. Oestradiol mediates its effects through several intracellular or membrane-associated receptor proteins. In the rodent cerebral cortex, oestrogen receptor (ER) expression, as demonstrated by hormone binding studies, is high early in postnatal life and declines precipitously as the animal approaches puberty. This decline is caused by a decreased expression of ERalpha mRNA. An understanding of the mechanisms involved in the regulation of ERalpha gene expression is critical for understanding the developmental as well as postpubertal expression of the ER. Despite recent data indicating the current hormone replacement therapies can be detrimental in older women, numerous animal studies have shown that the endogenous oestrogen, 17beta-oestradiol, is neuroprotective. Specifically, low levels of oestradiol protect the cortex from cell death caused by middle cerebral artery occlusion (MCAO). The attenuation of cell death by oestradiol in this model is mediated through an ERalpha-dependent mechanism. To this end, ERalpha expression is rapidly increased after MCAO, suggesting a return to the developmental programme of gene expression within neurones. One mechanism of suppressing gene expression is by the epigenetic modification of the promoter regions, which results in gene silencing. Of the epigenetic modifications utilised by cells, DNA methylation has been intensively studied as a mechanism by which genes are both permanently and reversibly silenced. Little is known about the mechanisms of ER gene regulation in the brain; however, in breast cancer cells, both ERalpha and ERbeta are down-regulated by promoter methylation, and subsequent binding of the methyl-CpG-binding protein, MeCP2. Data from our laboratory demonstrate that the promoters of the ERalpha gene are also methylated during development and after neuronal injury, suggesting a role of DNA methylation in regulating ER expression in the brain.

Epigenetic regulation of the estrogen receptor alpha promoter in the cerebral cortex following ischemia in male and female rats.

Permanent middle cerebral artery occlusion (MCAO) causes neuronal cell death in the striatum and cortex. In rodents, estradiol treatment protects the cortex from cell death in an estrogen receptor alpha (ERalpha) dependent manner. ERalpha is only transiently expressed in the cortex during neonatal development and is very low in uninjured adult cortex. Following MCAO, ERalpha mRNA expression is upregulated in the cortex of female rats, but the mechanism of this increase is still unknown. It is also unknown whether a similar increase in ERalpha expression in seen in males. In the following studies, male and vehicle or estradiol-treated ovariectomized (OVX) female rats underwent MCAO to investigate the regulation of ERalpha expression after ischemia. Twenty-four hours after surgery, mRNA or genomic DNA was collected from 1 mm micropunches taken from 300 mum brain sections for quantitative reverse transcription-polymerase chain reaction (RT-PCR) or methylation-specific (MSP) PCR, respectively. Additionally, adjacent 20 mum sections were processed for ERalpha immunohistochemistry. In OVX females, ERalpha mRNA and protein were increased in the ischemic cortex, but unchanged in males. We hypothesized that this increase in ERalpha in females is due to a reversal of gene silencing by DNA methylation. Using MSP targeting of CpG islands within the 5' untranslated region (UTR) of the rat ERalpha gene, we found that ischemia decreased methylation in the ischemic cortex of both groups of females, but there was no change in methylation in males. Using chromatin immunoprecipitation, we found that MeCP2 associates with ERalpha 5'UTR corresponding with the methylation status of the promoter. These data are the first to demonstrate a difference in the regulation of ERalpha expression in response to MCAO between males and females and that methylation of the ERalpha gene corresponds with mRNA levels in the brain.

Ultrasound-guided follicular aspiration in squirrel monkeys.

The objective of this study was to test whether ultrasound-guided oocyte retrieval is an effective mechanism for collecting oocytes in squirrel monkeys. Although ultrasound-guided follicular aspiration has been described in Old World primates, oocyte retrieval in New World primates is typically performed via laparoscopy or laparotomy. However, these procedures, especially the first, can be invasive. Ultrasound has been used for pregnancy monitoring in multiple species of primates including Saimiri spp. Transabdominal ultrasound as a diagnostic tool is non-invasive. Transabdominal ultrasound was utilized to visualize ovarian follicles during aspiration under light anesthesia. This procedure resulted in collection of a total of 29 oocytes from six animals with minimal post-procedural pain. Manipulated animals were returned to the social group the same day.

Glucocorticoid resistance in squirrel monkeys results from a combination of a transcriptionally incompetent glucocorticoid receptor and overexpression of the glucocorticoid receptor co-chaperone FKBP51.

Squirrel monkeys have high cortisol compared to Old World primates to compensate for glucocorticoid resistance. Glucocorticoid resistance in squirrel monkeys may result from mutations in the glucocorticoid receptor (GR) that render it less transcriptionally competent, or expression of the co-chaperone FKBP51 that reduces ligand binding. The goal of this study was to reconcile the contribution of each mechanism. Responsiveness of squirrel monkey GR in COS-7 cells was reduced compared to human GR, but induction of GR activity by maximum dexamethasone concentrations was similar. Also, expression of squirrel monkey FKBP51 reduced responsiveness of both squirrel monkey and human GR in T-REx-293 cells. The EC(50) for dexamethasone was 100-fold higher in cells expressing squirrel monkey GR and excess FKBP51 compared to cells expressing only human GR. Effects of FKBP51 expression and treatment with FK506 were also determined in squirrel monkey SQMK-FP cells that naturally express high levels of FKBP51. Overexpression of FKBP51 in SQMK-FP cells had little effect on GR responsiveness, but treatment with FK506 that blocks the effect of FKBP51 increased GR responsiveness. Thus, glucocorticoid resistance in squirrel monkey cells results from both expression of GRs that are less responsive and overexpression of FKBP51 that further reduces GR responsiveness.