It is well established in mammals that chronic, long-term elevations in sex steroids are associated with improvements in spatial cognition. It is less clear the extent to which short to medium term elevations in sex steroids improve spatial cognition and change hippocampal morphology, particularly in birds. The avian hippocampus expresses both androgen receptors (AR) and oestrogen receptor alpha (ERα) and high levels of the enzyme aromatase that converts testosterone to oestrogen. I began by comparing spatial cognition, hippocampal sex steroid receptor and aromatase expression between males and females. There were no differences in spatial or visual cognition or in hippocampal sex steroid receptor expression between the sexes, although hippocampal aromatase mRNA expression was higher in males. I then addressed the effects of acute and medium-term sex steroid treatment on spatial cognition and hippocampal aromatase and sex steroid receptor expression. A single treatment of testosterone 30 minutes or four hours prior to cognitive testing improved spatial performance. Additionally, when testosterone and oestrogen were given daily for five days spatial cognition in both sexes was improved. The testosterone-induced improvement was blocked when testosterone was administered in conjunction with the aromatase inhibitor fadrozole but not when administered with saline. These findings suggest that spatial cognition is improved by an oestrogenic effect. Thirty minutes following acute testosterone treatment, plasma testosterone levels, hippocampal AR and ERα mRNA expression all increased. Five days of oestrogen treatment increased plasma oestrogen levels, hippocampal ERα mRNA and Nmethyl- D-aspartate (NMDA) receptor levels in males and females; all were positively correlated with enhanced spatial cognition on day five of treatment. Finally, I determined which genes were differentially expressed as a result of five days of oestrogen treatment. Nineteen genes, identified as being involved in learning and memory were differentially expressed in the hippocampus, eleven of which were up-regulated and eight were down-regulated. Taken together these results demonstrate that oestrogen can improve spatial cognition in birds. It is plausible that oestrogen acts to improve spatial memory in the hippocampus through upregulation of genes that control neurotransmitter release, reuptake and receptor levels.

Xenoestrogens are compounds, foreign from the body, that can enter cells and interact with the estrogen receptors (ER) to produce an estrogenic response. Many additives used in plastics are compounds with estrogenic activity. Some of these additives are known to slowly leach from the plastics. When using plastic containers as lunchboxes for reheating or food storage, these additives can leach from the plastics and end up in the food. In this project, food simulates were cooked in six different thermoplastic containers, made of polypropylene, in an oven at 100 °C for 15 minutes. Three of the thermoplastic containers were lunchboxes marketed to be able to withstand cooking in a microwave. The other three were provisional lunchboxes made from various food storing containers originally made for refrigeration purposes. The estrogenic activity in the different samples was measured using an ER-CALUX in vitro assay. The results were measured in 17β-estradiol equivalent (Bio-EEQ) values in pg/ml. The purpose of this project was to investigate whether or not these plastic containers leach xenoestrogens that can be measured with an ER-CALUX assay, and compare the results with the results from other existing toxicological studies, and also to see if there is a difference in Bio-EEQ levels between the plastic containers made for microwave usage and those made for refrigerated purposes. The results from this project indicate that most of these plastic containers do leach estrogenic compounds that can be detected in the ER-CALUX, even the ones made for microwave usage. Fortunately, compared to other toxicological studies, the Bio-EEQ levels in these food samples cooked in plastic containers are low. However the potential adverse effects in prenatally exposed children cannot be ignored as other studies have shown that very low levels of xenoestrogens are enough to potentially cause a disturbance in the reproductive development and fertility.

No / Reactive oxygen species (ROS) are produced by a wide variety of chemicals and physiological processes in which enzymes catalyse the transfer of electrons from a substrate to molecular oxygen. The immediate products of such reactions, superoxide anion radicals and hydrogen peroxide can be metabolised by enzymes such as superoxide dismutase (SOD) and catalase (CAT), respectively, and depending on its concentration by Vitamin C (Vit C). Under certain circumstances the ROS form highly reactive hydroxyl radicals. We examined human sperm and lymphocytes after treatment with six oestrogenic compounds in the Comet assay, which measures DNA damage, and observed that all caused damage in both cell types. The damage was diminished in nearly all cases by catalase, and in some instances by SOD and Vit C. This response pattern was also seen with hydrogen peroxide. This similarity suggests that the oestrogen-mediated effects could be acting via the production of hydrogen peroxide since catalase always markedly reduced the response. The variable responses with SOD indicate a lesser involvement of superoxide anion radicals due to SOD-mediated conversion of superoxide to hydrogen peroxide generally causing a lower level of DNA damage than other ROS. The variable Vit C responses are explained by a reduction of hydrogen peroxide at low Vit C concentrations and a pro-oxidant activity at higher concentrations. Together these data provide evidence that inappropriate exposure to oestrogenic compounds could lead to free-radical mediated damage. It is believed that the observed activities were not generated by cell free cell culture conditions because increased responses were observed over and above control values when the compounds were added, and also increasing dose¿response relationships have been found after treatment with such oestrogenic compounds in previously reported studies.

A plethora of naturally-produced steroid hormones, or artificial homologues of them, are being introduced into the aquatic and terrestrial environments each year. Two examples of these are the natural oestrogen 17-oestradiol (E2) and the oestrogen receptor antagonist, Bisphenol A (BPA), both of which target the ribonucleoprotein telomerase through upregulation of its telomerase reverse transcriptase component, TERT. The main objectives of this study were firstly to isolate and characterize the actual mRNA sequence for the telomerase catalytic subuninit, Tert, in rainbow trout (Oncorhynchus mykiss) (Walbaum, 1792) and European purple sea urchin (Paracentrotus lividus) (Lamarck, 1816), with the aim of developing qPCR assays for the amplification and quantification of Tert. Further objectives were to use these assays in controlled exposure studies to establish whether and to what extent the aforementioned chemicals regulate Tert transcription and by doing so further understand the mechanism of Telomerase gene expression and the extent to which environmental oestrogen can interfere. The initial step of sequence characterization and assay devlopment was successful in the case of rainbow trout where two possible splice variants of Tert mRNA are identified, omTertShort and omTertLong. Two qPCR assays were developed for the relative quantification of both of these splice variants in rainbow trout samples, the latter of these successfully amplifying its target in test samples. In order to demonstrate in vitro and in vivo modulation of telomerase activity and mRNA expression, early life-stages of rainbow trout and purple sea urchin, as well as rainbow trout hepatocytes, were exposed to a range of concentrations of E2 and BPA. Purple sea urchin embryos were exposed to 200, 20 and 2 ng E2/ml for 28 hours until they had reached the stage of pluteus larvaes. Rainbow trout embryos were exposed to 500, 20 and 0.1 ng E2/ml and 600 and 150 ng BPA/ml for 167 days from immediately after fertilization. Rainbow trout hepatocytes were exposed to 20 and 2 ng E2/ml for 48 hours. The results from this study show that telomerase activity as well as TERT mRNA expression can be significantly modulated by exposure to oestrogens and other oestrogenic chemicals. E2 concentrations as low as 20 ng/ml lead to an increase in telomerase activity early-life stages of purple sea urchin and upregulation in the transcription of Tert mRNA in unhatched rainbow trout embryos. BPA induced similar response (600 ng/ml) in hatched rainbow trout alevins larvae. Very high exposures to E2 (500 ng/ml) do however lead to downregulation of Tert mRNA in hatched alevins larvae. Differential regulatory response can be observed between different tissue types of 167 day old fry, with an upregulatory response observed at 0.1 ng E2/ml in liver and muscle tissues, but not in brain. Similarly, brain tissues were observed expressing significantly less mRNA than liver and muscle samples when exposed to BPA (150 ng/ml). It is evident that the previously observed link between environmental oestrogens and telomerase is also present in the two test species examined; purple sea urchin and rainbow trout.