Faculty of Science School of Anatomical Sciences 9901061h karrenthomson@yahoo.co.uk / Clomiphene citrate (CC), a synthetic estrogen, is an efficient superovulator used in infertility treatment. However pregnancy rates resulting from CC treatment are low. Research has suggested that this may be due to an aberrant effect on implantation; CC binds to estrogen receptors (ER) and may affect estrogen responsive gene expression and thus implantation. This study investigates the effect of CC on ERa, 90kDa heat shock protein (Hsp90) and Hoxa10 expression in the rat uterus. Hsp90 binds to ERa in the absence of ligand and is involved in inducing a high affinity ligand binding conformation in the ER and in transactivation of the ER. Hoxa10 has been shown to be essential for uterine receptivity to implantation. CC (0.25mg) was given to ovariectomized rats, either alone or prior to a hormonal regime known to induce uterine receptivity for implantation. Expression of ERa, Hsp90 and Hoxa10 was determined by Western blotting, fluorescence immunocytochemistry and reverse transcription polymerase chain reaction. The single dose CC treated rats were compared to the controls as well as to ovariectomized rats treated with 0.5mg 17b estradiol (E2). The CC treated pseudopregnant rats (CCPPPE treated) were compared to 5½ day pregnant and pseudopregnant rats without CC (PPPE treated), to determine CCs effect at implantation. E2 upregulated ERa and Hsp90 expression in the rat uterus compared to controls (p<0.05). The finding for ERa was unexpected as other studies have shown that E2 decreases ERa levels a few hours after administration in the uterus. The present study therefore suggests a biphasic effect of E2 on ERa expression in the rat uterus. The effect of E2 on Hsp90 and ERa also proposes a balance between the levels of these two proteins in the uterus, to keep ERa in its optimal state and suggests that too high and too low a concentration of Hsp90 may both be inhibitory to ERa functioning. No significant difference was found in ERa and Hsp90 expression between the non-receptive (vehicle treated) and the receptive (PPPE treated) rat uteri, suggesting that these two genes are not markers for receptivity. However E2 is known to induce implantation of donor blastocysts in progesterone (P4) primed uteri. Therefore it is still essential for ERa to be present at implantation. It is of interest that CC downregulated ERa levels both in ii the absence of ovarian hormones and at implantation in the rat uterus. It is therefore proposed that this antiestrogenic effect would render the uterus less sensitive to the E2 required to induce implantation, thus accounting for low pregnancy rates with CC use. Although CC did not alter the expression of Hsp90 in this study, the reduction in ERa levels in response to CC may also upset the balance in the expression of these two genes, which may affect the transcriptional activity of ERa, and further prevent implantation. No clear results were obtained for Hoxa10 expression with the Western blots. However based on the ICC results, CC did not appear to affect Hoxa10 expression. Since P4 and not E2 is known to have the predominant effect on Hoxa10 expression, it is likely that E2 analogs, such as CC, would also not affect Hoxa10 expression to a significant degree. Future work will aim to separate the different uterine compartments and to determine the effects of CC on the expression of other implantation specific genes in the uterus.