Estrogen Localization and Local Synthesis of Estrogen by Cataractous LEC

Abstract

Abstract: : Purpose: Our laboratory has identified overexpression of estrogen receptor alpha (ERa) in cataractous LEC. We know that ERa can function in both an estrogen–dependent and –independent manner. Furthermore, sulfatase and aromatase enzyme activity are necessary for the synthesis of active estrogen (17 beta estradiol). We hypothesized that in order for ERa to function in an estrogen–dependent manner in cataractous LEC, estrogen would have to be not only present in the LEC or aqueous humor, but there had to be local production of 17 beta estradiol within the anterior segment of the eye. Methods: Samples included normal whole lenses collected from dogs euthanized at a local animal shelter for overpopulation reasons, anterior capsulotomy samples from dogs with naturally occurring cataracts, and aqueous humor collected from both populations prior to dissection or surgery, respectively. Immunohistochemical staining was performed using antibodies against 17 beta estradiol and sulfatase. In addition, real time RT–PCR was used to measure aromatase transcription, an accepted method to evaluate the final step in the synthesis of 17–beta estradiol. Results: Immunohistochemistry found that cataractous LEC had more intense immunostaining for 17 beta estradiol and sulfatase in the cytoplasm and nucleus than normal LEC, which had primarily cytoplasmic immunolocalization of both proteins. RT–PCR found significantly up–regulated aromatase transcription in cataractous LEC and only negligible levels in normal LEC. Conclusions: The synthesis of 17 beta estradiol requires the activity of at least one of 2 enzymes, sulfatase and aromatase. This is the first evidence of 17 beta estradiol production by cataractous LEC supported by the positive immunostaining for 17–beta estradiol and sulfatase, and up–regulated transcription of aromatase by RT–PCR. These findings, combined with overexpression of ERa in cataractous LEC, suggest that LEC may become estrogen–responsive during or due to cataractogenesis. Perhaps, the presence of estrogen in normal LEC, without local its production or ERa overexpression, is a protective mechanism against cataractogenesis, but the up–regulation of ERa and local production of estrogen result in cataractogenesis.

Keywords: cataract • immunohistochemistry • transcription