β-Estradiol-6-one 6-(O-carboxymethyloxime)

Specific plasma membrane binding sites for 17 beta-estradiol were shown to exist on avian osteoclasts by the use of a fluorescent conjugated estrogen, 17 beta-estradiol-6-carboxymethyloxime- bovine serum albumin-fluorescein isothiocyanate. Binding was dose and time dependent, as well as specific, since 17 beta-estradiol blocked the binding of the complex. Tamoxifen was also an effective blocking agent. Rapid responses to 17 beta-estradiol included decreased acid production (41.5%) and distinct cell shape changes.

With greater utilization of histochemical methods for detecting estrogen binding (ER) in tumor cells, there is an increasing need to quantitate objectively these fluorescently stained cells. This study utilizes flow cytometry (FCM) to examine the binding specificity and kinetics of 17 beta-estradiol-6-CMO-BSA-FITC(E-BSA-FITC) in two human mammary carcinoma cell lines, MCF-7 and 47-DN. Cells are rendered permeable to this ligand by freeze-thawing, a process analogous to the routine staining of frozen tumor sections with E-BSA-FITC for the clinical detection of ER. FCM quantification of E-BSA-FITC binding intensity demonstrates a saturable dose-response that is specifically reduced in the presence of diethylstilbestrol (DES) in doses known to saturate Type I ER. Scatchard analysis suggests that E-BSA-FITC binding occurs with receptors of varying affinities (Kd). Lineweaver-Burk plots show that the DES inhibition is competitive for a high-affinity receptor binding to E-BSA-FITC with a Kd of approximately 50 nM. This report also compares both FCM and biochemical methods of quantitating ER under two conditions of tumor cell growth potentially encountered in clinical specimens: quiescent versus actively proliferating cells, and cells pretreated with the antiestrogen tamoxifen. By FCM analysis, the cells with greater proliferating activity contain tenfold more specifically bound E-BSA-FITC, and tamoxifen pretreatment reduces this specific binding by 50%. These FCM measurements correlate well with biochemical results and suggest that this new methodology may supplement the detection of E-BSA-FITC binding by fluorescence microscopy.

The mechanism of progesterone action within the ovarian follicle was investigated in Rana dybowskii, by using immobilized progesterone. Fluorescein isothiocyanate-labeled progesterone 3-O-carboxymethyloxime-BSA (P-BSA) was localized on the outside surface of the denuded oocyte, which indicated that P-BSA did not cross the barrier of cell surface. Progesterone-BSA induced germinal vesicle breakdown (GVBD) of denuded oocytes in a dose-dependent manner but failed to induce GVBD of follicle wall-enclosed oocytes. The time course of P-BSA-induced GVBD in denuded oocytes was similar to that observed with progesterone. Furthermore, both P-BSA and progesterone induced oocyte maturation in the presence of RU486, a well-known nuclear progesterone receptor antagonist. Treatment of denuded oocytes with P-BSA resulted in a threefold increase in inositol triphosphate (IP3) and a fourfold increase in diacylglycerol levels within 10 min. Additionally protein kinase C (PKC) activity was markedly increased by 30 min of incubation following exposure to P-BSA. Such changes were not observed in denuded oocytes exposed to beta-estradiol-6-O-carboxymethyloxime-BSA, which failed to induce GVBD. These results suggest that progesterone acts initially at the oocyte surface where it triggers generation of membrane-mediated second messengers during oocyte maturation in amphibians.