Phyto- and endogenous estrogens differently activate intracellular calcium ion mobilization in bovine endometrial cells

The main purpose of this study was to check whether phyto- and endogenous estrogens influence calcium ion mobilization [Ca²⁺]_i in bovine endometrial cells and whether this action is connected with biological effects i.e. prostaglandin (PG)F_2α production. In our study we used two calcium measurement methods by comparing the microscopic method with widely used quantitative - spectrofluorometric method of [Ca²⁺] _i measurement. We also wanted to confirm whether visualization of calcium ion [Ca²⁺]_i in cells using microscopic method supported by micro image analysis (Micro Image Olympus system) reflects real, qualitative changes in the ion concentration. In both methods a cell-permeable form of fluorescent [Ca²⁺]_i indicator Fura-2 was used. Cultured bovine endometrial epithelial and stromal cells influenced by phorbol-2-myristate-13-acetate (PMA; positive control), estradiol 17-β (E₂; endogenous estrogen) and active metabolites of phytoestrogens (environmental estrogens) were used as a model to study PGF_2α secretion and [Ca²⁺]_i mobilization in the cells. Equol and para-ethyl-phenol in doses of 10-8-10-6 M increased PGF_2α concentration both in epithelial and stromal cells (P<0.05). In both methods, equol and para-ethyl-phenol did not cause intracellular [Ca²⁺] _i mobilization in epithelial and stromal cells (P>0.05). Both methods revealed that only E₂ and PMA induced intracellular [Ca ²⁺]_i mobilization in epithelial and stromal cells (P<0.05). The results of both methods were highly correlated (P<0.001; r=0.82 for epithelial cells and r=0.89 for stromal cells). In conclusion, both methods gave approximately the same results and showed that phytoestrogens, in contrast to PMA and E₂, did not cause intracellular [Ca ²⁺]_i mobilization in endometrial cells. The obtained results proved that the [Ca²⁺]_i visualization method supported by micro image analysis can produce similar results to the spectrofluorometric method.