Demonstration of receptor sites with electron microscope autoradiography —distribution of receptors for estradiol and its ligands in the mouse and rat uteri—

A large number of developed silver grains accumulated on the nuclei and some were scattered in the cell organelles of mouse endometrial, interstitial, and myometrial cells, 30 min after 3H-17-β-6,7-estradiol administration. 2 hr later, they showed distinct active reactions in their cytoplasmic fine structures, although developed silver grains were still located in their nuclei. A single fixation method with 2 to 4% osmium tetroxide seemed very effective to preserve the carrier bound estradiol. Female rats were perfused with PLP (periodate-lysine-paraformaldehyde) or PG (paraformaldehyde and glutaraldehyde) before incubation in radioactive estradiol or its ligands. The perfused uteri were cut in small pieces and incubated in 3H-estradiol or 3H-moxestrol with a 10 μCi/ml in phosphate buffer for 2 hr at 37°C. They were post-fixed with a mixture of glutaraldehyde and osmium tetroxide for 2 hr, then embedded in Epon after dehydration. In both cases, the developed silver grains were homogenously on the cell surfaces, in the cytoplasm, and on the nuclear membranes of endometrial, interstitial, and myometrial cells. No nuclear accumulation was observed in those experiments. However, a very strong accumulation of labeled compounds was observed in the granules of eosinophilic leucocytes and in the lysosomal granules of tissue macrophages. These data suggest that the receptors for the estradiol or ligands in the endometrial cells, especially in the nuclei, cannot accept directly without carrier proteins, except in the eosinophilic leucocyte or macrophages. The results may support facts obtained by many biochemical studies. Furthermore, it is possible that the primary receptors for estradiol or its ligands are distributed in the cytoplasm and on the membrane surface of cells, with only a small number in the nuclei. In this case, secondary receptors should exist in the nuclei, which only accept the carrier-bound estradiol or its ligands.