Female sex steroids control the induction and maintenance of the differentiated state of most cells in the female reproductive tract. The morphological details of cellular differentiation are well documented particularly for epithelial cells in the uterus and oviduct, and these changes correlate well with the number of nuclear estrogen receptors (ERn) measured biochemically. I have recently shown with immunocytochemistry that, in general, this fundamental correlation of ER numbers with biological responsiveness holds true for various target tissues of the reproductive tract of hormone-treated cynomolgus macaques. However, we have observed that in tissue from many spayed animals, target cell nuclei are intensely stained for ER. Also, during early estrogen action, immunostainable ER is observed in stromal but not epithelial cells. Studies in progress indicate that the distribution of intranuclear ER is heterogeneous, and that mitotic cells do not stain positively for ER. One must conclude that acceptor sites may vary significantly during differentiation, and that epithelial and stromal components contain different arrays of ER acceptor sites. The experiments proposed in this application address these issues by asking - What is the intranuclear distribution of estrogen receptors in the target cells of reproductive tissues of macaques? To examine the subcellular localization of ER, an indirect immunocytochemical procedure employing colloidal gold, currently being developed, will be validated. In this method, tissues are fixed in picric-acid containing glutaraldehyde and embedded in Lowicryl K4M or araldite. In situ localization of ER will be conducted on specimens from spayed, hormone and antiestrogen-treated and normally cycling macaques so as to evaluate an array of differentiated states. In addition, the sensitivity of intranuclear receptor populations to sequential nuclear extraction by detergent, DNAase, varying ionic strength and RNAase will be evaluated at the light and electron microscopic level. Frozen or Lowicryl sections, isolated nuclei and etched araldite sections of uterus will be immunocytochemically stained following each step of extraction with a variety of monoclonal antiestrophilins used alone and in combination. The immunocytochemical results will be correlated with measurements of receptor numbers after in vivo labeling with 3H-R2858. These data should provide new and useful information about the pools of ER within the nucleus and help to establish methodology for detecting these pools.
| McClellan, M C; Rankin, S; West, N B et al. (1990) Estrogen receptors, progestin receptors and DNA synthesis in the macaque endometrium during the luteal-follicular transition. J Steroid Biochem Mol Biol 37:631-41 |
| Crandall, R; McClellan, M; Arch, S et al. (1987) Inverse diffusion methods for data peak separation. Anal Biochem 167:15-22 |
