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This article reviews the effects of estradiol (E(2)), progesterone (P) and P receptor antagonists (PA) on the rhesus macaque endometrium. Ovariectomized macaques can be treated with implants of estradiol (E(2)) and P to induce precisely controlled, artificial menstrual cycles. During these cycles, treatment with E(2) alone induces an artificial proliferative phase marked by extensive endometrial epithelial cell proliferation and increased expression of stromal and epithelial estrogen receptor (ER) and P receptor (PR). Androgen receptor (AR) is also upregulated by E(2) but is expressed only by the endometrial stroma. Progesterone acts on the E(2) primed endometrium to induce secretory differentiation and causes suppression of epithelial and stromal ER, epithelial PR, and stromal AR in the functionalis zone. However, epithelial ER and PR are retained in the basalis zone during the secretory phase. When potent P antagonists (PA) are administered acutely at the end of an E(2) + P induced cycle, menses typically ensues similar to P withdrawal at the end of the menstrual cycle. When PAs are administered chronically there is significant blockage of all P- dependent effects including upregulation of ER, PR and AR and suppression of glandular secretory function. However, chronic PA administration also inhibits estrogen-dependent endometrial cell proliferation and growth. This endometrial antiproliferative effect is the basis of the clinical use of PA to control various diseases such as endometriosis.

Estrogen action is dependent upon the presence of specific ligand-activated receptors in target tissues. The aim of the present experiments was to compare the spatial and temporal pattern of expression of estrogen receptor beta (ERbeta) with that of ERalpha in full thickness endometrial samples (from the superficial to the basal zone) obtained from both women and rhesus macaques. Immunohistochemical localization with specific antibodies revealed that ERalpha and ERbeta were both expressed in nuclei of the glands and stroma. Consistent with previous studies, expression of ERalpha declined in the glands and stroma of the functionalis during the secretory phase. The luminal epithelium also displayed positive immunoreactivity for ERbeta. Expression of ERbeta declined in glandular cell nuclei, but not stroma, within the functionalis during the late secretory phase. Levels of expression of ERalpha and ERbeta in all cellular compartments remained unchanged in the basalis. Both receptor subtypes were detected on Western blots using proteins extracted from uterine samples obtained throughout the menstrual cycle. There was a striking contrast between the pattern of expression of ERalpha and ERbeta in the vascular endothelium and the perivascular cells surrounding endometrial blood vessels; only ERbeta was present in the endothelial cell population, although both forms of ER were expressed in perivascular cells. We conclude that estrogen action(s) within the vascular endothelium in the endometrium may be mediated via direct binding to the ERbeta isoform and that these cells could therefore be a target for agonists or antagonists that selectively target the beta form of the ER.

We have found that microwave (MW) stabilization greatly improves detection of the estrogen receptor (ER) in frozen sections of rhesus monkey oviduct by immunocytochemistry (ICC). Fresh samples of fimbriae were MW-irradiated, frozen, and then cryosectioned. The frozen sections were also MW-treated and then fixed in a paraformaldehyde-based fixative before ICC processing. A parallel set of samples from each monkey were frozen, sectioned and processed for ICC without any MW treatment. MW stabilization clearly increased immunostaining intensity with either of two ER-specific monoclonal antibodies, namely, H222 and 1D5. The greatest increase was noted in tissues collected from spayed or progesterone-treated animals. An antibody dilution series indicated that MW stabilization increased the sensitivity approximately 20- to 40-fold. In addition, we incubated spayed macaque fimbriae at 4 C in the presence of 10 nM [3H]Moxestrol and then either froze the tissues immediately (non-MW) or treated them with MW. Slide-mounted cryosections of non-MW and MW-treated tissue were then incubated with either a Tris-EDTA buffer (low salt) or the same buffer containing 4 M KCl (high salt). The quantity of [3H]Moxestrol-occupied ER extracted from the frozen sections by each buffer was determined by a sucrose gradient shift assay. The low salt buffer extracted significantly more radiolabeled ER from non-MW sections than from MW-treated sections (P < 0.01), whereas the high salt buffer extracted equal amounts of ER from both the MW-treated and non-MW sections. MW-irradiation enhanced ICC detectability of ER in frozen sections by greatly reducing the amount of ER extracted during the various washes used during normal ICC processing.

Previous immunocytochemical studies indicate that receptor regulation varies in different uterine cell types. In primates, progesterone (P) suppresses estrogen receptor (ER) in glandular epithelial cells in the functionalis, but fails to suppress ER in the glandular epithelial (GE) cells of the basalis. P also fails to suppress ER in the perivascular stromal and smooth muscle cells of the spiral arteries in the functionalis. We used nonradioactive in situ hybridization to determine whether similar cell type differences occur at the ER mRNA level. We used digoxigenin-labeled oligodeoxynucleotides (oligo-DNAs; 45-mer) as probes and detected the hybrids immunocytochemically with horseradish peroxidase-labeled antidigoxigenin antibody. This technique can discriminate between positive and negative cells in closely packed histological associations. In spayed monkeys, most of the GE cells as well as endometrial stromal cells were positive for ER mRNA, while all vascular smooth muscle, endothelium, and perivascular stromal cells were negative. Estradiol treatment for 14 days markedly increased ER mRNA staining in the GE cells, most stromal cells, and the vascular smooth muscle and perivascular stromal cells of spiral arteries in the functionalis. However, in the basalis, these components of the spiral arteries were negative as were the small basal arteries of the basalis. In most positive cells, ER mRNA was not homogeneously distributed in the cytoplasm, but, rather, was concentrated in their perinuclear regions. The GE cells in the basalis had especially intense concentrations of perinuclear signal at their apical poles. After sequential estradiol plus P treatment, the signal was greatly reduced in the GE cells of the functionalis, but not in the GE cells of the basalis or in the vascular smooth muscle or perivascular stromal cells of the spiral arteries of the functionalis. In myometrium, ER mRNA was localized to the perinuclear region of smooth muscle cells, but the staining intensity was not dramatically affected by hormonal manipulation. Unexpectedly, we observed clusters of stromal cells characterized by extremely high positive signals for ER mRNA ("hot cells") at the endometrial/myometrial border and deeper in the connective tissue of the myometrium, although such cells did not express high levels of ER protein. In general, however, the cellular distribution of ER mRNA and its hormonal regulation paralleled those of ER protein.

Conceptus secretion of oestrogen on Day 11 of gestation is involved with establishment of pregnancy in the pig. Changes in oestrogen receptor (ER) protein, mRNA and cellular localization in the endometrium were evaluated during the oestrous cycle and early pregnancy of the gilt. In nonpregnant gilts, concentration of nuclear ER in the endometrium increased from Days 0 to 12 followed by a decline on Day 15 of the oestrous cycle. In pregnant gilts, changes in endometrial nuclear ER during Days 10, 12, 15 and 18 were similar to that in cyclic pigs. Analysis of endometrial ER mRNA expression did not detect any difference between cyclic and pregnant pigs between Days 10 and 15 postoestrus. Expression of ER mRNA in endometrium of cyclic and pregnant gilts was greatest on Day 10 followed by a decline on Day 15. Endometrial ER mRNA increased on Day 18 of the oestrous cycle, but remained low during pregnancy. Immunocytochemical localization of ER in the endometria of cyclic and pregnant gilts indicated that there was intense staining for ER in stromal cells and moderate to strong staining in surface and glandular epithelial cells during oestrus (Day 0) and Day 18 of the oestrous cycle. However, stromal ER staining was absent from Days 5 to 15 of the oestrous cycle and continued to be suppressed on Day 18 of pregnancy. Immunocytochemical staining of ER in the surface and glandular epithelium was readily detectable from Days 0 to 12 of the oestrous cycle and during pregnancy. Intensity of staining for ER declined in surface epithelial cells on Day 15 in both cyclic and pregnant pigs whereas positive staining for ER in glandular epithelium was absent. Staining for ER on uterine surface epithelial cells increased during pro-estrus (Day 18) of cyclic gilts but remained similar to Day 15 in pregnant gilts. Changes in endometrial ER protein, mRNA and localization in surface epithelium are consistent with a physiological role for conceptus oestrogen secretion in uterine function and maternal recognition of pregnancy in the pig.

Sex steroid hormone receptors have been identified in the adrenal glands of rodents and may have a role in adrenal function. The highly estrogenic environment during pregnancy has been proposed to influence steroidogenesis by the fetal zone of the primate fetal adrenal gland. In order to determine whether these effects involve receptor-mediated mechanisms, we have examined the concentration and distribution of estrogen receptor (ER), androgen receptor, and progesterone receptor (PR) in the adrenal glands of fetal, immature, and adult rhesus monkeys. Monoclonal antibodies were used for immunocytochemistry (ICC), and in a gradient shift assay, for determination of receptor distribution and concentrations, respectively. There was no difference between the concentrations of ER in the adrenal glands from male and female adult animals (12.4 +/- 2.2, n = 3 and 13.2 +/- 2.0 fmol/mg DNA, n = 7; respectively); however, the concentration of ER in the fetal adrenal glands was markedly lower than in the adults (combined adult 12.7 +/- 1.6, n = 10, and fetal 0.9 +/- 0.4 fmol/mg DNA, n = 7; P less than 0.01). The concentration of ER in the adrenal glands of immature animals was also lower compared to adult animals (6.1 +/- 1.6, n = 6, P less than 0.05). In the adult, ICC revealed that staining for ER was restricted to the cell nucleus and was most dense in the zone fasciculata, with lesser staining in the zona glomerulosa and zona reticularis, and with no detectable staining in the medulla. ER staining was virtually absent in the fetal zone which comprises the major portion of the fetal gland; however, some staining was observed in the narrow definitive zone. The distribution of androgen receptor was similar to that of ER, whereas there was no detectable staining for PR in the adrenals of either adult or fetal animals. We conclude: 1) that the lower concentration of ER in fetal adrenal glands is due to the absence of ER in the fetal zone; 2) the lack of ER and PR in the fetal zone suggests that estrogens and progestins do not influence the growth or function of the fetal zone by receptor-mediated mechanisms; 3) estrogens and androgens may influence the function of the adult adrenal cortex.

A study was conducted to determine if a monoclonal antibody (MAB), H222, prepared against human breast cancer estrogen receptors (ER) would recognize ER in the oviduct and liver of garter snakes (Thamnophis sirtalis parietalis). Using sucrose gradient analysis of antibody-ER complexes bound to [3H]estradiol we have determined that the MAB H222 binds to an ER in the cytosolic and nuclear extracts of snake tissues. The snake ER is not bound by nonspecific MABs in the sucrose gradient analysis. Further, the snake ER does not bind to other steroids, including a synthetic progestin, R5020, or the androgens 5 alpha dihydrotestosterone and R1881. The quantity of ER in the snake oviduct (200-700 fmol/mg DNA) is within an order of magnitude of that found in the oviduct of the nonhuman primate. These results suggest that the MAB H222 and 17 beta-estradiol bind to an ER in the snake that shares common properties with mammalian ER.

We have suggested that in the nonhuman primate endometrium, stromal cells might play a role in mediating the effects of estrogen on the epithelium, especially during the luteal-follicular transition (LFT) when target cells normally escape from the inhibitory influence of progesterone (P). We now report that like estrogen receptors (ER), endometrial progestin receptors (PR) are detectable only in stromal cells until the fifth day of the LFT. With a technique that combined immunocytochemistry and autoradiography on the same sections, we characterized the cellular distribution of ER or PR coincidentally with the localization of [3H]thymidine taken up in vitro by endometria from monkeys undergoing an LFT. DNA synthesis in the glands of the upper endometrium was E2-dependent, but the distribution of [3H]thymidine was not positively correlated with the presence of ER or PR. Readministration of P to animals on days 3 or 4 of the LFT significantly reduced the [3H]thymidine labeling index of the glandular epithelium and caused stromal ER to decline, but P did not block the eventual appearance of ER in epithelial cells on day 5 of the LFT. Thus, E2 stimulated DNA synthesis in epithelial cells that lacked ER, and P suppressed DNA synthesis in these cells even though PR was only detected in the stroma when P treatment began. These data are consistent with a role for endometrial stromal cells in mediating the effects of E2 and P on the epithelium during the LFT.

Using biochemical methods we established that estrogen receptor content and distribution and progesterone receptor content in female and male baboon myocardium did not differ between sexes. In contrast, myocardial androgen receptor distribution between cytosolic and nuclear compartments was sexually dimorphic. Female baboon myocardial androgen receptors were restricted to the cytosolic compartment, whereas male myocardial androgen receptors were distributed between the cytosolic and nuclear compartments. Using human estrogen receptor cDNA we showed that baboon aorta, myocardium and uterus contain a 6.3 kb estrogen receptor transcript. Analyses performed with human progesterone receptor cDNA established that baboon aorta and uterus contain an 8 kb progesterone receptor transcript; however, progesterone receptor transcripts were not demonstrable in baboon myocardial RNA preparations. Because relative hybridization signal intensity reflected known uterine and aortic progesterone receptor content, failure to detect progesterone receptor transcripts in myocardial preparations may reflect sensitivity limitations and the fact that aortic progesterone receptor content is 5-fold greater than that of myocardium. Immunocytochemical analyses demonstrated that baboon myocardial progesterone receptors were present in greater than 25% of myocytes and generally absent from other myocardial cells. Our studies establish that: (1) gonadal steroid hormone receptor gene transcription occurs in cells of the baboon cardiovasculature, (2) these steroid hormone receptors may be physiologically functional, and (3) gonadal steroid hormone receptors may be restricted to specialized cells of the cardiovasculature.

We obtained the testes, ductuli efferentes, and epididymides from adult rhesus and cynomolgus macaques and examined these tissues for estrogen receptors (ER) with immunocytochemistry (ICC) and a sucrose gradient assay. Both techniques employed monoclonal antibodies prepared against ER, and both showed that high concentrations of ER were present OFFy in the ductuli efferentes. Moreover, all specific staining was confined to the nuclei of the nonciliated, absorptive epithelial cells. The quantity of salt-extractable ER in the ductuli efferentes (834 +/- 161 [SEM] fmol/mg DNA [n = 8]) did not differ significantly from the amounts measured with the identical assay in oviducts and endometrium of estrogenized female macaques. Testes and epididymides of macaques had no specific staining by ICC and barely detectable amounts by biochemical analysis (7 +/- 4 [n = 3], 8 +/- 2 [n = 5], 33 +/- 16 [n = 3], and 6 +/- 3 [n = 8] fmol/mg DNA for testis and caput, corpus, and cauda epididymis, respectively). The functional significance of the high levels of ER in the ductuli efferentes of macaques remains to be determined.

We measured tightly bound nuclear estrogen receptors (ER) in sex skin biopsies obtained from pig-tailed macaques (Macaca nemestrina) which were previously ovariectomized and treated with an estradiol-progesterone regimen. Incubation of fresh tissue slices with a saturating concentration of [3H]estradiol (E2) was done to determine the capacity of nuclear acceptor sites to bind activated ER with high affinity. The radiolabeled ER was extracted from nuclei with 0.5 M KCl, complexed with an anti-ER monoclonal antibody, and quantitated by analysis on sucrose gradients. Even though serum E2 levels were unchanged, 7 and 14 days of sequential progesterone (P) treatment decreased ER amounts below those found after 7, 14 and 21-23 days of E2 treatment. ER regulation in sex skin of this species is similar to that found in macaque reproductive tract; P suppresses ER levels even in the presence of continuous E2. The tissue responses of sex skin to the hormone treatments correlated well with the measured fluctuations of tightly bound nuclear ER, which suggests the functional significance of this ER component.

PRL increases during pregnancy in primates with rising levels of placental estradiol (E) and progesterone (P). However, while E will increase PRL secretion in monkey pituitary cell cultures, P has no effect. We recently localized progestin receptors (PR) to gonadotropes, but not lactotropes, with an immunocytochemical technique to double stain monkey pituitary cell cultures. The following studies were performed to confirm the immunocytochemical localization of PR in intact pituitary tissue and to determine the effect of E and P on the levels of estrogen receptors (ER) and PR in the pituitary. ER and PR levels were determined in the endometrium of the same animals for an internal comparison. Thirteen adult cycling female cynomolgus monkeys were ovariectomized and treated for 28 days with 1) an empty Silastic capsule (Spay), 2) a 2-cm E-filled capsule (E), or 3) a 2-cm E-filled capsule for 14 days plus a 6-cm P-filled capsule implanted for an additional 14 days (E + P). Blood samples were drawn daily for assay of serum E, P, and PRL levels. Serum PRL was not significantly affected by E, but the sequential addition of P significantly increased serum PRL levels over those observed in Spray animals. The anterior pituitary and endometrium were removed for measurement of ER and PR levels by a sucrose gradient shift assay incorporating monoclonal antibodies against ER and PR. Pituitary ER levels did not vary significantly with steroid treatment (158.2 +/- 33.6, 135.5 +/- 24.9, 104.3 +/- 13.4 fmol/mg DNA in Spay, E, and E + P animals, respectively). Pituitary PR levels were undetectable in Spay animals, were induced by E (393.3 +/- 53.4 fmol/mg DNA), and were suppressed to undetectable levels by the addition of P. A portion of the pituitary was frozen for immunocytochemical single staining for ER, PR, PRL, and LH and double staining for PRL + PR and LH + PR. ER staining was observed in many parenchymal cells, but there was no apparent change with steroid treatment. PR staining was absent in the Spay animals; many PR-positive cells were observed in E-treated females, and only a small number of faintly staining cells were detected in the E + P animals. Double staining for PRL + PR and LH + PR revealed PR in gonadotropes, but not lactotropes. In conclusion, PR, but not ER, are regulated by E and P in the monkey pituitary. Importantly, PR is regulated within gonadotropes, but not lactotropes. Therefore, P probably increases PRL secretion through a hypothalamic action.

With the aid of monoclonal antibodies specific to the estrogen and progestin receptors, we have examined the cellular localization of these proteins in the reproductive tract of male and female macaques. Two striking findings have resulted from our work with these new reagents. First, these receptors are detectable only in cell nuclei, regardless of hormonal treatment, and second, they are often detectable in stromal, but not epithelial cells when the epithelial cells undergo various estrogen or progestin-dependent events. The latter observation has led us to conclude that stromal cell-epithelial cell interactions may play previously unappreciated roles in the hormonal control of the primate reproductive tract. The lines of evidence that have drawn us to this conclusion will be reviewed.

Progesterone withdrawal as a mechanism for parturition in primates is controversial. The progesterone antagonist RU486, given in late pregnancy to rhesus monkeys at a dose of 47 mmol/kg.day (20 mg/kg.day), causes an increase in uterine activity, but not the expected increase in amniotic fluid prostaglandins or cervical dilatation. We, therefore, studied the effect of RU486 on estrogen receptor (ER) localization and concentration in reproductive tract tissues in rhesus monkeys during late gestation and after spontaneous labor at term. Distribution of ER in pregnant uterine tissues was studied by immunocytochemical techniques and quantified by a biochemical assay, both of which employed a monoclonal antibody specific for ER. ER was not present in amnion and chorion by immunocytochemical investigation; however, a significant increase in receptor staining was seen in decidua and myometrium after RU486 treatment compared to that in both pregnant control tissues and parturient tissues. Sucrose gradient assay of nuclear (n) and cytosolic (c) ER revealed a low level of ER (expressed as fmol of estradiol bound/mg of DNA) in pregnant and parturient decidua (pregnant: nER = 7.3 +/- 2.4, cER = 17.1 +/- 6.4; parturient, nER = 7.7 +/- 3.1, cER = 16.4 +/- 8.8) and myometrium (pregnant: nER = 21.7 +/- 4.1, cER = 20.8 +/- 5.3; parturient: nER = 30.0 +/- 2.8, cER = 10.7 +/- 6.7). In contrast, tissues collected from RU486-treated animals contained high levels of ER in decidua (nER = 52.3 +/- 16.8, cER = 240.5 +/- 145.3) and myometrium (nER = 77.0 +/- 19.2; cER = 66.5 +/- 31.6). We conclude that 1) the increase in ER in decidua and myometrium after RU486 treatment is the result of a decrease in the inhibitory action of progesterone on ER and documents the progesterone receptor antagonism by RU486 during induced myometrial contractility in late pregnant rhesus monkeys; 2) the absence of ER from amnion and chorion indicates that the normally observed increase in prostaglandin production by rhesus fetal membranes during labor is not mediated by ER; and 3) the absence of a change in the concentration of ER in decidua and myometrium from pregnant control monkeys and those in spontaneous labor indicates that an increase in ER (and, by inference, a withdrawal of receptor-mediated progesterone inhibition) is not part of the normal events in preparation for parturition in primates.

Sequential steroid administration of estradiol (E2) and progesterone (P) in spayed pigtailed macaques was used to precisely control the time course of sex skin swelling. After removal of the P implant, the sex skin swelled considerably and the water content of the sex skin increased manyfold over that of back skin. During the swelling phase, hyaluronate biosynthesis in sex skin increased dramatically compared with back skin of the same animals. Collagen synthesis also increased but to a lesser extent. Estrogen receptor levels were undetectable in back skin and very low in spayed animals that had been treated with both E2 and P. After removal of the P implant, both the level of estrogen receptor and the rate of hyaluronate biosynthesis increased. Immunocytochemistry with monoclonal antibodies against the estrogen receptor showed that the dermal fibroblast was the only cell type to stain positively for estrogen receptor. We conclude that the sex skin swelling that follows P withdrawal in pigtailed macaques bearing E2 implants is mediated by estrogen receptors in dermal fibroblasts and is a result of increased hyaluronic acid synthesis by these cells.

We used sequential treatment with implants of estradiol (E2) and progesterone (P) to create varied hormonal states in a group of spayed cynomolgus macaques. The reproductive tracts were removed, and nuclear and cytosolic estrogen receptors were analyzed in the cervical mucosa, endometrium, and oviducts. Nuclear receptor quantities were greater in tissues of E2-treated monkeys than in tissues of spayed animals. Sequential P treatment, even in the presence of continuous E2, decreased the amounts of nuclear and cytosolic E2 receptors. In the oviduct and endometrium, the P-mediated suppression of receptors occurred within 1 or 2 days. In the cervix, suppression occurred only if the serum P:E2 ratio was elevated to twice the amount (approximately 100:1) usually found during the luteal phase of the menstrual cycle (approximately 50:1) in this species. Of these three reproductive tract tissues, the cervix had the highest threshold for suppression by P of E2 receptors in the presence of E2.

Immunocytochemical, biochemical, and histologic analysis of endometriotic lesions and endometria from rhesus macaques with endometriosis revealed several distinctions between ectopic and eutopic endometrium. In lesions, unlike endometrium, neither the mean percentages of estrogen receptor positive (ER+) cells nor the total ER content changed significantly during the menstrual cycle. In eutopic endometria, ER staining in both stromal and epithelial cells increased and decreased synchronously during the cycle, but in endometriotic lesions, such synchrony was lacking. Moreover, in lesions, unlike endometria, the percentage of ER+ cells was low in the stroma and highly variable in epithelium throughout the cycle. These data, taken together, indicate a defect in the hormonal regulation of ER in endometriotic lesions of monkeys.

The distribution of estrogen receptors (ER), progesterone receptors (PR), PRL, and gonadotropins in different cell types of the monkey pituitary was examined by immunocytochemical (ICC) labeling of pituitary cell cultures. Dispersed monkey pituitary cells were cultured on extracellular matrix and in serum-free medium for 6-14 days. Individual cultures were singly stained for ER, PR, PRL, LH, and FSH or double labeled for PR and one of the protein hormones. ICC reaction product localizes over the nuclei of cells that are positive for the steroid receptors, whereas reaction product localizes over the cytoplasm of cells that are positive for the protein hormones. Sixty-one percent of the parenchymal cells were positive for PRL, while 1-3% were positive for LH or FSH. Sixty-two percent of the parenchymal cells were ER positive. ER staining was localized over the nuclei of two morphologically distinct cell types. One cell type is smaller and more prevalent than the second cell type. Based on single staining for each of the protein hormones, we propose that the smaller cells are lactotropes, and the larger cells are gonadotropes. PR-positive cells averaged 7.7% of the parenchymal cells. Double ICC staining for PR and the protein hormones demonstrated that PR localize in the nuclei of gonadotropes, but not lactotropes, of monkey pituitary cell cultures. The absence of PR in lactotropes is consistent with our observation that progesterone has no direct effect on PRL secretion in monkey pituitary cell cultures. In contrast, the presence of PR in gonadotropes suggests that progesterone may act directly at the pituitary to modulate gonadotropin secretion in the primate. In conclusion, ER are present in both lactotropes and gonadotropes. PR are present in gonadotropes, but not lactotropes, of the primate pituitary.

Uteri were removed and blood was drawn from hamsters during the estrous cycle, on the eighth day of pregnancy, and after different hormonal treatments. Serum estradiol (E2) and progesterone (P) levels were determined by RIA. Portions of the uteri were prepared for light and electron microscopy. Endogenous uterine nuclear E2 receptor was measured by exchange assay. Large increments of nuclear E2 receptor occurred when the level of serum E2 was rising and that of serum P was falling (diestrus day 2 through proestrus morning); decrements occurred when the serum E2 level was falling and the serum P level was rising (proestrus evening through diestrus day 1). Ovariectomy on proestrus morning led to a decline in the amount of nuclear E2 receptor, and this decline was prevented by administration of E2 at the time of ovariectomy. P depleted nuclear E2 receptor in the presence of high levels of serum E2. The uterine luminal epithelium passed through a phase of mitosis and hypertrophy (diestrus day 2 through proestrus morning), a brief phase suggestive of secretion (proestrus evening), a phase of degeneration (estrus), and a phase of quiescence (diestrus day I). Ovariectomy on proestrus morning led within 24 h to degenerative changes identical to those seen during estrus, and these changes were prevented by treatment with E2 at the time of ovariectomy. P induced these degenerative changes in the presence of high levels of serum E2. After 8 days of pregnancy, the epithelium resembled that seen during diestrus day 1. Elevated uterine nuclear E2 receptor levels were associated with growth and hypertrophy of the luminal epithelium, and severely depressed levels were associated with the degenerative and quiescent phases of the epithelium.

We sampled oviducts and endometria of 27 cynomolgus macaques during the menstrual cycle and measured the concentration of nuclear and cytoplasmic estrogen receptors in these tissues by exchange assay. We assessed the stage of the cycle by correlating serum estradiol (E2) and progesterone (P), as measured by radioimmunoassay, with the morphological condition of the ovaries, oviducts and endometrium of each animal. We have previously identified a series of oviductal stages that reflected the orderly sequence of cytological changes in the oviduct during the cycle, and we normalized receptor measurements to these stages. The amounts of nuclear and cytoplasmic estrogen receptor in both the oviduct and the endometrium were approximately twofold greater in the follicular phase than in the luteal phase. In the follicular phase, elevated receptor levels were associated with oviductal proliferation and differentiation, as well as with endometrial proliferation. During the luteal phase, lowered levels were correlated with atrophy and dedifferentiation in the oviduct, but with hypertrophy and progestational development in the endometrium. When the luteal phase of one cycle ends and the follicular phase of the next begins, it is a decline in serum P, not a rise in serum E2, that precedes the elevation in estrogen receptor level and the onset of proliferation in the oviduct and endometrium. Proliferation of the reproductive tract and elevations in nuclear estrogen receptor levels during the early follicular phase can therefore be viewed as consequences of the release of the system from antagonism by P.

Monoclonal antibodies to the estrogen receptor (anti-ER) were used to develop an immunocytochemical method to detect ERs in frozen sections of the macaque reproductive tract. Specific nuclear, but not cytoplasmic, staining occurred with two different methods: direct, in which an antiestrophilin -horseradish peroxidase conjugate was used as the first antibody, and indirect, in which a mixture of antiestrophilins was used in the first incubation step. Nuclear staining was absent when various control antibodies replaced the anti-ER. In uteri from spayed monkeys treated with estradiol (E2) for 14 days, nuclear staining was always present. In uteri from similar animals treated for an additional 14 days with E2 and progesterone, nuclear staining was almost completely absent. Mean endometrial nuclear ER levels, measured by an exchange assay, were 5-fold greater in the E2-treated than in the E2-plus progesterone-treated group. In addition, when samples of estrogenized uterus and oviduct were incubated for 60 min in vitro with 100 nM E2, the intensity of nuclear staining increased in parallel with an increase in the concentration of nuclear ER. The nuclei of stromal, smooth muscle, and epithelial cells of the estrogenized oviduct, cervix, and vagina as well as smooth muscle cells of the estrogenized myometrium were also receptor positive. Nontarget tissues, such as duodenum, colon, esophagus, and skeletal muscle, contained no cells that showed specific nuclear staining. Some staining of cytoplasmic and extracellular components occurred in all preparations. These latter reactions were nonspecific, because they were present in many nontarget tissues or when control antibodies replaced the anti-ER. With current methods, only nuclear ERs can be reliably localized in frozen sections of monkey tissues with monoclonal antiestrophilins .

We studied the antibody-combining properties of 3 forms of the estrogen receptor found in buffers of high ionic strength. Shifts to a faster sedimenting peak on sucrose gradients or a faster eluting peak on a gel filtration column with antibody addition allowed us to determine whether a given form contained one, two or more antibody-binding sites. The monomeric cytosolic estrogen receptor, ERC, contained one antibody binding site for each of 2 monoclonal antiestrophilins (H222 and H165, provided by Abbott Laboratories). Both the heat-transformed cytosolic estrogen receptor, ERC*, and a major fraction of the estrogen receptor extracted from nuclei, ERN, contained two sites for H165, but only one for H222. A minor fraction of ERN had only one site for each antibody. The kinetics of transformation of ERC to a species with two H165 binding sites were appropriate to a dimerization of ERC*. Addition of H222, but not H165, before the onset of the heat-induced transformation blocked the formation of ERC to ERC. These data suggest that ERC* and a major form of ERN are comprised of two immunologically similar subunits identical to ERC. Also, the antigenic determinant for H222, but not H165, appears to be located close to the dimerization domain. The minor form of ERN appears to contain an altered or dissimilar subunit.

Four weeks or more after being spayed, cats were either left untreated or treated with various regimens of estradiol (E2) and progesterone (P) in silastic implants. Oviducts and uteri were then removed and examined for morphological changes as well as for the amount and compartmentalization of the estrogen receptor. E2 treatment restored a full complement of ciliated and secretory cells to the oviduct, and subsequent P treatment (with or without continuous E2) of an E2-restored oviduct produced an atrophied epithelium equivalent to that resulting from E2 withdrawal. In the uterus, P treatment after E2 priming did not lead to E2-withdrawal symptoms but rather to a new stage of differentiation characterized by altered and increased secretory activity. In both organs E2 caused dramatic elevations in the E2-receptor content in the nuclear as well as in the cytoplasmic compartments. Also (in both organs) P treatment after E2 priming reduced the E2-receptor content in the nuclear and cytoplasmic compartments to the levels present in spayed cats. The degree of suppression caused by P in both organs was similar to that induced by E2 withdrawal. Suppression of the E2-receptor system by P correlated with suppressed function inthe oviduct but with increased growth and secretory activity in the uterus.

We examined changes in estrogen receptors (ERs) in endometrial stromal and epithelial cells in cynomolgus macaques during artificially induced menstruation and repair. We used Silastic implants filled with either estradiol (E2) or progesterone (P) to treat spayed animals for 14 days with E2 followed by 14 days of E2 plus P. We then withdrew the P (but not the E2) implants and removed uteri 0, 0.5, 1, 2, 3, 4, 5, 7, and 14 days later. Uterine tissues were assayed biochemically for ER content, fixed for histology and frozen for immunocytochemistry of ER with monoclonal antiestrophilins. On day 0, ER levels in endometrium were low [1330 +/- 201 (n = 9) fmol/mg DNA]. An increase in total receptor was evident by 3-4 days of P withdrawal 2762 +/- 190 (n = 6) fmol/mg DNA; P less than 0.001]. Total receptor concentrations increased linearly with time from 0.5-7 days of P withdrawal (r = 0.88). On day 0, staining for nuclear ER in the glandular epithelium and stroma of zones I, II, and III of the endometrium was negative. Beginning at 12-24 h and continuing through 4 days of P withdrawal, nuclear staining became detectable and increased in intensity only in endometrial stromal fibroblasts and myometrial smooth muscle cells. The glandular epithelium of the endometrium did not develop nuclear staining until 5-7 days of P withdrawal, coincident with a 10-fold increase in the mitotic index of the epithelium of the upper zones. Thus, the increase in endometrial ER levels that occurred during the first 5 days of an induced luteal-follicular transition took place almost exclusively in stromal fibroblasts.

We used immunocytochemistry (ICC) with monoclonal antibodies to the estrogen receptor (ER) to localize ER in the oviducts, uteri, and cervix of untreated, estrogen-treated, and estrogen-progestin-treated spayed macaques. We also used binding assays with labeled estrogens to quantify nuclear and cytosolic ER levels in parallel samples of the same tissues. In untreated spayed animals, cytosolic ER levels were much higher than nuclear ER levels, but all specific staining was nuclear. After treatment for 14 days with estradiol (E2), the degree of staining for ER in cell nuclei in the oviduct, cervix, and endometrium had increased, and there were significant increases in both nuclear and cytosolic ER levels. In the myometrium, ER levels and ICC staining of nuclei increased minimally with E2 treatment. In animals treated for 2 weeks with E2 followed by 2 weeks with E2 and progesterone (P; sequential P treatment) the degree of nuclear ER staining in the oviduct, endometrium, and cervix greatly decreased, and cytosolic and nuclear levels of ER declined significantly. In the myometrium of such animals there was a minimal decrease in the degree of staining and a nonsignificant decline in cytosolic and nuclear ER levels. Sequential P treatment reduced the degree of nuclear staining in the oviduct and endometrium below that found in spayed animals; however, such treatment only lowered the amount of cytosolic, not nuclear, ER significantly below spayed levels in those same tissues. Some animals were treated sequentially with P and sampled 1, 3, 12, and 24 h after the onset of P treatment. By 1 h, nuclear ER levels in the endometrium were significantly suppressed, but cytosolic levels were not lowered until 3 h of treatment; ICC staining was also not substantially reduced until 3 h of P treatment. In the oviduct, nuclear ER levels were significantly reduced by 1 h of P treatment, but cytosolic levels were not lowered until after 12-24 h of P treatment; the degree of nuclear staining in the oviduct was also not substantially reduced until 12-24 h of P treatment. In myometrium, there was no significant decline in ER in nuclear or cytosolic fractions or any substantial decrease in the degree of nuclear staining at any time during this treatment. These observations suggest that the ER detected by ICC in the nuclei of target cells in frozen sections represents the total ER detectable by binding assays in cytosolic and nuclear fractions.(ABSTRACT TRUNCATED AT 400 WORDS)

We examined nuclear estrogen receptors (ER) and progestin receptors (PR) in the rhesus monkey prostate. Tissues were obtained from six intact males, five untreated castrates, six castrates treated with testosterone (T) for 6 weeks, and four castrates treated with estradiol (E2) for 6 weeks. Samples of the caudal lobe were either assayed biochemically for ER or stained immunocytochemically (ICC) with monoclonal antibodies against the ER or PR. Prostates from untreated castrates had significantly more ER than tissues from intact or T-treated castrates. In E2-treated castrates, ER number increased compared to that in intact and T-treated castrates. With ICC, ER was found only in the nuclei of the fibroblasts and smooth muscle cells of the stroma, not the glandular, ductal, or urethral epithelial cells. Intact and T-treated castrates had a very small number of positive cells, while untreated and E2-treated castrates had a significantly increased number of positive ER cells in the fibromuscular stroma. With ICC, PR was absent in intact or T-treated animals and barely evident in untreated castrates, but was significantly increased in the fibromuscular stroma of E2-treated castrates. The histological preparations indicated there was no stromal hypertrophy in the E2-treated castrates, but the E2 treatment did cause dilation of the glandular acini. Aromatase activity was measured in prostatic microsomes with a radiometric assay. Levels were low (3-30 fmol/h.mg protein) compared to those in brain and placenta, and no differences in activity were seen between castrates and T-treated castrates. Our data demonstrate that androgens can suppress the level of nuclear ER in the rhesus prostate, and that E2 treatment of castrates can induce PR in the same cells as those that contain ER. Thus, under appropriate conditions, estrogens could affect the rhesus prostate through a receptor-mediated pathway.