Localization of the endogenous benzodiazepine ligand octadecaneuropeptide in the rat testis.

Diazepam binding inhibitor (DBI) is the precursor of a family of peptides, including an octadecaneuropeptide (ODN), which share with DBI the ability to specifically displace benzodiazepines (BZD) from their receptors. BZD receptors have been found not only in the brain, but also in a variety of peripheral tissues, including the testis. To clarify the role of ODN in the testis, we have investigated the localization of ODN in the rat testis using two different cytochemical approaches: immunocytochemistry and in situ hybridization. Immunocytochemical localization was achieved using rabbit antibodies developed against rat ODN. At the light microscopic level, immunostaining was exclusively located in interstitial cells; the seminiferous tubules were totally unlabeled. In the developing rat, immunostaining in the interstitial cells was first detected in an 18-day-old fetus. The immunolabeling increased as a function of age to reach a plateau at 40 days of age. The ultrastructural localization of ODN was achieved by immunogold staining. The gold particles were exclusively found in the cytoplasm of Leydig cells. HPLC analysis performed in adult rat testicular extracts revealed that immunoreactive material was detected in a peak eluted later than synthetic rat ODN. The cellular distribution of ODN was also studied by in situ hybridization using a 35S-labeled single stranded RNA probe complementary to DBI mRNA. Hybridization signal obtained at the light microscopic level was only detected over interstitial cells. The data obtained clearly indicate that in the rat, Leydig cells synthesize ODN and accumulate ODN-like immunoreactivity. Since Leydig cells have been shown to contain BZD receptors, it might be hypothesized that ODN and/or other DBI-related peptides can play a role in Leydig cell regulation.

Localization of cytoplasmic collagen mRNA in human aortic coarctation: mRNA enhancement in high blood pressure-induced intimal and medial thickening.

Enhanced synthesis and deposition of extracellular matrix components, including collagen, contribute significantly to arteriosclerotic changes in the arterial vessel wall. We localized cells actively synthesizing collagen by hybridizing 35S-labeled RNA probes complementary to type I and III collagen mRNA with cytoplasmic mRNA in frozen sections of surgically removed aortic coarctations. These were chosen as a model for comparing mRNA levels in areas of high blood pressure-induced wall thickening and in unaffected post-stenotic areas. In situ hybridization revealed increased expression of type I and III collagen mRNA in intimal cells and in cells adjacent to the medial-adventitial border in the pre-stenotic part of the coarctation. In contrast, cells of the post-stenotic area showed only a very low signal. No immunohistologically detectable macrophages were seen in the pre-stenotic subendothelial areas where mRNA levels were enhanced. Higher collagen mRNA levels therefore occur in particular regions of high blood pressure-induced arterial wall thickening in the absence of macrophages. The results suggest that in situ hybridization is suitable for detection of locally occurring transcriptional activation of cells for collagens in the vessel wall.

Studying single living cells and chromosomes by confocal Raman microspectroscopy.

Many indirect methods have been developed to study the constitution and conformation of macromolecules inside the living cell. Direct analysis by Raman spectroscopy is an ideal complement to techniques using directly labelled fluorescent probes or of indirect labelling with mono- and polyclonal antibodies. The high information content of Raman spectra can characterize biological macromolecules both in solution and in crystals. The positions, intensities and linewidths of the Raman lines (corresponding to vibrational energy levels) in spectra of DNA-protein complexes yield information about the composition, secondary structure and interactions of these molecules, including the chemical microenvironment of molecular subgroups. The main drawback of the method is the low Raman scattering cross-section of biological macromolecules, which until now has prohibited studies at the level of the single cell with the exception of (salmon) sperm heads, in which the DNA is condensed to an exceptionally high degree. Ultraviolet-resonance Raman spectroscopy has been used to obtain single cell spectra (and F. Sureau and P. Y. Turpin, personal communication), but in this method absorption of laser light may impair the integrity of the sample. We have avoided this problem in developing a novel, highly sensitive confocal Raman microspectrometer for nonresonant Raman spectroscopy. Our instrument makes it possible to study single cells and chromosomes with a high spatial resolution (approximately less than 1 micron 3).

Sixteen discrete RNA components in the cytoplasmic ribosome of Euglena gracilis.

We have isolated cytoplasmic ribosomes from Euglena gracilis and characterized the RNA components of these particles. We show here that instead of the four rRNAs (17-19 S, 25-28 S, 5.8 S and 5 S) found in typical eukaryotic ribosomes, Euglena cytoplasmic ribosomes contain 16 RNA components. Three of these Euglena rRNAs are the structural equivalents of the 17-19 S, 5.8 S and 5 S rRNAs of other eukaryotes. However, the equivalent of 25-28 S rRNA is found in Euglena as 13 separate RNA species. We demonstrate that together with 5 S and 5.8 S rRNA, these 13 RNAs are all components of the large ribosomal subunit, while a 19 S RNA is the sole RNA component of the small ribosomal subunit. Two of the 13 pieces of 25-28 S rRNA are not tightly bound to the large ribosomal subunit and are released at low (0 to 0.1 mM) magnesium ion concentrations. We present here the complete primary sequences of each of the 14 RNA components (including 5.8 S rRNA) of Euglena large subunit rRNA. Sequence comparisons and secondary structure modeling indicate that these 14 RNAs exist as a non-covalent network that together must perform the functions attributed to the covalently continuous, high molecular weight, large subunit rRNA from other systems.

Use of 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (5FBAPTA) in the measurement of free intracellular calcium in the brain by 19F-nuclear magnetic resonance spectroscopy.

We have applied the 19F-nuclear magnetic resonance (NMR) calcium indicator 1,2-bis(2-amino-5-fluoro-phenoxy)ethane-N,N,N',N'-tetraacetic acid (5FBAPTA) to the measurement of the free intracellular calcium concentration [( Ca2+]i) in superfused brain slices. A mean +/- SD control value of 380 +/- 71 nM (n = 18) was obtained at 37 degrees C using 2.4 mM extracellular Ca2+. Subcellular fractionation studies using [3H]5FBAPTA showed that after loading of its tetraacetoxymethyl ester, approximately 55% was de-esterified, with the other 45% remaining as the tetraester bound to membranes. Of the de-esterified 5FBAPTA, greater than 90% was in the cytosolic fractions, with less than 1% in the mitochondria or microsomes. The NMR-visible de-esterified 5FBAPTA slowly disappeared from the tissue with a t1/2 of 4 h. A time course after loading confirmed that the calculated [Ca2+]i was constant over a 5-h period, although the scatter of individual results was +/- 20%. The [Ca2+]i was increased by a high extracellular K+ concentration ([K+]e), by a low extracellular concentration of Na+, and by the calcium ionophore A23187. On recovery from high [K+]e, the [Ca2+]i "overshot" to values lower than the original control value. The [Ca2+]i was surpisingly resistant to changes in extracellular Ca2+ concentration.

Spontaneous calcium influx and its roles in differentiation of spinal neurons in culture.

Stimulation of embryonic amphibian spinal neurons has been shown to produce calcium-dependent action potentials of long duration at early stages of development. These impulses become brief and sodium-dependent upon further differentiation. The neurons are now shown to exhibit spontaneous, transient elevations of intracellular calcium in culture during the early developmental period when activity produces greatest calcium influx. Removal of extracellular calcium during this period alone is sufficient to perturb differentiation, and influx through voltage-dependent calcium channels is shown to be required for standard development of neuronal phenotypes. No large changes in steady-state calcium levels occur in the cytoplasm during the maturation of cultured neurons despite a reduction of the calcium-dependent component of the impulse. Transient elevation of intracellular calcium is necessary for standard cytodifferentiation and may provide a link between electrical activity and gene expression.

Nuclear and "cytoplasmic" estrogen and progesterone receptors in squamous cell carcinoma of the cervix.

Biopsies from 131 women with squamous cell carcinoma of the cervix diagnosed between May 1983 and July 1986 were assayed for nuclear and "cytoplasmic" estrogen receptors (NER and CER). Progesterone receptors (PR) were also assayed in 45 of these tumors. About a third of the tumors contained both CER and NER. One or the other fraction contained ER in 76.9% of cases and PR in 66.6%. Although there was a trend for those women whose tumors contained CER or NER to have a better prognosis, this was not significant. There was no evidence that PR status affected the prognosis.

Acridine orange flow cytometric analysis of renal cell carcinoma. Clinicopathologic implications of RNA content.

To explore the potential role that ribonucleic acid (RNA) content analysis may have in the assessment of primary renal cell carcinomas (RCC), biparametric flow cytometric (acridine orange) measurements for DNA/RNA were obtained on 108 fresh neoplastic specimens. RNA content was divided into low and high groups, based on the average RNA content in normal kidney controls. High RNA content was significantly correlated with aneuploidy, high proliferative index, high nuclear grade, cytoplasmic granularity, and large tumor size. No correlation was found between RNA content and patients' sex, race, and clinical stage of the carcinomas. When diploid RCCs were separately analyzed, high RNA content was correlated with high nuclear grade, large tumor size, high clinical stage, and cytoplasmic granularity. There was no correlation between RNA content and the patient's sex or race or the neoplasm's proliferative index. Of the 16 patients that relapsed (5 diploid and 11 aneuploid), four of the diploid and all 11 aneuploid neoplasms displayed high RNA content. The authors' data show that RNA may be a valuable objective and quantitative parameter in the clinicopathologic assessment of RCC.

Immunogold probing of platelet factor 4 in different ploidy classes of rat megakaryocytes sorted by flow cytometry.

Different ploidy classes of rat megakaryocytes were sorted by flow cytometry from highly purified perfusion-fixed megakaryocyte cell suspensions prepared by sequential centrifugal elutriation and Percoll gradient centrifugation. Sorted cell populations were studied for the localization of platelet factor 4 (PF-4) probed with the monoclonal antibody 2E7 in order to clarify the relevance of PF-4 localization to the cytoplasmic and nuclear development of megakaryocytes. The relative numbers of labeled alpha granules and labeled alpha granule-related small vesicular structures (AGR-SVS) were quantitated using the gold-labeled antibody detection method and correlated with DNA content and cytoplasmic maturation in individual megakaryocytes. We determined that the stage of cytoplasmic maturation exerted a significant effect on the proportion of labeled alpha granules and labeled AGR-SVS. A significant interaction effect of stage and ploidy class resulted in the stage effect on proportion of labeled alpha granules being significant only in two of the three ploidy classes. The least mature cells present within each ploidy group exhibited PF-4 labeling mostly in SVS that were not related to alpha granules. During subsequent cytoplasmic maturation, more of the labeled SVS were seen related to alpha granules, with more of the mature alpha granules themselves becoming labeled. Polyploidization also affected the proportion of labeled AGR-SVS. Our data suggest that SVS play a role in the intramegakaryocytic transport of PF-4 into alpha granules. These data provide evidence of the complexity of megakaryocytic differentiation involving both cytoplasmic maturation and nuclear endoreduplication as reflected in PF-4 expression.

Ubiquitin in Chlamydomonas reinhardii. Distribution in the cell and effect of heat shock and photoinhibition on its conjugate pattern.

Ubiquitin, a highly conserved 76-amino-acid protein, is involved in the response of many types of eukaryotic cells to stress but little is known about its role in lower plants. In the present study we have investigated the distribution of ubiquitin in the unicellular alga Chlamydomonas reinhardii as well as the effect of heat and light stress on its conjugation to cellular proteins. Immunoelectron microscopy shows that ubiquitin is located in the chloroplast, nucleus, cytoplasm, pyrenoid and on the plasma membrane. The location of ubiquitin within chloroplasts has not been observed previously. In immunoblots of whole cell extracts with an antibody to ubiquitin a prominent conjugate band with an apparent molecular mass of 29 kDa and a broad region of high-molecular-mass conjugates (apparent molecular mass greater than 45 kDa) were observed. Exposure of cells to a 41.5 degrees C heat shock in both the dark and light caused the disappearance of the 29-kDa conjugate and an increase in the high-molecular-mass conjugates. After step down to 25 degrees C the 29-kDa conjugate reappeared while the levels of high-molecular-mass conjugates decreased. In light, the recovery of the 29-kDa band was more rapid than in the dark. Photoinhibition alters the ubiquitin conjugation pattern similarly to heat shock, but to a lesser degree. These observations imply that, in Chlamydomonas, ubiquitin has a role in the chloroplast and in the response to heat and light stress.

Polarization of gelsolin and actin binding protein in kidney epithelial cells.

Vasopressin regulates transepithelial osmotic water permeability in the kidney collecting duct and in target cells in other tissues. In the presence of hormone, water channels are inserted into an otherwise impermeable apical plasma membrane and the apical surface of these cells is dramatically remodelled. Because cytochalasin B and D greatly reduce the response of these cells to vasopressin, actin filaments are believed to participate in the events leading to an increase in transepithelial water permeability. Modulation of the actin filamentous network requires the concerted action of specific actin regulatory proteins, and in the present study we used protein A-gold immunocytochemistry to localize two important molecules, gelsolin and actin binding protein (ABP), in epithelial cells of the kidney inner medulla. Gelsolin and, to a lesser extent, ABP were concentrated in clusters in the apical cell web of principal cells of the collecting duct. Aggregates of gold particles were often associated with the cytoplasmic side of plasma membrane regions forming surface extensions or microvilli. The basolateral plasma membrane was labeled to a much lesser extent than the apical plasma membrane. In the thin limbs of Henle, ABP was localized over the apical plasma membrane in ascending limbs, but gelsolin labeling was weak in these cells. In thin descending limbs, the pattern of labeling was completely reversed, with abundant apical gelsolin labeling but only weak ABP immunolabeling. Although the significance of the distribution of actin regulatory proteins in thin limbs is unknown, the abundance and the predominantly apical polarization of both ABP and gelsolin in principal cells of the collecting duct is consistent with a role of the actin cytoskeleton in the mechanism of vasopressin actin.

Cytologic assessment of nuclear and cytoplasmic O-linked N-acetylglucosamine distribution by using anti-streptococcal monoclonal antibodies.

Recent studies have demonstrated the existence of single O-linked N-acetylglucosamine (O-GlcNAc) residues on cytoplasmic and nuclear glycoproteins. Labeled lectin and enzymatic techniques have been used to identify O-GlcNAc-bearing proteins, but no antibodies generally reactive with such O-linked GlcNAc moieties have been described. We have previously characterized monoclonal antibodies (mAbs) specific for the GlcNAc residues of streptococcal group A carbohydrate, which is composed of a polyrhamnose backbone with GlcNAc side chains. We now report that these mAbs recognize O-GlcNAc-bearing proteins. By immunofluorescence, the mAbs reacted strongly with the nuclear periphery and nucleoplasm of mammalian cells and stained the cytoplasm less intensely. The distribution was not consistent with labeling of the endoplasmic reticulum, Golgi complex, or plasma membrane. Furthermore, the staining pattern of a mutant cell line, which retains terminal GlcNAc residues on many N-linked glycans, was indistinguishable from that of wild-type cells. Nuclear and cytoplasmic staining were inhibited by free GlcNAc and were completely abolished by galactosylation of terminal GlcNAc residues. Indirect ELISA demonstrated GlcNAc- and galactosylation-inhibitable binding of the mAbs to a 65-kDa human erythrocyte cytosolic protein known to contain O-GlcNAc. Thus, these mAbs react with O-GlcNAc without apparent influence of peptide determinants, do not show detectable binding to N- or O-glycans, and, therefore, represent a valuable tool for the study of O-GlcNAc moieties. In addition, these mAbs provide the first cytologic analysis of the distribution of O-GlcNAc residues throughout the nucleus and the cytoplasm of mammalian cells.

Distribution of beta-human chorionic gonadotropin-positive cells in noncancerous gastric mucosa and in malignant gastric tumors.

The authors examined the localization and behavior of beta-human chorionic gonadotropin (HCG)-positive cells in human gastric noncancerous mucosa and in gastric malignant tumors, using immunohistochemistry and the anti-beta-HCG antibody. The beta-HCG-positive cells were located mainly in the antral mucosa and were generally restricted to the neck portion of the pyloric glands, although a few were present in fundic glands of the gastric body. The beta-HCG-immunoreactive cells were found in gastric carcinomas in 53% of the 92 cases examined. These cells were observed more often in advanced carcinomas that were histologically poorly differentiated than in early carcinomas or in well-differentiated tumors, but this prevalence had no statistical significance. The presence of the beta-HCG-positive cells in the gastric carcinomas suggested no appreciable prognostic significance, even quantitatively. In the syncytiotrophoblast-like tumor cells seen in four gastric tumor samples with histologic features of a choriocarcinoma, immunoreactivity to the beta-HCG was striking. There was, however, no recognizable dominance in the number of beta-HCG-reactive cells in the noncancerous mucosa around the tumor.

Early and late responses to induction of rasT24 expression in Rat-1 cells.

We have used a series of Rat-1 cell lines carrying a Zn-inducible human c-Ha-ras oncogene construction (MTrasT24) to evaluate the effect of varied ras oncogene expression on the expression of genes and proteins related to morphologic transformation in vitro. In response to the expression of the ras oncogene, at least two different classes of events occur. These events, referred to as 'early and late' events, are dependent on distinctively different accumulated levels of the ras oncoprotein. Relatively low levels of activated c-Ha-ras p21 protein (1.5-2.5 times the proto-oncogene level) stimulate rapid entry of quiescent (G0) cells into the cell cycle and result in increased steady state c-myc and glucose transporter mRNA levels which are detectable as early as 3-6 h after zinc addition. In contrast, morphologic transformation develops more slowly and does not appear until 72-96 h after Zn++ stimulation in cells with very low basal levels of activated p21 (MR4 cells) and 24-48 h in cells with higher basal levels (MR5 cells). These morphologic changes depend on the accumulation of significant amounts of the ras oncoprotein (greater than 4 to 5 times the proto-oncogene level) and are accompanied by large increases in the steady state mRNA levels of transin and TGF-alpha and decreases in PDGF-receptor mRNA and fibronectin protein and mRNA levels. In addition, the level of a novel cytoplasmic protein species (referred to as p29), which is stained by a monoclonal antibody for ras, is dramatically reduced in response to these levels of activated ras protein. Thus changes in morphology and gene expression induced by rasT24 occur sequentially and are quantitatively dependent on activated ras expression.

Microtubule-associated protein 2 (MAP2) in Purkinje cell dendrites: evidence that factors other than binding to microtubules are involved in determining its cytoplasmic distribution.

We have studied the distribution of microtubule-associated protein 2 (MAP2) in the Purkinje cell dendrites of rats whose cerebella were exposed to X-irradiation during the second postnatal week. The Purkinje cells of such animals have abnormally elongated apical primary processes that branch in the other molecular layer rather than close to the cell body as in normal tissue. The results show that in these distorted dendrites the MAP2 distribution is "shifted" distally relative to the normal pattern, in which MAP2 is distributed evenly throughout the dendritic tree. Tubulin and other microtubule-associated proteins, such as MAP1, are not affected and remain evenly distributed throughout the dendritic tree despite the anatomical distortion. We conclude that the distribution of MAP2 in Purkinje cells is not determined solely by its binding to tubulin. Other factors must be involved and these appear to be related to dendritic morphology and possibly to branching.

Continuous fluorimetric assessment of the changes in cytoplasmic calcium concentration during exposure of rat isolated myocardium to conditions of simulated ischaemia.

1. The cytoplasmic calcium concentration ([Ca]c) of rat isolated atrial myocardium was assessed with the dye indo-1. Dye-loaded atria were superfused with physiological salt solution and excited with radiation at 360 nm, while epifluorescence emissions were collected simultaneously at 400 nm and 500 nm. The ratio of these emissions was used as a measure of [Ca]c. 2. Dye-loaded atria showed a phasic rise and fall in [Ca]c with each applied electrical pacing stimulus. The amplitudes of systolic increments in tension and [Ca]c were augmented by the presence of isoprenaline. 3. Atria superfused with a solution the composition of which resembled that found extracellularly in regions of ischaemia rapidly lost systolic increments in tension and [Ca]c, while end-diastolic [Ca]c and tension gradually rose. 4. The presence of lactate (20 mM) or flufenamate (5 microM) in the superfusate during simulated ischaemia aggravated the rises in both end-diastolic tension and end-diastolic [Ca]c. Inclusion in the superfusate of sulphinpyrazone (50 microM) or glucose (20 mM) protected against some of the deleterious effects of lactate seen during simulated ischaemia.

Myxoid meningioma.

A mucosubstance-rich myxoid meningioma that recurred twice is reported. This rare form of meningioma is a potential source of confusion with other myxoid neoplasms such as metastatic adenocarcinoma or chordoma. In addition to the usual stigmata of meningial cell differentiation, ultrastructural examination revealed spaces delineated by a network of cellular processes and enclosing loose granular and fibrillar material. This neoplasm is probably linked to the so-called microcystic meningioma but has an overt production of acid mucosubstance.

Cytoplasmic components of natural killer cells limit the growth of Cryptococcus neoformans.

Murine natural killer (NK) cell-mediated inhibition of growth of a yeast-like target cell, Cryptococcus neoformans, was completely abrogated by blocking the effector cell secretory process with monensin. Therefore, further studies were performed to determine the ability of various cytoplasmic fractions of NK cells to mediate inhibition of cryptococcal growth. Percoll-fractionated homogenates of rat LGL tumor cells demonstrated that the granule-containing fractions plus three additional sets of less dense cytoplasmic fractions displayed anti-cryptococcal activity; whereas only the cytoplasmic granule-containing fractions had cytotoxic activity against YAC-1 tumor cell and sheep erythrocyte targets. Maximal cryptococcal growth inhibition induced by LGL granules occurred after a 1 h incubation, required the presence of Ca2+ (1.0 mM) or Mg2+ (0.5 mM or 5.0 mM), and was completely abrogated in the presence of rabbit anti-LGL granule IgG. Cytolysin, the granule component which mediates tumor cell and sheep erythrocyte lysis, effectively limited the growth of cryptococci. Since Percoll gradient fractionation of the LGL homogenates demonstrated three separate peaks of anti-cryptococcal activity other than the granule peak, it is possible that the cytolysin-containing granules are not the only subcellular component of NK cells playing a role in inhibition of C. neoformans growth.

Expression of platelet glycoprotein Ib by cultured human megakaryocytes: ultrastructural localization and biosynthesis.

Glycoprotein Ib (GPIb), the receptor for von Willebrand factor, is a two-chain member constituent of the platelet/megakaryocytic lineage. Studies on its expression have been hampered by the difficulties in obtaining purified megakaryocytes in a sufficient number. We report a suspension liquid culture procedure that allowed isolation of more than 1 x 10(6) megakaryocytes with a purity ranging from 3% to 88% from the blood of patients with chronic myeloid leukemia, from fetal liver or from normal human bone marrow. GPIb was detected on the plasma membrane of all maturing megakaryocytes and also of promegakaryoblasts devoid of demarcation membranes. GPIb was detected on demarcation membranes of maturing megakaryocytes but was absent from all other organelles, including alpha granules. Biosynthesis of 35S-methionine labeled megakaryocytes showed that GPIb with similar electrophoretic mobility to the platelet molecule was synthesized and that it was also composed of two chains, since its molecular weight shifted in reducing conditions from 170 Kd to 145 Kd. The beta chain remained undetectable after methionine metabolic labeling, but it was immunoprecipitated after 3H-leucine metabolic labeling, confirming that this subunit is devoid of methionine. GPIb was associated with GPIX, as it is in platelets, since anti-GPIb antibodies coprecipitated a 17 Kd polypeptide.

Autoradiographic localization of [3H]RU 486 and [3H]progesterone in the uterus, pituitary and hypothalamus of the rat.

Twenty-one castrated oestrogen-primed Wistar rats, which were 2-months-old, were injected via the jugular vein with 100 mu Ci/100 g body weight of [3H]RU 486 or [3H]progesterone. Some of these received unlabelled compounds for competition studies. Samples of reproductive tract, pituitary and hypothalamus were excised after 15 min. The 4-microns frozen sections were processed for thaw-mounted autoradiography. The exposure time of the autoradiogram was approximately 6 months. After the injection of [3H]RU 486 and [3H]progesterone, the nuclear concentration of radioactivity was most distinct in muscular and stromal cells of the uterus, and the epithelial nuclei of lumina and glands showed weak labelling. Nuclear localization was also observed in muscle cells of the vagina, cervix and oviduct. After injection of [3H]progesterone, the radioactivity was found in the nuclei and cytoplasm of anterior pituitary cells and some cells showed a preferential nuclear concentration of radioactivity. The distribution of [3H]RU 486 in the anterior pituitary was more extensive than that of [3H]progesterone. In the hypothalamus, specific localization of [3H]RU 486 and [3H]progesterone existed in neurones accumulated in the preoptic nucleus, preoptic suprachiasmatic nucleus and the periventricular nucleus. No localization was found in the diaphragm. Pretreatment with RU 486, but not with dexamethasone, reduced the nuclear concentration of radioactivity of [3H]progesterone in the vagina, uterus, oviduct, pituitary and hypothalamus. The nuclear concentration of radioactivity after injection of [3H]RU 486 was also decreased by preinjection with progesterone. The autoradiographic results suggest that RU 486 and progesterone competed for the specific binding site (possibly a progesterone receptor) in the target cells at the levels of the uterus, pituitary and hypothalamus in vivo.