The cell kinetics of the adaptation of the human esophagus to organ culture

Summary The adaptation of normal human esophageal explants to organ culture for the first 33 d of in vitro growth was evaluated using histomorphology and [³H]TdR autoradiography combined with mitotic blockade. On the 3rd d in culture, extensive desquamation of superficial cells reduced the epithelium to about four cell layers. Thereafter, the epithelium remained atrophic, with a relative increase in basal and suprabasal cells. The percentage of cells synthesizing DNA was greatest from Day 4 through 8, just after desquamation, and reached a maximum on Day 4 (24 h [³H]TdR labeling index of 62%). The labeling index (LI) fluctuated, thereafter, but remained high (26% on Day 33). During the last 6 h of each [³H]TdR labeling interval, mitosis was blocked by colcemid. The 6 h mitotic rate (MR) was a reasonably constant fraction of the LI (maximum at 4 d: MR=1.44%), but was much lower than predicted by [³H]TdR labeling indicating the loss of large numbers of cells after DNA synthesis but before or during mitosis. Unlabeled mitotic figures appeared between Days 1 to 3 and 6 to 33, suggesting that the epithelium initially contained a considerable population of cells arrested or delayed in G₂ and continued to generate cells that remained in premitosis longer than 24 h. These results indicate that the atrophy observed in vitro is characterized by a relative increase in the basal and suprabasal cell category, a high replication rate, initial recruitment of cells arrested in premitosis, and rapid cell turnover with significant loss of cells at the premitotic or mitotic step, or both. Thus it seems that human esophageal epithelium grown in organ culture is a satisfactory substrate for experimentation (for example, in vitro carcinogenesis) that requires cell replication. However, there are major differences between the kinetics of esophageal epithelium in vivo and in vitro. Supported in part by Contract NOI-CP-75909 and NOI-CP-25604-59 from the National Cancer Institute, Bethesda, MD.     

Changes of colon epithelium proliferation due to individual aging with cyclin proliferating cell nuclear antigen (PCNA/cyclin) immunostaining compared to [3H]-thymidine radioautography

We report a change in the proliferative activity of mouse colonic epithelium due to development and aging. In order to measure the proliferative activity, colonic epithelium was immunostained for cyclin proliferating cell nuclear antigen (PCNA/cyclin), which appears from the Gl to the S phase of the cell cycle, and compared with labeling obtained by [³H]-thymidine radioautography. Litter mice of six age groups from the fetal period (embryonic day 19), newborn period (postnatal day 1), suckling period (postnatal day 5), weaning period (postnatal dy 21), adult period (2 month old) to the senescent period (11 month old) were examined by immunohistochemistry. The descending colons were fixed in methacarn (method-Carnoy) and embedded in paraffin. Sections were stained for PCNA/cyclin activity using 19A2 monoclonal antibody and the avidin-biotin peroxidase complex (ABC) technique. For radioautography, litter mice of nine age groups using in vivo intraperitoneal administration of [³H]-thymidine. The labeling indices of colonic epithelial cells in the proliferative zone were then analyzed and compared between the two investigative methods. Our results show that the prliferative activity of mice colon was high in the fetal and newborn periods and almost constant from the suckling period to senescence, as demonstrated by both PCNA/cyclin immunohistochemistry and [³H]-thymidine radioautography. The labeling index seen by PCNA/cyclin immunohistochemistry was, however, higher than that seen by [³H]-thymidine radioautography.     

Effects of cordycepin on the synthesis of nuclear and cytoplasmic RNAs in embryonic cells of Xenopus laevis

Effects of cordycepin on the incorporation of [3H] guanosine into embryonic Xenopus cells were examined. Cordycepin inhibited the labeling not only of poly(A) + RNA, but of all the other major classes of RNAs. Cellular fractionation showed that this inhibition was much stronger in the labeling of cytoplasmic RNAs than of nuclear RNAs. [3H]Cordycepin was incorporated into both poly(A) + RNA and other RNA species.     

Autoradiographic localization of specific [3H]dexamethasone binding in fetal lung

The cellular and subcellular localization of specific [3H]dexamethasone binding was examined in fetal mouse lung at various stages of development and in human fetal lung at 8 weeks of gestation using a rapid in vitro steroid incubation technique followed by thaw-mount autoradiography. Competition studies with unlabeled steroids demonstrate the specificity of [3H]dexamethasone labeling, and indicate that fetal lung mesenchyme is a primary glucocorticoid target during lung development. Quantitative binding studies, involving incubation of intact tissue with competing ligand and subsequent subcellular fractionation, show this to be specific, nuclear binding characteristic of glucocorticoid receptors. Autoradiographs of [3H]dexamethasone binding in lung tissue at early stages of development demonstrate that the mesenchyme directly adjacent to the more proximal portions of the bronchiolar network is heavily labeled. In contrast, the epithelium which will later differentiate into bronchi and bronchioles, is relatively unlabeled. Distal portions of the growing epithelium, destined to become alveolar ducts and alveoli, do show nuclear localization of [3H]dexamethasone. Because of the known importance of the mesenchyme in controlling lung development and the ability of glucocorticoids to stimulate lung development, these results suggest that many of the growth-promoting effects of glucocorticoids may be mediated through the mesenchyme. In addition, by utilizing a technique which allows the simultaneous examination of extracellular matrix components and [3H]dexamethasone binding, a relationship is observed between extensive mesenchymal [3H]dexamethasone binding and extensive extracellular matrix accumulation. Since glucocorticoids stimulate the synthesis of many extracellular matrix components, these results suggest a role for these hormones in affecting mesenchymal-epithelial interactions during lung morphogenesis.     

Localization of the estrogen receptor in uterine cells by affinity labeling with [3H]tamoxifen aziridine

The possibility that estrogen receptors may exist in uterine plasma membranes was investigated by covalent labeling of estrogen receptors in mouse uterine cells with [3H]tamoxifen aziridine (TA). Isolated epithelial and stromal cells of immature mice were incubated with [3H]TA in the presence or absence of unlabeled tamoxifen, homogenized and separated into nuclear, cytosolic and microsomal fractions by differential centrifugation. These fractions were subjected to SDS-polyacrylamide gel electrophoresis and the proteins labeled covalently with TA were visualized by autoradiography. Proteins labeled specifically with [3H]TA were observed almost exclusively in the nuclear fraction of both epithelial and stromal cells. In contrast, very little labeled protein was detected in the cytosolic or microsomal fraction. Although these data do not preclude the possibility that estrogen binding sites are present in plasma membranes of uterine cells, this cellular fraction is definitely not labeled to a significant extent by [3H]TA. Thus, if membrane estrogen binding sites exist, their structural conformations may be different from that of nuclear estrogen receptors.     

No evidence for aromatization of [3H]testosterone in oestrogen receptor containing cells of the epididymis

Oestrogen receptors are found in the principal cells of the caput and in apical and clear cells of the epididymis of the mouse. The distribution pattern of oestrogen receptors is different from that of androgen receptors and suggests a physiological role for oestrogens in the epididymis. We examined by competition experiments and thaw-mount autoradiography to see whether aromatization of [3H]testosterone is the source of oestrogens in the epididymis. After injection of [3H]testosterone we found the same labeling pattern as after non-aromatizable [3H]dihydrotestosterone. In particular, apical and clear cells showed a low or no nuclear concentration of radioactivity as with [3H]dihydrotestosterone. Competition with oestradiol had no effect on the binding pattern of [3H]testosterone in the epididymis in contrast to its effects in the brain of the same animals. Competition with dihydrotestosterone abolished labeling in contrast to the brain, where no effect was observed. Thus no aromatization of [3H]testosterone to oestrogens but conversion to dihydrotestosterone seems to occur in the epididymis.     

An autoradiographic study of the origin of intestinal blastemal cells in the newt, Notophthalmus viridescens.

Fifty adult newts were used in this investigation; in 44 animals, the intestine was transected perpendicular to its longitudinal axis approximately midway between pylorus and rectum. The free ends of the intestine were held in apposition with a single suture and replaced into the coelom. The animals were injected intraperitoneally with [³H]thymidine from 0 to 35 days after transection of the intestine and killed 6 hr later. In nontransected, control intestines, the only tissue that incorporated [³H]thymidine was the mucosal epithelium. In transected intestines, only the mucosal epithelium labeled in animals which had been injected with [³H]thymidine from 0 to 4 days after the intestine was incised. Later on, serosal cells and smooth muscle cells of the intestinal stump underwent morphological alteration, initiated the incorporation of [³H]thymidine into DNA, and began replication. At 6 days after transection, serosal cells adjacent to the plane of transection were incorporating [³H]thymidine and, at 12 days, smooth muscle cells at the transected surface were labeling. It seems probable that they both furnished cells to the intestinal blastema; the lining epithelium of the mucosa, however, did not appear to contribute to the blastema proper.     

Kinetics of cell replacement in the stratum granulosum of mouse tongue epithelium

Sheet preparations of the stratum granulosum from the epithelium of the ventral surface of mouse tongue permit examination of cell replacement of this maturation compartment of the tissue. The cell transit rate/day is related to the cell desquamation rate and the cell production rate. The latter is approximately 6500-8000 cells/mm2/day, suggesting a 4-5-fold greater turnover compared with mouse dorsal skin epithelium. The use of [3H]IUdR and [3H]TdR at different times of day provides evidence for a reutilization of label from [3H]TdR released during nuclear degradation in the stratum granulosum. Flooding with unlabelled thymidine is not effective in suppressing this reutilization.     

Patterns of cell differentiation revealed by L-[3H]fucose incorporation in Dictyostelium

A study of the incorporation of l-[6-³H]fucose and d-[6-³H]glucosamine hydrochloride was conducted during the development of the cellular slime mold Dictyostelium discoideum 1-H. Autoradiographs revealed that pulse-labeled vegetative amoebae incorporated [³H]fucose intracytoplasmically within 15 min. The majority of the cells had randomly scattered silver grains but the remainder were distinguished by a dense localized labeling which suggested that oligo or polysaccharide synthesis was occurring. The localized pattern of labeling attributed to active synthesis declines at aggregation and early conus formation. As the pseudoplasmodium makes the developmental transition from the conus to the culmination stages the localized pattern of [³H]fucose labeling was restricted to the prespore cells while the prestalk cells were devoid of label. Prespore vacuoles were not present at the onset of this transition and consequently [³H]fucose incorporation occurred in the cells prior to their differentiation into prespore cells. In contrast to cells composing earlier stages, mature spores exhibited [³H]fucose-containing substances at the cell surface. At appropriate stages certain cells actively synthesize slime and stalk sheath which were labeled with either [³H]fucose or [³H]glucosamine.Prestalk isolates were obtained by transecting migrating slugs. [³H]Fucose was incorporated within 10 min among the basal cells of the isolate in the localized pattern typically found in prespore cells. The incorporation of [³H]fucose occurred prior to prespore differentiation as certain preparations were devoid of prespore vacuoles. Prespore isolates differentiate prestalk cells which have lost the capacity to incorporate [³H]fucose. This investigation suggests that cell contacts and interactions may affect the incorporation of [³H]fucose.     

Differential distribution of dihydrotestosterone and estradiol binding sites in the epididymis of the mouse. An autoradiographic study

The distribution of androgen and estrogen binding sites in the mouse epididymis was assessed by autoradiography with 3H dihydrotestosterone (3H DHT) and 3H estradiol (3H E2). Nuclear labeling with 3H DHT in principal cells of the epithelium is high in the caput, low in the corpus, and high again in the cauda. 3H E2 also binds to the nuclei of principal cells. The pattern is distinct from 3H DHT: nuclear labeling is highest in the ductulus efferens and high in the caput, but low or absent in corpus and cauda. Apical cells in caput and clear cells in corpus and cauda are moderately labeled with 3H DHT but heavily labeled with 3H E2. Connective tissue cells show variable labeling with both hormones, being more pronounced with 3H E2. Smooth muscle cells are also labeled to varying degrees with both hormones. The different binding patterns of 3H DHT and 3H E2 and the results of the competition studies with unlabeled compounds demonstrate that in the epididymis besides the specific nuclear receptors for androgen also estrogen receptors are present.     

Estrogen receptors, progestin receptors and DNA synthesis in the macaque endometrium during the luteal-follicular transition

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.     

Angiotensin II increases diacylglycerol in calf adrenal glomerulosa cells by activating de novo phospholipid synthesis

Effects of angiotensin II (AII) on diacylglycerol (DAG) synthesis were examined in calf adrenal glomerulosa cells. AII provoked rapid increases in [3H]glycerol-labeling and content of DAG. Effects on [3H]glycerol-labeling of DAG were observed both in cells prelabeled with [3H]glycerol for 60 minutes, and when AII and [3H]glycerol were added simultaneously. Increases in [3H] DAG labeling were associated with increases in total glycerolipid labeling, and in simultaneous addition experiments, were preceded by increased [3H] phosphatidic acid (PA) labeling. Labeling of glycerol-3-PO4, on the other hand, was not increased by AII, suggesting that increases in lipid labeling were not due to prior increases in precursor specific activity. ACTH, which does not increase the hydrolysis of inositol-phospholipids appreciably in this tissue, provoked increases in content and [3H]glycerol-labeling of DAG, which were only slightly less than those provoked by AII. Thus, part of the AII-induced increase in DAG may also be derived from sources other than inositol-phospholipids. Moreover, AII-induced increases in DAG appear to be at least partly derived from increased de novo synthesis of PA.     

Endocytosis of exogenous GM1 ganglioside and cholera toxin by neuroblastoma cells.

Cholera toxin (CT) covalently linked to horseradish peroxidase (HRP) is a specific cytochemical marker for its receptor, the monosialoganglioside GM1. The binding and endocytosis of exogenous [3H]GM1 by cultured murine neuroblastoma cells (line 2A [CCl-131] ), which contain predominantly GM3, was examined by quantitative electron microscope autoradiography. The relationship between exogenous receptor, [3H]GM1, and CT HRP was studied in double labeling experiments consisting of autoradiographic demonstration of [3H]GM1 and cytochemical visualization of HRP. Exogenous [3H]GM1 was not degraded after its endocytosis by cells for 2 h at 37 degrees C. Quantitative studies showed similar grain density distributions in cells treated with [3H]GM1 alone and in cells treated with [3H]GM1 followed by CT-HRP. Qualitative studies conducted in double labeling experiments showed autoradiographic grains over the peroxidase-stained plasma membrane, lysosomes, and vesicles at the trans aspect of the Golgi apparatus. The findings indicate that exogenous glycolipid is associated with the plasmid membrane of deficient cells and undergoes endocytosis. The quantitative ultra-structural autoradiographic studies are consistent with the hypothesis that the spontaneous endocytosis of exogenous [3H]GM1 controls the subsequent uptake of CT-HRP.     

Stimulation of facilitated [3H]uridine transport by thyroid hormone in GH1 cells. Evidence for regulation by the thyroid hormone nuclear receptor

We have previously shown that 3,5,3'-triiodo-L-thyronine (L-T3) stimulates cell growth and a 4- to 8-fold increase in growth hormone mRNA in GH1 cells. These effects appear to be mediated by a thyroid hormone nuclear receptor with an equilibrium dissociation constant for L-T3 of 0.2 nM and an abundance of about 10,000 receptors per cell nucleus. In this report, we show that L-T3 exerts a pleiotypic effect on GH1 cells to rapidly (within 2 h) stimulate [3H]uridine uptake to a maximal value of 2.5- to 3-fold after 24 h. This results from an increase in the number of functional uridine "transport sites" as shown by studies documenting an increase in the apparent Vmax with no change in the Km, 17 microM. Although the labeling of the cellular uridine pool and pools of all phosphorylated uridine derivatives was increased by L-T3, there was no change in the relative amounts of the individual pools in cells incubated with or without hormone. The intracellular concentration of [3H]uridine was estimated to be similar to that of the medium, suggesting that facilitated transport mediates [3H]uridine uptake. That this increase in [3H]uridine transport was nuclear receptor-mediated is supported by the excellent correspondence of the L-T3 dose-response curve for [3H]uridine uptake and that for L-T3 binding to receptor. Finally, inhibition of protein synthesis by cycloheximide and RNA synthesis by actinomycin D demonstrated that the L-T3 effect required continuing protein and RNA synthesis. These results are consistent with an effect of the L-T3-nuclear receptor complex to increase uridine uptake in GH1 cells by altering the expression of gene(s) essential for the transport process.     

Localization of the lipid intermediate pathway of protein glycosylation in oviduct cell types

Oviduct tissue slices were incubated with [3H]-leucine or [3H]-mannose in the presence and absence of tunicamycin, a specific inhibitor of lipid-mediated protein glycosylation. Conditions were established where tunicamycin had maximal effect on [3H]-mannose incorporation (greater than 90% inhibition) but a minimal effect on [3H]-leucine incorporation (less than 10% inhibition) into total TCA-insoluble products. Analysis of incubated tissues by SDS-polyacrylamide gel electrophoresis revealed that in the absence of tunicamycin, [3H]-mannose was incorporated into only a few proteins, of which ovalbumin represented the major radiolabeled component. Tunicamycin markedly reduced the incorporation of [3H]-mannose into ovalbumin and other oviduct glycoproteins. In contrast, analysis by SDS-polyacrylamide gel electrophoresis showed that [3H]-leucine was incorporated into a variety of proteins in the absence of tunicamycin. The radioactivity profile of some of these proteins was shifted toward lower Mr when oviduct slices were incubated in the presence of tunicamycin, with only a minimal decrease in protein labeling. Light microscopic autoradiograms of tissue incubated with [3H]-leucine in either the presence or absence of tunicamycin exhibited extensive labeling of tubular gland and epithelial cells. In the absence of tunicamycin, these cell types also become markedly labeled with [3H]-mannose; however, incorporation of label in both cell types was substantially reduced in the presence of tunicamycin. Qualitatively, labeling of tubular gland cells appeared greater than that of epithelial cells, largely due to the concentration of silver grains over the dense population of secretory vesicles in the tubular gland cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

BrdUrd labeling of S-phase cells in testes and small intestine of mice, using microwave irradiation for immunogold-silver staining: an immunocytochemical study.

In this study, BrdUrd labeling of S-phase cells in the small intestine and testes was accomplished using microwave irradiation. In this way crypt cells, spermatogonia, and Leydig cells could be labeled using removable plastic-embedded sections and immunogold-silver staining (IGSS). By using short periods of microwave irradiation for incubation of the monoclonal antibodies and the protein A-colloidal gold solution, the detection of BrdUrd-labeled cells could be remarkably enhanced. A comparative study of BrdUrd labeled spermatogonia in the testis of a Cpb-N mouse that received both [3H]-thymidine and BrdUrd proved that 90% of the BrdUrd-labeled cells also showed [3H]-thymidine labeling. The radioactive [3H]-thymidine labeling was a time-consuming method of 4 weeks' duration, whereas the BrdUrd-labeled cells could be labeled, fixed, enhanced, and counterstained in less than 3 hr. This investigation proves that BrdUrd labeling of S-phase cells can be a reliable, reproductive, rapid, and non-radioactive alternative method for [3H]-thymidine labeling of proliferating cells.     

Role of RNA and protein synthesis and turnover in the heat-induced increase in nuclear protein

The proteins which become associated with nuclei during hyperthermic exposure were characterized by labeled amino acid incorporation. Actinomycin-D (Act-D) or cycloheximide (CHM) pretreatment was used to determine whether concurrent RNA or protein synthesis is required for hyperthermia to induce the increase in nuclear protein content. Prior to heat exposure exponentially growing HeLa cells were (i) pulse labeled for 1 h, (ii) labeled for 36 h, or (iii) labeled for 24 h followed by 17 h chase. The nuclear specific activity (CPM/microgram protein) of [3H]lysine-labeled proteins did not change under any of the labeling conditions, whereas that of [3H]leucine-containing proteins increased significantly with (i) but not with (ii) or (iii), while that of [3H]tryptophan-labeled protein increased significantly with (i) and (ii) but not with (iii). Act-D treatment 1 h prior to and during heating did not affect nuclear protein increase, while CHM-treated cells showed generally less nuclear protein content (70% of control at 60 min) but nevertheless significant nuclear protein increase upon heating (60% increase at 60 min from 0 min). These results suggest that those proteins associated with nuclei following heat exposure are nonhistones with a high turnover rate, and the process dose not require the synthesis of RNA or proteins.     

The glucocorticoid antagonist 17 alpha-methyltestosterone binds to the 10 S glucocorticoid receptor and blocks agonist-mediated dissociation of the 10 S oligomer to the 4 S deoxyribonucleic acid-binding subunit

The glucocorticoid antagonist 17 alpha-methyltestosterone inhibits binding of the agonist [3H]triamcinolone acetonide ot the glocucorticoid receptor in cytosol prepared from rat pituitary tumor GH1 cells. Competitive binding studies indicate that the dissociation constant for 17 alpha-methyltestosterone is about 1 microM. After incubation of intact GH1 cells with 10 nM [3H]triamcinolone acetonide at 37 C and subsequent cell fractionation at 4 C, three glucocorticoid receptor forms are observed: cytosolic 10 S receptor, cytosolic 4 S receptor, and nuclear receptor. Concurrent incubation with 17 alpha-methyltestosterone reduces the amount of [3H]triamcinolone acetonide bound to each of these receptor forms. Ligand-exchange assays performed at 0 C in intact cells using [3H]triamcinolone acetonide show that the exchangeable antagonist is associated predominantly with cytosolic 10 S receptor. Immunochemical analysis using monoclonal antibody BuGR2 indicates that 17 alpha-methyltestosterone does not cause substantial accumulation of glucocorticoid receptors in GH1 cell nuclei and, when present together with agonist, reduces nuclear accumulation of receptor seen with agonist alone. Results from dense amino acid labeling studies show that unlike [3H]triamcinolone acetonide, 17 alpha-methyltestosterone does not reduce the total amount of cellular glucocorticoid receptor and does not reduce receptor half-life. These results are consistent with a model for glucocorticoid receptor transformation in which binding of agonist promotes the dissociation of an oligomeric 10 S cytosolic receptor protein to its DNA-binding 4 S subunit. The antagonist 17 alpha-methyltestosterone competes with agonist for binding to the 10 S cytosolic receptor but does not appear to promote dissociation of the oligomer, thus inhibiting agonist-mediated nuclear actions of the glucocorticoid receptor.     

Histological,histochemical, and ultrastructural investigation of the male copulatory apparatus of Haminoea navicula (Gastropoda,Cephalaspidea)

Due to its biological and systematic importance, the morphology and function of the male copulatory apparatus of Haminoea navicula, a Cephalaspidea gastropod mollusk, was investigated by light and electron microscopy. These systems are poorly understood in haminoids, but are often used in the taxonomy of the genus. In H. navicula, the male copulatory apparatus comprises the penis within a penial sheath, a seminal duct and the prostate with two lobes. The penis is a muscular structure with a tip covered by spikes formed by muscular cells. The penial sheath consists of muscular tissue folds lined by an epithelium. Below this epithelium, polysaccharide‐secreting cells and pigment cells were observed. A large number of vacuolar cells were found below the ciliated epithelium of the seminal duct. The proximal lobe of the prostate was formed by tubules that could be divided in basal, middle and apical zones, containing cells that secrete polysaccharides and proteins. The tubules of the prostate distal lobe contained a single type of secretory cells with vesicles that were stained by histochemical techniques for detection of polysaccharides and proteins. Ciliated cells were present along the tubules in both lobes of the prostate. This study revealed a complex prostate with five types of secretory cells, which secrete substances that should be involved in the maintenance and eventually also in the maturation of spermatozoa. A comparison with previous publications, shows that the male copulatory apparatus can differ substantially among cephalaspideans, even between H. navicula and non‐European species attributed to this genus.     

Effects of Sulfhydryl Reagents on Uterine Nuclear Estrogen Receptors Labeled by in Vitro Exchange with [3H]Estradiol or [3H]4-Hydroxytamoxifen

Abstract

We have attempted to convert 4 S uterine nuclear estrogen receptors obtained after in vitro labeling with [³H]antiestrogens to 3 S, the form observed after in vitro exchange with [³H]estradiol, in order to examine the possible relationship between these forms. Treatment of nuclear extracts labeled with the high affinity antiestrogen, [³H]4-hydroxytamoxifen, with a variety of nucleases, phosphatases, or proteases either had no effect on the 4 S antiestrogen-receptor complex or led to loss of ligand binding. The sulfhydryl reducing agents, cysteine or reduced glutathione, on the other hand, brought about conversion of 4 S estrogen receptors to components sedimenting at about 3 S. Conversely, when oxidized glutathione was included in all buffers used for preparation and labeling of nuclear estrogen receptors with [³H]estradiol, more rapidly sedimenting (?4.6 S) forms of estrogen-receptor complex predominated. Cysteine still effected the 4 S to 3 S conversion when nuclear estrogen receptors, partially purified by sucrose gradient centrifugation, were used as substrate, suggesting a direct action of the sulfhydryl reagents on receptor molecules. From these results we propose that nuclear estrogen and antiestrogen-receptor complexes may differ in conformation such that the former may be more sensitive to the action of an endogenous reducing agent which contributes to formation of 3 S [³H]estradiol-receptor complexes.