REGENERATION OF UTERINE EPITHELIUM AFTER EXPERIMENTAL ABLATION IN THE RAT

Regeneration of the uterine luminal epithelium was studied after its mechanical removal in progesterone-primed rats, leaving one control horn intact. Pulse labelling with [³H]TdR during regeneration, showed a rapid peak of labelling index in remaining glands. A differentiated and highly labelled luminal epithelium reappeared at 34 hr, thereafter showing a rapidly declining LI. After initial depletion, the glandular cell population size was restored within 64 hr, whereas luminal epithelium cell numbers became stabilized at about half normal level. Grain counts after prelabelling showed more rapid dilution in gland cells of stripped uterine horns, indicating accelerated cycling of previously dividing cells. Thymidine labelling indices also showed that, after removal of the epithelium, almost all gland cells became rapidly committed to divide. On average, less than two cell cycles were necessary to restore stable glandular and epithelial population sizes. Numbers of labelled cells were also drastically increased in myometrium and serosa of treated horns. This suggests a non-specific mechanism for stimulation of mitotic activity after ablation of epithelium.     

Autoradiographic localization of platelet-activating factor (PAF) binding sites in the rabbit endometrium during the peri-implantation period

Summary This communication describes the use of in-vivo and in-vitro autoradiography to map specific platelet-activating factor (PAF) receptors in the rabbit uterus. Specific [³H]PAF uptake was predominantly localized on epithelial, but not on stromal or myometrial cells. Very few silver grains were associated with the luminal epithelial cells in the uterus of the estrous rabbit, primarily because of the non-differentiated state of the epithelium. In the differentiated pregnant uterus, significantly more [³H]PAF was bound to the glandular epithelial cells, with the stromal cells binding consistently significantly less. The highest density of silver grains was observed at the implantation sites on day 7 of pregnancy. There was no apparent difference in [³H]PAF C18:0 uptake between the epithelial cells at the inter-implantation zone on day 7 and on day 6. Bound [³H]PAF was displaceable by lyso-PAF, U66985, CV3988, but not U66982, L652,731, SRI 63,441 or the inactive PAF isomer, oleoyl PAF. Bovine serum albumin (BSA) significantly inhibited tissue uptake of [³H]PAF C18:0. Intraluminally administered [³H]PAF C18:0 and intravenously injected [³H]methylcarbamyl-PAF, a non-metabolizable PAF analog, penetrated the implanted blastocyst and bound to the embryoblast. This event was reproducible in vitro with pre-implantation blastocysts from day-6 pregnant rabbits, which suggests that uterine-derived PAF may translocate into the blastocyst after attachment.     

Autoradiographic localization of transported neutral amino acids in epithelia of cat submandibular and sublingual salivary glands

Summary Light-microscopic autoradiography was used to localize the cellular sites for neutral amino acid uptake in submandibular and sublingual salivary gland epithelia. The vasculature of isolated glands was perfused for 3–5 min with either L-(3-³H)serine or L-(4-³H)phenylalanine and then fixed by perfusion with buffered glutaraldehyde. In the submandibular gland the small neutral amino acid L-serine and the aromatic amino acid L-phenylalanine were localized to central acinar cells, demilunar cells and ductal cells. In the sublingual gland silver grains associated with each of these tritiated amino acids were localized to central acinar and ductal cells. Perfusion of both submandibular and sublingual glands with unlabelled L-serine (25 mM) or L-phenylalanine (30 mM) resulted in a significant decrease in the silver grain density associated with each labelled amino acid. The absence of silver grains in the lumina of acinar and ductal cells and the presence of tight junctions near the apical surface of the epithelium strongly suggest that the initial uptake of these amino acids was mediated by basolateral plasma membrane carriers.     

Estradiol receptor binding to the epithelium of uterine lumen and glands: region- and time-related changes during preimplantation and periimplantation periods studied by autoradiography

The presence and changes of estradiol nuclear binding and related functions in uterine luminal and glandular epithelium were studied before and after blastocyst implantation using receptor autoradiography with ³H-estradiol-17 in association with ³H-thymidine incorporation and immunocytochemical binding of antibody to estrogen receptor ER-. ³H-estradiol nuclear binding is present but variable during days 1.5–7.5 of pregnancy. Sites of strong nuclear binding of ³H-estradiol exhibit strong immunocytochemical staining with ER- antibody. Qualitative and quantitative evaluation of autoradiograms reveal that there is a general increase of nuclear ³H-estradiol binding during the first 3 days after fertilization in both luminal and glandular epithelium. The binding of estradiol is stronger in glandular epithelium from day 2.5 to day 7.5, paralleled by a rise in ³H-thymidine incorporation on day 2.5. By comparison, in the epithelium of the uterine lumen ³H-estradiol nuclear binding is low, but relatively high in epithelial cells at lateral branching of the lumen where the increase in ³H-estradiol binding corresponds to an increased labeling index with ³H-thymidine. A highly differentiated binding of ³H-estradiol to luminal and glandular epithelium was demonstrated with region- and time-specific changes of related effects on cell proliferation, differentiation, and secretion, probably involving involution and remodeling. The strong ³H-estradiol binding to glandular epithelium suggests that estradiol exerts pronounced effects on glandular activities in the periimplantation period.     

Studies on the release of barley aleurone cell proteins: Autoradiography

Summary Both uptake and incorporation of radioactivity from [³H]l-leucine into gibberellic-acid (GA₃)-treated aleurone layers of barley (Hordeum vulgare L.) was enhanced by pretreatment with 5 mM potassium bromate. The effect of 5 mM KBrO₃ on amino-acid incorporation was quantitative rather than qualitative and could be partly reversed by the addition of neutralized casein hydrolysate at 10 mg/ml. Autoradiographs of GA₃-treated aleurone cells pulsed with [³H]leucine showed distribution of silver grains predominantly over the endoplasmic reticulum (ER) and aleurone grains. After chasing with carrier l-leucine for 60 min, fewer silver grains were associated with the ER and aleurone grains while nearly half of the silver was associated with the ground cytoplasm of the cell. Autoradiographs were prepared from aleurone cells previously stratified by ultracentrifugation. After a 10-min pulse of label, the silver grains were found over the central ER zone of centrifuged cells; however, with an increase in duration of the chase, label was found distributed throughout the aleurone grain and spherosome region of the cell. The silver grains which were located over the central zone of centrifuged cells at the end of the pulse were almost exclusively associated with the ER. There is no evidence for association of label with dictyosomes or with vesicles derived from dictyosomes. The experimental evidence indicates that labelled amino acids are incorporated into aleurone cells on the ER and are released from these cells without the participation of a membrane-bound vesicle.     

Distribution of progestin-binding cells in estrogen-treated and untreated neonatal mouse uterus and oviduct: autoradiographic study with [125I]progestin

Summary Progestin-binding sites in uteri and oviducts of estrogen-treated and untreated 8-day-old mice were studied by thaw-mount autoradiography with [¹²⁵I]progestin. In the untreated uteri, nuclear concentration of radiolabelled progestin was observed in all tissues of the uterus, with strongest nuclear labelling in luminal and glandular epithelia and in stroma. In the estrogen-treated uteri, the degree of labelling was markedly augmented in stroma and muscle, but much reduced in the luminal and glandular epithelia, compared to untreated uteri. In untreated oviducts, nuclear labelling was observed in stroma and muscle in all regions and in epithelium in the isthmic and uterine regions. The epithelium in infundibular and ampullar regions was only scarcely labelled. The estrogen-treatment augmented the labelling in stroma and muscle of the oviduct as in the uterus, but the labelling in epithelium was not affected. These results indicate that estrogen-treatment induces progesteron receptor differentially among tissue compartments both in the uterus and oviduct.     

The de novo and salvage pathways for the synthesis of pyrimidine residues of RNA predominate in different locations within the mouse doudenal epithelium

Summary RNA synthesis was examined by radioautography in mouse doudenal epithelium using ³H-uridine as a tracer of the salvage pathway and ³H-orotic acid as a tracer of the de novo pathway. The incorporation of the two precursors was estimated by counting silver grains in light-microscopic and electron-microscopic radioautographs at successive levels of crypt and villus. With both precursors, silver grains were found over all epithelial nuclei, but in numbers varying by location. Thus, after ³H-uridine injection, the number of grains was high over nucleolus and nucleoplasm in the base of the crypt, declined gradually in the middle and top of the crypt, and was low along the villus. After ³H-orotic acid, the number of grains was fairly low throughout, but peaked over the nucleoplasm in lower villus cells. The ³H-uridine reaction over nucleolus and nucleoplasm in crypt cells was interpreted as synthesis by the salvage pathway of ribosomal RNA and heterogeneous RNA, respectively, whereas the ³H-orotic acid reaction over the nucleoplasm of some villus cells indicated that these cells synthesized heterogeneous RNA by the de novo pathway.     

Autoradiographic localization of 3H-histamine accumulation by the visual system of the locust

Summary The uptake of [³H]-histamine into the retina and optic lobe of the locust, Schistocerca americana gregaria was studied by means of autoradiography at the light- and electron-microscopic levels. Light-microscopic autoradiography showed a significant accumulation of [³H]-histamine in several regions of the optic lobe. Dense accumulations of silver grains were concentrated along the medial border of the medullary neuropil and around the entire periphery of the lobula. No significant accumulations of grains were present within the retina or the neuropil zones of the lamina, medulla or lobula.Electron-microscopic autoradiography showed histamine-accumulating cells along the border of the medulla to exhibit electron density and morphology typical of glial cells. Labelled histamine was present within both glial cell bodies and their processes. In the region surrounding the neuropil of the lobula, [³H]-histamine was concentrated within fine glial processes wrapped around neuronal cell bodies and their axons. No neuronal cell bodies or axons showed accumulation of silver grains above background.These results are consistent with previous studies showing the glial uptake of amino acid and biogenic amine putative neurotransmitters. However, the lack of a demonstration of a specific uptake of histamine in neuropil zones makes it difficult to assess the role of histamine uptake in the inactivation of neurally released histamine in the locust visual system.     

Uptake and accumulation of tritiated uteroglobin by day-6 rabbit blastocysts

Summary Uptake of uteroglobin (UGL) by day-6 rabbit blastocysts and the intracellular fate of this protein were studied by light- and electron-microscopic autoradiography, immunocytochemistry and acid-phosphatase cytochemistry. UGL, labelled with N-succinimidyl-(2-3-³H)-propionate, was administered to embryos in vitro for 15 min to 4 h. The kinetics, determined from light-microscopic autoradiographs, showed a continuous uptake of the labeled protein over a 4-h period of incubation. At the ultrastructural level, increasing numbers of silver grains and an intense UGL immunoreaction in protein vacuoles and crystalloid bodies of trophoblast cells indicated that ³H-UGL had accumulated in these organelles. The presence of crystalloid inclusions in protein vacuoles suggests their origin by a condensation of the protein content, including UGL. Lysosomes containing radioactivity were rarely found, suggesting a very low degradation rate of the ³H-UGL. Protein vacuoles and crystalloid bodies exhibited no acid phosphatase reaction. The enzyme was mainly found outside the basal and lateral cell membranes of trophoblast cells, and on the rough endoplasmic reticulum of endoderm cells.     

Light and electron microscopic radioautography of DNA synthesis in the endometria of pregnant-ovariectomized mice during activation of implantation window.

Pregnant mice were ovariectomized at pre-implantation stage and exogenous nidatory estradiol was administered to evaluate the DNA synthesis of the endometrial cells during activation of uterine receptivity for blastocyst implantation. After 0, 3, 6, 12 and 18 hrs. of estradiol treatment, the animals received 3H-thymidine injection, sacrificed 1 hr. later, and the uteri were prepared for light and electron microscopic radioautography. At time 0, no labelled stromal or epithelial cells was found in the endometrium. According to the time-lapse after estradiol induction, a gradual increase of labelled stromal and endothelial cells was seen in the endometrium. The highest labeling index was observed at the antimesometrial side of the implantation sites and the lowest value was found at the interimplantation site. The cells found at mesometrial side of the implantation site showed an intermediate labeling index. Eighteen hrs. after estradiol treatment, the labelled stromal cells found near the implantation chamber resembled the morphology of decidual cells while those labelled cells localized at the interimplantation sites were similar to the fibroblast. The uterine luminal epithelial cells showed low DNA synthesis after estradiol treatment resulting in only a few labelled cells at the interimplantation sites and no labelled cells at the implantation sites. A similar labeling pattern was seen in the glandular epithelium. The distribution of labelled cells seen among the regions of pregnant endometrium under estradiol effect suggest that DNA synthesis related to uterine activation for blastocyst implantation is a focal reaction, where the luminal epithelium does nt proliferate while the stromal and endothelial cells around the conceptus increase the DNA synthesis to prepare the endometrial decidualization.     

The uterovaginal sperm host glands of the quail (Coturnix coturnix japonica)

Summary Ultrastructural and ultrahistochemical studies were performed on the uterovaginal sperm host glands of the quail (Coturnix coturnix japonica). The proximal parts of the glandular necks are lined by a pseudostratified epithelium, consisting of high columnar ciliated cells and small, irregular shaped, basal cells.The true glandular epithelium is composed only of columnar cells with microvilli on their luminal end. A characteristic luminal feature is a large lipid droplet in the perinuclear region. In the subplasmalemmal region numerous tubular profiles are seen which could represent a cellular resorption system.To evaluate the absorptive capacity of the Uterovaginal sperm host glands, tracer studies with HRP, ferritin, lanthanum and ruthenium red were undertaken. Since between 5 min and 3 h after injection no absorption could be found with the techniques mentioned, it is suggested that phagocytosis of spermatozoa by the glandular epithelium is not likely to occur.     

Degeneration and apoptosis of endometrial cells in the bitch

The relationships between changes in plasma progesterone concentrations, degeneration of the luminal epithelium, the occurrence of apoptosis of endometrial cells and endometrial leucocyte populations in the bitch were determined. Mature bitches (n = 15) were euthanized and necropsied when in diestrus (Days 7-75, n = 12) or in anestrus (Days 10, 32 and 53). Degeneration of the luminal epithelium was observed in bitches in late diestrus (Days 38-75, n = 5) when plasma progesterone concentrations were decreasing and in anestrus (Days 10 and 32, n = 2) when plasma progesterone concentrations were < 0.5 ng/mL. Endometrial leucocyte populations increased after degeneration of the luminal epithelium (around Day 42 of diestrus). Apoptosis was mainly observed in the basal glandular epithelial cells and endothelial cells of blood capillaries in all except anestrous bitches. Very few apoptotic cells were found in the superficial glandular epithelial cells and stromal cells. Higher apoptotic indices were detected in the basal glandular epithelium on Days 12-42 of diestrus than at other stages. Therefore, apoptosis of glandular basal epithelial cells occurred mainly in early diestrus, degeneration of cells of the luminal epithelium occurred from mid-diestrus to early anestrus, and the increase in leucocyte numbers may have been a consequence and not a cause of luminal epithelial degeneration.     

‘3H-testosterone distribution and binding in RAT thymus cells in vivo’

Summary Autoradiography and biochemical investigations showed that [³H]-testosterone where injected intraperitoneally into male white rats was incorporated rapidly into thymus lymphocytes. Thymic cortex contained more silver grains than medulla, and larger lymphocytes were more labelled than medium or small lymphocytes.Cytosol fraction of thymus cells labelledin vivo with [³H]-testosterone, contained the largest quantity of labelled hormone. A 4S cytosol fraction binds [³H]-testosterone. This could be separated by Sephadex chromatography or by linear sucrose gradient centrifugation. Nuclear extract contained also a small quantity of the labelled hormone.     

Quantitative electron microscopic autoradiography on the mouse thyroid gland after administration of 3H-L-DOPA

Summary The cellular and subcellular distribution of radioactivity in the mouse thyroid gland different times (20 min — 8 hours) after intravenous administration of ³H-L-DOPA was studied by means of quantitative electron microscopic autoradiography.High concentrations of autoradiographic silver grains occur over parafollicular cells and adrenergic nerves while the labelling of follicular cells and lumina is low or absent and similar to the labelling of connective tissue cells at all observation times.Over the parafollicular cells high levels of radioactivity can be recorded already 20 min after administration of the labelled amino acid. The grain counts are highest at 1 hour and decrease then at 2.5 and 8 hours.The intracellular distribution of label is similar at all observation times; thus, the concentration of silver grains over the typical cytoplasmic granules of the parafollicular cells is 4–5 times higher compared to the concentration over the remainder of the cytoplasm and the nucleus.Treatment with a decarboxylase inhibitor prior to the injection of ³H-L-DOPA results in a low and uniform labelling of all thyroid cells. This finding, taken together with the observation that also pretreatment with reserpine abolishes the autoradiographic reaction over the cytoplasmic granules, gives strong support to the idea that the great majority of silver grains observed over parafollicular cells represents dopamine formed by decarboxylation of the labelled precursor.This study was supported by grant K71-12X-3352-01 from the Swedish Medical Research Council. The author wishes to express his gratitude to Mrs. Gunnel Bokhede and Miss Dala Sjögren for expert technical assistance.     

Binding of 125I-epidermal growth factor in human uterus

Summary Quantitative light-microscopic autoradiography was used on five human uteri at two different phases of the menstrual cycle to ascertain the cell types with binding sites for epidermal growth factor (EGF). The results revealed that stromal cells, glandular epithelium of endometrium, elongated and circular muscle cells of myometrium, smooth muscle and endothelial cells of arterioles in the basal endometrium and myometrium contained numerous silver grains following incubation with ¹²⁵I-EGF. Coincubation with 100-fold excess unlabeled EGF resulted in a complete disappearance of silver grains from all cell types. Quantitative grain analysis indicated that stromal cells contained the highest number of EGF-binding sites (P<0.05) with no significant differences among the others (P>0.05). There was no cyclic variation of EGF-binding to any of the uterine cell types. The present data demonstrate that all the cell types of human uterus, including arterioles, contain EGF-binding sites. This suggests that all the cells in human uterus subserving different functions are targets of EGF action.     

Selenium-induced autometallographic demonstration of endogenous zinc in organs of the rainbow trout,Salmo gairdneri

Summary Autometallographic (AMG) silver enhancement of endogenous zinc was studied in seven organs of the rainbow trout Salmo gairdneri. Groups of trout were injected intraperitoneally with sodium selenite in doses ranging from 0.08 to 25 ppm, administered 1 h before being killed. The concentration of selenium obtained by each organ was determined by gamma-spectrometry, and compared with the autometallographic deposition of silver grains. The relative accumulation of selenium in the organs was: liver > spleen > kidney > intestine > gills > brain > muscle. In the fish labelled with 10 and 25 ppm Se, AMG-deposits were found (1) within lysosomes of liver cells, (2) within the granules and on the nuclear membrane of melanophores in the spleen, (3) on the microvilli and in the apical cytoplasm of renal proximal tubular cells, (4) within the granules and along the plasma membrane of intestinal eosinophilic granule cells, and in the apical portion of the intestinal epithelium, and (5) in the gills, within granule cells and on the surface of the ionocytes. In the trouts injected with 5 ppm Se, silver grains were still observed in the liver, the intestine, and the gills, whereas, no such grains were found in preparations from fish having received 1 ppm Se. The use of selenium for the histochemical demonstration of endogenous zinc versus exogenous metals is discussed. Also, consideration is given to the question of which part of the total tissue zinc that is histochemically reactive.     

Localization of 3H-estradiol in the uterus of pregnant and non-pregnant armadillos

Summary The uptake and retention of radiolabeled estradiol by the uterus was examined in the armadillo. One pregnant and two non-pregnant armadillos were treated with 1.4 g/kg body weight of ³H-estradiol (E₂) by injection into the left ventricle, and one non-pregnant animal was injected with both the labeled hormone and 140 g/kg body weight of unlabeled E₂. One and a half hour after injection, the animals were sacrificed and the uteri were removed and processed for autoradiography. In the non-pregnant animals, nuclear localization was observed in the interstitial cells and glandular epithelium of the endometrium and the connective tissue cells and smooth muscle of the myometrium. Additionally, there was a gradation of uptake in the epithelial cells of the endometrium in that the glandular cells of the basal region were heavily labeled, while those cells in the sinusoidal, and luminal regions contained successively less label. The luminal cells were poorly labeled. In the pregnant female, the smooth muscle and glandular cells hypertrophied and their nuclei contained less label than was observed in the non-pregnant animals. The arteries of the myometrium were more easily distinguished in the pregnant animals and the nuclei of the endothelial cells and smooth muscle were more consistently labeled than those of the non-pregnant armadillos.     

Ultrastructural relationship between monoamine- and TRH-containing axons in the rat median eminence as revealed by combined autoradiography and immunocytochemistry in the same tissue section

Summary The correlation of dopamine (DA)-, noradrenaline (NA)- or serotonin (5HT)-containing neurons and thyrotropin releasing hormone (TRH)-containing neurons in the median eminence of the rat, as well as the coexistence of monoamines (MA) and TRH in the neurons, were examined by subjecting ultrathin sections to a technique that combines MA autoradiography and TRH immunocytochemistry. The distribution and localization of silver grains after ³H-MA injection were examined by application of circle analysis on the autoradiographs.TRH-like immunoreactive nerve terminals containing the immunoreactive dense granular vesicles were found to have an intimate contact with monoaminergic terminals labeled after ³H-DA, ³H-NA or ³H-5HT infusion in the vicinity of the primary portal capillaries in the median eminence. Synapses between TRH-like immunoreactive axons and MA axons labeled with silver grains, however, have not been observed to date. Findings suggesting the coexistence of TRH and MA in the same nerve terminals or the uptake of ³H-MA into TRH-like immunoreactive nerve terminals, where silver grains after ³H-MA injection were concurrently localized in TRH-like immunoreactive nerve terminals, were rarely observed in the median eminence. Percentages of the nerve terminals containing both immunoreactive granular vesicles and silver grains after ³H-MA injection to total nerve terminals labeled after ³H-MA infusion silver grains were equally very low in ³H-DA, ³H-NA or ³H-5HT, amounting to less than 6.1%.This work was supported in part by grant-in-aid for scientific research from the Japan Ministry of Education (No. 557018).     

The fine structure of the proliferative cells of the mouse intestine as revealed by electron microscopic autoradiography with 3H-thymidine

Summary The duodenal and colonic epithelia in mice were observed with electron microscopic autoradiography 2, 5 and 24 hours after a single injection of ³H-thymidine. After 2 hours, in the duodenum, silver grains are found in many undifferentiated cells, in a few young goblet cells, in some crystal-containing cells, and in some lymphocytes. In the colon after 2 hours silver grains are seen in some undifferentiated cells, and in many young goblet cells. Undifferentiated cells are characterized by a few short microvilli, poorly developed rough-surfaced endoplasmic reticulum, abundant free ribosomes, and a few apical moderately dense granules. In normal animals, absorptive cells seem to arise from undifferentiated cells, and goblet cells — from younger goblet cells. Undifferentiated cells could also become young goblet cells. Crystal-containing cells, which may not be of epithelial origin, proliferate in the epithelium in the adult animal.     

Proceedings: Heterochromatin and chromosome aberrations: a comparative study of normal and tumour cells of mouse with various distributions of heterochromatin.

Seedlings of Crepis capillaris were irradiated after pulse-labelling with tritiated thymidine ([³H]TdR), and both chromosomal aberrations and presence of silver grains were recorded in the same metaphase cells at various intervals throughout the whole mitotic cycle. The following results were obtained: (a) irradiated roots were homogeneous with respect to the number of aberrations, and heterogenous with respect to labelling index (LI); (b) time-effect curves for labelled (L) and unlabelled (U) cells showed no significant difference from one another; (c) no significant quantitative difference of aberration spectra produced in S and G₂ stages was found. These results support the view that the major factor which determines both quantitative and qualitative variation in the production of chromosomal aberrations by radiation is the time lapse between irradiation and fixation rather than relation of the time of irradiation to the time of DNA synthesis. In addition, it was found that labelling with [³H]TdR modifies the effect of radiation on chromosomes.