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.

Elemental composition of polyphosphate-containing vacuoles and cytoplasm of Leishmania major.

Leishmania major promastigotes contain electron-dense vacuoles. The elemental composition of these vacuoles and of the cytoplasm was measured by electron probe X-ray microanalysis, using rapid cryopreservation techniques to prevent alterations in composition due to diffusion. The electron-dense vacuoles are rich in P, presumably present as polyphosphate (poly P). Mg is present at about 9 times its cytoplasmic level. There is sufficient Mg to largely neutralize most of the negative charge of the Poly P. The electron-dense vacuoles also contain appreciable amounts of Ca and Zn, which are not detectable in the cytoplasm, as well as Na, K, and Cl, the latter two at concentrations below that of the cytoplasm. These results suggest that the vacuolar membranes have at least one cation transport system. Incubation of the promastigotes for 1 h in the absence of phosphate in the presence or absence of glucose did not cause significant changes in the vacuolar contents of P, Mg, or Zn, but changes in K and Cl content were observed in both the electron-dense vacuoles and in the cytoplasm.

Ultrastructural demonstration of increased sulfated proteoglycans and calcium associated with chondrocyte cytoplasmic processes and matrix vesicles in rat growth plate cartilage.

A monoclonal antibody (3-B-3) to chondroitin 6-sulfated proteoglycan was used with immunoperoxidase electron microscopy to study the relationship of chondrocyte cytoplasmic processes and matrix vesicles in rat epiphyseal growth plate cartilage. Immunoperoxidase staining of the chondrocyte plasmalemma was found at all levels in the growth plate and was most prominent in the hypertrophic zone. The plasmalemma and matrix of the cytoplasmic process often demonstrated stronger reactivity than the remainder of the cell surface. Matrix vesicles showed weak to strong surface or internal reactivity. The majority of them stained very similarly to the cytoplasmic process. X-ray microanalysis of specimens processed by rapid freezing and freeze substitution confirmed that both sulfur and calcium were localized within or in close association with both the cytoplasmic process and the matrix vesicle, suggesting a chemical combination of calcium with sulfated proteoglycans at both sites. These results indicate that there is a selective increase in the concentration of membrane-associated sulfated proteoglycan and calcium in the cell process, from which matrix vesicles may be released into the extracellular matrix.

Immunochemical and immunocytochemical identification of a myosin heavy chain polypeptide in Nicotiana pollen tubes.

A myosin heavy chain polypeptide has been identified and localized in Nicotiana pollen tubes using monoclonal anti-myosin antibodies. The epitopes of these antibodies were found to reside on the myosin heavy chain head and rod portion and were, therefore, designated anti-S-1 (myosin S-1) and anti-LMM (light meromyosin). On Western blots of the total soluble pollen tube proteins, both anti-S-1 and anti-LMM label a polypeptide of approximately 175,000 Mr. Immunofluorescence microscopy shows that both antibodies yield numerous fluorescent spots throughout the whole length of the tube, often with an enrichment in the tube tip. These fluorescent spots are thought to represent vesicles and/or organelles in the pollen tubes. In addition to this common pattern, anti-S-1 stains both the generative cell and the vegetative nuclear envelope. The different staining patterns of the nucleus between anti-S-1 and anti-LMM may be caused by some organization and/or anchorage state of the myosin molecules on the nuclear surface that differs from those on the vesicles and/or organelles.

[Ultrastructural localization of mRNA coding for oxytocin by in situ hybridization. Study by high resolution autoradiography using a tritiated oligonucleotide probe]. / Localisation ultrastructurale d'ARNm codant pour l'ocytocine par hybridation in situ. Etude par radioautographie à haute résolution à l'aide d'une sonde oligonucléotidique tritiée.

In situ hybridization (ISH) using a 25 mer tritiated oligonucleotide probe has been performed to study at the electron microscopic level the subcellular localization of the oxytocin mRNA in the rat hypothalamic magnocellular neurons. After high resolution radioautography, silver grains appeared to be localized over the cytoplasm of some magnocellular neurons of the supra-optic nucleus and frequently overlapped the ergastoplasmic "cisternae" of the Nissl bodies. These results demonstrate the possible application of ISH at a subcellular level using high resolution radioautography and a tritiated probe.

Thyroid status affects the regulation of prolactin mRNA accumulation by tri-iodothyronine and thyrotrophin-releasing hormone in cultured rat anterior pituitary cells.

The effects of tri-iodothyronine (T3) and TRH on prolactin mRNA accumulation in monolayer pituitary cell cultures prepared from both euthyroid and hypothyroid rats were investigated. Basal prolactin mRNA concentrations and prolactin release into culture medium were increased in hypothyroid cultures, the increase being related to the duration of hypothyroidism in vivo. The inhibitory effects of T3 seen in euthyroid cells were preserved in cells derived from hypothyroid animals, and the degree of inhibition was greater in cells from the most severely hypothyroid rats. However, the stimulation of prolactin synthesis and secretion induced by TRH in euthyroid cultures was not found in the hypothyroid cells. Hypothalamic and anterior pituitary TRH content were measured in similarly hypothyroid and euthyroid rats. A large hypothalamic pool of TRH was found, which was unchanged in hypothyroidism, whereas anterior pituitary TRH content was increased in the hypothyroid rats. The consequent down-regulation of anterior pituitary TRH receptors may explain the poor response of prolactin to TRH seen in vitro.

Elemental concentrations within types 2 and 3 epithelial cells of the rat thymus.

The elemental composition (Na, Mg, P, S, Cl, K, Fe and Zn) of epithelial cells of rat thymus cortex has been determined using X-ray microanalysis of freeze-dried frozen sections. The cytoplasm contained highly significantly lower levels of potassium and phosphorus than the nuclei. When the results were compared with a previous study of the elemental levels in cortical thymocytes (using the same material), only differences in the P concentration were found. This is probably attributable to the greater degree of leptochromasia usually observed in epithelial cells compared with thymocytes. Since thymocyte maturation depends very closely on the microenvironment created by the epithelial cells, the great similarity in elemental levels observed in this study could be important in the control of cell division and maturation events.

Electron probe analysis, X-ray mapping, and electron energy-loss spectroscopy of calcium, magnesium, and monovalent ions in log-phase and in dividing Escherichia coli B cells.

The elemental composition of individual cells of rapidly frozen and cryosectioned Escherichia coli B was measured with electron optical microanalytic methods. The Ca content was high (26.2 mmol/kg) in a 10-nm-wide region of the cell envelope. Amounts of cytoplasmic Ca in actively dividing cells were significantly higher (32.6 mmol/kg [dry weight]) than in the log-phase (1.5 mmol/kg) cells. Cellular Mg was 205 mmol/kg (dry weight) and it was uniformly distributed throughout the cell. Cells washed in distilled water before freezing lost monovalent ions (Na, Cl, and K), but the membrane-bound Ca and cellular Mg were not reduced, indicating that cellular Mg and membrane Ca are more tightly bound.

[Cytoplasmic and nuclear receptors of estradiol in the hypothalamus and cerebral cortex of female rats in the neonatal period]. / Tsitoplazmaticheskie i iadernye retseptory éstradiola v gipotalamuse i kore golovnogo mozga samok krys v neonatal'nom periode.

The content of estradiol receptors (E2) was studied in the cytoplasmic and nuclear fractions of the female rat hypothalamus and cortex during neonatal development. In the cytosol the E2-binding proteins, having a high capacity, include both true estradiol receptors and proteins identical with alpha-fetoprotein. True E2 receptors were found in the nuclear fraction: their concentration underwent almost no changes in hypothalamus and decreased from the 1st to the 5th day of postnatal development in cortex.

Vertical distribution of elements in cells and matrix of epiphyseal growth plate cartilage determined by quantitative electron probe analysis.

Quantitative electron probe analysis was performed on chick epiphyseal growth cartilage prepared by two anhydrous methods, ultrathin cryosections and freeze-dried epoxy-embedded tissue. Levels of Na, Mg, P, S, Cl, K, and Ca were determined in cytoplasm, mitochondria, extracellular matrix, matrix vesicles, and mineral nodules in four zones of the cartilage--proliferative, prehypertrophic, early hypertrophic, and early calcification. The exceptionally high levels of Na and K (up to 550 and 200 mmol/kg wet wt, respectively) found in the matrix are believed to be largely bound to fixed anions. Within cells, Na was higher than K (140 versus 20-34 mmol/kg wet wt), a condition that may reflect hypoxia. Ca and P were low in cells and unmineralized matrix. Ca and P were high in mitochondrial granules of the early hypertrophic zone and diminished in amount in the calcifying zone; the converse occurred in matrix vesicles. Mg was low to undetectable except in heavily mineralized structures (i.e., mitochondrial granules, matrix vesicles, and mineral nodules). S levels were high in matrix (approximately 400 mmol/kg wet wt) and increased slightly with maturation. The amount of S present greatly exceeds Ca levels and implies that sulfate, the predominant form of sulfur in proteoglycans, may serve as an ion-exchange mechanism for the passage of Ca through the matrix to sites where Ca and phosphate are precipitated.

Cerebral insulin-like immunoreactivity in rats and mice. Drastic decline during postnatal ontogenesis.

We studied the behavior of the insulin-like immunoreactivity in brains of rats and mice during the first 20 d post natum by immunohistochemistry and radioimmunoassay. It was found that a dramatic decline in the concentration of the peptide, accompanied by a strong reduction of immunoreactive cells, takes place during this period. A possible role of cerebral insulin as a promoter of nerve cell growth and development is briefly discussed.

Sequential appearance of fibronectin and collagen fibres in experimental arthritis in rabbits.

The sequential changes in the presence of fibronectin in the synovial membrane during the development of antigen-induced arthritis in rabbits were studied using an indirect immunoperoxidase technique on the tissue specimens fixed in formaldehyde, embedded in paraffin and pre-treated with pepsin and testicular hyaluronidase. The relation to the distribution of fibronectin and connective tissue fibres, demonstrated as either argyrophilic or red by van Gieson method, was studied. Initial after the induction of the arthritis the synoviocytes became increased in size and number. The subsynoviocytial tissue was invaded by granulocytes and the number of vessels was increased. Fibronectin in increased amount was seen around the lining cells. After 2-4 weeks a markedly reduced amount of granulocytes were seen together with an increase in the number of macrophages. At this stage, fibronectin was also found together with argyrophilic fibres in the subsynoviocytial connective tissue. After 8-13 weeks the synovial membrane was found hypertrophic and folded. The lining layer was unchanged, but in the subsynoviocytial tissue lymphocytes and plasma cells were more focally arranged. At that time fine fibres, stained by the van Gieson method, were present together with fibronectin and argyrophilic fibres in the subsynoviocytial tissue. The morphological change and the distribution of fibronectin in experimentally induced arthritis correlated temporally to the morphological change and the presence of fibronectin found in experimentally induced granulation tissue.

The sulphur content of chondrocyte nuclei.

The sulphur content as a measure of glycosaminoglycan content of growth plate cartilage was determined by energy dispersive x-ray analysis on fresh freeze dried unstained, unfixed ultra thin sections of rat growth plate. In the resting and proliferative zones, quantities of sulphur were found in the nuclei equal to that of the matrix. Less sulphur was present in the cytoplasm. In areas of cell degeneration nuclear and cytoplasmic content of sulphur fell to levels a fraction of that seen in the matrix. It was presumed that most of the sulphur was in glycosaminoglycans. Although glycosaminoglycans have been reported in small amounts in the nuclei of cells, no study of the glycosaminoglycan content of chondrocyte nuclei has been reported. The use of freeze dried unstained, unfixed sections presumably prevented the migration of sulphur and glycosaminoglycans from compartment to compartment.

Quantification of receptors for estradiol-17 beta and progesterone in the pituitary and hypothalamus of gilts during the estrous cycle and early pregnancy.

Nuclear and cytoplasmic exchange assays were utilized to quantify receptors for estradiol-17 beta (E2) and progesterone (P4) in hypothalamic and pituitary tissues from 4-6 gilts each on Days 1, 5, 10, 15 and 18 of the estrous cycle and from 4-5 gilts each on Days 5, 10, 15, 21 and 30 of pregnancy. No differences in the number of cytoplasmic E2 or P4 receptors in the pituitary were found from Days 1 to 15 of the estrous cycle (P greater than 0.05). However, on Day 18, the quantities of E2 and P4 receptors were 64-fold and 25-fold lower (P less than 0.01) than those found during Days 1 to 15 of the estrous cycle. No differences in the number of nuclear receptors for E2 in the pituitary were observed from Days 1 to 18 of the estrous cycle, but nuclear receptors for P4 were 2-fold higher (P less than 0.01) on Day 1 than Days 5 to 18. In hypothalamic tissue, the numbers of cytoplasmic and nuclear receptors for E2 and P4 were lower (P less than 0.05) on Day 18 than Day 10 of the cycle. The quantity of most steroid receptors decreased between Days 15 and 18 in nonpregnant gilts as luteolysis occurred and a new follicular phase was initiated. Pregnant pigs on Days 5, 10 and 15 had decreased pituitary receptors for E2 and P4 when compared with cycling animals on these days. In general, numbers of receptors in hypothalamic tissue did not differ between pregnant and nonpregnant pigs except for decreased (P less than 0.01) nuclear P4 receptors on Day 15.(ABSTRACT TRUNCATED AT 250 WORDS)

X-ray microanalysis of proximal and distal tubule cells in the mouse kidney, and the influence of cadmium on the concentration of natural intracellular elements.

Quantitative X-ray microanalysis of frozen freeze-dried sections of mouse cortex have been used to determine the concentrations of Na, Mg, P, S, Cl, K, Ca and Cd in normal mice and those subjected t 0.7 mumol of cadmium chloride in two subcutaneous injections. These injections result in tissue levels of approximately 100 mg Cd/kg dry weight (less than 1 mM) in whole kidney when analysed by atomic absorption spectrometry. There were distinct and characteristic differences -- 'fingerprints' -- in the elemental composition of both cytoplasm and mitochondria in proximal and distal tubules of normal mice that were distributed by the cadmium treatment. The most significant effect of the cadmium injections was a highly significant increase in the sulphur content of the cytoplasm and mitochondria of distal tubules and a loss in concentration of Mg, P, Cl, K, and particularly Na, from the mitochondria. These results are discussed in the light of current concepts of metallothionein induction (metallothionein is a sulphur-rich protein that acts to bind, amongst other metals, cadmium) and the lack of damage observed in the distal tubules.

X-ray microanalysis of HeLa S3 cells. II. Analysis of elemental levels during the cell cycle.

HeLa S3 cells were synchronized using hydroxyurea. Cryoultramicrotomy and X-ray microanalysis were used to study changes occurring in concentrations of elements during the cell cycle of the synchronized cells. Three subcellular compartments were studied : cytoplasm, nucleus and nucleolus. Potassium concentrations showed little fluctuation in all of the cell compartments during the cell cycle. Sodium concentrations increased during S. and M phases, returning to lower levels in the G1 phase. Chlorine concentrations were highest during the S and G2 phases. At all stages of the cell cycle respective concentrations of potassium, sodium, sulphur and chlorine were similar in the cytoplasm and nucleus. Concentrations of phosphorus increased in the nucleus during S, G2 and M, and also showed fluctuations in the nucleolus during the cycle; these were not seen in the cytoplasm. In S, M and M/G1 sodium concentrations were highest in the nucleolus compared with the other compartments. In the cytoplasm these changes resulted in an increase in total monovalent cation concentration (i.e. sodium + potassium) during S, G2 and M, which returned to base levels after mitosis. This increase in monovalent cation concentration is due almost entirely to the increase in sodium, with little change occurring in the concentration of potassium.

Mitochondrial modifications associated with the cytoplasmic male sterility in faba beans.

Isolated mitochondria of faba beans carrying two different determinisms of the cytoplasmic male sterility (cytoplasms 447 and 350) have been compared to fertile lines. 1. In addition to the major mitochondrial DNA, five small DNA species (in the range of 1000-2000 base pairs) were detected by agarose gel electrophoresis in the four cytoplasms. An additional small DNA species was found specifically in the cytoplasm 350. After endonuclease restriction of the mitochondrial DNA, the patterns obtained for both male-sterile cytoplasms were identical to each other but distinct by two to four fragments from the patterns obtained for male-fertile cytoplasms. 2. [35S]Methionine labeling in situ of the mitochondrial protein synthesis revealed an additional polypeptide (Mr = 25000) detected only in the two male-sterile cytoplasms. 3. The male-sterile cytoplasm 350 showed a decrease of the respiratory state 3 of oxygen uptake during oxidation of NADH or malate + pyruvate. This decrease is thought to reflect a smaller capacity of the respiratory chain. These specific mitochondrial modifications support the hypothesis of a mitochondrial localization of the cytoplasmic male sterility determinant in faba beans.

Purification and characterization of cytoplasmic protamine messenger ribonucleoprotein particles from rainbow trout testis cells.

Poly(A)-containing protamine messenger ribonucleoprotein particles [poly(A+) pmRNP particles] have been isolated from the polysomal and free cytoplasmic subcellular fractions of trout testis cells by a two-step isolation procedure. Ethylenediaminetetraacetic acid (EDTA) treated particles from both cytoplasmic fractions were first fractionated by sucrose gradient centrifugation and the putative pmRNP particles localized by utilizing 3H-labeled protamine complementary DNA (pcDNA) probes. In addition, particles present in these fractions were characterized by their translational activity in the heterologous, rabbit reticulocyte cell-free system and the protein components of crude mRNP complexes analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoesis. The final purification step involved affinity chromatography of pooled gradient fractions on oligo(dT)-cellulose from which intact pmRNP could be eluted with distilled water at 40 degrees C. Highly purified particles from both polysomal and free cytoplasmic fractions prepared by this procedure had buoyant densities of 1.35-1.37 g/cm3 in CsCl or a protein content of approximately 82%. Particles isolated from EDTA-dissociated polysomes were actively translated in vitro, while their free cytoplasmic counterparts were not. High salt washed pmRNP particles or the RNA extracted from pmRNP preparations, however, directed the synthesis of trout protamines in this system. A model of the activation of stored pmRNP particles in vitro and in vivo is presented.

Quantitative x-ray microanalysis of spleen lysosomes after acid phosphatase reaction.

A quantitative X-ray microanalytical study of 80 spleen lysosomes after histochemical reaction for acid phosphatase was carried out. Lysosomal Fe concentrations showed a skewed distribution, with most lysosomes having relatively low concentrations. The concentration of Pb in the lysosomes (indicative of acid phosphatase activity) showed a close to normal distribution. Although the plot of lysosomal Pb concentrations against Fe concentrations showed a marked scattering of the points, there is a statistically highly significant correlation between accumulated metal and acid phosphatase activity as determined from the Pb concentrations. The relation between concentrations of P and those of Pb in the lysosomes points to the possibility that Pb(H2PO4)2 is the main reaction product of the histochemical acid phosphatase reaction.