Tissue specificity of testosterone fixation: demonstrated by ultrastructural immunocytochemistry after cryoultramicrotomy]

Testosterone can be detected by immunocytochemistry on ultrathin slices obtained by cryoultramicrotomy. Testosterone detected by this method is likely bound on a binding site having a high affinity. In order to study the tissue specificity of this binding, testosterone was tested in Rat pituitary gland, liver and adrenal glands. Immunoreactive testosterone was detected in the gonadotropic cells of the pituitary gland, in the hepatocytes. Testosterone was not detected by immunocytochemistry in the pituitary cells other than the gonadotropic cells and in the cells of the medulla of adrenal glands. These data testify in favour of tissue specificity of the testosterone binding.     

Visualization of opiate receptors in the locus coeruleus of rats: high resolution radioautography after administration of a tritiated met-enkephalin analog]

Opiate receptor sites were visualized by high resolution radioautography in the locus coeruleus of the Rat following intraventricular injection of the met-enkephalin analogue FK 33-824 tritiated with a high specific activity (3H-FK). After administration of a tracer dose of 3H-FK, more than 75% of the radioactivity detected in the locus coeruleus is specifically bound to opiate receptor sites. These are distributed both between and inside neuronal perikarya. After administration of 3H-FK at high concentration, electron microscope radioautography shows the presence of specific opiate binding sites at the level of axo-somatic and axo-dendritic synaptic junctions. These synaptic binding sites could correspond to receptor sites for endogenous enkephalins.     

Ecdysteroid receptors in the neuroendocrine-endocrine axis of a moth

With a combination of thaw-mount autoradiography using a tritiated 20-hydroxyecdysone agonist, ponasterone A, and immunocytochemistry with a monoclonal antibody to 29 K-prothoracicotropic hormone, high affinity binding sites for ecdysteroids were identified in the tissues of the neuroendocrine-endocrine axis inManduca sexta larvae. At specific times during larval-pupal development in fifth stadium larvae, nuclear ecdysteroid binding sites were present in the cerebral prothoracicotropes, the corpora allata and prothoracic glands, the main axis for the regulation and production of ecdysteroids. A stage-specific appearance of ecdysteroid receptors also occurred in cells of fat body, midgut and Malpighian tubules, tissues which convert ecdysone into 20-hydroxyecdysone. Our data identify new target tissues for ecdysteroids and suggest that ecdysteroids could affect their own production at the genomic level via long and short feedback loops.     

Proliferation of adipose cells and their lipid accumulation in the rat at the perinatal stage; histoautoradiographic study]

After thymidine-3H injection into pregnant Rat, histoautoradiographic studies showed that adipose cell proliferation in different subcutaneous and visceral sites, was very important in Rat foetuses about 24 h before birth and that lipid accumulation in the labelled cells was very fast during perinatal 48 h.     

Localisation ultrastructurale de l'œstradiol et du moxestrol dans les cellules gonadotropes du rat par immunocytologie après cryo-ultramicrotomie: Caractérisation de la spécificité hormonale

Summary Estradiol (E₂) was specifically localized by immunocytochemistry in the cytoplasm and in the nucleus of the gonadotropic cells. The immunocytochemical reaction was not observed after injection of moxestrol, but it was not modified by injection of testosterone, progesterone, or dexamethasone. These data suggest that E₂ might be bound to a high-affinity binding-site which could also have a hormonal specificity.

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Ce travail a bénéficié d'une aide de l'I.N.S.E.R.M., ATP 58 78 90 contrat 48 (P.M. Dubois).     

Demonstration of specific receptors for angiotensin III in rat adrenal glands]

Distinct and specific binding sites for 3H-angiotensin II (A II) AND 3H-angiotensin III (A III) have been demonstrated in Rat adrenal gland preparations. A III binding sites have the highest affinity (KD29C1-2.10(-10)M) for the 2-8 heptapeptide. This suggests the possibility of a separate and distinct physiological role for the 2-8 heptapeptide, mediated via such A III binding sites.     

Existence of several non-specific recognition systems in macrophages]

Rat peritoneal cells were made to bind five particle species: immunoglobulin-coated Sheep red cells, glutaraldehyde-treated Sheep red cells, latex beads, leishmania and tumor cells. The dependence of binding on various physico-chemical parameters was studied. The binding of latex beads or Leishmania was not inhibited by cold (4 degrees C), sodium azide, cytochalasin B and ethyleneglycol or dimethylsulphoxide. The binding of immunoglobulin-coated Sheep red cells was unaffected by cold and azide, but it was inhibited by cytochalasin B, ethyleneglycol and dimethylsulphoxide. The binding of glutaraldehyde-treated Sheep red cells was inhibited by cold, azide and ethyleneglycol, but it resisted cytochalasin B and dimethylsulphoxide. The binding of tumor cells was inhibited by azide, cytochalasin B, ethyleneglycol and dimethylsulphoxide. It is concluded that: (a) macrophages are endowed several sets of non-specific binding structures that are differently affected by physico-chemical parameters, which provides a simple way of characterizing them; (b) the expression of a given binding structure on the macrophage membrane is modulated by metabolic inhibitors; (c) some lymphocytes were able to bind tumor cells or Leishmania. Thus, lymphocytes and macrophages might share some non-specific adhesive structures.     

Immunocytochemical localization of neurons containing ACTH 17-39 in the rat brain]

Immunoreactive neurons and fibers were localized in the Rat brain by immunocytochemistry using an anti-ACTH 17-39 antiserum. Neither adrenalectomy nor water deprivation affected intensity of distribution of reaction of labelled neurons whereas colchicine increased labelling of hypothalamic perikarya. These results support the neuronal origin of cerebral ACTH.     

Histochemical and ultrastructural modifications of mice endometrium, vagina and pituitary following zeranol treatment

The histochemical and ultrastructural changes produced by zeranol treatment on the endometrium and the gonadotropic FSH cells of the adenohypophysis have been investigated in mice at the prepuberal and virginal stages. Modifications similar to those induced by estrogen treatment were observed. It is concluded that both estrogens and zeranol share the same activity on the tissues examined.     

Histochemical and ultrastructural modifications of mice endometrium,vagina and pituitary following zeranol treatment

Summary The histochemical and ultrastructural changes produced by zeranol treatment on the endometrium and the gonadotropic FSH cells of the adenohypophysis have been investigated in mice at the prepuberal and virginal stages. Modifications similar to those induced by estrogen treatment were observed. It is concluded that both estrogens and zeranol share the same activity on the tissues examined.     

Formation of the neuromuscular junction in cultures of rat embryonic cells]

The morphological evidence of the primary nerve muscle contacts are described. They consist of areas of cholinesterase activity (detected histochemically) localized on the myotube membranes and of mutiple clusters of ACh receptors whose 125I-alpha-bungarotoxin binding sites are revealed by radio-autography. After the stage of the primary nerve muscle contacts, some of which seem transient, characteristic neuromuscular junctions appear. These neuromuscular junctions which possess subneural infoldings are similar to the end-plates of the Rat in vivo.     

Species-specific differences in the mitogenic activity of heparin-binding growth factors in the sera of various mammals

Sera from different mammalian species displayed great differences in mitogenic activity, as measured by stimulation of DNA synthesis in BALB/c 3T3 cells (3T3 cells). Among the sera examined, fetal bovine serum was least active, and increasing activity was detected in calf serum, human serum, rat serum and mouse serum, in that order. Rat and mouse sera exhibited extremely high mitogenic activity with 3T3 cells, but when TIG-1 human fetal lung fibroblasts were used for the DNA assay instead, the activity levels of all of the sera were lower, and the differences between them were smaller. To determine the reasons for these differences, the heparin-binding growth factors in each serum were separated on a heparin affinity column. Five peaks of DNA-stimulating activity were obtained. Three of these were found in all sera examined, with both 3T3 cells and TIG-1 cells. Two other peaks were found only with 3T3 cells; one was peculiar to rat and mouse sera, with extremely high activity in the rat, and the other was specific to fetal serum. The dependence of the activity of these peaks on the cells used for the test was confirmed using normal rat lung fibroblasts and immortalized rat kidney cells. These findings adequately explain the species-specific differences in mitogenic activity of whole sera, and the variation in activity depending on the cells used for assay of DNA synthesis.     

Species-specific differences in the mitogenic activity of heparin-binding growth factors in the sera of various mammals.

Sera from different mammalian species displayed great differences in mitogenic activity, as measured by stimulation of DNA synthesis in BALB/c 3T3 cells (3T3 cells). Among the sera examined, fetal bovine serum was least active, and increasing activity was detected in calf serum, human serum, rat serum and mouse serum, in that order. Rat and mouse sera exhibited extremely high mitogenic activity with 3T3 cells, but when TIG-1 human fetal lung fibroblasts were used for the DNA assay instead, the activity levels of all of the sera were lower, and the differences between them were smaller. To determine the reasons for these differences, the heparin-binding growth factors in each serum were separated on a heparin affinity column. Five peaks of DNA-stimulating activity were obtained. Three of these were found in all sera examined, with both 3T3 cells and TIG-1 cells. Two other peaks were found only with 3T3 cells; one was peculiar to rat and mouse sera, with extremely high activity in the rat, and the other was specific to fetal serum. The dependence of the activity of these peaks on the cells used for the test was confirmed using normal rat lung fibroblasts and immortalized rat kidney cells. These findings adequately explain the species-specific differences in mitogenic activity of whole sera, and the variation in activity depending on the cells used for assay of DNA synthesis.     

Mucosal effector memory T cells: the other side of the coin

Immunological memory allows for rapid and effective protective immunity to previously encountered pathogens. New insights in understanding specific memory differentiation and function have now indicated that in addition to providing enhanced immunity, an important purpose of immunological memory is to provide immediate protection at all sites of the body, including non-lymphoid tissues. Effector memory CD8 T cells have the capacity to reside long-term at epithelial surfaces, where they allow for rapid containment of the invading pathogens at the local entry site and prevent systemic spreading and excessive immune responses. The accumulation of tissue-specific memory T cell subsets, together with cross-reactivity of these antigen-experienced T cells even to unrelated pathogens, provides flexibility and expansion of their specificity repertoire that over time greatly surpasses that of the declining na?ve T cell populations. This review will discuss new insights into T cell memory. We will focus in particular on the generation and function of effector memory CD8 T cells at the intestinal mucosa, which represents one of the largest entry sites for pathogens.     

The enteric neural receptor for 5-hydroxytryptamine

An enteric neural receptor for serotonin (5-HT) has been characterized. This receptor was assayed, using 3H-5-HT as a radioligand, by rapid filtration of isolated enteric membranes and by radioautography. In addition, intracellular recordings were made from ganglion cells of the myenteric plexus. High affinity, saturable, reversible, and specific binding of 3H-5-HT was demonstrated both to membranes of the dissected longitudinal muscle with adherent myenteric plexus and the mucosa-submucosa. Radioautographs showed these 3H-5-HT binding sites to be in myenteric ganglia and in a broad unresolved band at the mucosal-submucosal interface. Antagonists active at receptors for other neurotransmitters than 5-HT, at either of the two known types of CNS 5-HT receptor, and at 5-HT uptake sites on serotonergic neurons failed to inhibit binding of 3H-5-HT. The structural requirements of analogues for binding to the enteric 5-HT receptor matched the known pharmacology of M or neural 5-HT receptors. A novel 5-HT antagonist was found. This compound, N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP), antagonized the action of 5-HT on type II/AH cells of the myenteric plexus but did not affect the release or actions of acetylcholine (nicotinic or muscarinic) or substance P. 5-HTP-DP was also an equally potent displacer of 3H-5-HT from its binding sites on enteric membranes. It is concluded that the sites responsible for specific binding of 3H-5-HT are enteric M or neural 5-HT receptors. These receptors differ from those now known to be present in the CNS.     

In vivo occupation of estrogen receptors by hydroxylated metabolites of tamoxifen]

We report that after in vivo administration of (3H) tamoxifen, the cytosol and nuclear estrogen receptor sites of Rat uterus and Chicken oviduct are mostly occupied by polar metabolites. One of the major metabolites is 4-hydroxy-tamoxifen which we have identified by cocrystallisation withe non radioactive compound and which is known to display a high affinity for the estrogen receptor. In the Rat uterus, the proportion of the metabolites versus tamoxifen, increases with time with a maximum at 8 hrs. for the 4-hydroxy-tamoxifen. Other hydroxylated metabolites (M2) became predominant after 24 hrs. We propose that in vivo, the synthetic antiestrogens act mostly via their transformation into hydroxylated metabolites.     

The enteric neural receptor for 5-hydroxytryptamine

Summary An enteric neural receptor for serotonin (5-HT) has been characterized. This receptor was assayed, using³H-5-HT as a radiologand, by rapid filtration of isolated enteric membranes and by radioautography. In addition, intracellular recordings were made from ganglion cells of the myenteric plexus. High affinity, saturable, reversible, and specific binding of³H-5-HT was demonstrated both to membranes of the dissected longitudinal muscle with adherent myenteric plexus and the mucosa-submucosa. Radioautographs showed these³H-5-HT binding sites to be in myenteric ganglia and in a broad unresolved band at the mucosal-submucosal interface. Antagonists active at receptors for other neurotransmitters than 5-HT, at either of the two known types of CNS 5-HT receptor, and at 5-HT uptake sites on serotonergic neurons failed to inhibit binding of³H-5-HT. The structural requirements of analogues for binding to the enteric 5-HT receptor matched the known pharmacology of M or neural 5-HT receptors. A novel 5-HT antagonist was found. This compound, N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP), antagonized the action of 5-HT on type II/AH cells of the myenteric plexus but did not affect the release or actions of acetylcholine (nicotinic or muscarinic) or substance P. 5-HTP-DP was also an equally potent displacer of³H-5-HT from its binding sites on enteric membranes. It is concluded that the sites responsible for specific binding of³H-5-HT are enteric M or neural 5-HT receptors. These receptors differ from those now known to be present in the CNS.     

Increase in spontaneous beating frequency in rat heart cells cultured in homologous serum]

Rat serum at different concentrations added to cultured Rat heart cells medium increased spontaneous beating frequency of myoblasts. This increase occurred instantaneously and lasted more than 24 hrs. It was related to the serum concentration used. Electrophysiological recording showed a decrease in the iso-electric phase duration of membrane potential, with no change in action potential. In spite of the beating frequency increase, cells remained sensitive to the chronotrope positive action induced by Isoprenaline. There was no difference between male and female sera. The global results could partly be explained by the action of Ca++ in Rat serum.     

High affinity binding of labeled androgens in the androgen-target tissues of the male rhesus monkey

Summary High affinity testosterone (T)-specific and dihydrotestosterone (DHT)-specific binding sites exist in a 12 ratio in the cytosol fractions of the caput epididymidis, prostate, seminal vesicles and the ductus deferens of the rhesus monkey. The number of androgen-binding sites in the caput epididymidis is 3 times greater than that of the other 3 tissues.This investigation was supported by financial assistance from the Indian Council of Medical Research and the University Grants Commission.     

Peroxisomes in the apocrine sweat glands of the human axilla and their putative role in pheromone production

The products of the human apocrine axillary glands contain volatile steroids which act as pheromones. The steroidal structure of these pheromones implies that the axillary glands should be able to synthesize cholesterol which is the essential precursor of these molecules. Since important steps in cholesterol synthesis are localized within peroxisomes, we investigated the occurrence and the putative role of peroxisomes in the axillary glands at protein and mRNA levels by immunocytochemistry, Western blotting, and RT-PCR. Numerous peroxisomes were localized in the cells of the apocrine glands by immunocytochemistry, and the presence of catalase was confirmed by Western blotting and RT-PCR. Additionally, RT-PCR revealed the presence of mRNAs of two peroxisome-associated enzymes of the cholesterol biosynthetic pathway, mevalonate kinase and farnesyl diphosphate synthase. The results suggest that the peroxisomes in the human apocrine axillary glands may play a pivotal role in the biosynthesis of pheromones.