Primary tissue culture of human adult adrenocortical cells. Methodology and electron microscopic observations on ACTH-deprived and ACTH-treated cortical cells

Summary A method of primary tissue culture involving both disaggregation of cells by repeated exposure of small tissue fragments to a solution of trypsin, collagenase and hyaluronidase and explantation of the residual tissue fragments intermingled with isolated cells onto polyethylene discs, has been shown to be adequate for the prolonged maintenance (up to 30 days) in vitro of cells arising from decapsulated adult human adrenocortical tissue. The technique and its critical points are discussed.Adrenocortical cells were organized both as outgrowing columns from microexplants or as variously sized islets of monolayered cells. The ultrastructural features of ACTH-deprived adrenocortical cells (i.e., mitochondria with laminar cristae, endoplasmic reticulum mainly consisting of rough profiles, abundance of lipid droplets and -glycogen particles) suggest that the cells dedifferentiate and retain practically no steroidogenic activity.After 2 days of ACTH-treatment, cultured parenchymal cells were found to be quite similar to the zona fasciculata elements of the normal human adrenal cortex. They were grouped in islets of about 50–100 cells. Rough endoplasmic reticulum had decreased, but smooth endoplasmic reticulum showed focal proliferation. The pleomorphic mitochondria with laminar cristae, transformed into a homogeneous population of round or ovoid mitochondria containing tubulo-vesicular cristae. Lipid droplets and glycogen particles were decreased in number.After 7 days of daily treatment with ACTH, the cortical elements, whose nucleus and cytoplasm seemed to be enlarged, were arranged in clusters formed by up to 300 monolayered elements, in which dividing cells were consistently observed. Their cytoplasm was filled with a meshwork of smooth reticulum tubules, in which scantly ribosome-studded profiles and occasional small stacks of granular cisternae were embedded. Mitochondria were similar to those of the 2 days ACTH-treated cultures. Lipid droplets and glycogen particles were absent. The functional significance of these structural changes as well as the possible mechanism underlying the differentiative effect of ACTH are discussed.Primary cultures of human adult adrenals are proposed as a new tool for studies into the physiopathology of the adrenocortical cells under carefully controlled experimental conditions.mis|It is a pleasure to acknowledge our thanks to Drs. F. Mantero and C. Eccher for kindly supplying the normal human adrenocortical tissue. Thanks are also due to Mr. G. Gottardo for his excellent technical assistance.mis|This work was partly supported by a contract with the CNR-Italy (C.T. 73.00663.04).A preliminary report on part of this work was given at the Annual Meeting of the European Tissue Culture Society, Genua, May 1974, and at the Annual Meeting of the Société Française de Microscopie Electronique, Rennes, May 1974, and published as short communication in The Journal of Endocrinology 63, 247, 1974.     

Restitution and genetic differentiation of salmon populations in the southern Baltic genotyped with the Atlantic salmon 7K SNP array

Background

Native populations of Atlantic salmon in Poland, from the southern Baltic region, became extinct in the 1980s. Attempts to restitute salmon populations in Poland have been based on a Latvian salmon population from the Daugava river. Releases of hatchery reared smolts started in 1986, but to date, only one population with confirmed natural reproduction has been observed in the Slupia river. Our aim was to investigate the genetic differentiation of salmon populations in the southern Baltic using a 7K SNP (single nucleotide polymorphism) array in order to assess the impact of salmon restitution in Poland.

Methods

One hundred and forty salmon samples were collected from: the Polish Slupia river including wild salmon and individuals from two hatcheries, the Swedish Morrum river and the Lithuanian Neman river. All samples were genotyped using an Atlantic salmon 7K SNP array. A set of 3218 diagnostic SNPs was used for genetic analyses.

Results

Genetic structure analyses indicated that the individuals from the investigated populations were clustered into three groups i.e. one clade that included individuals from both hatcheries and the wild population from the Polish Slupia river, which was clearly separated from the other clades. An assignment test showed that there were no stray fish from the Morrum or Neman rivers in the sample analyzed from the Slupia river. Global F_ST over polymorphic loci was high (0.177). A strong genetic differentiation was observed between the Lithuanian and Swedish populations (F_ST = 0.28).

Conclusions

Wild juvenile salmon specimens that were sampled from the Slupia river were the progeny of fish released from hatcheries and, most likely, were not progeny of stray fish from Sweden or Lithuania. Strong genetic differences were observed between the salmon populations from the three studied locations. Our recommendation is that future stocking activities that aim at restituting salmon populations in Poland include stocking material from the Lithuanian Neman river because of its closer geographic proximity.

Electronic supplementary material

The online version of this article (doi:10.1186/s12711-015-0121-9) contains supplementary material, which is available to authorized users.     

Mobilization of vacuolar iron by AtNRAMP3 and AtNRAMP4 is essential for seed germination on low iron

Iron (Fe) is necessary for all living cells, but its bioavailability is often limited. Fe deficiency limits agriculture in many areas and affects over a billion human beings worldwide. In mammals, NRAMP2/DMT1/DCT1 was identified as a major pathway for Fe acquisition and recycling. In plants, AtNRAMP3 and AtNRAMP4 are induced under Fe deficiency. The similitude of AtNRAMP3 and AtNRAMP4 expression patterns and their common targeting to the vacuole, together with the lack of obvious phenotype in nramp3-1 and nramp4-1 single knockout mutants, suggested a functional redundancy. Indeed, the germination of nramp3 nramp4 double mutants is arrested under low Fe nutrition and fully rescued by high Fe supply. Mutant seeds have wild type Fe content, but fail to retrieve Fe from the vacuolar globoids. Our work thus identifies for the first time the vacuole as an essential compartment for Fe storage in seeds. Our data indicate that mobilization of vacuolar Fe stores by AtNRAMP3 and AtNRAMP4 is crucial to support Arabidopsis early development until efficient systems for Fe acquisition from the soil take over.     

Microrestes de Vertébrés du Siluro-Dévonien d'Algérie,de Turquie et de Thaïlande

Acanthodian («Shark-like spiny fishes) scaleshave been collected in three Uppermost Silurian-Lowermost Devonian localities from Algeria, southeastern Turkey and Thailand, in association with agnathan remains (heterostracans in Algeria, thelodonts in Thailand). These vertebrate microremains are the oldest ones for those three countries. They were associated with marine invertebrate faunas and are supposed to have lived in littoral environments. Their world-wide biogeographical distribution by the Silurian-Devonian boundary leads to analyse the palaeogeographical relations of north-Atlantis and Siberia on one hand, with southern Europe, southeastern Asia and Gondwanaland on the other hand.     

Immuno-electron microscopic localization of estradiol receptor in cells of male and female reproductive and non-reproductive organs

Summary— The localization of estradiol receptor (ER) in various tissues and their distribution in sub-cellular compartments were studied by means of immunogold-electron microscopic methods using a site-directed polyclonal antibody developed against a peptide from the DNA binding site of ER. This method was used to determine the presence and localization of ER in tissues and cells of male and female reproductive and non-reproductive organs. In the female reproductive tract, endometrial cells and the cells of the corpus luteum were found to contain ER. In non-reproductive organs of both sexes the following cell types showed significant labeling: hepatocytes, epithelial duodenal cells, striated muscle fibers, cells of the proximal convoluted tubules of the kidney, lymphocytes, neurons, and adipose cells. Alveolar epithelial cells were studied only in female specimens and were labeled by the anti-ER. Prostatic and epididymal epithelial cells were found to be labeled in the male reproductive organs. In all these cells a higher density of label was found in the nucleus, especially in the space between the clumps of compact chromatin, as was previously found in epithelial endometrial cells. These results suggest that estradiol exerts its effects through a common nuclear mechanism in cells of male and female reproductive and non-reproductive organs.     

HPV-DNA integration and carcinogenesis: putative roles for inflammation and oxidative stress

HPV-DNA integration into cellular chromatin is usually a necessary event in the pathogenesis of HPV-related cancer; however, the mechanism of integration has not been clearly defined. Breaks must be created in both the host DNA and in the circular viral episome for integration to occur, and studies have shown that viral integration is indeed increased by the induction of DNA double strand breaks. Inflammation generates reactive oxygen species, which in turn have the potential to create such DNA strand breaks. It is plausible that these breaks enable a greater frequency of HPV-DNA integration, and in this way contribute to carcinogenesis. Consistent with this idea, co-infections with certain sexually transmitted diseases cause cervical inflammation, and have also been identified as cofactors in the progression to cervical cancer. This article examines the idea that inflammation facilitates HPV-DNA integration into cellular chromatin through the generation of reactive oxygen species, thereby contributing to carcinogenesis.     

Drought survival in Dactylis glomerata and Festuca arundinacea under similar rooting conditions in tubes

Drought survival in perennial forage plants involves different adaptative responses such as delay of dehydration through water uptake, limitation of water loss and tolerance of tissues to dessication. To compare the importance of these responses in contrasting cultivars of forage grasses at the whole plant level, we carried out two experiments under glasshouse conditions. Plants of cocksfoot (Dactylis glomerata L.) cultivars, cvs. Currie, Medly (both of Mediterranean origin) and Lutetia (of continental origin), and of tall fescue (Festuca arundinacea L.) cv. Centurion (Mediterranean) were grown in 60 cm-deep cylinders to eliminate the effect of differences of root depth on water availability whilst allowing severe drought to be imposed at a realistic rate. In both experiments, the cvs. were ranked similarly for plant survival, with high mortality for Centurion, low for the Mediterranean cocksfoots Currie and Medly, and intermediate for Lutetia. These differences could not be ascribed to water use during most of the drought period since water uptake and decrease in leaf extension were not significantly different between species and cultivars. However, resistant cvs. of cocksfoot were able to extract water for a longer period and at a lower soil water potential (s) than other cvs. The critical s at plant death was –3.8 and –3.6 MPa for Medly and Currie and –3.0-,–2.6 MPa for Lutetia and Centurion. Moreover, at a low soil water reserve (15–2%), membrane stability and water content were maintained for longer in enclosed immature leaf bases of cocksfoots cultivars, whereas the fescue Centurion exhibited accelerated lamina senescence and steady increase of membrane damage in surviving tissues. Therefore, it is proposed that the drought resistance of tall fescue in the field can mainly be ascribed to its ability to develop a deep root system. In cocksfoot, dehydration tolerance in surviving tissues and the ability of roots to extract water at low soil water potentials may, in addition to root depth, contribute significantly to plant survival under severe drought.