Jacques Hourdry

Investigations of endocrine cells in the gastrointestinal tract and pancreas during the metamorphosis of an anuran (Alytes obstetricans L.): histochemical detection of APUD cells

Endocrine cells were detected at premetamorphosis, prometamorphosis, climax, and juvenile stages using an amine-inducing fluorescence technique with or without previous L-3,4-dihydroxyphenylalanine (L-DOPA) treatment. At premetamorphosis, serotonin cells exhibited yellow fluorescence in the gut primary epithelium of the L-DOPA untreated animals. In the treated animals, green fluorescent APUD cells could be seen in addition to the serotonin cells. In the pancreas, numerous clusters of fluorescent APUD cells were observed. At prometamorphosis the number of fluorescent cells increased in the intestinal primary epithelium and, close to the basal membrane, numerous small regenerative buds devoid of fluorescent cells appeared. In the pancreas of L-DOPA-treated animals, two types of APUD cells could be distinguished by their different fluorescence intensities. At the climax stage, the stomach developed and APUD cells were detectable in the gastric glandular buds. The degenerated primary intestinal epithelium was progressively removed in the intestinal lumen. At this stage, the regenerative buds of the secondary epithelium exhibited APUD cells. In the disorganized pancreas, the induced fluorescence decreased strongly. At the juvenile stage, cords of APUD cells displayed a cytoplasmic green fluorescence in the pancreas. In the stomach and intestine, serotonin and APUD cells were numerous.

Hormonal control of the intestinal brush border enzyme activities in developing anuran amphibians: I. Effects of hydrocortisone and insulin during and after spontaneous metamorphosis

The effects of hydrocortisone and insulin on the intestinal brush border membrane enzymatic activities in an anuran amphibian, Alytes obstetricans, were investigated at the end of spontaneous metamorphosis and 2 weeks after its completion. At the end of metamorphosis, the brush border is differentiating in the apical region of a developing neoformed epithelium. Two weeks after the completion of metamorphosis, this epithelium is entirely formed. The animals received one hormone injection per day for 2 or 3 days running (hydrocortisone: 1, 5, or 25 micrograms/g body wt/day; insulin: 0.5, 1, or 5 mU/g body wt/day). The hydrolases studied were three glucosidases (maltase, glucoamylase, trehalase), gamma-glutamyl-transferase and alkaline phosphatase. In animals reaching the end of metamorphosis, hormonal treatments rarely modify the three glucosidase activities. Two weeks after metamorphosis, a 5 microgram/g body wt/day hydrocortisone injection usually results in a significant increase of the three glucosidase activities. Conversely, a 0.5 mU/g body wt/day insulin injection induced a marked decrease in these activities. At the end of metamorphosis, hydrocortisone has variable effects on gamma-glutamyl-transferase activity; insulin, however, does not significantly modify this activity. Two weeks later, insulin and sometimes hydrocortisone inhibit gamma-glutamyl-transferase activity. Whatever the developmental stage is, hydrocortisone is able to stimulate alkaline phosphatase activity. At the end of metamorphosis, insulin has no influence on this activity, but 2 weeks after metamorphosis, low doses of the hormone (0.5 mU/g body wt/day) significantly reduce it. These results emphasize the possibility that after spontaneous metamorphosis the enzymatic activities of the new intestinal brush border are hormone controlled. This control could be related to the development of the interrenal and pancreatic islet functions.

Proto-oncogenes and embryonic development

The role of proto-oncogenes in embryonic development was investigated using one of the most characterized vertebrates, the amphibian Xenopus laevis. Genes which belong to the major proto-oncogene families have been detected in Xenopus genome. The developmental control of the myc gene was assayed using a characterized Xenopus myc probe and specific antibodies. The myc gene is highly expressed as a stable maternal mRNA in oocyte, and an unfertilized egg contains 5 X 10(5)-fold the myc RNA content of a proliferative somatic cell. The myc RNA store is evenly distributed in the oocyte and the egg. Fertilization triggers a post-transcriptional control of the gene and the RNA store is progressively degraded to a constitutive value of 10 to 30 myc RNA copies registered per gastrula embryonic cell. The 62K myc protein is accumulated late in oogenesis. This uncoupling of myc expression and cell proliferation appears as a specific developmental regulation of the myc gene, adapted to the series of rapid cell cleavages occurring after fertilization.

Dosage des activités de quelques hydrolases lysosomiques intestinales, au cours du développement larvaire deDiscoglossus pictus Otth, Amphibien Anoure

The whole specific activities of 5 lysosomal enzymes (acid phosphatase, N-acetylβ-glucosaminidase,β-glucuronidase, cathepsin and aryl sulphatase) were measured in the guts of Discoglossus tadpoles, before and during metamorphosis.For each hydrolase, the activities show the same variation pattern. They significantly increase at the end of prometamorphosis and the beginning of the climax, while there is histolysis of the first (primary) epithelium of the gut.The measured activities decrease at the end of the climax, while a secondary, enzyme-negative epithelium is developing.The free specific activity of the acid phosphatase was measured in these same guts. It significantly increases at the end of prometamorphosis and the beginning of the climax, during the epithelial histolysis. The total activity increases more slowly than the free one.In the same way, an increase is obtained for the specific activity of the bound (intralysosomal) acid phosphatase.The changes in whole enzymatic activities may result from accelerated synthesis of lysosomal hydrolases due to the primary epitheliocytes, and induced by the simultaneous increase in the plasmatic level of the thyroid hormones. Furthermore the free activity of the acid phosphatase may produce an increased permeability or a breaking of the lysosomal membrane, by the same hormones.

C- Myc Proto-Oncogene Expression During Newt Limb Regeneration

Utilizing the method of in situ hybridization, myc-homologous RNA transcripts were detected in histological sections of the newt limb regenerate. A radiolabeled Xenopus myc-cDNA was used as probe. Autoradiographic grains representing myc-homologous RNAs were observed in cells of the thickened wound epidermis, irrespective of the layer examined. Further, mesenchymatous-like cells which form the blastema also contained multiple grains, indicating that these cells possess myc-like RNA molecules. The presence of RNAs, homologous to the myc proto-oncogene in cells of the regenerating newt limb indicate that a myc-related gene is expressed in these cells during the regenerative process and, therefore, may play an important regulatory role, as myc gene expression has been implicated in the control of cellular proliferation in other systems. Our results are discussed against the background of literature on c-myc proto-oncogene expression.

Hormonal control of the intestinal brush border enzyme activities in developing anuran amphibians: II. Effects of glucocorticoids and insulin during experimental metamorphosis

The effects of glucocorticoids (hydrocortisone, dexamethasone) and insulin on enzymatic activities of the intestinal brush border membrane were investigated in an anuran amphibian, Alytes obstetricians, before and during experimental metamorphosis produced by immersion into a thyroxine solution. During experimental metamorphosis, a new epithelium (secondary epithelium) replaces the degenerating primary epithelium. The enzymes studied were three glucidases (maltase, glucoamylase, trehalase) and alkaline phosphatase. In tadpoles reaching the end of premetamorphosis, hormones were injected every day (hydrocortisone, dexamethasone: 25 micrograms/g body wt/day; insulin: 5 mU/g body wt/day, for 3 and occasionally 6 consecutive days. Under such conditions, most of the activities in the primary epithelium increased or remained stable. In animals which completed experimental metamorphosis, the secondary epithelium formed. Hydrocortisone (25 micrograms/g body wt/day) and insulin (5 mU/g body wt/day) treatments significantly decreased the enzymatic activities of the new brush border membrane in animals which received one hydrocortisone and/or insulin injection per day, during 3 consecutive days. Such results, which previously had not been obtained systematically in spontaneously metamorphosing tadpoles (El Maraghi-Ater, Mesnard, and Hourdry (1986). Gen. Comp. Endocrinol. 61, 53-63), emphasize the relative independence of the intestinal metabolism during experimental and spontaneous metamorphosis.

Phosphorylated proteins from anuran intestinal microvilli membranes—I. Relations with alkaline phosphatase

The degree of phosphorylation of intestinal microvilli membrane proteins in an adult amphibian, Rana esculenta, was investigated under various experimental conditions. The microvilli protein phosphorylation rate rapidly increases during the first 4 min of incubation in a medium containing [gamma-32P]ATP. This increase is slower afterwards. Cyclic nucleotides (cyclic AMP, cyclic GMP) and sorbitol do not modify the microvilli protein phosphorylation rate. On the contrary, this phosphorylation rate significantly decreases in the presence of L-lysine, when its concentration in the incubation medium is greater than 25 mM. The time course of phosphorylation confirms the inhibitory effects of L-lysine (100 mM). The microvilli membrane proteins were distinguished by polyacrylamide gel electrophoresis. In heated samples, electrophoresis followed by an radioautograph systematically reveals the existence of a very phosphorylated protein with a mol. wt of 86 kDa. The phosphorylation of this protein is partially inhibited by L-lysine (100 mM). The very phosphorylated protein could be the monomer of alkaline phosphatase. The dimer (170 kDa) is visualized on electrophoretograms by its catalytic activity. In mammals, several authors have established a correlation between phosphorylation of the microvilli membrane proteins and the intensity of intestinal calcium absorption. Such a control is presently being investigated in adult Rana esculenta.