Growth hormone in neural tissues of the chick embryo

Growth hormone (GH) gene expression predominantly occurs in the pituitary gland, although it also occurs in many extrapituitary sites, including the brain. The cellular location and ontogeny of neural GH production is, however, largely unknown. This has therefore been determined during chick embryogenesis. In chicks, the brain develops from the neural tube at embryonic day (ED) 3. At this age, the divisions of the brain (the telencephalon, diencephalon, mesencephalon, metencephalon and myelencephalon) have intense GH immunoreactivity (GH-IR) (detected by two polyclonal antibodies and a monoclonal antibody for chicken GH). The otic and optic vesicles were also strongly GH immunoreactive, as were the Vth (semi-lunar), VIIth (facial), VIIIth (acoustic) and IXth (glossopharyngeal) nerve ganglia. This GH-IR was specific for GH and was lost when the antibodies were preabsorbed with recombinant chicken GH. The widespread distribution of GH-IR in the neural tissues of ED 3 embryos was mirrored by the distribution of GH receptor (GHR) immunoreactivity, detected by an antibody raised against the chicken GHR. In ED 6/ED 7 embryos, the neural retina of the eye and the epithelial and lens fiber cells were intensely stained for GH-IR, as was Rathke's pouch and the wall of the diencephalon. In contrast, only a few scattered cells were immunoreactive in the surrounding mesoderm. At ED 14, the GH-IR in the brain was restricted to specific tissues and cells. For instance, immunoreactive cells were present in the molecular and pyramidal layers of the cerebral cortex, in the gray matter of the cerebellum, in the choroid plexus, and in the walls of the ventricles. In summary, GH- and GHR-like proteins are abundant in neural tissues of the chick during the first third of incubation, becoming discretely localized to specific tissues and cells during later incubation. The localization of GH and GHR in these tissues, prior to the ontogeny of plasma GH, suggests autocrine or paracrine roles for GH during early embryogenesis.     

Hemodynamic characteristics in neural crest cell-excised chick embryo

Summary Damage to premigratory cranial neural crest cells results in cardiovascular anomalies of so-called conotruncal anomaly. In one report, it was suggested that hemodynamic alteration would precede abnormal cardiovascular morphogenesis. We repeated this hemodynamic study in neural crest cell-excised chick embryos and found that there was no difference in heart rate and blood pressure between the treated embryos (n=11; 164±3 beats per min (BPM) and 0.75±0.03 mmHg, respectively) and control embryos (n=7; 160±6 BPM and 0.72±0.07 mmHg, respectively). The response of heart rate to acetylcholine was less (P<0.05) in the treated embyos (–13% ±2%) than in the control (–20%±2%). Thus, the present data do not support the hypothesis that alterations in blood pressure and heart rate are causally related to abnormal cardiovascular morphogenesis. The developmental significance of subtle functional changes in neural crest-extirpated embryos in response to cholinergic challenge is unclear.     

Cyproterone-mediated stimulation of delta-aminolevulinic acid synthetase in chick embryo liver cells.

In cultured chick embryo liver cells, cyproterone and cyproterone acetate, synthetic anti-androgenic steroids, were found to be potent inducers of mitochondrial delta-aminolevulinic acid (ALA) synthetase, the rate-limiting enzyme in the heme biosynthetic pathway. Both steroids produced a detectable increase in enzyme activity at 5 muM and a maximal stimulation, 36-fold for cyproterone and 29-fold for cyproterone acetate, at 55 muM. The dose-response curves of the steroids differed, however, in that cyproterone acetate produced a greater mean stimulation of the enzyme at concentrations less than approximately 25 muM, whereas, at higher concentrations, cyproterone was the more effective inducer. Increased activity of ALA synthetase was not apparent until about 12 hours after the addition of cyproterone, and maximal activity was not achieved until 20-24 hours. The induction of ALA synthetase by these anti-androgens was prevented by actinomycin D, cordycepin, anisomycin, cycloheximide, and puromycin. These results suggest that new RNA and protein synthesis are necessary for enzyme induction. The cyproterone-mediated induction of the enzyme was inhibited 50% by 2 muM heme, the putative physiological inhibitor of ALA synthetase. These antiandrogens, unlike other potent steroid inducers of this enzyme in chick embryo liver, do not possess either a 5beta-pregnane or 5beta-androstane nucleus. The stimulation of hepatic ALA synthetase represents the first example of a direct effect of these steroids on enzyme induction.     

Distinct calcium-independent and calcium-dependent adhesion systems of chicken embryo cells.

Three criteria have been used to distinguish among different systems of embryonic cell adhesion: dependence on Ca2+, involvement of particular cell-surface molecules, and binding specificity. The characterization of the adhesion with respect to cell-surface molecules was carried out by using specific antibodies against the neural and liver cell adhesion molecules (N-CAM and L-CAM) and antibodies raised against retinal cells prepared by limited trypsinization in the presence of Ca2+ (called "T/Ca cells"). Aggregation of cells prepared from retina or brain without Ca2+ did not require Ca2+ and was inhibited by anti-(N-CAM) antibodies but not by anti-(L-CAM) or anti-T/Ca cell antibodies. In contrast, cells obtained from the same tissues in the presence of Ca2+ did require Ca2+ to aggregate. This aggregation was inhibited by anti-T/Ca cell antibodies but not by anti-(N-CAM) or anti-(L-CAM) antibodies. Hepatocyte aggregation also required Ca2+ and was inhibited only by anti-(L-CAM) antibodies. These results define three antigenically distinct cell adhesion systems in the embryo and raise the possibility that additional systems will be found. The neural Ca2+-independent system displayed a limited tissue specificity, mediating binding to neural but not liver cells. In contrast, the Ca2+-dependent systems of both neural and liver cells caused binding to all cell types tested. The Ca2+-dependent system was most active in retinal cells from 6-7 day embryos, whereas the Ca2+-independent system was most active at later times during development. In addition, treatments that inhibited the Ca2+-independent or Ca2+-dependent systems had very different effects on the fasciculation of neurites from dorsal root ganglia. All of the results suggest that Ca2+-independent and Ca2+-dependent adhesion systems play different functional roles during embryogenesis.     

Nonspecific nature of the stimulus to DNA synthesis in cultures of chick embryo cells.

The rate of DNA synthesis in chick embryo cultures deprived of serum is stimulated 5- to 20-fold by a large variety of substances, including subtoxic concentrations of certain metal ions such as Zn++, Cd++, and Hg++. The stimulatory concentrations of Zn++ and Cd++ have sharp optima, which are just below the concentrations that produce frank morphological damage in each case. A much wider gap exists between stimulatory and morphologically damaging concentrations of Hg++. These metal ions also stimulate RNA synthesis, and the uptake of 2-deoxy-D-glucose. The stimulatory effects of Zn++, but not those of Hg++, are prevented by treatment with EDTA. Although medium from cultures stimulated by Zn++ or Hg++ retains its stimulatory capacity for a new set of cultures, the capacity in the case of Zn++-treated cultures is almost entirely lost upon addition of EDTA. It is also lost upon dialysis of conditioned medium from cultures treated with either Zn++ or Hg++. It is concluded that the stimulatory effect is the direct result of interaction between metal ions and cells, and not to the release of growth-stimulatory materials from the cells. The stimulation is thus seen as a non-specific event resulting in an integrated, metabolic response by the cells.     

FSH- and TSH-binding cells in the ovary of the developing chick embryo

The numerical density (Nv) and location of thyroid stimulating hormone (TSH)- and follicle stimulating hormone (FSH)-binding cells in the left ovary of the chick embryo were determined on Days 6.5 through 19.5 of incubation. From Days 6.5 to 11.5, TSH- and FSH-positive cells were located in the medullary cords and there was no statistically significant difference in the Nv of these two cellular populations. However, beginning on Day 12.5 and continuing through Day 19.5, TSH and FSH were bound principally to cells of the cortical cords and there was a significant difference (P less than 0.05) between the Nv of TSH-positive and FSH-positive cells. Results are discussed in terms of a prevalent hypothesis that throughout chick embryo development the left ovary lacks specific receptors for TSH and FSH.     

Actin microheterogeneity in chick embryo fibroblasts.

Secondary chick embryo fibroblasts contain three distinct actin species--alpha, beta, and lambda, in the approximate ratio of 1:6:3--with the same molecular weights but different isoelectric points. The most acidic of these components, alpha, comigrates on isoelectric focusing gels with the major actin of cardiac and skeletal muscle, while lambda, the most basic of the actins, comigrates with smooth muscle actin. The three components have overlapping methionine-containing tryptic peptides. All three actins are found to be present in actomyosin and cytoskeleton preparations from chick embryo fibroblasts. Identification of alpha-actin as the major actin from sarcomere-containing cells is confirmed by comparing embryonic chicken pre- and post-fusion myoblast cultures. Following myoblast fusion, the relative amount of alpha-actin increases until it changes from a minor actin component to the predominant actin species in the culture.     

A neural cell adhesion molecule from human brain.

We have used a crossreactive monoclonal antibody against a mouse neural cell adhesion molecule (N-CAM) to characterize and purify an antigen from embryonic and neonatal human brain. This antigen was identified as human N-CAM according to several criteria, including molecular weight, sialic acid content, amino acid composition, analysis of peptide fragments, reactivity with a variety of anti-CAM antibodies, and activity of the antigen in assays of cell and membrane vesicle aggregation. The findings make feasible an exploration of the possible role of N-CAM in various neurological disorders.     

Mechanisms of serum-enhanced adhesion of human alveolar macrophages to epithelial cells.

Adhesive interactions between macrophages and epithelial cells in the pulmonary alveoli may be important in the pathogenesis of inflammatory lung diseases, such as those induced by cigarette smoking. Potential mechanisms controlling the interactions between these cells were investigated using human alveolar macrophages (AM) and MDCK or A549 epithelial cells. Five percent human serum enhanced the adhesion of AM to MDCK cells by approximately 6-fold and to A549 cells by approximately 1.7-fold. This enhancement was reduced by heating the serum for 30 min at 55 degrees C. Treating normal human serum with methylamine to inactivate C3, substituting C3-deficient serum, or pretreating serum-exposed MDCK cells with anti-C3 F(ab')2 all significantly diminished the adhesion of AM, suggesting that complement is involved. With the use of flow cytometry to examine complement receptors on AM, both CD11b/CD18 and CD11c/CD18 were detected but CR1 was not evident. Preincubating AM with a monoclonal antibody to CD18 reduced the adhesion of AM to MDCK cells by 40% while a significant reduction could not be demonstrated after preincubation with antibodies to either CD11b or CD11c. These data suggest that in the presence of serum C3bi is deposited on the surface of MDCK cells and that AM may attach to these cells, at least in part, through interactions between C3bi and one or more receptors in the CD11/CD18 family, which are present on AM.     

Experimental creation of univentricular heart in the chick embryo.

An experimental study on the heart of the chick embryo demonstrates that the atrio-ventricular canal does not migrate towards the right, but that it is the inter-ampullar ring which almost completely migrates to the left. Consequently: 1. absence of migration of the interampullar ring will be responsible for the development of type A univentricular heart with an accessory chamber; 2. incomplete migration will result in a special type in which the right atrioventricular orifice overrides the septum; 3. excessive migration may be the explanation for the development of type B univentricular heart; 4. lacking or incomplete development of the inter-ampullar ring is the cause for the development of type C or D univentricular heart. In most of the cases, the univentricular heat can be considered the result of arrest of the normal embryogenesis at a more or less early state.     

Thyroidal iodine metabolism during the development of the chick embryo.

Stable iodine was measured in the thyroid gland of the chick embryo from day 9 to day 20 of incubation in order to evaluate quantitatively the functional development of the gland. Total iodine content increased progressively during incubation. From day 9 to day 17 of incubation, this increase resulted from the increases of pellet-bound iodine and of soluble iodine. Afterwards, it essentially paralleled the increase of the soluble thyroglobulin-bound iodine which reflected the increase in both thyroglobulin content and the degree of iodination of the thyroglobulin. The total iodine, thyroglobulin-bound iodine and thyroglobulin (TG) content, increased as power functions of time during incubation, with critical times on days 11 and 15. Their concentrations also increased during the whole incubation period, while the iodide concentration remained roughly constant (25 ng/mg) from day 13 to day 19. Only one iodoprotein, 19.5 S TG, was found, and its heterogeneity of iodination was demonstrated during the whole period of incubation studied (from day 11 to day 20). The degree of dissociation with sodium dodecyl sulfate (SDS) of the TG into 12 S subunits decreased as the degree of iodination of the TG increased. Throughout embryonic development, iodine was bound more and more to TG molecules, which were resistant to dissociation with SDS. While the average iodine content of the TG increased, no appreciable changes were found in iodotyrosine and iodothyronine percentages of TG-bound iodine: monoiodotyrosine, 26%; diiodotyrosine, 43%; thyroxine 12%; 3,5,3'-triiodothyronine, 2.5%. As a consequence, a linear relationship existed for each iodoamino acid between the number of its residues per mole of TG and the iodine content of TG (127I atoms per mole)-- about 30 atoms of iodine was required to form 1 mole of T4. The low efficiency of the TG of the chick embryo as a thyroidal hormone-forming protein was compensated for by its high degree of iodination.     

Pathogenesis of persistent truncus arteriosus and dextroposed aorta in the chick embryo after neural crest ablation

To investigate the contribution of cranial neural crest cells to the developing cardiovascular system in the chick embryo, cauterization of various regions of cranial neural crest was performed. Five regions may be distinguished, each of which contributes mesenchyme to pharyngeal (branchial) arches 1 through 4 and 6. Ablation of arch 3, 4, and 6 regions resulted in a high incidence of persistent truncus arteriosus (PTA) associated with anomalies of the aortic arch. Dextroposed aorta (DPA) or anomalies of the inflow tract were found in all ablation groups. Although anomalies of the aortic arch arteries were induced in all ablation groups and were usually associated with intracardiac anomalies, those of the third and right fourth aortic arch were most frequent in the arch 4 and arch 4 + 6 groups. Anomalies of the sixth aortic arch were most frequent after extensive ablations that included the arch 6 region. We speculate that PTA is a direct result of the decreased population of mesenchymal cells derived from the arch 3 through 6 neural crest regions. DPA or anomalies of the inflow tract may be related to altered hemodynamics due to anomalies induced by neural crest ablation. Anomalies of the aortic arch arteries may be caused by either the direct or indirect process.     

Follicle-stimulating hormone regulates steroidogenic enzymes in cultured cells of the chick embryo ovary

This investigation addresses the potential regulation of enzymes involved in the biosynthesis of steroid hormones during early stages of gonadal development by follicle-stimulating hormone (FSH). Gonadal cells of 10-day-old chick embryo and cells of the left ovary of 18-day-old chick embryo were cultured for 60 h in a defined medium with or without the addition of FSH (2.0 IU/ml). At the end of the culture, cells were recovered and evaluated by biotransformation of tritiated steroid precursors and mRNA levels were evaluated by RT-PCR. The production of estrone from androstenedione was increased in the FSH-treated cells, both human FSH (hFSH) and recombinant human FSH (rhFSH), indicating a stimulatory effect on aromatase (P450arom). Similarly, the intensity of the band corresponding to P450arom mRNA was higher in hFSH and rhFSH than in control and chorionic gonadotropin (hCG) groups. The P450arom stimulation was observed in the ovary of 10- and 18-day-old chick embryo. The transformation of dehydroepiandrosterone to androstenedione was taken as evidence of 3beta-hydroxysteroid dehydrogenase function. This enzyme was stimulated in the cultured ovarian cells of 18-day-old chick embryos treated with hFSH and rhFSH compared with controls. The production of pregnenolone in the mitocondrial fraction of 18-day-old chick embryo ovary was increased when cultured with hFSH and rhFSH. This observation together with the increase in the band intensity corresponding to mRNA of P450 cholesterol side-chain cleavage indicates stimulation by FSH treatment; hCG produced a similar effect. Somatic cells of the medullary cords are proposed to be FSH target cells in the ovary of the chick embryo.