Histones from chick embryo, chick and chicken liver nuclei

Histones were prepared and purified from chick embryo, chick and chicken liver nuclei. The comparative analysis of these histone preparations, fractionated by polyacrylamide gel electrophoresis, indicates that histone fractions of chick embryo, chick and chicken livers are respectively identical and they comigrate with calf thymus histones.     

Cytochemical study of the ovary cells in the chick embryo

In the present work, cytochemical studies of mucosubstances, 3-beta-hydroxysteroid dehydrogenase and lipids in the different cellular types of chick embryo female gonads were performed, at 7, 11, 15, and 19 days of embryonal development. Oocytes from the cortical zone of the left ovary were characterized by the presence of a juxtanuclear cytoplasmic cap, which was basophilic, PAS positive, alcianophilic and acidophilic and was related to Balbiani's vitelline body. These staining characteristics either decreased or disappeared from 15 days in oocytes of the normal left ovary medulla as well as in oocytes of the atrophic right ovary.     

Lectin binding in the ovary of the chick embryo and newborn.

The content, distribution and changes of the glycoconjugates sugar residues in the ovaries of chick embryos, from the 8th day of incubation to hatching and in 1-day old chick, were investigated. For this purpose, a battery of seven HRP-conjugated lectins was used (DBA, SBA, PNA, ConA, WGA, LTA and UEA I). Our data showed that SBA was a marker of the most immature oogonia in the ovarian cortex and medulla. The reactivity with ConA appeared to characterize the cells immediately prior to as well as during the meiotic division, as demonstrated by the presence of alpha-D-mannose at the "Balbiani bodies" in the oogonia of the ovarian cortex. The detection of Con A and SBA reactivity corresponded to maturative stages of the early oogonia in different cortical zones of the chick ovary. Our data also revealed that PNA seemed to be a marker of the degenerating oogonia located in the ovarian medulla. Moreover, PNA binding was a characteristic finding in the endothelial cells of the vessels located in the compact portion of the medulla in the left ovary, from the 8th to the 21st day of incubation and after hatching; PNA reactivity was only seen from the 16th day onwards in the endothelial cells of the cortex. During the whole considered period of incubation and after hatching, reactivity with UEAI, LTA and DBA was never detected.     

Solubilization and partial purification of aromatase from chicken ovary

Aromatase, the cytochrome P-450 that converts androgen to estrogen, has been solubilized from chicken ovarian microsomes with the nonionic detergent Emulgen 913. Following chromatography on gel filtration, anion exchange, dye affinity, and hydrophobic media, ovarian aromatase is purified up to 27-fold with 10-15% recovery. Separation of the cytochrome P-450 aromatase from NADPH cytochrome P-450 reductase is achieved during the purification. The partially purified enzyme is stable for as long as 6 months when frozen in liquid nitrogen in buffer containing dithiothreitol, glycerol, Emulgen and 150 mM KCl.     

Selective 19-hydroxylase inhibition by an aromatase inhibitor, 4-hydroxyandrostenedione

19-Nor-deoxycorticosterone is a newly recognized mineralocorticoid which has been associated with some forms of genetic, experimental, and human hypertension. To further examine this relationship, specific inhibitors of 19-nor-deoxycorticosterone biosynthesis must be developed. Since 19-hydroxylation is the pivotal step in both 19-nor-deoxycorticosterone biosynthesis and aromatization of androgens to estrogens, we evaluated an aromatase inhibitor, 4-hydroxyandrost-4-ene-3,17-dione on the inhibition of 19-hydroxylation in both rat and human adrenal mitochondria in vitro and 19-nor-deoxycorticosterone production and blood pressure in spontaneously hypertensive rats in vivo. Adrenal mitochondria from 48 male Sprague-Dawley rats and 1 patient with an aldosterone-producing adenoma were incubated in the presence of deoxycorticosterone substrate both with and without 4-hydroxyandrost-4-ene-3,17-dione. 4-Hydroxyandrost-4-ene-3,17-dione produced significant inhibition of 19-hydroxy-deoxycorticosterone production in both rat and human adrenal mitochondria, with a smaller and not significant inhibition of corticosterone and 18-hydroxy-corticosterone. 4-Hydroxyandrost-4-ene-3,17-dione given subcutaneously to spontaneously hypertensive rats lowered 19-nor-deoxycorticosterone by 69% and completely abolished hypertension compared to Wistar-Kyoto controls. These data demonstrate that 4-hydroxyandrost-4-ene-3,17-dione is a specific inhibitor of 19-hydroxylase, that it lowers 19-nor-deoxycorticosterone production and prevents hypertension in the spontaneously hypertensive rat. These studies reinforce the possible pathogenic significance of 19-nor-deoxycorticosterone in hypertension in spontaneously hypertensive rats.     

Inhibiting effect of the testis on the ovary of the chick embryo]

The testicular hormone responsible for the retrogression of the mullerian ducts in the female chick embryo submitted to an embryonic testicular graft, causes an atrophy of the two, cortical and medullary, parts of this gonad, which is more marked at the level of the cortex.     

Ultrastructural localization of acid phosphatase in germ cells of chick embryo left ovary

The ultracytochemical localization of acid phosphatase was studied in oogonia and oocytes of the chick embryo left ovary. The reaction products are evident in lysosomes of various types and, in some cells, in the GERL as well. Furthermore, from the onset of the meiotic prophase, the enzymatic reaction also appears in the rough endoplasmic reticulum. Non-incubated sections of the same stages were observed, with the aim of identifying and describing the structure of the organelles, in particular lysosomes which appeared positive in incubated sections. The significance of the presence of the enzyme is discussed.     

Fate of egg white trypsin inhibitor and start of proteolysis in developing chick embryo and newly hatched chick

Trypsin inhibitor and proteolytic activities were studied in incubated eggs, embryos, and newly hatched chicks. After rupture of the secondary seroamniotic suture at 11 days, the trypsin inhibitor content of the albumen gradually passes into the amniotic cavity; from there it is taken up orally by the chick embryo. It is supposed that between 11 and 18 days of embryonic development the trypsin inhibitor passes from the gut to the yolk sac through the vitellointestinal duct. The thin yolk contained only traces of trypsin inhibitor, and the allantoic fluid was entirely free from it. The amylase activity demonstrable in the liquid intestinal contents of the chick embryo indicates the presence of pancreatic secretion. The trypsin inhibitor probably suppresses the proteases not only directly, but also through prevention of the activation of zymogens. Enterocytes of chick embryos showed no morphological indication of the absorption of undigested proteins on histological examination. The cloacal membrane of the newly hatched chick ruptures shortly after the bird has dried up, and the trypsin inhibitor is subsequently eliminated along with the intestinal contents. The intestinal proteolytic enzymes appear immediately afterward. The proteolytic activity appeared regardless of whether the birds were or were not fed. Maximum proteolytic activity was measured in the small intestine of chicks that were fasted for 2 days after hatching. The pattern of proteolytic enzymes as well as their sensitivity to protease inhibitors did not notably differ from that of mammals.     

A photoaffinity inhibitor of aromatase

Several 7-substituted 4-androstene-3, 17-diones are potent inhibitors of the biosynthesis of estrogens, with the most effective being 7-(4'-amino)phenylthio-4-androstene-3, 17-dione. An azide derivative of this 7-thioether compound has been prepared as a potential photoaffinity inhibitor. The enzyme kinetics of the azide analog were examined under both dark conditions and UV irradiation. In the dark, the azide was a very potent competitive inhibitor, with an apparent K_i of 1.3 nM. Under UV-irradiation, a time-dependent loss of aromatase activity was also observed. These studies indicate that the 7-substituent enhances the affinity of the steroidal analogs for the enzyme site.     

Studies on porphyrinogen carboxy-lyase from chick embryo liver

The purpose of the present work was to find the optimal conditions for the assay of chick embryo liver porphyrinogen carboxy-lyase. The enzyme activity was studied as a function of protein and substrate concentrations, time, pH value and incubation temperature. The effects of reduced glutathione (GSH), ethylenediaminetetra-acetate (EDTA), oxygen and several chemical compounds such as pyridoxal phosphate, sodium and potassium halogenated salts, sulph - hydryl reagents, chelating agents and ferrous iron were also investigated. The following results were found: (1) The optimal protein concentration was 1.25-2.5 mg/ml. For the substrate uroporphyrinogen III, the best concentration was 2 mumol/l; a slight inhibition was found at higher substrate concentrations. (2) The optimal pH value was 6.8 for both stages of uroporphyrinogen III decarboxylation ( octacarboxylic first stage----heptacarboxylic second stage---- tetracarboxylic porphyrinogen). (3) The activity increased with the incubation temperature (25-60 degrees C). (4) The enzyme activity was not enhanced by the addition of GSH or other sulph - hydryl reagents (cysteine and dithiothreitol), nor by the incorporation of EDTA or other chelating agents (Na-diethyldithiocarbamate, alpha,alpha'-bipyridyl and Na-pyrophosphate). (5) Oxygen diminished the second stage of decarboxylation. (6) Pyridoxal phosphate seems not to be a cofactor necessary in the decarboxylation. (7) NaCl or KCl diminished the activity in higher degree than did NaF or KF; the second stage was, in all cases, more affected than the first. (8) FeSO4 slightly diminished the second stage of uroporphyrinogen III decarboxylation at concentrations of 0.05 and 0.15 mmol/l, but both stages were significantly decreased at 0.3 mmol/l.     

Isolation of a developmentally regulated lectin from chick embryo

A lectin is isolated from the microsomal fraction of chick embryo kidney after initial extraction with 1 M urea and 0.3 M lactose. To exhibit hemagglutination activity, the lectin in the microsomal fraction requires prior activation by solublization with deoxycholate or by treatment with trypsin, chymotrypsin or phospholipase c. The lectin is partially purified by hydrophobic interaction chromatography about 200 fold from the microsomal fraction. The lectin binds strongly to de-sialated embryonic carbohydrates and shows low affinity toward glucosamine, galactosamine and mannosamine, as judged by the inhibition of hemagglutination. Comparison of the lectin activity from kidneys of embryos at different ages shows that the lectin is developmentally regulated.     

Chromatin of primitive erythroid cells from the chick embryo

Differentiation of primitive erythroid cells derived from the yolk sac of the chick embryo is accompanied by changes in the morphology of and in the physicochemical properties of the nucleus. Microfluorimetry of individual nuclei stained with acridine orange was performed on thermally denatured cells. Measurements were made at 530 nm (green fluorescence) and 590 nm (redfluorescence). The ratio of these two measurements was used to monitor the susceptibility of chromatin to thermal denaturation. Differences were found (a) between mature erythrocytes and dividing erythroblasts, and (b) between dividing erythroblasts from successive cell generations of the erythroid series. There were differential characteristics of AO binding during thermal denaturation as signified by F₅₃₀ and F₅₉₀ measurements. The temperature at which the increase of the ratio (F₅₉₀/F₅₃₀) was 50% of its maximum was approximately 70° C for erythroblasts from the fifth generation (day 4), 80–85° C for the sixth generation (day 5), and 85–90° C for the nondividing erythrocytes (day 8). Interpretation of these differences may be complicated by changes in the sensitivity of nuclear proteins to the interactive effects of 0.15 M NaCl and thermal denaturation.     

Development of an embryonic thyroid grafted into a chick embryo

A morphological and physiological study of an embryonic thyroid grafted in a chick embryo showed that it developed according to the endocrine status of the host. Its relative age appreciated at various stages of embryonic life is different from that of a gland developing normally during similar lengths of time.     

Action of testicular graft on the ovary of chick embryo having undergone a partial decapitation]

The absence of the hypothalamo-hypophyseal complex had neither influence on the ovarian atrophy, nor on the mullerian retrogression induced by an embryonic testicular graft in the female embryo. So, the hormone responsible of the mullerian retrogression had an inhibiting action on the embryonic ovary of the chick which is independant of the hypothalamo-hypophyseal complex.