Death of myocardiocytes and destruction of their structures during embryonic histogenesis

By means of light and electron microscopy, death of cardiomyocytes and their structures has been studied in the myocardium of chick (the 4th-20th days of incubation) and in rat (the 14th-20th days of embryogenesis) embryos. The same processes have been studied after a single and repeated injections of insulin and 5-fluorauracyl to the chick embryos (into the air chamber of the egg). In the myocardium of the chick and rat embryos atrophy of some muscle cells takes place as a demonstration of histogenetic (physiological) death characterized by changes in nucleo- and cytoplasm according to the "dark" type. Under the effect of 5-fluorauracyl the same type of cardiomyocyte death is observed; differences with the control are but quantitative ones. When the chick embryo is affected with cytotoxic concentrations of insulin, together with histogenetic death of the cardiomyocytes (the "dark" type) the death according to the "light" type also takes place. Under the effect of both insulin and 5-fluorauracyl the destructive processes in the cardiomyocytes are manifested greater than in the control. This is a morphological manifestation of certain disturbances in processes of physiological restoration at the cellular level.     

CHOLINE ACETYLTRANSFERASE (ChAc) ACTIVITY IN DEVELOPING CHICK OPTIC CENTRES AND THE EFFECTS OF MONOLATERAL REMOVAL OF RETINA AT AN EARLY EMBRYONIC STAGE AND AT HATCHING

The choline acetyltransferase (ChAc) activity was measured in the optic centres of chick embryos after early removal of the optic cup and of young chicks after monolateral extirpation of the right eyeball after hatching. The contralateral optic lobes were thus deprived of their complement of retinal fibres. The following results were obtained: in chick embryos the ChAc was slightly lower in the deafferented lobe between the 10th and the 14th day of incubation; between the 14th and the 17th day a critical fall in activity was observed leading to a significant ChAc loss of 71 per cent. In eye deprived chicks no significant change in total ChAc activity occurred during the first postoperative month; significant changes were found only in the second month. The results reached so far suggest that removal of retinal fibres does not cause short term changes in optic centre ChAc in either the embryo or the chick. ChAc contained in nerve cell bodies seems independent of synapses and its behaviour is interpreted as a reflection of metabolic disturbance of the centre.     

Use of Horseradish Peroxidase Conjugated Lectins for Detection of Glycoconjugate Changes in Developing Lingual Epithelium of the Chick Embryo

A battery of six different horseradish peroxidase-labelled lectins (SBA, DBA, PNA, WGA, ConA and LTA) was used to study the distribution of carbohydrate residues in glycoconjugates during the development of the chick embryo tongue anlage (7th-21st day of incubation) and in the tongue of 3 day-old chicken, at the dorsal and ventral epithelium. From the 7th to the 16th day β-D-galactose, D-galactose-(β1 → 3)-N-acetyl-D-galactosamine, D-mannose and β-N-acetyl-D-glucosamine show the same epithelial distribution at two lingual aspects. From the 17th day to hatching the epithelium of the dorsal and ventral surfaces is characterized by a different distribution of sugar residues. The functional adaptation of the epithelium, as far as the saccharidic moletie distribution is concerned, seems to be completed at the 21st day of incubation.     

Action of the phenothiazine derivative methophenazine on prenatal development in rats.

Single doses of 100-400 mg/kg or multiple doses of 10 or 50 mg/kg of the phenothiazine derivative methophenzaine were given per osto Wistar rats at various times on the 7th-14th days fo gestation and the fetuses examined near term. Results indicated that methophenazine was mainly embryolethal when administered on the 8th-11th days, and was teratogenic at later times, producing types of malformations that depended on the day of treatment, the most susceptible period being the 13th and 14th days of gestation. Teratogenicity occurred only when the dosages were highly toxic to the pregnant rats. Ribovlavin given ip on the 14th day significantly reduced the embryolethal but not the teratogenic action of methophenazine.     

State of Dogiel type II neurons in the wall of the cat small intestine following a single exposure to gravitational stress]

Under study was the nervous apparatus of the small intestine in 22 cats subjected to a single gravitation stress by rotation in a centrifuge of 1,5 m radius. The stress was equal to 10 units, duration from 2,5 to 3,5 min., and was of head-pelvis direction. The material was treated after Nissl and Gomori in Chilingarian's modification. Within 1-3 days after exposure to gravitation stresses in the Auerbach plexus there appeared changes in the shape of the body of the type II Dogiel cells, such as pericellular oedema, vacuoles in the cytoplasm, chromatolysis and varicous thickenings of nerve fibres. Terminal structures of the dendrites of the type II Dogiel cells are thickened. The changes are most pronounced within the 4th-7th days after exposure to gravitation and are observed as late as the 14th day, being sharply diminished by the 30th day.     

The development of connections in the tecto-thalamo-telencephalic visual system of the brain in 13-day-old chick embryos

Light and electron microscopic studies have been made on degenerative changes in the nervous tissue induced by experimental destruction of the median brain bulb at the 5th day of incubation, in parts of the tecto-thalamo-telencephalic visual system in 13-day chick embryos (in the visual tectum, round nucleus of the thalamus and ectostriatum of the telencephalon). It was shown that to this period tecto-thalamic connections are already formed in the visual system, whereas thalamo-telencephalic connections are, presumably, indirect ones.     

Early development of the primitive cranial vault in the chick embryo.

The calcified tissues involved in the early morphogenesis of the cranial vault were studied by microradiographic analysis and histological techniques in 12 chick embryos on the 9th, 12th, and 14th days of incubation. On the 9th day, the frontal, parietal, and squamosal bones are comprised of a thin lamina of chondroid tissue deposited at a short distance from the fibers of the dura mater. Woven bone formation takes place in the calvarial mesenchyme only after the 12th day of incubation and occurs mainly on the external side of the chondroid primordium. The present data obviously indicate that the primitive desmocranium of the chick embryo, which is usually known to be formed by intramembranous ossification, consists first of chondroid tissue. This tissue represents thus the initial modality of skeletogenic differentiation within the cephalic mesenchyme of the cranial vault.     

Cell differentiation of the head ectoderm in mouse embryos in vitro

Cell differentiation has been studied in the explants of head ectoderm of 8, 9 and 10 day old mouse (CBA) embryos and of head epidermis of 13 day old embryos. Pieces of ectoderm were taken from the temporal region. It was established by indirect immunofluorescence that within 10, 15 and 20 days of cultivation spheroids with keratins or crystallins in some groups of fibres formed in the head ectoderm explants from 9 and 10 day old embryos. When cultivating the regions of head epidermis from 13 day old embryos, spheroids formed with keratin only in their cells. The data obtained suggest that there appear to be two clones of cells determined to the synthesis of keratins or crystallins in the head ectoderm of early mouse embryos. During embryogenesis, the number of cells determined to the synthesis of keratins appears to increase in the regions not related to the eye area. At the same time, the clone of cells determined to the synthesis of crystallins appears to be eliminated.     

Structural metabolic processes in the skeletal muscle tissue of chick embryos administered actinomycin D

The development of the skeletal muscle tissue has been studied cytophotometrically, electron microscopically and radioautographically at administration of actinomycin-D (0.2 mcg/g) to the 11- and 15-day-old chick embryos). Different character of restorative processes under the conditions when RNA synthesis is disturbed by actinomycin-D administration is noted: before morphologically distinguished myosatellites appear (before the 13th-14th day of embryogenesis) and after myosatellites appearance (from the 14th-15th day of development). Evidently, the myosatellites are the muscle cells resistive to certain external factors, they ensure an effective adaptation of the skeletal muscle tissue to unfavourable effects. When the satellite cells appear, the skeletal muscle tissue acquires a new quality as a dynamically stable cambial system.     

Spontaneous and benzo[a]pyrene-induced micronuclei in the embryos of the black-headed gull (Larus ridibundus L.)

The spontaneous levels of micronuclei in erythrocytes were established in embryos of the black-headed gull of two natural populations. In total 216 blood samples from the same number of individuals were examined. A statistically significant decrease in the number of spontaneous micronucleated erythrocytes was found after 13 days of incubation. We found no statistically significant difference in the spontaneous frequencies of micronucleated erythrocytes in the embryos of the two colonies studied, although they differed in anthropogenic load. Results of analysis of variance indicated that egg incubation time was the only variable significantly (P=0.0001) affecting spontaneous frequency of micronucleated erythrocytes in the embryos of black-headed gulls. We also took 78 eggs of different developmental stages from both colonies and exposed them for a further 24h to a dose of benzo[a]pyrene (30 microg per egg). After exposure to benzo[a]pyrene, the frequency of micronucleated erythrocytes was not increased in the embryos incubated for a total period of 13 days. A statistically significant increase in the number of micronucleated erythrocytes was recorded in the benzo[a]pyrene-treated embryos incubated for a total period of 14 days. Decrease in numbers of spontaneous micronucleated erythrocytes after the 13 day of incubation and increased levels of benzo[a]pyrene-induced micronuclei after the 13 day of incubation were discussed to be caused by changes in spleen and liver function in advanced developmental stages of the embryo.     

Histochemical analysis of goblet cell mucins in the chick embryo colon

The histochemical characteristics of colonic epithelial mucins were investigated in the chick embryo. At the 14th day of incubation it was possible to demonstrate the presence of glycogen. At the 15th day a few epithelial cells showed the presence of neutral and sialylated mucins. On the 16th day, also sulfated secretory material was detectable together with neutral and sialylated mucins in cells with the typical shape of goblet cells. From the 17th day to the 20th day of incubation the two types of acid mucins appeared in some cells to be placed in distinct zones of the supranuclear cytoplasm. At the 21st day, neutral, sialylated and sulfated mucins were all present in the majority of goblet cells, which were found mainly in the epithelium lining the crypts.     

Insulin synthesis in chick embryo retinas during development

Retinas of chick embryos contain insulin (1) and further, are capable of synthesizing it, as demonstrated by incubating retinas at different ages (7th–18th day) with [³H]leucine. The synthesized radioactive insulin was isolated and assayed by means of a HPLC procedure. The synthesis of insulin was found to be highest in the youngest retinas studied (day 7), afterwards it declined with age except for an increment found at 14–15 day. Explants of chick embryo retinas, cultured in vitro, rapidly degraded insulin. Nevertheless, the content of immunoreactive insulin in retinal explants diminished slowly with the age of culture, so that, after 8 days of incubation, it was about 60% of the content found in the retinas at the beginning of incubation. This was proof that cultured explants are capable of efficiently synthesizing insulin. The synthesized [³H]insulin was released from explants into the medium. This was evident also after 6–8 days in culture.     

Histochemical detection of sugar residues in the chick embryo mesonephros with lectin-horseradish peroxidase conjugates

Fragments of mesonephros were taken from chick embryos and studied from the 4th to the 21st day of incubation. A battery of seven different horseradish peroxidase-labelled lectins was used to study the distribution of carbohydrate residues in glycoconjugates along the mesonephric nephron during the period of excretory activity and the period of involution. ConA and WGA reacted at every site of the nephron thus showing the ubiquitous presence of alpha-D-mannose and N-acetyl-D-glucosamine. SBA was a good marker of the proximal tubule. Other lectins, such as PNA and LTA, reacted only for a short time at some sites during the considered period of incubation. The presence of sialic acid was detected in the podocytes, capillary wall and mesangial cells. From the 10th-11th day of incubation changes were noted in the proximal tubule as shown by PNA reactivity. This may be significant as regards the exact stage of incubation during which the involution of mesonephros begins.     

Developmental changes in free D-aspartic acid in the chicken embryo and in the neonatal rat

Free D-aspartic acid was measured in fertilized chicken eggs, chicken embryos, and neonatal rats. In each tissue examined a maximum value was found at a characteristic time of development. For the chicken embryo brain, the maximum was 9% D at 11 days of incubation; for the retina, 20% D at 13 days of incubation. In the neonatal rat, as in the chicken embryo, D-aspartic acid continued to increase in the retina after that in the brain and other tissues had begun to decline. The maximum, 29% D, was found 7 days after birth. Thus in two phylogenetically distant species, similar developmental patterns of D-aspartic acid change were observed. Some data on similarities between the D/L aspartic acid ratios of adult chicken and rat tissues are also reported. In addition, the total D-aspartic acid content of the egg, including the embryo, increased from 44 nmol at day 1 to 159 nmol at day 12, showing that release from a bound form or de novo synthesis is a continuing process during development.     

Time of origin of cells in the histiogenesis of the spinal cord]

In order to establish the moment of appearance of neuroblasts and ectoglia of the spinal cord the autoradiographic study with the use of H3-thymidine and C14-thmidine injected to pregnant mice with the intervals between injections 121/2 or 24 hours was undertaken. It was establised that spinal neurons were removed from the nervous tube beginning from the 10th up to 13th days of embroyogenesis. The motoneurons of the anterior horn were the first to appear (10th-12th days), the neurons of the intermideate zone were the next to appear (11th - 12th days) and the last were the neurons of posterior horn (13th day). Beginning from the 13th day of embryogenesis there appeared the ectoglia which migrated following meurblasts two days later. The saturation of the grey matter with glial cells and the saturation of the white matter with Schwann cells was brought about by means of additional multiplication at the site of the glioblasts removed from the nervous tube. The main function of the matrix layer neuroepithelium of the nervous tube as a provider of cells to the spinal cord terminated on the 15th day of embryogenesis.     

Enzymes involved in adenine nucleotide metabolism of developing chick embryo liver.

1. This paper describes the changes in the activity of adenylate deaminase, adenylate and inosinate phosphatase, and adenosine deaminase in the developing chick embryo liver. 2. The adenylate and inosinate phosphatase and adenosine deaminase activity appears considerably higher in chick embryo liver with respect to other chick embryo tissues previously examined. 3. During development the control exerted by ATP on AMP breakdown undergoes variations. Consequently, in the first period of incubation AMP is degraded by the direct pathway (AMP-IMP) and in the last period of incubation by the indirect pathway (AMP-adenosine). In the intermediate period (from the 12th to the 15th day of incubation) both pathways may be followed. 4. The ability to synthesize purine nucleotides through "salvage pathway" seems to be acquired by embryonic liver at least at the 15th day.     

PHYSIOLOGICAL ONTOGENY : A. CHICKEN EMBRYOS. XI. THE PH,CHLORIDE, CARBONIC ACID,AND PROTEIN CONCENTRATIONS IN THE TISSUES AS FUNCTIONS OF AGE.

Investigations of the chicken embryo during its incubation period show that: 1. The pH and the chloride concentration of the tissues decrease with age; the fall is most rapid between the 10th and the 13th days of incubation. 2. The concentration of total CO₂ increases with age. This fact is not considered inconsistent with a possible decrease in the concentration of active bicarbonate ions, since the increased CO₂ might well be the result of absorption of calcium carbonate from the shell and its precipitation as bone in the embryo. 3. The concentration of protein increases with age, especially between the 12th and the 16th days of incubation. The fact that the electrolytes change with the greatest rapidity at about 11½ days, the protein at 14 days, and the fat at 16½ days might be taken as a demonstration of the phenomenon of unequal development in the realm of biochemical differentiation and consequently that some notion of order, depending upon molecular reactivity and mobility would describe the process better than any concept of dynamic equilibrium.     

Histochemistry of mucosubstances in the gallbladder epithelium of the chick embryo

Summary Mucosubstances of epithelial cells of the chick embryo gallbladder were investigated by histochemical methods from days 12–21 of incubation (stages 38–46). On incubation day 14, only neutral mucins were detected; on day 15, neutral and sulfated mucosubstances were observed in the epithelial cells that invaginate the underlying mesenchyme. In the same sites, at day 16 of incubation, neutral, carboxylated and sulfated mucins were seen. From the 17th day of incubation until hatching, neutral, carboxylated and sulfated mucosubstances were present in the surface cells and in the cells lining the epithelial invaginations. During this period, the chemical characteristics of the secretory material are similar to those observed by Yamada and Hoshino (1972) in the fowl.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

Matrices containing glycosaminoglycans in the developing anterior chambers of chick and Xenopus embryonic eyes.

The fibrous matrix present in the anterior chamber of the chick eye on the 4th day of development has been shown by autoradiography and histochemistry to contain chondroitin sulfate, protein, neutral polysaccharides, and possibly hyaluronic acid (HA). Its synthesis, probably by the mesenchyme of the angle of the eye, is completed by around 10 days of development. In the scanning electron microscope (SEM) it can be seen that, although the matrix thins as the eye grows, it does not disappear until the 15th day. The development of the Xenopus cornea is described; this animal has a matrix in its anterior chamber from soon after the formation of the inner cornea (stage 41) until metamorphosis 7 weeks later. In the SEM, this material appears as a dense, featureless aggregate rather than as a matrix of thick fibres; in the transmission electron microscope, it is seen to be a network of fine filaments containing small dark-staining granules. Histochemistry shows that it contains HA, protein, and neutral polysaccharides. The morphological evidence is compatible with the matrix being made by the inner cornea. The probable major role of the matrix is to separate lens from cornea in establishing the anterior chamber. In the chick embryo, at least, the matrix is also likely to help stabilise the endothelium during its formation.