Quantitative morphologic studies on the myocardium in early stages of ontogenesis and after application of various cardioactive agents

The authors have morphometrically studied the differentiation of the myocardium in dynamic phases of the embryonic and postnatal development in chickens and Syrian Hamsters. Moreover, they investigated the action of the beta-adrenalytic substances Practolol and Trimepranol on ultrastructure of the cardiac muscle in adult animals. The volume of mitochondria in myocardial cells in 6-day old chicken embryos amounts to 5.65% of the total cell volume, in 12-day old embryos 14.35%, in 18-day old embryos 19.60%, in 1-day old chickens 23.24% which is nearly as much as in adult animals. The volume of myofibrils in 6-day old embryos is about 3.2%, in 12-day old embryos about 7.4%, in 18-day old embryos about 16.4% and in 1-day old chickens about 21.2%. The differences between individual groups are statistically significant. The dynamics of differentiation of the myocardium in Syrian Hamsters was studied in 5 phases, namely in 14-day old embryos and in postnatal phases on the 2nd, 5th, 14th and 21st days after birth. Most cells in 14-day old embryos are rather immature. Participation of the volume of mitochondria, myofibrils, equipment of mitochondria with cristae etc. considerably increase in postnatal phases. These findings suggest that the heart of mammals is rather immature at birth and will differentiate mainly in the postnatal developmental phases. Many morphometric findings, as regards the action of beta-adrenalytic drugs on the ultrastructure of the myocardium in adult rabbits, point to the fact that application of these substances will give rise to degenerative alterations in approximately 10% of myocardial cells. Theoretical explantation of these mechanisms is being discussed.     

The inotropic action of adrenergic agonists on the heart ventricles of the chick embryo]

The effects of adrenergic drugs on the twitch tension of the electrically driven (1.2-1.5 Hz) ventricular preparations from 2-20-day old chick embryos and hatched chicks were studied. Agonists evoked positive inotropic responses of 3-day embryonic ventricles and of ventricles from older animals. 2-day embryonic ventricles were unresponsive. 5-day embryonic ventricles were most sensitive to agonists (EC50 value of adrenaline = 4.5 x 10(-9) M), while ventricles from 14-20-day old embryos had a minimal sensitivity (1-2 x 10(-9) M), while ventricles from 14-20-day old embryos had a minimal sensitivity (1-2 x 10(-7) M). The order of agonists activity (isoproterenol greater than noradrenaline greater than adrenaline much greater than phenylephrine) and the high potency of propranolol as antagonist of adrenaline indicate that responses are mediated with beta-adrenoceptors. The role of GTP-binding protein for the regulation of adrenoreactivity in embryonic chick heart during ontogenesis is discussed.     

Fucose and sialic acid contents of intestinal microvillus membranes from different animal species

Fucose and sialic acid contents of intestinal microvillus membranes isolated from different animal species have been analysed. Expressed on protein basis, brush borders from fish contained considerably high amounts of sialic acid (298 +/- 16 nmole/mg protein), while rat, goat, sheep and guinea pig membranes showed 41-61 nmole/mg protein. Pig, frog, monkey rabbit and chicken membranes exhibited low levels of sialic acid (10-13 nmole/mg protein). Fucose content of the brush borders was quite high (203-212 nmole/mg protein) in frog and fish intestines. It was least in rabbit (54 +/- 3) and of intermediate levels (80-122 nmole/mg protein) in various other animal species analysed. Fucose to sialic acid molar ratio was less than 1 in fish microvillus membranes. In all other animal species, the ratio was however, greater than one and ranged between 1.65 and 15.20.     

Ontogeny of melatonin, Per2 and E4bp4 light responsiveness in the chicken embryonic pineal gland

The chicken pineal gland possesses the capacity to generate circadian oscillations, is able to synchronize to external light:dark cycles and can generate an hormonal output--melatonin. We examined the light responses of the chicken pineal gland and its effects on melatonin and Per2, Bmal1 and E4bp4 expression in 19-day old embryos and hatchlings during the dark phase, subjective light phase and in constant darkness. Expression of Per2 and E4bp4 were rhythmic under light:dark conditions, but the rhythms of E4bp4 and Bmal1 mRNA did not persist in constant darkness in 19-day old embryos. Per2 mRNA expression persisted in constant darkness, but with a reduced amplitude. Per2 expression was inducible by light only during the subjective day. Melatonin release was inhibited by light only at end of the dark phase and during the subjective light phase in embryos. Our data demonstrate that the embryonic avian pineal pacemaker is light sensitive and can generate rhythmic output, however the effects of light were diminished in chick embryos in compared to hatchlings.     

Ontogeny of melatonin, Per2 and E4bp4 light responsiveness in the chicken embryonic pineal gland.

The chicken pineal gland possesses the capacity to generate circadian oscillations, is able to synchronize to external light:dark cycles and can generate an hormonal output--melatonin. We examined the light responses of the chicken pineal gland and its effects on melatonin and Per2, Bmal1 and E4bp4 expression in 19-day old embryos and hatchlings during the dark phase, subjective light phase and in constant darkness. Expression of Per2 and E4bp4 were rhythmic under light:dark conditions, but the rhythms of E4bp4 and Bmal1 mRNA did not persist in constant darkness in 19-day old embryos. Per2 mRNA expression persisted in constant darkness, but with a reduced amplitude. Per2 expression was inducible by light only during the subjective day. Melatonin release was inhibited by light only at end of the dark phase and during the subjective light phase in embryos. Our data demonstrate that the embryonic avian pineal pacemaker is light sensitive and can generate rhythmic output, however the effects of light were diminished in chick embryos in compared to hatchlings.     

Comparative aspects of microvillus development in avian and mammalian enterocytes

1. Pieces of chicken mid-jejunum have been used to determine microvillus length in enterocytes located at different points along the crypt-villus axis to determine the positional dependence of microvillus elongation. 2. The rate at which enterocytes migrate along the crypt-villus axis has also been measured, by injecting tritiated thymidine into the same animals, to determine the time course of microvillus elongation. 3. In mammals it had been shown previously that the maximal rate of microvillus elongation correlated closely with the physical size of the intestinal crypt (Smith et al., 1984). Comparison of crypt size with maximal rate of microvillus growth in chickens showed this correlation to apply to both animal classes. 4. The maximal microvillus length of a chicken enterocyte was much longer (3.3 microns) and the rate of enterocyte migration much less (4.0 microns/hr) than values reported previously for mammalian intestines. 5. The possibility that some other factor in addition to crypt size is affecting microvillus growth is discussed.     

A comparative ontogenetic study of respiration and oxidative phosphorylation of chicken myocardium mitochondria

When adding alpha-ketoglutarate and glutamate the intensity of respiration by the myocardium mitochondria increases gradually from the 15th day of embryonic development till the chicken hatching out. In the presence of succinate respiration of mitochondria of 15- and 20-day embryos and 5-day chickens is almost the same and decreases noticeably in adult chickens. When the above-mentioned substrates are added the value of P/O gradually decreases during the chicken development.     

Testing the hypothesis that crypt size determines the rate of enterocyte development in neonatal mice

Pieces of mid-jejunum taken from 7-9 day old mice have been used to determine microvillus length in enterocytes located at different points along the crypt-villus axis to test the hypothesis that enterocyte development of structure is directly determined by the physical characteristics of the intestinal crypt. Parallel measurements of enterocyte migration rate were carried out using tritiated thymidine to determine the time course of microvillus elongation in neonatal mice. Microvillus length approximately doubled during early enterocyte migration from the crypt base to the lower part of the villus. Enterocyte migration rate was only 0.9 micron/hr at this stage of development, a value considerably less than that found in adult intestine. Results plotting the time dependency of microvillus elongation were fitted by a logistic curve giving a maximal rate for microvillus growth of 0.004 micron/hr. The corresponding estimate of crypt depth was 35 microns. Both these values are considerably less than those found in adult intestine. These results provide strong support for the general hypothesis that some factor associated with the physical length of the crypt, called crypt factor or CF, is directly responsible for controlling the way enterocytes organize subsequent structural differentiation of their surface membranes.     

Non-histone chromatin protein fractions associated with ‘active’ chromatin in embryonic chicken liver

Non-histone chromatin proteins synthesized during chicken embryonic liver development were labeled with [3H]tryptophan and [3H]methionine and characterized by electrophoresis. During embryonic development protein/DNA ratio in chromatin was low (1.30-1.62) but synthesis of non-histone protein was high. Especially one characteristic fraction K (MW 18 000), tightly bound with DNA was preferentially associated with DNAase II sensitive, active transcribed sequences. In 7-day old and adult chicken synthesis of all non-histone proteins was low, fraction K was absent or synthesized only in small amounts in association with non-active sequences, however protein/DNA ratio in chromatin was high (2.30-2.33).     

Topography of human placental receptors for epidermal growth factor

These studies were undertaken to determine whether term human placental microvillus plasma membranes, which are exposed to maternal blood, and basolateral plasma membranes, which are in close proximity to fetal blood capillaries, contain receptors for epidermal growth factor (EGF). These two highly purified membranes bound 125I-EGF with similar affinity (apparent dissociation constants, 0.07-0.12 nM, but the total number of available receptors was greater in microvillus (8.2 pmol/mg protein) compared to basolateral (4.9 pmol/mg protein) plasma membranes. Detailed characterization of 125I-EGF binding to these membranes revealed numerous similarities as well as differences. The two membranes contained two major (155 and 140 kDa) and at least three minor (115, 175, and 210 kDa) specific 125I-EGF binding proteins. The 115-kDa protein was only found in basolateral plasma membranes. The 155-kDa protein was predominantly labeled in microvillus, whereas the 140-kDa protein was labeled predominantly in basolateral plasma membranes. The addition of protease inhibitors did not alter the multiple 125I-EGF binding proteins pattern found in these membranes. EGF stimulated phosphorylation of 140- and 155-kDa proteins in both microvillus and basolateral plasma membranes. However, the 155-kDa protein was phosphorylated to a greater extent in microvillus, whereas both 140- and 155-kDa proteins were phosphorylated equally in basolateral plasma membranes. Light and electron microscope autoradiographic studies revealed that 125I-EGF preferentially associated with microvillus plasma membranes. The data demonstrates the presence of EGF receptors in outer cell membranes of syncytiotrophoblasts and suggests that maternal EGF may influence syncytiotrophoblast function by binding to receptors in microvillus plasma membranes, while fetal EGF may also influence syncytiotrophoblast function but via receptors in basolateral plasma membranes.     

Collagen synthesis by long-lived mRNA in embryonic chicken lens

Lens capsule collagen synthesis by epithelial and fiber cells was examined by immunoprecipitation and collagenase digestion in embryonic and posthatch chicken eye lens. Epithelial cells and lens fibers in the process of terminal differentiation produce alpha 1 and alpha 2 type IV collagen chains. At 6 days of embryonic development in addition to the alpha 1 (IV) and alpha 2 (IV) collagen chains, lens cells produce high molecular weight collagenase-sensitive proteins not immunologically related to type IV collagen. Lens capsule collagen components have been identified in central and outer fibers isolated from 18-day embryos and from 10-day posthatch chicken eyes. At these stages, fibers which have an increasing number of picnotic nuclei still show collagen synthesis due to long-lived mRNA. Analysis of collagen synthesis by lens cells incubated with actinomycin D suggests that stabilization of collagen mRNA occurs in lens fiber cells and to a lesser extent in epithelial cells as early as 6 days of embryonic development.     

Embryonic-like myosin heavy chains in regenerating chicken muscle

An antibody to chicken ventricular myosin was found to cross-react by enzyme immunoassay with myosin heavy chains from embryonic chicken pectorials, but not with adult skeletal myosins. This antibody, which was previously shown to label cultured muscle cells from embryonic pectoralis (Cantini et al., J cell biol 85 (1981) 903), was used to investigate by indirect immunofluorescence the reactivity of chicken skeletal muscle cells differentiating in vivo during embryonic development and muscle regeneration. Muscle fibers in 11-day old chick embryonic pectoralis and anterior latissimus dorsi muscles showed a differential reactivity with this antibody. Labelled fibers progressively decreasgd in number during subsequent stages and disappeared completely around hatching. Only rare small muscle fibers, some of which had the shape and location typical of satellite elements, were labelled in adult chicken muscle. A cold injury was produced with dry ice in the fast pectoralis and the slow anterior latissimys dorsi muscles of young chickens. Two days after injury a number of labelled cells was first seen in the intermediate region between the outer necrotic area and the underlying uninjured muscle. These muscle cells rapidly increased in number and size, thin myotubes were seen after 3 days and by 4–5 days a superficial layer of brightly stained newly formed muscle fibers was observed at the site of the injury. Between one and two weeks after the lesion the intensity of staining of regenerated fibers progressively decreased as their size further increased. These findings indicate that an embryonic type of myosin heavy chain is transitorily expressed during muscle regeneration.     

The pattern of methylation of RNA in peripheral nerve of the chick during development

The pattern of the methylation of RNA was investigated in organ cultures of the sciatic nerve of the chicken. Nerve tissue from 14-day embryos, 17-day embryos and 3-day- old chicks was incubated with [methyl-³H]methionine or with [2-¹⁴C]uridine and [methyl-³H]methionine simultaneously for various periods of time. Subsequently, RNA was extracted from the tissues and the purified preparations were fractionated by polyacrylamide gel electrophoresis. The electrophoretic patterns of the rapidly labelled RNA changed during the three developmental stages. The incorporation of both uridine and the methyl groups from methionine was highest in the‘heavy’RNA species of the 14-day embryonic nerve during the 0.5 and 1.0 h incubation periods. In contrast, in the nerves of 3-day-old chicks during a 0.5 h pulse with both precursors, methylation was almost entirely limited to the transfer RNA species. Furthermore, the incorporation of uridine in the nerves from 3-day-old animals revealed the presence of a heterogeneous population of rapidlylabelled, unmethylated species of RNA, most of which migrated between the smaller ribosomal RNA and transfer RNA components of the bulk RNA. The pattern of uridine incorporation and the methylation of the rapidly-labelled RNA of the 17-day embryonic nerve represented a transitional state between that of the 14-day embryos and that of the 3-day-old chicks. The 17-day embryonic stage of development corresponded to the phase of the onset of rapid deposition of myelin lipids in the sciatic nerve. Pulse-chase experiments on the embryonic nerves indicated that a number of methylated precursors of ribosomal RNA and labile, heterogeneous, probably DNA-like RNA were synthesized.     

Developmental changes in proteins of purified membranes of chicken lenses and evidence for contamination by cytoplasmic delta-crystallin.

Urea-washed membranes from embryonic chick lenses (15 days old) and from the cortical and nuclear regions of adult chicken lenses (1 year) have been prepared by repeated centrifugation through discontinuous density gradients. The protein components of the isolated membranes have been examined by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate and urea. Proteins with molecular weights of 75 000, 56 000, 54 000, 48 000, 34 000, 32 000, 25 000, and 22 000 were present in all the membrane preparations, although their proportions changed during development. One additional protein, molecular weight 70 000, was seen only in the embryonic lens membranes. The greatest developmental change was the increase in 25 000 molecular weight protein from 12% in the embryonic lens to about 45% in the adult lens. Since it has been suggested that this protein is associated with gap junctions, its increase during development may reflect a corresponding increase in the number of gap junctions in the lens. The 50 000 molecular weight protein of embryonic lens membranes and membranes of adult nuclear lens fibers consisted at least partly of delta-crystallin, since delta-crystallin peptides could be identified in tryptic peptide maps of the isolated protein after in vitro radioiodination. Peptide maps of the 50 000 molecular weight protein of cortical lens fiber membranes contained no identifiable delta-crystallin peptides, although it is possible that modified delta-crystallin peptides may be present. The level of cytoplasmic contamination of the membrane fraction was estimated by preparing lens membranes in the presence of added delta-[35S]crystallin. The results indicated that cytoplasmic contamination contributes significantly to the presence of delta-crystallin in lens membrane preparations.     

ONTOGENY OF CHICKEN HEMOGLOBIN II. CHROMATOGRAPHIC STUDY OF THE HETEROGENEITY OF HEMOGLOBIN IN DEVELOPMENT

Some properties of the carbonmonoxyhemoglobin (HbCO) from chicken embryos of ages 5, 10 and 15 days of incubation, from 1-day posthatching and from adult chickens have been investigated by chromatography on carboxymethylcellulose (CM-cellulose) column and by starch gel electrophoresis.
Chromatogram of the hemoglobin (Hb) from 5-day chicken embryos has shown that it consists of at least 6 components. Starch gel electrophoresis of each isolated component from the column in phosphate (pH 6.8), in borate (pH 8.6) and in formate buffer (pH 1.9) has shown later that there are 3–4 embryonic type Hb components in 5-day embryos.
Chromatogram of the hemoglobin from adult chickens has shown that it consists of at least 4 components, but the examination of each isolated component from the column by electrophoresis in phosphate (pH 6.8), in borate (pH 8.6) and in formate buffer (pH 1.9) has shown that there are 4–6 adult type Hb components in adults.
In ontogenic process, embryonic Hb type is detectable in embryos up to 15 days of incubation. Fetal Hb type, which is not detectable in adult chickens, can be first found in 10-day embryos.     

Synthesis of membrane glycoproteins in rat small-intestinal villus cells. Redistribution of l-[1,5,6-3H]-fucose-labelled membrane glycoproteins among Golgi, lateral basal and microvillus membranes in vivo

The biogenesis of plasmalemma glycoproteins of rat small-intestinal villus cells was studied by following the incorporation of l-[1,5,6-(3)H]fucose, given intraperitoneally with and without chase, into Golgi, lateral basal and microvillus membranes. Each membrane fraction showed distinct kinetics of incorporation of labelled fucose and was differently affected by the chase, which produced a much greater decrease in incorporation of label into Golgi and microvillus than into lateral basal membranes. The kinetic data suggest a redistribution of newly synthesized glycoproteins from the site of fucosylation, the Golgi complex, directly into both lateral basal and microvillus membranes. The observed biphasic pattern of label incorporation into the microvillus membrane fraction may be evidence for a second indirect route of incorporation. The selective effect of the chase suggests the presence of two different pools of radioactive fucose in the Golgi complex that differ in (1) their accessibility to dilution with non-radioactive fucose, and (2) their utilization for the biosynthesis of membrane glycoproteins subsequently destined for either the microvillus or the lateral basal parts of the plasmalemma. The radioactively labelled glycoproteins of the different membrane fractions were separated by sodium dodecyl sulphate/polyacrylamide-slab-gel electrophoresis and identified by fluorography. The patterns of labelled glycoproteins in Golgi and lateral basal membranes were identical at all times. At least 14 bands could be identified shortly after radioactive-fucose injection. Most seemed to disappear at later times, although one of them, which was never observed in microvillus membranes, increased in relative intensity. All but two of the labelled glycoproteins present in the microvillus membrane corresponded to those observed in Golgi and lateral basal membranes shortly after fucose injection. The patterns of labelled glycoproteins in all membrane fractions were little affected by the chase. These data support a flow concept for the insertion of most surface-membrane glycoproteins of the intestinal villus cells.     

Variations in dietary triacylglycerol saturation alter the lipid composition and fluidity of rat intestinal plasma membranes

Rats were maintained on nutritionally complete diets enriched in unsaturated (corn oil) or saturated (butter fat) triacylglycerols. After 6 weeks, significant differences in the lipid composition and fluidity of a number of intestinal membranes were observed. The corn oil diet (enriched mainly in linoleic acid) increased the overall unsaturation of the acyl chains and enhanced the lipid fluidity, as assessed by the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene, of enterocyte microvillus and basolateral membranes and of colonocyte basolateral membranes. Concomitantly, the cholesterol content and the cholesterol/phospholipid molar ratio were increased in the microvillus but not in the basolateral membranes. The increased cholesterol in ileal microvillus membranes can result from enhanced cellular biosynthesis, since ileal slices from rats fed the unsaturated diet incorporated [14C]octanoate more rapidly into digitonin-precipitable sterol. Increased fluidity of the enterocyte microvillus and basolateral membranes, respectively, enhanced the enzyme specific activities of p-nitrophenylphosphatase and (Na+ + K+)-dependent adenosine triphosphatase. The results indicate that the lipid composition, fluidity and enzyme activities of intestinal plasma membranes can be altered by dietary means. Moreover, rat enterocytes possess regulatory mechanisms which modulate the cholesterol content of the microvillus membranes so as to mitigate changes in lipid fluidity.     

Purification of microvilli and an analysis of the protein components of the microfilament core bundle

A fast and convenient method for the purification of microvilli from chicken intestinal brush borders is described. The microvilli appear morphologically very similar to those found on intact brush borders. Removal of the microvillus membrane from the microvilli by Triton X-100 treatment reveals compact bundles of microfilaments with small regularly spaced projections along their length. SDS-polyacrylamide gel analysis of the protein components of the brush border, the microvilli and the microvillus core bundles shows that little or no tropomyosin, myosin or filamin is found in the microvillus, whereas polypeptide chains with mobilities characteristic for these proteins are present in the whole brush border. The majority of the microvillus core protein is actin, and the other major protein present has a polypeptide molecular weight of 95 000. Total actin from both brush borders and microvilli, characterized by isoelectric focussing analysis, contained about 40% β actin and 60% γ actin. The presence of both the β and γ cytoplasmic actins in the highly ordered parallel arrays of microfilaments of the microvilli is discussed in light of hypotheses for different functional roles of these two actin species.     

Isolation and characterization of brush-border membrane from trout intestine. Regional differences

The isolation of brush-border membranes from trout enterocytes is described for both middle and posterior intestine. Both procedures are based on differential centrifugations combined with calcium precipitation. Classical marker enzymes are quantified and indicate a valuable purification of the membranes (13-18-fold). No difference appears when comparing the relative amounts of phospholipids, cholesterol and proteins in microvillus membranes isolated from either middle or posterior intestine. In contrast, the membranes isolated from middle intestine are more unsaturated than those from the posterior one, and their sphingomyelin/phosphatidylcholine ratio is lower. These differences are reflected by fluorescence anisotropy studies with diphenylhexatriene as lipid fluorophore which indicate a higher fluidity of the microvillus membranes from the middle intestine as compared with those from the posterior intestine. These results point out the importance of the fatty acyl chains and that of the relative amounts of phosphatidylcholine and sphingomyelin in controlling the fluidity of biological membranes in relation with their transport properties.