Some features of amino acid and lipid metabolism in embryos of meat-type fowl with different yolk weight

It has been proposed that variations in relative yolk weight in a population of meat-type fowl be used as a model of development of nidicolous and nidifugous birds. During development of the eggs with a high proportion of yolk, an excess of lipids is cleaved at a higher rate and oxidized until day 17 of incubation, while in the embryos developing from the eggs with a low relative yolk weight, amino acids are intensely cleaved during the period preceding the hatching. Significant differences in the body content of cystine were found in 17-day embryos and upon hatching, thus suggesting a delayed activity of the genes encoding keratins in the group corresponding to the seminidicolous type according to the egg content of lipids. These biochemical differences question the widespread concept on the occurrence of dichotomy by the end of embryogenesis and beginning of neonatal growth of nidifugous and nidicolous birds.Translated from Ontogenez, Vol. 36, No. 1, 2005, pp. 3–8.Original Russian Text Copyright © 2005 by Zhuravlev, Dolgorukova, Salamatin, Fisinin.     

Amino acid and lipid metabolism in embryos of flesh fowl with different yolk mass

It has been proposed that variations in relative yolk mass in a population of flesh fowl be used as a model of development of nidicolous and nidifugous birds. During development of the eggs with a high proportion of yolk, an excess of lipids is cleaved at a higher rate and oxidized until day 17 of incubation, while in the embryos developing from the eggs with a low relative yolk mass, amino acids are intensely cleaved during the period preceding the hatching. Significant differences in the body content of cystine were found in 17-day embryos and upon hatching, thus suggesting a delayed activity of the genes encoding keratins in the group corresponding to the seminidicolous type according to the egg content of lipids. These biochemical differences question the widespread concept on the occurrence of dichotomy by the end of embryogenesis and beginning of neonatal growth of nidifugous and nidicolous birds.     

The establishment of essential and metabolically derived amino acid profiles in developing chick embryo organs

The accumulation of certain essential and metabolically derived amino acids in the free amino acid pools of three excitable tissues has been studied in the chick embryo. Valine together with leucine are at the onset present in the yolk at higher concentrations than any of the other essential amino acids. By 15 days all the amino acids studied have accumulated in the embryonic pools at a higher rate than valine, although certain amino acids, such as phenylalanine or methionine, always remain at lower relative concentrations than valine. This reflects their low supply in the yolk, rather than a more rapid rate of disappearance (utilization). During early embryogenesis (E₂–E₄), tissues preferentially concentrate glutamic acid, besides taurine and phosphoethanolamine (6). The next distinct stage of development (E₄–E₇) is marked in the brain by a gradual rise in glutamic acid, glutamine and aspartic acid; the same three amino, acids do not demonstrate a further increase in the pool of the heart, while in the whole eye the amino acid profile begins to resemble the blood. Leucine in all three tissues declines rapidly, to reach isoleucine levels by day 7 of development; tyrosine increases slowly in apparent reciprocity to an equally gradual phenylalanine decrease. Into the second week of embryo growth (E₇–E₁₅), GABA appears in the mesencephalon (E₇) and the eye (E₉–E₁₀). In the mesencephalon, the free amino acid pool composition exhibits a rather sudden increase of most metabolically derived amino acids. Glutamic acid and glutamine in the brain increase in parallel; the rate of GABA and aspartic acid accumulation is slower, and for GABA stabilizes on day 14, as does glutamine. In the eye, by day 15, GABA levels are more closely aligned with the aspartic acid content. Finally, throughout embryogenesis serine fluctuations in blood and tissues are in parallel with those of threonine, and different from glycine or alanine which also change in tandem.     

Stimulatory Effect of Insulin and Epidermal Growth Factor on Activity of Protein Kinase A, Glucose-6-Phosphate Dehydrogenase, and Glycogen Synthase in Skeletal Muscles of Chick Embryos and Chickens

Insulin is able to produce two types of regulatory effects on muscles—metabolic and growth stimulating. Study of the effect of insulin and epidermal growth factor (EGF) on activity of cAMP-dependent protein kinase (PKA), glucose-6-phosphate dehydrogenase (G-6PDH), a starting enzyme of pentosephosphate pathway (PPP), and glycogen synthase (GS), a key enzyme of the glycogen synthesis, has shown that both types of the insulin effects do not arise simultaneously in the course of embryogenesis. The growth-stimulating effect mediated by adenylyl cyclase—cAMP is revealed since the 10th embryonal day. It was established for the first time that insulin could participate in growth stimulation by activating PKA in vivo and in vitro in muscles of the 10–14-day old embryos and the 8–10-day old chickens. The stimulating effect of insulin on G-6PDH activity is revealed since the same embryonal period and gradually increases. Insulin in vivo and in vitro simulates the glycogen synthase activity by increasing its conversion from non-active to active (GS-I) form only in the 15-day old embryos and in chickens. The stimulating effects of insulin and EGF on both G-6PDH activity (in embryos and chickens) and GS (in chickens) was shown to blocked by selective inhibitors of tyrosine kinases, thyrphostin 47 and genestein, in the dose-dependent manner, which indicates involvement of receptor of the tyrosine kinase type in these effects. The complex of the established facts permits concluding that insulin at early embryonal stages stimulates in the chicken muscles the PKA and G-6PDH activities involved in action of this hormone on growth, which is especially pronounced at the stage of myoblast proliferation. Meanwhile, the metabolic insulin effect (stimulation of the glycogen synthase system) appears in the second half of embryonal period and coincides in time with the period of muscle cell differentiation and active muscle contractures.     

Appearance and Differentiation of NADPH-D-Neurons in Ontogeny of Cerebral Cortex in Rats

The appearance of presumptive NO-ergic nerve cells and their differentiation in the rat neocortex were studied. For this purpose, a comparative analysis of the development and differentiation of NADPH-D-positive neurons in the neocortex transplants taken from the embryos of different ages and transplanted in the occipital cortex of adult rats and in the normally developing cerebral cortex was undertaken. The nervous tissue was stained histochemically for NADPH-D. The results we obtained suggest that no NADPH-D-containing neurons were found in the transplants from 15-day embryos, while they developed in those from 18-day embryos. Hence, precursors of NO-ergic neurons were still absent in the presumptive neocortex of 15-day embryos and appeared only on day 16–18 of embryogenesis. Expression of NADPH-D begins in them only within four to five days, but the neurons are differentiated during a relatively short period of time. Most NADPH-D-positive neurons reach their structural–functional maturity already by the end of the first week of postnatal development, while their complete maturation takes place by the end of the second week of postnatal development.     

Calcium regulation in the embryonic chick. II. Ultrastructure of the parathyroid glands in shell-less and in ovo embryos

The ultrastructure of the parathyroid glands was studied in chick embryos developing normally in ovo or in shell-less culture (after removal of the eggshell). Shell-less chick embryos are significantly hypocalcemic relative to their in ovo counterparts. At 12 days of incubation, the parathyroid glands of shell-less embryos contain more lipid and show evidence of increased protein synthetic activity relative to those grown in ovo (more rough endoplasmic reticulum, presence of some dense secretory granules). The glands from in ovo embryos do not contain secretory granules at this age. At 15 days of incubation, the in ovo glands have developed signs of protein synthetic activity similar to those of the 12-day shell-less embryos. However, the parathyroids of the 15-day shell-less embryos appear strikingly more active than at 12 days, containing stacks of concentric RER membranes and increased numbers of secretory granules. By 18 days of incubation, the ultrastructure of the glands of the two groups is indistinguishable, both appearing to be more active than the 15-day shell-less group. Thus, protein synthetic activity of the parathyroid glands, as detected by ultrastructural alterations of the chief cells, normally appears to be initiated during the latter part of embryogenesis (by approximately 15 days incubation) and its onset can be stimulated at least 3 days prematurely by hypocalcemia.     

Developmental fate of the yolk protein lipovitellin in embryos and larvae of winter flounder, Pleuronectes americanus.

The developmental fate of the vitellogenin-derived yolk protein, lipovitellin (Lv), was investigated in winter flounder embryos and yolk-sac larvae. Since Lv is present as only one major polypeptide in ovulated winter flounder eggs, unlike the multiple yolk polypeptides found in the mature eggs of most teleosts, this system is presented as a simpler model of yolk protein structure and utilization during teleostean development. Winter flounder Lv is cleaved during embryogenesis from a 94 kD polypeptide at fertilization to 67 kD and 26 kD polypeptides at hatching. The rate of this proteolytic processing is slow during early embryonic development, but enters a more rapid phase between days 8 and 12 post-fertilization in embryos reared at 4-5 degrees C, and approaches 50% completion at day 10. Lv processing is essentially complete 3 days before hatching; nevertheless, major degradation of the Lv peptide by the developing winter flounder does not occur until after hatching. The Stokes radius of Lv changes only moderately following processing, from 4.50 nm in unfertilized eggs to 4.19 nm in late embryos and newly hatched larvae, whereas the processed Lv retains its heat stability relative to other yolk polypeptides. Nearly 50% of its lipid content, however, is released from the Lv particle during embryogenesis, concomitant with cleavage of the Lv 94 kD polypeptide. Lv processing may thus render a portion of the yolk protein-associated lipid more accessible to the developing embryo, whereas other yolk components are retained for later use by the winter flounder larva. Alternately, removal of lipid may lead to proteolytic vulnerability of the Lv polypeptide. In either case, only a portion of the lipid moiety of the Lv particle appears to play a significant nutritive role for the embryo, whereas its protein component is reserved for larval use. J. Exp. Zool. 284:686-695, 1999.     

Induction of secondary somatic embryogenesis in hybrid larch (Larix x leptoeuropaea) as related to ethylene

Induction of secondary somatic embryogenesis was studied with hybridlarch (Larix x leptoeuropaea)cotyledonary somatic embryos obtained after 3, 4, 5 and 6 weeks of culture on amaturation medium supplemented with abscisic acid. Almost all 3-week maturedcotyledonary somatic embryos can develop embryonal masses whereas only 78, 27and 12% of them are able to do so after 4, 5 and 6 weeks of maturation,respectively. During the first week of culture on the induction medium, somaticembryos with high embryogenic potential (i.e. 3-weekmatured) release little ethylene (less than 1.5 nL h^–1g^–1 FW), whereas those which have almost completelylosttheir ability to induce embryonal masses (i.e. 6-weekmatured) produce much more ethylene. Thereafter, ethylene production by bothtypes of embryos is very similar at around 5–6 nLh^–1 g^–1 FW. Enrichment of theatmosphere with ethylene, or addition of 2-chloroethylphosphonic acid(ethephon)or ACC in the induction medium strongly reduced the induction of secondarysomatic embryogenesis. Moreover, inhibitors of ethylene action(AgNO₃and 2,5-norbornadiene) improved the development of embryonal masses fromsomaticembryos, particularly from the 6-week maturated ones. The results obtainedclearly suggest that ethylene is involved in the regulation of somaticembryogenesis in hybrid larch. The possible relationship between somaticembryogenic potential and ethylene biosynthesis by the explants or sensitivityof the latter to ethylene is discussed.     

Composition, accumulation and utilization of yolk lipids in teleost fish

Lipid reserves in teleost eggs are stored in lipoprotein yolk and, in some species, a discrete oil globule. Lipoprotein yolk lipids are primarily polar lipids, especially phosphatidylcholine and phosphatidylethanolamine (PE), and are rich in (n–3) polyunsaturated fatty acids, especially 22:6(n–3) (docosahexaenoic acid, DHA). Oil consists of neutral lipids and is rich in monounsaturated fatty acids (MUFA). Egg lipids are derived from dietary fatty acid, fatty acid mobilized from reserves and possibly fatty acid synthesized de novo. There is selective incorporation of essential fatty acids, particularly DHA, into yolk lipids and discrimination against incorporation of 22:1(n–11). Lipid is delivered to the oocyte by vitellogenin, which is rich in polar lipids, and likely also by other lipoproteins, especially very low density lipoprotein, which is rich in triacylglycerol (TAG). All classes of lipid may be used as fuel during embryonic and larval development and MUFA are preferred fatty acids for catabolism by embryos. Catabolism of oil globules is frequently delayed until latter stages of development. In some species, DHA derived from hydrolysis of phospholipid may be conserved by transfer to the neutral lipid. Recent work has expanded knowledge of the role of DHA in membrane structure, especially in neural tissue, and molecular species analysis has indicated that PE containing sn-1 oleic acid is a prime contributor to membrane fluidity. The results of this type of study provide an explanation for the selection pressures that influence yolk lipid composition. Future work ought to expand knowledge of specific roles of individual fatty acids in embryos along with knowledge of the ecological physiology of ovarian recrudescence, environmental influences on vitellogenin and yolk lipid composition, and the control of yolk lipid accumulation and utilization.     

Changes in amino acids and lipids during embryogenesis of European lobster, Homarus gammarus (Crustacea: Decapoda)

We studied the amino acid and lipid dynamics during embryogenesis of Homarus gammarus. Major essential amino acids (EAA) in the last stage of embryonic development were arginine, lysine and leucine; major nonessential amino acids (NEAA) were glutamic acid, aspartic acid, valine and glycine. The highest percent of utilization occurred in respect to EAA (27.8%), mainly due to a significant decrease (p<0.05) of methionine (38.3%) and threonine (36.0%). NEAA also decreased significantly (p<0.05, 11.4%), namely serine (38.1%), tyrosine (26.4%) and glutamic acid (25.7%). In contrast, the free amino acid content increased significantly (p<0.05) during embryonic development, especially the free nonessential amino acids (FNEAA). In the last stage, the most abundant FNEAA were glycine, proline, alanine and taurine, and the major free essential amino acids (FEAA) were arginine, lysine and leucine. Lipid content decreased significantly (p<0.05) during embryonic development. A substantial decrease in all neutral lipid classes was observed (>80% of utilization). Major fatty acids were 16:0, 18:0, 18:1n-9, 18:2n-6, 18:3n-3, 20:5n-3 and 22:6n-3. Unsaturated (UFA) and saturated fatty acids (SFA) were used up at similar rates (76.5% and 76.3%, respectively). Within UFA, monounsaturates (MUFA) were consumed more than polyunsaturates (PUFA) (82.9% and 67.5%, respectively).     

Development of spontaneous motility in chick embryos. Interaction of strychnine, glycine and GABA.

The interaction of strychnine (1 mg/kg egg weight), glycine (100 mg/kg egg weight) and GABA (103 mg/kg egg weight) on spontaneous motor activity recorded by the method of Kovach (1970) in intact eggs was studied in chick embryos from the 11th to 21st day of incubation. In 11- and 13-day embryos, neither of the amino acids influenced strychnine activation of spontaneous motility. From the 15th incubation day, strychnine activation was distinctly affected by both amino acids, but the maximum effect was observed on the 19th day. Glycine had a stronger inhibitory effect, since it prevented strychnine convulsions from developing, whereas GABA only modified them. It can be concluded from the results that glycine-sensitive and GABA-sensitive mechanisms of embryonal spontaneous motility do not begin to take effect in chick embryos until the 15th day of incubation.     

A continuous culture system of direct somatic embryogenesis in microspore-derived embryos of Brassica juncea

Summary An efficient culture system has been developed for repeated cycles of somatic embryogenesis in microspore-derived embryos of Brassica juncea without a callus phase. Haploid embryos produced through anther culture showed a high propensity for direct production of somatic embryos in response to 2 mgL^–1 BA and 0.1 mgL^–1 NAA. The embryogenic cultures which comprised the elongated embryonal axis of microspore-derived embryos when explanted and grown on the medium of same composition produced a large number of secondary embryos. These somatic embryos in turn underwent axis elongation and produced more somatic embryos when explanted and cultured. This cycle of repetitive somatic embryogenesis continued with undiminished vigour passage after passage and was monitored for more than a year. Somatic embryos from any passage when isolated at cotyledonary stage and grown on auxin-free medium for 5 days and then on a medium containing NAA (0.1 mgL^–1), developed into complete plants with a profuse root system and were easily established in the soil. The cytology of the root tips of these plants confirmed their haploid nature. The total absence of callus phase makes the system ideal for continuous cloning of androgenic lines, Agrobacterium-mediated transformation and mutation induction studies.     

Accumulation of calcium and phosphorus in pigeon (Columba livia) embryos

Summary Calcium and phosphorus were measured in the yolk and albumen of fertile pigeon (Columba livia) eggs incubated for 0–17 days, and in embryos and hatchlings. Shell provided most of the calcium for skeletal mineralization of the embryos, whereas phosphorus was derived from the yolk and albumen. Mobilization of calcium from the shell to the embryo commenced at approximately day 11 of incubation, accumulating both in the embryo and the yolk sac. There was 1.4 times more calcium in squab yolk sacs than that contained in newly laid egg yolks. The results suggest that whereas general patterns of calcium and phosphorus accumulation during embryogenesis in altricial birds closely resemble those of precocial birds, calcium mobilization from the shell begins later, proceeds at a slower rate and results in a less mineralized hatchling.CIDA/NSERC Visiting Research Associate Permanent address: Department of Animal Science, University of Peradeniya, Peradeniya, Sri Lanka     

Amino Acid Metabolism in Rat Hippocampus During the Period of Brain Growth Spurt

We studied protein synthesis, lipid synthesis and CO₂ production by oxidation of glycine, alanine and leucine by slices of rat hippocampus during the period of brain growth spurt. The metabolism of the three amino acids decreased with the age of the animals, A major reduction was observed in protein synthesis, which was 4 times higher at 7 days of age than at 21 days of age for all amino acids studied. Glycine oxidation to CO₂ was twice as high as alanine oxidation and ten times higher than leucine oxidation. The major pathway of leucine utilization was incorporation into proteins. Glycine was the amino acid that had the highest metabolic rate.     

Collagen and elastin synthesis in the developing chick aorta

Thoracic aortas from 8- to 18-day embryonic chicks were incubated in vitro for 30 min with [³H]glycine and the newly synthesized, labeled proteins were subjected to polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The gels were fractionated and the incorporation of label into procollagen (125,000 M_r) and tropoelastin (70,000 M_r) was estimated by summation of the radioactivity found in the appropriate regions of the gel. The analyses showed that at Day 8 approximately 14% of the incorporated [³H]glycine was found in procollagen and 22% in tropoelastin. In the following 6 days of development, there was a significant decline in the relative incorporation into procollagen and an increase into tropoelastin so that at Days 14–18 less than 10% of the label was found in collagen and 40% was now found in tropoelastin. Since glucocorticoids have been shown to alter the rate of synthesis of other proteins in the developing chick, 150 μg of hydrocortisone was injected into 8-day eggs and 24 h later the aortas were incubated and treated as described above. The pattern of protein synthesis exhibited by the hormone-treated aortas resembled that of 14- to 18-day embryos. Furthermore, incubation of 8-day aortas with 10^?8m hydrocortisone for 24 h produced a significant increase in the rate of elastin synthesis relative to that of other proteins. These results demonstrate that collagen and elastin synthesis vary during development of the chick aorta and they suggest that glucocorticoids may be involved in the control of their synthesis.     

Effects of 3-acetylpyridine on the levels of several amino acids in different CNS regions of the rat

The effects of one intraperitoneal injection of 60–65 mg/kg of 3-acetylpyridine (3-AP) on the levels of aspartate, glutamate, GABA, taurine, glycine, and alanine in the cerebellum, medulla, telencephalon, and diencephalon-mesencephalon of the rat were studied at various times (4–28 days) after injection. In the first 4–7 days, the levels of glutamate, GABA, glycine, and alanine in the cerebellum were 10–30% higher in the 3-AP-treated rats than in the control animals. By day 14, the levels of these four amino acids were normal (in the case of glutamate and glycine) or below normal (for GABA and alanine). By day 21, the values for GABA and alanine returned to normal. In the first 7 days, the level of aspartate in the cerebellum was the same in both the 3-AP- and saline-injected groups. From days 14 to 28, the level of aspartate in the cerebellum was 10–20% lower in the 3-AP-injected group than in the saline-treated animals. The level of taurine in the cerebellum was 15–30% lower in the 3-AP group than in the control group from days 7 to 28. The pattern of changes observed in the medulla in the first 7 days was similar to that found in the cerebellum for this period. However, unlike the data for the cerebellum, the level of aspartate in the medulla was unchanged by the 3-AP injection from day 14 to day 28, and the level of glutamate in the medulla remained higher (10–15%) from days 14 to 28 in the 3-AP-injected animals with respect to control values. The levels of taurine in the medulla were lower (10–15%) from day 7 to day 28 in the 3-AP injected group with respect to control values. The injection of 3-AP did not alter the levels of aspartate, glutamate, GABA, taurine, glycine, or alanine in the telencephalon on days 7, 14, 21, or 28 and in the diencephalon-mesencephalon on day 21 with respect to control levels.     

Free Amino Acid Content of Astroglial Cell Clones Derived from 8-Day Postnatal Mouse Cerebella

We have measured the free amino acid content of three distinct astroglial cell clones derived from permanent lines obtained after "spontaneous immortalization" of 8-day postnatal mouse cerebellar cultures; these clones show characteristics similar to the Golgi Bergmann glia cells, the fibrous astrocytes, and the velate protoplasmic astrocytes, i.e., the three main types of cerebellar astrocytes. The relative concentrations of amino acids that are thought to act as neurotransmitters were compared in confluent cultures of the different astroglial clones. The most striking result was a high concentration of glycine (20% of free amino acids), even in astroglial cells cultured in a glycine-free medium, a finding suggesting that glycine is synthesized by the astroglial clones. Furthermore, no gamma-aminobutyric acid (GABA) was detected. In contrast, a "neuron-like" clone derived from the same cerebellar culture contained GABA, whereas its glycine content was much lower than that of the astroglial clones. The present results, together with our previous finding of glycine synthesis in an astrocytic clone derived from 14-day postnatal mouse cerebella transformed by simian virus 40, indicate that a high glycine content may be characteristic of many cerebellar astroglial types.     

Environmental modulation of calcium and phosphorus metabolism in embryonic snapping turtles (Chelydra serpentina)

Summary Eggs of common snapping turtles (Chelydra serpentina) were incubated on wet (–150 kPa water potential) and dry (–950 kPa) substrates in a laboratory study assessing the effects of the hydric environment on patterns of mobilization of calcium and phosphorus by developing embryos. We found that embryos developing in wet environments withdrew nutrients from their yolk faster, grew more rapidly, and incubated longer than embryos exposed to dry environments. Turtles developing in both environments absorbed calcium from the yolk at similar rates and depleted the yolk of almost its entire reserve of calcium prior to hatching. Calcium withdrawn from the yolk was supplemented with calcium mobilized from the eggshell, but embryos in wet environments obtained substantially more calcium from the eggshell than did those in dry settings. Embryos obtained all of the phosphorus used in skeletogenesis from the yolk, but those incubating in wet environments mobilized phosphorus from this compartment more rapidly than did those in dry settings. Exposing embryonic snapping turtles to wet environments apparently allows them to make more efficient use of the transitory source of calcium in the eggshell than is possible in dry environments. However, the residual yolk in hatchlings from both wet and dry environments contains too little calcium to support the growth of hard and soft tissues in neonates at rates similar to those characterizing the growth phase of development in embryogenesis.     

High efficiency adventive embryogenesis on somatic embryos of anther,filament and immature proembryo origin in horse-chestnut (Aesculus hippocastanum L.) tissue culture

Adventive embryogenesis was successfully induced in cultures of zygotic and somatic embryos on MS medium supplemented with BA and NAA. A procedure has been proved successful for the in vitro multiplication of somatic embryos regenerated at low frequencies from filament and callus cultures. The occurrence and rate of adventive embryogenesis did not depend on the origin of the primary embryos (zygotic and somatic), but did depend on the developmental stage. Primary embryos are capable of embryogenesis in each of the different phases of embryogenesis, though the rate is different. BA concentrations of 22–44 M increased the rate of adventive embryogenesis and accelerated the development of embryos. The highest proliferation rate (22–25x/5 weeks) was achieved at hormone concentrations of 44 M BA and 5.4 M NAA.Abbreviations BA benzyladenine - CH casein hydrolysate - CM coconut milk - 2,4-D dichlorophenoxyacetic acid - MS Murashige & Skoog medium - WPM woody plant medium - NAA 1-naphtaleneacetic acid