- Email: [email protected]
Quantitative and Qualitative Protein Changes in the Developing Single Comb White Leghorn Chicken Embryo
313
FEED AND LITTER FUNGI Ray, M. L., and R. D. Child, 1965. Chicken litter as a supplement in wintering beef cows and calves on pasture. Arkansas Farm Res. 14: 5. Southwell, B. L., O. M. Hale and W. C. McCormick. 1958. Poultry house litter as a protein supplement in steer fattening rations. Mimeograph Series N. S. 55. Georgia Agricultural Experiment Station, Athens, Ga.
Verrett, J., J. Marliac and J. McLaughlin, Jr. 1964. Use of the chicken embryo in the assay of anatoxin toxicity. J. Assoc. Off. Agri. Chem. 47: 1003-1006. Wehunt, K. E., H. L. Fuller and H. M. Edwards, Jr., 1960. The nutritional value of hydrolyzed poutry manure for broiler chicks. Poultry Sci. 39: 1057-1063.
J. A. ASMAR, P. L. PELLETT, NUR HARIRI AND M. D. HARIRI Department of Animal Science and Food Technology & Nutrition, Faculty of Agricultural Sciences, American University of Beirut, Beirut, Lebanon (Received for publication June 21, 1971)
ABSTRACT Fertile Single Comb White Leghorn chicken eggs were incubated in an automatic incubator. Embryo weights, dry matter and protein content were determined at 6, 12 and 18 days of incubation. Embryos of the foregoing ages were homogenized and their tissue fluids electrophoresed in acrylamide gel. Amino acid composition was determined at 6 and 18 days. Embryo weight, total solids and protein content increased exponentially but at differing rates. Amino acids were incorporated at exponential rates which differed for different amino acids. With advancing incubation time soluble proteins increased in heterogeneity with new fractions appearing at the anodic end of the spectrum. POULTRY SCIENCE 5 1 : 313-320,
NDER suitable incubation conditions the chicken embryo grows within three weeks from the few blastocystic cells in the freshly laid egg to the large and composite population of differentiated cells and intercellular substances making up the tissues and organs of the fully formed chick. This fast sequence of morphogenetic events is made possible by rapidly occurring changes in metabolic processes and chemical composition affecting egg constituents and embryonic tissues at once. Various aspects of the chemistry of chicken embryo development have retained the attention of a number of investigators (Needham, 1931, 1950; Williams et al., 1954; Rupe and Farmer, 1955; Kaminski and Durieux,
U
1972
1956; Flickinger, 1957; Walter and Mahler, 1958; Romanoff, 1960; Fitzsimmons and Waibel, 1968). Here, as in other systems, the rapidly occurring morphogenic and chemical changes are genetically controlled. This is made possible by the synthesis of the appropriate amounts and quality of proteins mediating phenotypic expressions as directed by genome and affected by environment. Determining total amino acid and protein composition of the embryo in the course of incubation would be of interest to the experimenter studying responses of the developing embryo to genetic, chemical, nutritional or microbial treatments. The usual complexity of the required analyses, however,
Downloaded from http://ps.oxfordjournals.org/ at Northern Arizona University on June 4, 2015
Quantitative and Qualitative Protein Changes in the Developing Single Comb White Leghorn Chicken Embryo
314
J. A. ASMAR, P. L. PELLETT, N. HARIRI AND M. D. HARIRI
has in general deterred investigators whose primary concern was other than embryogenesis. In the present work a coordinated use was made of conventional or improved analytical procedures to determine some of the quantitative and qualitative protein changes. MATERIALS AND METHODS
Downloaded from http://ps.oxfordjournals.org/ at Northern Arizona University on June 4, 2015
Fertile Single Comb White Leghorn hatching, eggs within commercial hatching specifications were obtained from a private source and incubated at 37.8 ± 0.3° C. and 55-57% relative humidity. The eggs were set in the incubator not more than 36 hr. from oviposition time and were automatically tilted at 60 minutes intervals. Candling was done daily and infertile eggs or eggs with dead embryos were discarded. Fewer than nine percent of the eggs incubated were so excluded. Embryo samples were collected at 6, 12 and 18 days of incubation with ± 2 hours from time set in the incubator. To collect 6-day old embryos the egg shell and the attached membrane were removed from over the air cell (broad pole of the egg). The chorioallantoic membranes were excised, the embryos lifted with a pair of tweezers and severed from the extraembryonic tissues. Individually collected embryos were quickly dipped in three consecutive baths of physiological salt solution, drained and blotted with filter paper, then weighed to the nearest 0.1 mgm. Due to their larger size 12- and 18-day old embryos were easier to harvest as just described. In all cases extraembryonic tissues were excluded. Embryos used for total solids determinations were oven-dessicated at 100°C. until constant weight was reached and recorded to the nearest 0.1 mgm. Twelve and 18-day embryos were split open along the mid sa-
gittal ventral line for more effective dehydration. To prepare the hydrolysates for amino acid analysis individual whole six-day embryos minced with small scissors were introduced together with 10 ml. 6 N HC1 in vacuum hydrolysis tubes (Phoenix Precision Instruments Co., Philadelphia, Pa., U.S.A.) flushed twice with N 2 gas and hydrolyzed at 105°C. for 22 hours in vacuo. Because of their larger size 18-day embryos required a preliminary preparation. The embryos were first freeze dried, individually chopped in an electric grinder then homogenized in a ball mill. Samples of suitable size from the pulverized material were then individually hydrolyzed as just described. The hydrolysates were analyzed in an automatic amino acid analyzer (Phoenix Precision Instruments Co.) as described by Jamalian and Pellett (1968) but modified to use the new spherical resins for high speed resolution and completion of analysis in about 4 hours. The amino acid values were calculated in mgm. per gram nitrogen and as moles percent. The micro Kjeldahl procedure was used to determine nitrogen. For the 6-day embryos, measured amounts of the HC1 hydrolysates were individually digested and analyzed. For 12- and 18-day old embryos samples of the homogenized dried carcasses prepared as just described were digested and analyzed. Protein was expressed throughout as N X 6.25. Tissue soluble proteins used in electrophoretic analysis were prepared from freshly harvested embryos. Whole embryos were individually minced with scissors and homogenized in a mechanically driven teflon homogenizer maintained in an ice bath. The homogenates were then carried through several quick freezing and thawing cycles, centrifuged at 2000 X G and the supernatant fluids decanted for electrophoresis in acrylamide gel. The Microzone acryl-
315
PROTEIN CHANGES IN EMBRYO
amine gel electrophoresis procedure (Beckman Instruments, Inc., Fullerton, California) was used according to manufacturer's instructions (Anonymous, 1970). Sixty minutes was the duration of the separation. The finished electropherograms were mounted in plastic covers and scanned in an R 110 densitometer (Beckman Inst., Inc.) at 594 nm.
Embryo weights, total solids, N/100 gm. and protein/embryo are reported in Table 1. At six days of incubation the embryos contained an average 5.8% solids of which better than 71% was protein. At 12 days solid matter increased to 9.2% of embryo
TABLE 1.—Weight, solids, nitrogen and prot-ein values (gm.) of S.C.W.L. chicken embryos at three stages of incubation Days in incubator
Embryo Wt. Solids/embryo N/100 gm.0 Protein/embryo a b 0
0.3863 + 0.0094" (27) 0.0226±0.0029 (8) 0.642 +0.0064(11) 0.016 +0.0014(11)
b
12
18
5.0726+0.0704(20) 0.4645 + 0.0030 (12) 0.913 +0.0230 (7) 0.282 +0.0051 (7)
23.1672±0.2430(19) 4.4151±0.0865 (11) 1.733 +0.0327 (9) 2.535 ±0.0511 (9)
Means+ SE Figures between brackets = number of determinations. Wet weight basis.
Downloaded from http://ps.oxfordjournals.org/ at Northern Arizona University on June 4, 2015
RESULTS AND DISCUSSION The eggs used in this investigation weighed an average 55.5 ± 1.7 gm. and were within the range of egg weights considered suitable in the commercial hatching of Single Comb White Leghorn chicks. Eggs in this weight range are known to produce maximum embryo viability and hatchability and their embryos may be regarded as physiologically normal. Upward of 90 such embryos were used. Statistically analyzed egg and embryo weight values showed that within the egg size range selected there was no correlation between these two parameters. In other words egg weight in this range had no detectable effect on embryo growth through 18 days of incubation.
weight and although there was an approximate 17 fold increase in protein, the latter was not more than 60% of total solids. There was further loss of water between 12 and 18 days of incubation at which time the embryos contained an average 19% solids made of approximately 57% protein and a balance of non nitrogenous substances. Plotting weight, solids and protein values on a semilograrithmic chart (Fig. 1) shows that all three parameters increased exponentially between six and 18 days but at differing and slightly declining rates. Solid matter made the fastest gains whereas total weight gain was slowest. These differences in rates account not only for the gradual decline in water content but also indicate that increasing amounts of materials other than protein were being added to the embryonic tissues. Minerals deposited in the skeletal structures must represent part of these non nitrogenous solids. The gradual decline in water concentration and the appearance of increasing amounts of non nitrogenous substances indicate that metabolic processes were changing with advancing embryonic development. For these changes to take place increasing amounts of proteins with new enzymatic and physiological functions are required. This is reflected by the difference in amino acid composition of 6- and 18-day
316
J. A. ASMAR, P. L. PELLETT, N. HARIRI AND M. D. HARIRI FIG. 1. Plotted on a semilogarithmic scale total weight, total solids and protein content of S.C.W.L. chicken embryo rise exponentially but at differing rates between 6 and 18 days of incubation.
10 12 V DAYS IN INCUBATOR
TABLE 2.—Amino acid composition of S.C.W.L. chicken embryos at 6 and 18 days of incubation 18-day (4)a
6-day (5)a mgm./gm.N b Isoleucine Leucine Lysine Methionine Cystine Total Sulfur AA Phenylalanine Tyrosine Total aromatic AA Threonine Valine Arginine Histidine Alanine Aspartic Acid Glutamic Acid Glycine Proline Serine NH 3
368+19.6 497+ 6.3 490+16.6 125+ 5.8 70+ 9.1 195 + 10.7 264+ 9.8 252+ 4.6 516+10.3 297+ 7.5 328+ 6.0 497 + 23.2 177+12.4 342+ 6.5 575+ 6.7 891 + 11.0 323+ 0.9 257+ 8.7 309+16.1 116+ 6.6
iM%° 5.9 8.0 7.1 1.8 0.6 2.4 3.4 3.0 6.4 5.3 5.9 6.1 2.4 8.2 9.2 12.9 9.2 4.7 6.2 99.9
a
Figures between brackers = number of determinations. Means ± S E . c Does not include ammonia. d xP<0.05. xxxP<0.001. b
mgm./gm.N
fM%°
320 + 29.2 500+ 9.7 365+ 3.2,™"* 104+11.0 156+ 5.6*** 260+14.3 300± 3.7" 252+ 4.7 552 + 7.8* 310+ 7.1 352 + 10.8 507± 6.4 167+ 8.9 365± 4.4 552+ 4.7 799+ 8.6"1* 541 + 11.5™ 427+ 4.5*** 416+ l.C™* 103± 4.9
4.8 7.4 4.8 1.4 1.3 2.7 3.5 2.7 6.2 5.0 5.8 5.7 2.1 8.0 8.1 10.5 14.0 7.2 7.7 100.0
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embryos presented in Table 2. Results of amino acid analysis are expressed both as mg. per gram nitrogen and as micromoles percent. Naturally the high rate of protein synthesis occurring during development is accompanied by increasing levels of all amino acids. There are, however, significant changes in the proportions of certain of the amino acids. Thus very large and significant increases (P < 0.001) were observed for cystine, glycine and proline accompanied by smaller but still significant increases in serine (P < 0.001) and phenylalanine (P < 0.0S). These changes are
PROTEIN CHANGES IN EMBRYO
317
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probably related to increased synthesis in TABIE 3.—Milligrams of individual amino acids per S.C.W.L. chicken embryo at 6 and 18 days keratin and collagen which are high in sevof incubation eral of these amino acids. The foregoing inDays in creases are in proportions of the total amino acids found and must, therefore, be 6 18 compensated for by corresponding decreases in the proportions of other amino 0.960 129.6 128.6 13,396 Isoleucine 1.297 202.5 201.2 15,513 Leucine acids. Highly significant decreases (P < Lysine 1.278 147.8 146.5 11,463 0.001) were in fact observed for lysine and Methionine 0.326 42.1 41.7 12,791 Cystine 0.182 63.0 62.8 34,505 glutamic acid with smaller though not sigPhenylalanine 0.689 121.5 120.8 17,532 nificant (P > 0.05) falls for isoleucine, Tyrosine 0.657 102.1 101.4 15,433 0.775 125.5 124.7 16,090 Threonine methionine and aspartic acid. The other 0.856 142.5 141.6 16,542 Valine amino acids remained essentially at the Arginine 1.297 205.3 204.0 15,728 Histidine 0.461 67.6 67.1 14,555 same relative levels. Alanine 0.892 147.8 146.9 16,468 The changes observed here were in genAspartic Acid 1.500 223.5 222.0 14,800 Glutamic Acid 2.325 323.5 321.1 13,810 eral similar to those obtained by Fitzsim0.843 219.1 218.2 25,884 Glycine mons and Waibel (1968) for 6- and 12-day 0.670 172.9 172.2 25,701 Proline 0.806 168.4 167.5 20,780 Serine embryos though certain differences were noted. These were particularly in our large increase in proline concentration and the It is evident from Table 3 that with insmaller increase in serine while Fitzsim- creasing incubation time and embryo size mons and Waibel found a decrease in the considerable amounts of the respective serine and a constant level for proline. A amino acids were incorporated into embryfurther difference was also observed for onic tissues, but the relative amounts phenyalalanine and tyrosine. Although added differed for different amino acids. nothing is mentioned in their article some Plotting embryonic amino acids contents at failure must have occurred in the analysis six and 18 days on a semilogarithmic scale by Fitzsimmons and Waibel (1968) since showed that virtually each of them had its their phenylalanine data are impossibly low own average incorporation rate for the in(less than 10% of expected) and the tyro- cubation period under consideration. This sine data are missing, whereas we recorded is illustrated in Figure 2 which for clarity sizeable amounts for each of these amino purpose includes only a limited number of acids with a moderate increase for phenyl- the amino acids. alanine at 18 days. The present observation that different Pellett and Kaba (1971) have recently amino acids are incorporated into embryreported changes in carcass amino acid onic tissues at differing rates is in general composition of growing rats. This observa- agreement with the data of Fitzsimmons tion suggests that changes in amino acid and Waibel (1968) but stands in contracomposition may not be limited to the pre- diction with the conclusions of Rupe and Farmer (1955) who reported exponential natal phase of development. On the basis of the data in Tables 1 and but essentially equal incorporation rates for 2 the actual amounts of amino acids per all amino acids through 400 hours of incubaembryo were calculated for 6- and 18-day tion. It must be noted, however, that Rupe incubation. These values are listed in Table and Farmer included both embryos and 3 together with the respective total and rel- extra embryonic tissues in their amino acid ative gains made between six and 18 days. analyses. Moreover, their study was based
318 1000.0
J. A. ASMAR, P. L. PELLETT, N. HARIRI AND M. D. HARIRI 3
1 6
1 7
1 8
1 1 1 1 1 1 9 10 11 12 13 1*. DAYS IN INCUBATOR
1 15
1 16
1 17
1 18
FIG. 2. Between 6 and 18 days of incubation S.C.W.L. chicken embryos incorporated amino acids at exponential rates. Different amino acids showed differing rates. For reasons of clarity a limited number was included. See Table 3 for all amino acids determined.
on the microbiological assay method of determining amino acids. The dramatic rise of cystine from 0.182 mgm. at six days to 53.0 mgm. at 18 days of incubation seems to agree well with the passage from the featherless condition of the six-day old embryo to the fully formed feather coat of the 18-day embryo and lends strong support to our observation on differing amino acid incorporation rates. Of the various analytical procedures used in this study it is undoubtedly electrophoresis in acrylamide gel that gave the most direct evidence of the qualitative changes associated with the increases displayed by the soluble embryonic proteins at 6, 12 and 18 days of incubation. Typical densitometric tracings of protein electropherograms at the three ages tested are presented in Figure 3.
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nn\ J
Proteins from six-day embryos show a rather limited degree of heterogeneity and are confined mostly to the gamma and adjacent regions of the electrophoretic spectrum. At 12 days new protein populations have been added on the anodic side of the spectrum thus showing an increased electrophoretic diversity of proteins. Meanwhile the slower migrating fractions have gained in amounts leading their densitometric readings to higher values on the scale. The expansion of the electrophoretic spectrum of proteins is further accented at 18 days with new fractions of higher electrophoretic mobility appearing at the anodic end of the electropherogram. The changes observed in soluble protein patterns indicate that with advancing incubation increasingly larger proportions of acidic or less basic proteins were synthesized. It is of interest to note that the 18-day old embryos contained a higher proportion (66%) of the neutral amino acids than the six-day olds (59%) (Table 2). In contrast, their content of basic and acidic amino acids (respectively 13% and 21%) was lower than that of the six-day olds (respectively 16% and 25%) showing a comparable decline for both groups of amino acids with increasing development. Admittedly the procedures used here do not indicate whether the detected proteins are the result of de novo synthesis by the embryo or have been absorbed more or less intact from the egg. It has been previously demonstrated that some proteins do pass directly from egg to embryo (Kaminski and Durieux, 1956). With the possible exception of immunoglobulins, there seems to be virtually no information regarding the passage or role of egg proteins in embryonic tissues. However, since the absorption of intact egg proteins by the embryo seems to be a highly selective process (Kaminski and Durieux, 1956) it would be normal to
PROTEIN CHANGES IN EMBRYO
319
regard all proteins found in the embryo as integral parts of its system. REFERENCES Anonymous, 1970. Model 113 Acrylamide Gel Accessory for the Microzone System. Palo Alto: Spinco Division of Beckman Instruments Inc. Fitzsimmons, R. C , and P. E. Waibel, 1968. Free amino acid composition of the avian egg as related to embryonic development. Poultry Sci. 47: 219-224. Flickinger, R. A., 1957. Amino acid studies in relation to yolk utilization in the chick embryo. Experientia, 13: 248. Jamalian, J., and P. L. Pellett, 1968. Nutritive value of Middle Eastern foodstuffs. IV. Amino acid composition. J. Sci. Food Agric. 19: 378-382.
Kaminski, M., and J. Durieux, 1956. Etude comparative des serums de poule, de coq, de poussin, d'embryon et du blanc d'oeuf. Exptl. Cell. Res. 10: 590-618. Needham, J., 1931. Chemical Embryology. Cambridge University Press, London. Needham, J., 1950. Biochemistry and Morphogenesis. Cambridge University Press, London. Pellett, P. L., and H. Kaba, 1971. Carcass amino acids of the rat under conditions of determination of net protein utilization. J. Nut. (submitted for publication). Romanoff, A. L., 1960. The Avian Embryo. The Macmillan Company, New York. Rupe, C. O., and C. J. Farmer, 1955. Amino acid studies in the tranformation of the hen's egg to tissue proteins during incubation. J. Biol. Chem. 213: 899-906.
Downloaded from http://ps.oxfordjournals.org/ at Northern Arizona University on June 4, 2015
FIG. 3. Densitometric tracings of soluble proteins from 6- 12- and 18-day old S.C.W.L. embryos electrophoiesed in acrylamide gel. With advancing incubation time proteins increased in concentration and heterogeneity.
320
J. A. ASMAR, P. L. PELLETT, N. HARIRI AND M.
Walter, H., and H. R. Mahler, 1958. Biochemical studies on the developing avian embryo. I. Protein precursors in "vivo." J. Biol. Chem. 230: bryo. Nature, 173 : 490.
D.
HARIRI
Williams, M. A., W. A. DaCosta, L. H. Newman and L. M. Marshall, 1954. Free amino acids in yolk during the development of the chick em241-249.
85
Sr:45Ca Ratios from Intestine to Egg Shell During Perfusion with Ca and Sr1 MRAZ
(Received for publication June 23, 1971)
ABSTRACT The small intestine of 40 Leghorn hens (with an egg in utero) were perfused with 0, 5, 25 or 125 mM levels of Ca and/or Sr at a rate of 0.8 ml./min. After 25 ml. was perfused, ^Ca and 85Sr were added to the remaining 50 ml. of solution. Absorption and concentrations of 45Ca and ^Sr in blood plasma, egg shell and mucosal cell particulates of the small intestine and uterus were measured. The 85Sr :45Ca ratios decreased from 0.8 at absorption to 0.5 at the egg shell. The radionuclide content and ^Sri^Ca ratios in the cellular particulates generally decreased with particulate size as determined by ultracentrifugation. Differences between Ca and Sr do not stop at the intestinal wall but rather increase through the tissues, and are influenced by the availability of Ca and Sr. POULTKY SCIENCE 51:
C TRONTIUM has been of interest for ^ some time because of its chemical similarity to calcium, and because it normally follows metabolic routes similar to those of Ca. Wasserman (1960) ascertained from everted intestinal sacs that, under given conditions, Ca-Sr discrimination was dependent upon metabolically active membranes. Palmer and Thompson (1961) reported that the absorption of 85Sr and 45 Ca from the small intestines of rats was related inversely to the concentration of Ca in the perfusate and that the effect on 45Ca absorption was greater than the effect on 85 Sr. Mraz (1962a) concurred, adding that, while increasing concentrations of Sr 1 This manuscript is published with the permission of the Dean of The University of Tennessee Agricultural Experiment Station, Knoxville, Tennessee. 2 Operated by the Tennessee Agricultural Experiment Station for the U. S. Atomic Energy Commission under Contract No. AT-40-1-GEN-242.
320-325,
1972
also reduced 45Ca and 85Sr, no effect on the 85Sr:45Ca ratio was seen. However, the addition of Ca to the perfusate containing Sr increased the ratio back to that found with Ca alone. A difference between 45 Ca and 85Sr uptake in vitro by rat kidney and liver mitochondria that was increased by adding Ca to the substrate was also reported (Mraz, 1962b). The addition of Sr tended to reduce this difference. Hens preferentially deposited radiocalcium over radiostrontium in bones and egg shells (Edwards and Mraz, 1961; Monroe et al., 1961; Mraz et al., 1967). To ascertain if the preferences for Ca instead of Sr stopped at the small intestine or continued through to the egg shell of the hen, absorption and the concentrations of 45Ca and 85 Sr in blood plasma, egg shell and mucosal cell particulates of the small intestine and uterus were measured after perfusion with varying levels of Ca and/or Sr.
Downloaded from http://ps.oxfordjournals.org/ at Northern Arizona University on June 4, 2015
F R A N K R.
Agricultural Research Laboratory of the University of Tennessee, Oak Ridge, Tennessee* 37830
FEED AND LITTER FUNGI Ray, M. L., and R. D. Child, 1965. Chicken litter as a supplement in wintering beef cows and calves on pasture. Arkansas Farm Res. 14: 5. Southwell, B. L., O. M. Hale and W. C. McCormick. 1958. Poultry house litter as a protein supplement in steer fattening rations. Mimeograph Series N. S. 55. Georgia Agricultural Experiment Station, Athens, Ga.
Verrett, J., J. Marliac and J. McLaughlin, Jr. 1964. Use of the chicken embryo in the assay of anatoxin toxicity. J. Assoc. Off. Agri. Chem. 47: 1003-1006. Wehunt, K. E., H. L. Fuller and H. M. Edwards, Jr., 1960. The nutritional value of hydrolyzed poutry manure for broiler chicks. Poultry Sci. 39: 1057-1063.
J. A. ASMAR, P. L. PELLETT, NUR HARIRI AND M. D. HARIRI Department of Animal Science and Food Technology & Nutrition, Faculty of Agricultural Sciences, American University of Beirut, Beirut, Lebanon (Received for publication June 21, 1971)
ABSTRACT Fertile Single Comb White Leghorn chicken eggs were incubated in an automatic incubator. Embryo weights, dry matter and protein content were determined at 6, 12 and 18 days of incubation. Embryos of the foregoing ages were homogenized and their tissue fluids electrophoresed in acrylamide gel. Amino acid composition was determined at 6 and 18 days. Embryo weight, total solids and protein content increased exponentially but at differing rates. Amino acids were incorporated at exponential rates which differed for different amino acids. With advancing incubation time soluble proteins increased in heterogeneity with new fractions appearing at the anodic end of the spectrum. POULTRY SCIENCE 5 1 : 313-320,
NDER suitable incubation conditions the chicken embryo grows within three weeks from the few blastocystic cells in the freshly laid egg to the large and composite population of differentiated cells and intercellular substances making up the tissues and organs of the fully formed chick. This fast sequence of morphogenetic events is made possible by rapidly occurring changes in metabolic processes and chemical composition affecting egg constituents and embryonic tissues at once. Various aspects of the chemistry of chicken embryo development have retained the attention of a number of investigators (Needham, 1931, 1950; Williams et al., 1954; Rupe and Farmer, 1955; Kaminski and Durieux,
U
1972
1956; Flickinger, 1957; Walter and Mahler, 1958; Romanoff, 1960; Fitzsimmons and Waibel, 1968). Here, as in other systems, the rapidly occurring morphogenic and chemical changes are genetically controlled. This is made possible by the synthesis of the appropriate amounts and quality of proteins mediating phenotypic expressions as directed by genome and affected by environment. Determining total amino acid and protein composition of the embryo in the course of incubation would be of interest to the experimenter studying responses of the developing embryo to genetic, chemical, nutritional or microbial treatments. The usual complexity of the required analyses, however,
Downloaded from http://ps.oxfordjournals.org/ at Northern Arizona University on June 4, 2015
Quantitative and Qualitative Protein Changes in the Developing Single Comb White Leghorn Chicken Embryo
314
J. A. ASMAR, P. L. PELLETT, N. HARIRI AND M. D. HARIRI
has in general deterred investigators whose primary concern was other than embryogenesis. In the present work a coordinated use was made of conventional or improved analytical procedures to determine some of the quantitative and qualitative protein changes. MATERIALS AND METHODS
Downloaded from http://ps.oxfordjournals.org/ at Northern Arizona University on June 4, 2015
Fertile Single Comb White Leghorn hatching, eggs within commercial hatching specifications were obtained from a private source and incubated at 37.8 ± 0.3° C. and 55-57% relative humidity. The eggs were set in the incubator not more than 36 hr. from oviposition time and were automatically tilted at 60 minutes intervals. Candling was done daily and infertile eggs or eggs with dead embryos were discarded. Fewer than nine percent of the eggs incubated were so excluded. Embryo samples were collected at 6, 12 and 18 days of incubation with ± 2 hours from time set in the incubator. To collect 6-day old embryos the egg shell and the attached membrane were removed from over the air cell (broad pole of the egg). The chorioallantoic membranes were excised, the embryos lifted with a pair of tweezers and severed from the extraembryonic tissues. Individually collected embryos were quickly dipped in three consecutive baths of physiological salt solution, drained and blotted with filter paper, then weighed to the nearest 0.1 mgm. Due to their larger size 12- and 18-day old embryos were easier to harvest as just described. In all cases extraembryonic tissues were excluded. Embryos used for total solids determinations were oven-dessicated at 100°C. until constant weight was reached and recorded to the nearest 0.1 mgm. Twelve and 18-day embryos were split open along the mid sa-
gittal ventral line for more effective dehydration. To prepare the hydrolysates for amino acid analysis individual whole six-day embryos minced with small scissors were introduced together with 10 ml. 6 N HC1 in vacuum hydrolysis tubes (Phoenix Precision Instruments Co., Philadelphia, Pa., U.S.A.) flushed twice with N 2 gas and hydrolyzed at 105°C. for 22 hours in vacuo. Because of their larger size 18-day embryos required a preliminary preparation. The embryos were first freeze dried, individually chopped in an electric grinder then homogenized in a ball mill. Samples of suitable size from the pulverized material were then individually hydrolyzed as just described. The hydrolysates were analyzed in an automatic amino acid analyzer (Phoenix Precision Instruments Co.) as described by Jamalian and Pellett (1968) but modified to use the new spherical resins for high speed resolution and completion of analysis in about 4 hours. The amino acid values were calculated in mgm. per gram nitrogen and as moles percent. The micro Kjeldahl procedure was used to determine nitrogen. For the 6-day embryos, measured amounts of the HC1 hydrolysates were individually digested and analyzed. For 12- and 18-day old embryos samples of the homogenized dried carcasses prepared as just described were digested and analyzed. Protein was expressed throughout as N X 6.25. Tissue soluble proteins used in electrophoretic analysis were prepared from freshly harvested embryos. Whole embryos were individually minced with scissors and homogenized in a mechanically driven teflon homogenizer maintained in an ice bath. The homogenates were then carried through several quick freezing and thawing cycles, centrifuged at 2000 X G and the supernatant fluids decanted for electrophoresis in acrylamide gel. The Microzone acryl-
315
PROTEIN CHANGES IN EMBRYO
amine gel electrophoresis procedure (Beckman Instruments, Inc., Fullerton, California) was used according to manufacturer's instructions (Anonymous, 1970). Sixty minutes was the duration of the separation. The finished electropherograms were mounted in plastic covers and scanned in an R 110 densitometer (Beckman Inst., Inc.) at 594 nm.
Embryo weights, total solids, N/100 gm. and protein/embryo are reported in Table 1. At six days of incubation the embryos contained an average 5.8% solids of which better than 71% was protein. At 12 days solid matter increased to 9.2% of embryo
TABLE 1.—Weight, solids, nitrogen and prot-ein values (gm.) of S.C.W.L. chicken embryos at three stages of incubation Days in incubator
Embryo Wt. Solids/embryo N/100 gm.0 Protein/embryo a b 0
0.3863 + 0.0094" (27) 0.0226±0.0029 (8) 0.642 +0.0064(11) 0.016 +0.0014(11)
b
12
18
5.0726+0.0704(20) 0.4645 + 0.0030 (12) 0.913 +0.0230 (7) 0.282 +0.0051 (7)
23.1672±0.2430(19) 4.4151±0.0865 (11) 1.733 +0.0327 (9) 2.535 ±0.0511 (9)
Means+ SE Figures between brackets = number of determinations. Wet weight basis.
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RESULTS AND DISCUSSION The eggs used in this investigation weighed an average 55.5 ± 1.7 gm. and were within the range of egg weights considered suitable in the commercial hatching of Single Comb White Leghorn chicks. Eggs in this weight range are known to produce maximum embryo viability and hatchability and their embryos may be regarded as physiologically normal. Upward of 90 such embryos were used. Statistically analyzed egg and embryo weight values showed that within the egg size range selected there was no correlation between these two parameters. In other words egg weight in this range had no detectable effect on embryo growth through 18 days of incubation.
weight and although there was an approximate 17 fold increase in protein, the latter was not more than 60% of total solids. There was further loss of water between 12 and 18 days of incubation at which time the embryos contained an average 19% solids made of approximately 57% protein and a balance of non nitrogenous substances. Plotting weight, solids and protein values on a semilograrithmic chart (Fig. 1) shows that all three parameters increased exponentially between six and 18 days but at differing and slightly declining rates. Solid matter made the fastest gains whereas total weight gain was slowest. These differences in rates account not only for the gradual decline in water content but also indicate that increasing amounts of materials other than protein were being added to the embryonic tissues. Minerals deposited in the skeletal structures must represent part of these non nitrogenous solids. The gradual decline in water concentration and the appearance of increasing amounts of non nitrogenous substances indicate that metabolic processes were changing with advancing embryonic development. For these changes to take place increasing amounts of proteins with new enzymatic and physiological functions are required. This is reflected by the difference in amino acid composition of 6- and 18-day
316
J. A. ASMAR, P. L. PELLETT, N. HARIRI AND M. D. HARIRI FIG. 1. Plotted on a semilogarithmic scale total weight, total solids and protein content of S.C.W.L. chicken embryo rise exponentially but at differing rates between 6 and 18 days of incubation.
10 12 V DAYS IN INCUBATOR
TABLE 2.—Amino acid composition of S.C.W.L. chicken embryos at 6 and 18 days of incubation 18-day (4)a
6-day (5)a mgm./gm.N b Isoleucine Leucine Lysine Methionine Cystine Total Sulfur AA Phenylalanine Tyrosine Total aromatic AA Threonine Valine Arginine Histidine Alanine Aspartic Acid Glutamic Acid Glycine Proline Serine NH 3
368+19.6 497+ 6.3 490+16.6 125+ 5.8 70+ 9.1 195 + 10.7 264+ 9.8 252+ 4.6 516+10.3 297+ 7.5 328+ 6.0 497 + 23.2 177+12.4 342+ 6.5 575+ 6.7 891 + 11.0 323+ 0.9 257+ 8.7 309+16.1 116+ 6.6
iM%° 5.9 8.0 7.1 1.8 0.6 2.4 3.4 3.0 6.4 5.3 5.9 6.1 2.4 8.2 9.2 12.9 9.2 4.7 6.2 99.9
a
Figures between brackers = number of determinations. Means ± S E . c Does not include ammonia. d xP<0.05. xxxP<0.001. b
mgm./gm.N
fM%°
320 + 29.2 500+ 9.7 365+ 3.2,™"* 104+11.0 156+ 5.6*** 260+14.3 300± 3.7" 252+ 4.7 552 + 7.8* 310+ 7.1 352 + 10.8 507± 6.4 167+ 8.9 365± 4.4 552+ 4.7 799+ 8.6"1* 541 + 11.5™ 427+ 4.5*** 416+ l.C™* 103± 4.9
4.8 7.4 4.8 1.4 1.3 2.7 3.5 2.7 6.2 5.0 5.8 5.7 2.1 8.0 8.1 10.5 14.0 7.2 7.7 100.0
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embryos presented in Table 2. Results of amino acid analysis are expressed both as mg. per gram nitrogen and as micromoles percent. Naturally the high rate of protein synthesis occurring during development is accompanied by increasing levels of all amino acids. There are, however, significant changes in the proportions of certain of the amino acids. Thus very large and significant increases (P < 0.001) were observed for cystine, glycine and proline accompanied by smaller but still significant increases in serine (P < 0.001) and phenylalanine (P < 0.0S). These changes are
PROTEIN CHANGES IN EMBRYO
317
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probably related to increased synthesis in TABIE 3.—Milligrams of individual amino acids per S.C.W.L. chicken embryo at 6 and 18 days keratin and collagen which are high in sevof incubation eral of these amino acids. The foregoing inDays in creases are in proportions of the total amino acids found and must, therefore, be 6 18 compensated for by corresponding decreases in the proportions of other amino 0.960 129.6 128.6 13,396 Isoleucine 1.297 202.5 201.2 15,513 Leucine acids. Highly significant decreases (P < Lysine 1.278 147.8 146.5 11,463 0.001) were in fact observed for lysine and Methionine 0.326 42.1 41.7 12,791 Cystine 0.182 63.0 62.8 34,505 glutamic acid with smaller though not sigPhenylalanine 0.689 121.5 120.8 17,532 nificant (P > 0.05) falls for isoleucine, Tyrosine 0.657 102.1 101.4 15,433 0.775 125.5 124.7 16,090 Threonine methionine and aspartic acid. The other 0.856 142.5 141.6 16,542 Valine amino acids remained essentially at the Arginine 1.297 205.3 204.0 15,728 Histidine 0.461 67.6 67.1 14,555 same relative levels. Alanine 0.892 147.8 146.9 16,468 The changes observed here were in genAspartic Acid 1.500 223.5 222.0 14,800 Glutamic Acid 2.325 323.5 321.1 13,810 eral similar to those obtained by Fitzsim0.843 219.1 218.2 25,884 Glycine mons and Waibel (1968) for 6- and 12-day 0.670 172.9 172.2 25,701 Proline 0.806 168.4 167.5 20,780 Serine embryos though certain differences were noted. These were particularly in our large increase in proline concentration and the It is evident from Table 3 that with insmaller increase in serine while Fitzsim- creasing incubation time and embryo size mons and Waibel found a decrease in the considerable amounts of the respective serine and a constant level for proline. A amino acids were incorporated into embryfurther difference was also observed for onic tissues, but the relative amounts phenyalalanine and tyrosine. Although added differed for different amino acids. nothing is mentioned in their article some Plotting embryonic amino acids contents at failure must have occurred in the analysis six and 18 days on a semilogarithmic scale by Fitzsimmons and Waibel (1968) since showed that virtually each of them had its their phenylalanine data are impossibly low own average incorporation rate for the in(less than 10% of expected) and the tyro- cubation period under consideration. This sine data are missing, whereas we recorded is illustrated in Figure 2 which for clarity sizeable amounts for each of these amino purpose includes only a limited number of acids with a moderate increase for phenyl- the amino acids. alanine at 18 days. The present observation that different Pellett and Kaba (1971) have recently amino acids are incorporated into embryreported changes in carcass amino acid onic tissues at differing rates is in general composition of growing rats. This observa- agreement with the data of Fitzsimmons tion suggests that changes in amino acid and Waibel (1968) but stands in contracomposition may not be limited to the pre- diction with the conclusions of Rupe and Farmer (1955) who reported exponential natal phase of development. On the basis of the data in Tables 1 and but essentially equal incorporation rates for 2 the actual amounts of amino acids per all amino acids through 400 hours of incubaembryo were calculated for 6- and 18-day tion. It must be noted, however, that Rupe incubation. These values are listed in Table and Farmer included both embryos and 3 together with the respective total and rel- extra embryonic tissues in their amino acid ative gains made between six and 18 days. analyses. Moreover, their study was based
318 1000.0
J. A. ASMAR, P. L. PELLETT, N. HARIRI AND M. D. HARIRI 3
1 6
1 7
1 8
1 1 1 1 1 1 9 10 11 12 13 1*. DAYS IN INCUBATOR
1 15
1 16
1 17
1 18
FIG. 2. Between 6 and 18 days of incubation S.C.W.L. chicken embryos incorporated amino acids at exponential rates. Different amino acids showed differing rates. For reasons of clarity a limited number was included. See Table 3 for all amino acids determined.
on the microbiological assay method of determining amino acids. The dramatic rise of cystine from 0.182 mgm. at six days to 53.0 mgm. at 18 days of incubation seems to agree well with the passage from the featherless condition of the six-day old embryo to the fully formed feather coat of the 18-day embryo and lends strong support to our observation on differing amino acid incorporation rates. Of the various analytical procedures used in this study it is undoubtedly electrophoresis in acrylamide gel that gave the most direct evidence of the qualitative changes associated with the increases displayed by the soluble embryonic proteins at 6, 12 and 18 days of incubation. Typical densitometric tracings of protein electropherograms at the three ages tested are presented in Figure 3.
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nn\ J
Proteins from six-day embryos show a rather limited degree of heterogeneity and are confined mostly to the gamma and adjacent regions of the electrophoretic spectrum. At 12 days new protein populations have been added on the anodic side of the spectrum thus showing an increased electrophoretic diversity of proteins. Meanwhile the slower migrating fractions have gained in amounts leading their densitometric readings to higher values on the scale. The expansion of the electrophoretic spectrum of proteins is further accented at 18 days with new fractions of higher electrophoretic mobility appearing at the anodic end of the electropherogram. The changes observed in soluble protein patterns indicate that with advancing incubation increasingly larger proportions of acidic or less basic proteins were synthesized. It is of interest to note that the 18-day old embryos contained a higher proportion (66%) of the neutral amino acids than the six-day olds (59%) (Table 2). In contrast, their content of basic and acidic amino acids (respectively 13% and 21%) was lower than that of the six-day olds (respectively 16% and 25%) showing a comparable decline for both groups of amino acids with increasing development. Admittedly the procedures used here do not indicate whether the detected proteins are the result of de novo synthesis by the embryo or have been absorbed more or less intact from the egg. It has been previously demonstrated that some proteins do pass directly from egg to embryo (Kaminski and Durieux, 1956). With the possible exception of immunoglobulins, there seems to be virtually no information regarding the passage or role of egg proteins in embryonic tissues. However, since the absorption of intact egg proteins by the embryo seems to be a highly selective process (Kaminski and Durieux, 1956) it would be normal to
PROTEIN CHANGES IN EMBRYO
319
regard all proteins found in the embryo as integral parts of its system. REFERENCES Anonymous, 1970. Model 113 Acrylamide Gel Accessory for the Microzone System. Palo Alto: Spinco Division of Beckman Instruments Inc. Fitzsimmons, R. C , and P. E. Waibel, 1968. Free amino acid composition of the avian egg as related to embryonic development. Poultry Sci. 47: 219-224. Flickinger, R. A., 1957. Amino acid studies in relation to yolk utilization in the chick embryo. Experientia, 13: 248. Jamalian, J., and P. L. Pellett, 1968. Nutritive value of Middle Eastern foodstuffs. IV. Amino acid composition. J. Sci. Food Agric. 19: 378-382.
Kaminski, M., and J. Durieux, 1956. Etude comparative des serums de poule, de coq, de poussin, d'embryon et du blanc d'oeuf. Exptl. Cell. Res. 10: 590-618. Needham, J., 1931. Chemical Embryology. Cambridge University Press, London. Needham, J., 1950. Biochemistry and Morphogenesis. Cambridge University Press, London. Pellett, P. L., and H. Kaba, 1971. Carcass amino acids of the rat under conditions of determination of net protein utilization. J. Nut. (submitted for publication). Romanoff, A. L., 1960. The Avian Embryo. The Macmillan Company, New York. Rupe, C. O., and C. J. Farmer, 1955. Amino acid studies in the tranformation of the hen's egg to tissue proteins during incubation. J. Biol. Chem. 213: 899-906.
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FIG. 3. Densitometric tracings of soluble proteins from 6- 12- and 18-day old S.C.W.L. embryos electrophoiesed in acrylamide gel. With advancing incubation time proteins increased in concentration and heterogeneity.
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Walter, H., and H. R. Mahler, 1958. Biochemical studies on the developing avian embryo. I. Protein precursors in "vivo." J. Biol. Chem. 230: bryo. Nature, 173 : 490.
D.
HARIRI
Williams, M. A., W. A. DaCosta, L. H. Newman and L. M. Marshall, 1954. Free amino acids in yolk during the development of the chick em241-249.
85
Sr:45Ca Ratios from Intestine to Egg Shell During Perfusion with Ca and Sr1 MRAZ
(Received for publication June 23, 1971)
ABSTRACT The small intestine of 40 Leghorn hens (with an egg in utero) were perfused with 0, 5, 25 or 125 mM levels of Ca and/or Sr at a rate of 0.8 ml./min. After 25 ml. was perfused, ^Ca and 85Sr were added to the remaining 50 ml. of solution. Absorption and concentrations of 45Ca and ^Sr in blood plasma, egg shell and mucosal cell particulates of the small intestine and uterus were measured. The 85Sr :45Ca ratios decreased from 0.8 at absorption to 0.5 at the egg shell. The radionuclide content and ^Sri^Ca ratios in the cellular particulates generally decreased with particulate size as determined by ultracentrifugation. Differences between Ca and Sr do not stop at the intestinal wall but rather increase through the tissues, and are influenced by the availability of Ca and Sr. POULTKY SCIENCE 51:
C TRONTIUM has been of interest for ^ some time because of its chemical similarity to calcium, and because it normally follows metabolic routes similar to those of Ca. Wasserman (1960) ascertained from everted intestinal sacs that, under given conditions, Ca-Sr discrimination was dependent upon metabolically active membranes. Palmer and Thompson (1961) reported that the absorption of 85Sr and 45 Ca from the small intestines of rats was related inversely to the concentration of Ca in the perfusate and that the effect on 45Ca absorption was greater than the effect on 85 Sr. Mraz (1962a) concurred, adding that, while increasing concentrations of Sr 1 This manuscript is published with the permission of the Dean of The University of Tennessee Agricultural Experiment Station, Knoxville, Tennessee. 2 Operated by the Tennessee Agricultural Experiment Station for the U. S. Atomic Energy Commission under Contract No. AT-40-1-GEN-242.
320-325,
1972
also reduced 45Ca and 85Sr, no effect on the 85Sr:45Ca ratio was seen. However, the addition of Ca to the perfusate containing Sr increased the ratio back to that found with Ca alone. A difference between 45 Ca and 85Sr uptake in vitro by rat kidney and liver mitochondria that was increased by adding Ca to the substrate was also reported (Mraz, 1962b). The addition of Sr tended to reduce this difference. Hens preferentially deposited radiocalcium over radiostrontium in bones and egg shells (Edwards and Mraz, 1961; Monroe et al., 1961; Mraz et al., 1967). To ascertain if the preferences for Ca instead of Sr stopped at the small intestine or continued through to the egg shell of the hen, absorption and the concentrations of 45Ca and 85 Sr in blood plasma, egg shell and mucosal cell particulates of the small intestine and uterus were measured after perfusion with varying levels of Ca and/or Sr.
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F R A N K R.
Agricultural Research Laboratory of the University of Tennessee, Oak Ridge, Tennessee* 37830