Comp. Biochem. PhysioL, 1976, Vol, 55B. pp. 145 to 149. Per~3amon Press. Printed in Great Britain
ONTOGENETIC CHANGES IN THE HAEMOGLOBINS OF GEESE, DUCKS, CHICKENS AND TURKEYS A. STRATIL AND M. VALENTA Czechoslovak Academy of Sciences, Institute of Animal Physiology and Genetics, Department of Genetics, 277 21 Lib~chov, Czechoslovakia (Received 6 October 1975) During ontogenetic development of the chicken, turkey, goose and duck distinct qualitative and quantitative changes take place in haemoglobins A, D, P, E and H. 2. Haemoglobin components are similar in all species investigated even though differences exist in the time of appearance and disappearance of individual haemoglobins in various species. 3. In early embryos of all four species a very weak haemoglobin zone, X, was detected, which migrates during electrophoresis more to the cathode than haemoglobin H. 4. In four cases out of 150, additional fractions were found in haemoglobins of adult ducks, which indicate the existence of polymorphism in haemoglobins A and D. Abstract--1.
INTRODUCTION
MATERIALS AND METHODS
Incubation of eggs, blood collection and the preparation of The majority of authors agree in that the haemoglohaemoglobin solutions bin of adult chickens is composed of two components, A and D, which occur in a ratio of approx 3:1. In The eggs of the chickens, Gallus gallus L. (White Legearly embryos haemoglobins P and E are present and horn breed), turkey, Meleagris gallopavo L. (White Broadrecently haemoglobin M has also been described. Breasted Turkey breed), duck, Arias platyrhynchos L. (Peking Duck breed) and goose, Anser anser L. (Roman Goose These differ from the haemoglobins of adults. In the breed) were used. The eggs were incubated in a thermostat case of older embryos and in chickens up to 90 days at 38°C and 65-70% relative humidity. The blood of the old haemoglobin H is also present which is electroembryos was taken with a Pasteur pipette after cutting phoretically similar to haemoglobin E. (For a more of the shell from the main blood vessels of the extraemdetailed review and list of references see Bruns & Inbryonic blood circulation. In the case of larger embryos gram, 1973; Brown & Ingram, 1974; Keane et al., the blood was taken from the heart. In the postincubation 1974). period it was taken from the wing vein. The erythrocytes In addition to these haemoglobins a number of were washed 3- to 5-times with a cold physiological soluminor haemoglobin components have been detected, tion and submitted to lysis by the addition of 1 vol of cold distilled water and half the vol of CC14. The contents both in embryos and in adult animals (e.g. Hashimoto of the test tubes was shaken thoroughly and then centri& Wilt, 1966; Moss & Thompson, 1969; Fraser et fuged at 1500 0 for 10 min. The haemoglobin solution was al., 1972; Schalekamp et al., 1972; Bruns & Ingram, transferred into clean test tubes and the haemoglobin 1973). These components could not be detected by samples stored, mostly for several days, at -20°C and anaall investigators and the authors also differ in their lysed as oxyhaemoglobin. Only when trace cathodic haeopinions regarding their actual existence. It is known moglobin was studied, freshly prepared samples of haethat avian haemoglobins are very unstable. Even mere moglobin solution were used which were converted to freezing and thawing leads to the appearance of adcyanmethaemoglobin by the addition of Drabkin's solution ditional haemoglobin zones and to a deteriorated (see Brown & Ingram, 1974) or to carboxyhaemoglobin electrophoretic separation (Manwell et al., 1966; (bubbling of CO). Bruns & Ingram, 1973). Starch gel electrophoresis Embryonic haemoglobins have been demonstrated in a number of avian species. In addition to the A horizontal arrangement of starch gel electrophoresis was employed. The gel troughs were cooled by tap water. chicken these are the turkey and chukar partridge (Manwell et al., 1963, 1966), as well as the duck (Bor- The buffer used was Tris-EDTA-boric acid, pH 8.9 (Gahne et al., 1960). The samples were applied into the gese & Bertles, 1965) and sparrow (Bush & Towngel after being absorbed into pieces of Whatman No. 1 send, 1971). However, haemoglobins E and H have filter paper; the diluted fractions obtained after chromatobeen differentiated in the case of the chicken only graphy on CM-Sephadex were absorbed into pieces of and that only by some investigators. Whatman No. 3 paper. The voltage gradient was 7-9 V/cm In this paper the results are given of the study of and the separation lasted 3 4 hr. The gels were stained developmental changes in haemoglobins during the with benzidine (Lathem & Worley, 1959) or amido black preincubation and postincubation period of chickens, solutions. turkeys, geese and ducks. The presence of an as yet Fractionation of haemoglobin undescribed trace cathodic haemoglobin in early embryos of all four species and a polymorphic haeThe haemoglobin of 5-day old embryos was fractionated moglobin in ducks is also demonstrated. as cyanmethaemoglobin on CM-Sephadex C-50 in 0.03 M 145
146
A. STRAT1LAND M. VALENTA
phosphate buffer (Na2HPO4 + KHzPO4), pH 6.9, containing 0.0015 M KCN. A freshly prepared solution of cyanmethaemoglobin from 80 5 day-old embryos was transferred into the starting buffer on Sephadex G-25 and absorbed on a 1.3 × 20 cm column of CM-Sephadex C-50. The haemoglobins were eluted by a linear salt gradient from 0.03 M to 0.3 phosphate, pH 6.9 (chamber vol 150ml). Flow rate was l l.4ml/hr and the fractions of 3.8 ml were collected. The fractions from the column were concentrated using DEAE-Sephadex and CM-Sephadex. The whole experiment took place at + 6°C and lasted no longer than 3 days.
5-day embryo
P+o
m
m
15-doy embryo
m
I-doy gosling
m
Adult
m
Origin
Nomenclature of haemoglobin zones The majority of authors investigating avian haemoglobins use a nomenclature of their own. Very often these nomenclatures are difficult to compare. In this paper the nomenclature used in Ingram's laboratory (Bruns & Ingram, 1973; Brown & Ingrain, 1974) is employed.
E
v////////~
a
v///////l
g//z/////]
d
r////////A
X ' RESULTS
Developmental changes in haemoglobins in preincubation and postineubation periods The changes in the number and in the intensity of single haemoglobin zones were studied from the fifth day of incubation, at 1-2 day intervals. The patterns of embryonic and adult haemoglobins after electrophoresis in starch gel are analogous in all four species. Among species differences exist in the mobilities of the haemoglobin zones. These differences are negligible in adult haemoglobins but distinct in the case of embryonic haemoglobins. The ontogenetic changes in the haemoglobins of chickens are virtually identical with those described by other authors. In five-day-old embryos two P components and zone E occur (Fig. 1). Besides, a cathodically localized, as yet undescribed trace zone is also present, designated as zone X (cf. also Fig. 6b). This band migrates much farther to the cathode than zone H. Haemoglobin X was detected in 3, 4, 5, 6 and 7 day-old embryos only. In view of its very low concentration samples had to be overloaded, causing other zones to become too intensive. Shortly after staining with benzidine the intensity of the zone X 5 - doy embryo ........
I- doy chick D
~
Adult
D
h-
~
P ~ Origin
E
V/////////A H v/////////A
X
Fig. 1. Schematic diagram of starch gel electrophoretic separation of embryonic and adult haemoglobins of chickens.
Fig. 2. Ontogenetic changes in haemoglobins of geese.
began to fade. Haemoglobin X was present in the samples of oxyhaemoglobin, cyanmethaemoglobin and carboxyhaemoglobin. All these samples were analysed about 18 hr after preparation and they were kept throughout at +5°C. The attempt at a partial isolation of haemoglobin X will be presented later. On the sixth day of incubation the zones of adult haemoglobin A and D began to appear in chickens, gradually increasing in intensity, while the zones of embryonic haemoglobins P and E weakened. On the ninth day a weak zone of haemoglobin H was detected which gradually became stronger. On the 15th day of incubation only zones A, D and H could be detected. We did not follow the postincubation changes in chicken haemoglobins, but according to literature data haemoglobin H disappears at about the 90th day of age (Washburn, 1968). Similar electrophoretic bands of haemoglobins were also observed in turkeys. However, the embryonic zone P did not seem doubled. On the 8th day of incubation the adult zones A and D began to appear, on the tenth day a weak zone H also appeared. After 17 days the zones P and E were no longer detectable. Zone H is distinct for about 80 days after hatching, after that it weakens and on the 100th day it is no longer detectable. In early embryos zone X is also present. Analogous patterns were observed in geese and ducks. In the case of geese (Fig. 2) the embryonic haemoglobin P was also doubled and the more anodically localized zone had the same mobility as zone D in adult animals. Zone A is detectable on the 9th day of incubation. Zone H begins to appear on the 10th day. After the 18th day of incubation haemoglobins P and E are no longer detectable. In 5 day-old embryos haemoglobin X was also observed. Haemoglobin H is distinct up to about 110 days; however, its traces can be detected up to about the 140th day after hatching. In the case of ducks (Fig. 3) the embryonic haemoglobin P did not seem doubled. On the 8th day zones A and D began to appear, on the 9th day zone H was also present and on the 17th day zones P and E were no longer present. Five to 6 day-old embryos
Ontogeny of avian haemoglobins 5-day embryo
I I-doy embryo
D
..........
I-day duckling ~ B m
147
Adull"
t
i !
P
I
I
v........, r T / / / / / / / / / l
1888888888N v/////////~,
A
~
A
Odgln
Odgin
Abnormot
Normal
-
Fig. 4. Normal and abnormal types of haemoglobins of adult ducks. H
X !
v///////A
[(Ill/IlIA
!
Fig. 3. Ontogeny of duck haemoglobins.
also contained zone X. Haemoglobin H disappeared after 90 days of age. The distance between the zones E and H in geese and ducks was larger than in hens and turkeys. In all species investigated several additional weak haemoglobin fractions were observed, the nature of which is unknown.
Polymorphism of haemoglobins in ducks During an electrophoretic analysis of haemoglobins of adult ducks the presence of additional haemoglobin fractions with distinctly higher anodic mobility than would correspond to haemoglobins A and D (Fig. 4) was observed in 4 cases out of 150. The genetic control of this polymorphism is under investigation.
Fractionation of haemoglobins of 5 day-old chick embryos Since the component X occurs in the lysates of the erythrocytes of embryos at a very low concentration
only, we tried to carry out a partial fractionation of haemoglobin and the concentration of the fraction containing haemoglobin X. For this purpose we used chromatography on CM-Sephadex. In order to prevent too high a dilution of the haemoglobins during chromatography, which might lead to a loss of the trace component X, we used a relatively steep gradient. The result of the chromatography (Fig. 5) was analogous to the chromatography of haemoglobin of 5 day-old embryos described by Brown & Ingram (1974); nevertheless, some differences still existed. In Fraction I (Fig. 6) haemoglobin P, which is doubled, was evidently present. In Fraction II a zone was present the mobility of which was similar to that of the slow zone of haemoglobin P. In Fraction III a haemoglobin was present with a mobility similar to that of the fast moving zone of haemoglobin P and it probably represents haemoglobin M (cf. Brown & Ingram, 1974). In Fraction IV haemoglobin E was localized together with haemoglobin X. The latter is visible only when a relatively large sample volume was applied into the gel. In addition to these main haemoglobin zones other weak zones were also observed in all fractions (Fig. 6). It is difficult to decide whether they represent artifacts formed during separation or whether they are true haemoglobins.
1.4 --"
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1.2-i
0 I.¢-(M
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-
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50
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150
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Fig. 5. Chromatography of 5 day-old chick embryonic haemoglobin on CM-Sephadex. The details are given in "Materials and Methods". Fractions I and II were concentrated on DEAE-Sephadex, Fractions III-V on CM-Sephadex.
148
A. STRATILAND M. VALENTA
-I-
P+ M Origin E X
_Z
E
TIT
I~T
(a)
-~r
5day adult embryo (b)
Fig. 6. Starch gel electrophoresis of five-day-old chick embryonic haemoglobin. (a) Haemoglobin fractions obtained by chromatography shown in Fig. 5. Fraction IV was overloaded in order to make the zone of haemoglobin X visible. (b) For comparison, starch gel electrophoretic separations of carboxyhaemoglobins of 5 day-old chick embryo and adult chicken, respectively, are shown. DISCUSSION
Our results concerning ontogenetic changes in the haemoglobins of chickens are in many respects similar to those of a series of other authors (e.g. Manwell et al., 1963, 1966; Fraser et al., 1972; Schalekamp et al., 1972; Shimizu, 1972a, b; Bruns & Ingram, 1973; Cirotto et al., 1975). The differences observed in the time of appearance and disappearance of single haemoglobin components may be attributed to the differences in the techniques employed, but mainly in the differing concentration of haemoglobin solutions used for electrophoresis. In this paper we studied the ontogeny of the main haemoglobin components A, D, P, E, H and a trace haemoglobin X, only. Similarly to other authors (e.g. Moss & Thompson, 1969; Fraser et al., 1972; Schalekamp et al., 1972; Bruns & Ingrain, 1973) we also observed further weak fractions, but these were not always distinct. It is possible that some of them were artifacts produced by the freezing of the samples. Up to now various authors differ in their opinions regarding the existence or the number of weak fractions. Some consider them as artifacts only, formed by the preparation procedure, or by their storage (Manwell et at., 1966; Bruns & Ingram, 1973) while others do not doubt their actual existence (e.g. Hashimoto & Wilt, 1966; Fraser et al., 1972; Schalekamp et al., 1972). In our case the existence of the trace haemoglobin X is beyond doubt. We also believe that the weak zones present in Fractions III and V after CMSephadex chromatography may represent true haemoglobins.
The main aim of this work was the comparison of the developmental changes of the chicken haemoglobins with those of turkeys, ducks and geese. Detailed knowledge acquired with chickens has not yet been sufficiently correlated with other avian species. Our results indicate quite clearly that analogous changes in the presence of haemoglobins take place in all four species investigated and that the differences can be observed only in the time of appearance and disappearance of individual haemoglobins. Although some data on the embryonic haemoglobins of turkeys and ducks have already been published, the techniques used did not permit the distinction of haemoglobins E and H (Manwell et al., 1963; Borgese & Bertles, 1965). The ontogeny of goose haemoglobins has not yet been studied. The data on the disappearance of zone H during the postincubation development of turkeys and geese are also original. In ducks zone H disappears later labout the 90th day) than published by Borgese & Bertles (19651 (about 70 days). Evidently, this difference is caused by differing concentration of the haemoglobin samples used for analyses. Brown & lngram (1974) state that chicken haemoglobins A and D contain identical chains /3 in addition to differing chains ~ and ~ , respectively. The fact that in the polymorphic haemoglobin of ducks bands with a higher mobility than that of haemoglobins A and D are present indicates that the possible formation of additional bands is given by the polymorphism in chain fl and that the type observed by us is a heterozygote /3//3'. The chain fl' has a higher electrophoretic mobility towards the anode.
Ontogeny of avian haemoglobins Acknowledgements--We thank Miss J. Janatkov~ for her skillful technical assistance.
REFERENCES
BORGESET. A. & BER~ES J. F. (1965) Hemoglobin heterogeneity: Embryonic hemoglobin in the duckling and its disappearance in the adult. Science, N.Y. 148, 509-511. BROWN J. L. & INGRAMV. M. (1974) Structural studies on chick embryonic hemoglobins. J. biol. Chem. 249, 3960-3972. BRtrNS G. A. P. & INGRAMV. M. (1973) The erythroid cells and haemoglobins of the chick embryo. Phil. Trans R. Soc. Lond. 266, 225-303. BUSH F. M. & TOWNSENDJ. I. (1971) Ontogeny of hemoglobin in the house sparrow. J. Embryol. exp. Morph. 25, 3345. CmOTTO C., SCOTTODI TELLAA. & GERAC1G. (1975) The hemoglobins of the developing chicken embryos. Fractionation and globin composition of the individual component of total erythrocytes and of a single erythrocyte type. Cell Differentiation 4, 87-99. FRASER R., HORTON B., DUPOURQUED. & CHERNOFEA. (1972) The multiple hemoglobins of the chick embryo. J. Cell Physiol. 80, 79-88. GAHNE B., RENDEL J. & VENGE O. (1960) Inheritance of fl-globulins in serum and milk from cattle. Nature, Lond. 186, 907 908. HASmMOTO K. & WILT F. H. (1966) The heterogeneity of chicken hemoglobin. Proc. hath. Acad. Sci. U.S.A. 56, 1477-1483.
149
KEANE R. W., ABBOTTU. K., BROWN J. L. & INGRAM V. M. (1974) Ontogeny of hemoglobins: Evidence for hemoglobin M. Devl. Biol. 38, 229-236. LATHEM W. & WORLEYW. E. (1959) The distribution of extracorpuscular hemoglobin in circulating plasma. J. clin. Invest. 38, 474-483. MANWELLC., BAKERC. M. A. & BETZ T. W. (1966) Ontogeny of hemoglobin in the chicken. J. Embryol. exp. Morph. 16, 65-81. MANWELLC., BAKERC. M. A., ROSLANSKYJ. D. & FOGHT M. (1963) Molecular genetics of avian proteins--II. Control genes and structural genes for embryonic and adult hemoglobins. Proc. natn. Acad. Sci. U.S.A. 49, 496-503. Moss B. A. & THOMPSON E. O. P. (1969) Haemoglobins of the adult domestic fowl Gallus clomesticus. Aust. J. biol. Sci. 22, 1455-1471. SCHALEKAMP M., SCHALEKAMP i . , VAN GOOR D. & SLINGERLANDR. (1972) Re-evaluation of the presence of multiple haemoglobins during the ontogenesis of the chicken. Electrophoretic and chromatographic characterization, polypeptide composition and immunochemical properties. J. Embryol. exp. Morph. 28, 681-713. SHIMIZU K. (1972a) Ontogeny of chicken hemoglobin--I. Electrophoretic study of the heterogeneity of hemoglobin in development. Devl. Growth Differ. 14, 43-55. SHIMIZU K. (1972b) Ontogeny of chicken hemoglobin--II. Chromatographic study of the heterogeneity of hemoglobin in development. Devl. Growth Differ. 14, 281-295. WASHBURNK. W. (1968) Effects of age of bird and hemoglobin type on the concentration of adult hemoglobin components of the domestic fowl. Poult. Sci. 47, 1083-1089.
ONTOGENETIC CHANGES IN THE HAEMOGLOBINS OF GEESE, DUCKS, CHICKENS AND TURKEYS A. STRATIL AND M. VALENTA Czechoslovak Academy of Sciences, Institute of Animal Physiology and Genetics, Department of Genetics, 277 21 Lib~chov, Czechoslovakia (Received 6 October 1975) During ontogenetic development of the chicken, turkey, goose and duck distinct qualitative and quantitative changes take place in haemoglobins A, D, P, E and H. 2. Haemoglobin components are similar in all species investigated even though differences exist in the time of appearance and disappearance of individual haemoglobins in various species. 3. In early embryos of all four species a very weak haemoglobin zone, X, was detected, which migrates during electrophoresis more to the cathode than haemoglobin H. 4. In four cases out of 150, additional fractions were found in haemoglobins of adult ducks, which indicate the existence of polymorphism in haemoglobins A and D. Abstract--1.
INTRODUCTION
MATERIALS AND METHODS
Incubation of eggs, blood collection and the preparation of The majority of authors agree in that the haemoglohaemoglobin solutions bin of adult chickens is composed of two components, A and D, which occur in a ratio of approx 3:1. In The eggs of the chickens, Gallus gallus L. (White Legearly embryos haemoglobins P and E are present and horn breed), turkey, Meleagris gallopavo L. (White Broadrecently haemoglobin M has also been described. Breasted Turkey breed), duck, Arias platyrhynchos L. (Peking Duck breed) and goose, Anser anser L. (Roman Goose These differ from the haemoglobins of adults. In the breed) were used. The eggs were incubated in a thermostat case of older embryos and in chickens up to 90 days at 38°C and 65-70% relative humidity. The blood of the old haemoglobin H is also present which is electroembryos was taken with a Pasteur pipette after cutting phoretically similar to haemoglobin E. (For a more of the shell from the main blood vessels of the extraemdetailed review and list of references see Bruns & Inbryonic blood circulation. In the case of larger embryos gram, 1973; Brown & Ingram, 1974; Keane et al., the blood was taken from the heart. In the postincubation 1974). period it was taken from the wing vein. The erythrocytes In addition to these haemoglobins a number of were washed 3- to 5-times with a cold physiological soluminor haemoglobin components have been detected, tion and submitted to lysis by the addition of 1 vol of cold distilled water and half the vol of CC14. The contents both in embryos and in adult animals (e.g. Hashimoto of the test tubes was shaken thoroughly and then centri& Wilt, 1966; Moss & Thompson, 1969; Fraser et fuged at 1500 0 for 10 min. The haemoglobin solution was al., 1972; Schalekamp et al., 1972; Bruns & Ingram, transferred into clean test tubes and the haemoglobin 1973). These components could not be detected by samples stored, mostly for several days, at -20°C and anaall investigators and the authors also differ in their lysed as oxyhaemoglobin. Only when trace cathodic haeopinions regarding their actual existence. It is known moglobin was studied, freshly prepared samples of haethat avian haemoglobins are very unstable. Even mere moglobin solution were used which were converted to freezing and thawing leads to the appearance of adcyanmethaemoglobin by the addition of Drabkin's solution ditional haemoglobin zones and to a deteriorated (see Brown & Ingram, 1974) or to carboxyhaemoglobin electrophoretic separation (Manwell et al., 1966; (bubbling of CO). Bruns & Ingram, 1973). Starch gel electrophoresis Embryonic haemoglobins have been demonstrated in a number of avian species. In addition to the A horizontal arrangement of starch gel electrophoresis was employed. The gel troughs were cooled by tap water. chicken these are the turkey and chukar partridge (Manwell et al., 1963, 1966), as well as the duck (Bor- The buffer used was Tris-EDTA-boric acid, pH 8.9 (Gahne et al., 1960). The samples were applied into the gese & Bertles, 1965) and sparrow (Bush & Towngel after being absorbed into pieces of Whatman No. 1 send, 1971). However, haemoglobins E and H have filter paper; the diluted fractions obtained after chromatobeen differentiated in the case of the chicken only graphy on CM-Sephadex were absorbed into pieces of and that only by some investigators. Whatman No. 3 paper. The voltage gradient was 7-9 V/cm In this paper the results are given of the study of and the separation lasted 3 4 hr. The gels were stained developmental changes in haemoglobins during the with benzidine (Lathem & Worley, 1959) or amido black preincubation and postincubation period of chickens, solutions. turkeys, geese and ducks. The presence of an as yet Fractionation of haemoglobin undescribed trace cathodic haemoglobin in early embryos of all four species and a polymorphic haeThe haemoglobin of 5-day old embryos was fractionated moglobin in ducks is also demonstrated. as cyanmethaemoglobin on CM-Sephadex C-50 in 0.03 M 145
146
A. STRAT1LAND M. VALENTA
phosphate buffer (Na2HPO4 + KHzPO4), pH 6.9, containing 0.0015 M KCN. A freshly prepared solution of cyanmethaemoglobin from 80 5 day-old embryos was transferred into the starting buffer on Sephadex G-25 and absorbed on a 1.3 × 20 cm column of CM-Sephadex C-50. The haemoglobins were eluted by a linear salt gradient from 0.03 M to 0.3 phosphate, pH 6.9 (chamber vol 150ml). Flow rate was l l.4ml/hr and the fractions of 3.8 ml were collected. The fractions from the column were concentrated using DEAE-Sephadex and CM-Sephadex. The whole experiment took place at + 6°C and lasted no longer than 3 days.
5-day embryo
P+o
m
m
15-doy embryo
m
I-doy gosling
m
Adult
m
Origin
Nomenclature of haemoglobin zones The majority of authors investigating avian haemoglobins use a nomenclature of their own. Very often these nomenclatures are difficult to compare. In this paper the nomenclature used in Ingram's laboratory (Bruns & Ingram, 1973; Brown & Ingrain, 1974) is employed.
E
v////////~
a
v///////l
g//z/////]
d
r////////A
X ' RESULTS
Developmental changes in haemoglobins in preincubation and postineubation periods The changes in the number and in the intensity of single haemoglobin zones were studied from the fifth day of incubation, at 1-2 day intervals. The patterns of embryonic and adult haemoglobins after electrophoresis in starch gel are analogous in all four species. Among species differences exist in the mobilities of the haemoglobin zones. These differences are negligible in adult haemoglobins but distinct in the case of embryonic haemoglobins. The ontogenetic changes in the haemoglobins of chickens are virtually identical with those described by other authors. In five-day-old embryos two P components and zone E occur (Fig. 1). Besides, a cathodically localized, as yet undescribed trace zone is also present, designated as zone X (cf. also Fig. 6b). This band migrates much farther to the cathode than zone H. Haemoglobin X was detected in 3, 4, 5, 6 and 7 day-old embryos only. In view of its very low concentration samples had to be overloaded, causing other zones to become too intensive. Shortly after staining with benzidine the intensity of the zone X 5 - doy embryo ........
I- doy chick D
~
Adult
D
h-
~
P ~ Origin
E
V/////////A H v/////////A
X
Fig. 1. Schematic diagram of starch gel electrophoretic separation of embryonic and adult haemoglobins of chickens.
Fig. 2. Ontogenetic changes in haemoglobins of geese.
began to fade. Haemoglobin X was present in the samples of oxyhaemoglobin, cyanmethaemoglobin and carboxyhaemoglobin. All these samples were analysed about 18 hr after preparation and they were kept throughout at +5°C. The attempt at a partial isolation of haemoglobin X will be presented later. On the sixth day of incubation the zones of adult haemoglobin A and D began to appear in chickens, gradually increasing in intensity, while the zones of embryonic haemoglobins P and E weakened. On the ninth day a weak zone of haemoglobin H was detected which gradually became stronger. On the 15th day of incubation only zones A, D and H could be detected. We did not follow the postincubation changes in chicken haemoglobins, but according to literature data haemoglobin H disappears at about the 90th day of age (Washburn, 1968). Similar electrophoretic bands of haemoglobins were also observed in turkeys. However, the embryonic zone P did not seem doubled. On the 8th day of incubation the adult zones A and D began to appear, on the tenth day a weak zone H also appeared. After 17 days the zones P and E were no longer detectable. Zone H is distinct for about 80 days after hatching, after that it weakens and on the 100th day it is no longer detectable. In early embryos zone X is also present. Analogous patterns were observed in geese and ducks. In the case of geese (Fig. 2) the embryonic haemoglobin P was also doubled and the more anodically localized zone had the same mobility as zone D in adult animals. Zone A is detectable on the 9th day of incubation. Zone H begins to appear on the 10th day. After the 18th day of incubation haemoglobins P and E are no longer detectable. In 5 day-old embryos haemoglobin X was also observed. Haemoglobin H is distinct up to about 110 days; however, its traces can be detected up to about the 140th day after hatching. In the case of ducks (Fig. 3) the embryonic haemoglobin P did not seem doubled. On the 8th day zones A and D began to appear, on the 9th day zone H was also present and on the 17th day zones P and E were no longer present. Five to 6 day-old embryos
Ontogeny of avian haemoglobins 5-day embryo
I I-doy embryo
D
..........
I-day duckling ~ B m
147
Adull"
t
i !
P
I
I
v........, r T / / / / / / / / / l
1888888888N v/////////~,
A
~
A
Odgln
Odgin
Abnormot
Normal
-
Fig. 4. Normal and abnormal types of haemoglobins of adult ducks. H
X !
v///////A
[(Ill/IlIA
!
Fig. 3. Ontogeny of duck haemoglobins.
also contained zone X. Haemoglobin H disappeared after 90 days of age. The distance between the zones E and H in geese and ducks was larger than in hens and turkeys. In all species investigated several additional weak haemoglobin fractions were observed, the nature of which is unknown.
Polymorphism of haemoglobins in ducks During an electrophoretic analysis of haemoglobins of adult ducks the presence of additional haemoglobin fractions with distinctly higher anodic mobility than would correspond to haemoglobins A and D (Fig. 4) was observed in 4 cases out of 150. The genetic control of this polymorphism is under investigation.
Fractionation of haemoglobins of 5 day-old chick embryos Since the component X occurs in the lysates of the erythrocytes of embryos at a very low concentration
only, we tried to carry out a partial fractionation of haemoglobin and the concentration of the fraction containing haemoglobin X. For this purpose we used chromatography on CM-Sephadex. In order to prevent too high a dilution of the haemoglobins during chromatography, which might lead to a loss of the trace component X, we used a relatively steep gradient. The result of the chromatography (Fig. 5) was analogous to the chromatography of haemoglobin of 5 day-old embryos described by Brown & Ingram (1974); nevertheless, some differences still existed. In Fraction I (Fig. 6) haemoglobin P, which is doubled, was evidently present. In Fraction II a zone was present the mobility of which was similar to that of the slow zone of haemoglobin P. In Fraction III a haemoglobin was present with a mobility similar to that of the fast moving zone of haemoglobin P and it probably represents haemoglobin M (cf. Brown & Ingram, 1974). In Fraction IV haemoglobin E was localized together with haemoglobin X. The latter is visible only when a relatively large sample volume was applied into the gel. In addition to these main haemoglobin zones other weak zones were also observed in all fractions (Fig. 6). It is difficult to decide whether they represent artifacts formed during separation or whether they are true haemoglobins.
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2~
Elu~on volume, ml
Fig. 5. Chromatography of 5 day-old chick embryonic haemoglobin on CM-Sephadex. The details are given in "Materials and Methods". Fractions I and II were concentrated on DEAE-Sephadex, Fractions III-V on CM-Sephadex.
148
A. STRATILAND M. VALENTA
-I-
P+ M Origin E X
_Z
E
TIT
I~T
(a)
-~r
5day adult embryo (b)
Fig. 6. Starch gel electrophoresis of five-day-old chick embryonic haemoglobin. (a) Haemoglobin fractions obtained by chromatography shown in Fig. 5. Fraction IV was overloaded in order to make the zone of haemoglobin X visible. (b) For comparison, starch gel electrophoretic separations of carboxyhaemoglobins of 5 day-old chick embryo and adult chicken, respectively, are shown. DISCUSSION
Our results concerning ontogenetic changes in the haemoglobins of chickens are in many respects similar to those of a series of other authors (e.g. Manwell et al., 1963, 1966; Fraser et al., 1972; Schalekamp et al., 1972; Shimizu, 1972a, b; Bruns & Ingram, 1973; Cirotto et al., 1975). The differences observed in the time of appearance and disappearance of single haemoglobin components may be attributed to the differences in the techniques employed, but mainly in the differing concentration of haemoglobin solutions used for electrophoresis. In this paper we studied the ontogeny of the main haemoglobin components A, D, P, E, H and a trace haemoglobin X, only. Similarly to other authors (e.g. Moss & Thompson, 1969; Fraser et al., 1972; Schalekamp et al., 1972; Bruns & Ingrain, 1973) we also observed further weak fractions, but these were not always distinct. It is possible that some of them were artifacts produced by the freezing of the samples. Up to now various authors differ in their opinions regarding the existence or the number of weak fractions. Some consider them as artifacts only, formed by the preparation procedure, or by their storage (Manwell et at., 1966; Bruns & Ingram, 1973) while others do not doubt their actual existence (e.g. Hashimoto & Wilt, 1966; Fraser et al., 1972; Schalekamp et al., 1972). In our case the existence of the trace haemoglobin X is beyond doubt. We also believe that the weak zones present in Fractions III and V after CMSephadex chromatography may represent true haemoglobins.
The main aim of this work was the comparison of the developmental changes of the chicken haemoglobins with those of turkeys, ducks and geese. Detailed knowledge acquired with chickens has not yet been sufficiently correlated with other avian species. Our results indicate quite clearly that analogous changes in the presence of haemoglobins take place in all four species investigated and that the differences can be observed only in the time of appearance and disappearance of individual haemoglobins. Although some data on the embryonic haemoglobins of turkeys and ducks have already been published, the techniques used did not permit the distinction of haemoglobins E and H (Manwell et al., 1963; Borgese & Bertles, 1965). The ontogeny of goose haemoglobins has not yet been studied. The data on the disappearance of zone H during the postincubation development of turkeys and geese are also original. In ducks zone H disappears later labout the 90th day) than published by Borgese & Bertles (19651 (about 70 days). Evidently, this difference is caused by differing concentration of the haemoglobin samples used for analyses. Brown & lngram (1974) state that chicken haemoglobins A and D contain identical chains /3 in addition to differing chains ~ and ~ , respectively. The fact that in the polymorphic haemoglobin of ducks bands with a higher mobility than that of haemoglobins A and D are present indicates that the possible formation of additional bands is given by the polymorphism in chain fl and that the type observed by us is a heterozygote /3//3'. The chain fl' has a higher electrophoretic mobility towards the anode.
Ontogeny of avian haemoglobins Acknowledgements--We thank Miss J. Janatkov~ for her skillful technical assistance.
REFERENCES
BORGESET. A. & BER~ES J. F. (1965) Hemoglobin heterogeneity: Embryonic hemoglobin in the duckling and its disappearance in the adult. Science, N.Y. 148, 509-511. BROWN J. L. & INGRAMV. M. (1974) Structural studies on chick embryonic hemoglobins. J. biol. Chem. 249, 3960-3972. BRtrNS G. A. P. & INGRAMV. M. (1973) The erythroid cells and haemoglobins of the chick embryo. Phil. Trans R. Soc. Lond. 266, 225-303. BUSH F. M. & TOWNSENDJ. I. (1971) Ontogeny of hemoglobin in the house sparrow. J. Embryol. exp. Morph. 25, 3345. CmOTTO C., SCOTTODI TELLAA. & GERAC1G. (1975) The hemoglobins of the developing chicken embryos. Fractionation and globin composition of the individual component of total erythrocytes and of a single erythrocyte type. Cell Differentiation 4, 87-99. FRASER R., HORTON B., DUPOURQUED. & CHERNOFEA. (1972) The multiple hemoglobins of the chick embryo. J. Cell Physiol. 80, 79-88. GAHNE B., RENDEL J. & VENGE O. (1960) Inheritance of fl-globulins in serum and milk from cattle. Nature, Lond. 186, 907 908. HASmMOTO K. & WILT F. H. (1966) The heterogeneity of chicken hemoglobin. Proc. hath. Acad. Sci. U.S.A. 56, 1477-1483.
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KEANE R. W., ABBOTTU. K., BROWN J. L. & INGRAM V. M. (1974) Ontogeny of hemoglobins: Evidence for hemoglobin M. Devl. Biol. 38, 229-236. LATHEM W. & WORLEYW. E. (1959) The distribution of extracorpuscular hemoglobin in circulating plasma. J. clin. Invest. 38, 474-483. MANWELLC., BAKERC. M. A. & BETZ T. W. (1966) Ontogeny of hemoglobin in the chicken. J. Embryol. exp. Morph. 16, 65-81. MANWELLC., BAKERC. M. A., ROSLANSKYJ. D. & FOGHT M. (1963) Molecular genetics of avian proteins--II. Control genes and structural genes for embryonic and adult hemoglobins. Proc. natn. Acad. Sci. U.S.A. 49, 496-503. Moss B. A. & THOMPSON E. O. P. (1969) Haemoglobins of the adult domestic fowl Gallus clomesticus. Aust. J. biol. Sci. 22, 1455-1471. SCHALEKAMP M., SCHALEKAMP i . , VAN GOOR D. & SLINGERLANDR. (1972) Re-evaluation of the presence of multiple haemoglobins during the ontogenesis of the chicken. Electrophoretic and chromatographic characterization, polypeptide composition and immunochemical properties. J. Embryol. exp. Morph. 28, 681-713. SHIMIZU K. (1972a) Ontogeny of chicken hemoglobin--I. Electrophoretic study of the heterogeneity of hemoglobin in development. Devl. Growth Differ. 14, 43-55. SHIMIZU K. (1972b) Ontogeny of chicken hemoglobin--II. Chromatographic study of the heterogeneity of hemoglobin in development. Devl. Growth Differ. 14, 281-295. WASHBURNK. W. (1968) Effects of age of bird and hemoglobin type on the concentration of adult hemoglobin components of the domestic fowl. Poult. Sci. 47, 1083-1089.
