ABSTRACT
Differentiation of plasma proteins in avian embryology has been analysed by electrophoresis (Brandt, Clegg & Andrews, 1951; Schechtman, 1952; Heim & Schechtman, 1954; Weller & Schechtman, 1962). Information has been obtained on affinities between plasma and egg proteins by electrophoretic studies (Wise, Ketterer & Hansen, 1964) and by immunochemical studies (Kaminski & Durieux, 1956; Williams, 1962a; Stratil, 1967; Zaccheo & Grossi, 1967).
There is a certain orderliness in the appearance of proteins. Embryo specific proteins, distinct from adult proteins, occur in the developing chicken. These are considered unique on the basis of electrophoretic mobility and by virtue of their transitory nature. Examples include some alpha globulins and gamma globulin found initially by 5-12 days (Kaminski & Durieux, 1956; Weller & Schechtman, 1962), and pre-albumin, present by 8-11 days of incubation (Heim & Schecht-man, 1954; Kaminski &. Durieux, 1956; Weller & Schechtman, 1962; Wise et al. 1964). Furthermore, the precise time of’switchover’ from embryonic to adult forms is well established for some classes of proteins.
Adult proteins can also be synthesized by embryos; however, some others form after hatching. Vitelloid (yolk-like) albumin exists by the 3rd (Nace, 1953) or 4th (Zaccheo & Grossi, 1967) day of incubation, whereas liver synthesis of albumin occurs by the 7th embryonic day (Zaccheo & Grossi, 1967). Proteins having the same immunochemical character throughout the remainder of morphogenesis are β2 globulins and gamma globulin, present between 11 and 20 embryonic days (Heim & Schechtman, 1954; Kaminski & Durieux, 1956) and pre-albumin, alpha and beta1 globulins, present between 18 days embryonic and 2 days post-hatching (Kaminski & Durieux, 1956; Weller & Schechtman, 1962). Whether the β2 globulin, transferrin, exists earlier than 11 day of incubation (Wise et al. 1964) is unknown, but conalbumin, which has an identical protein moiety (Williams, 1962b), can be detected by 8 days of incubation (Kaminski & Durieux, 1956).
Details of the embryogeny of some altricial species, such as the house sparrow, Passer domesticus, are fairly well known (Romanoff, 1960), but little is known of changes in its plasma proteins during ontogeny. Recent studies have shown that the plasma of newly hatched young has half the proteins found in adult plasma (Bush, 1965), and non-specific esterase, cholinesterase and lipoprotein increase and amine oxidase decreases with maturation (Bush, 1967). This present paper reports the results of continued study on hatchling and adult plasma proteins, including reactions of their antisera with both embryonic plasma and egg proteins. Embryo and egg proteins referred to herein are not specifically confined to these stages, but instead are necessarily immunologically identical with the hatchling or adult proteins used to evoke the immune sera. This paper also discusses the degree of antigenic correspondence between house sparrow plasma proteins and those of chicken and human.
METHODS AND MATERIALS
Collection and preparation of samples
Heparinized blood was obtained by capillary pipette after cutting embryonic vitelline vessels; by syringe, from hatchling and adult sparrow hearts and from chicken axial and human median cubital veins. After centrifugation at 2000 g for 15 min, each plasma sample was used immediately or stored at -20°C without preservative. It was thawed only when used. Individual samples studied were from 50 females, 50 males, 91 hatchlings, 10 embryos and 5 eggs collected from the same population at Richmond, Virginia.
Fresh egg white was pipetted away from egg yolk. Yolk was washed three times with 0·9% saline without rupturing the vitelline membrane and then collected by pipette after rupture. Egg white was used undiluted and yolk was diluted with an equal volume of saline.
Embryonic stages were determined by comparison with chick developmental stages (Hamilton, 1952); age of hatchlings was assigned by weights and lengths and of older birds by the degree of skull ossification as used previously (Bush, 1965). ‘‘
Quantitation of plasma protein
Protein concentration was determined in duplicate by the biuret method (Colowick & Kaplan, 1957) using bovine serum albumin (Sigma) as a standard. Values were read at 550 mμ with a Bausch and Lomb Spectronic 20 colorimeter.
Production of antisera
Antisera against sparrow plasma were produced in mature rabbits of either sex. Two kinds of antisera were produced. They had to be prepared with pooled plasmas to elicit a satisfactory antibody response. One was produced against pooled adult plasmas (anti-A serum) and the other, against pooled hatchling plasmas (anti-H serum). Three rabbits were immunized with the adult plasmas and one rabbit with the hatchling plasmas.
Twelve ml of plasma from 20 adults (11 females: 9 males) were used to make each anti-A serum. A total of 50 hatchling plasmas, mean age, 18 days (range: 6-25 days) was used to make the anti-H serum. Antisera were prepared by modifying a procedure used previously (Proom, 1943). Pooled adult and pooled hatchling plasmas containing 40 mg protein each were mixed separately with 16 ml of distilled water and 18 ml of 10% potassium aluminum sulfate and the pH of the solution adjusted to 6·5 with 5 N sodium hydroxide. After centrifuga-tion at 4000 g for 30 min, the sediment was washed twice with saline and the final volume made to 20 ml in saline with addition of 0·01% merthiolate.
The schedule used for injection followed one described previously (Hirschfeld, 1960). Each rabbit was injected intramuscularly with 10 ml of antigen mixture; 14 days later the same quantity was injected and then 1 ml of fresh plasma was injected intraperitoneally on the 24th day. Blood was collected 10 days after the final injection and the antisera frozen after addition of 0·01% merthiolate.
Chicken and human antisera were purchased commercially (Lloyd Brothers: Cincinnati, Ohio).
Immunoelectrophoresis. Previous results showed that newly hatched sparrow chicks had so little plasma that the conventional starch gel technique yielded low resolution of total proteins. Such direct analysis proved even more difficult using embryonic plasma. Thus we employed the highly sensitive immunoelectro-phoretic analysis as described previously (Grabar & Williams, 1953), Micro-scopic slides coated with 1% agar (Nobel: Difco) in barbiturate buffer, 0·05 ionic strength, pH 8·6 were used. Samples were electrophoresed with the same buffer at 25°C for 4 h at 250 V using LKB equipment, type 3276. After electro-phoresis, antiserum was applied, slides were incubated at 4°C for 36 h, washed, stained with amidoblack, cleared and then photographed.
RESULTS
Typical immunoelectrophoretic resolution of adult plasma proteins is shown when reacted with anti-A serum (Text-fig. 1). Mobilities of precipitates are compared with specific migration rates of human plasma precipitates. Similarity in mobilities does not imply homologous proteins.
Schematic diagram of principal antigens detected in adult house sparrow plasma (above). Antibody: anti-adult sparrow serum. Schematic diagram of principal antigens detected in human plasma (below). Antibody: anti-human serum.
The mean number of precipitates is 15 for either sex (range: female, 12-17; male, 12-19). Neither sex exhibits any unique precipitate. Albumin, β2−oand gamma globulins exhibit a characteristic boat-shape (Text-fig. 2; Plate 1). β2-2 globulin migrates as tandem intersecting arcs. Double lines of precipitation occur for some patterns in the α1 and β2 regions indicating additional proteins. Their presence is not caused by denaturation. Plasmas heated to 100° C form smudged patterns; only albumin, α1−3, α2−1, and α2−3 globulins precipitate.
Schematic diagram of sparrow plasma and egg proteins reacted with anti-H (left) and anti-A sera (right).
The hatchling essentially attains its adult complement by 23 days. This is reflected by the gradual increase in number of preci pitates present after hatching. Pre-albumin, albumin, α1−1, β2−0 and probably α2−1 globulins exist by the time of hatching. Some minor precipitates can be regarded as provisional, since their precipitation varies depending upon the plasma sampled.
Most beta globulins usually react with anti-A serum by 3-5 days after hatch-ing, as shown by the frequency of precipitates in Table 1. Gamma globulin and pre-albumin precipitate weakly by 5 days and intensely by 11 and 14 days, respectively. Plasma obtained from hatchlings 7-14 days old exhibits the greatest variation. Albumin, β2−0 and gamma globulin assume their adult appearance by 11 days. In this hatching period, β2−0 and β2−2 globulins precipitate intensely; β2−0 globulin typically reacts in later post-hatching development. Probably the β2−2 globulin and the cathodal arc of the A-2 globulin are unique to anti-Aserum, which shows that they are the last proteins synthesized.
Female plasma forms 12 precipitates and male plasma, 13 precipitates (range: 11-14) when reacted with anti-H serum (Text-fig. 2; Plate 2). Some plasmas lack distinct α2−2 and β2−2 globulins. Hatchling plasmas form even fewer precipitates. Their number increases with advancing age. Gamma and β2−0 globulins are boat-shaped by 9 days. As shown in Table 1, α1−3 and β2−1 globulins precipitate intensely by 7-14 days as does β2−3, even until 23 days. α2−1 and β2−2 globulin stain intensely after 11 days, whereas most A globulins appear less intense.
Embryos of 8-12 days have plasma proteins that form distinct precipitates; however, their migration rates are comparable to those of adult plasma when electrophoresed at the same time. The principal difference is that embryonic arcs of precipitation are shorter. Albumin, α1−1 and β2−0 globulins react with both antisera. Pre-albumin is apparent at this time. Most α2 and β2 globulins are absent; gamma globulin is undetectable.
Both egg white and yolk precipitate the β2−0 globulin with both antisera. Albumin and β2−2 globulin of yolk stain distinctly with anti-H serum.
Sparrow and chicken plasmas cross-react with antisera produced against each species ‘plasma. Albumin, α2−1, β2−2 and probably α1−3 globulins are the common proteins. The mobility of sparrow albumin is slower than the mobility of chicken albumin. Sparrow and human plasmas fail to cross-react with antisera produced against their plasmas.
The protein content of sparrow plasma increases progressively after hatching (Text-fig. 3). The most rapid synthesis occurs from 1 to 10 days after hatching. Plasmas pooled from five embryos have slightly more protein than is found in 1-to 2-day hatchling plasma. Embryo plasma is yellow from the presence of yolk carotenoid. This pigment disappears a short time after hatching. Results of a /-test show no difference (P < 0·05) between the plasma protein content of mature individuals of either sex.
DISCUSSION
The results of our immunoelectrophoresis of house sparrow plasma proteins are generally in accordance with changes observed for the chicken. A greater variety of proteins is synthesized in the early hatching period of the house sparrow, a disparity explained by the shorter time of incubation. The mean length of incubation is 12 days for this species (Summers-Smith, 1963) and 21 days for the chicken. Because of the shorter time, a house sparrow embryo that has incubated for 5 days will have reached a developmental stage comparable to an 8-day chick embryo. Development of a 7-day hatchling would correspond to an 18-to 20-day chick embryo. Thus, sparrow embryo plasma still lacks some essential proteins just before hatching, which makes continual brooding of hatchlings obligatory. One may infer that precocity is advantageous because such essential proteins are synthesized by the time of hatching.
Relative mobilities of precipitates show that house sparrow embryos lack proteins specifically limited to stages examined in this study. The absence of pre-albumin between the 6th and 13th day after hatching may be interpreted as the end of an embryonic form followed by synthesis of an adult form having the same protein moiety. But such an interpretation must be accepted with reservation because of the degree of variability; found throughout the population (compare pre-albumin in male patterns, Plate 1). Some adult patterns have and some lack pre-albumin, its presence or absence appearing to depend on the individual bird as reported previously for the chicken (Williams, 1962a; Stratil, 1967) rather than being associated specifically-with hen plasma (Brandt et al. 1951; Heim & Schechtman, 1954; Kaminski & Durieux, 1956). But there are several proteins produced only after hatching; for example, most a2 and globulins, particularly α2−2 and the cathodal arc of β2−2 globulins found only with anti-A serum.
Immunoelectrophoretic patterns differ slightly from individual to individual. The extent of variability is unrelated to the technique employed. Non-immunized rabbit serum also fails to react with house sparrow plasma. The number of precipitates is within the range determined for the chicken (Kaminski & Durieux, 1956; Croisille, 1962; Williams, 1962a; Peetoom et al. 1963; Stratil, 1967) and also confirmed for this species in the present study. This variability is also consistent with the variability found using starch gel electro-phoresis (Bush, 1967). Therefore, these variable patterns reflect genetic differences in this random sample from the Richmond sparrow population.
The developmental pattern of proteins synthesized in the house sparrow and in the chicken is comparable with changes reported in plasma proteins for both the developing rat and the human. The processes of differentiation are funda-mentally similar for all of these species. Proteins formed by these birds are essentially the same general classes present shortly before or shortly after parturition in these mammal species. Pre-albumin, albumin, alpha globulins, a major β2 globulin (probably transferrin) and gamma globulin comprise the initial complement. Other remarkable similarities include the remainder of proteins necessary to complete the adult complement. As in the sparrow and in the chicken, some a2 and beta globulins form in rat plasma 3 days after birth (Kelleher & Villee, 1962) and in human plasma during the first to third trime-sters after birth (Hitzig, 1964).
Sparrow yolk has only four proteins found in adult plasma. This egg yolk appears to lack gamma globulin, which is prominent in chicken egg yolk. There is also less variety of alpha and beta globulins in sparrow egg yolk than has been reported for the chicken (Stratil, 1967). Although nothing is known of the iron binding properties of egg or plasma proteins in the house sparrow, the presence of globulin in egg white and yolk suggests that these may be a conalbumin and a transferrin, respectively, on the basis of their identical precipitation.
Lack of cross-reactivity between sparrow and human plasmas with their antisera shows that although mobilities may be similar for some proteins, their moieties are unique to each species. Cross-reactivity of sparrow and chicken plasmas with their antisera shows that the moieties of albumin and some alpha and β2−0 globulins are also immunologically different between the chicken and the human.
The protein content of adult plasma is slightly higher than that determined for young Passer (species ?), mean 2·2 g/100 ml plasma (Lustig & Ernst, 1937). This difference should not be considered significant because of the variation observed in chicken plasma protein content. While no systematic study has been made of the protein content during ontogeny of the chicken, evidence shows that embryos have the lowest and laying hens, the highest per ml plasma. Embryos of 11 days have 0·86 g/100 ml which increases to 1·08 and 2·40 by 14 and 17 days respectively (Kaminski & Durieux, 1956). Hatchlings between 2 and 58 days have from 3·36 to 3·64 g/100 ml (Brandt et al. 1951; Patterson, Youngnen, Weigle & Dixon, 1962). Greater viariation has been reported for adult plasma, from 1·96 (Kaminski & Durieux, 1956) to 6·11 g/100 ml (Brandt et al. 1951).
Therefore the difference between this altricial species and the chicken is the slower appearance of proteins during house sparrow embryogenesis. Whether there are any specifically embryonic proteins at earlier stages cannot be deter-mined from these present data, but the more rapid incubation period coupled with the relatively labile system after hatching suggests less likelihood of an earlier ‘switchover’. This pattern of differentiation coincides with a slower ‘switchover’ from embryonic to adult hemoglobins for this species (Bush & Farrar, 1967) than reported for the chicken (Manwell, Baker & Betz, 1966) and for the red-winged blackbird, Agelaius phoeniceus, whose two major adult hemoglobins are present from the beginning of ontogeny (Manwell, Baker, Roslansky & Foght, 1963). The quantitative increase in protein content for the house sparrow parallels the quantitative increase in total hemoglobin only to 23 days after hatching. There is a significant increase in hemoglobin between fledging and adult stages (Bush & Farrar, 1967).
Studies, such as these, of avian plasma proteins make it desirable to investi-gate further the significance of specifically embryonic plasma proteins, genetic controls restricting the initial and the final complements and why variability exists in time of appearance of some classes of proteins such as alpha globulins.
SUMMARY
Plasma and egg proteins of the developing house sparrow have been examined by immunoelectrophoresis.
Quantitative and qualitative changes occur in the plasma proteins during morphogenesis. Albumin, α1−1, β2−0 and β2−1 globulins characteristically occur after the 8th day of incubation. Pre-albumin is present in embryos, diminishes soon after hatching and increases by 14 days after hatching. Gamma globulin forms by the 5th day after hatching; most alpha globulins, by 9 days; and β1 globulins, by 14 days. β2 globulins complete the complement by 23 days.
Total protein increases over two-fold within the period between hatching and fledging.
Both egg white and yolk form the same β2−0 globulin when reacted with antisera against sparrow plasma. Yolk forms the albumin precipitate. Both egg white and yolk seem to lack gamma globulin.
The protein moieties of house sparrow and human plasmas are immuno-logically distinct. Albumin, α1−3, α2−1 and β2−2 globulins are not species-specific for either house sparrow or chicken plasmas.
RÉSUMÉ
Analyse immunoélectrophorétique des protéines de l’œuf et du plasma pendant le développement du moineau domestique, Passer domesticus
Ce travail est consacré à une étude immunoélectrophorétique des pro-téines du plasma et de l’œuf pendant le développement du moineau domestique.
Pendant le développement on observe des variations quantitatives et qualitatives dans les différentes protéines du plasma, L’albumine et les globulines α1−1, β2−0, et β2−1 sont détectables à partir du 8ème jour de l’incubation. La préalbumine est présente chez l’embryon, diminue peu après l’éclosion, et augmente de nouveau vers le 14ème jour après l’éclosion. La gamma-globuline est détectable au moins à partir du 5ème jour après l’éclosion. La plupart des alpha-globulines sont décelables aux environs du 9ème jour; les globulines & sont détectables à partir du I4ème jour et les globulines β2 font leur apparition vers le 23ème jour après l’éclosion.
La quantité de protéines totales augmente considérablement (elle est plus que doublée) pendant la période qui s’écoule entre l’éclosion et le moment de abandonnement du nid.
Le sérum anti-plasma de moineau révèle la présence de la même β2−0 globuline dans le blanc d’œuf et dans le jaune d’œuf. L’albumine est présente dans le jaune d’œuf alors que la gamma-globuline semble absente du jaune d’œuf et du blanc d’œuf.
Les protéines du plasma de moineau et du plasma humain sont immunologiquement distinctes. L’étude des plasmas de poulet et de moineau révèle que l’albumine et les globulines α1−3, α2−1 et β2−2 ne sont pas spécifiques d’espèce.
Acknowledgements
Our appreciation is expressed to Drs J. I. Townsend, Department of Biology and Genetics and J. D. Burke, Department of Anatomy, Medical College of Virginia, for criticisms on this paper and J. R. Price, Medical College of Virginia, for help with photographs and diagrams. We acknowledge the technical assistance of C. G. Shcppek, Medical College of Virginia and the assistance with the arrangement of photographs by J. Ahlquist, Department of Biology, Yale University. This work is supported by NIH Grant GM 13649-01.
REFERENCES
Plate 1
immunoelectrophoretic pattern obtained for sparrow egg and plasma proteins using anti-A serum during development and (he immunoelectrophoretic patterns for chicken (below) and sparrow plasmas using anti-chicken serum.
Tmmunoelectrophoretic pattern obtained for sparrow egg and plasma proteins using artti-H serum during development.