Ontogeny of hemoglobin in the house sparrow

By FRANCIS 2 M. BUSH 2 J. 1 2 YES TOWNSEND 0 2 0 Author's address: Department of Genetics, Health Sciences Division, Virginia Common- wealth University , Richmond, Virginia, 23219 , U.S.A 1 Author's address: Department of Anatomy, Health Sciences Division, Virginia Common- wealth University , Richmond, Virginia, 23219 , U.S.A 2 From the Department of Anatomy and Department of Genetics A 'switchover' from synthesis of predominantly embryonic hemoglobin to synthesis of predominantly adult hemoglobin occurs during differentiation of the house sparrow. This 'switchover' is demonstrated by a change in electrophoretic pattern generally comparable with that of the chicken, slightly different from those of the duck and man, and, perhaps, dissimilar to that of the red-wing blackbird. Differences in electrophoretic mobility show that 10-day embryos possess one hemoglobin not present in early hatchlings and two hemoglobins not present in adults. Major adult hemoglobin is synthesized by the tenth embryonic day; and minor adult hemoglobin, between the tenth embryonic day and the seventh day after hatching. The major increase in concentration of hemoglobin occurs between 2 and 11 days after hatching, a time when the two predominant embryonic and two predominant adult hemoglobins are being synthesized. The percentage of packed cells (hematocrit) also increases significantly within this period. Similarities of peptide maps for the house sparrow and other species, such as the owl, indicate the presence of a-and/?-polypeptide chains in adult house sparrow hemoglobin. The shift to hemoglobins with faster anodic mobilities and the presence of a higher concentration of hemoglobin in adults than in early hatchlings suggest that this synthesis is predominantly in the /?-chains. - Changes in hemoglobin patterns during morphogenesis are documented for some species of birds. Early embryonic hemoglobins, distinct from hemoglobins of late embryos (fetuses), hatchlings, and adults, exist in a number of precocial species, such as turkey, Meleagris gallopavo, and chuckar partridge, Alectoris graeca (reviewed by Man well, Baker & Betz, 1966), chicken, Gallus gallus Anas domesticus (Borgese & Bertles, 1965). Little is known of the ontogeny of hemoglobins in small altricial species; the only published study is limited to an analysis of embryonic stages in the red-wing blackbird, Agelaiusphoeniceus. In this species, no time-specific alteration occurs during embryonic stages, for the hemoglobin patterns of 2-day and 14-day embryos are identical (Manwell, Time-specific changes vary for different precocial species. The appearance of some new adult hemoglobins occurs by the sixth day in chicken (Hashimoto & Wilt, 1966; Manwell et al. 1966; Godet, 1967), by the sixth, seventh and eighth days, in chuckar partridge, bantam chicken and turkey, respectively (Manwell et al. 1966), and by the eighth day in Pekin duck (Borgese & Bertles, 1965). The ' switchover' from embryonic to hatchling or to adult hemoglobins is not entirely perfect because synthesis of the new component can occur before synthesis of the embryonic component ceases. Ontogenetic changes show that some hemoglobins formed early are transient and others are newly synthesized during either fetal or hatchling stages, or during both. Starch gel patterns reveal that an acidic component and a basic component of chick embryos appear by the seventh and tenth days, respectively, while another component, a major acidic hemoglobin, disappears by the ninth embryonic day (Godet, 1967). The hemoglobin appearing by the seventh day persists in the adult, and that appearing on the tenth day disappears during the first month after hatching; on the other hand two other acidic components develop by the fourth day after hatching, and both persist in the adult. The 7-day embryonic Pekin duck forms one acidic component that disappears by the tenth week after hatching (Borgese & Bertles, 1965). The observed differences between precocial and altricial species and the paucity of information about small species of feral birds prompted the present study, one of the continuing series (Bush, 1967; Bush & Seibert, 1968; Bush, Price & Townsend, 1970) to discover the biochemical properties of blood of the altricial species, house sparrow, Passer domesticus. This paper reports, correlates, and discusses in relation to what is known of other species, the quantity, composition and development of major and minor hemoglobins in embryo, hatchling and adult; the concentration of hemoglobin and measurement of hematocrit in hatchling and adult; and the mapping of peptides in the adult. MATERIALS AND METHODS The sample. The house sparrows, Passer domesticus, in our study were collected from the population at Richmond, Virginia. In all, 53 adult females and 69 adult males (older than 1 year), 127 hatchlings and juveniles (less than 50 days old), 60 first-year adults (50-124 days old), and 10 embryos (10-12 days old) were studied. Specimens were assigned relative ages based on criteria described elsewhere (Farrar & Bush, 1969). Collection of blood and determination of hematocrit. Blood was obtained by capillary pipette after embryonic vitelline vessels were cut, and by syringe from the hearts of hatchlings and adults. The samples were heparinized. Duplicate samples of hatchling and adult blood were centrifuged at 14000g for 10 min in a microhematocrit centrifuge (Clay-Adams), and the mean hematocrit value determined. Quantitation of hemoglobin. The modified Wong technique (MacFate, 1964) was used to determine the iron content in hemoglobin. This technique involves detachment of iron from the hemoglobin molecule by treatment with sulfuric acid and potassium persulfate, precipitation of proteins with sodium tungstate, and treatment of the supernatant fluid with potassium thiocyanate. Light transmission at 540 m/t by the supernatant fluid was then measured in a Bausch and Lomb Spectronic ' 2 0 ' colorimeter at 25 C. Each per cent transmittance value was compared with per cent transmittance values obtained from a calibration curve prepared by serially diluting a stock solution of ferrous iron (0-1 mg iron/ml). For each blood sample, the concentration was: g hemoglobin = Preparation of hemoglobin. After centrifugation of blood samples at 2000^ for 15 min, plasmas were drawn off. The cells remaining in the centrifuge tubes were washed three times with 1 % saline solution and then exposed to carbon monoxide to form the CO-hemoglobin derivative. Cells were hemolyzed in four volumes of distilled water and one volume of toluene, and then rotated for 2 h at 4 C. This mixture was centrifuged at lOOOOg for 1 h, and the clear red hemoglobin layer was filtered to remove any traces of cell debris. Starch gel electrophoresis. Samples were subjected to vertical starch gel electrophoresis (Smithies, 1959). Each gel slot contained approximately the same amount of hemoglobin solution. Gels were prepar (...truncated)