Re-evaluation of the presence of multiple haemoglobins during the ontogeny of the mouse

ByKOJI SHIMIZU 0 1 TOMOMASA 1 0 Authors' address: Institute for Developmental Research, Aichi Prefectural Colony , Kasugi, Aichi 480-03 , Japan 1 From the Department of Morphology and the Department of Biochemistry, Institute for Developmental Research, Aichi Prefectural Colony , Kasugai SUMMARY In mice, three alleles, Hbbs, HbbA and Hbbp, have been found at the /?-chain locus of haemoglobin. Analytical Polyacrylamide gel electrophoresis revealed the presence of almost the same four haemoglobin bands (E-T, E-IT, E-III and X) in all of the lysates of peripheral blood cells from 12-day-old foetuses of C57BL/6J (Hbb% A/He (Hbbd) and PON (Hbb"). All were embryonic-type haemoglobins; the most anodal band (X) seemed to be a product of polymerization of E-I. Electrophoretic patterns of the haemolysates from 12-day-old foetuses were not affected by alkylation with iodoacetamide or maleic anhydride. Almost the same electrophoretic patterns in subunit analysis, using acid starch and acid-urea Polyacrylamide gels, were obtained in 12-day-old foetuses of C57BL/6J, A/He and PON. E-I comprised aand x-chains, E-Il, a- and y-chains, and E-Ill, a- and z-chains. In adults, two haemoglobin bands, s and Y, were present in the haemolysate of C57BL/6J, while three, dmaj, dmin and Y, existed in A/He, and three, pmaj, pmin and Y, in PON. All were adult-type haemoglobins. Haemoglobin Y, which was not demonstrable after alkylation of the haemolysates, seemed to be a product of polymerization of major haemoglobins (s, dmaj and pmaj). Each haemoglobin possessed common a-chains, while the /?-chains of major haemoglobins were different from those of minor haemoglobins. Developmental changes of the haemolysates from foetuses of C57BL/6J, A/He and PON were similar to each other. Haemolysates of the liver cells of developing foetuses contained only adult-type haemoglobins. Our techniques used here were not able to demonstrate the presence of foetal-type haemoglobins. - WATANABE1 INTRODUCTION Electrophoretic studies of mouse haemolysates have revealed the existence of several haemoglobin patterns, classified as 'single' or 'diffuse'. These patterns are controlled by two alleles at the Ebb locus, Hbbs and Hbb, which determine the structure of the /?-chain of mouse haemoglobin. A third allele at the Hbb locus, Hbb9, was first found by Morton (1962, 1966). This allele was also demonstrated in Asian feral mice by Gilman (1974), and in two Japanese strains of mice, PON and Mol-A, by Watanabe, Shimizu, Goto & Ogasawara (1976). No other alleles have been reported (Hutton, 1969). As most vertebrates, mice have both embryonic and adult haemoglobins during the course of develop ment. The nomenclature of embryonic haemoglobins in mice is confusing (Schalekamp, Harrison & Paul, 1975), but three have been demonstrated in foetal haemolysates (Fantoni, Bank & Marks, 1967; Gilman & Smithies, 1968: Melderis, Steinheidev & Ostertag, 1974). Recently, Schalekamp et al. (1975) have reported foetal haemoglobins also in Porton white Swiss mice. The socalled 'single' adult haemoglobin has been split by Barker (1968) into two components through analytical Polyacrylamide gel electrophoresis. In this paper, we re-evaluate some features of multiple haemoglobins during the ontogeny of C57BL/6J (Hbbs), A/He (Hbb*) and PON (Hbb*) mice, using Polyacrylamide and starch gel electrophoresis, chromatography on DEAE-Sephadex column, and immunology. MATERIALS AND METHODS Animals. Mice of three strains, C57BL/6J (C57), A/He (A), and PON, were used. C57 and A were imported from the United States. PON is an inbred strain derived from Fx mice of C57 x Pink, which is a Japanese fancy mouse stock maintained in Laboratory of Animal Genetics, Nagoya University. Preparation of haemolysates. Adult mice were bled by decapitation. Foetuses from 12 to 17 days of gestation (the day on which vaginal plugs were observed being taken as day zero) were washed thoroughly with buffered saline (pH 7-2), and bled from the umbilical vessels for peripheral blood cells. Livers of foetuses were excised, washed thoroughly, and then disaggregated by pipetting. Citrated ice-cold buffered saline (pH 7-2) was used for washing and cell collection as follows. Blood cell suspensions were washed three times in at least a tenfold volume of isotonic ice-cold buffered saline (pH 7-2). Following the final wash ing, the cells were resuspended in a double volume of ice-cold deionized water, and left overnight at 4 C to haemolyse. The stroma was removed by centrifugation at 10000 g for 60 min, and clear supernatant was used as fresh haemolysate. Cyanmethemoglobin was obtained by mixing 1 vol. of haemoglobin solution (ca. 40 mg/ml) with 2 vol. of a solution containing 2 % K3Fe(CN6), 0-5 % KCN, and 0-1 % N a H C 0 3 (Moss & Ingram, 1968). Alkylation of mouse haemoglobin. Alkylation of mouse haemoglobin was carried out as described by Petras & Martin (1969). Fresh haemolysate {ca. 40 mg/ml) was mixed with equal volumes of iodoacetamide (6-84 mg/ml) and maleic anhydride (16-5 mg/ml, neutralized with NaOH). Analytical Polyacrylamide gel electrophoresis. Analytical Polyacrylamide gel electrophoresis in alkaline gels was carried out by the procedure of Barker (1968), but without KCN in the reservoir buffer (Tris-glycine buffer, pH 9-3). Elution of each haemoglobin from alkaline gels was performed (Bruns & Ingram, 1973). Analytical Polyacrylamide gel electrophoresis in acid-urea gels was carried out (Panyim & Chalkley, 1969). haemoglobins in mouse ontogeny Starch gel electrophoresis. Starch gel electrophoresis in alkaline gels was carried out by the method of Gilman & Smithies (1968). To separate each haemoglobin component into subunits (polypeptide chains), starch gel electro phoresis in 1-4 M formate buffer (ju, = 0-05 in gels, JU, = 0-2 in bridges) at pH 1-9 was performed (Muller, 1960). DEAE-Sephadex column chromatography. Column chromatography was carried out on diethylaminoethyl (DEAE)-Sephadex (Pharmacia A-50) column (2-0x21 cm), using 0 0 8 M trihydroxymethylaminomethane (Tris)-HCl buffer in a linear gradient from pH 8-6 to 7-4. About 100 mg haemoglobin from fresh haemolysates or cyanmethemoglobin forms in starting buffer was placed on the column, and elution was accomplished at 20 C. The optical density (o.D.) of the effluent fractions was measured at 280 nm, using LKB Uvicord III 2089 UV-absorptio-meter. Immunology. Male rabbits were immunized by five subcutaneous injections of mouse haemoglobin at weekly intervals (1 ml of about 50 mg/ml haemoglobin solution in buffered sahne, pH 7-2) mixed with equal volume of Freund's com plete adjuvant (Difco). Complement inhibition of antisera was performed at 56 C for 30 min. The maximum dilution of antisera which gave positive reactions in ring precipitin tests was about 64. Antiserum against the major (minor) haemoglobin component of adult A mice was obtained by absorbing antiserum to haemoglobin of adult A mice with the min (...truncated)