Erythropoiesis and haemoglobin ontogeny in the turtle Emys orbicularis L.

SUMMARY The erythropoietic sites and developmental patterns of haemoglobins have been investigated during ontogeny of Emys orbicularis. The yolk-sac blood islands seem to be the unique erythropoietic site during most of embryonic life. Bone marrow haemopoiesis is first observed in young turtles aged one year. The cortical haemopoietic layer of the liver appears involved mainly in granulopoiesis. There is no morphologically well-defined series of primitive or definitive erythrocytes. Rather there is a gradual shift in size from a mean length of 17-4 /tm in embryos to 19-9 /im in the adult. However the size of erythrocytes is highly variable at all stages. Three haemoglobins of adult type and three haemoglobins of embryonic type have been identified by electrophoretic separation. It seems that one haemoglobin is synthesized during the whole life. Embryonic haemoglobins persist for more than a year after hatching while the typically adult haemoglobins appear shortly before hatching. - Little is known about the ontogeny of erythropoiesis in reptiles. Morphological data were collected some years ago: Riickert & Mollier (1906) in Lacerta, Dantschakoff (1916) in Tropidonotus have described the differentiation of blood islands in the yolk sac, at the time when the first somites are laid down. Thereafter erythropoiesis remains active in the yolk sac during most of embryonic development. In the turtle Chelydra serpentina, bone-marrow sets in shortly before hatching (Jordan & Flippin, 1913). In Lacerta muralis, diffuse haematopoiesis has been found in the embryonic mesenchyme and in the bone marrow long before hatching (Schmekel, 1962). On the other hand, haemoglobin changes during development in that class of vertebrates have hardly been studied. The existence of embryonic haemoglobins, distinct from adult ones, has been inferred in Malaclemys centrata (McCutcheon, 1947) and in the garter snake Thamnophis sirtalis (Manwell, 1960; Pough, 1969, determinations of oxygen affinity. Separation of haemoglobin components by electrophoresis has been carried out in the garter snake (Pough, 1971, 1977), in the loggerhead Caretta caretta and the green sea turtle Chelonia mydas (Isaacks, Harkness & Witham, 1978). Pough has observed a continuous change in the electrophoretically separable haemoglobin components of garter snake blood with increasing body size; at birth, most of the haemoglobin moved as one slow-migrating band; faster migrating fractions appeared progressively in larger snakes. In the two species of sea turtles studied, Isaacks et al. (1978) have demonstrated a shift from embryonic to adult haemoglobins during development. No previous attempts have been made with reptiles to relate development of red-cell series, sites of erythropoiesis and sequential synthesis of different haemoglobins, as has been done in amphibians or higher vertebrates. MATERIAL AND METHODS Fresh-water turtles of the species Emys orbicularis L. were collected from the ponds of the Brenne region, near Chateauroux (France) and eggs incubated as described previously (Vasse, 1973, 1974). Twenty-seven stages were distinguished during embryonic development. In the present study, the staging was based on age of the embryo (number of days of incubation at 25 C). Blood was studied in the embryos starting from the 33-somite stage (stage 11 obtained after 12 days of incubation at 25 C) to hatching (75-80 days of incubation), in young turtles (seven individuals aged 8 days to 2 years) and in four adult individuals. Young turtles and adults were raised at a temperature of 20 C approximately. Haemopoietic organs were fixed in Maximow's or Zenker's fluid, embedded in paraffin, cut into 7-5 /im thick sections and stained by the May-Griinwald technique. Young embryos have been stained by dimethoxybenzidine embedded in paraffin, cut into 7-5 ^in-thick sections. Blood was collected from the embryos by rupturing one of the extraembryonic vessels, and from young or adult animals by cutting oif the tip of the tail. Smears and electrophoresis were always performed from the blood of individual animals. Smears were stained according to the May-Grunwald-Giemsa technique. For electrophoresis the erythrocytes were collected and washed in isotonic buffer then lysed in about five volumes of lysis buffer (Brans & Ingram, 1973). Analytical polyacrylamide gel electrophoresis was performed according to the method of Ornstein and Davis as modified by Moss and Ingram at pH 10-3 (Moss & Ingram, 1968). All haemoglobins extracts were electrophoresed as Fig. 1, 2. Yolk-sac blood islands of a 14-somite embryo turtle. Fig. 1. Thickening of the splanchnopleure with basophilic cells (arrows). Fig. 2. Free cells in a newly formed extraembryonic vessel. Fig. 3. Demonstration of haemoglobin in erythroid cells following benzidine staining: b+: benzidine-positive cells; b~: benzidine-negative cells. Fig. 4. Liver of a young turtle aged two years: granulocytes (arrow) accumulated around a vessel. Fig. 5. Cell population diagrams showing the change in length of the major axis of turtle erythrocytes , stage-18 embryo (35 days of incubation at 25 C); adult; Abscissa, lengths (/*m) measured photomicrographically on fixed smears. Ordinate, % number of erythrocytes in each class. I. Development of erythropoietic organs At the 14-somite stage (stage 8: 6 days of incubation at 25 C) which was the earliest stage studied, blood islands were present in the splanchnic layer of the extraembryonic mesoderm, in contact with the endodermal layer (Fig. 1). The cells in these thickenings had a basophilic cytoplasm. Some were dividing. In the blood islands, spaces appeared between the cells. Some of these were free and peripheral cells formed a limiting endothelium (Fig. 2). Between the 17and 25-somite stages (stages 9 and 10: respectively 7 and 9 days of incubation) extraembryonic vessels connected the yolk sac with the embryo. The cells, lying free in the vessels, were more elongated than at the primitive stage. Differentiation stages towards mature erythrocytes were found. In embryos at stages 11-20 (12-42 days of incubation) in which kidney, spleen and liver were formed, no haemopoietic activity was observed in these organs. At stages 20-25, haemopoiesis appeared in the superficial cortex of the liver. This haemopoietic layer was granulopoietic rather than erythropoietic. Thus Fig. 6-8. Erythrocytes of turtle at various stages (smears). Fig. 6. Stage-19 embryo (38 days of incubation at 25 C). Fig. 7. Stage-25 embryo (66 days of incubation at 25 C). Fig. 8. Adult turtle. G, eosinophil granulocyte; M, mast cell (with basophilic granules). Fig. 9. Electrophoretic separation of haemoglobins fractions of turtle blood at various stages: St. 11, Stage-11 embryo (12 days of incubation at 25 C); St. 18, Stage-18 embryo (35 days of incubation at 25 C); St. 25, Stage-25 embryo (66 days of incubation at 25 C); Ji-J2, Young turtles respectively aged one and two years (...truncated)