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 17·4μm in embryos to 19·9μm 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: Rückert & 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 erythropoiesis 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, 1971) from 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.

Fresh-water turtles of the species Emys orbicularis L. were collected from the ponds of the Brenne region, near Châteauroux (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 μm thick sections and stained by the May-Grünwald technique. Young embryos have been stained by dimethoxybenzidine embedded in paraffin, cut into 7·5 μm-thick sections.

Blood was collected from the embryos by rupturing one of the extraembryonic vessels, and from young or adult animals by cutting off the tip of the tail. Smears and electrophoresis were always performed from the blood of individual animals. Smears were stained according to the May-Grünwald-Giemsa technique. For electrophoresis the erythrocytes were collected and washed in isotonic buffer then lysed in about five volumes of lysis buffer (Bruns & 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 cyanomethemoglobins. The gels were stained with diaminobenzidine or Coo-massie blue.

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 17- and 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.

Fig. 1

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. 1

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. 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. 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.

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 the yolk sac is the main erythropoietic organ for most of embryonic development. Erythropoiesis occurs in the lumen of blood islands, and granulopoiesis is extravascular. Some erythroblasts are released in the blood where they pursue their maturation processes. The benzidine technique has been used to establish the identity of erythroid cells in presumed erythropoietic sites. Benzidine positive cells have been observed in the blood islands of yolk sac (Fig. 3) but not in the embryonic area.

Fig. 3

Demonstration of haemoglobin in erythroid cells following benzidine staining: b+: benzidine-positive cells; b: benzidine-negative cells.

Fig. 3

Demonstration of haemoglobin in erythroid cells following benzidine staining: b+: benzidine-positive cells; b: benzidine-negative cells.

After hatching, bone marrow was found in the leg bones. Granulopoiesis and erythropoiesis were observed there, and the granulopoietic layer of liver remained functional in the young turtle (Fig. 4).

Fig. 4

Liver of a young turtle aged two years: granulocytes (arrow) accumulated around a vessel.

Fig. 4

Liver of a young turtle aged two years: granulocytes (arrow) accumulated around a vessel.

II Morphological evolution of circulating erythrocytes

Turtle erythrocytes are nucleated cells, variable in size. Some appear very different from the mean population being either small or large (Fig. 58). During early embryonic stages red cells are slightly more rounded than in the young and the adult where they usually are elongated with an elliptical shape (Fig. 68). The mean length is slightly larger in the adult (Fig. 5). Chromatin is arranged in a network containing small scattered masses of denser material. The homogeneously acidophilic cytoplasm is typical of haemoglobin-rich cells.

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 (μm) measured photomicrographically on fixed smears. Ordinate,% number of erythrocytes in each class.

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 (μm) measured photomicrographically on fixed smears. Ordinate,% number of erythrocytes in each class.

Fig. 6

Fig. 68. Erythrocytes of turtle at various stages (smears). Stage-19 embryo (38 days of incubation at 25°C).

Fig. 6

Fig. 68. Erythrocytes of turtle at various stages (smears). Stage-19 embryo (38 days of incubation at 25°C).

Immature cells, i.e. basophilic and polychromatophilic erythroblasts, are frequent in the blood of the embryo. These cells are round or oval with diverse sizes. Their chromatin network displays larger masses of dense material than in erythrocytes. Mitoses are frequent, especially in the embryo (Fig. 6).

Granulocytes first seen around stage 14 (21 days of incubation), are very frequent after stage 25 (Fig. 78).

Fig. 7

Stage-25 embryo (66 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. 8

Adult turtle. G, eosinophil granulocyte; M, mast cell (with basophilic granules).

One striking feature of turtle blood is erythrocyte size variations at all stages. Despite the fact that the adult blood smear differs in general appearance from the embryo’s, it is not possible to identify specific forms typical of embryonic or adult stages. This progressive evolution is probably due to the co-existence of maturation stages rather than to the sequential inflow of cells belonging to different series.

III Ontogeny of haemoglobins (Fig. 9)

The haemoglobins in the blood of four adult animals have been analysed by polyacrylamide gel electrophoresis. In three animals (Ad1 in Figure 9), two major components Ax and A3 make up respectively 50 to 40% of the total haemoglobin content of erythrocytes, while a minor component A2 accounts for 10%. These three components are also found in the haemolysate of a fourth adult animal (Ad2), but the proportions of the components are rather different in this individual, the A2 band representing 30% of the total haemoglobin.

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); J1J2, Young turtles respectively aged one and two years; Adi-Ada, Adult individuals.

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); J1J2, Young turtles respectively aged one and two years; Adi-Ada, Adult individuals.

The erythrocytes of 41 embryos analysed between stage 11 and stage 20 (42 days of incubation at 25°C) yielded identical haemoglobin patterns, different from the adult one. Two major embryonic haemoglobins Ex and E3 have electrophoretic mobilities distinct from that of adult. The Ex band, very wide, which accounts for 75–80% of the material is probably composed of several molecular forms which have very similar electrophoretic mobilities in the experimental conditions used. A third band, E2, less important, moves to the same position as A2.

At stage 25 (66 days of incubation at 25°C), that is shortly before hatching, the adult bands A2 and A3 appear clearly.

During a very long period, adult and embryonic haemoglobins coexist in young turtle blood: one year after hatching, the haemolysates yielded about as much embryonic as adult haemoglobins (JJ. In a 2-year-old individual, the Ex band had disappeared (J2). In this same animal, A1, A2 and A3 were present in approximately equal proportions, thus the haemolysate was very similar to that of the fourth adult (Ad2) described above. In some adult or embryonic haemo-lysates, faint supernumerary bands have been found (e1, e2, a15 a2). In view of the variations also observed in major adult bands, it is likely that these components correspond to individual variations rather than to artifacts.

The data reported here show that erythropoiesis in Emys orbicularis evolves rather differently from that of other classes of vertebrates. The first point is the importance of the yolk-sac blood islands which seems to be the unique progenitor of erythrocytes during most of embryonic life. We have not been able to detect with certainty an intraembryonic organ supplementing the yolk sac in erythropoiesis during embryonic life. The liver has a cortical haemopoietic layer which appears involved mainly in granulopoiesis. This function goes on after hatching and still exists in the 2-year-old animal at a time when haemopoiesis is found in bone marrow. It is difficult to observe bone marrow immediately after hatching. This should be related to observations of Salvatorelli, Gulinati & Anzanel (1973), who could not find bone marrow in young turtles (Emys orbicularis and Testudo graeca).

The second point is the absence of a morphologically defined series of primitive erythrocytes. The highly variable size of red cells described previously in adult Emys (Saint-Girons & Duguy, 1964, Saint-Girons & Saint-Girons, 1969; Saint-Girons, 1970; Duguy, 1967, 1970; Frair, 1977a) and in other species as well (Jordan & Flippin, 1913; Frair, 1977b) is also observed during embryonic life, but at no stage does a well defined new recognizable cell line appear in the peripheral blood. The evolution of embryonic toward adult blood suggests either a progressive maturation of the cells or a very slow replacement of the first line by the next ones rather than an abrupt shift in the erythroid population.

The third point is the finding of adult haemoglobins (A1 and A3) at stage 25, two weeks before hatching, that is, independently of that event. The change from embryonic to adult haemoglobin is not correlated with either the replacement of erythrocytes in peripheral blood, or with the appearance of other erythropoietic site, as observed in other classes of vertebrates. The electro-phoretic analysis of adult Emys erythrocyte haemolysates reveals the presence of three haemoglobins, the proportions of which seem to vary from one individual to the next. The existence of two major and two minor components has been described in several other turtle species, in particular among the Emydiid family (Dozy, Reynolds, Still & Huisman, 1964; Sullivan & Riggs, 1967; Dessauer, 1970). During embryonic development of Emys orbicularis we find at least two components differing from the adult haemoglobins. In all individuals examined, whether they are embryos, young turtles or adults, one band is found in position E2 A2. Further biochemical studies are necessary to find out whether this component found at very different times of life is the same molecule or not. For comparison, it should be recalled that in the chick aA and aD globin chains are synthesized throughout life. In man, a foetal haemoglobin, and in Xenopus a tadpole haemoglobin persist throughout adult life but these haemoglobins are present only in very small amounts in adults (Jurd & Maclean. 1970).

The fourth point is the slow disappearance of embryonic haemoglobins observed in Emys in agreement with scattered data from the literature. In Malaclemys centrata, McCutcheon (1947), studying the blood oxygen affinity during embryonic, postnatal and adult life, postulated the existence of an embryonic haemoglobin which would be completely replaced by adult ones only two years after hatching. In the sea turtles Caretta caretta and Chelonia mydas, Isaacks et al. (1978) found that adult-type haemoglobin appears late, indeed after hatching; however the replacement is completed in a few weeks. On the other hand, Pough’s data (1977) based on electrophoretic analysis of haemoglobins in the snake Thamnophis may reflect the persistence of embryonic haemoglobins long after hatching. In other classes of vertebrates a comparable phenomenon has been described only in the toad Bufo bufo (Salvatorelli & Turpin, 1971). In any case, two sets of haemoglobins, embryonic and adult, are simultaneously present in the peripheral blood during a relatively long period of the development of Emys. It remains to determine if each set is confined to specialized lines or if all the haemoglobins may be synthesized by the same erythrocyte. In the first case either embryonic erythrocytes are long lived (the life span of adult erythrocytes of Terrapene Carolina has been estimated by Altland and Brace (1962) to be 600-800 days) or erythrocytes of embryonic type are produced after hatching. The presence in circulation of erythrocytes possessing both embryonic and adult haemoglobins has been recently demonstrated in the chick embryo (Chapman & Tobin, 1979) and in the mouse embryo (Brotherton, Chui, Gauldie & Patterson, 1979).

This work was supported by CNRS and by Grant no. 79–7–1224 from DGRST. The authors wish to express their grateful thanks to Dr F. Dieterlen for valuable comment and criticism during the course of this work.

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