An electron microscopic study of hematopoiesis in the liver of the fetal rabbit

An Electron Microscopic Study of Hematopoiesis in the Liver of the Fetal Rabbit'

GEORGE D. SORENSON" D e p a r t m e n t of A n a t o m y , W a s h i n g t o n U n i v e r s i t y School of M e d i c i n e , S t . L o u i s , Mis sour i

In mammalian fetuses hematopoiesis occurs successively in three principal sites. These are (1 ) yolk sac ( 2 ) liver and (3) bone marrow. Hematopoiesis in the yolk sac has previously been studied by electron microscopy (Sorenson, '60). Blood cell formation begins in the liver at the time that hematopoietic activity in the yolk sac is already diminishing. Because of its size and the duration of its function as a hematopoietic organ the liver may be considered the most important source of fetal blood cells. When hematopoiesis is at its peak the liver contains many more blood cells than hepatic cells. The liver forms in the fetus by an extension of the epithelium from the primitive foregut into mesodermal tissue within the septum transversum. The epithelial cells infiltrate the mass of mesenchyme and become enmeshed and surrounded by mesodermal cells. The development of blood cells from the mesodermal tissue surrounding the hepatic epithelium has been studied by light microscopy and described in detail by Saxer (1896), Maximow ('07, '09), Mollier ('09) and Neumann ('14). These investi- gators delineated the formation of erythrocytes, granulocytes and megakarocytes in the fetal liver and agreed concerning the predominant extravascular origin and development of the blood cells. In the rabbit fetus the liver forms on the 10th day of gestation and almost immediately the dif- ferentiation of hematopoietic cells begins (Bloom, '39). Hematopoiesis continues within the liver throughout gestation although lessening prior to birth.

MATERIAL AND METHODS

Rabbit fetuses of various ages from 13, to 30 days gestation were utilized in this study. Length of gestation was determined

from known breeding dates. Small pieces of liver were placed in Dalton's osmium- dichromate fixative at a pH of 7.4 or 7.6 (Dalton, '55). The tissue was fixed at room temperature for one-half to one hour, dehydrated in graded solutions of ethanol and infiltrated in methacrylate. Tissue was embedded in partially prepolymerized methacrylate to which 0.2% benzol perox- ide had been added as a catalyst. Poly- merization occurred at 60°C. Sections were cut on glass knives in a Porter-Blum micro- tome (Porter, '53). Sections approximately 1 in thickness were mounted on slides for identification by phase microscopy of the area to be sectioned for electron microscopy. Thin sections were mounted on coI- lodion coated copper grids and examined in either a RCA EMU 2B or 2E electron microscope at an initial magnification of 1500 to 8500 diameters.

OBSERVATION

Erythrocytes, heterophilic granulocytes and megakarocytes of varying stages of maturation as well as platelets and macro- phages were observed in the fetal liver. The developing blood cells were predom- inantly in extravascular locations, either just external to sinusoidal endothelium, i.e., in the space of Disse, or more often deeper within the liver cell cords and apparently farther away from the vascular spaces (fig. 1 ) . At the latter site the hepatic cells and hematopoietic cells were in immediate apposition. Some erythroblasts of all stages of maturation were also within the sinuses. Although these cells may

This investigation was supported in part by United States Public Health Service Grants RG- 3784 and CRT-5009.

2This work was done during the tenure of a National Cancer Institute Traineeship in Anatomy.

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28 GEORGE D. SORENSON

Fig. 1 Electron micrograph of liver from a fetal rabbit a t 17 days gestation. The erythroblasts (E) are closely surrounded by hepatic cells (H) . x 7500.

mature further while in the sinuses they were probably passing into the fetal circu- lation.

S t e m cells. The hematopoietic stem cells in the liver of the fetal rabbit (fig. 2 ) were similar to stem cells previously described in the yolk sac (Sorenson, '60). Within their cytoplasm there were fre-

quent free dense granules interpreted as ribonucleo-protein as well as occasional short, flattened ergastoplasmic sacs with associated ribonucleo - protein granules. Mitochondria were moderate in number and contained parallel lamellar cristae arranged perpendicular to their long axes. A few small cytoplasmic vesicles with smooth

E. M. OF FETAL HEMATOPOIESIS 29

Fig. 2 Hematopoietic stem cell in fetal liver. The large nucleus with prominent nucleoli and the abundant pale cytoplasm are characteristic features. Portions of two hepatic cells ( H ) can be seen at the lower margin of the electron micrograph. M, mitochondria. X 11,000.

profiles were observed, particularly in the periphery of the cell near the plasma membrane. The Golgi zone was simple in pattern and composed largely of small round smooth vesicles interspersed with a few elongated smooth profiles. Nuclei were round to ovoid and had moderately dense nucleoplasm with one or occasionally two prominent nucleoli.

Erythroblasts. Erythrocytic development in the fetal liver appeared the same as definitive erythropoiesis in the yolk sac

(Sorenson, '60). The transition from stem cell to the earliest erythroblastic stage was gradual and not easily recognized. The earliest identifiable erythroblast was smaller and had denser cytoplasm than the stem cell and with maturation both the cytoplasm and nucleus became extremely electron dense (fig. 3 ) . The increased cytoplasmic density was due to two com- ponents, ( 1 ) dense granules comparable in appearance to ribonucleo-protein granules and (2) a fine stippling. The nccleus


Fig. 3 Late erythroblast with increased electron density of nucleus and cytopIasm associated with maturation and hemoglobin accumulation. A part of a less mature erythroblast is visible a t the lower margin of the electron micrograph (EE). Note the invaginations in the plasma membranes of the erythroblasts ( I ) . X 16,000.

of the late erythroblast appeared similar to the cytoplasm and in an even later stage the nucleus could no longer be distinguished from cytoplasm except by recog- nition of its membrane. Mitochondria decreased as the erythroblasts matured. Groups of tightly clustered dense ferritin particles were observed in the cytoplasm of late erythroblasts but a membrane could not be identified about them. Usually only one cluster of ferritin was seen within an erythroblast and this was almost always near the periphery of the cell. Identical clumps of ferritin have been described in erythroblasts from human bone marrow (Bessis and Breton-Gorius, '57) and from guinea pig yolk sac (Sorenson, '60).

Granulocytes. Heterophilic (pseudo- eosinophilic) granulocytes were the only type of myeloid cell observed in the fetal rabbit liver. Immature forms were round and their cytoplasm contained a moderate

amount of ergastoplasm as well as numerous scattered groups of free ribonucleo- protein particles. The ovoid to elongated mitochondria contained lamellar cristae. The Golgi zone consisted of closely arranged parallel membranes as well as smooth round profiles. Even the least mature granulocytes contained a few round to ovoid cytoplasinic granules about 0.5 CI in greatest dimension (fig. 4). The granules were moderately dense and fairly well defined although at this early stage a limiting membrane could not be distinctly recognized. The irregular nuclei contained finely granular chromatin. During maturation of the granulocytes the nucleus decreased in size and the ergastoplasm increased concomitantly with the increase in granules. Granules in the more mature granulocytes were distributed throughout the cytoplasm although they were often more concentrated on the side of the cell

E. M. OF FETAL HEMATOPOIESIS 31

in which the Golgi zone was located. Oc- casionally small granules were also seen in the Golgi area (fig. 5 ) . The cytoplasmic granules had a well developed smooth lini- iting membrane from which the dense portion of the granule was often separated by a narrow pale zone possibly due to separation of the granule from the surrounding membrane. Most of the indi- vidual granules were homogeneous in appearance but some did vary in electron density and some possessed one large or several smaller vacuoles or holes.

Megakarocytes. Megakarocytes were present in the fetal rabbit liver on the 14th day of gestation and increased in frequency later in gestation but diminished prior to birth. The earliest recognized megakarocytes were ovoid, about 15 II in diameter, and had abundant cytoplasm and multilobed irregular nuclei (fig. 6). The cytoplasm contained a moderate number of ovoid to elongated mitochondria, frequent small cytoplasmic vesicles and occasional ergastoplasmic membranes. Fine nucleo- protein partides were present both free in the cytoplasm and attached to membranes.

The more mature megakarocytes were round and approximately 20 in diameter (fig. 7). The plasma membrane usually showed some irregularity in the form of either infolding or outpocketing ,with a resultant dovetailing of hepatic cells and megakarocytes. Nuclear-cytoplasmic ratio, nuclear outline and mitochondria were similar irrespective of the stage of maturation. The older megakarocytes contained small round cytoplasmic granules enclosed in a smooth membrane. Associated with the appearance of granules and probably related to their development there was an increase in cytoplasmic ribonucleo-protein particles. The cytoplasmic granules were smaller than mitochondria and the latter were not observed to be involved in granule formation. Cytoplasmic vesicles, often elongated, were increased in the mature megakarocytes. The elongated type of vesicles were described by Yamada ('57) in megakarocytes of mouse spleen and considered to be demarcation vesicles, i.e., lines of cleavage for the future separation of portions of the megakarocyte-cytoplasm as platelets. Small rounded vesicles, often

Fig. 4 Early heterophilic myelocyte containing a few homogeneous granules (G). Two parts of the nucleus ( N ) can be seen. M, mitochondria; GG, Golgi zone. x 14,000.


Fig. 5 Portions of two late granulocytes with numerous granules ( G ) and increased ergastoplasm ( E ) . The granules are surrounded by a single limiting membrane (L) . Smaller granules (SG) are in the region of the Golgi zone (GG). A portion of a hepatic cell is visible at the lower margin of the photograph. X 11,500. Insert in lower right corner of figure 5 illustrates granules from another late heterophilic myelocyte. x 23,000.

in rows, were arranged circumferentially about the nucleus.

Platelets. Platelets developed early so that small groups of apparently mature platelets were present by the 14th day of gestation (fig. 8). The appearance of the fetal platelets was similar to that of cir- culating platelets as described by Good- man, Reilly and Moore ('57). They were irregularly round to ovoid, averaged 3 CI in greatest dimension, and did not contain nuclei. Scattered ribonucleo-protein particles and granules of the same size and appearance as those in megakarocytes were present. The granules were dense and appeared homogenous. They were enclosed by a well defined membrane immediately within which in some instances there was a less dense rim surrounding the granule proper, an appearance which may be due to retraction of the granule from its surrounding membrane. Platelets also con-

tained large, often elongated, vesicles or vacuoles, small vesicles, and mitochondria similar to those in megakarocytes.

Phagocytic endothelial cells. Elongated endothelial cells lined the sinusoidal vascular spaces of the liver. At some places the endothelial cells were separated from the underlying hepatic cells by the space of Disse. In other places the endothelial cells abutted against the parenchymal cells. Sinusoidal endothelial cells had abundant cytoplasm, a moderate number of mitochondria and rather prominent ergastoplasm. Their nuclei were pale and elongated with rare nucleoli. These cells were highly phagocytic, a potential which was evident on the 14th day of gestation. The substances engulfed by the cytoplasm of these cells were both prominent and extremely variable in appearance. Erythro- cytes, often apparently hemolysed were present within some of the phagocytic cells

E. M. O F FETAL HEMATOPOIESIS

Fig. 6 Immature megakarocyte with large multilobed nucleus and abundant cytoplasm containing scattered mitochondria ( M ) . x 14,500.

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Fig. 7 Megakarocyte containing granules ( G ) , mitochondria ( M ) , ergastoplasmic membranes ( E ) and free ribonucleoprotein particles (P) . V, vesicles. X 14,500.

Fin. 8 Platelets in fetal rabbit liver at 14 davs gestation. The datelets exhibit the same I

cytoplasmic features as the megakarocytes. x 12,000.

(fig. 9 ) . The appearance of membranous formation (myelin figures) at the periphery of the phagocytized red cells was cominon and probably related to the high lipoprotein content (Policard, Bessis and Breton-Gorius, '57). Other material within the endothelial cell cytoplasm had a variegated non-specific electron density and although much of it may have been derived from engulfed red blood cells this could not be proven.

, I

G , granules; M, mitochondria; V, vesicles.

Hepatic cells. The hepatic cells could be readily distinguished from hematopoietic cells by (1) size (2 ) amount of ergastoplasm ( 3 ) lipid inclusions and ( 4 ) characteristic mitochondria (fig. 10). Liver parenchymal cells were larger than blood elements except megakarocytes. He- patic cells had abundant ergastoplasm, large cytoplasmic lipid inclusions and prominent rotund mitochondria with deli- cate villous cristae. Ferritin particles were

E. M. O F FETAL HEMATOPOIESIS

Fig. 9 Phagocytic endothelial cell containing several engulfed erythrocytes (E ) which are apparently hemolysed. Myelin figure formation is initially observed at the margin of the engulfed cell (MI) and later becomes more extensive (M?) . N, nucleus of endothelial cell. x 18,500.

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Fig. 10 Portions of two hepatic cells from fetus at 29 days gestation. The amount of ergastoplasm ( E ) , the rotund mitcchondria ( M ) , the cytoplasmic lipid (L) and the diffuse distribution of ferritin (F) are characteristic. x 31.000.

diffusely distributed in the cytoplasm of hepatic cells particularly during the latter part of gestation. The parenchymal cells often also contained clumps of ferritin or hemosiderin granules (fig. 11 ). In the latter the ferritin was arranged in an orderly pattern as observed in sectioned crystals of ferritin (Richter, '58). Bile canaliculi were frequently formed at the junction of several hepatic cells. Rarely a canaliculus was surrounded by the cytoplasm of a single parenchymal cell. Microvilli projected into the canaliculi.

Hematopoietic cells outnumbered the parenchymal cells in the liver during mid gestation. The hepatic cells were often elongated or otherwise deformed by pres-

sure from maturing erythroblasts. As a result the erythroid cells were partially or completely surrounded by hepatic cells and the plasma membranes of the two types of cells were usually immediately adjacent to each other. The plasma membranes of erythroblasts frequently had shallow invaginations (fig. 12). The appearance of the latter was quite uniform although some were deeper than others. The membrane about the tip of the invagination, i.e., margin of deepest penetration, was more prominent than the balance of the limiting membrane. This increased prominence was due to two characteristics (1) an increased width of the membrane and (2 ) an apparent increase in electron density

E. M. OF FETAL HEMATOPOIESIS

Fig. 11 Hemosiderin within a hepatic cell a t 29 days gestation. Note the orderly arrangement of the ferritin in some locations (F,) as well as the scattered ferritin particles (F2). Scattered ferritin particles are also present within the adjacent cytoplasm (F:?). X 60,000.

Fig. 12 Erythroblasts with invaginations (I) of their plasma membranes. These invaginations probably represent stages of pinocytosis. x 15,000.

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Fig. 13 Two erythroblasts ( E ) separated by a small portion of hepatic cell cytoplasm ( H ) which contains ferritin (F) . Finger-like process of hepatic cell cytoplasm (P ) is within an invagination of the erythroblastic plasma membrane. Vesicles (V) within the erythroblasts probably represent a later step in this process of intercellular transfer of material (ropheocytosis). X 45,000.

of the membrane at this site. Similar invaginations were less commonly seen at the margins of hepatic cells and endothelial cells. At times a small finger-like process appeared to extend from a hepatic cell into an erythroblast causing an invagination in the erythroblastic plasma membrane identical with that previously described (fig. 13). When merely a small portion of the cell next to the erythroblast was visualized it could still be determined that the cytoplasmic process was from an adjacent liver cell since ferritin was diffusely distributed in the cytoplasm as observed only in hepatic cells. Rounded, smooth walled vesicles were frequently observed in the peripheral cytoplasm of erythroblasts and sometimes also in the other cells of the fetal liver, and it appeared that the invaginations and the vesicles beneath the plasma membrane were successive steps of the same process.

DISCUSSION

Hematopoietic cells within the fetal liver develop and mature enmeshed among hepatic cells. The close contact of these two cellular types would appear to furnish an ideal arrangement for intercellular

transfer of substances. That the latter may occur is further supported by certain ultrastructural features at junctions between hepatic and erythroid cells. While many of the plasma membrane indenta- tions are probably part of pinocytosis, it appears that at least certain invaginations of the periphery of erythroblasts are finger- like processes of hepatic cell cytoplasm. Apparently a later step is the pinching off of the cytoplasmic process of the liver cell with the formation of an intracytoplasmic vesicle within the erythroid cell.

Intercellular transfer of substances from reticular (phagocytic) cells to erythroblasts has been observed by Policard and Bessis ('58) in human bone marrow. They delineated the transmission of ferritin be- between these two types of cells by a process which was likened to pinocytosis but named ropheocytosis. Somewhat similar observations of intercellular pinocytosis or ropheocytosis have also been made in the guinea pig yolk sac (Sorenson, '60). In the fetal liver of rabbits evidence of ropheocytotic transfer from hepatic cells to erythroblasts is much more prominent than evidence of transfer from phagocytic endothelial cells to erythroblasts although the

E. M. O F FETAL HEMATOPOIESIS 39

latter may also occur. It appears that ferritin is probably transported directly from hepatic cells to immature erythroid cells by this process of ropheocytosis.

The appearance that ropheocytotic transfer does occur between fetal hepatic cells and erythroblasts suggests that intercellular transfer of material is a more wide- spread process than has previously been described. In both the bone marrow and yolk sac the transfer of material was between cells of mesodermal origin, i.e., phagocytes to erythroblasts, while in the fetal liver the transfer is apparently from cells of entodermal derivation, i e., liver parenchymal cells, to cells of mesodermal origin, i.e., erythroblasts.

In 1955 Rinehart reported electron microscopic observations indicating that specific granules in white blood cells and platelets were derived from mitochondria. This concept was not supported by our observations on granulocytes, megakarocytes and platelets in the fetal liver. Granules in the early granulocyte were easily recognized. At the same time typical mitochondria were observed and transition stages between mitochondria and granules were not apparent. The difference between granules and mitochondria was also dis- tinct in late granulocytes. The observation of small granules within the Golgi zone suggests this region as the site of granule development. Megakarocytic granules were similar in that they also were surrounded by a single limiting membrane. Mitochon- dria in megakarocytes and platelets were larger than any of the granules and transition stages between these two cytoplasmic organelles were not observed.

SUMMARY

Hematopoiesis was studied in the fetal rabbit liver from the 12th to the 30th day of gestation. Erythrocytes, granulocytes and megakarocytes of varying stages of development as well as platelets and phagocytic endothelial cells were observed. As erythroid cells matured they decreased in size and developed increased electron density which was concomitant with hemoglobin formation. In developing heterophilic granulocytes an increase in ergastoplasm was associated with the increase in granules and the latter were often more

numerous in the region of the Golgi zone. Platelets appeared identical to portions of megakarocytic cytoplasm. Phagocytic endothelial cells were active in the fetal liver and much of their engulfed material was derived from erythrocytes. Maturing erythrocytes were closely surrounded by hepatic cells and intercellular transfer (ropheocytosis) appeared to occur between hepatic cells and erythroblasts. It appeared prob- able that ferritin was transported directly from hepatic cells to erythroblasts by this process.

LITERATURE CITED

Bessis, M. C., and J. Breton-Gorius 1957 Iron- particles in normal erythroblasts and pathologic erythroblasts. J. Biophys. Biochem. Cytol., 3: 503-504.

Bloom, W. 1939 The embryogenesis of mammalian blood. In: Handbook of Hematology, H. Downey, ed. Hoeber, New York, vol. 2, chap. xiii, pp. 863-922.

Dalton, A. J. 1955 A chrome-osmium fixative for electron microscopy. Anat. Rec., 121: 281.

Goodman, J. R., E. B. Reilly and R. E. Moore 1957 Electron microscopy of formed elements of normal human blood. Blood, 12: 428-442.

Maximow, A. 1907 uber die Entwicklung der Blut-und Bindegewebszellen beim Saugetier- embryo. Fol. Haematol., 4: 611-626.

1909 Untersuchungen uber Blut und Bindegewebe 1. Die frdhesten Entwicklungs- studien Blut und Bindegewebezellen beim Sangetierembryo, his zum Anfang der Blutbil- dung in der Leber. Arch. Mikr. Anat., 73: 444- 561.

Mollier, S. 1909 Die Blutbildung in der embryonalen Leber des Menschen und der Saugetiere. Ibid., 74: 474-524.

Neumann, E. 1914 Neuer Beitrag zur Kenntnis der embryonalen Leber. Ibid., 85: 480-520.

Policard, A., M. Bessis and J. Breton-Gorius 1957 Structures myeliniques observees au microscope electronique sur des coupes de globules rouges en voie de lyse. Ex?. Cell. Res., 13: 184-186.

Policard, A, , and M. Bessis 1958 Sur un mode d'incorporation des macromol6cules par la cel- lule, visible au microscope electronique: la ropheocytose. C. R. Acad. Sci., 246: 3194- 3197.

Porter, K. R., and J. Blum 1953 A study in microtomy for electron microscopy. Anat. Rec., 117: 685-712.

Richter, G. W. 1958 Electron microscopy of hemosiderin. J. Biophys. Biochem. Cytol., 4 : 55-58.

Rinehart, J. F. 1955 Electron microscopic studies of sectioned white blood cells and platelets : with observations on the derivation of specific granules from mitochondria. Am. J. Clin. Path., 25: 605-619.

Saxer, F. 1896 uber die Entwicklung und den Bau der normalen Lymphdriisen und die Ent- stehung der roten und weissen Blutkorperchen. Anat., 6: 347-532.


Sorenson, G . D. 1960 An electron microscopic Yamada, E. 1957 The fine structure of the study of hematopoiesis in the yolk sac. Lab. megakarocyte in the mouse spleen. Acta Anat., Invest., in press. 29: 267-290.

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An electron microscopic study of hematopoiesis in the liver of the fetal rabbit