The primordial germ cells (PGC’s) of the early mouse embryo have been identified in semi-thin and ultra-thin plastic sections on the combined bases of location and a distinctive set of morphological features. These cells originate extra-gonadally and their path of migration during the stages investigated agrees with results of previous histological and experimental studies. At 8–9 days of development, PGC’s are observed among the endoderm cells of the mid- and hindgut and the yolk stalk; at 10–11 days, PGC’s are seen in the dorsal mesentery, the dorsal coelomic lining, and in the rudimentary genital ridge; by 13 days, the gonad is abundantly populated with germ cells. During the migratory period the PGC’s appear as small (10–12 pm diameter), oval, basophilic cells which have a large nucleo-cytoplasmic ratio. These cells possess numerous free ribosomes and polysomes, a filamentous ground cytoplasm, and few profiles of endoplasmic reticulum. Mitochondria display small oval and thread-like profiles. A Golgi complex is not prominent in the PGC’s. The densely granular and fibrillar nucleus is often oval in outline but sometimes shows irregular contours. A large nucleolus is present. Although located among groups of cells that exhibit extensive cell junctions, the PGC’s have not been found to share such junctions with neighboring cells. Furthermore, the PGC’s possess small cytoplasmic processes that contain numerous microfilaments. These observations are interpreted as morphological correlates to the migratory activity of these cells.

In the early PGC’s located in the gut, regions of the endoplasmic reticulum where membranes are closely apposed, perhaps fused, often present the appearance of annulate cisternae. In addition, compact aggregates of granulofibrillar material are found in the cytoplasm of these same cells. Neither of these structures is detected in the mesenteric PGC’s, the gonadal germ cells, or any other cell of the embryo at the stages studied. It is suggested that these two cytoplasmic elements may be related to further differentiation of the germ cells.

The germ cells of the developing gonad are large, oval cells which possess a nucleus of very round contour. Both the nucleus and the cytoplasm retain a densely granular appearance. The expanded cytoplasm contains a large Golgi complex but still few profiles of endoplasmic reticulum. Free ribosomes and polysomes are abundant. The germ cells share extensive gap junctions with one another and with adjacent stromal cells.

It has been securely demonstrated that in vertebrate embryos the primordial germ cells originate extragonadally (Willier, 1937; Everett, 1943; Witschi, 1948; Blackler, 1958) and lodge in the genital ridge after an extensive migratory tour.

The earliest identification in the mouse embryo of primordial germ cells has been made by histological and histochemical methods at eight days of gestation, when organogenesis is just underway. The time-course of migration of these cells in the mouse embryo is as follows: at 8 days gestation, primordial germ cells (PGC’s) are found in the primitive streak, allantoic stalk, yolk sac, and hindgut endoderm; at 9–10 days gestation, PGC’s are found in the midgut endoderm and the dorsal mesentery; at 10–11 days gestation, PGC’s are found in the genital ridge (Everett, 1943; Chiquoine, 1954; Mintz & Russell, 1955, 1957; Bennett, 1956; Ozdzenski, 1967).

It is the object of the present study to describe the fine structure of the PGC’s during their migration and differentiation in the 8-to 13-day mouse embryo, to provide evidence, by the continuity of morphological features, for the direct lineage of the PGC’s of the primitive gut and of the gonad, and to attempt to correlate the morphology of the PGC’s with their unique activity and fate.

Mouse embryos were obtained from stocks maintained in our own colony. Age of embryos was predicted by the vaginal plug method and was precisely determined by counting somites at the time the embryos were dissected and fixed. Pregnant females were killed by cervical dislocation and their uteri were immediately removed and placed in fixative. The uterus was cut into transverse segments each containing one embryo in its decidua. The embryos in fixative were freed of deciduae and yolk sacs under the dissecting microscope; the gonadal primordia themselves were dissected from the older (13-day) specimens. Fixation was from 15–45 min, at room temperature, in 2% glutaraldehyde, buffered in 0·05 M Sorenson phosphate, with traces of calcium chloride, at pH 7·4 (3 drops from a Pasteur pipet of TO% calcium chloride solution were added to 80 ml of fixative); followed by 45 min, at room temperature, in 1·5% osmium tetroxide, 0·05 M Sorenson phosphate, and 0·035 M sodium chloride, with traces of calcium chloride, at pH 7·4 (3 drops from a Pasteur pipet of TO% calcium chloride solution were added to 66 ml of fixative). (These fixative compositions were suggested by Dr Irwin Spiegelman and are approximately 300 m-osmolar.) Tissue was dehydrated in a graded series of ethanol and propylene oxide and embedded in Epon (Luft, 1961).

Sections were cut by glass or diamond knives on Sorvall ultramicrotome models MT-1 and MT-2. Thin sections were mounted on parlodion- and carbon-coated copper grids and stained for 10 min in uranyl acetate and in lead citrate (Venable & Coggeshall, 1965). Preparations were viewed in a Philips 200 electron microscope.

Epon sections 1 μ m thick were mounted on glass slides and stained on a hot plate at 60°C for about 1 min, or until vapors just start to rise, with alkaline toluidine blue (1·0% toluidine blue in a 1·0% solution of borax, approximately pH 11, according to Pease, 1964, p. 260). Sections were examined in a Zeiss photomicroscope.

This is the first report to identify and describe the PGC’s of a mammalian embryo in plastic sections 1 μm thick at the light-microscopic level and in ultra-thin sections at the electron microscopic level. We will therefore present a detailed account here of each of the two categories of observations, in order to establish criteria for recognizing these cells by these methods.

Light microscopy

Small, oval, darkly staining cells interspersed among the endoderm cells of the yolk sac and primitive gut of the posterior half of the trunk of 8- and 9-day (from 2 to 20 somites) mouse embryos can be detected in plastic sections 1 μm thick stained with alkaline toluidine blue (Figs. 1–5). These cells correspond in their basophilia (Bennett, 1956) and location (Chiquoine, 1954; Mintz & Russell, 1957; Ozdzenski, 1967) to PGC’s identified in earlier studies and are likewise identified here as PGC’s. Moreover, cells of similar distinctive morphology are seen within the dorsal mesentery, the coelomic lining, and the genital ridge in 10-day embryos (Figs. 6–8), while such cells are no longer found in the primitive gut. In 13-day embryos the definitive germ cells of the gonad (Figs. 9, 10) are similar both in form and in density of cytoplasm and nucleus to the cells identified as PGC’s in younger embryos. The constellation of morphological features seen at the light-microscopic level in primordial germ cells at these three stages is summarized here.

Fig. 1–5

All figures are light micrographs of plastic sections, 1 μm thick, through mouse embryos of 8μ5–9 days gestation. Toluidine blue 0 stain.

Transverse section at level of closed neural tube (NT), closed hindgut (G) and allantoic vein (K). The three darkly stained cells within the rectangle (2) are primordial germ cells (PGC’s) among the endoderm cells of the ventral wall of the primitive gut. × 200.

Fig. 2. Inset of Fig. 1 depicting the PGC’s. The PGC’s do not border directly on gut lumen. Arrows, nuclear particulate material; Nu, nucleolus; L, lipid droplet in endoderm cell. ×1100.

Fig. 3. Transverse section at level of open midgut. Darkly stained cells within the rectangles (4, 5) are PGC’s among the endoderm cells of the primitive gut (G) and yolk stalk ( Y), × 200.

Fig. 4. Inset of Fig. 3 depicting the gut PGC’s. Arrows, nuclear particulate material; C, cytoplasmic ‘crescent’; L, lipid in endoderm cell, × 1100.

Fig. 5. Inset of Fig. 3 depicting the yolk stalk PGC’s. Arrows, cytoplasmic flanges of PGC at edge of splanchnic mesoderm layer; C, cytoplasmic ‘crescent’; L, lipid in endoderm cell. ×1100.

Fig. 1–5

All figures are light micrographs of plastic sections, 1 μm thick, through mouse embryos of 8μ5–9 days gestation. Toluidine blue 0 stain.

Transverse section at level of closed neural tube (NT), closed hindgut (G) and allantoic vein (K). The three darkly stained cells within the rectangle (2) are primordial germ cells (PGC’s) among the endoderm cells of the ventral wall of the primitive gut. × 200.

Fig. 2. Inset of Fig. 1 depicting the PGC’s. The PGC’s do not border directly on gut lumen. Arrows, nuclear particulate material; Nu, nucleolus; L, lipid droplet in endoderm cell. ×1100.

Fig. 3. Transverse section at level of open midgut. Darkly stained cells within the rectangles (4, 5) are PGC’s among the endoderm cells of the primitive gut (G) and yolk stalk ( Y), × 200.

Fig. 4. Inset of Fig. 3 depicting the gut PGC’s. Arrows, nuclear particulate material; C, cytoplasmic ‘crescent’; L, lipid in endoderm cell, × 1100.

Fig. 5. Inset of Fig. 3 depicting the yolk stalk PGC’s. Arrows, cytoplasmic flanges of PGC at edge of splanchnic mesoderm layer; C, cytoplasmic ‘crescent’; L, lipid in endoderm cell. ×1100.

Figures 6–8

All figures are light micrographs of plastic sections, 1 μm thick, through the gonad of the mouse embryo of 13 days gestation. Toluidine blue 0 stain.

Transverse section at level of gut (6), fused dorsal aorta (DA), mesonephros (N), and gonad primordia (go). PGC’s are located in the gonad primordium (7) and dorsal mesentery (8) at this time, × 200.

All figures are light micrographs of plastic sections, 1 μm thick, through the mouse embryo of 10-5 days of gestation. Toluidine blue 0 stain.

Fig. 7. Inset of Fig. 6 illustrating PGC at locus of future gonad. The cell and the nucleus are quite round in outline and darkly staining. Nu, Nucleolus, × 1100.

Fig. 8. Inset of Fig. 6 illustrating PGC in the mesoderm of the dorsal mesentery. Nucleus and cytoplasm stain darkly. Arrows, nuclear particulate material, × 1100.

Figures 6–8

All figures are light micrographs of plastic sections, 1 μm thick, through the gonad of the mouse embryo of 13 days gestation. Toluidine blue 0 stain.

Transverse section at level of gut (6), fused dorsal aorta (DA), mesonephros (N), and gonad primordia (go). PGC’s are located in the gonad primordium (7) and dorsal mesentery (8) at this time, × 200.

All figures are light micrographs of plastic sections, 1 μm thick, through the mouse embryo of 10-5 days of gestation. Toluidine blue 0 stain.

Fig. 7. Inset of Fig. 6 illustrating PGC at locus of future gonad. The cell and the nucleus are quite round in outline and darkly staining. Nu, Nucleolus, × 1100.

Fig. 8. Inset of Fig. 6 illustrating PGC in the mesoderm of the dorsal mesentery. Nucleus and cytoplasm stain darkly. Arrows, nuclear particulate material, × 1100.

Figures 9, 10

Fig. 9. Transverse section through gonad; sexual dimorphism is not yet evident. Many cells are non-germinal stromal cells. At this time the germ cells are disposed in clusters of morphologically similar cells, reflecting their clonal origin and synchronous development. These clusters may vary one from the other although here most of the germ cells are dark staining and are quite round in outline with very round nuclei. Rectangle (10) encloses several germ cells, ×200.

Fig. 10. Inset of Fig. 9 illustrating a number of the dark staining round germ cells (c) typical of this stage. Nucleoli (Nu) are less prominent in these germ cells than in the earlier PGC’s. Arrows, nuclear particulate material, × 1100.

Figures 9, 10

Fig. 9. Transverse section through gonad; sexual dimorphism is not yet evident. Many cells are non-germinal stromal cells. At this time the germ cells are disposed in clusters of morphologically similar cells, reflecting their clonal origin and synchronous development. These clusters may vary one from the other although here most of the germ cells are dark staining and are quite round in outline with very round nuclei. Rectangle (10) encloses several germ cells, ×200.

Fig. 10. Inset of Fig. 9 illustrating a number of the dark staining round germ cells (c) typical of this stage. Nucleoli (Nu) are less prominent in these germ cells than in the earlier PGC’s. Arrows, nuclear particulate material, × 1100.

In the 8-to 9-day embryo the PGC’s are usually oval, with smooth contours in section, and are denser and smaller than are the adjacent endoderm cells; in these respects the PGC’s bear some resemblance to splanchnic mesoderm cells (Figs. 1–5). Although they are located within the single layer of endoderm cells of the primitive gut, the PGC’s do not border on the lumen (Figs. 2, 4). Some cells of the 8-day embryo, possessing the features of PGC’s as outlined here, appear to be partially in the endoderm layer and partially in the splanchnic mesoderm (Figs. 3, 5). These ‘interzonal’ cells often exhibit somewhat irregular surface outlines with several short, narrow, cytoplasmic projections (Fig. 5). The contours of these cells, coupled with their disposition and frequent occurrence in the yolk stalk, suggest that they may be PGC’s migrating from a mesodermal to an endodermal location.

In the 10-day embryo the mesenteric PGC’s (Figs. 6‐8) have the same oval form and cellular density that are typical of the younger gut PGC’s.

The size of the PGC’s ranges from 10 to 13 μm in diameter and does not vary during the period of migration. Once these cells become resident in the gonad, however, they apparently begin a period of growth, because most germ cells found here at 13 days are about 12–13 μm in diameter, with some as large as 20 μm in diameter. Furthermore, the germ cells in the gonad occur as clusters of morphologically similar cells. The structural basis of this synchronized activity of each cell group probably rests in the cytoplasmic bridges between the interphase germ cells (Fawcett, 1961; Gondos & Zamboni, 1969). These bridges are not detected in the migratory PGC’s prior to arrival in the gonad.

The nuclei of the gut and mesenteric PGC’s are somewhat irregular in outline and are about as dense as the cytoplasm, with much particulate nuclear material apparent (Figs. 2, 4, 8). In addition, some compact masses of chromatin, usually near the nuclear envelope, are obvious in many nuclei and a well-developed nucleolus is often situated close to the nuclear envelope (Figs. 2, 7). The nuclei of the germ cells in the gonad are very round in section and slightly larger, about 9 //m in diameter. In a few cell clusters, the nuclei possess a pale nucleoplasm and several large chromatin masses, suggesting that the nuclei of such a group of cells may be in a phase of karyokinesis. The nuclei of most of the gonadal germ cells are very dense, however, and contain small masses of evenly dispersed, dark, particulate material (Fig. 10), as in the nuclei of the earlier PGC’s.

Mitochondria appear as small oval bodies and their distribution does not suggest any cellular polarization in the PGC’s or the germ cells. In many sections through the young PGC’s one or more colorless, crescent-shaped zones can be seen in the cytoplasm (Figs. 4, 5), a feature absent from other cell types observed. In the mesenteric PGC’s, some perinuclear clear zones are evident (Figs. 7, 8). The PGC’s, unlike the endoderm cells (Figs. 2, 5), do not usually contain prominent lipid droplets. In this respect, PGC’s resemble the splanchnic mesoderm cells. The gonadal germ cells do not contain lipid droplets either. The older PGC’s sometimes display small cytoplasmic ‘vacuoles’ which are interpreted as dilated or disrupted organelles.

Electron microscopy

It is at once apparent in electron micrographs that the great cytoplasmic density of the PGC’s of all stages is due to the close packing of large numbers of free ribosomes and polysomal arrays. (Figs, 11, 13, 16, 20, 24). This finding correlates with the basophilia of these cells at the light-microscopic level. The opacity of the cells is further enhanced by a finely granular and filamentous ground cytoplasm. This characteristic appearance of the cytoplasm in the germ cells of all stages studied is not encountered in the cells of the endoderm or mesoderm surrounding the migrating PGC’s nor in the supporting cells of the gonad.

In the germ cells of all stages the darkly staining nucleus observable in light micrographs is seen at low magnifications in the electron microscope to consist of abundant, compact masses of electron-opaque granules and fibrils (Figs. 11, 13, 16, 20, 24). A few small, dense clumps of chromatin are sometimes visible near the nuclear envelope (Figs. 11, 20) and numbers of large nuclear granules are readily detected in all nuclei. Nuclear pores are numerous but probably not more so than in any other cell type in these embryos. The nucleolus, as in other cells of the embryo, is large and reticulate.

Figures 11, 12

Electron micrographs; 8μ5-day mouse embryo.

PGC surrounded by endoderm cells (end) of the primitive gut. The PGC cytoplasm contains an abundance of free ribosomes and polysomes as well as a dense background. The nucleus contains a dense mass of granules and fibrils. Np, Nuclear pores; M, mitochondria; C, chromatin; Nu, nucleolus; ac, annulate cisternae; ER, endoplasmic reticulum; J, cell junctions; L, lipid in endoderm cell, × 6800.

Fig. 12. Profiles of annulate cisternae (ac) typical of the young PGC’s of the gut. These structures are often continuous with membranes of endoplasmic reticulum (ER) in these cells. N, nucleus, × 52000.

Figures 11, 12

Electron micrographs; 8μ5-day mouse embryo.

PGC surrounded by endoderm cells (end) of the primitive gut. The PGC cytoplasm contains an abundance of free ribosomes and polysomes as well as a dense background. The nucleus contains a dense mass of granules and fibrils. Np, Nuclear pores; M, mitochondria; C, chromatin; Nu, nucleolus; ac, annulate cisternae; ER, endoplasmic reticulum; J, cell junctions; L, lipid in endoderm cell, × 6800.

Fig. 12. Profiles of annulate cisternae (ac) typical of the young PGC’s of the gut. These structures are often continuous with membranes of endoplasmic reticulum (ER) in these cells. N, nucleus, × 52000.

Figures 13–15

Electron micrographs; 9-day mouse embryos.

PGC among endoderm cells of the primitive gut. The cytoplasm is densely packed with free ribosomes and polysomes. The equally dense nucleus is filled with fibrils and granules. The endoplasmic reticulum (ER) often displays a disrupted membrane at one side of the cisterna and is dilated. Rectangle (14) encloses cytoplasmic dense bodies. Np, Nuclear pores; M, mitochondrion; Nu, nucleolus; ac, annulate cisternae. × 8200.

Fig. 14. Tnset of Fig. 13 illustrating some of the many dense bodies present in the cytoplasm of the early PGC’s. Large dense bodies (DB) consist of a compact aggregate of granulofibrillar material. Granules of the dimensions of ribosomes are often apparent (arrows) at the periphery of the dense body. Numerous smaller dense bodies (db) are present throughout the cytoplasm and these cannot be resolved into granular or fibrillar elements. N, Nucleus; ac, annulate cisternae. × 50000.

Fig. 15. Another section through the cell pictured in Fig. 13 and in the region bounded by the rectangle. The dense body (DB) seen here may be contiguous with the one seen in Fig. 14. Ribosome-like granules are evident (arrows) at the margin of the dense body. N, Nucleus; M, mitochondrion; ac, annulate cisternae. × 50000.

Figures 13–15

Electron micrographs; 9-day mouse embryos.

PGC among endoderm cells of the primitive gut. The cytoplasm is densely packed with free ribosomes and polysomes. The equally dense nucleus is filled with fibrils and granules. The endoplasmic reticulum (ER) often displays a disrupted membrane at one side of the cisterna and is dilated. Rectangle (14) encloses cytoplasmic dense bodies. Np, Nuclear pores; M, mitochondrion; Nu, nucleolus; ac, annulate cisternae. × 8200.

Fig. 14. Tnset of Fig. 13 illustrating some of the many dense bodies present in the cytoplasm of the early PGC’s. Large dense bodies (DB) consist of a compact aggregate of granulofibrillar material. Granules of the dimensions of ribosomes are often apparent (arrows) at the periphery of the dense body. Numerous smaller dense bodies (db) are present throughout the cytoplasm and these cannot be resolved into granular or fibrillar elements. N, Nucleus; ac, annulate cisternae. × 50000.

Fig. 15. Another section through the cell pictured in Fig. 13 and in the region bounded by the rectangle. The dense body (DB) seen here may be contiguous with the one seen in Fig. 14. Ribosome-like granules are evident (arrows) at the margin of the dense body. N, Nucleus; M, mitochondrion; ac, annulate cisternae. × 50000.

Figures 16–19

Electron micrographs; 10·5-day mouse embryos.

Fig. 16. PGC among the mesoderm cells of the dorsal mesentery. Unlike the endoderm cells, the surrounding mesoderm cells are almost as dense as the PGC. The sparsely distributed endoplasmic reticulum (ER) continues to display defective membranes. M, Mitochondrion; Np, nuclear pores; Nu, nucleolus; bl, basal lamina of coelomic epithelium; Figs. 17–19 indicated by rectangles, 17, 18, 19. × 6700.

Figs. 17, 18. Insets of Fig. 16 illustrating subsurface microfilaments in cytoplasmic flanges of these migratory PGC’s. The microfilaments (arrow) are oriented parallel to the long axis of the cytoplasmic process and normal to the tip of the process. 17, × 49000. 18, × 62000.

Fig. 19. Inset of Fig. 16 illustrating one of the few small focal junctions between the PGC and adjacent mesoderm cells. The intermembrane space is about 9 nm wide; a small amount of filamentous material is accumulated in the cytoplasm at the apposing membranes, × 111000.

Figures 16–19

Electron micrographs; 10·5-day mouse embryos.

Fig. 16. PGC among the mesoderm cells of the dorsal mesentery. Unlike the endoderm cells, the surrounding mesoderm cells are almost as dense as the PGC. The sparsely distributed endoplasmic reticulum (ER) continues to display defective membranes. M, Mitochondrion; Np, nuclear pores; Nu, nucleolus; bl, basal lamina of coelomic epithelium; Figs. 17–19 indicated by rectangles, 17, 18, 19. × 6700.

Figs. 17, 18. Insets of Fig. 16 illustrating subsurface microfilaments in cytoplasmic flanges of these migratory PGC’s. The microfilaments (arrow) are oriented parallel to the long axis of the cytoplasmic process and normal to the tip of the process. 17, × 49000. 18, × 62000.

Fig. 19. Inset of Fig. 16 illustrating one of the few small focal junctions between the PGC and adjacent mesoderm cells. The intermembrane space is about 9 nm wide; a small amount of filamentous material is accumulated in the cytoplasm at the apposing membranes, × 111000.

Figures 20–23

Electron micrographs; 13-day mouse embryos.

Section through the gonad. The presence of a cytoplasmic bridge (B) establishes the identity of the darker cells as germ cells; the neighboring paler cells are the stromal cells of the gonad. The germ cell cytoplasm continues to contain large amounts of free ribosomes and polysomes and the very round nucleus contains a great mass of granules and fibrils. The Golgi complex (G) is well developed in these cells. Endoplasmic reticulum (ER) usually appears as small vesicular profiles. Np, Nuclear pores; M, mitochondria; Nu, nucleolus, × 4900.

Fig. 21. Typical parallel apposition of cell membranes between adjacent germ cells and between germ cell and stromal cell. The intermembrane space is usually 20 nm wide. Some regions of membrane apposition suggest incipient junctions (J) with the appearance of denser membranes, an accumulation of subsurface filamentous material, and moderately dense material in the intermembrane space at these places, ×120000.

Figs. 22, 23. Extensive cellular junctions shared by very closely apposed membranes of adjacent germ cells and of adjacent germ cell and stromal cell. The neighboring membranes converge suddenly in close apposition (c). The outer leaflet of the membrane appears very dense while the inner leaflet is pale. The intermembrane cleft (arrows) is no more than 2-5 nm and, in several places, the outer leaflets appear fused (f), giving the complex a pentalaminar form. There are no subsurface filamentous accumulations evident at these junctions, × 120000.

Figures 20–23

Electron micrographs; 13-day mouse embryos.

Section through the gonad. The presence of a cytoplasmic bridge (B) establishes the identity of the darker cells as germ cells; the neighboring paler cells are the stromal cells of the gonad. The germ cell cytoplasm continues to contain large amounts of free ribosomes and polysomes and the very round nucleus contains a great mass of granules and fibrils. The Golgi complex (G) is well developed in these cells. Endoplasmic reticulum (ER) usually appears as small vesicular profiles. Np, Nuclear pores; M, mitochondria; Nu, nucleolus, × 4900.

Fig. 21. Typical parallel apposition of cell membranes between adjacent germ cells and between germ cell and stromal cell. The intermembrane space is usually 20 nm wide. Some regions of membrane apposition suggest incipient junctions (J) with the appearance of denser membranes, an accumulation of subsurface filamentous material, and moderately dense material in the intermembrane space at these places, ×120000.

Figs. 22, 23. Extensive cellular junctions shared by very closely apposed membranes of adjacent germ cells and of adjacent germ cell and stromal cell. The neighboring membranes converge suddenly in close apposition (c). The outer leaflet of the membrane appears very dense while the inner leaflet is pale. The intermembrane cleft (arrows) is no more than 2-5 nm and, in several places, the outer leaflets appear fused (f), giving the complex a pentalaminar form. There are no subsurface filamentous accumulations evident at these junctions, × 120000.

Figures 24, 25

Electron micrographs; 13-day mouse embryos.

Section through the gonad. The large, pale stromal cells contain lipid droplets (L) and numerous profiles of endoplasmic reticulum (ER). The cytoplasmic density of the germ cells continues to be due to an abundance of free ribosomes and polysomes. Few profiles of endoplasmic reticulum (ER) are present in the germ cells and these are sometimes long and very flattened. M, Mitochondrion. ×4900.

Fig. 25. Profile of endoplasmic reticulum in germ cell of Fig. 24. One membrane surface (arrow) of the flattened cisterna appears very fragile, interrupted at places, and rarely bears any ribosomes. Organelles and ribosomes do not usually occupy the cytoplasm immediately adjacent to this membrane. Np, Nuclear pore, × 28000.

Figures 24, 25

Electron micrographs; 13-day mouse embryos.

Section through the gonad. The large, pale stromal cells contain lipid droplets (L) and numerous profiles of endoplasmic reticulum (ER). The cytoplasmic density of the germ cells continues to be due to an abundance of free ribosomes and polysomes. Few profiles of endoplasmic reticulum (ER) are present in the germ cells and these are sometimes long and very flattened. M, Mitochondrion. ×4900.

Fig. 25. Profile of endoplasmic reticulum in germ cell of Fig. 24. One membrane surface (arrow) of the flattened cisterna appears very fragile, interrupted at places, and rarely bears any ribosomes. Organelles and ribosomes do not usually occupy the cytoplasm immediately adjacent to this membrane. Np, Nuclear pore, × 28000.

In the PGC’s located in the gut or mesenteries, few profiles of granular endoplasmic reticulum are seen and these occur principally as short flattened cisternae (Figs. 11, 13, 16). By the gonadal stage, large segments of endoplasmic reticulum are occasionally encountered in the germ cells (Fig. 24) but they remain few in number (Fig. 20). In comparison with the neighboring cells, the various germ cells have fewer profiles of endoplasmic reticulum.

Some profiles of endoplasmic reticulum in the gut PGC’s are of special note; the morphology of these elements consists of a ‘cisterna’ in which one membrane surface of the usual bilamellar element is lacking, thus, a ‘cisternal space’ appears open to the cytoplasm on one side (Figs. 11, 13). This characteristic appearance of the endoplasmic reticulum in these cells is represented at the light-microscopic level by the colorless crescent-shaped areas described above (Figs. 4, 5). Although this configuration is very likely a result of artifact, it is observed only in these PGC’s, and repeatedly; therefore, it probably indicates some structural or physiological state typical only of PGC’s at this time. Endoplasmic reticulum elements of this kind are rarely encountered in the mesenteric PGC’s but distended cisternae are sometimes noted in these cells. Some of the longer profiles of endoplasmic reticulum occasionally found in the gonadal germ cells have an unusual appearance which may be related to the type of structure seen in the gut PGC’s. As in the younger cells, one membrane of the bilamellar unit appears normal and studded with ribosomes (Figs. 24, 25). The membrane at the opposite side of the cisterna is usually thinner, interrupted at several points, and bears few ribosomes (Fig. 25). The cytoplasm adjacent to this atypical cisternal surface is paler than the rest of the cytoplasm and contains few ribosomes. Thus, to varying degrees, the endoplasmic reticulum exhibits a fragility or susceptibility to disruption in this cell line.

Structures uniquely present in the gut PGC’s are annulate cisternae of the endoplasmic reticulum. Profiles of these elements are short bilamellar units usually exhibiting two or three regions of close membrane apposition having an annular form (Figs. 11–15). The annulate cisternae characteristically bear a striking resemblance to those regions of the nuclear envelope possessing nuclear pore complexes (Fig. 11, 13). An electron-dense ‘diaphragm’ is seen where the apposing membranes appear to meet, and perhaps fuse. On either side of this diaphragm there is moderately dense fibrillar material. Contiguous portions of the membranes appear as typical ribosome-studded endoplasmic reticulum. Annulate cisternae have not been detected in the later migrating PGC’s or in the gonadal germ cells.

Other cytoplasmic elements seen only in the young PGC’s, although with less regularity than annulate cisternae, are dense bodies which appear to consist of small granules embedded in a fibrillar matrix (Figs. 13–15). These bodies are variable in size and outline but are generally small, ranging from 200 to 250 nm in diameter. Some larger granules at the margin of these bodies have the dimensions of ribosomes. Additional dense bodies, which may be related to the perinuclear granulofibrillar bodies, are widely distributed throughout the cytoplasm (Figs. 13, 14). These bodies, however, are smaller (about 80 nm in diameter), more compact, and component particulate elements cannot be discerned. Each of these types of dense body has been observed only in PGC’s at this developmental stage and therefore, with the annulate cisternae, may constitute fine structural identifying features of the earliest PGC’s.

Mitochondria of the young PGC’s generally present oval or rod-shaped profiles and possess irregularly disposed cristae and a dense intercristal matrix (Figs. 11,13, 15). Mitochondria of the mesenteric PGC’s have a swollen appearance; the cristae are usually short and reduced in number and the intercristal matrix is very transparent (Fig. 16). Mitochondria of the gonadal germ cells also exhibit round profiles, but the cristae are more distinct and extensive and the matrix is moderately dense (Figs. 20, 24).

The Golgi apparatus is not a prominent feature in the young PGC’s, and it is evidently so small that in most sections of these cells it is not detected at all. It is possible, however, to detect a small Golgi complex in many mesenteric PGC’s, while the Golgi apparatus of the gonadal germ cells typically consists of numerous saccules and small associated vesicles (Fig. 20). Although the PGC’s are migratory cells, only a few pseudopod-like regions of the cell surface have been detected. Subsurface microfilaments are apparent within the cytoplasmic processes and under the sinuous membrane contours which have been seen in some of these cells (Figs. 16–18).

A few short regions of close apposition of cell membranes are seen between the migratory PGC’s and neighboring endoderm and mesoderm cells (Fig. 19). These appositional zones do not resemble any of the cellular junctions typically found in various mature cells. The small focal junctions encountered here usually have an intermembrane space of 9 nm and resemble those between mesenchyme cells of young embryos (Trelstad, Hay & Revel, 1967). The plasma membrane of adjacent cells in the gonad are closely parallel over most of their extent, having an intermembrane space of about 20 nm (Fig. 21). In addition, many of the gonadal germ cells share extensive close, or gap, junctions with both germinal and stromal cells adjacent to them (Figs. 22, 23); in some sections these junctions extend for as long as 1 μ m. A narrow cleft, about 2-5 nm wide, often separates the apposing membranes, but in some places the outer leaflets appear fused (Figs. 22, 23). The outer leaflets, whether fused or not, are more electronopaque than they are at other regions of the plasmalemma (Figs. 21–23). Subsurface cytoplasmic filaments are not detected in the zone of these gap junctions.

In summary, the germ cells of all stages inspected display several common characteristics which are not shared by other cell types during this period. These features include, chiefly, a densely fibrillar and granular nucleus, an abundance of ribosomes and polysomes, a paucity of endoplasmic reticulum, and a markedly filamentous cytoplasm. Moreover, alterations among several cellular components during these developmental stages have been noted, for example, the augmentation of the Golgi complex, the appearance of cell junctions and changes in the form of the mitochondria and of the endoplasmic reticulum. Finally, annulate cisternae and dense, granulofibrillar bodies have been discovered as structures distinctive to the early PGC’s among the diverse cells of the 8-day embryo.

Identification of PGC’s

Cells located among the endodermal cells of the primitive gut of the young mammalian embryo have exhibited several distinctive properties upon treatment with a variety of techniques; these cells have been accepted as a single cell type, the primordial germ cell. Thus, the PGC’s have been described as large, round cells with clear cytoplasm (hematoxylin and eosin preparations, Everett, 1943, mouse;Witschi, 1948, human;Chrétien, 1966, rabbit); as having cytoplasmic alkaline phosphatase activity (McKay, Hertig, Adams & Danziger, 1953, human;Chiquoine, 1954, mouse;Bennett, 1956, mouse;Mintz & Russell, 1957, mouse;Jost & Prepin, 1966, calf; Ozdzeñski, 1967, mouse); as moderatesized, actively migrating cells which possess pseudopods (Blandau, White & Rumery, 1963, mouse, in vitro); and as moderate-sized (about 11 μ m in diameter), oval cells which are decidedly basophilic (Bennett, 1956) when stained with the azure A method for nucleic acids (Flax and Pollister, 1949). The basophilia of PGC’s in human embryos is eradicated by RNase incubation (McKay et al. 1953). The distribution of the cells discriminated by all of the above methods is the same; the cells denoted as PGC’s in the mouse embryo, for example, are found, at 8 – 9 days, among cells of the primitive streak, the allantoic diverticulum, the yolk stalk, and the endoderm of the hind- and mid-gut and, at 10–11 days, in the dorsal mesentery and genital ridges. We have identified cells as PGC’s in Epon-embedded tissues on the combined bases of location, size, shape, nucleo-cytoplasmic ratio, and marked basophilia, and have been able to show, at the fine structural level, that these cells have several further characteristics that are not prominent in the surrounding population of cells of the embryo at these stages. These features include abundant polysomes, a moderately dense filamentous cytoplasm, annulate cisternae, and dense granulofibrillar bodies. Moreover, the PGC’s display few profiles of the endoplasmic reticulum and the Golgi complex whereas these structures are regularly observed in the diverse somatic cells. The sparse endoplasmic reticulum of the PGC’s seems to be especially fragile, often appearing disrupted. Finally, although the PGC’s are often situated within an epithelial layer, the gut or coelomic lining, they are never seen to share cell junctions, which regularly occur between the epithelial cells, with adjacent cells. We believe that a continuity of morphological features in the germ cell lineage from the early migratory cell to the gonadal germ cell is demonstrated in this study and the identity and fate of the young PGC’s is established. In view of the heterogeneity of former methods in characterizing PGC’s, we suggest that this present set of cellular features, observed by conventional light and electron microscopy, may serve as important criteria for recognizing the cells of the germ line.

Migration

It has been presumed that the PGC’s actively migrate to the genital ridge since no extensive endodermal or mesodermal displacement occurs that could account for the transposition of the PGC’s from the posterior gut to the genital ridge. An in vitro study of the tissues of the hindgut, midgut, and genital ridge of the young mouse embryo has revealed a class of cells, designated as PGC’s, which displays amoeboid activity (Blandau et al. 1963). For such cellular activity, it may be expected that cytoplasmic flanges of the PGC’s would contain a subsurface microfilament network as in glial cells (Spooner, Yamada & Wessells, 1971). The present observations haveuncoveredafew, small, pseudopodlike elements in which fine filaments can be discerned. That but few of these cytoplasmic projections have been recorded in this study may be due to the fact that most of the sections examined were transverse to the antero-posterior axis of the embryo, and thus transverse to the expected path of migration of the PGC’s within the endoderm as well. The chances are slim, therefore, of recognizing the leading edge of a migrating cell. It may be pointed out that there is a poverty of fine structural studies of migrating cells in situ to discover clues to the mechanisms of motility. Thus, there are no observations entirely comparable to these on the mouse PGC’s.

On the other hand, firm cell junctions and large bundles of tonofilaments would not be expected in PGC’s or other actively migrating cells. Such structures are indeed absent from migratory PGC’s which instead exhibit several small focal junctions; they resemble those typical of temporary junctions seen in migrating mesenchymal cells of the young chick embryo (Trelstad, Hay & Revel, 1967).

The extensive close junctions seen in the gonadal germ cells perhaps reflect the permanence of location of these cells by this time. The extreme electron-opacity of the outer leaflets of the apposed membranes has no counterpart in sections through diverse cell junctions of a number of mature tissues (Farquhar & Palade, 1965; Brightman & Reese, 1969; McNutt & Weinstein, 1970). The outer leaflet of the cell membranes may seem to be fused or separate within a single cell junction. In spite of the apparent fusion of the outer leaflets, the measurement across the membrane complex, about 17 nm, suggests that this junction is a gap junction (McNutt & Weinstein, 1970). These dense gap junctions of the germ cells have not been studied further and their full significance is unclear.

It has been previously suggested that the mobility of PGC’s between the closely apposed cells of the primitive gut, mesenteries, and gonad depends on a lytic destruction of the surrounding cells by the PGC’s (Witschi, 1948; Cuminge & Dubois, 1971). We have not, however, seen any evidence of cell death in the immediate vicinity of PGC’s.

Germ plasm

The germ plasm is considered to be a special cytoplasmic region of the egg which, during cleavage, becomes segregated into a small number of cells of the embryo, and determines the fate of this cell lineage as gonocytes (Wilson, 1925; Nieuwkoop, 1949; Davidson, 1968). In oocytes and fertilized eggs of various animals, cytoplasmic determinants have been detected and they have been traced into PGC’s of the embryo (for reviews, see Wilson, 1925, and Davidson, 1968; also Bounoure, Aubrey & Huck, 1954; Blackler, 1958; Geyer-Duszinska, 1959; Niklas, 1959; Mahowald, 1971 a). These determinants have been characterized in some eggs as dense aggregates of RNA- and protein-containing granules and fibrils (Bounoure et al. 1954; Smith, 1966; Czolowska, 1969; Mahowald, 1971a, b, c; Mahowald & Hennen, 1971). No such elements have been reported thus far in the mouse oocyte or egg (Weakley, 1968; Odor & Blandau, 1969; Zamboni, 1970,1971); however, it is not apparent that studies of these cells have been directed to uncovering germ cell determinants.

We hoped to examine the question of germ cell determinants in the mouse by first identifying the distinctive cytoplasmic structure of the PGC’s and then, with this information, turning both to earlier stages and to the embryonic gonad to seek a continuity of these markers. Our initial observations reveal annulate cisternae and dense granulofibrillar bodies as cytoplasmic elements unique to the early PGC’s. These structures have not been detected in the mouse egg (Weakley, 1968; Odor & Blandau, 1969; Zamboni, 1970, 1971); however, until a systematic investigation of the egg and early cleavage stages is presented, the question remains open whether the annulate cisternae and granulofibrillar bodies have a direct descent from the egg cytoplasm.

On the other hand, these cytoplasmic elements may be new formations in the PGC’s required for differentiation. The annulate cisternae of the endoplasmic reticulum bear a resemblance to annulate lamellae which have been frequently reported in several germ cells (Kessel, 1968; Weakley, 1968; Barton & Hertig, 1972). Both annulate lamellae and dense granulofibrillar bodies have been judged by some investigators as structures involved in future synthetic activities in a number of differentiating cells (Rebhun, 1961; Kessel, 1968; Wischnitzer, 1970). Presumably, the same function may be ascribed to these elements in the PGC’s. It has been suggested that annulate lamellae may give rise to endoplasmic reticulum (Hruban, Swift & Rechcigl, 1965; Wischnitzer, 1970; Barton & Hertig, 1972) and, in fact, in the PGC’s closely apposed membranes of endoplasmic reticulum often exhibit an annulate configuration.

Perhaps the continuity of the germ plasm may be said to reside in the retention by the germ cell line of a so-called ‘embryonic state’. The most persistent features of the early PGC’s and the gonadal germ cells are the great abundance of ribosomes and polysomes and a concomitant paucity of endoplasmic reticulum. In addition, the assumption that nuclear metabolic activity of the PGC is high, evidenced by the compact mass of granules and fibrils in the nucleus, corresponds to the notion that a continuing high production of ribosomes and polysomes occurs in these cells.

This work was supported by research grants from the National Science Foundation (GB 3380 4× and GB 27364) and by a Genetics Training Grant from the National Institutes of Health (5 T01 GM 01918-03). The authors are grateful to Miss Victoria Neufeld for technical assistance. We thank Dr Roy C. Swan, Dr Leonard Ross, and Dr Rosemary Bachvarova for reading the manuscript.

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