The process of primordial germ cell formation in the normal course of development of Xenopus laevis was examined with a light microscope on paraffin and Epon sections of embryos or tadpoles, extending over the period from the gastrula to the feeding tadpole stage. Positional changes of gonocytes with development were nearly the same as those reported on the same species by Blackler (1958) and Whitington & Dixon (1975). The following points were newly demonstrated. Gonocytes which have been located in a deep endodermal position till mid tail-bud stage come to be located in a rather peripheral region of the endoderm cell mass at stage 31 (late tail-bud), suggesting that the initial step of migration of the gonocytes towards the future genital ridge has already begun at this stage. Gonocytes at stages 33/34 and 35/36 were observed in a more dorsal part of the endoderm than at stage 31. Gonocytes which seem to have begun their migration are roundish in external shape and have a large intercellular space around them. At stage 40 gonocytes were located in the dorsal endodermal crest, and at stage 41 gonocytes were found with cell bodies extending over both the dorsal endoderm crest and the dorsal mesentery, indicating that the separation of the gonocytes from the endoderm was in progress at this stage. The present results seem to indicate that gonocytes migrate not passively but actively from the deep endodermal position to the genital ridge, passing through the dorsal mesentery. Counting the number of gonocytes at successive stages of development revealed that gonocytes proliferated exponentially throughout the developmental stages from gastrula to tadpole.

A cytoplasm named ‘germinal plasm’ which shows specific stainability in histological sections has been found in cells of the germ line of anuran eggs of various species; Rana temporaria (Bounoure, 1929), R. pipiens (DiBernardino, 1961), R. esculenta (Hammer, cited by Blackler, 1966), Xenopus laevis (Blackler, 1958; Nieuwkoop & Faber, 1967), Bufo bufo (Blackler, 1958) and Discoglossus pictus (Gipouloux, 1962). Thus, the ‘germinal plasm’-bearing cells may either be called gonocytes or presumptive primordial germ cells (pres. GCs).

Recently Whitington & Dixon (1975) reported, using a paraffin section method, quantitative studies on the ‘germinal plasm’ and gonocytes during early embryogenesis of X. laevis. According to our experience, however, identification of the gonocytes becomes more difficult in paraffin sections as development proceeds, especially after gastrulation, and this difficulty seems to result from a quantitative reduction of the plasm per gonocyte.

In the present study, Epon sections were examined with the light microscope, extending over the period from the gastrula to the tadpole stage. With this method, the ‘germinal plasm’ is seen as granular cytoplasm which is not found in other somatic endoderm cells. As compared with the paraffin section method, the structure of cells and tissues can be well preserved and the borders of cells are distinctly seen. Furthermore, gonocytes having a tiny amount of ‘germinal plasm’ which are probably overlooked in paraffin sections can be detected in Epon sections. This point is important particularly in determining the number of gonocytes.

Freshly laid fertilized eggs were obtained from X. laevis by injecting 200 and 300 i.u. of gonadotropic hormone, respectively, into sexually matured males and females. Embryos were staged after Nieuwkoop & Faber (1967).

All specimens at stages 10–41 were fixed with 7 % glutaraldehyde and 4 % paraformaldehyde in 04 M phosphate buffer, pH 7·3 for 7–24 h. They were postfixed with 1·0 % osmium tetroxide in the same buffer in an ice-bath for 4–6 h. After fixation they were embedded in Epon (Luft, 1961). Sections were cut on a Porter-Blum MT-2B ultramicrotome. To identify the gonocytes accurately, from every eight sections two successive sections, about 0·5–0·7 μm thick, were mounted on slides. Sections of embryos or tadpoles at stages 10, 18, 31, 33/34, 35/36, 40 and 41, all of which were obtained from the same batch, were stained with 0·5 % toluidine blue in 0·5 % borax solution and mainly used for observation of the location of gonocytes. In order to examine the number of gonocytes, sections of embryos at stages 12, 17, 23, 28 and 33/34, which were obtained from another egg-batch, were deprived of Epon by treating them for 20 min with potassium hydroxide and were stained with toluidine blue for 1 min, and aniline blue and orange G for 1 h, a slight modification of the method previously reported (Kotani, Ikenishi & Tanabe, 1973). In embryos at stages 10–41 gonocytes were identified by the possession of granular cytoplasm, i.e. ‘germinal plasm’ (see Czolowska, 1972; Ikenishi & Kotani, 1975). Since the granular cytoplasm was not observed in about half the number of gonocytes at stage 40 and in almost all gonocytes at stage 41, cells which were located in the dorsal endodermal crest, or within the dorsal mesentery or beneath the dorsal aorta and were rich in yolk platelets and had a roundish shape in sections were also identified as gonocytes at these two stages. The triple staining method mentioned above is superior to the single staining by toluidine blue for accurate identification of gonocytes, because according to the former the ‘germinal plasm’ is seen as an aggregate of mitochondria staining pale blue, which are distinguishable from small yolk platelets staining light yellow.

Tadpoles at stages 43 and 46 were fixed in Bouin’s fluid and embedded in paraplast. Serial sections, about 5 μm thick, were stained with Mayer’s hematoxylin and eosin. PGCs lodged in the genital ridges and dorsal mesentery were identified and counted according to the characteristics reported earlier (Tanabe & Kotani, 1974).

To analyse the features of gonocytes during migration, an embryo at stage 33/34 was serially sectioned and a few of the gonocytes and of the somatic endoderm cells in the embryo were reconstructed three-dimensionally by mounting up the photographic images of the cells in every other section.

Location of gonocytes

Gastrula, neurula and mid tail-bud stages

Embryos at stages 10,17,18, 23 and 28 were examined. The location of the gonocytes at these stages was the same as described for approximately the same stages by Whitington & Dixon (1975). At stage 10 gonocytes usually exist in the lower part of the endodermal cell mass between the floor of the blastocoel and the vegetal pole. At stages 17, 18, 23 and 28 gonocytes are ordinarily situated deeply within the endodermal cell mass below the archenteron cavity. Each gonocyte usually contains a single island of ‘germinal plasm’ of slightly varying size.

In embryos at stages from 17-28, the majority of gonocytes are closely aggregated forming one or two clusters in an embryo (Fig. 1).

Fig. 1.

Key to abbreviations in Figs. 14: a, archenteron; c, coelom; dm, dorsal mesentery; g, gonocyte; gp,germinal plasm; me, mesenchymal cell; n, nucleus; s, somite; y, yolk platelet. Location of gonocytes at stage 18. Epon transverse sections stained with toluidine blue. (A) Cluster of gonocytes situated deeply within the endodermal cell mass. (B) Higher magnification of gonocytes in the area marked in (A). The germinal plasm is seen in six out of eight gonocytes (arrows).

Fig. 1.

Key to abbreviations in Figs. 14: a, archenteron; c, coelom; dm, dorsal mesentery; g, gonocyte; gp,germinal plasm; me, mesenchymal cell; n, nucleus; s, somite; y, yolk platelet. Location of gonocytes at stage 18. Epon transverse sections stained with toluidine blue. (A) Cluster of gonocytes situated deeply within the endodermal cell mass. (B) Higher magnification of gonocytes in the area marked in (A). The germinal plasm is seen in six out of eight gonocytes (arrows).

Late tail-bud stage (stage 31)

Although some gonocytes are still situated deeply within the median part of the endodermal cell mass as seen at the previous stages, the majority of them are found in the lateral part of the endodermal cell mass more adjacent to the splanchnopleure than in the centre (Fig. 2A). This seems to indicate that the initial step of migration of gonocytes towards the future genital ridge has already begun at this stage, since at stage 28 gonocytes were observed closely aggregated to each other in the deep endodermal position.

Fig. 2.

Diagrammatic representations of distribution of gonocytes in embryos of stages 31 (A), 33/34 (B) and 35/36 (C). All specimens were obtained from the same batch. Five specimens were examined at each stage and the positions of all the gonocytes of the five specimens were projected onto the same drawing of the section. Gonocytes are seen more dorsally during stages from 31–35/36. The initial step of the migration of the gonocytes from the endodermal position to the future genital ridge seems to have already begun at stage 31.

Fig. 2.

Diagrammatic representations of distribution of gonocytes in embryos of stages 31 (A), 33/34 (B) and 35/36 (C). All specimens were obtained from the same batch. Five specimens were examined at each stage and the positions of all the gonocytes of the five specimens were projected onto the same drawing of the section. Gonocytes are seen more dorsally during stages from 31–35/36. The initial step of the migration of the gonocytes from the endodermal position to the future genital ridge seems to have already begun at stage 31.

Just hatching (stage 33/34) and shortly after hatching (stage 35/36)

Almost all gonocytes are located in the dorsal region of the endoderm around the archenteron on both sides and in the dorsal crest of the endoderm (Fig. 2B, C). Considerable numbers of these gonocytes are adjacent to the splanchnopleure. On the whole gonocytes at stage 35/36 are located more dorsally than at stage 33/34 as seen in Fig. 2C.

Tadpole (stages 40–46)

At stage 40 gonocytes are still located in the dorsal endoderm (Fig. 3 A). The coelomic cavity and the dorsal mesentery are not yet formed at this time. At stage 41 gonocytes are observed in three different regions, i.e. the dorsal endoderm region; the boundary region between the dorsal endodermal crest and the dorsal mesentery, which was just being formed at this time (Fig. 3B), and the nephric region near the dorsal root of the dorsal mesentery (Fig. 3C). Mesenchymal cells, which have differentiated from the lateral plate mesoderm, are observed between the outermost layer of splanchnic mesoderm and the endoderm proper. The mesenchymal cells were observed on both lateral sides of a gonocyte which was located in the boundary region between the endodermal crest and the dorsal mesentery, but not on the ventral side (Fig. 3B). And in other transverse sections through the anterior and posterior parts of the same gonocyte, where the gonocyte is seen located within the dorsal mesentery, mesenchymal cells are observed between the gonocyte and the dorsal endoderm cells (Fig. 3C). At stage 43 the gonocytes are observed in both the dorsal mesentery and in the paired genital ridges, and at stage 46 almost all gonocytes lie in the paired genital ridges.

FIGURE 3

Histological sections showing separation of gonocytes from the endoderm. Epon transverse sections stained with toluidine blue. (A) Stage 40; a gonocyte (arrow) is located in the uppermost part of the dorsal endoderm. Gonocytes leaving the endoderm were not observed at this stage. The coelomic cavity and the dorsal mesentery are not yet formed at this time. (B) Stage 41; a gonocyte (arrow) lies partly in the dorsal mesentery being formed at this time and partly in the dorsal endodermal crest, indicating that the gonocyte is separating from the endoderm. Mesenchymal cells are not found between the gonocyte and the endoderm cells. Another gonocyte is seen at the dorsal root of the mesentery, having already left the endoderm. (C) Stage 41; section through the anteriormost part of the same gonocyte as indicated by arrow in Fig. 3 B. Note the mesenchymal cells between the gonocyte (arrow) and the dorsal endoderm cell, indicating that the gonocyte is breaking through the mesenchymal sheet surrounding the endoderm.

FIGURE 3

Histological sections showing separation of gonocytes from the endoderm. Epon transverse sections stained with toluidine blue. (A) Stage 40; a gonocyte (arrow) is located in the uppermost part of the dorsal endoderm. Gonocytes leaving the endoderm were not observed at this stage. The coelomic cavity and the dorsal mesentery are not yet formed at this time. (B) Stage 41; a gonocyte (arrow) lies partly in the dorsal mesentery being formed at this time and partly in the dorsal endodermal crest, indicating that the gonocyte is separating from the endoderm. Mesenchymal cells are not found between the gonocyte and the endoderm cells. Another gonocyte is seen at the dorsal root of the mesentery, having already left the endoderm. (C) Stage 41; section through the anteriormost part of the same gonocyte as indicated by arrow in Fig. 3 B. Note the mesenchymal cells between the gonocyte (arrow) and the dorsal endoderm cell, indicating that the gonocyte is breaking through the mesenchymal sheet surrounding the endoderm.

Number of gonocytes

Gonocytes were counted in embryos obtained from the same batch at stages 12, 17, 23, 28, 33/34 and 46. The average number of gonocytes per embryo is shown in Table 1. The number increases about four-fold during the period from gastrula to feeding tadpole stage (stages 12–46), suggesting that (on the average) about two divisions per gonocyte take place during this period. As to the proliferative pattern of gonocytes, the results seem to indicate that the divisions of gonocytes are taking place at about a constant rate throughout the stages 12–33/34 and then stop.

Table 1.

Average number of gonocytes in animals at different stages*

Average number of gonocytes in animals at different stages*
Average number of gonocytes in animals at different stages*

Morphological characteristics of gonocytes during migration

Gonocytes which have begun the migration from the deep endodermal position to the genital ridge exhibit some morphological characteristics. Such gonocytes are roundish in external shape (Fig. 4A, B, C), being different from the surrounding somatic endoderm cells whose shape is rather polygonal. And the intercellular space between gonocytes and neighbouring endoderm cells is larger than that between somatic endoderm cells. Furthermore, in the former the width of the space is not uniform and is sometimes larger at the hollow of the gonocyte surface (Fig. 4B, C), whereas that of the latter is mostly uniform. Subsequently the gonocyte appears to be isolated from the surrounding endoderm cells (Fig. 4A, B, C). All gonocytes at stages 17–28 and a few gonocytes at stages 31 and 33/34, which were situated deeply within the median part of the endoderm cell mass, were morphologically not different from somatic endoderm cells except for the inclusion of ‘germinal plasm’ (Fig. 1B).

FIGURE 4

Morphological characteristics of gonocytes during migration. (A) Cross-section from a tadpole at stage 35/36, showing gonocyte (arrow) in which a chromosome-like structure is seen in a bilobed nucleus whose plasm is stained very weakly. The ‘germinal plasm’ is somewhat indistinct, being dispersed, but is clearly seen in other sections of the same cell. Toluidine blue staining. (B) Cross-section of tadpole at stage 33/34, showing gonocytes (arrows) situated in the peripheral region of the endoderm. Note their roundish shape and large intercellular space around them. Toluidine blue staining. The gonocyte indicated by the large arrow was reconstructed three-dimensionally and is shown in Fig. 4F. (C) Cross-section from tadpole at stage 33/34, showing elongated gonocyte (arrow) located in the peripheral part of the endoderm, suggesting amoeboid motility. Note the large intercellular space contiguous with a dorsal half of the cell body. Triple staining with toluidine blue, aniline blue and orange G after removal of Epon. (D) Cross-section from tadpole at stage 33/34. A somatic endoderm cell (arrow) adjacent to the splanchnopleure was reconstructed three-dimensionally, and is shown in Fig. 4E. (E) and (F) Photographs of three-dimensional reconstruction of somatic endoderm cell of Fig. 4D and gonocyte of Fig. 4B respectively. The dorsal side of the cell is directed upward in photographs. (E) Somatic endoderm cell showing an angular form and sharply-angled cytoplasmic parts. (F) Gonocyte showing that its form is not angular; the large cytoplasmic protrusions are directed towards the dorsal side of the animal. The nucleus is located in the lower portion of the cell.

FIGURE 4

Morphological characteristics of gonocytes during migration. (A) Cross-section from a tadpole at stage 35/36, showing gonocyte (arrow) in which a chromosome-like structure is seen in a bilobed nucleus whose plasm is stained very weakly. The ‘germinal plasm’ is somewhat indistinct, being dispersed, but is clearly seen in other sections of the same cell. Toluidine blue staining. (B) Cross-section of tadpole at stage 33/34, showing gonocytes (arrows) situated in the peripheral region of the endoderm. Note their roundish shape and large intercellular space around them. Toluidine blue staining. The gonocyte indicated by the large arrow was reconstructed three-dimensionally and is shown in Fig. 4F. (C) Cross-section from tadpole at stage 33/34, showing elongated gonocyte (arrow) located in the peripheral part of the endoderm, suggesting amoeboid motility. Note the large intercellular space contiguous with a dorsal half of the cell body. Triple staining with toluidine blue, aniline blue and orange G after removal of Epon. (D) Cross-section from tadpole at stage 33/34. A somatic endoderm cell (arrow) adjacent to the splanchnopleure was reconstructed three-dimensionally, and is shown in Fig. 4E. (E) and (F) Photographs of three-dimensional reconstruction of somatic endoderm cell of Fig. 4D and gonocyte of Fig. 4B respectively. The dorsal side of the cell is directed upward in photographs. (E) Somatic endoderm cell showing an angular form and sharply-angled cytoplasmic parts. (F) Gonocyte showing that its form is not angular; the large cytoplasmic protrusions are directed towards the dorsal side of the animal. The nucleus is located in the lower portion of the cell.

At stages 31, 33/34 and 35/36 figures suggesting the prophase of mitosis were noticed in some gonocytes (Fig. 4 A). The nucleus was large and the nucleoplasm was stained very weakly and a chromosome-like structure resembling a slender thread was observed in such a nucleus.

The typical three-dimensional forms of gonocyte and somatic endoderm cell at stage 33/34 (Fig. 4B, D) are illustrated in Fig. 4E and F. In general, gonocytes are not angular in form and have the cell body protruding towards the dorsal side of the animal. Somatic endoderm cells, on the other hand, are usually angular in form and frequently have a large and sharply-angled process which is not always facing the dorsal side.

The cephalo-caudal range of distribution of gonocytes

Length of the cephalo-caudal extent within which gonocytes were localized was estimated from the total thickness of the cross-sections in which all the gonocytes were found. Along the cephalo-caudal body axis, gonocytes are found within a range of length (with standard error) or 254·2 ± 44·3 μm at stage 18, 555·3 ± 88·1 μm at stage 31, 687·0 ± 57·1 μm at stage 33/34, and 840·0 ± 32·8 μm at stage 35/36. These values indicate that the fertile region enlarges with development. On the other hand, the numbers (with standard error) of the somites corresponding to the fertile region at stages 31, 33/34 and 35/36 were 4·2 (± 0·6), 4·8 (± 0·6) and 4·5 (± 0·2) respectively, i.e. the numbers do not increase significantly. Therefore it may be concluded that the enlargement of the fertile region is correlated with elongation of the endodermal cell mass as a result of the cephalo-caudal stretching of the body taking place at these stages.

The displacement of the gonocytes from a deep endodermal position at the neurula stage to the dorsal endodermal crest at the tadpole stage appears to be due to an active movement of the gonocytes since, as has been often presumed previously, no extensive endodermal or mesodermal displacement which could account for the translocation was observed during the relevant stages. The roundish or amoeboid shape of the gonocytes as manifested by a three-dimensional reconstruction, and the large intercellular spaces around the gonocytes during migration, also seem to support this interpretation. As to the course of migration, the distribution of gonocytes during migration (Fig. 2) suggests that they migrate first towards the lateral side and then migrate dorsalwards. On the other hand, Gipouloux (1964) concluded from the extirpation and implantation experiments in B. bufo that diffusible substance(s) produced by the dorsal axial mesodermal organs attract the gonocytes, and this conclusion has recently been supported by Giorge (1974). Therefore it may be thought, for instance, that a more rapid diffusion of such substance(s) could occur in the endodermal region near the lateral plate mesoderm (splanchnopleure) and this could thus play a role in determining the course of gonocyte migration suggested above.

The histological figures (Fig. 3 A, B, C) suggest that the separation of the gonocytes from the endodermal cell mass may be accomplished in a manner different from those suggested by Humphrey (1925), Blackler (1958) and Whitington & Dixon (1975). Humphrey claimed, from study of three species of Rana and B. americanus, that the gonocytes included in the roof of the archenteron were ‘pinched off’ from the endoderm by the penetration of the lateral mesoderm between the gonocytes and the endoderm proper. From studies mainly in R. temporaria Blackler speculated that the manner of separation was a passive one attending the formation of the mesentery. Whitington & Dixon concluded from their observations in X. laevis that gonocytes left the endoderm, were situated along the endodermal crest just inside the lateral plate mesoderm, and were carried and/or migrated towards the top of the dorsal mesentery when the mesentery was formed. In the present study, at stage 41 a gonocyte was distinctly observed, the dorsal part of which was located in the dorsal mesentery which was just being formed, while the rest was within the region of the dorsal endodermal crest (Fig. 3B). This gonocyte, at the same time, was breaking through the mesenchymal sheet surrounding the endoderm. These facts seem to indicate that at least in some cases of Xenopus the separation of gonocytes from the endodermal crest is achieved by their active migration through a sheet of mesenchymal cells and the dorsal mesentery which is just being formed.

The result of counting gonocytes seems to indicate that they have almost finished their proliferation by the hatching stage (stage 33/34). However, the possibility exists that gonocytes divide as well after stage 33/34, perhaps until they leave the endoderm, because figures suggestive of mitosis were observed in some gonocytes at stages 33/34 and 35/36. A comparison between the pattern of increase in the number of gonocytes found in this study and that of the endoderm cells reported by Woodland & Gurdon (1968) shows that the number of cells of both kinds increases at about a constant rate from gastrula till tadpole stages, and that gonocytes have a higher rate of increase than do the endoderm cells. This might mean that gonocytes have their own control mechanism in cell division different from that of the endoderm cells.

We are grateful to Drs Kenzo Yamagata and Kenjiro Wake, Osaka City University Medical School, for their kind permission to use the ultramicrotome and for their useful advice in the course of this study.

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