/. Embryol. exp. Morph. Vol. 36, 1, pp. 197-207, 1976
Printed in Great Britain
197
Observations on the migration and proliferation
of gonocytes in Xenopus laevis
ByMICHIKO KAMIMURA, 1 KOHJI IKENISHI, 2
MINORU KOTANI 3 AND TORU MATSUNO 4
From the Department of Biology, Osaka City University, Japan
SUMMARY
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 Bladder (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.
INTRODUCTION
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 (Bladder,
1958; Nieuwkoop & Faber, 1967), Bufo bufo (Blackler, 1958) and Discoglossus
1
Author's address: Department of Anatomy, Osaka City University Medical School,
Abeno-ku, Osaka 545, Japan.
2
Author's address: Laboratory of Biology, Gifu College of Dentistry, Takano 1851,
Hozumi-cho, Motosu-gun, Gifu Prefecture, Japan.
3
Author's address: Department of Biology, Faculty of Science, Osaka City University,
Sugimoto-cho, Sumiyoshi-ku, Osaka 558, Japan.
4
Author's address: Institute of Biology, Kyoto Prefectural University of Medicine,
Kawaramachi, Kamikyo-Ku, Kyoto, Japan.
198
MICHIKO KAMIMURA AND OTHERS
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.
MATERIALS AND METHODS
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 0-1 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/*m
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
Migration and proliferation of gonocytes in Xenopus
199
Key to abbreviations in Figs. 1-4: a, archenteron; c, coelom; dm, dorsal mesentery;
g, gonocyte; gp,germinal plasm; me, mesenchymal cell; n, nucleus; s, somite; y, yolk
platelet.
Fig. 1. 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).
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 /on 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.
RESULTS
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
200
M I C H I K O KAMIMURA AND OTHERS
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.
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).
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.
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 archen-
Migration and proliferation of gonocytes in Xenopus
201
Table 1. Average number of gonocytes in animals at different stages*
Stage
Stage no.
No. of
animals
Average no. of
gonocytes per animal
with standard error
Mid-gastrula
12
2
Mid-neurula
17
5
Late-neurula
23
5
Tail-bud
28
6
Just hatching
33/34
4
Feeding tadpole
46
16
* All animals examined were obtained from the same
13-5 ±3-5
14-6 ± 1 1
27-8 ±0-6
37-3 ±4-1
600 ±7-5
59-6 ±2-2
batch.
teron 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.
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.
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MICHIKO KAMIMURA AND OTHERS
Migration and proliferation of gonocytes in Xenopus
203
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. IB).
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
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.
204
MICHIKO KAMIMURA AND OTHERS
Migration and proliferation of gonocytes in Xenopus
205
found within a range of length (with standard error) or 254-2 ± 44-3 [im. at stage
18, 555-3 + 88-1 /an at stage 31, 687-0±57-l jum at stage 33/34, and 84O0±
32-8 jLtm 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.
DISCUSSION
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
FIGURE 4
Morphological characteristics of gonocytes during migration. (A) Cross-section
from a tadpole at stage 35/36, showing gonocyte (arrow) in which a chromosomelike 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 sharplyangled 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.
206
MICHIKO KAMIMURA AND OTHERS
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), Bladder (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 Bladder 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 ultramicrotomeand for their useful advice
in the course of this study.
Migration and proliferation of gonocytes in Xenopus
207
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