/ . Embryo!, exp. Morph. Vol. 72, pp. 183-195, 1982
Printed in Great Britain © Company of Biologists Limited 1982
\ 83
Germ line of Tetrodontophora
bielanensis (Insecta, Collembola). Ultrastructural
study on the origin of primordial
germ cells
By JERZY KLAG 1
From the Department of Zoology, Jagiellonian University, Poland
SUMMARY
In the early embryo of Tetrodontophora bielanensis (up to the stage of 500 blastomeres)
nuage granules occur in two different locations: (1) in areas where the invaginating cleavage
furrows have pushed fragments of the oosome into the yolk mass, and (2) in the oosome
proper. In the first areas the granules are few in number and certain cells that have enclosed
them in their cytoplasm eventually degenerate. The remaining cells arising in these areas are
devoid of any nuage granules and differentiate into yolk cells. A different situation is observed
in the other areas, where certain cells resulting from tangential divisions of the superficial
blastomeres contain many nuage granules and represent primordial germ cells (PGCs). The
incipient PGCs differ from the other cells of the embryo in possessing nuage granules
associated with mitochondria and in lacking any annulate lamellae.
INTRODUCTION
In many animals the fate of primordial germ cells (PGCs) is determined
earlier than that of the remaining cells of the body (Beams & Kessel, 1974). This
is why these cells, particularly in those species whose eggs contain the oosome,
are intensively studied in the hope of learning the mechanism of cellular
differentiation (Mahowald, 1968, 1971a, b, c; Mahowald, Illmensee & Turner,
1976; Mahowald et al 1979; Schwalm, 1974; Schwalm, Simpson & Bender,
1971). Although the biochemical basis of their determination may be similar
in all species, there is considerable morphological variation in the mode of
formation and differentiation of PGCs in different animal groups.
Among the Insecta, the process of PGCs formation has been examined in
many species whose eggs are furnished with the oosome (Counce, 1973; Beams
& Kessel, 1974; MacMorris-Swanson & Poodry, 1980), and also in a number of
those lacking this structure (Klag, 1977; Cavallin & Hajji, 1979). Until a few
years ago, it had been thought that the eggs of Collembola possess no oosome
1
Author's address: Jagiellonian University, Department of Zoology, 30-060 Krakow,
Poland.
184
J. KLAG
(Jura, 1967#, 1972), however, light and electron microscopic studies by Tamarelle (1972, 1979; Garaudy-Tamarelle, 1970) on the formation of PGCs in
Anurida maritima demonstrated the presence of an aggregate of basophilic
material, which she called the oosome, located on the pole opposite the site of
polar body extrusion. According to Tamarelle, (1972, 1979), as soon as the first
cleavage furrow divides the egg of Anurida maritima (holoblastic cleavage) the
oosome is transported to the 'geometric centre' of the embryo. The oosome
includes germ-line determinants in the form of electron-opaque bodies which
occur in clusters and are not bounded by a membrane. After the cell nuclei
have populated this region PGCs arise in the egg centre.
The embryonic development of Tetrodontophora bielanensis has been studied
in some detail by light microscopy (Jura, 1965, 1966, 1967 a, b, 1972;
Gancarzewicz, 1975; Tyszkiewicz, 1976). Both the type of cleavage and the
mode of embryo formation are very similar to those observed in other collembolan species (Jura, 1972). The egg of T. bielanensis contains a large amount of
yolk distributed uniformly except in an area of cortical layer where islets of
basophilic cytoplasm are observed; histochemical staining has revealed the
presence of RNA in this area (Gancarzewicz, 1975). Unlike the situation in
A. maritima, this material, probably representing the oosome, is not carried
upon cleavage to the egg centre but remains at the surface. Some RNA accumulates in the centre of the embryo undergoing cleavage; however, it does not
come from the cortical layer (Gancarzewicz, 1975). This apparent difference in
oosome behaviour between the eggs of A. maritima and T. bielanensis has
suggested also that PGCs of the two species arise in different ways and, in fact,
this appears to be the case.
In the course of my electron microscopic studies of germ-line cells in T.
bielanensis I paid special attention to those cytoplasmic areas of cells that
contained nuage, assuming that here as in many other animals this material
constitutes a typical element of the germ cells.
MATERIALS AND METHODS
Specimens of Tetrodontophora bielanensis were collected in the vicinity of
Krakow, especially on the Bielany Hills, where they can be found in great
numbers in the forest under loose bark of trees, dead leaves, on moss, or on
mushrooms. In nature oviposition occurs during the first days of November.
The animals collected at that time and kept under laboratory conditions laid
eggs in glass dishes filled with fragments of decaying wood, moss and leaves.
The embryos developed in cool chamber at 4 °C. The eggs were examined daily
under the dissecting microscope and fixed in appropriate stage of development.
Before fixation they were dechorionated with 2 % sodium hypochloride. After
a rinse in tap water they were placed in the fixative. They had to be pricked to
facilitate penetration of the fixative into the egg. Best results were obtained by
Fig. 1. Diagram illustrating how portions of the cortical layer are carried into the yolk by the first cleavage furrow and how yolk
cell formation areas are created. (A) Section through an undivided egg showing lenticular thickenings of the cortical layer. (B) The
invaginating cleavage furrows push the cortical layer inside the yolk mass. (C) The newly formed membrane splits the submerged
fragments of cortical layer without pushing them any deeper. Large dots indicate area with nuage granules.
oo
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ft
H
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Fig. 2. Diagram illustrating the formation of primordial germ cells. Large dots indicate area with nuage granules. Arrows point
to the yolk cell formation areas. (A) 32, blastomere stage. (B) 64, blastomere stage. (C) 128, blastomere stage. The cells that
arise within the area where nuage granules occur in large numbers are primordial germ cells.
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Origin ofPGCs in Tetrodontophora
187
pricking with a fine tungsten needle and removing the rest of the chorion with
watchmakers forceps after the primary fixation. The embryos were fixed in
2 % OsO4 in phosphate buffer, pH 7-4, with the addition of sucrose to produce
a final molarity of 0-037 M. After dehydration in a graded series of ethanol
followed by acetone, the material was embedded in Epon 812. Blocks were cut
on a Tesla BS490A ultramicrotome. Ultrathin sections were contrasted with
uranyl acetate and lead citrate and examined with a Tesla BS613 or Philips 300
electron microscopes. Semithin sections were stained with methylene blue. In
order to determine the amount of nuage granules in the embryos, selected
regions were cut into 100 ultrathin sections and nuage granules were counted
in every fifth section. Thus, 20 sections of each cell were analysed in detail.
RESULTS
In the eggs of T. bielanensis the cells containing nuage appear at two separate
sites and consequently, their ultimate fates are different.
/. Cells arising from material transported to the egg centre
With the first cleavage divisions, which in this species are total, small strands
of the cortical cytoplasm showing lenticular thickenings in places (Fig. 1 A) are
driven inside the embryo. This comes about as the invaginating cleavage furrow
pushes into the yolk a thickened portion of the cortical layer, which stands out
in the new surroundings as a clear cytoplasmic islet (Fig. 1B, 3). Such islets are
never carried to the egg centre but remain a short distance from the surface
(Fig. 1C). In all likelihood, the period of cell membrane invagination is followed
by its rapid growth, at which stage the bottom of the furrow splits the islets
instead of pushing it deeper (Fig. 1C). Fragments of the cortical layer are
transported not only from the egg poles but also from any other sites through
which the initial cleavage furrows pass. The islets (Fig. 3) include the smallest
spheres of proteid yolk, lipid droplets, dictyosomes, oval or slightly elongate
cisternae of the endoplasmic reticulum, sparse ribosomes and stacks of annulate
lamellae composed of two to three short cisternae. This cytoplasm also contains
spherical or ovoid mitochondria. The yolk spheres present in cytoplasmic islets
differ from those in other regions of the embryo in that they are very small,
irregular in shape and similar in appearance to lysosomes (Fig. 3). Some of the
cytoplasmic islets examined contained granules made up of fibrogranular material,
unbounded by a membrane, which I called the nuage granules (Fig. 6). There
are two to four such islets in one embryo; they measure 150 by 75 /im and never
reach deeper than half way down the egg radius (Fig. 5). I have called them
yolk-cell formation (YCF) areas.
At a stage of about 64 blastomeres, nuclei appear in the areas of YCF
(Fig. 2C) and rapidly multiply. A striking feature of these nuclei is their small
size; they are more than 10 times smaller as compared with the nuclei of super-
J. KLAG
*&:
>v
Origin of PGCs in Tetrodontophora
189
ficial or internal blastomeres. These nuclei are characterized by smooth outlines
and homogeneous contents similar in density to the cytoplasm, which renders
them difficult of detection under both light and electron microscopes (Fig. 4).
At the stage of about 150 blastomeres the YCF areas contain already many
nuclei, which then become separated from one another by cell membranes;
as a result, 10-15 cells arise in each such area (Fig. 5). Any nuage granules
present in a given YCF area are then included in the cytoplasm of one or several
cells (Fig. 6). Twelve YCF areas were scrutinized by the quantitative method
described above. In five of them none of the 19 cells examined contained any
nuage granules; in the remaining seven YCF areas 10 out of the 28 cells examined contained no granules whereas the other 18 cells possessed from one to six
nuage granules.
At that time two types of cells arise within the YCF areas. The cells that
contain nuage granules become abortive germ cells and those lacking nuage
granules differentiated into yolk cells of type I.
Abortive germ cells
The nuclei of the cells containing nuage granules possess a clear karyoplasm
as the chromatin is condensed in various places (Fig. 7). Thus the nuclei
resemble in appearance those of the germ cells of the adult insect (Bilinski,
1975). The other cells in the group remain unchanged. The transformed nuclei
described above soon begin to show degenerative changes, which then affect
the cytoplasm as well (Fig. 8). Such a cell is surrounded by one of the adjacent
cells of the group and digested (Fig. 8).
Yolk cells of type I
In the cells where no nuage granules were found, no degenerative changes
were observed, either. At a later stage, when the embryo consisted of more than
500 blastomeres, the cells from the YCF areas migrated among the segmented
reserve material and assumed the appearance of yolk cells of type I. Therefore
the main function of YCF area seems to be the production of yolk cells of
type I. These cells eventually differentiate into true vitellophags.
//. Cells resulting from tangential divisions
In an embryo consisting of 64 cells the blastomeres begin to divide tangentially, but the divisions are highly irregular (Fig. 2). As a result of this process
Fig. 3. Fragment of the yolk cell forming area with lysosome-like yolk spheres (y),
lipid droplets (1), a dictyosome (d) and oval mitochondria (m). x 20000.
Fig. 4. A similar area already populated by the nuclei (n), which are homogeneous
and do not differ in density from the surroundings. Besides the organelles seen in
Fig. 3, also annulate lamellae (al) are present here, x 5800.
Fig. 5. A cytoplasmic area derived from the egg surface (ycf), containing many
nuclei similar in density to the cytoplasm. Superficial blastomeres (sb); internal
blastomeres (ib). x 200.
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190
J. KLAG
Origin of PGCs in Tetrodontophora
191
large internal blastomeres filled with yolk spheres and lipid droplets appear
within the yolk mass. A closer examination of these cells has revealed structural
differences which make it possible to distinguish two categories of blastomeres:
one including the primordial germ cells, the other the yolk cells of type II
(Fig. 2C).
Primordial germ cells
The internal blastomeres subjacent to the developing blastoderm are, as the
others, enclosed by cell membranes, possess large, lobate nuclei devoid of
nucleoli and their cytoplasm contains many lipid droplets and yolk spheres
of typical structure (Fig. 9). The endoplasmic reticulum is poorly developed,
assuming the form of short cisternae and vesicles studded with a few ribosomes.
Also the dictyosomes are few in number but show a typical structure and consist
of four to six cisternae. In this stage no secretory vesicles were found accompanying them. The mitochondria are numerous and appear in two forms; those
less frequent (about 40 %) are spherical, the majority (60 %) being cup-shaped
(Fig. 9). Since the mouth of the cups is very narrow, the cups usually appear
ring-shaped in section, the interior of the rings being much lighter than the
cytoplasm. Both types of mitochondria are accompanied by numerous nuage
granules (Fig. 9). The latter are found in close proximity to the nucleus or less
frequently, some distance off and occur in such large numbers that in almost
every section including a fragment of nucleus one or several granules can be
seen. In 25 such cells examined in the above-described manner the number of
nuage granules was found to range between 11 and 21. In this stage, the lysosomes are either very small or absent altogether. No annulate lamellae were
observed in these cells. I consider the above-described cells to be primordial
germ cells (PGCs).
The PGCs are located in the vicinity of the differentiating blastoderm,
sometimes in close contact with this layer or even partly wedged between its
cells (Fig. 10). In two instances PGCs were found to be the constituent elements
of the differentiating blastoderm, with their broad surface facing the periembryonic space. The PGCs lying beneath the blastoderm are loosely disposed
and seldom come into contact with one another, their longer axis being usually
oriented in the direction of the yolk interior.
Fig. 6. Fragment of a cell formed in the YCF area with single nuage granule (ng)
and annulated lamellae (al). x 23000.
Fig. 7. A cell formed in the YCF area and containing few nuage granules (not
present in the micrograph). The nucleus with condensed chromatin as in the germ
cells of T. bielanensis. x 10000.
Fig. 8. Another such area as in Fig. 7. A degenerating cell appears completely
surrounded (arrows) by the cytoplasm of a neighbouring cell (yc). Nucleus of the
degenerating cell (n). x 10000.
7-2
192
J. KLAG
Fig. 9. UndifTerentiated primordial germ cell. The lobate nucleus (n) is surrounded
by a large amount of lipid droplets (1). In close proximity to the nucleus nuage granules (ng) are seen attached to mitochondria, many of which are ring shaped (arrows).
Dictyosome (d). x 11000.
Fig. 10. Primordial germ cell (pgc) wedged between the cells of the developing
blastoderm. x500.
Origin of PGCs in Tetrodontophora
193
Yolk cells of type II
When viewed under the light microscope, the other cells scattered throughout
the yolk mass do not differ at all from those described above; however, the
electron microscopic examination reveals some slight differences. First of all,
these cells lack any nuage granules; their mitochondria are mostly spherical,
the cup-shaped ones amounting to about 20% of all the mitochondria; and, in
addition, the cytoplasm contains stacks of two to four short, parallel cisternae
of annulate lamellae. It should be emphasized that the yolk cells resulting from
tangential divisions (yolk cells of type II) differ from those originating in the
YCF areas (yolk cells of type I). Yolk cells of type II are endoderm cells and
by the end of embryonal development they form the midgut epithelium.
DISCUSSION
The nuage granules of Tetrodontophora bielanensis are strikingly similar in
appearance to polar granules of various insects (Mahowald, 1962; Schwalm
et al. 1971; Schwalm, 1974) or to the germ plasm of amphibians (Czolowska,
1972; Mahowald & Hennen, 1971; Williams & Smith, 1971). The appearance
and behaviour of nuage granules in T. bielanensis suggest that they represent
germ-cell determinants or organelles containing such determinants. The function
of germ-cell determinants probably consists in synthesis, based on their own
m-RNA (Mahowald et al. 1979), of a specific protein which, in turn, acts on
the genes present in the cell nucleus (Ijiri & Egami, 1976). The studies by Ijiri &
Egami (1976) as well as experiments involving centrifugation of Drosophila
eggs (Jazdowska-Zagrodziriska, 1966) suggest that a sufficient amount of germcell determinants must be present in a given cell in order to determine its fate
as a gonocyte. When the egg of T. bielanensis undergoes the first cleavage
divisions, a certain amount of cortical cytoplasm is carried to the egg interior.
It happens often, though not always, that such cytoplasmic areas include
nuage granules, which, if at all present, are few in number and scattered at
random. This is why in the material studied they never occurred in concentrations
high enough to determine any cells as gonocytes; however, their amount and
effect appeared sufficient to prevent the cells from developing into the somatic
cell lineage. The contrary stimuli reaching the cell nucleus apparently impede
any further development and the cell degenerates. The remaining cells become
yolk cells. The areas in question probably represent sites of yolk cell formation,
whereas the nuage granules brought there by chance cause disturbances resulting
in degeneration of the cells that contain these structures. It is therefore clear
that in T. bielanensis the invagination of cortical cytoplasm, which accompanies
the progress of the first cleavage furrows, does not lead to the formation of
PGCs. The latter arise prior to blastoderm formation in an area of the superficial layer of ooplasm containing large amounts of nuage granules, i.e
194
J. KLAG
within the oosome. If the first cleavage furrow carries the entire contents of
the oosome inside the embryo, as is the case in Anurida maritima (Tamarelle,
1979), then the amount of germ cell determinants is indeed large enough effectively to influence the genome of the cells that contain them. From the evolutionary point of view, the formation of PGCs seems to be a rather conservative
process. In many species showing similar patterns of embryonic development
the PGCs arise in the same way. It is therefore strange that within one and the
same order, the Collembola - with a uniform type of embryonic development
(Jura, 1972; Tamarelle, 1972; Tyszkiewicz, 1976), the PGCs should arise in so
diverse a manner.
The author is grateful to Dr F. Kaczmarski of the Medical Academy, Krakow, for the use
of EM facilities in his laboratory. This work was supported by Government Problem Grant
11-1,3.13.
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{Revised version received 14 April 1982)