Download Differentiation of primordial germ cells in the

/ . Embryo/, exp. Morph. Vol. 38, pp. 93-114, 1977
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93
Differentiation of primordial
germ cells in the embryonic development of
Thermobia domestica. Pack. (Thysanura):
an ultrastructural study
ByJERZY KLAG1
From the Zoological Institute, Jagellonian University
Krakow, Poland
SUMMARY
The primordial germ cells (PGCs) of Thermobia domestica undergo some morphological
changes during the embryonic development. Most conspicuous are the changes in the ultrastructure of the nucleus, whose envelope shows a high degree of activity. Two types of vesicles
bleb off from the nucleus; the ones with the light interior are called the accessory nuclei, the
others, with electron-opaque contents, have been termed the dense bodies. The nucleolus,
initially clustered at the nucleus centre, undergoes dispersion and assembles again towards
the end of embryonic development. At the same time, the sex differentiation of PGCs takes
place. It is preceded by an increase in the activity of Golgi complexes and in the volume of
lysosomes and lamellar bodies, the latter giving rise to lipid droplets. At the early stages of
postembryonic development, preoogonia and prespermatogonia can readily be distinguished.
Preoogonia have a wavy-surfaced nucleus and their cytoplasm contains dense bodies. In prespermatogonia, the nucleus is spherical with smooth envelope and there are no dense bodies
in the cytoplasm. Throughout the period studied there occur nucleolus-like bodies and nuage
material considered to be the germ-cell determinants in this species.
INTRODUCTION
Fine structural studies of the differentiation of primordial germ cells (PGCs)
in the course of embryogenesis are scarce and chiefly deal with either single
instars (Brisson, 1973) or short developmental periods (Starke, 1971; Luchtel
1972a, b). Some more complete accounts are concerned with the ultrastructure of
differentiating PGCs of reptiles (Hubert, 1968 a, b, 1970), amphibians (Mahowald & Hennen, 1971; Al Mukhtar & Webb, 1971; Coggins, 1973; Kalt, 1973)
birds (Dubois & Cuminge, 1967; Cuminge &Dubois, 1971; Fujimoto, Ukeshima
& Kiyofugi, 1975, 1976), and mammals (Franchi & Mandl, 1962, 1964;
Gondos & Conner, 1973; Gondos, Renston & Conner, 1973; Jeon & Kennedy,
1973; Spiegelman & Bennett, 1973; Zamboni & Merchant, 1973; Clark &
Eddy, 1975). As regards studies on insects, the investigators have paid special
1
Author's address: Zoological Institute. Jagellonian University, 30-060 Krakow, Poland.
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attention to clusters of basophilic granules, called the polar granules or oosome,
which are found at the posterior pole of the egg in the Diptera, Coleoptera,
Hymenoptera (reviewed by Nelsen, 1934) and Collembola (Tamarelle, 1972,
1974). Many studies carried out over the last 100 years on the role of polar
granules have shown that they act as germ cell determinants (for review see
Counce, 1973). Because the PGCs of these insects differentiate earlier than other
cells, and the process is accompanied by conspicuous structures, namely the
polar granules, the first reports on the ultrastructure of insect PGCs were mainly
concerned with these structures (Mahowald, 1962; Ullman, 1965). Working on
various species of the genera Drosophila and Miastor, Mahowald (1962, 1968,
1971 #, b, 1975) has traced the fate of these determinants during an almost complete developmental cycle of the female germ cells.
However, eggs of the majority of insect species, including Thermobia domestica
do not contain any visible germ cell determinants (Nelsen, 1934). So far, there
have been no electron microscopic studies of the PGCs in such species, although
observations on the differentiation of germ cells at the ultrastructural level might
provide some comparative information which would help to elucidate the question of how the PGCs arise when there are no determinants, and how they
behave throughout the embryonic and larval development.
MATERIALS AND METHODS
Embryos and larvae were fixed in 2% or 4% osmium tetroxide in a phosphate buffer at pH 7-3 for 1-5 h at the temperature of melting ice. In addition
the material was fixed for 3 h in glutaraldehyde in cacodylate buffer, pH 7-4, at
room temperature and, after a rinse in buffer was postfixed for 2 h in 2 % osmium tetroxide in phosphate buffer at pH 7-4. Following dehydration in a graded
series of alcohols and in acetone, the specimens were embedded in Epon. The
accelerator DMP-30 was included in the Epon-acetone mixture to secure adequate infiltration of the yolk material.
RESULTS
The embryonal development of Thermobia domestica begins with oviposition
and last 14 days at 36 °C.
I. The populating of coelomic sacs
(4-6 days after oviposition)
At 4 days of development the primordial germ cells (PGCs) of Thermobia
domestica are in the process of penetration into the coelomic sacs. Their nuclei
are enclosed in a double membrane forming numerous protrusions, the chromatin is packed in clumps, the nucleoli are of irregular shape and consist of several
parts (Fig. 1). The cytoplasm is poor in organelles. Mitochondria usually occur
Differentiation of PGCs in Thermobia embryo
95
2"
Fig. 1. Four-day embryo. A PGC entering the mesoderm. The few organelles
(m, mitochondria, er, endoplasmic reticulum) are assembled in a limited region of
cytoplasm. The nucleolus (nu) is of irregular shape, x 11200.
Fig. 2. Five-day embryo. The cytoplasm of a PGC contains nuage material (ng),
nuceolus-like body (nib), lysome (ly), mitochondria, lipid (/), ribosomes and polyribosomes. x 10000.
7-2
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at one pole of the cell only, where they fill the space between the nucleus and
cell membrane. Few simple cisternae of the endoplasmic reticulum are densely
covered by ribosomes. The Golgi complexes, which comprise four to six cisternae, appear only sporadically in the cytoplasm. The cytoplasm also contains
sparse lysosomes and polyribosomes as well as many free ribosomes. At a short
distance from the nucleus aggregates of material, not bounded by a membrane,
are encountered. This material appears in two forms. Some aggregates are
spherical and show a high electron density, similar to that of nucleoli. These
have been termed 'nucleolus-like bodies'. The others are of irregular shape and
less electron opaque than the nucleolus-like bodies. Such aggregates have been
termed 'nuage material' (Fig. 2).
At 5 days of development all PGCs have lodged in the walls of coelomic sacs.
Now their nuclei are oval, the envelope being much less corrugated than it was
during the migration to the coelomic sacs. Small fat droplets appear in the cytoplasm (Fig. 2).
At 6 days, the nuclear envelope becomes strongly corrugated and forms digital
protrusions into the cytoplasm (Fig. 3). The nucleolus is dispersed throughout
the nucleus, its fragments varying in size and shape, often come close to the
nuclear envelope but never make an immediate contact with it. There is always
some chromatin between the nuclear envelope and fragments of nucleolus (Fig.
4). The outer nuclear membrane, i.e. that facing the cytoplasm, is thickly studded
with ribosomes. The cytoplasm contains many free ribosomes and polyribosomes. Cytoplasmic organelles are usually assembled in. one cell pole, filling
the space between cell membrane and nucleus. The Golgi complexes remain few
in number. A characteristic feature of the cytoplasm at 6 days of development is
the presence of annulate lamellae whose continuity with the endoplasmic
reticulum is easily noticeable, They occur as either complexes of up to four cisternae or single cisternae (Fig. 4) and are found only near the nucleus. These
structures have not been found in the cytoplasm of any other stage dealt with in
this work.
II. Resting period (8-12 days after oviposition)
During the resting period the appearance of chromatin in the nucleus does
not change. The nucleolar substance remains dispersed but the nucleus surface
is more deeply corrugated than it was in the preceding period and forms numerous processes (Fig. 5). Some images suggest that the processes detach from the
nucleus (Figs. 5, 6, 8) giving rise to double membrane-bounded vesicles of
nuclear origin. Two different types of these vesicles have been identified.
Vesicles of the one type generally resemble the nucleus in having a lighter centre
and chromatin associated with the membrane. Their outer membrane is thickly
covered by ribosomes (Fig. 6). Such formations have been called the 'accessory
nuclei' but their subsequent fate is not known. Nucleus-derived vesicles of the
other type contain homogenous, electron-opaque material, the enclosing membrane resembles that of the nucleus except that no ribosomes are ever attached
Differentiation of PGCs in Thermobia embryo
Fig. 3. On the 6th day of development finger-like processes appear at the nucleus
surface, x 10000.
Fig. 4. Six-day embryo. The nucleolus disintegrates and its fragments draw near the
nuclear envelope, but remain separated from it by a layer of chromatin (arrows).
Annulate lamellae (al), characteristic of this developmental stage, appear in the
cytoplasm, x 16000.
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Fig. 5. The formation of 'dense bodies' (db) in a PGC is accompanied by bundles
of microtubules (mt). x 36000.
Fig. 6. Eight-day embryo. An accessory nucleus (an) is seen as it separates from
the nucleus of a PGC. x 50300.
Fig. 7. Ten-day embryo. The contents of a nuclear process (nb) are more electronopaque than chromatin. The Golgi complex (d) is encircled by an endoplasmic
reticulum cisterna (er). Also present are oval mitochondria, polyribosomes, nuage
material (ng), nucleolus-like body (nib), and a lamellar body (Ib). x 11000.
Fig. 8. Eight-day embryo. A dense body (db), enclosed in a double membrane, is
seen adjacent to the nuclear process (nb). x 31000.
Differentiation of PGCs in Thermobia embryo
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to it. These vesicles are termed the 'dense bodies'. They arise from nuclear processes filled with material of a much higher electron density than that of the
nucleus interior (Fig. 8). Microtubules are involved in the separation of nuclear
fragments (Fig. 5). The initially irregular shape of the dense bodies becomes
spherical or oval (Fig. 8) as the development progresses, and the enclosing
double membrane is replaced by a single membrane (Figs. 18, 21).
Mitochondria in the PGCs are oval, sometimes slightly elongated (Fig. 7).
Lysomes as well as the nucleolus-like bodies and nuage material undergo no
changes. During the period in question there appear some more single cisternae
of the rough endoplasmic reticulum. Some of them partly surround the dictyosomes forming characterstic complexes (Fig. 7). Also encountered are centrioles,
each constructed of nine pairs of fibrils, very seldom arranged perpendicular
to each other in a pair, a linear arrangement being most frequent (Fig. 10). One
centriole is usually covered on one side by a cisterna of smooth endoplasmic
reticulum forming a sort of cap, and connected with the other one by fine
filaments (Fig. 10).
Towards the end of the resting period (at 12 days of development) many
small vesicles appear at the periphery of Golgi complexes, and particularly between the outer dictyosomal cisterna and that of endoplasmic reticulum (Fig. 9).
The electron-dense lamellar bodies increase in volume and some of them, especially in their central part, assume the appearance of lipid droplets (Fig. 12).
III. The earliest manifestation of sex differences
(13-15 days after oviposition)
Sex differentiation of the PGCs becomes visible in all embryos of this age. At
the ultrastructural level, one cell type resembles future oogonia, the other spermatogonia.
PGCs of the oogonial type have a nucleus with sinuous outline (Fig. 13),
their mitochondria are oval or round with numerous cristae. A number of
vesicles of the smooth endoplasmic reticulum and some rough endoplasmic
reticulum cisternae are present in the cytoplasm. The Golgi complexes, which
consist of straight cisternae, are surrounded by vesicles (Fig. 15). There also
occur dense bodies, now enclosed in a single membrane, with electron-opaque
inclusions inside (Fig. 18).
PGCs of the spermatogonial type have oval nuclei with only slightly wrinkled
envelope (Fig. 16). The mitochondria are oval or round, seldom elongated,
with very few cristae. The smooth endoplasmic reticulum is far less abundant
than that of the oogonial type PGCs. The rough endoplasmic reticulum forms
short, branching cisternae (Fig. 16) with occasional dilations. The Golgi complexes are composed of either straight or arched cisternae.
Besides the above differences, there are some similarities between the two cell
types. Nucleolar substance is confined to small areas in the nuclei. The cytoplasm
contains lysosomes of different sizes (Figs. 11, 17). Also present are electron-
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Differentiation ofPGCs in Thermobia embryo
101
dense lamellar bodies, varying greatly in size (Figs. 17, 19), and lipid droplets
but in some cases the two structures are hard to distinguish. Images of the structures whose periphery is made up of electron-opaque lamellae and the interior
filled up with lipid suggest that lamellar bodies may turn into lipid droplets
(Figs. 12, 19). In addition, both cell types contain nucleolus-like bodies, nuage
material (Fig. 17) and centrioles. Degenerating PGCs, phagocytosed by mesodermal cells, are found in the gonad primordium. In a cell that is on the point of
degeneration, lamellar bodies increase in both number and size, while the amount
of other organelles decreases. When its nucleus becomes pyknotic, the degenerating PGC is gradually encircled and then digested by a somatic cell of the gonad.
Many lamellar bodies and fat droplets remain as residual bodies (Fig. 19).
IV. Transformation of PGCs into prespermatogonia or preoogonia
(early stages of larval development)
In the third-instar larva it is possible to discriminate between male and female
gonads using just a light microscope. There are striking differences in the ultrastructure of the male and female cells.
The prespermatogonia have spherical nuclei (Fig. 20), the nucleolar substance
being condensed into distinct nucleoli. The nuclei of the early spermatogonia may
contain double membrane-bounded vesicles and short cisternae of the annulate
lamellae (Fig. 14). The outer nuclear membrane is very active, blebbing off
many vesicles of various sizes. The smallest are of the size of vesicles associated
with the Golgi complexes, the largest, which take up the shape of elongated
cisternae, may be as long as a mitochondrion's diameter. The mitochondria are
usually gathered at one cell pole, filling the space between the nucleus and
plasmalemma, and seldom occur at other sites. The large Golgi complexes, constructed of many cisternae, are uniformly distributed throughout the cell. In
some sections the Golgi complexes are seen in the form of enclosed concentric
rings. Each Golgi complex is on one side surrounded by a single endoplasmic
reticulum cisterna, and its vicinity, particularly the space between the cisterna and
the Golgi complex, is occupied by a large number of small vesicles (Fig. 20).
The cytoplasm of prespermatogonia in this stage contains lysosomes, lamellar
FIGURES 9-12
Fig. 9. Twelve-day embryo. The Golgi complex becomes more active. Numerous
vesicles appear between the Golgi complex and the surrounding endoplasmic
reticulum cisterna. x 22000.
Fig. 10. The centrioles in the PGCs of the species studied most frequently appear in
a linear arrangement and are interconnected by fine fibrils. One centriole is usually
surrounded by a cisterna of the smooth endoplasmic reticulum (a) x 54000.
Fig. 11. Fourteen-day embryo. The oogonial line. A large lysosome(/y) appears to
contain contours of mitochondria and many vesicles, x 26000.
Fig. 12. In close proximity to the lamellar body (Ib), a lipid droplet is seen (/), its
peripheral portions resembling a lamellar body, x 11200.
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14
Fig. 13. Fourteen-day embryo. The oogonial line. Nuclear membranes of the PGCs
(gc) have a sinuous outline and the mitochondria a large number of cristae. A
somatic cell (sc) is also seen, x 11100.
Fig. 14. Prespermatogonium of the third-instar larva. Smooth membranes and single
cisternae of the annulate lamellae occur in the nucleus interior, x 16000.
Fig. 15. Fourteen days of development - the oogonial line. The Golgi complex (d)
is formed from straight cisternae with a number of accompanying small vesicles,
x 10000.
Differentiation of PGCs in Thermobia embryo
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Fig. 16. Fourteen days of development. The onset of spermatogonial line differentiation. Oval nuclei of PGCs (gc) with smooth contours contain compact nucleolar
substance (nu). Typical of this type of cell, mitochondria with few cristae and endoplasmic reticulum (er) with distended cisternae occur in the cytoplasm. A somatic
cell (sc) is also seen, x 12000.
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19
Fig. 17. Spermatogonial line at 14 days of development. Lamellar bodies (Ib), nuage
material (ng), lysome (ly), and a nucleolus-like body (nib) are seen in the PGCs.
x11300.
Fig. 18. Oogonial line at 14 days of development. A dense body containing electronopaque inclusions is bounded by a single membrane, x 17000.
Fig. 19. Fourteen days of development. A degenerating PGC (rb) has been engulfed
and is being digested by the gonad's somatic cell (sc). Numerous lamellar bodies and
lipid droplets are visible in the residual body (rb). The arrow points to lamellar body
(Ib) undergoing transformation into lipid (/). x 9500.
Differentiation of PGCs in Thermobia embryo
105
Fig. 20 Prespermatogonium of the third-instar larva. The nucleolar substance
appears condensed (nu). The outer nuclear membrane is apparently active - arrows
point to the sites where vesicles are blebbing from the nuclear membrane. A Golgi
complex (d) is encircled by many small vesicles, x 9700.
bodies, lipid droplets and many polyribosomes as well as nucleolus-like bodies
and nuage material. No cytoplasmic bridges were found between prespermatogonia in this period.
In the period of early larval development, the preoogonia are characterized
by a nucleus with diversified surface configuration. The nuclear envelope forms
deep invaginations and long processes, varying in size and shape (Fig. 21). The
chromatin remains condensed as in the previous stages. The nucleolus, which
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Differentiation of PGCs in Thermobia embryo
107
consists of many parts, is situated in the centre of the nucleus. Near the inner
nuclear membrane and inside the nucleus one can see double membranes,
smooth cisternae and like in prespermatogonia, cisternae of the annulate
lamellae (Fig. 22). Vesicles blebbing off from the outer nuclear envelope were
very rarely observed. The cytoplasm contains numerous vesicles and cisternae of
the smooth endoplasmic reticulum and few, somewhat longer cisternae of the
rough endoplasmic reticulum. Just as in prespermatogonia the Golgi complexes
are partly surrounded by the endoplasmic reticulum cisternae but the dictyosomal cisternae are fewer in number and are not accompanied by as many
vesicles. The cytoplasm abounds in polyribosomes. The nuage material and
nucleolus-like bodies are also encountered (Fig. 22). Preoogonia are interconnected by fusomes (Fig. 22). The 'dense bodies', enclosed in a single membrane,
always lie near the nucleus (Fig. 21). Several lipid droplets usually appear at one
pole of the preoogonial cell (Fig. 21). In gonads of both sexes, the degenerating
cells, phagocytosed by the gonad's somatic cells, are found far more often than
in the previous stage.
DISCUSSION
The nucleus
During the embryonic development, the nuclei of primordial germ cells, as
distinct from those of the somatic cells, undergo conspicuous changes. From
the 14th day of development there appear clear differences between the male
and female lines of PGCs. In the male line the digital processes of the nuclear
envelope become smaller and finally the nucleus assumes a smooth outline,
while in the female line the configuration of nuclear surface becomes still more
diversified as compared with the initial stage. The differences in configuration
of the nuclear surface in Thermobia domestica larvae of opposite sex are striking.
As in Thermobia domestica, the nuclei of the oogonia in Chironomus larvae have a
rich surface configuration (Wulker & Winter, 1970). Wulker & Winter (1970)
interpret these images as depicting the process of formation of the accessory
nuclei, which detach from the parent nucleus and pass into the cytoplasm. A
similar process probably takes place in Thermobia domestica.
An interesting problem is the formation in the nuclei of vesicles and cisternae
FIGURES
21-22
Fig. 21. Preoogonium of the third-instar larva. The nucleus has a conspicuously irregular shape, the nucleolar substance being compact (nu). In the cytoplasm, a dense
body (db) is seen abutting on the nuclear membrane. Lipid droplets (/) are concentrated within a small area near the nucleus, x 10000.
Fig. 22. Preoogonium of the third-instar larva. The nucleus contains smooth
membranes and annulate lamellae (al). Short endoplasmic reticulum cisternae, nuage
material (ng), an active Golgi complex (d), and polyribosomes occur within the
cytoplasm. A cytoplasmic bridge (/), connecting two adjacent preoogonia, is seen at
the bottom of the micrograph, x 29000.
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of the annulate lamellae complexes. These structures occur in cells with highly
active outer nuclear membranes. Intranuclear annulate lamellae have been
observed in oocytes, eggs, young embryos and in some embryonic tissues of the
soma (Gulyas, 1971). In contradiction to the cytoplasmic annulate lamellae,
the intranuclear annulate lamellae almost always occur in the form of single cisternae (Boyer, 1972; Gulyas, 1972; Norberg, 1973). Their role is not yet known.
The nucleolus
In T. domestica, the nucleoli of the migrating PGCs are of irregular shape.
During the resting period the nucleolar substance is scattered throughout the
nucleus in the form of spheres and threads of varying size. The nucleoli in the
oogonia of Chironomus are of similar form (Wiilker & Winter, 1970). With the
onset of sex differentiation of the T. domestica gonocytes the nucleolar substance
becomes assembled in a smaller region of the nucleus and takes on a form that
is often seen in the gonocytes of other species, as well as in the somatic cells of
the gonad and in the ectoderm of T. domestica (Klag, unpublished material).
In many animal species the nucleolar substance frequently occurs in a state
of dispersal at vitellogenesis. This condition is believed to augment the potentialities for RNA synthesis due to an increased surface (Herbaut, 1972). The
dispersal of the nucleolar substance has often been found accompanied by the
penetration of a dense, granular substance through the nuclear pores into the
cytoplasm (Kessel, 1969; Cave & Allen, 1971; Osaki, 1971; Eddy & Ito, 1971).
No such phenomenon has been seen in T. domestica. Kalt & Gall (1974) maintain that replication of genes coding for the ribosomal RNA (rDNA) in the
PGCs of Xenopus is a two-phase process. The first phase of replication takes
place at the beginning of sex differentiation of the PGCs, the second phase
occurs only in female cells at the meiotic prophase. In the nuclei of the PGCs of
T. domestica the dispersal of the nucleolar substance falls in an analogous developmental period and may well reflect a similar process going on in both
types of gonocytes in this species. The high activity of the nucleolus during this
period may be related to the formation of the so-called 'dense bodies' from
electron-opaque nuclear processes. The relation between the proliferation of
nucleolar substance and the amplification of genes coding for ribosomal RNA
has been ascertained during oogenesis in Amphibia (Brown & David, 1968,
cited by Hinsch, 1970; Gall, 1968, cited by Hinsch, 1970) and in Echiurida
(Perkowska, 1968, cited by Hinsch, 1970).
Vesicles of nuclear origin (the dense bodies)
The formation of nucleus-derived vesicles has been observed in T. domestica
throughout the resting period, from 6th to 12th day of development.
Microtubules have often been reported to be actively involved in cell movement as well as in shape alteration of cells and nuclei (Granholm & Baker, 1970;
Fullilove & Jacobson, 1971; Handel & Roth, 1971; Karfunkel, 1971). It is
Differentiation of PGCs in Thermobia embryo
109
therefore supposed that in T. domestica microtubules help in the separation of
nuclear processes from the nucleus surface. Electron density of the nuclear
processes, which give rise to the 'dense bodies', is comparable to that of the
nucleoli rather than chromatin. The dispersal of the nucleolus coincides in
time with the formation of electron-opaque nuclear processes. It seems likely
that information in the form of RNA-protein, or rDNA-protein complexes
(Hinsch, 1970; Kalt & Gall, 1974) is being transferred to the cytoplasm by
means of the double membrane-bounded vesicles. During the resting period,
double membrane enclosing the dense bodies becomes substituted by a single
one. A more electron-opaque substance appears within these bodies at the time
of sex differentiation of the gonads. The dense bodies derived from nuclear
vesicles are only present in preoogonia, and not in prespermatogonia. Moreover,
during the resting period, when sex cannot be recognized, these formations do not
occur in all gonads. The problem requires more detailed studies, but it is conceivable that the occurrence of dense bodies will prove to be a very early indication of sex differentiation of the PGCs in T. domestica. The function of these
bodies, however, remains obscure.
Nuage material and nucleolus-like bodies
The nuage material and nucleolus-like bodies (regarded by many investigators
as one and the same substance varying in density, e.g. Fawcett, 1972) are common structures in the cytoplasm of PGCs of nearly all animal groups (Fawcett,
1972; Beams & Kessel, 1974).
The origin of nuage material and nucleolus-like bodies in T. domestica is not
known. Both these structures are already present in the migrating PGCs. It has
to be emphasized that in T. domestica they occur exclusively in the PGCs, and
not in somatic cells. An intimate association of the nuage material with mitochondria is a very frequent phenomenon in many species (Fawcett, 1972). No
such association was observed in T. domestica during the developmental
period studied. Thermobia domestica is one of those species whose eggs have not,
as yet, been found to contain any germ cell determinants. Light microscopic
studies conducted so far on the embryonic development of this species (Woodland, 1957) and oogenesis (Wojewoda, 1973) have revealed no germ cell
determinants. The electron micrographs presented in this work show that both
nucleolus-like bodies and aggregates of nuage material are of a size similar
to that of mitochondria, and so it is not possible to demonstrate them using the
light microscope. The detection by means of the electron microscope of these
structures in the earliest PGCs and subsequent observations during the entire
development allow us to suppose that they exist already in the ooplasm and act
as germ cell determinants.
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Mitochondria and Golgi complexes
The concentration of mitochondria at one cell pole of the PGCs in T. domestica is similar to that in other species (Hubert, 1968a, 1970; Wiilker & Winter,
1970; Al Mukhtar & Webb, 1971; Boyer, 1972; Herbaut, 1972; Brisson, 1973;
Guraya, Stegner & Pape, 1974). The alteration in shape from elongated to oval
exhibited by mitochondria in the course of development is presently difficult to
interpret. Also the changes in density of mitochondrial cristae in prespermatogonia and preoogonia are not understood.
The images of Golgi complexes after the 10th day of development clearly indicate their low activity in the initial stages of PGC development. Along with the
start of sex differentiation of the PGCs appear images suggesting an increased
activity of Golgi complexes. In many animal species the role of the Golgi complex becomes more prominent with the onset of gametogenesis (Sandoz, 1972;
Bilinski, 1976). In T. domestica the increased activity of these structures in the
PGCs occurs earlier than in other species. Cytochemical investigations on the
early oogonia (Herbaut, 1972) and spermatogonia (Descamps, 1971) of
Lithobius forficatus have demonstrated acid phosphatase activity in cisternae
and vesicles of Golgi complexes. The concurrent increase in activity of both
lysosomes and Golgi complexes in T. domestica would suggest that the Golgi
complexes in such an early stage of embryonic development are involved in the
production of lytic enzymes. The enzymes would subsequently be engaged in the
autolysis of some of the organelles in PGCs or in the digestion of degenerating
germ cells by the gonad's somatic cells. Both these processes can be observed
during this period.
Lysosomes, multivesicular bodies and lamellar bodies
Lysosomes, multivesicular bodies and electron-opaque lamellar bodies are
visible in the PGCs as early as the migration period. Multivesicular bodies are
encountered far less frequently than the other two organelles.
In this paper, qualified as lysosomes are structures with dark or polymorphic
contents in which particular cytoplasmic organelles being digested can sometimes be recognized, as well as larger cytoplasmic regions surrounded by an endoplasmic reticulum cisterna and filled with disintegrating organelles such as
mitochondria, reticulum membranes and vesicles. The multivesicular and lamellar bodies are reckoned by many authors among lysosomes (Mahowald, 1962,
1968; Flickinger, 1971; Bournier & Louis, 1971; Delachambre, 1971; Herbaut,
1972; Bottke, 1972; Zamboni & Merchant, 1973), but they have not been so
classified in this study because the images of these structures in T. domestica
suggest that they may have a different role, nevertheless their lysosomal character cannot be excluded. Little can be said about the multivesicular bodies in
T. domestica since they occur extremely rarely and only before the beginning of
resting period (8th day). They could therefore be a kind of yolk-digesting lysosome (Zissler & Sander, 1973; Osaki, 1971).
Differentiation of PGCs in Thermobia embryo
111
Lamellar bodies are conspicuous structures due to their great electron density. Analysis of their appearance in various developmental stages leads to the
supposition that they are lipid precursors. Such transformations of lamellar
bodies into lipid droplets have been observed in the oocytes of Gromphadorrhina
brauneri (Ksiazkiewicz-Ilijeva, personal communication). Describing similar
bodies in a dipteran, Smittia sp., Zissler & Sander (1973) consider them as yolk
material but do not exclude the possibility that they are a kind of lysosome. In
his study of vitellogenesis in the spider Plexippus paykuli, Osaki (1972) unequivocally qualifies the electron-opaque lamellar bodies as lipid precursors.
Studying by the freeze-etching technique the oocyte yolk of a dipteran Liu &
Davies (1972) have demonstrated lamellar structure of lipid in that species.
In T. domestica, the lamellar bodies occur in large numbers in the degenerating,
phagocytosed cells, which would point to their lysosomal character but, on the
other hand, what remains within the gonad's somatic cells of the degenerated
PGCs is lipid. An analogous condition has been reported in other species (Gondos, 1972). Although it would seem that the opinions on the character of lamellar
bodies are contradictory, the above considerations allow of the conclusion that
these organelles may be both lysosomes and structures that produce or accumulate reserve materials. Bluemink (1969) discusses in more detail the question of
identity of the yolk spheres and lysosomes and presents convincing evidence,
based on many examples, for the identity of these organelles in the embryonic
cells of various animals.
REFERENCES
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