J. Cell Set. I, 201-216 (1966)
Printed in Great Britain
201
AN ELECTRON-MICROSCOPICAL AND HISTOCHEMICAL STUDY OF THE OOCYTE
PERIPHERY IN BOMBUS
TERRESTRIS
DURING VITELLOGENESIS
C.R.HOPKINS* AND P. E. KING
Zoology Department, University College of Swansea, Singleton Park, Swansea
SUMMARY
The developing ovarian oocyte of Bombus terrestris has been studied using electron-microscopical, histochemical and autoradiographical techniques. In this publication only the events
taking place in the peripheral ooplasm and in the surrounding follicular epithelium are described.
During vitellogenesis three forms of yolk arise. Most of the lipid yolk, together with large
numbers of cytoplasmic organelles, is derived from the accompanying trophocyte cells during
the earlier phases of yolk synthesis. Later, the larger albuminous yolk spheres which form the
major part of the yolk arise at the oocyte periphery as a result of micropinocytotic activity.
Yolk precursor materials taken up in this way are probably derived from the haemolymph.
Glycogen yolk particles appear in the ooplasm in large numbers during the later phases of yolk
synthesis and arise from groups of small vesicles that have been derived largely from the
trophocytes.
Two kinds of follicular epithelial cells occur. All the evidence suggests that the larger, more
common form is extremely active during synthesis of albuminous yolk, although its precise role
remains unclear. The narrower, less common form of follicular epithelial cell occurs only before
albuminous yolk synthesis when its protein secretion is probably transferred to the oocyte.
After yolk synthesis the vitelline membrane is laid down in the intercellular space between the
follicular epithelium and the oocyte, both of which contribute towards its formation. Next the
chorion is laid down. Although only the earlier stages of its formation have been studied, it is
apparent that the chorion is laid down in the intercellular space immediately adjacent to the
vitelline membrane, and that it is formed by the follicular epithelium alone.
INTRODUCTION
Palm (1948) carried out a detailed histological investigation of ovarian structure and
vitellogenesis in the hymenopteran Bombus terrestris and suggested that the auxiliary
cells, and in particular the cells of the follicular epithelium, provided an important
contribution towards yolk formation. In view of recent work on other polytrophic
ovarioles, which has shown that the oocyte obtains a large part of its protein yolk
directly from the haemolymph, it was thought worth while to reinvestigate the processes involved in yolk synthesis in B. terrestris, using electron-microscopical, histochemical and autoradiographical techniques. The present publication is concerned only
with the processes occurring at the oocyte periphery.
* Present address: Histology Department, The University, Brownlow Hill, Liverpool 3
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MATERIALS AND METHODS
Ovaries of B. terrestris were dissected from laying queens or from workers that
had been isolated and fed on sucrose for 2 weeks to promote ovarian development.
For histochemical procedures, ovaries were fixed in a variety of fixatives. On
Bouin-fixed material, mercuric bromophenol blue (Bonhag, 1955) and ninhydrinSchiff (Yasuma & Itchikawa, 1953) were used as general protein tests. On material
fixed for 2 h in formol-calcium-acetate (Cowden, 1962) the Baker modification of the
Millon reaction for tyrosine (Pearse, i960) and the dimethylaminobenzaldehydenitrite method for tryptophan (Adams, 1957) were carried out. Also on material
fixed in this way Baker's (1947) modification of the Sakaguchi reaction was used as a
test for arginine and the 2,2'-dihydroxy-6,6'-dinaphthyl disulphide procedure of
Barrnett & Seligman (1952) was used to demonstrate protein-bound sulphydryl
groups. The method of Adams & Sloper (1955, 1956) was used as a test for proteinbound disulphide groups.
The periodic acid/Schiff (PAS) technique was used as a general test for carbohydrates (Hotchkiss, 1948); 1% diastase was employed to remove and thus detect
glycogen, and methanol/chloroform treatment (12 h at 60 °C) used to remove PASpositive lipids (Bonhag, 1956).
For use with lipid techniques tissue was fixed in formol-calcium/cadmium and
postchromed (Nath, i960), before being embedded in gelatin. Frozen sections were
stained with Sudan black B in 70% ethanol (Baker, 1946, 1956) in order to demonstrate phospholipid. The Nile blue sulphate method of Cain (1947,1948), in accordance
with Nath (i960), was used to detect triglycerides.
The Gomori (1939) method for alkaline phosphatase and the Gomori acid phosphatase procedure as modified by Holt (1959) and Bitensky (1963) were used on
formol-calcium-fixed frozen sections.
Autoradiographic experiments were carried out with laying queens using adenine8-14C sulphate (Radiochemical Centre, Amersham); 0-05 ml (0-5 c) of {14C]adenine in
0-75 % saline was injected into the abdominal cavity and ovaries were subsequently
removed at 5-min intervals, fixed in Carnoy and embedded in paraffin. As a control,
and to determine whether the label had been incorporated into RNA or DNA, RNA
was selectively removed by treatment with N HC1 at 60 °C for 6 min (Lajtha, 1954),
and both RNA and DNA were removed with 4 % trichloroacetic acid at 90 °C for
15 min (Pearse, i960). Extracted and non-extracted slides were washed and coated
with Kodak AR 10 stripping film at 25 °C and exposed in a light-tight box at 5 °C
for 14 days. The slides were developed in D19 B Kodak developer (4 min), rinsed in
distilled water and fixed for 10 min. Without drying the slides were stained with
pyronin/methyl green (Brachet, 1953) at 5 °C for 3 h.
For electron-microscope work individual oocytes were fixed for 1 h at o °C in
1 % osmium tetroxide buffered with phosphate at pH 7-8 and with the addition of
4-5 % sucrose. After rapid dehydration in a series of cold ethanols, the tissue was
embedded in Araldite (Luft, 1961). Sections were cut with a Huxley microtome and
Oocyte development in Bombus
Fig. i. Diagram of a single ovariole, showing the stages of oocyte development referred
to in the text. Stage i shows the germarial region (gr) and the enlarging basiphilic
oocyte (po). Stage 2 shows a discrete follicle, in which the oocyte nucleus (oori) is
prominent. In stage 3 the follicle has enlarged, the trophocyte nuclei (tn) are irregular in outline, and the oocyte prismatic process (pp) is well developed. Stage 4:
the follicle has further enlarged, but the prismatic process is reduced to a nutritive
pore (np). In stage 5 the trophocytes rapidly degenerate to a vestigial group (vgi)
surrounded by enlarged trophocyte follicular epithelial cells. The vitelline membrane
(vm) is laid down around the oocyte. Stage 6: the chorion (ch) is laid down outside the
vitelline membrane, (c, ovariole calyx; os, ovariole sheath.)
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C. R. Hopkins and P. E. King
stained for 15 min with lead citrate (Reynolds, 1963) before being viewed with an
AEI EM 6 electron microscope
Stages of development
During development the oocyte passes towards the base of the ovariole accompanied
by a group of 48 trophocytes and enveloped by a follicular epithelium. Vitellogenesis
proceeds throughout this time so that on arrival at the base of the ovariole the
oocyte is enveloped by the vitelline and chorionic membranes and contains its
full complement of yolk. In order to simplify the description of yolk synthesis,
development of the oocyte has been divided into six arbitrary stages (see Fig. 1),
as follows:
Stage 1. Trophocytes and oogonia/oocytes are in a germarial condition and separate
follicles are distinguishable only in the later phases. The nuclei of the trophocytes and
oocyte are spherical and central.
Stage 2. Trophocytes and oocyte form distinct follicles, the lengths of which are
equal. The prismatic process of the oocyte projects into the trophocyte follicle and the
follicular epithelial cells surround the follicles as a multiple epithelium.
Stage 3. The length of the trophocyte follicle is greater than that of the oocyte
follicle. The prismatic process is restricted to the nutritive pore and the oocyte follicular epithelium forms a single-layered columnar epithelium.
Stage 4. The lengths of the trophocyte and oocyte follicles increase and are equal.
The trophocyte nuclei are irregular in outline.
Stage 5. The trophocytes rapidly degenerate and the oocyte length continues to
increase. The vitelline membrane is laid down around the oocyte.
Stage 6. The oocyte attains its maximum length and only the debris of the degenerate
trophocyte cells remains. The chorion is laid down.
OBSERVATIONS
Stage 1
During this phase the oocyte and its attendant trophocytes arise by incomplete
division from a single cell (Bonhag, 1958). Thus, throughout vitellogenesis these cells
remain interconnected by cytoplasmic bridges through which the trophocytes contribute cytoplasmic organelles and yolk components to the ooplasm (King, i960;
Meyer, 1961; Brown & King, 1964; Hopkins, 1966). At this early stage the strongly
basiphilic nature of the ooplasm enables the oocyte to be distinguished from the trophocytes. Study of the fine structure of the ooplasm shows that its basiphilic nature is
associated with large numbers of free ribosomes; no elements of endoplasmic reticulum
are present.
The germinal vesicle is large and spherical, but only faintly Feulgen-positive. In
addition to a large RNA-positive nucleolar complex there are many smaller RNA
bodies distributed throughout the karyoplasm; these are associated with the formation
of accessory nuclei which are believed to arise in large numbers at this time (Hopkins,
1964).
Oocyte development in Bombus
205
During the later phases of stage 1 the follicular epithelial cells envelop the oocyte
and trophocytes as a multiple layer of undifferentiated cells.
Stage 2
In the early part of this stage, the cytoplasmic bridges which interconnect the
trophocytes and oocyte allow the transfer of yolk constituents and cytoplasmic
organelles to the ooplasm (Fig. 4). This transfer, evident at both light- and electronmicroscope levels, continues throughout the greater part of yolk synthesis and provides an important contribution towards the rapid increase in number of mitochondria and ribosomes and towards the appearance of lipid and glycogen yolk in the
ooplasm (Hopkins, 1966). High concentrations of free ribosomes are present within
the cytoplasmic bridges and in the ooplasm adjacent to them. During stage 3 these
concentrations, which coincide with the extreme basophilia evident with the light
microscope, become distributed at the oocyte periphery, where they remain throughout
yolk synthesis.
Initially, large numbers of mitochondria are concentrated in the ooplasm close to
the cytoplasmic bridges but during the later phases of this stage they become distributed in groups throughout the ooplasm.
During stage 2 the first lipid yolk appears in the ooplasm. This yolk arises in the
trophocytes and is transferred, in particulate form, via the cytoplasmic bridges to the
ooplasm, where it becomes distributed at the periphery. The lipid spheres, which are
approximately 0-5 /i in diameter, stain homogeneously blue-black with Sudan black B,
and blue with Nile blue sulphate. In the electron microscope they appear as spherical,
homogeneous, electron-dense structures (Fig. 6).
During the latter phases of stage 1, groups of small vesicles are present in the trophocyte cytoplasm which, during stage 2, become distributed throughout the ooplasm.
The vesicles are irregular and approximately 50 m/i in diameter. They vary in electron
density; usually, in a group, the vesicles are all electron dense or electron translucent,
but occasionally both types occur in the same group. During stage 3 larger irregular
particles (0-4 /i in diameter) are present at the periphery of each compact group of
vesicles, often surrounding the group in rosette fashion (Fig. 2). Where the larger
particles lie adjacent to the small vesicles, profiles indicate that they are increasing in
size as the small vesicles fuse to their periphery (Fig. 3). The larger particles are
morphologically identical with the glycogen yolk particles which appear in large
numbers during the later stages of yolk synthesis, but it has not been possible
with the light microscope to confirm this observation directly by histochemical
means.
The follicular epithelial cells at this time form a single layer and are slightly columnar, resting on a well-developed basement membrane. Adjacent cells adhere closely
and at their apices they lie against the ooplasmic membrane.
Stage 3
Ribosomes and mitochondria continue to pass from the trophocytes to the ooplasm,
the ribosomes becoming increasingly concentrated at the oocyte periphery. During the
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later phases of this stage mitochondria also become distributed predominantly in the
peripheral region.
In this stage, in addition to the form of lipid yolk deposited during stage 2, a
second kind of lipid yolk is formed. With Sudan black B staining, this form of lipid
yolk particle has a crescentic outline. Following treatment with phenol (Gupta, 1958)
for 24 h, however, it is stained homogeneously. It stains blue with Nile blue sulphate
but is extracted with acetone at 60 °C. In the more central regions of the ooplasm,
during the later phases of stage 3, a third form of lipid yolk particle appears which is
irregular in outline, and has a diameter of between 0-5 and 5 ji. These lipid yolk
particles stain homogeneously blue-black with Sudan black B, pink with Nile blue
sulphate and are extracted with acetone at room temperature. With the electron microscope crescent-shaped structures, which presumably correspond to the second form
of lipid particle, are readily distinguished from the previously formed lipid particles
by their greater electron density (Fig. 6). The fine structure of the third form of lipid
particle appears to vary considerably, probably because of the action of the lipid
solvents used during embedding.
The follicular epithelial cells, which are now columnar, adhere closely together
and two kinds of cells occur. The majority are wide and truncated apically, outnumbering the other narrower, darker-staining cells in a 3:1 proportion. The more
common follicular epithelial cells have large, central and spherical nuclei in which
the numbers of nucleoli and membrane annuli rapidly increase. The basal cytoplasmic regions contain well-developed endoplasmic reticulum and Golgi systems.
Large numbers of free ribosomes are distributed throughout the apical regions
of the cytoplasm and are responsible for the intense basiphilia of these areas.
Few cytoplasmic membrane elements occur in the more apical regions of the cytoplasm.
The narrower, darker follicular epithelial cells stain intensely with protein and
RNA procedures. The cytoplasm surrounding the small irregular nucleus contains
high concentrations of free ribosomes, and in the more apical regions numerous
protein-positive particles are present (Fig. 7).
During the later phases of stage 3 numerous microvilli form at the ooplasmic membrane, they are 3-4 fi long, 0-25-0-75 fi in diameter, and lie adjacent and firmly
adpressed to the membrane of the follicular epithelial cells (Fig. 7).
Stage 4
At the onset of this stage the transfer of ribosomal and mitochondrial elements to
the ooplasm ends and the flow of lipid particles is reduced. Glycogen yolk particles
which appear in large numbers in the trophocyte cytoplasm at this time are, however,
contributed to the ooplasm throughout this stage.
At the onset of albuminous yolk synthesis an intercellular space appears between the
oocyte and the follicular epithelium and at the same time intercellular spaces develop
between adjacent follicular epithelial cells (Fig. 5). The oocyte microvilli bend in all
directions and are seldom seen in longitudinal section along their entire length. Thus
the spaces between them are extensive and irregular, and frequently extensions of the
Oocyte development in Bombus
207
intercellular space occur in the ooplasm as much as 10/t away from the main intercellular region.
In actively laying females the intercellular spaces between the oocyte and the follicular epithelium, and between the adjacent follicular epithelial cells, contain a
densely packed fibrous material which gives positive protein and carbohydrate
reactions (Figs. 8, 9).
At the bases of the pits which are present between the oocyte microvilli, expanded,
vesicular structures occur (Fig. 8). They are about 50 m/t in diameter, and their
limiting membrane, which is confluent with the ooplasmic membrane, is bounded on
its ooplasmic surface by a well-defined array of radiating rod-like structures 12 m/i
in length. Similar vesicular structures, but without apparent connexion to the intercellular space, occur in the peripheral ooplasm (Figs. 8, 9). While some of these apparently free vesicles may be connected with the intercellular space out of the plane
of section, the limiting membranes of the majority are probably not an integral part of
the ooplasmic membrane, although they were probably derived from it.
During stage 4, the microvilli and their associated pits are distributed at the ooplasmic membrane in all regions of the oocyte. The free vesicles are numerous and are
distributed throughout the peripheral ooplasm within 4 fi of the outer limits of the
ooplasmic membrane.
The peripheral ooplasm contains many spherical structures which appear as intermediate forms between the free vesicles and the smaller albuminous yolk bodies
(Fig. 9). The smaller forms of these structures have an electron-dense central region,
a definite limiting membrane, and an outer rod-like coat, similar to those of the expanded vesicular structures present in the pits between the microvilli. The larger
bodies have no similar outer layer of rod-like structures, but usually retain a limiting
membrane. The material at the centre of these bodies is electron-dense, but appears
less compact than in the smaller vesicles. There are frequent profiles which suggest
that these structures may increase in size by the fusion of the smaller vesicles to those
of intermediate size (Fig. 9), or by two or more of the larger bodies coming together.
During this process the limiting membranes fuse and then open, allowing the contents
of the adjoining vesicles to become confluent.
The larger yolk spheres associated with the peripheral activity just described react
positively with general protein, carbohydrate and lipid procedures. More specifically,
they contain tyrosine and arginine, but lack cysteine and tryptophan. Although PASpositive, they do not contain glycogen or acid mucopolysaccharide components. The
lipid is a phospholipid.
Throughout the formation of albuminous yolk the peripheral ooplasm contains
high concentrations of free ribosomes, and more centripetally mitochondria and
accessory nuclei occur in large numbers. This region is intensely RNA- and carbohydrate-positive. Acid phosphatase activity is localized in small (0-2-0-5 /.i) bodies
distributed throughout the peripheral ooplasm. From their size and acid phosphatase
content these bodies are similar to lysosomes (de Duve, 1959), but because of the known
variation in lysosome ultrastructure, identification with the electron microscope from
morphology alone is uncertain.
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The Gomori alkaline phosphatase procedure produces a strong reaction in the
region of the ooplasmic membrane during albuminous yolk synthesis.
During stage 4, large numbers of glycogen yolk particles rapidly accumulate in the
peripheral ooplasm; they are irregular, intensely PAS-positive and malt diastase labile.
Only one kind of follicular epithelial cell occurs during stage 4 although vacuolated,
rather degenerate, dark cells are occasionally observed. The intercellular space
between adjacent follicular epithelial cells allows the tunica propria region to be confluent with the intercellular space surrounding the oocyte. Throughout stage 4 the intercellular regions continue to contain densely packed fibrous material which, as seen with
the light microscope, reacts positively to general protein and carbohydrate procedures.
The cytoplasm of the follicular epithelial cells contains extensive endoplasmic reticulum in the basal regions at this time, although the more apical regions, proximal to the
oocyte, continue to contain mainly free ribosome concentrations. Multivesicular
bodies and lysosome-like structures lie close to the Golgi region, which is perinuclear
and faintly PAS-positive. Towards the end of this phase the endoplasmic reticulum and
Golgi elements become more extensive in the basal regions of the cell, while in the
apical cytoplasm many small (o-1 fi) electron-dense particles appear.
The nucleus of the follicular epithelial cell is large and spherical and up to ten
nucleoli may be cut in one thin section. [14C]adenine is taken up within 5 min after
injection by these cells and is incorporated into the DNA and RNA components of the
nucleus. The cytoplasm also has a concentrated RNA autoradiograph but contains no
labelled DNA material. The intercellular regions and peripheral ooplasm have no
autoradiograph.
During the latter phases of stage 4, the plasma membrane of the follicular epithelial
cells proximal to the oocyte becomes elaborated into long microvilli which protrude
into the intercellular space and interdigitate with the smaller, more numerous ooplasmic microvilli. At the height of albuminous yolk synthesis it is in the regions where
the microvilli of the follicular epithelial cells penetrate most deeply into the ooplasmic
invaginations that the majority of the expanded vesicular structures occur (Fig. 8).
Stage 5
During the earlier phases of this stage the ooplasmic microvilli become reduced and
few expanded vesicular structures are observed. In addition, the follicular epithelial
cells adhere along their adjacent cell membranes, occluding the intercellular spaces
between them. The electron-dense intercellular region which lies between the oocyte
and the follicular epithelium, however, increases in size and a distinct layer, the vitelline membrane, appears in it. The outer surface of the vitelline membrane becomes
clearly defined earlier than the inner, and bears a thin layer of electron-dense material
similar to that described in Calliphora erythrocephala by Wigglesworth & Salpeter
(1962). The inner surface of the vitelline membrane later becomes clearly defined
immediately outside the region of oocyte microvilli which are reduced and closely
adpressed together.
Of the large numbers of small electron-dense particles present in the apical cytoplasm of the follicular epithelial cell during stage 4, only a few remain (Fig. 12). Their
Oocyte development in Bombus
209
rapid disappearance suggests that they represent part of the follicular epithelial cell
contribution towards the formation of the vitelline membrane. Similarly, small
vesicles occurring in the peripheral ooplasm immediately below the ooplasmic microvilli form part of the oocyte contribution towards the synthesis of this membrane.
Okada & Waddington (1959) observed similar bodies in the Drosophila oocyte and
termed them 'vitelline vesicles'.
The plasma membrane of the follicular epithelial cells proximal to the oocyte is
difficult to identify during the earlier phases of stage 5 as the appearance of the electrondense vitelline membrane obscures all evidence of the follicular epithelial-cell microvilli (Fig. 12). In the later phases, however, the plasma membrane of the follicular
epithelial cells becomes identifiable as a convoluted structure outsidethe vitelline and
chorionic membranes. Therefore both these structures are laid down in the intercellular space (Fig. 11).
Following the formation of the vitelline membrane, another electron-dense layer,
0-1-0-2 //, thick, appears in the region between the follicular epithelium and the vitelline membrane (Fig. 11). This layer may be regarded as a delamination of the vitelline
membrane, the two parts of the membrane then being termed the inner and outer
subchoral membranes (Hinton, 1963); or it may represent the innermost layer of the
chorion. Our electron micrographs suggest that in B. terrestris the outermost layer is
part of the inner chorion.
A thicker, less well-defined layer is subsequently formed outside the electrondense layer just described, the intervening region between the two layers being
traversed by numerous trabeculae. These strut-like trabeculae vary widely in thickness
and occasionally branch (Fig. 11).
Stage 6
During chorion formation the follicular epithelial cells decrease in height and their
adjacent cell walls become folded. Their nuclei, originally spherical, become ellipsoidal
and granular. In contrast to these apparently degenerate features, numerous whorls of
endoplasmic reticulum occur in the perinuclear cytoplasm (Fig. 13). In addition,
many Golgi elements are distributed throughout the cytoplasm and numerous translucent vesicles occur below the plasma membrane, in the regions proximal to the
chorion.
In the ooplasm during stage 6 the lipid and glycogen particles occur in the interstices between the densely packed albuminous yolk globules. Lipid yolk is distributed
throughout the ooplasm, but few particles of the L2 form (Nath, i960) are present.
Glycogen yolk particles are concentrated in the peripheral ooplasm, centripetally to an
outer homogeneous layer of ooplasm which appears at this time (Hopkins, 1964).
DISCUSSION
During stage 2 a rapid increase in the number of ribosomes, mitochondria and lipid
yolk particles occurs in the peripheral ooplasm. The trophocytes contribute significantly towards this increase, but the fine structure of the follicular epithelial cells
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suggests that it is unlikely they are concerned with these earlier vitellogenic processes.
The lipid bodies which appear during yolk synthesis are similar to the L2, L2 and L 3
bodies described by Nath (i960) from other insect oocytes. Histochemical procedures
show that the Lx bodies are homogeneously phospholipid in nature; the L2 bodies may
be interpreted as consisting of a phospholipid sheath surrounding a triglyceride core,
and the L3 bodies appear as larger homogeneous triglyceride structures. Nath (i960)
and Arggarwal (1962) have suggested that the Lx bodies become transformed into L2
and L 3 bodies. In B. terrestris, however, no transitional forms between L1} L2 and L3
bodies have been observed with the light- or electron-microscope.
Following the earlier description by Palm (1948) of a second form of follicular
epithelial cell in B. terrestris, similar dark-staining cells have been described in other
insect species (Cruickshank, 1964; King, i960; Lusis, 1963). Palm (1948) stated that
only the dark form of cell contributed towards yolk formation and that it did so by
passing deeply staining particles directly into the ooplasm. Cruickshank (1964) described similar basiphilic cells in Anagaster kuhniella and suggested that they transfer
RNA-positive secondary or accessory nuclei directly into the ooplasm. The present
work suggests that in B. terrestris the large particles occurring in the apical regions of
the dark follicular epithelial cells represent the secretory products of these cells but,
although they react positively to protein-staining procedures, they do not contain
ribonucleoprotein. Further, from ultrastructural observation there is no evidence to
suggest that these particles are passed directly into the ooplasm. The stage of development during which the dark follicular epithelial cells are active indicates that they are
associated with yolk synthesis, and the proteinaceous nature of their secretion suggests
that they are involved with the earlier stages of albuminous yolk synthesis. Their
precise function, however, remains uncertain.
The two forms of follicular epithelial cell are quite distinct, for in the absence of
intermediary stages during the earlier phases, it is unlikely that the narrower dark cells
arise from the wider, more numerous, follicular epithelial cells. In addition, the presence of degenerating dark-staining follicular epithelial cells during the later stages
suggests that, following the completion of their activity, these cells break down.
Although this degeneration may result in the wider intercellular spaces which occur
between adjacent follicular epithelial cells during albuminous yolk synthesis, the
majority of these spaces are only between immediately adjacent follicular epithelial cells.
The fine structure of the wider, more numerous, follicular epithelial cells during
stage 3 indicates that at this time they are synthetically active, but there is nothing
to suggest that they make any direct contribution towards yolk formation in the oocyte
at this time.
The increase in the number of nucleoli and the incorporation of [14C] into DNA
throughout stages 2, 3 and 4 shows an increase in degree of polyteny in the nucleus of
the follicular epithelial cell similar to that occurring in Drosophila melanogaster
(Schultz, 1956). This, together with the rapid labelling of the RNA in the follicular
epithelial cell cytoplasm also illustrates the high metabolic activity of these cells during
yolk synthesis. The high concentrations of basiphilic material in the apical cytoplasm
of the follicular epithelial cells proximal to the oocyte occurs in other insect species
Oocyte development in Bombus
211
(Mulnard, 1948; von Kraft, i960; Lusis, 1963). In B. terrestris they are associated
with large numbers of free ribosomes which occur in this region.
During yolk synthesis in the cecropia moth, Telfer (1961) and Telfer & Melius
(1963) have shown that labelled proteins taken up from the haemolymph are transferred to the oocyte across the follicular epithelium. They suggest that blood proteins
pass across the tunica propria and between the adjacent follicular epithelial cells, to be
taken up at the oocyte plasma membrane by a form of pinocytosis. Recently Roth &
Porter (1962, 1964) have described the ultrastructure of the oocyte periphery in the
mosquito, Aedes aegypti, during yolk synthesis, and in the light of Telfer's work they
have suggested that in this species too, haemolymph material traverses the interfollicular spaces before being taken up at the oocyte periphery. Our observations have
shown that the ultrastructure of the oocyte periphery in B. terrestris is very similar to
that of A. aegypti and serve to confirm the original observations of Telfer et al. and
Roth & Porter. They are also in general agreement with the more recent reports
of Kessell & Beams (1963), Anderson (1964) and Bier & Ramamurty (1964). In
B. terrestris the material present within the intercellular spaces is histochemically
similar to that of the albuminous yolk bodies in that it contains protein and carbo-:
hydrate components.
The free vesicles formed by micropinocytosis form larger bodies, either by fusing
with one another or by fusing with much larger, partly formed, yolk spheres. This process appears to be haphazard, and if the contents of the vesicles vary, as has been
indicated by the work of Wigglesworth (1943) and Telfer (1965), reflecting changes in
the composition of the haemolymph protein, the composition of the individual albuminous yolk spheres will also vary. In A. aegypti the albuminous yolk spheres acquire
a microcrystalline structure as they develop, and thus Roth & Porter (1964) have
suggested that following uptake, the material contained within the micropinocytotic
vesicles may be further modified perhaps by a form of selective hydrolysis. In B.
terrestris it is not known if the changes observed in the developing yolk spheres as they
move away from the oocyte plasma membrane are true or artifactual, but it seems
likely that some such changes may occur. The large number of lysosomes present in
the peripheral ooplasm of B. terrestris may be associated with the form of selective
hydrolysis suggested by Roth & Porter (1946) and there are many examples in our
electron micrographs of lysosome-like spheres fusing with the smaller albuminous
yolk spheres. Until further work employing electron-histochemical methods is completed, more definite conclusions cannot be made.
Concerning the number of lysosomes in the peripheral ooplasm it is also of interest
to note that in the Hymenoptera under a number of conditions (King & Hopkins,
1963) resorption of the developing oocyte occurs. This process also involves high acid
phosphatase activity (Hopkins & King, 1964a, b).
In addition to the material being transferred to the interfollicular spaces from the
haemolymph, it seems likely that in B. terrestris the follicular epithelial cells themselves secrete into these surrounding spaces. The fine structure of these cells at this
time together with their nucleic acid metabolism as shown by autoradiography and
uptake of precursor amino acids (Bier, 1962; Ramamurty, 1964) suggest that they are
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extremely active. The extensive development and distribution of the apical microvilli
also supports such a suggestion. In the absence of direct contact between the follicular
epithelial cells and the oocyte at this time, these cells must secrete into the intercellular space. Roth & Porter (1964) have suggested this secretion by the follicular
epithelial cells may be associated with bringing about a change in the haemolymph
material after it has passed across the tunica propria. In B. terrestris, however, the close
association of the follicular epithelial cell microvilli with the infoldings of the ooplasmic
membrane suggests that the activity of the follicular epithelial cells is more likely to
be associated with conditions at the surface of this membrane; possibly within the
expanded vesicles which arise in the vicinity of the follicular epithelial cell microvilli.
The histochemical procedures carried out on the formed albuminous yolk spheres
show that they consist of a protein/carbohydrate/lipid complex. Bier (1954), working
on other Hymenoptera, was unable to detect lipid in the large yolk spheres of the
species he studied. In addition, although Bonhag (1955) has shown that a phospholipid
occurs during a transitionary phase in the large yolk spheres of Oncopeltus fasciatus,
only King & Aggarwal (1965) working on Hyalophora cecropia have found a permanent lipid component in these spheres.
In agreement with most previous work (Bonhag, 1958) the large yolk spheres also
consist of a protein/carbohydrate complex, the PAS-positive component of which is
labile to malt diastase and therefore is not glycogen. The positive tyrosine and negative
trytophan and cysteine reactions of the protein moiety agree with those obtained on
Bombyx mori by Aggarwal (1963).
Although glycogen yolk can be detected in large quantities only during the later
phases of yolk synthesis the mechanism for its formation is present during the earliest
stages of vitellogenesis. The groups of small vesicles from which the glycogen yolk
particles arise are initially present in the nurse cell cytoplasm, but are transferred in
large numbers to the ooplasm during stages 2 and 3. As a result of their distribution
and later activity, glycogen occurs in large amounts in the trophocyte and oocyte cytoplasm during stage 4. By histochemical means, glycogen has also been shown to occur
in the trophocytes and oocytes in other species during the later stages of yolk synthesis
(Bonhag, 1956; Krainska, 1961; Aggarwal, 1962).
In Hyalophora cecropia, King & Aggarwal (1965) have associated the occurrence
of glycogen with groups of small vesicles, some of which are derived from the trophocytes. In this species, however, the vesicles do not occur in compact groups and do
not give rise to larger glycogen particles.
In the absence of micropinocytosis at the trophocyte plasma membrane (Hopkins,
1966) it is unlikely that the glycogen present within the trophocyte cytoplasm has been
derived, unchanged, from the haemolymph. Thus the groups of vesicles must be able
to synthesize glycogen from substances of small molecular size that are able to traverse
the plasma membrane. Engels & Drescher (1964) using another hymenopteran, Apis
mellifera, have shown that labelled glycogen appears in the oocyte within minutes of the
follicle being immersed in a solution of tritiated glucose. Such rapid uptake and synthesis can occur only if the glucose proceeds directly to the oocyte: the autoradiograph
rules out participation by the follicular epithelial cells.
Oocyte development in Botnbus
213
The large number of oocyte microvilli constituting a brush border, and the high
alkaline phosphatase activity in their vicinity indicates that extensive molecular transport
may occur across the ooplasmic membrane (Danielli, 1952). This transport is not
associated with micropinocytotic activity and provides a pathway along which smallmolecule precursors may pass, crossing the ooplasmic membrane by simple absorption.
Perhaps the uptake of glucose as shown by Engels & Drescher (1964) occurs in this way.
The site of origin of the vitelline membrane in B. terrestris agrees with that described in Drosophila melanogaster by King & Koch (1963) and in Hyalophora cecro-
pia by King & Aggarwal (1965), but the formation of precursor vitelline bodies prior to
the appearance of the vitelline membrane as observed in Drosophila by Okada &
Waddington (1959) does not occur in the Bombus oocyte. Here there is a gradual
transition from the phase of yolk synthesis, when the intercellular space contains
electron-dense material, to the phase of vitelline membrane formation, when a distinct
membrane structure can be identified.
King & Koch (1963) suggest that from their observations on the ovaries of Drosophila mutants, tiny and female sterile, that the follicular epithelium will produce a
vitelline membrane against cells other than the oocyte but that this structure has
altered chemical properties. In B. terrestris the evidence supports this suggestion,
indicating that while the follicular epithelial cells provide the major contribution
towards vitelline membrane formation, there is evidence of an oocyte contribution.
Although the structures described outside the vitelline membrane are part of the
chorion, detailed interpretation is not possible as we have been unable to obtain an
oviposited egg.
The whorls of endoplasmic reticulum and extensive Golgi areas present in the
follicular epithelial cell cytoplasm during chorion formation suggest that these cells
are actively synthesizing chorionic material which is passed across the convoluted
plasma membrane into the intercellular space. Although the compact profiles of endoplasmic reticulum are perhaps comparable with the epithelial bodies described in
Drosophila by King (i960) and King & Koch (1963), they are dissimilar in that they
are not associated with lipid.
We are indebted to Professor E. W. Knight-Jones, in whose Department the work was
carried out, and to the D.S.I.R. for financial assistance.
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216
C.R. Hopkins and P. E. King
Fig. 2. Electron micrograph of the peripheral ooplasm during stage 2. Groups of
electron-dense vesicles (ai-w,) are present in the ooplasm; groups v± and u2 are
surrounded by larger glycogen particles (arrows), {an, accessory nucleus; fc, follicular epithelial cells.)
Fig. 3. Higher magnification electron micrograph of a similar group of vesicles
in the ooplasm during stage 2. The vesicles (v) in this group are not electron-dense,
but note that at the periphery of the group (arrows) profiles suggest that they are
fusing with the larger glycogen particles (g). (m, mitochondria.)
Fig. 4. Electron micrograph showing mitochondria within the protoplasmic connexion (arrows) between a trophocyte (t) and the oocyte prismatic process (pp)
during stage 2. A group of electron-translucent vesicles (v) is present in the trophocyte cytoplasm and in the ooplasm. (g, glycogen particles.)
Journal of Cell Science, Vol. i, No. 2
C. R. HOPKINS AND P. E. KING
(Facing p. 216)
Fig. 5. Light micrograph of the oocyte periphery during stage 4. The follicular
epithelium (fe) stands on a thin basement membrane (bm) and intercellular channels
(large arrows) occur between adjacent cells. In the space between the follicular
epithelium and the ooplasmic membrane, fine follicular epithelial cell microvilli are
present (small arrows). In the most peripheral ooplasm small albuminous yolk spheres
are forming, while more centripetally larger spheres (ays) and an accessory nucleus
(an) are present, (eo, extra-ovarial space, the ovariole sheath having been removed;
00, ooplasm.)
Fig. 6. Electron micrograph of the ooplasm during stage 3. Note the two types of
lipid particle, Lx and L3.
Fig. 7. Electron micrograph showing follicular epithelial cells and a portion of the
peripheral oocyte (00). The central narrow 'dark' follicular epithelial cell differs
from the adjoining cell in containing high concentrations of free ribosomes (arrows)
and large numbers of proteinaceous particles (p). (n, nucleus; nil, nucleolus.)
Journal of Cell Science, Vol. i, No. 2
C. R. HOPKINS AND P. E. KING
Fig. 8. Electron micrograph showing the peripheral ooplasm (oo) and part of the
follicular epithelium (fc) during stage 4. Between the oocyte and the follicular epithelium lies an intercellular space (ics) containing fine fibrous material. At the base of
the pits between the oocyte microvilli (oom) there are a number of vesicular invaginations (small arrows). A limiting membrane (large arrow) may often be seen surrounding
the albuminous yolk spheres {ays).
Fig. 9. Electron micrograph taken at a higher magnification of the portion of the
section outlined in Fig. 8. The particles 1-4 outline the proposed pathway along
which the intercellular material passes before being incorporated into the albuminous
yolk spheres (ays). Note the long follicular epithelial cell microvillus (mo) which projects across the intercellular space (ics), and into an invagination of the ooplasmic
membrane, (fc, follicular epithelial cell; 00, ooplasm.)
Fig. 10. Electron micrograph of the follicular epithelium and the most peripheral
region of the oocyte (00) during stage 4. The follicular epithelial cells contain large
nuclei (n) within which there are numerous nucleoli (mi). Golgi elements (") are present
in the perinuclear cytoplasm. There are a large number of mitochondria and ribosomes
in the apical cytoplasm, but only a few elements of endoplasmic reticulum (er) occur.
The arrows indicate the electron-dense intercellular regions between adjacent follicular
epithelial cells, and between these cells and the oocyte. (mv, follicular epithelial cell
microvillus.)
Journal of Cell Science, Vol. i, No. 2
C. R. HOPKINS AND P. E. KING
Fig. I I . Electron micrograph showing part of the chorion being laid down outside
the vitelline membrane (vm) during stage 6. The cytological preservation of this
preparation is poor because it is extremely difficult to obtain adequate penetration of the
fixative and embedding medium without either disrupting the egg membranes or removing the follicular epithelium. Note the presence of a thin membrane adjacent to
the vitelline membrane (arrows) and the trabeculae (i) which are regarded as part of
the inner chorion. Numerous microvilli (uw) of the follicular epithelial cell can be
seen adjacent to the chorionic material, (fc, follicular epithelial cell; n, nucleus.)
Fig. 12. Electron micrograph of the developing vitelline membrane (vm) during
stage 5. Electron-dense particles (arrows), which are believed to represent the contribution of the follicular epithelial cells towards vitelline membrane formation, are
present in the apical cytoplasm of the follicular epithelial cells (fc). The innermost
margin of the vitelline membrane at the tips of the oocyte microvilli (oom) has not yet
become well defined. (00, ooplasm.)
Fig. 13. Electron micrograph showing one of the whorls of endoplasmic reticulum
(er) which occur in the perinuclear cytoplasm during chorion formation, (g, Golgi
elements ; m, mitochondrion ; «, nucleus.)
Journal of Cell Science, Vol. i, No. z
C. R. HOPKINS AND P. E. KING