AM. ZOOLOGIST, 3:185-191 (1963).
THE MECHANISM OF BLOOD PROTEIN UPTAKE
BY INSECT OOCYTES
WILLIAM H. TEI.FER AND MELVIN E. MELII/S, JR.
Zoological Laboratory
University of Pennsylvania
One does not normally think of chickens
and silkworms as kindred spirits. In the
process of yolk formation, however, there
are some remarkable similarities between
what was described by Dr. Schjeide (this
symposium) and the role of blood proteins
in yolk formation by the moth of the
Cecropia silkworm. By immunological procedures, it has been possible to show that
most, and possibly all proteins present in
the blood at the time of yolk formation
are also detectable in the yolk (Telfer,
1954). As in the experiments of Knight
and Schechtman (1954), even foreign proteins which have been injected into the
blood are detectable in the yolk (Telfer,
1960). As if in imitation of serum lipovitellin in the chicken, a protein which
circulates in the blood of female moths is
accumulated by the oocyte, apparently in
preference to many other blood proteins.
Furthermore, the protein in question is
not normally produced in significant
amounts by the male, although the male
clearly has the ability to synthesize it.
Blood proteins which have entered the
moth oocyte are laid away in discrete cytoplasmic particles, the protein yolk spheres
(Telfer, 1961). To complete'the list of
similarities, the surface of both the moth
oocyte and chick oocyte (Brambell, 1926),
at the time of yolk formation, is thrown
into the configuration of a brush border.
During the years when these similarities
were being demonstrated, it was difficult
to conceive of a decisive experiment for
which a previous experiment by Dr.
Schechtman or one of his students did not
serve as a model. It is thus with a deep
appreciation that this paper is submitted
to a symposium in his honor.
Supported by research grants from Phi Beta Psi
Sorority and from the U. S. Public Health Service
(RG-8977).
The application of immunochemical
techniques to the study of developing systems which Dr. Schechtman so effectively
encouraged has revealed many instances
of the interchange of proteins between
cells. In few cases is the interchange as
blatant as it is in yolk formation, where
gross amounts of blood proteins appear to
be deposited in the oocyte. We will examine some of the cytological correlations
of the process, and discuss their implications for the mechanisms of protein transmission through the tissues of the ovary
and into the yolk spheres within the
oocyte.
AUTORADIOGRAPHIC STUDIES
The moth oocyte is separated from the
blood by at least two layers of cells. It
is directly surrounded by a layer of follicle
cells, and these are in turn surrounded by
the wall of the ovariole, the tubular sheath
which contains a linear series of up to
fifty oocytes. At the anterior end of each
oocyte is a cluster of five nurse cells which
are included within the layer of follicle
cells. The tiurse cells have not as yet been
implicated in blood protein uptake or yolk
formation in the Cecropia moth. All of
the evidence has thus far indicated that
the protein yolk spheres are made in the
oocyte cytoplasm adjacent to the follicle
cells.
Experimental confirmation of this suggestion has emerged in a preliminary report by Bier (1962) concerning his autoradiographic studies of the oocytes of the
blowfly, Calliphora. Injection of tritiated
histidine into female flies led to the appearance of radioactivity in the ovary in
two phases. The first phase was characterized by a fairly rapid incorporation
leading to a maximum radioactivity within
fifteen minutes, especially in the cytoplasm
(185)
186
W. H. TELFER AND M. E. MELIUS, JR.
INSECT OOCYTES
187
FIG. 1. A follicle preserved by free-substitution
and low temperature osmium fixation, and stained
with PAS and hematoxylin. The follicle cell layer
with its intercellular spaces is at the top, and is
separated from the yolky oocyte by a brush border.
(The dark material between the yolk spheres is
PAS-stained glycogen.)
FIG. 2. Autoradiograph of a follicle cultured in
labeled blood proteins (for explanation see text).
The silver grains are concentrated in the ovariole
wall (on the right), between the follicle cells, and
in the peripheral regions of the oocyte (on the left).
FIG. 3. Autoradiograph of a follicle cultured for
30 minutes in blood containing free tritiated leucine.
The silver grains are concentrated over the follicle
cells.
FIG. 4. A whole mount of the ovariole wall in
surface view. The meshwork consists of striated
muscle fibers. Occasional tracheae and nuclei are
seen (phase contrast).
of the nurse and follicle cells. This result
might be provisionally interpreted as indicating that all the free injected histidine
had been utilized by this time. After forty
minutes, a second and more concentrated
radioactivity appeared at the periphery of
the oocyte adjacent to the follicle cells.
By eighty minutes after the injection, the
second zone of radioactivity had broadened significantly and was resolvable at
its inner surface into individual yolk
spheres.
Bier interpreted the slower and more
intensive appearance of radioactivity as
resulting from the influx of blood proteins into the yolk spheres. Its tardiness
could be attributed to the fact that the
injected histidine had first to be incorporated into proteins elsewhere in the insect,
and then secreted into the blood before
finally crossing the follicle cell layer and
reaching the oocyte.
Our first concern here is with events
which occur prior to the appearance of a
blood protein in a yolk sphere. Specifically,
what is the mechanism of transmission of
the protein through the ovariole wall and
the follicle cell layer?
It was noted earlier (Telfer, 1961) that
the follicle cells do not form a continuous
layer of cells in the Cecropia moth, but
that during yolk formation there are substantial intercellular spaces between them.
These can be seen after several kinds of
fixation, but the best preservation (Fig. 1)
is after freeze-substitution in combination
with low-temperature osmium fixation
(Feder and Sidman, 1958). It was previously noted that fluorescein-labeled antibodies against blood proteins stain the
interfollicular cell spaces in the same manner that they stain a droplet of blood
which adhered to the surface of the ovary
when it was being frozen. The same can
now be reported for a variety of stains, including PAS and bromphenol blue, both
of which combine with fixed blood proteins. Thus, the intercellular spaces contain fixable materials whose character and
concentration are similar to the proteins
in the blood.
A recent procedure has been to follow
the interchange of proteins between blood
and intercellular spaces with autoradiography. Blood proteins were labeled for this
purpose by injecting female pupae with
tritiated leucine. The pupae were bled
several days later and the blood was dialyzed to remove labeled small molecules.
Ovaries were immersed in the preparation
for varying periods of time and then fixed
and sectioned for autoradiography. Significant concentrations of silver grains were
seen in these preparations over the wall
of the ovariole, in the intercellular spaces
of the follicle cells layer (Fig. 2) and, in
favorable cases, within the oocyte in and
around the small yolk spheres close to its
periphery.
Under the conditions of these experiments, the labeled proteins reached the
oocyte surface between 30 to 60 minutes.
When the oocyte was transferred back to
unlabeled blood, the label disappeared
from the intercellular spaces in the same
period of time. The results have thus
suggested that there is a fairly ready twoway interchange of proteins between the
blood and the interfollicular cell spaces.
That the ovaries were viable during
these short-term incubations was suggested
by the fact that labeled blood proteins
appeared in some of the peripheral yolk
spheres, thus indicating that yolk forma-
188
W. H. TELFER AND M. E. MELIUS, JR.
tion had continued during the incubation.
In addition, ovarioles cultured under similar conditions in blood containing free
tritiated leucine, quite unlike the ovarioles
in labeled proteins, incorporated the label
into the cytoplasm of the follicle cells
(Fig. 3), nurse cells, and to a lesser extent
into the oocyte cytoplasm between the yolk
spheres.
While the experiments are subject to
all the limitations as well as the advantages
of in vitro studies, they strongly suggest
that blood proteins can travel in two directions across the wall of the ovariole.
The ovariole thus does not transmit and
trap proteins in the manner that they
appear to be trapped in the protein yolk
spheres.
STRUCTURE OF THE OVARIOLE WALL
We should therefore consider the manner in which proteins cross the wall of the
ovariole. The latter is actually a twolayered structure consisting of an outer
cellular envelope and an inner, PAS-positive basement membrane which is tightly
apposed to the surface of the follicle cells.
Both of these layers continuously surround the inner core of follicle cells, nurse
cells, and oocytes, and thus all proteins
en route to the oocyte must cross them.
The cellular envelope, where it is attenuated by the expansion of the growing
oocyte, is frequently less than 2-3 ju, thick.
In a light microscope view of sectioned
ovaries, little structure other than an occasional nucleus or trachea can be made out.
A more fruitful approach to the analysis
of its structure was that used by Bonhag
and Arnold (1961) in their study of the
ovariole of the cockroach. This entails observing the ovariole wall in surface view.
Oocytes, with their accompanying follicle
cells and the basement membrane which
adheres to the outer surface of the latter,
were dissected out of the ovariole. A hair
loop with a diameter approximating that
of the oocyte was then inserted into the
tubular sheath, and the preparation was
then fixed and examined in surface view
as a whole mount. Its most striking fea-
FIG. 5. A follicle stained by the PAS method, following digestion with salivary amylase. The stain
is particularly pronounced in the yolk spheres (left)
and in the basement membrane between the follicle
cells and ovariole wall (right).
ture is a sieve-like structure, with the mesh
being constructed of fine, striated muscle
fibers (Fig. 4). Tracheae and tracheoles
are occasionally seen running along the
outer surface of the ovariole in an unoriented fashion, and there is frequently
a suggestion of finer, randomly-oriented
fibers in the square and rectangular interstices between muscle fibers. Knowledge
that proteins readily penetrate this structure leads one to suspect that there is little,
if anything, in the way of a continuous
barrier to the diffusion of proteins in the
interstices.
The basement membrane which lies between the cellular sheath of the ovariole
and the follicle cells appears, in the
light microscope, as a continuous and homogeneous sheath of PAS-positive material
about 1-2 a thick (Fig. 5). While this
INSECT OOCYTES
structure could substantially reduce the
rate of diffusion of proteins from the blood
into the ovary, it is an empirical fact that
proteins penetrate it fairly readily in both
directions. We therefore anticipate that
the basement membrane will be shown to
possess a fine-structure which is porous
enough to permit proteins to diffuse across
it.
Everything seen thus far in the structure
and behavior of the layers surrounding
the oocyte is consistent with the proposal
that diffusion through intercellular spaces
and porous membranes accounts for the
transmission of blood proteins from the
hemocoel to the oocyte surface.
TRANSMISSION ACROSS THE OOCYTE SURFACE
The next question concerns the manner
in which blood proteins are handled at
the surface of the oocyte. The fact that
proteins cross the oocyte surface and are
finally present within discrete cytoplasmic
bodies—the protein yolk spheres—has made
it tempting to think in terms of pinocytosis as the mechanism of protein incorporation. If this is in fact the case, the pinocytotic vacuoles formed at the oocyte surface are small enough to be at or below
the resolving power of the light microscope, and reliance must be placed on electron microscopy for further evidence.
This approach is being taken in several
laboratories, and one preliminary report
concerning the mosquito occyte has already
emerged (Roth and Porter, 1962). An essential feature of this work is the study of
oocytes which one knows are in the process
of yolk formation at the time of fixation—
an obvious precaution which is frequently
overlooked. It was achieved in Roth and
Porter's work by fixing the ovaries at measured times after a blood meal which activates the endocrine system essential for yolk
formation.
Roth and Porter's micrographs show a
structure which is entirely consistent with
the occurrence of pinocytosis. The surface
of the oocyte is pitted by in-pocketings of
about 0.1 to 0.2/* diameter. The pits vary
in depth up to nearly 0.5/n, and their tips
189
frequently have the appearance of pinching
off to form small cytoplasmic vesicles. The
adjacent cytoplasm contains many such
vesicles. Thus, all intermediate stages in
what is very probably pinocytosis are observed in the surface of the mosquito oocyte.
The outer surface of the oocyte, and the
linings of the pits and vesicles are all coated
with an electron-dense material with a
thickness of about 0.2/n. The position of
this material is reminiscent of the mucoid
coat which is similarly distributed in
amoebae undergoing pinocytosis. In view
of the demonstration that the adsorption of
proteins on the mucoid coat of amoebae is
an essential prerequisite to pinocytosis
(Brandt, 1958; Marshall, Schumaker, and
Brandt, 1959), it is tempting to ascribe a
similar function to the material demonstrated by Roth and Porter. We are tempted
to speculate even further that the selectivity
of protein uptake by the insect oocyte will
be attributed during the next few years to
the relative avidity of different proteins for
the surface coat of the oocyte.
FORMATION OF THE YOLK SPHERES
We must finally consider the question of
how the protein yolk spheres are assembled.
If one accepts the proposal that blood proteins enter the oocyte by pinocytosis, the
most direct mechanism for the formation
of yolk spheres would entail the fusion of
the minute pinocytotic vesicles. The fusion
of pinocytotic vesicles has been observed
with time-lapse photographs in other systems (Rose, 1957). Whether it also occurs
in the growth of the yolk spheres in oocytes
has not yet been directly demonstrated. We
have found a number of conditions, however, in which the mature yolk spheres of
the Cecropia oocyte can be made to fuse
experimentally. In general, the condition
of fusion is a matter of pushing two yolk
spheres together with sufficient force. This
can be achieved in isolated yolk spheres by
placing them adjacent to each other on a
microscope slide and pressing down gently
on the cover glass so that they flatten
slightly and thus push against each other.
When the pressure is released, they remain
190
W. H. TELFER AND M. E. MELIUS, JR.
FIG. 6. Centrifugal pole of an oocyte centrifuged
at ten thousand gravities for one minute. The
follicle cells are below.
FIG. 7. Centripetal pole of the oocyte shown in
Fig. 6. The follicle cells are on the right and sedimented yolk spheres are on the left. The undisplaced yolk spheres at the oocyte surface are visible
next to the follicle cells.
attached as a single spherical structure. The
same result can be achieved by centrifuging
the intact oocyte at ten thousand gravities.
After one minute, many of the yolk spheres
which have been packed into the centrifugal
pole appear to have formed irregular fusion
bodies (Fig. 6).
That fusion occurs naturally in the
growth of the yolk spheres is suggested by
some of the figures of Roth and Porter (personal communication). The yolk spheres
of the mosquito oocyte have a fine structure indicative of an orderly arrangement,
as if the proteins were in a crystalline form.
In one micrograph a single yolk sphere
contains three discrete regions which differ
from each other in the orientation of the
crystalline axes. While there are several
possible explanations of this structure, one
would certainly be that fusion occurred between yolk spheres in which the crystalline
arrangement of proteins had already been
established.
One of the characteristics of the peripheral zone of blood protein uptake in the
moth oocyte is the presence of yolk spheres
running the full size range from less than
]/j. to diameters of 20^ and more (Fig. 5).
In the deeper layers of the oocyte, the yolk
spheres appear to have achieved a stable
size. This arrangement suggests that yolksphere growth is limited to the peripheral
cytoplasm. If we adopt the hypothesis that
growth occurs by fusion, then yolk spheres
and pinocytotic vesicles must be able to fuse
only in the peripheral cytoplasm. The
forces which bring about fusion are simply
not generated in the deeper layers of
cytoplasm.
In this connection, it is instructive to look
at the centripetal pole (Fig. 7) of the centrifuged oocyte whose centrifugal pole was
191
INSECT OOCYTES
depicted in Fig. 6. Here a cap of lipochondria and a band of cytoplasm containing
finely granular material had formed in the
region from which the large yolk spheres
had been sedimented. At the periphery of
the oocyte—the zone of yolk sphere growth—
the small- and intermediate-size yolk spheres
remained undisplaced. In other cases centrifugation at 20,000 g for one minute failed
to sediment them, although five minutes
exposure to this force finally did. It is presumed that we are concerned here with a
classical cortical gel phenomenon. If the
factors which operate to hold the growing
yolk spheres to the cell surface also serve to
pull small yolk spheres together with forces
of similar magnitude, the growth of yolk
spheres in the peripheral cytoplasm could
be accounted for. Whether this is, indeed,
the mechanism of yolk-sphere growth is a
matter of speculation, but the possibility
seems worthy of consideration.
Ward (1962) has published a number of
electron micrographs clearly demonstrating
that yolk platelets in the oocytes of amphibian tadpoles are contained within the
structure of mitochondria. While the origin
of this association is at the present time
obscure, two possibilities deserve comment.
Ward favors the suggestion that mitochondrial synthesis is responsible for the origin
of yolk platelets. There is of course nothing
to militate against other organisms forming
yolk by mechanisms quite different from
what appears to be the dominant mechanism in chickens and moths. A second
possibility is that the association arises by
the fusion of mitochondria and yolk platelets. It will be of great interest to learn if
the association is equally prominent in the
oocytes of mature frogs which are producing
yolk at a significant rate at the time of
fixation.
SUMMARY
The evidence is reviewed concerning the
mechanism by which blood proteins are incorporated into the yolk spheres of insect
oocytes. It is suggested that blood proteins
reach the surface of the oocyte from the
hemocoel by diffusion through the intercellular spaces of the ovary. The structure
of the oocyte surface is consistent with the
proposal that blood proteins are accumulated by pinocytosis. Finally, it is proposed
that large yolk spheres (up to 20/t in diameter) are formed by the fusion of smaller
yolk spheres and pinocytotic vesicles. The
forces which bring about fusion appear to
be generated in a cortical gel layer at the
surface of the oocyte.
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