Published February 1, 1964

YOLK PROTEIN
OF THE

UPTAKE

MOSQUITO

IN THE

AEDES

OOCYTE

AEGYPTI.

T H O M A S F. R O T H and K E I T H

L.

R. P O R T E R ,

Ph.D

From the Biological Laboratories, Harvard University, Cambridge, Massachusetts

ABSTRACT

In 1932 Jukes and Kay (21) suggested that yolk

protein of the egg of the domestic fowl is chemically
related to the serum proteins of the laying hen.
Since then, several investigators (7, 19, 20, 24,
25, 38, 42, 43, 45) have confirmed this finding,
and the concept has grown that chicken yolk
protein is made in the liver and transported via
the circulation to the egg for storage. Similar
studies with the frog (14--18) and rat (27) have
also shown egg protein to be nearly identical with
protein species found in the maternal serum. Of
further significance in this regard is the finding
that foreign proteins introduced into the circulation appear in the yolk protein substantially
unchanged (4, 24).
These observations on oogenesis in the vertebrates as well as Teller's work with an invertebrate, a saturniid moth (48-50) demonstrate
conclusively that, in many instances of egg pro-

tein production and deposition, the protein

species is made outside of the ovary, probably in
the liver or an analogous organ, is secreted into
the extracellular spaces, and is then removed from
the circulating blood by the developing egg.
There are exceptions to this general scheme, one
of which is found in the crayfish, which appears
to have a highly developed synthetic mechanism
in the oocyte cytoplasm for the formation of yolk
protein (3).
In the case of the mosquito, which is the object
of this study, there are reasons to suppose that
yolk deposition in the developing oocyte is accomplished also by removal of the protein from
the blood of the insect. In the first place, yolk
develops rapidly. In fact, synthesis and storage
are essentially completed in as little as 25 hours
after a blood meal. Concomitantly, none of the
usual structural mechanisms associated with

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Yolk proteins are thought to enter certain eggs by a process akin to micropinocytosis but
the detailed mechanism has not been previously depicted. In this study the formation of
protein yolk was investigated in the mosquito Aedes aegypti L. Ovaries were fixed in phosphate-buffered osmium tetroxide, for electron microscopy, before and at intervals after a
meal of blood. The deposition of protein yolk in the oocyte was correlated with a 15-fold
increase in 140 m# pit-like depressions on the oocyte surface. These pits form by invagination of the oocyte cell membrane. They have a 20 m# bristle coat on their convex cytoplasmic side. They also show a layer of protein on their concave extracellular side which
we propose accumulates by selective adsorption from the extraoocyte space. The pits, by
pinching off from the cell membrane become bristle-coated vesicles which carry the adsorbed protein into the oocyte. These vesicles lose the coat and then fuse to form small
crystalline yolk droplets, which subsequently coalesce to form the large proteid yolk bodies
of the mature oocyte. Preliminary radioautographs, and certain morphological features of
the fat body, ovary, and midgut, suggest that the midgut is the principal site of yolk protein
synthesis in the mosquito.

Published February 1, 1964

r a p i d synthesis of p r o t e i n or lipoprotein, especially

for segregation a n d storage in granules, a p p e a r in
the fine s t r u c t u r e of the follicle cells or the oocyte
c y t o p l a s m . I n d e e d , only one u n u s u a l structural
feature does a p p e a r , w h i c h seemingly m i g h t be
involved in yolk deposition, a n d t h a t is a n elaborate d e v e l o p m e n t of pits or wells in the surface of
the oocyte. It is the m a j o r p u r p o s e of this r e p o r t
to describe these pits a n d to p r e s e n t reasons for
i n t e r p r e t i n g t h e m as surface differentiations designed specifically for p r o t e i n u p t a k e a n d t r a n s p o r t
into the oocyte.
MATERIALS

AND

METHODS

314

OBSERVATIONS
T h e structure of the m o s q u i t o o v a r y has been
described in detail in several light m i c r o s c o p e
studies (8, 31-33, 36, 41) a n d is p e r h a p s best
s u m m a r i z e d in C h r i s t o p h e r s ' m o n o g r a p h (9). I t is
i m p o r t a n t to review first this w o r k a n d c u r r e n t
observations o n the structure of t h e ovary at the
time w h e n its d e v e l o p m e n t in the a d u l t has r e a c h e d
a steady state. This p l a t e a u occurs by 3 days after
e m e r g e n c e a n d persists until the a d u l t feeds on
blood. This act of f e e d i n g initiates a s e c o n d stage
in o v a r i a n d e v e l o p m e n t w h i c h c u l m i n a t e s in the
f o r m a t i o n of the m a t u r e egg.

The Resting Stage

T h e ovaries of the m o s q u i t o lie in the a b d o m e n
b e t w e e n the f o u r t h a n d sixth segments. E a c h o v a r y
(Fig. 1), consisting of a p p r o x i m a t e l y sixty ovarioles, is c o n t a i n e d b y a m u s c u l a r m e s o t h e l i u m
b e n e a t h w h i c h lie m a n y t r a c h e a e (Fig. 2). E a c h
ovariole in t u r n is e n w r a p p e d by a thin s q u a m o u s
m c s o t h e l i u m of cells s h o w i n g a kind o f striated
myofibril (Fig. 3). T h e ovariolcs c o n t a i n a m a t u r ing follicle, a p r e s u m p t i v e follicle, a n d a g e r m a r ium, a n d e a c h of the m e r o i s t i c - p o l y t r o p h i c follicles
is c o m p r i s e d of seven nurse cells a n d one oocyte
w i t h i n a follicular e p i t h e l i u m .

THE JOURNAL OF CELL BIOLOGY VOLUME~0, 1954

T h e cells of the follicular e p i t h e l i u m are at this

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T h e ovary of the adult mosquito Aedes aegypti L. was

studied. T h e insects were raised by routine methods
(51) at 27C, and the adult form maintained on
raisins until needed. T h e full development a n d
m a t u r a t i o n of oocytes was initiated by feeding 1-weekold adults on the shaved back of a rabbit. Thereafter
the mosquitoes were sacrificed at intervals as indicated in the next section. To facilitate handling them,
the mosquitoes were chilled to 4C just prior to
fixation.
I n preparation for light microscopy, entire mosquitoes sacrificed at various intervals after a blood
meal were fixed for 6 hours at 4C in 10 per cent
aqueous acrolein (according to the methods of Feder
(12, 13) as modified below). T h e mosquitoes were
then dehydrated in a graded series of 1:1 methyl
alcohol/1-methoxyethanol, and the abdomens were
removed a n d infiltrated under vacuum for 3 days in
six changes of a mixture of 95 per cent glycol methacrylate, 5 per cent Carbowax, a n d 0.3 per cent
~-azodiisobutyronitrile as a catalyst. T h e final
change of the infiltration mixture was allowed to
polymerize for 3 days at 60C before 1.5/z sections
were cut from the blocks. T h e sections were stained
with aqueous 0.5 per cent toluidine blue and 1 per
cent acid fuchsin.
For electron microscopic examination, the mosquitoes were fixed with 2 per cent osmium tetroxide,
buffered at p H 6.8 and 7.3 according to Millonig
(29). A b d o m e n s were removed at predetermined
intervals and the cuticles slit to allow rapid penetration of the fixative; fixation was continued for a
period of 3 to 4 hours at 4C. The tissues were thereafter rapidly dehydrated in increasing concentrations
of alcohol, with interruption only in 50 per cent
alcohol to permit dissection of the ovaries from the
abdomen. After dehydration and exposure to two
5-minute changes of propylene oxide, the tissues were
infiltrated overnight in a 1:1 mixture of propylene
oxide and the embedding m o n o m e r of Epon. After a
fresh change of monomer, the tissues were flat embedded and polymerized at 60 C for 2 days. T h e era-

bedding m o n o m e r was a 1 : 1 mixture of methyl nadic

anhydride ( M N A ) / E p o n 812 accelerated with 0.5 per
cent 2,4,6-tri(dimethylaminomethyl)phenol (26),
which gives very hard blocks.
Sections were cut for the most part on a Cambridge
(Huxley) microtome. T h e "silver" sections were
m o u n t e d on either carbon stabilized celloidin or
uncoated grids. Lead staining according to Millonig
(30), or Karnovsky (22) at a dilution of 20:1, gave
excellent contrast relatively free of contamination.
The sectioned material was examined in an E M U 3
(RCA) or a Philips E M 200.
T h e mosquitoes that were used for the experiment
with labeled leucine were fed on the legs and tail of a
14 g m rat that had been injected intraperitoneally
30 hours previously with 6 mc of L-leucine-H 3
(specific activity 5.0 c/raM) in 0.5 cc water. At intervals of 0.5, I, 2, 4.5, 16, and 30 hours thereafter, the
mosquitoes were sacrificed and e m b e d d e d for light
microscopy. Sections 1.5# thick were cut on a
Porter-Blum Servall microtome and prepared for
radioautography, following the methods of Caro
and van T u b e r g e n (6). Twelve weeks later, the seetions were developed, and the n u m b e r of silver grains
compared with the aid of a phase microscope.

Published February 1, 1964

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FIGVRE 1 A light micrograph of a cross-section through a mosquito abdomen at a time just prior to yolk
deposition. The section was stained with toluidine blue and acid fuchsin. The ovaries consist of two clusters
of ovarioles (OV) situated centrally with respect to the spongy fat body (F) which encircles the abdomen
beneath the cuticle (C). A presumptive follicle (PO) is joined to each maturing follicle in the region of the
nurse cells (NC). The oocyte (OOC) is easily identified by the clear, non-staining lipid inclusions in its
cytoplasm. In the Malplligian tubules (MT) the refractile inclusions, polytene chromosomes, and the
lumen are especially evident. An undistended, scalloped midgut (MG) is situated in the central portion of
the hemocoel just above a fold in the section. X 150.
The inset depicts a single follicle at greater magnification. Two nurse cells (NC), at the lower half of the
follicle, exhibit intense basophifia in the cytoplasm. Numerous refractile lipid droplets surround the nucleus
(N) of the oocyte (OOC).A light region (arrow heads) at the periphery of the oocyte, just within the follicular epithelium (FE), is the zone of yolk protein uptake. X 570.
stage cuboidal with large nuclei and little cytoplasm. They are closely applied to each other and
joined, neighbor to neighbor, at their apical
margins by desmosomes. Their basal surfaces
rest on a basement membrane (Fig. 3). At higher
magnifications provided by electron microscopy,
the nuclei of these cells are seen to have a
prominent polymorphic nucleolus enveloped
by distinctly granular chromosomal elements
superimposed on a lighter nucleoplasm (Fig.
2). The nuclear envelope possesses numerous

pores. The cytoplasm, which is relatively sparse in

amount, is rich in ribosomes but poor in elements
of the endoplasmic reticulum (ER). These flattened cisternae are rough surfaced and occur
most especially in the basal halves of the follicle
cells. Mitochondria are numerous and possess
well ordered, parallel arrays of cristae and several
dense granules which in other types of cells are
binding sites for divalent cations (37). Occasional
Golgi elements and infrequent lysosomes are the
other major inclusions.

THOMAS F. ROTIt AND KEITH R. PORTER Yolk Protein

Uptake

315

Published February 1, 1964

different from that of the surrounding ceils (Fig.

2). Mitochondria here are larger with well ordered
cristae; dense lysosome-like bodies are more
numerous and many multilobed lipid droplets
are common among the prominent background of
free ribosomes. No yolk granules or precursors are
evident.
The cortex of the "resting" oocyte is also remarkable in a number of respects. The surface
adjacent to the follicular epithelial cells is covered
by many unoriented villi. Then, just within this
border of microvilli, there are many pit-like
invaginations (140 m# in diameter) of the oocyte
membrane and m a n y vesicles with diameters
similar to those of the pits (Fig. 2, insert). These
structures represent the first manifestation of a
cortical differentiation which, as will be described
below, appears to prepare the oocyte for the ingestion of large quantities of yolk protein. Although quite numerous even in this early resting
stage, before the insect obtains a blood meal, the
vesicles and pits are uniformly small and relatively
undeveloped as compared with those present
later when yolk deposition begins. This uniformity
of size and structure is an important point in
interpreting subsequent changes in this region. By
contrast, that part of the oocyte facing the nurse
cells shows relatively few microvilli, pits, and pitvesicles.
The structure of the "resting" follicle constitutes the base-line on which the substantial alterations that occur in the oocyte of the blood-fed
mosquito may be superimposed. A meal of blood
or a controlled diet (44) sets in motion a series of
changes in cell structure that quickly result in the
formation of the mature egg. By 4 hours after the
meal, these cytological changes are already in evidence, and after 7 hours they are striking.

FIGTSRE ~ This micrograph shows part of a follicle fixed before feeding, with a portion
of two adjacent nurse cells (NC) and an oocyte (OOC) enclosed by a follicular epithelial
layer (FE). A granular, ribosome-filled cytoplasm and scattered mitochondria characterize the nurse cells, while lipid droplets (L), and some denser droplets (LY), clumped
chromatin (CH), and complex nucleolus (Nucl) are typical of the oocyte (OOC) at this
stage. Great numbers of small, dense vesicles are present in the cortex of the oocyte (see
insert). Peripheral to the ovariole is the discontinuous muscular sheath (MS). Traeheoles
and trachea (T) lie exterior to this sheath tissue, but within that which surrounds the entire ovary. X 3,700.
The insert depicts at higher magnification a small segment of the vesiculated interface
between the ooeyte (OOC) and the follicular epithelium (FE). Bristle-coated vesicles (V)
and elements of the endoplasmie reticulum (ER) are common in the ooeyte cortex immediately beneath the interface. X ~0,000.

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The single oocyte and seven nurse cells reside

beneath the follicular epithelium. The nurse
cells, to be described more fully in a subsequent
paper, are relatively large (27 #), the nuclei
alone having the width of three follicular epithelial cells. The granular nucleoplasm, with
several discrete nucleoli, is limited by a nuclear
envelope perforated by a large number of pores.
Immediately outside the nuclear envelope is an
accumulation of dense granular material which
probably corresponds to the localized thickening
of the nuclear envelope reported earlier in Drosophila (23). This material at some places extends
about 0.5 # into the "sea" of ribosomes which
represents, in part, the matrix of the cytoplasm.
The nurse cells also have many rounded mitochondria, occasional lipid droplets, and a few
elements of a rough-surfaced endoplasmic reticulum scattered throughout the cytoplasm. Although the nurse cells are closely applied to each
other and to the oocyte and to the follicular
epithelium, no evidence exists of cytoplasmic
bridges.
The oocyte, as noted above, is readily identified
by its uncommon nucleus in which two distinct
bodies stand out against the relatively clear nucleoplasm (Fig. 2). One of these, the nucleolus, is a
dense, meandering, polymorphic structure which
sends long branches out into the nucleoplasm.
T h e other, the chromosomal mass, consists of a
unique configuration of synaptic chromosomes
which have migrated from their previous peripheral position in the nucleus. This structure will be
described in detail elsewhere. The enveloping
nuclear membrane, regularly perforated with
pores, provides a distinct separation from the
rest of the oocyte.
The cytoplasm of the oocyte is characteristically

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THOMAS F. ROTH AND KEITtt R. PORTER Yolk Protein Uptake

317

Published February 1, 1964

Structural Changes Associated With Yolk

Formation

extremities of the microvilli from the oocyte

surface are relatively free.
B. THE OOCYTE SURFACE

W i t h the onset of yolk deposition after the

insect has a blood meal, some striking changes
a p p e a r in the follicular epithelium. Most obvious
are the d e v e l o p m e n t of large intercellular spaces
(Fig. 3) a n d a c o n c o m i t a n t decrease in desmosomal
connections w h i c h m a y be associated with this
separation. In some instances, the spaces encountered were large a n d extended from the basem e n t m e m b r a n e of the follicle to the oocyte surface.
Continuity of the epithelium is, however, not lost
a n d there is a suggestion in the micrograph in
Fig. 3 t h a t contact with the basement m e m b r a n e
is retained by a fine reticulation of thin cell extensions. Obviously this behavior of the follicle
cells produces open channels between the extrafollicular space a n d the surface of the o o c y t e - open, that is, except for the basement m e m b r a n e .
Some note should be taken of the material
t h a t fills these extracellular spaces. I t is finely particulate a n d somewhat flocculent in appearance.
Clearly the same material is not present in the
interstitial spaces beyond the basement m e m b r a n e
of the ovariole. T h r o u g h o u t the intercellular
spaces the distribution of this material is essentially uniform. At the oocyte surface it is continuous with a more condensed layer of similar texture, representing apparently a n adsorbed layer
of the same material. This tendency to concentrate on this surface is shown only in relation to
the oocyte; adjoining surfaces of follicle cells a n d
nurse cells are not so covered (Fig. 4). Even the

Thus the surface of the oocyte in the m a t u r i n g

ovariole faces on a greatly e x p a n d e d space u n d e r
a n d between the cells of the follicular epithelium
(Figs. 3 a n d 4). It is especially in this cortical
region of the m a t u r i n g oocyte t h a t a series of
i m p o r t a n t alterations occurs, which are the particular interest of this report. T h e area of the
oocyte facing the follicular epithelium is covered
with great numbers of microvilli (also somewhat
evident in the resting oocyte) which, by 7 hours
after the blood meal, push into the extracellular
spaces created by the separation of the follicular
epithelial cells (Fig. 3). These microvilli vary
somewhat in length a n d are not in any sense
regularly disposed like those, for example, of the
typical intestinal epithelium. T h e y seem, however,
to achieve a uniform d i a m e t e r (60 m~) especially
in their greatest extension from the surface. O n e
gets the impression from the variation in length
a n d orientation t h a t the population m a y in life
be actively changing, some retracting a n d others
forming.
I n between the microvilli, a n d also disposed
irregularly, are n u m e r o u s dense vesicles which
are morphologically similar to the dense vesicles
present in the resting oocyte (Figs. 2, 3, a n d 4).
These connect in some instances with the oocyte
surface a n d a p p e a r in profiles as small pits or
wells (140 m # in diameter). In other cases they
are free a n d e m b e d d e d in the cortex u p to 0.5 /~
from the surface (Fig. 4).

FIGURE 3 This micrograph shows parts of two adjacent follicular epithelial cells (FE)
and an associated oocyte (OOC)fixed 7 hours after a blood meal. The follicle cells, separated by large spaces (FES), rest upon a typical basement membrane (BM). At some
points along the basement membrane extremely attenuated extensions of the epithelial
cells appear as a row of circular profiles (black arrows). The openings between these provide easy access to the intercellular space for any materials that traverse the basement
membrane. The ovariole is covered by a thin reticular sheath of mesothelial cells (MS)
which contain muscle fibrils. The oocyte at this stage in its development contains large
proteid granules (PG), lipid droplets (L), mitochondria (M), and elements of the endoplasmic reticulum in a cytoplasmic matrix rich in ribosomes. The cortical zone, depicted
here, possesses other features of special interest. Numerous microvilli (MV) extend from
the oocyte surface into the intercellular space. Between the microvilli are several small
dense pits (white arrows) still continuous with the cell surface. Small plt-derived vesicles
(V) of the same size, density, and structure as the pits reside in the cortex of the oocyte. A
part of a follicle cell in the center shows some profiles of the endoplasmic reticulum (ER)
and a prominent Golgi component (G). X 15,500.

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A. THE FOLLICULAR EPITHELIUM

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Published February 1, 1964

about 60 m# in diameter, varies greatly in length.

It may be as long as 150 m# or essentially nonexistent. In the latter case, the pit is a shallow
depression in the oocyte surface, identified, however, as a pit of this type by the characteristic coat
on the cytoplasmic side. Apparently these variants
represent stages in the development of deep pits
from shallow ones and the eventual pinching off
of the deepest part of the pit to form a coated
vesicle. Ordinarily the bristle coating is not present
on the neck of the pit but only over its deepest
part, the diameter of which exceeds that of the
neck.
The individual cortical vesicles in the cortical
zone of a maturing oocyte are no longer of uniform
size as they were prior to the blood meal. They
display, in fact, a broad spectrum of sizes crowded
into the region immediately beneath the membrane and slightly deeper in the cytoplasm (Figs.
3, 4, and 6). Close to the surface the majority are
140 to 150 m# in diameter and still have the
bristle coating. Farther in, there are larger vesicular units with a content of the same density as
that of the coated vesicles, but without the characteristic coating. And deeper still, there are
vesicles whose content is similar to that of both
pits and yolk granules (Fig. 6). Apparently through
a fusion of these various units the larger proteid
bodies of the mature oocyte gradually develop
(Figs. 6, 7, and 8). The yolk material, which first
appears as a layer adsorbed onto the oocyte
surface, is therefore thought to be taken into the
cell via the coated pit and vesicle, possibly changed
in character as the vesicles coalesce, and finally
incorporated into yolk granules.
It should be noted that profiles of flattened

FIGURE 4' Micrograph of the interface (FES) between the oocyte (OOC) and the follicular epithelium (FE) 7 hours after a blood meal. A few mitoehondria (M) and a multivesicular body or lysosome (MB) are among the organdies in this region of the follicular
epithelial cytoplasm. The nuclei (N) show the usual nuclear envelope with pores (NP).
Between the mierovilli (MV), which arise from the oocyte surface, are frequent pits (P)
filled with a dense content. Pit-derived vesicles (V), lipid (L), mitoehondria (M), elements
of the endoplasmie retieulum (ER), and graded stages in proteid droplet (PR) formation
crowd the oocyte cortex. A uniform layer of dense material (arrows) adheres to the oocyte
plasma membrane and is similar in density and texture to that found in the pits, vesicles,
and proteid droplets. X 34,500.
The inset at the lower right shows at greater enlargement a portion of the oocyte cortex.
The materials contained in the pit (P), bristle-coated vesicles (V), naked vesicle (NV),
and proteid droplet (PR) all have the same density and granularity as that on the oocyte
surface (arrow). Where a vesicle membrane is cut in vertical section, it has the same
dimensions as the plasma membrane. X 50,000.

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During this period of active yolk deposition

there are roughly 300,000 pits on the surface of
the oocyte facing the follicle cells. This figure is
nearly 15 times the number of pits on the resting
oocyte fixed only 7 hours earlier.
The pits and vesicles have structural features in
common which help to relate them. It is, for
example, characteristic for a cortical vesicle near
the surface to be about 140 m # in over-all diameter
and to show, morphologically, 3 distinct layers.
Of these, the middle one is most dense and the
thinnest (ca. 75 A). This layer is obviously equivalent in all its characteristics to the plasma membrane of the oocyte (Fig. 4, insert, and Fig. 5). Internal to this line there is a layer of material 250 to
400 A thick, which by its density and texture can
be identified with the layer of material on the external surface of the oocyte cell membrane. External to the middle layer, and continuous with
the cytoplasmic matrix, there is a layer or coat
about 200 A thick which seems to be made up of
small bristles or hairs radiating outward from the
dense line. We shall refer to this as the "bristle
coat" and to the vesicles as "coated."
It is not difficult to see a close structural relationship between the coated vesicles and the pits.
Both show the same 3-layered structure, and it is
evident in the pits in Fig. 5 that the middle one of
the layers has the thickness and molecular structure
of a unit membrane. In the same image, the
structure of the inner, adsorbed layer and the
outer bristle coat are shown to better advantage
than elsewhere.
In other respects the pits seem to represent
simply a coated vesicle connected with the surface
of the oocyte by a neck. The neck, which is usually

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ThoMAs F. ROTH AND KEITH R. PORTER Yolk Protein Uptake

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vesicles often a p p e a r a t t a c h e d to the limiting

m e m b r a n e of the larger proteid droplets a n d show
open continuity with the content of the droplet
(Fig. 6). According to one interpretation, these
m a y represent the r e m n a n t of a recently emptied
" e x c o a t e d " vesicle t h a t has fused with a droplet.
T h e y m i g h t equally well represent small elements
of the endoplasmic reticulum or Golgi system
possibly c o n t r i b u t i n g some material, m a y b e a n
enzyme, to the droplet.
T h e larger, dense bodies present in the egg
cytoplasm, the yolk granules, display a crystalline
p a t t e r n in their content, a n d this m a y exhibit
various orientations in a single plane of section
(Figs. 6 a n d 8). This shift in orientation pre-

sumably arises from the fusion of several smaller

units which h a d adopted crystalline order before
fusion. These large, highly ordered, dense bodies
give positive protein a n d PAS histochemical
tests, a n d thus are considered to be identical with
the protein yolk granules observed b y the light
microscopists (32, 33).

Site of Yolk Synthesis

T h e implication from the above observations
a n d the literature references on yolk synthesis is
t h a t these cortical pits take u p material from the
extracellular space of the follicle a n d contribute it
to yolk granule formation. T h e question of immediate interest is where in the insect are the

I~[GURE 6 At the juncture shown here between the follicular epithelium (FE), nurse
cells (NC) and the oocyte (OOC),microvilli extend into the widening follicular epithelial
space (FES) of the maturing ovary. Pits (P) along the oocyte cell membrane are filled
with a dense material similar to that in the developing proteid droplets (Pit). In some
cases out pocketings appear on the membrane of the droplet (arrow) which are interpreted
as vesicles (V) that have just emptied their contents into the membrane-limited space
(also see Fig. 7). Small cisternae (C) are also continuous with the membrane of the droplet.
These may represent the membrane remnant of large vesicles that have recently coalesced
with the droplet. The content of the yolk protein body assumes a crystalline-like configuration when it matures (PC). ER, endoplasmic reticulum; M, mitochondria. X 41,500.

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FIGUaE 5 Several pits are shown in greater detail. A uniform layer of dense material (DM) adheres to
the 75 A unit membrane (U) on the free surface of the pit, while on the cytoplasmic side of the membrane
the 200 A bristles (arrows) radiate into the oocyte cortex. The unit membrane has the same thickness and
3-layered structure as that limiting the oocyte. The bristles, in some images, appear joined near their outer
ends. OOC, oocyte; FES, interface between oocyte and follicular epithelium. )< 106,000.

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yolk proteins etc., synthesized. To obtain some

clues to this, we made a survey of the cells in the
mosquito's abdomen, paying special attention
to the occurrence of systems at the fine structure
level usually associated with protein synthesis for
export.
The cells of the Malpighian tubules, for
example, do not possess the ergastoplasmic form
of the ER, and show little evidence of an enlarged

200 A in diameter, designed presumably to increase the extent of the cell surface. In the subcortical zone, under the free surface of these cells,
numerous mitochondria are crowded into a layer
2 to 4/~ deep which blends into a cytoplasm that
is characterized by arrays of rough ER, free
ribosomes, and extensive infoldings from the basal
and lateral surfaces of the cell. The nucleus, at
this posffeeding phase, contains a prominent

Golgi component or of secretory granules. Cells

of the fat body do show rough E R in substantial
amounts, but no secretory granules. From the
survey, only the ceils of the midgut epithelium
emerged as likely candidates for the role of
synthesizing yolk protein.
Two types of cells are found in the midgut of the
mosquito, one a regenerative and the other an
absorptive type (Fig. 9). The apical pole of the
latter is covered with closely packed microvilli,
each about 140 m# in diameter and of undetermined length. These have, in turn, upon them a
number of tiny microvilli (micromicrovilli) about

324

nucleolus. The prominent development of roughsurfaced ER, and the numerous Golgi profiles
with some evidence of secretory granules, encourage the thought that these cells, beside being
absorptive, are active in synthesis and secretion.
In their fine structure they remind one of vertebrate liver cells.
AUTORADIOGRAPHIC

EXPERIMENTS

In an attempt to determine the pathway by

which the proteins, formed after a blood meal,
enter the different tissues of the abdomen, the
mosquitoes were fed on an anesthesized rat made

THE JOURNALOF CELL BIOLOGY VOLUME~0, 1964

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FIGURE7 This mierograph of the cortex of the oocyte shows what we interpret as a naked vesicle (NV) in
the act of fusing (arrow) with the membrane of the yolk droplet (PR). Smaller irregularly shaped droplets
(PR) and coated vesicles (V) are abundant in the area immediately beneath the pit-studded (P) plasma
membrane (PM). Lipid bodies (L) and Golgi components (G) are other common inclusions of the egg cytoplasm at this period of yolk formation. )< 56,500.

Published February 1, 1964

radioactive by an intraperitoneal injection of DLleucine-H3. Although the results are quite preliminary, and are now being repeated at the resolution
offered by the electron microscope, they give some
information of possible significance to this study.
In all samples, silver grains were located in
high concentration over the blood meal in the
midgut. Above the midgut epithelium, however,
the number of silver grains increased from just

Malphighian tubules exhibit a pattern of labeling

similar to that observed above the midgut cells.
DISCUSSION
The proteid droplets in Aedes aegypti constitute
the major form of yolk protein in the egg (31,
32). By studying the formation of these bodies,
we have sought to shed some light on the site of

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FmURE8 The different orientation of the crystal lattice planes in the small granules that fuse to form
the yolk proteid body is shown in this micrograph. A unit membrane (UM) limits the droplets. X 108,000.
above background at 0.5 hours after the blood
meal to a plateau of about triple background at
4.5 hours, and then were maintained near that
level in all samples taken later in yolk formation.
In the ovary a similar rise in radioactivity occurred in the nurse cells and oocyte during the
early 0.5 to 4.5 hours after a blood meal. Later, in
the 16- and 30-hour samples, the number of silver
grains increased notably above the protein yolk
bodies.
The abdominal fat body does not appear to
show a significant increase above background
labeling until the 16- and 30-hour samples. The

synthesis, transport, and method of uptake of

yolk protein by the oocyte.

Sites of Yolk Synthesis

The possible sites of synthesis of yolk protein
in the mosquito ovary can be considered, on
morphological grounds, to be restricted to three.
One could be the follicular epithelial cells; their
proximity to the oocyte, for example, suggests a
possible involvement in the synthesis of material
for oocyte growth and differentiation (Fig. 3).
However, these cells contain almost none of the
machinery usually associated with intracellular

THOMASF. ROTHANDKEITHR. PORTER Yolk Protein Uptake

325

Published February 1, 1964

that the same pattern is true for the insects he has

studied. Particularly significant for the interpretation of our observations is Telfer's demonstration, by immunohistochemical studies on the
saturniid moth, that certain foreign proteins are
taken into the oocyte. He also demonstrated that
naturally occurring proteins in the hemolymph
are preferentially concentrated in the ovary, and
that no yolk protein could be detected in the
follicular epithelial cells, the nurse cells, or in the
cytoplasm of the oocyte apart from the yolk
droplets. He concludes justifiably that yolk proteins are synthesized at some extraovarian point
in the insect and transported to the ovary for
deposition. Though no similar studies have been
made on the mosquito, it seems reasonable to
expect that the same mechanisms would be operative in this form.
If the ceils of the ovary are not involved, where
does yolk synthesis take place? Thus far, as reported here, our studies of the tissues of the mosquito abdomen have identified only certain cells
of the midgut as having the structural equipment
needed for this function. This consists of ribosome-studded cisternae in parallel arrays and
other elements of fine structure usually associated
with secretion. These cells at their apical pole are
in contact with the contents of the intestine following a blood meal, and on their other side are in a
favorable position to secrete into the hemocoel.
Furthermore, the autoradiographs in the experiments which included tritiated leucine in the
blood, while preliminary, showed definite accumulation and concentration of label over these
cells. It seems highly probable, therefore, that
these units of the midgut are at least one site of
yolk synthesis.
The only other candidates for this role are the

FIGURE 9 This micrograph shows the greater part of an epithelial cell from the midgut
of the mosquito. The apical pole is covered with microvilli (MV) about 140 m# in diameter, which appear here in both longitudinal and cross-section. Smaller microvilli (VB),
ca. ~O m#, bud off from the larger ones. In the subapieal region, immediately beneath a
relatively clear cortex, is a zone densely populated with mitochondria (M). At other levels
in the cytoplasm, profiles of the endoplasmic reticulum (ER) dominate the picture. These
profiles describe the system as being constructed in part of large lamellar cisternae, their
surfaces heavily enerusted with ribosomes, typical for ceils active in protein synthesis.
Sections through the Golgi complex ((7) are scattered about in the supranuelear zone.
Prominent also are extensive infoldings of the plasma membrane along the lateral surfaces
of the cell, and most especially the basal smface. These infoldings reach in some instances
almost to the apical pole of the cell and show irregular dilations (arrows). D marks the
separation between adjacent cells. )< approximately 21,600.

326

TnE JOURNALOF CELL BIOLOGY " VOLUME20, 1964

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synthesis of protein for secretion (35). They have

little of the rough form of the endoplasmic reticulure, infrequent Golgi bodies, and, at this stage, no
evidence of secretory granules or other secretory
activity at the cell surface. Only after yolk deposition is essentially completed do the cells of this
epithelium show any proliferation of the E R and
Golgi complex. When this occurs, these cells are
engaged in producing the chorion of the egg (31,
33) and most yolk deposition has already been
completed.
The nurse cells and the oocyte itself are the
other two possible sites of synthesis, but here, as
in the follicular epithelium, the machinery (cytoplasmic inclusions and secretory granules) usually
present for such purposes is absent during the short
period of yolk deposition. The presence of great
numbers of fi-ee ribosomes in the cytoplasm of
both cells suggests active protein synthesis. This
is, however, the pattern found in rapidly growing
undifferentiated cells in which the components of
the cytoplasmic matrix are being formed, and
there is no associated mechanism for the transport
and assembling of the protein into yolk droplets.
Therefore, unlike the crayfish oocyte in which
the E R is clearly involved in the synthesis of
yolk protein (3), or the frog oocyte (52) where the
mitochondria are associated with yolk deposition,
or the snail oocyte where Favard and Carasso
(11) implicate both mitochondria and E R in
yolk formation, the mosquito oocyte shows no
obvious involvement in the manufacture of its
own yolk.
This conclusion is in no way surprising. The
uptake and deposition in the oocyte of yolk proteins from extraovarian sources appears to be the
common pattern among the vertebrates. And
even more pertinent here are the findings of Telfer

Published February 1, 1964

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THOMAS F. ROTH AND KEITH R. PORTER Yolk Protein Uptake

327

Published February 1, 1964

cells of the fat body which likewise show rich

developments of the rough ER. Here, however,
the accumulation of label following the feeding of
tritiated leucine is delayed until late in yolk
deposition.
The suggestion that the midgut is the principal
site of protein synthesis for yolk formation is at
variance with a long standing bias among insect
physiologists toward the fat body for this role.
It is well established that most insects contain
protein stored in droplets in the fat body, and as
proteins or amino acids are needed this source is

328

mobilized. Our autoradiographic results may be

due to the absence of protein in the fat bodies of
the 1-week-old mosquitoes we used, and the
resultant absence of this potential protein source
during oogenesis.
The mechanisms of yolk transport to and uptake by the ovariole, and subsequent concentration in the oocyte, have been considered only
briefly by previous investigators except Telfer.
In a studious examination of the question, he
implicates the oocyte surface and the basement
membrane in the selective uptake. The surface

T~E JOURNALOF CELL BIOLOGY VOLUME20, 1964

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FIGURE 10 This schematic drawing interprets the mierographs examined in this study. It depicts
changes and events involving the cortical pits and coated vesicles of the mosquito oocyte. At (1) is shown
the first stage of invagination into the oocyte of the protein-coated plasma membrane from the intercellular
space. The fully developed pit (2), by pinching off, forms the coated vesicle (3). These vesicles lose their
bristles to form dense spheres of similar size (4) which then fuse with other dense spheres (5). Often a
flattened empty sac is attached to the droplet (7). This sac may be the membrane remnant of a vesicle or
perhaps some element of the Golgi complex that has recently fused with the droplet. The larger droplets
(6) coalesce to form the large crystalline proteid yolk bodies (8) of the oocyte. Other conspicuous and
characteristic inclusions and organelles of the oocyte cytoplasm are mitochondria, vesicles of the rough
surfaced cndoplasmic reticulum (ER), lipid (L) and ribosomes. At the top of the drawing, microvilli project
into the intercellular space fronting on the follicular epithelium. Note the absence of adhering material
on the membranes of the follicular epithelial cells (also see Fig. 4). )< approximately 60,000.

Published February 1, 1964

tein," coprecipitate at a lowered pH and/or

lowered ionic strength with the release of proteinbound calcium. If either or both of these conditions were to be met in vivo, coprecipitation would
explain the aggregates encountered in the interfollicular space.
The same inward migration of yolk protein is
doubtlessly influenced as well by the condensation
of material on the oocyte surface and its incorporation into the egg. Just what it is, in or on the oocyte
surface, that encourages the development of such
a layer is likewise not known. That it represents
yolk protein is indicated by the observation that
this oocyte-associated material in the Cecropia
moth, for example, is immunologically similar to
the yolk protein within the oocyte and the serum
proteins in the hemocoel (50).

The Uptake Mechanism

It has been shown here that, in the region of the
oocytc surface adjoining the follicular epithelial
cells, numerous small depressions or pits extend
into the egg cortex. Similar differentiations have
been noted by Wartcnbcrg (53) in the oocytc of
several amphibians, but without attention to their
characteristic fine structure. He interpreted these
as engaged in pinocytosis associated with the
uptake of materials for yolk formation. We find
that the cortical pits number approximately
300,000 in the stage of oocytc development
achieved 7 hours after feeding. This represents a
15-fold increase over the number of pits found in
the resting stage preceding feeding. Their structure is uniform. Each pit contains a quantity of
dense material having the same texture as that
adsorbed on the oocytc surface at other points,
which in turn seems related to the flocculent material in the follicular spaces. On their inner
convex surfaces facing the cytoplasm, the pits
are covered with a border of fine bristles about
200 A long.
Several significant reasons arc apparent for
associating these pits with yolk protein uptake
and the formation of proteid droplets and granules
of the maturing egg. The first one is the fact that
uptake is in progress when the pit development
reaches its most prominent expression. One
could argue further that other mechanisms of
uptake are not evident and that simple diffusion of
yolk through the plasma membrane is highly improbable. Then, it is obvious that vesicles deeper
in the cortex are related to the pits and are de-

THOMAS F. ROTH AND KZlTH R, :PORTER Yolk Protein Uptake

329

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of the oocyte, he speculates, might possess a

mechanism for selective absorption akin to that
demonstrated for the surface of the ameba (5).
Uptake and transport might be achieved by a
process similar to pinocytosis.
The observations derived from the present
study bear mostly on these questions and, in
general, support Telfer's speculations.
The spaces between the follicular epithelial
cells begin to widen by 4 hours after a blood meal,
and by 7 hours have opened sufficiently to allow
unobstructed communication from the basement
layer to the oocyte surface by this intercellular
route (Fig. 3). Before yolk proteins can enter this
newly formed space they must pass through the
basement layer enclosing the ovariole. Although
not all investigators agree that a basement layer
is permeable to whole proteins, there is good evidence that certain foreign and maternal proteins
present in the blood can penetrate this layer in
certain tissues (10, 24, 28, 47, 49).
Again, with respect to the present study, Telfer's
findings are especially significant, for they demonstrate that proteins in the hemocoel do traverse
the basement layer and are present in the interfollicular space just within. Some device probably
exists capable of holding the yolk proteins inside
the basement membrane and continuing their
inward diffusion. The micrographic evidence
bearing on this question describes a coarsely
granular material on the inside of the basement
membrane, and no resolvable material outside.
Furthermore, it suggests that once inside, the
protein is complexed into relatively large aggregates which are too large to diffuse out through
the membrane. Thus the conditions required for
the continuing inward diffusion of the smaller
molecular species are maintained with the basement membrane functioning as part of a diffusion pump. What serves to aggregate the proteins
into larger complexes is not known, but a factor or
factors synthesized in either the follicular epithelial cells or the oocyte could be secreted into
the extracellular space to perform the task.
An equally valid possibility is that the serum
proteins coprecipitate in the interfollicular space
and thus fulfill the same requirement for inward
diffusion. In this connection, it is worth noting
that Schjeide and Urist (43) have shown that two
serum and yolk proteins of the chicken are very
similar and that the two components of each,
the X1 phosphoprotein and the X2 "glycolipopro-

Published February 1, 1964

obvious from other observations, however, that

cortical pits of this general character form and
apparently separate from the surface without this
apparatus (34). Perhaps this proposal has less
merit than that which associates the specificity of
materials adsorbed with characteristics of the
bristle border. It is not possible at the moment
to be other than vague in these suggestions.
Pits of similar structure have been reported in
many cell types (1, 2, 39, 40, 46). Their occurrence
is indeed widespread, as will be reported in a
subsequent paper. They are not to be confused
with the simpler pits found especially in smooth
muscle cells and blood vascular endothelial cells
(34, 53). The latter have simple, clean limiting
membranes and a smaller diameter. At this time
we present the working hypothesis that pits of the
type found here in the mosquito are, in general,
involved in selective uptake of materials (proteins
and perhaps non-proteins) from the cell's environment. Since the mechanism evinces Some selectivity (39), it is reasonable to propose that different
cell types may manifest different specificities,
with some cells, such as those of the reticuloendothelial system, showing a broader spectrum of
interests than others.
This investigation was supported in part by a Public
Health Service Training Grant 2G-707 from The
National Institutes of Health, United States Public
Health Service.
Received for publication, May 28, 1963.

BIBLIOGRAPHY
1. ANDERSON,E., The ovary of Periplaneta americana,
Abstracts of the 2nd Annual Meeting of the
American Society for Cell Biology, San Francisco, 1962, 2.
2. ASHFORD,T., PORTER, K. R., and BADENHAUSEN,
S., Modulations in the fine structure of rat
liver cells under conditions of organ isolation
and perfusion, 1964, in press.
3. BEAMS, H. W., and KESSEL, R. G., Endoplasmic
reticulum and yolk formation in crayfish,
Abstracts of the 2nd Annual Meeting of the
American Society for Cell Biology, San Francisco, 1962, 13.
4. BRAMBELL, F. W. R., and HEMMINOS, W. A.,
Active transport through embryonic membranes, Syrup. Soc. Exp. Biol., 1954, 8, 476.
5. BRANDT, P. W., A study of the mechanism of
pinocytosis, Exp. Cell Research, 1958, 15, 300.
6. CARO, L. G., and VAN TUBERGEN, R. P., High-

330

7.
8.
9.

10.

II.

12.

ThE JOURNALOF CELL BIOLOGY " VOLUMEg0, 1964

resolution a utoradiogn-aphy.
I. Methods,
J. Cell Biol., 1962, 15, 173.
CHARGAFF,E., The formation of the phosphorus
compounds in egg yolk, J. Biol. Chem., 1942,
142,505.
CHRISTOPHERS, S. R., The development of the
egg follicle in An@helines, Paludism, 1911, 2, 73.
CHRISTOPHERS, S. R., Aedes aegypti L. Its life
history, Bionomics and Structure, London,
Cambridge University Press, 1961, 676.
FAR~UHAR,M. G., and PALADE,G. E., Glomerular capillary wall in nephritic rats, J. Exp.
Med., 1961, 114, 699.
FAVARD, P., and CARASSO, N., Origin et ultrastructure des plaquettes vitellines de la
Planorbe, Arch. Anat. Micro. et Morph. Exp.,
1958, 47, 211.
FEDER, N., Some modifications in conventional

Downloaded from on October 20, 2016

rivafives of them. These vesicles possess the same

content and the same border of bristles.
To this point the animation of this uptake
mechanism is not hard to picture and is essentially
that which characterizes pinocytosis. W h e n the
process of yolk absorption is at its peak, the formation of pits and their derivatives is probably
quite rapid. The subsequent fate of the pit-vesicles
is somewhat less clear. T h e y apparently lose the
bristle border, fuse to form larger vesicles, pick up
other satellite vesicles, and progressively grow
into "proteid droplets" several times greater in
diameter than the pit-vesicles. This interpretation
is depicted in Fig. 10.
The content of these larger dense yolk bodies
also changes. From a beginning of homogenous
density, there gradually emerges evidence of
order in the arrangement of the particles, a
crystallinity of structure common to yolk granules.
This could depend on a process of selective hydrolysis within the vesicle, resulting finally in a
high degree of purity in the residual yolk. Therefore, even though the pits and the oocyte surface
may be selective, to a degree, in what they adsorb
from the perioocyte lymph, subsequent purification is not unlikely.
The role of the bristle border in all of this is
hard to imagine. It may have a mechanical function, giving, by virtue of a natural repulsion of
the outer ends of the bristles, the spherical form
to the base of the pit and the pit-vesicles. It is

Published February 1, 1964

13.
14.

15.

16.

17.

18.

20.

21.
22.

23.

24.

25.
26.
27.

28.

29.

30. MILLONIO, G., A modified procedure for lead

staining of thin sections, or. Biophysic. and Biochem. Cytol., 1961, 11, 736.
31. NATH, V., Egg-Follicle of Culex, Quart. or.
Micr. Sc., 1924, 69, 151.
32. NATH, V., Studies on the shape of the Golgi
apparatus. I. The egg follicle of Culex, Z.
Zellforsch., 1929, 8, 655.
33. NICHOLSON, A. J., The development of the
ovary and ovarian egg of a mosquito, Anopheles
maculipennis, Quart. J. Micr. Sc., 1920, 65, 395.
34. PALADE, G. E., Transport in quanta across the
endothelium of blood capillaries, Anat. Rec.,
1960, 136, 254.
35. PALADE, G. E., and SIEKEVlTZ, P., Liver microsomes. An integrated morphological and biochemical study, J. Biophysic. and Biochem.
Cytol., 1956, 2, 171.
36. PARKS, J. J., An anatomical and histological
study of the female reproductive system and
follicular development in Aedes aegypti L.,
Master of Science Thesis, University of
Minnesota, 1955.
37. PEACHEY, L. D., Accumulation of divalent ions
in mitochondrial granules of intact cells, in
5th International Congress for Electron
Microscopy, Philadelphia, 1962, (S. S.
Breese, Jr., editor), New York, Academic
Press, Inc., 1962, 2, 00-3.
38. ROEPKE, R. R., and BUSHNELL, L. D., A serological comparison of the phosphoproteins of
the serum of the laying hen and the vitellin
of the egg yolk, J. Immunol., 1936, 30, 109.
39. ROTH, T. F., and PORTER, K. R., Specialized
sites on the cell surface for protein uptake, in
5th International Congress for Electron
Microscopy, Philadelphia,
1962, (S. S.
Breese, Jr., editor), New York, Academic
Press, Inc., 2, LL-4.
40. ROTH, T. F., and PORTER, K. R., Membrane
differentiation for protein uptake, Fed. Proc.,
1963, 22, No. 2, abstract.
41. RoY, D., and MAJUMDAR, S., On mating and
egg formation in Culex fatigans Weid, J.
Malaria Inst., India, 1939, 2, 243.
42. SCHJEIDE, O. A., and URIST, M. R., Proteins
and calcium in serums of estrogen treated
roosters, Science, 1956, 124, 1242.
43. SCHJEIDE, O. A., and URIST, M. R., Proteins
and calcium in egg yolk. Exp. Cell Research,
1959, 17, 84.
44. SINCH, K. R. P., and BROWN, A. W. A., Nutritional requirements of Aedes aegypti L., J.
Insect Physiol., 1957, 1, 109.
45. SMITH, A. H., Follicular permeability and yolk
formation. Poultry Sc., 1959, 38, 1437p.
46. STAY, B., personal communication on pits in the
Cecropia silkworm.

THOMASF. tlK)THAND KEITIt R. PORTER Yolk Protein Uptake

331

Downloaded from on October 20, 2016

19.

techniques of tissue preparation, J. Histochem.

and Cytochem., 1960, 8, 309.
FEBER, N., personal communication.
FLICmNGER, R. A., Formation, biochemical
composition and utilization of amphibian egg
yolk, in Symposium on Germ Cells and Development, Institut Internationale d'Embryologie and Fondazione A. Baselli, Pavia,
Italy, Premiata Tipografia Successore Fratelli
Fusi, 1960, 29.
FLICKINGER, R. A., and ROUNDS, D. E., The
maternal synthesis of egg yolk proteins as
demonstrated by isotopic and serological
means, Biochim. et Biophysic. Acta, 1956, 22, 38.
FLICKINGER,R. A., and SCHJEIDE, O. A., The
localization of phosphorus and the site of
calcium binding in the yolk proteins of the
frog's egg, Exp. Cell Research, 1957, 13, 312.
GLASS, L. E., Immuno-histological localization
of serum-like molecules in frog oocytes, J.
Exp. Zool., 1959, 141, 257.
GRANT,P., Phosphate metabolism during oogenesis in Rana temporaria, J. Exp. Zool., 1953, 124,
513.
HAHN, L., and HEVESY, G., Origin of yolk
lecithin, Nature, 1937, 140, 1059.
HOSODA, T., KANEKO, Z., MOGI, K., and ABE,
T., Serological Studies on Egg Production in
the Fowl. I. On the locus of serum vitellin
production, Poultry Sc., 1955, 34, 9.
JUKES, T. H., and KA', H. D., Egg yolk protein,
or. Nutrition, 1932, 5, 81.
KARNOVSKY,M. J., Simple methods for "staining
with lead" at high pH in electron microscopy,
J. Biophysic. and Biochem. Cytol., 1961, 11, 729.
KING, R. C., Oogenesis in adult Drosophila
melanogaster. IX. Studies on the cytochemistry
and ultrastructure of developing oocytes,
Growth, 1960, 24, 265.
KNmHT, P. F., and SCHECHTMAN,A. M., The
passage of heterologons serum proteins from
the circulation into the ovum of the fowl, or.
Exp. Zool., 1954, 127, 271.
LmKOWSKI,M., Darstellungsmethode des Serumvitelline, Biochem. Z., 1935, 278, 345.
LEDBETTER, M. C., unpublished observations.
MANCINI, R. E., VILAR, O., HEINRICH, J. J.,
DAVIDSON, O. W., and ALVAREZ, B., Transference of circulating labeled serum proteins
to the follicle of the rat ovary, J. Histochem.
and Cytochem., 1963, 11, 80.
MILLER, F., Hemoglobin absorption by the
cells of the proximal convoluted tubule in
mouse kidney, J. Biophysic. and Biochem. Cytol.,
1960, 8, 689.
MILLONIO,G., Advantages of a phosphate buffer
for OsO4 solutions in fixation, J. Appl. Physics,
1961, 32, 1637.

Published February 1, 1964

47. STRAUS, W,, Cytochemieal investigation of

phagosomes and related structures in cryostat
sections of the kidney and liver of rats after
intravenous administration of horseradish
peroxidase, Exp. Cell Research, 1962, 27, 80.
48. TELFER, W. H., Immunological studies of insect
metamorphosis. II. The role of a sex-limited
blood protein in egg formation by the Cecropia
silkworm, J. Gen. Physiol., 1954, 37, 539.
49. TELFER, W. H., The selective accumulation of
blood proteins by the oocytes of saturniid
moths, Biol. Bull., 1960, 118, 338.
50. "rEEFER, W. H., The route of entry and localization of blood proteins in the oocytes of saturniid moths. J. Biophysic. and Biochem. Cytol.,
1961, 9, 747.

51. TREMBLEY, H. L., Mosquito culture techniques

and experimental procedures, Bull. No. 3,
American Mosquito Control Association Inc.,
Berkeley, Calitornia; Lederer, Street, and Zeus
Co., Inc. 1955.
52. WARD, R. T., The origin of protein and fatty
yolk in Rana pipiens. II. Electron microscopical
and cytochemical observations on young and
mature oocytes, J. Cell Biol., 1962, 14, 309.
53. WArtTENBERC, H., Electronenmikroskopische
und Histochemische Studien uber die Oogenese der Amphibieneizelle, Z. Zellforsch.,
1962, 58, 427.
54. Wissio, S. L., An E M study of the permeability
of capillaries in muscle, Anat. Rec., 1957, 130,
467.

Downloaded from on October 20, 2016

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