/ . Embryol. exp. Morph., Vol. 17, 2, pp. 267-281, April 1967
With 10 plates
Printed in Great Britain
267
Aspects of the
development of yolk spheres in the hen's oocyte,
studied by electron microscopy
BY RUTH BELLAIRS 1
Department of Anatomy and Embryology, University College London
INTRODUCTION
The yolk of the hen's egg is composed mainly of proteins, lipids and water (see
reviews by Bellairs, 1964; Williams, 1966). It consists essentially of yolk spheres
floating in an aqueous protein medium (Grodzinski, 1939; Bellairs, 1961).
The raw materials from which the yolk is formed are synthesized in the liver
of the laying hen and pass from there in the blood to the ovary (see reviews by
Romanoff, 1960; Bellairs, 1964). Each oocyte is enclosed in a capsule of follicle
cells, and all the raw materials pass through this capsule before they enter the
oocyte. The morphological changes that take place within the oocyte as the
yolk spheres form have been described previously by light microscopists who
have produced a variety of theories to explain their observations. Formerly, it
was supposed that yolk arose in the so-called 'yolk nucleus', or Balbiani body,
which lies alongside the cell nucleus, but few would subscribe to this theory
now. Some authors have suggested that yolk may form by direct transformation
of mitochondria or of Golgi apparatus, but in view of the present electronmicroscopical investigation this hypothesis must also be abandoned.
The formation of the yolk spheres in the hen's oocyte has not previously been
studied by electron microscopy, though certain aspects of the problem have
been considered by Schjeide, McCandless & Munn (1964,1965 a, b). The present
paper is the first of a series devoted to elucidating the mechanism by which
yolk spheres form.
MATERIALS AND METHODS
Fourteen laying hens were used in the present investigation and oocytes were
obtained that ranged in size from about 7 0 ^ to about 35 mm maximum
diameter. The hens were killed with chloroform and pieces of ovary were
removed immediately afterwards. The following fixatives have been used for
electron microscopy: osmium tetroxide (Palade, 1952; Millonig, 1961), gluta1
Author's address: Department of Anatomy and Embryology, University College,
Gower Street, London, W.C.I, England.
268
R. BELLAIRS
raldehyde (Sabatini, Bensch & Barrnett, 1963), paraformaldehyde (Pease, 1964),
or formaldehyde and glutaraldehyde (Karnofsky, 1965), and all were buffered
with phosphate or cacodylate to a pH of 7-0-7-6. Fixation was at about 4 °C for
periods varying from \ to 2 h. Those specimens that were not initially treated
with osmium tetroxide were washed in buffer solution after the fixation and then
immersed in osmium tetroxide (Palade, 1952) for periods up to £ h.
Dehydration of all specimens was at room temperature with graded ethanols
and some specimens were stained in bulk with uranyl acetate in 100 % ethanol
(Westrum, 1965). The material was embedded in Araldite (Glauert & Glauert,
1958) and sectioned with a Porter-Blum Mark I ultra-microtome. The sections
were stained with either potassium permanganate (Lawn, 1959), uranyl acetate
(Watson, 1958) or lead citrate (Reynolds, 1963). They were examined with a
Siemens Elmiskop 1 b electron microscope.
Large thick sections were also prepared from the same blocks of tissue and
examined by phase-contrast microscopy. In this way it was possible to identify
the regions of the oocytes seen by electron microscopy.
Other material examined by light microscopy was fixed in Carnoy's fluid or in
alcoholic formalin, dehydrated in ethanols, embedded in paraffin wax, sectioned
and stained with haematoxylin and eosin. Some sections were stained with PAS
technique and others with Best's carmine (both techniques as given by Pearse,
1953).
RESULTS
Different investigators have recognized a variety of stages in the development
of the hen's oocyte. In the present study these have been grouped into two main
phases, i.e. stages before the appearance of yolk, and stages when yolk is present.
I. Stages prior to the appearance of yolk
(a) The Balbiani stage
The individual oocytes measure between about 70 and 150/* in maximum
diameter and are surrounded by a single layer of follicle cells about 4-20/* deep.
The nucleus of the oocyte is about 30/* in diameter and excentrically placed.
The Balbiani body lies near the middle of the cell (Plate 1, fig. A; Text-fig. 1)
and is surrounded by large numbers of lipid drops and mitochondria.
These gross elements have been recognized and described by light microscopists, so that only the electron-microscopical appearances will be considered
here.
The cell membrane of the oocyte at the Balbiani stage has already been described (Bellairs, 1965). It lies close beneath the follicle cell membrane but in
places it is raised into folds. These folds are at the junction of two adjacent
follicle cells. Occasionally, small pinocytotic-like vesicles can be seen beneath
the oocyte cell membrane.
Yolk sphere development
269
The non-Balbiani regions. The ground substance consists mainly of small
granules, individually about 100-120 A in diameter, and fine filaments, but
patches of glycogen are also present. Mitochondria are scarce but correspond
in structure with those of the Balbiani region. Lining body vesicles (see below)
are occasionally seen. Lipid drops surround the Balbiani body and extend
around the nucleus (see Text-fig. 1). They are sometimes irregular in shape and
usually about \-% ju, in diameter.
The Balbiani body (see Text-fig. 1). The most conspicuous feature of this
region of the oocyte is the large number of vesicles (Plate 2, fig. A). The largest
of these are patches of Golgi apparatus (Plate 2, fig. B) which are scattered
Follicle'cell
Pinocytotic
vesicle
Mitochondrion
Multivesicular
body
Granulefilled
vesicle
Balbiani
body
Perforated
nuclear
membrane
600 A
vesicles
Annulate
lamellae
Text-fig. 1. Interpretative diagram of the structures present in the oocyte at the
Balbiani stage. There is a high concentration of organelles in the Balbiani region
and this is surrounded by a region of lipid drops.
throughout the Balbiani body. The individual Golgi vesicles are irregular in
shape, may measure as much as 1 ju, in maximum length and have an 'empty'
appearance. Similar large 'empty' vesicles are also scattered apparently as
separate units in this region of the oocyte, though it is possible that they may
be in communication with other parts of the Golgi system. Other vesicles that
are present contain a fine granular matrix and are oval or round in section (Plate
2, inset). These are vesicles which vary in size, but the large ones are about
0-1-0-3 f.i in diameter, and the smaller ones are about 005/* in diameter. The
granular contents are less electron-dense in the large vesicles than in the small
ones.
Multivesicular bodies are also present in the Balbiani body but are relatively
uncommon. They contain smaller vesicles of varying size, as well as small,
densely osmiophilic granules (Plate 2, figs. A, B).
270
R. BELLAIRS
The smallest vesicles, however, are about 600 A or less in diameter and are
most common lying closely packed among the Golgi vesicles. All these vesicles
have in common the fact that they are each surrounded by a unit membrane.
In addition to being richly supplied with vesicles, the Balbiani region possesses
many mitochondria. Typically, these mitochondria are elongated, sections as
long as 3 fi being seen. They have well-preserved cristae and appear to be
scattered randomly among the vesicles (Plate 2, fig. A).
The ground substance is composed of small granules each about 100-120 A
together with occasional fine filaments that are individually about 50 A thick.
A few lipid drops are also present, and they resemble those found in other
parts of the oocyte.
It is generally considered by light microscopists that a centriole is situated at
the centre of the Balbiani body. Centrioles have not been seen in the present
investigation although the fact that they are present in the Balbiani bodies of
newly hatched chicks (Greenfield, 1966) suggests that they may have been overlooked in the adult bird. Annulate lamellae have however been seen in this
region in three specimens (Plate 3, figs. A, B). These structures appear to consist
of a series of membranous channels with pore-like constrictions at intervals
that resemble nuclear pores, both in appearance and in dimensions. In places
the tubular membranes of the annulate lamellae swell out into vesicles which so
closely resemble the granule-filled vesicles of the Balbiani region that it seems
possible that some at least of the latter are derived in this way. Plate 4, fig. A
illustrates an additional structure seen in the Balbiani body of one specimen.
Similar material has also been seen in larger oocytes on two occasions. It is
included here because superficially it resembles the crystalline structure of
amphibian yolk, although the size of the individual components is about 110—
120 A, whereas that of amphibian yolk is about 40 A (Ward, 1962; Karasaki,
1963).
ABBREVIATIONS
B., Balbiani body; /., follicle cell; G.v., Golgi vesicles; /., lipid drops; l.b., lining body;
m., mitochondrial layer; m.v.b., multivesicular body; «., nucleus; n.m., nuclear membrane;
p., pinocytotic vesicle; v.m., vitelline membrane.
PLATE 1
Light micrograph sections through three oocytes of diiferent sizes all taken from the same
ovary. Fixed in Carnoy's fluid and stained with haematoxylin and eosin.
Fig. A. Balbiani stage (see Text-fig. 1), oocyte is 70/* in diameter, x 600.
Fig. B. Dispersed Balbiani stage (see Text-fig. 2), oocyte is 350/* in diameter, x 150.
Fig. C. The stage with large vesicles. Oocyte is 2 mm in diameter. Many of the vesicles contain subdroplets and have therefore begun to change into yolk spheres. The nucleus has
already shifted to one side of the oocyte. x 50.
J. Embryol. exp. Morph., Vol. 17, Part 2
A
^f
R. BELLAIRS
facing p. 270
J. Embryo I. exp. Morph., Vol. 17, Part 2
PLATE 2
Fig. A. Electron micrograph of a section through the Balbiani body, x 25000.
Fig. B. Electron micrograph of part of the same section showing the nuclear membrane and
the edge of the Balbiani body. Note the small vesicles lying in clusters between the Golgi
vesicles, x 20000.
Inset: Enlargement of part of Balbiani body to show two sizes of granule-filled vesicles,
x 25000.
R. BELLAIRS
/. Embryol. exp. Morph., Vol. 17, Part 2
PLATE 3
Electron micrograph of two serial sections through the annulate lamellae lying in a Balbiani
body. The arrows infig.B indicate that the annulate lamellae may be sites of vesicle formation.
Figs. A and B x 32500 and 35000 respectively.
R. BELLA1RS
J. Embryol. exp. Morph., Vol. 17, Part 2
PLATE 4
Fig. A. Electron micrograph of a crystalline lattice structure seen in the Balbiani body of a
single specimen, x 70000.
Fig. B. Electron micrograph of the cortical region of an oocyte at the dispersed Balbiani stage.
The oocyte cell membrane is raised into projections. Note the pinocytotic vesicles and
(bottom left) the larger, granule-filled vesicles, x 55000.
R. BELLA1RS
facing p. 271
Yolk sphere development
271
(b) The dispersed-Balbiani stage {150 fi-1-5 mm.)
In oocytes about 150 /.i in diameter the components of the Balbiani body scatter
and some spread around the nucleus. Eventually, however, both the mitochondria and Golgi components come to be in a subcortical position (Raven,
1961). Meanwhile the nucleus shifts into a more centrally situated region of the
oocyte (Plate 1, fig. B). At this stage the oocyte consists of several fairly welldefined regions (see Text-fig. 2).
The cortical layer consists essentially of vesicles of various types together with
Follicle cell
nucleus
Desmosome
Granule-filled
vesicles
Lining bodies
indenting
oocyte (T.S.
.and L.S.)
Cortical
region
Mitochondrion
Golgi body
Lipid drop
Lining body
vesicle
Vesicles of
various types
Perinuclear
region
Yolk spindles
Perforated
nuclear membrane
Text-fig. 2. Interpretative diagram of the structures present in the oocyte at the
dispersed Balbiani stage. Three main 'layers' are recognized here. For details of the
structure of the follicle cells see Bellairs (1965).
272
R. BELLAIRS
a layer of mitochondria and patches of Golgi apparatus. It is bounded by the
oocyte cell membrane.
Small vesicles he immediately beneath the oocyte cell membrane and these are
probably pinocytotic (Plate 4, fig. B). Individually, they measure between about
500 and 700 A in diameter and are each surrounded by a unit membrane. As
the oocyte grows in size, the cell membrane becomes raised into villi which form
the zona radiata (for a fuller description see Bellairs, 1965; Press, 1964; Wyburn,
Aitken & Johnston, 1965). With this modification, the number of pinocytotic
vesicles increases (Plate 5).
Larger vesicles are also present in the cortex. These are between about 1000
and 4000 A in diameter and are each surrounded by a unit membrane. They
contain granules that are each about 50-100 A in diameter, and fibrillae (Plate 6,
fig. A). Similar vesicles were seen both in the cytoplasm of smaller oocytes (see
above) and in the follicle cells (see Bellairs, 1965). Beaded filaments lie in the
cytoplasm of the oocyte between the vesicles.
Another important component of the cortical region is the lining bodies (for a
full discussion see Bellairs, 1965). These structures are modifications of the
follicle cell membrane (Plates 5, 7) that project down and indent the oocyte cell
membrane. Several are seen in section in Plate 7 that are probably still attached
to the follicle parent cell. In some instances (Plate 7), extensive patches of
follicular cytoplasm are included in the projection. As I have previously pointed
out, however (Bellairs, 1964,1965), the structure of these lining bodies so closely
resembles that of certain vesicles found deep within the oocyte that it seems
likely that some of the lining bodies lose contact with the follicle cells and
become totally engulfed by the oocyte. These lining body vesicles are a general
component of the cytoplasm of this stage, being especially common near the
nucleus (see below).
The mitochondria lie as a continuous layer about 5-15 [i beneath the surface
of the oocyte (Text-fig. 2). The Golgi patches in some specimens are immediately
proximal to the mitochondria whereas in others they are situated within the
mitochondrial band. The mitochondrial profiles are generally elongated, the
cristae are well defined and the osmiophilic patches conspicuous. The Golgi
apparatus appears to possess a high proportion of vesicles, but no dense material
has been seen within them. Annulate lamellae have been found in this region in
two oocytes.
The lipid drops lie deep to the cortical region and form a wide band encircling the perinuclear region (Text-fig. 2). They lie among ground cytoplasm
comparable with that in the rest of the oocyte, namely granules measuring
100 A in diameter, fibrillae, and glycogen granules. A few mitochondria are also
found in this region.
The perinuclear region. The main components are vesicles of several types.
Some of these closely resemble certain vesicles seen in the earlier stage; in
particular, multivesicular bodies are, present. In addition, a further series of
J. Embryo I. exp. Morph., Vol. 17, Part 2
PLATE 5
Electron micrograph through an oocyte measuring 2-5 mm in diameter. Note the villi projecting from the oocyte surface, and the many pinocytotic vesicles in the oocyte cortex. The
vitelline membrane is beginning to form in the gap between follicle and oocyte. The follicle
cell also possesses pinocytotic vesicles, x 68000. (Abbreviations: see legend to Plate 1.)
R. BELLA1RS
facing p. 272
J. Embryo!, exp. Morph., Vol. 17, Part 2
PLATE 6
Fig. A. Electron micrograph of the large granule-filled vesicles lying in the cortex of anoocyte
at the dispersed Balbiani stage, x 120000.
Fig. B. Electron micrograph through a vesicle that is partially a lining body vesicle and
partially a multivesicular body, x 150000.
Fig. C. Electron micrograph of a multivesicular body, x 120000.
R. BELLA'IRS
/. Embryol. exp. Morph., Vol, 17, Part 2
PLATE 7
Electron micrograph through the cortical region of an oocyte at the dispersed Balbiani
stage to show lining bodies and pinocytotic vesicles. These lining bodies are probably still
attached to the follicle cell. A substantial amount of follicle cell cytoplasm including pinocytotic vesicles has been included in the lining body projection shown at the bottom left of the
plate, x 68000.
R. BELLAIRS
J. Embryo/, exp. Morph., Vol. 17, Part 2
PLATE 8
A
R. BELLAIRS
facing p. 273
Yolk sphere development
273
vesicles are found, some of which so closely resemble lining bodies that there
can be little doubt that they are derived from them (Plate 6, fig. B). No attempt
has been made to classify these lining body vesicles or to place them in a developmental series, for it is suggested that the differences between them reflect local
variations in the conditions of the environment. It even seems possible that some
of them give rise to multivesicular bodies (Plate 6, figs. B, C).
Mitochondria are scarce in the perinuclear region, and Golgi bodies have not
been seen here. The ground cytoplasm consists mainly of granules about
100 A in diameter, and these may occur singly or in clusters.
By this stage of development, however, two new structural types have appeared
in the perinuclear region. The first is osmiophilic granules each about 300 A in
diameter"(Plate 8, fig. C). The second is a structure that I have named 'yolk
spindle' on account of its spindle-shape and its apparent function in yolk sphere
formation (see Discussion).
Each yolk spindle is about 700 A in transverse diameter (Plate 8, fig. B). The
longest longitudinal section of one that I have seen is about 20,000 A. Each yolk
spindle is bounded by a unit membrane and contains within it a rod that is
about 50 A in diameter. Fine granular material adheres to the outside of the unit
membrane (Plate 8,figs.A, B). The yolk spindles are frequently found in clusters
and are sometimes constricted (Plate 8, fig. C).
II. Stages in which yolk is present
(a) The stage with large vesicles
This corresponds with the first part of the second phase of van Durme (1914)
and is found in oocytes between about 1-5 and 2-5 mm. Each oocyte is surrounded by a layer of follicle cells that is either stratified or pseudo-stratified.
The main feature of this phase is the appearance of large vesicles (Plate 1,fig.C).
These have been described many times by light microscopists and have sometimes been called 'vacuoles' by them. They probably arise from smaller vesicles
in the oocyte (see Discussion).
Individually, the large vesicles may measure as much as 20 [i in diameter,
though many are considerably smaller. They first appear deep to the lipid layer
PLATE 8
Fig. A. Electron micrograph of a section through a yolk spindle showing the unit membrane
with its adherent granular material, and the rod lying in the axis of the spindle, x 270000.
Fig. B. Electron micrograph of a section passing transversely through a yolk spindle, x 140000.
Fig. C. Electron micrograph of a section through a constricted yolk spindle. Note the 300 A
particles, visible as black spots, x 50000.
Fig. D. Electron micrograph of a section through an oocyte at the large vesicle stage. Note
the yolk spindles clustered around the large vesicles, x 25000.
274
R. BELLAIRS
but subsequently are found also in the subcortical mitochondrial layer. The
mitochondria then become displaced and appear to penetrate deeper into the
oocyte.
Several regions can be recognized in the oocyte at this stage. The cell membrane is raised into a series of villi (Bellairs, 1965) and beneath it lies the cortical
region which contains many pinocytotic vesicles but no large vesicles. Deep to
this region is a band of vesicles interspersed with mitochondria and many yolk
spindles. The centre of the oocyte contains large vesicles and some small ones.
Generally the nucleus is central but in Plate 1, fig. C, it is peripheral (see
Discussion).
The large vesicles. When the tissue is examined by electron microscopy, each
large vesicle is seen to be bounded by a unit membrane and thus it resembles
other vesicles within the cell. The large vesicles appear to be relatively empty,
however, though some enclose granules (Plate 8, fig. D) and some contain
structures that appear to be derived from lining bodies. Indeed, some large
vesicles contain as many as ten or twelve lining bodies.
The yolk spindles appear to have increased in number. Individually they have
often become longer and the spine has become more conspicuous. Some of
them still lie freely in the cytoplasm. Others are clustered around certain large
vesicles in such a way that there appears to be a continuity between the unit
membrane of yolk spindle and vesicle (Plate 8, fig. D; Plate 9, fig. A).
Primordial yolk spheres. The fine structure of the yolk spheres in the laid egg
was considered by Bellairs (1961). As the population of large vesicles in the
oocyte increases some of them begin to possess contents that have led me to
identify them as yolk spheres (Plate 9, fig. B). These are densely osmiophilic
subdroplets, together with a granular, less osmiophilic material. These primordial yolk spheres vary in size from about 2 to 3 /i. In addition, the primordial
yolk spheres frequently possess dense particles, each about 300 A in diameter,
that closely resemble similar particles present in the ground substance. Some at
least of the primordial yolk spheres also contain lining bodies.
Lipid drops and ground substance. The lipid drops have now almost entirely
disappeared from the cytoplasm. The ground substance consists mainly of
granules about 100 A in diameter, though in some regions, especially in the
cortical zone, large granules (200-300 A) are present in clusters. They are
probably glycogen for they correspond in appearance to structures shown to be
glycogen in adult tissues (Plate 9, inset) and are found here in a region of the
hen's oocyte that stains intensely with PAS stain. The dense osmiophilic
particles, each about 300 A in diameter, that were described in the previous
stage are also present at this stage, and appear to have increased in number.
The mitochondria are frequently arranged in clusters interspersed among the
large vesicles.
J. Embryo I. exp. Morph., Vol. 17, Part 2
PLATE 9
A
Fig. A. Higher magnification of part of Plate 8,fig.D. The arrow indicates a region where the
yolk spindle is continuous with the vesicle, x 50000.
Fig. B. Electron micrograph of a primordial yolk sphere containing granular material and a
densely osmiophilic subdroplet. x 140000.
Inset: glycogen granules, x 50000.
R. BELL AIRS
facing p . 274
J. Embryol. exp. Morph., Vol. 17, Part 2
PLATE 10
A*.
Fig. A. Electron micrograph of a section through a yolk sphere from an oocyte measuring
7 mm in diameter. An enclosed lining body vesicle is indicated by the arrow, x 18000.
Fig. B. Enlargement of the lining body vesicle shown in fig. A. x 180000.
Fig. C. Electron micrograph of a section through the edge of a yolk sphere from an oocyte
measuring 30 mm in diameter. Note that a membrane surrounds the yolk sphere and that the
remains of many lining bodies are present within the yolk sphere, x 12000.
R. BELLA IRS
facing p. 275
Yolk sphere development
275
(b) The stage of well-defined yolk
This stage corresponds with the final stages of van Durme (1914); that is, with
the second part of her second phase as well as with her third phase. It is found
in oocytes larger than about 2-0 mm. At this stage the oocyte is surrounded by a
single layer of follicle cells and is packed with primordial yolk spheres, each
containing one or several subdroplets. These yolk spheres are not surrounded
by yolk spindles.
When these yolk spheres were examined by electron microscopy (Plate 10,
fig. A) the highly osmiophilic subdroplets were found to consist of particles
each about 30-60A in diameter. In addition, each yolk sphere contained less
densely staining material composed of granules each about 250 A, as well as the
densely osmiophilic 300 A granules found in earlier stages. Lining bodies were
found within the yolk spheres (Plate 10, figs. A, B, C) even in those obtained
from oocytes of 35 mm diameter, just prior to ovulation. Certain differences
were, however, observed in the yolk spheres obtained from oocytes of 2-0 and
3-0 mm diameter. A unit membrane surrounded each yolk sphere, but although
this was well preserved in oocytes of about 2-0 mm diameter it was less densely
defined in oocytes of about 30 mm diameter. Furthermore, the size of the yolk
spheres varied with growth of the oocyte. The yolk spheres of 2 mm oocytes
seldom exceeded 20 fi in diameter, whereas those of 36 mm oocytes were
frequently as large as 150/*.
In the 2 mm oocytes each yolk sphere was surrounded by cytoplasmic constituents, but by 3 mm each yolk sphere was floating in a fluid which resembled
that of the laid egg.
DISCUSSION
During the last 8-10 days before ovulation the hen's oocyte grows from about
6 mm in diameter to its maximum size of about 35 mm. This growth occurs
largely because most of the yolk is laid down during this period. It seems likely,
therefore, that many of the changes that take place in the young oocyte are in
preparation for this event.
The mechanism by which the raw materials enter the oocyte is of especial
interest for there is evidence that some at least of the proteins may pass into the
oocytes in their final form. Williams (1962) showed that certain of the serum
proteins of the hen were identical with the a, /?- and y-livetins of the yolk. These
proteins have a high molecular weight (e.g. y-livetin has a molecular weight of
150000 according to Martin & Cook, 1958) and therefore require a special
mechanism for passing through the oocyte cell membrane. This mechanism
appears to be provided by the pinocytotic vesicles which are described in this
paper (see also Bellairs, 1964, 1965; Press, 1964; Wyburn et al 1965).
The entry of these materials into the oocyte, however, does not in itself cause
yolk spheres to form, for although the pinocytotic vesicles appear to enter the
276
R. BELLAIRS
oocyte in the greatest number during the period of yolk formation, they have
also been seen in the smallest oocytes examined. They have even been reported
in the oocytes of the chick soon after hatching (Greenfield, 1966). Indeed they
appear to be commonplace in oocytes with widely different modes of yolk
formation (for a review of the subject see Norrevang, 1965; Williams, 1966). It
seems possible that the pinocytotic vesicles give rise to the larger granule-filled
vesicles shown in Plate 6, fig. A, though there is no direct evidence on this
point.
In the hen there is another mechanism by which raw materials enter the oocyte
—by means of the lining bodies. Like the pinocytotic vesicles they are found at
all stages, even in the 5-day-old-chick (Greenfield, 1966), but unlike the pinocytotic vesicles the lining bodies have not been reported in any other class of
vertebrates. The lining bodies tend to undergo changes that appear to be due
to the breaking down of their original structure, and they come to resemble the
multivesicular bodies that are so prominent a feature of many types of oocyte.
The function and origin of the multivesicular bodies are not known. Multivesicular bodies are found in many types of cell, however, and it seems possible
that their role may vary. Sotelo & Porter (1959) have suggested that they may
be a mechanism by which new vesicles are formed and added to the cytoplasm.
The way in which the oocyte deals with the materials that enter it and converts
them into yolk will now be considered. Several changes take place just before
yolk formation. The first is the migration of the mitochondria and Golgi
apparatus from the Balbiani body to the periphery. This is a common feature
of most oocytes that possess a Balbiani body (Raven, 1961). Its effect is to place
these structures in a strategic position for intercepting the incoming pinocytotic
vesicles. Wallace (1964) has shown that in Ranapipiens a mitochondrial enzyme,
a protein kinase, plays an important role in yolk synthesis. It seems possible
therefore that in the hen's oocyte also the mitochondria may produce enzymes
that influence yolk formation. In the past, both mitochondria and Golgi
apparatus have been attributed with the power of either transforming into or
directly secreting yolk, and there is indeed evidence from electron-microscopical
studies that yolk may be formed within mitochondria in amphibians (Lanzavecchia & Coultre, 1958; Ward, 1962; Balinsky & Devis, 1963, Karasaki, 1963).
It has not been possible however to find any support for these transformation
theories in the fowl in the present investigation.
The second change that takes place in the oocyte of the hen before yolk
formation is the reduction in the numbers of free lipid drops in the cytoplasm.
The third change is the appearance of two new structures within the cytoplasm,
the 300 A particles and the yolk spindles.
There can be little doubt that many if not all of the yolk spheres are derived
from the large vesicles in the oocyte, but the source of the latter is not understood (see Text-fig. 3). It is possible they arise directly from the pinocytotic
vesicles or from the granule-filled vesicles which are present in enormous
Yolk sphere development
111
numbers and which probably contain much of the raw materials for the yolk.
On the other hand the Golgi bodies appear to be producing many vesicles, and
it is also possible that the annulate lamellae do so. Other workers have suggested
that the annulate lamellae may be responsible for producing vesicles in oocytes
(Rebhun, 1961; Swift, 1961; Balinsky & Devis, 1963). Yet another possibility is
that the large vesicles are derived from the multivesicular bodies which themselves appear to be formed from lining body vesicles (see above).
A. Types of small vesicle
B. Conversion of large vesicles into yolk spheres
Yolk spindles
Pinocytotic vesicles
Granule-filled vesicles
Primordial
yolk
sphere
Lining body vesicle and
multivesicular bodies
Golgi body vesicles
Vesicles from
annulate lamellae
300 A
particles
Text-fig. 3. Interpretative diagram to show some of the events of yolk sphere formation. A, Several different types of small vesicle are present and it seems probable that
one or more of these give rise to the large vesicles. B, After the yolk spindles have
clustered around the large vesicles the latter grow in size and acquire contents
such as granular material, 300 A particles, lining body vesicles and subdroplets. The
large vesicles may then be considered to be yolk spheres.
Whatever the ultimate source of the large vesicles they must grow in size, and
they start to do this at about the time they become associated with the yolk
spindles. It seems probable therefore that they either incorporate the yolk
spindles or receive some influence from them. It is tempting to conjecture that
the yolk spindles may be structures containing specialized enzymes necessary for
the reorganization of the raw materials into yolk, although there is no evidence
on this point. It may be significant that they tend to be clustered around the
large vesicles and not around the smaller ones. Similar clustering' of tubules
around a vesicle has been reported in pericardial cells of aphids by Bowers (1964).
278
R. BELLAIRS
At first the large vesicles are relatively empty in electron micrographs. Subsequently with increased growth they acquire contents that are more electrondense and which give them the characteristic appearance of yolk spheres (see
Text-fig. 3). These new materials are mainly the yolk lipids and proteins, and the
manner in which they enter the yolk spheres is not known. However, any
explanation of the process must also account for the fact that the lining bodies
and the dense 300 A particles also enter some of the yolk spheres. It seems unlikely that they enter by pinocytosis for no pinocytotic-like vesicles have been
seen within developing yolk spheres. Similarly there is no evidence to suggest
that certain vesicles engulf their neighbours.
An interesting finding is that whereas the primordial yolk spheres are each
surrounded by a well-defined unit membrane, it is difficult to demonstrate this
at the time the egg is laid (Bellairs, 1961). The reason is probably associated
with the fact that a unit membrane is a living structure that needs to be maintained by cytoplasm. When the oocyte is about 2-5 mm diameter, however, the
nucleus and cytoplasmic components stream up to the animal pole of the oocyte.
Thus it is likely that the unit membrane around each yolk sphere undergoes
chemical and physical changes which make it difficult to see by routine electron
microscopy.
SUMMARY
1. Yolk-sphere formation in the hen's oocyte has been studied by electron
microscopy using the following stages: Balbiani body present, Balbiani body
dispersed, large vesicles visible, well-defined yolk present. A detailed description
is given of each stage.
2. Raw materials from which the yolk is formed appear to enter the oocyte
in two ways. The first is probably by pinocytosis, the second by specialized pieces
of follicle cell apparently becoming engulfed by the oocyte. These pieces of
follicle cell have been described in detail elsewhere under the name of ' lining
bodies'.
3. Prior to yolk formation the mitochondria and Golgi bodies migrate to the
periphery of the oocyte, the numbers of free lipid drops in the cytoplasm become
reduced and two new structures appear in the oocyte. These are respectively
electron-dense particles measuring 300 A in diameter and specialized structures
that may be termed' yolk spindles'. The latter are about 700 A in T.S. and contain
within them a rod that is about 50 A in diameter.
4. The yolk spheres develop from large, membrane-bounded vesicles that
appear in the ooplasm. There is no direct evidence as to the origin of these
vesicles though several alternative suggestions are made.
5. Yolk spindles cluster around some of the large vesicles and appear to
become incorporated into them. It is suggested that the yolk spindles influence
the large vesicles to transform into primary yolk spheres, though the mechanism
is not understood.
Yolk sphere development
279
6. As the large vesicles become recognizable as primordial yolk spheres, they
acquire osmiophilic subdroplets as well as more granular material. Frequently
they also contain many 300 A dense particles, as well as degenerating lining
bodies. The mechanism by which these materials enter the primordial yolk
spheres is unknown.
7. Each yolk sphere within the oocyte is surrounded by a well-defined unit
membrane. By the time the egg is laid, however, it has become difficult to
demonstrate this membrane. It is suggested in this paper that the unit membrane
of the yolk sphere retains its normal appearance so long as it remains in contact
with living cytoplasmic structures. This contact is retained only until the oocyte
is about 2-5 mm in diameter, for at about that time the nucleus and cytoplasmic
components stream up to the animal pole where they remain.
RESUME
Aspects du developpement des spherules vitellines dans Voocyte de poule,
etudiees au microscope electronique
1. On a etudie au microscope electronique la formation des spherules de
vitellus dans l'oeuf de poule, en utilisant les stades suivants: presence du corps
de Balbiani, corps de Balbiani disperse, grandes vesicules visibles, presence de
vitellus bien defini. On donne une description detaillee de chaque stade.
2. Les materiaux bruts a partir desquels se forme le vitellus penetrent dans
l'oocyte de deux manieres. La premiere est probablement par pinocytose, la
seconde par l'absorption apparente, par l'oocyte, de fragments specialises de
cellules folliculaires. Ces fragments ont de] a ete decrits en detail sous le nom de
'corps limitants'.
3. Avant la formation du vitellus, les mitochondries et les corps de Golgi
migrent a la peripherie de l'oocyte, le nombre de gouttelettes lipidiques libres
dans le cytoplasme se reduit et deux structures apparaissent dans l'oocyte. Ce
sont, respectivement, des particules denses aux electrons mesurant 300 A de
diametre, et des structures specialises qu'on peut nommer des fuseaux vitellins.
Ces derniers ont environ 700 A de longueur totale et renferment une baguette
d'environ 50 A de diametre.
4. Les spherules vitellines se developpent a partir de grandes vesicules limitees
par des membranes, qui apparaissent dans l'ooplasme. Bien qu'on fasse diverses
hypotheses, il n'y a pas d'observations directes indiquant clairement l'origine
de ces vesicules.
5. Les fuseaux vitellins s'agglomerent autour de certaines des grandes vesicules
et s'y incorporent. On suggere que les fuseaux vitellins agtssent sur les grandes
vesicules pour les faire se transformer en spherules vitellines primaires, quoique
le mecanisme n'en soit pas compris.
6. Quand les grandes vesicules deviennent reconnaissables en tant que
spherules vitellines primaires, elles acquierent des sousgouttelettes osmiophiles
l8
J E EM 17
280
R. BELLAIRS
ainsi que du materiel plus granulaire. Frequemment elles contiennent aussi de
nombreuses particules denses de 300 A, ainsi que des corps limitants en degenerescence. Le mecanisme par lequel ces materiaux penetrent dans les spherules
vitellines primordiales est inconnu.
7. A l'interieur de l'oocyte, chaque spherule vitelline est entouree d'une
membrane unitaire bien dennie. Neanmoins, quand l'oeuf est pondu, il devient
difficile de mettre cette membrane en evidence. On suggere dans cet article que
la membrane unitaire de la spherule vitelline conserve son apparence normale
aussi longtemps qu'elle demeure en contact avec des structures cytoplasmiques
vivantes. Ce contact n'est conserve que jusqu'a ce que l'oocyte ait environ
2,5 mm de diametre car a peu pres a ce moment-la, le noyau et les constituants
cytoplasmiques s'ecoulent vers le pole animal ou ils demeurent ensuite.
I am most grateful to Mr Raymond Moss for his skilled technical help, Mrs J. Astafiev for
drawing the text-figures and to Professor J. Z. Young, F.R.S., for his helpful criticisms.
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{Manuscript received 1 August 1966, revised 11 October 1966)
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