/. Embryol. exp. Morph., Vol. 15, 3, pp. 297-316, June 1966
With 6 plates
Printed in Great Britain
297
The oocyte of the domestic chicken shortly after
hatching, studied by electron microscopy
By M. L. GREENFIELD 1
From the Department of Anatomy and Embryology, University College, London
INTRODUCTION
The cytoplasm of oocytes is highly complex. This has been demonstrated by
light-microscopists not only in birds but in most other classes of animals (see
review by Raven, 1961), although the various authors have not always agreed
as to the nature of the cytoplasmic components nor as to their significance. For
instance, in birds it has often been reported that certain structures pass from the
follicle cells into the oocyte, but these have been identified as Golgi bodies,
as mitochondria or as lipid drops by different authors. Recently, however, it has
been demonstrated by electron microscopy that in the oocytes of adult birds the
structures are instead a new type of organelle formed by a modification of the
follicle-cell membrane and they have been termed 'lining bodies' (Bellairs, 1964,
1965). The role of these structures is not understood and it is clear that more
information is needed about them. It has been one of the objects of the present
investigation to see if 'lining bodies' are also present in immature birds.
The nature of the Balbiani body of young oocytes has also been a matter of
controversy among light-microscopists. This region has therefore received
particular attention in the present electron-microscopical study.
The nucleus of the oocyte is of especial interest in that it carries genetic
material from one generation to the next. It has long been known that the
chromosomes may remain in meiotic prophase for extended periods, at least in
the young bird (Romanoff, 1960), though the significance of this phenomenon
is not clearly understood. In the present investigation the fine structure of the
chromosomes has been described and it has been found that the morphological
appearance varies according to the size and age of the oocyte.
MATERIALS AND METHODS
Sixty sex-linked female chickens (from Light Sussex hens by Rhode Island
Red Cocks) of varying ages from one day to three months after hatching were
used in this investigation. Ovaries were selected at daily intervals until the
1
Author's address: Department of Zoology, The University College, Dar-es-Salaam,
Tanzania.
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M. L. GREENFIELD
seventh post-hatching day. After this, ovaries of 3-, 6- and 11-week-old chickens
were examined.
For a preliminary light-microscope investigation twenty ovaries were fixed
in Bouin's fixative or formal saline and dehydrated in graded ethanols. Paraffin
blocks were prepared and sections were cut at 10 ji, some of which were stained
with haematoxylin and eosin. Others were stained with periodic acid-Schiff's
reagent to test for carbohydrates (Pearse, 1953), control sections being first
treated with saliva. Fixation for the electron microscope was carried out with
ice-cold fixatives at pH ranges between 7-2 and 7-5. The fixatives used were
osmium tetroxide buffered with acetate (Palade, 1952) or with phosphate
(Millonig, 1961); and glutaraldehyde (Sabatini, Bensch & Barrnett, 1963)
buffered with phosphate. Fixation time was from 30-40 min in the osmium
tetroxide and from 6-24 h in the glutaraldehyde. Glutaraldehyde fixation was
usually followed by a further 15 min of fixation in osmium tetroxide. The material was dehydrated in graded ethanols, stained with 1 % phosphotungstic acid
made up in absolute ethanol, and embedded in Araldite (Glauert & Glauert,
1958). Sections were again stained with 2 % potassium permanganate (Lawn,
1959), or with a saturated solution uranyl acetate in 50% or 70% ethanol
(Watson, 1958), or with lead citrate (Reynolds, 1963). The sections were
examined with a Siemens Elmiskop 1 b electron microscope.
The distribution and general appearance of the oocytes were studied with the
light-microscope. In addition to paraffin sections thick sections from Araldite
blocks were employed. In the early stages of development the oocyte can be
identified by the large size of the cell, the greatly enlarged nucleus and in particular the presence of a juxta-nuclear specialized region (Balbiani body). The
following criteria were adopted for distinguishing atretic from normal oocytes:
unevenly distributed chromatin in the nucleus, crinkled nuclear' membrane,
swollen cytoplasmic organelles and the presence of lamellated structures. For
a discussion of these and other ways in which degenerating cells can be identified
see Bellairs (1961), Ingram (1962), Franchi & Mandl (1962) and Pollie (1964).
RESULTS
Immediately after hatching, the ovary of the chicken contains many small
oocytes which have similar dimensions and are at much the same stage of
development as each other. These oocytes do not all grow at the same rate,
however, so that during the next few weeks the ovary comes to contain oocytes
which vary not only in size but also in degree of development. For this reason it
has been found convenient to set out the results in four sections according to
the stage of oocyte development rather than to the age of the ovary. A fifth
section is devoted to a description of lining bodies and microbodies.
Electron microscopy of chick oocyte
299
I.
The Balbiani stage (see Text-fig. 1)
This stage is the only one present in the youngest ovaries examined. In the
ovaries of 1- to 2-day-old individuals the oocytes are extra-follicular and are
present in clusters, but are often partially separated from one another by
projections from adjacent somatic cells. These projections may be the primordia
of the follicle cells. These oocytes are about 8-20 /i in diameter.
Text-fig. 1. Diagrammatic distribution of cytoplasmic particles in an extrafollicular oocyte about 8-20/* in diameter (the 'Balbiani stage'). The Balbiani
body lies near the nucleus and consists of centriole, Golgi apparatus and mitochondria.
Key to text-figures
1
2
3
4
5
6
Nucleus
Chromosome filaments 7
8
Nucleolus
9
Centriole
10
Golgi apparatus
11 Lipid drop
Mitochondrion
Cytoplasmic vesicle 12 Folded oocyte surface
Cytoplasmic granule 13 Follicle cell
14 Glycogen granule
Lining body
Macrobody
This stage is also found in the smallest oocytes of all the other stages examined,
though from about 4 to 7 days post-hatching the individual oocytes become
encapsulated by follicle cells. In the older animals the oocytes of this stage may
measure as much as 50 jn in diameter.
(a) The nucleus. The nucleus is large in relation to the size of the cell, measuring as much as 7 /i in some extra-follicular oocytes, and is sometimes irregular
in outline. Pores of about 600-800 A diameter are a common feature of the
nuclear envelope (Plate 1, fig. B). A nucleolus is sometimes present at this stage
(Plate 1, fig. A). It is composed of granules (100-120 A diameter) which vary in
density so that a number of less electron-dense patches are visible within the
mass.
Chromosome filaments are found in the nuclei of many oocytes at this stage,
at least during the first few days after hatching. In material fixed in osmium
tetroxide and stained with potassium permanganate each group of filaments
appears to be a ribbon-like structure consisting basically of two lateral thick
300
M. L. GREENFIELD
filaments and a median thin filament (Plate 1, fig. C; Plate 3, fig. A). In some
sections the chromosome appears to be spirally twisted (Plate 2, fig. A). The
entire structure of the three filaments is about 190-200 m/i across. Each lateral
filament is about 45 m/i thick, and the space between median and lateral
filaments is about 30-45 m/i. At higher magnifications each lateral filament
appears to consist of two longitudinal sub-units (Plate 1, fig. C). Furthermore,
the outer border of each lateral filament seems to be at least partially composed
of fibrils that run transversely to the long axis of the ribbon.
In 15-20 pi oocytes areas of dense chromatin are found along the outer margin
of the lateral filaments. Thus the three filaments now appear to form a central
core which is surrounded by an outer mass of large granules (Plate 2, fig. A).
Other patches of dense chromatin in the nucleus surround short pieces of triple
filaments, and in effect seem to represent the chromosome sectioned transversely. Chromosome filaments are sometimes found close to the nuclear
membrane and at times seem to be attached by one end to the inner membrane,
which is thickened in that region (Plate 3, fig. A).
(b) Balbiani body. The small extra- and intra-follicular oocytes have a
specialized region next to the nucleus. The region has been described by many
light-microscopists and is frequently referred to as the Balbiani body (see
Discussion). The region is oval or crescentic in section (Plate 3, fig. A). In the
centre are two electron-dense bodies which in favourable sections can be
resolved into parallel cylinders, each of which has the appearance that is characteristic of a centriole. Their fine structure will not be considered in detail here.
The area surrounding the centrioles has been called the centrosphere by lightmicroscopists. Outside the centrosphere and surrounding it is the Golgi region,
consisting of a mass of vesicles and lamellae. The vesicles are usually about
0-05-0-10 jLt in diameter, though they vary in size from 0-02 to 0-2 fi. The lumena
of the larger vesicles appear empty while those of the smaller vesicles seem to
contain some finely granular material. The remaining element of the Balbiani
body is the mitochondrial cloud which lies at the periphery of the Golgi apparatus. Both elongated and spherical mitochondrial profiles are present. Vesicles
PLATE 1
Fig. A. Section through portion of the nucleus from 2-day-old oocyte of diameter 12/*.
The nucleolus (n) is not homogeneous and contains a number of clear areas. (Fixed in
glutaraldehyde, post-fixed in osmium tetroxide, and stained with phosphotungstic acid and
potassium permanganate, x 15000.)
Fig. B. Portion of nuclear membrane taken from day-old oocyte of diameter 10 p. (Fixed
in acetate-buffered osmium tetroxide and stained with phosphotungstic acid and potassium
permanganate, x 70000.)
Fig. C. Chromosome filament from 2-day-old oocyte of diameter 15 ii. Each lateral filament
consists of two sub-units. (Fixed in glutaraldehyde, post-fixed in osmium tetroxide, and
stained with phosphotungstic acid and uranyl acetate, x 60000.)
/. Embryol. exp. Morph., Vol. 15, Part 3
PLATE 1
A
M. L. GREENFIELD
facing p. 300
J. Embryol. exp. Morph., Vol. 15, Part 3
M. L. GREENFIELD
PLATE 2
facing p. 301
Electron microscopy of chick oocyte
301
similar to those described in the Golgi region are also found among the mitochondria. At this stage of development the mitochondria are confined to this
region and are not visible elsewhere in the oocyte.
(c) Other components of the ooplasm. In oocytes 8-20 fi in diameter the ground
cytoplasm is mainly granular and some of the granules are linked together to
form short branching filaments of low electron density. The granules are from
80 to 120 A in diameter. Numerous small smooth-surfaced vesicles of 60-200 mjn
are present in the ooplasm. They contain short fibrils and granules in their
lumen. Thick strands of linked granules (80-120 A) also occur in the ooplasm of
most of these oocytes (Plate 3, fig. A, arrow). They are sometimes found close
to the nucleus but are present also in other areas of the ooplasm. Structures that
are probably lipoprotein bodies are observed in the young oocytes (Plate 2,
fig. B). Frequently these are visible as dense osmiophilic patches associated with
lamellae. The latter consist of a number of unit membranes each about 80 A
thick and often arranged concentrically. (A unit membrane consists of two dense
lines separated by a light interzone, the whole structure measuring about 75 A
(Robertson, 1959,1964).) Both osmiophilic patches and lamellae may, however,
occur separately. The structures are considered to represent two phases of
lipoprotein, the first characterized by its osmiophilia, the second by being
hydrated to the myelin-form-like lamellae (see Discussion). These structures are
most common during the first few days after hatching, and are less common in
oocytes of this stage taken from older chicks.
(d) The follicle cells. Many oocytes at this stage are not enveloped by follicle
cells although from the day after hatching somatic cells are visible between some
of the slightly larger oocytes. These are usually attenuated cells with large nuclei.
Each nucleus has a granular nucleoplasm with one or more areas of dense
granules. The cells of the follicle primordia are packed with organelles. They
contain mitochondria and numerous small vesicles which resemble closely those
found in the oocyte itself. Some of the vesicles have granular membranes and
are therefore considered to be part of the granular endoplasmic reticulum, which
is poorly represented in both oocyte and follicle primordia at this stage.
(e) A few oocytes appear to be undergoing degeneration. The oocyte shown
in Plate 3, fig. B is partially enclosed by the arms of developing follicle cells.
PLATE 2
Fig. A. Section through portion of nucleus of 2-day-old oocyte of diameter 16 /*. Note twisted
chromosome filament (arrows) and the masses of dense chromatin (c) present. (Fixed in
glutaraldehyde, post-fixed in osmium tetroxide, and stained with potassium permanganate,
x 42000.)
Fig. B. Section through a portion of the ooplasm of a day-old oocyte of diameter 12/*,
showing osmiophilic structures (os) and lamellated bodies (La). A number of cytoplasmic
vesicles (V) are also present. (Fixed in acetate-buffered osmium tetroxide and stained with
phosphotungstic acid and potassium permanganate, x 50000.)
302
M. L. GREENFIELD
The oocyte membrane itself is still present but the nuclear membrane, although
also present, is much folded, and there is a clumping of chromatin within the
nucleus. Several clumps of granules of high electron-density are scattered in the
ooplasm. The mitochondria appear to be swollen and are massed together near
the nuclear membrane. The rest of the ooplasm contains a number of empty
vesicles and lamellar bodies which vary in the numbers and arrangement of their
lamellae. By contrast, the cells in the tissues around this oocyte appeared to be
normal. Not more than about 1 % of the oocytes examined showed any marked
signs of atresia, and the incidence was even lower in older ovaries.
II.
Dispersed Balbiani stage (see Text-fig. 2)
This stage of development is first seen after about the fifth post-hatching day.
It is found in oocytes measuring between about 20-80 /i in diameter.
(a) The nucleus. The diameter of the nucleus is usually between about 15 and
40 /£, but its condition varies with the size of the oocyte. Chromosome filaments
are still present in the nuclei of oocytes about 20 fi in diameter although only
single filaments (about 60 mju, in width) have been observed. In some oocytes
above 40 ft in diameter the nuclei contains small patches of dense granules and
a single nucleolus. A nucleolus is also visible in some 50-80 JLC oocytes.
(b) The Balbiani body. The region is not the same as it was in the smaller
oocytes. The Golgi apparatus is no longer a single compact mass, but small
groups of it are found near the nucleus, and the mitochondria are now scattered
throughout the ooplasm (Plate 4, fig. A; Text-fig. 2).
(c) Other components of the cytoplasm. Associated with the groups of vesicles
of the Golgi apparatus are clusters of granules. Individual granules measure
about 100-120 A in diameter, about the same size as some classes of ribosomes. Cytoplasmic vesicles of both the smooth- and rough-surfaced variety
are visible in these oocytes. A few smooth-surfaced vesicles are as large as
0-3 fi in diameter, but those between 0-1 and 0-2 /i are most common. The latter
appear to be nearly empty, but the majority of vesicles contain either granules
PLATE 3
Fig. A. Chromosome filaments (Ch) are present in the nucleus of a day-old oocyte (diameter
14 ii). The Balbiani body, which lies mainly to the middle and right of the picture, contains
centrioles (C) surrounded by Golgi vesicles (G) and mitochondria (M). Granular filamentous
structures (arrow) lie near to the nucleus. (Fixed in acetate-buffered osmium tetroxide and
stained with phosphotungstic acid and potassium permanganate, x 22000.)
Fig. B. Day-old oocyte (diameter 12 /i) in advanced stage of atresia. Swollen nucleus (TV)
contains clumps of chromatin. A mass of mitochondria (M) is next to the nucleus. The
ooplasm also contains osmiophilic patches (os) and lamellated bodies (La). (Fixed in acetatebuffered osmium tetroxide and stained with phosphotungstic acid and potassium permanganate, x 13000.)
/. Embryo!. exp. Morph., Vol. 15, Part 3
PLATE 3
M. L. GREENFIELD
facing p. 302
/. Embryol. exp. Morph., Vol. 75, Part 3
PLATE 4
A
M. L. GREENFIELD
facing p. 303
Electron microscopy of chick oocyte
303
(100-120 A) or some filamentous material. The cytoplasm contains also a few
lining bodies and macrobodies (to be described later).
(d) Follicle cells. Most oocytes above 20/* in diameter have a complete
follicular layer, at this stage it varies in thickness between about 6 and 15 /i.
Plate 4, fig. A shows a portion of a 25 fi oocyte and its follicular layer. The tips
of the follicle cells either stretch for long distances along the oocyte surface or
project over adjacent follicle cells so that in section the follicular epithelium
13
10
Text-fig. 2. Diagram to show the structure of an oocyte about 20-70 /* in diameter
(the 'dispersed Balbiani stage'). The Balbiani body is dispersed but the Golgi
apparatus still lies near to the nucleus; the mitochondria are scattered in the
ooplasm. Oocytes at this stage are intra-follicular.
often has a pseudo-stratified appearance. The follicle cells are frequently
flattened with long narrow nuclei, and the inner nuclear membrane generally
has masses of granules along its margin (Plate 4; fig. B); a nucleolus is often
visible. Mitochondrial profiles are not numerous in the follicle cells at this stage
and only small groups of Golgi apparati are visible. The endoplasmic reticulum
PLATE 4
Fig. A. Section through 6-day-old oocyte of diameter 25 ft. The nucleus (A0 contains a nucleolus (n). Golgi apparatus (G) is dispersed but still near the nucleus. Mitochondria (M) are
scattered in the ooplasm. Follicle cells (F) enclose the oocyte. (Fixed in glutaraldehyde,
post-fixed in osmium tetroxide, and stained with phosphotungstic acid and potassium
permanganate. x9000.)
Fig. B. Section through follicle cells of a 7-day-old oocyte of diameter 60 [i. Lining bodies
(Lb) are in the follicle cells and in the expanded intercellular space between the follicle cells.
Those lying within the cell are each surrounded by a membrane. The cytoplasm also contains
small vesicles (V) and clumps of granules (G). (Fixed in acetate-buffered osmium tetroxide
and stained with potassium permanganate, x 24000. Inset: Higher magnification of one of
the lining bodies surrounded by a membrane, x 42000.)
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M. L. GREENFIELD
is also poorly developed and is represented by a few granule-studded sacs and a
number of smooth-surfaced vesicles. Lipid drops are frequently observed in
follicle cells at this stage of development. They appear as large irregularly shaped
areas with contents of very low electron-scattering power. A few follicle cells
contain beaded filaments 40-50 A thick. They run in all directions in the cell.
A number of lining bodies and macrobodies are sometimes present in the cell
(Plate 4, fig. B). These will be described later.
III.
The stage of the cortical mitochondrial layer (see Text-fig. 3)
This stage of development is seen in oocytes about 3 weeks post-hatching and
older. It is found in oocytes measuring between about 80 and 100 /* in diameter.
(a) The nucleus. Chromosome filaments are no longer visible but a nucleolus
can usually be seen. Each nucleolus consists of a network of strands made up of
particles 120-200 A in diameter. These strands are embedded in a less dense
matrix.
(b) The Balbiani body. In oocytes at this stage the Balbiani body has dispersed
and the mitochondria form a cortical layer.
11
14
Text-fig. 3. A later stage of development found mainly in oocytes between about
80 and 100/* in diameter (the 'cortical stage'). The mitochondria form a cortical
layer, and macrobodies and lining bodies are now visible. (These also occur infrequently in oocytes represented by Text-fig. 2 but are not characteristic.)
(c) Other components of the ooplasm. Nearly all oocytes of this size are rich
in granules (100-120 A) which are often arranged in clusters; similar granules
are found on small profiles of endoplasmic reticulum. Smooth-surfaced cortical
vesicles are arranged in a definite pattern, giving the appearance of linked sacs
(Plate 5, fig. A). They appear to contain the same finely granular material found
in the rest of the oocyte cortex. The ooplasm also contains more lipid patches
than were found in the younger stages (Text-fig. 3), though lamellar bodies are
Electron microscopy of chick oocyte
305
less common. Lining bodies and macrobodies are however, more common.
In oocytes more than 90 /i in diameter small areas of the cell membrane are
thrown into folds, these folds being restricted to the region where two follicle
cell surfaces and the oocyte surface meet. The cell membranes of the follicle
cells are not folded at this stage.
(d) The follicle cells. The follicle cells are arranged in a single layer and they
do not differ greatly from those described in the previous stage. There is an
increase in the amount of granule-studded sacs and lipid droplets at this stage.
12
11
14
Text-fig. 4. Stage of oocyte development that is found in oocytes of approximately
100/* diameter ('surface-folding stage'). Note the increased number of macrobodies in the ooplasm and the appearance of folds in the oocyte surface.
IV.
The stage of surface folding (see Text-figs. 4 and 5)
This stage of development is seen in oocytes about 6 weeks post-hatching and
older. Oocytes of this stage measure between about 100 and 400 fi in diameter.
(a) The nucleus. The nuclei of these oocytes are about 80-100 /* in diameter
and have fine granules making up the bulk of the nucleoplasm. No large
aggregations of granules corresponding to a nucleolus have been observed.
(b) The Balbiani body. It is not present.
(c) Other components of the ooplasm. In some oocytes a cortical mitochondrial
layer is visible but the ooplasm also contains lining bodies and macrobodies,
vesicles and lipid drops.
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M. L. GREENFIELD
Oocytes above 100 /i in diameter have a folded surface. A number of slender
projections and crypts appear at the surface. Large numbers of vesicular
structures, which may represent either processes of the oocyte surface seen in
cross-section or vesicles which have been nipped off into the ooplasm, form a
narrow cortical border (Plate 5, fig. C; Text-fig. 5).
(d) The follicle cells. The follicle layer now appears to be three or four cells
deep and may measure as much as 35 /i. It is not clear, however, whether it has
13
12
10
14
Text-fig. 5. Oocytes of this stage are usually above 250/A in diameter ('surfacefolding stage'). The stage is characterized by complete folding of the oocyte surface,
an increased number of macrobodies in the oocyte and the presence of glycogen
granules in the follicle cells.
PLATE 5
Fig. A. Section through portion of the follicle cell and cortex of an oocyte of diameter 70 fi.
The follicle cell contains granules (g), granule-studded sacs (er) and lining body (Lb). There
is an interruption of the follicle membrane (arrow), though this may be an artifact. The
oocyte cortex contains strings of vesicles (V). (Fixed in acetate-buffered osmium tetroxide
and stained with phosphotungstic acid and lead citrate, x 39000.)
Fig. B. Section through follicle cell of an oocyte of diameter 350/i, showing clumps of
granules (g) believed to be glycogen. Note also large lipid drop (L) in this area. (Fixed in
phosphate-buffered osmium tetroxide and stained with phosphotungstic acid and uranyl
acetate, x 42 500.)
Fig. C. Section through folded cortex and cell membrane of a 6-week-old oocyte of diameter
350/*. Cortex of oocyte contains a number of vesicles (V) near to the folds. Mitochondria
(M) are visible beneath the vesicles. (Fixed in phosphate-buffered osmium tetroxide and
stained with phosphotungstic acid and lead citrate, x 36000.)
J. Embryo/, exp. Morph., Vol. 15, Part 3
PLATE 5
M. L. GREENFIELD
facing p. 306
/. Embryol. exp. Morplu, Vol. 15, Part 3
M. L. GREENFIELD
PLATE 6
facing p. 307
Electron microscopy of chick oocyte
307
become stratified OF merely pseudo-stratified. The region of the follicle cells
next to the oocyte is packed with clumps of granules (Plate 5, fig. B). They are
larger than the Palade granules described in the oocytes and measure between
140 and 300 A in diameter. The granules all occur in clusters similar to those
described in glycogen-rich cells (Drochmans, 1962). This region stains intensely
with periodic acid-Schiff 's reagent and indeed is the only area to give a definite
reaction, although this technique has been employed at all stages.
V.
Lining bodies and macrobodies
Lining bodies are a conspicuous feature of the adult oocyte and follicle (see
Discussion). They have not however been previously recorded in juvenile ovaries.
Each lining body consists essentially of a modification of a patch of cell membrane, generally of follicle cell membrane where it abuts on to the oocyte (Plate
6, fig. A). The modification is a thickened' lining' layer or plaque on the proximal
side of the membrane, and to this lining layer are attached a number of large
granules each about 150-350 A in diameter. The lining body may be as much as
5 f.i long in section. The fine structure of the lining body corresponds with that
described for the adult (Bellairs, 1965) and will not be considered here.
Lining bodies are found at the surface of many different types of cell in the
ovary of the young fowl, namely at the junction of follicle cells with other follicle
cells, of follicle cells with the oocyte, and at the junction of neighbouring stroma
cells in both the cortex and medulla. They were found at all stages examined
though they were least common in the day-old ovary.
There is some evidence that in the adult bird ovary the lining bodies from one
cell may become engulfed by a neighbouring cell. Such a lining body then
becomes surrounded by a capsule formed from the cell membrane of the
engulfing cell (see Text-fig. 6). Such a situation probably also exists in the juvenile
ovary for lining bodies have been seen lying in the intercellular space between
adjacent cells, and also enclosed within the cells (Plate 4, fig. B). In the latter
PLATE 6
Fig. A. Lining body (Lb) with large granules (g) near the inner surface of the lining plaque.
Compare the membrane arrangement with that in Text-fig. 6. (Fixed in acetate-buffered
osmium tetroxide and stained with potassium permanganate, x 86000.)
Fig. B. Macrobodies (Ma) and lipid drops (L) occupy the interior of an 11-week-old oocyte
(diameter 300 fi). (Fixed in phosphate-buffered osmium tetroxide and stained with phosphotungstic acid and lead citrate, x 18000.)
Fig. C. A macrobody composed of a group of five lining bodies enclosed in a membrane.
(Fixed in phosphate-buffered osmium tetroxide and stained with phosphotungstic acid and
potassium permanganate, x 94000.)
Fig. D. Large macrobody from interior of 6-week-old oocyte of diameter 250 [i. (Fixed in
phosphate-buffered osmium tetroxide and stained with phosphotungstic acid and lead citrate,
x 24000.)
308
M. L. GREENFIELD
case, each lining body is surrounded by a unit membrane which is probably
derived from the recipient cell.
In addition to the lining bodies, however, structures that I have termed
'macrobodies' are also present in the juvenile ooplasm (Plate 6, figs. B, C, D),
and these do not appear to be a feature of the adult organ. Each macrobody is
composed of a cluster of lining bodies surrounded by a common membrane
(Plate 6, figs. C, D). It is suggested by analogy with the lining bodies that this
membrane is derived from the cell membrane of the oocyte. A mechanism by
which a macrobody could be formed is suggested in Text-fig. 6.
Text-fig. 6. Diagram illustrating a possible developmental sequence of macrobodies
from lining bodies. 1, Lining body at surface of follicle cell; a, b are the membranes
of adjacent cells, c is the double-layered lining plaque. 2, Lining body indents cell
surface of cell a. 3, Lining body protrudes into cell a. 4, Section through the tip of
such a projection. 5, Suggested origin of macrobodies, i.e. the projection of a group
of lining bodies into the surface of another cell. Such a section has not been seen and
is conjectural. It is suggested that a macrobody is formed when a cluster of lining
bodies is engulfed together by the oocyte. 6, Section through the tip of such a
projection; sections of this type have been seen frequently. 7-10, Degenerative
stages observed in macrobodies.
A marked feature of the macrobodies is that it is often difficult to distinguish
the components of the individual lining bodies clearly (Plate 6, fig. D). It is
unlikely that this effect is the result of poor fixation, for it may be seen in a
well-fixed oocyte. It thus appears that the macrobodies undergo some process of
modification or breakdown within the oocyte.
Electron microscopy of chick oocyte
309
More than twenty lining bodies have been seen within a macrobody (Plate 6,
fig. D). Small macrobodies containing only two or three lining bodies have been
found in many cells of the developing ovary, but large macrobodies have only
been seen within oocytes larger than about 50 fi diameter.
^
,
DISCUSSION
The nucleus
It has been shown in this investigation that the morphological appearance of
the chromosomes varies according to the size and age of the oocyte. For instance,
in oocytes about 15 fi in diameter, and at 24 h after hatching, the filaments are
visible as triple structures, whereas in oocytes about 20 fi in diameter at 4 or
5 days after hatching the filaments are single, and in the later stages at about
40 fi they are no longer visible. Franchi & Mandl (1962) noted similar changes
in the number of chromosome filaments in rat oocytes of different ages examined
by the electron microscope. It has also been shown in the present work that the
amount of dense material associated with the filaments varies. It seems possible
that this dense material is derived from the nucleoli, since during the period
when the filaments are embedded in the chromatin mass the nucleolus is not
visible, and conversely when the chromatin mass disappears the nucleolus
reappears. It is tempting to speculate that the nucleolus is reformed from the
dense material when the chromosome filaments disappear.
There has been some discussion in the literature as to the relationship that
these structures seen by electron microscopy bear to the chromosome components seen by light-microscopy. Sotello & Porter (1959) suggested that the
triple filament seen by electron microscopy corresponded with a chromatid.
This theory has however received little support, for, as the authors themselves
pointed out, if it were true, then six parallel threads should be visible at synapsis
and no one has yet reported seeing so many associated filaments. The alternative
hypothesis, which is generally considered more convincing, is that each filament
seen by electron microscopy represents a chromatid, and that the triple structure
is visible only when pairing has occurred (Moses, 1958; Franchi & Mandl, 1962).
The term 'synaptinemal complex' was introduced by Moses to describe the
triple structure. The evidence is based on correlated light and electron-microscopy : thus at synapsis a triple structure is seen by electron microscopy, whereas
at the diplotene stage single filaments only are visible by electron microscopy.
It is, however, not understood why pairing should lead to three filaments being
present instead of two, and it has been suggested that the central strand is a
'condensation product' (Moses, 1958).
The Balbiani body
The Balbiani body is found in oocytes less than 50 ju, in diameter in both
juvenile and adult birds. This region of the bird's oocyte has been described many
times by light-microscopists, although they have frequently disagreed in their
20
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310
M. L. GREENFIELD
interpretations. Most workers have, until recently, believed that the region had
something to do with the formation of yolk, and this is reflected in the multiplicity of terms applied to it, for instance 'yolk body of Balbiani' (d'Hollander,
1904); 'yolk body' (Loyez, 1906); 'yolk-forming layer' or 'yolk bed' (Van
Durme, 1914); 'mitochondrial yolk body' (Brambell, 1926) and 'early yolk'
(Konopacka, 1933). It now seems unlikely that the region manufactures yolk
(see discussion by Raven, 1961; Bellairs, 1964).
With the use of electron microscopy it has been possible to identify the
components as consisting essentially of paired centrioles surrounded by Golgi
vesicles and mitochondria. A Balbiani body has been found in most oocytes
examined by light-microscopy at least in the early stages (Raven, 1961), although
the fine structure is not necessarily the same in all species. For instance, in
amphibian oocytes it contains predominantly masses of mitochondria (Wischnitzer, 1964), whereas in spider oocytes it consists mainly of stacks of lamellae
and mitochondria (Sotello & Trujillo-Cenoz, 1958). In molluscs (Rebhun, 1956)
and echinoderms (Afzelius, 1956) the region contains stacks of annulate
lamellae resembling nuclear membranes.
In the present investigation it was found that the Balbiani body usually
disappeared as such when the oocytes were more than 50 ft in diameter. About
the same time the mitochondria and Golgi apparatus migrated to the cortical
region. These findings confirm a number of observations made by Brambell
(1926), who investigated the same material by light-microscopy. The dispersal
of the Balbiani body has also been reported in oocytes from a wide variety of
animals examined by light-microscopy (Raven, 1961), and in oocytes of certain
mammals (Sotello, 1959; Blanchette, 1961; Adams & Hertig, 1964) and amphibians (Kemp, 1956; Wischnitzer, 1964) examined by electron microscopy.
The ground substance
Smooth-surfaced cytoplasmic vesicles have been seen by electron microscopy
in the oocytes of adult birds (Bellairs, 1964, 1965; Press, 1964), of mammals
(Sotello & Porter, 1959; Blanchette, 1961; Adams & Hertig, 1964) and of
amphibians (Wischnitzer, 1964). In the present investigation it was found that
their number and distribution varied with the size of the oocyte. Thus they
increased in number with the dispersal of the Golgi region when the oocyte
was about 50/^, but seemed to decrease in certain 80 fi oocytes, and were
subsequently found mainly in the cortex. The smooth-surfaced cytoplasmic
vesicles present in the oocytes of the young chickens resemble those found in
the oocytes of adult birds (Bellairs, 1965) in that they usually contain granules,
each about 100 A.
It is not known if there is any relationship between the granules enclosed in
the smooth-surfaced vesicles and some of those lying freely in the cytoplasm.
The latter vary in size from about 80-300 A and it seems possible that the smaller
ones are ribonucleoprotein granules. The larger ones are, from their size, more
Electron microscopy of chick oocyte
311
likely to be glycogen, and furthermore they are usually grouped together in a
manner characteristic of glycogen granules (see Luft, 1956; Drochmans, 1962).
Thus it seems possible that both ribonucleoprotein granules and glycogen
granules lie freely in the cytoplasm. The glycogen is unlikely to be present in
large amounts, for this region stained only weakly with the PAS technique.
Rough-surfaced endoplasmic reticulum, which is very scarce in embryonic
tissue, was found only in small amounts in oocytes of adult birds (Bellairs, 1965),
of mammals (Sotello & Porter, 1959; Anderson & Beams, 1960) and of amphibians (Wischnitzer, 1964).
Lamellar bodies
Lamellar bodies and the dense osmiophilic masses found in the cytoplasm of
oocytes in the young chicken have not been reported in oocytes of the adult hen
(Press, 1964; Bellairs, 1965). Similar structures have, however, been seen in
oocytes and spermatocytes of rats by Franchi & Mandl (1962, 1964), who
suggested that they were lysosomal bodies concerned with autolysis. Their
evidence was based on the fact that, although the structures were present in most
gonocytes in the foetal and early post-natal period, they were particularly
numerous in degenerating gonocytes. Similarly, in the present investigation
lamellar bodies have been seen in a high proportion of normal cells, both
oocytic and stromal, but some of the bodies, especially the osmiophilic ones,
were found especially in autolytic cells, It is, however, not easy to be certain
that the structures described by Franchi & Mandl (1962, 1964) are strictly
comparable with those seen in the present investigation.
The lamellar bodies of the chicken oocyte resemble myelin figures in appearance. The latter can be produced experimentally by hydration of lipids (Stoeckenius, 1959), and therefore it seems likely that the lamellar bodies are formed in
a similar way. This suggestion is supported by the fact that degenerating oocytes
characteristically contain increased amounts of lipids (see review by Ingram,
1962).
Degenerating oocytes
The incidence of degenerating oocytes observed in this study is low, in contrast
to the findings of Brambell (1926) that all oocytes in the chicken above 100 [i in
diameter exhibited marked signs of atresia. It was further observed that many
oocytes exhibiting marked signs of osmiophilia under the light-microscope were
found on examination by the electron microscope to be packed with macrobodies, some of which had lost their membranes. It is not suggested here that
loss of the membrane from the macrobody is a sign of degeneration of the
oocyte but rather that this is the means whereby the macrobody discharges its
contents into the ooplasm (see below).
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M. L. GREENFIELD
Lining bodies and macrobodies
Lining bodies were not seen in oocytes smaller than about 50 fi although they
were found in the stroma before this time. Structures of this type have previously
been reported in the ovaries of adult birds, as a result of electron-microscopical
studies. They have been termed 'pre-mitochondria' (Schjeide & McCandless,
1962), 'transosomes' (Press, 1964) or lining bodies (Bellairs, 1964, 1965). In
the present study the lining bodies have not been examined at high resolution,
but the results obtained appear to support the interpretation of Bellairs.
There has been some disagreement about the fate of the lining bodies.
According to Press they remain permanently attached to the follicle cells from
which they arise. His evidence was based on the fact that he failed to find these
structures deeper than the most superficial layers of the oocyte. According to
Schjeide, McCandless & Mann (1963) and Bellairs (1964, 1965), these lining
bodies become detached from the follicle cells and become engulfed by the
oocyte. They thus come to lie within the oocyte. Bellairs's evidence was based
on the fact that structures closely resembling lining bodies were seen deep
within the oocyte, and that each of them was surrounded by a vesicle that
appeared to have been derived from the oocyte cell membrane. In the present
investigation also, vesicles containing lining bodies have been seen deep within
the oocyte and it seems possible that they are derived from the lining bodies of
the follicle cells (see Text-fig. 6).
The oocytes of the juvenile bird contain macrobodies, whereas those of the
adult apparently do not. Structures resembling macrobodies do not appear to
have been described previously by electron microscopists. From their distribution in the oocyte as well as from their marked osmiophilia, however, the
macrobodies appear to correspond with certain structures previously described
by light-microscopists. Brambell (1926) concluded that they were Golgi bodies
and he gave them the name 'Golgi Type 2', but in the present study this interpretation has been shown to be invalid. The macrobodies now appear to be
composed of clusters of lining bodies enclosed by a unit membrane (see Textfig. 6).
There have been many suggestions by light-microscopists that cytoplasmic
organelles pass from the follicle cells into the oocyte. The organelles have been
described as mitochondria (Varma, 1954), as fat drops (Loyez, 1906; Konopacka, 1933) or as Golgi apparatus (Guraya, 1957). From the present electronmicroscopical investigation it appears that the organelles described are macrobodies and lining bodies. The period of this apparent transport is limited in
duration since the number of bodies decreases with increasing size and age of
the oocyte. Furthermore, many macrobodies observed in the older oocytes
appear to be discharging their contents into the ooplasm, since many have lost
their limiting membranes and have a disrupted lining layer.
Electron microscopy of chick oocyte
313
A consideration of the relationship between the oocyte and ovarian stroma
In the extra-follicular phase the groups are probably nourished from the
surrounding stroma cells. The individual oocytes are relatively small and it
seems likely that most of the materials enter by diffusion for few pinocytic
vesicles are visible. In the intra-follicular phase each oocyte becomes larger in
volume, and its absorptive surface is increased by the production of villi. The
development of villi in the oocyte is accompanied by the appearance of pinocytic
vesicles and it therefore seems probable that more material is entering the oocyte.
The apparent passage of lining bodies and macrobodies into the oocyte is of
interest for two reasons. First, the repeated injection of large masses of cytoplasm from one cell into another is an unusual occurrence in biology; secondly,
it is a transference of cytoplasmic material from one generation to the next. The
cytochemical nature of the material is not known and its significance is not
understood although it is apparently restricted to birds, and possibly reptiles.
It has been suggested that it may have some importance for the oocyte in
relation to yolk formation in large eggs (Bellairs, 1964).
In the adult the fine structure of the follicle cells strongly suggests secretory
activity at certain stages (Bellairs, 1965); that is, these cells contain extensive
amounts of granular endoplasmic reticulum arranged in stacks, secretion granules and Golgi apparati. In the infant bird, the follicle cells do not have this
appearance and it seems probable that they are not synthesizing and secreting
material into the oocyte. In the adult the follicle cells also appear to have a
selective action on the materials that pass through them into the oocyte (Bellairs,
1965) and a similar situation possibly exists in the infant. The role played by the
glycogen granules in the physiology of the follicle cells is not understood, but it
is possible that they provide a source of energy for some of the follicular
activities.
SUMMARY
1. Oocytes taken from chickens from 1 day to 11 weeks after hatching have
been examined by electron microscopy.
2. Early extra-follicular oocytes have chromosome filaments in the nucleus at
the prophase of meiosis. The fine structure of these filaments is described and is
found to vary according to the size and age of the oocyte.
3. The Balbiani body present in oocytes of about 8-20 /i in diameter has been
shown to consist of paired centrioles surrounded by Golgi vesicles and an outer
border of mitochondria.
4. Golgi vesicles and mitochondria were dispersed in larger oocytes, and in
those larger than about 80 /i the mitochondria formed a cortical layer.
5. In oocytes with a diameter greater than about 100 /i macrobodies (clusters
of lining bodies) were mainly confined to the periphery of the oocyte, whereas in
others they were present, together with lipid drops, mainly in the interior.
314
M. L. GREENFIELD
6. Folding of the oocyte membrane was observed in many oocytes measuring
about 100 /i in diameter, but was much more pronounced in oocytes above
250 (i. In these oocytes cortical vesicles in close proximity to the crypts of the
folds appear to have been formed by pinocytosis.
7. In the follicle cells of oocytes 250 /JL in diameter and over, large masses of
glycogen granules occur in the cytoplasm near the oocyte membrane.
RESUME
Etude au microscope electronique de Voocyte du poulet peu apres Veclosion
1. On a examine au microscope electronique des oocytes preleves du lcr jour
a la l l e semaine apres l'eclosion.
2. Les jeunes oocytes extrafolliculaires ont des filaments chromosomiques
dans le noyau en prophase meiotique. On decrit la structure fine de ces filaments,
qui varie selon la taille et l'age de l'oocyte.
3. On a montre que le corps de Balbiani present dans les oocytes d'environ
8 a 20 ii de diametre consiste en une paire de centrioles entoures par des vesicules
golgiennes et une bordure externe de mitochondries.
4. Les vesicules golgiennes et les mitochondries se trouvent dispersees dans
les oocytes plus gros et les mitochondries forment une couche corticale dans les
oocytes d'un diametre superieur a 80 fi environs.
5. Dans les oocytes d'un diametre superieur a environ 100 fi, des macroinclusions (groupes de corps alignes) etaient surtout confinees a la peripherie de
l'oocyte, tandis que dans d'autres, elles se trouvaient surtout a l'interieur,
associees a des gouttelettes lipidiques.
6. Le plissement de la membrane oocytaire a ete observe dans de nombreux
oocytes mesurant environ 100 pi de diametre, mais etait beaucoup plus accentue
dans les oocytes d'environ 250 /*. Dans ces oocytes, il apparait que des vesicules
corticales, tres rapprochees des cryptes des replis, se sont formees par pinocytose.
7. Dans les cellules folliculeuses d'oocytes de 250 ju de diametre et au-dessus,
de grandes quantites de granules de glycogene se trouvent dans le cytoplasme
pres de la membrane de l'oocyte.
This work was carried out whilst I held the Jamaican Government Independence PostGraduate Award. I am very much indebted to Professor J. Z. Young, F.R.S., for allowing
me to work in his department and to him and to Dr Ruth Bellairs for their encouragement
and advice. I should like to thank Miss Judy Spillman for technical assistance and Mrs J.
Astafiev for the drawings.
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