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Morphometry of capillaries in three zones of rabbit lungs fixed by vascular perfusion

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THE ANATOMICAL RECORD 244:165-174 (1996)
Form and Function of Lacunae in the Ovary of the Laying Hen
HASSAN NIL1 AND W. ROGER KELLY
Department of Veterinary Pathology, The University of Queensland, Queensland, Australia
ABSTRACT
Background: Spontaneous atresia of large, yolky, ovarian
follicles occurs in the chicken and in other species when the stimulus for
ovulation ceases. This means that as much as 40 ml of yolk must be resorbed by the ovary if normal ovarian morphology is to be regained. This
study examines the process of yolk resorption in the ovarian stroma following bursting atresia.
Method: Light, transmission, and scanning electron microscopy were
used to study the morphology of lacuna spaces and their changes in ovaries
of broiler and layer breeds of domestic chicken, both in actively laying
birds and in birds undergoing spontaneous or induced follicular atresia. A
comparative study was made of the ultrastructural differences between
lacuna-lining cells, blood-vessel endothelium, and germinal epithelium of
the ovary.
Results: Transformation of lacuna-lining cells and peritoneal cells into
macrophages occurred after bursting atresia and exposure of these cells to
released yolk. Complete atresia of large yolky follicles occurred very
quickly.
Conclusion: The resorption of this large volume of yolk, at least in part,
seems to be mediated by the transformed lacuna-lining cells.
0 1996 Wiley-Liss, Inc.
Key words: Avian ovary, Ovarian lacunae, Bursting atresia, Yolk resorption
Avian and reptilian ovaries differ from those of mammals in one important aspect, which is the large volume
of yolk that is associated with the ovum in the former
phyla. Although much study has been conducted on the
mechanisms of yolk secretion, follicle development, and
ovulation in birds, little attention has been paid in recent years to the mechanisms whereby yolk is removed
and disposed of in birds whose large yolky follicles are
undergoing atresia instead of ovulation. Gupta and
Maiti (1986)described atresia in large yolky follicles as
a process of internal bursting and escape of yolk into the
ovarian stroma, but they did not describe the process of
yolk disposal in detail. There are extensive chambers
(lacunae) in the stroma of the avian ovary (Callebaut,
1979),and this report describes the role of these lacunae
in the process of yolk disposal.
MATERIALS AND METHODS
Laying Hens
Twenty-eight actively laying Leghorn hens (14 Siro
CB and 14 Tegel; 59 weeks of age) were divided into
control and treatment groups that each consisted of
seven birds of each strain. In the treatment groups,
ovarian follicular atresia (“pause”) was induced by
withholding the standard ration for 7 days and feeding
whole barley. The control groups were fed a balanced
layer ration. Seven days after the diet change, the birds
were killed by intravenous injections of barbiturate,
and the gross morphological features of the ovarian
0 1996 Wiley-Liss, Inc.
follicles were recorded in both control and treatment
groups. Then, the ovaries were fixed whole in neutralbuffered formalin. Ovarian volumes were determined
by immersing the fixed organs in a vessel full of water
and measuring the total volume of water displaced.
Broiler Breeders
Hens were culled from a commercial flock soon after
exhibiting behavioural and other signs of going out of
lay. A total of 53 birds of the same strain from two
flocks were examined postmortem; they ranged from
38 to 54 weeks of age. A total of 19 birds were found to
be affected with systemic diseases; these included six
cases of lymphoid leucosis and other myeloproliferative
disorders, two cases of bacterial oophoritis, and ten
cases of wasting of unknown cause. These diseased
birds were excluded from the study, and the morphological studies were performed on 17 otherwise normal
birds whose ovaries were in the process of undergoing
spontaneous atresia. Birds were killed by intravenous
injections of barbiturate.
Haematoxylin and eosin-stained sections of ovarian
tissue were prepared from birds in both of these studies
using routine histological procedures. Seven actively
laying Tegel strain laying hens between 48 and 55
Received May 18, 1995; accepted September 27, 1995.
Address reprint requests to Dr. W.R. Kelly, Department of Veterinary Pathology, The University of Queensland, Queensland 4072,
Australia.
166
H. NIL1 AND W.R. KELLY
Fig. 1. Stromal lacunae (L) between two growing follicles (F)in the ovary of actively laying hen.
Stromal smooth muscle (arrowheads) and blood vessels (V). x40. Haematoxylin and eosin (H & E).
Fig. 2. Lacuna spaces (L) that were filled with phagocytic cells 36 hours after bursting atresia. B, blood
vessel; Y, yolk proteins. X400. H & E.
OVARIAN LACUNAE IN T H E LAYING H E N
167
Fig. 3. A Lacuna spaces (L) that were filled within less than 12 hours with yolk (Y) and several
phagocytic cell layers (arrowheads) breaking up yolk components are shown. The flat and elongated
germinal epithelial layer is indicated by the arrows. B: Bursting atresia of large yolky follicle (F).Lacuna
spaces filled with yolk components that are paler than the yolk in original follicular wall. S, stigma; W,
follicular wall; P, peritoneal cavity; H, haemorrhage inside follicle. x400 in A, X40 in B. H & E.
weeks of age were used for the electron microscopic
studies.
mounted on aluminium stubs, sputter coated with gold,
and examined in a Jeol 6400F scanning electron microscope.
Transmissionand Scanning Electron Microscopy
(TEM and SEM)
Birds were anaesthetised by intramuscular injection
of ketamine (25 mg/kg) and xylazine (20 mg/kg). Fixation was achieved by intraaortic perfusion of normal
saline followed by glutaraldehyde (Karnovsky’s) solution (Perry et al., 1978). Blocks of about one cubic millimetre of tissue were removed from the ovarian stroma1 tissue, and fixation was continued by immersion
for 1 hour in the same fixative. Tissues were then
washed in phosphate-buffer solution, pH 7.2, postfixed
in 1%osmium tetroxide, dehydrated in ascending concentrations of methanol, and embedded in medium
Spurr’s resin. Survey sections (1 Fm) were cut and
stained with toluidine blue; ultrathin section were cut,
stained with uranyl acetate and lead citrate (Perry et
al., 1978), and examined in a Phillips 400T electron
microscope.
For SEM, small blocks of stromal tissue from perfusion-fixed ovaries were dehydrated in ascending concentrations of methanol. They were critical-point dried,
Ultrasonography
The onset of pause was monitored daily using ultrasound examination in order to follow the response of
ovarian follicles. The birds were anaesthetised, feathers were removed to allow application of ultrasound
gel, and an ultrasound probe (Advanced Technology
Laboratories, Bothell WA) was placed on the left abdominal wall between last rib and the pubic bone on
the left side.
RESULTS
Morphological Changes
Laying hens
There were between five and seven large yolky follicles on the ovaries of laying hens in the control group;
however, all of the follicles in the treatment group were
undergoing atresia by 8 days following diet restriction.
In about 50% of the cases, follicular atresia was associated with both intra- and extrafollicular haemor-
H. NIL1 AND W.R. KELLY
168
rhage. In three cases, there was free yolk in the peritoneal cavity.
Broiler breeders
There were no follicular hierarchies in the ovaries of
broiler breeders that had spontaneously gone out of
lay. Follicular atresia, either partial or complete, was a
prominent feature in all of these ovaries. Generally,
there was no difference between the process of follicular atresia of large yolky follicles in broiler breeders
and that in laying breed hens, nor were there any obvious differences in the configuration of stromal lacunae between the breeds. Therefore, this configuration
and the process of follicular atresia in both breeds are
described together.
Stromal Lacunae in Normal Actively Laying Ovaries
In histological sections of the normal active ovary of
the laying hen, the ovarian stroma between the follicles and other cortical structures presented an extremely loose appearance due to the presence of large
irregular, but sharply demarcated, spaces (Figs. 1, 5).
The extensive disposition of these spaces was also obvious in SEM preparations (Fig. 4).These spaces (lacunae) were lined by flattened cells that appeared
identical to vascular endothelium in neighbouring
blood vessels (Fig. 5). In routinely prepared paraffin
sections, the lacunae were usually completely empty;
rarely, some faintly stained eosinophilic material was
seen inside lacunae in active ovaries. The lacunae,
therefore, were differentiated from veins primarily by
the fact that they were consistently free of blood cells
and by their extremely irregular outline (Figs. 1, 5).
They were judged to be thicker walled than lymphatics,
but, apart from the extensive irregular profiles of the
lacunae and the absence of blood in their lumens, clear
distinction between lacunae, veins, and lymphatics in
normal active ovaries was often difficult. In perfused
preparations, lacunae tended to maintain their shape,
whereas arteries and veins were fixed in distension
(Fig. 5). The lacuna spaces were present in both the
medulla and the cortex of the avian ovary, and the only
part of the ovarian stroma that was free of lacuna
spaces in both growing and regressing follicles was the
stigma. Communications between the lacunae and the
peritoneal space were not seen.
Follicular Atresia
Atresia in follicles with a large amount of yolky ooplasm (diameter >500 pm) was of the bursting type,
whereas smaller follicles were involuting without
bursting. When the large follicle started to regress, its
contents were extruded into the surrounding lacuna
spaces through a rupture site that appeared in the follicular wall, usually on the stalk side of the follicle
(Figs. 6, 7). A few hours after the commencement of
bursting atresia, the follicular diameter became noticeably smaller as the follicular contents were extruded
into the lacuna spaces. Then, the follicular wall started
to shrink and, finally, collapse. Consequently, lacuna
spaces which were originally narrow and collapsed, expanded very rapidly to accommodate the escaped yolk
(Fig. 3B). In cross sections of fixed ovary, it appeared
that the extruded follicular contents were paler and
were relatively soft in texture (Fig. 3B) compared to
Fig. 4. Scanning electron micrograph (SEM) of normal active ovary
of laying hen demonstrating the extensiveness of lacunae spaces (L).
The arrow indicates germinal epithelium. V, blood vessels. X60.
the yolk in the original follicle, which was more deeply
coloured and more dense. In some sections that included the site of rupture of the follicular wall, continuity could be seen between the yolk in the follicular
lumen and the yolk in the lacunae. In the early stages
of atresia, the lacunae were lined by a thin single or
double layer of flattened endothelial-type cells. A few
hours after discharge of yolk from the follicle, the lacuna walls were lined by three t o four layers of rounded
phagocytic cells that were distended with yolk droplets
(Fig. 3A). In later stages, lacuna spaces become filled
with phagocytic cells (Fig. 2). Mitotic figures were
sought in these cells but were difficult to discern, because the nuclei of these cells tended to be obscured and
distorted by the cytoplasmic content. During the phase
of rapid filling with yolk, the external lacunar wall
(comprising the original germinal epithelium, the lacuna-lining cells, and the interstitial stroma) became
extremely thin and fragile (Fig. 3A,B). In the few cases
where yolk had escaped into the peritoneal cavity from
regressing ovaries, we observed transformation of peritoneal-lining cells into macrophage-like cells in the
same manner of the transformation of lacuna-lining
cells when exposed to yolk. During all stages of atresia,
OVARIAN LACUNAE IN THE LAYING HEN
Fig. 5. Plastic section (1 pm) of perfused avian ovary showing lacuna space (L) and blood vessels (V)
adjacent to a growing follicle (F) in a laying (nonatretic) ovary. Flat and endothelial-like cells lining the
lacuna are indicated by arrowheads. The arrow indicates germinal epithelium. B, basement membrane;
G, granulosa cells; P, peritoneal cavity; LU, luteal gland. X550. Toluidine blue.
Fig. 6. The rupture site (arrow) in the follicular wall (W) of a bursting atresia of yolky follicle.
Ooplasmic contents escape through the rupture site and are seen moving into ovarian stromal lacunae
(L). x220. H & E.
169
170
H. NIL1 AND W.R. KELLY
Fig. 7. Diagram (not to scale) of the preovulatory follicle before (A) and during (B) bursting atresia,
when the yolk passes through a defect in the follicular wall and escapes into the stromal lacunae.
the only structure in the original follicular wall that
appeared to maintain contact with the germinal epithelium was the stigma: the rest of the follicular wall
became separated from the germinal epithelium of the
ovary by extension and expansion of the lacunae as
they progressively filled with yolk from the emptying
follicle. All cellular components of the follicular wall
showed a variety of degenerative changes ranging from
the separation of granulosa cells from the basement
membrane to the vacuolation of cytoplasm and frequent apoptosis. There was some infiltration of heterophils through the follicular wall into the follicular lumen. In later stages, haemorrhage into the follicular
lumen was a common finding in regressing follicles,
mainly due to breakdown of capillaries following separation of granulosa cells and the underlying basement
membrane from the theca layer (Fig. 3B).
The degenerative changes seen in most of the atretic
follicular wall were not seen in the stigma, which, in
the maturing follicle, had been identified merely as an
avascular band around the free part of the follicular
circumference. However, with the onset of bursting
atresia, the stigma rapidly became thick and contracted, thus forming a barrier to the escape of follicu-
lar contents by this route. The mean reduction in total
ovarian volume in the paused birds was 36 ml.
Ultrastructural Findings
TEM study of lacuna-lining cells, blood-vessel endothelium, and the germinal epithelium of avian ovary
revealed differences between these three cell kinds as
well as similarities in the ultrastructural components.
Some of the more important similarities and differences are presented in Table 1.
In active (nonatretic) ovaries, the differences between the lacuna-lining cells and the blood-vessel endothelial cells were relatively minor. The number of
mitochondria were more numerous in the endothelial
cells; also, the nuclei in the endothelial cells were more
prominent. The intercellular junctions between the endothelial cells were denser than in the lacuna cells
(Fig. 8). The mesothelial (germinal epithelium) cells
were triangular and sometimes cuboidal in shape and
were situated on a very well-defined continuous basement membrane in contrast the lacuna-lining cells,
which were mostly elongated, sometimes had very thin
cytoplasm, and had basement membranes that were
171
OVARIAN LACUNAE I N THE LAYING HEN
TABLE 1. Ultrastructural Differences and Similarities Between Germinal Epithelial,
Lacunae-Lining,and Endothelial Cells of Active (Nonatretic)Ovaries
Ultrastructural characteristics
Cell type
Germinal epithelium
Lacuna-lining cell
Vascular endothelium
MIV'
-
+
+
Pinocytotic
Basement
vesicles
membrane
Intercellular
junctions
+++
+
++
+++
+
++
++
++
+++
Nuclear Mitochondria1
size
numbers
+++
+
++
++
+
+++
'Microvillus frequency.
comparatively thinner and did not show continuity.
Variable numbers of collagen fibres were seen in the
matrix of the lacuna walls; however, collagen fibres in
the matrix under the basement membrane of the mesothelial layer were much more extensive. Appreciable
numbers of pinocytotic vesicles were seen in all three
kinds of cell studied. Microvilli were observed in both
blood-vessel endothelial cells and lacuna-lining cells.
The number of these process was more variable on the
surface of lacuna-lining cells.
When the lacuna-lining cells became phagocytically
active, they lost their intercellular junctions, became
almost free in the lumen, and became markedly engorged with yolk particles (Fig. 9). However, when the
germinal epithelial cells were exposed to yolk, they
maintained their attachment and appeared to be less
phagocytically active than lacuna-lining cells. Especially in lacuna-lining cells and in cells floating free in
the lacunae, the yolk droplets appeared be being broken down very rapidly, because they could be seen in
many stages of breakdown within phagolysosomes in
the one cell (Fig. 10).
Fig. 8. Transmission electron micrograph of a lacuna-lining (L)cell (arrowhead) and blood-vessel (V)
endothelium in an actively laying ovary. The lacuna-lining cells are flat and resemble endothelial cells.
F, fibroblast; D, lipid droplets in stromal macrophage; C, collagen fibers. X9,800.
H. NIL1 AND W.R. KELLY
172
Fig. 9. A SEM of cells lining a lacuna in the stroma of the nonatretic ovary of a n actively laying hen.
The cells form a thin layer with variable number of microvillous projections. B SEM of cells lining a
lacuna in the stroma of an ovary near a preovulatory follicle undergoing bursting atresia shows the
transformation from endothelial-like cells to macrophage-type cells (MI engorged by phagocytosis of yolk
globules (arrowheads). E, erythrocyte. x5,400 in A, x4,600 in B.
Ultrasonography
During ultrasonographic monitoring of ovaries, the
large mature follicles appeared as spherical structures,
with a central hyperechoic sphere about 8 mm in diameter (Fig. 11A). After withholding the usual ration
for 3-4 days, the follicles lost both their contrast and
their spherical shape during a space of 24 hours; thus,
they became indiscernible (Fig. 11B). Also, the echorich ring structure in the centre disappeared. These
changes were consistent with sudden onset of bursting
atresia.
DISCUSSION
The process by which yolk components are transported to the growing follicles from the ovarian circulation has been studied extensively (Wyburn et al.,
1965, Gilbert et al., 1980). However, the mechanisms
involved in the resorption of yolk following follicular
atresia have been neglected in the past. Davis (19421,
Gilbert (1979), and Gupta and Maiti (1986) described
resorption of yolk in the ovarian stroma; however, they
did not relate this process to the ovarian lacunae. Earlier, Betz (1963) described the same pattern of bursting
follicular atresia in the ovary of the diamond-backed
water snake but did not describe lacunae in this species. Callebaut (1979) demonstrated the existence of
spaces in the ovaries of immature chickens and of adult
quails and, in fact, named them lacunae. Since then,
not much research has been focused on the nature,
morphology, and function of these spaces in the ovary
of adult domestic chickens. Prochazkova and Komarek
(1970) cited earlier workers, who had variously named
these spaces: Koch (1925) called them lymphatic
spaces, and Techver (1965) called them lagoons or fissures of the reticular part of the medulla. Prochazkova
and Komarek (1970) believed that the lacuna spaces
are part of the vascular system. The complexity of ovarian blood vessels as described by these workers could be
partly due to the confusion of lacuna spaces with the
venous system.
In our study, the patterns of follicular atresia were
generally similar to the patterns reported by Gupta et
al. (1988), who studied the histological features of follicular atresia in the ovary of the domestic hen and, for
the first time, described the importance of the internal
bursting phenomenon during atresia of large yolky follicles. However, they did not describe the role of lacuna
spaces in this phenomenon. Gilbert (1979) overlooked
the preexistence of lacunae and believed that escaped
yolk from the atretic follicles eventually forms these
spaces.
The use of ultrasound on successive days during the
induction of atresia demonstrated in vivo the speed of
shrinkage of the large yolky follicles in a manner that
OVARIAN LACUNAE IN T H E LAYING H E N
173
Fig. 10. Transmission electron micrograph of lacuna-lining cells (arrow) involved in the breakdown of
yolk proteins (PI. Protein globules can be seen in different stages of degradation (arrowheads).Pseudopodia (asterisks) are much more prominent than in the resting lacuna-lining cells (compare to Fig. 8).
x12,ooo.
the static histological methods could not duplicate. Our
work has shown that the main function of the system of
lacunae seems to be to aid in the disposal of yolk from
large atretic follicles in laying hens by providing voluminous spaces in which phagocytic cells can break
down yolk into simpler, presumably soluble, units that
can be easily removed and metabolised. Our findings
also support Sturkie’s (1955)experiment, which demonstrated the rapid disappearance of free yolk from the
peritoneal cavity in the laying hens. Thus, it seems
that hens have a “back-up” mechanism for disposing of
yolk in the event of escape of follicular contents into
the abdominal cavity. This escape in our birds seemed
most likely to have occurred by the breakdown of the
extremely thin external wall of the lacuna after filling
with yolk.
The ultrastructural features of vascular endothelium
and of lacuna-lining cells in the laying (nonatretic)
ovary were very similar, differing only in quantitative
features such as pinocytotic activity and numbers of
microvilli and mitochondria. The vascular endothelial
cells did not appear to participate in phagocytosis of
yolk, but this may be because they did not have access
to it.
The ultrastructural study revealed that yolk protein
droplets in lacuna-lining cells were present in various
stages of digestion. Because these cells had access to
yolk for only about 8 hours, it follows that yolk protein
H. NIL1 AND W.R. KELLY
174
Fig. 11. A Ultrasonograph of two preovulatory follicles (the larger is mature) in the ovary (nonatretic)
of a n actively laying hen. Arrows indicate the hyperechoic zone in the centre of each ovum. B: Ultrasonograph of ovarian follicles (arrowheads) 24 hours after the onset of bursting atresia. The follicles have
become shrunken and have lost their biphasic structure.
breakdown is extremely rapid in these cells. The rate of
lipid droplet breakdown cannot be estimated, but it is
probably slower, because, in late stages of atresia, lacunae contain macrophages with lipid droplets and
with no discernible protein.
The extreme distensibility and very extensive disposition of these spaces within the ovary may provide for
the extremely rapid expansion of maturing follicles
that takes place shortly before ovulation in normal laying hens. A typical follicle may expand from 8 mm to 37
mm in diameter over 7 days (Johnson, 1986), and, with
several such follicles maturing a t the same time, the
lacunae may well be necessary to allow the stroma to
accommodate this rapid increase in volume.
Origin and Function of the Lacuna-Lining Cells
Blood monocytes may migrate into the lacunar
spaces from the adjacent blood vessels following bursting atresia and could be one of the sources of the phagocytic cells that fill the lacunae so rapidly after they fill
with yolk. If this happens, it was not observed in our
material. In other experiments, we have shown that
avian monocytes in vitro are able to respond morphologically and phagocytically to the presence of yolk in a
relatively short period of time (Nili and Kelly, 1995).
At this stage, we cannot determine the relative contribution, if any, of blood monocytes to the phagocytic
lacuna cells in the regressing ovary. The other possibility is that the large numbers of phagocytic cells in
the lacunae are the result of proliferation of lacunalining cells. In preliminary experiments, we have observed that avian monocytes proliferate in vitro in response to yolk exposure (Nili and Kelly, unpublished
observations).
ACKNOWLEDGMENTS
We thank Mr. D. Robinson from the Poultry Research Centre of Queensland. This work was supported
in part by the Ministry of Culture and Higher Educa-
tion of Iran and by grants from the Australian Chicken
Meat and the Australian Egg Industry Research and
Development Councils (grants UQ22CM and UQ25E,
respectively).
LITERATURE CITED
Betz, T.W. 1963 The ovarian histology of the diamond-backed water
snake, NurtL rhombiferu during the reproductive cycle. J Morphol., 113:245-254.
Callebaut, M. 1979 The avian ovary is an open organ. Anat. Embryol.,
158t103-119.
Davis, D.E. 1942 Bursting of avian follicles a t the beginning of
atresia. Anat. Rec., 82:153-161.
Gilbert, A.B. 1979 Female genital organs. In: Form and Function in
Birds, Vol. 1. A.S. King and J. Mclelland, eds. Academic Press,
London, pp. 237-360.
Gilbert, A.B., M.A. Harie, M.M. Perry, H.R. Dick, and J.W. Wells
1980 Cellular changes in the granulosa layer of the maturing
follicle of the domestic fowl. Br. Poultry Sci., 21t257-263.
Gupta, S.K., and B.R. Maiti 1986 Study of atresia in the ovary during
the annual reproductive cycle and nesting cycle of the Pied Myna.
J . Morphol., 19Ot285-296.
Gupta, S.K., A.B. Gilbert, and M.A. Walker 1988 Histological study of
follicular atresia in the ovary of the domestic hen (Gullus domestzcus). J . Reprod. Fertil., 82219-225.
Johnson, A.L. 1986 Reproduction in the female. In: Avian Physiology,
4th Ed. P.D. Sturkie, ed. Springer-Verlag, New York, pp. 403431.
Koch, W. 1925: Untersuchungen iiber die Entwicklung des Eierstockes der Vogel. Inaug. disert. Vet. Med. fak. Univ. Munchen. S.
1-52. (cited by Prochazkova and Komarek, 1970)
Nili, H., and W.R. Kelly 1995 Yolk recycling in the ovary ofthe laying
hen: A critical feature of pause. Proc. Aust. Poultry Sci. Symp.,
7t72-78.
Perry, M.M., A.B. Gilbert, and A.J. Evans 1978 Electron microscope
observations on the ovarian follicle of the domestic fowl during
the rapid growth phase. J. Anat., 125t481-497.
Prochazkova, E., and V. Komarek 1970 Growth and differentiation of
the ovarian follicles in the postnatal development of the chicken.
Acta Vet. Bra., 39t11-16.
Sturkie, P.D. 1955 Absorption of egg yolk in the body cavity of the
hen. Poultry Sci., 34:736-737.
Techver, J.T. 1965: Gistologija domasnych ptic. Eston. selskochoz.
akad. sci. gistol. laborat., Tartu. (cited by Prochazkova and Komarek, 1970)
Wyburn, G.M, R.N.C. Aitken, and H.S. Johnston 1965 The ultrastructure of the zona radiata of the ovarian follicle of the domestic
fowl. J. Anat., 99:469-484.
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