/. Embryo/, exp. Morph. Vol. 44, pp. 31-43, 1978
Printed in Great Britain © Company of Biologists Limited 1978
3\
Crystalline inclusion bodies in rabbit embryos
By JOSEPH C. DANIEL, J R . 1 AND JOHN R. KENNEDY 1
From the Department of Zoology, University of Tennessee
SUMMARY
Crystalline inclusion bodies (CIB) may be found as prominent ultrastructural components
of the trophoblast cells of rabbit blastocysts and of progestational uterine endometrium. In
the work reported here we have sought to describs developmental steps in crystal formation,
to correlate these events with embryonic age and to determine if the uterus is essential
(either as a source or an environment) for crystal formation in the embryo.
CIB, which are of a size and periodicity to make them appear to be clusters or packages
of microtubules, are first detectable in embryos 4 days 6 h post coitum and by A% days are
well established in significant numbers. Another structural component, granular vesicles,
may bs seen in embryos as early as 2\ days post coitum, and decrease in number during the
same time the CIB are increasing. We believe that the CIB originate from the pre-existing
granular vesicles and present electron micrograph evidence of crystal formation progressing
from such vesicles.
CIB formation does not occur in 4-^- to 5-day-old embryos which have been locked in the
oviduct by a suture around the utero-tubal junction. However, when such tube-locked
embryos are transplanted into the uterus, they develop crystals within 36 h thereafter. We
conclude that the uterus is essential for CIB formation to occur in the rabbit embryo.
INTRODUCTION
Ultrastructural studies of the blastocysts of rabbits show the trophoblast cells
to contain prominent crystalline inclusion bodies (Hadek & Swift, 1960; Enders
& Schlafke, 1965; Tyndale-Biscoe, 1965; Steer, 1970; Hesseldahl, 1971).
Similar bodies have been reported in preimplantation mouse embryos (Enders,
1971; Calarco & Szollosi, 1973), and in placental tissue of rats (Ollerich, 1968)
and possibly sheep (Davies & Wimsatt, 1966). The bodies appear to be absent
in rabbit embryos of cleavage or morula stages (Merchant, 1970) or blastocysts
that have been grown in vitro (Van Blerkom, Manes & Daniel, 1973), and are
relatively rare in mouse parthenotes (Van Blerkom & Runner, 1976).
Crystalline inclusion bodies (CIB) are also found in the glandular cells of the
uterine epithelium of both humans (Nakao, Meyer & Noda, 1971) and rabbits
(Hoffman & Davies, 1973). Hoffman, Davies & Long (1975) reported the
presence of CIB in cells of the uterine lining between days 10 and 20 of pregnancy and 4-16 of pseudopregnancy in the rabbit. The bodies were absent in
the estrous uterus and prior to day 4 of pseudopregnancy. Thus CIB are
1
Authors'1 address: Department of Zoology, University of Tennessee, Knoxville, Tennessee
37916.
3-2
32
J. C. DANIEL AND J. R. KENNEDY
observed during the secretory and fusion stages of the endometrium, a time
'associated with rising or maximal progesterone secretion' (Davies & Hoffman,
1975). They also appear in ovariectomized rabbits after administration of
progestins but not estrogens (Nakao et al. 1971; Hoffman et al. 1975). These
observations implicate the hormonally activated uterus either as the source of
the component material, or as an environment critical for crystal formation in
the embryo.
In the work reported here, we have sought to describe developmental steps
in crystal formation, to refine the temporal correlations of their appearance
and to determine if the uterus is essential (either as a source or an environment)
for CI.B formation in the embryo.
METHODS AND MATERIALS
Animals. New Zealand white rabbits were used. Does were held for at least
3 weeks after delivery to assure against disease, pregnancy or pseudopregnancy,
and maintained on a standard diet. Pregnancy was initiated by natural mating
to two males; the matings marked the beginning of day 1 post coitum. Killing
was by cervical dislocation.
Embryos. Cleavage-stage embryos were isolated by flushing the oviducts
with 1 ml each of F10 culture medium (Ham, 1965) supplemented with 5 %
rabbit serum and collecting the flushings in large sterile watch glasses. About
10 ml of the flushing medium were used to isolate blastocysts from the uterine
horns by the same procedure. Embryos were photographed and then either
studied directly by phase-contrast microscopy, fixed and prepared for study by
electron microscopy or transplanted to the uterus of the same animal or that
of a foster mother. Animals were opened mid-ventrally after anesthetization
with sodium pentobarbital and ether; embryos were transplanted by the use of
standard surgical procedures (see Staples, 1971).
Tissue preparation
For electron microscopic examination, embryos werefixedin a glutaraldehydeparaformaldehyde mixture in cacodylate buffer (Karnovsky, 1965) for 2 h,
washed overnight in 0-1 M cacodylate buffer and post-fixed in 1 % osmium
tetroxide in 0-1 M cacodylate buffer. Embryos were stained in 0-5% uranyl
acetate in 0-1 M sodium maleate (pH 5-2) in the dark (Van Blerkom et al. 1973)
prior to dehydration. Dehydration was carried out in ethanol and propylene
oxide and embryos were embedded in Epon 812. Sections were cut on a
Porter-Blum MT-1 microtome with diamond knives, stained with uranyl
acetate and lead citrate (Venable & Coggeshall, 1965) and examined in an
RCA-EMU-3H microscope.
Crystalline inclusion bodies in rabbit embryos
33
Experimental protocol
Two studies were conducted.
(1) Temporal relationship of CIB formation. Embryos, collected on days 2\,
4, 4 plus 6 h, 4 plus 9 h, 4%, 5, 6, 7 and 9 post coitum, were studied by electron
microscopy to identify the time of crystal formation and to examine the
sequence of events in their development. A minimum of six embryos was
examined for each stage.
(2) Tube-locking of embryos. The utero-tubal junctions were ligated in does
on the third day of pregnancy so as to prevent embryos from getting to the
uterus. On day 4^- or 5 these tube-locked embryos were isolated and studied for
the presence of CIB. In each case only one side was ligated so that the embryos
on the other side were free to migrate into the uterine horn at the normal time
and thus serve as controls.
In a variation of this experiment, 4^-day-old embryos, which had been tubeblocked since 2\ days post coitum, were flushed from the oviducts and transplanted back into the uterine horn on the same side. For controls, to negate the
effect of handling, other 4^-day-old embryos were flushed from the uterus and
immediately transplanted back into it. For each repeat of this experiment, at
least two of the embryos were prepared for EM studies at the time the others
were being transplanted. On day 6 post coitum the transplanted embryos were
isolated from the uterus and studied for crystalline inclusion bodies.
RESULTS
Structure of crystalline inclusion bodies. Crystalline inclusion bodies in rabbit
trophoblast cells are variable in number. Some random sections show no inclusions, others only one or two and still others have clusters of inclusions
(Fig. 1). The crystalline bodies have rounded ends and are needle-like in shape
when observed in full longitudinal section (Fig. 11) and in the light microscope.
Size is also variable, up to 10 /tm in length according to Hadek & Swift (1960).
In cross-section a well developed inclusion has a highly ordered crystalline
appearance (Fig. 2). The major periodicity in crystalline appearance is about
20 nm when cut either in tangential (Fig. 2) or longitudinal (Fig. 4) sections.
When the crystalline inclusions are loosely or diffusely packaged they have a
cross-sectional appearance comparable to microtubules (Fig. 3), with a diameter
of 20 nm similar to the distance between major dense lines seen in both cross
and longitudinal sections of the mature or well developed crystal. A loosely
organized crystal when viewed in longitudinal section (Fig. 6) also has a
microtubule-like composition, although in this plane it is often difficult to
demonstrate such an organization because of the absence of clearly separate
microtubular structure. Rather they appear as frayed ends of microtubules,
possibly suggesting a loose cross-linkage between adjacent tubules in this
34
J. C. DANIEL AND J. R. KENNEDY
Crystalline inclusion bodies in rabbit embryos
35
condition. The crystals, both in their highly organized condition (Fig. 5) and
their more loosely microtubule-like state (Fig. 6), seem to be composed of
granular subunits with a 7-5 nm size.
Formation of crystalline inclusion bodies. In order to ascertain the origin of
crystalline inclusion bodies and the factors controlling their formation, two
types of embryos were examined. Normal embryos were examined from
2\ days to 9 days post coitum. A second group of embryos which had been
experimentally altered with respect to their time periods in the oviduct and
uterus were also examined and compared with normally developed embryos.
As indicated previously, embryos were retained unilaterally in a ligated oviduct
for 4-2—5 days, the opposite oviduct and uterine horn serving as a control. Some
of the oviduct-retained embryos were examined with the electron microscope
and others were transferred to the uterine horn of the same side for an additional
1-2- days before being examined.
The 2^-day embryos lacked crystalline inclusions, as was expected from the
earlier literature. The general cytoplasmic organization at this time was not
significantly different from that observed in the 4-day embryo. At day 4 the
embryos contained large vesicles filled with a fine granular material (Figs. 7-8).
The cytoplasm was filled with ribosomes and a few short segments of granular
endoplasmic reticulum. The mitochondria from 4-day uterine embryos (Fig. 7)
had undergone differentiation as described by Van Blerkom et a/. (1973). In
general, the mitochondria from the oviduct-retained 4-day embryos had not
undergone differentiation (Fig. 8) although exceptions were occasionally seen.
Crystalline inclusions were not present in either the 4-day embryos from the
uterus or those retained in the oviduct. The first appearance of crystalline
inclusions was at 4 days 6 h post coitum in uterine embryos. Crystals in these
embryos were infrequent and scattered among the granule-containing vesicles.
On occasion one could find what appeared to be an elongating granular vesicle
which had begun to take on a crystalline shape. By 4 days 9 h the frequency of
CIB increased with an apparent decrease in the number of granular vesicles,
although at this time it would be difficult to quantitate such a change. Trophoblast cells were becoming flattened. At day 4^- uterine embryos contained well
developed crystalline inclusion bodies. Tube-locked embryos, on the other
FIGURES
1-3
Fig. 1. Section through a portion of trophoblast cell, containing several well formed
crystalline bodies (CB), mitochondria (M), granular endoplasmic reticulum (GR)
and numerous ribosomes in the cytoplasm, x 26000.
Fig. 2. The cross-sectional structure of a well developed crystalline body (CB).
x 50000.
Fig. 3. A cross-section through a partially organized crystalline inclusion body.
The microtubular substructure is indicated by arrows. Also present in tangential
section is a complete crystal (CB). x 47000.
36
J. C. DANIEL AND J. R. KENNEDY
Crystalline inclusion bodies in rabbit embryos
37
Table 1. Relationship between embryo location and crystal formation
No. of
embryos
Uterine
Oviduct retained
Uterine
Oviduct/uterus
(4±-5 days)
(4*-5 days)
(6 days)
(4i/lidays)
14
8
9
7
Crystals
Present
Absent
Present
5 Present
2 Absent*
* Embryos degenerating.
hand, lacked C1B (Table 1), although generally the mitochondria were differentiated as compared with 4-day oviduct-retained embryos (Fig. 8). Retention of
embryos for A\ days within the oviduct apparently stressed the embryos,
because some signs of cellular abnormality were observed. However, it did not
affect crystalline inclusion formation when the embryos were transferred into
the uterine horn.
All uterine embryos from day 4% to 6 contained well developed CIB in their
trophoblast cells. By day 4\ very few granular vesicles were present in trophoblast cells but numerous crystals with enlarged granular regions could be
observed. By day 6 no granular vesicles were present. However, occasionally
what appeared to be partially formed crystals were observed (Fig. 9). The
crystal usually projected from one side of a large granular vesicle. On one end
the crystalline organization, enclosed in a unit membrane, was evident when
the section cut completely through the longitudinal axis of the crystal (Fig. 9).
The microtubule-like substructure of the developing crystal projected into the
granular portion of the vesicle which tended at this stage to be more irregular
in shape than spherical. This type of organization of a crystal within a granular
body was comparable to that observed in the early (4 days 6 h) trophoblasts.
However, at the earlier time periods the crystalline configuration was not as
well developed.
Of seven embryos retained in the oviduct for 4\ days and then transferred to
the uterus, five developed crystals (Table 1). These embryos showed more
FIGURES 4-6
Fig. 4. Longitudinal section through a crystalline inclusion body showing the
enclosing unit membrane (arrows) to be comparable in size to the unit membrane
of the granular endoplasmic reticulum (GRM). Periodicity of the crystal is also
evident, x 65000.
Fig. 5. In tangential section the granular composition of the major dark lines in the
crystalline inclusion body is evident, x 55000.
Fig. 6. Longitudinal section through a crystalline inclusion body which is partially
organized. The microtubular substructure is further suggested (arrows), x 47000.
38
J. C. DANIEL AND J. R. KENNEDY
Crystalline inclusion bodies in rabbit embryos
39
variation in content of granular vesicles and developing crystals than did the
6-day uterine embryos. The granular bodies tended to be larger in size than in
younger embryos and varied in shape from round to elongate. In some cases
the crystals seemed to project from a round granular vesicle (Fig. 10) and when
cut tangentially the unit membrane was not evident. Fusion of granular bodies
was not particularly evident in these embryos. However, examination of
embryos grown in vitro (Kennedy & Daniel, unpublished) where the number of
granular vesicles was considerably greater, showed extensive fusion of granular
bodies. As formation of a crystalline inclusion body continued, the granular
vesicle associated with that crystal became smaller. Frequently residual components of the granular body could be seen in the mid-region of a nearly
completed crystal (Fig. 11). This latter condition was observed in the five
tube-locked embryos after transfer to the uterus but was not observed in
normal 6-day uterine embryos. Thus by retaining the embryos in the oviduct
until day 4-^—5 and then transferring them to the uterus a more synchronous
crystal formation within the granular bodies may have been attained. This
would increase the chances of identifying various stages in the formation of
crystalline inclusion bodies.
Some of the oviduct-retained embryos showed signs of cellular degeneration
(Fig. 11), but this apparently did not inhibit crystal formation. Two transferred
embryos which were clearly degenerating failed to form crystals (Table 1). At
no time was there any evidence of endocytotic activity which might account for
crystalline inclusion formation.
DTSCUSSION
Crystalline inclusion bodies may be seen in the rabbit embryo as early as
4i days post coitum and are well established by 4^ days. This observation agrees
with the time of first appearance as noted by Hadek & Swift (1960), Hesseldahl
(1971) and Van Blerkom et ah (1973). We contend however that precursors to
CIB may be seen in 2^-day-old embryos (the earliest stage studied) in the form
of granular vesicles. Suitable sections (e.g. Figs. 9 and 10) show crystal formation
progressing from such vesicles.
That CIB do not develop in cultured embryos has been clearly established
by Van Blerkom et al. (1973). This fact, plus the appearance of comparable
bodies in the lining of the uterus on day 4 (Hoffman & Davies, 1973), strongly
supports the hypothesis that the presence of a uterine environment is essential
FIGURES 7 AND 8
Fig. 7. Section of trophoblast cells of a 4-day uterine embryo. Granular bodies
(GB) are numerous and mitochondria (M) are differentiated. The nucleus (N) and
lipid granules (L) are indicated. Crystalline inclusion bodies are absent, x 8500.
Fig. 8. Portion of a trophoblast cell from a 4-day tube-locked embryo, containing
many granular bodies (GB), but undifferentiated mitochondria (DM), x 22500.
40
J. C. DANIEL AND J. R. KENNEDY
Crystalline inclusion bodies in rabbit embryos
41
for the formation of crystalline inclusions. When we tested this by restricting
embryos to the oviduct by ligation of the utero-tubal junction, embryos of
4\ days of age did not develop CIB. When such embryos were transferred to
their respective uteri and thus became exposed to the environment provided by
a normal, hormonally activated uterus, they showed significant crystal formation
within 36 h. Nakao et at. (1971) thought that embryonic CIB might be acquired
by endocytosis. For substantial crystal inclusion formation to occur in 1-j days
through uptake from the uterus, substantial endocytotic activity would be
required, unless the crystal precursor were already present in trophoblast cells.
We observed no evidence for endocytotic uptake of CIB and Davies & Hoffman
(1975) found no 'evidence for the release of crystals into the glandular lumina'.
This in conjunction with the changes in earlier embryos leads us to believe that
crystalline inclusion bodies are formed from pre-existing granular vesicles
present as early as 2\ days in the developing embryos. The uterine environment,
however, is essential for the conversion of the granular material into crystalline
inclusion bodies.
Hoffman et at. (1975) discuss the available evidence to show that CIB are,
at least in part, proteinaceous and suggest the possibility that they may be
composed of blastokinin (Krishnan & Daniel, 1967). This is an appealing idea
because the temporal appearance and localization of blastokinin and the
hormonal regulation of its existence seem to parallel the same conditions for
CIB. However, studies by Van Blerkom (personal communication) with SDS
electrophoresis show that the material composing CIB is a much heavier
molecule than blastokinin. We feel that the characteristics of crystalline inclusion bodies suggest that they may be packages of microtubules, contained
in a membrane comparable in average thickness to that of the membranes of
the granular endoplasmic reticulum. Bensch & Malawista (1969) induced the
formation of crystals of comparable structure from microtubule protein in
L-strain fibroblasts utilizing periwinkle alkaloids. The organization appears
comparable to that of a CIB; however, their crystals were not membrane bound.
FIGURES
9-11
Fig. 9. A developing crystalline inclusion body from a 6-day embryo, exposed to
the uterine environment in normal sequence but removed and reintroduced back
to the uterus at day A\ (mock-transferred). A partially formed crystal in a granular
body is evident, x 39000.
Fig. 10. A region of a trophoblast cell from an embryo which had been oviductretained for 4 | days and then transferred to the uterus for \\ days. A crystalline
inclusion body (CB) forming in a granular body is visible as are differentiated
mitochondria (M) and profiles of granular endoplasmic reticulum (GR). x 27500.
Fig. 1.1. Trophoblast cell from an embryo handled as in Fig. 10. Some cellular
degeneration has occurred (arrows). Two crystalline inclusion bodies (CB) are
visible, one which shows the remnants of a granular body (GB), at its midpoint,
x 7000.
42
J. C. DANIEL AND J. R. KENNEDY
Steer (1970) notes that certain of the CIB found in trophoblastic knobs of
implanting 7-day-old rabbit blastocysts are 'in process of disintegrating into
tubules'. Hoffman et al. (1975), although agreeing to a superficial resemblance
between uterine CIB and microtubule crystals, note that the former differ in
being membrane-bound and in the periodicity of subunits.
Moskalewski, Sawicki, Gabara & Koprowski (1972) found that treatment of
unfertilized mouse eggs, oocytes and two-celled embryos with cytochalasin B
'evoked formation of large crystalloid bodies' in the cytoplasm. These bodies
stained positive for proteins but their formation was not prevented by puromycin. The authors suggested that the bodies were formed by crystallization of
material already present in the eggs. No preliminary stages of formation were
found, however, so it was assumed that formation occurred very rapidly.
Calarco & Szollosi (1973) note an association between the crystalloid aggregates they see in mouse embryos with certain virus-like particles and comment
on similar associations between crystalline arrays in tissues infected with viruses.
Both the blastocyst (Manes, 1974) and uterine endometrium (Daniel, 1973;
Yang, Tyndall & Daniel, 1976) of the rabbit also contain virus-like particles
during the period when CIB are present. It is impossible at this time to assign
any significance to this coincidence. Similarly, we do not know what relationship these CIB may have to the crystalline bodies found in arterial endothelia
(Weibel & Palade, 1964) or to those sometimes associated with regenerating
tissue (e.g. Leeb, 1975).
We conclude that the crystalloid inclusion bodies found in rabbit embryos
originate from pre-existing granular vesicles and from some substance or
condition provided by the progestational uterus.
This work was supported by N1H grant no. NIH 5R01 BD-06226. The authors also wish
to express gratitude to Patricia Allen, Phyllis Bice, Carolyn Booher and David Wise for their
technical help.
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