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Protein uptake by rat preimplantation stages.

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Protein Uptake
R a t Preimplant at ion Stages
Department of Anatomy, Washington University School of Medicine,
St. Louis, Missouri 631 10
The protein tracers, horseradish peroxidase and ferritin, are
demonstrable in the subzonal space of all preimplantation stages within ten
minutes when incubated in vivo or in vitro. However, there is very little uptake
of these proteins by ova and two-cell stages. By the blastocyst stage there is
greatly increased uptake of exogenous protein. The proteins appear in coated
micropinocytotic vesicles and tubules, larger vacuoles, and more complex bodies.
Blastocysts from the period of lactationally delayed implantation show an even
greater amount of uptake, especially in the supranuclear region. Peroxidase reaction product can be demonstrated in the cavity of day 5 blastocysts in 30
minutes, and in the cavity and basal lamina of the blastocysts during delayed
implantation in ten minutes. Ferritin was more sparsely distributed, and was
not seen in the blastocyst cavity in any of the time periods. Peroxidase is apparently transported via an intracellular pathway, since it is not seen in the elaborate intercellular spaces between trophoblast cells. Acid phosphatase activity
is demonstrable in vacuoles, dense bodies and Golgi cisternae in all stages, indicating that the potential for degradation of ooplasm and phagocytized material
by a lysosomal system is present in all of the preimplantation stages examined.
Although much information is still
needed concerning protein distribution in
the lumina of the female reproductive
tract, several general features appear to be
emerging. The proteins and glycoproteins
found are products of both transudation
from the blood and specific synthetic and
secretory activity of cells (Fredricsson,
'69; Daniel, '69; Feigelson and Kay, '72).
Consequently, in addition to serum proteins some proteins specific to the uterus
are also found (Krishnan and Daniel, '67;
Beier, '68). Variations both in total protein content and in composition of protein
during the preimplantation period can be
demonstrated (Daniel, '68, '69; Gwatkin,
'69; Urzua et al., '70). Proteins present in
the uterine lumen can also be demonstrated in blastomeres of the preimplantation stage of at least some species a s has
been shown by immunofluorescence (Glass,
'63) and biochemical methods (Beier, '70;
Hamana and Hafez, '70).
In in vitro systems, serum proteins have
a stimulatory effect on trophoblast outgrowth in the mouse (Gwatkin, 'SS), increase blastocyst metabolism as judged by
carbon dioxide output i n this species
ANAT. REC., 175: 539-560.
(Menke and McLaren, '70), and promote
blastocyst growth in the rabbit (Krishnan
and Daniel, '67; Gulyas and Krishnan, '71;
Maurer, Onuma and Foote, '70; El-Banna
and Daniel, '72), and rat (Daniel and
Krishnan, '69).
In a comparative cytological study it
was demonstrated that blastocysts of a
number of different species can ingest
exogenous proteins (Enders, '7 1). The
present study was designed to determine
whether proteins are transported into blastomeres during different stages of the preimplantation period, whether such substances can reach the cavity of the
blastocyst, and the cellular basis of this
protein ingestion and transport. Since some
of the protein entering preimplantation
stages is probably digested, we also wanted
to determine whether a lysosomal system
could be identified which could serve a s a
component of the degradative pathway.
Two exogenous proteins were used:
horseradish peroxidase, which has a molecular weight of 40,000 and a n approximate
Received Aug. 21, '72. Accepted Oct. 11, '72.
1 This study was supported by grant HD 04962 from
the National Institute of Child Health and Human
size of 40 A, and ferritin, which has a
molecular weight of greater than 500,000
and a size of at least 100 A. Hastings et
al. ('72) have recently demonstrated that
both of these proteins readily penetrate
the zona pellucida of a number of species
including the rat.
Young female rats of the Holtzman
strain were placed with males, and the day
on which sperm were found in the vagina
designated day 1 of pregnancy. Preimplantation stages were collected on day 1
( 4 rats), day 2 ( 3 rats), day 4 ( 3 rats)
and day 5 (14 rats), and from five rats
during lactationally delayed implantation
(day 7 and day 11).
In most instances, preimplantation
stages were flushed from the reproductive
tracts in physiological saline. Two to six
zygotes from each animal were then placed
either in .5% horseradish peroxidase
(Sigma Type 11) in saline or in ferritin
(Pentex cadmium-free, 50 mg/ml saline)
for 10, 30 or 60 minutes. In five instances
(including all 60 minute incubations) the
ferritin or peroxidase solutions were introduced directly into the uterine lumen for
the length of time desired. No differences
were seen between the stages incubated
in utero and comparable stages incubated
in vitro.
All preimplantation stages were subsequently rinsed in saline and placed in fixative containing 2% formaldehyde and
2.5% glutaraldehyde in 0.1 M phosphate
buffer (pH 7. 3). Ferritin-treated material
was rinsed in 0.1 M phosphate buffer, postfixed in 2 % osmium tetroxide in 0.1 M
phosphate buffer, dehydrated and embedded in araldite epoxy resin (Durcupan).
Peroxidaseexposed preimplantation stages
were rinsed in 0.1 M phosphate buffer
overnight, incubated in diaminobenzidine
and peroxide (Graham and Karnovsky,
' 6 6 ) ,then postfixed in osmium, dehydrated
and embedded.
Control blastocysts which had been exposed to peroxidase were incubated in a
medium lacking diaminobenzidine, or alternatively in a medium lacking HZ02. In
addition, blastocysts which had not been
exposed to peroxidase were incubated in
the complete medium to indicate whether
or not any endogenous peroxidase activity
was demonstrable with this incubation
time. None of these control blastocysts
showed any peroxidase activity or deposits
which could be mistaken for the osmiumdiaminobenzidine complex.
Two-cell stages from one rat on day 2,
and blastocysts from three rats on day 5
were fixed in 3% glutaraldehyde in 0.1 M
cacodylate buffer, rinsed in cacodylate buffer, and incubated for acid phosphatase
activity by the method of Barka and Anderson ('62) for 30 and 60 minutes. These
tissues were then rinsed, postfixed in 1%
osmium tetroxide in 0.1 N s-collidine buffer, dehydrated and embedded.
Sections 1-2
thick of the plastic embedded material were stained with Azure
Blue B to confirm stage of development,
The peroxidase reaction product could
often be seen by light microscopy in these
preparations. Sections for electron microscopy were stained with lead citrate before
examination in an RCA EMU 3-G electron
Ovum, two-cell stage
Both peroxidase reaction product and
ferritin particles can be seen throughout
the zona pellucida of the ovum and twocell stages with ten minutes exposure to
tracer. Both proteins appear to accumulate
in the subzonal space, where they are often
in a clumped rather than dispersed form.
The cell surface of the ovum and twocell stage has widely and irregularly distributed short microvilli (figs. 1, 2).
Coated micropinocytotic vesicles (caveolae) are found both continuous with the
surface membrane and subjacent to this
membrane. However such vesicles are usually several hundred millimicrons apart.
Although the thin discontinuous rim of
reaction product found after peroxidase
exposure is present in those vesicles continuous with the surface, i t is largely absent from those that are separated from
the surface when ten minutes exposure
to tracer is used (figs. 3-5). Ferritin is
infrequent even in those micropinocytotic
vesicles communicating with the surface
(fig. 2). After 30 minutes, occasional iso-
lated vesicles containing peroxidase activity are found in the apical cytoplasm.
Both tracer proteins completely permeate the space between blastomeres of the
two-cell stage, illustrating the lack of restrictive junctions between the blastomeres
at this stage (fig. 6 ) . Vesicles with demonstrable peroxidase activity are more frequent in association with the intercellular
surface of the two-cell stage than they are
in relationship to the subzonal surface.
However, even after 60 minutes of exposure, the number of vesicles containing
tracer that can be observed within the
blastomeres is small, and no accumulation
into large vacuoles is seen.
oriented limiting epithelial layer separating
the external and internal environments of
the blastocyst. In addition, within this
epithelium the intercellular spaces between
the apical junctional complexes and the
bases of the juxtaposed trophoblast cells
constitute a series of potential intercellular compartments external to the blastocyst
As in earlier stages, both ferritin and
peroxidase penetrate the zona pellucida
and appear in the subzonal space within
ten minutes. Peroxidase reaction product
coats the surface of the trophoblast cells
unevenly (fig. 9). Slightly concave regions show an especially heavy reaction
(figs. 9, lo), and are apparently incipient
Eight-cell stage
coated vesicles similar to those associated
The eight-cell stage of the rat is unusual with protein uptake in other tissues (Roth
in that the blastomeres are usually in and Porter, '64; Fawcett, '65). Ferritin, on
either a planula or a triradiate arrange- the other hand, does not coat the surface
ment and, although no cavity formation is of the cells and only rarely appears to be
seen, rudimentary junctional complexes adsorbed to specialized regions of the surare found between cells near the surface face (figs, 11, 13). Both peroxidase and,
adjacent to the zona pellucida. On the sub- to a lesser extent, ferritin are found in
zonal surfaces some of the coated micro- coated micropinocytotic vesicles subjacent
pinocytotic vesicles contain small amounts to the surface of the cells and in similarly
of ingested protein (fig. 7 ) . Unlike earlier situated small tubules (figs. 9, 10, 13).
stages the apposed surfaces of the adjacent Large vacuoles containing peroxidase or
cells are generally smooth with few micro- ferritin are seen within ten minutes of
villi and relatively little intercellular space. exposure to the protein (figs. 11, 12).
Nevertheless tracer proteins readily pene- Often such vacuoles are surrounded by
trate these intercellular spaces (fig. 8). As vesicles and tubules, and confluence of
in the two-cell stage, isolated vesicles tubules with vacuoles can be demonwithin the cytoplasm containing tracer pro- strated. Although isolated micropinocyteins are numerous near the apposed sur- totic vesicles can be found anywhere along
faces of the blastomeres and less common the surface, accumulations of vacuoles are
near the subzonal surface.
largely restricted to the supranuclear region of cytoplasm (fig. 9). This area is
Blas tocys t stage
the region of concentration of cell orIn the blastocyst stage, micropinocytotic ganelles.
When specimens are taken 30 to 60 minvesicles are more numerous than at earlier
stages. In addition, the segregation of the utes after introduction of the tracer, peroxcytoplasm into areas rich in organelles and idase is present not only apically in the
plaque-filled zones so conspicuous in the structures described but, in addition, is
eight-cell stage is continued into the blasto- found in similar structures near the base
cyst stage (fig. 9). Further evidence of of the cells (fig. 14). However, in this
maturation of the cytoplasm is the forma- region occasional large vacuoles contain
tion of rod-shaped mitochondria, as op- both peroxidase activity and variable granposed to the earlier spherical germ-cell ules, membranes and other unidentifiable
type mitochondria with peculiar cristae. inclusions. These vacuoles consequently
Distinct junctional complexes now com- may be degradative in nature (see below).
pletely border the apical margins of the In vacuoles containing ferritin the tracer
trophoblast cells (Schlafke and Enders, is usually peripherally distributed, and the
'67), marking their differentiation into an central regions of the vacuoles are devoid
of structured elements (fig. 11). Neither
peroxidase reaction product nor ferritin is
seen in the intercellular spaces between
trophoblast cells or between cells of the
embryonic cell mass (figs. 9, 14). However small amounts of peroxidase reaction
product can be seen scattered throughout
the cavity of the blastocyst as well as in
small vesicles in some of the embryonic
cell mass cells (fig. 14).
Blastocysts during delayed implantation
Blastocysts during lactationally delayed
implantation show the greatest amount of
uptake of exogenous proteins. Numerous
vesicles, tubules and large vacuoles containing peroxidase are concentrated in the
supranuclear region of the cytoplasm (figs.
15-17). Apparent fusion of vesicles and
tubules to the periphery of large vacuoles
is common. Peroxidase is also seen in
small vesicles in the basal region of the
trophoblast cells. Ferritin is taken up in
moderate amounts and appears in the same
types of vesicles, tubules and vacuoles as
the peroxidase (fig. 18). However, it cannot be followed beyond the apical cytoplasm, and has not been found in the
blastocyst cavity.
By day 7 of lactational delay a basal
lamina is present on the inner surface of
the trophoblast surrounding the cavity of
the blastocyst. This basal lamina is heavily labeled with peroxidase reaction product (but not with ferritin) within ten
minutes after exposure to tracer (figs. 16,
17). The elaborate intercellular spaces
characteristic of blastocysts during delay
of implantation do not contain either protein (figs. 15-17). The penetration of
tracers must therefore be not only blocked
by the apical junctional complex but also
limited by a basally situated junction.
Although small amounts of peroxidase
are seen in embryonic cell mass cells, i t is
not demonstrable in the basal lamina
which underlies trophoblast adjacent to
the embryonic cell mass or in the intercellular spaces between embryonic cell mass
cells (fig. 15).
Acid phosphatase activity
In the two-cell stage, the Golgi elements
are generally situated at the periphery of
complex membranous areas composed of
agranular endoplasmic reticulum and various associated membrane-bound structures
(Schlafke and Enders, '67). Acid phosphatase activity is demonstrable in some
of the small vacuoles of the complex, and
in some of the cisternae of the Golgi elements (fig. 19). The degenerating sperm
mid-piece and tail-piece are devoid of acid
phosphatase activity.
At the blastocyst stage, acid phosphatase activity is most conspicuous in many
of the large vacuoles which have previously
been termed degradative bodies (figs. 2023). The lead deposits indicative of acid
phosphatase activity are frequently peripheral to the varied content of these vacuoles.
Since some of the vacuoles contain recognizable organelles in addition to phosphatase activity, at least part of the population
of these secondary lysosomes is derived
from autophagic processes (figs. 22, 23).
Golgi elements at this stage are isolated
stacks of cisternae with peripheral vesicles.
One or two cisternae commonly display
moderate acid phosphatase activity (fig.
The study of uptake of tracer proteins
and of acid phosphatase activity in preimplantation stages, when combined with
our earlier descriptive studes of the
changes in polyribosomes and plaques in
these stages in the rat, provides cytological
information suggesting changing patterns
of protein metabolism during cleavage. The
presence in the one- and two-cell stages of
numerous degradative bodies, some of
which contain acid phosphatase, the
absence of appreciable numbers of polyribosomes (Schlafke and Enders, '67), and
the limited uptake of exogenous protein
suggest a dependence on the stored proteins during early stages. The dramatic
increase in areas rich in polyribosomes in
the eight-cell through blastocyst stages, the
diminution of plaques, and the apparent
increase in capacity for ingesting protein
suggest not only increased dependence on
synthesis as opposed to utilization of stored
materials, but also the ingestion of protein
from the environment to provide substrates
for protein synthesis.
How does this picture, derived from
cytological studies of rat preimplantation
stages, compare with a closely related
species, the mouse, in which more information concerning metabolism of protein is
available? Brinster ('67) has shown that
during the first three days of development
there is a diminution in total protein, and
that protein content increases at the blastocyst stage. Since there is a relative absence of protein synthesis during the early
stages (Weitlauf and Greenwald, '67; Weitlauf, '71) loss from degradation of intrinsic
proteins is not counter-balanced by synthesis. [Both mouse and rat ova have morphologically unique storage materials whose
fixation properties indicate that they are
largely protein in content (Enders and
Schlafke, '65; Enders, '71).] In the mouse
the increase in protein synthesis at the
blastocyst stage has been demonstrated by
incorporation of radioactively labeled exogenous amino acids (Weitlauf, '69). The
evidence on stimulation of trophoblast outgrowth mentioned previously (Gwatkin,
'66; Menke and McLaren, '70) suggests
that proteins may be a satisfactory source
of amino acids, although no direct incorporation experiments using radioactively
tagged proteins as substrate have been reported.
A comparison of these observations on
the rat with those of Glass ('63, '69) on the
mouse is more difficult. Glass presented
evidence that homologous serum proteins
and bovine plasma albumin could be demonstrated in oviducal ova of the mouse. Her
immunofluorescence methods have indicated appreciable transfer of these proteins
at the ovum and two-cell stages. The evidence presented here indicates not only
that the exogenous proteins are taken into
the rat ovum in extremely small amounts,
but that the cytological apparatus for protein uptake (coated micropinocytotic vesicles) is minimal at this time. The appearance of tracer in contiguous regions of
blastomeres at the two-cell stage is insufficient in amount to explain the heavy nonlocalized fluorescence observed in comparable stages by Glass ('69). Although
differences in species, tracer and method
all contribute to the differences in observation, the results of the two types of investigation are still in rather striking contrast.
Considering that blastocysts from delayed implantation have a reduced metabolic rate, at least in the mouse (McLaren
and Menke, '71; Weitlauf, '69), it might
be surprising that the rat blastocysts from
delay very actively ingest exogenous protein. However, numerous tubules and vesicles in the apical cytoplasm were reported
by Schlafke and Enders ('63) in unincubated blastocysts from lactational delay,
indicating abundant ingestive activity even
in the absence of introduced exogenous
protein. The depletion of oval stores of material in the form of plaques probably
makes the blastocyst particularly dependent on sources of protein from the uterine
environment during delay of implantation.
Since there is a reduction in amount of
protein present in the luminal fluid of the
rat during delay as opposed to the time of
normal implantation (Daniel, '69), the
high level of ingestive activity by the
blastocyst may constitute a mechanism of
partial compensation.
The presence of peroxidase activity
within the cavity of the blastocyst shows
that not all of the entering tracer is degraded. Since the reaction product can be
readily demonstrated in vesicles and tubules within the cytoplasm, but cannot be
demonstrated either within the junctional
complexes or in intercellular spaces between trophoblast cells, the pathway of
transport of the undegraded tracer must be
intracellular. Such a mechanism of intracellular transport would permit the accumulation of materials in the cavity of the
blastocyst that are not necessarily synthesized by the cells of the blastocyst.
It is difficult directly to compare results
obtained using peroxidase with those from
ferritin, since in one instance the iron core
of the molecule is visualized directly and
in the other the reaction product seen is the
result of the amplifying effect of the enzymatic activity. Nevertheless the relatively
sparse distribution of ferritin adhering to
the surface coat of the blastocyst and the
absence of ferritin from the cavity of the
blastocyst probably indicate genuine differences in the response of the trophoblast
to these tracers. In contrast to the situation
in the rat blastocyst, the initial stages of
uptake of the two tracers are similar in the
Feigelson, M., and E. Kay 1972 Protein patterns of rabbit oviducal fluid. Biol. Repro., 6 :
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In this micrograph of a fertilized ovum from day 1 of pregnancy,
horseradish peroxidase reaction product appears in the subzonal space
and coats part of the cell surface. No reaction product is seen within
the ovum. The outer fibers and fibrous sheath of the principal piece
of the fertilizing sperm tail are equally dense in control ova. Ten
minutes exposure. x 25,600.
2 Ferritin particles are dispersed throughout the zona of this day 1
ovum after ten minutes exposure. However the tracer neither coats
the surface of the ovum nor appears within the ovum, even when
coated micropinocytotic vesicles are present (arrow). x 30,200.
Two-cell stages have occasional micropinocytotic vesicles which contain peroxidase reaction product. Note especially the unusual double
vesicle in the left center. Day 2, 30 minutes. x 22,800.
4 After 60 minutes exposure to peroxidase, the distribution of reaction
product in two-cell stages is similar to that with short exposures. The
protein does not accumulate in larger vacuoles within the cytoplasm.
Note the numerous plaques within the cytoplasm at this stage. Day 2.
X 23,100.
Sandra Schlafke and Allen C . Enders
5 Ferritin appears throughout the zona in this two-cell stage, but only
a few particles are adjacent to the cell surface. None of the protein
is in the few small micropinocytotic vesicles (arrows). G, Golgi
zone. Day 2, ten minutes. x 36,700.
6 Peroxidase reaction product is demonstrable between blastomeres at
the two-cell stage within 30 minutes of exposure. The protein is also
seen in vesicles and small vacuoles within the cells in this region
(arrows). Day 2. x 20,600.
Sandra Schlafke and Allen C. Enders
At the eight-cell stage, ferritin is clumped in the subzonal space but
is not seen in adjacent coated micropinocytotic vesicles (arrows).
Day 4, ten minutes. X 28,200.
Peroxidase penetrates between contiguous blastomeres of the eightcell stage, despite the reduced intercellular space. Some uptake of
protein by micropinocytotic vesicles is seen in this region (arrows).
Day 4, 30 minutes. x 18,200.
At the blastocyst stage, peroxidase is adsorbed to portions of the
cell surface (brackets). The numerous apical vesicles and tubules
containing reaction product are concentrated in the region of normal
organelles (right) as apposed to that of plaques (left). Day 5, ten
minutes. x 20,100.
The smaller vesicles and tubules that contain peroxidase surround or
directly communicate (arrows) with large vacuoles ( V ) . Day 5, ten
minutes. x 21,800.
Sandra Schlafke and Allen C. Enders
11 Ferritin particles sometimes appear to be absorbed in areas of presumptive micropinocytotic vesicle formation (large arrow), as well
as in vesicles within the cytoplasm (small arrows). The protein
accumulates at the periphery of large vacuoles. Day 5 , ten minutes.
X 57,500.
12 Occasional large bodies are densely packed with ferritin molecules,
while others may include vesicles as well (arrow). Day 5, 60 minutes.
X 55,000.
13 After 60 minutes exposure at the blastocyst stage, ferritin is abundant
in the subzonal space. It is seen in coated micropinocytotic vesicles
and in tubules (arrows), but is not present in the apical junctional
complex or in the intercellular spaces between trophoblast cells. Zona
pellucida (ZP). Day 5. X 90,500.
14 In this day 5 blastocyst, which was exposed to peroxidase for 60
minutes, a small amount of reaction product is seen in the cavity of
the blastocyst but not in the intercellular spaces (ICS). Peroxidase is
concentrated in two of the large secondary lysosomes (SL). Similar
bodies in the same region of cytoplasm have not accumulated exogenous protein. Lipid droplets (L) are also seen in the trophoblast
cells, as well as regions consisting almost solely of plaques and
regions with numerous polyribosomes. x 13,000.
Sandra Schlafke and Allen C. Enders
15 Peroxidase is seen in numerous vesicles, tubules and vacuoles in the
apical cytoplasm of the trophoblast from a blastocyst during delayed
implantation. Note that the reaction product does not appear in the
intercellular spaces (ICS), the basal lamina (BL), nor in the underlying embryonic cells mass cells. Day 7, ten minutes. x 22,400.
16 There is a heavy accumulation of tubules containing peroxidase in
the supranuclear region of this trophoblast cell from a blastocyst on
day 11 of delayed implantation. Although reaction product is absent
from the elaborate intercellular spaces, it has accumulated in the
basal lamina (BL) which underlies trophoblast adjacent to the cavity
of the blastocyst. Day 11, ten minutes. x 21,800.
17 The supranuclear cytoplasm of this delay blastocyst exhibits the
typical accumulation of vesicles and tubules containing peroxidase.
In addition peroxidase is present in the basal lamina lining the cavity
but is absent in intercellular spaces. Day 11, ten minutes. x 16,700.
Sandra Schlafke and Allen C. Enders
18 The apical vesicles and tubules (arrows) of the delay blastocyst contain numerous ferritin particles. The particles also line the periphery
of vacuoles. Day 7, ten minutes. x 50,000.
In this two-cell stage, acid phosphatase activity is demonstrable in
a few of the vacuoles (V), and in Golgi cisternae ( G and inset).
Acid phosphatase does not appear in relation to the areas of plaques.
Day 2, 30 minute incubation. x 17,700.
Sandra Schlafke and Allen C. Enders
At the blastocyst stage, acid phosphatase activity is seen in some of
the Golgi cisternae ( G ) and in large irregular bodies (V), containing
a variety of granules, membranes and vesicles. Note that plaques are
shorter and less distinct than i n early stages, and that polyribosomes
are numerous. Day 5, 30 minutes incubation. x 23,700.
The large dense body (above) contains acid phosphatase reaction
product, while the one below does not. Day 5, 30 minutes incubation.
x 16,500.
In this blastocyst acid phosphatase activity is seen in an autophagic
vacuole (secondary lysosome) containing a mitochondrion ( M ) and
a lipid droplet. Day 5. x 24,300.
A n autophagic vacuole similar to that seen in figure 22 from a day 5
blastocyst which did not undergo incubation for acid phosphatase
activity. It contains a mitochondrion and a few ribosomes, in addition
to amorphous material. X 24,300.
Sandra Schlafke and Allen C. Enders
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stage, protein, rat, uptake, preimplantation
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