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Nuclear and cytoplasmic annulate lamellae in trophoblast giant cells of rat placenta.

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Nuclear and Cytoplasmic Annulate Lamellae in
Trophoblast Giant Cells of R a t Placenta'
Department of Anatomy, Tulane University School of Medicine,
New Orleans, Louisiana 701 12
Single stranded profiles of nuclear annulate lamellae were identified
in giant cells of rat trophoblast from the day when the chorioallantoic placenta first
becomes vascularized, viz., day 12 post cdtum, until the day before term, viz., day 22.
Cytoplasmic annulate lamellae were observed only in giant cells from placentas at day
12. Occasionally cytoplasmic annulate lamellae were found in parallel array. Often
the lamellar membranes were continuous with both granular and agranular membranes
of endoplasmic reticulum; they closely resembled doubled outer nuclear membrane.
Nuclear annulate lamellae resembled doubled inner nuclear membrane; and often the
two were found in continuity. In addition, at later gestational ages ( 1 7 and 22 days),
nuclear lamellae often were related anatomically to the variety of nuclear inclusions
which characterize giant trophoblast cells during late pregnancy. A possible relationship
of annulate lamellae to the synthesis of DNA, RNA and protein i s considered.
Cytoplasmic annulate lamellae have
been observed in a variety of germ cells,
both invertebrate (Afzelius, '57; Hsu, '67;
Kessel, '65; Rebhun, '56; Swift, '56) and
vertebrate (Kessel, '63; Wischnitzer, '58,
'60), including man (Adams and Hertig,
'65; Baca and Zamboni, '67); in certain
cancer cells (Binggeli, '59; Chambers and
Weiser, '64; Eliot and Arhelger, '66;
Hoshino, '63; Maul, '68; Schulz, '57;
Wessel and Bernhard, '57); in a few embryonic body cells (Merkow and Leighton,
'66; Ross, '62); and in adult body cells
which have been exposed to certain drugs
(Hruban et al., '65; Krishan, Hsu and
Hutchins, '68; Ma and Webber, '66; Merkow et al., '67). Though reported less frequently, nuclear annulate lamellae have
been identified in both oocytes, (Afzelius,
'55; Everingham, '68; Hsu, '63, '67; Kessel,
'64, '65) and fertilized ova (Zamboni and
Mastroianni, '66) ; in the relatively undifferentiated cells of several early embryos
(Harris, '61); and in certain fully differentiated somatic cells, notably of glandular
parenchyma (Gross, '66; Harrison, '66).
From such a survey, one might infer
that under normal conditions, both varieties of annulate lamellae either are more
apparent, or are better devloped, in cells
which are young and undifferentiated, and
either rapidly proliferating, or otherwise
undergoing elevated metabolism. It has
been suggested that the lamellae reflect a
specialized type of nuclear-cytoplasmic inANAT. REC.. 165: 1-14.
teraction which is related to differentiation
(Kessel, '65 and '68).
In the present report, both nuclear and
cytoplasmic annulate lamellae are described in trophoblast giant cells of the rat
at various stages of placental maturation.
Giant cells are the first trophoblastic elements to differentiate from the implanting
blastocyst (Alden, '48), They are actively
phagocytic through day 14 of pregnancy
(Jollie, '65). Although they are probably
not fully differentiated until day 16
(Jollie, '65), and although they are known
to synthesize DNA until this gestational
age (Jollie, '64), they have never been
seen to divide, mitotically or otherwise.
They do not synthesize DNA after the
sixteenth day of pregnancy (Jollie, '64).
RNA synthesis ceases by the nineteenth
day (Dorgan and Schultz, '68). Aside from
the synthesis of protein, (Dorgan and
Schultz, '68) the role of these cells in the
terminal stages of pregnancy is unknown.
Sprague-Dawley rats were used for this
investigation. Gestational ages were reckoned as follows: when typically cycling
females exhibited a vaginal lavage characteristic of early estrus, they were caged
Received March 5, '69. Accepted May 9, '69,
1 Supported by a Research Grant from the National
Institutes of Health (HD-00569).
2 Present address: Department of Anatomy. Medical
College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23219.
with breeding males at 5 : 00 PM. A second
lavage was taken at 9 : O O AM the following morning. If spermatozoa were present
in this lavage, the females were considered to be one day pregnant until the
evening of the day when spermatozoa
were identified.
Females were autopsied at three gestational ages, viz., when 12, 17 and 22
days pregnant. As will be subsequently
discussed, these 3 gestational ages are
believed to represent giant cells respectively at undifferentiated, mature, and
senescent stages. Placentas from at least
three animals at each gestational age were
Under nembutal anesthesia, the pregnant animals were laparotomized; and
the uteri were incised. Entire placentas
were surgically removed and transferred
to fixative (0.1 M phosphate buffered, 3%
glutaraldehyde at pH 7.4) at 0-4" C. From
the maternal surface of each chorioallantois, the placental junctional zone
was excised, immersed in another drop
of cold fixative, and cut into 1 X 1 X 2
mm tissue blocks. These were fixed at
4" C for two hours, washed several times
in 5% sucrose (0.1 M phosphate buffer,
pH 7.4), post-fixed for two hours in 2%
osmium tetroxide (5% sucrose, same
buffer), rapidly dehydrated in ethanol and
embedded in epoxy resin ("Durcupan":
Fluka). Thin sections were cut on an LKB
Ultratome 11, mounted on naked 300 X
300 mesh copper grids, and double-stained
with uranyl acetate (saturated aqueous
solution for 15 minutes) and lead citrate
(Reynolds, '63). They were examined in
an RCA EMU 3-G.
In 12-day placental giant cells, cytoplasmic annulate lamellae appeared both
in normal section (fig. 1 ) and in grazing
surface view (fig. 3). In the latter orientation, parallel lamellae occasionally were
arranged in stacks (fig. 3 ) . For the most
part, the related, periodically apposed
membranes of each lamella were smooth.
Occasionally, however, they were observed
in continuity with both granular and
agranular membranes of endoplasmic
reticulum (figs. 2, 3). In profile view the
annuli were often marked by a fuzzy
density on the cytoplasmic surface of the
apposing smooth membranes and by the
interposition of a dense diaphragm (fig.
1). In both these respects, the annuli resembled closely the pores of the nuclear
envelope, save that both membranes of
the lamellae were like the outer membrane
of the envelope. Furthermore, lamellar
annuli and nuclear pores were both about
800A in diameter and were both spaced
at about 800A (fig. 1). In surface view,
both appeared roughly circular (figs. 2,
Single strands of nuclear annulate
lamellae were observed at each gestational
age which was examined. At the earliest
stage, in which giant-cell nuclei were
relatively free of inclusions, the lamellae
appeared as isolated profiles which extended randomly throughout a largely
vesiculate nuclear background (figs. 4, 5,
6). Occasionally they were observed in
continuity with the nuclear envelope (fig.
4). In each case, the annuli duplicated
nuclear pores, both in periodic spacing
and in morphologic detail. Both membranes of the lamellae, however, were
like the inner nuclear membrane, in that
they were related closely to a fibrous
lamina and to occasional small clumps
of heterochromatin (figs. 4, 6 ) . Again, in
surface view, the annuli appeared circular
with both a diameter and an interannulate
distance of about 800A (fig. 6).
The nuclei of giant cells from 17- and
22-day placentas were characterized by
inclusions which consisted of small,
smooth vesicles, larger granular profiles,
free granules, and occasional myeloid
figures, or dense bodies (figs. 7-13). In
giant cells at all stages of pregnancy, the
enlarged nucleus was vesiculate and was
characterized by a pronounced fibrous
lamina against the inner surface of the
inner membrane of the nuclear envelope
and by a smooth outer nuclear membrane
(figs. 7, 8, 10). In larger measure, this
structure of the nuclear membrane was
duplicated, but reversed, about each nuclear inclusion. However, occasionally,
along one side of the perimeter of an
inclusion, the outer membrane of the inclusion also was smooth (figs. 7-13).
Annulate lamellae were observed in continuity with the nuclear envelope (figs. 7,
11), with the envelope of the nuclear
inclusions (figs. 7, 10, 13), or with both
(figs. 7, 8). For the most part, the strands
of nuclear lamellae had both membranes
associated with a fibrous lamina and, in
this respect, duplicated the inner membrane of the nuclear envelope (figs. 7, 8,
10). Such fibrous laminar reinforcement
of annulate lamellae was particularly evident in instances where a lamella was in
continuity with the fibrous laminar membrane of a nuclear inclusion (figs. 8, 10).
Occasionally, however, the membranes of
the nuclear annulate lamellae were continuous with smooth intranuclear membranes which, in turn, formed the smooth
portion of the outer membrane of the
envelope of an inclusion (fig. 11). Infrequently nuclear annulate lamellae appeared to be formed of apposed membranes, only one of which was related to
a fibrous lamina, the other membrane
appearing smooth (fig. 12). Moreover, in
serial sections of a given portion of a
giant-cell nucleus, it was not uncommon
to observe a configuration which suggested
that the nuclear annulate lamellae were
highly variable membranes which were
formed as inward extensions of the inner
nuclear membrane and which incompletely enveloped the characteristic nuclear inclusions (figs. 11-13).
Previous investigations have shown that
until the sixteenth day following copulation, the giant-cells of trophoblast which
are interposed between the chorioallantoic
placenta and the underlying decidua of
the rat were engaged in the synthesis of
DNA, RNA and protein (Dorgan and
Schultz, '68; Jollie, '64). After the sixteenth day, giant cells no longer synthesize DNA; and by the twenty-second day,
they no longer synthesize RNA (Dorgan
and Schultz, '68). The entire chorioallantois is delivered as an afterbirth
typically late on the 22nd or early on
the twenty third day (Jollie, '65). As a
result of this developmental sequence,
giant cells of placentas at the three gestational ages which were selected for the
present investigation (viz. 12, 17, 22 days)
represent 3 successive stages in the functional life of these elements, viz., a stage
when they synthesize DNA, RNA and protein; a stage when they synthesize RNA
and protein alone; and a stage when they
synthesize only protein.
Single strands of nuclear annulate
lamellae were identified in giant cells at
each selected stage. In each case, the
nuclear lamellae consisted of apposed, annulated membranes which were occasionally observed in continuity with the inner
nuclear membrane. By virtue of a closely
related fibrous lamina and clumps of
heterochromatin, the nuclear lamellae
closely resembled this inner membrane.
Furthermore, in both membranes, interannular spacing and annular diameters
were alike. Often nuclear lamellae were
close to the nuclear inclusions which
characterize giant cells at later gestational
ages. Occasionally membranes of nuclear
lamellae were continuous with those which
delimited these inclusions. Although no
statistical study of lamellar distribution
was attempted, the nuclear annulate
lamellae seemed more abundant at later
gestational ages, i.e., at stages when the
cells are known to be losing their synthesizing capabilities. The observation that
nuclear lamellae are abundant at times
when the cells are becoming quiescent or
senescent, suggests that in trophoblast
giant cells, nuclear annulate lamellae are
not associated with elevated cellular synthesis.
Cytoplasmic annulate lamellae, on the
other hand, were identified only at the
earliest stage, i.e., when cellular functions
were most diversified (12 days). A lack
of the membranes in the micrographs of
placentas at the two later stages does not,
of course, necessarily mean that this organelle is indeed absent; it simply may
not have been encountered in the arbitrary
sectioning of the tissues. On the limited
evidence that is presented, therefore, great
significance cannot be attached to the observation that cytoplasmic lamellae were
identified only at the earliest stage of the
three which were examined. This is the
only stage of the three, however, during
which giant trophoblast cells synthesize
DNA; and the possibility remains that
there may be a relationship between such
synthetic function and the presence of
annulate lamellae as a cytoplasmic organelle. Moreover, although DNA synthesis occurs at 12 days (and earlier), giant-
primary follicles. J. Cell Biol., 27: 119A (abstract).
Afzelius, B. A. 1955 The ultrastructure of the
nuclear membrane of the sea urchin oocyte as
studied with the electron microscope. Exp. Cell
Res., 8: 147-158.
1957 Electron microscopy on the basophilic structures of the sea urchin egg. Z.
Zellforsch., 45: 666675.
Alden, R. H. 1948 Implantation of the rat egg.
111. Origin and development of primary trophoblast giant cells. Am. J. Anat., 83: 143-181.
Baca, M.,and L. Zamboni 1967 The h e structure of human follicular oocytes. J. Ultrastruct.
Res., 19: 354-381.
Binggeli, M. F. 1959 Abnormal intranuclear
and cytoplasmic formations associated with a
chemically induced, transplantable chicken
sarcoma. J. Biophys. Biochem. Cytol., 5: 143152.
Chambers, V. C., and R. S . Weiser 1964 Annulate lamellae in Sarcoma I cells. J. Cell Biol.,
21: 133-139.
Dorgan, W. J., and R. L. Schultz 1968 DNA,
RNA, and protein synthesis in the giant cells
of the rat placenta in vitro. Teratol., 1: 213
Eliot, R.L., and R. B. Arhelger 1966 Fine structure of parathyroid adenomas, with special
reference to annulate lamellae and septate
desmosomes. Arch. Path., 81: 200-212.
Everingham, J. W. 1968 Intranuclear annulate
lamellae in ascidian embryos. J. Cell Biol., 37:
Gross, B. G. 1966 Annulate lamellae in the
axillary apocrine glands of adult man. J. Ultrastruct. Res., 14: 64-73.
Harris, P. 1961 Electron microscope study of
mitosis in sea urchin blastomeres. J. Biophys.
Biochem. Cytol., 11: 419-431.
Harrison, G. 1966 Some observations on the
presence of annulate lamellae in alligator and
sea gull adrenal cortical cells. J. Ultrastruct.
Res., 14: 158-166.
Hoshino, M. 1963 Submicroscopic characteristics of four strains of Yoshida ascites hepatoma
of rats: a comparative study. Cancer Res., 23:
Hruban, Z. H., H. Swift, F. W. Dunn and D. E.
Lewis 1965 Effect of beta-3-furylalanine on
the ultrastructure of the hepatocytes and pancreatic acinar cells. Lab. Invest., 14: 70-80.
Hsu, W. S. 1963 The nuclear envelope in the
developing oocytes of the tunicate, Boltenia
villosa. Z. Zellforsch., 58: 660-678.
1967 The origin of annulate lamellae
in the oocyte of the ascidian, Boltenia willosa
Stimpson. Z. Zellforsch., 82: 376-390.
Jollie, W. P. 1964 Radioautographic observations on variations in desoxyribonucleic acid
synthesis i n rat placenta with increasing gestational age. Am. J. Anat., 114: 161-171.
1965 Fine structural changes i n the
junctional zone of the rat placenta with increasing gestational age. J. Ultrastruct. Res.,
12: 420438.
Kessel, R. G. 1963 Electron microscope studies
on the origin of annulate lamellae i n oocytes
Adams, E. C., and A. T. Hertig 1965 Annulate
of Necturus. J. Cell Biol., 19: 391-414.
lamellae in human oocytes in primordial and
cell nuclei have never been seen to divide.
Consequently, cytoplasmic annulate lamellae in rat giant trophoblast cells cannot
be regarded as isolated fragments of nuclear membrane which, following mitoses,
failed to be reincorporated into the nuclearcytoplasmic interfacial membrane, as has
been suggested for the sea urchin oocyte
(Afzelius, '55). Although, in the present
study, no continuity of cytoplasmic annulate lamellar membranes and nuclear
membranes was observed, and despite recent evidence that in certain cells they
may derive from fenestrated dictyosomal
membranes (Maul, '68), there is a striking
similarity between cytoplasmic annulate
lamellae and the outer nuclear membrane :
both membranes are agranular, and the
annular diameters and the interannular
spacing of both membranous systems are
A continuity of annulate lamellae with
the membranous cisterns of both granular
and agranular endoplasmic reticulum also
was noted. Such continuity supports the
evidence that the lamellae, the nuclear
envelope, and the endoplasmic reticulum
may be one continuous membrane system
(Kessel, '68). Whether the system is continuous in time as well as in space is, of
course, unknown. However, it is difficult
to imagine how, in this placental tissue,
such a membranous relationship necessarily is a morphological expression of
nuclearcytoplasmic interactions which are
functionally related to differentiation.
Since it has been shown previously that
there is an increase in abundance of endoplasmic reticulum in trophoblast giant cells
until the sixteenth day of pregnancy
(Jollie, ' 6 5 ) , cytoplasmic annulate lamellae observed before this stage may represent a method by which additional membranes of both granular and agranular
reticulum are "spun off from the nuclear
membrane. According to this line of speculation, the presence of such annulated
cytoplasmic membranes might result from
rapid membrane formation. Therefore,
they may be related only indirectly to the
function of the fully differentiated reticulum, that is, to protein synthesis.
1964 Electron microscope studies on
oocytes of an echinoderm, Thyone bTiareus,
with special reference to the origin and structure of the annulate lamellae. J. Ultrastruct.
Res., 10: 498-514.
1965 Intranuclear and cytoplasmic annulate lamellae i n tunicate oocytes. J. Cell Biol.,
24: 471487.
1968 Annulate lamellae. J. Ultrastruct.
Res., Suppl. 10.
Krishan, A.D., D. Hsu and P. Hutchins 1968
Hypertrophy of granular endoplasmic reticulum and annulate lamellae in Earle’s L cells
exposed to vinblastine sulfate. J. Cell Biol., 39:
Ma, M. H., and A. J. Webber 1966 Fine structure of liver tumors induced in the rat by
3’-methyl-4-dimethylaminoazobenzene. Cancer
Res., 26: 935-946.
Maul, G. 1968 On the origin of annulate lamellae i n human melanoma cells in vitro. J.
Cell Biol., 39: 88A (abstract).
Merkow, L. P., S. M. Epstein, B. J. Caito and B.
Bartus 1967 The cellular analysis of liver
carcinogenesis: Ultrastructural alterations within hyperplastic liver nodules induced by 2fluorenylacetamide, Cancer Res., 27: 1712-1721.
Merkow, L. P., and J. Leighton 1966 Increased
numbers of annulate lamellae in myocardium
of chick embryos incubated a t abnormal temperature. J. Cell. Biol., 28: 127-137.
Rebhun, L. I. 1956 Electron microscopy of
basophilic structures of some invertebrate
oocytes. I. Periodic lamellae and the nuclear
envelope. J. Biophys. Biochem. Cytol., 2: 93104.
Reynolds, E. S. 1963 The use of lead citrate
a t high pH as an electron-opaque stain i n electon microscopy. J. Cell Biol., 17: 208-212.
Ross, M. H. 1964 Annulate lamellae in the
adrenal cortex of the fetal rat. J. Ultrastruct.
Res., 7: 373-382.
Schulz, H. 1957 Elektronenmikroskopische Untersuchungen eines Mammakarzinoms der
Ratte. Oncologia, 10: 307-329.
Swift, H. 1956 The fine structure of annulate
lamellae. J. Biophys. Biochem. Cytol., 2
(Suppl.): 415-418.
Wessel, W. and W. Bernhard 1957 Vergleichende elektronenmikroskopische Untersuchung
von Erlich-und Yoshida-Ascitestumorzellen. Z.
Krebsforsch., 62: 140-162.
Wischnitzer, S. J. 1958 An electron microscope
study of the nuclear envelope of Amphibian
oocytes. J. Ultrastruct. Res., 1: 201-222.
1960 Observations on the annulate
lamellae of immature amphibian oocytes. J.
Biophys. Biochem. Cytol., 8: 558-563.
Zamboni, L. and L. Mastroianni, Jr. 1966
Electron microscopic studies on rabbit ova. 11.
The penetrated tubal ovum. J. Ultrastruct. Res.,
14: 118-132.
In a 12-day trophoblast giant cell, a portion of juxtanuclear cytoplasm is depicted
which contains a single strand of annulate lamella i n profile view (arrow). At
either end of this strand, the lamella is continuous with what appears to be
agranular endoplasmic reticulum. Note that in profile view, about the annuli and
about the pores of the nuclear envelope, both of which are of similar diameter,
there is an amorphous density, and that both annuli are closed by diaphragmata.
X 31,000.
2 In another 12-day giant cell, a strand of cytoplasmic annulate lamella is sectioned
obliquely (arrow). At the right of this strand, there appears to be continuity with
granular reticulum. The spacing of the lamellar annuli is comparable to that of
the nuclear pores which are seen a t the bottom of the figure both in profile view
(left) and in the surface view (right). X 27,000.
At the large arrows, stacks of cytoplasmic annulate lamellae in a 12-day giant cell
are sectioned in a grazing plane. The small arrow indicates a lamella cut in
profile view showing membranous continuity with agranular and granular reticula.
x 18,000.
William P. Jollie
In a very small segment of the nucleus ( N ) of a 12-day giant cell,
annulate lamellae are shown in continuity with the nuclear envelope.
Note that both membranes of the lamellae are like this inner nuclear
membrane in that they are related to clumps of heterochromatin.
Annular spacing on both the nuclear lamellae and the nuclear membrane appears comparable at about 8 0 0 A X 16,000.
5 A large portion of the nucleus of a 12-day giant cell is depicted.
Isolated strands of annulate lamellae are randomly distributed in the
largely vesiculate nucleus. These membranes are seen at higher
magnification in the next figure. x 12,000.
At higher magnification, nuclear annulate lamellae are seen in both
normal and grazing section. In normal view, the membranes exhibit
a fibrous lamina and clumps of heterochromatin applied to the outer
surface. In surface view, the annuli appear circular and measure
about 800 A in diameter. X 30,000.
William P. Jollie
A small portion of a 17-day giant-cell nucleus ( N ) is depicted. Note the similarity
between the inner nuclear membrane (large arrow) and the strand of nuclear
annulate lamellae (small arrow). The nuclear inclusions appear to be enclosed
in membranes which are similar save that in many half of the perimeter is made
up of doubled smooth membrane. A continuity between such smooth membranes
and a strand of annulate lamellae is shown at the arrowhead. x 22,500.
A portion of a 17-day giant-cell ( N ) illustrates a continuity between inner nuclear
membrane, nuclear annulate lamellae and a nuclear inclusion. Again the inclusions
are delimited by double membranes, the outer of which is related to a fibrous
lamina only about one side. X 22,500.
9 At low magnification, the complex structure of the nucleus in a 22-day giant cell
is apparent. The great irregularity of nuclear outline suggests that at least some
of the inclusions may be cytoplasmic invaginations. Though abundant, nuclear
annulate lamellae are difficult to recognize at this magnification. x 9,500.
10 At greater magnification, the same nucleus as in figure 9 is seen to contain strands
of annulate lamellae which are continuous with the nuclear inclusions. Though
the deeper membranes of the inclusions are similar to the outer nuclear membrane, the annulate lamellae appear to resemble more closely doubled inner
nuclear membrane. x 19,000.
William P. Jollie
Selected sections from a series through the same small segment
of a giant-cell nucleus at 22 days. The arrowhead is at the same
point in each figure. In figure 11, two strands of nuclear annulate
lamellae extend inward from the nuclear envelope. Though they
approximate a t one point, they are separate; and distally each is
continuous with smooth membranes. In figure 12, the two strands
have interconnected so that the distal ends are fused together and
the proximal ends (i.e., the segments related to the nuclear membrane) are similary conjoined. In figure 13, the same membranes
which previously were seen as separate strands, are now united
by a conjoining of the smooth membranous portion of the strands.
A continuous, double membranous circular profile, half the perimeter of which is smooth, is thus formed. The membranes of the
proximal segments have become continuous with the delimiting
membrane of a nuclear inclusion which appears a t the upper left
in each figure. All three figures. x 13,500.
William P. Jollie
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cytoplasmic, nuclear, giants, trophoblast, lamellae, rat, placental, annulate, cells
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