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The fine structure of granulosa cell nexuses in rat ovarian follicles.

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The Fine Structure of Granulosa Cell Nexuses in
R a t Ovarian Follicles
D e p a r t m e n t of Biology, Boston University, Boston, Massachusetts 0221 5
and Department of Pathology, Univel-sity Hospital, Boston,
Massachusetts 02118
An intercellular junction known as the nexus has been identified
in developing granulosa tissue of rat ovaries. Nexuses are common in Graafian
follicles of mature cycling rats and in similar follicles of immature animals
stimulated by exogenous gonadotrophins. The angle at which the plane of section intersects a nexus significantly governs its internal appearance. Within
obliquely cut nexuses there is often a cross-striated pattern of uniform lines in
specimens prepared by conventional fixation and staining procedures. These
lines could be significant because they may represent points of continuity between communicating cells.
Two forms of nexuses are observed. The abutment form is found at cell surfaces, is continuous with plasma membranes, and is frequently associated with
cell processes. The other form of nexus is ring-shaped and appears free within
the cytoplasm. These circular profiles probably represent transverse views through
nexus-girdled cell processes as well as nexus-bound spheres which may have
pinched off the processes. Since the profiles from the two sources are ordinarily
indistinguishable, both are designated "annular nexuses" even though some may
be cell inclusions. Lanthanum tracer can percolate into the central region of
abutment nexuses but is absent from the large majority of annular nexuses,
suggesting that many of them are isolated from the extracellular space. Reconstruction of serial sections reveals that some annuli are parts of cell inclusions.
Sphere formation could be a means of removing nexuses from cell surfaces.
When plasma membranes of adjacent
cells come into contact they sometimes
form a junction known as the nexus
(Dewey and Barr, '64) which is also referred to in the literature as "gap junction"
or "close junction." High resolution studies
of thin sections (Robertson, ' 6 3 ) and sections of tissue impregnated with lanthanum hydroxide tracer reveal that each
nexus contains closely packed subunits
which are thought to bridge the space between the two membranes (Revel and
Karnovsky, '67; Brightman and Reese, '69;
McNutt, '70). Studies employing negative
staining (Benedetti and Emmelot, '68) and
freeze-cleaving (Chalcroft and Bullivant,
'70; McNutt and Weinstein, '70) techniques have confirmed the presence of
globular substructures within the internal
aspect of each nexus membrane. In many
tissues the nexus has been demonstrated
ANAT. REC, 175: 107-126.
to electrically (ionically) couple adjacent
cells and is considered important in intercellular communication (Dreifuss et al.,
'66; Loewenstein, '66;Penn, '66; Bergman,
'68; Barr et al., '68; Sheridan, '71). Previous studies on the rat (Bjorkman, ' 6 2 ) ,bat
(Wimsat and Parks, '66),mouse (Byskov,
'69; Anderson, '71), and rabbit (Espey and
Stutts, '72) granulosa cells and human
luteal tissues (Carsten, '65; Van Lennep
and Madden, '65; Adams and Hertig, '69;
Crisp et al., '70) have shown junctions
similar to nexuses. These structures were
given a diverse nomenclature and several
possible functions were suggested.
Received Nov. 29, '71. Accepted Oct. 4, '72.
lThis work is part of a dissertation submitted by
the first author to the faculty of arts and sciences,
Boston University Department of Biology, in partial
fulfillment of the requirements for the Ph.D. degree,
a Present address: Department of Pathology University Hospital Boston University Medical Cen'ter, Boston, Massacdusetts 02118.
This report describes the fine structure
of rat granulosa cell nexuses prepared by
standard thin sectioning techniques. In addition, results obtained from lanthanum
tracer studies and serial sectioning suggest
that the two forms of nexuses may represent static images of nexus membranes involved in a dynamic turnover process.
Thirty-two Harvard strain rats, divided
into four groups were employed. One
group, consisting of sexually mature cycling animals approximately six months
old, was sacrificed during estrus (stage of
cycle determined by vaginal smear cytology). The rats had been individually caged
and maintained on a standard lab chow
diet. The remaining three groups were immature rats. One group served as control
and the other two groups were treated with
exogenous gonadotrophins. Gonadotrophin
treatment was undertaken in immature
animals to promote follicular development
in the absence of corpora lutea. At 18 days
of age the animals of both experimental
groups were injected subcutaneously with
30 I.U. pregnant mare serum gonadotrophin (PMSG) (Equinex, Ayerst Laboratories, Inc., New York, N.Y.) dissolved in
0.85% saline. Fifty-six hours later, one of
the experimental groups was injected with
40 I.U. human chorionic gonadotrophin
(HCG) (Upjohn Co., Kalamazoo, Mich.)
dissolved in 0.85% saline. All immature
rats were killed by ether anesthesia at 21
days (68 hours after PMSG injections in
the case of the experimental animals).
Fixation of ovarian tissue was carried
out at room temperature for six hours in a
2% glutaraldehyde, 2% paraformaldehyde
and 0.04-0.2% picric acid solution (It0
and Karnovsky, '68) with 0.1 M Sorensen
phosphate buffer (pH 7.2). Following a
buffer rinse at 4"C, the specimens were
postfixed for four hours in a 2 % OsOa
solution (Millonig phosphate buffer, 0.13
M, pH 7.2) at the same temperature. Dehydration was done in an ascending
ethanol series at 4°C. The extracellular
spaces, in tissue from some of the PMSG
treated animals, were impregnated with
colloidal lanthanum hydroxide. This electron opaque tracer was added to fixative
solutions prior to dehydration according
to the method of Revel and Karnovsky
('67). Tissues from all groups were embedded in Epon 812. Sections displaying
silver interference colors were cut on an
LKB Ultrotome with a diamond knife and
mounted on uncoated 300 mesh grids.
Serial sections displaying silver-gold interference colors were cut from specimens of
PMSG treated animals and mounted on
formvar coated slot grids. The sections
were stained for 90 minutes by floating
the grids on an aqueous 2.5% uranyl acetate solution in a chamber warmed to
45°C. After a thorough wash, they were
stained for 90-120 seconds on a drop of
lead citrate (Reynolds, '63) under a blanket of freon gas. The specimens were examined in a Siemens Elmiskop 1 or a
Philips 300 electron microscope using a
50 objective aperture.
During the course of this study, particular attention has been directed to enlarged
Graafian follicles. After exogenous gonadotrophin administration to immature rats,
the population of enlarged follicles is increased. No noticeable differences are
observed in nexus fine structure in any of
the untreated or hormone treated groups.
However, the number of nexuses per cell
appears to increase with follicular development which is somewhat more widespread
in ovaries of the hormone treated immature animals than in the adults. Comparatively few follicles with large antra are
present in the 21 day controls.
Granulosa tissue in developing rat follicles has two forms of nexuses which can
be distinguished on the basis of shape
and position in the cytoplasm. One form,
called abutment nexus, is a specialized
region of plasma membranes joining adjacent cells. These junctions often delimit
invaginations which are occupied by neighboring granulosa cell cytoplasm (fig. 1).
The other form, called annular nexus, has
no visible contact with plasma membranes
and appears in the cytoplasm as a ringshaped profile (figs. 2, 12, 13-18).
Both forms of nexuses appear to have
similar dimensions and substructure. In
the glutaraldehyde-osmic acid fixed preparations presented here, the total thickness
of nexuses is about 180-190 A which is
more than twice the distance across nonjunctional plasma membranes. An additional feature, common to both annular
arid abutment nexuses, is the frequent
presence of an electron lucent zone adjacent to their concave surface(s) (figs. 1,
2). This zone probably represents a fixation artifact. Nexuses usually appear fivelayered; i.e., three dense layers each separated by two electron lucent zones.
However, in thin sections the central layer
is sometimes observed to be composed of
two electron dense leaflets. The leaflets,
which are the outer lamellae of apposed
cell membranes, are separated by an irregular 20 A electron lucent “gap” giving
these nexuses a seven-layered appearance
(fig. 1, inset; fig. 2). When an area of
nexus has been cut somewhat obliquely, a
series of uniform short lines with a periodicity of 45-50 A is often found within
the nexus, producing a cross-striated pattern (fig. 3 ) .
Nexuses in granulosa tissue are more
frequently located at cell processes than
elsewhere along the plasma membrane.
These junctions are sometimes observed
along the lateral walls of shafts located
between cells (fig. 4) or at the head of
an invading process (fig. 3). Often the
entire surface of a process appears to
form a nexus with an adjacent cell (fig.
6 ) . If an invading process, girdled by a
nexus, is sectioned transversely an unattached annulus will be seen within the
cytoplasm of the host cell. Larger annuli
are seen if the plane of section passes
through a nexus at the broad base of a
process. If non-junctional plasma membranes are located anywhere at the level
of the cut, then the annulus appears incomplete (fig. 5). Annular nexuses, comrnonly found deep within the cytoplasm,
occasionally envelop another annulus (fig.
7),or intact (fig. 8), or degenerating (fig.
9) organelles. Nexuses belonging to longitudinally sectioned cell processes are observed much less frequently than are
annuli consisting of well defined nexus
membranes (i.e., cut in true cross-section).
When sectioned obliquely, the nexus membranes of processes are indistinct.
Specimens permeated with colloidal
lanthanum hydroxide reveal abundant
tracer in the extracellular space (fig. 10).
When viewed transversely, the “gap” of
abutment nexuses is observed to be filled
with lanthanum; in frontal section, an
array of electron lucent polygonal subunits is outlined by the tracer (fig. 11).
Lanthanum can percolate into the central
region of large annular nexuses (fig. 10)
but is rarely observed within nexuses of
smaller annuli even when situated close
to a cell boundary congested with tracer
(fig. 12).
The diameters of smaller annular nexuses range from about 3,000 A-8,000 A.
Some annuli are segments of cell inclusions which appear completely incorporated in short ribbons of consecutive serial
sections. Non-junctional plasma membranes are not observed to be part of the
spherical inclusion’s surface (figs. 13-18).
The nature of nexus ultrastructure has
been a matter of controversy (Robertson,
’63; Revel and Karnovsky, ’67; Brightman
and Reese, ’69; Chalcroft and Bullivant,
’70; McNutt and Weinstein, ’70). The
outer leaflets of apposed plasma membranes in nexuses usually appear more
distinct if the tissue has been stained en
bloc with aqueous uranyl acetate prior to
dehydration. Nexuses of specimens treated
in this manner often have a seven-layered
appearance since the outer leaflets appear
separated by a fairly regular 20-30 A electron lucent zone or “gap” (Revel and
Karnovsky, ’67; Brightman and Reese, ’69;
McNutt, ’70). In the present investigation
both five- and seven-layered nexuses are
observed although contrast has been enhanced only by a prolonged staining of
the sections with uranyl acetate followed
by lead citrate. The finding of sevenlayered junctions in tissue without e n bloc
staining is supported by the observations
of Matter et al. (’69) and Rosenbluth
(’65), but conflicts with conclusions
reached by McNutt (’70) and Brightman
and Reese (’69). The latter authors suggest that staining of sections (following
en bloc uranyl treatment) obliterates the
median “gap”, converting seven-layered
junctions into five-layered ones. Our study
supports the interpretation that the 20 A
“gap” is not an artifact produced by en
bloc exposure of tissue to heavy metal
The internal appearance of the nexus
is determined in part by the angle at which
the plane of section intersects its membranes, If any part of a nexus is oriented
precisely normal to the plane of section a
seven-layered structure may be seen (fig.
1, inset; fig. 2). A slightly oblique cut
results in increased density within the
“gap” to a level where the outer leaflets
of cell membranes are so superimposed
that the “gap” becomes indistinct. A
greater deviation of nexus membranes
from a n axis perperidicular to the plane
of section results in a striated pattern.
Since a continuity between cells is presumably required for efficient transfer of
ions, the presence of periodic lines across
nexuses may be significant. These striations appear at right angles to the midline
and extend toward the juxtacytoplasmic
boundaries of the nexus (fig. 3). Because
of their length, it seems unlikely that the
lines represent substances strictly confined
to either the “gap” or interiors of the
membranes. Electron opaque structures,
located within both regions and arranged
in register, may form the linear pattern.
This concept is supported by a model of
nexus ultrastructure derived from freezecleave data (McNutt and Weinstein, ’70).
The cross striations we observed in
nexuses resemble in part the periodic lines
found within goldfish Mauthner cell synapses (Robertson, ’ 6 3 ) , which have been
shown to be typical “gap junctions” or
nexuses (Brightman and Reese, ’69).
According to Robertson, frontally sectioned synapses from preparations fixed
or stained with permanganate, exhibit an
array of polygonal subunits each with a
central spot. Oblique views superimpose
the edges of aligned and overlapping subunits to produce a major period of 90 A.
This period is bisected by thin intraperiod
lines produced by superimposed central
spots. Although the distance between the
uniform periodic lines in our study corresponds well to the dimensions reported
by Robertson, absence of major and minor
strata may be explained by differences in
tissue source or method of specimen preparation. The affinity of various subunit
components for different electron stains
probably varies. A uniform periodicity
with similar measurements has been demonstrated in an obliquely sectioned nexus
impregnated with lanthanum hydroxide
(Revel and Karnovsky, ’67). The nexus
subunits have presumably been outlined
by tracer, but whether the impregnated
zones correspond to the electron lucent
or dense bands found in the present study
has not been determined.
Abutment and annular nexuses are probably interrelated. In some cases the two
forms must represent different planes of
section through the same type of nexus
located on a cell evagination. However,
many more annular nexuses are found
than can be accounted for as cross sections through the few nexus-girdled processes seen in longitudinal section. A
reasonable explanation is that another
source of annuli is present-probably intracellular spheres pinched off the processes. Figure 6 illustrates a static view
of what may be the pinching off phenomenon. The term “annular nexus” is used
because it accurately describes any ringshaped nexus profile, observed in a standard thin section, without implying sphere
or process origin.
Spherical inclusions, demonstrated in
the granulosa of the rabbit, have been
designated “spherae occlusae” in belief
that their boundaries are zonulae occludentes (Espey and Stutts, ’72). The junctions in question are not the occluding
type as indicated by our observation of a
central “gap” (figs. 1, 2) which is permeable to lanthanum (figs. 10, 11).
Penetration by lanthanum tracer into
the extracellular space, including the central region of abutment nexuses (fig. l l ) ,
is a useful tool to identify these junctions
(Revel and Karnovsky, ’67). In contrast,
tracer is rarely present in membranes of
the small annuli suggesting their isolation
from the extracellular space (Merk, ’71).
Similarly, Garant (’72) has demonstrated
that lanthanum does not penetrate into
the interior of some circular “gap” junctions found in the enamel organ of the
mouse. These junctions have a morphology strikingly similar to the annular
nexuses described here.
Although the existence of nexus spheres
is implied in single photographs (fig. 12),
the absence of tracer must be considered
negative evidence, particularly in view of
the capricious nature of colloidal lanthanum deposition. Furthermore, absence
of tracer from sphere membranes does not
exclude the possibility that pinching off
is a fixation artifact since both fixative
and lanthanum are applied in the same
solution. However, the presence of degenerating cytoplasmic organelles, sometimes
found within annuli (fig. 9) but not associated with abutment nexuses, suggests
that isolation of spheres from parent cell
processes and organelle degeneration probably occur prior to fixation.
If portions of a cell evagination, housed
by the cytoplasm of an adjacent cell, are
pinched off, they presumably lose contact
with the extracellular space and become
incorporated within the host cell. Positive
evidence for nexus inclusions is provided
by serial sections (figs. 13-18).
During the development of straight abutment nexuses, changes in surface tension
characteristics, cytoskeletal elements, or
other factors may contribute to cell process
formation. When granulosa cells of immature rats are deprived of estrogen (as the
result of hypophysectomy), there is a
considerable reduction in the number of
nexuses, particularly the annular form,
as well as nexus-girdled cell processes. The
population of annular nexuses is markedly
increased in hypophysectomized animals
following treatment with estrogen (Merk,
'71; Merk et al., '72). This finding suggests that the formation of nexus-girdled
processes represents a more advanced
stage in the development of these
The sphere membranes, which no longer
join cells in communication, have ceased
to be nexuses and must be regarded as cell
inclusions. The significance of the pinched
off spheres is not clear. Since it is likely
that new nexuses are continually being
formed in granulosa tissue, sphere formation could represent a means of removing
older junctions from the cell surface. The
ultimate fate of nexus membrane inclusions could not be determined in the present study. Breakdown of nexus spheres
is not observed during the follicular stage
but might be expected after further cell
development. Fragments of pentalaminar
structures, which may be nexus membrane inclusions, have been reported in
granulosa lutein cells of human corpus
luteum (Crisp et al., '70).
The authors are grateful to Dr. N. Scott
McNutt, Massachusetts General Hospital,
for generous aid with the lanthanum tracer
aspect of this study and for his critical
review of the manuscript. We wish to
thank Prof. Frederick L. Hisaw for the
supply of HCG and Dr. Linda P. Merk,
Miss Sheila A. Gill and Miss Deborah J.
Turling for editorial and typing assistance.
The help of Mr. George Duggan and Mr.
Leo Gavin, Harvard Biological Laboratories, is appreciated.
This investigation has been supported
by grants CH-HD-TO1 00217, HE 06214
and HE 05411-09 from the United States
Public Health Service.
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on the human corpus luteum. I. Observations
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the differentiating Graafian follicle of the
mouse. Anat. Rec., 169: 473.
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Electrical transmission at the nexus between
smooth muscle cells. J. Gen. Physiol., 51:
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fikers in castrate and estrogen treated rats.
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Granulosa cells from a gonadotrophin treated immature rat are
joined by two abutment nexuses. This form is continuous with plasma
membranes. One nexus appears to outline a cell protuberance. Arrow
indicates region of a nexus, also seen at high magnification in the
inset, where the midline is observed to be two electron dense leaflets.
This gives the junction a seven-layered appearance ( 4 dense zones
separated by 3 lucent ones). X 116,000; inset: X 232,000.
F. B. Merk, J. T. Albright and C. R. Botticelli
Annular nexus. The dimensions and internal architecture of this
structure closely resemble the abutment form except that there is no
visible contact with non-junctional plasma membranes. The midline
is composed of two electron dense leaflets (arrow). It is not possible
to determine whether this image represents a cross section through
a nexus-girdled cell process, or a sphere cut through the equator.
X 250,000.
Longitudinal view through a granulosa cell process revealing a nexus
at the extremity. A regular series of parallel lines 45-50 A apart, at
right angles to the membrane, is seen within obliquely sectioned
regions of this nexus. Total width of the nexus in obliquely cut areas
appears slightly greater than areas sectioned normal to the membranes. From a gonadotrophin treated animal. X 210,000.
F. B. Merk, J. T. Albright and C. R. Botticelli
Two abutment nexuses are revealed in this longitudinal section
through a process which is located between other granulosa cells.
Nexuses are frequently associated with cell processes. x 50,000.
A n invading cell process, probably sectioned near the base, is seen
in transverse view. The shaft is not completelv surrounded by nexus
membranes in the plane of section illustr&ed. From a mature -animal.
X 55,000.
F. B. Merk, J. T. Albright and C. R. Botticelli
6 An abutment nexus sheathes a longitudinally sectioned cell process.
The upper half of this process may have been separating from the
lower half at fixation. Nexus membranes in oblique view (arrows)
_ _ to isolate a relativelv clear zone within the head from remaining process cytoplasm. From a gonadotrophin treated animal.
X 98,000.
7 An annular nexus within the boundaries of a larger annulus.
Intact mitochondria within an annular nexus.
x 65,000.
x 72,000.
9 Degenerating mitochondrion within an annular nexus. The fact that
organelles inside annuli sometimes appear to lose their morphological
integrity, even in well fixed tissue, supports the concept that isolation
of spheres from parent cell processes takes place prior to fixation.
From a mature animal. x 58,000.
F. B. Merk, J. T. Albright and C. R. Botticelli
10 This specimen is permeated with lanthanum hydroxide (L), a n electron opaque tracer, which is found in the extracellular space. A large
annular nexus, which delimits a transversely sectioned process, is in
contact with the extracellular space as indicated by the presence of
lanthanum (arrows and inset). If sectioned at right angles, this
junction would have been termed a n abutment nexus. x 83,000;
inset: x 172,000.
F. B. Merk, J. T. Albright and C. R. Botticelli
Granulosa cell nexus infiltrated with lanthanum. Region of the junction sectioned transversely (near the X) reveals a congested midline;
whereas en. face views are characterized by polygonal subunits outl i e d by tracer (arrows). x 148,000.
12 The midline of this small annular nexus, located close to a cell border,
is free of lanthanum. The nearby extracellular space is well infiltrated with tracer ( L ) suggesting that membranes of the annulus
have separated from the cell surface. x 138,000.
F. B. Merk, J. T. Albright and C. R. Botticelli
13-18 Consecutive serial sections, (each section approx. 900 A thick)
from an ovary of a PMSG treated rat, reveal a small nexus inclusion which
is about 4,100 A in diameter. A large annular nexus is also present, but it
is not shown to be isolated from the cell surface by this series. x 44,000.
The cytoplasmic matrix adjacent to a mitochondrion (M), which
is used as a reference point, appears devoid of organelles.
14 A “halo” appears in the cytoplasm (arrows) where nexus membranes have been obliquely sectioned.
15-16 Plane of section passes near the sphere’s equator cutting the nexus
membranes almost transversely.
17 Sphere membranes are indistinct.
18 Inclusion is no longer apparent.
F. B. Merk, J. T. Albright and C. R. Botticelli
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structure, granulosus, ovarian, rat, nexuses, fine, follicle, cells
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